Molecular Biology of the Cell, 5th edition

Citation preview

MolecularBiologyof Fifth Edition

MolecularBiologyof Fifth Edition

BruceAlberts Johnson Alexander JulianLewis MartinRaff KeithRoberts PeterWalter

Withproblemsby JohnWilson TimHunt

GarlandScience Group Taylor& Francis

Garland Science Vice President:Denise Schanck Assistant Editor: Sigrid Masson Production Editor and Layout: Emma leffcock Senior Publisher: Jackie Harbor Illustrator: Nigel Orme Designer: Matthew McClements, Blink Studio, Ltc. Editors: Marjorie Anderson and Sherry Granum Copy Editor: Bruce Goatly Indexer: Merrall-Ross International, Ltd. Permissions Coordinator: Marv Disoenza Cell Biology Interactiue Artistic and Scientific Direction: PeterWalter Narrated by: Julie Theriot Production Design and Development: Michael Morales

@ 2008, 2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Rafi Keith Roberts, and PeterWalter. @ f 983, f 989, 1994 by Bruce Alberts, Dennis Bray, Iulian Lewis, Martin Raff, Keith Roberts, and lames D. Watson.

Bruce Alberts received his Ph.D. from Harvard university and is professor of Biochemistry and Biophysics at the university of california, san Francisco.For 12 years,he served as President ofthe u.s. NationalAcademy ofSciences (1993-2005). Alexander Johnson received his Ph.D. from Harvard University and is professor of Microbiology and Immunology and Director of the Biochemistry cell Biology, Genetics, and Developmental Biology Graduate Program at the University of california, San Francisco. Iulian Lewis received his D.Phil. from the University of Oxford and is a Principal Scientist at the London ResearchInstitute of Cancer ResearchUK. Martin Raffreceived his M.D. from McGill University and is at the Medical Research Council Laboratory for Molecular Cell Biology and the Biology Department at University College London. Keith Roberts received his Ph.D. from the University of Cambridge and is Emeritus Fellow at the John Innes Centre, Norwich. peterWalter received his ph.D. from The Rockefeller University in Newyork and is professor and chairman of the Department of Biochemistry and Biophysics at the University of california, san Francisco, and an Investigator of the Howard Hughes Medical Institute.

All rights reserved. No part of this book covered by the copyright heron may be reproduced or used in any format in any form or by any means-graphic, electronic, or mechanical, including photocopying, recording, taping, or information storage and retrieval systems-without permission of the publisher. Library of CongressCataloging-in-Publication Data Molecularbiology of the cell / BruceAlberts ... [et al.].-- 5th ed. p.cm ISBN 978-0-8153-4r05-5 (hardcover)---ISBN978-0-8f5 g-4t06_Z(paperback) L Cytology.2. Molecular biology. I. Alberts, Bruce. QHsB1.2.M642008 571.6--dc22 2007005475CIP Published by Garland science, Taylor & Francis Group, LLC, an informa business, 270 Madison Avenue, NewYork NY f 0016,USA, and 2 park Square,Milton park, Abingdon, OXl4 4RN, UK. Printed in the United States of America 15 14 13 12 lt

10 I

B 7 6 5 4 3 2 |

Preface In many respects,we understand the structure of the universebetter than the workings of living cells.Scientistscan calculatethe age of the Sun and predict when it will ceaseto shine,but we cannot explain how it is that a human being may live for eighty years but a mouse for only two. We know the complete genomesequencesof theseand many other species,but we still cannot predict how a cell will behaveif we mutate a previouslyunstudied gene.Starsmay be l0a3times bigger,but cells are more complex, more intricately structured,and more astonishingproductsof the laws of physicsand chemistry.Through heredity and natural selection,operating from the beginningsof life on Earth to the presentday-that is, for about 20Voof the ageof the universe-living cellshave been progressivelyrefining and extending their molecular machinery and recording the results of their experimentsin the genetic instructions they pass on to their progeny. With each edition of this book, we marvel at the new information that cell biologistshave gatheredin just a few years.But we are even more amazedand daunted at the sophisticationof the mechanismsthat we encounter.The deeper we probe into the cell,the more we reafizehow much remainsto be understood. In the daysof our innocence,working on the first edition, we hailed the identification of a singleprotein-a signalreceptol say-as a greatstep forward' Now we appreciatethat eachprotein is generallypart of a complexwith many others, working togetheras a system,regulatingone another'sactivitiesin subtleways, and held in specificpositionsby binding to scaffoldproteins that givethe chemical factory a definite spatial structure.Genomesequencinghas given us virtually complete molecular parts-listsfor many different organisms;geneticsand biochemistry have told us a great deal about what those parts are capableof individually and which ones interact with which others; but we have only the most primitive grasp of the dynamics of these biochemical systems,with all their interlocking control loops. Therefore,although there are great achievements to report, cell biologistsface evengreaterchallengesfor the future. In this edition, we haveincluded new material on many topics,rangingfrom epigenetics,histonemodifications,small RNAs,and comparativegenomics,to geneticnoise,cytoskeletaldlmamics,cell-cyclecontrol, apoptosis,stem cells, and novel cancer therapies.As in previous editions, we have tried aboveall to give readersa conceptualframework for the mass of information that we now have about cells.This meansgoing beyond the recitation of facts.The goal is to learn how to put the facts to use-to reason,to predict, and to control the behavior of living systems. To help readerson the way to an activeunderstanding,we have for the first time incorporatedend-of-chapterproblems,written by Iohn Wilson and Tim Hunt. Theseemphasizea quantitative approach and the art of reasoningfrom experiments.A companion volume, MolecularBiologyof the CelI,Fifth Edition: by the sameauthors,givescomTheProblemsBook0SBN978-0-8153-4110-9), plete answersto theseproblemsand also containsmore than 1700additional problemsand solutions. A further major adjunct to the main book is the attachedMedia DVD-ROM disc.This provideshundredsof moviesand animations,including manythat are new in this edition, showingcells and cellular processesin action and bringing the text to life; the disc alsonow includesall the figuresand tablesfrom the main

book,pre-loadedinto PowerPoint@ presentations. Otherancillariesavailablefor the book include a bank of test questionsand lectureoutlines,availableto qualified instructors,and a set of 200full-coloroverheadtransparencies. Perhapsthe biggestchange is in the physical structure of the book. In an effort to make the standard Student Edition somewhatmore portable, we are providing chapters 2r-25, covering multicellular systems,in electronic (pDF) form on the accompanyingdisc,while retaining in the printed volume chapters l-20, covering the core of the usual cell biology curriculum. But we should emphasizethat the final chaptershavebeen revisedand updated as thoroughly as the rest of the book and we sincerelyhope that they will be read!A Reference Edition (ISBN97s-0-8153-4r11-6), containingthe full set of chaptersasprinred pages,is also availablefor thosewho prefer it. Full details of the conventionsadopted in the book are given in the Note to the Readerthat follows this Preface.As explainedthere,we have taken a drastic approachin confronting the different rules for the writing of genenamesin different species:throughout this book, we use the same style, regardlessof species,and often in defianceofthe usualspecies-specific conventions. As always,we are indebted to many people. Full acknowledgmentsfor scientific help are given separately,but we must here singleout someexceptionally important contributions: Iulie Theriot is almost entirely responsiblefor chapters 16 (cytoskeleton)and 24 (Pathogens,Infection, and Innate Immunity), and David Morgan likewisefor chapter 17 (cell cycle).wallace Marshall and Laura Attardi provided substantialhelp with chapters 8 and 20, respectively,as did Maynardolson for the genomicssectionof chapter4, Xiaodongwangfor chapter 18,and NicholasHarberdfor the plant sectionof Chapter15. we also owe a huge debt to the staff of Garland science and others who helped convert writers' efforts into a polished final product. Denise schanck directed the whole enterpriseand shepherdedthe wayward authors along the road with wisdom, skill, and kindness.Nigel orme put the artwork into its final form and supervisedthe visualaspectsof the book,including the backcover,with his usual flair. Matthew Mcclements designedthe book and its front cover. Emma Jeffcocklaid out its pageswith extraordinaryspeedand unflappableefficiency,dealingimpeccablywith innumerablecorrections.MichaelMoralesmanagedthe transformationof a massof animations,video clips, and other materials into a user-friendly DVD-ROM. Eleanor Lawrence and sherry Granum updatedand enlargedthe glossary.JackieHarbor and SigridMassonkept us organized.Adam Sendroffkeptus awareofour readersand their needsand reactions. MarjorieAnderson,BruceGoatly,and sherry Granumcombedthe text for obscurities, infelicities, and errors.we thank them all, not only for their professional skill and dedication and for efficiencyfar surpassingour own, but also for their unfailing helpftrlnessand friendship:they havemadeit a pleasureto work on the book. Lastly,and with no less gratitude, we thank our spouses,families, friends and colleagues. without their patient,enduringsupport,we could not haveproducedany of the editionsof this book.

Contents Speci.al Features Detailed Contents Acknowledgments A Note to the Reader

PARTI

uiii ix xxui xxxi

I. 2. 3.

TOTHECELL INTRODUCTION Cellsand Genomes CellChemistryand Biosynthesis Proteins

I 45 t25

PARTII 4. 5. 6. 7.

MECHANISMS BASICGENETIC DNA, Chromosomes,and Genomes DNA Replication,Repair,and Recombination How CellsReadthe Genome:From DNA to Protein Controlof GeneExpression

195 263 329 41I

PARTIII B. 9.

METHODS Manipulating Proteins,DNA, and RNA VisualizingCells

50r

PARTIV 10. 11.

INTERNAL ORGANIZATION OFTHECELL Membrane Structure MembraneTransportof SmallMoleculesand the Electrical Propertiesof Membranes Intracellular Compartmentsand Protein Sorting IntracellularVesicularTraffic EnergyConversion:Mitochondriaand Chloroplasts Mechanismsof CellCommunication The Cytoskeleton The Cell Cycle Apoptosis

12. 13. 14. 15. 16. t7. tB. PARTV 19. 20. 2I, 22. 23, 24. 25. Glossary Index TabIes

CONTEXT CELLS INTHEIRSOCIAL Cell lunctions, Cell Adhesion,and the ExtracellularMatrix Cancer Chapters2I-25 availableon Media DVD-ROM SexualReproduction:Meiosis,Germ Cells,and Fertilization Developmentof MulticellularOrganisms Tissues,StemCells,and TissueRenewal Specialized Pathogens,Infection, and Innate Immunity The Adaptive Immune System

The Genetic Code,Amino Acids

579 6t7 651 695 749 Br3 879 965 1053 1115 1131 I205 1269 r305 L4l7 1485 1539 G-1 L1 T-1

SpecialFeatures Table l-l Table l-2 Table 2-1 Table2-2 Table 2-3 Table2-4 Panel 2-l Panel2-2 Panel 2-3 Panel2-4 Panel 2-5 Panel 2-6 Panel2-7 Panel 2-B Panel 2-9 Panel 3-l Panel 3-2 Table 3-1 Panel 3-3 Table 4-l Table 5-3 Table 6-l Panel B-l Table 10-l Thble 11-l Panel 1l-2 Panel ll-3 Table l2-l Table l2-2 Table l4-l Panel l4-l Thble r5-5 Panel 16-2 Panel 16-3 Table I7-2 Panel l7-l

SomeGenomesThat HaveBeenCompletelySequenced p. 18 The Numbersof GeneFamilies,classifiedby Function,That Are common to All ThreeDomainsof the LivingWorld p.24 Covalentand NoncovalentChemicalBonds p. 53 TheTypesof MoleculesThat Form a BacterialCell p.55 ApproximateChemicalCompositionsof a TypicalBacteriumand a Typical MammalianCell p.63 RelationshipBetweenthe StandardFree-Energy Change,AG, and the Equilibrium Constant p.77 ChemicalBondsand GroupsCommonlyEncounteredin BiologicalMolecules pp. 106-107 Waterand Its Influenceon the Behaviorof BiologicalMolecules pp. 108-109 The PrincipalTypesof weak NoncovalentBondsthat Hold Macromolecules Together pp. r 1 0 - 1 1 1 An Outline of Someof the Typesof SugarsCommonlyFoundin Cells pp. 1 1 2 - 1 1 3 FattyAcidsand Other Lipids pp. I l4-1 I5 A Surveyof the Nucleotides pp. I 16-1r7 FreeEnergyand BiologicalReactions pp. I IB-t 19 Detailsof the t0 Stepsof Glycolysis pp. r20-I2l The CompleteCitric Acid Cycle pp. I22-t23 The 20 Amino AcidsFoundin Proteins pp.tzg-729 Four DifferentWaysof Depictinga SmallProtein,the SH2Domain pp. 132-133 SomeCommonTypesof Enzymes p.159 Someof the MethodsUsedto StudyEnzymes pp. 162-163 SomeVitalStatisticsfor the Human Genome p.206 ThreeMajor Classesof Transposable Elements p.318 PrincipalTlpes of RNAsProducedin Cells p.336 Reviewof ClassicalGenetics pp. 554-555 ApproximateLipid Compositionsof DifferentCellMembranes p.624 A Comparisonof Ion ConcentrationsInsideand Outsidea TypicalMammalianCell p . 6 5 2 The Derivationof the NernstEquation p.670 SomeClassicalExperimentson the SquidGiantAxon p. 679 RelativeVolumes Occupiedby the Major IntracellularCompartmentsin a Liver Cell (Hepatocyre) p. 697 RelativeAmounts of MembraneTypesin Two Kinds of Eucaryoticcells p.697 ProductYieldsfrom the Oxidationof Sugarsand Fats p.824 RedoxPotentials p.830 The RasSuperfamilyof MonomericGTpases p.926 The Polymerizationof Actin and Tubulin pp. 978-979 AccessoryProteinsthat Controlthe Assemblyand positionof Cvtoskeletal Filaments pp. 994-995 Summaryof the Major Cell-CycleRegulatoryproteins p. 1066 The Princinle

Stases of M Phasp (Mitnsis nnrl Crrfnlrinpcic\ in qn Animal

/-oll

nn

rATo rA?a

DetailedContents Chapter 1 Cells and Genomes

1

THEUNIVERSAL FEATURES OF CELLSON EARTH

1

Information in the SameLinear AllCellsStoreTheirHereditary Chemical Code(DNA) byTemplated Hereditary Information AllCellsReplicateTheir Polymerization Portionsof TheirHereditary Informationinto All CellsTranscribe Form(RNA) the SameIntermediary All CellsUseProteinsasCatalysts RNAinto Proteinin the SameWay All CellsTranslate to One TheFragmentof GeneticInformationCorresponding ProteinlsOneGene LifeRequires FreeEnergy with the Factories Dealing AllCellsFunction asBiochemical BuildingBlocks SameBasicMolecular Across Which in a Plasma Membrane AllCellsAreEnclosed MustPass NutrientsandWasteMaterials A LivingCellCanExistwith FewerThan500Genes Summary

5

4 5 o

7 8 8 9 10 11

OF GENOMES AND THETREEOF LIFE THEDIVERSITY CellsCanBePoweredby a Varietyof FreeEnergySources SomeCellsFixNitrogenand CarbonDioxidefor Others Cells TheGreatest Biochemical DiversityExistsAmongProcaryotic Archaea, Bacteria, TheTreeof LifeHasThreePrimaryBranches: and Eucaryotes OthersAreHighlyConserved SomeGenesEvolveRapidly; Genes and ArchaeaHave1000-6000 MostBacteria from Preexisting Genes NewGenesAreGenerated of RelatedGenesWithin GiveRiseto Families GeneDuplications a SingleCell Bothin the BetweenOrganisms, GenesCanBeTransferred and in Nature Laboratory of GeneticInformation in HorizontalExchanges SexResults Withina Species TheFunctionof a GeneCanOftenBeDeducedfrom lts Sequence AreCommonto AllThreePrimary MoreThan200GeneFamilies Branches of the Treeof Life the Functions of Genes MutationsReveal HaveFocused a Spotlighton E coli MolecularBiologists Summary

11

IN EUCARYOTES GENETICINFORMATION CellsMayHaveOriginatedasPredators Eucaryotic CellsEvolvedfrom a Symbiosis ModernEucaryotic HaveHybridGenomes Eucaryotes Eucaryotic GenomesAreBig DNA GenomesAreRichin Regulatory Eucaryotic Development TheGenomeDefinesthe Programof Multicellular LiveasSolitaryCells:the Protists ManyEucaryotes A YeastServesasa MinimalModelEucaryote Levelsof AllTheGenesof An OrganismCanBe TheExpression MonitoredSimultaneously and Computers, To MakeSenseof Cells,We NeedMathematics, Information Quantitative Asa Model HasBeenChosenOut of 300,000Species Arabidopsis

26

Plant

12 13 14 t5 to

17 18 19 21

22 22

23 z)

24 26

26 30 30 31 31 5Z

33 34 35 JO

Bya Worm,a Fly, TheWorldof AnimalCellsls Represented anda Human a Mouse, Development Providea Keyto Vertebrate Studiesin Drosophila Duplication Genomels a Productof Repeated TheVertebrate Butlt Creates ls a Problemfor Geneticists, GeneticRedundancy for EvolvingOrganisms Opportunities asa Modelfor Mammals TheMouseServes Reporton TheirOwnPeculiarities Humans WeAreAll Differentin Detail Summory Problems References

Chapter2 CellChemistryand Biosynthesis OFA CELL COMPONENTS THECHEMICAL

36 37 38 39 5>

40 41 42 42 44

45 45

of Atoms CellsAreMadeFroma FewTypes DetermineHow Atomslnteract TheOutermostElectrons CovalentBondsFormby the Sharingof Electrons ThereAreDifferentTypesof CovalentBonds asif lt Hasa FixedRadius An AtomOftenBehaves in Cells Waterlsthe MostAbundantSubstance AreAcidsand Bases SomePolarMolecules AttractionsHelpBringMolecules of Noncovalent FourTypes Togetherin Cells A Cellls Formedfrom CarbonCompounds Molecules of SmallOrganic CellsContainFourMajorFamilies SugarsProvidean EnergySourcefor CellsandArethe Subunits of Polysaccharides asWellasa of CellMembranes, FattyAcidsAreComponents Sourceof EnergY AminoAcidsArethe Subunitsof Proteins of DNAandRNA Arethe Subunits Nucleotides with of Cellsls Dominatedby Macromolecules TheChemistry Properties Remarkable Shapeof a BondsSpecifyBoththe Precise Noncovalent anditsBindingto OtherMolecules Macromolecule Summary

45 46 48 50 51 51 52

AND THE USEOF ENERGYBY CELLS CATALYSIS by Enzymes CellMetabolismls Organized of HeatEnergy by the Release Orderls MadePossible Biological from Cells Organic UseSunlightto Synthesize Organisms Photosynthetic Molecules CellsObtainEnergyby the Oxidationof OrganicMolecules Transfers Oxidationand ReductionInvolveElectron ThatBlockChemicalReactions Lowerthe Barriers Enzymes Rapidityof TheEnormous FindTheirSubstrates: HowEnzymes MolecularMotions Whetherlt Changefor a ReactionDetermines TheFree-Energy CanOccur the Free-Energy Influences of Reactants TheConcentration Direction Changeand a Reaction's AG"ValuesAreAdditive Reactions, ForSequential for Biosynthesis AreEssential ActivatedCarrierMolecules of an ActivatedCarrierlsCoupledto an TheFormation Reaction Favorable Eneroeticallv

65

53 54 55 55 58 59 61 oz 63 65

66 66 68 70 71 72 74 75 76 77 78

ATPlsthe MostWidelyUsedActivatedCarrierMolecule EnergyStoredin ATPlsOftenHarnessed to JoinTwoMolecules Together NADHand NADPHAre lmportantElectronCarriers ThereAreManyOtherActivatedCarrierMolecules in Cells TheSynthesis of Biological Polymers ls Drivenby ATpHydrolysis Summary

80 81 82 83 84 87

HOW CELLSOBTAINENERGY FROMFOOD 88 pathway Glycolysis ls a CentralATP-producing 88 Fermentations ProduceATPin the Absenceof Oxygen Rq Glycolysis lllustrates How Enzymes CoupleOxidationto Energy Storage 91 Organisms StoreFoodMolecules in SpecialReservoirs 91 MostAnimalCellsDeriveTheirEnergyfrom FattyAcidsBetween Meals 95 Sugarsand FatsAreBothDegradedto AcetylCoAin Mitochondria vo TheCitricAcidCycleGenerates NADHby OxidizingAcetylGroups to CO2 o7 Electron TransportDrivesthe Synthesis of the Majorityof the ATp in MostCells 100 AminoAcidsand Nucleotides Are partof the NitrogenCycle 100 Metabolismls Organized and Regulated 101 Summary 103 Problems 103 References 124

Chapter3 Proteins THESHAPE ANDSTRUCTURE OFPROTEINS TheShapeof a Proteinls Specified by lts AminoAcidSequence ProteinsFoldinto a Conformation of LowestEnergy ThecrHelixand the B SheetAreCommonFoldingpatterns ProteinDomainsAreModularUnitsfrom whichLargerproteins AreBuilt Fewof the ManyPossible Polypeptide ChainsWillBeUsefur to Cells Proteins CanBeClassified intoManyFamilies Sequence Searches CanldentifyCloseRelatives SomeProteinDomainsFormpartsof ManyDifferentproteins CertainPairsof DomainsAreFoundTogetherin Manyproteins TheHumanGenomeEncodes a ComplexSetof proteins, Revealing MuchThatRemains Unknown LargerProteinMolecules OftenContainMoreThanOne Polypeptide Chain SomeProteinsFormLongHelicalFilaments ManyProteinMolecules HaveElongated, FibrousShapes ManyProteins Containa Surprisingly LargeAmountof Unstructured Polypeptide Chain proteins CovalentCross-Linkages OftenStabilize Extracellular ProteinMolecules OftenServeasSubunitsfor the Assembry of LargeStructures ManyStructures in CellsAreCapableof Self-Assembly AssemblyFactors OftenAidthe Formationof Comolex Biological Structures Summary

125 125 tzJ

130 131 tJ)

I Jt)

137 139 140 141 142 "t42 143 t4)

"146 147 148 149 151 't52

PROTEINFUNCTION 152 All Proteins Bindto OtherMolecules 153 TheSurfaceConformation of a ProteinDetermines lts Chemistrv 154 Sequence Comparisons BetweenproteinFamilyMembers Highlight Crucial Ligand-Binding Sites 155 ProteinsBindto OtherProteins ThroughSeveral Typesof Interfaces tf,o AntibodyBindingSitesAreEspecially Versatile 156 TheEquilibrium Constant Measures BindingStrength 157 Enzymes ArePowerfuland HighlySpecific Catalysts 158 Substrate Bindingls the FirstStepin EnzymeCatalysis i59 Enzymes SpeedReactions by Selectively Stabilizing Transitron States 160 Enzymes CanUseSimultaneous AcidandBaseCatalysis 160 Lysozyme lllustrates Howan EnzymeWorks 16"1 TightlyBoundSmallMolecules Add ExtraFunctions to prorerns 166

MolecularTunnels Channel Substrates in Enzymes with '167 MultipleCatalytic Sites Multienzyme Complexes Helpto Increase the Rateof Cell Metabolism 168 TheCellRegulates the Catalytic Activitiesof its Enzymes 169 AllostericEnzymes HaveTwoor MoreBindingSitesThatInteract 1 7 1 TwoLigandsWhoseBindingSitesAreCoupledMust Reciprocally AffectEachOther'sBinding 171 SymmetricProteinAssemblies ProduceCooperative Allosteflc Transitions 172 TheAllosteric Transition in Aspartate Transcarbamoylase ls Understood in AtomicDetail 173 ManyChangesin Proteins Are Drivenby Protein Phosphorylation 175 A Eucaryotic CellContainsa LargeCollectionof ProteinKinases and ProteinPhosphatases 176 TheRegulation of Cdkand SrcProteinKinases ShowsHowa ProteinCanFunctionasa Microchip 177 Proteins ThatBindand Hydrolyze GTPAreUbiquitousCellular Regulators 178 proteins Regulatory Proteins Controlthe Activityof GTP-Binding by Determining WhetherGTPor GDPls Bound 179 LargeProteinMovements CanBeGenerated FromSmallOnes 179 MotorProteinsProduceLargeMovementsin Cells 181 Membrane-Bound Transporters Harness Energyto Pump Molecules ThroughMembranes 182 ProteinsOftenFormLargeComplexes ThatFunctionasProtein Machines 184 ProteinMachines with Interchangeable PartsMakeEfficientUse of Geneticlnformation 184 TheActivationof ProteinMachines OftenInvolvesPositioning Themat SpecificSites 185 ManyProteinsAreControlledby MultisiteCovalentModification t 6 0 A ComplexNetworkof ProteinInteractions Underlies CellFunction 187 Summary 190 Problems 191 References 193

Chapter4 DNA,Chromosomes, and Genomes

195

THESTRUCTURE ANDFUNCTION OFDNA

197

A DNAMoleculeConsists of TwoComplementary Chains of Nucleotides TheStructureof DNAProvides a Mechanism for Heredity In Eucaryotes, DNAls Enclosed in a CellNucleus Summory

197 199 200 201

CHROMOSOMALDNA AND ITSPACKAGING IN THE CHROMATIN FIBER

202 Eucaryotic DNAls Packaged into a Setof Chromosomes 202 Chromosomes ContainLongStringsof Genes 204 TheNucleotide Sequence ofthe HumanGenomeShowsHow OurGenesAreArranged 205 GenomeComparisons RevealEvolutionarily Conserved DNA )equences 207 Chromosomes Existin DifferentStatesThroughout the Life ofa Cell 2OB EachDNAMoleculeThatFormsa LinearChromosome Must Containa Centromere, TwoTelomeres, and Replication Origins 2Og DNAMolecules Are HighlyCondensed in Chromosomes 210 Nucleosomes Area BasicUnitof Eucaryotic Chromosome Structure 211 TheStructureofthe Nucleosome CoreParticleReveals How DNAls Packaged Z'tZ Nucleosomes Havea DynamicStructure, and AreFrequentry Subjected to ChangesCatalyzed by ATp-Dependent ChromatinRemodeling Complexes 215 Nucleosomes AreUsuallyPacked Togetherinto a Compact Chromatin Fiber lto Summary 218

THEREGULATION OFCHROMATIN STRUCTURE SomeEarlyMysteries Concerning Chromatin Structure

219 220

Resistant Heterochromatin ls HighlyOrganized andUnusually 220 to GeneExoression Modifiedat ManyDifferentSites TheCoreHistonesAreCovalently ChromatinAcquiresAdditionalVarietythroughthe Site-Specific Variants lnsertion of a SmallSetof Histone andthe HistoneVariantsAct in TheCovalentModifications Concertto Producea "HistoneCode"ThatHelpsto Function Determine Biological andCode-Writer Proteins CanSpread A Comolexof Code-Reader Alonga for LongDistances Specific ChromatinModifications Chromosome Complexes Blockthe Spreadof Reader-Writer BarrierDNASequences Domains Separate Neighboring Chromatin andThereby How HistoneVariants Reveals TheChromatinin Centromeres zt6 CanCreateSpecialStructures 230 CanBeDirectlyInherited ChromatinStructures to Eucaryotic Add UniqueFeatures ChromatinStructures 231 Function Chromosome 233 Summary THEGLOBALSTRUCTURE OF CHROMOSOMES

233

AreFoldedinto LargeLoopsof Chromatin Chromosomes Chromosomes AreUniquelyUsefulfor Visualizing Polytene ChromatinStructures ThereAreMultipleFormsof Heterochromatin Whenthe GenesWithinThemAre ChromatinLoopsDecondense Exoressed ChromatinCanMoveto SpecificSitesWithinthe Nucleusto AlterTheirGeneExoression Forma Setof DistinctBiochemical Networksof Macromolecules insidethe Nucleus Environments AreFormedfrom Chromatinin lts Most MitoticChromosomes State Condensed Summary

234

EVOLVE HOW GENOMES

245

of the Norma AreCausedby Failures GenomeAlterations DNA for CopyingandMaintaining Mechanisms Differin Proportionto of TwoSpecies TheGenomeSequences Evolved the LengthofTimeThatTheyHaveSeparately of DNA from a Comparison TreesConstructed Phylogenetic of All Organisms Tracethe Relationships Sequences Shows of HumanandMouseChromosomes A Comoarison Howthe Structures of GenomesDiverge Ratesof GenomeReflects the Relative TheSizeof a Vertebrate DNAAdditionand DNALossin a Lineage the Sequence of SomeAncientGenomes WeCanReconstruct ldentifylmportantDNA Comparisons Multispecies Sequence Sequences of UnknownFunction Sequences Can Conserved Changesin Previously Accelerated HelpDecipher Critical Stepsin HumanEvolution an lmportantSourceof Genetic GeneDuplicationProvides NoveltyDuringEvolution GenesDiverge Duplicated TheEvolutionof the GlobinGeneFamilyShowsHow DNA of Organisms Contribute to the Evolution Duplications CanBeCreatedby the GenesEncodingNewProteins Recombination of Exons NeutralMutationsOftenSpreadto BecomeFixedin a Population, that Dependson PopulationSize with a Probability oftheVariation A GreatDealCanBeLearnedfrom Analyses AmongHumans Summary Problems References

Chapter5 DNA Replication,Repair,and Recombination OFDNASEQUENCES THEMAINTENANCE Low MutationRatesAre Extremely for LifeasWe Knowlt LowMutationRatesAreNecessary Summory

236 256

239 239 241 243 245

246 247 248 249 251 251 2s2 253 z>5 254

257 257 258 260 lou

262

263 263 205

265 265

266 MECHANISMS DNA REPLICATION too andDNARepair DNAReplication Underlies Base-Pairing 266 Forkls Asymmetrical TheDNAReplication Proofreading Several Requires TheHighFidelityof DNAReplication Mechanisms AllowsEfficientError in the 5'-to-3'Direction OnlyDNAReplication 27"1 Correction ShortRNA EnzymeSynthesizes A 5pecialNucleotide-Polymerizing 272 on the LaggingStrand PrimerMolecules Helpto OpenUpthe DNADoubleHelixin Front Proteins Special 273 Fork ofthe Replication 273 ontothe DNA A SlidingRingHoldsa MovingDNAPolymerase to Forma Replication ForkCooperate at a Replication TheProteins 275 Machine Replication MismatchRepairSystemRemoves A Strand-Directed 276 Machine from the Replication ErrorsThatEscape PreventDNATanglingDuringReplication z I d DNATopoisomerases and in Eucaryotes Similar ls Fundamentally DNAReplication 280 Bacteria 281 Summary OF DNA REPLICATION AND COMPLETION THEINITIATION 281 IN CHROMOSOMES 281 Origins Replication at Begins DNASynthesis TypicallyHavea SingleOriginof DNA Chromosomes Bacterial 26l Reolication ContainMultipleOriginsof Replication 282 Chromosomes Eucaryotic TakesPlaceDuringOnlyOnePart DNAReplication In Eucaryotes 284 of the cell cycle at Distinct Replicate on the SameChromosome DifferentRegions 285 Timesin S Phase Late,WhileGenesin ChromatinReplicates HighlyCondensed 285 Early Tendto Replicate Chromatin LessCondensed Originsin a ServeasReplication DNASequences Well-Defined 260 the BuddingYeast SimpleEucaryote, Originsof A LargeMultisubunitComplexBindsto Eucaryotic 287 Reolication ThatSpecifythe Initiationof TheMammalianDNASequences 288 HaveBeenDifficultto ldentify Replication 289 Fork Behindthe Replication AreAssembled NewNucleosomes DuplicationEnsure Chromosome of Eucaryotic TheMechanisms 290 CanBeInheriteo of HistoneModification ThatPatterns 292 the Endsof Chromosomes Replicates Telomerase zY5 by CellsandOrganisms Lengthls Regulated Telomere 294 Summary DNA REPAIR DNADamageWouldRapidly Spontaneous WithoutDNARepair, ChangeDNASequences TheDNADoubleHelixls ReadilyRepaired DNADamageCanBeRemovedby MoreThanOne Pathway Thatthe Cell'sMost Ensures CouplingDNARepairto Transcription Repaired lmportantDNAls Efficiently DamageDetection of the DNABasesFacilitates TheChemistry to RepairDNA AreUsedin Emergencies SpecialDNAPolymerases Repaired Are Efficiently Breaks Double-Strand of the CellCycle DNADamageDelaysProgression Summary

295

RECOMBINATION HOMOLOGOUS HasManyUsesin the Cell Recombination Homologous in All Cells HasCommonFeatures Recombination Homologous Recombination GuidesHomologous DNABase-Pairing TheRecAProteinand its HomologsEnablea DNA5ingleStrand Regionof DNADoubleHelix to Pairwith a Homologous or Regions BranchMigrationCanEitherEnlargeHetroduplex DNAasa SingleStrand NewlySynthesized Release DoubleRepair CanFlawlessly Recombination Homologous in DNA Breaks Stranded Recombination the Useof Homologous CellsCarefullyRegulate in DNARepair HollidayJunctionsAreOftenFormedDuringHomologous Events Recombination

304

296 296 297 299 300 302 302 303 304

304 305 305 307 308 308 310 311

MeioticRecombination Beginswith a programmed DoubleStrandBreak Homologous Recombination OftenResults in GeneConversron promiscuous MismatchProofreading Prevents Recombinatron BetweenTwoPoorlyMatchedDNASequences Summary TRANSPOSITION AND CONSERVATIVE SITE-SPECIFIC RECOMBINATION

312 314 315 5to

316

ThroughTransposition, MobileGeneticElements CanInsertlnto AnyDNASequence y7 DNA-OnlyTransposons Moveby BothCut-and-paste and Replicative Mechanisms 317 SomeVirusesUsea Transposition Mechanism to MoveThemselves intoHostCellChromosomes 319 Retroviral-like Retrotransposons Resemble Retroviruses, but Lacka ProteinCoat 320 A LargeFractionof the HumanGenomels Comoosedof Nonretroviral Retrotransposons 32,l predominate DifferentTransposable Elements in Different Organisms 322 GenomeSequences Reveal the Approximate Timesthat Transposable Elements HaveMoved 323 Conservative Site-Specific Recombination CanReversibly Rearrange DNA 323 Conservative Site-Specific Recombination WasDiscovered in Bacteriophage ), n+ Conservative Site-Specific Recombination CanBeUsedto Turn GenesOn or Off 324 Summary 326 Problems 327 References 328

Chapter6 How CellsReadthe Genome:From DNAto Protein

329

FROM DNATO RNA

331

Portionsof DNASequence AreTranscribed into RNA 552 Transcription Produces RNAComplementary to OneStrandof DNA 5 5 5 CellsProduceSeveral Typesof RNA 335 SignalsEncodedin DNATellRNApolymerase Whereto Startand Stop 336 Transcription Startand StopSignalsAreHeterogeneous in NucleotideSequence 338 Transcription Initiationin Eucaryotes Requires Manyproteins 339 RNAPolymerase ll Requires GeneralTranscription Factors 340 Polymerase ll AlsoRequires Activator, Mediator, andChromatinModifyingProteins 342 Transcription Elongation Produces Superhelical Tension in DNA 343 Transcription Elongationin Eucaryotes lsTightlyCoupledto RNA Processing 345 pre-mRNAs 346 RNACappinglsthe FirstModification of Eucaryotic RNASplicingRemoves IntronSequences from NewlyTranscribed Pre-mRNAs 347 Nucleotide Sequences SignalWhereSplicing Occurs 349 RNASplicingls Performedby the Spliceosome 349 TheSpliceosome UsesATPHydrolysis to producea ComplexSeries of RNA-RNA Rearrangements 351 OtherProperties of Pre-mRNA and lts Synthesis Helpto Explain the Choiceof ProperSpliceSites 352 A Second5et of snRNPs Splicea SmallFractionof IntronSequences in Animals andPlants 353 plasticity RNASplicingShowsRemarkable J55 Spliceosome-Catalyzed RNASplicingprobablyEvolvedfrom Self-Splicing Mechanisms 355 RNA-Processing Enzymes Generate the 3, Endof Eucaryotic mRNAs5 t / MatureEucaryotic mRNAsAreSelectively Exportedfrom tne Nucleus 358 ManyNoncodingRNAsAreAlsoSynthesized and processed in the Nucleus 360 TheNucleolus ls a Ribosome-producing Factory 502 TheNucleusContainsa Varietyof Subnuclear Structures 50J Summarv 366

FROMRNATO PROTEIN

366

An mRNASequence ls Decodedin SetsofThreeNucleotide IRNAMolecules MatchAminoAcidsto Codonsin mRNA tRNAsAreCovalently ModifiedBeforeTheyExitfrom the Nucleus SpecificEnzymes CoupleEachAminoAcidto ltsAppropriateIRNA Molecule Editingby RNASynthetases Ensures Accuracy AminoAcidsAreAddedto the C-terminal Endof a Growing Polypeptide Chain TheRNAMessage ls Decodedin Ribosomes ElongationFactorsDriveTranslation Forwardand lmprovelts Accuracy TheRibosome ls a Ribozyme NucleotideSequences in mRNASignalWhereto StartProtein Synthesis StopCodonsMarktheEndofTranslation Proteins AreMadeon Polyribosomes ThereAreMinorVariations in the StandardGeneticCode Inhibitorsof Procaryotic ProteinSynthesis AreUsefulas Antibiotics Accuracy in Translation Requires the Expenditure of FreeEnergy Actto Prevent QualityControlMechanisms Translation of Damaqed mRNAs SomeProteinsBeginto FoldWhileStillBeingSynthesized MolecularChaperones HelpGuidethe Foldingof Mostproteins ExposedHydrophobic RegionsProvideCritical5ignalsfor protein QualityControl TheProteasome lsa Compartmentalized Protease with Sequestered ActiveSites An Elaborate Ubiquitin-Conjugating SystemMarksProteins for Destruction ManyProteins AreControlledby Regulated Destruction AbnormallyFoldedProteins CanAggregateto CauseDestructive HumanDiseases ThereAreManyStepsFromDNAto Protein Summory

367 368 369

THERNAWORLDAND THEORIGINS OF LIFE LifeRequires StoredInformation Polynucleotides CanBothStoreInformationand Catalyze ChemicalReactions A Pre-RNA WorldMayPredatethe RNAWorld Single-Stranded RNAMolecules CanFoldintoHighlyElaborate Structures Self-Replicating Molecules UndergoNaturalSelection How Did ProteinSynthesis Evolve? All Present-Day CellsUseDNAasTheirHereditary Material Summary Problems References

Chapter7 Controlof GeneExpression AN OVERVIEW OF GENECONTROL TheDifferentCellTypesof a Multicellular OrganismContainthe SameDNA DifferentCellTypesSynthesize DifferentSetsof proteins ExternalSignalsCanCausea Cellto Changethe Expression of ItsGenes GeneExpression CanBeRegulated at Manyofthe Stepsin the Pathway from DNAto RNAto Protein Summary

370 371 5t5

373 377 379 379 381 391 392 383 385 385 387 388 390 391 393 39s 396 3gg 3gg 4OO 401 401 402 403 404 407 408 408 409 410

411 4'11 411 412 413 415 415

DNA-BINDING MOTIFS INGENE REGULATORY PROTEINS 416 GeneRegulatory Proteins WereDiscovered UsingBacterial Genetics TheOutsideof the DNAHelixCanBeReadby proteins ShortDNASequences Are Fundamental Components of Genetic Switches GeneRegulatory Proteins ContainStructuralMotifsThatCan ReadDNASeouences TheHelix-Turn-Helix Motif lsOneof the Simplestand Most CommonDNA-B|nding Motifs

416 416 418 418 419

Proteins Constitutea SpecialClassof Helix-TurnHomeodomain 420 HelixProteins 421 of DNA-B|nding ZincFingerMotifs ThereAreSeveralTypes p sheetsCanAlsoRecognize 422 DNA SomeProteinsUseLoopsThatEnterthe Majorand MinorGroove 423 to Recognize DNA TheLeucineZipperMotifMediatesBothDNABindingand Protein 423 Dimerization That Expands the Repertoire of DNASequences Heterodimerization 424 Proteins CanRecognize GeneRegulatory and DNA MotifAlsoMediatesDimerization TheHelix-Looo-Helix 425 Binding Recognized to Predictthe DNASequences It ls NotYetPossible 426 Proteins by All GeneRegulatory ShiftAssayReadilyDetectsSequence-Specific A Gel-Mobility 427 Proteins DNA-Binding of Facilitates the Purification DNAAffinityChromatography 428 Proteins DNA-Binding Sequence-Specific Protein Recognized by a GeneRegulatory TheDNASequence 429 CanBeDeterminedExperimentally Sequences FootprintingldentifiesDNARegulatory Phylogenetic 431 Genomics ThroughComparative ldentifiesManyof the Sites Chromatinlmmunoprecipitation 431 Proteins Occupyin LivingCells ThatGeneRegulatory 432 Summary 432 WORK HOW GENETICSWITCHES Genes That Turns Repressor ls a Simple Switch TheTryptophan 433 On and Off in Bacteria 435 Activators TurnGenesOn Transcriptional Repressor Activatorand a Transcriptional A Transcriptional 435 Controlthe LocOperon 437 GeneRegulation DNALoopingOccursDuringBacterial to Help RNAPolymerase Subunits Bacteria UseInterchangeable 438 GeneTranscription Regulate ComplexSwitchesHaveEvolvedto ControlGeneTranscription 439 in Eucaryotes of a PromoterPlus GeneControlRegionConsists A Eucaryotic 440 DNASequences Regulatory of RNA GeneActivatorProteinsPromotethe Assembly Eucaryotic at the Factors Polymerase and the GeneralTranscription 441 Startpointof Transcription AlsoModifyLocalChromatin GeneActivatorProteins Eucaryotic 442 Structure 444 WorkSynergistically GeneActivatorProteins ProteinsCanInhibitTranscription GeneRepressor Eucaryotic 445 in VariousWays ProteinsOftenBindDNA GeneRegulatory Eucaryotic 445 Cooperatively Development ThatRegulate Drosophila ComplexGeneticSwitches 447 Modules AreBuiltUp fromSmaller Controls 448 by Combinatorial EveGenels Regulated fhe Drosophila AreAlsoConstructed GeneControlRegions ComplexMammalian 450 Modules from SimpleRegulatory Gene ThatPreventEucaryotic Are DNASequences Insulators +)z from Influencing DistantGenes Proteins Regulatory 453 RapidlyEvolve GeneSwitches 453 Summary THATCREATE MECHANISMS GENETIC THEMOLECULAR 454 CELLTYPES SPECIALIZED 454 in Bacteria PhaseVariation Mediate DNARearrangements CellTypein a ProteinsDetermines A Setof GeneRegulatory 455 BuddingYeast the Determine EachOther! Synthesis Repress Two ProteinsThat Lambda HeritableStateof Bacteriophage CircuitsCanBeUsedto MakeMemory SimpleGeneRegulatory 458 Devices Allowthe Cellto CanyOut LogicOperations 459 Circuits Transcriptional Parts 460 Biological from Existing NewDevices BiologyCreates Synthetic Loopsin GeneRegulation 460 ClocksAre Basedon Feedback Circadian the Expression ProteinCanCoordinate A SingleGeneRegulatory of a Setof Genes

ProteinCanTrigger of a CriticalGeneRegulatory Expression Genes of a WholeBatteryof Downstream the Expression ManyDifferentCellTypes GeneControlCreates Combinatorial in Eucaryotes ProteinCanTriggerthe Formation A SingleGeneRegulatory of an EntireOrgan ThePatternof DNAMethylationCanBeInheritedWhen CellsDivide Vertebrate on DNAMethylation lmprintingls Based Genomic with ManyGenesin Mammals lslandsAreAssociated CG-Rich of ThatStablePatterns Ensure Mechanisms Epigenetic to DaughterCells CanBeTransmitted GeneExpression in ChromatinStructure Alterations Chromosome-Wide CanBeInherited Noisy is Intrinsically TheControlof GeneExpression Summary PTIONALCONTROLS POST-TRANSCRI Termination the Premature AttenuationCauses Transcription of SomeRNAMolecules AncientFormsof GeneControl MightRepresent Riboswitches AlternativeRNASplicingCanProduceDifferentFormsof a Proteinfrom the SameGene TheDefinitionof a GeneHasHadto BeModifiedSincethe RNASplicing of Alternative Discovery Dependson a Regulated Drosophilo SexDeterminationin Seriesof RNASplicingEvents and Poly-A Cleavage A Changein the Siteof RNATranscript of a Protein AdditionCanChangethe C-terminus the Meaningof the RNAMessage RNAEditingCanChange from the NucleusCanBeRegulated RNATransport of the Cytoplasm to SpecificRegions SomemRNAsAre Localized Control of mRNAs Regions The5'and3'Untranslated TheirTranslation Protein of an lnitiationFactorRegulates ThePhosphorylation Globally Synthesis Start lnitiationat AUGCodonsUpstreamof the Translation Initiation Translation CanRegulateEucaryotic for EntrySitesProvideOpportunities InternalRibosome Control Translation GeneExpression Changesin mRNAStabilityCanRegulate Poly-AAdditionCanRegulateTranslation Cytoplasmic ManyAnimaland Regulate RNATranscripts SmallNoncoding PlantGenes ls a CellDefenseMechanism RNAInterference Formation CanDirectHeterochromatin RNAInterference Tool HasBecomea PowerfulExperimental RNAlnterference Summory Problems References

464 465 467 468 470 471 473 476 477 477 477 478 479 480 481 482 483 485 486 488 488 489 491 492 493 493 495 496 497 497 497 499

Chapter8 ManipulatingProteins,DNA,and RNA 50r 501 THEMINCULTURE ANDGROWING CELLS ISOLATING CellsCanBelsolatedfrom IntactTissues CellsCanBeGrownin Culture CellLinesArea WidelyUsedSourceof Eucaryotic Cells Homogeneous Medicine StemCellsCouldRevolutionize Embryonic MayProvidea Wayto SomaticCellNuclearTransplantation StemCells Personalized Generate ThatProduceMonoclonal HybridomaCellLinesAreFactories Antibodies Summary

502 s02

PROTEINS PURIFYING intoTheirComponentFractions CellsCanBeSeparated to StudyCellFunctions Systems ProvideAccessible CellExtracts by Chromatography CanBeSeparated Proteins ExploitsSpecificBindingSiteson AffinityChromatography Proteins TagsProvidean EasyWayto Purify Genetically-Engineered Proteins

510

50s 505 s07 508 s10 510 511 512 513 514

PurifiedCell-Free Systems AreRequired for the preciseDissection of Molecular Functions Summory ANALYZING PROTEINS Proteins CanBeSeparated by SDSpolyacrylamide-Gel Electrophoresis SpecificProteins CanBeDetectedby Blottingwith Antibodies MassSpectrometry Provides a HighlySensitive Method for ldentifyingUnknownproteins Two-Dimensional powerful Separation MethodsareEspecially Hydrodynamic Measurements Reveal the SizeandShapeof a Proteincomolex Setsof InteractingProteins CanBeldentifiedby Biochemical Methods Protein-Protein Interactions CanAlsoBeldentifiedby a Two-Hybrid Technique in yeast produces CombiningDataDerivedfrom DifferentTechniques Reliable Protein-lnteraction MaDs OpticalMethodsCanMonitorProteinInteractions in RealTime SomeTechniques CanMonitorSingleMolecules ProteinFunctionCanBeSelectively Disruptedwith Small Molecules ProteinStructureCanBeDeterminedUsingX-RayDiffraction NMRCanBeUsedto DetermineproteinStructurein Solutron ProteinSequence and StructureprovideCluesAboutprotein Function Summory ANALYZING AND MANIPULATING DNA Restriction Nucleases Cut LargeDNAMolecules into Fragments GelElectrophoresis Separates DNAMolecules of DifferentSizes Purified DNAMolecules CanBeSpecifically Labeled with Radioisotopes or ChemicalMarkersin yitro providea Sensitive NucleicAcidHybridization Reactions Wayof DetectingSpecificNucleotideSequences Northernand SouthernBlottingFacilitate Hybridization with Electrophoretically Separated NucleicAcidMolecules GenesCanBeClonedUsingDNALibraries TwoTypesof DNALibraries ServeDifferentpurooses cDNAClones ContainUninterrupted CodingSequences GenesCanBeSelectively Amplifiedby pCR CellsCanBeUsedAs Factories to produceSoecificproteins Proteins and NucleicAcidsCanBeSynthesized Directlyby Chemical Reactions DNACanBeRapidly Sequenced Nucleotide Sequences AreUsedto predictthe AminoAcio Sequences of Proteins TheGenomesof ManyOrganisms HaveBeenFullySequenceo Summary STUDYING GENEEXPRESSION AND FUNCTION Classical Genetics Begins by Disrupting a Cellprocess by Ranoom Mutagenesis GeneticScreens ldentifyMutantswith Specific Abnormalirres MutationsCanCauseLossor Gainof proteinFunction Complementation TestsReveal WhetherTwoMutationsAre in the SameGeneor DifferentGenes GenesCanBeOrderedin Pathways by Epistasis Analysis Genesldentifiedby MutationsCanBeCloned HumanGenetics Presents 5pecialproblems andSpecial Opportunities HumanGenes AreInherited in Haplotype Blocks, WhichCan Aid in the Searchfor MutationsThat CauseDisease ComplexTraitsAre Influenced by MultipleGenes Reverse GeneticsBeginswith a KnownGeneand Determines WhichCellProcesses Requirelts Function GenesCanBeRe-Engineered in Several Ways Engineered GenesCanBeInsertedintothe GermLineof ManyOrganisms Animals CanBeGenetically Altered Transgenic PlantsAre lmportantfor BothCellBiologyand Agriculture

) to ) to

517 517 518 519 521

522 523 523 524 524 526 J2/ ill

529 530 )Jl

532 s32 534 s35 )J)

s38 540 541 544 544 546

548 548 550 551 ))z

553 553 556

558 558 s59 s60 561 s63

LargeCollections ofTaggedKnockouts Providea Toolfor Examining the Function of EveryGenein an Organism RNAInterference ls a Simpleand RapidWayto TestGeneFunction ReporterGenesand /n SituHybridization RevealWhen ano Wherea Genels Expressed Expression of Individual GenesCanBeMeasured Usino RT-PCR Quantitative Microarrays Monitorthe Expression of Thousands of Genesat Once 5ingle-Cell GeneExpression Analysis Reveals Biological"Noise" Summary Problems References Chapter 9 Visualizing Cells

569 571 572 573 574 575 576 576 579

579

LOOKING AT CELLSIN THELIGHTMICROSCOPE

579 TheLightMicroscope CanResolve Details0.2pm Apart s80 LivingCellsAreSeenClearlyin a Phase-Contrast or a DifferentialInterference-Contrast Microscooe 583 lmagesCanBeEnhanced andAnalyzed by DigitalTechniques 583 IntactTissues AreUsuallyFixedand SectionedbeforeMicroscopy 585 SpecificMolecules CanBeLocatedin Cellsby Fluorescence Microscopy 586 AntibodiesCanBeUsedto DetectSpecificMolecules 588 lmagingof ComplexThree-Dimensional ObjectslsPossible with the OpticalMicroscope 589 TheConfocalMicroscope Produces OpticalSectionsby Excluding Out-of-Focus Light 590 Fluorescent Proteins CanBeUsedtoTagIndividualproteinsin LivingCellsandOrganisms 592 ProteinDynamics CanBeFollowed in LivingCells 593 Light-Emitting Indicators CanMeasure Rapidly Changing Intracellular lonConcentrations 596 Several Strategies AreAvailableby WhichMembrane-lmpermeant Substances CanBeIntroducedinto Cells 597 LightCanBeUsedto ManipulateMicroscopic ObjectsAsWell Asto lmageThem 598 SingleMolecules CanBeVisualized by UsingTotalInternal Reflection Fluorescence Microscopy 5gg Individual Molecules CanBeTouched andMovedUsingAtomic ForceMicroscopy 600 Molecules CanBeLabeledwith Radioisotopes 600 Radioisotopes AreUsedtoTraceMolecules in CellsandOrganisms602 Summary 603 LOOKING AT CELLSAND MOLECULES IN THEELECTRON

MtcRoScoPE

604

TheElectronMicroscope Resolves the FineStructureofthe Cell 604 Biological Specimens Require Special Preparation for the Electron Microscope 605 Specific Macromolecules CanBeLocalized by lmmunogold Electron Microscopy 606 lmagesof Surfaces CanBeObtained by Scanning Electron Microscopy 607 MetalShadowing AllowsSurfaceFeatures to BeExamined at HighResolution byTransmission ElectronMicroscopy 60g NegativeStainingand Cryoelectron Microscopy BothAllow Macromolecules to BeViewedat HighResolution 6l O MultiplelmagesCanBeCombined to Increase Resolution 610 DifferentViewsof a SingleObjectCanBeCombinedto Givea Three-Dimensional Reconstruction 612 Summary ot2 Problems 614 References ot)

s63 564

Chapter10 MembraneStructure

565

THELIPIDBILAYER Phosphoglycerides, Sphingolipids, andSterols AretheMajor

617

L i p i d si n C e l lM e m b r a n e s P h o s p h o l i o i d sS o o n t a n e o u s l vF o r m B i l a v e r s

618

foo

)oaJ

6",7

A)6

TheLipidBilayer lsa Two-Dimensional Fluid TheFluidityof a LipidBilayerDependson lts Composition CanFormDomainsof DespiteTheirFluidity,LipidBilayers DifferentCompositions Monolayer LipidDropletsAreSurrounded by a Phospholipid lmportant TheAsymmetryof the LipidBilayerls Functionally Are Foundon the Surfaceof All PlasmaMembranes Glycolipids Summary

oll

MEMBRANE PROTEINS

629

with the LipidBilayerin MembraneProteins CanBeAssociated Various Ways of Some LipidAnchorsControlthe MembraneLocalization Proteins Signaling Proteins the Polypeptide ChainCrosses In MostTransmembrane the LipidBilayerin an o-HelicalConformation Transmembrane crHelicesOftenlnteractwith OneAnother FormLargeTransmembrane Channels Somep Barrels AreGlycosylated ManyMembraneProteins and Purifiedin Detergents MembraneProteins CanBeSolubilized lsa Light-Driven ProtonPumpThatTraverses Bacteriorhodopsin the LipidBilayerasSevens Helices OftenFunctionasLargeComplexes MembraneProteins ManyMembraneProteinsDiffusein the Planeof the Membrane and Lipidsto SpecificDomainsWithin CellsCanConfineProteins a Membrane Mechanical Strength GivesMembranes TheCorticalCytoskeleton and RestrictMembraneProteinDiffusion Summary Problems References

622 624 ot>

626 628 629

629 630 631 632 634 o5f

636 640 642 642 645 646 648 648 650

Chapter11 MembraneTransportof SmallMolecules 651 and the ElectricalPropertiesof Membranes PRINCIPLES OF MEMBRANETRANSPORT

651

to lons LipidBilayers AreHighlylmpermeable Protein-Free TransportProteins: of Membrane ThereAreTwo MainClasses and Channels Transporters Coupledto an ActiveTransportls MediatedbyTransporters EnergySource Summary

652

AND ACTIVEMEMBRANETRANSPORT TRANSPORTERS CanBeDrivenby lon Gradients ActiveTransport pH Cytosolic in the PlasmaMembraneRegulate Transporters Cells in Epithelial Distribution of Transporters An Asymmetric Transportof Solutes Underlies the Transcellular Pumps of ATP-Driven ThereAreThreeClasses P-typeATPase TheCa2+Pumplsthe Best-Understood the PumpEstablishes ThePlasmaMembraneP-typeNa+-K+ Na+GradientAcrossthe PlasmaMembrane Constitutethe LargestFamilyof Membrane ABCTransporters TransoortProteins Summary

654 656

652

6s3 654

o)t

658 oou 661 663 667

OF PROPERTIES ANDTHEELECTRICAL ION CHANNELS 667 MEMBRANES and FluctuateBetweenOpenand Arelon-Selective lon Channels 667 closedStates TheMembranePotentialin AnimalCellsDependsMainlyon K+Leak 669 and the K+GradientAcrossthe PlasmaMembrane Channels Pump the Na+-K+ TheRestingPotentialDecaysOnlySlowlyWhen 669 ls Stopped K+ChannelShows Structureof a Bacterial TheThree-Dimensional 671 CanWork Howan lonChannel 673 to lons to WaterButlmpermeable ArePermeable Aquaporins 675 Structure TheFunctionofa NeuronDependson lts Elongated in ActionPotentials Generate CationChannels Voltage-Gated 676 Excitable Cells Electrically of ActionPotential the Speedand Efficiency MyelinationIncreases o/6 in NerveCells Propagation

IndividualGatedChannels IndicatesThat Recording Patch-Clamp 680 Fashion Openin an All-or-Nothing and Structurally Are Evolutionarily CationChannels Voltage-Gated 682 Related ConvertChemicalSignalsinto lon Channels Transmittercated 682 Onesat ChemicalSynapses Electrical 684 or Inhibitory CanBeExcitatory ChemicalSynapses JunctionAre at the Neuromuscular Receptors TheAcetylcholine 684 CationChannels Transmitter-Gated for Psychoactive AreMajorTargets lon Channels TransmitterGated 686 Drugs Activation the Sequential Involves Transmission Neuromuscular 687 of FiveDifferentSetsof lon Channels 688 SingleNeuronsAreComplexComputationDevices of at Least a Combination NeuronalComputationRequires 689 ThreeKindsof K+Channels (LTP) in the MammalianHippocampus Potentiation Long-Term 691 Channels NMDA-Receptor Dependson Ca2+EntryThrough 692 Summary 693 Problems 694 References

and Compartments Chapter12Intracellular ProteinSorting OFCELLS THECOMPARTMENTALIZATION Setof MembraneBasic CellsHavetheSame AllEucaryotic Organelles Enclosed of Relationships OriginsExplainthe Topological Evolutionary Organelles in DifferentWays CanMoveBetweenCompartments Proteins to the CorrectCellAddress DirectProteins SignalSequences DeNovo:TheyRequire CannotBeConstructed MostOrganelles Informationin the Organelleltself Summary

695 695 695 697 699 701 702 704

THE NUCLEUS BETWEEN OF MOLECULES THETRANSPORT 704 ANDTHE CYTOSOL 705 Envelope Nuclear the Perforate Complexes Pore Nuclear to the Nucleus 705 SignalsDirectNuclearProteins NuclearLocalization Bindto BothNuclearLocalization NuclearlmportReceptors 707 andNPCProteins Signals 708 NuclearExportWorksLikeNuclearlmport,Butin Reverse Through on Transport lmposesDirectionality TheRanGTPase 708 NPCs by Controlling NPCsCanBeRegulated TransportThrough 709 MachinerY to the TransPort Access 7't0 Disassembles DuringMitosisthe NuclearEnvelope 712 Summary INTOMITOCHONDRIA OF PROTEINS THETRANsPORT AND CHLOROPLASTS Dependson SignalSequences into Mitochondria Translocation and ProteinTranslocators ArelmportedasUnfolded Proteins Precursor Mitochondrial Chains Polypeptide and a MembranePotentialDriveProteinlmport ATPHydrolysis Intothe MatrixSpace to lnsert Mechanisms UseSimilar andMitochondria Bacteria 2 PorinsintotheirOuterMembran Membraneand Intothe InnerMitochondrial TransDort SpaceOccursViaSeveralRoutes Intermembrane to the Thylakoid DirectProteins TwoSignalSequences in Chloroplasts Membrane Summary PEROXISOMES to UseMolecularOxygenand HydrogenPeroxide Peroxisomes PerformOxidativeReactions Directsthe lmportof Proteinsinto A ShortSignalSequence Peroxisomes Summary

713 7'13 715 716 717 717 719 720 721 721 722 t25

THEENDOPLASMIC RETICULUM 723 TheERlsStructurally and Functionally Diverse 724 SignalSequences WereFirstDiscovered in proteinslmoorteo into the RoughER 726 A Signal-Recognition Particle(SRp)DirectsERSignalSequences to a SpecificReceptorin the RoughERMembrane 727 porein the ThePolypeptide ChainPasses Throughan Aqueous Translocator 730 Translocation Acrossthe ERMembraneDoesNot AlwaysRequire OngoingPolypeptide ChainElongation 731 In Single-Pass Transmembrane Proteins, a SingleInternal ERSignal Sequence Remains in the LipidBilayer asa Membrane-spanning o Helix 732 Combinations of Start-Transfer and Stop-Transfer SignalsDetermine proteins the Topologyof Multipass Transmembrane 734 Translocated Polypeptide ChainsFoldandAssemble in the Lumen of the RoughER n6 MostProteins Synthesized in the RoughERAreGlycosylated by the Additionof a CommonN-Linked Oligosaccharide 736 Oligosaccharides Are UsedasTagsto Markthe Stateof protein Folding 738 lmproperlyFoldedProteins Are Exportedfrom the ERand Degradedin the Cytosol 739 MisfoldedProteinsin the ERActivatean Unfoldedprotein Resoonse 740 SomeMembraneProteins Acquirea Covalently Attached Glycosylphosphatidylinositol (Gpl)Anchor 742 TheERAssembles MostLipidBilayers 743 Summory 745 Problems 746 References 748

Chapter 13 Intracellular VesicularTraffic THEMOLECULAR MECHANISMS OFMEMBRANE TRANSPORT ANDTHEMAINTENANCE OF COMPARTMENTAL DIVERSITY There AreVarious Types of Coated Vesicles TheAssembly of a Clathrin CoatDrives Vesicle Formation NotAllCoats FormBasket-like Structures Phosphoinositides MarkOrganelles andMembrane Domarns Cytoplasmic Proteins Regulate thepinching-Off andUncoarrng of CoatedVesicles MonomericGTPases ControlCoatAssembly NotAllTransport Vesicles AreSpherical RabProteinsGuideVesicle Targeting SNAREs MediateMembrane Fusion InteractingSNAREs Needto BepriedApartBeforeThey Can Function Again ViralFusionProteins andSNAREs MayUseSimilar Fusion Mechanisms Summary TRANSPORT FROMTHEERTHROUGH THEGOLGI APPARATUS ProteinsLeavethe ERin COPII-Coated Transport Vesicles OnlyProteins ThatAre properlyFoldedand Assembled CanLeave the ER Vesicular TubularClusters MediateTransportfrom the ERto the GolgiApparatus TheRetrieval Pathwayto the ERUsesSortingSignals ManyProteins AreSelectively Retainedin the Compartments in WhichTheyFunction TheGolgiApparatus Consists of an OrderedSeriesof Compartments Oligosaccharide Chains AreProcessed in the GolgiApparatus Proteoglycans AreAssembled in the GolgiApparatus Whatlsthe Purpose of Glycosyfationt Transport Throughthe GolgiApparatus MayOccurbyVesicular Transportor Cisternal Maturation GolgiMatrixProteinsHelpOrganize the Stack Summory

749

750 751 754 757 t)I

758 760 760 toz

764 764 766

766 767 767 768 769 771 771 773 775 776 777 77g 77g

TRANSPORT FROMTHE IRANSGOLGINETWORK TO LYSOSOMES

779

Lysosomes Arethe Principal Sitesof Intracellular Digestion Lysosomes AreHeterogeneous Plantand FungalVacuoles Are Remarkably Versatile Lysosomes MultiplePathways DeliverMaterials to Lysosomes A Mannose6-Phosphate Receptor Recognizes Lysosomal Proteins in the lronsGolgiNetwork TheM6PReceptor ShuttlesBetweenSpecificMembranes A SignalPatchin the Hydrolase Polypeptide ChainProvides the Cuefor M6PAddition Defectsin the GlcNAcPhosphotransferase Causea Lysosomal Storage Disease in Humans SomeLysosomes UndergoExocytosis Summary

779 780 781 792 783 784 785 785 786 786

TRANSPORT INTOTHECELLFROMTHEPLASMA MEMBRANE: ENDOCYTOSIS

787 Specialized Phagocytic CellsCanIngestLargeParticles 787 Pinocytic Vesicles Formfrom CoatedPitsin the PlasmaMemorane 789 Not All Pinocytic Vesicles AreClathrin-Coated 790 CellsUseReceptor-Mediated Endocytosis to lmportSelected Extracellular Macromolecules 791 Endocytosed Materials ThatAreNot Retrieved from Endosomes EndUp in Lysosomes 792 SpecificProteins AreRetrieved from EarlyEndosomes and Returned to the PlasmaMembrane 793 Multivesicular BodiesFormon the Pathway to LateEndosomes 795 Transcytosis Transfers Macromolecules AcrossEpithelial CellSheets 797 Epithelial CellsHaveTwoDistinctEarlyEndosomal Compartments but a CommonLateEndosomal Comoartment 798 Summory 799 TRANSPORT FROMTHE IRANSGOLGINETWORK TO THECELLEXTERIOR: EXOCYTOSIS 799 ManyProteins and LipidsSeemto BeCarriedAutomaticallv from the GolgiApparatus to the CellSurface 800 Secretory Vesicles Budfrom thefuonsGolgiNetwork 801 Proteins AreOftenProteolytically Processed Duringthe Formationof Secretory Vesicles 803 Secretory Vesicles WaitNearthe PlasmaMembraneUntil Signaled to Release TheirContents 803 Regulated Exocytosis CanBea Localized Response ofthe Plasma Membrane andltsUnderlying Cytoplasm 804 Secretory VesicleMembraneComponents AreeuicklyRemoved from the PlasmaMembrane 805 SomeRegulated Exocytosis EventsServeto Enlarge the plasma Membrane 80s Polarized CellsDirectProteins from the lransGolgiNetworkto the Appropriate Domainof the Plasma Membrane 805 DifferentStrategies GuideMembraneProteins and LipidsSelectively to the CorrectPlasmaMembraneDomains 806 Synaptic Vesicles CanFormDirectlyfrom Endocytic Vesicles 807 >ummary 809 Problems 810 References 812

Chapter14 EnergyConversion:Mitochondria and Chloroplasts T H EM I T O C H O N D R I O N TheMitochondrion Contains an OuterMembrane, an Inner Membrane, andTwoInternal Compartments TheCitricAcidCycleGenerates High-Energy Electrons A Chemiosmotic Process ConvertsOxidationEnergyinto ATp NADHTransfers its Electrons to OxygenThroughThreeLarge Respiratory Enzyme Complexes As Electrons MoveAlongthe Respiratory Chain,Energyls Stored asan Electrochemical ProtonGradientAcrossthe lnner Membrane TheProtonGradientDrivesATPSynthesis

813 815 916 817 917 gl9

820 a2'l

TheProtonGradientDrivesCoupledTransportAcrossthe Inner Membrane ProduceMostof the Cell'sATP ProtonGradients Mitochondria Maintain a HighATP:ADP Ratioin Cells MakesATP A LargeNegativeValueof AGfor ATPHydrolysis Usefulto the Cell to Hydrolyze ATPand ATPSynthase CanFunctionin Reverse PumoHr Summary ELECTRON-TRANSPORT CHAINSAND THEIRPROTOI' PUMPS

822 822 823 824 826 827

827

827 ProtonsAre Unusually Easyto Move 828 TheRedoxPotentialls a Measureof ElectronAffinities 829 EfectronTransfers Release LargeAmountsofEnergy in the MethodsldentifiedManyElectronCarriers Spectroscopic 829 Respiratory Chain TheRespiratory ChainIncludesThree LargeEnzyme Complexes 831 in the InnerMembrane Embedded Efficient An lron-CopperCenterin Cytochrome OxidaseCatalyzes 832 02 Reduction Transfers in the InnerMitochondrial MembraneAreMediated Electron 834 Tunneling duringRandom Collisions by Electron A LargeDropin RedoxPotentialAcrossEachoftheThreeRespiratory 835 EnzymeComplexes Provides the Energyfor H+Pumping in theThreeMajor by DistinctMechanisms TheH+PumpingOccurs 835 EnzymeComplexes Transport from ATPSynthesis 836 H+lonophores UncoupleElectron ElectronFlowThrough Respiratory ControlNormallyRestrains 837 the Chain in BrownFatinto NaturalUncouolers Convertthe Mitochondria 838 Heat-Generating Machines PlaysManyCriticalRolesin CellMetabolism 838 TheMitochondrion Mechanisms to Harness Bacteria AlsoExploitChemiosmotic 839 Energy 840 Summary 840 AND PHOTOSYNTHESIS CHLOROPLASTS ls OneMemberof the PlastidFamilyof TheChloroplast 841 Organelles Resemble Mitochondria ButHavean Extra Chloroplasts 842 Compartment fromSunlight andUselt to Fix CaptureEnergy Chloroplasts 843 Carbon by Ribulose Bisphosphate CarbonFixationlsCatalyzed 844 Carboxylase ThreeMolecules EachCO2MoleculeThatls FixedConsumes 845 ofNADPH ofATPandTwoMolecules to Facilitate CarbonFixationin SomePlantslsCompartmentalized 846 Growthat LowCO2Concentrations of Chlorophyll Dependson the Photochemistry Photosynthesis 847 Molecules Reaction CenterPlusan AntennaComplex A Photochemical 848 Forma Photosystem In a Reaction Center,LightEnergyCapturedby Chlorophyll 849 Creates a StrongElectronDonorfrom a WeakOne BothNADPHand ATP 850 Produces NoncyclicPhotophosphorylation CanMakeATPby CyclicPhotophosphorylation Chloroplasts 853 WithoutMakingNADPH and AlsoResemble I and ll HaveRelatedStructures, Photosystems 8s3 Photosystems Bacterial and Forcelsthe Samein Mitochondria TheProton-Motive 6fJ Chloroplasts Control in the Chloroplast InnerMembrane Proteins Carrier 854 with the Cytosol MetaboliteExchange 855 AlsoPerformOtherCrucialBiosyntheses Chloroplasts 855 Summary AND OF MITOCHONDRIA THEGENETIC SYSTEMS 85s PLASTIDS ContainCompleteGeneticSystems856 Mitochondria and Chloroplasts the Numberof Determine GrowthandDivision Organelle 857 in a Cell Mitochondria andPlastids

859 HaveDiverseGenomes and Chloroplasts Mitochondria ProbablyBothEvolvedfrom and Chloroplasts Mitochondria 859 Bacteria Endosymbiotic CodonUsageand CanHavea Havea Relaxed Mitochondria 861 VariantGeneticCode Known 862 Containthe SimplestGeneticSystems AnimalMitochondria 863 GenesContainIntrons SomeOrganelle About Genomeof HigherPlantsContains TheChloroplast 863 120Genes by a Non-Mendelian GenesAreInherited Mitochondrial 864 Mechanism 866 in ManyOrganisms Inherited GenesAreMaternally Organelle the Overwhelming Demonstrate PetiteMutantsin Yeasts Biogenesis 866 for Mitochondrial of the CellNucleus lmportance that Proteins ContainTissue-Specific and Plastids Mitochondria 867 in the CellNucleus AreEncoded Make Chloroplasts lmportMostof TheirLipids; Mitochondria 867 Mostof Theirs MayContributeto the Agingof CellsandOrganisms 606 Mitochondria HaveTheirOwn Genetic and Chloroplasts WhyDo Mitochondria 868 Systems? 870 Summary 870 CHAINS OF ELECTRON-TRANSPORT THE EVOLUTION 870 ATP to Produce Fermentation CellsProbablyUsed TheEarliest to Use ChainsEnabledAnaerobicBacteria Electron-Transoort 871 asTheirMajorSourceof Energy Molecules Nonfermentable Sourceof ReducingPower, ByProvidingan Inexhaustible a MajorEvolutionary Overcame Bacteria Photosynthetic 872 Obstacle Chainsof Cyanobacteria Electron-Transport ThePhotosynthetic Oxygenand PermittedNewLife-Forms 873 Atmospheric Produced 875 Summary 877 Problems 878 References Chapter 15 Mechanisms of Cell Communication

879

879 OF CELLCOMMUNICATION PRINCIPLES GENERAL 880 Receptors Bindto Specific SignalMolecules Extracellular CanAct OverEitherShortor Long SignalMolecules Extracellular 881 Distances Cellsto ShareSignaling AllowNeighboring GapJunctions 884 lnformation of Combinations to Specific to Respond EachCellls Programmed 884 SignalMolecule5 Extracellular to the Same DifferentTypesof CellsUsuallyRespondDifferently 885 SignalMolecule Extracellular CellsDependson TheirPositionin TheFateof SomeDeveloping 886 MorphogenGradients Molecule of an lntracellular A CellCanAlterthe Concentration 886 QuicklyOnlylf the Lifetimeof the Moleculels Short the Activityof NitricOxideGasSignalsby DirectlyRegulating 887 SpecificProteinsInsidetheTargetCell GeneRegulatory AreLigand-Modulated NuclearReceptors 889 Proteins ProteinsArelonReceptor of Cell-Surface TheThreeLargestClasses and Enzyme-Coupled G-Protein-Coupled, Channel-Coupled, 891 Receptors ViaSmall RelaySignals Receptors MostActivatedCell-Surface 893 SignalingProteins and a Networkof Intracellular Molecules Switches asMolecular Function Proteins Signaling ManyIntracellular 895 or GTPBinding ThatAreActivatedby Phosphorylation the Speed,Efficiency, Enhance Complexes Signaling Intracellular 897 ofthe Response and Specificity Between ModularInteractionDomainsMediatelnteractions 897 SignalingProteins Intracellular Abruptlyto to Respond CellsCanUseMultipleMechanisms Signal 899 ofan Extracellular Concentration Increasing a Gradually MakeUseof Usually Networks Signaling Intracellular 901 Loops Feedback 902 to a Signal Sensitivity CellsCanAdjustTheir 903 Summary

SIGNALING THROUGH G-PROTEIN-COUPLED CELL(GPCRs) sURFACE RECEPTORS ANDSMALL INTRACELLULAR MEDIATORS Trimeric GProteins Relay Signals fromGpCRs

904

SomeG ProteinsRegulate the Production of CyclicAMp Cyclic-AMP-Dependent ProteinKinase(pKA)MediatesMosr of the Effectsof CyclicAMP SomeG Proteins Activate An InositolPhospholipid Signaling Pathwayby ActivatingPhospholipase C-p Ca2+ Functions asa Ubiquitous Intracellular Mediator TheFrequency of Ca2+Oscillations lnfluences a Cell! Response proteinKinases Ca2+/Calmodulin-Dependent (CaM-Kinases) MediateManyof the Responses to Ca2+ Signals in AnimalCells SomeG ProteinsDirectlyRegulatelon Channels SmellandVisionDependon GPCRs ThatRegulate CyclicNucleotide-Gated lonChannels Intracellular Mediatorsand Enzymatic Cascades Amplify Extracellular Signals phosphorylation GPCR Desensitization Dependson Receptor Summory

90s 90s 908 909 912 912 914 916 917 919 920 921

SIGNALING THROUGHENZYME-COUPLED CELL-SURFACE RECEPTORS 921 phosphorylate ActivatedReceptorTyrosine (RTKs) Kinases Themselves 922 Phosphorylated Tyrosines on RTKsServeas DockingSitesfor Intracellular Signaling Proteins 923 Proteins with SH2DomainsBindto phosphorylated Tyrosines 924 RasBelongsto a LargeSuperfamily of MonomericGTpases 926 RTKs ActivateRasViaAdaptorsand GEFs: Evidence from the Developing Drosophila Eye 927 RasActivates a MAPKinase Signaling Module 928 ScaffoldProteinsHelpPreventCross-Talk BetweenparallelMAp Kinase Modules 930 RhoFamilyGTPases Functionally CoupleCell-Surface Receptors to the Cytoskeleton 931 Pl3-Kinase Produces LipidDockingSitesin the plasmaMemorane 932 ThePl-3-Kinase-Akt SignalingPathwayStimulates AnimalCellsto Surviveand Grow 934 TheDownstream SignalingPathways ActivatedBy RTKs and GpCRs Overlao v5) Tyrosine-Kinase-Associated Receptors Dependon Cytoplasmic Tyrosine Kinases 935 CytokineReceptors Activatethe JAK-STAT Signalingpathway, Providinga FastTrackto the Nucleus 937 phosphorylations 9 3 8 ProteinTyrosine Phosphatases ReverseTyrosine SignalProteinsof the TGFBSuperfamily ActThroughReceptor Serine/Threonine Kinases andSmads 939 proteinKinases Serine/Threonine andTyrosine AreStructurally Related 941 Bacterial Chemotaxis Dependson a Two-Component Signaling PathwayActivatedby Histidine-Kinase-Associated Receptors 941 Receptor Methylationls Responsible for Adaptationin Bacterial Chemotaxis 943 Summory 944 SIGNALING PATHWAYS DEPENDENT ON REGULATED PROTEOLYSIS OF LATENTGENEREGULATORY PROTEINS protein TheReceptorProteinNotchls a LatentGeneRegulatory Wnt ProteinsBindto Frizzled Receptors and Inhibitthe Degradation of p-Catenin Hedgehog Proteins Bindto patchedRelieving lts Inhibition of Smoothened ManyStressful and Inflammatory StimuliActThroughan NFrB-Dependent Signaling Pathway Summory

946 946 948 950 952 954

SIGNALING IN PLANTS 955 Multicellularity andCellCommunication Evolved Independently in PlantsandAnimals 955 Receptor Serine/Threonine Kinases Arethe LargestClassof Cell-Surface Receptors in Plants vf,o

EthyleneBlocksthe Degradation of SpecificGeneRegulatory Proteinsin the Nucleus Regulated Positioning of AuxinTransporters Patterns PlantGrowth Phytochromes DetectRedLight,andCryptochromes DetectBlue Light Summory Problems References

Chapter 16 The Cytoskeleton

957 959 960 961 964

965

THESELF-AssEMBLY AND DYNAMICSTRUCTURE OF CYTOSKELETAL FILAMENTS

965 Cytoskeletal Filaments Are Dynamicand Adaptable 966 TheCytoskeleton CanAlsoFormStableStructures 969 EachTypeof Cytoskeletal Filamentls Constructed from Smaller ProteinSubunits 970 Filaments Formedfrom MultipleProtofilaments Have Advantageous Properties 971 Nucleationlsthe Rate-Limiting Stepin the Formationof a Cytoskeletal Polymer 973 TheTubulin andActinSubunits Assemble Head-to-Tailto CreatePolarFilaments 973 Microtubules andActinFilaments HaveTwoDistinctEnds ThatGrowat DifferentRates 975 Filament Treadmilling andDynamicInstability AreConsequences of NucleotideHydrolysis byTubulinand Actin 976 Treadmilling and DynamicInstability Aid RapidCytoskeletal Rearrangement 980 TubulinandActinHaveBeenHighlyConserved During Eucaryotic Evolution 982 Intermediate FilamentStructureDependson TheLateral Bundling andTwisting of CoiledCoils 983 Intermediate Filaments lmpartMechanical Stability to AnimalCells 985 DrugsCanAlterFilamentPolymerization 987 Bacterial CellOrganization andCellDivision Dependon Homologsofthe Eucaryotic Cytoskeleton 999 Summary 991 HOWCELLSREGULATETHEIRCYTOSKELETAL FILAMENTS 992 A ProteinComplexContaining yTubulinNucleates Microtubules 992 Microtubules Emanate fromthe Centrosome in AnimalCells 992 ActinFilaments AreOftenNucleated at the PlasmaMembrane 996 TheMechanism of NucleationInfluences Large-Scale Filament Organization 999 Proteins ThatBindto the FreeSubunitsModifyFilamentElongation999 SeveringProteinsRegulate the Lengthand KineticBehaviorof ActinFilaments andMicrotubules 1000 Proteins ThatBindAlongthe Sidesof Filaments CanEitherStabilize or DestabilizeThem 1OO1 ProteinsThat Interact with Filament EndsCanDramatically Change Filament Dynamics 1OO2 DifferentKindsof ProteinsAlterthe Properties of RapidlyGrowing Microtubule Ends 1003 Filaments AreOrganized into Higher-Order Structures in Cells 1005 Intermediate Filaments AreCross-Linked and Bundledlnto StrongArrays 1005 Cross-Linking Proteins with DistinctProperties OrganizeDifferent Assemblies of ActinFilaments 1006 Filaminand SpectrinFormActinFilamentWebs l OOg Cytoskeletal Elements MakeManyAttachments to Membrane 1009 Summary l0l0 MOLECULARMOTORS Actin-Based MotorProteins AreMembersof the Mvosin Superfamily ThereAreTwoTypesof MicrotubuleMotorProteins: Kinesins and Dyneins TheStructural Similarity of MyosinandKinesin Indicates a CommonEvolutionaryOrigin MotorProteins Generate Forceby CouplingATPHydrolysis to Conformational Chanqes

1010 1 0 11

rc14 1015 1016

AreAdaptedto CellFunctions MotorProteinKinetics Transport of MembraneMediatethe Intracellular MotorProteins Organelles Enclosed Localizes SpecificRNAMolecules TheCytoskeleton CellsRegulateMotorProteinFunction Summary

1020 1021 1022 1023 1025

1025 AND CELLBEHAVIOR THE CYTOSKELETON Muscles to Causes Slidingof Myosinll andActinFilaments 1026 Contract InitiatesMuscle Ca2+ Concentration A SuddenRisein Cytosolic 1028 Contraction 10 3 1 Engineered Machine HeartMusclelsa Precisely AreMotileStructures Builtfrom Microtubules Ciliaand Flagella 1031 andDyneins Microtubule of the MitoticSpindleRequires Construction 1034 of ManyMotorProteins Dynamics and the Interactions 1036 ManyCellsCanCrawlAcrossA SolidSubstratum 1037 DrivesPlasmaMembraneProtrusion ActinPolymerization CellAdhesionandTractionAllowCellsto PullThemselves 1040 Forward Membersof the RhoProteinFamilyCauseMajorRearrangements 1041 of the ActinCytoskeleton Extracellular SignalsCanActivatethe ThreeRhoProtein 1043 FamilyMembers 1045 ExternalSignalsCanDictatethe Directionof CellMigration Betweenthe Microtubuleand ActinCytoskeletons Communication 1046 and Locomotion Whole-Cell Polarization Coordinates of NeuronsDepends Specialization TheComplexMorphological 1047 on the Cytoskeleton 1050 Summary 1050 Problems 1052 References

Chapter17 The CellCycle OFTHECELL CYCLE OVERVIEW CellCyclels Dividedinto FourPhases TheEucaryotic Cell-Cycle Controlls Similarin All Eucaryotes by Analysis of Genetically Cell-Cycle ControlCanBeDissected YeastMutants in Animal ControlCanBeAnalyzedBiochemically Cell-Cycle Embryos Cells Cell-Cycle ControlCanBeStudiedin CulturedMammalian Progression CanBeStudiedin VariousWays Cell-Cycle Summary THE CELL-CYCLE CONTROLSYSTEM Triggersthe MajorEventsof the TheCell-Cycle ControlSystem CellCycle Activated ControlSystemDependson Cyclically TheCell-Cycle (Cdks) ProteinKinases Cyclin-Dependent and CdkInhibitoryProteins(CKls) InhibitoryPhosphorylation CdkActivity CanSuppress Proteolysis ControlSystemDependson Cyclical TheCell-Cycle Regulation ControlAlsoDependson Transcriptional Cell-Cycle asa Networkof ControlSystemFunctions TheCell-Cycle Switches Biochemical Summaty 5 PHASE OncePerCycle S-CdkInitiatesDNAReplication Duplication of Chromatin DuplicationRequires Chromosome Structure Together HelpHoldSisterChromatids Cohesins Summory

1053 1054 1054 1056 1056 1057 1059 1059 1060 1060 1060 1062 1063 1064 I uof,

1065 't067 't067 1067 1069 1070 1071

MITOSIS

1071

M-CdkDrivesEntryInto Mitosis M-Cdkat the Onsetof Mitosis Activates Dephosphorylation for Chromosomes HelpsConfigureDuplicated Condensin Separation Machine TheMitoticSpindlels a Microtubule-Based

1071 1074 1075 1075

GovernSpindle MotorProteins Microtubule-Dependent and Function Assembly of a BipolarMitotic in the Assembly Collaborate TwoMechanisms Soindle OccursEarlyin the CellCycle Duplication Centrosome in Prophase M-CdkInitiatesSpindleAssembly in AnimalCellsRequires TheCompletionof SpindleAssembly Breakdown NuclearEnvelope Greatlyin Mitosis MicrotubuleInstabilityIncreases PromoteBipolarSpindleAssembly MitoticChromosomes to the Spindle AttachSisterChromatids Kinetochores ls AchievedbyTrialand Error Bi-Orientation on the Spindle MultipleForcesMoveChromosomes andthe Separation TriggersSister-Chromatid TheAPC/C Completionof Mitosis Separation: BlockSister-Chromatid Chromosomes Unattached CheckPoint TheSpindleAssemblY A and B in Anaphase Segregate Chromosomes in DaughterNucleiat ArePackaged Chromosomes Segregated Teloohase Meiosisls a SoecialFormof NuclearDivisionInvolvedin Sexual Reproduction Summory

1077 1077 l 078 1078 1079 1080 1081 1082 1083 1085 1087 1088 1089 1o9o 1090 1092

1092 CYTOKINESIS for the Force Ring Generate Actinand Myosinll in the Contractile 1093 Cytokinesis of the andContraction LocalActivationof RhoATriggersAssembly 1094 Ring Contractile the Planeof of the MitoticSpindleDetermine TheMicrotubules 1095 AnimalCellDivision 1097 in HigherPlants GuidesCytokinesis ThePhragmoplast to Daughter MustBeDistributed Organelles Membrane-Enclosed 1098 CellsDuringCytokinesis TheirSpindleto DivideAsymmetrically 1099 SomeCellsReposition 1099 MitosisCanOccurWithoutCytokinesis 1100 TheG1Phasels a StableStateof Cdklnactivity 11 0 1 Summary CONTROLOF CELLDIVISIONAND CELLGROWTH MitogensStimulateCellDivision Nondividing CellsCanDelayDivisionby Enteringa Specialized State Activities MitogensStimulateGr-Cdkand GrlS-Cdk TheDNADamageResponse DNADamageBlocksCellDivision: on the Number ManyHumanCellsHavea Built-lnLimitation of TimesTheyCanDivide Arrestor SignalsCauseCell-Cycle AbnormalProliferation Exceptin CancerCells Apoptosis, OrganismandOrganGrowthDependon CellGrowth TheirGrowthand Division CellsUsuallyCoordinate Proliferating SignalProteins CellsCompetefor Extracellular Neighboring CellMassby UnknownMechanisms AnimalsControlTotal Summary Problems References

Chapter18 APoPtosis

11 0 1 11 0 2 1103 1103 1105 1oo7 1107 r 108 11 0 8 1110 1111 1112 1112 1113

11 1 5

1115 UnwantedCells CellDeathEliminates Programmed 1117 Recognizable ApoptoticCellsAreBiochemically Cascade Proteolytic ApoptosisDependson an Intracellular 1118 Thatls MediatedbYCasPases Pathway Activatethe Extrinsic DeathReceptors Cell-Surface 1120 ofApoptosis 1121 TheIntrinsicPathwayof ApoptosisDependson Mitochondria 1121 the IntrinsicPathwayof Apoptosis Bcf2ProteinsRegulate 1124 lAPsInhibitCaspases '1126 Ways Various in Apoptosis Inhibit Factors Survival Extracellular to Disease1127 CanContribute Apoptosis or Insufficient EitherExcessive 1128 Summary 1128 problems 1129 References

Chapter19 CellJunctions,CellAdhesion,and the Extracellular Matrix CADHERINS ANDCELL-CELL ADHESION

I 131 11 3 3

Cadherins MediateCa2+-Dependent Cell-Cell Adhesion in AllAnimals TheCadherinSuperfamily in Vertebrates IncludesHundredsof Different Proteins, Including Manywith Signaling Functions Cadherins MediateHomophilic Adhesion 5electiveCell-CellAdhesionEnables Dissociated Vertebrare Cellsto Reassemble into Organized Tissues Cadherins Controlthe Selective Assortment of Cells TwistRegulates Epithelial-Mesenchyma I Transitions CateninsLinkClassical Cadherins to the ActinCytoskeleton Adherens Junctions Coordinate the Actin-Based Motilityof AdjacentCells Desmosome Junctions GiveEpithelia Mechanical Strenqth Cell-Cell Junctions SendSignals intothe CellInterior Selectins Mediate Transient Cell-Cell Adhesions in the Bloodstream Members of the lmmunoglobulin 5uperfamily of proteins MediateCa2+-lndependent Cell-Cell Adhesion ManyTypes of CellAdhesionMolecules Act in parallelto Create a Synapse ScaffoldProteins OrganizeJunctionalComplexes Summary

1147 11 4 8 1149

TIGHTJUNCTIONS AND THEORGANIZATION OF EPITHELIA

11 5 0

11 3 5

tt50

1137 139 140 141 142

1142 1143 1"t45 1145 1146

TightJunctionsForma SealBetweenCellsand a FenceBetween Membrane Domains playa KeypartIn ScaffoldProteinsin JunctionalComplexes the Controlof CellProliferation Cell-CellJunctions andthe Basal LaminaGovernAoico-Basal Polarity in Epithelia planarCellpolarity A Separate Signaling System Controls Summary

11 5 5 1157 11 5 8

PASSAGEWAYS FROMCELLTO CELL:GAp JUNCT|ONS AND PLASMODESMATA

11 5 8

11 5 0 11 5 3

GapJunctions CoupleCellsBothElectrically andMetabolically A Gap-Junction Connexon lsMadeUp of SixTransmembrane Connexin Subunits GapJunctions HaveDiverse Functions CellsCanRegulate the Permeability of TheirGapJunctions performManyof the SameFunctions In Plants,Plasmodesmata asGapJunctions Summary

1162 11 6 3

THEBASALLAMINA

^t't64

Basal Laminae Underlie All Epithelia andSurround Some Nonepithelial CellTypes Lamininlsa Primary Component of the BasalLamina TypelV CollagenGivesthe BasalLaminaTensileStrenoth BasalLaminae HaveDiverse Functions Summary INTEGRINS AND CELL-MATRIX ADHESION IntegrinsAreTransmembrane Heterodimers ThatLinkto tne Cytoskeleton IntegrinsCanSwitchBetweenan Activeand an Inactive Conformation IntegrinDefectsAre Responsible for ManyDifferentGenetic Diseases Integrins Cluster to FormStrongAdhesions Extracellular MatrixAttachments ActThroughIntegrinsto ControlCellProliferation and Survival proteins Integrins Recruit Intracellular Signaling at Sitesof CellSubstratum Adhesion IntegrinsCanProduceLocalized Intracellular Effects Summory

THEEXTRACELLULAR MATRIX OFANIMALCONNECTIVE TlsSuES 1178 TheExtracellular Matrixls Madeand Orientedby the Cells j179 Withinlt (GAG)ChainsOccupyLargeAmountsof Glycosaminoglycan SpaceandFormHydrated Gels 1179 Hyaluronan Actsasa SpaceFilleranda Facilitator of CellMigration DuringTissue Morphogenesis andRepair 1180 Proteoglycans AreComposed of GAGChainsCovalently Linked to a CoreProtein 11 8 1 Proteoglycans CanRegulate the Activitiesof Secreted Proterns 1182 Cell-Surface Proteoglycans Act asCo-Receptors 11 8 3 Collagens Arethe MajorProteins of the Extracellular Matrix 1184 CollagenChainsUndergoa Seriesof Post-Translational Modifications 11 8 6 Propeptides AreClippedOff Procollagen Afterlts Secretion to AllowAssembly of Fibrils 1187 't187 Secreted Fibril-Associated Collagens HelpOrganize the Fibrils CellsHelpOrganize the CollagenFibrilsTheySecreteby ExertingTensionon the Matrix 11 8 9 ElastinGivesTissues TheirElasticitv 11 8 9 Fibronectin ls an Extracellular ProteinThatHelpsCellsAttach to the Matrix 1191 TensionExertedby CellsRegulates Assemblyof Fibronectin 't't91 Fibrils Fibronectin Bindsto IntegrinsThrough an RGDMotif 11 9 3 CellsHaveto BeAbleto DegradeMatrix,asWellasMakeit 11 9 3 MatrixDegradation ls Localized to the Vicinityof Cells 1194 Summary 1195 THEPLANTCELLWALL TheComposition of the CellWallDependson the CellType TheTensileStrengthof the CellWallAllowsPlantCellsto DevelopTurgorPressure ThePrimary CellWallls BuiltfromCellulose Microfibrils Interwovenwith a Networkof PecticPolysaccharides OrientedCell-Wall DepositionControlsplantCellGrowth Microtubules OrientCell-Wall Deposition Summary Problems References

11 9 5 11 9 5 't't97 1197 1199 1200 1202 1202 1204

11 5 8 11 5 9 11 6 1 11 6 1

1164 11 6 5 't166 1167 1169 1169 1170 1"170 "1172 1174 1175 1176 11 7 7 1178

Chapter20 Cancer CANCER A5A MICROEVOLUTIONARY PROCESS CancerCellsReproduce WithoutRestraint and Colonize OtherTissues MostCancers Derivefrom a SingleAbnormalCell Cancer CellsContainSomatic Mutations A SingleMutationls Not Enoughto Cause Cancer Cancers Develop Gradually fromIncreasingly Aberrant Cells Cervical Cancers Are Prevented by EarlyDetection TumorProgression InvolvesSuccessive Roundsof Random InheritedChangeFollowedby NaturalSelection TheEpigenetic Changes ThatAccumulate in CancerCellsInvolve Inherited Chromatin Structures andDNAMethylation HumanCancer CellsAreGenetically Unstable Cancerous GrowthOftenDependson Defective Controlof CellDeath,CellDifferentiation, or Both CancerCellsAre UsuallyAlteredin TheirResponses to DNA Damageand OtherFormsof Stress HumanCancer CellsEscape a Built-lnLimitto Cellproliferation A SmallPopulation of Cancer StemCellsMaintains Many Tumors How Do CancerStemCellsArise? To Metastasize, MalignantCancerCellsMustSurviveand Proliferate in a ForeignEnvlronment TumorsInduceAngiogenesis TheTumorMicroenvironment Influences Cancer Development ManyProperties Typically Contributeto Cancerous Growth Summary

1205 1205 1206 1207 I 208 1209 1210 1211 1212 1213 1214 1215 1216 12'.t7 1217 1218 1220 1220 1222 1223 1223

CAU5E5OF CANCER THEPREVENTABLE

1224

AgentsDamage DNA Many,ButNotAll,Cancer-Causing Do Not Damage DNA;TumorPromoters TumorInitiators Contribute to a Significant Viruses andOtherInfections of HumanCancers Proportion Reveals Waysto Avoid ldentification of Carcinogens Cancer Summary

1225 1226 1227 1229 1230

1230 GENES FINDINGTHECANCER-CRITICAL and Loss-of-Function of Gain-of-Function Theldentification 1231 MutationsRequires DifferentMethods ThatAlter CanAct asVectorsfor Oncogenes Retroviruses 1232 CellBehavior on the for Oncogenes HaveConverged DifferentSearches 1233 SameGene-Ras Firstldentified Cancer Syndromes of RareHereditary Studies 1234 Genes TumorSuppressor fromStudies Genes CanAlsoBeldentified TumorSuopressor Il5> of Tumors Tumor Mechanisms CanInactivate andEpigenetic BothGenetic 1235 Genes Suppressor in Many CanBeMadeOveractive GenesMutatedin Cancer 1237 Ways 1239 GenesContinues TheHuntfor CancerCritical 1240 Summary 1240 BEHAVIOR BASISOF CANCER-CELL THEMOLECULAR Embryos andGenetically of BothDeveloping Studies of the Function MiceHaveHelpedto Uncover Engineered 1241 Genes Cancer-Critical 1242 CellProliferation GenesRegulate ManyCancer-Critical of Cell-Cycle MayMediate the Disregulation DistinctPathways of CellGrowthin andthe Disregulation Progression 1244 Cells Cancer Cells AllowCancer ThatRegulate Apoptosis in Genes Mutations 1245 WhenTheyShouldNot to Survive Cellsto Survive in thep53GeneAllowManyCancer Mutations 1246 DespiteDNADamage and Proliferate Blockthe Actionof KeyTumorSuppressor DNATumorViruses 1247 Proteins AreStill ThatLeadto Metastasis in TumorCells TheChanges 1249 Largelya Mystery of Visible a Succession Evolve SlowlyVia Colorectal Cancers 1250 Changes AreCommonto a LargeFractionof A FewKeyGeneticLesions 1251 Colorectal Cancers Repair 1254 in DNAMismatch Cancers HaveDefects SomeColorectal with CanOftenBeCorrelated TheStepsof TumorProgression 1254 Mutations SDecific by lts Own Arrayof Genetic EachCaseof CancerlsCharacterized I z)o Lesions Iz>o Summary AND FUTURE PRESENT TREATMENT: CANCER but Not Hopeless Cures ls Difficult for Cancer TheSearch andLossof Instability Exploit the Genetic TraditionalTherapies in Cancer Cells Responses Checkpoint Cell-Cycle Genetic of a Tumor's Cause the Specific NewDrugsCanExploit Instability More BecomeProgressively GeneticInstabilityHelpsCancers Resistant to Therapies of Cancer AreEmergingfrom Our Knowledge NewTherapies Biology Oncogenic to InhibitSpecific CanBeDesigned SmallMolecules Proteins AreLogicalTargetsfor CancerTherapy TumorBloodVessels the lmmune by Enhancing MayBeTreatable ManyCancers Tumor Againsta Specific Response Has with Several DrugsSimultaneously Patients Treating for CancerTherapy PotentialAdvantages into Cancers Profiling CanHelpClassify GeneExpression Subgroups Meaningful Clinically

1256 1257 1257 1257 1259 1260 1260 I loz

| 202

| 205

1264

1264 1265 1265 1267

Therels StillMuchMoreto Do Summory problems References

Chapters21-25 availableon Media DVD-ROM

Meiosis, Chapter2t SexualReproduction: Fertilization GermCells,and

1269

OF SEXUALREPRODUCTIOII OVERVIEW ls Brief TheHaploidPhasein HigherEucaryotes Diversity Genetic Creates Meiosis Advantage a Competitive GivesOrganisms SexualReproduction Summary

1269 1269 1271 1271 1272

1272 ME|OS|S 1272 byTwoMeioticCellDivisions AreProduced Gametes PairDuringEarly (andSexChromosomes) Homologs Duplicated 1274 proohase 1 a Synaptonemal of Formation in the Culminates Pairing Homolog 1275 Complex KinetochoreDependson Meiosis-Specific, HomologSegregation 1276 Proteins Associated 1278 GoesWrong Frequently Meiosis 1279 GeneticReassortment Enhances Crossing-Over 1280 ls HighlyRegulated Crossing-Over 1280 Mammals in MaleandFemale Differently lsRegulated Meiosis 1281 Summary IN GERMCELLSAND sEXDETERMINATION PRIMORDIAL MAMMALS Signalsfrom NeighborsSpecifyPGCsin MammalianEmbryos Gonads Migrateintothe Developing PGCS Gonadto TheSryGeneDirectsthe DevelopingMammalian Becomea Testis VaryGreatlybetween ManyAspectsof SexualReproduction AnimalsPecies Summary

1282 1282 1283 1283 1285 1286

EGGS

1287

for IndependentDevelopment An Eggls HighlySpecialized EggsDevelopin Stages to Growto TheirLargeSize OocytesUseSpecialMechanisms MostHumanOocytesDieWithoutMaturing Summary

1287 1288 1290 1291 1292 't292

SPERM TheirDNAto an Egg SpermAre HighlyAdaptedfor Delivering Testis in the Mammalian Continuously SpermAreProduced SpermDevelopasa SYncYtium Summary

1292 1293 1294 1296

1297 FERTILIZATION in the FemaleGenitalTract 1297 SpermBecomeCapacitated Ejaculated and Undergoan Pellucida Zona to the Bind Sperm Capacitated 1298 AcrosomeReaction 1298 of Sperm-EggFusionls StillUnknown TheMechanism Ca2+in the Cytosol 1299 the Eggby Increasing SpermFusionActivates OnlyOneSpermFertilizes Ti'reCorticalReactionHelpsEnsureThat 1300 the Egg asWellaslts Genometo the Zygote1301 Centrioles TheSoermProvides theTreatmentof Human IVFand lCSlHaveRevolutionized 1301 Infertility 1303 Summary 1304 References

of Multicellular Chapter22 Development Organisms

1305

OFANIMALDEVELOPMENT 1305 MECHANISMS UNIVERSAL 1307 Features Anatomical Basic Some Share Animals

Multicellular Animals AreEnriched in proteins Mediatino Cell Interactions andGeneRegulation 1308 Regulatory DNADefines the program of Development 1309 Manipulation of the EmbryoReveals the Interactions Between ItsCells 13 1 0 Studies of MutantAnimalsldentifythe Genes ThatControl Developmental Processes 131 A CellMakesDevelopmental Decisions LongBeforelt Shows a Visible Change 131 CellsHaveRemembered Positional Values ThatReflect Their Locationin the Body 1312 InductiveSignalsCanCreateOrderlyDifferences Between Initially ldentical Cells 13 1 3 SisterCellsCanBeBornDifferentby an Asymmetric Cell Division 1313 PositiveFeedback CanCreateAsymmetryWhereThereWas NoneBefore 1314 patterns, PositiveFeedback Generates Creates All-or-None Outcomes, and Provides Memory t5t) A SmallSetof SignalingPathways, UsedRepeatedly, Controls Developmental Patterning 13 1 6 Morphogens AreLong-Range Inducers ThatExertGradedEffects 13 1 6 Extracellular Inhibitors of SignalMolecules Shapethe Response to the Inducer 1317 Developmental Signals CanSpread Through Tissue in Severar DifferentWays 13 1 8 Programs ThatAreIntrinsicto a CellOftenDefinethe Time-Course of its Develooment 1319 InitialPatterns AreEstablished in SmallFields of Cellsano Refined by Sequential Induction asthe EmbrvoGrows 13 1 9 Summory 1320 CAENORHABDITIS ELEGANS: DEVELOPMENT FRoM THE PERSPECTIVE OFTHEINDIVIDUAL CELL Caenorhabditis elegans ls Anatomically Simple CellFatesin the Developing NematodeAreAlmostperfectly Predictable Productsof Maternal-Effect GenesOrganize the Asymmetric Division of the Egg Progressively MoreComplexpatternsAreCreatedby Cell-Cell Interactions Microsurgery andGenetics Reveal the Logicof Developmental Control; GeneCloningandSequencing Reveal ltsMolecular Mechanisms CellsChangeOverTimein TheirResponsiveness to Developmental Signals Heterochronic GenesControlthe Timingof Development CellsDo NotCountCellDivisions in TimingTheirInternal Programs Selected CellsDie by Apoptosisaspartof the proqramof Development Summary

13 2 1 't321 1322 1323 1324

| 5l)

1325 1326 1327 1327 1328

DROSOPHILA AND THEMOLECULAR GENETICS OF PATTERN FORMATION: GENESIS OFTHEBODYPLAN 1328 TheInsectBodylsConstructed asa Series of Segmental Units 1329 Drosophilo Beginslts Development asa Syncytium 1330 GeneticScreens DefineGroupsofGenesRequired for Specific Aspectsof EarlyPatterning 1332 Interactions of the OocyteWith lts Surroundings Definethe Axesof the Embryo:the Roleof the Egg-polarity Genes 13 3 3 TheDorsoventral Signaling GenesCreate a Gradient of a protern Nuclear GeneRegulatory 1334 DppandSogSetUp a Secondary Morphogen Gradient to Refinethe Patternof the Dorsalpartof the Embrvo 1336 TheInsectDorsoventral AxisCorresponds to the Veriebrate Ventrodorsal Axis 1336 ThreeClasses of Segmentation GenesRefinethe Anterior_posterior MaternalPatternand Subdivide the Embrvo 1336 TheLocalized Expression of Segmentation Genesls Regulated by a Hierarchy of Positional Signals 1337 TheModularNatureof Regulatory DNAAllowsGenesto Have MultipleIndependently Controlled Functions I 339

Egg-Polarity, Gap,andPair-Rule Genes Create aTransient Pattern ThatlsRemembered bvOtherGenes Summary HOMEOTIC SELECTOR GENES ANDTHEPATTERNING OF THEANTEROPOSTERIOR AXIS The Hox Code SpecifiesAnterior-PosteriorDifferences

proteins Homeotic Selector GenesCodefor DNA-Binding That lnteractwith OtherGeneRegulatory Proteins TheHomeoticSelectorGenesAreExpressed Sequentially Accordingto TheirOrderin the HoxComplex TheHoxComplexCarries a Permanent Recordof Positional Information TheAnteroposterior Axisls Controlledby HoxSelectorGenesIn vertebrates Also Summary

1340 1341 1341 1342 1342 1343 1344 1344 1347

ORGANOGENESIS AND THEPATTERNING OF APPENDAGES

1347 Conditional andInducedSomatic Mutations Makeit possible to AnalyzeGeneFunctionsLatein Development 1348 BodyPartsof the Adult FlyDevelopFromlmaginalDiscs 1349 HomeoticSelectorGenesAre Essential for the Memoryof Positional Information in lmaginal DiscCells tSfl SpecificRegulatory GenesDefinethe CellsThatWillForman Appendage 13 5 1 TheInsectWing DisclsDividedintoCompartments 1352 FourFamiliar Signaling Pathways Combine to Pattern the WingDisc:Wingless, Hedgehog, Dpp,and Notch I 353 TheSizeof EachCompartment ls Regulated by Interactions AmongltsCells 13 s 3 Similar Mechanisms Pattern the Limbsof Vertebrates 1355 Localized Expression of Specific Classes of GeneRegulatory ProteinsForeshadows CellDifferentiation 1356 Lateral Inhibition Singles Out Sensory MotherCellsWithin Proneural Clusters 1357 Lateral Inhibition Drives the Progeny of the Sensory MotherCell TowardDifferentFinalFates 857 Planar Polarity of Asymmetric Divisions isControlled by Signaling viathe ReceptorFrizzled 1359 Asymmetric Stem-Cell Divisions Generate AdditionalNeurons in the CentralNervousSystem 1359 Asymmetric Neuroblast Divisions Segregate an Inhibitorof Cell Division intoJustOneof the Daughter Cells 1361 patternof NotchSignaling Regulates the Fine-Grained Differentiated CellTypesin ManyDifferentTissues 1362 SomeKeyRegulatory GenesDefinea CellType;OthersCan Activatethe Programfor Creationof an EntireOrgan 1362 Summary 1363 CELLMOVEMENTS AND THESHAPING OFTHE VERTEBRATE BODY 1363 ThePolarityof the AmphibianEmbryoDependson the polarity of the Egg 1364 Cleavage Produces ManyCellsfrom One l 365 Gastrulation Transforms a HollowBallof Cellsinto a Three-Lavered Structurewith a PrimitiveGut I JO) predictable TheMovements of Gastrulation ArePrecisely | 500 Chemical Signals Trigger the Mechanical Processes 1367 ActiveChanges of CellPackingProvidea DrivingForcefor Gastrulation 1368 Changing Patterns of CellAdhesion Molecules ForceCells IntoNewArrangements 1369 TheNotochord Elongates, Whilethe NeuralplateRollsUp ro Formthe NeuralTube 1370 A Gene-Expression Oscillator ControlsSegmentation of the 't371 Mesodermlnto Somites DelayedNegativeFeedback MayGenerate the Oscillations of the Segmentation Clock 1373 Embryonic Tissues AreInvadedin a Strictly Controlled Fashion by Migratory Cells 1373 TheDistribution of MigrantCellsDepends on Survival Factors asWellasGuidance Cues 1375

Left-RightAsymmetryof theVertebrateBodyDerivesFrom in the EarlyEmbryo Molecular Asymmetry Summary

1376

T H EM O U S E

1378

Preamble Begins Witha Specialized Mammalian Development Embryols HighlyRegulative TheEarlyMammalian TotipotentEmbryonicStemCellsCanBeObtainedFroma Embryo Mammalian Generate Between Epithelium andMesenchyme Interactions TubularStructures Branching Summary

1378 1380

NEURALDEVELOPMENT

13 8 3

ltt I

1380 13 8 1 1382

Accordingto the NeuronsAreAssignedDifferentCharacters 1383 Timeand PlaceWhereTheyAreBorn the Assigned to a Neuronat lts BirthGoverns TheCharacter 1385 lt WillForm Connections EachAxonor DendriteExtendsby Meansof a GrowthConeat 1386 ItsTip TheGrowthConePilotsthe DevelopingNeuriteAlonga Precisely 1387 DefinedPath/n Vlvo 1389 asTheyTravel Sensibilities GrowthConesCanChangeTheir Neurotrophic Factors ThatControlNerve TargetTissues Release 1389 CellGrowthand Survival Guidesthe Formationof OrderlyNeural NeuronalSpecificity 1391 Maps AxonsFromDifferentRegionsof the RetinaRespondDifferently | sYZ in theTectum of Reoulsive Molecules to a Gradient AreSharpened by of SynapticConnections DiffusePatterns 1393 Remodeling Activity-Dependent in the Moldsthe Patternof SynapticConnections Experience 1395 Brain May Synapse Remodeling AdultMemoryandDevelopmental 1396 Mechanisms Dependon Similar 1397 Summary PLANTDEVELOPMENT

1398

Arabidopsis Servesasa ModelOrganismfor PlantMolecular 1398 Genetics Contro Genomels Richin Developmental fhe Arabidopsis 1399 Genes a Root-Shoot Development Startsby Establishing Embryonic 1400 AxisandThenHaltsInsidethe Seed 1403 by Meristems Sequentially ThePartsof a PlantAreGenerated '1403 Signals on Environmental of the Seedling Depends Development Events Coordinate Developmental Hormonal Signals Long-Range 1403 in Separate Partsofthe Plant TheShapingof EachNewStructureDependson Oriented 1406 andExoansion CellDivision Setof Primordia EachPlantModuleGrowsFroma Microscopic 1407 in a Meristem AuxinTransportControlsthe Patternof Primordia Polarized 1408 in the Meristem 1409 the Meristem Maintains CellSignaling PlantTopologyby MutationsCanTransform Regulatory 1410 in the Meristem AlteringCellBehavior TheSwitchto FloweringDependson Pastand Present 1412 Environmental Cues 1413 HomeoticSelectorGenesSpecifythe Partsof a Flower 1415 Summary 1415 References

Tissues,StemCells, Chapter23 Specialized and TissueRenewal BYSTEM CELLS ANDIT5RENEWAL EPIDERMIS

'a417 1417

1419 Barrier Waterproof Epidermal CellsForma Multilayered of Different CellsExpress a Sequence Epidermal Differentiating 1420 GenesasTheyMature StemCellsin the BasalLayerProvidefor Renewalof the Epidermis1420 of a StemCellDo Not AlwaysHaveto TheTwo Daughters 1421 BecomeDifferent

TheBasalLayerContainsBothStemCellsandTransitAmplifying Cells ArePartof the Strategyof Growth TransitamplifyingDivisions Control DNA Original Retain Selectively StemCellsof SomeTissues Strands Dramatically DivisionCanIncrease TheRateof Stem-Cell WhenNewCellsAre NeededUrgentlY Renewal GovernEpidermal Signals ManyInteracting and Cyclesof Development TheMammaryGlandUndergoes Regression Summary EPITHELIA SENSORY Replaced OlfactorySensoryNeuronsAreContinually AuditoryHairCellsHaveto Lasta Lifetime CellsRenewTheirParts:the Photoreceptor MostPermanent Cellsof the Retina Summary

1422 1423 1424 1425 1426 1426 1428 1429 1429 1430 't432 1433

1434 ANDTHEGUT THEAIRWAYS 1434 Lungs of the in the Alveoli AdjacentCellTypesCollaborate to Collaborate andMacrophages Cells, Ciliated GobletCells, 1434 Keepthe AirwaysClean ltselfFasterThan Renews TheLiningof the SmallIntestine 1436 AnyOtherTissue 1438 Compartment the GutStem-Cell Maintains WntSignaling 1439 GutCellDiversification Controls NotchSignaling of GutEpithelial the Migrations Controls Signaling Ephrin-Eph 1440 Cells Combine Pathways andBMPSignaling PDGF, Wnt,Hedgehog, 1441 Niche to Delimitthe Stem-Cell Tract asan InterfaceBetweenthe Digestive TheLiverFunctions 1442 andthe Blood "t443 LiverCellProliferation LiverCellLossStimulates InsulinDoesNot Haveto Dependon StemCells: Renewal Tissue 1444 Cellsin the Pancreas Secreting 1445 Summary AND ENDOTHELIAL LYMPHATICS, BLOODVEsSEL5, 1445 CELLS 1445 andLymphatics CellsLineAll BloodVessels Endothelial 1446 Angiogenesis TipCellsPioneer Endothelial ofVessel 1447 CellsFormDifferentTypes of Endothelial DifferentTypes NotchSignaling VEGF; a BloodSupplyRelease Requiring Tissues 1448 the Response CellsRegulates Endothelial Between of Pericytes CellsControlRecruitment from Endothelial Signals 1450 Wall and SmoothMuscleCellsto Formthe Vessel 1450 Summary BLOODCELL STEMCELLS: BYMULTIPOTENT RENEWAL 1450 FORMATION Granulocytes, Are Cells of WhiteBlood TheThreeMainCategories 1451 and LYmPhocytes Monocytes, of EachTypeof BloodCellin the BoneMarrowls TheProduction 1453 Controlled Individually 1454 StemCells Hemopoietic BoneMarrowContains 1456 of BloodCells A MultipotentStemCellGivesRiseto All Classes |+)o Process Commitmentls a StePwise of Number the Amplify Cells Progenitor of Committed Divisions 1457 BloodCells Specialized 1458 StemCellsDependon ContactSignalsFromStromalCells CanBeAnalyzedin Culture 1459 Hemopoiesis ThatRegulate Factors 1459 Dependson the HormoneErythropoietin Erythropoiesis Production 1460 andMacrophage Neutrophil lnfluence MultipleCSFs 1461 CellDependsPartlyon Chance of a Hemopoietic TheBehavior of Cell lsaslmportantasRegulation of CellSurvival Regulation 1462 Proliferation 1462 Summary OF AND REGENERATION MODULATION, GENESIS, MUSCLE SKELETAL Fuseto FormNewSkeletalMuscleFibers Myoblasts

1463 1464

MuscleCellsCanVaryTheirProperties by Changing the protein fsoforms TheyContain 1465 Skeletal MuscleFibersSecrete Myostatin to LimitTheirOwnGrowth 1465 SomeMyoblasts Persist aseuiescentStemCellsin the Adult :|466 Summary 1467

FIBROBLASTS ANDTHEIRTRANSFORMATTONS: THE CONNECTIVE-TISSUE CELLFAMILY

1467

F i b r o b l a s tC s h a n g eT h e i rC h a r a c t e ri n R e s p o n s et o C h e m i c a l Signals T h e E x t r a c e l l u l aMr a t r i x M a y I n f l u e n c eC o n n e c t i v e - T i s s uCee l l Differentiation by Affecting Cell Shape and Attachment OsteoblastsMake Bone Matrix M o s t B o n e sA r e B u i l t A r o u n d C a r t i l a g eM o d e l s B o n e l s C o n t i n u a l l yR e m o d e l e db y t h e C e l l sW i t h i n l t OsteoclastsAre Controlled by SignalsFrom Osteoblasts Fat CellsCan Develop From Fibroblasts Leptin Secretedby Fat CellsProvidesFeedbackto Requlate

1467 1468 i46g 1470 lr472 1473 1474

Eating Summary

1475 1476

S T E M - C E LELN G I N E E R I N G

1476

Hemopoietic StemCellsCanBeUsedto Replace Diseased Blood Cellswith Healthy Ones 1477 Epidermal StemCellPopulations CanBeExpanded in Culturefor Tissue Repair 1477 NeuralStemCellsCanBeManipulated in Culture ir478 NeuralStemCellsCanRepopulate the CentralNervousSystem 147g StemCellsin the Adult BodyAreTissue-Specific 1479 ESCellsCanMakeAnyPartofthe Body 1480 Patient-Specific ESCellsCouldSolvethe problemof lmmune Rejection 1481 ESCellsAreUsefulfor DrugDiscovery and Analysis of Disease 14g2 Summary lr4g2 References l4g3

Chapter 24 Pathogens, Infection, andInnate lmmunity INTRODUCTION TOPATHOGENS PathogensHave EvolvedSpecificMechanismsfor Interacting with Their Hosts T h e S i g n sa n d S y m p t o m so f I n f e c t i o nM a y B e C a u s e db y t h e Pathogen or by the Host! Responses PathogensAre PhylogeneticallyDiverse BacterialPathogensCarry SpecializedVirulenceGenes Fungal and ProtozoanParasitesHave Complex Life Cycleswith MultipleForms Alf AspectsofViral PropagationDepend on Host Cell Machinery PrionsAre Infectious Proteins Infectious DiseaseAgents Are Linked To Cancer,Heart Disease,

andOtherChroniclllnesses Summary

1485 1486 1486 1487 1488 1489 1494 1496 14gA

1499 '1501

CELLBIOLOGY OF INFECTION 15 0 1 Pathogens CrossProtective Barriers to Colonizethe Host r5 0 1 Pathogens ThatColonize Epithelia MustAvoidClearance bv the Host 1502 Intracellular Pathogens HaveMechanisms for BothEnterinq and Leaving HostCells I 504 VirusParticles Bindto Molecules Displayed on the HostCell Surface 1505 poreFormation, Virions EnterHostCellsby Membrane Fusion, or Membrane Disruotion 1506 Bacteria EnterHostCellsby phagocytosis 1507 Intracellular Eucaryotic Parasites ActivelyInvadeHostCells 1508 ManyPathogens AlterMembraneTrafficin the HostCell 151 Viruses and Bacteria Usethe HostCellCytoskeleton for Intracellular Movement 1514 ViralInfections TakeOverthe Metabolism of the HostCell 1517 PathogensCan Alter the Behaviorof the Host Organism to Facilitate the Spreadofthe Pathogen

Pathogens EvolveRapidly AntigenicVariationin Pathogens Occursby Multiple Mechanisms Error-Prone Replication Dominates ViralEvolution Drug-Resistant Pathogens Area GrowingProblem Summary

15 1 8 15',19 1520 t)zl

1524

B A R R I E R S TI N OF E C T I OANN D T H EI N N A T E IMMUNE

5YsTEM

1524

Epithelial Surfaces and Defensins HelpPreventInfection HumanCellsRecognize Conserved Features of Pathogens Complement ActivationTargetsPathogens for Phagocytosis or Lysis Toll-likeProteins and NODProteins Arean AncientFamilyof PatternRecognition Receptors Phagocytic CellsSeek,Engulf,and DestroyPathogens ActivatedMacrophages Contributeto the Inflammatory Response at Sitesof Infection Virus-lnfected CellsTake DrasticMeasures to PreventViral Replication NaturalKillerCellsInduceVirus-lnfected Cellsto KillThemselves Dendritic CellsProvide the LinkBetween the Innateand AdaptivelmmuneSystems Summary References

1525 1526

Chapter25 The Adaptivelmmune System

152g 1530 15 3 1 | )55

1534 1535 1536 1537 |53t

1539

LYMPHOCYTES AND THECELLULAR BA5I5OF ADAPTIVE IMMUNITY 1540 Lymphocytes AreRequired for Adaptivelmmunity 1540 TheInnateand AdaptivelmmuneSystems WorkTogether 154j B Lymphocytes Developin the BoneMarrow;TLymphocytes Developin theThymus 1543 TheAdaptivelmmuneSystemWorksby ClonalSelection 1544 MostAntigensActivateManyDifferentLymphocyte Clones 1545 lmmunological MemoryInvolves BothClonalExpansion and jS45 Lymphocyte Differentiation lmmunologicalTolerance Ensures ThatSelfAntigens AreNot NormallyAttacked 1547 Lymphocytes Continuously Circulate ThroughPeripheral Lymphoid Organs 1549 Summary 15 5 1 B CELLSAND ANTIBODIES 15 5 1 B CellsMakeAntibodiesas BothCell-Surface AntigenReceptors and Secreted Proteins 1552 A TypicalAntibodyHasTwoldenticalAntigen-Binding Sites 1552 An AntibodyMolecule lsComposed of HeavyandLightChains 1552 ThereAre FiveClasses of AntibodyHeavyChains, Eachwitn DifferentBiological Properties 15 5 3 TheStrengthofan Antibody-Antigen InteractionDependson Boththe Numberand the Affinityof the Antigen-Binding Sites 15s7 AntibodyLightandHeavyChains Consist of Constant andVariable Regions 15 5 8 The Light and Heavy ChainsAre Composed of Repeatinglg Domains 1559 An Antigen-Binding Site ls Constructedfrom HypervariableLoops 1s60 't561 Summary

THEGENERATION OFANTIBODY DIVERSITY AntibodyGenesAreAssembled FromSeparate GeneSegments DuringB CellDevelopment lmprecise Joiningof GeneSegments GreatlyIncreases the Diversityof V Regions TheControlof V(D)JRecombination EnsuresThat B CellsAre Monospecific Antigen-Driven SomaticHypermutation Fine-Tunes Antibooy Responses B CellsCanSwitchthe Classof AntibodyTheyMake Summary

1562 1562 1564 I 565 1566 1567 1569

T CELLSAND MHC PROTEINS

1569

(TCRs) AreAntibodylikeHeterodimers T CellReceptors by DendriticCellsCanEitherActivate AntigenPresentation T Cells orTolerize T CellsInduceInfectedTargetCellsto EffectorCytotoxic KillThemselves EffectorHelperT CellsHelpActivateOtherCellsof the Innate and AdaptivelmmuneSystems the Activityof OtherT Cells Regulatory T CellsSuppress ForeignPeptides Boundto MHCProteins T CellsRecognize Reactions Wereldentifiedin Transplantation MHCProteins WereKnown BeforeTheirFunctions Similar AreStructurally ll MHCProteins Class I andClass Heterodimers with a An MHCProteinBindsa Peptideand Interacts T CellReceptor Targets MHCProteinsHelpDirectT Cellsto TheirAppropriate Bindto InvariantPartsof MHC CD4and CD8Co-Receptors Proteins Fragments of ForeignCytosolic T CellsRecognize Cytotoxic with ClassI MHCProteins Proteinsin Association Foreign of Endocytosed HelperTCellsRespondto Fragments with Classll MHCProteins ProteinAssociated in the Thymus Selected Potentially UsefulT CellsArePositively

1570 1571 tJ/t

1573 1574 1575 1575 1576 1577 1579 1580 15 8 1 1583 1585

Cytotoxicand HelperT CellsThatCould MostDeveloping AreEliminated Complexes BeActivatedby Self-Peptide-MHC 1586 in theThymus in the Expressed AreEctopically Proteins SomeOrgan-specific 1587 ThymusMedulla TheirPolymorphism1588 HelpsExplain of MHCProteins TheFunction 1588 Summary

ACTIVATION ANDLYMPHOCYTE T CELLS HELPER to CellsUseMultipleMechanisms Dendritic Activated ActivateT Cells TheActivationof T CellslsControlledby NegativeFeedback the Nature of EffectorHelperT CellDetermines TheSubclass lmmuneResPonse of the Adaptive and StimulateAn Tu1CellsActivateInfectedMacrophages ResPonse lnflammatory (BCRs) ls OnlyOneStepin AntigenBindingto B CellReceptors B CellActivation for ActivatingMost HelperTCellsAreEssential Antigen-specific B Cells Antigens T-Cell-lndependent of B CellsRecognize Class A Special Belongto theAncientlg Molecules lmmuneRecognition Superfamily Summary References

1589 1590 15 9 1 | )YZ

1594 1595 1597 1598 1599 1600 1600

Acknowledgments In writing this book we have benefited greatly from the advice of many biologists and biochemists. We would like to thank the following for their suggestions in preparing this edition, as well is those who helped in preparing the first, second, third and fourth editions' (Those who helped on this edition are listed first, those who^helpedwith the first, second, third and fourth editions follow.) Chapter1: W.FordDoolittle(Dalhousie University, Canada), (Exploratorium@, Jennifer Frazier SanFrancisco), DouglasKellogg (University of California, SantaCruz),EugeneKoonin(National Institutes of Health), MitchellSogin(WoodsHoleInstitute) Chapter2: MichaelCox(University of Wisconsin, Madison), Christopher Mathews(OregonStateUniversity), DonaldVoet (University of Pennsylvania), JohnWilson(Baylor Collegeof Medicine) Chapter3: DavidEisenberg (University of California, Los Angeles), Louise Johnson(University of Oxford), SteveHarrison (Harvard University), GregPetsko(Brandeis University), Robert Stroud(University of California, SanFrancisco), JanetThornton (European Bioinformatics Institute, UK) Chapter4: DavidAllis(TheRockefeller University), AdrianBird (Wellcome TrustCentre, (National UK),GaryFelsenfeld Institutes of Health), (University SusanGasser of Geneva, Switzerland), Eric

(Massachusetts Instituteof Technology), JoanSteitz(yale (Harvard University), JackSzostak MedicalSchool, Howard HughesMedicalInstitute), (University DavidTollervey of Edinburgh, (California UK).Alexander Varshavsky Instituteof Technology), (University Jonathan Weissman of California, San Francisco) Chapter7: RaulAndino(University of California, SanFrancisco), DavidBartel(Massachusetts Instituteof Technology), Michael Bulger(University of Rochester MedicalCenter), MichaelGreen (University of Massachusetts MedicalSchool), CarolGross (University of California, SanFrancisco), FrankHolstege (University MedicalCenter, TheNetherlands), RogerKornberg (Stanford University), HitenMadhani(University of California, San Francisco), Barbara Panning(University of California, San Francisco), (Memorial MarkPtashne Sloan-Kettering Center), Ueli (University Schibler of Geneva, Switzerland), AzimSurani (University of Cambridge, Chapter8: Wallace (University Marshall [majorcontribution] of California, SanFrancisco)

Washington) Chapter5: Elizabeth (University Blackburn of California, San Francisco), JamesHaber(Brandeis University), NancyKleckner (Harvard University), JoachimLi (University of California, San Francisco), ThomasLindahl(Cancer Research, UK),Rodney (Columbia Rothstein University), (University AzizSancar of North Carolina, ChapelHill),BruceStillman(ColdSpringHarbor Laboratory), StevenWest(CancerResearch, UK),RickWood (University of Pittsburgh)

Chapter9: WolfgangBaumeister (MaxplanckInstituteof Biochemistry, Martinsried), KenSawin(TheWellcome TrustCentre for CellBiology,UK),PeterShaw(JohnInnesCentre,UK),Werner (MaxPlanckInstitute KLlhlbrandt of Biophysics, Frankfurt am Main),Ronald Vale(University of California, SanFrancisco), Jennifer (National Lippincott-Schwartz Institutes of Health) (Swiss Chapter10:Ari Helenius Federal Instituteof Technology Ztjrich,Switzerland), (MaxplanckInstituteof WernerKtjhlbrandt Biophysics, Frankfurt (Maxplanck am Main),DieterOsterhelt Instituteof Biochemistry, Martinsried), KaiSimons(Maxplanck Instituteof Molecular CellBiologyandGenetics, Dresden)

Chapter1l: Wolfhard Almers(OregonHealthand Science Chapter6: RaulAndino(University of California, SanFrancisco), University), (University Robert Edwards of California, San DavidBartel(Massachusetts Instituteof Technology), Richard Francisco), (University Bertil Hille of Washington), Lily Jan Ebright(Rutgers University), DanielFinley(Harvard University), (University of California, SanFrancisco), RogerNicoll(University of JosephGall(Carnegie Institution of Washington), MichaelGreen California, 5an Francisco), (University Robert Stroud (University of California, of Massachusetts MedicalSchool), CarolGross 5anFrancisco), (University Patrick Williamson of Massachusetts, (University of California, SanFrancisco), Christine Guthrie Amherst) (University of California, SanFrancisco), Art Horwich(yale University Schoolof Medicine), (Stanford RogerKornberg Chapterl2tLarry Gerace(TheScrippsResearch Institute), University), Reinhard (MaxplanckInstituteof Lrjhrman Ramanujan Hegde(National Institutes of Health), Nikolaus Biophysical Chemistry, Gottingen), (University Pfanner euinn Mitrovich(University of of Freiburg, Germany), DanielSchnell California, SanFrancisco), (HarryNoller(University (University of California, of Massachusetts, Amherst),KarstenWeis(University SantaCruz), (University RoyParker of Arizona), RobertSauer of California, Berkeley), Susan Wente(Vanderbilt University

rlnarts lallelal)oU) Nl)lU)lq)laleMsolesew'$1 lalseqrueyl;o {tlstan;u1) Inpdrue!lllM'(rQtslantul JosalnlrlsulleuorteN) 6uaq5ueblo6l a}nll}sulspasnqlesseW) seUeq)'1{6olouqre1;o 6rervrzuessnp leerl)rW'(a6p;rqr.ue3 ;o fi;slen1ul)ta6laqnay 'ralua)q)lPesau ells6aayuay '({6o1ouq)alJoa}nlllsul puele)!pawqslMafleuolleN) '(Alsrenlunalel5 ue61qr;y1) leeq)lw'(re^uac sau{gpieq)lU'$n 1a}saq)ueW }o ^}lsla^lun) sDesnq)PsseW) ues'elulo1lle) ;o {r1sten1u6) l)eueW eddr;rq6'(o)st)uerl q6nouepoog '(a6plrqruel;ofirsran1ul) sa;tqdungulueW'(loot{)S le)lpewple^JeH) uotea3se;6no6 rarue'lsrma-l '$n 'lelsaq)uew;o{lrsranru6) porre9pl^eq'({llslantuq '(looq)S lalueC leulLlaog uo^ pleleH:97raldeql le)rpawpJe^reH) '(o)sr)uerl 'eluloJllef ues q6lqa-l) ;o ftlstantu1) Ilel selqlrew (luoula4 lo {ttslanlu6) ueuq)no) uqof'(ll!H eurlore3'()n'a6allof {>lsureq le1radu1) 'eu1;ore)qlroN firsranlu6)e6pprngqlla) '(^ueulag pre6 {re9'({lrsrenru6 eqf) ueuulelsqdleu'()n rallaJal)ou Jo ;eder43 'elulorlle) '({e1a1tag 'q)reasau /aulllpew ouelae3 esnos a stau lale/l Ja)uel) Jo reln)olowloj .ralua]l)nlqlao-xew)lalaulq)J!8 ueS'eturoJ!lef {oulrod;a1ueq'(orsouelj {1rsran1u61) ,o Illstanluq) '(lalual {ilstanlunploJuels)pollaxv,(ar;;a;:61 raldeql le)tpaw ueS'etuloJtle) {a;s1ro1preq)lu'(o)st)uell }o ^}lsla^lun) orolY) (uedel,Qlsrenru61 '(a6prrquel;oIlrsran;u1) uotealse;6no6 raruplsrma-l '(lerrdsop se;6no6 apnf'lS)uaa.r9 s,ualplrq) '({lrsranrul ele6eNnze1a61q5 uo}a)ulld)lslnbul pro}ue}S) MoIlel ue}S'({l;slantu6 '(o)sl)uell '(etleJlsnv'r{)leasau le)lpawJoalnlllsullleHezlll puelalleMaLl! uu{1'(1ooqr5 le)lpawple^leH)lotuqeo8uo^ pleleH jo t$lstantul sexef suepy,fura;'(looq)5 le)lpawulalsaMqlnos ueS'eruroJrleJ ;o ft rstanrul)doqslglaeql;y1'(,Qtstan;uq reldeql 6ueM6uopoelX:91 eqf) [uo!]nqU]uol lPlluelsqnsl ploluels) ro[eu]loUaLlIallnf:tZ re$eq1 [uorlnquluo) (gergdsog tuarplrq)apn|ls) laqs soUeq)'(a6p;tque1 ()n 'alnlllsulq)rpasau ra)ue))DeMeuorl'()n 'elnlrlsuluoprng)sautdueqleuof'(pJolxgJo^llsle^lun) 'qOlnqulpl 'ralsaq)ueW JoIlrsrantuleqf) llnallssapeq)'()n oleloqe1(6o1o;g I eln)elow qv(usep u ry'((uetutag',fu (ltstantul) 1oftrsranruq)qllulsullsnv'(allleas'uot6urqseMJo p;eep1 rluasle):1r3'({e;a1leg/eluloJlle)JoI11stan1u61) qeuseuoql'(looq)Sle)lpawple^leH)ulllo uenls '(loor'l)S ueadorn3) '(al}}eas'le}ua) laz}ol9 e))aqeU'(o6er1q3;o{1rsren1u61) laeq)lW uas;gurofg'(a)uprl'eun)lnlllsul)pJe^nol le)tpawpre^reH) ue5 parj)re6p3a>nrg',(o6a16 le)ue) uosu!Ll)lnH q)leasou tl)leasau sOu;y) seqbnguoluls'(alnlrlsul larueC'()n'aba;1o1 /erurojtle) ueS'e!uloJ!lel jo {ltstanlun)leseopeLlsrv'(o)sl)ueU 'lS'rQ!stentun uot6ulqsenn) uopuol)lprPqregre6;og'(srno1 ;o Il;sten;u6)[uollnquluo)rofeul ue6roylpl^eq:Zl raldeql aql'alnillsullq)elqnH)sle^el) uoplog(er;;a;'(spue;raqleN alenpleuou jleu :97raldeql q)leasauuopuol)sLuepv sueH'(alnlllsul (o)sDuellues'elulojllef;o A;sten;u1) '()fl 'rueqbu;tut1g '(looq)S le)!pawpre^leH)uos!q)llw{qloulf uopuol)uodelselor1l'()n'alnlllsuluopjnD 's)lleua9 (alnlllsulrl)reeseu elnel'(uapsalC ;o {ltslantu6aq1)I1saqre61 loj allua] leuolleN )!J!lual)s aqf) rll!rusutr'(e)uPll 'tl)leasau pue{6o;o19lla) leln)elo4}o oln}llsull)ueldxew) pleMoH ueulnHlo, ellua) q)uau) q1n6s1amg>s slofuell',()n',s)lreuag reln)olopro; Itoletoqel 35y11) aof '()n [6o1o;9lla) pue16o1o19 '(oba16ueS'eluloJ!le) lsnrl auro)llaMeLlI)uosllaqouLllaqezlll /eluroJlle);o {tte1 u1a1sp;o9 {1;sren;u61) llpHuelv'(o6alqueS sluu!9)Wue;11;nn'({6olouqlal I Jooln}l}su ;o 11;sranrul; '(o)st)uerlues'elulo;llP);o,tlsranlun)aulno€ftuag '({tlstan1u6 zlrmola{a6; eluroJrlef) }}o!lll'$n'aln}t}suluoplnDaql) uosutlof a!lnf:91 raldeql ploluels) ]ollaqf [uoltnqll]uo)JofeLul 'tS sra6lnu)aul^llqlauuo)'({11slantu6 latuec'(ftrsren;u61 '(uopuo'la6a1;o1 'eluroJlle1 'atnlllsul ual suaqdar5 aq1) uleqeJqeg ()n a1n6)ue6ogpr6ug'({ala1tag Jo,(rtstanlul) 'q)unZ l(ltstantuq) '(pueUezllMS ra;seg prer.l)lu puel.reH laq)lldellnf'(oluolol';e11dsop1 leu!StunoW)uosmed Jo ftrsran;u1) '1{6olouqra1;oe}n}l}sul (uof '(ArstenlunploJuels)assnN peruo)'()n 'arnllsuluoprngeq1)ra6urrqyollnf:zz reldeql laou Dollll'(o6el6 ueS'eluloJ! |e) ;o {llstantu1) etulojrIe)) zlrlnote{ayy '(uapaMs (au!]|pawjo filstan;u1)aul^llulqou uue) laeqrlW'(a6prrqr.ue1lo '()n 'alnlllsuluopln9eq1) 'q)leosa5 Ined looq)Sreurslunow)ueullessPM Je)ue)loJelnlllsul6ttnpnl)u!plaHlUuaH-lle) ualleq)Splelag ruernS urzv'(lalua) tuaudolana6q6tnqsp14) '(relue) uelV'(aul)!pew 6ulteDe)-ueolS letue) lleH leuoLuaW) '(o)sr)uerlueS'etulojtle) {lrsten;u61) '(lalue) o[ragaeuaS'({6o;ouq>a1 ;o le)lpaw ]o looq)s,firstantulele ) qsoqgrelues '{6o1o;9 ,fute1'(s;ne6'eluloJllel ra^eeM-lrO jo alnlllsulsuasnq)esseW) sexalJo{ltstantulaql) ueulllgpalilv'()n ulalsamqlnos u61eduel '(,tlstenlunptoluer5) sal(y1euer6'(a6puque3'etnltlsul ;o I1;srenru6) Meqlle6 ueuiaell leln)aloW Jofuo1eroqe1) u1 q)reaseuletue] /a[uo)llaM)uelel]W auuy'(rttstant 'Un 'qlleaseu le)lpawloJelnlllsul leuolleN) lleilal sauef 'eluloJllef pJe^reH) roul)el) {tuel '1srne6 }o fi;slentu6) {etg stuua6'(olsl)uell ao)sugsarue6'(abplrqule) Jo{11slan1u1) '()n 'al}ua)sauul ralunHllaN'(^rl) sPsue)'qlreosaulellpaw loj alntllsulsJaMolS) ueS'eluro,tle);o{llsra4un)aulnog^luaH 'eluoj!lef fttstan1ul)6tnqula6 Ia;rvreg}}or5 '(,{a;a1iag }o plaqleHseloqllN:91 raldeql uqof)luot]nqll]uollel]uelsqnsl '(o)sl)uell {qqy '(uopuola6e11o1 fttstantul;lloJre)uqof (obe16ue5 ueS'elu.roJllef ;o {tlstenlun)o)tele)erlllled:17 reldeql '(eruenl(suue6;o 'e!uroJrlpf {ilstanru6) lllstantul a#e leeLl)lW ]o suasnq)esseW) ({6o1ouq>a1 'alnltlsultl)Jeasau 1oa}nlt}sul uosurelqy)uosduoql la)ue) r(;rure3 'q)leasau uosulltuof 'sr1s,(qdo1g le)ue)) 6taqutann llaqou 'Un ule 61er3'(u;e61 unpluell Jo elnlllsull)ueld 'lolsll8 uosduoql 6;er1'(abp;tquel ue1'(eruenl{suua6 clellsaleH jo ;o r$tstantul) ^l!sle^!un) xew) rpuerqlqu laula A'()n lepuodarnrg '({toleroqelroqte;16ut.td5 :91raldeql 1oftrsran1u6) {er9 ;aeq>161 elsnoqleC) MalpuV'(I11slan1u61 plo)) eMol llo)s '(aul)lpewJo looq)s(trsrantunploluers)l)lsd!l (utewuleun}lueu 's)llaue9pue{6o1o19 1|e1 sel6noq ueqeueH qdesol'(o)st)ue4 ues'elulo1lle) 1oI11s.ranru61 '({11slan1u1 a1e1) '(abpqrquel lltslanlu6)spieMpl Ined'(epeue)'oluolol leln)alow,oalnlltsulI)ueld xew)lellazoulrPW 1o '()n 'q)leasau uopuol)o^elq)sotlaldruelD q)Jeesau uleqelg'(alnlllsul uaileM re)ue) Joelnlllsul ;o lltsrantul;IttC uqof '(a6ppque)'ll)uno) q)reasau lerlpawaq1)rueqla6q6ng suqor)zun€pell '(o)sl)ueJl or.lf)ouoS-acuueqor'(sulldoH '(6o;ouqral;o ele ) ueullawPll'(puelrazllMs'q)UnZ doqslglaeq>1y1'(spueUaqloN'(Ilrs.ranlun ueS'elurojtle) ;o Iltstantu6l) snlualappy'(o6er1q3r 1r1;9 ;o fitsten1u6) sula€uoluv'(ftlslantu6 alnlrlsulleJepalss!MS) otll'aln]!lsulle)ueJspueueqlaN) uag'(o6a;6ues'etu.loJllel ;o {lrsrenrul)ltul llo)S :g 1 reldeql projuels)[uot]nqUluo)lel]uetsqnsl !pJeDVelnel :67 reldeql (lsJaquV 'sllasnq)esseW{}rsranrul)uosulellllM led'(le}ua)le)!pal/! ;o (qlleeH aqo) epeue qlauua)'(uedel'alnlllsul leuolreN) ]o selnlrlsul

lunH utI'()n lalseq)ueW serrqdungur1re61 '(se;e6uy sol'etulojtle);o {lrsranrul)6teques13 Jofirsrenru6) pt^e6,(o)st)uell '(uopuolaba11o1 s,6ury) saq6nguotur5,({lrsrantun ralla;el)ou ueS'etuloJ!le) jo ^ltsla^tun) spremplyeqog,(e6prrquel aq1)qradspngseuef ,(allleas,uol6ulr.lse6Jo firsrarrrul)preMoH spleMplIned,()n ,q)tMloN,alnltlsulsauul Jo^ltsle^tun) ueqlpuof'(16olouqta1 ueulsnoH uqof)llaMunCurtf'(uopuo'l,1ru1-1 ;o alnltlsulsDasnq)esseW) s>rs{qdotg lla) fUW)uunC prne6'({6o;ouqlel}o ,()n ,qrreesag atnltlsulsgasnq)esseW) z}t^loH rueqerg'(uopuo'l a6a;;o1q6ury; UeqoU qllo) {a1pnq '(ft rsranr ,(elDeas,i(6o1or u uola)u plagdo ud) g qof u g 1 sr.uels{5 pJeMuMoO .ro1 Jalup3) uer;n;,(o6er6 ues,etuloJtle1 1oftrsrenruq) atnlllsul)poo;1{ore1'110019 {uot5 ,uo^ MeN;o {lrsran;u1a1e15) al]l!looc;1essn5'(a6prrqLUp) Jo{t;stenrul;uosqoc.raqdolsrrq) ,qlleaH salnltlsul '(o)st)uel3 quornsbur;;op {ruey ,(epsaqlag ueS'etulo;tlp) tsrenruq) Jo o)uellaC IuoqluV leuotteN) ft ;o ,uo16urqse14;o '()n 'lsnrl q)snqauutH ,(alpeos '(uopuol uelV lgrslenlu6) alltH auo)llaM eqf) lauac a6e11o1 ltyeg laeq)lW '(pasealep) zltMolsloH ell ,(qeln;ofirsranruq) r(lrslenluq) (pue1-11n3 aleCatlsal'(uopuola6a11o1 ,Ols.renrun) l)ulaH uualg '(abprrquel'{6o;org ,({r;sren;u6 reln)aloW uoslapueH Uenls'(uostpeW'utsuo)stMro {lrsrengul) Jo,toletoqel )UW) Mor) saulef p r e q ) t u ' ( ^ l r s l a ^ teul e n l )s n t u a l eU .rS,oul)lpow HV , ( ) n , u e q 6 u r u r r 6 su;1dogsuqop)6rerl{ruep ,(srno1 Jo loor.l)S ,1ru1,{6o;o19 pup,{6o1o19 {lls.renruq qleaHuqof ,(uopuo-.| 1o{lrslearu6) uol6ulqsen4) .redooluqof ,(o)st)uellues,eturo;tle) llo) ,({ueureg,6raq;aprag lla) leln)aloryrol {roreioqelfUW)poomlpHupupv,(elneas JoIltsre^tun)alool te6og lgWJ)ueqo) 'uot6urqsetr4 {lrsranr un) |la/v\U eHpuelel,(Itrsra^t un pl e^reH) ;o uaqdals'(olst)upllueS'elulo;tlp);ollrslarrul) ueqo)lleqou ,o6etg;o,tlsranlun)slrreH '(puellols'eepunC;o uosuleHuaqdal5,(pueleaz ana51 firslanrul)uaqol drIq6,Un ,Ll)tMroN ,eturortle) 'elnltlsul uqof 'Un 'q)reasau '(6rnoqsel15;o ra)ue)) suleH ueupV,(i(a;e>Fag sauul uqof) ueo) oluuf r{llstenru6) ,aapunC;o pleqltu,(puplro)S 1oIlrstanrul)pueUeH uoqueq) arretd,(uopuola6a;;o1q6ul)) qllurs_ratle^e) ftrs.ranru6) ulol atpleHuleqel9,(yn,q)tMtoN,arlua)sauuluqof)pleqleH '(obalCueS'eruro;!le);o laluadrelaptelapv,(se;a6uy ftrsranruq) seloq)tN'(e6puquel '(qe}n (are1 aIueHpr^e6,(o)sr)uelj sol /erulojtle);o{1;vanru61) ;o {lrstan;uq) {1rs.renlu6) ;eeq)y1 Jo ,(y1,uoldueqlno5 ues'erulo1rleJ l(1rsienlul) q)ez rqt>adel 1o oueyy'1uo6erg;or(1rsrenru61) lleH ;p1ede1 l)uepou '(I1rsianru61 stapuelg) {ar;;a1,(uopuol ;o firsranruq) uqof ,(rQrsranrul lleH etqulnlo))lotueJsalleqJ,(looqtsle]|pawple^leH) 1;eg l(6o1o19 reln)elow.ro;{roleioqel)UW)llpH llof pup{6o1o;9 Iellue3 sranal'({a;e1rag'etulo}tlef}o {lrslenru6)apue) sneq)ez '(ploJXO 'tueutrr;u; ue;y'({grsranrun ple^reH)6regprne6,(q)UnZ uajpH leltsle^tun) e#!l)peu) sulte)uqof,1llrgledeq3,eurlole) ,(sle)!lne)eujleqd lsulf '(o)st)uellueS,eturoJtlel ^lrs.renrun) auqfng eullsuq) quoN ^l!sle^lun) ebprllng qlle) 1o Jo XgS) '(elloqup)'{lrslanlul uetletlsnv) 6uruungueug,(la}ue) {e;tngueqdals'(laseg lpuotteN rabln6xeyl'(eur)tpew Jo^lrs.renru6) Jo re)ueJ6uua11ay-ueolS leuoueW)rautqulng{rreg,(se;a6uy Ilrstanruplro MaN)uepJnge^als,(ploJXO ;o {1slenru1)uanolg ,(spueuoqlaN '(uopuo-l sol 'eruroJtle) ^ltsra^tun) ,(uopuol ulolsunrD ar{l Jo ebe;1o1 laeq)tW {rreg laeq)rW a6aJ;o1 E6ury)unnorg 'lrailsla^run ,(o)st)uell ,etuJoJtle) '(I6olouq)af snLlsell)pla^solg ueS qoorg luelJ jo rJoqou olntltsul etulojtle)) Ebury) leseJl '1e6prrquel,(6o;orglelnleloW ssorglole] /(spuelJoqleN -reuuorgeuuelreW ;o ^lrsranruq) aqf ,ureple]sruv Jo Jo{roteroqel ^}rsranru g) lla^u9etlsal'(lslequV,s]lasnq)essew ;o ^}rsranruq) o}nllsullelapel )UW)loq)slel€lleW,(Ll)unz,{6o;ouqrelyo ueelg ple^leH) ,(a6prrqLuel pleMoH,(uopuol uaalg laeLl)tW,(&rslanlun sst^ tlpuelg olpuv,(pesealep) uapuerg S) ;re) abal;o1s,6ul))ieaeig lalleM'(plojxo;o i(1rs.ranru6) ,(uopuo-l ua1er9 {lrslenru6) puer€ utue4 e6eg1o3 Ilrsranlu6) 1o ,loorl)s uelv'(uopuo-l xasalpptw) plnoglalad ap{oguely'(o)sl)uplJ le)tpaw;el;dso;1 ups,eturoJtle) }o Atsto^tun)aurnog(tueg '(raplnog /opelolof r.llupoogultr,(looq)sle)tpaw '(uepralsuv Jo{lrsranrul,aln}tlsululeplauuleMsupf)lsroglatd Jor(lrsranru6) ,(uopuol,looLl)S pre^reH) '(aur^rl'etuloJrle) qbnouepoog ,q)leesau latueC epo€sueH,(uopuo-.| lellpeW ;elldsog Jo{Usranrun) a6a11o1 llstenlul) suadr.uog uotlspg,(o6e16 ueS,etuloJtlpJ sstlgLlltf'(olst)uellues,erujojtle) le)paw toJalnltlsulleuotleN) ro ,(eulsny,euuetn {lrsranru6) utolsplog ,(lootl)S rt.re1 l(}rsranrul) lez]olg l(1rsranru6) doqsrg }o 1o ;eeqrr61 le)tpawuosuqofpoo4 uole)uud)6ren1r9 leeq)lW'(llrsrenruq selleq),(pesee)ep) elnltg rraqou-fCNWn)lltg pt^eo,(a6prrqueS,otnlllsul r,!eqplqeg arurog'(lsleqrlv,sllesnq)esse61 {lrsranrun) ejoultDptau aqf a6prrreg ple^rpH) ;o plouureguouaw leeq)tW'(loor.tls le)tpaw '(auprpe4 '(o6alqueS'etuto;tleJ;o {lrslanrupale ) qsoqgleIueS'(^6olouqlaf ,(eut)tpew Jo loor.l)S 6legurnnre6 firsranruq) ,({6o;ouqrel lalUagluell ,(pausutUpW ,o alnlllsulsllesnLl)psseW) ulalsulluaqlv)Dauueglaeq)rW ;o a6e1;o1 ',tls[uaq:ol€ ,(Atsla^tun Joalntrlsull)ueld xew)q)sua9laqlung ,({e;a1reg ploruels) Joalntrtsulsuesnq)essew) ;auegpt^ec 'eturoJtle) ^}rslenru6) Ueq.lagur]of,(a]n]tlsul r.llleoseu saJleguag'(o)st)uell ,etuJoIle);o Jo ueS {1rs.ranru6) uueu6reg ,lo^or.leu ,a)uet)Sjo alnulsul sddrrr5eq1)o)elag &Je1,(1aers1 Prlaulo)'(o)st)uerlues,etulojtle) epupglaeq)lw Jo^llsra^tun) ,unJluazotg) '({lrsranru6 uueuztel ) le6legr(uuag,(leseg prolue}S)utMplegpt^ec ,(paseo)ap) JoIlrslenlu6l releg lalod /q)leeseu '(o)sr)uerl 'etuloJtle);o 6ulrqeglolleM,(uopuo-l ,(uopuol lo)ue) elnltlsul) ueS {pqenny Jo ftrslenrul) eu{e1 puepPD ielad'(uopuolabel;o1{lrslenrup) ut/v\paW-rauplpDa6a;1o1 {l;sranru61) elouqsVueqteuof,(e6p;rqruel ;o ft rstenruq) ^uoqluv'(plolxoJo{trsranlun) lauplegplpqltu,({lgsranguq a;e1) taurnqqsvlapq)tW,(looq)Sle)tpowple^leH)seuolesf ,(o6errq3 ,(olsr)uelJ ,(o)st)uell ,etulo;tle) -slue^euV sq)nJ ;1e9qdaso; Alsranlun) autell ueS 1o sol^ds ueS JoIlrslenru6) 'elulojlle) ^rlsran!un) '(epeue),ueMaq)lplses,o '(eruenl{suua6 pualrl Jo pneurv eLlUeW laluec Illsle^lun) 6uor}stu.rv Iel) Jo ,(loorlls '(o)sr)uerlueS'etuloJtle) Ilrs.ranru6) Ilrsranguq) a1ano3,(ire1 lp)lpowpre^lpH)ueu)loj qepnf outpuv;neg,(a6puque1 1o '(r(grsranru6 '(6o1o19 sUnI) ueLuoll {enreg ,(o)souellues/etulo;tleJ leln)alowJo{role.roqel)UW)soulv epur-l,(uo}sog 'q)leesou ,(a6prrquel;o ;o {ltstentul) lreqog'(Ilrste^run etqunlo))r.lleqq)srJ l)ua}lall loJ alnlltsul pat3 le)tpaulolg llv Ug)) ,etulo}tle) p;era9'({e;e1tog ,(o)st)uellueS,eturo;tle);o {re9 Jo{lrslenlu6)auo}satrl firsranrul)uelv laeLl)tW A;slen;up) pte6y prne6suolllpe qyno, pue,prrql ,puo)es,lsrrl (epeup)'oluorofJofirsranru6) seutqeqS eac ,(epeue) 'otuolof ,(eulsnv,euuatn jo {t;stanru6; ltzpnul)lN Jorerstenru6l) ,(firslenru6alnC) snleqsrypl^e6 lptol) lanueuu3 srapeag '(uopuol'tl)reasau ,(fttsla^tun la)ue) Joolnttlsul)ranu3buel unuerg r(Iaq5 ,eruermelloueell {resso;g a)tn6)Mopul ufueq5,(o6arqupS,etuJo;tle);o l(1rsranlul) ,(atnlrlsul lurl llo)s '(eru16rr410 r(tls.ran!un) uoslaulf '(^l!sla^tun selleq) lles eql) uosloul {;laneg,()n ,uoffns,q)leasaule)ue) ,()n ,q)leaseu o}ntllsul) rolleja))ou aql) qdlpU ueululors Jo lo)ue)) esnos ueurlllqlnu '(slnol.lS,,(llslanru6 uol6u!rlse4) ur6;3qere5 o sreuouelae)'(looq)S ,fismefeg snely,(rllleoH le)!powpre^leH)

(Cancer Hurst(University of Bath,UK), Research, UK),Laurence TonyHyman(Max CollegeLondon), JeremyHyams(University Dresden), CellBiology& Genetics, PlanckInstituteof Molecular Philip Instituteof Technology), Hynes(Massachusetts Richard UK),Normanlscove(Ontario Ingham(University of Sheffield, (Cancer Research, Toronto), Davidlsh-Horowicz CancerInstitute, (University Charles 5an Francisco), Lily Jan of California, UK), (Columbia (deceased), Arthur University), TomJessell Janeway AndyJohnston(JohnInnes A & M University), Johnson(Texas College, Norwich,UK),E.G. Jordan(QueenElizabeth Institute, (University Ray LosAngeles), of California, London), RonKaback DouglasKellogg of California, Berkeley), Keller(University (University of of California, SantaCruz),RegisKelly(University (MRCLaboratory JohnKendrick-Jones SanFrancisco), California, of Biology, Cambridge), CynthiaKenyon(University of Molecular (University of RogerKeynes 5anFrancisco), California, Madison), of Wisconsin, JudithKimble(University Cambridge), (Massachusetts Marc Hospital), Kingston General Robert (National (Harvard Klausner University), Richard Kirschner (Harvard Mike University), NancyKleckner of Health), Institutes (University Boulder), KellyKomachi of Colorado, Klymkowsky (University EugeneKoonin(National of California, SanFrancisco), (University 5an of California, JuanKorenbrot lnstitutes of Health), (University 5anFrancisco), of California, TomKornberg Francisco), (Washington Daniel 5t.Louis), University, StuartKornfeld (University MarilynKozak of California, Berkeley), Koshland (Stanford (University University), MarkKrasnow of Pittsburgh), (MaxPlancklnstitutefor Biophysics, Frankfurt WernerKrlhlbrandt (University Robert Berkeley), of California, am Main),JohnKuriyan Peter London), CellBiology, for Molecular Kypta(MRCLaboratory (MRCCenter, Cambridge), UlrichLaemmli(University Lachmann of Cambridge), TrevorLamb(University Switzerland), of Geneva, of Research, UK),DavidLane(University HartmutLand(Cancer (University JayLash of Oxford), JaneLangdale Dundee, Scotland), of (University PeterLawrence(MRCLaboratory of Pennsylvania), (MountSinaiSchool PaulLazarow Biology, Cambridge), Molecular (DukeUniversity), Michael RobertJ.Lefkowitz of Medicine), WarrenLevinson of California, Berkeley), Levine(University (Hebrew (University AlexLevitzki SanFrancisco), of California, (University of York, UK),Joachim Leyser lsrael), Ottoline University, TomasLindahl(Cancer SanFrancisco), of California, Li (University (University San of California, Research, UK),VishuLingappa (National of Institutes JenniferLippincott-Schwartz Francisco), Schoolof DanLittman(NewYorkUniversity Health,Bethesda), UK),Richard Norwich, CliveLloyd(JohnInnesInstitute, Medicine), (National Institute RobinLovell-Badge University), Losick(Harvard of London), ShirleyLowe(University for MedicalResearch, (University of LauraMachesky 5anFrancisco), California, Medical of Colorado UK),iamesMaller(University Birmingham, (Harvard ColinManoil(Harvard University), TomManiatis School), (National JewishMedicaland Marrack Philippa MedicalSchool), of Cancer MarkMarsh(lnstitute Denver), Research Center, San of California, GailMartin(University London), Research, Joan CollegeLondon), PaulMartin(University Francisco), (Memorial Center), Brian Cancer Sloan-Kettering Massagu6 (University McCarty lrvine),Richard of California, McCarthy (University (Cornell of California, WilliamMcGinnis University), (Wellcome/Cancer Campaign Research Anne McLaren Davis), of California, FrankMcNally(University Cambridge), Institute, Institut,Basel), Miescher Meins(Freiderich Freiderick Davis), lraMellman 5anDiego), Mel(University of California, Stephanie (YaleUniversity). of California, Meyer(University Barbara Instituteof Technology), ElliotMeyerowitz(California Berkeley), of RobertMishell(University University), ChrisMiller(Brandeis (University CollegeLondon), UK),AvrionMitchison Birminoham,

(University TimMitchison CollegeLondon), N.A.Mitchison (TheRockefeller (Harvard PeterMombaerts MedicalSchool), DavidMorgan MarkMooseker(YaleUniversity), University), MichelleMoritz (University SanFrancisco), of California, Moses(Duke Montrose (University 5anFrancisco), of California, (University SanFrancisco), of California, Mostov Keith University), HansM0ller-Eberhard CollegeLondon), AnneMudge(University of AlanMunro(University Institute), (Scripps Clinicand Research (Harvard Richard University), Mitchison J.Murdoch Cambridge), of California, DianaMyles(University University), Myers(Stanford MarkE.Nelson University), AndrewMurray(Harvard Davis), MichaelNeuberger (University Urbana-Champaign), of lllinois, Walter Cambridge), Biology, (MRCLaboratory of Molecular DavidNicholls of Munich,Germany), Neupert(University of Noble(University Suzanne (University Scotland), of Dundee, (University of California, HarryNoller 5anFrancisco), California, Paul Davis), of California, JodiNunnari(University SantaCruz), Patrick UK),DuncanO'Dell(deceased), Research, Nurse(Cancer Olson Maynard (University 5anFrancisco), of California, O'Farrell (Children's Orkin Stuart (University Seattle), Washington, of (Massachusetts Instituteof TerriOrr-Weaver Hospital,Boston), WilliamOtto ErinO'Shea(HarvardUniversity), Technology), of Birmingham, (Cancer UK),JohnOwen(University Research, Palade (University George Michigan), of Oxender UK),Dale (University San of California, (deceased), Panning Barbara WilliamW. (University Tucson), of Arizona, RoyParker Francisco), TerencePartridge Seattle), of Washington, Parson(University WilliamE.Paul(National (MRCClinical London), Centre, Sciences (MountSinaiHospital, Toronto), TonyPawson of Health), Institutes Cambridge), Biology, of Molecular HughPelham(MRCLaboratory Greg Philadelphia), Research, of Cancer RobertPerry(lnstitute (Cancer Research, GordonPeters University), Petsko(Brandeis JeremyPickettUniversity), UK),DavidPhillips(TheRockefeller JuliePitcher Australia), of Melbourne, Heaps(TheUniversity JeffreyPollard(AlbertEinstein (University CollegeLondon), BrucePonder TomPollard(YaleUniversity), of Medicine), College of California, DanPortnoy(University (University of Cambridge), (University Seattle), Washington, of JamesPriess Berkeley), (Duke (Tulane DalePurves University), DarwinProckop JordanRaff EfraimRacker(CornellUniversity), University), (University KlausRajewsky (Wellcome/CRC Institute,Cambridge), (University Elio Oxford)' of Ratcliffe George Germany), of Cologne, (University MartinRechsteiner (Harvard MedicalSchool), Raviola Institutefor Medical of Utah,SaltLakeCity),DavidRees(National (University San of California, Reichardt Louis London), Research, (YaleUniversity), ConlyRieder FredRichards Francisco), (Massachusetts Robbins Phillips (Wadsworth Albany), Center, of Reading, ElaineRobson(University Instituteof Technology), Rosenbaum Joel (The University), Rockefeller UK),RobertRoeder Toronto), (MountSinaiHospital, (YaleUniversity), JanetRossant JimRothman(Memorial of Health), Institutes JesseRoth(National (LaJollaCancer ErkkiRuoslahti Center), Cancer Sloan-Kettering General GaryRuvkun(Massachusetts Foundation), Research (NewYorkUniversity), AlanSachs DavidSabatini Hospital), of AlanSachs(University Berkeley), (University of California, (University North of Salmon Edward Berkeley), California, Peter University), ChapelHill),JoshuaSanes(Harvard Carolina, LisaSatterwhite(DukeUniversity Sarnow(StanfordUniversity), (University of California, HowardSchachman MedicalSchool), of Basel), (Biozentrum, University Schatz Gottfried Berkeley), Richard (University Berkeley), of California, RandySchekman (Cancer Schiavo Giampietro (Stanford University), Scheller (NewYorkUniversity Medical UK),JosephSchlessinger Research, (HebrewUniversity), RobertSchreiber MichaelSchramm Center), (Columbia JamesSchwartz lnstitute), (Scripps Clinicand Research

University), RonaldSchwartz (National Institutes of Health), Franqois (ENS, Schweisguth Paris), JohnScott(University of Manchester, UK),JohnSedat(University of California, San rJK),ZviSellinger Francisco), PeterSelby(CancerResearch, (HebrewUniversity, lsrael), (JohnsHopkins GreggSemenza University), peter PhilippeSengel(University of Grenoble, France), Shaw(JohnInnesInstitute, Norwich,UK),MichaelSheetz (Columbia University), DavidShima(Cancer Research, UK), SamuelSilverstein (Columbia University), KaiSimons(Maxplanck Instituteof Molecular CellBiologyandGenetics, Dresden), Melvin l. Simon(California Instituteof Technology), Jonathan Slack (Cancer Research, UK),AlisonSmith(JohnInnesInstitute, Norfolk, UK),JohnMaynardSmith(University of Sussex, UK),Frank Solomon(Massachusetts Instituteof Technology), Michael (University Solursh of lowa),BruceSpiegelman (Harvard Medical School), (Harvard TimothySpringer MedicalSchool), Mathias Sprinzl(University of Bayreuth, Germany), ScottStachel (University of California, Berkeley), (University AndrewStaehelin of Colorado, Boulder), (University DavidStandring of California, SanFrancisco), (University Margaret Stanley of Cambridge), MarthaStark(University of California, SanFrancisco), WilfredStein (HebrewUniversity, lsrael), (princeton MalcolmSteinberg University), PaulSternberg(California Instituteof Technology), ChuckStevens(TheSalkInstitute),MurrayStewart(MRC Laboratory of Molecular Biology, Cambridge), Monroe (University Strickberger of Missouri, St.Louis),RobertStroud (University of California, SanFrancisco), MichaelStryxer (University of California, SanFrancisco), WilliamSullivan (University of California, SantaCruz),DanielSzollosi (lnstitut Nationalde la Recherche Agronomique, France), JackSzostak (Massachusetts General Hospital), (Kyoto Masatoshi Takeichi University), CliffordTabin(HarvardMedicalSchool),Diethard Tautz(University of Cologne,Germany), JulieTheriot(Stanford University), RogerThomas(University of Bristol,UK),Vernon Thornton(King's CollegeLondon), (University CheryllTickle of Dundee, Scotland), JimTill(Ontario CancerInstitute, Toronto), LewisTilney(University of Pennsylvania), NickTonks(ColdSpring HarborLaboratory), (lnstitute AlainTownsend of Molecular

Medicine, JohnRadcliffe (Anthony Hospital, Oxford), PaulTravers NolanResearch Institute, (UMDNJ, London), RobertTrelstad RobertWoodJohnsonMedicalSchool), AnthonyTrewavas (Edinburgh University, Scotland), NigelUnwin(MRCLaboratory of Molecular (University Biology, Cambridge),Victor Vacquier of California, 5anDiego),HarryvanderWesten(Wageningen, The Netherlands), TomVanaman(University of Kentucky), Harold Varmus(Sloan-Kettering Institute), Alexander Varshavsky (California Instituteof Technology), MadhuWahi(University of California, 5anFrancisco), VirginiaWalbot(StanfordUniversity), FrankWalsh(Glaxo-Smithkline-Beecham, UK),TrevorWang(John InnesInstitute, Norwich, UK),Yu-Lie Wang(Worcester Foundation for Biomedical Research), AnneWarner(University College London),GrahamWarren(YaleUniversitySchoolof Medicine), (MountSinaiSchoolof Medicine), PaulWassarman FionaWatt (CancerResearch, (TheScripps UK),ClareWaterman-Storer Research Institute),FionaWatt(CancerResearch, UK),JohnWatts (JohnInnesInstitute, Norwich, UK),KlausWeber(MaxPlanck Institutefor Biophysical Chemistry, Gottingen), MartinWeigert (lnstitute of Cancer Research, Philadelphia), HaroldWeintraub (deceased), KarstenWeis(University of California, Berkeley), lrving (StanfordUniversity), Weissman (University JonathanWeissman (Stanford of California, SanFrancisco), NormanWessells University), JudyWhite(University of Virginia), StevenWest (Cancer Research, UK),WilliamWickner(Dartmouth College), Michael (ChironCorporation), Wilcox(deceased), LewisT.Williams KeithWillison(Chester BeattyLaboratories, London),JohnWilson (BaylorUniversity), AlanWolffe(deceased), RichardWolfenden (University of NorthCarolina, ChapelHill),Sandra Wolin(yale UniversitySchoolof Medicine), LewisWolpert(University College London),RickWood(CancerResearch, UK),AbrahamWorcel (University of Rochester), NickWright(Cancer Research, UK), JohnWyke(Beatson Institutefor CancerResearch, Glasgow), KeithYamamoto(University of California, 5anFrancisco), Charles Yocum(University of Michigan, AnnArbor),peter (UMDNJ, Yurchenco RobertWoodJohnsonMedicalSchool), Rosalind Zalin(University CollegeLondon), Patricia Zambryski (University of California, Berkeley).

A Noteto the Reader Structure of the Book Although the chapters of this book can be read independently of one another, they are arranged in a logical sequence of five parts. The first three chapters of Part I cover elementary principles and basic biochemistry. They can serve either as an introduction for those who have not studied biochemistry or as a refresher course for those who have. Part II deals with the storage, expression and transmission of genetic information. Part III deals with the principles of the main experimental methods for investigating cells. It is not necessary to read these two chapters in order to understand the later chapters, but a reader will find it a useful reference. Part IV discusses the internal organization of the cell. Part V follows the behavior of cells in multicellular systems, starting with cell-cell junctions and extracellular matrix and concluding with tvvo chapters on the immune system. Chapters 2l-25 can be found on the Media DVD-ROM which is packaged with each book, providing increased portability for students. End-of-Chapter Problems A selection of problems, written by Iohn Wilson and Tim Hunt, now appears in the text at the end of each chapter. The complete solutions to these problems can be found in Molecular Biology of the CelI, Fifth Edition: The Problems Book. References A concise list of selectedreferencesis included at the end of each chapter. These are arranged in alphabetical order under the main chapter section headings. These references frequently include the original papers in which important discoveries were first reported. Chapter 8 includes several tables giving the dates of crucial developments along with the names of the scientists involved. Elsewhere in the book the policy has been to avoid naming individual scientists. Media Codes Media codes are integrated throughout the text to indicate when relevant videos and animations are available on the DVD-ROM. The four-letter codes are enclosed in brackets and highlighted in color, like this .The interface for the CeII Biology Interactiue media player on the DVD-ROM contains a window where you enter the 4-letter code. lVhen the code is typed into the interface, the corresponding media item will load into the media player. GlossaryTerms Throughout the book, boldface type has been used to highlight key terms at the point in a chapter where the main discussion of them occurs. Italic is used to set off important terms with a lesser degree of emphasis. At the end of the book is the expanded glossary, covering technical terms that are part of the common currency of cell biology; it is intended as a first resort for a reader who encounters an unfamiliar term used without explanation. Nomenclature for Genes and Proteins Each species has its own conventions for naming genes; the only common feature is that they are always set in italics. In some species (such as humans)' gene names are spelled out all in capital letters; in other species (such as zebrafish),

case and rest in lower case; or (as in Drosophila) with different combinations of upper and lower case,according to whether the first mutant allele to be discovered gave a dominant or recessivephenotype. conventions for naming protein products are equally varied. This typographical chaos drives everyone crazy. lt is not just tiresome and absurd; it is also unsustainable. we cannot independently define a fresh convention for each of the next few million species whose genes we may wish to study. Moreover, there are many occasions, especially in a book such as this, where we need to refer to a gene generically,without specifliing the mouse version, the human version, the chick version, or the hippopotamus version, because they are all equivalent for the purposes of the discussion. \.A/hatconvention then should we use? We have decided in this book to cast aside the conventions for individual species and follow a uniform rule: we write all gene names, like the names of people and places, with the first letter in upper case and the rest in lower case,but all in- italics, thus: Apc, Bazooka, cdc2, Disheuelled,Egll. The corresponding protein, where it is named after the gene, will be written in the same way, but in roman rather than italic letters:Apc, Bazooka, cdc2, Dishevelled,Egll. lvhen it is necessary to specify the organism, this can be done with a prefix to the gene name. For completeness,we list a few further details of naming rules that we shall follow In some instances an added letter in the gene name is traditionally used to distinguish between genes that are related by function or evolution; foi those geneswe put that letter in upper case if it is usual to do so (LacZ,RecA,HoxA4). we use no hyphen to separate added letters or numbers from the rest of the name. Proteins are more of a problem. Many of them have names in their own right, assigned to them before the gene was named. such protein names take many forms, although most of them traditionally begin with a lower-case letter (actin, hemoglobin, catalase), Iike the names of ordinary substances (cheese, nylon), unless they are acronyms (such as GFB for Green Fluorescent protein, or BMP4, for Bone Morphogenetic Protein #4).To force all such protein names into a uniform style would do too much violence to established usages,and we shall simply write them in the traditional way (actin, GFB etc.). For thl corresponding gene names in all these cases,we shall nevertheless follow our standard rule: Actin, Hemoglobin, catalase, Bmp4, G/p. occasionally in our book we need to highlight a protein name by setting it in italics for emphasis; the intention will generally be clear from the context. For those who wish to know them, the Table below shows some of the official conventions for individual species-conventions that we shall mostlv vioIate in this book, in the manner shor.tm.

Mouse

Human Zebrafish Coenorhabditis Drosophila

Yeast Socch aromyces cerevisiae (budding yeast) Schizosacch aromyces pombe(fissionyeast) Arabidopsis E.coli

Hoxo4 Bmp4 integrinu-|, ltgal HOXA4 cyclops,cyc unc-6 sevenless, sey(named afterrecessive mutant phenotype) Defarmed,Dfd (named afterdominantmutant phenotype) CDC28 Cdc2 GAI uvrA

Hoxa4 BMP4 integrincr1 HOXA4 Cyclops, Cyc UNC-6 Sevenless, SEV

Deformed,DFD

Deformed, Dfd

Deformed, Dfd

Cdc28, Cdc28p Cdc2,Cdc2p GAI UvrA

Cdc28 Cdc2

Cdc28 Cdc2 GAI UvrA

HoxA4

HoxA4

Bmp4 lntegrin d,l,ltgal

BMP4 i n t e g r i na 1

HoxA4

HoxA4

Cyclops,Cyc Unc6

Cyclops,Cyc Unc6

Sevenless,Sev

Sevenless, Sev

Gai UvrA

Ancillaries Molecular Biolagy of the Cell,Fifih Edition:The ProblemsBook by Iohn Wilson and Tim Hunt (ISBN:978-0-8I 53-4f 10-9) The ProblemsBook is designedto help students appreciatethe ways in which experimentsand simple calculationscan lead to an understandingof how cells work. It providesproblemsto accompanyChaptersI-20 of MolecularBiologyof the Cell. Each chapter of problems is divided into sectionsthat correspondto those of the main textbook and review key terms, test for understandingbasic problems.MolecularBiologyof the Cell,Fifth concepts,and poseresearch-based Bookshould be useful for homework assignmentsand as Edition: TheProblem.s a basisfor classdiscussion.It could evenprovide ideasfor examquestions.Solutions for all of the problems are provided on the CD-ROMwhich accompanies the book. Solutionsfor the end-of-chapterproblemsin the main textbookare also found in TheProblemsBook. MBoCSMediaDVD-ROM The DVD included with everycopy of the book contains the figures,tables,and presentations, one for micrographsfrom the book,pre-loadedinto PowerPoint@ eachchapter.A separatefolder containsindividual versionsof eachfigure,table, and micrograph in JPEGformat. The panels are availablein PDF format. There arealsoover 125videos,animations,molecularstructuretutorials, and high-resolution micrographson the DVD.The authors have chosento include material that not only reinforcesbasicconceptsbut alsoexpandsthe contentand scope of the book.The multimedia can be accessedeither asindividual files or through the Cell BiologyInteractiuemedia player.As discussedabove,the media player has been programmedto workwith the Media Codesintegratedthroughout the book. A completetable of contentsand overviewof all electronicresourcesis contained in the MBoCSMedia Viewing Guide,a PDF file located on the root level of the DVD-ROMand in the Appendix of the media player.The DVD-ROM also containsChapters21-25which covermulticellularsystems.The chapters arein PDFformat and can be easilyprinted or searchedusingAdobe@Acrobat@ Readeror other PDF software. TeachingSupplements Upon request,teaching supplements for MolecularBiologt of the Cell are available to qualified instructors. MBoC1TransparencySet Provides200 frrll-color overheadacetatetransparenciesof the most important figuresfrom the book. MBoCSTestQuestions A selection of test questions will be available.Written by Kirsten Benjamin (AmyrisBiotechnologies,Emeryville,California)and Linda Huang (Universityof Boston),thesethoughtquestionswill teststudents'understandMassachusetts, ing of the chapter material. MBoCSLecture Outlines Lectureoutlines createdfrom the conceptheadsfor the text are provided. Garlnnd ScienceClasswirerM All of the teachingsupplementson the DVD-ROM(theseinclude figuresin PowerPointand JPEGformat;Chapters2l-25 in PDFformat; 125videos,animations, and movies)and the test questionsand Iectureoutlines areavailableto qualified instructorsonline at the GarlandScienceClasswire'"Web site.GarlandScience Classwire'"offersaccessto other instructional resourcesfrom all of the Garland Sciencetextbooks,and providesfreeonline coursemanagementtools. For addior tional information, pleasevisit http://www.classwire.com/garlandscience Inc.) (Classwire of ChalKree, is a trademark e-mail [email protected]. Adobe and Acrobat are either registeredtrademarks or trademarks of Adobe SystemsIncorporated in the United Statesandlor other countries PowerPoint is either a registeredtrademark or trademark of Microsoft Corporation in the United Statesand/or other countries

INTRODUCTION TOTHECELL

Chapter 1

Cells and Genomes The surface of our planet is populated by living things—curious, intricately organized chemical factories that take in matter from their surroundings and use these raw materials to generate copies of themselves. The living organisms appear extraordinarily diverse. What could be more different than a tiger and a piece of seaweed, or a bacterium and a tree? Yet our ancestors, knowing nothing of cells or DNA, saw that all these things had something in common. They called that something “life,” marveled at it, struggled to define it, and despaired of explaining what it was or how it worked in terms that relate to nonliving matter. The discoveries of the past century have not diminished the marvel—quite the contrary. But they have lifted away the mystery as to the nature of life. We can now see that all living things are made of cells, and that these units of living matter all share the same machinery for their most basic functions. Living things, though infinitely varied when viewed from the outside, are fundamentally similar inside. The whole of biology is a counterpoint between the two themes: astonishing variety in individual particulars; astonishing constancy in fundamental mechanisms. In this first chapter we begin by outlining the universal features common to all life on our planet. We then survey, briefly, the diversity of cells. And we see how, thanks to the common code in which the specifications for all living organisms are written, it is possible to read, measure, and decipher these specifications to achieve a coherent understanding of all the forms of life, from the smallest to the greatest.

1 In This Chapter THE UNIVERSAL FEATURES OF CELLS ON EARTH

1

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

11

GENETIC INFORMATION IN EUCARYOTES

26

THE UNIVERSAL FEATURES OF CELLS ON EARTH It is estimated that there are more than 10 million—perhaps 100 million—living species on Earth today. Each species is different, and each reproduces itself faithfully, yielding progeny that belong to the same species: the parent organism hands down information specifying, in extraordinary detail, the characteristics that the offspring shall have. This phenomenon of heredity is central to the definition of life: it distinguishes life from other processes, such as the growth of a crystal, or the burning of a candle, or the formation of waves on water, in which orderly structures are generated but without the same type of link between the peculiarities of parents and the peculiarities of offspring. Like the candle flame, the living organism consumes free energy to create and maintain its organization; but the free energy drives a hugely complex system of chemical processes that is specified by the hereditary information. Most living organisms are single cells; others, such as ourselves, are vast multicellular cities in which groups of cells perform specialized functions and are linked by intricate systems of communication. But in all cases, whether we discuss the solitary bacterium or the aggregate of more than 1013 cells that form a human body, the whole organism has been generated by cell divisions from a single cell. The single cell, therefore, is the vehicle for the hereditary information that defines the species (Figure 1–1). And specified by this information, the cell includes the machinery to gather raw materials from the environment, and to construct out of them a new cell in its own image, complete with a new copy of the hereditary information. Nothing less than a cell has this capability.

1

2

Chapter 1: Cells and Genomes

(A)

(B)

(E)

(C)

50 mm

50 mm

100 mm

(D)

(F)

Figure 1–1 The hereditary information in the fertilized egg cell determines the nature of the whole multicellular organism. (A and B) A sea urchin egg gives rise to a sea urchin. (C and D) A mouse egg gives rise to a mouse. (E and F) An egg of the seaweed Fucus gives rise to a Fucus seaweed. (A, courtesy of David McClay; B, courtesy of M. Gibbs, Oxford Scientific Films; C, courtesy of Patricia Calarco, from G. Martin, Science 209:768–776, 1980. With permission from AAAS; D, courtesy of O. Newman, Oxford Scientific Films; E and F, courtesy of Colin Brownlee.)

All Cells Store Their Hereditary Information in the Same Linear Chemical Code (DNA) Computers have made us familiar with the concept of information as a measurable quantity—a million bytes (to record a few hundred pages of text or an image from a digital camera), 600 million for the music on a CD, and so on. They have also made us well aware that the same information can be recorded in many different physical forms. As the computer world has evolved, the discs and tapes that we used 10 years ago for our electronic archives have become unreadable on present-day machines. Living cells, like computers, deal in information, and it is estimated that they have been evolving and diversifying for over 3.5 billion years. It is scarcely to be expected that they should all store their information in the same form, or that the archives of one type of cell should be readable by the information-handling machinery of another. And yet it is so. All living cells on Earth, without any known exception, store their hereditary information in the form of double-stranded molecules of DNA—long unbranched paired polymer chains, formed always of the same four types of monomers. These monomers have nicknames drawn from a four-letter alphabet—A, T, C, G—and they are strung together in a long linear sequence that encodes the genetic information, just as the sequence of 1s and 0s encodes the information in a computer file. We can take a piece of DNA from a human cell and insert it into a bacterium, or a piece of bacterial DNA and insert it into a human cell, and the information will be successfully read, interpreted, and copied. Using chemical methods, scientists can read out the complete sequence of monomers in any DNA molecule—extending for millions of nucleotides—and thereby decipher the hereditary information that each organism contains.

THE UNIVERSAL FEATURES OF CELLS ON EARTH

3

All Cells Replicate Their Hereditary Information by Templated Polymerization The mechanisms that make life possible depend on the structure of the doublestranded DNA molecule. Each monomer in a single DNA strand—that is, each nucleotide—consists of two parts: a sugar (deoxyribose) with a phosphate group attached to it, and a base, which may be either adenine (A), guanine (G), cytosine (C) or thymine (T) (Figure 1–2). Each sugar is linked to the next via the phosphate group, creating a polymer chain composed of a repetitive sugarphosphate backbone with a series of bases protruding from it. The DNA polymer is extended by adding monomers at one end. For a single isolated strand, these can, in principle, be added in any order, because each one links to the next in the same way, through the part of the molecule that is the same for all of them. In the living cell, however, DNA is not synthesized as a free strand in isolation, but on a template formed by a preexisting DNA strand. The bases protruding from the existing strand bind to bases of the strand being synthesized, according to a strict rule defined by the complementary structures of the bases: A binds to T, and C binds to G. This base-pairing holds fresh monomers in place and thereby controls the selection of which one of the four monomers shall be added to the growing strand next. In this way, a double-stranded structure is created, consisting of two exactly complementary sequences of As, Cs, Ts, and Gs. The two strands twist around each other, forming a double helix (Figure 1–2E).

(A)

building block of DNA

(D)

double-stranded DNA

phosphate sugar

+ sugar phosphate

(B)

G G

A

C

T

G

G

C

A

A

T

G

nucleotide

T

G

A

C

C

G

T

T

A

C

base

DNA strand

G

T

A

A

C

G

G

sugar-phosphate backbone

A

C

T

(E) (C)

hydrogen-bonded base pairs

DNA double helix

templated polymerization of new strand nucleotide monomers

C C

C

A

A G

G

T

T

A

G C

G

G

T

T G

T

T A

G G

C

A

A

A

G C

T

C

A

C

G A

C

C

A

Figure 1–2 DNA and its building blocks. (A) DNA is made from simple subunits, called nucleotides, each consisting of a sugar-phosphate molecule with a nitrogen-containing sidegroup, or base, attached to it. The bases are of four types (adenine, guanine, cytosine, and thymine), corresponding to four distinct nucleotides, labeled A, G, C, and T. (B) A single strand of DNA consists of nucleotides joined together by sugarphosphate linkages. Note that the individual sugar-phosphate units are asymmetric, giving the backbone of the strand a definite directionality, or polarity. This directionality guides the molecular processes by which the information in DNA is interpreted and copied in cells: the information is always “read” in a consistent order, just as written English text is read from left to right. (C) Through templated polymerization, the sequence of nucleotides in an existing DNA strand controls the sequence in which nucleotides are joined together in a new DNA strand; T in one strand pairs with A in the other, and G in one strand with C in the other. The new strand has a nucleotide sequence complementary to that of the old strand, and a backbone with opposite directionality: corresponding to the GTAA... of the original strand, it has ...TTAC. (D) A normal DNA molecule consists of two such complementary strands. The nucleotides within each strand are linked by strong (covalent) chemical bonds; the complementary nucleotides on opposite strands are held together more weakly, by hydrogen bonds. (E) The two strands twist around each other to form a double helix—a robust structure that can accommodate any sequence of nucleotides without altering its basic structure.

4

Chapter 1: Cells and Genomes template strand

new strand

Figure 1–3 The copying of genetic information by DNA replication. In this process, the two strands of a DNA double helix are pulled apart, and each serves as a template for synthesis of a new complementary strand.

new strand parent DNA double helix

template strand

The bonds between the base pairs are weak compared with the sugar-phosphate links, and this allows the two DNA strands to be pulled apart without breakage of their backbones. Each strand then can serve as a template, in the way just described, for the synthesis of a fresh DNA strand complementary to itself—a fresh copy, that is, of the hereditary information (Figure 1–3). In different types of cells, this process of DNA replication occurs at different rates, with different controls to start it or stop it, and different auxiliary molecules to help it along. But the basics are universal: DNA is the information store, and templated polymerization is the way in which this information is copied throughout the living world.

All Cells Transcribe Portions of Their Hereditary Information into the Same Intermediary Form (RNA) To carry out its information-bearing function, DNA must do more than copy itself. It must also express its information, by letting it guide the synthesis of other molecules in the cell. This also occurs by a mechanism that is the same in all living organisms, leading first and foremost to the production of two other key classes of polymers: RNAs and proteins. The process (discussed in detail in Chapters 6 and 7) begins with a templated polymerization called transcription, in which segments of the DNA sequence are used as templates for the synthesis of shorter molecules of the closely related polymer ribonucleic acid, or RNA. Later, in the more complex process of translation, many of these RNA molecules direct the synthesis of polymers of a radically different chemical class—the proteins (Figure 1–4). In RNA, the backbone is formed of a slightly different sugar from that of DNA—ribose instead of deoxyribose—and one of the four bases is slightly different—uracil (U) in place of thymine (T); but the other three bases—A, C, and G—are the same, and all four bases pair with their complementary counterparts in DNA—the A, U, C, and G of RNA with the T, A, G, and C of DNA. During transcription, RNA monomers are lined up and selected for polymerization on a template strand of DNA, just as DNA monomers are selected during replication. The outcome is a polymer molecule whose sequence of nucleotides faithfully represents a part of the cell’s genetic information, even though written in a slightly different alphabet, consisting of RNA monomers instead of DNA monomers. The same segment of DNA can be used repeatedly to guide the synthesis of many identical RNA transcripts. Thus, whereas the cell’s archive of genetic information in the form of DNA is fixed and sacrosanct, the RNA transcripts are mass-produced and disposable (Figure 1–5). As we shall see, these transcripts function as intermediates in the transfer of genetic information: they mainly serve as messenger RNA (mRNA) to guide the synthesis of proteins according to the genetic instructions stored in the DNA. RNA molecules have distinctive structures that can also give them other specialized chemical capabilities. Being single-stranded, their backbone is flexible, so that the polymer chain can bend back on itself to allow one part of the

DNA synthesis (replication) DNA

RNA synthesis (transcription) RNA

protein synthesis (translation) PROTEIN

amino acids

Figure 1–4 From DNA to protein. Genetic information is read out and put to use through a two-step process. First, in transcription, segments of the DNA sequence are used to guide the synthesis of molecules of RNA. Then, in translation, the RNA molecules are used to guide the synthesis of molecules of protein.

THE UNIVERSAL FEATURES OF CELLS ON EARTH

5 RNA MOLECULES AS EXPENDABLE INFORMATION CARRIERS

DOUBLE-STRANDED DNA AS INFORMATION ARCHIVE TRANSCRIPTION

strand used as a template to direct RNA synthesis many identical RNA transcripts

molecule to form weak bonds with another part of the same molecule. This occurs when segments of the sequence are locally complementary: a ...GGGG... segment, for example, will tend to associate with a ...CCCC... segment. These types of internal associations can cause an RNA chain to fold up into a specific shape that is dictated by its sequence (Figure 1–6). The shape of the RNA molecule, in turn, may enable it to recognize other molecules by binding to them selectively—and even, in certain cases, to catalyze chemical changes in the molecules that are bound. As we see in Chapter 6, a few chemical reactions catalyzed by RNA molecules are crucial for several of the most ancient and fundamental processes in living cells, and it has been suggested that more extensive catalysis by RNA played a central part in the early evolution of life.

Figure 1–5 How genetic information is broadcast for use inside the cell. Each cell contains a fixed set of DNA molecules—its archive of genetic information. A given segment of this DNA guides the synthesis of many identical RNA transcripts, which serve as working copies of the information stored in the archive. Many different sets of RNA molecules can be made by transcribing selected parts of a long DNA sequence, allowing each cell to use its information store differently.

All Cells Use Proteins as Catalysts Protein molecules, like DNA and RNA molecules, are long unbranched polymer chains, formed by stringing together monomeric building blocks drawn from a standard repertoire that is the same for all living cells. Like DNA and RNA, they carry information in the form of a linear sequence of symbols, in the same way as a human message written in an alphabetic script. There are many different protein molecules in each cell, and—leaving out the water—they form most of the cell’s mass. The monomers of protein, the amino acids, are quite different from those of DNA and RNA, and there are 20 types, instead of 4. Each amino acid is built around the same core structure through which it can be linked in a standard way to any other amino acid in the set; attached to this core is a side group that gives each amino acid a distinctive chemical character. Each of the protein molecules, or polypeptides, created by joining amino acids in a particular sequence folds into a precise three-dimensional form with reactive sites on its surface (Figure

G U A U

G C C A G U U A G C C G

C A U A

C

A G C U U A A A

CC U

G GG

(A)

A

U C G A A U U U

A U G C A U

U A C G U A

AAA UU

U (B)

Figure 1–6 The conformation of an RNA molecule. (A) Nucleotide pairing between different regions of the same RNA polymer chain causes the molecule to adopt a distinctive shape. (B) The three-dimensional structure of an actual RNA molecule, from hepatitis delta virus, that catalyzes RNA strand cleavage. The blue ribbon represents the sugarphosphate backbone; the bars represent base pairs. (B, based on A.R. Ferré D’Amaré, K. Zhou and J.A. Doudna, Nature 395:567–574, 1998. With permission from Macmillan Publishers Ltd.)

6

Chapter 1: Cells and Genomes polysaccharide chain + + catalytic site lysozyme molecule (B)

(A) lysozyme

Figure 1–7 How a protein molecule acts as catalyst for a chemical reaction. (A) In a protein molecule the polymer chain folds up to into a specific shape defined by its amino acid sequence. A groove in the surface of this particular folded molecule, the enzyme lysozyme, forms a catalytic site. (B) A polysaccharide molecule (red)—a polymer chain of sugar monomers—binds to the catalytic site of lysozyme and is broken apart, as a result of a covalent bond-breaking reaction catalyzed by the amino acids lining the groove.

1–7A). These amino acid polymers thereby bind with high specificity to other molecules and act as enzymes to catalyze reactions that make or break covalent bonds. In this way they direct the vast majority of chemical processes in the cell (Figure 1–7B). Proteins have many other functions as well—maintaining structures, generating movements, sensing signals, and so on—each protein molecule performing a specific function according to its own genetically specified sequence of amino acids. Proteins, above all, are the molecules that put the cell’s genetic information into action. Thus, polynucleotides specify the amino acid sequences of proteins. Proteins, in turn, catalyze many chemical reactions, including those by which new DNA molecules are synthesized, and the genetic information in DNA is used to make both RNA and proteins. This feedback loop is the basis of the autocatalytic, self-reproducing behavior of living organisms (Figure 1–8).

All Cells Translate RNA into Protein in the Same Way The translation of genetic information from the 4-letter alphabet of polynucleotides into the 20-letter alphabet of proteins is a complex process. The rules of this translation seem in some respects neat and rational, in other respects strangely arbitrary, given that they are (with minor exceptions) identical in all living things. These arbitrary features, it is thought, reflect frozen accidents in the early history of life—chance properties of the earliest organisms that were passed on by heredity and have become so deeply embedded in the constitution of all living cells that they cannot be changed without disastrous effects. The information in the sequence of a messenger RNA molecule is read out in groups of three nucleotides at a time: each triplet of nucleotides, or codon, specifies (codes for) a single amino acid in a corresponding protein. Since there are 64 (= 4 ¥ 4 ¥ 4) possible codons, all of which occur in nature, but only 20 amino acids, there are necessarily many cases in which several codons correspond to the same amino acid. The code is read out by a special class of small RNA molecules, the transfer RNAs (tRNAs). Each type of tRNA becomes attached at one end to a specific amino acid, and displays at its other end a specific sequence of three nucleotides—an anticodon—that enables it to recognize, through base-pairing, a particular codon or subset of codons in mRNA (Figure 1–9). For synthesis of protein, a succession of tRNA molecules charged with their appropriate amino acids have to be brought together with an mRNA molecule and matched up by base-pairing through their anticodons with each of its successive codons. The amino acids then have to be linked together to extend the growing protein chain, and the tRNAs, relieved of their burdens, have to be released. This whole complex of processes is carried out by a giant multimolecular machine, the ribosome, formed of two main chains of RNA, called ribosomal RNAs

THE UNIVERSAL FEATURES OF CELLS ON EARTH

7

amino acids

Figure 1–8 Life as an autocatalytic process. Polynucleotides (nucleotide polymers) and proteins (amino acid polymers) provide the sequence information and the catalytic functions that serve—through a complex set of chemical reactions—to bring about the synthesis of more polynucleotides and proteins of the same types.

nucleotides

catalytic function

sequence information

proteins

polynucleotides

(rRNAs), and more than 50 different proteins. This evolutionarily ancient molecular juggernaut latches onto the end of an mRNA molecule and then trundles along it, capturing loaded tRNA molecules and stitching together the amino acids they carry to form a new protein chain (Figure 1–10).

The Fragment of Genetic Information Corresponding to One Protein Is One Gene DNA molecules as a rule are very large, containing the specifications for thousands of proteins. Individual segments of the entire DNA sequence are transcribed into separate mRNA molecules, with each segment coding for a different protein. Each such DNA segment represents one gene. A complication is that RNA molecules transcribed from the same DNA segment can often be processed in more than one way, so as to give rise to a set of alternative versions of a protein, especially in more complex cells such as those of plants and animals. A gene therefore is defined, more generally, as the segment of DNA sequence corresponding to a single protein or set of alternative protein variants (or to a single catalytic or structural RNA molecule for those genes that produce RNA but not protein). In all cells, the expression of individual genes is regulated: instead of manufacturing its full repertoire of possible proteins at full tilt all the time, the cell adjusts the rate of transcription and translation of different genes independently, according to need. Stretches of regulatory DNA are interspersed among the segments

Figure 1–9 Transfer RNA. (A) A tRNA molecule specific for the amino acid tryptophan. One end of the tRNA molecule has tryptophan attached to it, while the other end displays the triplet nucleotide sequence CCA (its anticodon), which recognizes the tryptophan codon in messenger RNA molecules. (B) The three-dimensional structure of the tryptophan tRNA molecule. Note that the codon and the anticodon in (A) are in antiparallel orientations, like the two strands in a DNA double helix (see Figure 1–2), so that the sequence of the anticodon in the tRNA is read from right to left, while that of the codon in the mRNA is read from left to right.

amino acid (tryptophan)

specific tRNA molecule tRNA binds to its codon in mRNA A

C

C

anticodon

A

C

C

U

G

G

base-pairing

anticodon

codon in mRNA (A)

NET RESULT: AMINO ACID IS SELECTED BY ITS CODON

(B)

8

Chapter 1: Cells and Genomes Figure 1–10 A ribosome at work. (A) The diagram shows how a ribosome moves along an mRNA molecule, capturing tRNA molecules that match the codons in the mRNA and using them to join amino acids into a protein chain. The mRNA specifies the sequence of amino acids. (B) The threedimensional structure of a bacterial ribosome (pale green and blue), moving along an mRNA molecule (orange beads), with three tRNA molecules (yellow, green, and pink) at different stages in their process of capture and release. The ribosome is a giant assembly of more than 50 individual protein and RNA molecules. (B, courtesy of Joachim Frank, Yanhong Li and Rajendra Agarwal.)

growing polypeptide chain incoming tRNA loaded with amino acid

STEP 1 2

1

2

3

4

P

A

3

4

STEP 2

that code for protein, and these noncoding regions bind to special protein molecules that control the local rate of transcription (Figure 1–11). Other noncoding DNA is also present, some of it serving, for example, as punctuation, defining where the information for an individual protein begins and ends. The quantity and organization of the regulatory and other noncoding DNA vary widely from one class of organisms to another, but the basic strategy is universal. In this way, the genome of the cell—that is, the total of its genetic information as embodied in its complete DNA sequence—dictates not only the nature of the cell’s proteins, but also when and where they are to be made.

two subunits of ribosome

3

2 1

mRNA

4

2

P 3

A 4

STEP 3 2

3

1

4

Life Requires Free Energy 2

A living cell is a dynamic chemical system, operating far from chemical equilibrium. For a cell to grow or to make a new cell in its own image, it must take in free energy from the environment, as well as raw materials, to drive the necessary synthetic reactions. This consumption of free energy is fundamental to life. When it stops, a cell decays towards chemical equilibrium and soon dies. Genetic information is also fundamental to life. Is there any connection? The answer is yes: free energy is required for the propagation of information. For example, to specify one bit of information—that is, one yes/no choice between two equally probable alternatives—costs a defined amount of free energy that can be calculated. The quantitative relationship involves some deep reasoning and depends on a precise definition of the term “free energy,” discussed in Chapter 2. The basic idea, however, is not difficult to understand intuitively. Picture the molecules in a cell as a swarm of objects endowed with thermal energy, moving around violently at random, buffeted by collisions with one another. To specify genetic information—in the form of a DNA sequence, for example—molecules from this wild crowd must be captured, arranged in a specific order defined by some preexisting template, and linked together in a fixed relationship. The bonds that hold the molecules in their proper places on the template and join them together must be strong enough to resist the disordering effect of thermal motion. The process is driven forward by consumption of free energy, which is needed to ensure that the correct bonds are made, and made robustly. In the simplest case, the molecules can be compared with spring-loaded traps, ready to snap into a more stable, lower-energy attached state when they meet their proper partners; as they snap together into the bonded arrangement, their available stored energy—their free energy—like the energy of the spring in the trap, is released and dissipated as heat. In a cell, the chemical processes underlying information transfer are more complex, but the same basic principle applies: free energy has to be spent on the creation of order. To replicate its genetic information faithfully, and indeed to make all its complex molecules according to the correct specifications, the cell therefore requires free energy, which has to be imported somehow from the surroundings.

3

STEP 4 2

3

1

4

3

4

4

new tRNA bringing next amino acid 5

4

5

STEP 1 2

3

1

3

(A)

All Cells Function as Biochemical Factories Dealing with the Same Basic Molecular Building Blocks Because all cells make DNA, RNA, and protein, and these macromolecules are composed of the same set of subunits in every case, all cells have to contain and

4

(B)

THE UNIVERSAL FEATURES OF CELLS ON EARTH

9

manipulate a similar collection of small molecules, including simple sugars, nucleotides, and amino acids, as well as other substances that are universally required for their synthesis. All cells, for example, require the phosphorylated nucleotide ATP (adenosine triphosphate) as a building block for the synthesis of DNA and RNA; and all cells also make and consume this molecule as a carrier of free energy and phosphate groups to drive many other chemical reactions. Although all cells function as biochemical factories of a broadly similar type, many of the details of their small-molecule transactions differ, and it is not as easy as it is for the informational macromolecules to point out the features that are strictly universal. Some organisms, such as plants, require only the simplest of nutrients and harness the energy of sunlight to make from these almost all their own small organic molecules; other organisms, such as animals, feed on living things and obtain many of their organic molecules ready-made. We return to this point below.

All Cells Are Enclosed in a Plasma Membrane Across Which Nutrients and Waste Materials Must Pass There is, however, at least one other feature of cells that is universal: each one is enclosed by a membrane—the plasma membrane. This container acts as a selective barrier that enables the cell to concentrate nutrients gathered from its environment and retain the products it synthesizes for its own use, while excreting its waste products. Without a plasma membrane, the cell could not maintain its integrity as a coordinated chemical system. The molecules forming this membrane have the simple physico-chemical property of being amphiphilic—that is, consisting of one part that is hydrophobic (water-insoluble) and another part that is hydrophilic (water-soluble). Such molecules placed in water aggregate spontaneously, arranging their hydrophobic portions to be as much in contact with one another as possible to hide them from the water, while keeping their hydrophilic portions exposed. Amphiphilic molecules of appropriate shape, such as the phospholipid molecules that comprise most of the plasma membrane, spontaneously aggregate in water to form a bilayer that creates small closed vesicles (Figure 1–12). The phenomenon can be demonstrated in a test tube by simply mixing phospholipids and water together; under appropriate conditions, small vesicles form whose aqueous contents are isolated from the external medium. Although the chemical details vary, the hydrophobic tails of the predominant membrane molecules in all cells are hydrocarbon polymers (–CH2–CH2–CH2–), and their spontaneous assembly into a bilayered vesicle is but one of many examples of an important general principle: cells produce molecules whose chemical properties cause them to self-assemble into the structures that a cell needs. The cell boundary cannot be totally impermeable. If a cell is to grow and reproduce, it must be able to import raw materials and export waste across its plasma membrane. All cells therefore have specialized proteins embedded in their membrane that transport specific molecules from one side to the other (Figure 1–13). Some of these membrane transport proteins, like some of the proteins that catalyze the fundamental small-molecule reactions inside the cell,

Figure 1–11 Gene regulation by protein binding to regulatory DNA. (A) A diagram of a small portion of the genome of the bacterium Escherichia coli, containing genes (called LacI, LacZ, LacY, and LacA) coding for four different proteins. The protein-coding DNA segments (red) have regulatory and other noncoding DNA segments (yellow) between them. (B) An electron micrograph of DNA from this region, with a protein molecule (encoded by the LacI gene) bound to the regulatory segment; this protein controls the rate of transcription of the LacZ, LacY, and LacA genes. (C) A drawing of the structures shown in (B). (B, courtesy of Jack Griffith.)

site of protein binding shown in micrograph (B) below LacI

LacZ

noncoding DNA segments LacY LacA 2000 nucleotide pairs

(A)

(B)

protein bound to regulatory segment of DNA (C)

segment of DNA coding for protein

10

Chapter 1: Cells and Genomes

have been so well preserved over the course of evolution that we can recognize the family resemblances between them in comparisons of even the most distantly related groups of living organisms. The transport proteins in the membrane largely determine which molecules enter the cell, and the catalytic proteins inside the cell determine the reactions that those molecules undergo. Thus, by specifying the proteins that the cell is to manufacture, the genetic information recorded in the DNA sequence dictates the entire chemistry of the cell; and not only its chemistry, but also its form and its behavior, for these too are chiefly constructed and controlled by the cell’s proteins.

phospholipid monolayer

OIL

phospholipid bilayer

A Living Cell Can Exist with Fewer Than 500 Genes The basic principles of biological information transfer are simple enough, but how complex are real living cells? In particular, what are the minimum requirements? We can get a rough indication by considering a species that has one of the smallest known genomes—the bacterium Mycoplasma genitalium (Figure 1–14). This organism lives as a parasite in mammals, and its environment provides it with many of its small molecules ready-made. Nevertheless, it still has to make all the large molecules—DNA, RNAs, and proteins—required for the basic processes of heredity. It has only about 480 genes in its genome of 580,070 nucleotide pairs, representing 145,018 bytes of information—about as much as it takes to record the text of one chapter of this book. Cell biology may be complicated, but it is not impossibly so. The minimum number of genes for a viable cell in today’s environments is probably not less than 200–300, although there are only about 60 genes in the core set shared by all living species without any known exception.

H+

plasma membrane OUTSIDE INSIDE

sugars (13)

(A)

amino acids, peptides, amines (14)

ions (16)

other (3)

(B)

Figure 1–13 Membrane transport proteins. (A) Structure of a molecule of bacteriorhodopsin, from the archaeon (archaebacterium) Halobacterium halobium. This transport protein uses the energy of absorbed light to pump protons (H+ ions) out of the cell. The polypeptide chain threads to and fro across the membrane; in several regions it is twisted into a helical conformation, and the helical segments are arranged to form the walls of a channel through which ions are transported. (B) Diagram of the set of transport proteins found in the membrane of the bacterium Thermotoga maritima. The numbers in parentheses refer to the number of different membrane transport proteins of each type. Most of the proteins within each class are evolutionarily related to one another and to their    counterparts in other species.

WATER

Figure 1–12 Formation of a membrane by amphiphilic phospholipid molecules. These have a hydrophilic (water-loving, phosphate) head group and a hydrophobic (water-avoiding, hydrocarbon) tail. At an interface between oil and water, they arrange themselves as a single sheet with their head groups facing the water and their tail groups facing the oil. When immersed in water, they aggregate to form bilayers enclosing aqueous compartments.

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE Figure 1–14 Mycoplasma genitalium. (A) Scanning electron micrograph showing the irregular shape of this small bacterium, reflecting the lack of any rigid wall. (B) Cross section (transmission electron micrograph) of a Mycoplasma cell. Of the 477 genes of Mycoplasma genitalium, 37 code for transfer, ribosomal, and other nonmessenger RNAs. Functions are known, or can be guessed, for 297 of the genes coding for protein: of these, 153 are involved in replication, transcription, translation, and related processes involving DNA, RNA, and protein; 29 in the membrane and surface structures of the cell; 33 in the transport of nutrients and other molecules across the membrane; 71 in energy conversion and the synthesis and degradation of small molecules; and 11 in the regulation of cell division and other processes. (A, from S. Razin et al., Infect. Immun. 30:538–546, 1980. With permission from the American Society for Microbiology; B, courtesy of Roger Cole, in Medical Microbiology, 4th ed. [S. Baron ed.]. Galveston: University of Texas Medical Branch, 1996.)

11

(A)

5 mm

Summary Living organisms reproduce themselves by transmitting genetic information to their progeny. The individual cell is the minimal self-reproducing unit, and is the vehicle for transmission of the genetic information in all living species. Every cell on our planet stores its genetic information in the same chemical form—as double-stranded DNA. The cell replicates its information by separating the paired DNA strands and using each as a template for polymerization to make a new DNA strand with a complementary sequence of nucleotides. The same strategy of templated polymerization is used to transcribe portions of the information from DNA into molecules of the closely related polymer, RNA. These in turn guide the synthesis of protein molecules by the more complex machinery of translation, involving a large multimolecular machine, the ribosome, which is itself composed of RNA and protein. Proteins are the principal catalysts for almost all the chemical reactions in the cell; their other functions include the selective import and export of small molecules across the plasma membrane that forms the cell’s boundary. The specific function of each protein depends on its amino acid sequence, which is specified by the nucleotide sequence of a corresponding segment of the DNA—the gene that codes for that protein. In this way, the genome of the cell determines its chemistry; and the chemistry of every living cell is fundamentally similar, because it must provide for the synthesis of DNA, RNA, and protein. The simplest known cells have just under 500 genes.

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE The success of living organisms based on DNA, RNA, and protein, out of the infinitude of other chemical forms that we might conceive of, has been spectacular. They have populated the oceans, covered the land, infiltrated the Earth’s crust, and molded the surface of our planet. Our oxygen-rich atmosphere, the deposits of coal and oil, the layers of iron ores, the cliffs of chalk and limestone and marble—all these are products, directly or indirectly, of past biological activity on Earth. Living things are not confined to the familiar temperate realm of land, water, and sunlight inhabited by plants and plant-eating animals. They can be found in the darkest depths of the ocean, in hot volcanic mud, in pools beneath the frozen surface of the Antarctic, and buried kilometers deep in the Earth’s crust. The creatures that live in these extreme environments are generally unfamiliar, not only because they are inaccessible, but also because they are mostly microscopic. In more homely habitats, too, most organisms are too small for us to see without special equipment: they tend to go unnoticed, unless they cause a disease or rot the timbers of our houses. Yet microorganisms make up most of the

(B)

0.2 mm

12

Chapter 1: Cells and Genomes

total mass of living matter on our planet. Only recently, through new methods of molecular analysis and specifically through the analysis of DNA sequences, have we begun to get a picture of life on Earth that is not grossly distorted by our biased perspective as large animals living on dry land. In this section we consider the diversity of organisms and the relationships among them. Because the genetic information for every organism is written in the universal language of DNA sequences, and the DNA sequence of any given organism can be obtained by standard biochemical techniques, it is now possible to characterize, catalogue, and compare any set of living organisms with reference to these sequences. From such comparisons we can estimate the place of each organism in the family tree of living species—the ‘tree of life’. But before describing what this approach reveals, we need first to consider the routes by which cells in different environments obtain the matter and energy they require to survive and proliferate, and the ways in which some classes of organisms depend on others for their basic chemical needs.

Cells Can Be Powered by a Variety of Free Energy Sources Living organisms obtain their free energy in different ways. Some, such as animals, fungi, and the bacteria that live in the human gut, get it by feeding on other living things or the organic chemicals they produce; such organisms are called organotrophic (from the Greek word trophe, meaning “food”). Others derive their energy directly from the nonliving world. These fall into two classes: those that harvest the energy of sunlight, and those that capture their energy from energy-rich systems of inorganic chemicals in the environment (chemical systems that are far from chemical equilibrium). Organisms of the former class are called phototrophic (feeding on sunlight); those of the latter are called lithotrophic (feeding on rock). Organotrophic organisms could not exist without these primary energy converters, which are the most plentiful form of life. Phototrophic organisms include many types of bacteria, as well as algae and plants, on which we—and virtually all the living things that we ordinarily see around us—depend. Phototrophic organisms have changed the whole chemistry of our environment: the oxygen in the Earth’s atmosphere is a by-product of their biosynthetic activities. Lithotrophic organisms are not such an obvious feature of our world, because they are microscopic and mostly live in habitats that humans do not frequent—deep in the ocean, buried in the Earth’s crust, or in various other inhospitable environments. But they are a major part of the living world, and are especially important in any consideration of the history of life on Earth. Some lithotrophs get energy from aerobic reactions, which use molecular oxygen from the environment; since atmospheric O2 is ultimately the product of living organisms, these aerobic lithotrophs are, in a sense, feeding on the products of past life. There are, however, other lithotrophs that live anaerobically, in places where little or no molecular oxygen is present, in circumstances similar to those that must have existed in the early days of life on Earth, before oxygen had accumulated. The most dramatic of these sites are the hot hydrothermal vents found deep down on the floor of the Pacific and Atlantic Oceans, in regions where the ocean floor is spreading as new portions of the Earth’s crust form by a gradual upwelling of material from the Earth’s interior (Figure 1–15). Downward-percolating seawater is heated and driven back upward as a submarine geyser, carrying with it a current of chemicals from the hot rocks below. A typical cocktail might include H2S, H2, CO, Mn2+, Fe2+, Ni2+, CH2, NH4+, and phosphorus-containing compounds. A dense population of microbes lives in the neighborhood of the vent, thriving on this austere diet and harvesting free energy from reactions between the available chemicals. Other organisms—clams, mussels, and giant marine worms—in turn live off the microbes at the vent, forming an entire ecosystem analogous to the system of plants and animals that we belong to, but powered by geochemical energy instead of light (Figure 1–16).

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

13

SEA dark cloud of hot mineral-rich water hydrothermal vent

anaerobic lithotrophic bacteria invertebrate animal community

chimney made from precipitated metal sulfides

2–3°C

sea floor

Figure 1–15 The geology of a hot hydrothermal vent in the ocean floor. Water percolates down toward the hot molten rock upwelling from the Earth’s interior and is heated and driven back upward, carrying minerals leached from the hot rock. A temperature gradient is set up, from more than 350°C near the core of the vent, down to 2–3°C in the surrounding ocean. Minerals precipitate from the water as it cools, forming a chimney. Different classes of organisms, thriving at different temperatures, live in different neighborhoods of the chimney. A typical chimney might be a few meters tall, with a flow rate of 1–2 m/sec.

350°C contour

percolation of seawater

hot mineral solution

hot basalt

Some Cells Fix Nitrogen and Carbon Dioxide for Others To make a living cell requires matter, as well as free energy. DNA, RNA, and protein are composed of just six elements: hydrogen, carbon, nitrogen, oxygen, sulfur, and phosphorus. These are all plentiful in the nonliving environment, in the Earth’s rocks, water, and atmosphere, but not in chemical forms that allow easy incorporation into biological molecules. Atmospheric N2 and CO2, in particular, are extremely unreactive, and a large amount of free energy is required to drive the reactions that use these inorganic molecules to make the organic compounds needed for further biosynthesis—that is, to fix nitrogen and carbon dioxide, so as to make N and C available to living organisms. Many types of living cells lack the biochemical machinery to achieve this fixation, and rely on other classes of cells to do the job for them. We animals depend on plants for our supplies of geochemical energy and inorganic raw materials

bacteria

multicellular animals e.g., tubeworms

1m

Figure 1–16 Living organisms at a hot hydrothermal vent. Close to the vent, at temperatures up to about 120°C, various lithotrophic species of bacteria and archaea (archaebacteria) live, directly fuelled by geochemical energy. A little further away, where the temperature is lower, various invertebrate animals live by feeding on these microorganisms. Most remarkable are the giant (2-meter) tube worms, which, rather than feed on the lithotrophic cells, live in symbiosis with them: specialized organs in the worms harbor huge numbers of symbiotic sulfur-oxidizing bacteria. These bacteria harness geochemical energy and supply nourishment to their hosts, which have no mouth, gut, or anus. The dependence of the tube worms on the bacteria for the harnessing of geothermal energy is analogous to the dependence of plants on chloroplasts for the harnessing of solar energy, discussed later in this chapter. The tube worms, however, are thought to have evolved from more conventional animals, and to have become secondarily adapted to life at hydrothermal vents. (Courtesy of Dudley Foster, Woods Hole Oceanographic Institution.)

14

Chapter 1: Cells and Genomes Figure 1–17 Shapes and sizes of some bacteria. Although most are small, as shown, measuring a few micrometers in linear dimension, there are also some giant species. An extreme example (not shown) is the cigar-shaped bacterium Epulopiscium fishelsoni, which lives in the gut of a surgeonfish and can be up to 600 mm long.

2 mm spherical cells e.g., Streptococcus

rod-shaped cells e.g., Escherichia coli, Vibrio cholerae

the smallest cells e.g., Mycoplasma, Spiroplasma

spiral cells e.g., Treponema pallidum

organic carbon and nitrogen compounds. Plants in turn, although they can fix carbon dioxide from the atmosphere, lack the ability to fix atmospheric nitrogen, and they depend in part on nitrogen-fixing bacteria to supply their need for nitrogen compounds. Plants of the pea family, for example, harbor symbiotic nitrogen-fixing bacteria in nodules in their roots. Living cells therefore differ widely in some of the most basic aspects of their biochemistry. Not surprisingly, cells with complementary needs and capabilities have developed close associations. Some of these associations, as we see below, have evolved to the point where the partners have lost their separate identities altogether: they have joined forces to form a single composite cell.

The Greatest Biochemical Diversity Exists Among Procaryotic Cells From simple microscopy, it has long been clear that living organisms can be classified on the basis of cell structure into two groups: the eucaryotes and the procaryotes. Eucaryotes keep their DNA in a distinct membrane-enclosed intracellular compartment called the nucleus. (The name is from the Greek, meaning “truly nucleated,” from the words eu, “well” or “truly,” and karyon, “kernel” or “nucleus”.) Procaryotes have no distinct nuclear compartment to house their DNA. Plants, fungi, and animals are eucaryotes; bacteria are procaryotes, as are archaea—a separate class of procaryotic cells, discussed below. Most procaryotic cells are small and simple in outward appearance (Figure 1–17), and they live mostly as independent individuals or in loosely organized communities, rather than as multicellular organisms. They are typically spherical or rod-shaped and measure a few micrometers in linear dimension. They often have a tough protective coat, called a cell wall, beneath which a plasma membrane encloses a single cytoplasmic compartment containing DNA, RNA, proteins, and the many small molecules needed for life. In the electron microscope, this cell interior appears as a matrix of varying texture without any discernible organized internal structure (Figure 1–18). Figure 1–18 The structure of a bacterium. (A) The bacterium Vibrio cholerae, showing its simple internal organization. Like many other species, Vibrio has a helical appendage at one end—a flagellum—that rotates as a propeller to drive the cell forward. (B) An electron micrograph of a longitudinal section through the widely studied bacterium Escherichia coli (E. coli). This is related to Vibrio but has many flagella (not visible in this section) distributed over its surface. The cell’s DNA is concentrated in the lightly stained region. (B, courtesy of E. Kellenberger.) plasma membrane

DNA

cell wall

flagellum

1 mm

ribosomes (A)

(B)

1 mm

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

H

S

15

V

10 mm

Figure 1–19 The phototrophic bacterium Anabaena cylindrica viewed in the light microscope. The cells of this species form long, multicellular filaments. Most of the cells (labeled V) perform photosynthesis, while others become specialized for nitrogen fixation (labeled H), or develop into resistant spores (labeled S). (Courtesy of Dave G. Adams.)

Procaryotic cells live in an enormous variety of ecological niches, and they are astonishingly varied in their biochemical capabilities—far more so than eucaryotic cells. Organotrophic species can utilize virtually any type of organic molecule as food, from sugars and amino acids to hydrocarbons and methane gas. Phototrophic species (Figure 1–19) harvest light energy in a variety of ways, some of them generating oxygen as a byproduct, others not. Lithotrophic species can feed on a plain diet of inorganic nutrients, getting their carbon from CO2, and relying on H2S to fuel their energy needs (Figure 1–20)—or on H2, or Fe2+, or elemental sulfur, or any of a host of other chemicals that occur in the environment. Many parts of this world of microscopic organisms are virtually unexplored. Traditional methods of bacteriology have given us an acquaintance with those species that can be isolated and cultured in the laboratory. But DNA sequence analysis of the populations of bacteria in samples from natural habitats—such as soil or ocean water, or even the human mouth—has opened our eyes to the fact that most species cannot be cultured by standard laboratory techniques. According to one estimate, at least 99% of procaryotic species remain to be characterized.

The Tree of Life Has Three Primary Branches: Bacteria, Archaea, and Eucaryotes The classification of living things has traditionally depended on comparisons of their outward appearances: we can see that a fish has eyes, jaws, backbone, brain, and so on, just as we do, and that a worm does not; that a rosebush is cousin to an apple tree, but less similar to a grass. As Darwin showed, we can readily interpret such close family resemblances in terms of evolution from common ancestors, and we can find the remains of many of these ancestors preserved in the fossil record. In this way, it has been possible to begin to draw a family tree of living organisms, showing the various lines of descent, as well as branch points in the history, where the ancestors of one group of species became different from those of another. When the disparities between organisms become very great, however, these methods begin to fail. How do we decide whether a fungus is closer kin to a plant or to an animal? When it comes to procaryotes, the task becomes harder still: one microscopic rod or sphere looks much like another. Microbiologists have therefore sought to classify procaryotes in terms of their biochemistry and nutritional requirements. But this approach also has its pitfalls. Amid the bewildering variety of biochemical behaviors, it is difficult to know which differences truly reflect differences of evolutionary history. Genome analysis has given us a simpler, more direct, and more powerful way to determine evolutionary relationships. The complete DNA sequence of an organism defines its nature with almost perfect precision and in exhaustive detail. Moreover, this specification is in a digital form—a string of letters—that can be entered straightforwardly into a computer and compared with the corresponding information for any other living thing. Because DNA is subject to random changes that accumulate over long periods of time (as we shall see shortly), the number of differences between the DNA sequences of two organisms can provide a direct, objective, quantitative indication of the evolutionary distance between them. This approach has shown that the organisms that were traditionally classed together as “bacteria” can be as widely divergent in their evolutionary origins as

6 mm

Figure 1–20 A lithotrophic bacterium. Beggiatoa, which lives in sulfurous environments, gets its energy by oxidizing H2S and can fix carbon even in the dark. Note the yellow deposits of sulfur inside the cells. (Courtesy of Ralph W. Wolfe.)

16

Chapter 1: Cells and Genomes

A R CH A EA EU

A RI

BA

CT

E

Sulfolobus

human Haloferax

Aeropyrum cyanobacteria

maize

Methanothermobacter

Bacillus

Methanococcus

yeast

CA

RY O

TE

Paramecium

S

Dictyostelium Euglena

E. coli

Thermotoga Aquifex

common ancestor cell

Trypanosoma Giardia 1 change/10 nucleotides

Trichomonas

Figure 1–21 The three major divisions (domains) of the living world. Note that traditionally the word bacteria has been used to refer to procaryotes in general, but more recently has been redefined to refer to eubacteria specifically. The tree shown here is based on comparisons of the nucleotide sequence of a ribosomal RNA subunit in the different species, and the distances in the diagram represent estimates of the numbers of evolutionary changes that have occurred in this molecule in each lineage (see Figure 1–22). The parts of the tree shrouded in gray cloud represent uncertainties about details of the true pattern of species divergence in the course of evolution: comparisons of nucleotide or amino acid sequences of molecules other than rRNA, as well as other arguments, lead to somewhat different trees. There is general agreement, however, as to the early divergence of the three most basic domains—the bacteria, the archaea, and the eucaryotes.

is any procaryote from any eucaryote. It now appears that the procaryotes comprise two distinct groups that diverged early in the history of life on Earth, either before the ancestors of the eucaryotes diverged as a separate group or at about the same time. The two groups of procaryotes are called the bacteria (or eubacteria) and the archaea (or archaebacteria). The living world therefore has three major divisions or domains: bacteria, archaea, and eucaryotes (Figure 1–21). Archaea are often found inhabiting environments that we humans avoid, such as bogs, sewage treatment plants, ocean depths, salt brines, and hot acid springs, although they are also widespread in less extreme and more homely environments, from soils and lakes to the stomachs of cattle. In outward appearance they are not easily distinguished from bacteria. At a molecular level, archaea seem to resemble eucaryotes more closely in their machinery for handling genetic information (replication, transcription, and translation), but bacteria more closely in their apparatus for metabolism and energy conversion. We discuss below how this might be explained.

Some Genes Evolve Rapidly; Others Are Highly Conserved Both in the storage and in the copying of genetic information, random accidents and errors occur, altering the nucleotide sequence—that is, creating mutations. Therefore, when a cell divides, its two daughters are often not quite identical to one another or to their parent. On rare occasions, the error may represent a change for the better; more probably, it will cause no significant difference in the cell’s prospects; and in many cases, the error will cause serious damage—for example, by disrupting the coding sequence for a key protein. Changes due to mistakes of the first type will tend to be perpetuated, because the altered cell has an increased likelihood of reproducing itself. Changes due to mistakes of the second type—selectively neutral changes—may be perpetuated or not: in the competition for limited resources, it is a matter of chance whether the altered cell or its cousins will succeed. But changes that cause serious damage lead nowhere: the cell that suffers them dies, leaving no progeny. Through endless repetition of this cycle of error and trial—of mutation and natural selection—

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

17

organisms evolve: their genetic specifications change, giving them new ways to exploit the environment more effectively, to survive in competition with others, and to reproduce successfully. Clearly, some parts of the genome change more easily than others in the course of evolution. A segment of DNA that does not code for protein and has no significant regulatory role is free to change at a rate limited only by the frequency of random errors. In contrast, a gene that codes for a highly optimized essential protein or RNA molecule cannot alter so easily: when mistakes occur, the faulty cells are almost always eliminated. Genes of this latter sort are therefore highly conserved. Through 3.5 billion years or more of evolutionary history, many features of the genome have changed beyond all recognition; but the most highly conserved genes remain perfectly recognizable in all living species. These latter genes are the ones we must examine if we wish to trace family relationships between the most distantly related organisms in the tree of life. The studies that led to the classification of the living world into the three domains of bacteria, archaea, and eucaryotes were based chiefly on analysis of one of the two main RNA components of the ribosome—the so-called smallsubunit ribosomal RNA. Because translation is fundamental to all living cells, this component of the ribosome has been well conserved since early in the history of life on Earth (Figure 1–22).

Most Bacteria and Archaea Have 1000–6000 Genes Natural selection has generally favored those procaryotic cells that can reproduce the fastest by taking up raw materials from their environment and replicating themselves most efficiently, at the maximal rate permitted by the available food supplies. Small size implies a large ratio of surface area to volume, thereby helping to maximize the uptake of nutrients across the plasma membrane and boosting a cell’s reproductive rate. Presumably for these reasons, most procaryotic cells carry very little superfluous baggage; their genomes are small, with genes packed closely together and minimal quantities of regulatory DNA between them. The small genome size makes it relatively easy to determine the complete DNA sequence. We now have this information for many species of bacteria and archaea, and a few species of eucaryotes. As shown in Table 1–1, most bacterial and archaeal genomes contain between 106 and 107 nucleotide pairs, encoding 1000–6000 genes. A complete DNA sequence reveals both the genes an organism possesses and the genes it lacks. When we compare the three domains of the living world, we can begin to see which genes are common to all of them and must therefore have been present in the cell that was ancestral to all present-day living things, and which genes are peculiar to a single branch in the tree of life. To explain the findings, however, we need to consider a little more closely how new genes arise and genomes evolve.

     

   

   

   

          

   

              

   

   

   

   

   

           

human Methanococcus E. coli human

Figure 1–22 Genetic information conserved since the days of the last common ancestor of all living things. A part of the gene for the smaller of the two main RNA components of the ribosome is shown. (The complete molecule is about 1500–1900 nucleotides long, depending on species.) Corresponding segments of nucleotide sequence from an archaean (Methanococcus jannaschii), a bacterium (Escherichia coli) and a eucaryote (Homo sapiens) are aligned. Sites where the nucleotides are identical between species are indicated by a vertical line; the human sequence is repeated at the bottom of the alignment so that all three two-way comparisons can be seen. A dot halfway along the E. coli sequence denotes a site where a nucleotide has been either deleted from the bacterial lineage in the course of evolution, or inserted in the other two lineages. Note that the sequences from these three organisms, representative of the three domains of the living world, all differ from one another to a roughly similar degree, while still retaining unmistakable similarities.

18

Chapter 1: Cells and Genomes

Table 1–1 Some Genomes That Have Been Completely Sequenced SPECIES

SPECIAL FEATURES

HABITAT

GENOME SIZE (1000s OF NUCLEOTIDE PAIRS PER HAPLOID GENOME)

ESTIMATED NUMBER OF GENES CODING FOR PROTEINS

has one of the smallest of all known cell genomes photosynthetic, oxygen-generating (cyanobacterium) laboratory favorite causes stomach ulcers and predisposes to stomach cancer causes anthrax lithotrophic; lives at high temperatures source of antibiotics; giant genome spirochete; causes syphilis bacterium most closely related to mitochondria; causes typhus organotrophic; lives at very high temperatures

human genital tract

580

468

lakes and streams

3573

3168

human gut human stomach

4639 1667

4289 1590

soil hydrothermal vents

5227 1551

5634 1544

soil human tissues lice and humans (intracellular parasite) hydrothermal vents

8667 1138 1111

7825 1041 834

1860

1877

hydrothermal vents

1664

1750

hydrothermal vents

2178

2493

hydrothermal and volcanic hot vents

491

552

minimal model eucaryote

grape skins, beer

12,069

~6300

model organism for flowering plants simple animal with perfectly predictable development key to the genetics of animal development most intensively studied mammal

soil and air

~142,000

~26,000

soil

~97,000

~20,000

rotting fruit

~137,000

~14,000

houses

~3,200,000

~24,000

BACTERIA Mycoplasma genitalium Synechocystis sp. Escherichia coli Helicobacter pylori Bacillus anthracis Aquifex aeolicus Streptomyces coelicolor Treponema pallidum Rickettsia prowazekii Thermotoga maritima ARCHAEA Methanococcus jannaschii Archaeoglobus fulgidus Nanoarchaeum equitans

lithotrophic, anaerobic, methane-producing lithotrophic or organotrophic, anaerobic, sulfate-reducing smallest known archaean; anaerobic; parasitic on another, larger archaean

EUCARYOTES Saccharomyces cerevisiae (budding yeast) Arabidopsis thaliana (Thale cress) Caenorhabditis elegans (nematode worm) Drosophila melanogaster (fruit fly) Homo sapiens (human)

Genome size and gene number vary between strains of a single species, especially for bacteria and archaea. The table shows data for particular strains that have been sequenced. For eucaryotes, many genes can give rise to several alternative variant proteins, so that the total number of proteins specified by the genome is substantially greater than the number of genes.

New Genes Are Generated from Preexisting Genes The raw material of evolution is the DNA sequence that already exists: there is no natural mechanism for making long stretches of new random sequence. In this sense, no gene is ever entirely new. Innovation can, however, occur in several ways (Figure 1–23): 1. Intragenic mutation: an existing gene can be modified by changes in its DNA sequence, through various types of error that occur mainly in the process of DNA replication. 2. Gene duplication: an existing gene can be duplicated so as to create a pair of initially identical genes within a single cell; these two genes may then diverge in the course of evolution.

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

ORIGINAL GENOME

19

GENETIC INNOVATION INTRAGENIC MUTATION

mutation

1 gene

GENE DUPLICATION +

2

gene A

DNA SEGMENT SHUFFLING +

3

+

gene B

organism A

4

+

HORIZONTAL TRANSFER

organism B organism B with new gene

3.

Segment shuffling: two or more existing genes can be broken and rejoined to make a hybrid gene consisting of DNA segments that originally belonged to separate genes. 4. Horizontal (intercellular) transfer: a piece of DNA can be transferred from the genome of one cell to that of another—even to that of another species. This process is in contrast with the usual vertical transfer of genetic information from parent to progeny. Each of these types of change leaves a characteristic trace in the DNA sequence of the organism, providing clear evidence that all four processes have occurred. In later chapters we discuss the underlying mechanisms, but for the present we focus on the consequences.

Gene Duplications Give Rise to Families of Related Genes Within a Single Cell A cell duplicates its entire genome each time it divides into two daughter cells. However, accidents occasionally result in the inappropriate duplication of just part of the genome, with retention of original and duplicate segments in a single cell. Once a gene has been duplicated in this way, one of the two gene copies is free to mutate and become specialized to perform a different function within the same cell. Repeated rounds of this process of duplication and divergence, over many millions of years, have enabled one gene to give rise to a family of genes that may all be found within a single genome. Analysis of the DNA sequence of procaryotic genomes reveals many examples of such gene families: in Bacillus subtilis, for example, 47% of the genes have one or more obvious relatives (Figure 1–24). When genes duplicate and diverge in this way, the individuals of one species become endowed with multiple variants of a primordial gene. This evolutionary

Figure 1–23 Four modes of genetic innovation and their effects on the DNA sequence of an organism. A special form of horizontal transfer occurs when two different types of cells enter into a permanent symbiotic association. Genes from one of the cells then may be transferred to the genome of the other, as we shall see below when we discuss mitochondria and chloroplasts.

20

Chapter 1: Cells and Genomes 283 genes in families with 38–77 gene members 764 genes in families with 4–19 gene members

2126 genes with no family relationship

273 genes in families with 3 gene members

Figure 1–24 Families of evolutionarily related genes in the genome of Bacillus subtilis. The biggest family consists of 77 genes coding for varieties of ABC transporters—a class of membrane transport proteins found in all three domains of the living world. (Adapted from F. Kunst et al., Nature 390:249–256, 1997. With permission from Macmillan Publishers Ltd.)

568 genes in families with 2 gene members

process has to be distinguished from the genetic divergence that occurs when one species of organism splits into two separate lines of descent at a branch point in the family tree—when the human line of descent became separate from that of chimpanzees, for example. There, the genes gradually become different in the course of evolution, but they are likely to continue to have corresponding functions in the two sister species. Genes that are related by descent in this way—that is, genes in two separate species that derive from the same ancestral gene in the last common ancestor of those two species—are called orthologs. Related genes that have resulted from a gene duplication event within a single genome—and are likely to have diverged in their function—are called paralogs. Genes that are related by descent in either way are called homologs, a general term used to cover both types of relationship (Figure 1–25). The family relationships between genes can become quite complex (Figure 1–26). For example, an organism that possesses a family of paralogous genes (for example, the seven hemoglobin genes a, b, g, d, e, z, and q) may evolve into two separate species (such as humans and chimpanzees) each possessing the entire set of paralogs. All 14 genes are homologs, with the human hemoglobin a orthologous to the chimpanzee hemoglobin a, but paralogous to the human or chimpanzee hemoglobin b, and so on. Moreover, the vertebrate hemoglobins (the oxygen-binding proteins of blood) are homologous to the vertebrate myoglobins (the oxygen-binding proteins of muscle), as well as to more distant ancestral organism

ancestral organism

early ancestral organism

gene G

gene G

SPECIATION TO GIVE TWO SEPARATE SPECIES

gene G

GENE DUPLICATION AND DIVERGENCE

GENE DUPLICATION AND DIVERGENCE

gene G1 species A

species B

gene GA

gene GB

later ancestral organism gene G2

gene G1

SPECIATION

gene G2 genes GA and GB are orthologs (A)

genes G1 and G2 are paralogs (B)

species A

species B

gene G1A

gene G1B

gene G2A

gene G2B

all G genes are homologs

Figure 1–25 Paralogous genes and orthologous genes: two types of gene homology based on different evolutionary pathways. (A) and (B) The most basic possibilities. (C) A more complex pattern of events that can occur.

G1A is a paralog of G2A and G2B but an ortholog of G1B (C)

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

21 Drosophila globin shark myoglobin

ancestral globin

human myoglobin chick myoglobin shark Hb b chick Hb b chick Hb e chick Hb r human Hb b human Hb d human Hb e human Hb Ag human Hb Gg shark Hb a human Hb q-1 chick Hb a-A human Hb a1 human Hb a2 chick Hb a-D chick Hb p human Hb z

genes that code for oxygen-binding proteins in invertebrates, plants, fungi, and bacteria. From the DNA sequences, it is usually easy to recognize that two genes in different species are homologous; it is much more difficult to decide, without other information, whether they stand in the precise evolutionary relationship of orthologs.

Genes Can Be Transferred Between Organisms, Both in the Laboratory and in Nature Procaryotes also provide examples of the horizontal transfer of genes from one species of cell to another. The most obvious tell-tale signs are sequences recognizable as being derived from bacterial viruses, also called bacteriophages (Figure 1–27). Viruses are not themselves living cells but can act as vectors for gene transfer: they are small packets of genetic material that have evolved as parasites on the reproductive and biosynthetic machinery of host cells. They replicate in one cell, emerge from it with a protective wrapping, and then enter and infect another cell, which may be of the same or a different species. Often, the infected cell will be killed by the massive proliferation of virus particles inside it; but sometimes, the viral DNA, instead of directly generating these particles, may persist in its host for many cell generations as a relatively innocuous passenger, either as a separate intracellular fragment of DNA, known as a plasmid, or as a sequence inserted into the cell’s regular genome. In their travels, viruses can accidentally pick up fragments of DNA from the genome of one host cell and ferry them into another cell. Such transfers of genetic material frequently occur in procaryotes, and they can also occur between eucaryotic cells of the same species. Horizontal transfers of genes between eucaryotic cells of different species are very rare, and they do not seem to have played a significant part in eucaryote evolution (although massive transfers from bacterial to eucaryotic genomes have occurred in the evolution of mitochondria and chloroplasts, as we discuss below). In contrast, horizontal gene transfers occur much more frequently between different species of procaryotes. Many procaryotes have a remarkable capacity to take up even nonviral DNA molecules from their surroundings and thereby capture the genetic information these molecules carry. By this route, or by virus-mediated transfer, bacteria and archaea in the wild can acquire genes from neighboring cells relatively easily. Genes that confer resistance to an

Figure 1–26 A complex family of homologous genes. This diagram shows the pedigree of the hemoglobin (Hb), myoglobin, and globin genes of human, chick, shark, and Drosophila. The lengths of the horizontal lines represent the amount of divergence in amino acid sequence.

22

Chapter 1: Cells and Genomes

antibiotic or an ability to produce a toxin, for example, can be transferred from species to species and provide the recipient bacterium with a selective advantage. In this way, new and sometimes dangerous strains of bacteria have been observed to evolve in the bacterial ecosystems that inhabit hospitals or the various niches in the human body. For example, horizontal gene transfer is responsible for the spread, over the past 40 years, of penicillin-resistant strains of Neisseria gonorrheae, the bacterium that causes gonorrhea. On a longer time scale, the results can be even more profound; it has been estimated that at least 18% of all of the genes in the present-day genome of E. coli have been acquired by horizontal transfer from another species within the past 100 million years.

Sex Results in Horizontal Exchanges of Genetic Information Within a Species Horizontal exchanges of genetic information are important in bacterial and archaeal evolution in today’s world, and they may have occurred even more frequently and promiscuously in the early days of life on Earth. Such early horizontal exchanges could explain the otherwise puzzling observation that the eucaryotes seem more similar to archaea in their genes for the basic information-handling processes of DNA replication, transcription, and translation, but more similar to bacteria in their genes for metabolic processes. In any case, whether horizontal gene transfer occurred most freely in the early days of life on Earth, or has continued at a steady low rate throughout evolutionary history, it has the effect of complicating the whole concept of cell ancestry, by making each cell’s genome a composite of parts derived from separate sources. Horizontal gene transfer among procaryotes may seem a surprising process, but it has a parallel in a phenomenon familiar to us all: sex. In addition to the usual vertical transfer of genetic material from parent to offspring, sexual reproduction causes a large-scale horizontal transfer of genetic information between two initially separate cell lineages—those of the father and the mother. A key feature of sex, of course, is that the genetic exchange normally occurs only between individuals of the same species. But no matter whether they occur within a species or between species, horizontal gene transfers leave a characteristic imprint: they result in individuals who are related more closely to one set of relatives with respect to some genes, and more closely to another set of relatives with respect to others. By comparing the DNA sequences of individual human genomes, an intelligent visitor from outer space could deduce that humans reproduce sexually, even if it knew nothing about human behavior. Sexual reproduction is widespread (although not universal), especially among eucaryotes. Even bacteria indulge from time to time in controlled sexual exchanges of DNA with other members of their own species. Natural selection has clearly favored organisms that can reproduce sexually, although evolutionary theorists dispute precisely what the selective advantage of sex is.

The Function of a Gene Can Often Be Deduced from Its Sequence Family relationships among genes are important not just for their historical interest, but because they simplify the task of deciphering gene functions. Once the sequence of a newly discovered gene has been determined, a scientist can tap a few keys on a computer to search the entire database of known gene sequences for genes related to it. In many cases, the function of one or more of these homologs will have been already determined experimentally, and thus, since gene sequence determines gene function, one can frequently make a good guess at the function of the new gene: it is likely to be similar to that of the already-known homologs. In this way, it is possible to decipher a great deal of the biology of an organism simply by analyzing the DNA sequence of its genome and using the information we already have about the functions of genes in other organisms that have been more intensively studied.

(A) 100 nm

(B) 100 nm

Figure 1–27 The viral transfer of DNA from one cell to another. (A) An electron micrograph of particles of a bacterial virus, the T4 bacteriophage. The head of this virus contains the viral DNA; the tail contains the apparatus for injecting the DNA into a host bacterium. (B) A cross section of a bacterium with a T4 bacteriophage latched onto its surface. The large dark objects inside the bacterium are the heads of new T4 particles in course of assembly. When they are mature, the bacterium will burst open to release them. (A, courtesy of James Paulson; B, courtesy of Jonathan King and Erika Hartwig from G. Karp, Cell and Molecular Biology, 2nd ed. New York: John Wiley & Sons, 1999. With permission from John Wiley & Sons.)

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

More Than 200 Gene Families Are Common to All Three Primary Branches of the Tree of Life Given the complete genome sequences of representative organisms from all three domains—archaea, bacteria, and eucaryotes—we can search systematically for homologies that span this enormous evolutionary divide. In this way we can begin to take stock of the common inheritance of all living things. There are considerable difficulties in this enterprise. For example, individual species have often lost some of the ancestral genes; other genes have almost certainly been acquired by horizontal transfer from another species and therefore are not truly ancestral, even though shared. In fact, genome comparisons strongly suggest that both lineagespecific gene loss and horizontal gene transfer, in some cases between evolutionarily distant species, have been major factors of evolution, at least among procaryotes. Finally, in the course of 2 or 3 billion years, some genes that were initially shared will have changed beyond recognition by current methods. Because of all these vagaries of the evolutionary process, it seems that only a small proportion of ancestral gene families have been universally retained in a recognizable form. Thus, out of 4873 protein-coding gene families defined by comparing the genomes of 50 species of bacteria, 13 archaea, and 3 unicellular eucaryotes, only 63 are truly ubiquitous (that is, represented in all the genomes analyzed). The great majority of these universal families include components of the translation and transcription systems. This is not likely to be a realistic approximation of an ancestral gene set. A better—though still crude—idea of the latter can be obtained by tallying the gene families that have representatives in multiple, but not necessarily all, species from all three major domains. Such an analysis reveals 264 ancient conserved families. Each family can be assigned a function (at least in terms of general biochemical activity, but usually with more precision), with the largest number of shared gene families being involved in translation and in amino acid metabolism and transport (Table 1–2). This set of highly conserved gene families represents only a very rough sketch of the common inheritance of all modern life; a more precise reconstruction of the gene complement of the last universal common ancestor might be feasible with further genome sequencing and more careful comparative analysis.

Mutations Reveal the Functions of Genes Without additional information, no amount of gazing at genome sequences will reveal the functions of genes. We may recognize that gene B is like gene A, but how do we discover the function of gene A in the first place? And even if we know the function of gene A, how do we test whether the function of gene B is truly the same as the sequence similarity suggests? How do we connect the world of abstract genetic information with the world of real living organisms? The analysis of gene functions depends on two complementary approaches: genetics and biochemistry. Genetics starts with the study of mutants: we either find or make an organism in which a gene is altered, and examine the effects on the organism’s structure and performance (Figure 1–28). Biochemistry examines the functions of molecules: we extract molecules from an organism and then study their chemical activities. By combining genetics and biochemistry and examining the chemical abnormalities in a mutant organism, it is possible to find those molecules whose production depends on a given gene. At the same time, studies of the performance of the mutant organism show us what role those molecules have in the operation of the organism as a whole. Thus, genetics and biochemistry together provide a way to relate genes, molecules, and the structure and function of the organism. In recent years, DNA sequence information and the powerful tools of molecular biology have allowed rapid progress. From sequence comparisons, we can often identify particular subregions within a gene that have been preserved nearly unchanged over the course of evolution. These conserved subregions are likely to be the most important parts of the gene in terms of function. We can test their individual contributions to the activity of the gene product by creating in

23

24

Chapter 1: Cells and Genomes

Table 1–2 The Numbers of Gene Families, Classified by Function, That Are Common to All Three Domains of the Living World GENE FAMILY FUNCTION

NUMBER OF “UNIVERSAL” FAMILIES

Information processing Translation Transcription Replication, recombination, and repair Cellular processes and signaling Cell cycle control, mitosis, and meiosis Defense mechanisms Signal transduction mechanisms Cell wall/membrane biogenesis Intracellular trafficking and secretion Post-translational modification, protein turnover, chaperones Metabolism Energy production and conversion Carbohydrate transport and metabolism Amino acid transport and metabolism Nucleotide transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Inorganic ion transport and metabolism Secondary metabolite biosynthesis, transport, and catabolism Poorly characterized General biochemical function predicted; specific biological role unknown

63 7 13 2 3 1 2 4 8 19 16 43 15 22 9 8 5 24

For the purpose of this analysis, gene families are defined as “universal” if they are represented in the genomes of at least two diverse archaea (Archaeoglobus fulgidus and Aeropyrum pernix), two evolutionarily distant bacteria (Escherichia coli and Bacillus subtilis) and one eucaryote (yeast, Saccharomyces cerevisiae). (Data from R.L. Tatusov, E.V. Koonin and D.J. Lipman, Science 278:631–637, 1997, with permission from AAAS; R.L. Tatusov et al., BMC Bioinformatics 4:41, 2003, with permission from BioMed Central; and the COGs database at the US National Library of Medicine.)

the laboratory mutations of specific sites within the gene, or by constructing artificial hybrid genes that combine part of one gene with part of another. Organisms can be engineered to make either the RNA or the protein specified by the gene in large quantities to facilitate biochemical analysis. Specialists in molecular structure can determine the three-dimensional conformation of the gene product, revealing the exact position of every atom in it. Biochemists can determine how each of the parts of the genetically specified molecule contributes to its chemical behavior. Cell biologists can analyze the behavior of cells that are engineered to express a mutant version of the gene. There is, however, no one simple recipe for discovering a gene’s function, and no simple standard universal format for describing it. We may discover, for example, that the product of a given gene catalyzes a certain chemical reaction, and yet have no idea how or why that reaction is important to the organism. The functional characterization of each new family of gene products, unlike the description of the gene sequences, presents a fresh challenge to the biologist’s ingenuity. Moreover, we never fully understand the function of a gene until we learn its role in the life of the organism as a whole. To make ultimate sense of gene functions, therefore, we have to study whole organisms, not just molecules or cells.

Molecular Biologists Have Focused a Spotlight on E. coli Because living organisms are so complex, the more we learn about any particular species, the more attractive it becomes as an object for further study. Each

5 mm

Figure 1–28 A mutant phenotype reflecting the function of a gene. A normal yeast (of the species Schizosaccharomyces pombe) is compared with a mutant in which a change in a single gene has converted the cell from a cigar shape (left) to a T shape (right). The mutant gene therefore has a function in the control of cell shape. But how, in molecular terms, does the gene product perform that function? That is a harder question, and needs biochemical analysis to answer it. (Courtesy of Kenneth Sawin and Paul Nurse.)

THE DIVERSITY OF GENOMES AND THE TREE OF LIFE

25 Figure 1–29 The genome of E. coli. (A) A cluster of E. coli cells. (B) A diagram of the genome of E. coli strain K-12. The diagram is circular because the DNA of E. coli, like that of other procaryotes, forms a single, closed loop. Proteincoding genes are shown as yellow or orange bars, depending on the DNA strand from which they are transcribed; genes encoding only RNA molecules are indicated by green arrows. Some genes are transcribed from one strand of the DNA double helix (in a clockwise direction in this diagram), others from the other strand (counterclockwise). (A, courtesy of Dr. Tony Brain and David Parker/Photo Researchers; B, adapted from F.R. Blattner et al., Science 277:1453–1462, 1997. With permission from AAAS.)

origin of replication

(A)

Escherichia coli K-12 4,639,221 nucleotide pairs

terminus of replication

(B)

discovery raises new questions and provides new tools with which to tackle general questions in the context of the chosen organism. For this reason, large communities of biologists have become dedicated to studying different aspects of the same model organism. In the enormously varied world of bacteria, the spotlight of molecular biology has for a long time focused intensely on just one species: Escherichia coli, or E. coli (see Figures 1–17 and 1–18). This small, rod-shaped bacterial cell normally lives in the gut of humans and other vertebrates, but it can be grown easily in a simple nutrient broth in a culture bottle. It adapts to variable chemical conditions and reproduces rapidly, and it can evolve by mutation and selection at a remarkable speed. As with other bacteria, different strains of E. coli, though classified as members of a single species, differ genetically to a much greater degree than do different varieties of a sexually reproducing organism such as a plant or animal. One E. coli strain may possess many hundreds of genes that are absent from another, and the two strains could have as little as 50% of their genes in common. The standard laboratory strain E. coli K-12 has a genome of approximately 4.6 million nucleotide pairs, contained in a single circular molecule of DNA, coding for about 4300 different kinds of proteins (Figure 1–29). In molecular terms, we know more about E. coli than about any other living organism. Most of our understanding of the fundamental mechanisms of life— for example, how cells replicate their DNA, or how they decode the instructions represented in the DNA to direct the synthesis of specific proteins—has come from studies of E. coli. The basic genetic mechanisms have turned out to be highly conserved throughout evolution: these mechanisms are therefore essentially the same in our own cells as in E. coli.

26

Chapter 1: Cells and Genomes

Summary Procaryotes (cells without a distinct nucleus) are biochemically the most diverse organisms and include species that can obtain all their energy and nutrients from inorganic chemical sources, such as the reactive mixtures of minerals released at hydrothermal vents on the ocean floor—the sort of diet that may have nourished the first living cells 3.5 billion years ago. DNA sequence comparisons reveal the family relationships of living organisms and show that the procaryotes fall into two groups that diverged early in the course of evolution: the bacteria (or eubacteria) and the archaea. Together with the eucaryotes (cells with a membrane-enclosed nucleus), these constitute the three primary branches of the tree of life. Most bacteria and archaea are small unicellular organisms with compact genomes comprising 1000–6000 genes. Many of the genes within a single organism show strong family resemblances in their DNA sequences, implying that they originated from the same ancestral gene through gene duplication and divergence. Family resemblances (homologies) are also clear when gene sequences are compared between different species, and more than 200 gene families have been so highly conserved that they can be recognized as common to most species from all three domains of the living world. Thus, given the DNA sequence of a newly discovered gene, it is often possible to deduce the gene’s function from the known function of a homologous gene in an intensively studied model organism, such as the bacterium E. coli.

GENETIC INFORMATION IN EUCARYOTES Eucaryotic cells, in general, are bigger and more elaborate than procaryotic cells, and their genomes are bigger and more elaborate, too. The greater size is accompanied by radical differences in cell structure and function. Moreover, many classes of eucaryotic cells form multicellular organisms that attain levels of complexity unmatched by any procaryote. Because they are so complex, eucaryotes confront molecular biologists with a special set of challenges, which will concern us in the rest of this book. Increasingly, biologists meet these challenges through the analysis and manipulation of the genetic information within cells and organisms. It is therefore important at the outset to know something of the special features of the eucaryotic genome. We begin by briefly discussing how eucaryotic cells are organized, how this reflects their way of life, and how their genomes differ from those of procaryotes. This leads us to an outline of the strategy by which molecular biologists, by exploiting genetic information, are attempting to discover how eucaryotic organisms work.

Eucaryotic Cells May Have Originated as Predators By definition, eucaryotic cells keep their DNA in an internal compartment called the nucleus. The nuclear envelope, a double layer of membrane, surrounds the nucleus and separates the DNA from the cytoplasm. Eucaryotes also have other features that set them apart from procaryotes (Figure 1–30). Their cells are, typically, 10 times bigger in linear dimension, and 1000 times larger in volume. They have a cytoskeleton—a system of protein filaments crisscrossing the cytoplasm and forming, together with the many proteins that attach to them, a system of girders, ropes, and motors that gives the cell mechanical strength, controls its shape, and drives and guides its movements. The nuclear envelope is only one part of a set of internal membranes, each structurally similar to the plasma membrane and enclosing different types of spaces inside the cell, many of them involved in digestion and secretion. Lacking the tough cell wall of most bacteria, animal cells and the free-living eucaryotic cells called protozoa can change their shape rapidly and engulf other cells and small objects by phagocytosis (Figure 1–31). It is still a mystery how all these properties evolved, and in what sequence. One plausible view, however, is that they are all reflections of the way of life of a

GENETIC INFORMATION IN EUCARYOTES

27

microtubule centrosome with pair of centrioles

5 mm

extracellular matrix chromatin (DNA) nuclear pore nuclear envelope vesicles

lysosome

actin filaments nucleolus peroxisome ribosomes in cytosol

Golgi apparatus

intermediate filaments

plasma membrane

nucleus

primordial eucaryotic cell that was a predator, living by capturing other cells and eating them (Figure 1–32). Such a way of life requires a large cell with a flexible plasma membrane, as well as an elaborate cytoskeleton to support and move this membrane. It may also require that the cell’s long, fragile DNA molecules be sequestered in a separate nuclear compartment, to protect the genome from damage by the movements of the cytoskeleton.

Modern Eucaryotic Cells Evolved from a Symbiosis

endoplasmic reticulum

mitochondrion

Figure 1–30 The major features of eucaryotic cells. The drawing depicts a typical animal cell, but almost all the same components are found in plants and fungi and in single-celled eucaryotes such as yeasts and protozoa. Plant cells contain chloroplasts in addition to the components shown here, and their plasma membrane is surrounded by a tough external wall formed of cellulose.

A predatory way of life helps to explain another feature of eucaryotic cells. Almost all such cells contain mitochondria (Figure 1–33). These small bodies in the cytoplasm, enclosed by a double layer of membrane, take up oxygen and harness energy from the oxidation of food molecules—such as sugars—to produce most of the ATP that powers the cell’s activities. Mitochondria are similar in size to small bacteria, and, like bacteria, they have their own genome in the form of a circular DNA molecule, their own ribosomes that differ from those elsewhere in the eucaryotic cell, and their own transfer RNAs. It is now generally accepted that mitochondria originated from free-living oxygen-metabolizing (aerobic) bacteria that were engulfed by an ancestral eucaryotic cell that could otherwise make no such use of oxygen (that is, was anaerobic). Escaping digestion, these bacteria evolved in symbiosis with the engulfing cell and its progeny,

10 mm

Figure 1–31 Phagocytosis. This series of stills from a movie shows a human white blood cell (a neutrophil) engulfing a red blood cell (artificially colored red) that has been treated with antibody. (Courtesy of Stephen E. Malawista and Anne de Boisfleury Chevance.)

28

Chapter 1: Cells and Genomes Figure 1–32 A single-celled eucaryote that eats other cells. (A) Didinium is a carnivorous protozoan, belonging to the group known as ciliates. It has a globular body, about 150 mm in diameter, encircled by two fringes of cilia—sinuous, whiplike appendages that beat continually; its front end is flattened except for a single protrusion, rather like a snout. (B) Didinium normally swims around in the water at high speed by means of the synchronous beating of its cilia. When it encounters a suitable prey, usually another type of protozoan, it releases numerous small paralyzing darts from its snout region. Then, the Didinium attaches to and devours the other cell by phagocytosis, inverting like a hollow ball to engulf its victim, which is almost as large as itself. (Courtesy of D. Barlow.)

(A) 100 mm (B)

receiving shelter and nourishment in return for the power generation they performed for their hosts (Figure 1–34). This partnership between a primitive anaerobic eucaryotic predator cell and an aerobic bacterial cell is thought to have been established about 1.5 billion years ago, when the Earth’s atmosphere first became rich in oxygen.

(B)

(C)

(A) 100 nm

Figure 1–33 A mitochondrion. (A) A cross section, as seen in the electron microscope. (B) A drawing of a mitochondrion with part of it cut away to show the three-dimensional structure. (C) A schematic eucaryotic cell, with the interior space of a mitochondrion, containing the mitochondrial DNA and ribosomes, colored. Note the smooth outer membrane and the convoluted inner membrane, which houses the proteins that generate ATP from the oxidation of food molecules. (A, courtesy of Daniel S. Friend.)

GENETIC INFORMATION IN EUCARYOTES

29

ancestral eucaryotic cell internal membranes

early eucaryotic cell nucleus

Figure 1–34 The origin of mitochondria. An ancestral eucaryotic cell is thought to have engulfed the bacterial ancestor of mitochondria, initiating a symbiotic relationship.

mitochondria with double membrane

bacterium

Many eucaryotic cells—specifically, those of plants and algae—also contain another class of small membrane-enclosed organelles somewhat similar to mitochondria—the chloroplasts (Figure 1–35). Chloroplasts perform photosynthesis, using the energy of sunlight to synthesize carbohydrates from atmospheric carbon dioxide and water, and deliver the products to the host cell as food. Like mitochondria, chloroplasts have their own genome and almost certainly originated as symbiotic photosynthetic bacteria, acquired by cells that already possessed mitochondria (Figure 1–36). A eucaryotic cell equipped with chloroplasts has no need to chase after other cells as prey; it is nourished by the captive chloroplasts it has inherited from its ancestors. Correspondingly, plant cells, although they possess the cytoskeletal equipment for movement, have lost the ability to change shape rapidly and to engulf other cells by phagocytosis. Instead, they create around themselves a tough, protective cell wall. If the ancestral eucaryote was indeed a predator on other organisms, we can view plant cells as eucaryotes that have made the transition from hunting to farming. Fungi represent yet another eucaryotic way of life. Fungal cells, like animal cells, possess mitochondria but not chloroplasts; but in contrast with animal cells and protozoa, they have a tough outer wall that limits their ability to move

chloroplasts

chlorophyllcontaining membranes

inner membrane outer membrane

(A)

10 mm

(B)

Figure 1–35 Chloroplasts. These organelles capture the energy of sunlight in plant cells and some single-celled eucaryotes. (A) A single cell isolated from a leaf of a flowering plant, seen in the light microscope, showing the green chloroplasts. (B) A drawing of one of the chloroplasts, showing the highly folded system of internal membranes containing the chlorophyll molecules by which light is absorbed. (A, courtesy of Preeti Dahiya.)

30

Chapter 1: Cells and Genomes

early eucaryotic cell

photosynthetic bacterium

early eucaryotic cell capable of photosynthesis

chloroplasts with double membrane

rapidly or to swallow up other cells. Fungi, it seems, have turned from hunters into scavengers: other cells secrete nutrient molecules or release them upon death, and fungi feed on these leavings—performing whatever digestion is necessary extracellularly, by secreting digestive enzymes to the exterior.

Eucaryotes Have Hybrid Genomes The genetic information of eucaryotic cells has a hybrid origin—from the ancestral anaerobic eucaryote, and from the bacteria that it adopted as symbionts. Most of this information is stored in the nucleus, but a small amount remains inside the mitochondria and, for plant and algal cells, in the chloroplasts. The mitochondrial DNA and the chloroplast DNA can be separated from the nuclear DNA and individually analyzed and sequenced. The mitochondrial and chloroplast genomes are found to be degenerate, cut-down versions of the corresponding bacterial genomes, lacking genes for many essential functions. In a human cell, for example, the mitochondrial genome consists of only 16,569 nucleotide pairs, and codes for only 13 proteins, two ribosomal RNA components, and 22 transfer RNAs. The genes that are missing from the mitochondria and chloroplasts have not all been lost; instead, many of them have been somehow moved from the symbiont genome into the DNA of the host cell nucleus. The nuclear DNA of humans contains many genes coding for proteins that serve essential functions inside the mitochondria; in plants, the nuclear DNA also contains many genes specifying proteins required in chloroplasts.

Eucaryotic Genomes Are Big Natural selection has evidently favored mitochondria with small genomes, just as it has favored bacteria with small genomes. By contrast, the nuclear genomes of most eucaryotes seem to have been free to enlarge. Perhaps the eucaryotic way of life has made large size an advantage: predators typically need to be bigger than their prey, and cell size generally increases in proportion to genome size. Perhaps enlargement of the genome has been driven by the accumulation of parasitic transposable elements (discussed in Chapter 5)—“selfish” segments of DNA that can insert copies of themselves at multiple sites in the genome. Whatever the explanation, the genomes of most eucaryotes are orders of magnitude larger than those of bacteria and archaea (Figure 1–37). And the freedom to be extravagant with DNA has had profound implications. Eucaryotes not only have more genes than procaryotes; they also have vastly more DNA that does not code for protein or for any other functional product molecule. The human genome contains 1000 times as many nucleotide pairs as the genome of a typical bacterium, 20 times as many genes, and about 10,000

Figure 1–36 The origin of chloroplasts. An early eucaryotic cell, already possessing mitochondria, engulfed a photosynthetic bacterium (a cyanobacterium) and retained it in symbiosis. All present-day chloroplasts are thought to trace their ancestry back to a single species of cyanobacterium that was adopted as an internal symbiont (an endosymbiont) over a billion years ago.

GENETIC INFORMATION IN EUCARYOTES

Mycoplasma BACTERIA AND ARCHAEA

31 Figure 1–37 Genome sizes compared. Genome size is measured in nucleotide pairs of DNA per haploid genome, that is, per single copy of the genome. (The cells of sexually reproducing organisms such as ourselves are generally diploid: they contain two copies of the genome, one inherited from the mother, the other from the father.) Closely related organisms can vary widely in the quantity of DNA in their genomes, even though they contain similar numbers of functionally distinct genes. (Data from W.H. Li, Molecular Evolution, pp. 380–383. Sunderland, MA: Sinauer, 1997.)

E. coli yeast FUNGI

Amoeba

PROTISTS

Arabidopsis PLANTS Drosophila INSECTS

bean

lily

fern

MOLLUSKS

shark CARTILAGINOUS FISH Fugu zebrafish BONY FISH

newt

AMPHIBIANS REPTILES BIRDS

human

MAMMALS

105

106

107 108 109 1010 number of nucleotide pairs per haploid genome

1011

1012

times as much noncoding DNA (~98.5% of the genome for a human is noncoding, as opposed to 11% of the genome for the bacterium E. coli).

Eucaryotic Genomes Are Rich in Regulatory DNA Much of our noncoding DNA is almost certainly dispensable junk, retained like a mass of old papers because, when there is little pressure to keep an archive small, it is easier to retain everything than to sort out the valuable information and discard the rest. Certain exceptional eucaryotic species, such as the puffer fish (Figure 1–38), bear witness to the profligacy of their relatives; they have somehow managed to rid themselves of large quantities of noncoding DNA. Yet they appear similar in structure, behavior, and fitness to related species that have vastly more such DNA. Even in compact eucaryotic genomes such as that of puffer fish, there is more noncoding DNA than coding DNA, and at least some of the noncoding DNA certainly has important functions. In particular, it regulates the expression of adjacent genes. With this regulatory DNA, eucaryotes have evolved distinctive ways of controlling when and where a gene is brought into play. This sophisticated gene regulation is crucial for the formation of complex multicellular organisms.

The Genome Defines the Program of Multicellular Development The cells in an individual animal or plant are extraordinarily varied. Fat cells, skin cells, bone cells, nerve cells—they seem as dissimilar as any cells could be. Yet all these cell types are the descendants of a single fertilized egg cell, and all (with minor exceptions) contain identical copies of the genome of the species. The differences result from the way in which the cells make selective use of their genetic instructions according to the cues they get from their surroundings in the developing embryo. The DNA is not just a shopping list specifying the molecules that every cell must have, and the cell is not an assembly of all the items on the list. Rather, the cell behaves as a multipurpose machine, with sensors to receive environmental signals and with highly developed abilities to call different sets of genes into action according to the sequences of signals to which the cell has been exposed. The genome in each cell is big enough to accommodate the information that specifies an entire multicellular organism, but in any individual cell only part of that information is used. A large fraction of the genes in the eucaryotic genome code for proteins that regulate the activities of other genes. Most of these gene regulatory proteins act by

Figure 1–38 The puffer fish (Fugu rubripes). This organism has a genome size of 400 million nucleotide pairs— about one-quarter as much as a zebrafish, for example, even though the two species of fish have similar numbers of genes. (From a woodcut by Hiroshige, courtesy of Arts and Designs of Japan.)

32

Chapter 1: Cells and Genomes receptor protein in cell membrane detects environmental signal

gene-regulatory protein is activated... ...and binds to regulatory DNA...

...provoking activation of a gene to produce another protein...

Figure 1–39 Controlling gene readout by environmental signals. Regulatory DNA allows gene expression to be controlled by regulatory proteins, which are in turn the products of other genes. This diagram shows how a cell’s gene expression is adjusted according to a signal from the cell’s environment. The initial effect of the signal is to activate a regulatory protein already present in the cell; the signal may, for example, trigger the attachment of a phosphate group to the regulatory protein, altering its chemical properties.

...that binds to other regulatory regions... protein-coding region regulatory region

...to produce yet more proteins, including some additional gene-regulatory proteins

binding, directly or indirectly, to the regulatory DNA adjacent to the genes that are to be controlled (Figure 1–39), or by interfering with the abilities of other proteins to do so. The expanded genome of eucaryotes therefore not only specifies the hardware of the cell, but also stores the software that controls how that hardware is used (Figure 1–40). Cells do not just passively receive signals; rather, they actively exchange signals with their neighbors. Thus, in a developing multicellular organism, the same control system governs each cell, but with different consequences depending on the messages exchanged. The outcome, astonishingly, is a precisely patterned array of cells in different states, each displaying a character appropriate to its position in the multicellular structure.

Many Eucaryotes Live as Solitary Cells: the Protists Many species of eucaryotic cells lead a solitary life—some as hunters (the protozoa), some as photosynthesizers (the unicellular algae), some as scavengers (the unicellular fungi, or yeasts). Figure 1–41 conveys something of the variety of forms of these single-celled eucaryotes, or protists. The anatomy of protozoa,

Figure 1–40 Genetic control of the program of multicellular development. The role of a regulatory gene is demonstrated in the snapdragon Antirrhinum. In this example, a mutation in a single gene coding for a regulatory protein causes leafy shoots to develop in place of flowers: because a regulatory protein has been changed, the cells adopt characters that would be appropriate to a different location in the normal plant. The mutant is on the left, the normal plant on the right. (Courtesy of Enrico Coen and Rosemary Carpenter.)

GENETIC INFORMATION IN EUCARYOTES

33

I.

especially, is often elaborate and includes such structures as sensory bristles, photoreceptors, sinuously beating cilia, leglike appendages, mouth parts, stinging darts, and musclelike contractile bundles. Although they are single cells, protozoa can be as intricate, as versatile, and as complex in their behavior as many multicellular organisms (see Figure 1–32). In terms of their ancestry and DNA sequences, protists are far more diverse than the multicellular animals, plants, and fungi, which arose as three comparatively late branches of the eucaryotic pedigree (see Figure 1–21). As with procaryotes, humans have tended to neglect the protists because they are microscopic. Only now, with the help of genome analysis, are we beginning to understand their positions in the tree of life, and to put into context the glimpses these strange creatures offer us of our distant evolutionary past.

A Yeast Serves as a Minimal Model Eucaryote The molecular and genetic complexity of eucaryotes is daunting. Even more than for procaryotes, biologists need to concentrate their limited resources on a few selected model organisms to fathom this complexity. To analyze the internal workings of the eucaryotic cell, without the additional problems of multicellular development, it makes sense to use a species that is unicellular and as simple as possible. The popular choice for this role of minimal model eucaryote has been the yeast Saccharomyces cerevisiae (Figure 1–42)—the same species that is used by brewers of beer and bakers of bread. S. cerevisiae is a small, single-celled member of the kingdom of fungi and thus, according to modern views, at least as closely related to animals as it is to plants. It is robust and easy to grow in a simple nutrient medium. Like other fungi, it has a tough cell wall, is relatively immobile, and possesses mitochondria but not chloroplasts. When nutrients are plentiful, it grows and divides almost as

Figure 1–41 An assortment of protists: a small sample of an extremely diverse class of organisms. The drawings are done to different scales, but in each case the scale bar represents 10 mm. The organisms in (A), (B), (E), (F), and (I) are ciliates; (C) is a euglenoid; (D) is an amoeba; (G) is a dinoflagellate; (H) is a heliozoan. (From M.A. Sleigh, Biology of Protozoa. Cambridge, UK: Cambridge University Press, 1973.)

34

Chapter 1: Cells and Genomes

nucleus

cell wall

Figure 1–42 The yeast Saccharomyces cerevisiae. (A) A scanning electron micrograph of a cluster of the cells. This species is also known as budding yeast; it proliferates by forming a protrusion or bud that enlarges and then separates from the rest of the original cell. Many cells with buds are visible in this micrograph. (B) A transmission electron micrograph of a cross section of a yeast cell, showing its nucleus, mitochondrion, and thick cell wall. (A, courtesy of Ira Herskowitz and Eric Schabatach.)

mitochondrion (B)

(A) 10 mm

2 mm

rapidly as a bacterium. It can reproduce either vegetatively (that is, by simple cell division), or sexually: two yeast cells that are haploid (possessing a single copy of the genome) can fuse to create a cell that is diploid (containing a double genome); and the diploid cell can undergo meiosis (a reduction division) to produce cells that are once again haploid (Figure 1–43). In contrast with higher plants and animals, the yeast can divide indefinitely in either the haploid or the diploid state, and the process leading from the one state to the other can be induced at will by changing the growth conditions. In addition to these features, the yeast has a further property that makes it a convenient organism for genetic studies: its genome, by eucaryotic standards, is exceptionally small. Nevertheless, it suffices for all the basic tasks that every eucaryotic cell must perform. As we shall see later in this book, studies on yeasts (using both S. cerevisiae and other species) have provided a key to many crucial processes, including the eucaryotic cell-division cycle—the critical chain of events by which the nucleus and all the other components of a cell are duplicated and parceled out to create two daughter cells from one. The control system that governs this process has been so well conserved over the course of evolution that many of its components can function interchangeably in yeast and human cells: if a mutant yeast lacking an essential yeast cell-division-cycle gene is supplied with a copy of the homologous cell-division-cycle gene from a human, the yeast is cured of its defect and becomes able to divide normally.

The Expression Levels of All The Genes of An Organism Can Be Monitored Simultaneously The complete genome sequence of S. cerevisiae, determined in 1997, consists of approximately 13,117,000 nucleotide pairs, including the small contribution (78,520 nucleotide pairs) of the mitochondrial DNA. This total is only about 2.5 times as much DNA as there is in E. coli, and it codes for only 1.5 times as many distinct proteins (about 6300 in all). The way of life of S. cerevisiae is similar in many ways to that of a bacterium, and it seems that this yeast has likewise been subject to selection pressures that have kept its genome compact. Knowledge of the complete genome sequence of any organism—be it a yeast or a human—opens up new perspectives on the workings of the cell: things that once seemed impossibly complex now seem within our grasp. Using techniques Figure 1–43 The reproductive cycles of the yeast S. cerevisiae. Depending on environmental conditions and on details of the genotype, cells of this species can exist in either a diploid (2n) state, with a double chromosome set, or a haploid (n) state, with a single chromosome set. The diploid form can either proliferate by ordinary cell-division cycles or undergo meiosis to produce haploid cells. The haploid form can either proliferate by ordinary cell-division cycles or undergo sexual fusion with another haploid cell to become diploid. Meiosis is triggered by starvation and gives rise to spores—haploid cells in a dormant state, resistant to harsh environmental conditions.

2n

2n

proliferation of diploid cells 2n meiosis and sporulation (triggered by starvation) 2n n n mating (usually immediately after spores hatch)

n

n

spores hatch n n

n

proliferation of haploid cells n

BUDDING YEAST LIFE CYCLE

GENETIC INFORMATION IN EUCARYOTES

35

ACE2 FKH1 FKH2 MBP1 MC M NDD 1 RM E 1 SK 1 ST N7 S B1 SWWI4 S I5 A WI AS 1 6 H1

YJL206C UGA3 THI2 STP21 STP 4 SIP 1 SFPL1 SF G3 RT 1 G RT GT1 R U T3 P

G1 DI MS1 H E4 IM OT3 M D1 PH 101 RIM K2 SO 12 STE 1 SUM ABF1 DOT6 FHL1 HIR1 HIR2 RAP1 REB1 RGM CAD 1 CIN 1 CRZ 5 CU 1 G P9 H TS1 HA AA1 H L9 MA SF1 C1

CH C A4 BA BF1 S A AR ZF1 1 AR O80 ARGG81 8 ADR 0 ZMS11 ZAP1 YFL044C YAP7 YAP6 YAP5

N1 MS SN2 4 M N MS DR1 P CS1 R X1 RF 1 RLMX1 RO 1 RPH 1 SKO SMP1 YAP1 YAP3

P P HO N HO 4 MS RG1 2 S M 11 M IG1 METET4 MAL 31 MAL133 3 LEU3 IXR1 INO4 INO2 HAP5 HAP4 HAP3 HAP2 GLN3 GCR21 GCRN4 GC T3 GA T1 GA L4 GA ZF1 F L82 1 DA AL8 D

DNA/RNA/protein biosynthesis cell cycle

environmental response

developmental processes

metabolism

to be described in Chapter 8, it is now possible, for example, to monitor, simultaneously, the amount of mRNA transcript that is produced from every gene in the yeast genome under any chosen conditions, and to see how this whole pattern of gene activity changes when conditions change. The analysis can be repeated with mRNA prepared from mutants lacking a chosen gene—any gene that we care to test. In principle, this approach provides a way to reveal the entire system of control relationships that govern gene expression—not only in yeast cells, but in any organism whose genome sequence is known.

Figure 1–44 The network of interactions between gene regulatory proteins and the genes that code for them in a yeast cell. Results are shown for 106 out of the total of 141 gene regulatory proteins in Saccharomyces cerevisiae. Each protein in the set was tested for its ability to bind to the regulatory DNA of each of the genes coding for this set of proteins. In the diagram, the genes are arranged in a circle, and an arrow pointing from gene A to gene B means that the protein encoded by A binds to the regulatory DNA of B, and therefore presumably regulates the expression of B. Small circles with arrowheads indicate genes whose products directly regulate their own expression. Genes governing different aspects of cell behavior are shown in different colors. For a multicellular plant or animal, the number of gene regulatory proteins is about 10 times greater, and the amount of regulatory DNA perhaps 100 times greater, so that the corresponding diagram would be vastly more complex. (From T.I. Lee et al., Science 298:799–804, 2002. With permission from AAAS.)

To Make Sense of Cells, We Need Mathematics, Computers, and Quantitative Information Through methods such as these, exploiting our knowledge of complete genome sequences, we can list the genes and proteins in a cell and begin to depict the web of interactions between them (Figure 1–44). But how are we to turn all this information into an understanding of how cells work? Even for a single cell type belonging to a single species of organism, the current deluge of data seems overwhelming. The sort of informal reasoning on which biologists usually rely seems totally inadequate in the face of such complexity. In fact, the difficulty is more than just a matter of information overload. Biological systems are, for example, full of feedback loops, and the behavior of even the simplest of systems with feedback is remarkably difficult to predict by intuition alone (Figure 1–45); small Figure 1–45 A very simple gene regulatory circuit—a single gene regulating its own expression by the binding of its protein product to its own regulatory DNA. Simple schematic diagrams such as this are often used to summarize what we know (as in Figure 1–44), but they leave many questions unanswered. When the protein binds, does it inhibit or stimulate transcription? How steeply does the transcription rate depend on the protein concentration? How long, on average, does a molecule of the protein remain bound to the DNA? How long does it take to make each molecule of mRNA or protein, and how quickly does each type of molecule get degraded? Mathematical modeling shows that we need quantitative answers to all these and other questions before we can predict the behavior of even this single-gene system. For different parameter values, the system may settle to a unique steady state; or it may behave as a switch, capable of existing in one or other of a set of alternative states; or it may oscillate; or it may show large random fluctuations.

regulatory DNA

gene coding region

mRNA

gene regulatory protein

36

Chapter 1: Cells and Genomes

changes in parameters can cause radical changes in outcome. To go from a circuit diagram to a prediction of the behavior of the system, we need detailed quantitative information, and to draw deductions from that information we need mathematics and computers. These tools for quantitative reasoning are essential, but they are not allpowerful. You might think that, knowing how each protein influences each other protein, and how the expression of each gene is regulated by the products of others, we should soon be able to calculate how the cell as a whole will behave, just as an astronomer can calculate the orbits of the planets, or a chemical engineer can calculate the flows through a chemical plant. But any attempt to perform this feat for an entire living cell rapidly reveals the limits of our present state of knowledge. The information we have, plentiful as it is, is full of gaps and uncertainties. Moreover, it is largely qualitative rather than quantitative. Most often, cell biologists studying the cell’s control systems sum up their knowledge in simple schematic diagrams—this book is full of them—rather than in numbers, graphs, and differential equations. To progress from qualitative descriptions and intuitive reasoning to quantitative descriptions and mathematical deduction is one of the biggest challenges for contemporary cell biology. So far, the challenge has been met only for a few very simple fragments of the machinery of living cells—subsystems involving a handful of different proteins, or two or three cross-regulatory genes, where theory and experiment can go closely hand in hand. We shall discuss some of these examples later in the book.

Arabidopsis Has Been Chosen Out of 300,000 Species As a Model Plant The large multicellular organisms that we see around us—the flowers and trees and animals—seem fantastically varied, but they are much closer to one another in their evolutionary origins, and more similar in their basic cell biology, than the great host of microscopic single-celled organisms. Thus, while bacteria and eucaryotes are separated by more than 3000 million years of divergent evolution, vertebrates and insects are separated by about 700 million years, fish and mammals by about 450 million years, and the different species of flowering plants by only about 150 million years. Because of the close evolutionary relationship between all flowering plants, we can, once again, get insight into the cell and molecular biology of this whole class of organisms by focusing on just one or a few species for detailed analysis. Out of the several hundred thousand species of flowering plants on Earth today, molecular biologists have chosen to concentrate their efforts on a small weed, the common Thale cress Arabidopsis thaliana (Figure 1–46), which can be grown indoors in large numbers, and produces thousands of offspring per plant after 8–10 weeks. Arabidopsis has a genome of approximately 140 million nucleotide pairs, about 11 times as much as yeast, and its complete sequence is known.

The World of Animal Cells Is Represented By a Worm, a Fly, a Mouse, and a Human Multicellular animals account for the majority of all named species of living organisms, and for the largest part of the biological research effort. Four species have emerged as the foremost model organisms for molecular genetic studies. In order of increasing size, they are the nematode worm Caenorhabditis elegans, the fly Drosophila melanogaster, the mouse Mus musculus, and the human, Homo sapiens. Each of these has had its genome sequenced. Caenorhabditis elegans (Figure 1–47) is a small, harmless relative of the eelworm that attacks crops. With a life cycle of only a few days, an ability to survive in a freezer indefinitely in a state of suspended animation, a simple body plan, and an unusual life cycle that is well suited for genetic studies (described in Chapter 23), it is an ideal model organism. C. elegans develops with clockwork precision from a fertilized egg cell into an adult worm with exactly 959 body cells

Figure 1–46 Arabidopsis thaliana, the plant chosen as the primary model for studying plant molecular genetics. (Courtesy of Toni Hayden and the John Innes Foundation.)

GENETIC INFORMATION IN EUCARYOTES

37 Figure 1–47 Caenorhabditis elegans, the first multicellular organism to have its complete genome sequence determined. This small nematode, about 1 mm long, lives in the soil. Most individuals are hermaphrodites, producing both eggs and sperm. The animal is viewed here using interference contrast optics, showing up the boundaries of the tissues in bright colors; the animal itself is not colored when viewed with ordinary lighting. (Courtesy of Ian Hope.)

0.2 mm

(plus a variable number of egg and sperm cells)—an unusual degree of regularity for an animal. We now have a minutely detailed description of the sequence of events by which this occurs, as the cells divide, move, and change their characters according to strict and predictable rules. The genome of 97 million nucleotide pairs codes for about 19,000 proteins, and many mutants and other tools are available for the testing of gene functions. Although the worm has a body plan very different from our own, the conservation of biological mechanisms has been sufficient for the worm to be a model for many of the developmental and cell-biological processes that occur in the human body. Studies of the worm help us to understand, for example, the programs of cell division and cell death that determine the numbers of cells in the body—a topic of great importance in developmental biology and cancer research.

Studies in Drosophila Provide a Key to Vertebrate Development The fruitfly Drosophila melanogaster (Figure 1–48) has been used as a model genetic organism for longer than any other; in fact, the foundations of classical genetics were built to a large extent on studies of this insect. Over 80 years ago, it provided, for example, definitive proof that genes—the abstract units of hereditary information—are carried on chromosomes, concrete physical objects whose behavior had been closely followed in the eucaryotic cell with the light microscope, but whose function was at first unknown. The proof depended on one of the many features that make Drosophila peculiarly convenient for genetics—the

Figure 1–48 Drosophila melanogaster. Molecular genetic studies on this fly have provided the main key to understanding how all animals develop from a fertilized egg into an adult. (From E.B. Lewis, Science 221:cover, 1983. With permission from AAAS.)

38

Chapter 1: Cells and Genomes

giant chromosomes, with characteristic banded appearance, that are visible in some of its cells (Figure 1–49). Specific changes in the hereditary information, manifest in families of mutant flies, were found to correlate exactly with the loss or alteration of specific giant-chromosome bands. In more recent times, Drosophila, more than any other organism, has shown us how to trace the chain of cause and effect from the genetic instructions encoded in the chromosomal DNA to the structure of the adult multicellular body. Drosophila mutants with body parts strangely misplaced or mispatterned provided the key to the identification and characterization of the genes required to make a properly structured body, with gut, limbs, eyes, and all the other parts in their correct places. Once these Drosophila genes were sequenced, the genomes of vertebrates could be scanned for homologs. These were found, and their functions in vertebrates were then tested by analyzing mice in which the genes had been mutated. The results, as we see later in the book, reveal an astonishing degree of similarity in the molecular mechanisms of insect and vertebrate development. The majority of all named species of living organisms are insects. Even if Drosophila had nothing in common with vertebrates, but only with insects, it would still be an important model organism. But if understanding the molecular genetics of vertebrates is the goal, why not simply tackle the problem head-on? Why sidle up to it obliquely, through studies in Drosophila? Drosophila requires only 9 days to progress from a fertilized egg to an adult; it is vastly easier and cheaper to breed than any vertebrate, and its genome is much smaller—about 170 million nucleotide pairs, compared with 3200 million for a human. This genome codes for about 14,000 proteins, and mutants can now be obtained for essentially any gene. But there is also another, deeper reason why genetic mechanisms that are hard to discover in a vertebrate are often readily revealed in the fly. This relates, as we now explain, to the frequency of gene duplication, which is substantially greater in vertebrate genomes than in the fly genome and has probably been crucial in making vertebrates the complex and subtle creatures that they are.

The Vertebrate Genome Is a Product of Repeated Duplication Almost every gene in the vertebrate genome has paralogs—other genes in the same genome that are unmistakably related and must have arisen by gene duplication. In many cases, a whole cluster of genes is closely related to similar clusters present elsewhere in the genome, suggesting that genes have been duplicated in linked groups rather than as isolated individuals. According to one hypothesis, at an early stage in the evolution of the vertebrates, the entire genome underwent duplication twice in succession, giving rise to four copies of every gene. In some groups of vertebrates, such as fish of the salmon and carp families (including the zebrafish, a popular research animal), it has been suggested that there was yet another duplication, creating an eightfold multiplicity of genes. The precise course of vertebrate genome evolution remains uncertain, because many further evolutionary changes have occurred since these ancient events. Genes that were once identical have diverged; many of the gene copies have been lost through disruptive mutations; some have undergone further rounds of local duplication; and the genome, in each branch of the vertebrate family tree, has suffered repeated rearrangements, breaking up most of the original gene orderings. Comparison of the gene order in two related organisms, such as the human and the mouse, reveals that—on the time scale of vertebrate evolution—chromosomes frequently fuse and fragment to move large blocks of DNA sequence around. Indeed, it is possible, as we shall discuss in Chapter 7, that the present state of affairs is the result of many separate duplications of fragments of the genome, rather than duplications of the genome as a whole. There is, however, no doubt that such whole-genome duplications do occur from time to time in evolution, for we can see recent instances in which duplicated chromosome sets are still clearly identifiable as such. The frog

20 mm

Figure 1–49 Giant chromosomes from salivary gland cells of Drosophila. Because many rounds of DNA replication have occurred without an intervening cell division, each of the chromosomes in these unusual cells contains over 1000 identical DNA molecules, all aligned in register. This makes them easy to see in the light microscope, where they display a characteristic and reproducible banding pattern. Specific bands can be identified as the locations of specific genes: a mutant fly with a region of the banding pattern missing shows a phenotype reflecting loss of the genes in that region. Genes that are being transcribed at a high rate correspond to bands with a “puffed” appearance. The bands stained dark brown in the micrograph are sites where a particular regulatory protein is bound to the DNA. (Courtesy of B. Zink and R. Paro, from R. Paro, Trends Genet. 6:416–421, 1990. With permission from Elsevier.)

GENETIC INFORMATION IN EUCARYOTES Figure 1–50 Two species of the frog genus Xenopus. X. tropicalis, above, has an ordinary diploid genome; X. laevis, below, has twice as much DNA per cell. From the banding patterns of their chromosomes and the arrangement of genes along them, as well as from comparisons of gene sequences, it is clear that the large-genome species have evolved through duplications of the whole genome. These duplications are thought to have occurred in the aftermath of matings between frogs of slightly divergent Xenopus species. (Courtesy of E. Amaya, M. Offield and R. Grainger, Trends Genet. 14:253–255, 1998. With permission from Elsevier.)

genus Xenopus, for example, comprises a set of closely similar species related to one another by repeated duplications or triplications of the whole genome. Among these frogs are X. tropicalis, with an ordinary diploid genome; the common laboratory species X. laevis, with a duplicated genome and twice as much DNA per cell; and X. ruwenzoriensis, with a sixfold reduplication of the original genome and six times as much DNA per cell (108 chromosomes, compared with 36 in X. laevis, for example). These species are estimated to have diverged from one another within the past 120 million years (Figure 1–50).

Genetic Redundancy Is a Problem for Geneticists, But It Creates Opportunities for Evolving Organisms Whatever the details of the evolutionary history, it is clear that most genes in the vertebrate genome exist in several versions that were once identical. The related genes often remain functionally interchangeable for many purposes. This phenomenon is called genetic redundancy. For the scientist struggling to discover all the genes involved in some particular process, it complicates the task. If gene A is mutated and no effect is seen, it cannot be concluded that gene A is functionally irrelevant—it may simply be that this gene normally works in parallel with its relatives, and these suffice for near-normal function even when gene A is defective. In the less repetitive genome of Drosophila, where gene duplication is less common, the analysis is more straightforward: single gene functions are revealed directly by the consequences of single-gene mutations (the singleengined plane stops flying when the engine fails). Genome duplication has clearly allowed the development of more complex life forms; it provides an organism with a cornucopia of spare gene copies, which are free to mutate to serve divergent purposes. While one copy becomes optimized for use in the liver, say, another can become optimized for use in the brain or adapted for a novel purpose. In this way, the additional genes allow for increased complexity and sophistication. As the genes take on divergent functions, they cease to be redundant. Often, however, while the genes acquire individually specialized roles, they also continue to perform some aspects of their original core function in parallel, redundantly. Mutation of a single gene then causes a relatively minor abnormality that reveals only a part of the gene’s function (Figure 1–51). Families of genes with divergent but partly overlapping functions are a pervasive feature of vertebrate molecular biology, and they are encountered repeatedly in this book.

The Mouse Serves as a Model for Mammals Mammals have typically three or four times as many genes as Drosophila, a genome that is 20 times larger, and millions or billions of times as many cells in their adult bodies. In terms of genome size and function, cell biology, and molecular mechanisms, mammals are nevertheless a highly uniform group of organisms. Even anatomically, the differences among mammals are chiefly a matter of size and proportions; it is hard to think of a human body part that does not have a counterpart in elephants and mice, and vice versa. Evolution plays freely with quantitative features, but it does not readily change the logic of the structure.

39

40

Chapter 1: Cells and Genomes

gene G1

gene G1

gene G1

gene G1

gene G gene G2 ancestral organism (A)

gene G2

modern organism

EVOLUTION BY GENE DUPLICATION

loss of gene G1 (B)

gene G2 loss of gene G2

gene G2 loss of genes G1 and G2

MUTANT PHENOTYPES OF MODERN ORGANISM

For a more exact measure of how closely mammalian species resemble one another genetically, we can compare the nucleotide sequences of corresponding (orthologous) genes, or the amino acid sequences of the proteins that these genes encode. The results for individual genes and proteins vary widely. But typically, if we line up the amino acid sequence of a human protein with that of the orthologous protein from, say, an elephant, about 85% of the amino acids are identical. A similar comparison between human and bird shows an amino acid identity of about 70%—twice as many differences, because the bird and the mammalian lineages have had twice as long to diverge as those of the elephant and the human (Figure 1–52). The mouse, being small, hardy, and a rapid breeder, has become the foremost model organism for experimental studies of vertebrate molecular genetics. Many naturally occurring mutations are known, often mimicking the effects of corresponding mutations in humans (Figure 1–53). Methods have been developed, moreover, to test the function of any chosen mouse gene, or of any noncoding portion of the mouse genome, by artificially creating mutations in it, as we explain later in the book. One made-to-order mutant mouse can provide a wealth of information for the cell biologist. It reveals the effects of the chosen mutation in a host of different contexts, simultaneously testing the action of the gene in all the different kinds of cells in the body that could in principle be affected.

Humans Report on Their Own Peculiarities As humans, we have a special interest in the human genome. We want to know the full set of parts from which we are made, and to discover how they work. But even if you were a mouse, preoccupied with the molecular biology of mice, humans would be attractive as model genetic organisms, because of one special property: through medical examinations and self-reporting, we catalog our own genetic (and other) disorders. The human population is enormous, consisting today of some 6 billion individuals, and this self-documenting property means that a huge database of information exists on human mutations. The complete human genome sequence of more than 3 billion nucleotide pairs has now been determined, making it easier than ever before to identify at a molecular level the precise gene responsible for each human mutant characteristic. By drawing together the insights from humans, mice, flies, worms, yeasts, plants, and bacteria—using gene sequence similarities to map out the correspondences between one model organism and another—we enrich our understanding of them all.

Figure 1–51 The consequences of gene duplication for mutational analyses of gene function. In this hypothetical example, an ancestral multicellular organism has a genome containing a single copy of gene G, which performs its function at several sites in the body, indicated in green. (A) Through gene duplication, a modern descendant of the ancestral organism has two copies of gene G, called G1 and G2. These have diverged somewhat in their patterns of expression and in their activities at the sites where they are expressed, but they still retain important similarities. At some sites, they are expressed together, and each independently performs the same old function as the ancestral gene G (alternating green and yellow stripes); at other sites, they are expressed alone and may serve new purposes. (B) Because of a functional overlap, the loss of one of the two genes by mutation (red cross) reveals only a part of its role; only the loss of both genes in the double mutant reveals the full range of processes for which these genes are responsible. Analogous principles apply to duplicated genes that operate in the same place (for example, in a single-celled organism) but are called into action together or individually in response to varying circumstances. Thus, gene duplications complicate genetic analyses in all organisms.

GENETIC INFORMATION IN EUCARYOTES

98 84 86

Cretaceous

pig/whale pig/sheep human/rabbit human/elephant human/mouse human/sloth

77 87 82 83 89 81

Jurassic

human/kangaroo

81

Triassic

bird/crocodile

76

human/lizard

57

human/chicken

70

human/frog

56

human/tuna fish

55

human/shark

51

human/lamprey

35

Tertiary 50

100

100

human/orangutan mouse/rat cat/dog

time in millions of years

150

200

250 Permean 300 Carboniferous 350 Devonian 400 Silurian 450

% amino acids identical in hemoglobin α chain

human/chimp

0

41

Ordovician 500 Cambrian 550

Proterozoic

We Are All Different in Detail What precisely do we mean when we speak of the human genome? Whose genome? On average, any two people taken at random differ in about one or two in every 1000 nucleotide pairs in their DNA sequence. The Human Genome Project has arbitrarily selected DNA from a small number of anonymous individuals for sequencing. The human genome—the genome of the human species—is, properly speaking, a more complex thing, embracing the entire pool of variant genes that are found in the human population and continually exchanged and reassorted in the course of sexual reproduction. Ultimately, we can hope to document this variation too. Knowledge of it will help us understand, for example, why some people are prone to one disease, others to another; why some respond well to a drug, others badly. It will also provide new clues to our history—the population movements and minglings of our ancestors, the infections they suffered, the diets they ate. All these things leave traces in the variant forms of genes that have survived in human communities.

Figure 1–52 Times of divergence of different vertebrates. The scale on the left shows the estimated date and geological era of the last common ancestor of each specified pair of animals. Each time estimate is based on comparisons of the amino acid sequences of orthologous proteins; the longer a pair of animals have had to evolve independently, the smaller the percentage of amino acids that remain identical. Data from many different classes of proteins have been averaged to arrive at the final estimates, and the time scale has been calibrated to match the fossil evidence that the last common ancestor of mammals and birds lived 310 million years ago. The figures on the right give data on sequence divergence for one particular protein (chosen arbitrarily)—the a chain of hemoglobin. Note that although there is a clear general trend of increasing divergence with increasing time for this protein, there are also some irregularities. These reflect the randomness within the evolutionary process and, probably, the action of natural selection driving especially rapid changes of hemoglobin sequence in some organisms that experienced special physiological demands. On average, within any particular evolutionary lineage, hemoglobins accumulate changes at a rate of about 6 altered amino acids per 100 amino acids every 100 million years. Some proteins, subject to stricter functional constraints, evolve much more slowly than this, others as much as 5 times faster. All this gives rise to substantial uncertainties in estimates of divergence times, and some experts believe that the major groups of mammals diverged from one another as much as 60 million years more recently than shown here. (Adapted from S. Kumar and S.B. Hedges, Nature 392:917–920, 1998. With permission from Macmillan Publishers Ltd.)

Figure 1–53 Human and mouse: similar genes and similar development. The human baby and the mouse shown here have similar white patches on their foreheads because both have mutations in the same gene (called Kit), required for the development and maintenance of pigment cells. (Courtesy of R.A. Fleischman.)

42

Chapter 1: Cells and Genomes

Knowledge and understanding bring the power to intervene—with humans, to avoid or prevent disease; with plants, to create better crops; with bacteria, to turn them to our own uses. All these biological enterprises are linked, because the genetic information of all living organisms is written in the same language. The new-found ability of molecular biologists to read and decipher this language has already begun to transform our relationship to the living world. The account of cell biology in the subsequent chapters will, we hope, prepare you to understand, and possibly to contribute to, the great scientific adventure of the twenty-first century.

Summary Eucaryotic cells, by definition, keep their DNA in a separate membrane-enclosed compartment, the nucleus. They have, in addition, a cytoskeleton for support and movement, elaborate intracellular compartments for digestion and secretion, the capacity (in many species) to engulf other cells, and a metabolism that depends on the oxidation of organic molecules by mitochondria. These properties suggest that eucaryotes may have originated as predators on other cells. Mitochondria—and, in plants, chloroplasts—contain their own genetic material, and evidently evolved from bacteria that were taken up into the cytoplasm of the eucaryotic cell and survived as symbionts. Eucaryotic cells have typically 3–30 times as many genes as procaryotes, and often thousands of times more noncoding DNA. The noncoding DNA allows for complex regulation of gene expression, as required for the construction of complex multicellular organisms. Many eucaryotes are, however, unicellular—among them the yeast Saccharomyces cerevisiae, which serves as a simple model organism for eucaryotic cell biology, revealing the molecular basis of conserved fundamental processes such as the eucaryotic cell division cycle. A small number of other organisms have been chosen as primary models for multicellular plants and animals, and the sequencing of their entire genomes has opened the way to systematic and comprehensive analysis of gene functions, gene regulation, and genetic diversity. As a result of gene duplications during vertebrate evolution, vertebrate genomes contain multiple closely related homologs of most genes. This genetic redundancy has allowed diversification and specialization of genes for new purposes, but it also makes gene functions harder to decipher. There is less genetic redundancy in the nematode Caenorhabditis elegans and the fly Drosophila melanogaster, which have thus played a key part in revealing universal genetic mechanisms of animal development.

Which statements are true? Explain why or why not. 1–1 The human hemoglobin genes, which are arranged in two clusters on two chromosomes, provide a good example of an orthologous set of genes. 1–2 Horizontal gene transfer is more prevalent in singlecelled organisms than in multicellular organisms. 1–3 Most of the DNA sequences in a bacterial genome code for proteins, whereas most of the sequences in the human genome do not.

Discuss the following problems. 1–4 Since it was deciphered four decades ago, some have claimed that the genetic code must be a frozen accident, while others have argued that it was shaped by natural selection. A striking feature of the genetic code is its inherent resistance to the effects of mutation. For example, a change in the third position of a codon often specifies the same amino acid or one with similar chemical properties. The natural code

resists mutation more effectively (is less susceptible to error) than most other possible versions, as illustrated in Figure Q1–1. Only one in a million computer-generated “random” codes is more error-resistant than the natural genetic code. Does the extraordinary mutation resistance of the genetic code argue in favor of its origin as a frozen accident or as a result of natural selection? Explain your reasoning. number of codes (thousands)

PROBLEMS

25 20 15 10

natural code

5 0

0

5 10 15 susceptibility to mutation

Figure Q1–1 Susceptibility of the natural code relative to millions of computergenerated codes (Problem 1–4). Susceptibility measures the average change in amino acid properties caused by random mutations. A small value indicates that mutations tend to cause 20 minor changes. (Data courtesy of Steve Freeland.)

1–5 You have begun to characterize a sample obtained from the depths of the oceans on Europa, one of Jupiter’s moons. Much to your surprise, the sample contains a lifeform that grows well in a rich broth. Your preliminary analysis

END-OF-CHAPTER PROBLEMS

43

shows that it is cellular and contains DNA, RNA, and protein. When you show your results to a colleague, she suggests that your sample was contaminated with an organism from Earth. What approaches might you try to distinguish between contamination and a novel cellular life-form based on DNA, RNA, and protein?

GENE RNA mt nuc mt nuc

1–6 It is not so difficult to imagine what it means to feed on the organic molecules that living things produce. That is, after all, what we do. But what does it mean to “feed” on sunlight, as phototrophs do? Or, even stranger, to “feed” on rocks, as lithotrophs do? Where is the “food,” for example, in the mixture of chemicals (H2S, H2, CO, Mn+, Fe2+, Ni2+, CH4, and NH4+) spewed forth from a hydrothermal vent?

ratory gene Cox2, which encodes subunit 2 of cytochrome oxidase, was functionally transferred to the nucleus during flowering plant evolution. Extensive analyses of plant genera have pinpointed the time of appearance of the nuclear form of the gene and identified several likely intermediates in the ultimate loss from the mitochondrial genome. A summary of Cox2 gene distributions between mitochondria and nuclei, along with data on their transcription, is shown in a phylogenetic context in Figure Q1–2. A. Assuming that transfer of the mitochondrial gene to the nucleus occurred only once (an assumption supported by the structures of the nuclear genes), indicate the point in the phylogenetic tree where the transfer occurred. B. Are there any examples of genera in which the transferred gene and the mitochondrial gene both appear functional? Indicate them. C. What is the minimal number of times that the mitochondrial gene has been inactivated or lost? Indicate those events on the phylogenetic tree. D. What is the minimal number of times that the nuclear gene has been inactivated or lost? Indicate those events on the phylogenetic tree. E. Based on this information, propose a general scheme for transfer of mitochondrial genes to the nuclear genome. 1–11 When plant hemoglobin genes were first discovered in legumes, it was so surprising to find a gene typical of animal blood that it was hypothesized that the plant gene arose

+

+

+

Tephrosia Galactia Canavalia

+ + +

+ + +

Lespedeza

+

+

+

+

Eriosema Atylosia Erythrina

+ + +

+ + +

Ramirezella Vigna Phaseolus

+ + +

+ + +

+

+

+

Calopogonium + Pachyrhizus +

+ +

+ +

+ + + +

+ +

+ + + +

Cologania Pueraria Pseudeminia Pseudovigna

1–8 The genes for ribosomal RNA are highly conserved (relatively few sequence changes) in all organisms on Earth; thus, they have evolved very slowly over time. Were ribosomal RNA genes “born” perfect?

1–10 The process of gene transfer from the mitochondrial to the nuclear genome can be analyzed in plants. The respi-

+

Clitoria

Dumasia

1–7 How many possible different trees (branching patterns) can be drawn for eubacteria, archaea, and eucaryotes, assuming that they all arose from a common ancestor?

1–9 Genes participating in informational processes such as replication, transcription, and translation are transferred between species much less often than are genes involved in metabolism. The basis for this inequality is unclear at present, but one suggestion is that it relates to the underlying complexity. Informational processes tend to involve large aggregates of different gene products, whereas metabolic reactions are usually catalyzed by enzymes composed of a single protein. Why would the complexity of the underlying process—informational or metabolic—have any effect on the rate of horizontal gene transfer?

Pisum

+

+

+ + +

Ortholobium Psoralea Cullen Glycine

+

+

Neonotonia Teramnus Amphicarpa

+ + +

+

+ + + + + + +

+

+ +

Figure Q1–2 Summary of Cox2 gene distribution and transcript data in a phylogenetic context (Problem 1–10). The presence of the intact gene or a functional transcript is indicated by (+); the absence of the intact gene or a functional transcript is indicated by (–). mt, mitochondria; nuc, nuclei.

by horizontal transfer from an animal. Many more hemoglobin genes have now been sequenced, and a phylogenetic tree based on some of these sequences is shown in Figure Q1–3. A. Does this tree support or refute the hypothesis that the plant hemoglobins arose by horizontal gene transfer? B. Supposing that the plant hemoglobin genes were originally derived from a parasitic nematode, for example, what would you expect the phylogenetic tree to look like? Whale Rabbit Cat VERTEBRATES CobraChicken Human Salamander Cow Frog Goldfish

Barley

Lotus

Earthworm

Alfalfa Bean

Insect

PLANTS

Clam Nematode INVERTEBRATES

Chlamydomonas Paramecium

PROTOZOA

Figure Q1–3 Phylogenetic tree for hemoglobin genes from a variety of species (Problem 1–11). The legumes are highlighted in red.

44

Chapter 1: Cells and Genomes

1–12 Rates of evolution appear to vary in different lineages. For example, the rate of evolution in the rat lineage is significantly higher than in the human lineage. These rate differences are apparent whether one looks at changes in protein sequences that are subject to selective pressure or at

changes in noncoding nucleotide sequences, which are not under obvious selection pressure. Can you offer one or more possible explanations for the slower rate of evolutionary change in the human lineage versus the rat lineage?

REFERENCES

Genetic Information in Eucaryotes

General Alberts B, Bray D, Hopkin K et al (2004) Essential Cell Biology, 2nd ed. New York: Garland Science. Barton NH, Briggs DEG, Eisen JA et al (2007) Evolution. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Darwin C (1859) On the Origin of Species. London: Murray. Graur D & Li W-H (1999) Fundamentals of Molecular Evolution, 2nd ed. Sunderland, MA: Sinauer Associates. Madigan MT & Martinko JM (2005) Brock’s Biology of Microorganisms, 11th ed. Englewood Cliffs, NJ: Prentice Hall. Margulis L & Schwartz KV (1998) Five Kingdoms: An Illustrated Guide to the Phyla of Life on Earth, 3rd ed. New York: Freeman. Watson JD, Baker TA, Bell SP et al (2007) Molecular Biology of the Gene, 6th ed. Menlo Park, CA: Benjamin-Cummings.

The Universal Features of Cells on Earth Andersson SGE (2006) The bacterial world gets smaller. Science 314:259–260. Brenner S, Jacob F & Meselson M (1961) An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190:576–581. Fraser CM, Gocayne JD, White O et al (1995) The minimal gene complement of Mycoplasma genitalium. Science 270:397–403. Harris JK, Kelley ST, Spiegelman et al (2003) The genetic core of the universal ancestor. Genome Res 13:407–413. Koonin EV (2005) Orthologs, paralogs, and evolutionary genomics. Annu Rev Genet 39:309–338. Watson JD & Crick FHC (1953) Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature 171:737–738. Yusupov MM,Yusupova GZ, Baucom A et al (2001) Crystal structure of the ribosome at 5.5 Å resolution. Science 292:883–896.

The Diversity of Genomes and the Tree of Life Blattner FR, Plunkett G, Bloch CA et al (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474. Boucher Y, Douady CJ, Papke RT et al (2003) Lateral gene transfer and the origins of prokaryotic groups. Annu Rev Genet 37:283–328. Cole ST, Brosch R, Parkhill J et al (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544. Dixon B (1994) Power Unseen: How Microbes Rule the World. Oxford: Freeman. Kerr RA (1997) Life goes to extremes in the deep earth—and elsewhere? Science 276:703–704. Lee TI, Rinaldi NJ, Robert F et al (2002) Transcriptional regulatory networks in Saccharomyces cerevisiae. Science 298:799–804. Olsen GJ & Woese CR (1997) Archaeal genomics: an overview. Cell 89:991–994. Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740. Woese C (1998) The universal ancestor. Proc Natl Acad Sci USA 95:6854–6859.

Adams MD, Celniker SE, Holt RA et al (2000) The genome sequence of Drosophila melanogaster. Science 287:2185–2195. Andersson SG, Zomorodipour A, Andersson JO et al (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133–140. The Arabidopsis Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815. Carroll SB, Grenier JK & Weatherbee SD (2005) From DNA to Diversity: Molecular Genetics and the Evolution of Animal Design, 2nd ed. Maldon, MA: Blackwell Science. de Duve C (2007) The origin of eukaryotes: a reappraisal. Nature Rev Genet 8:395-403. Delsuc F, Brinkmann H & Philippe H (2005) Phylogenomics and the reconstruction of the tree of life. Nature Rev Genet 6:361–375. DeRisi JL, Iyer VR & Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278:680–686. Gabriel SB, Schaffner SF, Nguyen H et al (2002) The structure of haplotype blocks in the human genome. Science 296:2225–2229. Goffeau A, Barrell BG, Bussey H et al (1996) Life with 6000 genes. Science 274:546–567. International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409:860–921. Kellis M, Birren BW & Lander ES (2004) Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428:617–624. Lynch M & Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 290:1151–1155. Mulley J & Holland P (2004) Comparative genomics: Small genome, big insights. Nature 431:916–917. National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/ Owens K & King MC (1999) Genomic views of human history. Science 286:451–453. Palmer JD & Delwiche CF (1996) Second-hand chloroplasts and the case of the disappearing nucleus. Proc Natl Acad Sci USA 93:7432–7435. Pennisi E (2004) The birth of the nucleus. Science 305:766–768. Plasterk RH (1999) The year of the worm. BioEssays 21:105–109. Reed FA & Tishkoff SA (2006) African human diversity, origins and migrations. Curr Opin Genet Dev 16:597–605. Rubin GM, Yandell MD, Wortman JR et al (2000) Comparative genomics of the eukaryotes. Science 287:2204–2215. Stillman B & Stewart D (2003) The genome of Homo sapiens. (Cold Spring Harbor Symp. Quant. Biol. LXVIII). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. The C. elegans Sequencing Consortium (1998) Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018. Tinsley RC & Kobel HR (eds) (1996) The Biology of Xenopus. Oxford: Clarendon Press. Tyson JJ, Chen KC & Novak B (2003) Sniffers, buzzers, toggles and blinkers: dynamics of regulatory and signaling pathways in the cell. Curr Opin Cell Biol 15:221–231. Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291:1304–1351.

9n sq8ra^auoJlnau Jo uoloJd euo 'Jeleuqlrru B ueds ol 'eurl ]qBIeJls e ul lno p1el 'rueqlJo uorTlrrug tnoqe e{Bl plno.^ tl }eqt os teteuerp ur uru 7'g,(lqBnor sr ruo}e uoqJ€f, lenprlrpu uV'ezrs rraq] aurBeurr o] preq sr 1rluql IIEIUsos eJp sruolv 'ruole uaSorpdq e go sseru eq] o1 pnba ,(laleruxordde 11unsspru cnuolp ue Suraq uolpp euo 'suoilDp ur pagrcadsuego sr elnJaloru e Jo ruole ue Jo sseru agl'Orr su uailrJm sr pue 7I go lq8raa,rJlruole uE seq uoqJeJ Jo adotosr elqelsun cruole ue seq uoqrec 3o adol uE seaJaqm')71 se pezrToqufs sr pue ZI ;o lq8rarvr. -osr rofetu eql snqJ'lplol eql o1Surqlou tsorule a]nqrJluoJ pue ralq8q qonru ere suoJlJeleaql ecurs 'sureluoc alnceloru Jo ruole eql leql suoJlneu snld suolord;o raqunu aql o] pnba rtlepuassa q slqJ'urole uaSorp,(q u 1o leql ol elrleleJ ssetu slr sr 'alnoeloru u Jo fq8laiu JBInJeIoru eql Jo 'ruole up go rqtgam Jlurolu eql

sruolv Jo sad^I ru\ale urorl apew aJVslla) 'peruJoJ eJp selnf,elotu ur raqleSol sruote ploq ]Eql spuoq leJrrueqJ eql Jo IIB ^aoq A\ODI ol [BrJnJJ sJlr 'aJoJaJaq]tellEur elprurueur ruoq llrnq are srusrueSro 3utntl llroq puelsJepun ot JepJo uI 'salnJalou ruJoJ ol sdnor8 ur raqlaSol pe{url eJpsruolp rraql ,(ea,raq1uo puadap-aperu eJe s11ac 8ur -^I qclqm ruo4 slerJaleuraq] SurpnlJur-slueuala arnd ueql Jaqto seJuelsqns '11-7 arnt;g) Iaolv ue st saqradord Jo scrlsrJa]f,eJeqJeql tananoll lecrrueqc elrl 'superu -cuDSIpslr suplal Ilrls ]eql lueuele ue;o aycqredlsell"rus eqJ Iecrueqf, ,{.q sacuelsqns Jaqlo o}ur paueluoJ Jo rrmop ue{oJq eq touueJ teql uoqrpo ro uaSorp^,tqse qons saouplsqns-sluautap Jo suouuurqruoo Jo epuru sr Japehtr

r1) v ro slNtNodwo) rv)rwlH)rHr_

88

COOIWOUIADU]N] Ntvt_80 sttt) MoH slltf A8A9UlNtlO

s9 ]SN ]HI CNVSISAIVIVf 5V

ttlf v lo -tv)lt slNtNodtJo) tHf tHI

raldeq) sFlI ul

'eJIIJorltslratrereqr aru leql s8ulql reqlo eq] ile op o] sB se-acnpordeJ pue,r,ror8 o] srusrueBro pue slleJ alqeue leql salnJalou IIe^ -oJceruaseqlJo sapradord anbrun eql sl lI 'pue-ol-pua peluq slrunqns leJrrueqc Jo surpqc-salnlalou c1tarut1od snoruroue olur paterodJoJur eJEslleo ur sruole uoqJpo eqt Jo lsolu 'salnJeloru SurumluoJ-uoqJec Ilerus Jo ,fiagen e ureluoc slac q8noqllv'umou{ uralsrts [EcruaqJ raqto rtue ueql ,fu]sluaqJ slr ul patecrld -tuoc aroru dllsel sr :xalduroc dlsnor.uJouasr drlsnuaqc 1ac eqt uela lsalduns 1ac 'lueUodurr 'pJrql'uonnlos snoanbe u1 aceyde{Bi lsoru pue luql suorlf,eal IeJr -uaqc uo ,(1a8re1spuadap eJIIpue leluru lueJJad 64 are sllal 'puoras 'ttlslutaqc uunBn sE u,r,rornleJoJeJeqlsr r(pn1sesoqm 'spunodruoc uoqJec uo ,{punulaq,rn -relo peseq sl ]l 'lsJIC 'prcads s aytgo rfulsrueqc aql tele,r,roH 'sira.e1 pcrsdqd puu IeorrueqJ sLaqosrp ]eql susrue8ro 3urn1 ur 3urqlou sr eJaqt lr,rorDl^ ou a A 'sauradord alrlf, urlsrp Jreql ro; alqrsuodseJ sem lpql-,,snrurup,, uE-acJoC 1etr1 e ureluof, ol peleqaq aJelrlr sprutue drnluec qluee]auru eq] []un 'paepq 'saqrrcsapl.lpurrou drlsrueqf, ]eql sase8pue 'sprnbu 'sprlosJo plrom ar{} ruoq pede ruaql tas ol readde acnpordar pue aror8 o] rqilqe Jlaqt pup torlpqaq pyasodrnd [18uruaas 4aqt 'surro;3unr1 ;o rlrsranrp alqrpeJJuraq;'ualsds leJrruaqc e flararu sl 1 raldeq3 ur peqrJcsep sarnleaJf, Surnq aql Jo qrea ]eq] eapr eql tdecJe 01 llnorJJrp rr{8ls rsrg 1u sf lI

s!seHlu{solg pue Atlsltueq)Ila)

'aur!] Joluelsutua^t6Iue le sr uorl)alaue alaqm{l}texa 6urDrpard Jo (em ou sraraq]pue /slrueq)auuinluenb Jo sltnelaqt {q pauranobst lotneqaq rlaqt^trlearur 'saprlredlpnpr^rpul se alaq uMoqsaresuoll)elaaq1q6noqrle 'pnolr uor})elaaq} {1;eu13 Joleqt,_0t tnoqer(;uosrsnal)nuaql Jolalauetp aql'uor]rppeu1'araqpelera66exa ,(;lear6srozrsraql ialoqMp se uole aqi ol uortplarur elnuruJI1r;earur ate suorlralapue'suo1o.rd'suollneu aqf 's eualeur I >rue6.ro 1ourbrro;oar.u!leql autLulalap ot {boloaeqtreut pesnst q)tqM '6u!rep tI uoqre)se urvroulanbruqral e roJsrseqeqt sLuro]srql'alel ,{pea1s 1nq Molse lp {etap enrl>eorpe.r sao6tapuny1 uoqre)'suollnau1q6rapue suolo.rd xrs a^eLlsuJoleasoqMitt uoqre)a^tl)eotpel oql'aoolosralqelsunueJo stunoue xts lleuisoslealp atoL]l'suollnau pue suolordxrsr1llM'Z t uoqlef adolosr alqetsaLllse slsrxaqlrel uo uoqle) lsout alrqM'alotUpxa lol 'alqelsunalp lsqt auos 6urpn;rur'{1;ernleu ln)fo sluaulola oql llelsorulpJo sadolosra;d1t;ny1 'suolord;olaquJnuaulpsaql lnq suollnau,olaqulnu luata;Jtpe 6urneq adolosr\)ea'sadolos t palle)'sr.!loj le)!]uep!{1;e>tuaql1nqalqeqsrnburlsrp Illerrs{qdlelanasut }stxaue) }uauale ue 'suorlnauJo asne)a8'tlotp aql Jo saryadordle)ruraq)oql lalle lou op {eql lnq-^e)ap a^rl)eorpelIq alet6elursrp Ieu sna;lnu eqr MaJool ro ,(ueu.r ool arearaqljr-snal)nu aql Jo ^ttltqets leJn])nrlsaql ol alnquluor{aq1'suolord sesseu auuesaq1{;;erluassa Jo sa;rrlred pableq)unaJesuollnoN lr,l-toleqns 'laqIIJnu]il.llole aures aq] a^eqtuauale uanr6e;o suroleaql Jo llP'ruoleue,o lot^eqaqlP)tulaq)aql eururalapleql suorl)elaaql srlr asnplag 'e6reqr 1auou spqulolp aql lpql os '(Jaqwnu )twolDaql) suotoldJo laqunu slrol lenDasruiole uP ur suoll)elaJo loqurnu atll'suotlnau;erlnau,{;lerulta;a pue suolotdpa6teqt {;anrlsod qloq Jo slsrsuo)ue6orpr(qlda)xa uole llane lo snapnuaq1'ua6orplq ro urole ue pue uoqre) Jo ruolp ue,o suopleluaserde.r )lleulaq)s(1q6191-7 arnbr3

uoJlJele auo,{1uo qlr^ 'lspJ}uoc[q 'uaSorprtg 'sase8uaul ilP eJEasaql 1g+ g + 7 qll-M uoBJE pue 'B + Z qlyv\ uoau (suoJlJala Z qlF runlTeq eru saldluuxg 'o^Ilce -aJun,li.llerrluaqraroJaraqtpue alqEtsf.lprcadsa sI suorlJale q}l \ palllJ dlarpua sr ilaqs lsoruJelno asoq ^ Iuole uv 'uo os puE '.pJlql eq] eJoJaqpuoces eql 'puoses eq] aroJaq lleqs ]srrJeql-Jepro uI slEllqro eq] ilu ruolP uP Jo suorlcele aq] 'sluole JaBJEIaqt u suolldeJxa ul€lJec qlFu 'aroJaJeqJ'sllaqs lsoureuul eql ,,(dnccodaqt uaqarr'sl ]Eql-alqlssod eJe]eq] salpls punoq dFqBI] tsoru eql uI ere suoJlrale aq] ile ueq^a alq€ls ]solu sI luolP uE Jo luetuaSuBJJPuoJlsela eqJ 'salnJelolu 't{JEe suoDcele Ief,rSolorq ur aJpJ ,{ran aru sfieqs rnoJ ueq] eJolu qll^\ surolv 8I ploq uBr slleqs qulJ puE qunoJ aqJ'suol]cele 1q3le ol dn sploq osp lI :punoq dpq8u ssel uale are tpqt suorlcale suletuoJ llaqs prlql eql 'suorlcala lq8le ol dn sploq ilaqs puoJas srql 'punoq fpq8p ssel are suorlcala sll pue 'snelJnu eq] tuo4 d.e.vreraqpeJ sr Ileqs puosas eql 'suoJ]cale o^\l Jo lunlurxBlu P sploq 'ileqs 'Jsolurauul ,{dncco pue ]l o1 flSuorls punoq eql dllq8p Jsoru fiaqs sIqI uo lsesolJ suoJlJala aqL'Ip|.F aJEsnalJnu a^rlrsod aql ol eSeJaAe ]sotl'rpe13eJ11p uoJpala palles-os E-ad,{} ua^r3 e Jo l€lrqJo ue uI pa}Pporuruof,Jeeq uPc }eq} suoJ]f,eleJo Jeqrunu eq] ol llurll l3rJls e sI aJaq] leql pue 'spllqJo pellEJ 'selEls eleJcsrpureual ur,{luo lsue ueJ ruole ue ur suoJlcala}uql elBlJIp srltelasaql 'aJIT depLrala ur JeIIrueJ asoql ruo4 s,lrel ]uaJaJJIpfrarr daqo apcs cldocsoJJlruqns slql uo suorloru lnq'snalsnu eql punoJBuollotu snonunuoJ uI aJesuoJlselg 'salnJalou ruJoJo] eulquoJ sruole qJIqM,,(q.,tr1sruraq3 selnJ Jo eqf .{JIJads pue ruo}e ue Jo JoIJe}xa aql uJoJ ^,(aq; 'sluaure8ueJJeer oBrapun 'sanssl] SuntT uI 'rolJear JBelcnu e ]Bql ruole uE Jo suoJlJele eql dpo st lI Jo Jo uns aq] Jo JorJaluraq] ur Jo 'aldurexa ro; ,{ecap eAI}JEoIpeJSutrnp-suoplpuoc arueJlxeJepun .{1uosraulred aSueqc puE snelJnu eq} uI Jaqlouu euo ol dFqSll pepla,/!\aJEsuorlnau pup suoloJd 'suoJlsalerleq] uo snJoJe^\'srusrueSro3unr1 dn aleu leql salnJelou aql {uJoJ ot JaqleSol puoq stuo}P ^ oq pue}sJapun oJ

l)eJalul stuolv /vloHau!uJaloc suoll)elf lsotltlolno eql Lrlsnuaqc;o ad,rt a,ulcupslp e Jo eoueplla sI pue (t-Z arn8lg) lua(uuoJllue '1q8raa,r Jlue8rour Sur.trluou eq] Jo ]Pql tuoq ^lpe{Jelu sJaJJIpuolllsodtuol srql s.usrup8ro ue Jo %9'96 dn a{Eur-(O) ue8dxo pue '(N) uaSorllu '(H) ueSorpdq (sluaruele ' (3) esaqt uoqrec-qcrq ^ Jo uol]f,elas llstus E ,,tpo ;o aperu eJe Jo JnoJ 're^e,{i\oq'sursrue8roSuyrtt 'sluole sll uI suoJ]3elapue suoloJd Jo Jeqrunu aql uI sJaqlo aq] uro4 Surra;;rp qJea 'slualuele SuuJnoJo -,(1urnleu 68 aJe eJaqJ '(z-z arnElc) eruBlsqns aql Jo elolu auo palpc sr fi11 -uenb srql 'sruer8XJo sseruE aneq IIIM ]l Jo selnoelorug70I x 9 'XJo tq8la,vrrEIn -Jelou e seq eJuulsqns eJI 'selnJelou Jo sruole PnpIAIpulJo s[uJal uI peJnsEau sapnuunb pup sarlrtuenb fepI-rana uaaMleq dtqsuouelar eqt Sulquf,sep JolceJ 'sluolE eleJs ^e{ eql sI (Jaqlunu s.oJpBSo^vpa11ec'g70I x 9) Jequnu aSnq srq; ez0l x 9 suleluoc ueSoJp^q;o ruur8 auo os'luer8 (sz0l x 9)/I ,{lateurxordde

! = ]q6ta^^ lil-uole I = laqulnu )!ulole urole ua6olpfq

7 1 = 1 q 6 r a m: r u o 1 e )lulole 9 = JAqLUnU r.,lrole uoqre)

uoJpala

pue^rlsruaq) llo) :z ieldeqf srsaqlu^so!g

9V

47

THECHEMICAL COMPONENTS OF A CELL

and only a half-filled shell, is highly reactive. Likewise, the other atoms found in living tissues have incomplete outer electron shells and can donate, accept, or share electrons with each other to form both molecules and ions (Figure 2-4). Becausean unfilled electron shell is less stable than a filled one, atoms with incomplete outer shells tend to interact with other atoms in a way that causes them to either gain or lose enough electrons to achieve a completed outermost shell. This electron exchange occurs either by transferring electrons from one atom to another or by sharing electrons between two atoms. These two strategies generate two types of chemical bonds between atoms: an ionic bond is formed when electrons are donated by one atom to another, whereas a coualent bondis formed when two atoms share a pair of electrons (Figure 2-5). Often, the pair of electrons is shared unequally, with a partial transfer between two atoms that attract electrons differently-one more electronegatiuethanthe other: this intermediate strategy results in a polar coualentbond, as we shall discusslater. An H atom, which needs only one electron to fill its shell, generally acquires it by electron sharing, forming one covalent bond with another atom; often this bond is polar-meaning that the electrons are shared unequally. The other common elements in living cells-C, N, and O, with an incomplete second shell, and P and S, with an incomplete third shell (see Figure 2-4)-generally share electrons and achieve a filled outer shell of eight electrons by forming several covalent bonds. The number of electrons that an atom must acquire or lose (either by sharing or by transfer) to fill its outer shell is knorm as irs ualence. The crucial role of the outer electron shell in determining the chemical properties of an element means that, when the elements are listed in order of their atomic number, there is a periodic recurrence of elements with similar properties: an element with, say, an incomplete second shell containing one electron will behave in much the same way as an element that has filled its second shell

A mole is X grams of a substance, where X is its relative molecularmass (molecularweight).A mole will contain 5 x 102m 3 o l e c u l e so f t h e s u b s t a n c e . 1 m o l e o f c a r b o nw e i g h s1 2 g 1 m o l e o f g l u c o s ew e i g h s 1 8 0g 1 m o l e o f s o d i u mc h l o r i d ew e i g h s5 8 g Molar solutions have a concentration of 1 mole of the substancein 1 liter of s o l u t i o n .A m o l a r s o l u t i o n( d e n o t e da s 1 M ) o f g l u c o s e{,o r e x a m p l e ,h a s , h i l e a m i l l i m o l asr o l u t i o n 1 8 0g / 1 w (1 mM) has 180mg/|. The standardabbreviationfor gram is g; the abbreviationfor liter is l.

Figure2-2 Molesand molar solutions.

:,4.4:lpri',1:ll:rllr:ai:':ir:il

!

numan oodv Earth'scrust

o c 6

Ef s o !

a @ a o

E20 o o

I.: ano Mg

NaP ano K

Figure2-3 The abundancesof some chemicalelementsin the nonliving world (the Earth'scrust)comparedwith their abundancesin the tissuesof an animal.The abundanceof eachelement is expressedas a percentageof the total numberof atomspresentincluding water.Thus,becauseof the abundanceof water,more than 600loof the atoms in a livingorganismarehydrogenatoms.The relativeabundanceof elementsis similar i n a l l l i v i n gt h i n g s .

48

Chapter2: CellChemistryand Biosynthesis tomic number

I

e l e c t r o ns h e l |-

Figure2-4 Filledand unfilledelectron shellsin somecommonelements.All the elementscommonlyfound in living organismshaveunfilledoutermostshells (red)andcan thus participatein chemical reactions with otheratoms.For comparison, someelementsthat have only filled shells(yellow)areshown;these arechemicallyunreactive.

&ae&&& &&&&&s e&8*a€ &***** &&&&&& e e e * | & # * t * , s & & & ee

and has an incomplete third shell containing one electron. The metals, for example, have incomplete outer shells with just one or a few electrons,whereas, as we have just seen,the inert gaseshave full outer shells.This pattern gives rise to the famous periodic table of the elements, presented in Figure 2-6 with the elements found in living organisms highlighted.

CovalentBondsFormby the Sharingof Electrons All the characteristics of a cell depend on the molecules it contains. A molecule is defined as a cluster of atoms held together by covalent bonds; here electrons are shared between atoms to complete the outer shells,rather than being transferred between them. In the simplest possible molecule-a molecule of hydrogen (H2)-two H atoms, each with a single electron, share two electrons, which is the number required to fill the first shell. These shared electrons form a cloud of negative charge that is densestbetween the two positively charged nuclei and helps to hold them together, in opposition to the mutual repulsion between like charges that would otherwise force them apart. The attractive and repulsive forces are in balance when the nuclei are separatedby a characteristic distance, called the bond length. Another property of any bond-covalent or noncovalent-is its bond strength, which is measured by the amount of energy that must be supplied to break that bond. This is often expressedin units of kilocalories per mole (kcal/mole), where a kilocalorie is the amount of energy needed to raise the temperature of one liter aroms

a

U

Y-,;

ELECTRoNS

I

molecule

covalent bond

positive ron

negative ton

i o n i cb o n d

Figure2-5 Comparisonof covalentand ionicbonds.Atomscanattaina more stablearrangement of electronsin their outermostshellby interactingwith one another.An ionicbond isformedwhen electronsare transferredfrom one atom to the other.A covalentbond isformed when electronsare sharedbetween atoms.The two casesshown represent extremes;often,covalentbonds form with a partialtransfer(unequalsharingof electrons), resultingin a polarcovalent bond (seeFigure2-43).

49

THECHEMICAL COMPONENTS OFA CELL a t o m i cn u m b e r a t o m i cw e i g h t 6789

CNOF 12

11

12

14

23

28

24 20

23

24

25

26

21

KCa

VCrMnFeCoNi

39

51

40

16

siPScl

Na Mg 19

16

14 15

52 42

55

56

59

2A

29

11

64

35

34

30

5e

CuZn 59

32

19 11

19

65

53

Mo

I

96

127

of water by one degreeCelsius(centigrade).Thus if 1 kilocalorie must be supplied to break 6 x 1023bonds of a specific type (that is, I mole of these bonds), then the strength of that bond is I kcal/mole. An equivalent, widely used measure of energy is the kilojoule, which is equal to 0.239kilocalories. To understand bond strengths, it is helpful to compare them with the average energiesof the impacts that molecules are constantly experiencing from collisions with other molecules in their environment (their thermal, or heat, energy), as well as with other sources of biological energy such as light and glucose oxidation (Figure 2-7).Typical covalent bonds are stronger than the thermal energies by a factor of 100, so they resist being pulled apart by thermal motions and are normally broken only during specific chemical reactions with other atoms and molecules. The making and breaking of covalent bonds are violent events, and in living cells they are carefully controlled by highly specific catalysts, called enzymes.Noncovalent bonds as a rule are much weaker; we shall see later that they are important in the cell in the many situations where molecules have to associateand dissociate readily to carry out their functions. \Mhereasan H atom can form only a single covalent bond, the other common atoms that form covalent bonds in cells-O, N, S, and B as well as the allimportant C atom-can form more than one. The outermost shell of these atoms, as we have seen, can accommodate up to eight electrons, and they form covalent bonds with as many other atoms as necessary to reach this number. Oxygen, with six electrons in its outer shell, is most stable when it acquires an extra two electrons by sharing with other atoms and therefore forms up to two covalent bonds. Nitrogen, with five outer electrons, forms a maximum of three covalent bonds, while carbon, with four outer electrons, forms up to four covalent bonds-thus sharing four pairs of electrons (seeFigure 2-4). \.Vhen one atom forms covalent bonds with several others, these multiple bonds have definite arrangements in spacerelative to one another, reflecting the orientations ofthe orbits ofthe shared electrons.The covalent bonds ofsuch an atom are therefore characterized by specific bond angles as well as by bond lengths and bond energies (Figure 2-B). The four covalent bonds that can form around a carbon atom, for example, are arranged as if pointing to the four corners of a regular tetrahedron. The precise orientation of covalent bonds forms the basis for the three-dimensional geometry of organic molecules.

average t h e r m a lm o t i o n s E NE R G Y CONTENT ( k c a l / m o l e0 ) .1

1 noncovalentbond breakagein water

ATPhydrolysis in cell

Figure2-6 Elementsorderedby their atomicnumberform the periodictable. Elements fall into groupsthat show similarpropertiesbasedon the number in its of electronseachelementpossesses outershell.Forexample,Mg and Catend to giveawaythe two electronsin their outershells;C, N,and O completetheir The secondshellsby sharingelectrons. four elementshighlightedin red constitute99oloof the total number of atomspresentin the humanbody.An additionalsevenelements,highlightedin of b/ue,together representabout 0.9olo the total.Otherelements,shownin green, arerequiredin traceamountsby humans. It remainsunclearwhetherthose in elementsshownin yellowareessential humansor not.Thechemistryof life,it the seems,is thereforepredominantly chemistryof lighterelements. Atomicweights,givenby the sum of the orotonsand neutronsin the atomic will varywith the particular nucleus, isotopeof the element.Theatomic weightsshownherearethoseof the mostcommonisotopeof eachelement.

C-C bond breakage

[ ::.,tr,,+;:.1]*rrg|.f+€*#*#Ei#fflff 100 10 green light

1000

complete glucoseoxidation

Figure2-7 Someenergiesimportant for cells.Notethat theseenergiesare comparedon a logarithmicscale

50

Chapter2: CellChemistryand Biosynthesis

-ooxygen (A)

-N-

I nitrogen

I

-c I caroon

Figure2-8 The geometry of covalent bonds.(A)Thespatialarrangement of the covalentbondsthat can be formed by oxygen,nitrogen,and carbon. (B)Moleculesformed from theseatoms havea precisethree-dimensional structure,as shown here by ball-and-stick modelsfor waterand propane. A structurecan be specifiedby the bond anglesand bond lengthsfor each covalentlinkage. Theatomsarecolored accordingto the following,generally usedconvention:H, white;C, block; O, red; N, blue.

water (H2O) (B)

p r o p a n e( C H 3 - C H 2 - C H 3 )

ThereAre DifferentTypesof CovalentBonds Most covalent bonds involve the sharing of two electrons, one donated by each participating atom; these are called single bonds. Some covalent bonds, however, involve the sharing of more than one pair of electrons. Four electrons can be shared, for example, two coming from each participating atom; such a bond is called a double bond. Double bonds are shorter and stronger than single bonds and have a characteristic effect on the three-dimensional geometry of molecules containing them. A single covalent bond between tvvo atoms generally allows the rotation of one part of a molecule relative to the other around the bond axis. A double bond prevents such rotation, producing a more rigid and less flexible arrangement of atoms (Figure 2-9 and Panel 2-1, pp. 106-107). In some molecules, electrons are shared among three or more atoms, producing bonds that have a hybrid character intermediate between single and double bonds. The highly stable benzene molecule, for example, consists of a ring of six carbon atoms in which the bonding electrons are evenly distributed (although usually depicted as an alternating sequence of single and double bonds, as shown in Panel 2-1). \ivhen the atoms joined by a single covalent bond belong to different elements, the two atoms usually attract the shared electrons to different degrees. compared with a c atom, for example, o and N atoms attract electrons relatively strongly, whereas an H atom attracts electrons more weakly. By definition, a polar structure (in the electrical sense)is one with positive charge concentrated toward one end (the positive pole) and negative charge concentrated toward the other (the negative pole). covalent bonds in which the electrons are shared unequallyinthiswayarethereforeknown aspolarcoualentbonds(Figure2-10). For example, the covalent bond between oxygen and hydrogen, -O-H, or between nitrogen and hydrogen, -N-H, is polar, whereas that between carbon and hydrogen, -C-H, has the electrons attracted much more equally by both atoms and is relatively nonpolar. Polar covalent bonds are extremely important in biology because they create permanent dipolesthat allow molecules to interact through electrical forces. Any large molecule with many polar groups will have a pattern of partial positive and negative chargeson its surface.\Ay'hen such a molecule encounters a second molecule with a complementary set of charges, the two molecules will be attracted to each other by electrostatic interactions that resemble (but are weaker than) the ionic bonds discussedoreviouslv.

(A) ethane

(B) ethene Figure2-9 Carbon-carbondouble bonds and singlebondscompared.(A)The ethanemolecule,with a singlecovalent bond betweenthe two carbonatoms, illustrates the tetrahedral arrangement of singlecovalentbondsformedby carbon. One of the CH3groupsjoined by the covalentbond can rotaterelativeto the otheraroundthe bond axis.(B)The doublebond betweenthe two carbon atomsin a moleculeof ethene(ethylene) altersthe bond geometryof the carbon atomsand bringsall the atomsinto the sameplane(blue);thedoublebond preventsthe rotationof one CH2group relativeto the other.

THECHEMICAL COMPONENTS OFA CELL

51

An AtomOftenBehaves asif lt Hasa FixedRadius \.Vhena covalent bond forms between two atoms, the sharing of electrons brings the nuclei of these atoms unusually close together. But most of the atoms that are rapidly jostling each other in cells are located in separate molecules. \A/hat happens when two such atoms touch? RoshanKeab 02I-66950639 For simplicity and clarity, atoms and molecules are usually represented schematically-either as a line drawing of the structural formula or as a balland-stick model. Space-fiIling models,however, give us a more accurate representation of molecular structure. In these models, a solid envelope represents the radius of the electron cloud at which strong repulsive forces prevent a closer approach of any second, non-bonded atom-the so-called uan derWaals radius for an atom. This is possible because the amount of repulsion increases very steeply as two such atoms approach each other closely.At slightly greater distances, any two atoms will experience a weak attractive force, knor,rryras a uan der Waalsattraction. As a result, there is a distance at which repulsive and attractive forces precisely balance to produce an energy minimum in each atom's interaction with an atom of a second, non-bonded element (Figure Z-tl). Depending on the intended purpose, we shall represent small molecules as Iine drawings, ball-and-stick models, or space-filling models. For comparison, the water molecule is represented in all three ways in Figure 2-l2.lMhenrepresenting very large molecules, such as proteins, we shall often need to further simplifu the model used (see,for example, Panel 3-2, pp. 132-133).

6-

ls

Figure2-10 Polarand nonpolar covalentbonds.Theelectron distributions in the oolarwatermolecule (H:O)and the nonpolaroxygenmolecule (Oz)are compared(6+,partialpositive charge;6-, partialnegativecharge).

Waterls the MostAbundantSubstance in Cells Water accounts for about 70% of a cell'sweight, and most intracellular reactions occur in an aqueous environment. Life on Earth began in the ocean, and the conditions in that primeval environment put a permanent stamp on the chemistry of living things. Life therefore hinges on the properties of water. In each water molecule (HzO) the two H atoms are linked to the O atom by covalent bonds (seeFigure 2-12). The two bonds are highly polar becausethe O is strongly attractive for electrons, whereas the H is only weakly attractive. Consequently,there is an unequal distribution of electrons in a water molecule, with a preponderance of positive charge on the two H atoms and of negative charge on the O (see Figure 2-10). 'vVhen a positively charged region of one water molecule (that is, one of its H atoms) approaches a negatively charged region (that is, the O) of a secondwater molecule, the electrical attraction between them can result in a weak bond called a hydrogenbond (seeFigure 2-15). These bonds are much weaker than covalent bonds and are easily broken by the random thermal motions due to the heat energy of the molecules, so each bond lasts only a short time. But the combined effect of many weak bonds can be profound. Each water molecule can form hydrogen bonds through its two H atoms to two other water molecules, producing a network in which hydrogen bonds are being continually broken and formed (Panel 2-2, pp.f0B-109). It is only because of the

. (+) I E U

z

U

(-)

v a n d e r W a a l sf o r c e e q u i l i b r i u ma t t h i s p o i n t

Figure2-1 1 The balanceofvan der Waalsforces between two atoms. As the nucleiof two atomsapproach eachother,they initiallyshowa weak due to their bondinginteraction fluctuatingelectriccharges.However,the sameatomswill stronglyrepeleachother if they are brought too closetogether. The balanceof thesevan derWaals forcesoccursat attractiveand reoulsive the indicatedenergyminimum.This minimumdetermines the contact distancebetweenany two noncovalently bondedatoms;this distanceis the sum of theirvan der Waalsradii.By definition, zero energy(indicatedby the dotted red line)is the energywhen the two nuclei areat infiniteseparation.

52

Chapter2: CellChemistryand Biosynthesis van derWaals

radiusofO=t+A

o HH (A)

(B)

van derWaals r a d i u so f H i=1.24

(c)

";i.ill3l?ll,"l'

hydrogen bonds that link water molecules together that water is a liquid at room temperature, with a high boiling point and high surface tension-rather than a gas. Molecules, such as alcohols, that contain polar bonds and that can form hydrogen bonds with water dissolve readily in water. Molecules carrying plus or minus charges (ions) likewise interact favorably with water. Such molecules are termed hydrophilic, meaning that they are water-loving. A large proportion of the molecules in the aqueous environment of a cell necessarilyfall into this category including sugars, DNA, RNA, and most proteins. Hydrophobic (waterhating) molecules, by contrast, are uncharged and form few or no hydrogen bonds, and so do not dissolve in water. Hydrocarbons are an important example (see Panel 2-I, pp. 106-107). In these molecules the H atoms are covalently linked to C atoms by a largely nonpolar bond. Becausethe H atoms have almost no net positive charge, they cannot form effective hydrogen bonds to other molecules. This makes the hydrocarbon as a whole hydrophobic-a property that is exploited in cells,whose membranes are constructed from molecules that have long hydrocarbon tails, as we shall see in Chapter I0.

Some PolarMoleculesAre Acidsand Bases One of the simplest kinds of chemical reaction, and one that has profound significance in cells,takes place when a molecule containing a highly polar covalent bond between a hydrogen and a second atom dissolvesin water. The hydrogen atom in such a molecule has largely given up its electron to the companion atom and so resembles an almost naked positively charged hydrogen nucleus-in other words, a proton (H+).\A/henwater molecules surround the polar molecule, the proton is attracted to the partial negative charge on the O atom of an adjacent water molecule and can dissociate from its original partner to associate instead with the oxygen atoms of the water molecule to generate a hydronium ion (H3O+)(Figure 2-f 3A). The reversereaction also takes place very readily, so one has to imagine an equilibrium state in which billions of protons are constantly flitting to and fro from one molecule in the solution to another. The same tlpe of reaction takes place in a solution of pure water itself. As illustrated in Figure 2-13B, water molecules are constantly exchanging protons with each other. As a result, pure water contains an equal, very low concentration of H3O+and OH- ions, both being present at 10-7M. (The concentration of H2O in pure water is 55.5M.) Substancesthat releaseprotons to form H3O+when they dissolve in water are termed acids. The higher the concentration of HsO*, the more acidic the solution. As H3O* rises, the concentration of OH- falls, according to the equilibrium equation for water: [HsO*][OH-] = 1.0 x 10-la, where square brackets denote molar concentrations to be multiplied. By tradition, the H3O+ concentration is usually referred to as the H+ concentration, even though nearly all H+ in an aqueous solution is present as H3O+.To avoid the use of unwieldy numbers, the concentration of H+ is expressedusing a logarithmic scale called the pH scale, as illustrated in Panel 2-2 (pp.108-109). Pure water has a pH of 7.0, and is neutral-that is, neither acidic (pH < 7.0) nor basic (pH > 7.0).

Figure2-1 2 Threerepresentations of a water molecule.(A)The usualline formula,in drawingof the structural whicheachatom is indicatedby its standardsymbol,and eachline represents a covalentbondjoiningtwo model,in atoms.(B)A ball-and-stick by spheres whichatomsarerepresented of arbitrarydiameter,connectedby sticks representing covalentbonds.Unlike(A), represented bond anglesareaccurately in thistype of model(seealsoFigure model,in which 2-8).(C)A space-filling both bond geometryand van derWaals represented. radiiareaccurately

THECHEMICAL COMPONENTS OFA CELL

o

53 HOH

cH3-C

o-H 66acetic acid

|| !:!l HrO

hydronium ton

acetate ton

(A)

HrO

_-

proton moves from one m o l e c u l et o the other

(B)

HrOt

OH-

hydronium ton

hydroxyl ton

Becausethe proton of a hydronium ion can be passedreadily to many types of molecules in cells, altering their character,the concentration of H3O+inside a cell (the acidi$ must be closely regulated. The interior of a cell is kept close to neutrality, and it is buffered by the presence of many chemical groups that can take up and releaseprotons near pH 7. The opposite of an acid is a base. Just as the defining property of an acid is that it donates protons to a water molecule so as to raise the concentration of H3O+ions, the defining property of a base is that it acceptsprotons so as to lower the concentration of H3O+ions, and thereby raise the concentration of hydroxyl ions (OH-). A base can either combine with protons directly or form hydroxyl ions that immediately combine with protons to produce H2O. Thus sodium hydroxide (NaOH) is basic (or alkaline) because it dissociatesin aqueous solution to form Na+ ions and OH- ions. Other bases,especially important in living cells, contain NH2 groups. These groups directly take up a proton from water: -NH2 + H2O -+ -NHs* + OH-. All molecules that accept protons from water will do so most readily when the concentration of H3O* is high (acidic solutions). Likewise, molecules that can give up protons do so more readily if the concentration of H3O+in solution is low (basic solutions), and thev will tend to receive them back if this concentration is high.

FourTypesof Noncovalent AttractionsHelpBringMolecules Togetherin Cells In aqueous solutions, covalent bonds are 10-100 times stronger than the other attractive forces between atoms, allowing their connections to define the boundaries of one molecule from another. But much of biology depends on the specific binding of different molecules to each other. This binding is mediated by a group of noncovalent attractions that are individually quite weak, but whose energies can sum to create an effective force between two separate molecules. We have previously introduced three of these attractive forces: electrostatic attractions (ionic bonds), hydrogen bonds, and van der Waals attractions. Table 2-l compares the strengths of these three types of noncoualent bonds with that of a typical covalent bond, both in the presence and in the Table2-1 Covalentand NoncovalentChemicalBonds

Covalent Noncovalent:ionicx hydrogen van derWaalsattraction (peratom)

0.15 0.2s 0.30 0.35

90 80 4 0.1

*An ionicbond isan electrostatic attraction betweentwo fullvcharoedatoms

90 3 1 0.1

Figure2-13 Acids in water. (A)The reactionthat takesolacewhen a moleculeof aceticaciddissolves in water. (B)Watermolecules arecontinuously exchangingprotonswith eachotherto form hydroniumand hydroxylions.These ionsin turn rapidlyrecombineto form watermolecules.

54

Chapter2: CellChemistryand Biosynthesis

absenceof water. Becauseof their fundamental importance in all biological systems, we summarize their properties here: Electrostatic attractions. These result from the attractive forces between oppositely charged atoms. Electrostatic attractions are quite strong in the absence of water. They readily form between permanent dipoles, but are greatestwhen the two atoms involved are fully charged (ionic bonds).However,the polar water molecules cluster around both fully charged ions and polar molecules that contain permanent dipoles (Figure 2-14). This greatly reduces the attractivenessof these charged species for each other in most biological settings. Hydrogen bonds. The structure of a typical hydrogen bond is illustrated in Figure 2-15. This bond represents a special form of polar interaction in which an electropositive hydrogen atom is partially shared by two electronegative atoms. Its hydrogen can be viewed as a proton that has partially dissociated from a donor atom, allowing it to be shared by a second acceptor atom. Unlike a typical electrostatic interaction, this bond is highly directional-being strongest when a straight line can be drawn between all three of the involved atoms. As already discussed,water weakens these bonds by forming competing hydrogen-bond interactions with the involved molecules. van der Waals attractions. The electron cloud around any nonpolar atom will fluctuate, producing a flickering dipole. Such dipoles will transiently induce an oppositely polarized flickering dipole in a nearby atom. This interaction generates a very weak attraction between atoms. But since many atoms can be simultaneously in contact when two surfaces fit closely,the net result is often significant. Water does not weaken these socalled van der Waals attractions. The fourth effect that often brings molecules together in water is not, strictly speaking, a bond at all. However, a very important hydrophobic force is caused by a pushing of nonpolar surfaces out of the hydrogen-bonded water network, where they would otherwise physically interfere with the highly favorable interactions between water molecules. Bringing any two nonpolar surfaces together reduces their contact with water; in this sense,the force is nonspecific. Nevertheless, we shall see in Chapter 3 that hydrophobic forces are central to the proper folding of protein molecules. Panel 2-3 provides an overview of the four types of attractions just described. And Figure 2-16 illustrates schematically how many such interactions can sum to hold together the matching surfaces of two macromolecules, even though each interaction by itself would be much too weak to be effective in the face of thermal motions.

tr_

Figure2-14 How the dipoleson water moleculesorientto reducethe affinity of oppositelychargedionsor polar groups for each other.

A Cellls Formedfrom CarbonCompounds

Figure2-15 Hydrogenbonds.(A)Ball-and-stick modelof a typical hydrogenbond.Thedistancebetweenthe hydrogenand the oxygenatom hereis lessthan the sum of theirvanderWaalsradii,indicatinga partial (B)The mostcommonhydrogenbondsin cells. sharingof electrons.

"o uH

(A)

Having looked at the ways atoms combine into small molecules and how these molecules behave in an aqueous environment, we now examine the main classesof small molecules found in cells and their biological roles.We shall see that a few basic categoriesof molecules, formed from a handful of different elements, give rise to all the extraordinary richness of form and behavior shown by living things. If we disregard water and inorganic ions such as potassium, nearly all the molecules in a cell are based on carbon. Carbon is outstanding among all the elements in its ability to form large molecules; silicon is a poor second. Becauseit is small and has four electrons and four vacancies in its outermost shell, a carbon atom can form four covalent bonds with other atoms. Most important, one carbon atom can join to other carbon atoms through highly stable covalent C-C

u6-

u,

h y d r o g e nb o n d - 0 . 3 n m l o n g oonor atom

accepror atom

.ou-rf"na UonO - 0 . 1n m l o n g (B)

o-Hililililililro o-Hililililililtoo o - H ilililililil| - H ililililililr o H ililililililr O - H ilililililil|

T H EC H E M I C AC L O M P O N E N TOSF A C E L L

55

bonds to form chains and rings and hence generatelarge and complex molecules with no obvious upper limit to their size (seePanel 2-1, pp. 106-107).The small and large carbon compounds made by cells are called organic molecules. Certain combinations of atoms, such as the methyl (-CHs), hydroxyl (-OH), carboxyl (-COOH), carbonyl (-C=O), phosphate (-POs2-),sulfhydryl (-SH), and amino (-NHz) groups, occur repeatedly in organic molecules. Each such chemical group has distinct chemical and physical properties that influence the behavior of the molecule in which the group occurs. The most common chemical groups and some of their properties are summarized in Panel 2-1, pp. 106-107.

CellsContainFourMajorFamilies of SmallOrganicMolecules The small organic molecules of the cell are carbon-based compounds that have molecular weights in the range 100-1000 and contain up to 30 or so carbon atoms. They are usually found free in solution and have many different fates. Some are used as monomer subunits to construct the giant polymeric macromolecules-the proteins, nucleic acids, and large polysaccharides-of the cell. Others act as energy sources and are broken down and transformed into other small molecules in a maze of intracellular metabolic pathways. Many small molecules have more than one role in the cell-for example, acting both as a potential subunit for a macromolecule and as an energy source. Small organic molecules are much less abundant than the organic macromolecules, accounting for only about one-tenth of the total mass of organic matter in a cell (Table 2-Z). As a rough guess,there may be a thousand different kinds of these small molecules in a typical cell. All organic molecules are slmthesized from and are broken down into the same set of simple compounds. Both their slmthesis and their breakdown occur through sequences of limited chemical changes that follow definite rules. As a consequence, the compounds in a cell are chemically related and most can be classified into a few distinct families. Broadly speaking, cells contain four major families of small organic molecules: lhe sugars, the fatty acids, the amino acids, and the nucleotides (Figure 2-17). Although many compounds present in cells do not fit into these categories,these four families of small organic molecules, together with the macromolecules made by linking them into long chains, account for a large fraction of cell mass (seeTable 2-2).

SugarsProvidean EnergySourcefor Cellsand Arethe Subunitsof Polysaccharides The simplest sugars-the monosaccharides-are compounds with the general formula (CH2O)2,where n is usually 3, 4, 5, 6,7 , or 8. Sugars,and the molecules made from them, are also called carbohydratesbecause of this simple formula. Glucose,for example, has the formula C6H1206@igure 2-18). The formula, however,does not fully define the molecule: the same set of carbons, hydrogens, and Table2-2 TheTypesof MoleculesThat Forma BacterialCell

Water I n o r g a n i co n s Sugars and precursors Aminoacidsand precursors Nucleotides and precursors Fattyacidsand precursors O t h e rs m a lm l olecules (protei Macromolecules ns, nucleicacids,and polysaccharides)

70 1 1 0.4 0.4 1 0.2 26

1 20 250 100 100 50 -300 -3000

il

Figure2-16 Schematicindicatinghow with two macromolecules complementarysurfacescan bind tightly to one anotherthrough noncovalentinteractions,

56

Chapter2:CellChemistryand Biosynthesis

b u i l d i n gb l o c k s of the cell

l a r g e ru n i t s of the cell

-laltaaclg:---J+ _AUlIgASlps"_-"__l+

PROTEINS NUCLEIC ACIDS

___NUcIi-oJlPSl**.-I+ !w

oxygens can be joined together by covalent bonds in a variety ofways, creating structures with different shapes.As shown in Panel 2-4 (pp.1l2-113), for example, glucose can be converted into a different sugar-mannose or galactosesimply by switching the orientations of specific OH groups relative to the rest of the molecule. Each of these sugars,moreover, can exist in either of two forms, called the D-form and the l-form, which are mirror images of each other. Setsof molecules with the same chemical formula but different structures are called isomers,and the subset of such molecules that are mirror-image pairs are called optical isomers.Isomers are widespread among organic molecules in general, and they play a major part in generating the enormous variety of sugars. Panel 2-4 presents an outline of sugar structure and chemistry. Sugarscan exist as rings or as open chains. In their open-chain form, sugars contain a number of hydroxyl groups and either one aldehyde ( > C : O) or one ketone H (> C: O) group. The aldehyde or ketone group plays a special role. First, it can react with a hydroxyl group in the same molecule to convert the molecule into a ring; in the ring form the carbon of the original aldehyde or ketone group can be recognized as the only one that is bonded to two oxygens.Second, once the ring is formed, this same carbon can become further linked, via oxygen, to one of the carbons bearing a hydroxyl group on another sugar molecule. This creates a disaccharide such as sucrose,which is composed of a glucose and a fructose unit. Larger sugar polymers range from the oligosaccharldes(trisaccharides, tetrasaccharides,and so on) up to giant polysaccharides,wlr'ich can contain thousands of monosaccharideunits. The way that sugars are linked together to form poly'rnersillustrates some common features of biochemical bond formation. A bond is formed between an -OH group on one sugar and an -OH group on another by a condensation reaction, in which a molecule of water is expelled as the bond is formed (Figure 2-19). Subunits in other biological polymers, such as nucleic acids and proteins, are also linked by condensation reactions in which water is expelled.The bonds created by all of these condensation reactions can be broken by the reverseprocessof hydrolysis, in which a molecule of water is consumed (seeFigure 2-19).

CH,OH ta -a) "\()H H i lH \r CC H ,/l l\ OH l/ Ho\l n C-C L]

r\H

(c)

Figure2-17 Thefour main familiesof smallorganicmoleculesin cells.These form the monomeric smallmolecules buildingblocks,or subunits,for mostof the macromolecules and other of the cell.Some,suchasthe assemblies sugarsand the fatty acids,arealsoenergy 50urce5.

Figure2-18 The structureof glucose,a previously simplesugar.As illustrated for water(seeFigure2-12),any moleculecan in severalways.In the be represented structuralformulasshownin (A),(B)and (C),the atomsareshownas chemical symbolslinkedtogetherby lines representing the covalentbonds.The thickenedlineshereare usedto indicate the planeof the sugarring,in an attempt to emphasize that the -H and -OH groupsarenot in the sameplaneasthe ring.(A)Theopen-chain form of this sugar,which is in equilibriumwith the morestablecyclicor ringform in (B). (C)Thechairform is an alternative way to drawthe cyclicmoleculethat reflectsthe geometrymoreaccurately than the structuralformulain (B).(D)A spacefillingmodel,which,aswell as depicting the three-dimensional arrangement of the atoms,alsousesthe van derWaals radiito representthe surfacecontoursof the molecule.(E)A ball-and-stick model in whichthe three-dimensional arrangement of the atomsin spaceis shown.(H,white;C,black;O, red;N, blue.)

IHE CHEMICAL COMPONENTS OFA CELL monosaccharide

57 monosaccharide

CONDENSATION

Figure2-19 The reactionof two monosaccharidesto form a Thisreactionbelongsto a disaccharide. generalcategoryof reactions termed reactions, in which two condensation join togetheras a resultof the molecules The reverse lossof a watermolecule. reaction(in whichwateris added)is termed hydrolysrs.Note that the reactive carbonat whichthe new bond is formed (on the monosaccharide on the /efthere) is the carbonjoinedto two oxygensasa resultof sugarringformation(seeFigure this commontype of 2-18),As indicated, covalentbond betweentwo sugar bond moleculesis known as a glycosidic (seealsoFigure2-20).

HYDROLYSIS

H:O

H,O

water expelled

water consumed

'""1,]"0 j" ^o flY.'."n'.?,'j Because each monosaccharide has several free hydroxyl groups that can form a link to another monosaccharide (or to some other compound), sugar polymers can be branched, and the number of possible polysaccharide structures is extremely large. Even a simple disaccharide consisting of two glucose units can exist in eleven different varieties (Figure 2-2O), while three different hexoses (CoHrzOo)can join together to make several thousand trisaccharides. For this reason it is a much more complex task to determine the arrangement of sugarsin a polysaccharide than to determine the nucleotide sequenceof a DNA molecule, where each unit is joined to the next in exactly the same way. The monosaccharide glucoseis a key energy source for cells. In a series of reactions, it is broken down to smaller molecules, releasing energy that the cell can harness to do useful work, as we shall explain later. Cells use simple polysaccharides composed only of glucose units-principally glycogenin animals and starchin plants-as energy stores.

p1*6 CH,OH

CH]OH

t_t_

q"q fo'"

,r-Q '

p 1 *4

CH,OH t-

io..\_,/

I

CH,OH I'

,/-o\ \-,/

CH,OH

,r-o\

o,.f\_/ |\i

(Il * o(l

Rl* 2

Figure2-20 Elevend isaccharides consistingof two D-glucoseunits. Althoughthesedifferonly in the type of linkagebetweenthe two glucoseunits, distinct.Sincethe they arechemically associated with proteins oligosaccharides and lipidsmay havesixor moredifferent kindsof sugarjoined in both linearand through branchedarrangements glycosidic bondssuchasthoseillustrated here,the numberof distincttypesof that can be usedin cells oligosaccharides is extremelylarge.Foran explanation seePanel2-4 of s and p linkages, (pp.112-113).Shortb/acklinesending (Redlines "blind"indicateOH positions. bond merelyindicatedisaccharide and'torners"do not imply orientations extraatoms.)

58

Chapter2: CellChemistryand Biosynthesis

Sugars do not function only in the production and storage of energy.They can also be used, for example, to make mechanical supports. Thus, the most abundant organic chemical on Earth-the cellulose of plant cell walls-is a polysaccharide of glucose.Becausethe glucose-glucoselinkages in cellulose differ from those in starch and glycogen, however, humans cannot digest cellulose and use its glucose. Another extraordinarily abundant organic substance, the chitin of insect exoskeletonsand fungal cell walls, is also an indigestible polysaccharide-in this case a linear polymer of a sugar derivative called ly'-acetylgl.,cosamine (see Panel 2-4). Other polysaccharides are the main components of slime, mucus, and gristle. Smaller oligosaccharidescan be covalently linked to proteins to form glycoproteins and to lipids to form glycolipids,both of which are found in cell membranes.As described in Chapter 10,most cell surfacesare clothed and decorated with glycoproteins and glycolipids in the cell membrane. The sugar side chains on these molecules are often recognized selectively by other cells. And differences between people in the details of their cell-surface sugars are the molecular basis for the different major human blood groups, termed A, B, AB, and O.

FattyAcidsAreComponents of CellMembranes, asWellasa Sourceof Energy A fatty acid molecule, such as palmitic acid,has two chemically distinct regions (Figure 2-21). One is a long hydrocarbon chain, which is hydrophobic and not very reactive chemically. The other is a carboxyl (-COOH) group, which behaves as an acid (carboxylic acid): it is ionized in solution (-COO-), extremely hydrophilic, and chemically reactive.Almost all the fatty acid molecules in a cell are covalently linked to other molecules by their carboxylic acid group. The hydrocarbon tail of palmitic acid is saturated: it has no double bonds between carbon atoms and contains the maximum possible number of hydrogens. Stearic acid, another one of the common fatty acids in animal fat, is also saturated. Some other fatty acids, such as oleic acid, have unsaturatedtails,with one or more double bonds along their length. The double bonds create kinks in the molecules, interfering with their ability to pack together in a solid mass. It is this that accounts for the difference between hard margarine (saturated) and liquid vegetable oils (polyunsaturated). The many different fatty acids found in cells differ only in the length of their hydrocarbon chains and the number and position ofthe carbon-carbon double bonds (seePanel2-5, pp.1l4-ll5). Fatty acids are stored in the cytoplasm of many cells in the form of droplets of triacylglycerol molecules, which consist of three fatty acid chains joined to a glycerol molecule (seePanel 2-5); these molecules are the animal fats found in meat, butter, and cream, and the plant oils such as corn oil and olive oil. \Mhen required to provide energy, the fatty acid chains are released from triacylglycerols and broken dor,rminto two-carbon units. These Wvo-carbonunits are identical to those derived from the breakdor,rrnof glucose and they enter the same energyyielding reaction pathways, as will be described later in this chapter.Triglycerides serve as a concentrated food reserve in cells, because they can be broken down to produce about six times as much usable energy,weight for weight, as glucose. Fatty acids and their derivatives such as triacylglycerols are examples of lipids. Lipids comprise a loosely defined collection of biological molecules that are insoluble in water, while being soluble in fat and organic solvents such as benzene. They typically contain either long hydrocarbon chains, as in the fatty acids and isoprenes,or multiple linked rings, as inthe steroids. The most important function of fatty acids in cells is in the construction of cell membranes. These thin sheets enclose all cells and surround their internal organelles. They are composed largely of phospholipids, which are small molecules that, like triacylglycerols, are constructed mainly from fatty acids and glycerol. In phospholipids the glycerol is joined to two fatty acid chains, however, rather than to three as in triacylglycerols. The "third" site on the glycerol is linked to a hydrophilic phosphate group, which is in turn attached to a small hydrophilic compound such as choline (see Panel 2-5). Each phospholipid

h y d r o p h i l i cc a r b o x y l i ca c i d h e a d

o \

h y d r o p h o b i ch y d r o c a r b o nt a i l (A)

(B)

(c)

Figure2-21 A fatty acid.A fatty acid is composedof a hydrophobichydrocarbon chainto which is attacheda hydrophilic carboxylic acidgroup.Palmiticacidis shown here.Differentfatty acidshave differenthydrocarbontails.(A)Structural formula.Thecarboxylic acidgroupis shownin its ionizedform.(B)Ball-andstickmodel.(C)Space-filling model.

THECHEMICAL COMPONENTS OFA CELL

rwo hydrophobic fatty acid tails

59 Figure2-22 Phospholipidstructure and the orientationof phospholipidsin membranes.In an aqueousenvironment, the hydrophobictailsof phospholipids packtogetherto excludewater.Here they haveformed a bilayerwith the hydrophilicheadof eachphospholipid facingthe water.Lipidbilayersarethe asdiscussed in basisfor cellmembranes, detailin Chapter10.

oa

00ltff00f000of

oo

p h o s p h o l i p i dm o t e c u t e

molecule, therefore, has a hydrophobic tail composed of the two fatty acid chains and a hydrophilic head, where the phosphate is located. This gives them different physical and chemical properties from triacylglycerols,which are predominantly hydrophobic. Molecules such as phospholipids, with both hydrophobic and hydrophilic regions, are termed amphiphilic. The membrane-forming property of phospholipids results from their amphiphilic nature. Phospholipids will spread over the surface of water to form a monolayer of phospholipid molecules, with the hydrophobic tails facing the air and the hydrophilic heads in contact with the water. TWo such molecular layers can readily combine tail-to-tail in water to make a phospholipid sandwich, or lipid bilayer. This bilayer is the structural basis of all cell membranes (Figure 2-22).

AminoAcidsArethe Subunitsof Proteins Amino acids are a varied class of molecules with one defining property: they all possessa carboxylic acid group and an amino group, both linked to a single carbon atom called the cr-carbon (Figure 2-23). Their chemical variety comes from the side chain that is also attached to the cx-carbon.The importance of amino acids to the cell comes from their role in making proteins, which are polymers of amino acids joined head-to-tail in a long chain that is then folded into a threedimensional structure unique to each type of protein. The covalent linkage between two adjacent amino acids in a protein chain forms an amide (seePanel 2-l), and it is called a peptide bond; the chain of amino acids is also known as a polypeptide (Figure 2-24). Regardlessof the specific amino acids from which it is made, the pollpeptide has an amino (NH2) group at one end (its N-terminus) and a carboxyl (COOH) group at its other end (its C-terminus).This givesit a definite directionality-a structural (as opposed to an electrical) polarity. Each of the 20 amino acids found commonly in proteins has a different side chain attached to the o-carbon atom (seePanel 3-1, pp. 128-129).All organisms, amtno group

c ar D o x yI group

s i d ec h a i n( R ) n o n i o n i z e df o r m (A)

i o n i z e df o r m (B)

(c)

Figure2-23 The amino acid alanine. (A)In the cell,wherethe pH is closeto 7, the freeaminoacidexistsin its ionized into a form;but when it is incorporated polypeptidechain,the chargeson the aminoand carboxylgroupsdisappear. (B)A ball-and-stick modeland (C)a modelof alanine(H,white; space-filling C, black;O, red;N,blue).

60

Chapter2:CellChemistryand Biosynthesis Figure 2-24 A small part of a protein molecule.The four amino acids shown are linkedtogether by three peptide bonds,one of which is highlightedin yellow.One of the amino acidsis shadedin gray.The amino acid sidechainsare shown in red.The two ends of a polypeptidechainare chemically distinct.Oneend,the N-terminus, terminatesin an amino group,and the other,the C-terminus,in a carboxylgroup.The sequenceis alwaysreadfrom the N-terminalend; hencethis sequenceis Phe-Ser-Glu-Lys.

N-terminal end o{ p o l y p e p t i d ec h a i n I N_H I H-C -CH? I

O=C I

N-H H-C -CHr

-oH

t-

whether bacteria,archea,plants, or animals,haveproteins made of the same20 amino acids.How this preciseset of 20 cameto be chosenis one of the mysteries of the evolution of life; there is no obviouschemicalreasonwhy other amino acidscould not haveservedjust aswell. But once the choicewas established,it could not be changed;too much dependedon it. Like sugars,all amino acids,exceptglycine,existasoptical isomersin D- and L-forms(seePanel3-l). But only L-forms€ueeverfound in proteins(althoughDamino acids occur as part of bacterial cell walls and in some antibiotics). The origin of this exclusiveuse of l-amino acids to make proteins is another evolutionarymystery. The chemicalversatility of the 20 amino acidsis essentialto the function of proteins.Five of the 20 amino acidshave side chainsthat can form ions in neutral aqueoussolution and thereby can carry a charge(Figure2-25). The others are uncharged;some are polar and hydrophilic, and some are nonpolar and hydrophobic.As we discussin Chapter3, the propertiesof the amino acid side chainsunderlie the diverseand sophisticatedfunctions of proteins.

O=C Glu

Lys

N-H ,,H H - C - C H , --C H ? - C H r - C H ) --N - H l | \H O=C

C-terminal end of p o l y p e p t i d ec h a i n

11

pH

aspartic acid pK-4.7

glutamic acid pK-4.7

histidine

lysine

argrnrne

pK-6.5

pK-10.2

pK-12

Figure2-25 The charge on amino acid side chainsdepends on the pH. The five differentsidechainsthat can carrya chargeare shown.Carboxylicacidscan readilyloseH+in aqueoussolutionto form a negativelychargedion, which is denoted by the suffix"-atei'asin aspartdteor glutamote.A comparable situationexistsfor amines.which in aqueoussolutioncan take up H+to form a positivelychargedion (whichdoes not havea specialname).Thesereactionsare rapidlyreversible, and the amountsof the two forms,chargedand uncharged, dependon the pH of the solution.At a high pH, carboxylicacidstend to be chargedand aminesuncharged. At a low pH,the oppositeis true-the carboxylic acidsare unchargedand aminesare charged.The pH at which exactlyhalf of the carboxylic acidor amineresidues are chargedis known as the pK of that amino acid sidechain (indicatedbyyellow stripe). In the cellthe pH is closeto 7, and almostall carboxylic acidsand aminesare in theirfullychargedform.

THECHEMICAL COMPONENTS OFA CELL

61

o() Pl"

o

Figure2-26Chemicalstructureof adenosinetriphosphate(ATP). (A)Structural formula.(B)Space-filling model.In (8)the colorsof the atomsare C, black;N, b/ue;H, white;O, red; and P,yellow.

H.a-N-ra-NH,

\ -"oo'in

ll

I

"\oto' \

o"'xozclt oX^,t-a.

)

H nlln OH OH

"rlJt'

(A)

(B)

Nucleotides Arethe Subunitsof DNAand RNA A nucleotide is a molecule made up of a nitrogen-containing ring compound linked to a five-carbon sugar, which in turn carries one or more phosphate groups (Panel2-6, pp.116-117). The five-carbon sugar can be either ribose or deoxyribose. Nucleotides containing ribose are known as ribonucleotides, and those containing deoxyribose as deoxyribonucleotides.The nitrogen-containing rings are generally referred to as basesfor historical reasons:under acidic conditions they can each bind an H+ (proton) and thereby increase the concentration of OH- ions in aqueous solution. There is a strong family resemblance between the different bases. Cyfosine (C), thymine (T), and uracil (U) are called pyrimidines becausethey all derive from a six-membered pyrimidine ring; guanine (G) and adenine (A) are purinecompounds, and theyhave a second, five-membered ring fused to the six-membered ring. Each nucleotide is named for the base it contains (seePanel 2-6). Nucleotides can act as short-term carriers of chemical energy.Above all others, the ribonucleotide adenosine triphosphate, or ATP (Figure 2-26), transfers energy in hundreds of different cell reactions. ATP is formed through reactions that are driven by the energy released by the oxidative breakdown of foodstuffs. Its three phosphates are linked in seriesby two phosphoanhydride bonds,whose rupture releaseslarge amounts of useful energy.The terminal phosphate group in particular is frequently split off by hydrolysis, often transferring a phosphate to other molecules and releasing energy that drives energy-requiring biosynthetic reactions (Figure 2-27). Other nucleotide derivatives are carriers for the transfer of other chemical groups, as will be described later. The most fundamental role of nucleotides in the cell, however, is in the storage and retrieval of biological information. Nucleotides serve as building blocks for the construction of nucleic aclds-long pol].rynersin which nucleotide subunits are covalently linked by the formation of a phosphodiester bond between the p h o s p h o a n h y d r i dbeo n d s TI

TI

oo-ortl

P -O-P-O-PP-O_ o P i l it l t l ol oo o

energy from s u n l i g h ot r from food

Hzo

o-

H*+

I o-P-o

H+

il

o Inorganrc p h o s p h a t e( P i )

e n e r g ya v a i l a b l e f o r c e l l u l a rw o r k and {or chemical synthesis

Figure2-27 The ATPmoleculeservesas an energycarrierin cells.Theenergyrequiringformationof ATPfrom ADPand inorganicphosphateis coupledto the oxidationof foodstuffs energy-yielding (in animalcells,fungi,and somebacteria) or to the captureof light energy(in plant The hydrolysis cellsand somebacteria). of this ATPbackto ADPand inorganic phosphatein turn providesthe energyto drivemanvcell reactions.

62

Chapter2: CellChemistryand Biosynthesis Figure2-28A smallpartof onechainof a deoxyribonucleic acid(DNA) molecule. Fournucleotides areshown. Oneof thephosphodiester bonds thatlinksadjacent nucleotide residues ishighlighted inyellow, andoneof thenucleotides isshaded in gray.Nucleotides arelinkedtogether bya phosphodiester linkage between specific carbonatomsof theribose, knownasthe5'and3'atoms. Forthisreason, oneendof a polynucleotide groupandtheother,the chain,the5' end,willhavea freephosphate group. 3' end,a freehydroxyl Thelinearsequence of nucleotides in a polynucleotide chainiscommonly abbreviated by a one-letter code,and thesequence isalways readfromthe5'end.Intheexample illustrated the sequence isG-A-T-C.

phosphate group attached to the sugar of one nucleotide and a hydroxyl group on the sugar of the next nucleotide (Figure 2-28). Nucleic acid chains are sgrthesized from energy-rich nucleoside triphosphates by a condensation reaction that releasesinorganic plrophosphate during phosphodiesterbond formation. There are two main types of nucleic acids, differing in the type of sugar in their sugar-phosphate backbone. Those based on the sugar ribose are knor,tmas ribonucleic acids, or RNA, and normally contain the basesA, G, C, and U. Those based on deoxyribose(in which the hydroxyl at the 2' position of the ribose carbon ring is replaced by a hydrogen are knor,rm as deoxyribonucleic acids, or DNA, and contain the bases A, G, C, and T (T is chemically similar to the U in RNA, merely adding the methyl group on the pyrimidine ring; see Panel2-6). RNA usually occurs in cells as a single polynucleotide chain, but DNA is virtually always a double-stranded molecule-a DNA double helix composed of two polynucleotide chains running antiparallel to each other and held together by hydrogen-bonding between the basesof the two chains. The linear sequence of nucleotides in a DNA or an RNA encodes the genetic information of the cell. The ability of the bases in different nucleic acid molecules to recognize and pair with each other by hydrogen-bonding (called base-pairing)-G with C, and A with either T or U-underlies all of heredity and evolution, as explained in Chapter 4.

5'end

I G

o I

o-

T

o I

o

TheChemistry of Cellsls Dominatedby Macromolecules with Remarkable Properties

I

3'end

By weight, macromolecules are the most abundant carbon-containing molecules in a living cell (Figure 2-29 and Table 2-3). They are the principal building blocks from which a cell is constructed and also the components that confer the most distinctive properties of living things. The macromolecules in cells are polymers that are constructed by covalently linking small organic molecules (called monomers) into long chains (Figure 2-3O). Yet they have remarkable properties that could not have been predicted from their simple constituents. Proteins are especially abundant and versatile. They perform thousands of distinct functions in cells. Many proteins serve as enzymes,the catalysts that i o n s ,s m a l l m o l e c u l e s( 4 % ) p h o s p h o l i p i d(s2 % ) D N A( 1 % ) R N A( 5 % )

^ 7

7Oo/o

Hzo

p r o t e i n s( 1 5 % )

o -m r) C

-

m

polysaccharide ( 2s% )

Figure2-29 Macromoleculesare abundantin cells.Theapproximate compositionof a bacterialcellis shown by weight.The compositionof an animal cellis similar(seeTable2-3).

T H EC H E M I C AC L O M P O N E N TOSF A C E L L

63

Table2-3 ApproximateChemicalCompositionsof a TypicalBacteriumand a TypicalMammalianCell

Hzo lnorganic ions(Na+,K*,Mg2*, ca2+,cl-, etc.) Miscellaneous smallmetabolites Proteins RNA DNA Phospholipids Otherlipids Polysaccharides Totalcellvolume Relativecellvolume

70 1

70 1

3 15 6 1 ')

3 18 1.1 0.25 ?

2

.\ 2 y 1 g - 1.2r 3 1

z

4 x 1 0 - ec m 3 2000

P r o t e i n s p, o i y s a c c h a r i d e sD,N A ,a n d R N Aa r e m a c r o m o l e c u l e sL i p i d sa r e n o t g e n e r a l l yc a s s e da s

b o t h m a m m a la n a n d b a c L e r r a ce ts

bles to make the cell'slong microtubules, or histones, proteins that compact the DNA in chromosomes. Yet other proteins act as molecular motors to produce force and movement, as in the caseof myosin in muscle. proteins perform many other functions, and we shall examine the molecular basis for many of them later in this book. Here we identifu some general principles of macromolecular chemistry that make such functions possible. Although the chemical reactions for adding subunits to each polyrner are different in detail for proteins, nucleic acids, and polysaccharides, they share important features. Each polymer grows by the addition of a monomer onto the end of a growing polymer chain in a condensation reaction, in which a

made from a set of monomers that are slightly different from one another-for example, the 20 different amino acids from which proteins are made. It is critical to life that the polymer chain is not assembled at random from these subunits; instead the subunits are added in a particular order, or sequence.T]ne elaborate mechanisms that allow this to be accomplished by enzymes are describedin detail in Chapters5 and 6.

NoncovalentBondsSpecifyBoth the preciseShapeof a Macromoleculeand its Bindingto Other Molecules Most of the covalent bonds in a macromolecule allow rotation of the atoms they join' giving the polymer chain great flexibility. In principle, this allows a macromolecule to adopt an almost unlimited number of shapes,or conformations, as

SUBUNIT

s u g ar

o

MACROMOLECULE

p o l y s a c c hr ai d e

amrno u.,0"

protern

nu c l e o t i d e

n u c l e i ca c i d

Figure2-30 Threefamiliesof macromolecules. Eachis a polymer formedfrom smallmolecules(called monomers)linkedtogetherby covalentbonds.

64

Chapter2: CellChemistryand Biosynthesis Figure2-31 Most proteinsand many RNAmoleculesfold into only one stable bonds conformation.lf the noncovalent are maintainingthis stableconformation disrupted, the moleculebecomesa flexiblechainthat usuallyhasno biologicalvalue.

many unstable conformations

one stable folded conformation

random thermal energy causesthe polymer chain to writhe and rotate. However, the shapesof most biological macromolecules are highly constrained becauseof the many we ak noncoualent bonds that form between different parts of the same molecule. If these noncovalent bonds are formed in sufficient numbers, the polyrner chain can strongly prefer one particular conformation, determined by the linear sequenceof monomers in its chain. Most protein molecules and many of the small RNA molecules found in cells fold tightly into one highly preferred conformation in this way (Figure 2-31). The four types of noncovalent interactions important in biological molecules were described earlier, and they are reviewed in Panel 2-3 (pp. 110-lll). Although individuallyveryweak, these interactions cooperate to fold biological macromolecules into unique shapes.In addition, theycan also add up to create a strong attraction between two different molecules when these molecules fit together very closely,like a hand in a glove.This form of molecular interaction provides for great specificity, inasmuch as the multipoint contacts required for strong binding make it possible for a macromolecule to select outthrough binding-just one of the many thousands of other types of molecules present inside a cell. Moreover, because the strength of the binding depends on the number of noncovalent bonds that are formed, interactions of almost any affinity are possible-allowing rapid dissociation when necessary. Binding of this type underlies all biological catalysis,making it possible for proteins to function as enzymes. Noncovalent interactions also allow macromolecules to be used as building blocks for the formation of larger structures. In cells, macromolecules often bind together into large complexes, thereby forming intricate machines with multiple moving parts that perform such complex tasks as DNA replication and protein synthesis (Figure 2-32).

SUBUNITS

MACROMOLECULES c o v a l e n tb o n d s

n o n c o v a l e nbt o n d s

MACROMOLECULAR ASSEMBLIES

e g , s u g a r sa, m i n o a c i d s , and nucleotides 30 nm eg,globularproteins and RNA

e 9., ribosome

Figure2-32 Smallmolecules,proteins,and a ribosomedrawn approximatelyto scale.Ribosomes area centralpart of the machinerythat the (proteinand RNAmolecules). cellusesto makeproteins:eachribosomeisformedasa complexof about90 macromolecules

CATALYSIS ANDTHEUsEOFENERGY BYCELLS

65

Summary Liuing organismsare autonomous, self-propagatingchemical systems.Theyare made from a distinctiue and restrictedset of small carbon-basedmoleculesthat are essentially the samefor eueryliuing species.Each of thesemoleculesis composedof a small set of atoms linked to each other in a preciseconftguration through coualent bonds. The main categoriesare sugars,fatty acids,amino acids,and nucleotides.Sugarsare a primary sourceof chemical energyfor cellsand can be incorporated into polysaccharides for energy storage.Fatty acids are also important for energy storage,but their most critical function is in the formation of cell membranes.Polymers consisting of amino acids constitute the remarkably diuerseand uersatilemacromoleculesknown as proteins. Nucleotidesplay a central part in energy transfer.They are also the subunits for the informational macromolecules,RNAand DNA. Most of the dry massof a cell consistsof macromoleculesthat hauebeenproduced as linear polymersof amino acids (proteinsl or nucleotides(DNA and RNA),coualently linked to each other in an exact ordex Most of the protein moleculesand many of the RNAsfold into a unique conformation that depends on their sequenceof subunits. This folding processcreatesunique surfaces,and it depends on a large set of weak attractions produced by noncoualentforces between atoms. Theseforces are of four types:electrostaticattrqctions, hydrogen bonds, uan der Waals attractions, and an interaction between nonpolar groups caused by their hydrophobic expulsion f'rom water. The same set of weak forcesgouernsthe specific binding of other moleculesto macromolecules,making possible the myriad associations between biological moleculesthat produce the structure and the chemistrv of a cell.

CATALYSIS ANDTHEUSEOFENERGY BYCELLS One property of living things above all makes them seem almost miraculously different from nonliving matter: they create and maintain order, in a universe that is tending always to greater disorder (Figure 2-33). To create this order, the cells in a living organism must perform a never-ending stream of chemical reactions. In some of these reactions, small organic molecules-amino acids, sugars, nucleotides, and lipids-are being taken apart or modified to supply the many other small molecules that the cell requires. In other reactions, these small molecules are being used to construct an enormously diverse range of proteins, nucleic acids, and other macromolecules that endow living systems with all of their most distinctive properties. Each cell can be viewed as a tiny chemical factory performing many millions of reactions every second.

Figure2-33 Order in biologicalstructures.Well-defined,ornate,and beautifulspatialpatternscan be size:(A)proteinmoleculesin In orderof increasing foundat everylevelof organization in livingorganisms. the coat of a virus;(B)the regulararrayof microtubulesseenin a crosssectionof a spermtail; (C)surface contoursof a pollengrain(a singlecell);(D)close-upof the wing of a butterflyshowingthe patterncreated by scales, eachscalebeingthe productof a singlecell;(E)spiralarrayof seeds,madeof millionsof cells,in the headof a sunflower.(A,courtesyof R.A.Grantand J.M.Hogle;B,courtesyof LewisTilney;C,courtesyof ColinMacFarlane and ChrisJeffree; D and E,courtesyof KjellB.Sandved.)

66

Chapter2:CellChemistryand Biosynthesis

otecue l

molecule

molecule

molecule

morecure

motecute

c a t a l y s ibs y e n z y m e1

5 A B B R E V I A T EAD

o-o-o-a-a-o e n z y m e2

e n z y m e3

e n z y m e4

e n z y m e5

Figure2-34 How a set ofenzyme-catalyzedreactionsgeneratesa metabolic pathway.Eachenzyme In this example,a setof enzymes catalyzes a particularchemicalreaction,leavingthe enzymeunchanged. actingin seriesconvertsmoleculeA to moleculeF,forminga metabolicpathway.

CellMetabolismls Organized by Enzymes The chemical reactions that a cell carries out would normally occur only at much higher temperatures than those existing inside cells. For this reason, each reaction requires a specific boost in chemical reactivity.This requirement is crucial, because it allows the cell to control each reaction. The control is exerted through the specialized proteins called enzymes,each of which accelerates,or catalyzes,just one of the many possible kinds of reactions that a particular molecule might undergo. Enzyme-catalyzedreactions are usually connected in series,so that the product of one reaction becomes the starting material, or substrate,for the next (Figure 2-34). These long linear reaction pathways are in turn linked to one another, forming a maze of interconnected reactions that enable the cell to survive, grow, and reproduce (Figure 2-35). TWoopposing streams of chemical reactions occur in cells: (l) Ihe catabolic pathways break down foodstuffs into smaller molecules, thereby generating both a useful form of energy for the cell and some of the small molecules that the cell needs as building blocks, and (2) the anabolic, or biosynthellq pathways use the energy harnessed by catabolism to drive the synthesis of the many other molecules that form the cell. Together these two sets of reactions constitute the metabolism of the cell (Figure 2-36). Many of the details of cell metabolism form the traditional subject of biochemistry and need not concern us here. But the general principles by which cells obtain energy from their environment and use it to create order are central to cell biology. We begin with a discussion of why a constant input of energy is needed to sustain living organisms.

Biological Orderls MadePossible by the Release of HeatEnergy from Cells The universal tendency of things to become disordered is a fundamental law of physics-the secondlaw of thermodynamics-which states that in the universe, or in any isolated system (a collection of matter that is completely isolated from the rest of the universe), the degreeof disorder only increases.This law has such profound implications for all living things that we restate it in severalways. For example,we can present the second law in terms of probability and state that systems will change spontaneously toward those arrangements that have the greatest probability. If we consider, for example, a box of 100 coins all lying heads up, a series of accidents that disturbs the box will tend to move the arrangement toward a mixture of 50 heads and 50 tails. The reason is simple: there is a huge number of possible arrangements of the individual coins in the mixture that can achieve the 50-50 result, but only one possible arrangement that keeps all of the coins oriented heads up. Becausethe 50-50 mixture is therefore the most probable, we say that it is more "disordered." For the same reason,

Figure2-35 Someof the metabolicpathwaysand their interconnections in a typicalcell,About500commonmetabolicreactions areshown diagrammatically, with eachmoleculein a metabolicpathwayrepresented by a filledcircle,as in the yel/owbox in Figure2-34.The pathwaythat is highlightedin this diagramwith largercirclesand connectinglinesisthe centralpathwayof sugarmetabolism, whichwill be discussed shortly.

CATALYSIS ANDTHEUSEOF ENERGY BYCELLS Figure2-36 Schematicrepresentationof the relationshipbetween catabolicand anabolicpathwaysin metabolism.As suggestedhere,since a major portion of the energystoredin the chemicalbondsof food moleculesis dissipated as heat,the massof food requiredby any organism that derivesall of its energyfrom catabolism is muchgreaterthan the mass of the molecules that can be oroducedbv anabolism.

67

it is a common experience that one's living space will become increasingly disordered without intentional effort: the movement toward disorder is a sponta-

neous process,requiring a periodic effort to reverse it (Figure 2-37). The amount of disorder in a system can be quantified and expressedas the entropy of the system: the greater the disorder, the greater the entropy. Thus, another way to express the second law of thermodynamics is to say that systems will change spontaneously toward arrangements with greater entropy. Living cells-by surviving, growing, and forming complex organisms-are generating order and thus might appear to defu the second law of thermodynamics. How is this possible?The answer is that a cell is not an isolated system: it takes in energy from its environment in the form of food, or as photons from the sun (or even, as in some chemosynthetic bacteria, from inorganic molecules alone), and it then uses this energy to generate order within itself. In the course of the chemical reactions that generateorder, the cell converts part of the energy it usesinto heat. The heat is dischargedinto the cell'senvironment and disorders it, so that the total entropy-that of the cell plus its surroundings-increases, as demanded by the laws of thermodlmamics. To understand the principles governing these energy conversions, think of a cell surrounded by a sea of matter representing the rest of the universe. As the cell lives and grows, it creates internal order. But it constantly releases heat energy as it synthesizes molecules and assembles them into cell structures. Heat is energy in its most disordered form-the random jostling of molecules. \iVhen the cell releasesheat to the sea, it increases the intensity of molecular motions there (thermal motion)-thereby increasing the randomness, or disorder, of the sea. The second law of thermodynamics is satisfied because the increase in the amount of order inside the cell is more than compensated for by an even greater decreasein order (increase in entropy) in the surrounding sea of matter (Figure 2-38). \Mhere does the heat that the cell releases come from? Here we encounter another important law of thermodynamics. The first law of thermodynamics statesthat energy can be converted from one form to another, but that it cannot

t h e m a n ym o l e c u l e s that form the cell

food molecules

CATABOLIC PATHWAYS

useful forms of energy + lost heat

t h e m a n y b u i l d i n gb l o c k s for biosynthesis

"SPONTANEOUS" REACTION a st i m e e l a p s e s

O R G A N I Z EE DF F O RR T E Q U I R I NEGN E R G Y INPUT

Figure2-37 An everydayillustrationof the spontaneousdrive toward disorder. Reversingthis tendencytoward disorder reouiresan intentionaleffort and an In inputof energy:it is not spontaneous. fact.from the secondlaw of we can be certainthat thermodynamics, the humaninterventionrequiredwill release enoughheatto the environment for the to morethan compensate reorderingof the items in this room.

68

Chapter2: CellChemistryand Biosynthesis sea of matter

o I

J \ a

t o

J -O

o.'. {

to

a\ o.* increaseddisorderincreasedorder Figure2-38A simplethermodynamic analysis of a livingcell.Inthediagram on theleftthe (theseaof matter) molecules of boththecellandtherestof theuniverse in a aredepicted relatively disordered Inthediagram state. on therightthecellhastakenin energy fromfood molecules andreleased heatbya reaction thatorders themolecules thecellcontains. Because theheatincreases thedisorder in theenvironment aroundthecell(depictedby thejagged arrows anddistorted molecules, indicating molecular theincreased motions caused by heat), thesecond lawof thermodynamics-which states thattheamountof disorder in theuniverse mustalways increase-is satisfied asthecellgrowsanddivides. Fora detailed discussion, see (pp.118-119). Panel2-7

be created or destroyed.Figure 2-39 illustrates some interconversions between different forms of energy. The amount of energy in different forms will change as a result of the chemical reactions inside the cell, but the first law tells us that the total amount of energy must always be the same. For example, an animal cell takes in foodstuffs and converts some of the energy present in the chemical bonds between the atoms of these food molecules (chemical bond energy) into the random thermal motion of molecules (heat energy).As described above,this conversion of chemical energy into heat energy is essentialif the reactions that create order inside the cell are to cause the universe as a whole to become more disordered. The cell cannot derive any benefit from the heat energy it releasesunless the heat-generating reactions inside the cell are directly linked to the processesthat generate molecular order. It is the tight coupling of heat production to an increase in order that distinguishes the metabolism of a cell from the wasteful burning of fuel in a fire. Later, we shall illustrate how this coupling occurs. For now it is sufficient to recognize that a direct linkage of the "burning" of food molecules to the generation of biological order is required for cells to create and maintain an island of order in a universe tending toward chaos.

Photosynthetic Organisms UseSunlightto Synthesize OrganicMolecules All animals live on energy stored in the chemical bonds of organic molecules made by other organisms,which they take in as food. The molecules in food also provide the atoms that animals need to construct new living matter. Some animals obtain their food by eating other animals. But at the bottom of the animal food chain are animals that eat plants. The plants, in turn, trap energy directly from sunlight. As a result, the sun is the ultimate source of the energy used by animal cells. Solar energy enters the living world through photosynthesis in plants and photosynthetic bacteria. Photosynthesis converts the electromagnetic energy in sunlight into chemical bond energy in the cell. Plants obtain all the atoms they need from inorganic sources: carbon from atmospheric carbon dioxide, hydrogen and oxygen from water, nitrogen from ammonia and nitrates in the

, :,

CATALYSIS ANDTHEUSEOF ENERGY BYCELLS f a l l i n g b r i c kh a s kinetic energy

r a i s e db r i c k h a sp o t e n t i a l e n e r g yo u e to pull of gravity

/'/'

\

heat isreleased w h e n b r i c kh i t s the floor

,,iri:iitirililrliii

1

potential energy due to position--+

.T

kinetic energy

. /c, 3G \\&",\)

\\

ll

a&

two hydrogen oxygen gas g a sm o l e c u l e s m o l e c u l e

\r*

r,

c h e m i c abl o n d e n e r g y

4

heat dispersedto s ur r o u n di n g s

heat energy

liSli.ilr1';'#]tr

kinetic energy

electricalenergy

+

-

sunlight

u,.' j; G&'

r a p i dv i b r a t i o n sa n d rotations of two newly f o r m e dw a t e r m o l e c u l e s

chemicat bondenersy inH2and02 +

3

heat energy

chlorophyll molecule

electromagnetic(light) energ! --+

c h l o r o p h y lm l olecule in excitedstate

high energy electrons --+

photosynthesis

chemicalbond energy

soil, and other elements needed in smaller amounts from inorganic salts in the soil. They use the energy they derive from sunlight to build these atoms into sugars, amino acids, nucleotides, and fatty acids. These small molecules in turn are converted into the proteins, nucleic acids, polysaccharides, and lipids that form the plant. All of these substances serve as food molecules for animals, if the plants are later eaten. The reactions of photosynthesis take place in two stages (Figure 2-4O).ln the first stage, energy from sunlight is captured and transiently stored as chemical bond energy in specializedsmall molecules that act as carriers of energy and reactive chemical groups. (We discussthese "activated carrier" molecules later.) Molecular oxygen (Oz gas) derived from the splitting of water by light is released as a waste product of this first stage. In the second stage,the molecules that serve as energy carriers are used to help drive a carbonfixallon process in which sugars are manufactured from carbon dioxide Bas (COz) and water (HzO), thereby providing a useful source of stored chemical bond energy and materials-both for the plant itself and for any animals that eat it. We describe the elegant mechanisms that underlie these two stagesofphotosynthesis in Chapter 14.

69

Figure2-39 Someinterconversions between different forms of energy. All energyformsare,in principle, interconvertible. ln all theseorocesses the total amountof energyis conserved. Thus,for example,from the heightand weightof the brickin (1),we can predict exactlyhow much heatwill be released when it hitsthe floor.In (2),notethat the largeamountof chemicalbond energy released when wateris formedis initially convertedto very rapidthermal motions in the two new watermolecules; but with other molecules almost collisions instantaneously spreadthis kinetic energyevenlythroughoutthe (heattransfer), makingthe surroundings from all new moleculesindistinguishable the rest.

70

Chapter2: CellChemistryand Biosynthesis

f.^..^> \1

'11

capture o{ light energy

I

---

H2O+ CO2

energy carflers SUGAR

) ( ) heat

heat

STAGE2

STAGE1

Figure2-40Photosynthesis. Theenergycarriers created in thefirst Thetwo stages of photosynthesis. stagearetwo molecules thatwediscuss shortly-ATP andNADPH. The net result of the entire process of photosynthesis, so far as the green plant is concerned, can be summarized simply in the equation light energy + CO2+ H2O -+ sugars + 02 + heat energy The sugarsproduced are then used both as a source of chemical bond energy and as a source of materials to make the many other small and large organic molecules that are essentialto the Dlant cell.

CellsObtainEnergyby the Oxidationof OrganicMolecules All animal and plant cells are powered by energy stored in the chemical bonds of organic molecules, whether they are sugarsthat a plant has photosynthesized as food for itself or the mkture of large and small molecules that an animal has eaten. Organisms must extract this energy in usable form to live, grow, and reproduce. In both plants and animals, energy is extracted from food molecules by a process ofgradual oxidation, or controlled burning. The Earth'satmosphere contains a great deal of oxygen, and in the presence of oxygen the most energeticallystable form of carbon is CO2and that of hydrogen is H2O.A cell is therefore able to obtain energy from sugarsor other organic molecules by allowing their carbon and hydrogen atoms to combine with oxygen to produce COz and H2O,respectively-a processcalled respiration. Photosynthesisand respiration are complementary processes(Figure 2-41). This means that the transactions between plants and animals are not all one way. Plants, animals, and microorganisms have existed together on this planet for so long that many of them have become an essentialpart of the others' environments. The oxygen releasedby photosynthesis is consumed in the combustion of organic molecules by nearly all organisms. And some of the COz molecules that are fixed today into organic molecules by photosynthesis in a green leaf were yesterday releasedinto the atmosphere by the respiration of an animal-or by that of a fungus or bacterium decomposing dead organic matter. We therefore seethat carbon utilization forms a huge cycle that involves the biosphere (all of the living organisms on Earth) as a whole, crossing boundaries PHOTOSYNT EH SIS COr+HrO+02+SUGARS 02

co,

RESPIRATION S U G A R+ S Or+ HrO+ CO,

co,

or

Figure2-41 Photosynthesis and respirationas complementaryprocesses in the livingworld. Photosynthesis uses the energyof sunlightto producesugars and otherorganicmolecules. These moleculesin turn serveasfood for other organisms. Manyof theseorganisms carryout respiration, a processthat uses 02 to form CO2from the samecarbon atomsthat had beentakenup asCO2and convertedinto sugarsby photosynthesis. In the process, the organisms that respire obtainthe chemicalbond energythat Thefirstcellson the they needto survive. Eartharethoughtto havebeencapable of neitherphotosynthesis nor respiration (discussed in Chapter14).However, photosynthesis musthavepreceded respiration on the Earth,sincethereis strongevidencethat billionsof yearsof photosynthesis were requiredbefore02 had beenreleased in sufficientquantity to createan atmosphere richin this gas, (TheEarth's atmosphere currently contains20o/o Ot.)

CATALYSIS ANDTHEUSEOF ENERGY BYCELLS

71

co2 tN ATMOSPHERE ANDWATER

\\ RESPIRATION

\

PHOTOSYNTHESIS

I PLANTS, ALGAE BACTERIA

Figure2-42 The carbon cycle.Individual into carbonatomsareincorporated of the livingworld by organicmolecules activityof bacteria the photosynthetic Theypassto and plants(includingalgae). and organic animals,microorganisms, materialin soiland oceansin cyclicpaths. when CO2is restoredto the atmosphere organicmolecules areoxidizedby cellsor burnedby humansasfuels.

H U M U SA N D D I S S O L V E D ORGANICMATTER

between individual organisms (Figare2-42). Similarly, atoms of nitrogen, phosphorus, and sulfur move between the living and nonliving worlds in cycles that involve plants, animals, fungi, and bacteria.

Oxidationand Reduction InvolveElectron Transfers The cell does not oxidize organic molecules in one step, as occurs when organic material is burned in a fire. Through the use of enzyme catalysts,metabolism takes the molecules through a large number of reactions that only rarely involve the direct addition of oxygen. Before we consider some of these reactions and their purpose, we discusswhat is meant by the process of oxidation, Oxidation does not mean only the addition of oxygen atoms; rather, it applies more generally to any reaction in which electrons are transferred from one atom to another. Oxidation in this senserefers to the removal of electrons, and reduction-the converse of oxidation-means the addition of electrons. Thus, Fe2*is oxidized if it loses an electron to become Fe3*,and a chlorine atom is reduced if it gains an electron to become Cl-. Since the number of electrons is conserved (no loss or gain) in a chemical reaction, oxidation and reduction always occur simultaneously: that is, if one molecule gains an electron in a reaction (reduction), a second molecule loses the electron (oxidation). \Mhen a sugar molecule is oxidized to CO2and HzO, for example, the 02 molecules involved in forming H2O gain electrons and thus are said to have been reduced. The terms "oxidation" and "reduction" apply even when there is only a partial shift of electrons between atoms linked by a covalent bond (Figure 2-43). (A)

e-

€p+

e

_

F O R M A T I OO NF A POLAR COVALENT gOttg

ry'Try

H

€-

e-

I H*C-H I H

I

./\

*) ATOM 1

ATOM 2

methane

u

MoLEcuLE

methanol I

H _ C" -' O H

I

H g Figure2-43 Oxidation and reduction.(A)Whentwo atoms form a polar covalentbond (seep. 50),the atom endingup with a greatershareof electronsis saidto be reduced, The whilethe otheratom acquires a lessershareof electronsand is saidto be oxidized. reducedatom hasacquireda partialnegativecharge(6-)asthe positivechargeon the atomicnucleusis now morethan equaledby the total chargeof the electrons surroundingit, and conversely, the oxidizedatom hasacquireda partialpositivecharge (6+).(B)Thesinglecarbonatom of methanecan be convertedto that of carbondioxide by the successive replacement of its covalentlybondedhydrogenatomswith oxygen atoms.With eachstep,electronsareshiftedawayfrom the carbon(asindicatedby the b/ueshading), and the carbonatom becomesprogressively moreoxidized.Eachof thesestepsis energetically favorableunderthe conditionspresentinsidea cell.

I formaldehyde

c:o Hl I I f o r m i ca c i d

H

C:O HOI I

I n

---n

c a r b o nd i o x i d e

72

Chapter2:CellChemistryand Biosynthesis

\Mhen a carbon atom becomes covalently bonded to an atom with a strong affinity for electrons, such as oxygen,chlorine, or sulfur, for example, it givesup more than its equal share of electrons and forms a polar covalent bond: the positive charge of the carbon nucleus is now somewhat greater than the negative charge ofits electrons, and the atom therefore acquires a partial positive charge and is said to be oxidized. Conversely,a carbon atom in a C-H linkage has slightly more than its share ofelectrons, and so it is said to be reduced (seeFigure 2-43). 'W/hena molecule in a cell picks up an electron (e), it often picks up a proton (H+) at the same time (protons being freely available in water). The net effect in this caseis to add a hydrogen atom to the molecule A+o+H+-+AH Even though a proton plus an electron is involved (instead ofjust an electron), such hydrogenation reactions are reductions, and the reverse, dehydrogenation reactions, are oxidations. It is especiallyeasyto tell whether an organic molecule is being oxidized or reduced: reduction is occurring if its number of C-H bonds increases,whereas oxidation is occurring if its number of C-H bonds decreases (see Figure 2-438). Cells use enzymes to catalyze the oxidation of organic molecules in small steps,through a sequenceof reactions that allows useful energy to be harvested. We now need to explain how enzymes work and some of the constraints under which they operate.

Enzymes Lowerthe Barriers ThatBlockChemical Reactions Considerthe reaction paper + 02 -+ smoke + ashes+ heat + CO2+ H2O The paper burns readily, releasing to the atmosphere both energy as heat and water and carbon dioxide as gases,but the smoke and ashesnever spontaneously retrieve these entities from the heated atmosphere and reconstitute themselves into paper.\Ahen the paper burns, its chemical energy is dissipated as heat-not lost from the universe, since energy can never be created or destroyed,but irretrievably dispersed in the chaotic random thermal motions of molecules.At the same time, the atoms and molecules of the paper become dispersed and disordered. In the language of thermodlmamics, there has been aloss of free energJ, that is, of energy that can be harnessedto do work or drive chemical reactions. This loss reflects a loss of orderliness in the way the energy and molecules were stored in the paper. We shall discuss free energy in more detail shortly, but the general principle is clear enough intuitively: chemical reactions proceed spontaneously only in the direction that leads to a loss of free energy;in other words, the spontaneous direction for any reaction is the direction that goes "dor.rmhill."A "doumhill" reaction in this senseis often said tobe energeticallyfauorable, Although the most energetically favorable form of carbon under ordinary conditions is COz, and that of hydrogen is HzO, a living organism does not disappear in a puff of smoke, and the book in your hands does not burst into flames. This is because the molecules both in the living organism and in the book are in a relatively stable state, and they cannot be changed to a state of Iower energy without an input of energy: in other words, a molecule requires activation energy-a kick over an energy barrier-before it can undergo a chemical reaction that leaves it in a more stable state (Figure 244).In the case of a burning book, the activation energy is provided by the heat of a lighted match. For the molecules in the watery solution inside a cell, the kick is delivered by an unusually energetic random collision with surrounding moleculescollisions that become more violent as the temperature is raised. In a living cell, the kick over the energy barrier is greatly aided by a specialized class of proteins-the enzymes. Each enzyme binds tightly to one or more molecules, called substrates, and holds them in a way that greatly reduces the activation energy of a particular chemical reaction that the bound substrates can undergo. A substance that can lower the activation energy of a reaction is

CATALYSIS ANDTHEUsEOF ENERGY BYCELLS

73

e n z y m er o w e r s activation e n e r g yf o r catalyzed reaction Y+X

I

I o o c o 6

(A)

ff'113'J""'1"*"v

(B)

:lzJffic;€attalf:i

y (areactant) Figure2-44Theimportantprinciple (A)compound of activation energy. isin a relatively stablestate, andenergyisrequired to convert it to compound X (aproduct), even thoughX isat a loweroverall energylevelthanY Thisconversion willnottakeplace, therefore, unlesscompound Y canacquireenoughactivationenergy(energy a minusenergy b)fromits surroundings to undergo thereaction thatconverts it intocompound X.This energymaybe provided by means of anunusually energetic collision withothermolecules. Forthereverse reaction, X -+ Y theactivation energywill be muchlarger(energy a minusenergy c);thisreaction willtherefore occurmuchmorerarely. Activation positive; energies arealways note,however, thatthetotalenergychange fortheenergetically favorable reaction Y -+ X isenergy c minus (B)Energy energy b,a negative number. barriers for specific reactions canbe loweredby catalysts, asindicated bythelinemarked d. Enzymes areparticularly effective catalysts because theygreatly reduce theactivation energy forthereactions theyperform.

termed a catalyst; catalystsincreasethe rate of chemical reactions becausethey allow a much larger proportion of the random collisions with surrounding molecules to kick the substratesover the energy barrier, as illustrated in Figure 2-45.Enzymes are among the most effective catalystsknown, capable of speeding up reactions by factors of 101aor more. They thereby allow reactions that would not otherwise occur to proceed rapidly at normal temperatures. Enzymes are also highly selective.Each enzyme usually catalyzesonly one particular reaction: in other words, it selectivelylowers the activation energy of only one of the several possible chemical reactions that its bound substrate molecules could undergo. In this way, enzymes direct each of the many different molecules in a cell along specific reaction pathways (Figure 2-46). The success of living organisms is attributable to a cell's ability to make enzymes of many types, each with precisely specified properties. Each enzyme has a unique shape containing an actiue site, a pocket or groove in the enzyme into which only particular substrates will fit (Figure z-42). Like all other catalysts, enzyme molecules themselves remain unchanged after participating in a reaction and therefore can function over and over again. In chapter 3, we discuss further how enzymes work.

I

m a n y m o l e c u l e sh a v e e n o u g he n e r g yt o u n d e r g o the enzyme-catalyzed c h e m i c arl e a c t i o n

I

E .=>

39 6o

a l m o s tn o m o l e c u l e s h a v et h e v e r y h i g h e n e r g yn e e d e dt o u n d e r g oa n uncatalyzed

F> o!

b9

m o l e c u l ew s ith a v e r a g ee n e r g y

chemical reaction

-.5

e n e r g yp e r m o l e c u l e+

activation e n e r g yf o r catalyzed reaction

activation e n e r g yf o r u ncatalyzed reactron

Figure2-45 Loweringthe activation energygreatlyincreases the probability of reaction.At any giveninstant,a populationof identicalsubstrate molecules will havea rangeof energies, distributedas shownon the graph.The varyingenergiescomefrom collisions with surroundingmolecules, which make jiggle,vibrate, the substratemolecules and spin.Fora moleculeto undergoa chemicalreaction, the energyof the moleculemustexceedthe activation energybarrierfor that reaction; for most biologicalreactions, this almostnever happenswithoutenzymecatalysis. Even with enzymecatalysis, the substrate moleculesmustexperience a particularly energeticcollisionto react(redshaded areo).Raisingthe temperaturecan also increase the numberof molecules with sufficientenergyto overcomethe activationenergyneededfor a reaction; but in contrastto enzymecatalysis, this effectis nonselective, speedingup all reactions.

74

Chapter2: CellChemistryand Biosynthesis

'i.

o(o

dry river bed

l a k ew i t h

n$,

&l)

\\

(*- .U '

\

flowing s t r e am

WAVES

a

u n c a t ay z e d r e a c t i o n - w a v e sn o t a r g e e n o u g ht o s u r m o u n b t arrler

catalyzedreaction-waves often surmount barrier

(A)

t

t

2^ I

) a c o

uncatalyzed (B)

e n z y m ec a t a l y s i s o f r e a c t i o n1

How Enzymes FindTheirSubstrates: TheEnormous Rapidity of MolecularMotions An enzyme will often catalyzethe reaction of thousands of substrate molecules every second. This means that it must be able to bind a new substrate molecule in a fraction of a millisecond. But both enzl'rnesand their substratesare present in relatively small numbers in a cell. How do they find each other so fast?Rapid binding is possible because the motions caused by heat energy are enormously fast at the molecular level. These molecular motions can be classified broadly into three kinds: (1) the movement of a molecule from one place to another (translational motion), (2) the rapid back-and-forth movement of covalently linked atoms with respect to one another (vibrations), and (3) rotations. All of these motions help to bring the surfacesof interacting molecules together. The rates of molecular motions can be measured by a variety of spectroscopic techniques.A large globular protein is constantly tumbling, rotating about its axis about a million times per second. Molecules are also in constant translational motion, which causesthem to explore the space inside the cell very efficiently by wandering through it-a process called diffusion. In this way, every molecule in a cell collides with a huge number of other molecules each second. As the molecules in a liquid collide and bounce off one another, an individual molecule moves first one way and then another, its path constituting a random walk (Figure 2-48). In such a walk, the average net distance that each molecule travels (asthe crow flies) from its starting point is proportional to the square root of the time involved: that is, if it takes a molecule I second on averageto travel 1 pm, it takes 4 secondsto travel 2 pm, 100 secondsto travel 10 pm, and so on. The inside of a cell is very crowded (Figure 2-49). Nevertheless,experiments in which fluorescent dyes and other labeled molecules are injected into cells

activesite

m o l e c u l eA (substrate)

enzymesubstrate comolex

enzymeproduct comolex

molecule B (product)

Figure2-46 Floatingball analogiesfor enzyme catalysis.(A)A barrier dam is loweredto representenzyme catalysis. The greenball representsa potentialreactant(compoundY) that is bouncingup and down in energylevel due to constantencounters with waves (ananalogyfor the thermal bombardmentof the reactantmolecule with the surrounding watermolecules). Whenthe barrier(activation energy)is loweredsignificantly, it allowsthe favorablemovementof the energetically ball(thereactant)downhill.(B)Thefour wallsof the box reoresent the activation energybarriers for four differentchemical reactions that areall energetically favorable, in the sensethat the products areat lowerenergylevelsthan the reactants.ln the left-handbox, none of thesereactions occursbecauseeventhe largestwavesarenot largeenoughto surmountany of the energybarriers. In the right-handbox, enzymecatalysis lowersthe activationenergyfor reaction number1 only;now the jostlingof the wavesallowspassage ofthe reactant moleculeoverthis energybanier, inducingreaction1.(C)A branchingriver with a set of barrierdams(yellowboxes) servesto illustratehow a seriesof enzyme-catalyzed reactions determines the exactreactionpathwayfollowedby eachmoleculeinsidethe cell.

Figure2-47 How enzymeswork. Each enzymehasan activesiteto whichone or more substrotemoleculesbind, formingan enzyme-substrate complex. A reactionoccursat the activesite, producingan enzyme-product complex. fhe productis then released, allowingthe enzymeto bind furthersubstrate mnlarr

rla
Y. If the ratio of Y to X increases,the AG becomes more negative for the transition Y -+ X (and more positive for the transition X -+ Y). How much of a concentration difference is needed to compensate for a given decreasein chemical bond energy (and accompanying heat release)?The answer is not intuitively obvious, but it can be determined from a thermodynamic analysis that makes it possible to separatethe concentration-dependent and the concentration-independent parts of the free-energy change.The AG for a given reaction can thereby be written as the sum of two parts: the first, called Ihe standard free-energychange,AGo,depends on the intrinsic charactersof the reacting molecules; the second depends on their concentrations. For the simple reactionY -+ X at 37'C, A G = A G + 0 . 6 1 6l n ] $ = A G + . r + 2 I o"e E lYl lYl where AG is in kilocalories per mole, [Y] and [X] denote the concentrations of Y and X, ln is the natural logarithm, and the constant 0.616 is equal to R7: the product of the gas constant, R, and the absolute temperature, Z. Note that AG equals the value of AG when the molar concentrations of Y and X are equal (log I = 0). As expected,AG becomes more negative as the ratio of X to Y decreases(the log of a number < I is negative). Inspection of the above equation reveals that the AG equals the value of AG when the concentrations of Y and X are equal. But as the favorable reactionY -+ X proceeds,the concentration of the product X increasesand the concentration of the substrate Y decreases.This change in relative concentrations will cause ffi / [Y] to become increasingly large, making the initially favorable AG less and less negative. Eventually, when AG = 0, a chemical equilibrium will be attained; here the concentration effect just balances the push given to the reaction by AG, and the ratio of substrate to product reaches a constant value (Figure 2-52). How far will a reaction proceed before it stops at equilibrium? To address this question, we need to introduce the equilibrium constant, K The value of K is different for different reactions, and it reflects the ratio ofproduct to substrate at equilibrium. For the reactionY -+ X: IX]

^=lfr

The equation that connects AG and the ratio tX / tYl allows us to connect AG directly to K Since AG = 0 at equilibrium, the concentrations of Y and X at this point are such that:

tG =-r.421"c j+

or,

LG =-L4ZIogK

this reactioncan occurspontaneously

ENERGETICALLY UNFAVORABLE REACTION

lf the reactionX*Y o c c u r r e dA , Gw o u l d be > 0, and the u n i v e r s ew o u l d b e c o m em o r e oroereo.

thisreaction canoccuronlyif it iscoupledto a second, favorablereaction energetically Figure2-50 The distinction between energetically favorableand energeticallyunfavorablereactions.

the energeticallyunfavorable reactionX*Y is driven by the energeticallyfavorable reaction C*D, becausethe net free-energychangefor the pair of coupled reactionsis less than zero Figure 2-51 How reaction coupling is used to drive energetically unfavorable reactions.

C

LYSIS ANDTHEUSEOFENERGY BYCELLS

77 Figure2-52 Chemicalequilibrium. the Whena reactionreachesequilibrium, forwardand backwardfluxesof reacting areequaland opposite. molecules

THEREACTION

Theformation of X isenergetically favoredin thisexampleIn otherwords,the A6 of Y -+ X isnegative andthe AGof X + Y ispositiveButbecause of thermal bombardments, therewill always besomeX converting to Y andviceversa. SUPPOSE WESTART WITHAN EQUAL NUMBER OFY ANDX MOLECULES

thereforethe ratioof X to " molecules will increase

y1Jh"

transition

EVENTUALLY therewill be a largeenoughexcess of X overY to just compensate for the slowrateof X -+ Y.Equilibrium willthenbeattained

Table2-4 Relationship Betweenthe StandardFreeEnergyChange,AG",and the EquilibriumConstant AT EQUILIBRIUM t h e n u m b e ro f Y m o l e c u l e sb e i n gc o n v e r t e dt o X m o l e c u l e s e a c hs e c o n di s e x a c t l ye q u a lt o t h e n u m b e ro f X m o l e c u l e sb e i n gc o n v e r t e dt o Y m o l e c u l e se a c hs e c o n ds. o t h a t t h e r e i s n o n e t c h a n o ei n t h e r a t i o o f Y t o X .

Using the last equation, we can see how the equilibrium ratio of X to Y (expressedas an equilibrium constant, K) depends on the intrinsic character of the molecules, as expressedin the value of AG (Ihble 2-4). Note that for every 1.4 kcal/mole (5.9 kJ/mole) difference in free energy at 37"C, the equilibrium constant changes by a factor of 10. \Alhen an enzyme (or any catalyst) lowers the activation energy for the reaction Y -+ X, it also lowers the activation energy for the reaction X -+ Y by exactly the same amount (see Figure 2-44).The forward and backward reactions will therefore be acceleratedby the same factor by an enzyme, and the equilibrium point for the reaction (and AG) is unchanged (Figure 2-53).

ForSequentialReactions, AGoValuesAre Additive We can predict quantitatively the course of most reactions.A large body of thermodlmamic data has been collected that makes it possible to calculate the standard change in free energy,AG, for most of the important metabolic reactions of the cell. The overall free-energy change for a metabolic pathway is then simply the sum of the free-energychangesin each of its component steps.Consider, for example, two sequential reactions X-+Y and Y -+Z whose AG values are +5 and -13 kcal/mole, respectively.(Recallthat a mole is 6 x 1023molecules of a substance.)If these two reactions occur sequentially, the AG for the coupled reaction will be -8 kcal/mole. Thus, the unfavorable reaction X -+ Y which will not occur spontaneously, can be driven by the favorable reactionY -+ Z, provided that this second reaction follows the first. Cells can therefore cause the energetically unfavorable transition, X -+ Y to occur if an enzyme catalyzing the X -+ Y reaction is supplemented by a second enzyme that catalyzes the energetically fauorable reaction,Y -->Z. In effect, the reaction Y -+ Z will then act as a "siphon' to drive the conversion of all of molecule X to molecule Y and thence to molecule Z (Figure 2-54) . For example,

10s 104 103 102 lor 1 10 10-2 10-3 1o-4 1o-s

-7.1(-29.7) -s.7 (-23.8) -4.3(-18.0) - 2 . 8( - 1 1 . 7 ) -1.4(-s.e) 0 (0)

'r.4(s.9)

2 . 8( 1 1 . 7 ) 4.3(18.0) s.7(23.8) 7.1(2s.7)

V a l u eosf t h e e q u i l l b r i ucmo n s t a n t for the simple werecalculated Y = X usingthe chemlcalreaction equationglvenin the text TheAG"givenhereis in kilocalories permoleat 37"C,with kilojoules (1 kilocalorie permolein parentheses s )s i se q u atl o 4 l 8 4 k i l o j o u l e A in the text,AG'represents explained under difference the free-energy (whereal1 conditions standard arepresenlar a components 'l of 0 mole/liter) concentration Fromthistable,we seethat if thereis change free-energy standard a favorable (AG")of -a 3 kca/mole(-l B0 kJlmole)for Y + X,therewill be 1000 the transitlon in stateX than timesmoremolecuJes (K= 1000), in stateY at equilibrium

chapter2:cellchemistry andBiosynthesis

78

XY

XY UNCATALYZED REACTION

ENZYME-CATALYZED REACTION

several of the reactions in the long pathway that converts sugars into CO2 and H2O would be energetically unfavorable if considered on their or,rm.But the pathway neverthelessproceeds becausethe total AG for the seriesof sequential reactions has a large negative value. But forming a sequential pathway is not adequate for many purposes. Often the desired pathway is simply X -+ Y without further conversion of Y to some other product. Fortunately, there are other more general ways of using enzymes to couple reactions together. How these work is the topic we discuss next.

Figure2-53 Enzymescannotchange the equilibriumpoint for reactions, Enzymes, likeall catalysts, speedup the forwardand backwardratesof a reaction by the samefactor.Therefore,for both the catalyzed and the uncatalyzed reactions shownhere,the numberof molecules undergoingthe transition X -+ Y is eoualto the numberof molecules undergoingthe transition Y -+ X when the ratioof Y molecules to X molecules is 3.5to 1. In otherwords,the two reactionsreacheouilibriumat exactlythe samepoint.

ActivatedCarrierMolecules Are Essential for Biosynthesis The energy released by the oxidation of food molecules must be stored temporarily before it can be channeled into the construction of the many other molecules needed by the cell. In most cases,the energy is stored as chemical bond energy in a small set of activated "carrier molecules,"which contain one or more energy-rich covalent bonds. These molecules diffuse rapidly throughout the cell and thereby carry their bond energy from sites of energy generation to the sites where energy is used for bioslnthesis and other cell activities (Figure 2-55). The activated carriers store energy in an easily exchangeable form, either as a readily transferable chemical group or as high-energy electrons, and they can serve a dual role as a source of both energy and chemical groups in biosynthetic reactions. For historical reasons,these molecules are also sometimes referred to as coenzymes.The most important of the activated carrier molecules are ATP and two molecules that are closely related to each other, NADH and NADPHas we discuss in detail shortly. We shall see that cells use activated carrier molecules like money to pay for reactions that otherwise could not take place.

z e q u i l i b r i u mp o i n t f o r X * Y r e a c t i o na l o n e

e q u i l i b r i u mp o i n t f o r Y*Z reactionalone

(c)

I

=-

/.-'

EDlr

2 x pyruvate

o. o\// C

I

HC:O

I

CH:

I

CH:

2 x acetaldehyde

H-, C l -OH CH:

2x

COz

2 x ethanol

Figure2-71 Two pathwaysfor the anaerobicbreakdownof pyruvate. (A)Whenthereis inadequate oxygen,for example,in a musclecellundergoing vigorouscontraction, the pyruvate producedby glycolysisis convertedto lactateas shown.Thisreactton regenerates the NADt consumedin step 6 of glycolysis,but the whole pathway yieldsmuch lessenergyoverallthan completeoxidation.(B)In some organisms that can grow anaerobically, suchasyeasts,pyruvateis convertedvia acetaldehyde into carbondioxideand ethanol.Again,this pathwayregenerates NAD+from NADH,as requiredto enable glycolysis to continue.Both(A)and (B) are exampfes of fermentations.

HOWCELLS OBTAIN ENERGY FROMFOOD

Glycolysis lllustrates HowEnzymes CoupleOxidationto Energy Storage Returning to the paddle-wheel analogy that we used to introduce coupled reactions (see Figure 2-56), we can now equate enzymes with the paddle wheel. Enzymes act to harvest useful energy from the oxidation of organic molecules by coupling an energetically unfavorable reaction with a favorable one. To demonstrate this coupling, we examine a step in glycolysis to see exactly how such coupled reactions occur. TWo central reactions in glycolysis (steps 6 and 7) convert the three-carbon sugar intermediate glyceraldehyde3-phosphate (an aldehyde) into 3-phosphoglycerate(a carboxylic acid; seePanel2-8, pp. 120-121).This entails the oxidation of an aldehyde group to a carboxylic acid group in a reaction that occurs in two steps.The overall reaction releasesenough free energy to convert a molecule of ADP to AIP and to transfer two electrons from the aldehyde to NAD* to form NADH, while still releasing enough heat to the environment to make the overall reaction energeticallyfavorable (AG for the overall reaction is -3.0 kcal/mole). Figure 2-72 otttlines the means by which this remarkable feat of energy harvesting is accomplished. The indicated chemical reactions are precisely guided by two enzymes to which the sugar intermediates are tightly bound. In fact, as detailed in Figure 2-72, the first enzyme (glyceraldehyde 3-phosphate dehydrogenase) forms a short-lived covalent bond to the aldehyde through a reactive -SH group on the enzyme, and catalyzes its oxidation by NAD+ in this attached state. The reactive enzyme-substrate bond is then displaced by an inorganic phosphate ion to produce a high-energy phosphate intermediate, which is released from the enzyme. This intermediate binds to the second enzyme (phosphoglycerate kinase), which catalyzesthe energetically favorable transfer of the high-energy phosphate just created to ADB forming AIP and completing the process of oxidizing an aldehyde to a carboxylic acid. We have shown this particular oxidation process in some detail because it provides a clear example of enzyme-mediated energy storage through coupled reactions (Figure 2-73). Steps 6 and 7 are the onlyreactions in glycolysis that create a high-energy phosphate linkage directly from inorganic phosphate. As such, they account for the net yield of two AIP molecules and two NADH molecules per molecule of glucose (seePanel 2-8, pp.l20-I2l). As we have just seen,AIP can be formed readily from ADP when a reaction intermediate is formed with a phosphate bond of higher-energy than the phosphate bond in AIP Phosphatebonds can be ordered in energy by comparing the standard free-energy change (AGl for the breakage of each bond by hydrolysis. Figure 2-74 compares the high-energy phosphoanhydride bonds in ATP with the energy of some other phosphate bonds, several of which are generated during glycolysis.

in SpecialReservoirs StoreFoodMolecules Organisms All organisms need to maintain a high ATP/ADP ratio to maintain biological order in their cells. Yet animals have only periodic accessto food, and plants need to survive overnight without sunlight, when they are unable to produce sugar from photosynthesis. For this reason, both plants and animals convert sugars and fats to special forms for storage (Figure 2-75). To compensate for long periods of fasting, animals store fatty acids as fat droplets composed of water-insoluble triacylglycerols,largely in the cytoplasm of specialized fat cells, called adipocltes. For shorter-term storage, sugar is stored as glucose subunits in the large branched polysaccharide glycogen, which is present as small granules in the cltoplasm of many cells,including liver and muscle. The synthesis and degradation of glycogen are rapidly regulated according to need. \.A/hencells need more AIP than they can generate from the food molecules taken in from the bloodstream, they break down glycogen in a reaction that produces glucose 1-phosphate,which is rapidly converted to glucose 6-phosphate for glycolysis.

91

92

Chapter2: CellChemistryand Biosynthesis

(A)

HO

\ /(_ /

I

H-C-OH

glyceraldehyde 3-phosphate

SH

A covalent bond is formed between glyceraldehyde3-phosphate(the substrate)and the -5H group of a cysteineside chain of the enzyme glyceraldehyde3-phosphate d e h y d r o g e n a s ew,h i c h a l s ob i n d s noncovalentlyto NAD+.

t-? I

H -C-OH

I

H_C_OH I

Oxidation of glyceraldehyde 3-phosphateoccurs,as two electronsplus a proton (a hydride ion, see Figure2-60) are transferredfrom glyceraldehyde 3-phosphateto the bound NAD+, forming NADH.Part of the energy releasedby the oxidation of the a l d e h y d ei s t h u s s t o r e di n N A D H , and part goes into convertingthe bond between the enzymeand its substrateglyceraldehyde 3-phosphateinto a high-energy thioester bond

cH2oo

(o c r F

I H-C-OH

HO-

A m o l e c u l eo f i n o r g a n i cp h o s p h a t e displacesthe high-energybond to the enzymeto create 1,3-bisphosphoglycerate,which contains a high-energyacyl-anhydride bond.

PIO

ti

rU- iAL J

1,3-bisphosphoglycerate

C:O

I

H-C-OH

l^

cH2o(L)

@@o F 4

H

r fr_A-r,

C

I

H- C-OH

l^

cH2o(L)

3 - p h o s p h olgy c e r a t e

The high-energybond to phosphate is transferredto ADP to form ATP.

(B) SUMMARYOF STEPS 6 AND 7 Much of the energy of oxidation has been stored in the activateo carriersATPand NADH.

Figure2-72 Energystoragein steps6 and 7 of glycolysis.In thesestepsthe oxidationof an aldehydeto a carboxylic acidis coupledto the formationof ATP and NADH.(A)Step6 beginswith the formationof a covalentbond between the substrate(glyceraldehyde 3-phosphate) and an -5H groupexposed on the surfaceof the enzyme (glyceraldehyde 3-phosphate dehydrogenase). Theenzymethen catalyzestransferof hydrogen(asa hydrideion-a protonplustwo electrons) from the bound glyceraldehyde 3-phosphate to a moleculeof NAD+.Partof the energy released in this oxidationis usedto form a moleculeof NADHand part is usedto convertthe originallinkagebetweenthe enzymeand its substrate to a highenergythioesterbond (shownin red.). A moleculeof inorganicphosphatethen displaces this high-energy bond on the enzyme,creatinga high-energy sugarphosphatebond instead(red).At this point the enzymehas not only stored energyin NADH,but alsocoupledthe energetically favorableoxidationof an aldehydeto the energetically unfavorable formationof a high-energy phosphate bond.Thesecondreactionhasbeen drivenby the first,therebyactinglikethe "paddle-wheel" couplerin Figure2-56. In reactionstep7, the high-energy just made, sugar-phosphate intermediate 1,3-bisphosphoglycerate, bindsto a secondenzyme,phosphoglycerate kinase.The reactivephosphateis transferredto ADP,forming a moleculeof ATPand leavinga freecarboxylic acid groupon the oxidizedsugar. (B)Summaryof the overallchemical changeproducedby reactions6 and 7.

93

HOWCELLSOBTAINENERGY FROMFOOD

o

ME

o c o a o

o

Figure 2-73 Schematicview of the coupledreactionsthat form NADHand ATPin steps 6 and 7 of glycolysis.The C-H bond oxidationenergydrivesthe formationof both NADHand a highenergyphosphatebond.The breakageof bond then drivesATP the high-energy formation.

\,/

C

I

\,, C I

C-H bond oxidation energy

+

STEP6

7 STEP

t o t a l e n e r g yc h a n g ef o r s t e p6 f o l l o w e d b y s t e p 7 i s a f a v o r a b l e- 3 k c a l / m o l e

o-o \//

CO

enol phosphate bond

rll

H'' C : C - O - . P

Hro

-O-

/ ,/lo-

p h o s p h o e n oply r u v a t e isee ianel Z-8,'pp. 120-121)

- 14'6 (-61 e)

oo

tltl c-c-o y'P o-

anhydride bondto carbon

o

Hro/

phosphate bond in creaTtne phosphate

anhydride bondto phosphate (phosphoanhydride bond)

a._a)-

ooo ililtl

i-"-i-o7i-"o-

o-

for example, 1,3-bisphosphoglycerate (seePanel2-8)

c r e a t i n eo h o s p h a t e ( a c t i v a t e dc a r r i e rt h a t storesenergy in muscle)

(-43.0)

for example, ATPwhen hydrolyzed tOADP

-7.3 (-306)

-10.3

/o-

Hzo HO phosphoester bond

lll , -i-"vP-o-

for example, g l u c o s e6 - p h o s p h a t e (seePanel2-8)

",/o-

Hzo type of phosphatebond

specificexamplesshowing the standardfree-energychange (AG') for hydrolysisof phosphatebond

Figwe 2-74 Phosphatebonds have different energies.Examplesof differenttypes of phosphatebondswith their sitesof hydrolysisare shown in the moleculesdepictedon the left.Thoseitarting with a gray catbonatom show only part of a molecule.Examplesof molecules (kilojoules transfer in parentheses)'The in kilocalories changefor hydrolysis containingsuchbondsaregivenon the right,with the free-energy (AG') the of for hydrolysis change free-energy if the standard phosfhate group favorable of a from one moleculeto anotheris energetically Thus,a phosphategroup of the phosphatebond in the second. phosphatebond of the firstmoleculeis more negativethan that for hydrolysis to ADPto form ATPThe hydrolysisreactioncan be viewedas the transferof the phosphate is readilytransferredfrom 1,3-bisphosphoglycerate group to water.

94

Chapter2: CellChemistyand Biosynthesis

9rycogen g r a n u l e isn the cytoplasm o f a l i v e rc e l l

b r a n c hp o i n t

g l u c o s es u b u n i t s

1pm

ql a 1,4-glycosidic bond in backbone

,,-

Figure2-75 The storageof sugarsand fats in animaland plant cells.(A)The structures of starchand glycogen, the storageform of sugarsin plantsand animals,respectively. Botharestorage polymersof the sugarglucoseand differ only in the frequencyof branchpoints (theregioninyellowisshownenlarged below).Therearemanymore branchesin glycogenthan in starch.(B)An electron micrographshowsglycogengranulesin the cytoplasmof a livercell.(C)A thin sectionof a singlechloroplast from a plantcell,showingthe starchgranules and lipid(fatdroplets)that have accumulated asa resultof the biosyntheses occurringthere.(D)Fat droplets(stainedred)beginningto accumulate in developingfat cellsof an animal.(8,courtesyof RobertFletterick and DanielS.Friend;C,courtesyof K.Plaskitt; D,courtesyof RonaldM. Evans and PeterTotonoz.)

o 1 , 6 - 9 l y c o s i dbi co n d at branch point

/ o-cH2

OH

l;------., Quantitatively, fat is far more important than glycogen as an energy store for animals, presumably becauseit provides for more efficient storage.The oxidation of a gram of fat releasesabout twice as much energy as the oxidation of a gram of glycogen. Moreover, glycogen differs from fat in binding a great deal of water, producing a sixfold difference in the actual mass of glycogen required to store the same amount of energy as fat. An averageadult human stores enough glycogen for only about a day of normal activities but enough fat to last for nearly a month. If our main fuel reservoir had to be carried as glycogen instead of fat, body weight would increase by an averageof about 60 pounds. Although plants produce NADPH and Arp by photosynthesis,this important process occurs in a specialized organelle, called a chloroplast, which is isolated from the rest of the plant cell by a membrane that is impermeable to both types of activated carrier molecules. Moreover, the plant contains many other cellssuch as those in the roots-that lack chloroplasts and therefore cannot produce their or,rmsugars.Therefore, for most of its ATP production, the plant relies on an

50u.

HOWCELLSOBTAINENERGY FROMFOOD

.95 Figure 2-76 How the ATPneeded for most plant cell metabolismis made.In plants,the chloroplasts and mitochondria to supplycellswith collaborate metabolitesand ATP.(Fordetails,see Chapter14.)

light

chloroplast

metabolites

export of sugars from its chloroplasts to the mitochondria that are located in all cells of the plant. Most of the AIP needed by the plant is synthesized in these mitochondria and exported from them to the rest of the plant cell, using exactly the same pathways for the oxidative breakdor,rrnof sugars as in nonphotosynthetic organisms (Figure 2-76). During periods of excessphotosynthetic capacity during the day, chloroplasts convert some of the sugars that they make into fats and into starch, a polgner of glucose analogous to the glycogen of animals. The fats in plants are triacylglycerols, just like the fats in animals, and differ only in the types of fatty acids that predominate. Fat and starch are both stored in the chloroplast as reservoirs to be mobilized as an energy source during periods of darkness (see Figure 2-75C). The embryos inside plant seedsmust live on stored sources of energy for a prolonged period, until they germinate to produce leaves that can harvest the energy in sunlight. For this reason plant seeds often contain especially large amounts of fats and starch-which makes them a malor food source for animals, including ourselves (Figare 2-7 7),

MostAnimalCellsDeriveTheirEnergyfrom FattyAcidsBetween Meals After a meal, most of the energy that an animal needs is derived from sugars derived from food. Excesssugars,if any, are used to replenish depleted glycogen stores,or to synthesizefats as a food store. But soon the fat stored in adipose tissue is called into play, and by the morning after an overnight fast, fatty acid oxidation generatesmost of the ATP we need. Low glucose levels in the blood trigger the breakdown of fats for energy production. As illustrated in Figure 2-78, the triacylglycerols stored in fat droplets in adipocl'tes are hydrolyzed to produce fatty acids and glycerol, and the fatty acids released are transferred to cells in the body through the bloodstream. \.\hile animals readily convert sugars to fats, they cannot convert fatty acids to sugars.Instead, the fatty acids are oxidized directly.

Figure2-77 SomePlant seedsthat serveas important foods for humans. Corn,nuts,and Peasall containrich storesof starchand fat that providethe youngplantembryoin the seedwith energyand buildingblocksfor (Courtesy of the JohnInnes biosynthesis. Foundation.)

96

Chapter 2:CellChemistry and Biosynthesis

stored fat bloodstream glycerol

MUSCLE CELL

fatty acids

o x i d a t i o ni n mitochondria

Figure 2-78 How stored fats are mobilized for energy production in animals.Low glucoselevelsin the blood triggerthe hydrolysisof the triacylglycerolmoleculesin fat droplets to free fatty acidsand glycerol,as illustrated.Thesefatty acidsenter the bloodstream,wherethey bind to the abundantblood protein,serumalbumin. Specialfatty acidtransportersin the plasmamembraneof cellsthat oxidize fatty acids,suchas musclecells,then pass thesefatty acidsinto the cytosol,from whichthey aremovedinto mitochondria for energyproduction(seeFigure2-80).

)

Sugarsand FatsAre Both Degradedto AcetylCoAin Mitochondria

The fatty acids imported from the bloodstream are moved into mitochondria, where all of their oxidation takes place ). Each molecule of fatty acid (as the activated molecule /a tty acyl coA) is broken down completely by a cycle of reactions that trims two carbons at a time from its carboxyl end, generating one molecule of acetyl coA for each turn of the cycle. A molecule of NADH and a molecule of FADH2 are also produced in this proces Sugars and fats are the major energy sources for most nonorganisms, including humans. However, most of the useful ene

8 t r i m e r so f lipoamide reductasetransacetylase

+6 dimersof dihydrolipoyl dehydrogenase

+ 1 2 d i m e r so f pyruvatedecarboxylase

o ,//

cH.c

si*ht{iiii acetyl coA (B)

Figure 2-79 The oxidation of pyruvate to acetylCoA and COz.(A)The structure of the pyruvatedehydrogenase complex, whichcontains60 polypeptidechains. Thisis an exampleof a large multienzymecomplexin which reaction intermediatesare passeddirectlyfrom one enzymeto another.In eucaryotic cellsit is locatedin the mitochondrion. (B)The reactionscarriedout by the pyruvatedehydrogenase complex.The complexconvertspyruvateto acetylcoA in the mitochondrial matrix;NADHis also producedin this reaction.A, B,and C are the three enzymespyruvate decarboxylase,Iipoam ide reductasetronsacetylose,and dihydrolipoyI dehydrogenase,respectively.These enzymesareillustrated in (A);their activities arelinkedas shown.

97

HOWCELL5OBTAINENERGY FROMFOOD

S u g a r sa n d polysaccharides

Fats+fatty acids CYTOSOL

Figure2-80 Pathwaysfor the production of acetyl CoAfrom sugarsand fats. The mitochondrionin lt is eucaryotic cellsis the placewhereacetylCoAis producedfrom both typesof majorfood molecules. occurand wheremostof its ATPis made. thereforethe olacewheremostof the cell'soxidationreactions in detailin Chapter14. arediscussed Thestructureand functionof mitochondria

extracted from the oxidation of both types of foodstuffs remains stored in the acetyl CoA molecules that are produced by the two t)?es of reactions just described. The citric acid cycle of reactions, in which the acetyl group in acetyl CoA is oxidized to CO2and H2O,is therefore central to the energy metabolism of aerobic organisms. In eucaryotesthese reactions all take place in mitochondria. We should therefore not be surprised to discover that the mitochondrion is the place where most of the ATP is produced in animal cells. In contrast, aerobic bacteria carry out all of their reactions in a single compartment, the cytosol, and it is here that the citric acid cycle takes place in these cells.

TheCitricAcidCycleGenerates NADHby OxidizingAcetylGroups to COz In the nineteenth century, biologists noticed that in the absence of air (anaerobic conditions) cells produce lactic acid (for example, in muscle) or ethanol (for example, in yeast), while in its presence (aerobic conditions) they consume 02 and produce CO2and H2O.Efforts to define the pathways of aerobic metabolism

Figure2-81 The oxidation of fatty acids to acetyl CoA.(A)Electronmicrographof a lipid droplet in the cytoplasm(top),and the structureof fats (bottom).Fatsare The glycerolportion,to triacylglycerols. whichthreefatty acidsarelinked throughesterbonds,is shownherein areinsolublein waterand form blue.Fats fat largelipiddropletsin the specialized in whichthey are cells(calledadipocytes) stored.(B)The fatty acid oxidationcycle. The cycleis catalyzedby a seriesof four Each enzymesin the mitochondrion. turn of the cycleshortensthe fattyacid chain by two carbons(shownin red)and generatesone moleculeof acetylCoA and one moleculeeachof NADHand The structureof FADHzis FADHz. presentedin Figure2-838.(4, courtesy of Daniel5. Friend.)

(B) fatty acyl CoA Rr,-CH2-CH2-

CH2-C

hydrocarbontail

fatty acyl CoA shortenedby .:fl,- CH2 - C two carDons

//

o

\s-coA / C H'?\ S -C

1pm

@

o C-

acetylCoA hydrirclrrbohtail

hydrocarbon tail

\? R-CH2-C-CH2-C.

,,o

OHH

r$,-CHr- Ct l- C HH

o tlC

e s t e rb o n d

6it

Hzo

o C-

//o -c cH i$tct-tr-cu: 's

hydrocarbontail

2 C

o

.5{6A

98

Chapter2: CellChemistryand Biosynthesis

eventually focused on the oxidation ofpyruvate and led in 1937to the discovery of the citric acid cycle, also knoum as the tricarboxylic acid cycle or the Krebs cycle.Thecitric acid cycle accounts for about two-thirds of the total oxidation of carbon compounds in most cells, and its major end products are CO2and highenergy electrons in the form of NADH. The CO2 is released as a waste product, while the high-energy electrons from NADH are passed to a membrane-bound electron-transport chain (discussedin Chapter 14), eventually combining with 02 to produce H2O. Although the citric acid cycle itself does not use 02, it requires 02 in order to proceed because there is no other efficient way for the NADH to get rid of its electrons and thus regeneratethe NAD+ that is needed to keep the cycle going. The citric acid cycle takes place inside mitochondria in eucaryotic cells. It results in the complete oxidation of the carbon atoms of the acetyl groups in acetyl CoA, converting them into CO2. But the acetyl group is not oxidized directly. Instead, this group is transferred from acetyl CoA to a larger, four-carbon molecule, oxaloacetate,to form the six-carbon tricarboxylic acid, citric acid, for which the subsequent cycle of reactions is named. The citric acid molecule is then gradually oxidized, allowing the energy of this oxidation to be harnessedto produce energy-rich activated carrier molecules. The chain of eight reactions forms a cycle because at the end the oxaloacetate is regenerated and enters a new turn of the cycle, as shown in outline in Figure 2-82. we have thus far discussed only one of the three types of activated carrier molecules that are produced by the citric acid cycle, the NAD+-NADH pair (see Figure 2-60). In addition to three molecules of NADH, each turn of the cycle also produces one molecule of FADH2 (reduced flavin adenine dinucleotide) from FAD and one molecule of the ribonucleotide GTP (guanosine triphosphate) from GDP The structures of these two activated carrier molecules are illustrated in Figure 2-83. GTP is a close relative of ATB and the transfer of its terminal phosphate group to ADP produces one ATP molecule in each cycle. Like NADH, FADHz is a carrier of high-energy electrons and hydrogen. As we discussshortly, the energy that is stored in the readily transferred high-energy electrons of NADH and FADH2will be utilized subsequently for Arp production through the process of oxidatiue phosphorylation, the only step in the oxidative catabolism of foodstuffs that directly requires gaseousoxygen (oz) from the atmosphere. Panel 2-9 (pp. 122-123)presents the complete citric acid cycle.Water, rather than molecular oxygen, supplies the extra oxygen atoms required to make co2 from the acetyl groups entering the citric acid cycle.As illustrated in the panel,

o -t - s-coA Hrc acetylCoA 2C

t

oxd toacelale

4C +H*

/ I 4C

STEP1

*{ srEP2

-)l

u4lllil-,,/

./

V

srEP8

5C

\

srEP 3

r*E!E-f.-

co,

I 5C

N E TR E S U L O T N ET U R NO FT H EC Y C L E P R O D U C ETSH R E EN A D H ,O N EG T BA N D O N EF A D H 2A, N D R E L E A S E TS W O M O L E C U L EOSF C O I

Figure2-82 Simpleoverviewof the citric acid cycle.The reactionof acetylcoA with oxaloacetatestartsthe cycleby producingcitrate(citricacid).In eachturn of the cycle,two molecules of CO2are producedas wasteproducts,plus threemolecules of NADH,one molecule of GTP, and one moleculeof FADH2. The numberof carbonatomsin each intermediateis shown in a yellowbox. Fordetails,see Panel2-9 (pp. 122-123).

guanine

o-

o ll c

2H-

???\'.,-i

2e

OH

ill - N - - c- t - r n c llrtll

HrC-9

t-r-t--N-C\O

H3C-C OH

OH

tl

,rN '' c,. ,rC'-N.' n

CH,

I' I H-C-OH I

H-C -OH

H-C-OH (B)

three moleculesof water are split in each cycle,and the orygen atoms of some of them are ultimately used to make CO2. In addition to pyruvate and fatty acids, some amino acids pass from the cytosolinto mitochondria, where they are alsoconvertedinto acetylCoAor one of the other intermediatesof the citric acid cycle.Thus,in the eucaryoticcell,the mitochondrion is the center toward which all energy-yieldingprocesseslead, whether they begin with sugars,fats,or proteins. Both the citric acid cycle and glycolysisalso function as starting points for important biosynthetic reactionsby producing vital carbon-containing intermediates,such as oxaloacetateand a-ketoglutarate.Someof these substances produced by catabolism are transferredback from the mitochondrion to the cytosol,where they servein anabolicreactionsasprecursorsfor the synthesisof many essentialmolecules,such as amino acids (Figure244).

H2C-O-

-O-

Figure2-83 The structuresof GTPand FADHz.(A)GTPand GDPare close relativesof ATPand ADP,respectively. (B)FADH2is a carrierof hydrogensand high-energyelectrons,like NADHand NADPH.lt is shown here in its oxidized form (FAD)with the hydrogen-canying atoms h ighlightedin yellow.

nucleotides glucose6-phosp nur. /

amrno sugars

+

fructose 6-phosphate

'

glycolipids glycoproteins

+

/\ I

+ + +

serine

d i h y d r o x y a c e t o n+e PhosPhate

3-phosphoglycerate

lipids

a m i n oa c i d s pyrimidines

phosphoenolpyruvate alanine .*.-

orrrlura"

andthe citric Figure2-84Glycolysis acidcycleprovidethe precursors manyimportant neededto synthesize Theaminoacids, biologicalmolecules. andother lipids,sugars, nucleotides, hereasproducts-in molecules-shown for the many turnserveasthe precursors the cell.Eachb/ack macromoleculesof arrowinthisdiagramdenotesa single thered reaction; enzyme-catalyzed with arrowsgenerallyrepresentpathways to produce manystepsthatarerequired products. the indicated

100

Chapter2:CellChemistryand Biosynthesis

ElectronTransportDrivesthe Synthesis of the Majorityof the ATP in MostCells Most chemical energy is released in the last step in the degradation of a food molecule. In this final process the electron carriers NADH and FADH2 transfer the electrons that they have gained when oxidizing other molecules to the electron-transport chain, which is embedded in the inner membrane of the mitochondrion (seeFigure 14-10).As the electrons pass along this long chain of specialized electron acceptor and donor molecules, they fall to successivelylower energy states.The energy that the electrons release in this process pumps H+ ions (protons) across the membrane-from the inner mitochondrial compartment to the outside-generating a gradient of H+ ions (Figure 2-85). This gradient servesas a source of energy,being tapped like a battery to drive a variety of energy-requiring reactions.The most prominent of these reactions is the generation of ATP by the phosphorylation of ADP At the end of this series of electron transfers, the electrons are passed to molecules of oxygen gas (Oz) that have diffused into the mitochondrion, which simultaneously combine with protons (H*) from the surrounding solution to produce water molecules. The electrons have now reached their lowest energy Ievel, and therefore all the available energy has been extracted from the oxidized food molecule. This process, termed oxidative phosphorylation (Figure 2-86), also occurs in the plasma membrane of bacteria. As one of the most remarkable achievements of cell evolution, it is a central topic of Chapter 14. In total, the complete oxidation of a molecule of glucose to H2O and CO2is used by the cell to produce about 30 molecules of ATP In contrast, only 2 molecules of ATP are produced per molecule of glucose by glycolysis alone.

AminoAcidsand Nucleotides ArePartof the NitrogenCycle So far we have concentrated mainly on carbohydrate metabolism and have not yet considered the metabolism of nitrogen or sulfur. These two elements are important constituents of biological macromolecules. Nitrogen and sulfur atoms pass from compound to compound and between organisms and their environment in a seriesof reversible cycles. Although molecular nitrogen is abundant in the Earth's atmosphere, nitrogen is chemically unreactive as a gas.Only a few living speciesare able to incorporate it into organic molecules, a process called nitrogen fixation. Nitrogen fixation occurs in certain microorganisms and by some geophysical processes, such as lightning discharge.It is essentialto the biosphere as a whole, for without it life could not exist on this planet. Only a small fraction of the nitrogenous compounds in today's organisms, however, is due to fresh products of nitrogen fixation from the atmosphere. Most organic nitrogen has been in circulation for

pyruvatefrom gl y c o l y s i s I

Coz I

N A D Hf r o m glycolysis I

Oz I

pyruvate

:*Hl * P

II

t

acetyl CoA

ctTRtc ACID CYCLE

CoA

2eI

u^rDAilvE

-PHOSPHORYLATION

t

MITOCHONDRION

Hzo

Figure2-85 The generationof an H+gradientacrossa membraneby electron-transportreactions. A high-energy electron(derived, for example,from the oxidationof a metabolite) is passedsequentially by carriers A, B,and C to a lowerenergy state.In this diagramcarrierB is arranged in the membranein sucha way that it takesup H+from one sideand releases it to the otherasthe electronpasses. The resultis an H+gradient.As discussed in Chapter14,this gradientis an important form of energythat is harnessed by other membraneoroteinsto drivethe formationof ATP.

Figure2-86 Thefinal stagesof oxidation of food molecules.Molecules of NADH (FADHz and FADH2 is not shown)are producedby the citricacidcycle.These activatedcarriers donatehigh-energy electrons that areeventuallyusedto reduceoxygengasto water. A majorportionof the energyreleased duringthe transferof theseelectrons alongan electron-transfer chainin the mitochondrial innermembrane(or in the plasmamembraneof bacteria)is harnessed to drivethe synthesis of ATPhencethe nameoxidative (discussed phosphorylation in Chapter14).

101

HOW CELLSOBTAINENERGY FROMFOOD

some time, passing from one living organism to another. Thus present-day nitrogen-fixing reactions can be said to perform a "topping-up" function for the total nitrogen supply. Vertebrates receive virtually all of their nitrogen from their dietary intake of proteins and nucleic acids. In the body these macromolecules are broken down to amino acids and the components of nucleotides, and the nitrogen they contain is used to produce new proteins and nucleic acids-or utilized to make other molecules. About half of the 20 amino acids found in proteins are essential amino acids for vertebrates (Figure 2-87), which means that they cannot be synthesizedfrom other ingredients of the diet. The others can be so synthesized, using a variety of raw materials, including intermediates of the citric acid cycle as described previously.The essentialamino acids are made by plants and other organisms, usually by long and energeticallyexpensivepathways that have been lost in the course of vertebrate evolution. Roshanl(eab 02l-66950639 The nucleotides needed to make RNA and DNA can be synthesized using specializedbiosynthetic pathways. All of the nitrogens in the purine and pyrimidine bases (as well as some of the carbons) are derived from the plentiful amino acids glutamine, aspartic acid, and glycine, whereas the ribose and deoxyribose sugars are derived from glucose. There are no "essential nucleotides" that must be provided in the diet. Amino acids not used in biosynthesis can be oxidized to generatemetabolic energy.Most of their carbon and hydrogen atoms eventually form COz or HzO, whereas their nitrogen atoms are shuttled through various forms and eventually appear as urea, which is excreted.Each amino acid is processeddifferently, and a whole constellation of enzymatic reactions exists for their catabolism. Sulfur is abundant on Earth in its most oxidized form, sulfate (SOaz-).To convert it to forms useful for life, sulfate must be reduced to sulfide (S2-),the oxidation state of sulfur required for the synthesis of essential biological molecules. These molecules include the amino acids methionine and cysteine,coenzymeA (seeFigure 2-62), and the iron-sulfur centers essentialfor electron transport (see Figure 14-23). The process begins in bacteria, fungi, and plants, where a special group of enzymes use ATP and reducing power to create a sulfate assimilation pathway. Humans and other animals cannot reduce sulfate and must therefore acquire the sulfur they need for their metabolism in the food that they eat.

Metabolismls Organized and Regulated One gets a sense of the intricacy of a cell as a chemical machine from the relation of glycolysis and the citric acid cycle to the other metabolic pathways sketched out in Figure 2-88. This type of chart, which was used earlier in this chapter to introduce metabolism, represents only some of the enzymatic pathways in a cell. It is obvious that our discussion of cell metabolism has dealt with only a tiny fraction of cellular chemistry. All these reactions occur in a cell that is less than 0.1 mm in diameter, and each requires a different enzyme. As is clear from Figure 2-88, the same molecule can often be part of many different pathways. Pyruvate,for example, is a substrate for half a dozen or more different enzymes,each of which modifies it chemically in a different way. One enzyme converts pyruvate to acetyl CoA, another to oxaloacetate;a third enzyrne changespyruvate to the amino acid alanine, a fourth to lactate, and so on. All of these different pathways compete for the same pyruvate molecule, and similar competitions for thousands of other small molecules go on at the same time. The situation is further complicated in a multicellular organism. Different cell tlpes will in general require somewhat different sets of enzymes. And different tissues make distinct contributions to the chemistry of the organism as a whole. In addition to differences in specialized products such as hormones or antibodies, there are significant differences in the "common" metabolic pathways among various types of cells in the same organism. Although virtually all cells contain the enzymes of glycolysis, the citric acid cycle, lipid synthesis and breakdown, and amino acid metabolism, the levels of

THEESSENTIAL AMINOACIDS

Figure?-87The nine essentialamino by acids.Thesecannotbe synthesized humancellsand so mustbe suppliedin the diet.

102

Chapter2:CellChemistryand Biosynthesis Figure2-88 Glycolysisand the citric acid cycleare at the center of metabolism.Some500 metabolic reactions of a typicalcellareshown with the reactions schematically of glycolysis and the citricacidcyclein red. Otherreactions eitherleadinto these two centralpathways-delivering small molecules to be catabolized with productionof energy-or they lead outwardand therebysupplycarbon compoundsfor the purposeof biosynthesis.

these processes required in different tissues are not the same. For example, nerve cells, which are probably the most fastidious cells in the body, maintain almost no reservesof glycogen or fatty acids and rely almost entirely on a constant supply of glucose from the bloodstream. In contrast, liver cells supply glucose to actively contracting muscle cells and recycle the lactic acid produced by muscle cells back into glucose.All types of cells have their distinctive metabolic traits, and they cooperate extensivelyin the normal state, as well as in response to stressand starvation. One might think that the whole system would need to be so finely balanced that any minor upset, such as a temporary change in dietary intake, would be disastrous. In fact, the metabolic balance of a cell is amazingly stable.\.A/henever the balance is perturbed, the cell reacts so as to restore the initial state. The cell can adapt and continue to function during starvation or disease.Mutations of many kinds can damage or even eliminate particular reaction pathways, and yet-provided that certain minimum requirements are met-the cell survives.It does so because an elaborate network of control mechanismsregulates and coordinates the rates of all of its reactions.These controls rest, ultimately, on the remarkable abilities of proteins to change their shape and their chemistry in response to changesin their immediate environment. The principles that underlie how large molecules such as proteins are built and the chemistry behind their regulation will be our next concern.

103

END-OF-CHAPTER PROBLEMS

Su m m a r y Glucoseand otherfood moleculesare broken down by controlled stepwiseoxidation to prouide chemical energy in the form of ATP and NADH. Thereare three main setsof reactions that act in series-the products of each being the starting material for the next:glycolysis(which occursin the cytosol),the citric acid cycle(in the mitochondrial matrix), and oxidatiue phosphorylation (on the inner mitochondrial membrane).The intermediate products of glycolysk and the citric acid cycleare usedboth as sourcesof metabolic energyand to produce many of the small moleculesusedas the raw materials for biosynthesis.Cellsstore sugar moleculesas glycogenin animals and starch in plants; both plants and animals also usefats extensiuelyas a food store.Thesestorage materials in turn serueas a major sourceof food for humans, along with the proteins that comprisethe majority of the dry massof most of the cellsin thefoods we eet.

PROBLEMS

TableQ2-1 Radioactiveisotopesand someof their properties(Problem2-12).

Whichstatementsare true?Explainwhy or why not. 2-1 Of the original radioactivityin a sample,only about 1/ 1000will remain after 10 half-lives. 2-2

A 10-BM solution of HCI has a pH of B.

2-3 Most of the interactions between macromolecules could be mediatedjust aswell by covalentbonds as by noncovalentbonds.

14c 36 35s 32P

B particle B particle B particle B particle

5730years 12.3years 87.4days 14.3days

0.062 29 1490 9120

2-4 Animals and plants use oxidation to extract energy from food molecules. 2-5 If an oxidation occurs in a reaction, it must be accompaniedby a reduction. 2-6 Linking the energetically unfavorable reaction A -+ B to a second,favorablereaction B -+ C will shift the equilibrium constantfor the first reaction. 2-7 The criterion for whether a reaction proceedsspontaneouslyis AG not AGo,becauseAG takesinto accountthe concentrationsof the substratesand products. 2-8 Becauseglycolysis is only a prelude to the oxidation of glucosein mitochondria, which yields l5-fold more AIB glycolysisis not really important for human cells. 2-9 The oxygen consumed during the oxidation of glucosein animal cellsis returned as COzto the atmosphere. Discussthe following problems. 2- 10 The organicchemistryof living cellsis said to be special for two teasons:it occurs in an aqueous environment and it accomplishessome very complex reactions.But do you suppose it is really all that much different from the organic chemistry carried out in the top laboratoriesin the world? \A/tryor why not? 2-11 The molecular weight of ethanol (CHgCHzOH)is 46 and its density is 0.789g/cm3. A. \A4ratis the molarity of ethanol in beer that is 5% ethanol by volume? [Alcohol content of beer varies from about 4Vo(lite beer) to B% (stout beer).1 B. The legal limit for a driver's blood alcohol content varies,but 80 mg of ethanol per 100 mL of blood (usually

referredto as a blood alcohollevel of 0.08)is t)?ical. \ /hat is the molarity of ethanol in a person at this legal limit? t. How many l2-oz (355-mL)bottles of 5% beer could a 70-kgpersondrink and remain under the legallimit? A 70-kg person contains about 40 liters of water. Ignore the metabolism of ethanol, and assumethat the water content of the person remains constant. D. Ethanol is metabolizedat a constant rate of about 120 mg per hour per kg body weight, regardlessof its concentration. If a 70-kg person were at twice the legal limit (160 mg/f 00 mL), how long would it take for their blood alcohol level to fall below the legal limit? 2-12 Specificactivity refers to the amount of radioactivity per unit amount of substance,usually in biology expressedon a molar basis,for example,as Ci/mmol. [One curie (Ci) corresponds to 2.22 x 1012disintegrations per minute (dpm;.1 As apparent in Table Q2-1, which lists properties of four isotopes commonly used in biology, there is an inverserelationship between maximum specific activity and half-life. Do you suppose this is just a coincidence or is there an underlying reason? Explain your answer. 2-13 By a convenientcoincidencethe ion product ofwater, K- = lH+l[OH-],is a nice round number: 1.0x 10-14M2. A. \AIhyis a solution at pH 7.0 said to be neutral? B. \A/tratis the H+ concentrationand pH of a I mM solution of NaOH? C. If the pH of a solution is 5.0,what is the concentration of OH- ions? 2-14 Suggesta rank order for the pKvalues (from lowestto highest)for the carboxylgroup on the aspartateside chain

104

Chapter2:CellChemistryand Biosynthesis

in the following environments in a protein. Explain your ranking. 1. An aspartateside chain on the surfaceof a protein with no other ionizable groupsnearby. 2. An aspartateside chain buried in a hydrophobic pocket on the surlaceof a protein. 3. An aspartateside chain in a hydrophobic pocket adjacent to a glutamateside chain. 4. An aspartateside chain in a hydrophobic pocket adjacent to a lysine side chain. 2-15 A histidine side chain is knol,rrnto play an important role in the cataly.ticmechanismof an enz).ryne; however,it is not clear whether histidine is required in its protonated (charged)or unprotonated (uncharged)state.To answerthis question you measureenzyrneactivity over a range of pH, with the resultssho\^Trin Figure Q2-1. \Ahich form of histidine is required for enz)ryneactivity? FigureQ2-1 Enzyme activityasa functionof pH(Problem 2-15).

c f

E o

E

o ,r_ ,_a, , ! C- O

FigureQ2-2 Threemolecules that illustrate the sevenmostcommonfunctionalgroupsin biology(Problem2-17).1,3-Bisphosphoglycerate and pyruvateareintermediates in glycolysis and cysteineis an aminoacid.

HO-CH

1 , 3 - b i s p h o s p h o g l y c e r a t e pyruvare

SH

cyslerne

Calculatethe instantaneousvelocity of a water molecule (molecularmass= 1Bdaltons),a glucosemolecule (molecular mass = lB0 daltons),and a myoglobin molecule (molecular mass = 15,000daltons) at 37"C. Just for fun, convert thesenumbers into kilometers/hour.Beforeyou do any calculations,try to guesswhether the moleculesare moving at a slow crawl ( 1), reactionswith a large increasein 5 (that is, for which A5 > 0) are favoredand will occurspontaneously. in Chapter2, heat energycausesthe random As discussed the transferof heat from an commotionof molecules.Because the number of enclosedsystemto its surroundingsincreases different arrangementsthat the moleculesin the outsideworld their entropy.lt can be shown that the can have,it increases releaseof a fixed quantity of heat energyhasa greaterdisordering effect at low temperaturethan at high temperature,and that the value of A5 for the surroundings.as defined above (ASr"u), is preciselyequalto h, the amount of heattransferredto the surroundingsfrom the system,dividedby the absolute temperature(f ):

T H EG I B B S FREE E N E R G YG, When dealingwith an enclosedbiologicalsystem,one would like to have a simpleway of predictingwhether a given reaction will or will not occurspontaneously in the system.We have seenthat the crucialquestionis whether the entropy changefor the universeis positiveor negativewhen that reactionoccurs. In our idealizedsystem,the cell in a box,there are two separate componentsto the entropy changeof the universe-the entropy changefor the systemenclosedin the box and the entropy changefor the surrounding"sea"-and both must be added together before any predictioncan be made.For example,it is possiblefor a reactionto absorbheat and therebydecreasethe entropy of the sea (A5r""< 0) and at the sametime to cause sucha large degreeof disorderinginsidethe box (A56o* > 0) = A5r"" + A56o,is greater than 0. In this that the total A5rn;u"rr" casethe reactionwill occurspontaneously, eventhough the seagivesup heat to the box during the reaction.An exampleof sucha reactionisthe dissolvingof sodiumchloridein a beaker containingwater (the "box"), which is a spontaneousprocess eventhough the temperatureof the water dropsasthe salt goesinto solution. Chemistshavefound it usefulto define a number of new "compositefunctions"that describecombinationsof physical propertiesof a system.The propertiesthat can be combined includethe temperature(f), pressure(P), volume (V), energy (E), and entropy (5). The enthalpy(H) is one suchcomposite function.But by far the most usefulcompositefunction for biologistsis the Gibbs free energy, G. lt servesas an accounting devicethat allowsone to deducethe entropy changeof the universeresultingfrom a chemicalreactionin the box, while avoidingany separateconsiderationof the entropychangein the sea.The definition of G is G=H-TS where, for a box of volume V, H is the enthalpydescribedabove (E + PV), r is the absolutetemperature,and 5 is the entropy. Eachof thesequantitiesappliesto the insideof the box only. The changein free energyduring a reactionin the box (the G of the productsminusthe G of the startingmaterials)is denoted asAG and, as we shallnow demonstrate,it is a direct measureof the amount of disorderthat is createdin the universewhen the reaction occurs.

At constant temperature the change in free energy (AG) during a reactionequalsAH - IA5. Rememberingthat AH = -h, the heat absorbedfrom the sea,we have

But h/f is equal to the entropy change of the sea (A5r""),and the A5 in the above equation is A56o^.Therefore

We concludethat the free-energychangeis a direct measure of the entropy changeof the universe.A reactionwill proceed in the directionthat causesthe changein the free energy(AG) to be lessthan zero, becausein this casethere will be a positive entropy changein the universewhen the reactionoccurs. For a complexset of coupledreactionsinvolvingmany the total free-energychangecan be comdifferent molecules, puted simplyby adding up the free energiesof all the different molecularspeciesafter the reactionand comparingthis value with the sum of free energiesbefore the reaction;for common the requiredfree-energyvaluescan be found from substances publishedtables.In this way one can predictthe directionof a reactionand thereby readilycheckthe feasibilityof any proposedmechanism. Thus,for example,from the observed proton gradient valuesfor the magnitudeof the electrochemical acrossthe inner mitochondrialmembraneand the AG for ATP hydrolysisinsidethe mitochondrion,one can be certainthat ATP synthaserequiresthe passageof more than one proton for each moleculeof ATPthat it synthesizes. The value of AG for a reactionis a direct measureof how far the reactionis from equilibrium.The large negativevaluefor ATP hydrolysisin a cell merelyreflectsthe fact that cellskeep the ATP hydrolysisreactionas much as 10 ordersof magnitude away from equilibrium.lf a reactionreachesequilibrium, AG = 0, the reactionthen proceedsat preciselyequal rates in the forward and backward direction. For ATP hydrolysis, equilibriumis reachedwhen the vast majorityof the ATP has been hydrolyzed,as occursin a dead cell'

F o r e a c hs t e p ,t h e p a r t o f t h e m o l e c u l et h a t u n d e r g o e sa c h a n g ei s s h a d o w e di n b l u e , a n d t h e n a m e o J t h e e n z y m et h a t c a t a l y z etsh e r e a c t i o ni s i n a y e l l o w b o x .

S T E P1 G l u c o s ei s phosphorylatedby ATPto f o r m a s u g a rp h o s p h a t e . T h e n e g a t i v ec h a r g eo f t h e phosphatepreventspassage of the sugar p h o s p h a t et h r o u g h t h e p r a s m am e m D r a n e , t r a p p i n gg l u c o s ei n s i d e the cell.

o. .H \,/

't-

!Ir

1CH?OH

H-C.-OH

l'

f r o m c a r b o n1 t o H O c a r o o nz , t o r m t n g a ketosefrom an a l d o s es u g a r ( S e e Panel2-4.)

*'-i? +

H.)-(--H

lH-

OH

H-C-OH 5 | -CH,OqP(openchainform)

H-C-OH

l5 .CH,OP ( o p e nc h a i nf o r m )

STEP3 The new hydroxyl .l g r o u p o n c a r b o n1 ' , phosphorylatedby ATP,in p r e p a r a t i o nf o r t h e f o r m a t i o n o f t w o t h r e e - c a r b o ns u g a r phosphates.The entry of sugars i n t o g l y c o l y s iiss c o n t r o l l e da t t h i s s t e p ,t h r o u g h r e g u l a t i o no f t h e enzyme p hosphof ru ctok i nase-

P O H-) |C, " - \o (

rLJ/lHr))

(ringform)

CH,O P

|

+

tt''

OH

f ructose 1,6-bisphosphate

CH,O P

STEP 4 The s i x - c a r b o sn u g a ri s c l e a v e dt o p r o d u c e two three-carbon m o l e c u l e sO n l y t h e glyceraldehyde 3-phosphate can p r o c e e di m m e d i a t e l y through glycolysis

I

a-A

I

HO

HO-C-H

I

+C

H-C-OH

I I

cH2o P

I

H-C-OH

I

cH2o

I

( o p e nc h a i nf o r m ) f r u c t o s e1 , 5 - b i s p h o s p h a t e

CH,OH

\//

I

H-C-OH

cH2o P

S T E P5 Theother product of step 4, i hyd d roxyacetone p h o s p h a t ei,s isomerized to form glyceraldehyde 3-phosphate.

ADP

P

glyceraldehyde ?-nhncnh:te

C

,ro

" I I

H-C-OH

cH2o P

g l y c e r a l d e h y d3e- p h o s p h a t e

t

ovo

+EEEE+u*

f

H-C-OH

l

cH2o P F i g u r e2 - 7 3 )

1,3-bisphosphoglycerate

g l y c e r a l d e h y d3e- p h o s p h a t e

S T E P7 T h et r a n s f e r t o A D Po f t h e h i g h - e n e r g yp h o s p h a t e groupthat was generated in step 6 forms ATP.

C

I I

+

H-C-OH

cH2o P 1,3-bisphosphoglycerate

o. .o \//

o. .o\// 'Cl

STEP 8 Theremaining p h o s p h a t ee s t e rl i n k a g ei n 3-phosphoglycerate, which has a relativelylow free energy of hydrolysis,is moved from carbon 3 to carbon 2 to form 2-phosphoglycerate.

C I

H-C-Oi*P

H-C-OH

,l

3-phosphoglycerate

C

C

I H-C-O

P

I

cH2oH 2 - p h o s p h olgy c e r a t e

P

cHz p h o s ph o e n oIp y r u v a t e

C

C

C-O

I

C-O

o. .o\./

o. .o\//

I

2-p hosphog lycerate

o. .o \./

o. .o \,/

STEP9 The removal of water from 2-phosphoglycerate c r e a t e sa h i g h - e n e r g ye n o l p h o s p h a t el i n k a g e .

STEP10 The transfer to ADP of the high-energy p h o s p h a t eg r o u p t h a t w a s generated in step 9 forms A T P ,c o m p l e t i n gg l y c o l y s i s .

t-

cH2oH

- C H r O ' .P .

P

cHz p h o s p h o e n oply r u v a t e

N E TR E S U LO T FG L Y C O L Y S I S

In addition to the pyruvate,the net productsare t w o m o l e c u l e so f A T Pa n d t w o m o l e c u l e so f N A D H

I I

CH:

HS-CoA

pyruvate

The completecitricacidcycle.The two carbonsfrom acetylCoA that enter this turn of the cycle(shadowedin ) will be convertedto CO, in subsequentturns of the cycle:it is the two carbons shadowed in blue that are convertedto CO, in this cycle.

(2c)

acetyl CoA

HS-CoA

eoo *", tHO-C-COO-

Step 1

(+a^)

in, \*'ioo\

Po

(6c) isocitrate fn, HC COO

t

citrate(6c)

no-tH Coo-

fumarate (4C)

(x-ketoglutarate(5C)

ffoo-

€oo-

2

t

fu

'

t' CH

s u c c i n a t e( 4 C ) ,rStep6

GOO-

Hzo

Coz

CH.

succinylCoA (4C)

t'

Ioo-

I

coo-

2

CH,

il*p_:,

t-

C=O I S-CoA

HS-CoA

EE*tcoz

HS-CoA

Detailsof the eight stepsare shown below. For eachstep,the part of the moleculethat undergoesa changeis shadowedin hlue and the name of the enzymethat catalyzesthe reactionis in a yellow box.

O:C -S-CoA

STEP1 After the enzyme removesa proton from the CH, group on acetyl CoA, t h e n e g a t i v e l yc h a r g e d C H r - f o r m sa b o n d t o a carbonylcarbon of oxaloacetate.The s u b s e q u e nlto s sb y h y d r o l y s ios f t h e c o e n z y m e A (CoA)drivesthe reaction strongly forward.

S T E P2 An isomerization reaction,in which water is f i r s t r e m o v e da n d t h e n added back, movesthe hydroxyl group from one c a r b o na t o m t o i t s n e i g h b o r

I

CH,

cooI CHr

t-

HO-C-COO

I 9H, l

coo-

t-

HO-C-COO

+ HS-CoA + H*

I

CH,

t-

coocitrate

coo-

Hzo H-

citrate

cooI C-H I c-cootl C-H I coo-

cls-aconitateintermediate

H-C

cooI-H

I

H-C -COO-

I

HO-C -H I

coo

isocitrate

STEP3 ln the first of f o u r o x i d a t i o ns t e p si n t h e cycle,the carbon carrying the hydroxyl group is convertedto a carbonyl g r o u p .T h e i m m e d i a t e p r o d u c ti s u n s t a b l e l,o s i n g C O ,w h i l e s t i l l b o u n d t o the enzyme.

cooI H-C -H I H-C -H I a-i I coo

cooI-H

H-C

H_C

I

HO-c-H

I coo-

(x-ketogIutarate

isocitrate

STEP4 The o-ketog/utarate dehyd ro gen asecomplex closely r e s e m b l etsh e l a r g ee n z y m e complexthat convertspyruvate to acetyl co{(pyruvate dehydrogenase).lt likewise catalyzesan oxidation that producesNADH,CO2,and a h i g h - e n e r g yt h i o e s t e rb o n d t o coenzymeA (CoA).

STEP5 A phosphate m o l e c u l ef r o m s o l u t i o n d i s p l a c etsh e C o A ,f o r m i n g a h i g h - e n e r g yp h o s p h a t e l i n k a g et o s u c c i n a t eT. h i s p h o s p h a t ei s t h e n p a s s e dt o G D Pt o f o r m G T P .( l n b a c t e r i a and plants,ATP is formed instead.)

cooI H-C-H I-H

cooI H-C -H I H-C-H I c:o I coo-

H-C

S

succinyl-CoA

(x-ketoglutarate

cooH-C

-H

H-C

I S-CoA s u c c ln a r e

H-C

coo I-H I

H-C-H

s u c cni a t e

cooI C-H H-C

I

coo f u marate

S T E P8 l n t h e l a s to f J o u r o x i d a t i o ns t e p si n t h e c y c l et,h e c a r b o nc a r r y i n gt h e h y d r o x y l group is converted t o a c a r b o n y lg r o u p , regeneratingthe oxaloacetate n e e d e df o r s t e p 1 .

,,

I coo-

I coo

S T E P7 T h e a d d i t i o no f water to fumarate placesa hydroxyl group next to a c a r b o n y lc a r b o n .

coo I-H

I ,, ) n-L-r

I H-C-H I

succinyl-CoA

S T E P6 ln the third oxidation step in the cycle,FAD removestwo hydrogen atoms from succinate.

cooI HO-C -H I H-C-H I coomalate

I I-CoA

succinatedehydrogenase

-

cooI

C-H

/\

rl

H-C

I coof umarate

cooI HO-C -H I H-C-H I coo malate

cooI c:o I CH,

I coo oxa loacetate

+ HS-CoA

124

Chapter2: CellChemistryand Biosynthesis

REFERENCES General Berg,JM,Tymoczko, JL& StryerL (2006)Biochemistry, 6th ed New York:WH Freeman GarrettRH& Grisham CM (2005)Biochemistry, 3rded philadelphia: ThomsonBrooks/Cole Hortonl-1R, MoranLA,Scrimgeour et a (2005)Princip esof Bioch-.mistry 4th ed UpperSaddleRiver, NJ:prenticeHall NelsonDL& CoxMM (2004)Lehnlnger Principles of Biochemistry, 4th ed NewYork:Worth NichollsDG& Ferguson S_l(2002)Bioenergerics,3rd ed Newyork: AcademicPress MathewsCK,van Ho de KE& AhernK G (2000)Biochemistry, 3rded 5 a ql r a r c , s c oB: e n j a rr C u m m i n g s MooreJA(1993)Sclence Asa Wayof KnowingCambridge, MA: Harvard University Press VoetD,Voet.lG& PrattCIV(2004)Fundamentals of Biochemistry, 2nd ed NewYork:Wiley The ChemicalComponentsof a Cell AbelesRH,FreyPA& Jencks WP(1992)Biochemistry Boston: Jones& Bartlett AtkinsPW('l996)Mo ecues NewYork:WH Freeman Branden C & ToozeJ (l 999) ntroduction to ProteinStructure, 2nd ed NewYork:Garland Scence Bretscher MS(,l985) Themolecules of the cel membrane5clAm 2 5 3 :01O I O 9 Burey 5K& Petsko GA(t 9BB)Weaklypolarinteractions in proteins,4dy PrateinChem39.125-1 89 De DuveC (2005)Singulanties: Landmarks on the pathways of Lif-. Cambridge: Cambridge University Press DowhanW (1997) Molecular phospholipid basisfor membrane diversity: Whyarethereso many ipids?AnnuRevBiochem66:j99-232 EsenbergD & Kauzman W (l 969)TheStructure and properties of WaterOxford:OxfordUnivers ty Press FershtAR(198/)Ihe hydrogenbond in molecular recognitionIrendj BiochemSci123A1-304 Franks F ('l993)WaterCambridge: RoyalSociety of Chemistry Henderson ll (1927) TheFitness of the Envronment,1958ed Boston: Beacon Neidhardt FC,Ingraham _lL& Schaechter M (t 990)physioiogy of the Bacterial Cel: A Mo ecularApproachSunderland, MA:Sinauer PaulingL (1960)Ihe Natureof the Chemical Bond,3rded thaca,Ny: CornellUniversity Press Saenger W (l 984)Princrples of NucleicAcidStructure, New yorx: S p rni g e r SharonN (1980)Carbohydrates 5ci,4,m 243.90116 Stillinger FH(1980) WarerrevisitedScience 2a9.45j-457 TanfordC ('1978) Thehydrophobtc effectandthe organization of living m a t t e rS c l e n c2e0 0 : , l 0 1l2O l 8 TanfordC (1980) ThelydrophobicEffectFormation of Micelesand BioogicalMembranes, 2nd ed Newyork.JohnWi ey Catalysisand the Use of Energy by Cells AtkinsPW(1994) Ihe SecondLaw:Energy, Chaosand Form Newyork: Scientif c American Books AtkinsPW& De PaulaiD (2006)Physical Chemistry for the Life press Sciences Oxford:OxfordUniversity BaldwinJE& KrebsH (1981)The Evolurion of Metabolic CyclesNciure 291:381-382 BergHC(1983)RandomWalksin B ology Princeton, NJ:princeton University Press Dickerson RE(,1969) MolecularThermodynamics Menlopark,CA: B e n j a m iCn u m m i n g s DillKA& Bromberg S (2003) Molecular DrivingForces: StatisticalThermodynamics in Chemistry and Bioogy Newyork:Garland Science Dressler D & PotterH (1991)DiscovelngEnzymes Newyork:Sclentific American L brary

Einstein A (1956)lnvestigations on the Theoryof Brownian Movement NewYork:Dover FrutonJS(1999)Proteins, Enzymes, Genes: The nterplayof Chemistry and Bioogy NewHaven:Yale University Press, GoodseI DS(1991)nsidea livingcell Trends BiochemSci16:203-206 Karplus M & McCammon JA (1986) Thedynamics of protens SclAm 254:42-51 ) o l e c u l adry n a m i cssi m u l a t i o ni ns K a r p l uM s & P e t s kG o A( 1 9 9 0M biology Nature347:631639 Kauzmann W (1967) Thermodynamics andStatistics: withApplications to GasesIn ThermalProperties of MatterVol2 NewYork:WA Benjamin, Inc Kornberg A (1989)Forthe Loveof Enzymes Cambridge, MA:Harvard University Press Lavenda BH(,1985) Brownian Motion5ci,4m252:7085 LawlorDW (2001)Photosynthesis, 3rded Oxford:BIOS L e h n i n g eArL ( 1 9 1 1T) h eM o l e c u l aBra s iosf B i o l o g i cE an l ergy Transformations, 2nd ed MenloPark, CA:Benjamin Cummings LipmannF (1941)Metabolic generation and uti izationof phosphate bondenergyAdvEnzymol 1:99-162 LipmannF (1971) Wanderings of a Biochemist NewYork:Wiley NisbetEE& SleepNH (2001)The habitatand narureof earlylife Nature 409:1081 3091 Racker E (l9BO)FromPasteur to Mitchell: a hundredyearsof n l n t r n t r r ^ o l r r c L o / 1p t ^ . t , ) : 2 l O - 2 I 5

Schrodinger E (1944& 1958)Whatis Life?: ThePhysicai Aspectof the L i v i n gC eI a n dM i n da n dM a t t e r1, 9 9 2c o m b i n e d ed Cambridge: Cambridge University Press van HoldeKE,JohnsonWC& Ho PS(2005)Principles of Physical Biochemistry, 2nd ed UpperSaddleRiver, NJ:Prentice Hal WalshC (2001)Enabling the chemistry of life Nature409.226-23i Westheimer FH(1982) Why naturechosephosphates Science 235.11/3-1t78 YouvanDC& MarrsBL(1987)Molecular mechanisms of photosynthesis SciAm 256:4249 How CellsObtain Energyfrom Food CramerWA & KnaffDB(1990)Energy Transduction in Bioogical Membranes, NewYork:Springer-Verlag, Dismukes GC,KlimovW, Baranov SVet al (2001) Theoriginof atmospheric oxygenon Earth: Theinnovation of oxygenic photosyntheis PracNatAcadSciUSA9821702175 Fel D (l 997)Understanding the Controlof MetabolismLondon: Portand Press F att JP(1995)Useand storageof carbohydrate and fat Am J ClinNutr 61,95259595. FriedmannHC(2004)FromButybacterium to E.coli:An essayon unity in biochemistryPerspect Biollrled47:47-66 Fothergill-Gilmore LA (,1986) Theevolutionof the glycolytic pathway Trends BiochemSci11:475l Heinrich R,Melendez-Hevia E,MonteroF et al (,1999) Thestructural designof glycolysis: An evo utionaryapproachBiochem SocTrans 27:294-298 HuynenMA,Dandekar T & BorkP (1999)Variarion and evolutionof the citricacidcycle:a genomicperspective, Trends Microbrol l:281-291 Kornberg HL(2000)Krebsand histrinityof cyclesNatureRevMolCell Biol1.225-228 KrebsHA& MartinA (1981)Reminiscences and Reflections Oxford/New York:Clarendon Press/Oxford University Press, KrebsHA (l 970)The historyof the tricarboxylic acidcycle perspect Biol Med14.154-17a MartlnBR(1987)Metabolic Regulation: A Molecular ApproachOxford: Blackwell Scientific McGilvery RW(,1983) Biochemistry: A Functional Approach, 3rded P h i l a d e l p hSi aa:u n d e r s MorowitzHJ(1993)Beginnings of Cellular Life:Metabolism Recapitulates Biogenesis NewHaven: YaleUniversity Press Newsholme EA& StarkC (1973)Regulation of Metabolism NewYork.Wiley,

gzl

'suorrerlaJqqe Jraq] srsr z-g a.rntlc pue seJnronJlsc..uore rreqt s^aoqs(6ZI-8ZI'dd) f-g larrud 'uo os pue 'spuoq tualeloc urrog ,{lrpear aruos 'pa8reqc dlanrlrsodro dyanrle8aueJesJeqlo '(,,3upeay-ra1e,,rn,,) crqoqdorpl.q pue reloduou eJe sureqo aprs asaq] Jo auros '(I-t arnt1fl suleqc epls prJe ourue lueJeJJrp0Z aql :saruadord anbrun slr prJEourrue qrea arr€ leqt pue puoq appdad u Suqeru ur pallolur lou aJp leql sprJp ourup aql Jo suoruod asoq] erp urcqr anppadar sq] ol peqJeDV'auoqlJuq eppdad,tod aql sB 01 parreJer sr ureqJ apltdaddlod eq] Jo eroo aqt Suop sruole;o acuanbas Surleadar aql 'acuanbas prJE ourrue relnrrged uir,ro sll qlp/r qf,Ea 'suralord luaragrp Jo spuesnoql dueur eJe ereq] pue 'spr3e ourrue yo acuanbas anbrun e seq uraloJd yo addr qteg'saptldadt1od se u,\i\oDl osp aJoJereqleJe sureloJd 'puoq apudad luapnoc e q8norq] roqq8rau slr ol pa{url qJpe 'sproeourrup asaqlJo uuqc Suoye tuo4 eperu sr elnJalou ugalord V'serua -dord pcruraqc lualaJJrpqllm qJEe 'suralord ur sprJeounuu;o saddl 0Z eJeaJaqJ

e)uenbesp!)V oulr,uvsll ^q palJ!)adssl ulelorde Jo adeqsaql 'lleJ e ur uorlJury slr seurru -Jelap alnJelour uralord qJee adeqs asrcardaql rtoq aqrJosepol Jo la^al Jnuole eqt te eJn]f,nr1suralord;o Surpuelsrapun srql esn alvrtaldeqc aqt ur Jale'I 'adeqs puolsuolulp-eerqt sll saurruJalep uralord p sruJoJleq] sprJe ourrue ;o 3uu1s 3uo1aql ur prJ€ ourrue qcea Jo uouelol eql ,lrroqJeprsuooa^ 'uorloes srq] uI 'Eurzeute ,{pr1 ruaas uec suralord;o flqqesran alqe{JeueJ eql 'suedxe ol uala '1atr'&o1srq ,{.reuor1n1ona ;o sreaf Jo suoilrq JeAopeun]-eug pue pado -la^ep uaeq seq uratord qJeaJo frlsruraqc pup eJnlJnJlsaql leq] ezlleat ellr eJuo 'Sursrrdrns 1ou sdeqrad sl sHI'umou) se1ncelotupalecqsrqdosl.lpuorlcurg pue xaldruoc rtlernpnrls lsotu eq] re; dq are suralord '^aet,tJolurod pcnuaqJ E ruoJC

SNI]]OUd IO ]UNDNU]SCNV]dVHS3HI

L)

I

NOtr)NnlNElOUd sNE1OUd lo tunl)nuts

SZL

C N V ] d V H S] H I

raldeq) s$ll ul

'suralord;o Surpuelsrapun daap B urpllp lsnur a^a'uorlJunJ serpoq Jno ,t,roqro 'dolanap so,,fuqrua^r,roq'dlrculcele lJnpuoc selreu ^ oq 'lJeJluoc salcsnur ntoq '{{Jomsaua8 anoq pue}sJepun o1 adoq uec e.nA aJoJag'aJuaJseurrunlJosaJJnosro'sadol 'sJeqgJIlsEIa'salnceloruezee4rlup 'sauoruJoq 'sulxol 'sarpoqque sE 'selnoeloru ]Je suralord pazqurcads Jaqlo VNCI palloul afuelun ue) asolautostodol iutseldofc aqr q8norql salaue8ro sladord 'aldruexa rc! 'u6au!q :sped Sulrour qlIM saurqcptu relncaloru Luq sE elJas sJeqlo la;'snalcnu ilac aq] ol eueJqruau eruseydaqt tuo4 pJE^ut spu -8rs;o ,te1ar pu8rs slas srolerSalur sB lJe Jo taqloue o] IIef, euo uror; sa8es leql -saur .d.rrucsuralord Jaqlo 'lleo aql Jo lno pue olut selnoeloru IIErusJo a8essed eq] IoJtuoJ teqt sdund pue slauueqJ ruJoJauerqruaru eruseld aqt ur peppaqua suralord 'suor]3eal IEJrueqJ r{ueru slr alourord tEq} IIaJ e ur seJeJJnsJEInJeloIu aleJrrlur aq] apnord saudzua 'snql 'suorlJunJ s,llal eql IIE dpeau elnJexa osp ,taql ls{Jolq Surplnq s,lleJ eql ,i.Iuo 1ou are .,(aq; 'ssuru 'fup s,llar e Jo lsou alnlrlsuoc suraloJd 'suralord Suurrasqo 'eJuesse ur 'eJe al'- ',Qnrloe IeJrruaqJ -orq Jo lef,rJlJelas1razdpue ro adocsorcnu e q8norql IIeJ u lp {ool a a uaq \

su!elold

126

Chapter3: Proteins

methionine (Met)

Tf

Ol

l/z

H-:N-C-C

|

H

(il

\i.

()

leucine(Leu)

(

f 'l t,/

o

()tl

(.'1

()

lH

oo

o

H

:N-c-c

\._o

I

\J

I

C.U, o

H

I

+

\^O

H

( |l,

I I -^N - c tl H

I

oC

\

H

o

I

( H,

('H

I

H,('

5

//\

tyrosine (Tyr)

Cl-JJ

(-H,

Hzo

H:O

( ) tl

p o l y p e p t i d eb a c k b o n e

s i d ec h a i n s I I

HHO

a m i n ot e r m i n u s or N-terminus

ov

^tttl

Hei-i-3 rl Hl

C

\

o

(H,

I

oono

(-H,

I

H,(-

5

( l-l I

(H, p o l y p e p t i d eb a c k b o n e

SCHEMATIC

SEQUENCE

Met

Asp

Tyr

As discussedin chapter 2, atoms behave almost as if they were hard spheres with a definite radius (their uan derwaals radius). The requirement that no two atoms overlap limits greatly the possible bond angles in a pollpeptide chain (Figure 3-3). This constraint and other steric interactions severely restrict the possible three-dimensional arrangements of atoms (or conformaflons). Nevertheless, a long flexible chain, such as a protein, can still fold in an enormous number of ways. The folding of a protein chain is, however, further constrained by many different sets of weak noncoualent bonds that form between one part of the chain and another. These involve atoms in the polypeptide backbone, as well as atoms in the amino acid side chains. There are three tlpes of weak bonds: hydrogen bonds, electrostatic attractions, and uan der waals .tttractions, as explained in chapter 2 (see p. 54). Individual noncovalent bonds are 30-300 times weaker than the tlpical covalent bonds that create biological molecules. But manyweak bonds acting in parallel can hold two regions of a polypeptide chain tightly together. In this way, the combined strength of large numbers of such noncovalent bonds determines the stability of each folded shape (Figure 3-4).

Figure3-1 The components of a protein. A protein consistsof a polypeptide backbonewith attachedsioe chains.Eachtype of protein differsin its sequenceand numberof aminoacids; therefore,it is the sequence of the chemically different sidechainsthat makeseach c a r b o x ytl e r m i n u s proteindistinct.Thetwo ends or C-terminus of a polypeptidechainare chemically different:the end carryingthe freeaminogroup (NH3+, alsowrittenNH2)isthe aminoterminus,or N-terminus, and that carrying the free carboxylgroup (COO-,alsowritten COOH)is the carboxylterminusor C-terminus. Theaminoacid sequenceof a proteinis alwayspresentedin the N-to-Cdirection,reading from left to rioht.

127

THESHAPEAND STRUCTURE OF PROTEINS

A M I N OA C I D Asparticacid G l u t a m i ca c i d Arginine Lysine Histidine Asparagine Glutamine Serine Threonine Tyrosine

Asp Glu Arg Lys His Asn Gln Ser Thr Tyr

S I D EC H A I N

A M I N OA C I D D E R K H N a S T Y

Ala Alanine Gly Glycine Val Valine Leu Leucine lle lsoleucine Pro Proline P h e n y l a l a n i n eP h e Met Methionine Trp Tryptophan Cys Cysteine

negative negative positive positive positive polar uncharged polar uncharged unchargep dolar polar uncharged polar uncharged

A G V L I P F M W C

nonpolar nonpolar nonpolar nonpolar nonpolar nonpolar nonpolar nonpolar nonpolar nonpolar

Figure3-2 The 20 amino acidsfound in proteins.Each aminoacidhasa three-letter and a one-letterabbreviation. Thereareequalnumbersof p o l a ra n d n o n p o l asr i d e chains;howevetsomeside chainslistedhereas polarare largeenoughto havesome non-polarproperties(for example,Tyr,Thr,Arg,Lys).For seePanet atomicstructures, 3-1 (pp.128-129).

A fourth weak force also has a central role in determining the shape of a protein. As described in Chapter 2, hydrophobic molecules, including the nonpolar side chains of particular amino acids, tend to be forced together in an aqueous environment in order to minimize their disruptive effect on the hydrogenbonded network of water molecules (see p. 54 and Panel 2-2, pp. f08-109). Therefore, an important factor governing the folding of any protein is the distribution of its polar and nonpolar amino acids.The nonpolar (hydrophobic) side chains in a protein-belonging to such amino acids as phenylalanine, leucine, valine, and tryptophan-tend to cluster in the interior of the molecule (just as hydrophobic oil droplets coalesce in water to form one large droplet). This enables them to avoid contact with the water that surrounds them inside a cell. In contrast, polar groups-such as those belonging to arginine, glutamine, and histidine-tend to arrange themselves near the outside of the molecule, where they can form hydrogen bonds with water and with other polar molecules (Figure 3-5). Polar amino acids buried within the protein are usually hydrogenbonded to other polar amino acids or to the polypeptide backbone.

(A)

(B) +180

a m i n oa c i d

o HC

. l- ' c i

R2

n

I

I

N

,,\,' I

-C

o

R1

p e p t i d eb o n d s

-180

0

+18(

ohl

threebonds Figure3-3 Stericlimitationson the bond anglesin a polypeptidechain.(A)Eachaminoacidcontributes (red)to the backboneofthe chain.Thepeptidebond is planar(grayshading)and doesnot permitrotation.By contrast, rotationcanoccuraboutthe Co-Cbond,whoseangleof rotationis calledpsi (V),and aboutthe N-Cobond,whoseangle an R group is often usedto denotean aminoacidsidechain(greencircles). of rotationis calledphi (Q).BVconvention, (B)Theconformation atomsin a proteinis determinedby one pairof Q and ry anglesfor eachaminoacid; of the main-chain betweenatomswithin eachaminoacid,most pairsof Q and ry anglesdo not occur.In this sobecauseof stericcollisions plot,eachdot represents an observedpairof anglesin a protein.Theclusterof dots in the bottom calledRamachandran (seeFigure3-7A)'(8,from quadrant that arelocatedin cr-helixstructures the amino acids left represents all of from AcademicPress.) 1981.Wlthpermission J. Richardson,Adv.Prot.Chem.34:174-175,

THEAMINO ACID

OPTICALISOMERS

Theo,-carbon atomisasymmetric, which allowsfor two mirrorimage(or stereo-) rsomers, LanoD.

T h e g e n e r a fl o r m u l ao f a n a m i n oa c i di s || ,/ c-carbonatom I t't' amtno ^ ^O^H. . c a r b o' x v l -CI -CO group H:N giouf R

group side-chain

R is commonlyone of 20 different sidechains. At pH 7 both the amino and carboxylgroups areionized.

Proteinsconsistexclusivelv of l-amino acids.

F A M I L I EO SF A M I N OA C I D S

BASIC S I D EC H A I N S histidine (Hiso , r H)

T h e c o m m o na m i n oa c i d s are grouped accordingto w h e t h e rt h e i r s i d ec h a i n s are

H

-N-C

tl

a c i di c basic u n c h a r g e dp o l a r nonpolar

H

HO

I-

CH,

I CH, I

CH, T h e s e2 0 a m i n o a c i d s are given both three-letter and one-letterabbreviations.

HO

ltl C-CI CH: I CH., I 9H, I

CH,

ttl

-N-C-CH

I

HN

./

I

NH,

T h u s :a l a n i n e= A l a = A

/tr

/\

C

t-

C.

//\

NH

NHr

CH,

HC:

CH NHt

Thesenitrogenshavea relativelyweak affinity for an H+and are only partly positive at neutral pH.

P E P T I DBEO N D S A m i n o a c i d sa r e c o m m o n l yj o i n e dt o g e t h e rb y a n a m i d el i n k a g e , c a l l e da p e p t i d eb o n d .

\l//

H

o -f

N-C-C

/l\

R

OH

\l//

R

Peptidebond: The four atoms in eachgray box form a rigid planar unit. There is no rotation around the C-N bond.

o

HOI'i

\llll// -c-\-c \-e /ll\

-C

N-C

/l\

OH

H

o -c

iiHH

;H Proteinsare long polymers o f a m i n oa c i d sl i n k e db y peptide bonds,and they are alwayswritten with the N-terminustoward the left. The sequenceof this tripeptide is histidine-cysteine-valine.

a m t n o -o r N-terminus

\

',f t'tt:

( ll {t ,

T h e s et w o s i n g l eb o n d sa l l o w r o t a t i o n ,s o t h a t l o n g c h a i n so f a m i n oa c i d sa r e v e r yf l e x i b l e .

SIDECHAINS NONPOLAR

A C I D I CS I D EC H A I N S

alanine (Val, or V)

(Ala,or A)

glutamicacid ( G l u ,o r E )

HO

HO

til

lll -N-C-C-

HO

ltl

-N-C-C-

-N-C-C-

(-llr

H

HCH

,/\

CH:

(F],

H

CH:

I

('tl,

I

( )/ \

leucine

(.

(Ile, or I)

(Leu,or L)

()

HO

HO

lll

ttl -N-C-C-

-N-C-C-

(tl ,

H

HCH

I

I

'('flr

(.llj

U N C H A R G EPDO L A RS I D EC H A I N S

CH,

CH.

( ' fI

CH:

proline (Phe,or F)

(Pro,or P)

HO

HO -N-C-C-

_N-C-C-

,/\

H

CH,

H

l-l

\

//\ oNHzc o

)n,

ll

n 9n,

#A

t . lI

( a c t u a lal yn i m i n oa c i d )

| //\

-N-C-C('ll,

( H,

CH? CHr

C

tll

lll -N-C-C-

/

methionine

di*ffiiiffift$ (Trp,or W)

(Met, or M)

HO

HO

til

lil -N-C-C-

Although the amide N is not chargedat neutral pH, it is polar.

ll

H

-N-C-C-

tl

( ll, C'H,

i

s-cll r

glycine

H

- cI I ('t

t,

I

('\ \-' I oH The -OH group is polar.

(Cys,or C)

(Gly,or G)

HO

HO

- N - c -lcl rll

lll -N-C-Cll

H

HH

CH,

I

SH

Disulfidebondscan form betweentwo cysteinesidechains in oroteins. --.u

-q-q-aH

--

130

Chapter3: Proteins g l u t a m i ca c i d

electrostatic attractions R

o

//

h y d r o g e nb o n d

H H

N -

CH, tCH,

van der Waalsattractions

t-

CHt

t-

t

Figure3-4 Threetypes of noncovalent bonds help proteinsfold. Althougha singleone of thesebondsis quiteweak, many of them often form togetherto createa strongbondingarrangement, as in the exampleshown.As in the previous figure,R is usedasa generaldesignation for an aminoacidsidechain.

n':

ProteinsFoldinto a Conformationof LowestEnergy As a result of all of these interactions, most proteins have a particular threedimensional structure, which is determined by the order of the amino acids in its chain. The final folded structure, or conformation, of any polypeptide chain is generally the one that minimizes its free energy. Biologists have studied protein folding in a test tube by using highly purified proteins. Treatment with certain

sequence contains all the information needed for specifying the three-dimensional shape of a protein, which is a critical point for understanding cell function. Each protein normally folds up into a single stable conformation. However, the conformation changes slightly when the protein interacts with other molecules in the cell. This change in shape is often crucial to the function of the protein, as we see later. Although a protein chain can fold into its correct conformation without outside help, in a living cell special proteins called.molecular chaperonesoften assist in protein folding. Molecular chaperones bind to partly folded polypeptide chains and help them progress along the most energetically ravoriute-rolaing

-* r1I;e1e1, eql sre;duexasrql'au{zua ue ro alls a^rl)e oql le p!)e oulruP anll)pa.r{l1ensnunuy gg-g arn6r3

: HJ- t)^.-

sat!S6urpu!8-pue6!l ler)nr)lt{6!lt{6!H sraquaw {1;ure1ulelorduea/n}e8suos!.leduo)e)uanbes drlsnuaqc Jreq] ur ,tltear8 ra;;rp,{eu apcelo{u ulelord aues eq}Jo suolleruJoJuoJluaJeJJIpr(pq311s ua^a o^^l 'uoseer srq] Joc 'JeqlouEeuo 01 eArlEIaJuopeluelJo lsexa Jleql uo osp

'::-o-

H- N.r ^7 N llllllllrlH 1

N\r-N-

NOrlfNnJ NETOUd

s9[

raqlaool suralordo^^tlu!l uauo sa)eJlnspr6u^Jelueualduo) oMf (l) 'lto)-palto)e ulloJol laqtabol purquel sa)tleqn oMl (B).utalold puo)ase uo (,,6uuls,, e) uteLl)eptldadllod Jo dool papuatxaup ot putq ue) utalord auo uo a)eJlnsp!6u v (v)'uMoqsale suraloldoM] aql Jo sued 6utl)elalutaql I1u6 raqto q)ea ol pu!q ue) sulalord o/vqq)!qm ur srtemealql gg-g eln6r1

ol {uI uP3 uraloJd eql teq} os pueSl E punoJrns o1 sdnoJSIef,rrueqf,Jo Jeqrunu a8rel e ,rlrolle,{teqJ'seln3alou raqlo Surdser8roJ papr ere puDI srq} Jo sdool 'arn]3nJls ureloJd JrsBq aqt Suuetle ]noqtrm 'sdoo1asaql Jo aJuenbes prf,e ourrue pue qfual aql {1uo 3u€ueqc ,r(qsals Surpurq-ua8pup Jo .{lrsranrp snoruJoua ue aleraua8 serpoq -ltue luereJJIC '(It-t arnt;g) sureruop uralord pasoderynf dlasolc;o rred e;o spua aql ruo4 apnrtord leqt ureqc apqdaddlodyo sdool pranas ruo4 peruroJ are ,,{aqlreql slEaAaJsarpoqrlueJo salrsSurpurq-ue8rlueeql Jo uonpuruexa pelre}ep 'alnJelotu ua8uue eql V Jo ecpJJnsaql Jo uorlrod IIBrus e o1 ,rfueluauayduroc eJplpqt salrsSurpurq leJrluepr oml qtlm selnoelorupadeqs-trare serpoqpuv 'serpoquuB ]ualaJ -JrpJo suoulrq acnpord ot alqe aq ot aAEqallr'relunoJua lq8nu sueunq ]eq] sua8 -ltue tueraJJrp suorpq dlequalod ere araql asnpoag ,{lrcgrcads alqelreuer Jo qrwr (uatpue up pelleo) la8rcl slr sazruSocar.{poqrlue uV 'uor}f,nJ}sep JoJ t1 Suqreur ro ,(pcarrp alnoalou le8re1 aqt Surle.,lrlreur raqlra .{qaraql 'epceloru 1a8re1 repcpred e o] l(llq8p spurq ,ri.poqrtup qceg 'rusrueBroorJrtu Surpplur ue Jo aceJrns eql uo asoql sE qJns 'salnJelour uBrarogol esuodseJur uralsr(s aunurtur aql dq pacnpord suralord ere 'surlnqolSounruur Jo 'salpoqpuv '(97 raldeq3 ur Irelep ur passno -srp) Surpurq a^rtJelas rq8rl ro; dllcedec slr JoJ alqelou sr dyruu;,rtpoqrlue aql 'suollcunJ snorJEArraql ]no drrec o] spue8l relncqred ol pulq lsnur suralord 1ry

elrlesren I;;errads3arv salrs6u;pu;g{poqlluy 'lle3 B ur punoJ suralord luara;;rp;o spuesnoql .,(ueuraq1uror; JeulJpd auo ]sn[ ]Jalas 01 uralord e Suqqeua 'crgrcadsdlauarga eq uec suor]3e -ralur eJBJJns-aJeJrns qJns 'uosBel alues aql Joc 'lla1!\q3]eru ]eq] saJeJJnso1!\] uaamlaq ruJoJUEJ spuoq Tea^ Jo Jequnu a8reye acurs '1q8p fral aq uec suor] -Jerelur qJnS '(10'-t arn8rg) reqloup Jo teq] qtlm aJeJJnsp€u auo go Surqcleu asrcard aql .{q sr 'Je^e^toq'lJpJalur o1 suralord ro; ,(e,vruoruuros lsolu aql 'z Jeloeql ur passnJsrp se 'suralord Irolep8ar aua8 go sarlrueJ IBJeAasur punoJ sr aJeJJalururalord go adfi sq1 '(S0?-g arn8rg) IroJ-pelroJ E ruroJ o1 raqtaSo] Jred 'urelord qJee ruorJ auo 'sacllaq rl oml uaqm sruJoJaJeJralururalord-uratord 3o adfi puocas V '(ntoleq aas) aleldroqdsoqd IIIr\ ll ]eq] suralord aql azluSocal o] aseuDluratord e selqeua osle ]r pue 'paqrJJsaplsnf se 'uralord puooes e uo dool apqdaddlod pa1e1,{roqdsoqde anuSocal ol ureruop ZHS eql smolle 'aldurexa JoJ'|uorlJpJa}ur 8urr1s-ace;rns e qons '(V07-t arn8gg) uralord puoces e uo (,,3uu1s,,p) ureqr apqda&t1od;o dool papualxe uB slJeluoc uralord euo Jo eoeJJnseql Jo uoq -rod e 'seser.{ueru u1 's,{emaarq} lseel le ur suralord Jeq}o o} purq uBJ suraloJd

sa)eJralul Jo sad^I lela^asq6norql sulalotdraqlo ol pu!g suratord 'uorlcunJ uralord raqdrcap o1Surdlaq dqaraqt ,{puel leqt Jo sraqruetu aq} JoJ salrs Surpurq aurruJa}ap o1 slsrSolorq s^^olp Surcerl druuorlnlona 'raqrueru ,r{11ue3 auo JoJ paururalep uaeq seq aln}cnrls 'u^\ou{un erB suorlsunJ asoqM peJa^ossrp ueeq IPuorsuarurp-ealq] e eJuo alerl serlrrueJuralord a,rau.,tueru'SurcuanbasaruouaSanrsualxaJo pJaslq] uI l)vruns-3)vJUns o)

x|EH-Xt'llH (8)

9NrUrS-3)VlUnS(v)

I x!laq

z a)e|ns I a)e+Jns

sulaloJd:€ Jaloeql

95l

PROTEIN FUNCTION

157

h e a v yc h a i n

lr: "fr

l o o p st h a t b i n d a n t i g e n Vs domain --...... NH,

lq \-"l

Vrdomain

v a r i a b l ed o m a i n o f l i g h t c h a i n( V r ) 5"rn

(A)

(B)

cooH

it with many weak bonds. For this reason, loops often form the ligand-binding sites in proteins.

TheEquilibrium ConstantMeasures BindingStrength Molecules in the cell encounter each other very frequently because of their continual random thermal movements. Colliding molecules with poorly matching surfaces form few noncovalent bonds with one another, and the two molecules dissociate as rapidly as they come together. At the other extreme, when many noncovalent bonds form between two colliding molecules, the association can persist for a very long time (Figure 3-42). Strong interactions occur in cells whenever a biological function requires that molecules remain associatedfor a long time-for example, when a group of RNA and protein molecules come together to make a subcellular structure such as a ribosome. We can measure the strength with which any tvvo molecules bind to each other. As an example, consider a population of identical antibody molecules that suddenly encounters a population of ligands diffusing in the fluid surrounding them. At frequent intervals, one of the ligand molecules will bump into the binding site of an antibody and form an antibody-ligand complex. The population of antibody-ligand complexes will therefore increase, but not without limit: over time, a second process, in which individual complexes break apart because of thermally induced motion, will become increasingly important. Eventually, any population of antibody molecules and ligands will reach a steady state, or equilibrium, in which the number of binding (association)events per second is precisely equal to the number of "unbinding" (dissociation)events (seeFigure 2-52). From the concentrations of the ligand, antibody, and antibody-ligand complex at equilibrium, we can calculate a convenient measure-the equilibrium constant (K)-of the strength of binding (Figure 3-43A.).The equilibrium constant for a reaction in which two molecules (A and B) bind to each other to form a complex (AB) has units of liters/mole, and half of the binding sites will be occupied by ligand when that ligand's concentration (in moles/liter) reaches a value that is equal to l/K This equilibrium constant is larger the greater the binding strength, and it is a direct measure of the free-energy difference

Figure3-41 An antibodymolecule, (A)A typicalantibodymoleculeis and hastwo identicalbinding Y-shaped sitesfor its antigen,one on eacharm of the Y.The protein is composedof four polypeptidechains(two identicalheavy chainsand two identicaland smallerlight chains)heldtogetherby disulfidebonds. Eachchainis madeup of severaldifferent immunoglobulindomains,hereshaded eitherb/ueor groy.fheantigen-binding siteisformedwherea heavy-chain variabledomain(VH)and a light-chain variabledomain(Vr)comeclose together.Thesearethe domainsthat differmost in their seouenceand structurein differentantibodies.Each domainat the end of the two armsof the antibodymoleculeformsloopsthat bind to the antigen.ln (B)we can seethese fingerlikeloops (red)contributedby the Vrdomain.

158

Chaoter3: Proteins

t h e s u r f a c e so f m o l e c u l e sA a n d B , a n d A a n d C , a r e a p o o r m a t c ha n d a r e c a p a b l eo f f o r m i n g o n l y a f e w w e a k b o n d s ;t h e r m a l m o t i o n r a p i d l y breaksthem aoart

((

m o l e c u l eA r a n d o m l ye n c o u n t e r s o t h e r m o l e c u l e s( 8 , C ,a n d D )

(

the surfacesof moleculesA and D match well and therefore can form enough weak bonds to withstand t h e r m a lj o l t i n g ;t h e y t h e r e f o r e stay bound to each other

between the bound and free states (Figure 3-438 and C). Even a change of a few noncovalent bonds can have a striking effect on a binding interaction, as shown by the example in Figure 3-44. (Note that the equilibrium constant, as defined here is also knor,rmas the association or affinity constant, Ku.) We have used the case of an antibody binding to its ligand to illustrate the effect of binding strength on the equilibrium state, but the same principles apply to any molecule and its ligand. Many proteins are enzymes, which, as we now discuss, first bind to their ligands and then catalyze the breakage or formation of covalent bonds in these molecules.

Figure3-42 How noncovalentbonds mediate interactionsbetween macromolecules,

Enzymes Are Powerfuland HighlySpecific Catalysts Many proteins can perform their function simply by binding to another molecule. An actin molecule, for example, need only associatewith other actin 1 dissociation AB-A+B

The relationshipbetween free-energydifferencesand equilibriumconstants(37"C)

d i s s o c i a t i o.n" 1 " = d i s s o c i a t i o n x c o n c e n t r a t i o n rate constant of AB

equilibrium constant

d i s s o c i a t i orna t e = k o r [ A B ]

A+lassociationrate =

AB assoclatlon rate constant

of AB minus o f A B m i n u s free enerqv f r e e e n e r g y tAllBl ofA+B-OTA+ts (liters/mole) (kcal/mole) (kJ/mole) lABl

association c o n c e n t r a t i o n concentration ofA ofB

1 10 102 103 104 1os 106 107 108 10e 1010 1oll

a s s o c i a t i o nr a t e = k o n [ A ] [ B l

AT EQUILIBRIUM: a s s o c i a t i orna t e = d i s s o c i a t i o rna t e kon[A] [B]

(A)

=

fre*energydifference

k"r [AB]

(B)

A l t h o u g hj o u l e sa n d k i l o j o u l e s( 1 0 0 0j o u l e s )a r e standard units of energy, c e l l b i o l o g i s t su s u a l l yr e f e r t o f r e e e n e r g yv a l u e si n t e r m so f c a l o r i e sa n d kilocalories.

K

0 -'t 4 -2.8 -4.3 -5.7 - 7. 1 -8.5 -9.9 - 11 3 -12.8

0 -5.9 -11.9 -17.8 -23.7 -29.7 -35.5 -41.5 47.4 -53 4

- 1 56

-55.3

-J9.4

O n e k i l o c a l o r i e( k c a l )i s e q u a lt o 4 . 1 8 4k i l o j o u l e s (kJ). T h e r e l a t i o n s h i pb e t w e e n the f ree-energychange, A G ,a n d t h e e q u i l i b r i u m constant is AG = -0.00458 r log K whereAGis in kilocalories a n d f i st h e a b s o l u t e t e m p e r a t u r ei n K e l v i n s ( 3 1 0K = 3 7 " C )

(c)

Figure3-43 Relatingbinding energiesto the equilibriumconstantfor an association reaction.(A)Theequilibrium betweenmolecules A and B and the complexAB is maintainedby a balancebetweenthe two opposingreactionsshownin panels1 and 2. Molecules A and B mustcollideif they areto react,and the association rateis thereforeproportionalto the productof their individualconcentrations As shownin panel3, the ratio [A]x [B].(Squarebracketsindicateconcentration.) of the rateconstantsfor the association and the dissociation reactionsis equalto the equilibriumconstant(K)for the reaction.(B)Theequilibriumconstantin panel3 is that for the reactionA + B + AB,and the largeritsvalue,the stronger the bindingbetweenA and B.Notethat for everyl.41 kcal/mole(5.91kJlmole)decrease in freeenergythe equilibrium constantincreasesby a factor of 10 at 37'C. Theequilibriumconstantherehasunitsof liters/mole: for simplebindinginteractions it is alsocalledthe affinityconstant ot association constant,denoted Ku.The reciprocalof Kuis calledthe dissociationconstant,K6(in units of moles/liter).

159

P R O T E IFNU N C T I O N

molecules to form a filament. There are other proteins, however, for which ligand binding is only a necessaryfirst step in their function. This is the casefor the large and very important class of proteins called enzyrnes. As described in Chapter 2, enzymes are remarkable molecules that determine all the chemical transformations that make and break covalent bonds in cells. They bind to one or more ligands, called substrates, and convert them into one or more chemically modified products, doing this over and over again with amazing rapidity. Enzymes speed up reactions, often by a factor of a million or more, without themselves being changed-that is, they act as catalysts that permit cells to make or break covalent bonds in a controlled way. lt is the catalysisof organized sets of chemical reactions by enzymes that createsand maintains the cell, making life possible. We can group enzymes into functional classesthat perform similar chemical reactions (Table 3-1). Each type of enzyme within such a classis highly specific, catalyzing onll, a single type of reaction. Thus, hexokinase adds a phosphate group to o-glucose but ignores its optical isomer t-glucose; the blood-clotting enzyme thrombin cuts one tlpe of blood protein between a particular arginine and its adjacent glycine and nowhere else, and so on. As discussed in detail in Chapter 2, enzymes work in teams, with the product of one enzvme becoming the substrate for the next. The result is an elaborate network of metabolic pathways that provides the cell with energy and generatesthe many large and small moleculesthat the cell needs (seeFigure2-35).

Substrate Bindingls the FirstStepin EnzymeCatalysis

C o n s i d e r1 0 0 0m o l e c u l e so f A a n d 1 0 0 0m o l e c u l e so f B i n a e u c a r y o t i c c e l l T h e c o n c e n t r a t i o no f b o t h w i l l b e a b o u t 1 0 - eM l f t h e e q u i l i b r i u m . c o n s t a( K n )t f o r A + B . - A B i s 1 0' ' , t h e n o n e c a n c a l c u l a t et h a t a t e q u i l i b r i u mt h e r e will be 270

270

730

ABAB molecules molecules molecules l f t h e e q u i l i b r i u mc o n s t a n ti s a l i t t l e w e a k e ra t 1 0 ' , w h i c h r e p r e s e n t s a l o s so f 2 8 k c a l / m o l eo f b i n d i n g e n e r g yf r o m t h e e x a m p l e a b o v e ,o r 2 - 3 f e w e r h y d r o g e n b o n d s ,t h e n t h e r e w i l l b e 915

915

85

ABAB molecules molecules molecules

Figure3-44 Smallchangesin the numberof weak bondscan havedrastic effectson a binding interaction.This the dramaticeffectof examoleillustrates or absenceof a few weak the presence noncovalent bondsin a bioloqical conIexI.

For a protein that catalyzesa chemical reaction (an enzyme), the binding of each substratemolecule to the protein is an essentialprelude.In the simplest case,if we denote the enzyme by E, the substrate by S, and the product by Il the basic reaction path is E + S -+ ES -+ EP -+ E + P From this reaction path, we see that there is a limit to the amount of substrate that a single enzyme molecule can process in a given time. An increase in the concentration of substrate also increasesthe rate at which product is formed, up to a maximum value (Figure 3-45). At that point the enzyme molecule is saturated with substrate, and the rate of reaction ( V-oJ depends only on how rapidly the enzyme can processthe substrate molecule. This maximum rate divided by the enzvme concentration is

Table3-1 SomeCommonTypesof Enzymes

ENZYME

REACTION CATALYZED

Hydrolases

andproteoses generaltermfor enzymesthat catalyzea hydrolyticcleavagereaction;nucleases aremorespecific namesfor subclasses of theseenzymes. breakdownnucleicacidsby hydrolyzing bondsbetweennucleotides. breakdownproteinsby hydrolyzing bondsbetweenaminoacids. together. two smallermolecules synthesize molecules by condensing in anabolicreactions catalyze the rearrangement of bondswithina singlemolecule. polymerization of DNAand RNA. catalyze reactions suchasthe synthesis arean importantgroup groupsto molecules. Proteinkinases catalyze the additionof phosphate groupsto proteins. of kinases that attachphosphate groupfroma molecule. catalyze removalof a phosphate the hydrolytic whilethe generalnamefor enzymes reactions in whichonemoleculeisoxidized that catalyze namedeitheroxidases, otheris reduced.Enzymes of this type areoften morespecifically reductases, or dehydrogeno ses. ATPase hydrolyzeATP. Manyproteinswith a wide rangeof roleshavean energy-harnessing activityaspart of theirfunction,for example,motor proteinssuchasmyosinandmembrane pump. transportproteinssuchas thesodium-potassium

Nucleases Proteases Synthases lsomerases Polymerases Kinases Phosphatases Oxido-Reductases

ATPases

that weredlscovered thrombinand lysozyme Enzymenamestyp ca ly end in " ase,"with the exc-apt trypsin, suchaspepsln, on of some-onzymes, centuryThecommonnameof an enzymeusually and namedb-.forethe convention becamegeneraly acceptedat the end of the nineteenth of citrateby a reaction catayzesthe synthests ndjcates the substrate citratesynthase andthe natureof the reactiorcatayzed Forexample, betweenacetvCoAandoxaloacetate

160

Chapter3: Proteins

I

C

o E 6 o o 0 5v.", o 6

K-

s u b s t r a t ec o n c e n t r a t i o n+

Figure3-45 Enzymekinetics,The rate of an enzymereaction(V)increases asthe substrateconcentration increases untila maximumvalue(Vr"r) is reached. At this point all substrate-binding siteson the enzymemolecules arefullyoccupied, and the rateof reactionis limitedby the rateof the catalyticprocesson the enzymesurface.Formostenzymes, the concentration of substrate(Kr) at which the reactionrate is half-maximal(black dot)is a measureof how tightlythe substrateis bound,with a largevalueof K. corresponding to weakbinding.

called the turnouer number. The turnover number is often about 1000 substrate molecules processedper second per enzyme molecule, although turnover numbers between 1 and 10,000are known. The other kinetic parameter frequently used to characterizean enzyme is its K-, the concentration of substrate that allows the reaction to proceed at onehalf its maximum rate (0.5 V-*) (seeFigure 3-45). A low K^value means that the enzyme reaches its maximum catalytic rate at a low concentration of substrate and generally indicates that the enzyme binds to its substrate very tightly, whereas a high K- value corresponds to weak binding. The methods used to characterize enzymes in this way are explained in Panel 3-3 (pp. 162-163).

Enzymes SpeedReactions by Selectively Stabilizing Transition States Enzymes achieve extremely high rates of chemical reaction-rates that are far higher than for any synthetic catalysts.There are several reasons for this efficiency. First, the enzyme increases the local concentration of substrate molecules at the catal)'tic site and holds all the appropriate atoms in the correct orientation for the reaction that is to follow. More importantly, however, some of the binding energy contributes directly to the catalysis. Substrate molecules must pass through a series of intermediate states of altered geometry and electron distribution before they form the ultimate products of the reaction. The free energy required to attain the most unstable transition state is called the actiuation energyfor the reaction, and it is the major determinant of the reaction rate. Enzymes have a much higher affinity for the transition state of the substrate than they have for the stable form. Becausethis tight binding greatly lowers the energies of the transition state, the enzyme greatly acceleratesa particular reaction by lowering the activation energy that is required (Figure 3-46). By intentionally producing antibodies that act like enzymes, we can demonstrate that stabilizing a transition state can greatly increase a reaction rate. Consider, for example, the hydrolysis of an amide bond, which is similar to the peptide bond that joins two adjacent amino acids in a protein. In an aqueous solution, an amide bond hydrolyzes very slowly by the mechanism shown in Figure 3-47A. In the central intermediate, or transition state, the carbonyl carbon is bonded to four atoms arranged at the corners of a tetrahedron. By generating monoclonal antibodies that bind tightly to a stable analog of this very unstable tetrahedral intermediate, one can obtain an antibody that functions like an enzyme (Figure 3-47F_).Becausethis catalytic antibodybinds to and stabilizes the tetrahedral intermediate, it increases the spontaneous rate of amide-bond hydrolysis more than 10,000-fold.

EnzymesCan Use5imultaneousAcid and BaseCatalysis Figure 3-48 compares the spontaneous reaction rates and the corresponding enzyme-catalyzed rates for five enzyrnes. Rate accelerations range from 109to 1023. Clearly, enzymes are much better catalysts than cata\tic antibodies.

a c t i v a t i o ne n e r g y for uncatalyzed reaction

I

A

o q c

o

EP progress of reaction acTrvaron energy for catalyzed reaction

Figure3-46 Enzymaticaccelerationof chemicalreactionsby decreasingthe activation energy.Often both the uncatalyzed reaction(A)and the enzymecatalyzed reaction(B)cango through severaltransitionstates.lt isthe transitionstatewith the highestenergy (Srand ESr)that determines tne activationenergyand limitsthe rateof p = product the reaction.(S= substrate; of the reaction;ES= enzyme-substrate complex;EP= enzyme-product complex.)

PROTEIN FUNCTION

161

( A ) H Y D R O L Y SO I SF A N A M I D EB O N D

oo-o

'H

l(

tetrahedral intermediate

water

( B )T R A N S I T I O N - s T AATNEA L O GF O RA M I D EH Y D R O L Y S I S

o\ D'

Noz

Figure3-47 Catalyticantibodies.The of a transitionstateby an stabilization antibodycreatesan enzyme.(A)The reactionpath for the hydrolysisof an amidebond goesthrougha tetrahedral transition the high-energy intermediate, statefor the reaction.(B)The moleculeon the left wascovalentlylinkedto a protein and usedasan antigento generatean antibodythat bindstightlyto the region of the moleculeshown in yellow.Because this antibodyalsoboundtightlyto the transitionstatein (A),it was found to functionasan enzymethat efficiently of the amide the hydrolysis catalyzed bond in the moleculeon the riqht.

Noz

o analog

Enz).rynes not only bind tightly to a transition state, they also contain precisely positioned atoms that alter the electron distributions in those atoms that participate directly in the making and breaking of covalent bonds. Peptide bonds, for example, can be hydrolyzed in the absence of an enzyme by exposing a polypeptide to either a strong acid or a strong base, as illustrated in Figure 3-49. Enzymes are unique, however, in being able to use acid and base catalysissimultaneously, since the rigid framework of the protein binds the acidic and basic residues and prevents them from combining with each other (as they would do in solution) (Figure 3-49D). The fit between an enzyme and its substrate needs to be precise. A small change introduced by genetic engineering in the active site of an enzyme can have a profound effect. Replacing a glutamic acid with an aspartic acid in one enz)ryne,for example, shifts the position of the catalytic carborylate ion by only I A (about the radius of a hydrogen atom); yet this is enough to decreasethe activity of the enzyme a thousandfold.

LysozymelllustratesHow an EnzymeWorks To demonstrate how enzymes catalyze chemical reactions, we examine an enzlrrne that acts as a natural antibiotic in egg white, saliva, tears, and other secretions.Lysozyme catalyzesthe cutting of polysaccharide chains in the cell walls of bacteria. Because the bacterial cell is under pressure from osmotic forces,cutting even a small number of polysaccharide chains causesthe cell wall to rupture and the cell to burst. Lysozl'rneis a relatively small and stable protein

h a l f - t i m ef o r r e a c t i o n 1 0 6y e a r s

1year

UNCATALYZED

1 sec

cnrnLvzeo

Figure3-48 The rate accelerations causedby five different enzymes, (Adaptedfrom A, Radzickaand 1995. R.Wolfenden,Science267'.90-93, from AAAS.) With permission

WHY ANALYZETHE KINETICS OF ENZYMES? Enzymesare the most selectiveand powerful catalystsknown. An understandingof their detailedmechanisms providesa criticaltool for the discoveryof new drugs,for the large-scale industrialsynthesis of usefulchemicals, and for appreciating the chemistryof cellsand organisms.A detailedstudyof the ratesof the chemicalreactionsthat are catalyzedby a purified enzyme-more specifically how theserateschangewith changesin conditionssuchasthe concentrations of substrates, products,inhibitors,and regulatory Iigands-allows

biochemists to figure out exactlyhow eachenzymeworks. For example,this is the way that the ATP-producing reactions of glycolysis, shown previouslyin Figure2-72, were deciphered-allowing us to appreciatethe rationalefor this criticalenzymaticpathway. In this Panel,we introducethe important field of enzyme kinetics,which hasbeen indispensable for derivingmuch of the detailedknowledgethat we now haveabout cell chemistry.

STEADY-sTATE ENZYM E KINETICS Many enzymeshaveonly one substrate,which they bind and then processto produceproductsaccordingto the scheme outlined in Figure3-504. In this case,the reactionis written as kr

E.S

*.

rate of ESbreakdown k-l [E5]+ kcat[Es]

Kr:t

Es -;

At this steadystate,[ES]is nearlyconstant,so that

rate of ESformation

kr tEltsl

E+p

K_l

Herewe haveassumedthat the reversereaction.in which E + P recombineto form EPand then ES,occursso rarelythat we can ignore it. In this case,EPneed not be represented,ano we can expressthe rate of the reaction- known as its velocity,V, as

or, sincethe concentrationof the free enzyme,[E],is equal to [Eo]- [E5],

r,,r= (;|;)

-,,,,),,, r,r'r = (-jr-_; (,,",

V= k'"t [ES] where IES]is the concentrationof the enzyme-substrate complex, Rearranging,and defining the constantKmas and k.". is the turnover number,a rate constantthat hasa value k-1 + k.", equal to the number of substratemoleculesprocessed per enzymemoleculeeachsecond. k1 But how doesthe value of IES]relateto the concentrations that we know directly,which are the total concentrationof the we get enzyme,IEo],and the concentrationof the substrate,[S]?When enzymeand substrateare first mixed,the concentrationIES]will lE,lIs] tEsl = riserapidlyfrom zero to a so-calledsteady-state lever,as K. + [5] illustratedbelow. or, rememberingthat V = kr"t [E5],we obtain the famous Michaelis-Mentenequation

I c

k."t IEo][S]

g

K. + [S]

c o c

As IS] is increasedto higher and higher levels,essentially all of the enzymewill be bound to substrateat steadystate;at this point, a maximumrate of reaction,V-"r, will be reachedwhere V = V^u, = k."1[E6J.Thus,it is convenientto rewrite the Michaelis-Mentenequationas time + pre-steady state: E Sf o r m i n g

steadystate: ESalmostconstant

164

Chapter3: Proteins

\ir, H

o SLOW

o FAsr ll

tl

c.

\H

oH

n

H

c-

,,/-

,/,

\H

-NC--N HOHHO

,/\

H

o ll

FAsr

\/FRV

FAST \H

/ \

HHH

C

cHHOH

-N

(B)

acid catalysis

(C)

II

,,/.- \H

/\-

o //

base catalysis

il

HH

H

\//

o

C

I (A) no catalysis

O

(

I

(D)

both acid and base catalyses

that can be easily isolated in large quantities. For these reasons, it has been intensively studied, and it was the first enzyme to have its structure worked out in atomic detail by x-ray crystallography. The reaction that lysozyme catalyzes is a hydrolysis: it adds a molecule of water to a single bond between two adjacent sugar groups in the polysaccharide chain, thereby causing the bond to break (seeFigure 2-19). The reaction is energetically favorable because the free energy of the severedpolysaccharide chain is lower than the free energy of the intact chain. However, the pure polysaccharide can remain for years in water without being hydrolyzed to any detectable degree.This is because there is an energy barrier to the reaction, as discussedin Chapter 2 (seeFigure 2-46). Acolliding water molecule can break a bond linking tvvo sugars only if the polysaccharide molecule is distorted into a particular shape-the transition state-in which the atoms around the bond have an altered geometry and electron distribution. Becauseof this distortion, random collisions must supply a very large activation energy for the reaction to take place. In an aqueous solution at room temperature, the energy of collisions almost never exceeds the activation energy. Consequently, hydrolysis occurs extremely slowly, if at all. This situation changes drastically when the polysaccharide binds to lysozyme.The active site of lysozyme,becauseits substrate is a polymer, is a long groove that holds six linked sugars at the same time. As soon as the polysaccharide binds to form an enzyme-substrate complex, the enzyme cuts the polysaccharide by adding a water molecule across one of its sugar-sugar bonds. The product chains are then quickly released,freeing the enzyme for further cycles of reaction (Figure 3-50). The chemistry of the binding of lysozl.rneto its substrate is the same as that for antibody binding to its antigen-the formation of multiple noncovalent

Figure3-50 The reactioncatalyzedby lysozyme.(A)The enzyme lysozyme(E)catalyzes the cuttingof a polysaccharide chain,which is its substrate(S).Theenzymefirstbindsto the chainto form an enzyme-substrate complex(ES)and then catalyzes the cleavageof a specificcovalentbond in the backboneof the polysaccharide, formingan enzyme-productcomplex(EP)that rapidlydissociates. Release of the severedchain(the productsP)leavesthe enzymefreeto act on another substratemolecule.(B)A space-filling modelof the lysozymemolecule boundto a shortlengthof polysaccharide chainbeforecleavage. (B,courtesyof RichardJ. Feldmann.)

(A)

)+E

E)

*

E+P

Figure3-49 Acid catalysisand base catalysis.(A)The start of the uncatalyzed reactionshownin Figure3-474,with b/ueindicatingelectrondistributionin the waterand carbonylbonds.(B)An acid likesto donatea proton(H+)to other atoms.By pairingwith the carbonyl oxygen,an acidcauseselectronsto move awayfrom the carbonylcarbon,making this atom much moreattractiveto the electronegative oxygenof an attacking watermolecule.(C)A baselikesto take up H+.By pairingwith a hydrogenof the attackingwatermolecule, a basecauses electronsto move toward the water oxygen/makingit a betterattacking groupfor the carbonylcarbon.(D)By positionedatoms havingappropriately on its surface, an enzymecan perform both acidcatalysis and basecatalysis at the sametime.

165

PROTEIN FUNCTION

bonds. However,lysozyme holds its polysaccharide substrate in a particular way, so that it distorts one of the two sugarsin the bond to be broken from its normal, most stable conformation. The bond to be broken is also held close to two amino acids with acidic side chains (a glutamic acid and an aspartic acid) within the active site. Conditions are thereby created in the microenvironment of the lysozyme active site that greatly reduce the activation energy necessaryfor the hydrolysis to take place. Figure 3-51 shows three central steps in this enzymatically catalyzed reaction. The enzyme stressesits bound substrate, so that the shape of one sugar more closely resembles the shape of high-energy transition states formed during the reaction. 2. The negatively charged aspartic acid reactswith the Cl carbon atom on the distorted sugar,and the glutamic acid donates its proton to the oxygen that links this sugar to its neighbor. This breaks the sugar-sugar bond and leaves the aspartic acid side chain covalently linked to the site of bond cleavage. 3. Aided by the negatively charged glutamic acid, a water molecule reacts with the Cl carbon atom, displacing the aspartic acid side chain and completing the process of hydrolysis. t.

Figure3-51 Eventsat the active site of lysozyme.The top left and top rightdrawingsshowthe freesubstrate and the freeproducts,respectively, whereasthe otherthreedrawingsshow eventsat the enzyme the sequential activesite.Notethe changein the of sugarD in the conformation complex;this shape enzyme-substrate the oxocarbenium changestabilizes ion-liketransitionstatesrequiredfor of the covalent formationand hydrolysis intermediate shownin the middlepanel. It is alsooossiblethat a carboniumion formsin step2, asthe intermediate shownin the covalentintermediate middlepanelhasbeendetectedonly with a syntheticsubstrate.(SeeD.J.Vocadloet 2001.) al.,Nature412:835-838,

The overall chemical reaction, from the initial binding of the polysaccharide on the surface of the enzyme through the final release of the severed chains, occurs many millions of times faster than it would in the absence of enzyme. Other enzymes use similar mechanisms to lower activation energies and speed up the reactions they catalyze.In reactions involving two or more reactants, the active site also acts like a template, or mold, that brings the substrates together in the proper orientation for a reaction to occur between them (Figure

PRODUCTS

SUBSTRATE T h i ss u b s t r a t ei s a n o l i g o s a c c h a r i doef s i xs u g a r s , l a b e l e dA - F .O n l y s u g a r sD a n d E a r e s h o w n i n d e t a i l

T h e f i n a l p r o d u c t sa r e a n o l i g o s a c c h a r i doef f o u r s u g a r s (/eft) and a disaccharide(dght), produced by hydrolysis.

cHzoH A B CrO

otF cH2oH

-o ---\i_' I H

,3

oV

\ (.

I n t h e e n z y m e - s u b s t r a tceo m p l e x( E 5 ) t, h e e n z y m ef o r c e ss u g a rD i n t o a s t r a i n e d c o n f o r m a t i o nw , i t h G l u 3 5 p o s i t i o n e dt o s e r v ea s a n a c i dt h a t a t t a c k st h e a d j a c e n ts u g a r - s u g a r b o n d b y d o n a t i n ga p r o t o n ( H + )t o s u g a rE ,a n d A s o 5 2 o o i s e dt o a t t a c kt h e C 1 c a r b o na t o m

T h e A s p 5 2 h a sf o r m e d a c o v a l e n tb o n d b e t w e e n t h e e n z y m ea n d t h e C 1c a r b o na t o m o f s u g a rD T h e G l u 3 5 t h e n p o l a r i z e sa w a t e r m o l e c u l e( r e d ) , s o t h a t i t s o x y g e nc a n r e a d i l ya t t a c kt h e C 1 c a r b o na t o m a n d d i s p l a c eA s o 5 2

T h e r e a c t i o no f t h e w a t e r m o l e c u l e( r e d ) c o m p l e t e st h e h y d r o l y s ias n d r e t u r n st h e e n z y m e t o i t s i n i t i a ls t a t e ,f o r m i n gt h e f i n a l e n z y m e o r o d u c tc o m p l e x( E P ) .

166

Chapter3: Proteins

Figure3-52 Somegeneralstrategiesof enzyme catalysis.(A)Holding substrates togetherin a precisealignment. (B)Chargestabilization of reaction (C)Applyingforcesthat intermediates. distortbondsin the substrate to increase the rateof a particularreaction. ( A ) e n z y m eb i n d st o t w o s u b s t r a t em o l e c u l e sa n d o r i e n t st h e m p r e c i s e ltyo e n c o u r a g ea r e a c t i o nt o o c c u rb e t w e e nt h e m

( B ) b i n d i n go f s u b s t r a t e ( C )e n z y m es t r a i n st h e ro enzyme rearranges bound substrate e l e c t r o n si n t h e s u b s t r a t e , m o l e c u l ef,o r c i n gi t c r e a t i n gp a r t i a ln e g a t i v e toward a transition a n d p o s i t i v ec h a r g e s state to favor a reaction that favor a reaction

3-524.).As we saw for lysozyme, the active site of an enzyme contains precisely positioned atoms that speed up a reaction by using charged groups to alter the distribution of electrons in the substrates (Figure 3-528). In addition, when a substrate binds to an enzyme, bonds in the substrate often bend, changing the substrate shape.These changes,along with mechanical forces, drive a substrate toward a particular transition state (Figure 3-52C). Finally, like lysozyme, many enzymes participate intimately in the reaction by briefly forming a covalent bond between the substrate and a side chain of the enzyme. Subsequent steps in the reaction restore the side chain to its original state, so that the enzyme remains unchanged after the reaction (seealso Figure 2-22).

TightlyBoundSmallMolecules Add ExtraFunctions to Proteins Although we have emphasized the versatility of proteins as chains of amino acids that perform different functions, there are many instances in which the amino acids by themselves are not enough. Just as humans employ tools to enhance and extend the capabilities of their hands, proteins often use small nonprotein molecules to perform functions that would be difficult or impossible to do with amino acids alone. Thus, the signal receptor protein rhodopsin, which is made by the photoreceptor cells in the retina, detects light by means of a small molecule, retinal, embedded in the protein (Figure 3-53A). Retinal changes its shape when it absorbs a photon of light, and this change causesthe protein to trigger a cascade of enzymatic reactions that eventually lead to an electrical signal being carried to the brain. Another example of a protein that contains a nonprotein portion is hemoglobin (see Figure 3-22). A molecule of hemoglobin carries four heme groups, ring-shaped molecules each with a single central iron atom (Figure 3-538). Heme gives hemoglobin (and blood) its red color. By binding reversibly to oxygen gas through its iron atom, heme enables hemoglobin to pick up oxygen in the lungs and releaseit in the tissues. sometimes these small molecules are attached covalently and permanently to their protein, thereby becoming an integral part of the protein molecule itself. we shall see in chapter l0 that proteins are often anchored to cell membranes through covalently attached lipid molecules. And membrane proteins exposed COOH

I

fn, 9Hz

COOH

I

T", CHz

ua

-e ,u, l La - u

aH-

H

CH: CH:

(A)

(B)

HC CHz

Figure3-53 Retinaland heme.(A)The structureof retinal,the light-sensitive moleculeattachedto rhodopsinin the eye.(B)The structureof a hemegroup. Thecarbon-containing hemering is red and the iron atom at its centeris orange. A hemegroup is tightlyboundto eachof the four polypeptidechainsin hemoglobin,the oxygen-carrying protein whosestructureis shownin Fiqure3-22.

167

PROTEIN FUNCTION Table3-2 ManyVitaminsProvideCritical Coenzymes for HumanCells

T h i a m i n e( v i t a m i nB r ) Riboflavin(vitaminBz) Niacin Pantothenicacid Pyridoxine Biotin Lipoicacid Folicacid V i t a m i nB r z

thiaminepyrophosphate FADH NADH,NADPH coenzymeA pyridoxal phosphate biotin lipoamide tetrahydrofolate cobalamin coenzymes

activationand transferof aldehydes oxidation-reduction oxidation-reduction acyl group activationand transfer amino acid activation;alsoglycogenphosphorylase CO2activationand transfer acyl group activation;oxidation-reduction activationand transferof singlecarbon groups isomerizationand methyl group transfers

on the surface of the cell, as well as proteins secreted outside the cell, are often modified by the covalent addition of sugars and oligosaccharides. Enzymes frequently have a small molecule or metal atom tightly associated with their active site that assistswith their catalytic function. Carboxypeptidase, for example, an enzyrne that cuts polypeptide chains, carries a tightly bound zinc ion in its active site. During the cleavageof a peptide bond by carboxypeptidase, the zinc ion forms a transient bond with one of the substrate atoms, thereby assisting the hydrolysis reaction. In other enzymes, a small organic molecule servesa similar purpose. Such organic molecules are often referred to as coenzymes. An example is biotin, which is found in enzymes that transfer a carboxylate group (-COO-) from one molecule to another (see Figure 2-63). Biotin participates in these reactions by forming a transient covalent bond to the -COO- group to be transferred, being better suited to this function than any of the amino acids used to make proteins. Because it cannot be synthesized by humans, and must therefore be supplied in small quantities in our diet, biotin is a uitamin. Many other coenzymes are produced from vitamins (Table3-2). Vitamins are also needed to make other types of small molecules that are essential components of our proteins; vitamin A, for example, is needed in the diet to make retinal, the light-sensitive part of rhodopsin.

with Multiple Molecular TunnelsChannelSubstrates in Enzymes CatalyticSites Some of the chemical reactions catalyzedby enzymes in cells produce intermediates that are either very unstable or that could readily diffuse out of the cell through the plasma membrane if released into the cltosol. To preserve these intermediates, enzymes have evolved molecular tunnels that connect tvvo or more active sites, allowing the intermediate to be rapidly processed to a final product-without ever leaving the enzyme. Consider, for example, the enzyme carbamoyl phosphate synthetase,which uses ammonia derived from glutamine plus two molecules of ATP to convert bicarbonate (HCO3-) to carbamoyl phosphate-an important intermediate in several metabolic pathways (Figure 3-54). This enzyme contains three widely separated active sites that are connected to each other by a tunnel. The reaction starts at active site 2, located in the middle of the tunnel, where AIP is used to phosphorylate (add a phosphate group to) bicarbonate, forming carbory phosphate. This event triggers the hydrolysis of glutamine to glutamic acid at active site 1, releasing ammonia into the tunnel. The ammonia immediately diffuses through the first half of the tunnel to active site 2, where it reacts with the carboxyphosphate to form carbamate. This unstable intermediate then diffuses through the second half of the tunnel to active site 3, where it is phosphorylated byATP to the final product, carbamoyl phosphate.

168

Chapter3: Proteins

Figure3-54 The tunnelingof reactionintermediatesin the enzyme carbamoylphosphatesynthetase.(A)Diagramofthe structureof the enzyme,in whicha redribbonhasbeenusedto outlinethe tunnelon the insideof the proteinconnectingits three activesites.Thesmalland largesubunitsof this dimericenzyme (B)The path of the are color codedyellow and blue,respectively. reaction.As indicated, activesite 1 producesammonia,which diffusesthroughthe tunnelto activesite2, whereit combines with carboxyphosphateto form carbamate. Thishighlyunstable intermediate then diffusesthroughthe tunnelto activesite3, whereit is phosphorylated by ATPto producethe finalproduct, (A,modifiedfrom F.M.Raushel, carbamoylphosphate. J.B.Thoden, and H.M.Holden,Acc.Chem.Res.36:539-548,2003.Witn permission from AmericanChemicalSocietv.)

I

L

]'l]]::]:,.']':]i]]'::]'.':l:','.|.i':.:],:].

oo

Hzo I

iltl

- o/ / bicarbonate

CP \

q - llu t a m i n e

l\

odo-

p,' *l

L+ I

q l u t a m i ca c i d V NHr

carboxyphosphate

II

t

NHj diffusion

I I

-",1.(B)

Severalother well characterized enzymes contain similar molecular tunnels. Ammonia, a readily diffusable intermediate that might otherwise be lost from the cell, is the substrate most frequently channeled in the examples thus far kno'nrm.

Multienzyme Complexes Helpto Increase the Rateof Cell Metabolism The efficiency of enzymes in accelerating chemical reactions is crucial to the maintenance of life. cells, in effect, must race against the unavoidable processes of decay, which-if left unattended-cause macromolecules to run downhill toward greater and greater disorder. If the rates of desirable reactions were not greater than the rates of competing side reactions, a cell would soon die. we can get some idea of the rate at which cell metabolism proceeds by measuring the rate of ArP utilization. A typical mammalian cell "turns over" (i.e.,hydrolyzes and restoresby phosphorylation) its entire ATp pool once every I or 2 minutes. For each cell, this turnover represents the utilization of roughly 107molecules of AIP per second (or, for the human body, about I gram of nfi, everv minute).

169

PROTEIN FUNCTION

The rates of reactions in cells are rapid because enzyme catalysisis so effective. Many important enzymes have become so efficient that there is no possibility of further useful improvement. The factor that limits the reaction rate is no Ionger the enzyme's intrinsic speed of action; rather, it is the frequency with which the enzyme collides with its substrate. Such a reaction is said to be diffusion-limited (seePanel3-3, p. 162-163). If an enzyme-catalyzed reaction is diffusion-limited, its rate depends on the concentration of both the enzyme and its substrate. If a sequence of reactions is to occur extremely rapidly, each metabolic intermediate and enzyme involved must be present in high concentration. However, given the enormous number of different reactions performed by a cell, there are limits to the concentrations that can be achieved. In fact, most metabolites are present in micromolar (10-6 M) concentrations, and most enzyme concentrations are much lower. How is it possible, therefore, to maintain very fast metabolic rates? The answer lies in the spatial organization of cell components. The cell can increase reaction rates without raising substrate concentrations by bringing the various enzJ,.rnes involved in a reaction sequence together to form a large protein assembly knor.vn as a multienzyme complex (Figure 3-55). Because this A to be passeddirectly to enzyme B, and so on, difallows the product of enzJ,ryne fusion rates need not be limiting, even when the concentrations of the substrates in the cell as a whole are very low. It is perhaps not surprising, therefore, that such enzyme complexes are very common, and they are involved in nearly all aspects of metabolism-including the central genetic processes of DNA, RNA, and protein slmthesis.In fact, few enzymes in eucaryotic cells diffuse freely in solution; instead, most seem to have evolved binding sites that concentrate them with other proteins of related function in particular regions of the cell, thereby increasing the rate and efficiency ofthe reactions that they catalyze. Eucaryotic cells have yet another way of increasing the rate of metabolic reactions: using their intracellular membrane systems.These membranes can segregateparticular substratesand the enzymes that act on them into the same membrane-enclosed compartment, such as the endoplasmic reticulum or the cell nucleus. If, for example, a compartment occupies a total of 10% of the volume of the cell, the concentration of reactants in that compartment may be increased by 10 times compared with a cell with the same number of enzymes and substrate molecules, but no compartmentalization. Reactions limited by the speed of diffusion can thereby be speeded up by a factor of 10.

the Catalytic Activitiesof its Enzymes TheCellRegulates many of which operate at the same A living cell contains thousands of enz).rynes, time and in the same small volume of the c1'tosol.By their catalytic action, these enzymes generate a complex web of metabolic pathways, each composed of chains of chemical reactions in which the product of one enzyme becomes the substrate of the next. In this maze of pathways, there are many branch points (nodes) where different enzymes compete for the same substrate.The system is so complex (see Figure 2-88) that elaborate controls are required to regulate when and how rapidly each reaction occurs.

8 t r i m e r so f l i p o a m i d er e d u c t a s e transacetylase

+ 1 2 m o l e c u l e so f dihydrolipoyl dehydrogenase

+24 moleculeo sf pyruvatedecarboxylase

Figure3-55 The structure of pyruvate Thisenzymecomplex dehydrogenase. catalyzesthe conversionof pyruvateto acetylCoA,as part of the pathwaythat oxidizessugarsto COzand HzO(seeFigure 2-79).lt is an exampleof a large multienzymecomplexin which reaction intermediatesare passeddirectlyfrom one enzymeto another.

17O

Chapter3: Proteins

Regulation occurs at many levels.At one level, the cell controls how many molecules of each enzyme it makes by regulating the expressionof the gene that encodes that enzyme (discussedin chapter 7). The cell also controls enzymatic activities by confining sets of enzymes to particular subcellular compartments, enclosed by distinct membranes (discussedin chapters 12 and 14). As will be discussed later in this chapter, enzymes are frequently covalently modified to control their activity. The rate ofprotein destruction by targeted proteolysis represents yet another important regulatory mechanism (seep. 395). But the most general process that adjusts reaction rates operates through a direct, reversible change in the activity of an enzyme in response to the specific small molecules that it encounters. The most common type of control occurs when a molecule other than one of the substrates binds to an enzyme at a special regulatory site outside the active site, thereby altering the rate at which the enzyme converts its substrates to products. For example, in feedback inhibition a product produced late in a reaction pathway inhibits an enzyme that acts earlier in the pathway. Thus, whenever large quantities of the final product begin to accumulate, this product binds to the enzyme and slows down its catalytic action, thereby limiting the further entry of substrates into that reaction pathway (Figure g-s6). \Mhere pathways branch or intersect, there are usually multiple points of control by different final products, each of which works to regulate its own synthesis (Figure 3-57). Feedback inhibition can work almost instantaneously, and it is rapidlv reversedwhen the level of the product falls.

Figure3-56 Feedbackinhibitionof a singlebiosyntheticpathway.TheendproductZ inhibitsthe firstenzymethat is uniqueto its synthesis and thereby controlsits own levelin the cell.Thisis an exampleof negativeregulation.

aspartate

I I

I I methionine

Figure3-57 Multiplefeedback inhibition.In this example,which shows the biosyntheticpathwaysfor four differentaminoacidsin bacteria, the red arrowsindicatepositionsat which productsfeed backto inhibitenzymes. Eachaminoacidcontrolsthe firstenzyme specificto its own synthesis, thereby controllingits own levelsand avoidinga wasteful,or evendangerous, buildupof intermediates. The productscanalso separately inhibitthe initialset of reactions commonto all the syntheses; in this case,three differentenzymes catalyze the initialreaction,each inhibitedbv a differentoroduct.

PROTEIN FUNCTION

Feedback inhibition is negatiueregulation: it prevents an enzyme from acting. Enzymes can also be subject to positiue regulation, in which a regulatory molecule stimulates the enzyme's activity rather than shutting the enzyme down. Positive regulation occurs when a product in one branch of the metabolic network stimulates the activity of an enzyme in another pathway. As one example, the accumulation of ADP activates several enzymes involved in the oxidation of sugar molecules, thereby stimulating the cell to convert more ADP to AIP

AllostericEnzymesHaveTwoor MoreBindingSitesThatInteract A striking feature of both positive and negative feedback regulation is that the regulatory molecule often has a shape totally different from the shape of the substrate of the enz].ryne.This is why the effect on a protein is termed allostery (from the Greekwords allos,meaning"other," andstereos,meaning"solid" or"three-dimensional"). As biologists learned more about feedback regulation, they recognized that the enzyrnes involved must have at least two different binding sites on their surface-an active site that recognizes the substrates, and a regulatory site that recognizes a regulatory molecule. These two sites must somehow communicate so that the catalytic events at the active site can be influenced by the binding of the regulatory molecule at its separatesite on the protein's surface. The interaction between separated sites on a protein molecule is now knor,tmto depend on a conformational changein the protein: binding at one of the sites causesa shift from one folded shape to a slightly different folded shape. During feedback inhibition, for example, the binding of an inhibitor at one site on the protein causesthe protein to shift to a conformation that incapacitates its active site, located elsewherein the protein. It is thought that most protein molecules are allosteric. They can adopt two or more slightly different conformations, and a shift from one to another caused by the binding of a ligand can alter their activity. This is true not only for enzymes but also for many other proteins, including receptors, structural proteins, and motor proteins. In all instances of allosteric regulation, each conformation of the protein has somewhat different surface contours, and the protein's binding sites for ligands are altered when the protein changes shape. Moreover as we discuss next, each ligand will stabilize the conformation that it binds to most strongly, and thus-at high enough concentrations-will tend to "switch' the protein toward the conformation that the ligand prefers.

TwoLigandsWhoseBindingSitesAreCoupledMustReciprocally AffectEachOther'sBinding The effects of ligand binding on a protein follow from a fundamental chemical principle knor.vnas linkage. Suppose,for example, that a protein that binds glucose also binds another molecule, X, at a distant site on the protein's surface. If the binding site for X changes shape as part of the conformational change induced by glucosebinding, the binding sites for X and for glucose are said to be coupled. Vy'henevertwo ligands prefer to bind to the same conformation of an allosteric protein, it follows from basic thermodynamic principles that each ligand must increasethe affinity of the protein for the other. Thus, if the shift of the protein in Figure 3-58 to the closed conformation that binds glucose best also causes the binding site for X to fit X better, then the protein will bind glucose more tightly when X is present than when X is absent. Conversely,linkage operates in a negative way if two ligands prefer to bind to dffirent conformations of the same protein. In this case,the binding of the first ligand discouragesthe binding of the second ligand. Thus, if a shape change caused by glucose binding decreasesthe affinity of a protein for molecule X, the binding of X must also decreasethe protein's affinity for glucose (Figure 3-59). The linkage relationship is quantitatively reciprocal, so that, for example, if glucose has a very large effect on the binding of X, X has a very large effect on the binding of glucose.

171

172

Chapter3: Proteins

INACTIVE

Figure3-58 Positiveregulation caused by conformationalcoupling between two distantbinding sites.In this example,both glucoseand moleculeX bind bestto the c/osedconformationof a proteinwith two domains.Becauseboth glucoseand moleculeX drivethe protein toward its closedconformation,each ligandhelpsthe otherto bind.Glucose and moleculeX arethereforesaidto bind cooperativelyto the protein.

molecule X

? I

positive r e gu l a t i o n

ACTIVE 10% active

100% active

The relationships sho'o.rnin Figures 3-58 and 3-59 apply to all proteins, and they underlie all of cell biology. They seem so obvious in retrospect that we now take it for granted. But the discovery of linkage in studies of a few enzymes in the 1950s,followed by an extensive analysis of allosteric mechanisms in proteins in the early 1960s, had a revolutionary effect on our understanding of biology. Since molecule X in these examples binds at a site on the enzyme that is distinct from the site where catalysis occurs, it need have no chemical relationship to glucose or to any other ligand that binds at the active site. Moreover, as we have just seen, for enzymes that are regulated in this way, molecule X can either turn the enzyme on (positive regulation) or turn it off (negative regulation). By such a mechanism, allosteric proteins serve as general switches that, in principle, allow one molecule in a cell to affect the fate of anv other.

SymmetricProteinAssemblies ProduceCooperative Allosteric Transitions A single-subunit enzyme that is regulated by negative feedback can at most decreasefrom 90% to about l0% activity in responseto a 1O0-foldincreasein the concentration of an inhibitory ligand that it binds (Figure 3-60, red line). Responsesof this type are apparently not sharp enough for optimal cell regulation, and most enzymes that are turned on or off by ligand binding consist of s).rynmetricassemblies of identical subunits. with this arrangement, the binding of a molecule of ligand to a single site on one subunit can promote an allosterii change in the entire assembly that helps the neighboring subunits bind the same ligand. As a result, a cooperatiue allosteric transition occurs (Figure 3-60, blue line), allowing a relatively small change in ligand concentration in the cell to switch the whole assembly from an almost fully active to an almost fully inactive conformation (or vice versa).

C

molecule X I

{ negative regu lation

100%active

1 0 %a c t i v e

Figure3-59 Negativeregulation caused by conformationalcoupling between two distant binding sites.The scheme hereresembles that in the orevious figure,but here moleculeX prefersthe open conformation,while glucoseprefers the c/osedconformation.Becauseglucose and moleculeX drivethe proteintoward oppositeconformations(closedand open, respectively), the presenceof eitherligandinterferes with the binding of the other.

173

PROTEIN FUNCTION

I I

o a ^_

EU) N c o o o o

5 i n h i b i t o rc o n c e n t r a t i o n-

The principles involved in a cooperative "all-or-none" transition are the same for all proteins, whether or not they are enzymes.But they are perhaps easiest to visualize for an enzyme that forms a s).rynmetricdimer. In the example sholtryrin Figure 3-61, the first molecule of an inhibitory ligand binds with great difficulty since its binding disrupts an energetically favorable interaction between the two identical monomers in the dimer. A second molecule of inhibitory ligand now binds more easily,however, because its binding restores the energetically favorable monomer-monomer contacts of a symmetric dimer (this also completely inactivates the enzyme). As an alternative to this inducedfirmodel for a cooperative allosteric transition, we can view such a symmetrical enzyme as having only two possible conformations, corresponding to the "enzyme on" and "enzyme off" structures in Figure 3-61. In this view, ligand binding perturbs an all-or-none equilibrium between these two states,thereby changing the proportion of active molecules. Both models represent true and useful concepts; it is the second model that we shall describe next.

Figure3-60 Enzymeactivity versusthe concentrationof inhibitoryligandfor single-subunitand multisubunit allostericenzymes.Foran enzymewith a singlesubunit (redline),a drop from 900/o activity(indicated enzymeactivityto 10o/o by the two dots on the curve)requiresa of in the concentration 10O-foldincrease Theenzymeactivityis inhibitor. from the simpleequilibrium calculated whereP is l( = tlPl/tlltPl, relationship activeprotein,I is inhibitor,and lP is the inactiveoroteinboundto inhibitor.An identicalcurveappliesto any simple bindinginteractionbetweentwo A and B.In contrast,a molecules, enzymecan multisubunitallosteric respondin a switchlikemannerto a the steep changein ligandconcentration: is causedby a cooperative response as bindingof the ligandmolecules, explainedin Figure3-61.Here,the green /ine representsthe idealizedresult expectedfor the cooperativebinding of to an two inhibitoryligandmolecules enzymewith two subunits,and allosteric the blueline showsthe idealized of an enzymewith four response subunits.As indicatedby the two dots on eachof thesecurves,the morecomplex activity enzymesdrop from 90o/oro10o/o overa much narrowerrangeof inhibitor than doesthe enzyme concentration composedof a singlesubunit.

ls Transcarbamoylase in Aspartate TheAllosteric Transition Understood in AtomicDetail One enzyme used in the early studies of allosteric regulation was aspartate transcarbamoylase from E coli. lt catalyzesthe important reaction that begins the synthesisof the pyrimidine ring of C, U, and T nucleotides: carbamoyl phosphate + aspartate -+ ly'-carbamoylaspartate.One of the final products of this pathway, cltosine triphosphate (CTP),binds to the enzyme to turn it off whenever CTP is plentiful. Aspartate transcarbamoylaseis a large complex of six regulatory and six catalyic subunits. The catalyic subunits form two trimers, each arranged in the shape of an equilateral triangle; the two trimers face each other and are held

Figure3-61 A cooperativeallosterictransitionin an enzymecomposed of how the conformation of two identicalsubunits.Thisdiagramillustrates The bindingof a single one subunitcan influencethat of its neighbor. moleculeof an inhibitoryligand (yellow)to one subunitof the enzyme of this subunit occurswith difficultybecauseit changesthe conformation and therebydisruptsthe symmetryof the enzyme.Oncethis the energygainedby changehasoccurred,however, conformational restoringthe symmetricpairinginteractionbetweenthe two subunits makesit especially easyfor the secondsubunitto bind the inhibitory the binding change.Because ligandand undergothe sameconformational the affinitywith whichthe other of the firstmoleculeof ligandincreases of the enzymeto changesin subunitbindsthe sameligand,the response of an of the ligandis much steeperthan the response the concentration enzymewith only one subunit(seeFigure3-60).

ON ENZYME

inhibitor

EASY TRANSITION

tr

174

Chapter3: Proteins

INACTIVE ENZYME: T STATE cata lytic s ub u n i t s

CTP

e

#

5nm ACTIVEENZYME:R STATE

together by three regulatory dimers that form a bridge between them. The entire molecule is poised to undergo a concerted, all-or-none, allosteric transition between two conformations, designated as T (tense) and R (relaxed)states (Figure 3-62). The binding of substrates (carbamoyl phosphate and aspartate) to the catalytic trimers drives aspartate transcarbamoylase into its catalytically active R state, from which the regulatory crP molecules dissociate. By contrast, the binding of crP to the regulatory dimers converts the enzyme to the inactive T state, from which the substrates dissociate. This tug-of-war between crp and substratesis identical in principle to that described previously in Figure 3-59 for a simpler allosteric protein. But because the tug-of-war occurs in a symmetric molecule with multiple binding sites, the enzyme undergoes a cooperative allosteric transition that will turn it on suddenly as substratesaccumulate (forming the R state) or shut it off rapidly when crp accumulates (forming the T state). A combination of biochemistry and x-ray crystallography has revealedmany fascinating details of this allosteric transition. Each regulatory subunit has two domains, and the binding of crP causes the two domains to move relative to each other, so that they function like a lever that rotates the two catalytic trimers and pulls them closer together into the T state (see Figure 3-62). \.4rhenthis occurs, hydrogen bonds form between opposing catal)'tic subunits. This helps widen the cleft that forms the active site within each catalytic subunit, thereby disrupting the binding sites for rhe substrates (Figure 3-63). Adding large amounts of substrate has the opposite effect, favoring the R state by binding in the cleft of each catalytic subunit and opposing the above conformational change. conformations that are intermediate between R and T are unstable, so that the enzyme mostly clicks back and forth between its R and T forms, producing a mixture of these two speciesin proportions that depend on the relitive concentrations of CTP and substrates.

Figure3-62 The transition between R and T statesin the enzyme aspartate transcarbamoylase.The enzyme consists of a complexof sixcatalytic subunitsand six regulatorysubunits,and the structures of its inactive(T state)and active(Rstate)forms havebeen determinedby x-raycrystallography. The enzymeis turned off by feedback inhibitionwhen CTPconcentrations rise. Eachregulatorysubunitcan bind one moleculeof CTP, which is one of the final productsin the pathway.By meansof this negativefeedbackregulation, the pathway is preventedfrom producingmore CTP than the cell needs.(Basedon K.L.Krause, K.W.Volzand W.N.Lipscomb, Proc.Natl Acad.Sci.U.5.A.82:1643-1647, 1985. With permission from NationalAcademy of Sciences.)

175

PROTEIN FUNCTION

Arg 167

Ws164

(g,rcs Arg229 rg 234 G l u2 3 9

T state (inactive)

in Proteins Are Drivenby ProteinPhosphorylation ManyChanges Proteins are regulated by more than the reversible binding of other molecules.A second method that eucaryotic cells use to regulate a protein's function is the covalent addition of a smaller molecule to one or more of its amino acid side chains. The most common such regulatory modification in higher eucaryotes is the addition of a phosphate group. We shall therefore use protein phosphorylation to illustrate some of the general principles involved in the control of protein function through the modification of amino acid side chains. A phosphorylation event can affect the protein that is modified in two important ways. First, because each phosphate group carries two negative charges,the enzyme- catalyzed addition of a phosphate group to a protein can cause a major conformational change in the protein by, for example, attracting a cluster of positively charged amino acid side chains. This can, in turn, affect the binding of ligands elsewhere on the protein surface, dramatically changing the protein's activity. \A/trena second enzyme removes the phosphate group, the protein returns to its original conformation and restoresits initial activity. Second, an attached phosphate group can form part of a structure that the binding sites of other proteins recognize.As previously discussed,certain protein domains, sometimes referred to as modules, appear very frequently as parts of larger proteins. One such module is the SH2 domain, described earliel which binds to a short peptide sequence containing a phosphorylated tyrosine side chain (seeFigure 3-398). More than ten other common domains provide binding sites for attaching their protein to phosphorylated peptides in other protein molecules, each recognizingaphosphorylated amino acid side chain in a different protein context. As a result, protein phosphorylation and dephosphorylation very often drive the regulated assembly and disassembly of protein complexes (seeFigure 15-22). Reversible protein phosphorylation controls the activity, structure, and cellular Iocalization of both enzymes and many other types of proteins in

Figure3-63 Part of the on-off switch in the catalyticsubunitsof aspartate Changesin the transcarbamoylase. interactions indicatedhydrogen-bonding for switchingthis arepartlyresponsible enzyme'sactivesite betweenactive (yellow)and inactiveconformations. Hydrogenbonds are indicatedby thin red /ines. Theaminoacidsinvolvedin the interactionin the T state subunit-subunit are shown in red,while thosethat form the activesite of the enzymein the R state areshownin blue.Thelargedrawings showthe catalyticsitein the interiorof the enzyme;the boxed sketchesshow the samesubunitsviewedfrom the enzyme's externalsurface.(Adaptedfrom E.R.Kantrowitzand W.N.Lipscomb,Irends 1990.With Biochem. Sci.15:53-59, permissionfrom Elsevier.)

'176

Chapter3: Proteins

eucaryotic cells. In fact, this regulation is so extensive that more than one-third of the 10,000or so proteins in a tlpical mammalian cell are thought to be phosphorylated at any given time-many with more than one phosphate. As might be expected, the addition and removal of phosphate groups from specific proteins often occur in responseto signals that specify some change in a cell'sstate. For example, the complicated series of events that takes place as a eucaryotic cell divides is largely timed in this way (discussedin Chapter 17), and many of the signals mediating cell-cell interactions are relayed from the plasma membrane to the nucleus by a cascadeofprotein phosphorylation events (discussed in Chapter 15).

A Eucaryotic CellContainsa LargeCollection of ProteinKinases and ProteinPhosphatases Protein phosphorylation involves the enzyme- catalyzedtransfer of the terminal phosphate group of an ATP molecule to the hydroxyl group on a serine, threonine, or tyrosine side chain of the protein (Figure 3-64). A protein kinase catalyzesthis reaction, and the reaction is essentiallyunidirectional because of the large amount of free energy released when the phosphate-phosphate bond in ATP is broken to produce ADP (discussedin chapt er 2) . Aprotein phosphatase catalyzesthe reversereaction of phosphate removal, or dephosphorylation. cells contain hundreds of different protein kinases, each responsible for phosphorylating a different protein or set of proteins. There are also many different protein phosphatases;some are highly specific and remove phosphate groups from only one or a few proteins, whereas others act on a broad range of proteins and are targeted to specific substratesby regulatory subunits. The state ofphosphorylation of a protein at any moment, and thus its activity, depends on the relative activities of the protein kinases and phosphatasesthat modiff it. The protein kinases that phosphorylate proteins in eucaryotic cells belong to a very large family of enzymes, which share a catal),'tic(kinase) sequence of about 290 amino acids. The various family members contain different amino acid sequences on either end of the kinase sequence (for example, see Figure 3-10), and often have short amino acid sequencesinserted into loops within it (red arrowheadsin Figure 3-65). Some of these additional amino acid sequences enable each kinase to recognize the specific set ofproteins it phosphorylates, or to bind to structures that localize it in specific regions of the cell. Other parts of the protein regulate the activity of each kinase, so it can be turned on and off in response to different specific signals,as described below. By comparing the number of amino acid sequence differences between the various members of a protein family, we can construct an "evolutionary tree" that is thought to reflect the pattern of gene duplication and divergence that gave rise to the family. Figure 3-66 shows an evolutionary tree of protein kinases.Kinases with related functions are often located on nearby branches of the tree: the protein kinases involved in cell signaling that phosphorylate tyrosine side chains, for example, are all clustered in the top left corner of the tree. The other kinases shor,m phosphorylate either a serine or a threonine side chain, and many are organized into clusters that seem to reflect their functionin transmembrane signal transduction, intracellular signal amplification, cellcycle control, and so on. Figure3-65 The three-dimensional structureof a proteinkinase. Superimposed on this structureareredarrowheads to indicatesiteswhere insertions of 5-100aminoacidsarefound in somemembersof the protein kinasefamily.Theseinsertions arelocatedin loopson the surfaceof the enzymewhereother ligandsinteractwith the protein.Thus,they distinguish differentkinasesand conferon them distinctiveinteractions with other proteins. TheATp(whichdonatesa phosphategroup)and the peptideto be phosphorylated areheld in the activesire,whichextends betweenthe phosphate-binding loop (yellow)and the catalyticloop (orange).Seealso Figure3-10. (Adaptedfrom D.R.Knightonet al.,Science 253:407-414, 1991.With permission from AAAS.)

olADP

O: P-OI

o

OH I

I

s e nn e CH s i d ec h a i n

CH,

(A)

--,

k in a s e

*--l

phosphatase k in a s e -_.

:--_-/

oFF

phosphatase

(B) Figure3-64 Proteinphosphorylation. Manythousandsof proteinsin a typical eucaryotic cellaremodifiedby the covalent additionof a phosphategroup. (A)Thegeneralreaction,shownhere, transfers a phosphategroupfrom ATPto an aminoacidsidechainof the targetprotein by a protein kinase.Removalof the phosphategroup is catalyzed by a second enzymera proteinphosphatase. In this example,the phosphateis addedto a serine sidechain;in othercases, the phosphateis insteadlinkedto the -OH groupof a threonineor a tyrosinein the protein. (B)Thephosphorylation of a proteinby a proteinkinasecan eitherincrease or decreasethe protein'sactivity,depending on the siteof phosphorylation and the structureof the protein.

177

PROTEIN FUNCTION

Figure3-66 An evolutionary tree of selectedprotein kinases.Although a cellcontainshundreds highereucaryotic and the human of suchenzymes, genomecodesfor morethan 500,onlY in this bookare someof thosediscussed snown.

CdcT

PDGF receptor EGF tyrosine recepror kinase subfamily

cyclic-AMPd e p e n d e n tk i n a s e cyclic-GMPd e p e n d e n tk i n a s e p r o t e i nk i n a s eC

IGFB receptor

Ca2*/calmodulin-

m y o s i nl i g h t dependent kinase c h a i nk i n a s e s

r e c e p t o rs e r i n e k i n a s es u b f a m i l y

As a result of the combined activities of protein kinases and protein phosphatases,the phosphate groups on proteins are continually turning over-being added and then rapidly removed. Such phosphorylation cyclesmay seem wasteful, but they are important in allowing the phosphorylated proteins to switch rapidly from one state to another: the more rapid the cycle, the faster a population of protein molecules can change its state of phosphorylation in responseto a sudden change in the phosphorylation rate (see Figure 15-11). The energy required to drive this phosphorylation cycle is derived from the free energy of ATP hydrolysis, one molecule of which is consumed for each phosphorylation event.

ShowsHowa TheRegulation of Cdkand SrcProteinKinases ProteinCanFunctionasa Microchip The hundreds of different protein kinases in a eucaryotic cell are organized into complex networks of signaling pathways that help to coordinate the cell's activities, drive the cell cycle, and relay signals into the cell from the cell's environment. Many of the extracellular signals involved need to be both integrated and amplified by the cell. Individual protein kinases (and other signaling proteins) serve as input-output devices, or "microchips," in the integration process. An important part of the input to these signal processing proteins comes from the control that is exerted by phosphates added and removed from them by protein kinases and protein phosphatases,respectively. In general, specific sets of phosphate groups serve to activate the protein, while other sets can inactivate it. A cyclin-dependent protein kinase (Cdk) provides a good example.Kinasesin this classphosphorylate serinesand threonines, and they are central components of the cell-cycle control system in eucaryotic cells,as discussedin detail in Chapter 17.In avertebrate cell, individual Cdk proteins turn on and off in succession, as a cell proceeds through the different phases of its division cycle.r'A/hena particular kinase is on, it influences various aspectsof cell behavior through effects on the proteins it phosphorylates. A Cdk protein becomes active as a serine/threonine protein kinase only when it is bound to a second protein called a cyclin. But, as Figure 3-67 shows, the binding of cyclin is only one of three distinct "inputs" required to activate the Cdk. In addition to cyclin binding, a phosphate must be added to a specific threonine side chain, and a phosphate elsewherein the protein (covalently bound to a specific tyrosine side chain) must be removed. Cdk thus monitors a specific set

INPUTS

OUTPUT Figure 3-67 How a Cdk protein acts as an integrating device.The of the functionof thesecentralregulators in Chapter17' cellcycleis discussed

178

Chapter3: Proteins

fatty acid

5 0 0a m i n o a c i d s

of cell components-a cyclin, a protein kinase, and a protein phosphatase-and it acts as an input-output device that turns on if, and only if, each of these components has attained its appropriate activity state. Some cyclins rise and fall in concentration in step with the cell cycle, increasing gradually in amount until they are suddenly destroyed at a particular point in the cycle. The sudden destruction of a cyclin (by targeted proteolysis) immediately shuts off its partner Cdk enzyme, and this triggers a specific step in the cell cycle.

Figure3-68 The domain structureof the Srcfamily of protein kinases,mapped alongthe amino acid sequence.Forthe three-dimensional structureof Src.see F i q u r e3 - 1 0 .

cated by the evolutionary tree in Figure 3-66, sequence comparisons suggest that tyrosine kinases as a group were a relatively late innovation that branihed off from the serine/threonine kinases, with the src subfamily being only one subgroup of the tyrosine kinases created in this way. The src protein and its relatives contain a short N-terminal region that becomes covalently linked to a strongly hydrophobic fatty acid, which holds the kinase at the c)'toplasmic face of the plasma membrane. Next come two peptide-binding modules, a Src homology 3 (sH3) domain and a sH2 domain, followed by the kinase catalytic domain (Figure 3-68). These kinasesnormally exist in an inactive conformation, in which a phosphorylated tyrosine near the c-terminus is bound to the SH2 domain, and the sH3 domain is bound to an internal peptide in a way that distorts the active site of the en4/me and helps to render it inactive. Turning the kinase on involves at least two specific inputs: removal of the c-

processing events that enable the cell to compute logical responsesto a complex set of conditions.

Proteins ThatBindand Hydrolyze GTpAre ubiquitousceilurar Regulators we have described how the addition or removal of phosphate groups on a protein can be used by a cell to control the protein's activity. In the examples discuised so

Figure3-69 The activation of a Src-type protein kinaseby two sequentialevents. (Adaptedfrom S.C.Harrisonet al.,Ceil 112:737-7 40,2003.With permission from Elsevier.)

a c t i v a t i n gl i g a n d

k i n a s ed o m a i n

PHOSPHATE REMOVAL LOOSENS STRUCTURE

K I N A S EC A NN O W PHOSPHORYLATE T Y R O S I NTEO SELF-ACTIVATE

179

PROTEIN FUNCTION

far, the phosphate is transferred from an AIP molecule to an amino acid side chain of the protein in a reaction catalyzedby a specific protein kinase. Eucaryotic cells also have another way to control protein activity by phosphate addition and removal. In this case,the phosphate is not attached directly to the protein; instead, it is a part of the guanine nucleotide GTB which binds very tightly to the protein. In general, proteins regulated in this way are in their active conformations with GTP bound. The loss of a phosphate group occurs when the bound GTP is hydrolyzed to GDP in a reaction catalyzed by the protein itself, and in its GDP-bound state the protein is inactive. In this way, GTP-binding proteins act as on-off switches whose activity is determined by the presence or absence of an additional phosphate on a bound GDP molecule (Figure 3-71). GTP-binding proteins (also called GTPasesbecause of the GTP hydrolysis they catalyze) comprise a large family of proteins that all contain variations on the same GTP-binding globular domain. !\4ren the tightly bound GTP is hydrolyzed to GDB this domain undergoes a conformational change that inactivates it. The three-dimensional structure of a prototypical member of this family, the monomeric GTPase called Ras, is shor.tmin Figure 3-72. The Ras protein has an important role in cell signaling (discussedin Chapter 15). In its GTP-bound form, it is active and stimulates a cascade of protein phosphorylations in the cell. Most of the time, however, the protein is in its inactive, GDP-bound form. It becomes active when it exchangesits GDP for a GTP molecule in responseto extracellular signals,such as growth factors, that bind to receptors in the plasma membrane (seeFigure 15-58).

INPUTS

src-tvpeproteinkinaseactivityturnson to all of the fuliyonlyi{ the answers areYes abovequestions OUTPUT Figure3-70 How a Src-tYPeProtein kinaseacts as an integrating device.The disruotionof the 5H3domaininteraction (green)involvesreplacingits binding to the indicatedred linkerregionwith a tighterinteractionwith an activating ligand,as illustratedin Figure3-69.

Proteins RegulatoryProteinsControlthe Activityof GTP-B|nding WhetherGTPor GDPls Bound by Determining GTP-binding proteins are controlled by regulatory proteins that determine whether GTP or GDP is bound, just as phosphorylated proteins are turned on and offby protein kinases and protein phosphatases.Thus, Rasis inactivated by a GTPase-actiuating protein (GAP),which binds to the Ras protein and induces it to hydrolyze its bound GTP molecule to GDP-which remains tightlyboundand inorganic phosphate (PJ, which is rapidly released.The Ras protein stays in its inactive, GDP-bound conformation until it encounters a guanine nucleotide exchangefactor (GEF),which binds to GDP-Rasand causesit to releaseits GDP Because the empty nucleotide-binding site is immediately filled by a GTP molecule (GTPis present in large excessover GDP in cells),the GEF activatesRas by indirectly adding back the phosphate removed by GTP hydrolysis' Thus, in a sense,the roles of GAP and GEF are analogous to those of a protein phosphatase and a protein kinase, respectively (Figure 3-73).

FromSmallOnes CanBeGenerated LargeProteinMovements The Ras protein belongs to a large superfamily of monomeric GTPases,each of which consists of a single GTP-binding domain of about 200 amino acids. Over the course of evolution, this domain has also become joined to larger proteins with additional domains, creating a large family of GTP-binding proteins. Family members include the receptor-associated trimeric G proteins involved in cell signaling (discussedin Chapter 15), proteins regulating the traffic of vesicles between intracellular compartments (discussed in Chapter 13), and proteins that bind to transfer RNA and are required as assembly factors for protein

ACTIVE

NACTIVE

NACTIVE

ACTIVE

Figure3-7 1 GTP-bindingproteinsas molecularswitches.The activityof a protein(alsocalleda GTP-binding generallyrequiresthe presence GTPase) of a tightlyboundGTPmolecule(switch 'bn").Hydrolysis of this GTPmolecule producesGDPand inorganicphosphate (Pi),and it causesthe proteinto convert to a different,usuallyinactive, conformation(switch'bff").As shown here,resettingthe switch requiresthe a slow tightlybound GDPto dissociate, step that is greatlyacceleratedby specific a oncethe GDPhasdissociated, signals; moleculeof GTPis quicklyrebound.

180

Chapter3: Proteins

Figure3-72 The structureof the Ras protein in its GTP-boundform. ThismonomericGTPase illustrates the structureof a GTP-binding domain,which is presentin a largefamilyof GTP-binding proteins. Theredregionschangetheir conformation when the GTPmoleculeis hydrolyzed to GDPand inorganic phosphateby the protein;the GDP remainsboundto the protein,whilethe inorganicphosphateis released. The specialroleof the "switchhelix"in proteinsrelatedto Rasis explainednext (seeFigure3-75).

synthesis on the ribosome (discussedin chapter 6). In each case,an important biological activity is controlled by a change in the protein's conformation that is caused by GTP hydrolysis in a Ras-like domain. The EF-Tu protein provides a good example of how this family of proteins works. EF-Tu is an abundant molecule that servesas an elongation factor (hence the EF) in protein synthesis, loading each aminoacyl tRNA molecule onto the ribosome. The tRNA molecule forms a tight complex with the GTp-bound form of EF-Tu (Figure 3-74). In this complex, the amino acid attached to the IRNA is improperly positioned for protein slmthesis. The IRNA can transfer its amino acid only after the GTP bound to EF-Tu is hydrolyzed on the ribosome, allowing the EF-Tu to dissociate. Since the GTp hydrolysis is triggered by a proper fit of the IRNA to the mRNA molecule on the ribosome, the EF-Tu serves as a factor that discriminates between correct and incorrect mRNA-IRNA pairings (seeFigure 6-67 for a further discussion of this function of EF-Tu). By comparing the three-dimensional structure of EF-Tu in its GTp-bound and GDP-bound forms, we can see how the repositioning of the IRNA occurs. The dissociation of the inorganic phosphate group (pJ, which follows the reaction GTP -+ GDP + Pi, causes a shift of a few tenths of a nanometer at the GTpbinding site, just as it does in the Rasprotein. This tiny movement, equivalent to rN. I 'IiGNAL r f

-

I,t GPP APP P APP

GPP P

srcrunl our l

\...',/

\?

S I G N A L I N GB Y P H O S P H O R Y L A T E D PROTEIN

S I G N A L I NB GY G T P - B I N D I NPGR O T E I N

Figure3-73 A comparisonof the two major intracellularsignaling mechanismsin eucaryoticcells.In both casesr a signalingproteinis activatedby the additionofa phosphategroupand inactivated by the removalof this phosphate. To emphasize the similarities in the two pathways,ATPand GTPare drawnas APPPand GPPP, and ADPand GDPasAPPand GPBrespectively. As shownin Figure3-64,the additionof a phosphateto a proteincanalsobe inhibitorv.

181

PROTEIN FUNCTION Thethree Figure3-74An aminoacyl boundto EF-Tu. tRNAmolecule proteinarecolored 3-75. to matchFigure differently, domains of theEF-Tu proteinexists protein; in however, a verysimilar Thisisa bacterial (Coordinates et by P.Nissen determined whereit iscalledEF-1. eucaryotes, fromAAA5.) 270:1464-1472, 1995. Withpermission al.,Science a few times the diameter of a hydrogen atom, causes a conformational change to propagate along a crucial piece of a helix, called Ihe switch helix, in the Raslike domain of the protein. The switch helix seems to serve as a latch that adheresto a specific site in another domain of the molecule, holding the protein in a "shut" conformation. The conformational change triggered by GTP hydrolysis causesthe switch helix to detach, allowing separatedomains of the protein to swing apart, through a distance of about 4 nm. This releasesthe bound IRNA molecule, allowing its attached amino acid to be used (Figure 3-75). Notice in this example how cells have exploited a simple chemical change that occurs on the surface of a small protein domain to create a movement 50 times larger.Dramatic shape changesof this type also causethe verylarge movements that occur in motor proteins, as we discuss next.

MotorProteinsProduceLargeMovementsin Cells We have seen that conformational changes in proteins have a central role in enzyrne regulation and cell signaling. We now discuss proteins whose major function is to move other molecules. These motor proteins generate the forces responsible for muscle contraction and the crawling and swimming of cells. Motor proteins also power smaller-scaleintracellular movements: they help to move chromosomes to opposite ends of the cell during mitosis (discussedin Chapter 17),to move organellesalong molecular tracks within the cell (discussed site of tRNA binding

GTPbinding site switch helix

(A)

(B)

(A)The three-dimensionalstructureof EF-Tuwith Figure3-75 The large conformationalchange in EF-Tucausedby GTPhydrolysis. is the switchhelix,which movesafterGTP lix protein, its and Ras GTPbound.The domainat the top hasa structuresimilarto the (B)Thechangein the conformation of the switchhelixin domain1 causesdomains2 and 3 to rotateas a singleunit by about90" hydrolysis. toward the viewer,which releasesthe IRNAthat was shown bound to this structurein Figure3-74. (A,adaptedfrom H. Berchtoldet al.,Noture Ltd.B,courtesyof MathiasSprinzland RolfHilgenfeld') from MacmillanPublishers 365:126-132,1 993.With permission

182

Chapter3: Proteins

in chapter 16), and to move enzyrnes along a DNA strand during the synthesis of a new DNA molecule (discussed in chapter 5). All these fundamental processesdepend on proteins with moving parts that operate as force-generating machines. How do these machines work? In other words, how do cells use shape changes in proteins to generate directed movements? If, for example, a protein is required to walk along a narrow thread such as a DNA molecule, it can do this by undergoing a series of conformational changes,such as those shor,rrnin Figure 3-76. But with nothing to drive these changes in an orderly sequence,they are perfectly reversible, and the protein can only wander randomly back and forth along the thread. we can look at this situation in another way. Since the directional movement of a protein does work, the laws of thermodynamics (discussed in chapter 2) demand that such movement use free energy from some other source (otherwise the protein could be used to make a perpetual motion machine). Therefore, without an input of energy,the protein molecule can only wander aimlessly. How can the cell make such a series of conformational changes unidirectional? To force the entire cycle to proceed in one direction, it is enough to make any one of the changes in shape irreversible. Most proteins that are able to walk in one direction for long distances achieve this motion by coupling one of the conformational changes to the hydrolysis of anATp molecule bound to the protein. The mechanism is similar to the one iust discussed that drives allosteric

Figure3-76 An allosteric"walking" protein. Although its three different conformationsallow it to wander randomlybackand forth while boundto a threador a filament,the protein cannot moveuniformlyin a singledirection.

In the model shorrrmin Figure 3-zz, Nlp binding shifts a motor protein from conformation I to conformation 2.The bound ATp is then hydrolyzed to produce ADP and inorganic phosphate (PJ, causing a change from conformation 2

Many motor proteins generate directional movement in this general way, including the muscle motor protein myosin, which walks along actin filamenis to generatemuscle contraction, and the kinesinproteins that walk along microtubules (both discussedin chapter l6). These movements can be rapid:iome of the motor proteins involved in DNA replication (the DNA helicises) propel themselves along a DNA strand at rates as high as 1000nucleotides p". second.

Membrane-Bound Transporters Harness Energyto pump Molecules ThroughMembranes

HYDROLYSIS

we have thus far seen how allosteric proteins can act as microchips (cdk and Src kinases),as assembly factors (EF-Tu),and as generatorsof mechanical force and motion (motor proteins). Allosteric proteins can also harness energy derived from ATP hydrolysis, ion gradients, or electron transport processesto pump specific ions or small molecules acrossa membrane. we consider one ex€rrnptetrere; others will be discussedin Chapter ll. The ABC transporters constitute an important class of membrane-bound pump proteins. In humans at least 48 different genesencode them. These transporters mostly function to export hydrophobic molecules from the cytoplasm, Figure3-77 An allostericmotor protein.The transitionbetweenthree differentconformations includesa stepdrivenby the hydrolysis of a bound ATPmolecule,and this makesthe entirecycleessentially irreversible. By repeatedcycles,the proteinthereforemovescontinuouslyto the right alongthe thread.

direction of movement

183

PROTEIN FUNCTION

m e m b r a n e - s p a n n i nsgu b u n i t s

lipid bilayer

CYTOSOL

Figure3-78 The ABC(ATP-binding cassette)transporter,a protein machine that pumps large hydrophobic molecules through a membrane.(A)The bacterial BtuCDprotein,whichimportsvitamin812 into E coli usingthe energyofATP of The bindingof two molecules hydrolysis. ATPclampstogetherthe two ATP-binding The structureis shownin its ADPsubunits. bound state,wherethe channelto the spacecan be seento be open extracellular but the gateto the cytosolremainsclosed. (B)Schematic of substrate illustration In bacteria, pumpingby ABCtransporters. the bindingof a substratemoleculeto the faceof the proteincomplex extracellular triggersATPhydrolysisfollowed by ADP gate; whichopensthe cytoplasmic release, the pump is then resetfor anothercycle.In eucaryotes,an oppositeprocessoccurs, to be pumped causingsubstratemolecules out ofthe cell.(A,adaptedfrom K.P.Locher, Curr. Ooin. Struct.Biol. 14:426-441,2004' from Elsevier.) With permission

A T P - b i n d i n sgu b u n i t s

ABCTRANSPORTER A EUCARYOTIC

(B) A BACTERIAL ABCTRANSPORTER s u b s t r a t em o l e c u l e

l:,

CI'TOSOL substrate molecule

,

\ 2P

{;

\ ATP-binding

serving to remove toxic molecules at the mucosal surface of the intestinal tract, for example, or at the blood-brain barrier. The study of ABC transporters is of intense interest in clinical medicine, because the overproduction of proteins in this class contributes to the resistance of tumor cells to chemotherapeutic drugs. And in bacteria, the same tlpe of proteins primarily function to import essential nutrients into the cell. The ABC transporter is a tetramer, with a pair of membrane-spanning subunits linked to a pair of ATP binding subunits located just below the plasma membrane (Figure 3-78A). As in other exampleswe have discussed,the hydrolysis of the bound ATP molecules drives conformational changes in the protein, transmitting forces that cause the membrane-spanning subunits to move their bound molecules acrossthe lipid bilayer (Figure 3-788). Humans have invented many different types of mechanical pumps, and it should not be surprising that cells also contain membrane-bound pumps that function in other ways. Among the most notable are the rotary pumps that couple the hydrolysis of ATP to the transport of H* ions (protons). These pumps resemble miniature turbines, and they are used to acidify the interior of lysosomes and other eucaryotic organelles.Like other ion pumps that create ion gradients, they can function in reverseto catalyzethe reactionADP + Pr-+ ATB if the gradient acrosstheir membrane of the ion that they transport is steep enough. One such pump, the ATP slrrthase, harnessesa gradient of proton concentration produced by electron transport processesto produce most of the AIP used in the living world. This ubiquitous pump has a central role in energy conversion, and we shall discussits three-dimensional structure and mechanism in Chapter 14.

+zri zi.:#Mi

184

Chapter3: Proteins

ProteinsOftenFormLargeComplexes ThatFunctionas protein Machines Large proteins formed from many domains are able to perform more elaborate functions than small, single-domain proteins. But large protein assemblies formed from many protein molecules perform the most impressive tasks. Now that it is possible to reconstruct most biological processesin cell-free systemsin the laboratory, it is clear that each of the central processesin a cell-such as DNA replication, protein synthesis,vesicle budding, or transmembrane signaling-is catalyzed by a highly coordinated, linked set of I0 or more proteins. In most such protein machines, an energetically favorable reaction such as the hydrolysis of bound nucleoside triphosphates (ATp or GTp) drives an ordered series of conformational changes in one or more of the individual protein subunits, enabling the ensemble of proteins to move coordinately. In this way, each enzyme can be moved directly into position, as the machine catalyzessuccessive reactions in a series.This is what occurs, for example, in protein synthesis on a ribosome (discussedin chapter 6)-or in DNA replication, where a large multiprotein complex moves rapidly along the DNA (discussedin chapter 5). cells have evolved protein machines for the same reason that humans have invented mechanical and electronic machines. For accomplishing almost any task, manipulations that are spatially and temporally coordinated through linked processesare much more efficient than the use of individual tools.

ProteinMachines with Interchangeable PartsMakeEfficientuse of Geneticlnformation To probe more deeply into the nature of protein machines, we shall consider a relatively simple one: the SCF ubiquitin ligase. This protein complex binds different "target proteins" at different times in the cell cycle, and it covalently adds multiubiquitin polypeptide chains to these proteins. Its c-shaped structure is formed from five protein subunits, the largest of which is a molecule that serves as a scaffold protein on which the rest of the structure is built. The structure underlies a remarkable mechanism (Figure 3-zg). At one end of the c is an E2 ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, a subunit knovrn as an F-box protein.These two subunits are separatedby a gap of about 5 nm. \Mhen this protein complex is acrivated, the F-box protein binds to a specific site on a target protein, positioning the protein in the gap so that some of its lysine side chains contact the ubiquitin-conjugating This enzyme can then catalyze the repeated addition of a ubiquitin "nry-e. polypeptide to these lysines (seeFigure 3-79c), producing a polyubiquitin chain that marks rhe target protein for rapid destruction in a proteasome (seep. 393). In this manner, specific proteins are targeted for rapid destruction in

PROTEIN FUNCTION

adaptor protein 2 1)

F-boxprotein ( s u b s t r a t e - b i n d i nagr m )

E 2u b i q u i t i n conjugating enzyme ubiquitin

I I two oJ many possible substrate-bind ing arms

(B)

p o l y u b i q ui t y l a t e d protein targeted lor destruction ,/r

(C)

u b i q u i t i nl i g a s e

in cells, inasmuch as new functions can evolve for the entire complex simply by producing an alternative version of one of its subunits.

OftenInvolvesPositioning TheActivationof ProteinMachines Themat SpecificSites As scientists have learned more of the details of cell biology, they have recognized increasing degreesof sophistication in cell chemistry. Thus, not only do we now know that protein machines play a predominant role, but it has recently become clear that most of these machines form at specific sites in the cell, being activated only where and when they are needed. Using fluorescent, GFP-tagged fusion proteins in living cells (see p. 593), cell biologists are able to follow the repositioning of individual proteins that occurs in response to specific signals. Thus, when certain extracellular signaling molecules bind to receptor proteins in the plasma membrane, they often recruit a set of other proteins to the inside surface of the plasma membrane to form protein machines that pass the signal on. As an example, Figure 3-804 illustrates the rapid movement of a protein kinase C (PKC)enzyme to a complex in the plasma membrane, where it associates with specific substrate proteins that it phosphorylates. There are more than 10 distinct PKC enzymes in human cells, which differ both in their mode of regulation and in their functions. When activated, these enz]rynesmove from the cytoplasm to different intracellular locations, forming specific complexes with other proteins that allow them to phosphorylate different protein substrates (Figure 3-808). The SCF ubiquitin ligases can also move to specific sites of function at appropriate times. As will be explained when we discuss cell signaling in Chapter 15, the mechanisms frequently involve protein phosphorylation, as well as scaffold proteins that link together a set of activating, inhibiting, adaptol and substrate proteins at a specific location in a cell. This general phenomenon is known as induced proximity, and it explains the otherwise puzzling observation that slightly different forms of enzymes with the same catalltic site will often have very different biological functions. Cells change the locations of their proteins by covalently modifying them in a variety of different ways, as part of a "regulatory code" to be described next.

Figure3-79 The structure and mode of actionof a SCFubiquitinligase.(A)The structureof the five-proteincomplexthat The includesan E2ubiquitinligase. proteindenoted hereas adapterprotein 1 is the Rbxl/Hrt1protein,adaPtor protein2 is the 5kp1protein,and the cullinis the Cull protein.(B)Comparison of the samecomplexwith two different arms,the F-box substrate-binding proteins Skp2 (top) and p-trCPl (bottom), (C)The binding and respectively. ubiquitylationof a target protein by the a SCFubiquitinligase.lf,as indicated, chainof ubiquitinmoleculesis addedto the samelysineof the target protein,that protein is markedfor rapid destructionby the proteasome.(A and B,adaptedfrom G.Wu et al.,Mol.Cell11:1445-1456,2003. With oermissionfrom Elsevier.)

r86

C h a p t e r3 : P r o t e i n s

0 min

3 min

(A)

,

I

(B) 2 0u m

1 0u m

unstructured regton , ,,,i

(C)

1 0m i n

rapid collisions +

structured d o m ar n

'fhese

rnoditicationscreate sites on proteins that bind them to particular scafftlld proteins,therebyclusteringthe proteins required for particular reactionsin specificregionsof the cell. Most biologicalreactionsare catalyzedby setsof 5 or more proteins, and such a clustering of proteins is often required for the reaction to occur. Scaffoldsthereby allow cells to compartmentalizereactionseven irr the absence of membranes. Although onty recently recognized as a widespread phenomenon, this tvpe of clustering is particularly obvious in the cell nucleus (seeFigure4-69). Many scaffolds appear to be quite different from the cullin illustrated previously in Figure 3-79: rather than holding their bound proteins in precisepositions lelative to each other, the interacting proteins are linked by unstructured regionsof polvpeptide chain. This tethersthe proteins together,causingthem to collicle frequently with each other in random orientations-some of which will lead to a productive reaction (Figure3-B0c). In essence,this mechanism greatly speeds reactions by creating a very high local concentration of the reacting species.For this reason,the use ofscaffold proteins representsan especiallyversatileway of controlling cell chemistry (seealso Figure l5-61).

Figure3-80 The assemblyof protein machinesat specificsitesin a cell. (A)In response to a signal(herea phorbol ester),the gammasubspecies of protein kinaseC movesrapidlyfrom the cytosol to the plasmamembrane. The protein kinaseis fluorescent in theselivingcells becausean engineered geneinsidethe cellencodesa fusionproteinthat links the kinaseto greenfluorescent protein (GFP). (B)Thespecificassociation of a differentsubspecies of proteinkinaseC (aPKC) with the apicaltip of a differentiating neuroblastin an early Drosophila embryo.The kinaseis stained red,andthe cellnucleusgreen. (C)Diagramillustrating how a simple proximitycreatedby scaffoldproteins cangreatlyspeedreactionsin a cell.In this example,long unstructured regions of polypeptidechainin a largescaffold proteinconnecta seriesof structured domainsthat bind a setof reacting proteins. The unstructured regionsserve asflexible"tethers"thatgreatlyspeed reactionratesby causinga rapid,random collisionof all of the proteinsthat are (Fora simple boundto the scaffold. exampleof tethering,seeFigure16-38.) (A,from N. Sakaiet al,J. CellBiol. 139:1465-1476, 1997.With permission from The Rockefeller University Press. B,courtesyof AndreasWodarz,Institute of Genetics, University of Dr.isseldorf, Germany.)

Many ProteinsAre controlled by Multisitecovalent Modification we have thus far described only one type of posttranslational modification of proteins-that in which a phosphate is covalentlv attached to an amino acid side chain (seeFigure3-64). But a largenumber of other such modifications also occLlr,rnore than 200 distinct types being known. To give a senseof the variety, lable 3-3 presents a subset of modifying groups with known regulatory roles.As Table3-3 SomeMoleculesCovalentlyAttachedto ProteinsRegulateProteinFunction MODIFYING GROUP

SOMEPROMINENT FUNCTIONS

Phosphateon Ser,Thr,or Tyr Methylon Lys

Drivesthe assembly of a proteininto largercomplexes (seeFigure15-,|9). Helpsto creates histonecodein chromatin throughformingeithermono-, di-,or tri-methyllysine(seeFigure4-38). Helpsto creates histonecodein chromatin(seeFigure4-38). Thisfattyacidadditiondrivesproteinassociation (see with membranes Figurel0-20). Controls enzymeactivityandgeneexpression in glucosehomeostasis. Monoubiquitin additionregulates the transportof membrane proteinsin vesicles (seeFigure13-58). A polyubiquitin chaintargetsa proteinfor degradation (seeFigure3-79).

Acetylon Lys Palmitylgroupon Cys N-acetylglucosamine on Seror Thr Ubiquitinon Lys

U b i q u i tsi na / 6 a m i n o a c i d p o y p e pt ht iedree;a r e e aa t stl0otherubiquitin-relatedproteins,suchasSUMo,thatmodifyproteinsins

187

PROTEIN FUNCTION

P

pl

iltl PP

P

P

P

Ac Ac

ttl

P

P

I

ubiquitin

w:"

P

T E T R A M E R I Z A T I OC N- T E R M I N A L REGULATORY DOMAIN DOMAIN

D N A - B I N D I NDGO M A I N

P R O T E Ip Ns 3

.e. phosphate SUMO

r,l P

TRANSACTIVATION DOMAIN (A)

{ti'

$P,' methyl

S I G N A L I N IGN P U T S

ts+J

/t+\

/

,

i

i

V

A

e

\/ VV ( B ) H I S T O NH E3

T H EC O D EI SR E A D

B I N DT O M O V ET O M O V ET O ^ r n \ / ET ^ pRoTEASoME pRoTErNsor or PLASMA ill;.];,',: or F O RD E G R A D A T I O N M E M B R A N E Y ANDZ

(c)

in phosphate addition, these groups are added and then removed from proteins according to the needs of the cell. A large number of proteins are now knor,vnto be modified on more than one amino acid side chain, with different regulatory events producing a different pattern of such modifications. A striking example is the protein p53, which plays a central part in controlling a cell'sresponseto adversecircumstances (seep. I 105). Through one of four different tlpes of molecular additions, this protein can be modified at 20 different sites (Figure 3-SfA). Becausean enormous number of different combinations of these 20 modifications are possible, the proteins behavior can in principle be altered in a huge number of ways. Moreover, the pattern of modifications on a protein can determine its susceptibility to further modification, as illustrated by histone H3 in Figure 3-BlB. Cell biologists have only recently come to recognize that each protein's set of covalent modifications constitutes an importanl combinatorial regulatory code' As specific modi$ring groups are added to or removed from a protein, this code causes a different set of protein behaviors-changing the activity or stability of the protein, its binding partners, and its specific location within the cell (Figure 3-8iC). This helps the cell respond rapidly and with great versatility to changes in its condition or environment.

Cell Underlies A ComplexNetworkof ProteinInteractions Function There are many challengesfacing cell biologists in this "post-genome" era when complete genome sequences are knor.tm.One is the need to dissect and reconstruct each one of the thousands of protein machines that exist in an organism such as ourselves. To understand these remarkable protein complexes, each must be reconstituted from its purified protein parts, so that we can study its detailed mode of operation under controlled conditions in a test tube, free from

Figure3-81 Multisiteprotein modification and its effects.A protein addition that carriesa post-translational to morethan one of its aminoacidside to carrya chainscan be considered regulatorycode.(A)The combinatorial oatternof known covalentmodifications to the proteinp53;ubiquitinand SUMO (seeTable3-3). arerelatedpolypeptides (B)The possiblemodifications on the first of 20 aminoacidsat the N-terminus histoneH3,showingnot onlYtheir locationsbut alsotheir activating(b/ue.) and inhibiting (red)effectson the additionof neighboringcovalent modifications.In additionto the effects and methylation shown,the acetylation of a lysinearemutuallyexclusive reactions(seeFigure4-38).(C)Diagram showingthe generalmannerin which areaddedto (and multisitemodifications removedfrom)a proteinthrough signalingnetworks,and how the regulatorycode resultingcombinatorial on the protein is readto alter its behavior in the cell.

188

Chapter3: Proteins

all other cell components. This alone is a massive task. But we now know that each of these subcomponents of a cell also interacts with other sets of macromolecules, creating a large network of protein-protein and protein-nucleic acid interactions throughout the cell. To understand the cell, therefore, we need to analyzemost of these other interactions as well. We can gain some idea of the complexity of intracellular protein netvvorks from a particularly well-studied example described in Chapter 16: the many dozens of proteins that interact with the actin cytoskeleton in the yeast saccharomycescereuisiae(seeFigure l6-18). The extent of such protein-protein interactions can also be estimated more generally. An enormous amount of valuable information is now freely available in protein databaseson the Internet: tens of thousands of three-dimensional protein structures plus tens of millions of protein sequencesderived from the nucleotide sequencesofgenes. Scientistshave been developing new methods for mining this great resource to increase our understanding of cells. In particular, computer-based bioinformatics tools are being combined with robotics and microarray technologies (seep. s74) to allow thousands of proteins to be investigated in a single set of experiments. proteomics is a term that is often used to describe such research focused on the large-scaleanalysis of proteins, analogous to the term genomics describing the Iarge-scaleanalysis of DNA sequencesand genes. Biologists use two different large-scalemethods to map the direct binding interactions between the many different proteins in a cell. The initial method of choice was based on genetics: through an ingenious technique known as the yeast two-hybrid screen (see Figure 8-24), tens of thousands of interactions between thousands of proteins have been mapped in yeast,a nematode, and the fruit fly Drosophila. More recently, a biochemical method based on affinity tagging and mass spectroscopy has gained favor (discussedin chapter 8), because it appears to produce fewer spurious results.The results of these and other analyses that predict protein binding interactions have been tabulated and organized in Internet databases.This allows a cell biologist studying a small set of proteins to readily discover which other proteins in the same cell are thought to bind to, and thus interact with, that set of proteins. \Arhendisplayed graphically as a protein interaction map, eachprotein is representedby a box or dot in a twodimensional network, with a straight line connecting those proteins that have been found to bind to each other. \Mhen hundreds or thousands of proteins are displayed on the same map, the network diagram becomes bewilderingly complicated, serving to illustrate how much more we have to learn before we can claim to really understand the cell. Much more useful are small subsections of these maps, centered on a few proteins of interest. Thus, Figure 3-82 shows a network of protein-protein interactions for the five proteins that form the SCFubiquitin ligase in a yeast cell (see Figure 3-79). Four of the subunits of this ligase are located at the bottom right of Figure 3-82. The remaining subunit, the F-box protein that serves as its substrate-binding arm, appears as a set of 15 different gene products that bind to adaptor protein 2 (the Skpl protein). Along the top and left of the figure are sets of additional protein interactions marked with yellow and green shading: as indicated, these protein sets function at the origin of DNA replication, in cell cycle regulation, in methionine slmthesis, in the kinetochore, and in vacuolar H+ArPase assembly.we shall use this figure to explain how such protein interaction maps are used, and what they do and do not mean. 1. Protein interaction maps are useful for identifuing the likely function of previously uncharacterized proteins. Examples are the products of the genes that have thus far only been inferred to exist from the yeast genome sequence,which are the six proteins in the figure that lack a simple threeletter abbreviation (white lettersbeginning withy). one, the product of socalled open readingframeYDRlg6c, is located in the origin of replication group' and it is therefore likely to have a role in starting new replication forks. The remaining five in this diagram are F-box proteins thai bind to Skpl; these are therefore likely to function as part of the ubiquitin ligase, serving as substrate-binding arms that recognize different target proteins.

189

P R O T E IFNU N C T I O N

However, as we discussnext, neither assignment can be considered certain without additional data. 2 . Protein interaction networks need to be interpreted with caution because, as a result of evolution making efficient use of each organism's genetic information, the same protein can be used as part of two different protein complexes that have different types of functions. Thus, although protein A binds to protein B and protein B binds to protein C, proteins A and C need not function in the same process.For example, we know from detailed biochemical studies that the functions of Skpl in the kinetochore and in vacuolar H+-ATPaseassembly (yellow shading) are separate from its function in the SCF ubiquitin ligase. In fact, only the remaining three functions of synthesis, cell cycle regulaSkpl illustrated in the diagram-methionine tion, and origin of replication (green shading)-involve ubiquitylation. 3 . In cross-speciescomparisons, those proteins displaying similar patterns of interactions in the two protein interaction maps are likely to have the same function in the cell. Thus, as scientists generate more and more highly detailed maps for multiple organisms, the results will become increasingly useful for inferring protein function. These map comparisons are a particularly powerful tool for deciphering the functions of human proteins. There is a vast amount of direct information about protein function that can be obtained from genetic engineering, mutational, and O R I G I NO F R E P L I C A T I O N CELLCYCLEREGULATORS

M E T H I O N I NSEY N T H E 5 I 5

KINETOCHORE Okpl

E 2u b i q u i t i n coniugating enzyme

Mit2

ctfl9

c Cep3

cbf2 .-..-

Mcm2l

Mckl

'/. adaptor protein1 Vm

Tfpl

'/'

Ram2 -

Vma2 VACUOLARH*-ATPase ASSEMBLY

adaptor protein 2

scaffoldprotein (cullin)

Figure3-82 A map of some protein- protein interactionsof the SCFubiquitin ligaseand other proteins in the yeast S.lerevisiae,Thesymbolsand/or colorsusedfor the 5 proteinsof the ligasearethose in Figure3-79. Note that 15 different with u/hitelettering(beginningwith Y) areonly knownfrom the genome F-boxproteinsareshown(purpte);those of PeterBowersand DavidEisenberg, sequenceasopen readingframes.Foradditionaldetails,seetext.(Courtesy UCLA.) UCLA-DOE Institutefor Genomicsand Proteomics,

190

Chapter3: Proteins Figure3-83 A networkof protein-bindinginteractionsin a yeastcell. Eachlineconnectinga pairof dots (proteins) indicates a protein-protein (FromA. Guimer6and M. Sales-Pardo, interaction. Mol.Syst. Biol.2:42,2006. With permission from MacmillanPublishers Ltd.)

genetic analyses in model organisms-such as yeast, worms, and fliesthat is not available in humans The available data suggestthat a typical protein in a human cell may interact with between 5 and 15 different partners. Often, each of the different domains in a multidomain protein binds to a different set of partners; in fact, we can speculate that the unusually extensivemultidomain structures observed for human proteins may have evolved to facilitate these interactions. Given the enormous complexity of the interacting networks of macromolecules in cells (Figure 3-83), deciphering their full functional meaning may well keep scientists busy for centuries.

Su m m a r y Proteins canform enormouslysophisticatedchemical deuices,whosefunctions largely depend on the detailed chemical properties of their surfaces.Binding sitesfor ligands areformed as surfacecauitiesin which preciselypositioned amino acid side chains are brought togetherby protein folding. In this way, normally unreactiueamino acid side chains can be actiuated to make and break coualentbonds.Enzymesare catalytic proteins that greatly speedup reaction rates by binding the high-energy transition states for a speciftcreaction path; they also perform acid catalysisand basecatalysissimultaneously.The ratesof enzymereactionsare often sofast thqt they are limited only by diffusion; ratescan befurther increasedif enzymesthat act sequentiallyon a substrate are joined into a single multienzyme complex, or if the enzymesand their substrates are confined to the same compartment of the cell. Proteins reuersiblychange their shape when ligands bind to their surface. The allosteric changesin protein conformation produced by one ligand affect the binding of a secondligand, and this linkage betweentwo ligand-binding sitesprouidesa crucial mechanism for regulating cell processes.Metabolic pathways, for example, are controlled by feedback regulation: some small moleculesinhibit and other small moleculesactiuate enzymesearly in a pathway. Enzymescontrolled in this way generally form symmetric assemblies,allowing cooperetiueconformational changesto reate a steepresponseto changesin the concentrationsof the ligands that regulatethem. The expenditure of chemical energy can driue unidirectional changesin protein shape.By coupling allosteric shape changesto ATp hydrolysis,for example, proteins can do useful work, such as generating a mechanical force or mouing for long distancesin a singledirection.The three-dimensionalstructuresof proteins,determined by x-ray crystallography,haue reuealedhow a small local change causedby nucleoside triphosphate hydrolysis is amplified to create major changes elsewherein the protein. By such means,theseproteinscan serueas input-output deuicesthat transmit information, as assemblyfactors, as motors, or as membrane-boundpumps. Highly efficient protein machines areformed by incorporating many dffirent protein moleculesinto larger assembliesthat coordinate the allosteric mouementsof the inttiuidual components.such machinesere now known to perform many of the most important reactionsin cells. Proteins are subjectedto mqny reuersiblepost-translational modifications, such as the coualentaddition of a phosphateor an acetylgroup to a specificamino acid side chain. The addition of thesemodifying groups is usedto regulate the actiuity of a protein, changing its conformation, its binding to other proteins and its location inside the cell.A ltpical protein in a celt will interact with more than fiue dffirent panners. using the new technologiesof proteomics,biologistscan analyze thousandsof proteins in one set of experiments.One important result is the production of detailed protein interaction maps, which aim at describingall of the binding interactions betweenthe thousandsof distinct proteins in a cell.

...,'*

t.{,

?1

191

END-OF-CHAPTER PROBLEMS

PROBLEMS

FigureQ3-1 The kelch repeatdomainof galactoseoxidasefrom D.dendroides(Problem 3-9).The seven individualB propellers The N- and areindicated. C-terminiareindicated by N and C.

6r*, f l ,

Whichstatementsare true? Explainwhy or why not. 3-1 Each strand in a B sheet is a helix with two amino acidsper turn. 3-2 Loops of polypeptide that protrude from the surface of a protein often form the binding sitesfor other molecules. 3-3 An enzymereachesa maximum rate at high substrate concentrationbecauseit has a fixed number of active sites where substratebinds. 3-4 Higher concentrationsof enzyrnegiverise to a higher turnover number. 3*5 Enz)rynessuch as aspartatetranscarbamoylasethat undergo cooperative allosteric transitions invariably contain multiple identical subunits. 3*6 Continual addition and removal of phosphates by protein kinases and protein phosphatasesis wasteful of energy-since their combined action consumesATP-but it is a necessaryconsequenceof effectiveregulation by phosphorylation. Discussthe following problems. 3-7 Consider the following statement. "To produce one molecule of each possible kind of polypeptide chain, 300 amino acidsin length, would require more atoms than existin the universe." Given the size of the universe,do you suppose this statement could possibly be correct?Since counting atoms is a tricky business,consider the problem from the standpoint of mass.The mass of the observableuniverseis estimated to be about l0B0grams, give or take an order of magnitude or so.Assumingthat the averagemassof an amino acid is I l0 daltons,what would be the massof one molecule of eachpossiblekind of pollpeptide chain 300 amino acidsin length?Is this greaterthan the mass of the universe? 3-8 A common strategyfor identifying distantly related proteins is to search the databaseusing a short signature sequenceindicative of the particular protein function. \A/hy is it better to searchwith a short sequencethan with a long sequence?Do you not have more chancesfor a'hit' in the databasewith a long sequence? 3-9 The so-calledkelch motif consistsof a four-stranded B sheet,which forms what is known as a B propeller. It is usually found to be repeatedfour to seventimes, forming a kelch repeat domain in a multidomain protein. One such kelch repeat domain is shor.tmin Figure Q3-1. Would you classifythis domain as an'in-line' or'plug-in type domain? 3-10 Titin, which has a molecular weight of 3 x 106daltons, is the largest polypeptide yet described. Titin moleculesextend from muscle thick filaments to the Z disc; they arethought to act as springsto keep the thick filaments centeredin the sarcomere.Titin is composedof a largenumber of repeatedimmunoglobulin (Ig)sequencesof 89 amino acids,each of which is folded into a domain about 4 nm in length (Figure Q3-2A). You suspectthat the springlikebehavior of titin is caused by the sequentialunfolding (and refolding) of individual Ig

domains. You test this hlpothesis using the atomic force microscope,which allowsyou to pick up one end of a protein molecule and pull with an accuratelymeasuredforce. For a fragment of titin containing seven repeats of the Ig domain, this experiment gives the sawtooth force-versusextension curve shourn in Figure Q3-28. \A4renthe experiment is repeatedin a solution of B M urea (a protein denaturant), the peaks disappear and the measured extension becomesmuch longer for a given force.If the experimentis repeated after the protein has been cross-linkedby treatment with glutaraldehyde,once again the peaks disappear but the extensionbecomesmuch smaller for a given force. A. Are the data consistentwith your hlpothesis that titin's springlike behavior is due to the sequential unfolding of individual Ig domains?Explainyour reasoning. B. Is the extension for each putative domain-unfolding event the magnitude you would expect? (In an extended polypeptide chain, amino acids are spaced at intervals of 0.34nm.) C. \Mhy is each successivepeak in Figure Q3-2B a little higher than the one before? D. \A/hydoesthe force collapseso abruptly after eachpeak? 3*11 It is often said that protein complexesare made from subunits (that is, individually slnthesized proteins) rather than as one long protein becausethe former is more likelyto give a correctfinal structure. A. Assuming that the protein synthesismachinery incorDoratesone incorrect amino acid for each 10,000it inserts, (A)

(B)

4oo ^ z o ;

c

300 200

,P loo 0

i . rr t:,

0

I r i i . i r , r at t , r i rr:. . . : r , , ' l i . r i r i , r i , , ' ' : . ' .

50

.,:,

150 100 (nm) extension

200

behaviorof titin (Problem3-10)'(A)The FigureQ3-2 Springlike versus structureof an individuallg domain.(B)Forcein piconewtons extensionin nanometersobtainedby atomicforce microscopy.

192

Chapter3: Proteins

calculatethe fraction of bacterial ribosomesthat would be assembledcorrectly if the proteins were synthesizedas one large protein versusbuilt from individual proteins?For the sake of calculation assumethat the ribosome is composed of 50 proteins, each 200 amino acids in length, and that the subunits-correct and incorrect-are assembledwith eoual likelihood into the completeribosome.IThe probability that a polypeptidewill be made correctly,Pc, equalsthe fraction correct for each operation,/6, raisedto a power equal to the number of operations, n: P6 = lfd". For an error rate of 1 / 1 0 , 0 0 0f r, . = 0 . 9 9 9 9 . 1 B. Is the assumption that correct and incorrect subunits assembleequally well likely to be true? \A4ryor why not? How would a changein that assumption affect the calculation in part A? 3-12 Roussarcomavirus (RSV)carriesan oncogenecalled Srq which encodes a continuously active protein tl,'rosine kinase that leadsto uncheckedcell proliferation. Normally, Src carries an attached fatty acid (myristoylate)group that allowsit to bind to the cy'toplasmicside of the plasmamembrane. A mutant version of Src that does not allow attachment of myristoylatedoesnot bind to the membrane.Infection of cells with RSV encoding either the normal or the mutant form of Src leads to the same high level of protein tyrosine kinase activity,but the mutant Src does not cause cell proliferation. A. Assumingthat the normal Srcis all bound to the plasma membrane and that the mutant Src is distributed throughout the cy.toplasm,calculatetheir relativeconcentrationsin the neighborhood of the plasma membrane. For the purposes of this calculation, assume that the cell is a sphere with a radius of l0 pm and that the mutant Srcis distributed throughout, whereasthe normal Src is confined to a 4-nmthick layer immediately beneath the membrane. [For this problem, assumethat the membrane has no thickness.The volume of a sphereis (4/3)rr3.l B. The target (X) for phosphorylationby Srcresidesin the membrane.Explainwhy the mutant Src does not causecell proliferation. 3-13 An antibody binds to anotherprotein with an equilibrium constant,K of 5 x lOeM-1.\A/henit binds to a second, relatedprotein, it forms three fewer hydrogenbonds,reducing its binding affinity by 2.8 kcal/mole.\Mhatis the Kfor its binding to the secondprotein?(Free-energy changeis related to the equilibrium constantby the equationAG" = -2.3 RTlog K whereR is t.9Bx 10-3kcal/(moleK) and Tis 310K.) 3-i 4 The protein SmpBbinds to a specialspeciesof tRNA, tmRNA, to eliminate the incomplete proteins made from truncated mRNAs in bacteria. If the binding of SmpB to tmRNA is plotted as fraction tmRNA bound versus SmpB concentration,one obtainsa symmetricalS-shapedcurve as shor.rnin Figure Q3-3. This curve is a visual displayof a very useful relationship between tr:i and concentration, which has broad applicability.The generalexpressionfor fraction of ligand bound is derived from the equation for K6 (trfr= lPrllll/ [Pr-L])by substituting([L]ror.- tL])for [pr-L] and rearranging.Becausethe total concentrationofligand ([L]ror) is equal to the free ligand (tll) plus bound ligand ([pr-L]),

ltmRNAlror = [SmpB]/([SmpB]+ rQ). Using this relationship, calculatethe fraction of tmRNA bound for SmpB concentrationsequal to 104Kd,103Kd,l02Kd,lOltra, Kd, lO-tIA, l0-2Kd,10-3^?,and 10rK4. 10

E 075 !

c l o c

05

E a

-

025

0 1 01 1

10-e

1 0s

10-7

(M) centration of SmpB FigureQ3-3Fraction of tmRNA boundversus SmpBconcentration ( P r o b l e3m- 1 4 ) .

3*15 Many enzymes obey simple Michaelis-Menten kinetics,which are summarizedby the equation rate = vmax[s]/([S] + K_) where V-* = maximum velociry [S]= concentrationof substrate,and Km= the Michaelisconstant. It is instructiveto plug a fewvaluesof [S]into the equation to seehow rate is affected.What are the ratesfor [S]equal to zero,equal to K-, and equal to infinite concentration? 3-16 The enzyme hexokinaseadds a phosphateto D-glucose but ignores its mirror image, L-glucose.Supposethat you were able to synthesizehexokinase entirely from Damino acids,which are the mirror image of the normal Lamino acids. A. Assuming that the 'D' enz).rnewould fold to a stable conformation,what relationshipwould you expectit to bear to the normal'l enzyme? B. Do you supposethe'D' en4/rnewould add a phosphate to L-glucose,and ignore D-glucose? 3-17 How do you supposethat a molecule of hemoglobin is ableto bind oxygenefficientlyin the lungs,and yet release it efficientlyin the tissues? 3-18 Synthesisof the purine nucleotidesAMP and GMP proceeds by a branched pathway starting with ribose 5phosphate (R5P),as shown schematicallyin Figure Q3-4. Using the principles of feedbackinhibition, proposea regulatory strategyfor this pathway that ensuresan adequate supply of both AMP and GMP and minimizes the buildup of the intermediates(,4-1)when suppliesof AMP and GMP are adequate. F +

G+AMP

H+

/ +GMP

,/ R5P+A+8+C+D+E

\

fraction bound = tll/ [L]ror = tprl/ (tprl + Ka) For SmpB and tmRNA, the fraction bound = [tmRNAl/

FigureQ3-4 Schematic diagramof the metabolicpathwayfor synthesis of AMPand GMPfrom R5P(Problem3*18).

REFERENCES

REFERENCES General Tymoczko Berg-1M, lL & StryerL (2006)Biochemistry, 6rh ed NewYork: WH Freeman Branden C &ToozeJ (1999)Introduction io ProteinStructure,2nd ed NewYork:GarlandScience Dickerson, RE(2005)Present at the FloodHowStructural Mo ecuar BiologyCameAbout Sunderland, MA:Slnauer KyteJ (2006)Structure in ProteinChemistryNewYork:Routledge Petsko GA& RingeD (2004)ProteinStructure and FunctionLondon: NewScience Press DroteinStrdclu.e: Pe'uLz M r 199..r) NewApp.oaches to Disease and TherapyNewYork:WH Freeman The Shapeand Structureof Proteins Anfinsen CB(1973)Principles that governthe foldingof proteinchains Science 181.2)3-230 peptidesandcytoplasmic BrayD (2005)Flexible Biol AelsGenome 6:106-I 09 P,Stetefe Burkhard d J & Strelkov SV(2001)Coiledcoils:a highly versatile protern fold ing r.laltf. Trends Ce|| Biol 11.82-BB principles CasparDLD& KlugA (1962)Physical in the construction of regularvrrusesColdSpringHarbSympQuantBiol27:1-24. DoolittleRF(1995) Themultiplicity of domainsin proteinsAnnuRev Biochem64.287-314 Eisenberg D (2003) Thediscovery ofthe alphahe ix and betasheet, theprincipe structural featuresofproteinsProcNatl AcadSct USA -11210 100.11207 Fraenkel ConratH & Williams RC(1955)Reconstitution of active tobaccomosaicvirusfrom itsinactiveproteinand nucleicacid componentsProcNatlAcadSciUSA41:69A-698 Goodsell DS& OlsonAJ (2000)Structural symmetryand protein function AnnuRevBiophys BtomolStruct29:105-1 53 HarrisonSC(1992)Yiuses CurrOpinStructBrol2.293-299 HarrisonSC(2004)Whitherstructuralbiology?NatureStructltlolBiol 11 : 1 2 -51 HudderA, Nathanson L & Deutscher MP (2003) Organization of mammaliancytoplasmMol CellBiol23.9318-9326 I n t e r n a t i o nHaul m a nG e n o m eS e q u e n c i nCgo n s o r t i u(m2 0 0 1l )n i t i a l sequencing and analysis of the humangenome/Vcfure 4A9.860-921 MeilerI & BakerD (2003)Coupledprediction of proteinsecondary and Tertiarv stnr.trrreProcNarlAcad5ciU5A100:12105 T2ll0 NomuraM (1973)Assembly of bacterial ribosomes Scrence 179. 864-873 OrengoCA& ThorntonJM (2005)Proteinfamilies andtheir perspective evolution a structural AnnuRevBiochem74.867-900 P a u l i n Lg& C o r e yR B( 1 9 5 1C) o n f i g u r a t i oonf sp o l y p e p t i dceh a i n w s ith favoredorientations aroundsinglebonds:two new pleatedsheets ProcNatlAcadSciUSA37:729-740 P a u l i n Lg ,C o r e yR B& B r a n s oHn R( 1 9 5 1 ) T hset r u c t u roef p r o t e i n tsw: o hydrogen-bonded helicalconfigurations of the polypeptide chain ProcNatlAcadSciUSA37.205-211 PontingCP,Schultz J,CopleyRRet al (2000)Evolution of domain familiesAdvProtetnChem54:185-244 T r i n i c k(J1 9 9 2U) n d e r s t a n d itnhge f u n c t i o nosf t i t i na n dn e b u l i n FEBS Leu3a7:44-48 VogelC,Bashton M, Kerrison NDet al (2004)Structure, functionand evolutionof multidomainproteinsCurrOpinStructBiol14.208-216 genomics: Tlang C & KimSH(2003)Overview from of structural structureto function,Curraptn ChemBiol7.28-32 Protein Function preparing AlbertsB (1998) Thecellasa collection of proteinmachines: the nextgeneration of molecular Cell92.291-294 biologists Benkovic| (1992)CatalyticantibodiesAnnuRevBtochem61:2954 BergOG& von HippelPH(1985)Diffusion-controlled macromolecular interactionsAnnu RevBiophys Biophys Chem14.131-1 60,

193 motifs, & LimWA (2006)Domains, RP,Remenyi A,YehB.J Bhattacharyya in the evolutionand and scaffods:Theroleof modularinteractions 75.655680 circuitsAnnuRevBiochem wiringof cellsignaling switches andclocks a familyof molecular BourneHR(1995)GTPases: PhilosTransR SocLondB 349:283-289 features ofthe reactions BradenBC& PoljakRJ(1995)Structural J 9.9-16 and proteinantigensFASEB betweenantibodies Structure, Function, RE& Geis| (1983)Hemoglobin: Dickerson CA:Benjamin Cummings and PathologyMenloPark, Evolution EnzymesNewYork:Scientific D & PotterH (1991)Discovering Dressler Library American | & Yeates TO (2000)Protein EM,Xenarios, D,Marcotte, Eisenberg functionin the post genomicera Nature405.823-826 in ProteinScience: Structure and Mechanisms FershtAR(,l999) NewYork.WH Freeman Catalysis A Guideto Enzyme Structural basisfor controlby LN& LewisRJ(200,1) Johnson, phosphorylation ChemRev101:2209-2242 WN (1988)Escheichia collaspartate ER& Lipscomb Kantrowitz andfunction the relationbetweenstructure transcarbamoylase: 4 Science 241:669-67 ModularenzymesNature409.247-252 Khosa C & HarburyPB(200,1) NatureRev KimE & ShengM (2004)PDZdomainproteinsof synapses Neurosci 5.771-781 DE,Jr (1984)Controlof enzymeactivityand metabolic Koshland pathwaysTrends 5cl9:,155-159 Btochem in enzyme D (2003)Challenges KrautDA,CarrollKS& Herschlag AnnuRevBiochen72.517-571 mechanism and energetics of protein KroganNJ,CagneyG,Yu H et al (2006)Globallandscape Nature cerevtstsae complexesin the yeastSaccharomyces 440:637-643 trace An evolutionary O, BourneHR& CohenFE(,1996) Lichtarge commonto proteinfamilles methoddefinesbindingsurfaces J ltlolBtol257.342-358 protein M, Ng HLet al (1999)Detecting MarcotteEM,Pellegrini fromgenomesequences interactions functionand protein-protein 285.751753, Science proteinsandcellular Allosteric JP& JacobF ('1963) MonodJ,Changeux controlsystemsJ ltlolBiol6.306-329 systems through of regulatory T & NashP (2003)Assembly Pawson 452 300'.445 proteininteraction domainsScience of cyclindependentkinaseregulation: NP(1999)Mechanisms Pavletich andCipand lNK4inhibitors of Cdks,theircyclinactivators, structures J l\lolBiol287.821-B2B in the lnteractions P & Kuriyan J (2006)Protein-protein Pellicena StructBrol regulation of proteinkinasesCurrOptn allosteric 16.702-709 Regulation and Allosteric of Cooperativity PerutzM (1990)Mechanisms Press University Cambrldge in ProteinsCambridge: with molecular F,Thoden,JB& HoldenHM (2003)Enzymes Raushel tunnels,4ccChemRes36.539548 Radzicka A & WolfendenR (1995)A proficientenzymeScience 267.9093, location: location, SatoTK,OverduinM & EmrS (200'])Location, targetingdirectedby PXdomainsScience Membrane 2 9 4 : 1 B B1l B B 5 state transition statesandtransition VL(1998)Fnzymatic Schramm 67:693-720 analogdesignAnnuRevBiochem to catalysis: diversity SchultzPG& LernerRA(1995)Frommolecular 269,1835-1842 fromthe immunesystemSclence lessons Thewaythingsmove:lookingunderthe ValeRD& MilliganRA(2000) 2BB,BB-95 hood of molecularmotor proteinsScience by hen Gl, LaineR& WithersSG(2001)Catalysis VocadloDJ,Davies /Vature proceeds viaa covalentintermedlate egg-whitelysozyme 412:835-838 of life Nature409:226-231 the chemistry WalshC (2001)Enabling and intramolecular YangXJ(2005)Multisiteproteinmodificatlon 24:16531662 signalingOncogene AnnuRevBtochem Zhu H,BilginM & SnyderM (2003)Proteomics 72:783812

DNA,Chromosomes, and Genomes Life depends on the ability of cells to store, retrieve, and translate the genetic instructions required to make and maintain a living organism. Tl:'is hereditary information is passed on from a cell to its daughter cells at cell division, and from one generation of an organism to the next through the organism's reproductive cells. These instructions are stored within every living cell as its genes, the information-containing elements that determine the characteristics of a species as a whole and of the individuals within it. As soon as genetics emerged as a science at the beginning of the twentieth century, scientists became intrigued by the chemical structure of genes. The information in genes is copied and transmitted from cell to daughter cell millions of times during the life of a multicellular organism, and it survives the process essentially unchanged.'What form of molecule could be capable of such accurate and almost unlimited replication and also be able to direct the development of an organism and the daily life of a cell?\A/hatkind of instructions does the genetic information contain? How can the enormous amount of information required for the development and maintenance of an organism fit within the tiny space of a cell? The answers to several of these questions began to emerge in the 1940s.At this time, researchers discovered, from studies in simple fungi, that genetic information consists primarily of instructions for making proteins. Proteins are the macromolecules that perform most cell functions: they serve as building blocks for cell structures and form the enzy'rnesthat catalyze the cell's chemical reactions (Chapter 3), they regulate gene expression (Chapter 7), and they enable cells to communicate with each other (Chapter 15) and to move (Chapter l6). The properties and functions of a cell are determined largely by the proteins that it is able to make. With hindsight, it is hard to imagine what other type of instructions the genetic information could have contained. Painstaking observations of cells and embryos in the late tgth century had led to the recognition that the hereditary information is carried on chromosomes,threadlike structures in the nucleus of a eucaryotic cell that become visible by light microscopy as the cell begins to divide (Figure 4-l). Later, as biochemical analysisbecame possible, chromosomes were found to consist of both deoxyribonucleic acid (DNA) and protein. For many decades, the DNA was thought to be merely a structural element. However, the other crucial advance made in the 1940swas the identification of DNA as the likely carrier of genetic information. This breakthrough in our understanding of cells came from studies

Figure4-l Chromosomes in cells.(A)Two adjacentplantcells photographed througha light microscope. The DNAhasbeenstainedwith a fluorescent dye (DAPI) that bindsto it.The DNAis presentin chromosomes, whichbecomevisibleasdistinctstructures in the light structures microscope onlywhen they becomecompact,sausage-shaped in preparation for celldivision,asshownon the left.Thecellon the right, which is not dividing,containsidenticalchromosomes, but they cannotbe clearlydistinguished in the light microscope at this phasein the cell'slife (B)Schematic cycle,becausethey are in a moreextendedconformation. diagramof the outlinesof the two cellsalongwith theirchromosomes. (A,courtesyof PeterShaw.)

In ThisChapter AND THESTRUCTURE FUNCTION OFDNA

197

DNA 202 CHROMOSOMAL IN AND ITSPACKAGING FIBER THECHROMATIN OF 219 THEREGULATION CHROMATIN STRUCTURE 233 THEGLOBALSTRUCTURE OFCHROMOSOMES EVOLVE245 HOWGENOMES

(A)

d i v i d i n gc e t l

n o n d i v i d i n gc e l l

10t.

196

Chapter4: DNA,Chromosomes, and Genomes

of inheritance in bacteria (Figure 4-2). But as the 1950sbegan, both how proteins could be specified by instructions in the DNA and how this information might be copied for transmission from cell to cell seemed completely mysterious. The mystery was suddenly solved in 1953,when the structure of DNA was correctly predicted by Iames Watson and Francis Crick. As outlined in Chapter 1, the double-helical structure of DNA immediately solved the problem of how the information in this molecule might be copied, or replicated.It also provided the first clues as to how a molecule of DNA might use the sequenceof its subunits to encode the instructions for making proteins. Today, the fact that DNA is the genetic material is so fundamental to biological thought that it is difficult to appreciate the enormous intellectual gap that was filled. In this chapter we begin by describing the structure of DNA. We see how despite its chemical simplicity, the structure and chemical properties of DNA make it ideally suited as the raw material of genes.We then consider how the many proteins in chromosomes arrange and packagethis DNA. The packing has to be done in an orderly fashion so that the chromosomes can be replicated and apportioned correctly between the two daughter cells at each cell division. It must also allow accessto chromosomal DNA for the enzymes that repair it when it is damaged and for the specialized proteins that direct the expression of its many genes.We shall also see how the packaging of DNA differs along the length of each chromosome in eucaryotes,and how it can store a valuable record of the cell's developmental history. In the past two decades,there has been a revolution in our ability to determine the exact sequence of subunits in DNA molecules. As a result, we now know the order of the 3 billion DNA subunits that provide the information for producing a human adult from a fertilized egg, as well as the DNA sequencesof thousands of other organisms. Detailed analysesof these sequenceshave provided exciting insights into the process of evolution, and it is with this subject that the chapter ends. This is the first of four chapters that deal with basic genetic mechanismsthe ways in which the cell maintains, replicates, expresses,and occasionally improves the genetic information carried in its DNA. This chapter presents a broad overview of DNA and how it is packaged into chromosomes. In the following chapter (Chapter 5) we discuss the mechanisms by which the cell accurately replicates and repairs DNA; we also describe how DNA sequencescan be

Sstrain

s m o o t h p a t h o g e n i cb a c t e r i u m c a u s e sD n e u m o n t a

S s t r a i nc e l l s

I noroovrMUTATToN t Rstrain

f r a c t i o n a t i o no f c e l l - rf e e extra(t into classesof p u r i f i e dm o l e c u l e s

roughnonpathogenic m u r a n tD a c t e n u m

C

o oo l i v e R s t r a i nc e l l sg r o w n i n p r e s e n c eo f e i t h e r h e a t - k i l l e d S s t r a i nc e l l so r c e l l - f r e e e x t r a c to f S s t r a i nc e l l s TRANSFORMATION

+

5 strain

S o m eR s t r a i nc e l l sa r e t r a n s f o r m e dt o S s t r a i n c e l l sw , h o s ed a u g h t e r s a r e p a t h o g e n i ca n d c a u s ep n e u m o n t a

C O N C L U S I OM N :o l e c u l e st h a t c a n c a r r yh e r i t a b l ei n f o r m a t i o na r e presentin 5 strain cells. (A)

protein

RNA

DNA

lipid carbohydrate

ttttl tttrl ttttl

moleculestested for transformationof R strain cells

ttttl ttttl

VVVVT

oc

oo

RRSRR strain strain

oo strain

strain

C O N C L U S I OTNh: e m o l e c u l et h a t c a r r i e st h e h e r i t a b l ei n f o r m a t i o n isDNA. (B)

strain

Figure 4-2 The first experimental demonstrationthat DNA is the genetic material.Theseexperiments, carriedout in the 1940s,showedthat addingpurified DNAto a bacteriumchangedits propertiesand that this changewas faithfullypassedon to subsequent generations. Two closelyrelatedstrainsof pneumoniae the bacteriumStreptococcus differfrom eachother in both their appearance underthe microscope and their pathogenicity. One strainappears smooth(5)and causesdeathwhen injectedinto mice,and the otherappears rough(R)and is nonlethal.(A)An initial experimentshowsthat a substance presentin the S straincanchange(or transform) the R straininto the S strain and that this changeis inheritedby generations subsequent of bacteria. (B)Thisexperiment, in whichthe R strain hasbeenincubatedwith variousclasses of biologicalmoleculespurifiedfrom the S strain,identifiesthe substance as DNA.

THESTRUCTURE AND FUNCTION OF DNA

rearranged through the process of genetic recombination. Gene expressionthe process through which the information encoded in DNA is interpreted by the cell to guide the synthesis of proteins-is the main topic of Chapter 6. In Chapter 7, we describe how this gene expression is controlled by the cell to ensure that each of the many thousands of proteins and RNA molecules encrlpted in its DNA are manufactured only at the proper time and place in the life of the cell.

THESTRUCTURE ANDFUNCTION OFDNA Biologists in the 1940s had difficulty in conceiving how DNA could be the genetic material because of the apparent simplicity of its chemistry. DNA was known to be a long poll.rner composed of only four types of subunits, which resemble one another chemically. Early in the 1950s,DNA was examined by xray diffraction analysis, a technique for determining the three-dimensional atomic structure of a molecule (discussedin Chapter 8). The early x-ray diffraction results indicated that DNA was composed of two strands of the polymer wound into a helix. The observation that DNA was double-stranded was of crucial significance and provided one of the major clues that led to the Watson-Crick model for DNA structure. But onlywhen this model was proposed in 1953 did DNAs potential for replication and information encoding become apparent. In this section we examine the structure of the DNA molecule and explain in general terms how it is able to store hereditary information.

A DNAMoleculeConsists of TwoComplementary Chainsof Nucleotides A deoxyribonucleic acid (DNA) molecule consists of two long polynucleotide chains composed of four types of nucleotide subunits. Each of these chains is knornm as a D.ly'Achain, or a DNA strand. Hydrogen bondsbetween the base portions of the nucleotides hold the two chains together (Figure 4-3). As we saw in Chapter 2 (Panel 2-6, pp. 116-117), nucleotides are composed of a five-carbon sugar to which are attached one or more phosphate groups and a nitrogen-containing base. In the case of the nucleotides in DNA, the sugar is deoxyribose attached to a single phosphate group (hence the name deoxyribonucleic acid), and the base maybe either adenine (A),cytosine(C),guanine (G),or thymine (T). The nucleotides are covalently linked together in a chain through the sugarsand phosphates, which thus form a "backbone" of alternating sugar-phosphatesugar-phosphate. Becauseonly the base differs in each of the four types of subunits, each polynucleotide chain in DNA is analogous to a necklace (the backbone) strung with four rypes of beads (the four basesA, C, G, and T). These same symbols (A, C, G, and T) are also commonly used to denote the four different nucleotides-that is, the baseswith their attached sugar and phosphate groups. The way in which the nucleotide subunits are linked together gives a DNA strand a chemical polarity. If we think of each sugar as a block with a protruding knob (the 5'phosphate) on one side and a hole (the 3'hydroxyl) on the other (see Figure 4-3), each completed chain, formed by interlocking knobs with holes, will have all of its subunits lined up in the same orientation. Moreover, the two ends of the chain will be easily distinguishable, as one has a hole (the 3'hydroxyl) and the other a knob (the 5'phosphate) at its terminus. This polarity in a DNA chain is indicated by referring to one end as tl:'e ! end and the other as the ! end. The three-dimensional structure of DNA-the double helix-arises from the chemical and structural features of its two polynucleotide chains. Because these two chains are held together by hydrogen bonding between the bases on the different strands, all the bases are on the inside of the double helix, and the sugar-phosphatebackbones are on the outside (seeFigure 4-3). In each case, a bulkier two-ring base (a purine; see Panel 2-6, pp. 116-l 17) is paired with a single-ring base (a pyrimidine); A always pairs with T and G with C (Figure

197

198

Chapter4: DNA, Chromosomes,and Genomes

$$iiiffiliiiii:ii:iii llilii:i:ilitffi

b u i l d i n gb l o c k so f D N A phosphate suqar \

';

+K-

sugar oase phosphate

ne

double-strandedDNA

3',

5'

D N A d o u b l eh e l i x

s',

Figure4-3 DNA and its building blocks. DNA is made of four types of nucleotides,which are linkedcovalently into a polynucleotide chain(a DNA strand)with a sugar-phosphate backbonefrom which the bases(A,C,G, andT) extend.A DNAmoleculeis composedof two DNA strandsheld together by hydrogenbonds between the pairedbases.Thearrowheadsatthe endsofthe DNAstrandsindicatethe polaritiesof the two strands,which run antiparallel to eachother in the DNA molecule.In the diagramat the bottom left of the figure,the DNA moleculeis shown straightenedout; in reality,it is twistedinto a doublehelix,as shownon the right.Fordetails,seeFigure4-5.

Y 3'

hydrogen-bonded b a s ep a i r s

4-4). This complementary base-pairlng enables the base pairs to be packed in the energetically most favorable arrangement in the interior of the double helix. In this arrangement, each base pair is of similar width, thus holding the sugarphosphate backbones an equal distance apart along the DNA molecule. To maximize the efficiency of base-pair packing, the two sugar-phosphate backbones

H

o \\

N - _CC '\

C -N

\/ -L

\lI

H-N

\\ C \\N sugar-phosphate backbone

H

C -C

C_

,-n, , ,o'l

adenine

[n,,

thymine

H

N -HilililililO

.ll

guanrne

ll / hydrogen DOnO

H

cytosine

Figure4-4 Complementarybasepairs in the DNAdouble helix.The shapesand chemicalstructureof the basesallow hydrogenbondsto form efficientlyonly betweenA and T and betweenG and C. where atomsthat are able to form hydrogen bonds(seePanel2-3, pp. 110-111)can be brought closetogetherwithout distorting the doublehelix.As indicated, two hydrogenbonds form betweenA and T, while three form betweenG and C.The basescan pairin thisway only if the two polynucleotide chainsthat containthem areantiparallel to eachother.

THESTRUCTURE AND FUNCTION OF DNA

199

5'end

\ .o-

d":, mtnor groove

3'end

L=o-l

\

do

I 0 . 3 4n m

o' o={-o 5'end (A)

/

'.. 3'end

Figure4-5 The DNAdouble helix. (A)A space-filling modelof 1.5turnsof the DNAdoublehelix.Eachturn of DNAis madeup of 10.4nucleotidepairs,and the center-to-centerdistancebetween adjacentnucleotidepairsis 3.4nm.The coilingof the two strandsaroundeach other createstwo groovesin the double helix:the widergrooveis calledthe major groove,and the smallerthe minorgroove. (B)A shortsectionof the doublehelix viewedfrom its side,showingfour base pairs.The nucleotides are linkedtogether bondsthat covalentlyby phosphodiester join the 3rhydroxyl(-OH)groupofone sugarto the 5rhydroxylgroup of the next strand sugar.Thus,eachpolynucleotide hasa chemicalpolarity;that is,itstwo The5' end of different. endsarechemically the DNApolymeris by conventionoften illustrated carryinga phosphategroup, whilethe 3rend is shownwith a hydroxyl.

(B)

wind around each other to form a double helix, with one complete turn every ten base pairs (Figure 4-5). The members of each base pair can fit together within the double helix only if the two strands of the helix are antiparallel-that is, only if the polarity of one strand is oriented opposite to that ofthe other strand (seeFigures 4-3 and 4-4). A consequence of these base-pairing requirements is that each strand of a DNA molecule contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand.

TheStructureof DNAProvides a Mechanism for Heredity Genescarry biological information that must be copied accurately for transmission to the next generation each time a cell divides to form two daughter cells. TWo central biological questions arise from these requirements: how can the information for specifying an organism be carried in chemical form, and how is it accurately copied?The discovery of the structure of the DNA double helix was a landmark in twentieth-century biology because it immediately suggested answers to both questions, thereby providing a molecular explanation for the problem of heredity. We discuss these answers briefly in this section, and we shall examine them in much more detail in subsequent chapters. DNA encodes information through the order, or sequence, of the nucleotides along each strand. Each base-A, C, T or G-can be considered as a Ietter in a four-letter alphabet that spells out biological messagesin the chemical structure of the DNA. As we saw in Chapter 1, organisms differ from one another because their respective DNA molecules have different nucleotide sequencesand, consequently,carry different biological messages.But how is the nucleotide alphabet used to make messages,and what do they spell out? As discussed above, it was known well before the structure of DNA was determined that genes contain the instructions for producing proteins. The DNA messagesmust therefore somehow encode proteins (Figure 4-6). This relationship immediately makes the problem easier to understand. As discussed in Chapter 3, the properties of a protein, which are responsible for its biological function, are determined by its three-dimensional structure. This structure is determined in turn by the linear sequenceof the amino acids of which it is composed. The linear sequence of nucleotides in a gene must therefore somehow spell out the linear sequence of amino acids in a protein. The exact correspondence between the four-letter nucleotide alphabet of DNA and the twenty-letter amino acid alphabet of proteins-the genetic code-is not obvious from the DNA structure, and it took over a decade after the discoverv of the double helix

| |

|

lorue

t

I

|

I

rrl lll tti

g e n eC

g e n eB T--t

g e n eA

|

gene expresslon

protein A

hi€ protein B

double

t'"tx

lffi protein c

Figure4-6 The relationshipbetween geneticinformationcarriedin DNAand proteins(discussed in Chapter1).

200

Chapter4: DNA,Chromosomes, and Genomes

before it was worked out. In Chapter 6 we will describe this code in detail in the course of elaborating the process,knoltm as geneexpresslon,through which a cell converts the nucleotide sequence of a gene first into the nucleotide sequenceof an RNA molecule, and then into the amino acid sequenceof a protein. The complete set of information in an organism'sDNA is called its genorne, and it carries the information for all the proteins and RNA molecules that the organism will ever synthesize. (The term genome is also used to describe the DNA that carries this information.) The amount of information contained in genomes is staggering: for example, a typical human diploid cell contains 2 meters of DNA double helix. Written out in the four-letter nucleotide alphabet, the nucleotide sequence of a very small human gene occupies a quarter of a page of text (Figure 4-7), while the complete sequence of nucleotides in the human genome would fill more than a thousand books the size of this one. In addition to other critical information, it carries the instructions for roughly 24,000distinct proteins. At each cell division, the cell must copy its genome to pass it to both daughter cells. The discovery of the structure of DNA also revealed the principle that makes this copying possible: because each strand of DNA contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand, each strand can act as a template, or mold, for the synthesis of a new complementary strand. In other words, if we designate the two DNA strands as S and S', strand S can serve as a template for making a new strand S', while strand S' can serve as a template for making a new strand S (Figure 4-8). Thus, the genetic information in DNA can be accurately copied by the beautifully simple process in which strand S separatesfrom strand S', and each separated strand then servesas a template for the production of a new complementary partner strand that is identical to its former partner. The ability of each strand of a DNA molecule to act as a template for producing a complementary strand enables a cell to copy, or replicate,its genome before passing it on to its descendants.In the next chapter we shall describe the elegant machinery the cell uses to perform this enormous task.

CCCTCTCCAGCCACACCCTAGGGTTGGCCA ATCTACTCCCAGGAGCAGGGAGGGCAGGAG r-Aaaaa4---! \.AgUUU f UU9UA

r ffiU

I UAUUUUAbAb

CCATCTATTGCTTACATTTGCTTCTGACAC AACTGTGTTCACTAGCAACTCAAACAGACA -i^taifr^ii uunrwrw6LU

FF

r uAv

rLU

r u69uAuffibt

CTGCCGTTACTGCCCTGTC,CGGCAAGGTGA nugr

ugArglsur

uudgu

r 1w

r\srwl96wuuulls 1A

!uftuu

I IAvlsuALauu

I

TTAAGGAGACCAATAGAAACTGGGCATGTG GAGACAGAGMGACTC TTGCCTTTCTGATA CGCACTGACTCTCTCTGCCTATTGGTCTAT T TTCCCACCC TTAGGCTCCTGGTGGTCTAC timnaalFFr ^vvr fuunvwvn96|Wl

IUI

I IUAUIUUI

I I

CGGGATCTGTCCACTCCTGCTGCTGT TATG uuuruLuu ffiur

rlu\srusuu!l'

u9 lluulwv

Iuuuffig I I tAul

tsI

uuuLlu

GCTCACCTGGACAACCTCAAGffi ACCTTT GCCACACTGAGTGACCTrcACTGTGACMG CTGCACGTCGATCCTGAGAACTTCAGGGTG ACTCTATGGGACCCTTGATGTTTTCTTTCC CCTTCTTTTCTATGGTTMGTTCATCTCAT AUUffiUUUUHUSU

I &UHUUU

t nLAb

I 1 1

AGAATGGGAAACAGACGAATGAT TGC.qTCA GTGTGGAACTCTCAGGATCGTTTTAGTTTC TTTTATTTGCTGTTCATMCAATTGTTTTC TTTTGTTTAATTCTTGCTTTCTTTTTTTTT CTTCTCCGCAATTTTTACTATTATACTTAA T GCCTTAACAT TGTGTATAACAAAAGGAAA TATCTCTGAGATACAT TAAGTAACTTAAAA ffiUI

I IAVAIdUIUIUU!IAUIAUAI

I

ACTATTTGGAATATATGTGTGCTTATTTGC ATATTCATAATCTCCCTACTTTATTTTCTT TTATTTTTAATTGATACATAATCATTATAC ATATTTATGGGTTAAAGTGTAATGTTTTAA I hIUI

In Eucaryotes, DNAls Enclosed in a CellNucleus As described in Chapter l, nearly all the DNA in a eucaryotic cell is sequestered in a nucleus, which in many cells occupies about l0% of the total cell volume. This compartment is delimited by a nuclear enuelopeformed by two concentric lipid bilayer membranes (Figure 4-9). These membranes are punctured at intervals by large nuclear pores, which transport molecules between the nucleus and the cytosol. The nuclear envelope is directly connected to the extensive membranes of the endoplasmic reticulum, which extend out from it into the cytoplasm. And it is mechanically supported by a network of intermediate filaments called the nuclear lamina, which forms a thin sheetlike meshwork just beneath the inner nuclear membrane (seeFigure 4-98). The nuclear envelope allows the many proteins that act on DNA to be concentrated where they are needed in the cell, and, as we see in subsequent Figure4-7 The nucleotidesequenceof the human p-globingene.By convention, a nucleotidesequenceis writtenfrom its 5'end to its 3'end, and it shouldbe readfrom left to right in successive linesdown the pageasthough it werenormalEnglishtext.Thisgenecarriesthe informationfor the aminoacidsequenceof one of the two typesof subunitsof the hemoglobinmolecule, the proteinthat carriesoxygenin the blood.A differentgene,the a-globingene,carriesthe informationfor the othertype of hemoglobinsubunit(a hemoglobinmoleculehasfour subunits, two of eachtype).Onlyone of the two strandsof the DNAdouble helixcontainingthe B-globingeneis shown;the otherstrandhasthe exact complementary sequence. The DNAsequences highlightedin yellowshow the threeregionsof the genethat specifythe aminoacidsequence for the B-globinprotein.We shallseein Chapter6 how the cellsplicesthesethree sequences togetherat the levelof messenger RNAin orderto synthesize a full-lenqthB-qlobinprotein.

U I6IA!AIAl

1 UHLUffi

lUAUWl

AATTTTGCATTTGTAATTTTAAAAAATGCT TTCTTCTTTTAATATACTTTTTTGTTTATC TTATTTCTAATACTTTCCCTAATCTCTTTC TTTCAGGGCAATAATGATACAATGTATCAT GCCTCTTTGCACCATTCTAAAGMTMCAG TGATAATTTCTGGGTTAAGGCAATAGCAAT Affluru\AfAr

l l9ruuAtAlls

ATTCTAACTGATGTAAGAGGTTTCATATTG TACAATCCAGCTACCAT CTAATAGCAGC TC TGCTTTTATTTTATGGTTGGGATAAGGCTG GATTATTCTGAGTCCAAGCTAGGCCCTTTT GCTAATCATGTTCATACCTCTTATCTTCCT TGTGCT rcCCCATCACTTTCCCAAAGMTT CACCCCACCAGTGCAG@T@CTATCAGAA AGTGGTffi TGGTGTGCC TAATGCCC TC,CC CCACMGTATCACTAAGCTCGCT TTCTTGC lulLu&t

I LLr6lrffiuul

rLuiIIull

CCCTAAGTCCAACTACTAAACTGGGGGATA TTATCAAGGGCCTTGAGCATCTGGATTCTG !!

f M]ffiLhI

I f dI

ru^rul6ttrffir hL

rrffiuuuM

r6l

f 1 IUAf

IULlg

I lulu/fiIhlttl

r u r uuuAuu

I uAu

IULA

TT T ry\'\i\CAT Ai\,\GAAATGATGAGCTGTTC ffiUUl

1l&Uffi

IAUAL

IAIAII-

I 1H

CTCCATCAAAGAAGGT GAGGC TGCAACCAG CTAATGCACATTGGCAACAGCCCCTGATGC !

] H I l*L

I I AI

IUAIU!!f

UAUffiUUAI

TCTTGTAGAGGC TTGATTTGCAGGTTAAAG TGTTTTAGCTGTCCTCATGAATGTCTTTTC

THESTRUCTURE AND FUNCTION OF DNA

201 t e m p l a t eS s t r a n d

Figure4-8 DNA as a template for its own duplication.As the nucleotideA pairsonly with I and G with successfully C,eachstrandof DNAcan act asa templateto specifythe sequenceof in its complementary strand. nucleotides In this way,double-helical DNAcan be with eachparentalDNA copiedprecisely, helixproducingtwo identicaldaughter DNAhelices.

5 strand new S'strand

new 5 strand S'strand p a r e n t a lD N A d o u b l e h e l i x t e m o l a t e5 ' s t r a n d d a u g h t e rD N A d o u b l e h e l i c e s

chapters, it also keeps nuclear and cytosolic enzymes separate,a feature that is crucial for the proper functioning of eucaryotic cells. Compartmentalization, of which the nucleus is an example, is an important principle of biology; it serves to establish an environment in which biochemical reactions are facilitated by the high concentration of both substrates and the enzymes that act on them. Compartmentalization also prevents enzymes needed in one part of the cell from interfering with the orderly biochemical pathways in another.

Su m m a r y Genetic information is caruied in the linear sequenceof nucleotides in DNA. Each molecule of DNA is a double helix formed from two complementery strands of nucleotidesheld togetherby hydrogen bonds betweenG-C and A-T basepairs. Dultlication of the geneticinformation occursby the useof one DNA strand as a templatefor theformation of a complementarystrand. Thegeneticinformation storedin an organism's DNA contains the instructionsfor all the proteins the organism will euersynthesize and is said to comprise its genome.In eucaryotes,DNA is contained in the cell nucleus,a largemembrane-boundcompartment. e n d o p l a s m i rce t i c u l u m

peripheral heterochromatin

DNA and associated p r o t e i n s( c h r o m a t i n ) , p l u sm a n y R N Aa n d p r o t e i nm o l e c u l e s

nucteotuS

nucleolus microtubule n u c l e a lra m i n a

\) n u c l e a rp o r e/

(B) z pm

membrane innernuclear

l

Figure4-9 A cross-sectional view of a typicalcell nucleus.(A)Electronmicrographof a thin sectionthroughthe nucleus the outer (B)Schematic of a humanfibroblast. drawing,showingthat the nuclearenvelopeconsistsof two membranes, one beingcontinuouswith the endoplasmic reticulummembrane(seealsoFigure12-8).The spaceinsidethe endoplasmic The lipid reticulum(theERlumen)is coloredyel/or4t it is continuouswith the spacebetweenthe two nuclearmembranes. networkof bilayersof the innerand outer nuclearmembranes areconnectedat eachnuclearpore.A sheet-like forminga special supportfor the nuclearenvelope, intermediate filaments(brown)insidethe nucleusprovidesmechanical nearthe laminacontains supportingstructurecalledthe nuclearlamina(fordetails,seeChapter12).The heterochromatin later. specially condensedregionsof DNAthat will be discussed

202

Chapter4: DNA,Chromosomes, and Genomes

CHROMOSOMAL DNAAND ITSPACKAGING INTHE CHROMATIN FIBER The most important function of DNA is to carry genes, the information that specifies all the proteins and RNA molecules that make up an organismincluding information about when, in what types of cells, and in what quantity each protein is to be made. The genomes of eucaryotesare divided up into chromosomes, and in this section we see how genes are typically arranged on each chromosome. In addition, we describe the specialized DNA sequencesthat are required for a chromosome to be accurately duplicated and passedon from one generation to the next. We also confront the serious challenge of DNA packaging. If the double helices comprising all 46 chromosomes in a human cell could be laid end-toend, they would reach approKimately 2 meters; yet the nucleus, which contains the DNA, is only about 6 pm in diameter. This is geometrically equivalent to packing 40 km (24 miles) of extremely fine thread into a tennis ball! The complex task of packaging DNA is accomplished by specializedproteins that bind to and fold the DNA, generating a series of coils and loops that provide increasingly higher levels of organization, preventing the DNA from becoming an unmanageable tangle. Amazingly, although the DNA is very tightly folded, it is compacted in a way that keeps it available to the many enz).rnes in the cell that replicate it, repair it, and use its genesto produce RNA molecules and proteins.

Eucaryotic DNAls Packaged into a Setof Chromosomes In eucaryotes,the DNA in the nucleus is divided between a set of different chromosomes. For example, the human genome-approximately 3.2 x 10e nucleotides-is distributed over 24 different chromosomes. Each chromosome consists of a single, enormously long linear DNA molecule associatedwith proteins that fold and pack the fine DNA thread into a more compact structure. The complex of DNA and protein is called chromatin (from the Greek chroma, "color," because of its staining properties). In addition to the proteins involved in packaging the DNA, chromosomes are also associated with many proteins and RNA molecules required for the processesof gene expression,DNA replication, and DNA repair. Bacteria carry their genes on a single DNA molecule, which is often circular (see Figure 1-29). This DNA is associatedwith proteins that package and condense the DNA, but they are different from the proteins that perform these functions in eucaryotes.Although often called the bacterial "chromosome," it does not have the same structure as eucaryotic chromosomes, and less is knoltm about how the bacterial DNA is packaged.Therefore, our discussion of chromosome structure will focus almost entirely on eucaryotic chromosomes. With the exception of the germ cells (discussed in Chapter 2l) and a few highly specialized cell types that cannot multiply and lack DNA altogether (for example, red blood cells),each human cell contains two copies of each chromosome, one inherited from the mother and one from the father. The maternal and paternal chromosomes of a pair are called homologous chromosomes (homologs). The only nonhomologous chromosome pairs are the sex chromosomes in males, where a Y chromosome is inherited from the father and an X chromosomefrom the mother. Thus, each human cell contains a total of 46 chromosomes-22 pairs common to both males and females, plus two so-called sex chromosomes (X and Y in males, two Xs in females). DNA hybridization is a technique in which a labeled nucleic acid strand servesas a "probe" that localizes a complementary strand, as will be described in detail in Chapter B. This technique can be used to distinguish these human chromosomes by "painting" each one a different color (Figure 4-f0). Chromosome painting is typically done at the stagein the cell cycle called mitosis, when chromosomes are especiallycompacted and easy to visualize (seebelow). Another more traditional way to distinguish one chromosome from another

CHROMOSOMAL DNAAND ITSPACKAGING FIBER IN THECHROMATIN

(A)

(B)

-r^

along each mitotic chromosome (Figure 4-f l). The structural bases for these banding patterns are not well understood. Nevertheless,the pattern of bands on each type of chromosome is unique, and it is these patterns that initially allowed each human chromosome to be identified and numbered. The display of the 46 human chromosomes at mitosis is called the human karyotype. If parts of chromosomes are lost or are switched between chromosomes, these changes can be detected by changes in the banding patterns or by changes in the pattern of chromosome painting (Figure 4-12). Cytogeneticists use these alterations to detect chromosome abnormalities that are associated with inherited defects, as well as to characterize cancers that are associated with specific chromosome rearrangementsin somatic cells (discussedin Chapter 20).

5

2

I5

203 Figure4-10 The completeset of human from chromosomes.Thesechromosomes, a male,wereisolatedfrom a cell and undergoingnucleardivision(mitosis) arethereforehighlycompacted.Each chromosomehasbeen"painted"a differentcolorto permitits unambiguous identification underthe light microscope. paintingis performedby Chromosome to a collection exposingthe chromosomes that havebeen of humanDNAmolecules coupledto a combinationof fluorescent derived dyes.Forexample,DNAmolecules from chromosome1 arelabeledwith one specificdye combination,those from chromosome2 with another,and so on. Because the labeledDNAcanform base pairs,or hybridize, only to the chromosomefrom which it was derived (discussed in Chapter8),each chromosomeis differentlylabeled.For the chromosomes are suchexperiments, subjectedto treatmentsthat separatethe DNAinto individualstrands, double-helical with the designedto permitbase-pairing labeledDNAwhile single-stranded keepingthe chromosomestructure relativelyintact.(A)Thechromosomes visualized asthey originallyspilledfrom the lysedcell.(B)Thesamechromosomes linedup in their numericalorder. artificially Thisarrangement of the full chromosome (FromE.Schrock set is calleda karyotype. With et al..Science273:494-497,1996. permissionfrom AAAS.)

Figure4-1 1 The bandingpatternsof Chromosomes human chromosomes. order 1-22 arenumberedin approximate of size.A typicalhumansomatic(nongerm-line)cellcontainstwo of eachof plustwo sex thesechromosomes, in a chromosomes-twoX chromosomes female,one X and oneY chromosomein a usedto make male.The chromosomes thesemapswerestainedat an earlystage are in mitosis,when the chromosomes incompletelycompacted.The horizontol redline representsthe positionof the centromere(seeFigure4-21),which appearsas a constrictionon mitotic The red knobson chromosomes. c h r o m o s o m e1s3 , ' l 4 ,1 5 , 2 1, a n d 2 2 indicatethe positionsof genesthat code in for the largeribosomalRNAs(discussed Chapter6).Thesepatternsareobtainedby with Giemsastain, stainingchromosomes and they can be observedunderthe light (Formicrographs, seeFigure microscope. 21-1 8; adaptedfrom U. Franke , Cytogenet. 1981.With CellGenet.31:24-32,

204

Chapter4: DNA,Chromosomes, and Genomes

l,?: f J#i,1lff:Tl1flT:ffl[S?ilil:;'1] 1,5# : ;i,en,w,h

ataxia,a diseasecharacterized by progressive deterioration of motor skills. poil, but one The patienthasa normalpairof chromosome 4s (left-hand

il?;|;;;:,T:"?;il::::ililiiil:,'""#i.1,ffi ff:iil: ;:JI:liffi (A)

(B)

aberrantchromosome12 (redbracket\was deduced,from its patternof bands,asa copyof part of chromosome4 that had becomeattachedto

:il:il:::il: l'"*:::l#lit",'*1" f :?il:Tl:T:T;:**il "

pairs,"painted"redfor chromosome4 DNA and bluefor chromosome12 DNA.Thetwo techniquesgive riseto the sameconclusionregardingthe natureof the aberrantchromosome12,but chromosomeoainting providesbetterresolution, allowingthe clearidentification of evenshort piecesof chromosomes that havebecometranslocated. However, Giemsa stainingis easierto perform.(Adaptedfrom E.Schrocket al.,Sclence 273:494-497,1996.With permissionf rom AAA5.)

Chromosomes ContainLongStringsof Genes Chromosomes carry genes-the functional units of heredity. A gene is usually defined as a segment of DNA that contains the instructions for making a particular protein (or a set of closely related proteins). Although this definition holds for the majority of genes, several percent of genes produce an RNA molecule, instead of a protein, as their final product. Like proteins, these RNA molecules perform a diverse set of structural and catalltic functions in the cell, and we discuss them in detail in subsequent chapters. As might be expected, some correlation exists between the complexity of an organism and the number of genes in its genome (see Table l-1, p. 1B). For example, some simple bacteria have only 500 genes, compared to about 25,000 for humans. Bacteria and some single-celled eucaryotes, such as yeast, have especially concise genomes; the complete nucleotide sequence of their genomes reveals that the DNA molecules that make up their chromosomes are little more than strings of closely packed genes (Figure 4-13). However, chromosomes from many eucaryotes (including humans) contain, in addition to genes, a large excessof interspersed DNA that does not seem to carry critical information. Sometimes called "junk DNA' to signify that its usefulness to the cell has not been demonstrated, the particular nucleotide sequence of most of this DNA may not be important. However, some of this DNA is crucial for the proper expression of certain genes,as we discuss elsewhere. Becauseof differences in the amount of DNA interspersed between genes, genome sizes can vary widely (see Figure l-37). For example, the human genome is 200 times larger than that of the yeast S. cereuisiae,but 30 times smaller than that of some plants and amphibians and 200 times smaller than that of a species of amoeba. Moreover, because of differences in the amount of excessDNA, the genomes of similar organisms (bony fish, for example) can vary severalhundredfold in their DNA content, even though they contain roughly the same number of genes.\.Vhateverthe excessDNA may do, it seems clear that it is not a great handicap for a eucaryotic cell to carry a large amount of it. How the genome is divided into chromosomes also differs from one eucaryotic species to the next. For example, compared with 46 for humans, somatic cells from a speciesof small deer contain only 6 chromosomes, while those from a species of carp contain over 100. Even ciosely related species with similar genome sizes can have very different numbers and sizes of chromosomes (Figure 4-14). Thus, there is no simple relationship between chromosome number,

0 5 % o f t h e D N A o f t h e y e a s tg e n o m e

3', 1 0 , 0 0 0n u c l e o t i d ep a i r s

Figure4-1 3 The arrangementof genes in the genome o'fS,cerevisiae. S.cerevisiae is a budding yeastwidely usedfor brewingand baking.The genomeof this yeastcellis distributed over 16 chromosomes. A smallregionof one chromosomehasbeenarbitrarily selectedto showthe high densityof genescharacteristic of this species. As indicatedby the light redshading,some genesare transcribedfrom the lower strand,whileothersaretranscribed from the upperstrand.Thereareabout 6300genesin the completegenome, whichcontainssomewhatmorethan 12 millionnucleotidepairs.(Forthe closelypackedgenesof a bacterium whosegenomeis 4.6millionnucleotides long,seeFigure1-29).

('pl-lsraqs!lqnd uellrul)eW urelord paplol @ uro4 uolssil,utao qllM. t002, l"z6_0ggt60b a.rnJDN'ulntuosuoJ 6ut)uanbasauJouag ueulnHleuotleuratul urolJpaldepv) UlalOlO mM 'uorsr^tp sa)uanoos Jo,uotlpJedald ut saulosouloJq) uo,ssa.rd*a aue6 slr 6urle)tldnplou uaqMsitedapttoal)nu uorlu! | I 'vNc .l ,.:,:.,,,:: i t t , r ,i i l i t l , : , , r , r , i : i t , I i l !t I I Jo lunouleptoldeqaqt 601x v'9lo rlr.ri,lillll:.1 i i r rl.::tli, r, i tiri i' i i' tt I I alrMl ^lq6nolsuteluo)snal)nulle) )tteuos s r r e da p r l o a ; : n u , O tx t € ; o a u a 6 a u o ueurnqe'ploldlp6urag.eupuoqrollu aqt pue sauosoutolq)leal)nutz aql u! VNOro aluenbasaplloallnu ega;du.ro> or1lol sraJaraluanbasawouabuownq urol eqf '(t t latdeq) u! llPlaput passn)stp pue'l raldeql ur pa)npollul)pupuoq)o]il,! s a u a 6y 6 u r u r e l u o la u r o s o r . x o r q + )o % L aql u! puno,sr(lla)rad sardote;d11;nr.u ur-stred eptloal)nu69S'9t)euoua6 ueurnqaql,o uotl)elJelnutLuv 'snal)nu aql ulqll/!^punol ( t [-t pue g 1-7 sarn6rl oos)sauosotuolLl)xas z pue saulosolne saua6gy- [!re auJosouorq)p VoOl ZZeql Ja^opalnqutstpsraurouab 'saoadslno o16ur6uo;aq slll Jo lle1sor,u;y uorlptrtroJur )llauablo Illlelol aq1sr(srred aprtoal)nu60l.x z'€)auouao ueunq aql '9 laldeqf ul llelapul passn)srp se'lueuodutunI;ant1e1al s1$ot61 suorluraqlJo atuanbasVNCaql ollqm 'ulalord aql uorlrod e )o! sapcD(pil) ;o 6uo; s.rredaprloal)nu e0[ x 8? q)ee 'seln]alourVNc orltr]]o pasodulo) uoxa Ll)el 'uorsueoxaploluol loqunj e toue 'uolleuijoluo) )!lol!ul sl! ul zz eurosol,jrolqlueulnq (v) umoqssrauo6le)rdIl e 1olueule6uele uoxo-uoltut aqf (C)'saua6lela^as 'Qt-V eJnBIil suleloJd JoJsapof, (luacrad lBtuosoruoJqcSurureuralaqlJo qonlN Jo rllbual arlua aql sMoqs(g)Jo uoruod ^{eJ e LIuo) ]l Jo allfl ^ oq sr eluoue8 ueunq eq] Jo erntBeJ3ut1u1s ]srIJ eqJ papuedxauy (1; 'sauabpal)!pard ale par 'Iooq sHl ur raldeqs ^,t-lana;o ul esoqlpue seua6umou) oJeu/AorqryDp lueluoJ ut asoqf'pale)rpursaue60t tnoqe qllm s]ueuuedxe 1v\eupe]Elnruus,{.pBeJIBsEq lI aql uo slceJJeJofptu peq e^eq ]eq] iZZaurosouolq>;ouorl.lode1o uolsuedxa 'paz,(lEuB,illry sr uorleuJoJur srq] eJoJeqsapecep zi.uuu aq lnq ill \ u )JolJ aq] plolualv (B)'tardeq)srrllu! ralel passn)stp punoJe puocas rad sapuoelcnu 000I Jo elPJP le seJuenbasaplloelcnu ^^eJpelE sr q)rqM'(ulteuolr.l)olalaq) utleuJolr..l) '{Eed -reuag stl }V'(I-? alqul pue 9I-? arntlg) 8uua83els sl l3eford 1ca[or4eq] l>eduor {;re;ntrlrede ur pabel>ed 1o r,u.ro1 'alqPlle^e are leql VNOJo sa)uanbaspaleada.r aruoueS uEunH aql .{q papr^ord uopBIuJoJuI Jo .&IluBnb reeqs aqJ sr saruosoruoJqc uErunq ile uI uouuuJoJur crleueS elql'v002 q ,,ecuanbasvNC lroqsJo slsrsuo)zz auosoruolq)Jo tllle 'euoua6 ueunq peqsrurJ,, altlua aqt puB I00z q auoueS uErunq aJuue eql Jo ,,ueJp ]sJg,,aql Jo uop aq] Uel aqrJo1soy1 r(lalerulxordde dn saleu pue s.rred -ecnqnd Joo/og'L qll^\ Lepol'(9I-? arnSlC) aluosoluorqr alerqeile^ eJllue ue SuolE aql aprloal)nue0t x It sureluo)'sauosorlojtl) pegueJJe are seue8^^oq.{Fcexeees o} arull IsJIJeql JoJelqlssod arueceq 1l '666I ueuJnqlsolleulsaql lo auo'zz uI '(t-I eJnBrCaes) suraloJd a>lpluol 'selElpauualul VNU q8norqt 'pasn pue lno aurosoruolq)(V)'aulosotuoJq)ueunq e peal sr VNCI ul uorlP{uJoJur eq} /\/\oq'sr.uJe}IeJauaSuI 'pessnsslp e,\/\I raldeq] uI uo saua6lo uoglezgue6,ro aq1 91-9 arn6r1

I

I

II

pobuerrv erv saueD rno /vloHs/v\orls auoueguerunHeql,o a)uanbaseplloel)nNoql ('0002'sraqs!lqnd llalupg g sauor:vy! [rnqpn5'pa pr€ /uorlnlon3'ra6req1trr15'n11'y1 'seluu.{Jeuor}nl r,uor;pardepy)'saua6;o taqurnu -ola Suol Je^osaJnsseJduollJales,{q uo pelcu 'sluala f,l}eua8ruopuer flSutruees repurse ureluo)seoadsom] asaql'leulue aq] uo l)aJJeloleu e burneq]noq];m'pasn; Jo ^Jolsrq enbrun e ,{.qpadeqs ueaq {1JEaaleq selJeds ,(ep-urapou Jo seuros -ouorr{J pue sauoueS aq} 'raqleg 'aZIs euoueS IPlo} puP salJads saurosourol r.lta1e.r edasI; ;erlrur'reflunu "fiIxalduoc uerpuleql uorlnlo^aaLllul 's,taqurnu Jo euosou,roltl)lueraggp{ran qllm raap :e[1unuasaurql > e [ u n u .u e r p u ; pale;ar {;aso1>orvrltr 1-y arn6r1 1o sag>eds tA I

l.x r(f

,!

rr

--

--

XZ

|l

v9

o,

9r

rrv

gla

(ro

trv

9r,

v(

uU

ba

UU

uu uu (r(, 0u

902

U]8II NIIVIAOUHf]HI NI 9NI9V))Vd SII CNV VNC IVWOSOWOUH)

pue leln])nrls'isuor6a.r po^.tasuol pue'svNUleuolt)unJ vNC aqt uo satrs6u purq-uralord pa^lasald pJlelsuerlun) sl1n ,€ pue,g 6urpoluaVNCapnputasaq]lsuot60lleuorl)unJ *** ,4dotauo ur aqt uror; suortetnLu 6urbeiuep;o uortelnunllpaql .(q poMollolauableuotDunle 1ouotlettldnp 6ututeluot1nq uorssardxa radordsll luanardleql suorlelnusnorautnu asueseuabopnasd 1so1,11 'eua6 y ** yp6 1oaruanbasapttoallnue sraua6opnasd elo leqt 6urlqurasar,(lasop leuorlrunJ lluapuel areleql ouo u.ror1 6uua;rpsraquJnu leedarq1t,a,r'paleader lxouaqt ol lenpr^rpur y5166uruteurar peleade;llqbrqlroqs1ostsrsuo) lluer-uud aql (saplloal)nu saruanbas OOO'OO L l r rJ U o l n o q e A l u o J o J l p r r o r a ) , { l a s r : a r d usM J po [uo)Jslr) n u , r o l l l l q q B 6 . 1 o a t u a n b a s e q l * (dor-q6tq ul oTogg {qaler"urxorddes}uarrrele anr}r}ader VNC }o a6e}ue)rad o/o9't ***SelUenOeS pa^rasuo)I;q6rq raqlo ul vNc,o abetua)red

o/o9'l 000'02ueql erou sredeprloel)nuItt srledaprloal)nugoL'LL t'0t 8LL l srledeprloel)nuooo'Lz sltedaptloel)nueOI x t'Z ggg'97Ilaleurxordde aptloallnu60l. x Z'€ +sJted

(sa>uenbas 6urpor urelord)suoxaura)uonbas VNCJoebelue)rod *xsoua60pnesd ;o requrnSl azrsuoxoueew azrsuoxalsa6lel aua6rad suoxaJoraqLUnu ueaw eua6radsuoxaJoraqunu lsablel euebrad suoxaro Jaqurnu ]salleus azrsaua6ueew auoblsa6re-l saua6JoraquinN !]6ua1YP6

ououaD ueuinHaql rol s)llsllels lelln euos [-t alqel

'dlssalarecrltuaredde 'r{.yaleururr.rcslpur sualr elqenp^ ,,'lnoq8norql paralleJs dliualed,r,ra; aql pue :pepreosrpra^e Sulqlou,!1en1rp 1(,1unf,sp pateqlulun aqt ^q ol peJJeJer)JallnlJ palepruncre qonru luoqpzrue8to Jo eJuaplle ell]{ l}duelun 'ralaure lano trrtsrlenpplpul dUBq :eJrT/roteJe8r4ar/uoorpaqTa8ure8rno/t alquasar lsnf ^luo ol dn ppe lnq plnoMaua6stLltut sa)uanbes6utpo)aql .4.eru11sitem auros u1,, 'auroua8 Jno paqrJcsap Joleluaruruoc auo sy ferresrp ;o ]nq 'LU0€ roJpuatxe plno^^aua6 a6ela^e uV 'tu 0€ t r(ranaaua6 6urpor-ura1o.rd alels SurruJpleup uI aq ol suaas ueunq e af,npoJd ol pepeeu uollBruJoJur lPcrlrJc aql leql paleelar seq aruoua8 uerunq aq] Jo aouenbas apnoalcnu eql dlpurC e'e6elaneuo 'aq plnoMelaql'ale)s 'Irorlr saJuanbasy11q Lrolep8ar,uoq raldeq3 ur ssnrsrp slql lv '(Bu aq par)sur6uoueunq rno 1o 2 alrsaql 'e)uJVjo ratua) aql ssol)e q)lells aM'sauoua8 esrJuoJqlyv\ susruESro ur passarduoc aroru eJBsaJuanbesdroleln o1q6nouareJ'(sapur 6697Ila1er-urxordde) -3er asaq] 'palradxa eq plnom sy 'srrcd epqoalf,nu Jo spuesnoql Jo sue] JaAolno Lul 002€pualxaplnoMeuoua6 ueunq peards aru eue8 pcldril e JoJ seJuenbas ,trolep8eJ er{l 'suerunq q 'lleJ Jo eddr aql'(v) ur se'eprloal)nuq)pa uaaMlaq JadoJdeq] ut I1uo puu 'la^el alerrdordde aql lE pessaJdxa'arur1JedoJdaql te JJo areds uut I e qllM umplpl .euoua6 Jo uo paurnl sr eue8 aq] teq] Surrnsua JoJelqrsuodseJ eJe qJIqM 'sacuanbasy6161 uetunqaqtro e1er591-tr arnbrl holaynBat qlyv\ pelprJossp sr eue8 qope 'suoxe pue suoJlur o] uonrppe uI 'saruoso(uoJqcJraql ul vNo SurpocSouorlJe4 raqSrqqcnu aql JoJ se IIeM se '(seua8 upunq Jo ]eq] qlapue^ l-euo lnoqP) sauaSrraql Jo azrs rallerus qJnru aql JoJslunocce srql'suoJlur {rEI saruoua8 asrcuoc qlylr susrue8Jo ruor; saua8 .&rroferu aq] 'lseJ]uoc uI 'suoJ]urJo 3ur]srsuoc eue8 eq] Jo lsotu q]lm ;o 'suoJlul pue suoxe Surleuralp go Surns Suoye Jo lsrsuoc snqt seue8 ueurnq;o dlr -rofeu eqJ'(I-t elqeJ pue g1-7 arn8rgaas)suonrq palpc ere saua8ur seruenbes (Surpocuou) Suruarrratureq] lsuoxe pelleJ ale saJuenbasSurpoc eq] 'g JeldeqJ ur 'uralord JoJepol ]Bqt VNC Jo stuetu8es uoqs /i.la^ue Irelep ur pessnf,srpaq III ^ sy -lal eqt Surpocuou ldnJJalur ]Eql VNCI ;o seqcleJls3uo1yo stsrsuoc eua8 E ul VNe Sutureruar aq] Jo lsolN '(sueunq ut sprJp ourrup gtt lnoqe) azrs aBpJaAE Jo urel -ord e epoJua ol parrnbal eJEsJredeppoelonu 00tI lnoqp dpg 'uralord e sprce Jo ourue er{l Jo aruanbas ruauq eql JoJ uopeuJoJur aqt sepnoelJnu go acuanbas 'sJred appoelJnu 'eloqe passnJsrp Jeeurl slr ur serJJec eue8 e sV 000'LZ Jo Imrd,{r azs aua8 aBpJeAEa8rel aql sr eruoue8 ueunq aql eJnl€eJelqelou puooas V Jo 'sraldeqc relel ur lrc]ap ur vuawap a1qasod (v) -su0Jl esarfl ssncsrp aM'etup dteuorlnloura JaAoaruosoruoJqJ ar{] ur sa^lesueql pauasur dlenperS a^Eq lpql VNC Jo sacard alqou 'iloqs Jo dn apeu sl VNCI

'vNo :t raldeql sauouaDpuP'sauosouroJqf

902

.: :..:.,,,1:;,1$illJ

CHROMOSOMAL DNAAND ITSPACKAGING INTHECHROMATIN FIBER percentage 0

10

20

30

40

LINEs SlNEs r e t r o v i r a l - l i ke l e m e n t s D N A - o n l yt r a n s p o s o n ' f o s s i l s '

50

50

70

80

90

100

protein-coding regions GENES

IRANSPOSONS

s r m p t es e q u e n c er e p e a t s s e g m e n t adl u p l i c a t i o n s R E P E A T ESDE O U E N C E S

non-repetitiveDNA that is n e i t h e ri n i n t r o n sn o r c o d o n s UNIQUE SEQUENCES

GenomeComparisons RevealEvolutionarily Conserved DNA Sequences A major obstacle in interpreting the nucleotide sequences of human chromosomes is the fact that much of the sequence is probably unimportant. Moreover, the coding regions of the genome (the exons) are typically found in short segments (average size about 145 nucleotide pairs) floating in a sea of DNA whose exact nucleotide sequence is of little consequence. This arrangement makes it very difficult to identify all the exons in a stretch of DNA sequence. Even harder is the determination of where a gene begins and ends and exactly how many exons it spans. Accurate gene identification requires approaches that extract information from the inherently low signal-to-noise ratio of the human genome. We shall describe some of them in Chapter 8. Here we discussonly one general approach, which is based on the observation that sequencesthat have a function are relatively conserved during evolution, whereas those without a function are free to mutate randomly. The strategy is therefore to compare the human sequence with that of the corresponding regions of a related genome, such as that of the mouse. Humans and mice are thought to have diverged from a common mammalian ancestor about 80 x 106years ago,which is long enough for the majority of nucleotides in their genomes to have been changed by random mutational events.Consequently,the only regions that will have remained closely similar in the two genomes are those in which mutations would have impaired function and put the animals carrying them at a disadvantage, resulting in their elimination from the population by natural selection. Such closely similar regions are known as conserued regions. The conserved regions include both functionally important exons and regulatory DNA sequences. In contrast, nonconserued regionsrepresent DNA whose sequenceis unlikely to be critical for function. The power of this method can be increased by comparing our genome with the genomes of additional animals whose genomes have been completely sequenced,including the rat, chicken, chimpanzee, and dog. By revealing in this way the results of a very long natural "experiment," lasting for hundreds of millions of years, such comparative DNA sequencing studies have highlighted the most interesting regions in these genomes.The comparisons reveal that roughly 5% of the human genome consists of "multi-species conserved sequences,"as discussed in detail near the end of this chapter. Unexpectedly, only about onethird of these sequences code for proteins. Some of the conserved noncoding sequences correspond to clusters of protein-binding sites that are involved in gene regulation, while others produce RNA molecules that are not translated into protein. But the function of the majority of these sequences remains unknor.tm. This unexpected discovery has led scientists to conclude that we understand much less about the cell biology of vertebrates than we had previously imagined. Certainly, there are enormous opportunities for new discoveries, and we should expect many surprises ahead. Comparative studies have revealed not only that humans and other mammals share most of the same genes,but also that large blocks of our genomes contain these genes in the same order, a feature calIed conseruedsynteny.As a result, Iarge blocks of our chromosomes can be recognized in other species. This allows the chromosome painting technique to be used to reconstruct the recent evolutionarv historv of human chromosomes (Fieure 4-18).

Figure 4-17 Representationof the nucleotidesequencecontent of the completelysequencedhuman genome. retroviral-like elements, The LlNEs, SlNEs, aremobile and DNA-onlytransposons geneticelementsthat havemultipliedin themselves our genomeby replicating and insertingthe new copiesin different positions. Thesemobilegeneticelements in Chapter5 (seeTable5-3, arediscussed p. 318).Simplesequencerepeatsare (lessthan 14 shortnucleotidesequences nucleotidepairs)that arerepeatedagain Segmental and againfor long stretches. arelargeblocksof the duplications genome(1000-200,000 nucleotidepairs) that are presentat two or more locations in the genome.The most highlyrepeated have blocksof DNAin heterochromatin not yet been completelysequenced; of human DNA thereforeabout 10olo in this arenot represented sequences diagram.(Datacourtesyof E.Margulies.)

208

Chapter4: DNA,Chromosomes, and Genomes

A N C E S T OC RH R O M O S O M E

a n c e s t o rD N A of human c h r o m o s o m e3

2 i n v e r soin 5

a n c e s t o rD N A of human c h r o m o s o m e2 1

f usion

I

(A)

"6'

B

-

ry lemur

orangutan

nu m a n

abcdabcdabcd (B)

ChromosomesExistin DifferentStatesThroughout the Life of a Cell

Figure4-18 A proposedevolutionary historyof human chromosome3 and its relativesin other mammals.(A)The orderof chromosome3 segments hypothesized to be presenton a chromosomeof a mammalianancestoris shown$rellowbox).The minimum changesin this ancestral chromosome necessary to accountfor the appearance of eachof the threemodern (The chromosomes areindicated. present-day chromosomes of humans and Africanapesareidenticalat this resolution.) The smallcirclesdepicted in the modernchromosomes reoresent the positionsof centromeres. A fissionand inversion that leadsto a changein chromosomeorganization is thoughtto occuronceevery5-10 x 106yearsin mammals.(B)Someof the chromosome paintingexperiments that led to the diagramin (A).Eachimageshowsthe chromosomemostcloselyrelatedto humanchromosome3, paintedgreenby hybridization with differentsegmentsof DNA,lettereda, b, c, and d alongthe bottom of the figure.Theseletters correspondto the coloredsegmentsof the diagramsin (A),as indicatedon the (From5. MLilleret ancestral chromosome. al.,Proc.NatlAcad.Sci.U.5.A.97:206-211, 2000.With permission from National Academyof Sciences.)

We have seen how genesare arranged in chromosomes, but to form a functional chromosome, a DNA molecule must be able to do more than simply carry genes: it must be able to replicate, and the replicated copies must be separatedand reliably partitioned into daughter cells at each cell division. This process occurs through an ordered series of stages,collectively known as the cell cycle, which provides for a temporal separation between the duplication of chromosomes and their segregation into two daughter cells. The cell cycle is briefly summarized in Figure 4-19, and it is discussed in detail in Chapter 17. Only certain

n u c t e a re n v e t o p e

MTTOSTS

In r e r p n a s e c hr o m o s o m e

m itotic

chromosome I NTERPHASE

M PHASE

INTERPHASE

Figure4-19 A simplifiedview of the eucaryoticcell cycle,Duringinterphase, the cellis activelyexpressing its genesano is thereforesynthesizing proteins. Also,duringinterphase and beforecelldivision,the DNAis replicated and each chromosomeis duplicatedto producetwo closelypaireddaughterchromosomes (a cellwith onlytwo chromosomes is illustrated here).OnceDNAreplication is complete,the cellcan enterM phase,when mitosisoccursand the nucleusis dividedinto two daughternuclei.Duringthis stage,the chromosomes condense, the nuclearenvelopebreaksdown,and the mitoticspindleformsfrom microtubules and other proteins. Thecondensedmitoticchromosomes arecapturedby the mitoticspindle,and one completesetof chromosomes is then pulledto eachend of the cellby separating eachdaughter chromosomepair.A nuclearenvelopere-formsaroundeachchromosomeset,and in the finalstepof M phase,the cell dividesto producetwo daughtercells,Mostof the time in the cellcycleis spentin interphase; M phaseis briefin comparison, occupyingonly aboutan hour in manymammaliancells.

CHROMOSOMAL DNAAND ITSPACKAGING INTHECHROMATIN FIBER

,:' l$9 ";,;:'"' Figure4-20 A comparisonof extended interphasechromatinwith the chromatin in a mitotic chromosome. (A)A scanningelectronmicrographof a mitoticchromosome: a condensed duolicatedchromosomein whichthe arestilllinked two new chromosomes together(seeFigure4-21).The regionindicatesthe position constricted describedin Figure of the centromere, 4-21.(B)An electronmicrograph showingan enormoustangleof chromatinspillingout of a lysed interphasenucleus.Note the differencein scales.(A,courtesyof TerryD. Allen; B,courtesyof VictoriaFoe.)

(A)

1 lr.

(B)

l0 pm

parts of the cycle concern us in this chapter. During interphase chromosomes are replicated, and during mitosis they become highly condensed and then are separated and distributed to the two daughter nuclei. The highly condensed chromosomes in a dividing cell are knorm as mitotic chromosomes (Figure 4-2OA).This is the form in which chromosomes are most easily visualized; in fact, the images of chromosomes shor,rmso far in the chapter are of chromosomes in mitosis. During cell division, this condensed state is important for the accurate separation of the duplicated chromosomes by the mitotic spindle, as discussedin Chapter 17. During the portions of the cell cycle when the cell is not dividing, the chromosomes are extended and much of their chromatin exists as long, thin tangled threads in the nucleus so that individual chromosomes cannot be easily distinguished (Figure 4-208).We shall refer to chromosomes in this extended state as interphasechromosomes.Since cells spend most of their time in interphase, and this is where their genetic information is being read out, chromosomes are of greatestinterest to cell biologists when they are least visible.

EachDNAMoleculeThatFormsa LinearChromosome Must Containa Centromere, Origins TwoTelomeres, and Replication A chromosome operates as a distinct structural unit: for a copy to be passed on to each daughter cell at division, each chromosome must be able to replicate, and the newly replicated copies must subsequently be separated and partitioned correctly into the two daughter cells.These basic functions are controlled by three types of specialized nucleotide sequencesin the DNA, each of which binds specific proteins that guide the machinery that replicates and segregates chromosomes (Figure 4-21) . Experiments in yeasts, whose chromosomes are relatively small and easy to manipulate, have identified the minimal DNA sequence elements responsible for each of these functions. One type of nucleotide sequenceacts as a DNA replication origin, the location at which duplication of the DNA begins. Eucaryotic chromosomes contain many origins of replication to ensure that the entire chromosome can be replicated rapidly, as discussedin detail in Chapter 5. After replication, the two daughter chromosomes remain attached to one another and, as the cell cycle proceeds, are condensed further to produce mitotic chromosomes. The presence of a second specialized DNA sequence, called a centromere, allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell when a cell divides. A protein

21O

Chapter4: DNA,Chromosomes, and Genomes INTERPHASE

telomere

r e p li c a t i o n oflgrn

A

MtTOStS

INTERPHASE

i!, E

HH

centromere

HH

portion of m i t o t i cs p i n d l e

tlLl

d u pl icated chromosomes i n s e p a r a t ec e l l s

complex called a kinetochore forms at the centromere and attaches the duplicated chromosomes to the mitotic spindle, allowing them to be pulled apart (discussedin Chapter I7). The third specializedDNA sequenceforms telomeres, the ends of a chromosome. Telomerescontain repeated nucleotide sequencesthat enable the ends of chromosomes to be efficiently replicated. Telomeresalso perform another function: the repeated telomere DNA sequences,together with the regions adjoining them, form structures that protect the end of the chromosome from being mistaken by the cell for a broken DNA molecule in need of repair. We discuss both this type of repair and the structure and function of telomeres in Chapter 5. In yeast cells, the three types of sequencesrequired to propagate a chromosome are relatively short (typically less than 1000base pairs each) and therefore use only a tiny fraction of the information-carrying capacity of a chromosome. Although telomere sequencesare fairly simple and short in all eucaryotes, the DNA sequencesthat form centromeres and replication origins in more complex organisms are much longer than their yeast counterparts. For example, experiments suggestthat human centromeres contain up to 100,000nucleotide pairs and may not require a stretch of DNA with a defined nucleotide sequence. Instead, as we shall discuss later in this chapter, they seem to consist of a large, regularly repeating protein-nucleic acid structure that can be inherited when a chromosome replicates.

DNAMolecules AreHighlyCondensed in Chromosomes All eucaryotic organisms have special ways of packaging DNA into chromosomes. For example, if the 48 million nucleotide pairs of DNA in human chromosome 22 could be laid out as one long perfect double helix, the molecule would extend for about 1.5 cm if stretched out end to end. But chromos ome 22 measures only about 2 pm in length in mitosis (seeFigures 4-10 and 4-ll), representing an end-to-end compaction ratio of nearly 10,000-fold.This remarkable feat of compression is performed by proteins that successivelycoil and fold the DNA into higher and higher levels of organization. Although much less condensed than mitotic chromosomes, the DNA of human interphase chromosomes is still tightly packed, with an overall compaction ratio of approximately 500-fold (the length of a chromosome's DNA helix divided by the end-to-end length of that chromosome). In reading these sections it is important to keep in mind that chromosome structure is dynamic. We have seen that each chromosome condenses to an unusual degree in the M phase of the cell cycle. Much less visible, but of enormous interest and importance, specific regions of interphase chromosomes

Figure4-21 The three DNAsequences requiredto producea eucaryotic chromosomethat can be replicatedand then segregatedat mitosis.Each chromosomehasmultipleoriginsof replication, one centromere, and two telomeres.Shownhere is the seouenceof eventsthat a typicalchromosome follows duringthe cellcycle.The DNAreplicates in interphase, beginningat the originsof replication and proceeding bidirectionally from the originsacrossthe chromosome. In M phase,the centromere attachesthe duplicatedchromosomes to the mitoticspindleso that one copyis distributedto eachdaughtercellduring mitosis. Thecentromerealsohelpsto hold the duplicatedchromosomes togetheruntilthey arereadyto be movedapart.The telomeresform special caosat eachchromosomeend.

CHROMOSOMAL DNA AND ITSPACKAGING INTHECHROMATIN FIBER

211

decondense as the cells gain access to specific DNA sequences for gene expression, DNA repair, and replication-and then recondense when these processesare completed. The packaging of chromosomes is therefore accomplished in a way that allows rapid localized, on-demand accessto the DNA. In the next sections we discuss the specialized proteins that make this type of packagingpossible.

Nucleosomes Area BasicUnitof Eucaryotic Chromosome Structure The proteins that bind to the DNA to form eucaryotic chromosomes are traditionally divided into two general classes:the histones and the nonhistone chromosomal proteins.The complex of both classesof protein with the nuclear DNA of eucaryotic cells is knor,rmas chromatin. Histones are present in such enormous quantities in the cell (about 60 million molecules of each t)?e per human cell) that their total mass in chromatin is about equal to that of the DNA. Histones are responsible for the first and most basic level of chromosome packing, the nucleosome, a protein-DNA complex discovered in 1974.\Mhen interphase nuclei are broken open very gently and their contents examined under the electron microscope, most of the chromatin is in the form of a fiber with a diameter of about 30 nm (Figure 4-22A).If this chromatin is subjected to treatments that cause it to unfold partially, it can be seen under the electron microscope as a series of "beads on a string" (Figure 4-228). The string is DNA, and each bead is a "nucleosome core particle" that consists of DNA wound around a protein core formed from histones. The structural organization of nucleosomes was determined after first isolating them from unfolded chromatin by digestion with particular enzymes (called nucleases) that break dor,rmDNA by cutting between the nucleosomes. After digestion for a short period, the exposed DNA between the nucleosome core particles, the linker Dl/A, is degraded. Each individual nucleosome core particle consists of a complex of eight histone proteins-two molecules each of histones H2A, HzB, H3, and H4-and double-stranded DNA that is 147 nucleotide pairs long. The histone octamer forms a protein core around which the double-stranded DNA is wound (Figure 4-23). Each nucleosome core particle is separated from the next by a region of linker DNA, which can vary in length from a few nucleotide pairs up to about 80. (The term nucleosometechnically refers to a nucleosome core particle plus one of its adjacent DNA linkers, but it is often used synonymously with nucleosome core particle.) On average,therefore, nucleosomes repeat at intervals of about 200 nucleotide pairs. For example, a diploid human cell with 6.4 x 10enucleotide pairs contains approximately 30 million nucleosomes.The formation of nucleosomes converts a DNA molecule into a chromatin thread about one-third of its initial length.

(A)

Figure 4-22 Nucleosomesas seenin the electron microscope,(A)Chromatin isolateddirectlyfrom an interphase nucleusappearsin the electron microscooe asa thread30 nm thick. (B)Thiselectronmicrographshowsa lengthof chromatinthat hasbeen or unpacked, experimentally afterisolationto showthe decondensed, (A,courtesyof Barbara nucleosomes. Hamkalo;B,courtesyof VictoriaFoe.)

212

Chapter4: DNA,Chromosomes, and Genomes Figure4-23 Structuralorganizationof the nucleosome. A nucleosome containsa proteincoremadeof eight histonemolecules. In biochemical experiments, the nucleosome coreparticlecan be released from isolated chromatinby digestionof the linkerDNAwith a nuclease, an enzymethat breaksdown DNA,(Thenuclease candegradethe exposedlinkerDNAbut cannotattackthe DNAwound tightlyaroundthe nucleosome core.)After dissociation of the isolatednucleosome into its proteincoreand DNA,the lengthof the DNAthat waswound aroundthe corecan be determined. Thislengthof 147nucleotidepairsis sufficientto wrap 1.7 timesaround the histonecore.

c o r eh i s t o n e s of nucleosome

nucleosome includes " beads-on-a-string " -200 nucleotide formof chromatin p a i r so f D N A

I NUCLEASE I DTGESTS I L I N K EDRN A I V

,.rd'..Q'...od TheStructureof the Nucleosome CoreParticleReveals How DNA ls Packaged The high-resolution structure of a nucleosome core particle, solved in 1997, revealed a disc-shaped histone core around which the DNAwas tightlywrapped 1.7 turns in a left-handed coil (Figure 4-24). All four of the histones that make up the core of the nucleosome are relatively small proteins (102-135 amino acids), and they share a structural motif, known asthe histonefold,formed from three cr helices connected by two loops (Figure 4-25).In assembling a nucleosome, the histone folds first bind to each other to form H3-H4 and H2A-H2B dimers, and the H3-H4 dimers combine to form tetramers. An H3-H4 tetramer then further combines with two HZA-H2B dimers to form the compact octamer core, around which the DNA is wound (Figure 4-26). The interface between DNA and histone is extensive: 142 hydrogen bonds are formed between DNA and the histone core in each nucleosome. Nearly half of these bonds form between the amino acid backbone of the histones and the phosphodiester backbone of the DNA. Numerous hydrophobic interactions and salt linkages also hold DNA and protein together in the nucleosome. For example, more than one-fifth of the amino acids in each of the core histones are either lysine or arginine (two amino acids with basic side chains), and their positive

I

t -^,^^-^r *(

w

octameric h i s t o n ec o r e

+

ilJ H2A

side view

€ t h i s t o n eH 2 A

bottom view

@ h i s t o n eH 2 B

@ h i s t o n eH 3

O

h i s t o n eH 4

r

"ill".'"?..&ll rir .. :;;;i:r

sL H2B

147-nucleotide-pair D N Ad o u b l eh e l i x

Jt'

H3

ar6 H4

Figure4-24 The structureof a nucleosome core particle,as determined by x-ray diffraction analysesof crystals.Each histoneis coloredaccordingto the scheme in Figure4-23,with the DNAdoublehelixin light gray.(From K. Luger et al.,Nature 389:251-260,1997.With permissionfrom MacmillanPublishers Ltd.)

CHROMOSOMAL DNAAND IT5PACKAGING INTHECHROMATIN FIBER

213

(A) H2A 4la6t

H2B

H3N

n

H4

m*"'-' N - t e r m i n atla i l

**ffi h i s t o n ef o l d

Figure4-25 The overallstructuralorganizationof the core histones. (A)Eachof the corehistonescontainsan N-terminal tail,which is subjectto severalformsof covalentmodification, and a histonefold region,as (B)Thestructureof the histonefold,whichis formedby allfour indicated. (C)Histones of the corehistones. 24 and 28 form a dimerthroughan interactionknownasthe "handshakei'Histones H3 and H4 form a dimer throughthe sametype of interaction.

charges can effectively neutralize the negatively charged DNA backbone. These numerous interactions explain in part why DNA of virtually any sequencecan be bound on a histone octamer core. The path of the DNA around the histone core is not smooth; rather, several kinks are seen in the DNA, as expected from the nonuniform surface of the core.The bending requires a substantial compression of the minor groove of the DNA helix. Certain dinucleotides in the minor groove are especiallyeasyto compress,and some nucleotide sequencesbind the nucleosome more tightly than others (Figure 4-27). This probably explains some striking, but unusual, casesof very precise positioning of nucleosomes along a stretch of DNA. For most of the DNA sequencesfound in chromosomes, however, the sequence preference of nucleosomes must be small enough to allow other factors to dominate, inasmuch as nucleosomes can occupy any one of a number of positions relative to the DNA sequence in most chromosomal regions. In addition to its histone fold, each of the core histones has an N-terminal amino acid "tail", which extends out from the DNA-histone core (see Figure 4-26). These histone tails are subject to several different types of covalent modifications that in turn control critical aspects of chromatin structure and function, as we shall discuss shortly. As a reflection of their fundamental role in DNA function through controlling chromatin structure, the histones are among the most highly conserved eucaryotic proteins. For example, the amino acid sequenceof histone H4 from a pea and from a cow differ at only 2 of the 102 positions. This strong evolutionary conservation suggeststhat the functions of histones involve nearly all of their amino acids, so that a change in any position is deleterious to the cell. This suggestion has been tested directly in yeast cells, in which it is possible to mutate a given histone gene in uitro andintroduce it into the yeast genome in place of the normal gene. As might be expected, most changes in histone sequences are lethal; the few that are not lethal cause changes in the normal pattern of gene expression,as well as other abnormalities. Despite the high conservation of the core histones, eucaryotic organisms also produce smaller amounts of specializedvariant core histones that differ in amino acid sequence from the main ones. As we shall see,these variants, combined with a surprisingly large variety of covalent modifications that can be added to the histones in nucleosomes, make possible the many different chromatin structures that are required for DNA function in higher eucaryotes.

c

214

Chapter4: DNA,Chromosomes, and Genomes Figure4-26 The assemblyof a histone octamer on DNA.The histoneH3-H4 dimerand the H2A-H2Bdimerare formed from the handshakeinteraction. An H3-H4tetramerformsand bindsto the DNA.Two H2A-H2Bdimersarethen added,to completethe nucleosome. The histonesarecoloredas in Figures4-24 and4-25. Notethat all eight N-terminal tailsof the histonesprotrudefrom the disc-shaped corestructure. Their conformations arehighlyflexible. lnsidethe cell,the nucleosome assemblyreactions shownhereare mediatedby histonechaperoneproteins, some specificfor H3-H4 and others specificfor H2A-H28.(Adaptedfrom figuresby J.Waterborg.)

H 3 - H 4d i m e r

I H3-H4 tetramer

two dimers bind to H3-H4 tetramer

/3

G-Cpreferred here (minor groove outside)

TT,and TA dinucleotides preferred here ( m i n o rg r o o v ei n s i d e ) histone core of nucleosome (histoneoctamer)

D N Ao f nucteosome

Figure4-27 The bending of DNA in a nucleosome. The DNAhelixmakes 1.7tight turnsaroundthe histone octamer.This diagramillustrates how the minorgrooveis compressed on the insideof the turn.Owingto certain structural featuresof the DNAmolecule, the indicateddinucleotides are preferentially accommodated in sucha narrowminorgroove,which helpsto explainwhy certainDNAsequences will bind moretightlythan othersto the nucleosomecore.

CHROMOSOMAL DNAAND ITSPACKAGING INTHECHROMATIN FIBER w r a p p e on u c r e o s o m e existsfor 250 milliseconds

u n w r a p p e dn u c l e o s o m e existsfor 10-50 milliseconds

rewrapped nucleosome

215

Figure4-28 Dynamicnucleosomes. showthat the DNA Kineticmeasurements is surprisingly in an isolatednucleosome dynamic,rapidlyuncoilingand then core. rewrappingaroundits nucleosome As indicated, this makesmostof its bound to otherDNADNAsequenceaccessible bindingproteins.(Datafrom G. Li and J.Widom, Nat.Struct.Mol.Biol.11:763-769, from Macmillan 2004.With oermission Ltd.) Publishers

Nucleosomes Havea DynamicStructure, and Are Frequently Subjected to Changes ChromatinCatalyzed by ATP-Dependent Remodeling Complexes For many years biologists thought that, once formed in a particular position on DNA, a nucleosome remains fixed in place because of the very tight association between its core histones and DNA. If true, this would pose problems for genetic readout mechanisms, which in principle require rapid accessto many specific DNA sequences,as well as for the rapid passageof the DNA transcription and replication machinery through chromatin. But kinetic experiments show that the DNA in an isolated nucleosome unwraps from each end at rate of about 4 times per second, remaining exposed for 10 to 50 milliseconds before the partially unr,trapped structure recloses.Thus, most of the DNA in an isolated nucleosome is in principle availablefor binding other proteins (Figure 4-28). For the chromatin in a cell, a further loosening of DNA-histone contacts is clearly required, because eucaryotic cells contain a large variety of ATP-dependent chromatin remodeling complexes. The subunit in these complexes that hydrolyzes ATP is evolutionarily related to the DNA helicases (discussed in Chapter 5), and it binds both to the protein core of the nucleosome and to the double-stranded DNA that winds around it. By using the energy of AIP hydrolysis to move this DNA relative to the core, this subunit changes the structure of a nucleosome temporarily, making the DNA less tightly bound to the histone core. Through repeated cycles of ATP hydrolysis, the remodeling complexes can catalyze nucleosomesliding, and by pulling the nucleosome core along the DNA double helix in this way, they make the nucleosomal DNA availableto other proteins in the cell (Figure 4-25). In addition, by cooperating with negatively ATP-dependent c h r o m a t i nr e m o d e l i n g

complex

/

4J

.s.Fl CATALYSIS OF NUCLEOSOM SL EI D I N G

Figure4-29 The nucleosomesliding catalyzedby ATP-dependentchromatin remodelingcomplexes.Usingthe the remodeling energyof ATPhydrolysis, complexis thoughtto pushon the DNA and loosenits of its bound nucleosome core.Each attachmentto the nucleosome and cycleof ATPbinding,ATPhydrolysis, release of the ADPand PiProducts therebymovesthe DNAwith respectto the histoneoctamerin the directionof the arrowin this diagram.lt requires manysuchcyclesto producethe slidingshown.(Seealso nucleosome Figure4-468.)

2'16

Chapter4: DNA,Chromosomes, and Genomes

Figure4-30 Nucleosomeremovaland histone exchangecatalyzedby ATP-dependent chromatinremodelingcomplexes.By cooperating with specifichistonechaperones, somechromatinremodelingcomplexes can removethe H2A-H2Bdimersfrom a (top seriesof reactions) nucleosome and replacethem with dimersthat containa variant histone,suchas the H2AZ-H2Bdimer (see Figure4-41).Otherremodelingcomplexes are attractedto specificsiteson chromatinto removethe histoneoctamercompletelyand/or to replaceit with a differentnucleosome core (bottomseriesof reactions)

h i s t o n ec h a p e r o n e

ATP-dependent chromatin remodeling complex

i2

"i+b

charged proteins that serve as histone chaperones,some remodeling complexes are able to remove either all or part of the nucleosome core from a nucleosome-catalyzing either an exchange of its HZA-H2B histones, or the complete removal of the octameric core from the DNA (Figure 4-90). Cellscontain dozensof differentATP-dependentchromatin remodeling complexes that are specializedfor different roles. Most are large protein complexes that can contain 10 or more subunits. The activity of these complexesis carefully controlled by the cell.As genesare turned on and off, chromatin remodeling complexes are brought to specific regions of DNA where they act locally to influence chromatin structure (discussedin Chapter 7; seealso Figure 4-46, below). As pointed out previously,for most of the DNA sequencesfound in chromosomes,experimentsshow that a nucleosomecan occupy any one of a number of positions relative to the DNA sequence.The most important influence on nucleosomepositioning appearsto be the presenceof other tightly bound proteins on the DNA. Some bound proteins favor the formation of a nucleosome adjacent to them. others create obstacles that force the nucleosomes ro move to positions between them. The exact positions of nucleosomes along a stretch of DNA therefore depends mainly on the presence and nature of other proteins bound to the DNA. Due to the presenceof ATP-dependentremodeling complexes, the arrangement of nucleosomes on DNA can be highly dynamic, changing rapidly accordingto the needs of the cell.

Nucleosomes Are UsuallyPacked Togetherinto a Compact Ch r o m a t i F n i b er Although enormously long strings of nucleosomes form on the chromosomal DNA, chromatin in a living cell probably rarely adopts the extended "beads on a string" form. Instead, the nucleosomes are packed on top of one anothe; generating regular arrays in which the DNA is even more highly condensed. Thus, when nuclei are very gently lysed onto an electron microscope grid, most of the chromatin is seen to be in the form of a fiber with a diameter of about 30 nm, which is considerably wider than chromatin in the "beads on a string" form (see Figure 4-22).

217

CHROMOSOMAL DNAAND IT5PACKAGING FIBER IN THECHROMATIN

F i g u r e 4 - 3 1A z i g z a g m o d ef lo r t h e 3 0 - n m c h r o m a t i n f i b e r . (TAh) e c o n f o r m a t i o n o f t w o o f t h e f o u r (B)Schematic of nucleosomes in a tetranucleosome, from a structuredeterminedby x-raycrystallography. is not visible,beingstackedon the bottom nucleosome the entiretetranucleosome; the fourth nucleosome of a possiblezigzagstructurethat couldaccount illustration and behindit in this diagram.(C)Diagrammatic 2005'With for the 30-nm chromatinfiber.(Adaptedfrom C.L.Woodcock,Ndf.Sttuct.Mol.Biol.12:639-640, permission Ltd.) from MacmillanPublishers

How are nucleosomes packed in the 30-nm chromatin fiber? This question has not yet been answered definitively, but important information concerning the structure has been obtained. In particular, high-resolution structural analyses have been performed on homogeneous short strings of nucleosomes, prepared from purified histones and purified DNA molecules. The structure of a tetranucleosome, obtained by X-ray crystallography,has been used to support a zigzag model for the stacking of nucleosomes in the 30-nm fiber (Figure 4-3f ). But cryoelectron microscopy of longer strings of nucleosomes supports a very different solenoidal structure with intercalated nucleosomes (Figure 4-32). \Arhatcauses the nucleosomes to stack so tightly on each other in a 30-nm fiber? The nucleosome to nucleosome linkages formed by histone tails, most notably the H4 tail (Figure 4-33) constitute one important factor. Another

(A)

(c)

1 0n m

Figure4-32 An interdigitatedsolenoidmodelfor the 30-nmchromatinfiber.(A)Drawingsin whichstringsof color(B)Schematic diagramof finalstructurein (A). codednucleosomes areusedto illustratehow the solenoidis generated. arrays imagesof nucleosome (C)Structuralmodel.The modelis derivedfrom high-resolution microscopy cryoelectron octamersand Bothnucleosome of specificlengthand sequence. reconstituted from purifiedhistonesand DNAmolecules a linkerhistone(discussed below)wereusedto produceregularlyrepeatingarrayscontainingup to 72 nucleosomes' 1, 2006.With (Adaptedfrom P.Robinson,L. Fairall, V. Huynhand D. Rhodes,Proc.NatlAcad.Sci.U.S.A.103:6506-651 permission from NationalAcademyof Sciences.)

218

Chapter4: DNA, Chromosomes,and Genomes

H 4t a i l H 2 At a i l

H 2 Bt a i l . . H 3t a i l

H4 tail

H3 tail

important factor is an additional histone that is often present in a l-to-1 ratio with nucleosome cores, knor,r,nas histone Hl. This so-called linker histone is larger than the individual core histones and it has been considerably less well conserved during evolution. A single histone Hl molecule binds to each nucleosome, contacting both DNA and protein, and changing the path of the DNA as it exits from the nucleosome. Although it is not understood in detail how Hl pulls nucleosomes together into the 30-nm fiber, a change in the exit path in DNA seems crucial for compacting nucleosomal DNA so that it interlocks to form the 30-nm fiber (Figure 4-34). Most eucaryotic organisms make several histone Hl proteins of related but quite distinct amino acid sequences. It is possible that the 30-nm structure found in chromosomes is a fluid mosaic of several different variations. For example, a linker histone in the Hl family was present in the nucleosomal arrays studied in Figure 4-32 but was missing from the tetranucleosome in Figure 4-31. Moreover, we saw earlier that the linker DNA that connects adjacent nucleosomes can vary in length; these differences in linker length probably introduce local perturbations into the structure. And the presenceof many other DNA-binding proteins, as well as proteins that bind directly to histones, will certainly add important additional features to any array of nucleosomes.

Figure4-33 A speculativemodel for the role playedby histonetailsin the formationof the 30-nmfiber.(A)This schematic diagramshowsthe approximate exit pointsof the eight histonetails,one from eachhistone protein.that extendfrom each nucleosome. Theactualstructureis shown to its right.In the high-resolution structure of the nucleosome, the tailsarelargely unstructured, suggesting that they are highlyflexible.(B)A speculative model showinghow the histonetailsmay helpto packnucleosomes togetherinto the 30-nmfiber.Thismodelis basedon (1) experimental evidencethat histonetails aid in the formationof the 30-nmfiber, and (2)the x-raycrystalstructureof the nucleosome, in whichthe tailsof one nucleosome contactthe histonecoreof an adjacentnucleosome in the crystallattice.

Su m m a r y A geneis a nucleotidesequencein a DNA moleculethat actsas a functional unit for the production of a protein, a structural RNA,or a catalytic or regulatory RNAmolecule.In eucaryotes,protein-codinggenesare usually composedof a string of alternating introns and exonsassociatedwith regulatory regionsof DNA. A chromosomeisformeclfrom a single,enormously long DNA moleculethat contains a linear array of many genes.The human genomecontains3.2x ]d DNA nucleotidepairs,diuidedbetween22 dffirent autosomesand 2 sexchromosomes.only a small percentageof this DNA codesfor proteins or functional RNAmolecules.A chromosomal DNA moleculealso contains three other filpes of functionally important nucleotide sequences:replication origins and telomeresallow the DNA molecule to be fficiently replicated, while a centromere attaches the daughter DNA moleculesto the mitotic spindle, ensuring their accurate segregationto daughter cellsduring the M phaseof the cell cycle.

Figure4-34 How the linkerhistone bindsto the nucleosome. The position and structureof the globularregionof histoneH1 areshown.As indicated, this regionconstrains an additional 20 nucleotidepairsof DNAwhereit exits from the nucleosome core.Thistype of bindingby H1 isthoughtto be important for formingthe 30-nmchromatinfiber. The long C-terminal tail of histoneH1 is alsorequiredfor the high-affinity binding of H1 to chromatin,but neitherits positionor that of the N-terminal tail is (B)structure. known.(A)Schematic, (8,from D. Brown,T. lzardand T. Misteli, Nat,Struct.Mol. Biol. 13:250-255,2006. With permission from Macmillan Publishers Ltd.)

219

THEREGULATION OF CHROMATIN STRUCTURE

The DNA in eucaryotesis tightly bound to an equal massof histones,which form repeatedarrays of DNA-protein particles called nucleosomes.The nucleosomeis composedof an octameric core of histone proteins around which the DNA double helix is wrapped.Nucleosomesare spacedat interuals of about 200 nucleotidepairs, and they are usually packed together (with the aid of histone Hl molecules)into quasi-regular arrays to form a 30-nm chromatin fiber. Despite the high degreeof compaction in chromatin, its structure must be highly dynamic to allow accessto the DNA. Thereis somespontaneousDNA unwrapping and rewrappingin the nucleosomeitself;how' euer,the general strategyfor reuersiblychanging local chromatin structure features ATP-driuen chromatin remodeling complexes.Cells contain a large set of such complexes,which are targeted to speciflc regionsof chromatin at appropriate times. The remodeling complexescollaborate with histone chaperonesto allow nucleosomecores to be repositioned,reconstitutedwith dffirent histones,or completelyremouedto exposethe underlying DNA.

THEREGULATION OFCHROM IN STRUCTURE Having described how DNA is packagedinto nucleosomesto create a chromatin fiber, we now turn to the mechanisms that create different chromatin structures in different regions of a cell's genome. We now know that mechanisms of this type are used to control many genesin eucaryotes.Most importantly, certain types of chromatin structure can be inherited; that is, the structure can be directly passed donm from a cell to its decendents.Becausethe cell memory that results is based on an inherited protein structure rather than on a change in DNA sequence,this is a form of epigenetic inheritance. The prefix epl is Greek for "on"; this is appropriate, becauseepigeneticsrepresentsa form of inheritance that is superimposed on the genetic inheritance based on DNA (Figure,t-35). In Chapter 7, we shall introduce the many different ways in which the expression of genes is regulated. There we discuss epigenetic inheritance in detail and present severaldistinct mechanisms that can produce it. Here, we are concerned with only one, that based on chromatin structure. We begin this section with an introduction to inherited chromatin structures and then describe the basis for them-the covalent modification of histones in nucleosomes.We shall see that these modifications serve as recognition sites for protein modules that bring specific protein complexes to the appropriate regions of chromatin, thereby producing specific effects on gene expressionor inducing other biological functions. Through such mechanisms, chromatin structure plays a central role in the development, growth, and maintenance of eucaryotic organisms' including ourselves.

G E N E T IIC NHERITANCE

E P I G E N E TI N I CH E R I T A N C E g e n eY o n

g e n eX o n

seeuerucr I orun

cHANGE IV cHnovnrtru

I CHANGE

CIITI!

gene X ofl

o e n eY o f f CELLS MULTTPLTCATTON OF SOMATTC /\

gene X off

*E*il

gene Y off

P R O D U C T I OONF G E R MC E L L S :ililiii:li:i.t, t:]it::]iltl::.lul

gene X off

I

I tiiilillii*:i iilisi:i:liitl

Figure4-35 A comparisonof genetic inheritancewith an epigenetic inheritancebasedon chromatin is based structures.Geneticinheritance of DNA on the directinheritance duringDNA nucleotidesequences DNAsequencechangesare replication. not only transmittedfaithfullyfrom a but somaticcellto all of its descendents, alsothroughgerm cellsfrom one generationto the next.Thefieldof genetics,reviewedin Chapter8, is based of thesechanges on the inheritance The type of betweengenerations. shownhereis epigeneticinheritance basedon other moleculesboundto the DNA,and it is thereforelesspermanent in than a changein DNAsequence; particular, epigeneticinformationis usually(but not always)erasedduring the formationof eggsand sPerm. that Onlyone epigeneticmechanism, of chromatin basedon an inheritance in this chapter. is discussed structures, are Otherepigeneticmechanisms presentedin Chapter7, whichfocuseson (see the controlof geneexpression Figure7-86).

220

Chapter4: DNA,Chromosomes, and Genomes

SomeEarlyMysteries Concerning ChromatinStructure Thirty years ago, histones were viewed as relatively uninteresting proteins. Nucleosomes were known to cover all of the DNA in chromosomes, and they were thought to exist to allow the enormous amounts of DNA in many eucaryotic cells to be packaged into compact chromosomes. Extrapolating from what was knor.m in bacteria, many scientists believed that gene regulation in eucaryotes would simply bypass nucleosomes, treating them as uninvolved bystanders. But there were reasons to challenge this view. Thus, for example, biochemists had determined that mammalian chromatin consists of an approximately equal mass of histone and non-histone proteins. This would mean that, on auerLge,every 200 nucleotide pairs of DNA in our cells is associated with more than 1000 amino acids of non-histone proteins (that is, a mass of protein equivalent to the total mass of the histone octamer plus histone Hl). We now know that many of these proteins bind to nucleosomes, and their abundance might suggestthat histones are more than just packaging proteins. A second reason to challenge the view that histones were inconsequential to gene regulation was based on the amazingly slow rate of evolutionary change in the sequences of the four core histones. The previously mentioned fact that there are only two amino acid differences in the sequence of mammalian and pea histone H4 implies that a change in almost any one of the 102 amino acids in H4 must be deleterious to these organisms.\iVhattype of process could make the life of an organism so sensitive to the exact structure of the nucleosome core that only two amino acids had changed in more than 500 million years of random variation followed by natural selection? Last but not least, a combination of genetics and cytology had revealed that a particular form of chromatin silencesthe genesthat it packageswithout regard to nucleotide sequence-and does so in a manner that is directly inherited by both daughter cells when a cell divides. It is to this subiect that we turn next.

Heterochromatin ls HighlyOrganized and Unusually Resistant to GeneExpression Light-microscope studies in the 1930sdistinguished two types of chromatin in the interphase nuclei of many higher eucaryotic cells: a highly condensed form, called heterochromatin, and all the rest, which is less condensed, called euchromatin. Heterochromatin representsan especially compact form of chromatin (see Figure 4-9), and we are finally beginning to understand important aspects of its molecular properties. Although present in many locations along chromosomes, it is also highly concentrated in specific regions, most notably at the centromeres and telomeres introduced previously (seeFigure 4-21). In a typical mammalian cell, more than ten percent of the genome is packaged in this way. The DNA in heterochromatin contains very few genes, and those euchromatic genes that become packaged into heterochromatin are turned off by this type of packaging. However, we know now that the term heterochromatin encompassesseveraldistinct types of chromatin structures whose common feature is an especially high degree of compaction. Thus, heterochromatin should not be thought of as encapsulating "dead" DNA, but rather as creating different tlpes of compact chromatin with distinct features that make it highly resistant to gene expression for the vast majority of genes. lvhen a gene that is normally expressedin euchromatin is experimentally relocated into a region of heterochromatin, it ceasesto be expressed,and the gene is said to be silenced.Thesedifferences in gene expression are examples of position effects, in which the activity of a gene depends on its position relative to a nearby region of heterochromatin on a chromosome. First recognized in Drosophila, position effects have now been observed in many eucarvotes, including yeasts,plants, and humans.

221

THEREGULATION OFCHROMATIN STRUCTURE 12345 b ar r i e r

I

I

genes _ a---123 45

heterochromatin euchromatin

12345 trI1T."I{tr;iffiTlTi_T: jl

12345

I

e a r l yi n t h e d e v e l o p i n ge m b r y o ,h e t e r o c h r o m a t i fno r m s a n d s p r e a d si n t o n e i g h b o r i n g euchromatinto different extents in different cells

I

r--r-I

CHROMOSOME TRANSLOCATION

12345

T:'t-Tr

I

12345 heterochromatin euchromatin

r--r[rT-r] c l o n eo f c e l l sw i t h gene 1 inactive (A)

c l o n eo f c e l l sw i t h g e n e s1 , 2 , a n d 3 i n a c t i v e

c l o n eo f c e l l sw i t h n o g e n e si n a c t i v a t e d

(B)

Figure4-36 The causeof position effect variegationin Drosophild.(A)Heterochromatin(green)is normallyprevented whichwe shalldiscussshortly. sequences, from spreadinginto adjacentregionsof euchromatin(red)by specialbarrierDNA this barrieris no longerpresent.(B)Duringthe early In fliesthat inheritcertainchromosomal however, rearrangements, for different DNA,proceeding developmentof suchflies,heterochromatin can spreadinto neighboringchromosomal patternof heterochromatin is inherited,so that distances in differentcells.Thisspreadingsoonstops,but the established and largeclonesof progenycellsareproducedthat havethe sameneighboringgenescondensedinto heterochromatin is (hencethe "variegated" therebyinactivated appearance of someof theseflies;seeFigure4-37).Although"spreading" the term may not be existingheterochromatin, usedto describethe formationof new heterochromatin closeto previously can"skipover"someregionsof chromatin, whollyaccurate. heterochromatin Thereis evidencethat duringexpansion, sparingthe genesthat lie withinthem from repressive effects

The position effects associated with heterochromatin exhibit a feature called position effectuariegation,which in retrospect provided critical clues concerning chromatin function. ln Drosophila, chromosome breakage events that directly connect a region of heterochromatin to a region of euchromatin tend to inactivate the nearby euchromatic genes.The zone of inactivation spreadsa different distance in different early cells in the fly embryo, but once the heterchromatic condition is established on a gene, it tends to be stably inherited by all of the cell's progeny (Figure 4-36). This remarkable phenomenon was first recognized through a detailed genetic analysis of the mottled loss of red pigment in the fly eye (Figure 4-37), but it shares many features with the extensive spread of heterochromatin that inactivates of one of the two X chromosomes in female mammals (seep. 473). Extensive genetic screenshave been carried out in Drosophila, as well as in fungi, in a search for gene products that either enhance or suppress the spread of heterochromatin and its stable inheritance-that is, for genes that when mutated serve as either enhancers or suppressorsof position effect variegation. In this way, more than 50 genes have been identified that play a critical role in these processes.In recent years, the detailed characterization of the proteins produced by these genes has revealed that many are nonhistone chromosomal proteins that underlie a remarkable mechanism for eucaryotic gene control, one

White gene at normal location

barner heterochromatin

rare cnromosome In v e r s t o n

Dar

Figure4-37 The discoveryof position effectson gene expression.TheWhite gene in the fruit fly Drosophilacontrols eyepigmentproductionand is named afterthe mutationthat firstidentifiedit. Wild-typeflieswith a normal Whitegene (White+) havenormalpigmentproduction, which givesthem red eyes,but if the White the geneis mutatedand inactivated, mutantflies(White-)makeno pigment and havewhiteeyes.Infliesin whicha normalWhite+gene has been moved near the eyesare a regionof heterochromatin, mottled,with both red and whltepatches. fhe white patchesrepresentcell lineages in which the White+gene hasbeen silencedby the effectsof the In contrast, the red heterochromatin. patchesrepresent celllineagesin which the White+gene is expressed.Earlyin when the heterochromatin develooment, isfirstformed,it spreadsinto neighboring to differentextentsin euchromatin differentembryoniccells(seeFigure 4-36).The presenceof largepatchesof red and whitecellsrevealsthat the stateof activity,asdeterminedby transcriptional the packagingof this geneinto chromatin in thoseancestorcells,is inheritedby all dauqhtercells.

222

Chapter4: DNA,Chromosomes, and Genomes

( A ) L Y S I NA EC E T Y L A T I OANN D M E T H Y L A T I OANR EC O M P E T I NR GE A C T I O N S

HO

-N-

I

H

HO

rtl C-C-

-N-

I

CH,

H

tau

t.-

lysrne

l\H

t-

fn' CHt

CH, I N-

,.+

I

CH, +

t-

I CHt

H:C

t-

CH,

I'

H^C

|

t t- Cl -

n fn, I

CHr t|

CHt

C:O

-N-C

CHt +

H

HO

tll C-CI I

I.

\cn.

/ \cn. | H,c -

H

H-

monomethyllysine

dimethyl lysine

CH:

CH: acetyllysine

Figure4-38 Someprominenttypesof covalentamino acid side-chain modificationsfound on nucleosomalhistones.(A)Threedifferentlevelsof lysinemethylationareshown;eachcan be recognized by a different bindingproteinand thuseachcan havea differentsignificance for the cell. Notethat acetylation removesthe pluschargeon lysine,and that,most importantly,an acetylatedlysinecannot be methylated,and viceversa. (B)Serinephosphorylation addsa negativechargeto a histone. Modifications not shownherearethe mono-or di-methylation of an arginine,the phosphorylation of a threonine,the additionof ADP-ribose to a glutamicacid,and the additionof a ubiquityl,sumoyl,or biotingroupto a lysine.

trimethyl lysine

( B ) S E R I NP EH O S P H O R Y L A T I O N

HO

HO

ttl

ttl -N-C-CH

-N-C-C-

CH?

lOH Senne

H

+

CH,

t-

o O-

I

P:O

I

that requires the precise amino acid sequencesof the core histones. This mechanism of gene control therefore helps to explain the remarkably slow change in the histones over time.

TheCoreHistones AreCovalently Modifiedat ManyDifferent Sites The amino acid side chains of the four histones in the nucleosome core are subjected to a remarkable variety of covalent modifications, including the acetylation of lysines, the mono-, di-, and tri-methylation of lysines, and the phosphorylation of serines (Figure 4-38). A large number of these side-chain modifications occur on the eight relatively unstructured N-terminal "histone tails" that protrude from the nucleosome (Figure 4-39). However, there are also specific side-chain modifications on the nucleosome'sglobular core (Figure 4-40). Ail of the above types of modifications are reversible.The modification of a particular amino acid side chain in a nucleosome is created by a specific enzyme, with most of these enzymes acting only on one or a few sites.A different enzyme is responsible for removing each side chain modification. Thus, for example, acetyl groups are added to specific lysines by a set of different histone acetyl transferases (FIATs)and removed by a set of histone deacetylase complexes (HDACs).Likewise,methyl groups are added to lysine side chains by a set of different histone methyl transferases and removed by a set of histone demethylases. Each enzwe is recruited to specific sites on the chromatin at defined times in each cell'slife history. For the most part, the initial recruitment of these enz).rnesdepends on gene regulatory proteins thatbind to specific DNA sequencesalong chromosomes, and these are produced at different times in the life of an organism, as described in chapter 7. But in at least some cases,the covalent modifications on nucleosomes can persist long after the gene regulatory proteins that first induced them have disappeared,thereby carrying a memory in the cell of its developmental history. very different patterns of covalent modifications are therefore found on different groups of nucleosomes, according to their exact position on a chromosome and the status of the cell.

o

phosphoserine

223

THEREGULATION OFCHROMATIN STRUCTURE

# 'ift- #W

|

H2A

s c n e x o c e r . q n a x a l s t R s s R A GL Q F P v G R V - i r 1315 9 1 5 119

PM

A

.A le l

I

r

iA 'f

I'

1.."4

l-r

H2B . "ffi' \ Y

prratIr\ou aAA'selppuereJJlpIIec p ueq ^ tsol ,{1enrlca1as saua8 leql palcadsns rtlpu€rro slsrSoyorq'elqrsJalaur uaryo sr uorlertueJeJJlpIIac asnef,eq pue 'uospeJ srql JoC '11-2 arnt;g) auroua8 erues eql uleluoJ sllec olvtl aql IEIII aulSeur ot ]lnJrJJrpsr tl teql eruaJlxa os eJBsecueJeJJlpeql 'aldruexa ro; 'ail.coqdud1 p qly\^ uoJnau uellerurupur e aruduroo errr.Jl'uorlJunJ pue eJnlJnJls qloq uI dgerqeuerp raJJrprusrueS;o relnlacllpru E u1 sed,'i1flac tualeJJrp eqJ

tour_No) lNl9 lo

ilAUtAoNV

'sursrue8roJelnlleJrtlnu ul IoJluoo auaS;o saldrculrd Jrseq eruos Jo ^ er,t"ralo ue ql1,trru€aq all'uJnl ur slelel tuaJeJJIpeql ssncsrp e,r,rpup 's1ana1 dueur le alerado sauaS;o uolssardxa eql IoJluoJ luql srusper{f,aru er{J 'llal qoee ur passardxa ^{1a,rqca1as sr saua8 eq} Jo }esqns E qf,rr.Imd.q sursrue -qJaru pue selnr aql-uelqoJd gogeq sql eql ssnJsrp am raldeqc srql uI lsrrJ 'puP iop ]r saop lBqM'epeul eJuo 'apeur lcnpord aua8 qcea sl suoplpuoJ teq ^ repun 'pasn aJBlsll eq] uo spJom eql Jo acuenbas VNO eql ur slueruala eqt ^\oq llrorDl o] sr uralqord eql 'saseJ qtoq q 'areadsa4eqg dq ,fu1de ]f,nrtsuocer ot sn selqeue sprom qqpug 3o 1s11 e uuqt ursrue8Jo eql lcnJlsuof,eJ ol sn salquue eJoru ou-uerunq B Jo sapuoelf,nu uoIIIq ,ry\alaql Jo runrratceq p Jo sappoelcnu uoIlru 1v\eJaqt ll aq-usr -uu8ro ue;o acuanbesVNC aql;o uo4drrcsap alaldruoc e la 'slleJ sll lJnDSuoO o1 parmbar sepceloru uratord pue VNU eql Jo IIp sepoJue VNC s.ursrueSrouV

uolsseldxleuaDJo lotluo)

412

Chapter7:Controlof GeneExpression Figure7-1 A mammalianneuronand a lymphocyte.The long branchesof this neuronfrom the retinaenableit to receiveelectrical signalsfrom many cellsand carrythosesignalsto manyneighboringcells.The lymphocyteis a white bloodcellinvolvedin the immuneresponse to infectionand movesfreelythroughthe body.Bothof thesecellscontainthe same genome/but they expressdifferentRNAsand proteins.(From B.B.Boycott,Essays on the NervousSystem[R.Bellairs and E.G.Gray,eds.]. Oxford,UK:ClarendonPress, 1974.)

Further evidence that large blocks of DNA are not lost or rearranged during vertebrate development comes from comparing the detailed banding patterns detectable in condensed chromosomes at mitosis (seeFigure 4-l l). By this criterion the chromosome sets of differentiated cells in the human body appear to be identical. Moreover, comparisons of the genomes of different cells based on recombinant DNA technology have confirmed, as a general rule, that the changes in gene expression that underlie the development of multicellular organisms do not rely on changes in the DNA sequencesof the corresponding genes. There are, however, a few cases where DNA rearrangements of the genome take place during the development of an organism-most notably, in generating the diversity of the immune system of mammals, which we discussin Chapter 25.

DifferentCellTypesSynthesize DifferentSetsof proteins As a first step in understanding cell differentiation, we would like to know how many differences there are between any one cell type and another. Although we still do not have a detailed answer to this fundamental question, we can make certain general statements. 1. Many processes are common to all cells, and any two cells in a single organism therefore have many proteins in common. These include the structural proteins of chromosomes, RNA polymerases, DNA repair enzymes, ribosomal proteins, enzymes involved in the central reactions of metabolism, and many of the proteins that form the cytoskeleton. 2. Some proteins are abundant in the specializedcells in which they function and cannot be detected elsewhere,even by sensitive tests.Hemoglobin, for example, can be detected only in red blood cells. 3. Studies of the number of different mRNAs suggestthat, at any one time, a typical human cell expresses30-60% of its approximately 25,000 genes. \.\4ren the patterns of mRNAs in a series of different human cell lines are compared, it is found that the level of expression of almost every active gene varies from one cell tlpe to another. A few of these differences are striking, like that of hemoglobin noted above, but most are much more subtle. Even genes that are expressedin all cell types vary in their level of expression from one cell type to the next. The patterns of mRNA abundance (determined using DNA microarrays, discussed in chapter g) are so characteristic of cell type that they can be used to type human cancer cells of uncertain tissue origin (Figure 7-3).

in gene expressionbetween cell types is through methods that directly display the levels of proteins and their post-translational modifications (Figure 7-4).

neuron

a lymphocyte

413

AN OVERVIEW OFGENECONTROL (A)

n o r m a le m b r y o

u n f e r t i l i z e de g g

n u c l e u sd e s t r o y e d by UV light

'tFt'

secuo n of carrot

p r o li f e r a t n i g c e l lm a s s

separated c e l l si n r i c h liquid medium

meiotic indle ++

single cell

organized c l o n eo f dividing

young embryo

young plant

carrot

cells

donorcell praceo N C XtIO Egg

D O N O RC E L L reconstructed wlTH To FUSE zygore ENUCLEATED E G GC E L L

e m b r' v o

e m b r y op l a c e di n foster mothers

unfertilized meioticspindle andassociated eggcell cnromo50mes removed Figure7-2 Evidencethat a differentiatedcell containsall the genetic instructionsnecessaryto direct into the formationof a completeorganism.(A)The nucleusof a skincellfrom an adultfrog transplanted egg can g ive riseto an entiretadpole.Thebrokenarrowindicatesthat,to g ivethe an enucleated a furthertransferstepis requiredin genometime to adjustto an embryonicenvironment, transplanted whichone of ihe nucleiis takenfrom the earlyembryothat beginsto developand is put backinto a cellsretainthe abilityto'dedifferentiate," egg.(B)In manytypesof plants,differentiated secondenucleated so that a singlecellcanform a cloneof progenycellsthat latergiveriseto an entireplant.(C)A egg from a differentcow can differentiated cellnucleusfrom an adultcow introducedinto an enucleated give riseto a calf.Differentcalvesproducedfrom the samedifferentiatedcell donor are genetically 1968. identicaland arethereforeclonesof one another.(A,modifiedfrom J.B.Gurdon,Sci.Am.2'19:24-35, American.) from Scientific With permission

of lts ExternalSignalsCanCausea Cellto Changethe Expression Genes Most of the specialized cells in a multicellular organism are capable of altering their patterns of gene expressionin responseto extracellularcues.If a liver cell is exposedto a glucocorticoid hormone, for example,the production of severalspecific proteins is dramatically increased.Glucocorticoids are releasedin the body during periods of starvation or intense exerciseand signal the liver to increasethe

414

Chapter7:ControlofGeneExpression unknown

prostate

leukemia

I

I lung

brain

stomach

I renal I

( A ) h u m a nb r a i n =-r '=

-

ovarian

breast

liver

( B )h u m a n l i v e r -3.

-' l t

_r4

'"*.dlr

I

ia '1..

o

.

-q

acidic

isoelectricpoint

basic

Figure7-4 Differencesin the proteins expressedby two human tissues.In each panel,the proteinsaredisplayedusingtwo-dimensional polyacrylamide-gel (see electrophoresis

spectrometry(seepp. 519-521\providemuch more detailedinformationand are thereforemore commonlyused.(Courtesy of Tim Myersand LeighAnderson,LargeScaleBiologyCorporation.)

Figure7-3 Differencesin mRNAexpression patternsamong different types of human cancercells.Thisfiguresummarizes a very largesetof measurements in whichthe mRNAlevelsof 1800selectedgenes (arrangedtop to bottom)were determined for 142differenthumantumor celllines (arrangedIeftto right),eachfrom a different patient.Eachsmallredbar indicates that the givengenein the giventumor is transcribed at a levelsignificantly higherthan the averageacrossall the celllines.Eachsmall greenbar indicatesa less-than-average expressionlevel,and eachblackbar denotes an expressionlevelthat is closeto average acrossthe differenttumors.The procedure usedto generatethesedata-mRNA isolationfollowed by hybridizationto DNA microarrays-isdescribedin Chapter8 figureshowsthat the @p.57a-575).The relativeexpressionlevelsof eachof the 1800 genesanalyzedvary among the different tumors(seenby followinga givengene from left to right acrossthe figure).This analysis alsoshowsthat eachtype of tumor hasa characteristic gene expressionpattern. Thisinformationcan be usedto "type" cancercellsof unknowntissueoriginby matchingthe gene expressionprofilesto those of known tumors.Forexample,the unknownsamplein the figurehasbeen identifiedas a lung cancer.(Courtesyof PatrickO. Brown,DavidBotstein,and the StanfordExpression Collaboration.)

AN OVERVIEW OFGENECONTROL

415

Figure7-5 Sixstepsat which eucaryotic gene expressioncan be controlled. Controlsthat operateat steps1 through in this chapter.Step6, the 5 arediscussed regulationof proteinactivity,occurs largelythroughcovalentpostincluding modifications translational and phosphorylation, acetylation, ubiquitylation(seeTable3-3, p. 186)and in manychaptersthroughout is discussed the book.

production of glucose from amino acids and other small molecules; the set of proteins whose production is induced includes enz).rnessuch as tyrosine aminotransferase,which helps to convert tyrosine to glucose.\.A/henthe hormone is no longer present, the production of these proteins drops to its normal level. Other cell types respond to glucocorticoids differently. Fat cells, for example, reduce the production of tyrosine aminotransferase,while some other cell types do not respond to glucocorticoids at all. These examples illustrate a general feature of cell specialization: different cell types often respond differently to the same extracellular signal. Underlying such adjustments that occur in response to extracellular signals,there are features ofthe gene expressionpattern that do not change and give each cell type its permanently distinctive character.

CanBeRegulated at Manyof the Stepsin the GeneExpression Pathwayfrom DNAto RNAto Protein If differences among the various cell types of an organism depend on the particular genes that the cells express,at what level is the control of gene expression exercised?As we saw in the previous chapter, there are many steps in the pathway leading from DNA to protein. We now know that all of them can in principle be regulated.Thus a cell can control the proteins it makes by (l) controlling when and how often a given gene is transcribed (transcriptional control), (2) controlling the splicing and processingof RNA transcripts (RNA processing control), (3) selectingwhich completed mRNAs are exported from the nucleus to the cytosol and determining where in the c1'tosol they are localized (RNA transport and localization control), (4) selecting which mRNAs in the cytoplasm are translated by ribosomes (translational control), (5) selectively destabilizing certain mRNA molecules in the c1'toplasm (mRNA degradation control), or (6) selectively activating, inactivating, degrading, or locating specific protein molecules after they have been made (protein activity control) (FigureT-5). For most genes transcriptional controls are paramount. This makes sense because, of all the possible control points illustrated in Figure 7-5, only transcriptional control ensuresthat the cell will not synthesizesuperfluous intermediates. In the following sections we discuss the DNA and protein components that perform this function by regulating the initiation of gene transcription' We shall return at the end of the chapter to the many additional ways of regulating gene expressron.

Su m m a r y The genome of a ceII contains in its DNA sequencethe information to make many thousands of dffirent protein and RNA molecules.A cell typically expressesonly a fraction of its genes,and the dffirent types of cells in multicellular organisms arise Moreover,cells can change the pattern of becausedffirent setsof genesare expressed. genesthey expressin responseto changesin their enuironment, such as signalsfrom other cells.Although all of the stepsinuolued in expressing& Senecan in principle be regulated,for most genesthe initiation of RNA transcription is the most important point of control.

416

Chapter7:ControlofGeneExpression

DNA-BINDING MOTIFS IN GENEREGUL ORY PROTEI NS How does a cell determine which of its thousands of genesto transcribe?As outlined in chapter 6, the transcription of each gene is controlled by a regulatory region of DNA relatively near the site where transcription begins. Some regulatory regions are simple and act as switches thrown by a single signal. Many others are complex and resemble tiny microprocessors, responding to a variety of signals that they interpret and integrate in order to switch their neighboring gene on or off. \.Vtrethercomplex or simple, these switching devices are found in all cells and are composed of two types of fundamental components: (l) short stretches ofDNA ofdefined sequence and (2) gene regulatory proteins that recognize and bind to this DNA. We begin our discussion of gene regulatory proteins by describing how they were discovered.

GeneRegulatory Proteins WereDiscovered UsingBacterial Genetics Genetic analysesin bacteria carried out in the 1950sprovided the first evidence for the existenceof gene regulatory proteins (often loosely called "transcription factors") that turn specific sets of genes on or off. one of these regulators, the lambda repressor, is encoded by a bacterial virus, bacteriophage lambtta. The repressor shuts off the viral genes that code for the protein components of new virus particles and thereby enables the viral genome to remain a silent passenger in the bacterial chromosome, multiplying with the bacterium when conditions are favorable for bacterial growth (seeFigure 5-78). The lambda repressor was among the first gene regulatory proteins to be characterized,and it remains one of the best understood, as we discuss later. other bacterial regulators respond to nutritional conditions by shutting off genes encoding specific sets of metabolic enzymes when they are not needed. The Lac repressor, the first of these bacterial proteins to be recognized, turns off the production of the proteins responsible for lactose metabolism when this sugar is absent from the medium. The first step toward understanding gene regulation was the isolation of mutant strains of bacteria and bacteriophage lambda that were unable to shut off specific sets of genes.It was proposed at the time, and later proven, that most of these mutants were deficient in proteins acting as specific repressorsfor these sets of genes. Because these proteins, like most gene regulatory proteins, are present in small quantities, it was difficult and time-consuming to isolate them. They were eventually purified by fractionating cell extracts. once isolated, the proteins were shor,unto bind to specific DNA sequencesclose to the genes that they regulate.The precise DNA sequencesthat they recognized were then determined by a combination of classical genetics and methods for studying protein-DNA interactions discussedlater in this chanter.

TheOutsideof the DNAHelixCanBeReadby proteins

Figure7-6 Double-helical structureof DNA.A space-filling modelof DNA showingthe majorand minorgrooveson the outsideof the doublehelix. The atomsare coloredasfollows:carbon,dark blue;nitrogen,tight blue; hydrogen,white;oxygen,red;phosphorus,yellow.

417

DNA-BINDING MOTIFSIN GENEREGULATORY PROTEINS rnator groove

rflalor 9rooVe

g

-{

g

6 ,g

.f

l?)inor groovc

rDinorgroo!e groovs rn31or

tnalor 9roovq

rDinorgroove

rTlinorgroo'Je

Figure7-7 How the different basepairs in DNA can be recognizedfrom their edgeswithout the need to open the of basepairsareshown,with potentialhydrogenbond donorsindicatedin double helix.Thefour possibleconiigurations asa seriesof short s in red,and hydrogenbondsof the basepairsthemselves b/ue,potentialhydrogenbond accepior yellow, hydrogenatomsthat are and in shown protuberances, are groups, form hydrophobic parallelredlines.Methyl wfrich for hydrogenbonding,arc white.(FromC. Brandenand J.Tooze, and arethereforeunavailable attachedto carbons, 1999.) 2nd ed. NewYork:GarlandPublishing, Introductionto ProteinStructure,

sequence and another. It is now clear, however, that the outside of the double helix is studded with DNA sequence information that gene regulatory proteins can recognize without having to open the double helix. The edge of each base pair is exposed at the surface of the double helix, presenting a distinctive pattern ofhydrogen bond donors, hydrogen bond acceptors, and hydrophobic patches for proteins to recognize in both the major and minor groove (Figure 7-7). But only in the major groove are the patterns markedly different for each of the four base-pair arrangements (Figure 7-8). For this reason, gene regulatory proteins generally make specific contacts with the maior groove-as we shall see. m a J o rg r o o v e

m r n o rg r o o v e

L

KEY: T

C=H-bondacceptor

o

e=H-bonddonor atom i,;r = hYdrogen

A

grouP ,,,. = methYl

Figure7-8 A DNA recognitioncode.The edgeof eachbasepair,seenherelooking directlyat the majoror minorgroove, containsa distinctivePatternof hydrogenbond donors,hydrogenbond acceptors,and methylgroups.Fromthe majorgroove,eachof the four base-pair projectsa uniquepattern configurations of features.Fromthe minor groove, however,the patternsare similarfor G-C and C-G as well as for A-T and T-A' The colorcodeis the sameasthat in Figure 7-7. (FromC. Brandenand J.Tooze, ed. lntroductionto ProteinStructure,2nd 1999.) NewYork:GarlandPublishing,

418

Chapter7: ControlofGeneExpression

ShortDNAsequences Are Fundamental componentsof Genetic Switches A specific nucleotide sequence can be "read" as a pattern of molecular features on the surface of the DNA double helix. particular nucleotide sequences,each typically less than 20 nucleotide pairs in length, function as fundamental com-

we now turn to the gene regulatory proteins themselves,the second fundamental component of genetic switches. we begin with the structural features that allow these proteins to recognize short, specific DNA sequencescontained in a much longer double helix.

GeneRegulatoryProteinsContainStructuralMotifsThatCan ReadDNASequences Molecular recognition in biology generally relies on an exact fit between the surfaces of two molecules, and the study of gene regulatory proteins has provided some of the clearest examples of this principle. A gene regulatory protein recognizes a specific DNA sequence because the surface of the protein is extensively

Table7-1 SomeGeneRegulatoryproteinsand the DNA sequencesThatThey Recognize

Lacrepressor

Yeast

Drosophila

CAP

TGTGAGTTAGCTCACT ||ltl ACACTCAATCGAGTGA

Lambdarepressor

TATCACCGCCAGAGGT rlllIi||l ATAGTGGCGGTCTCCAT

Gal4

CGAGGACTGTCCTCCG li l l ! GCCTCCTGACAGGAGGC

Mato2

CATGTAATT lt |tl GTACATTAA

Gcn4

ATGACTCAT |11 TACTGAGTA

Kruppel

AACGGGTTAA lllll mm^^^^n I f WUUffiI

Bicoid Mammals

^mm

]

GGGATTAGA ltli CCCTAATCT

5p1 rll

cccc,cc Octl Poudomain

ATGCAAAT |1 TACGTTTA

GATAl l|ll ACTATC

MyoD p53 *Forconvenience, onlyone recognition sequence, ratherthana consensus (seeFigure sequence 6 12),isgivenfor eachp.otein

DNA-BINDING MOT]F5IN GENEREGULATORY PROTEINS

419

complementary to the special surface features of the double helix in that region. In most cases the protein makes a series of contacts with the DNA, involving hydrogen bonds, ionic bonds, and hydrophobic interactions. Although each individual contact is weak, the 20 or so that are typically formed at the protein-DNA interface add together to ensure that the interaction is both highly specific and very strong (Figure 7-9). In fact, DNA-protein interactions include some of the tightest and most specific molecular interactions knor,rmin biology. Although each example of protein-DNA recognition is unique in detail, xray crystallographic and NMR spectroscopic studies of several hundred gene regulatory proteins have revealed that many of them contain one or another of a small set of DNA-binding structural motifs. These motifs generally use either a helices or p sheets to bind to the major groove of DNA; this groove, as we have seen, contains sufficient information to distinguish one DNA sequence from any other. The fit is so good that it has been suggestedthat the dimensions of the basic structural units of nucleic acids and proteins evolved together to permit these molecules to interlock.

Motif ls Oneof the Simplestand Most TheHelix-Turn-Helix Motifs CommonDNA-Binding The first DNA-binding protein motif to be recognized was the helix-turn-helix. Originally identified in bacterial proteins, this motif has since been found in many hundreds of DNA-binding proteins from both eucaryotes and procaryotes. It is constructed from two s helices connected by a short extended chain of amino acids,which constitutes the "turn" (Figure 7-10). The two helices are held at a fixed angle, primarily through interactions between the tvvo helices. The more C-terminal helix is called the recognition helixbecause it fits into the major groove of DNA; its amino acid side chains, which differ from protein to protein, play an important part in recognizing the specific DNA sequence to which the protein binds. Outside the helix-turn-helix region, the structure of the various proteins that contain this motif can vary enormously (Figure 7-ll). Thus each protein "presents" its helix-turn-helix motif to the DNA in a unique way, a feature thought to enhance the versatility of the helix-turn-helix motif by increasing the number of DNA sequencesthat the motif can be used to recognize.Moreover, in most of these proteins, parts of the polypeptide chain outside the helix-turn-helix domain also make important contacts with the DNA, helping to fine-tune the interaction.

sugar-phosphate backboneon outside of d o u b l eh e l i x

Figure7-9 The binding of a gene regulatory protein to the major groove of DNA.Onlya singlecontactis shown. Typically,the protein-DNAinterface would consistof 10-20 suchcontacts, involvingdifferentamino acids,each contributingto the strengthof the orotein-DNAinteraction.

420

Chapter7:Controlof GeneExpression

Figure7-10 The DNA-binding helix-turn-helix motif. The motif is shown in (A),where eachwhitecircle denotesthe centralcarbonof an amino acid.The C-terminalahelix (red is called the recognitionhelixbecauseit participates in sequence-specific recognitionof DNA.As shownin (B),this helixfits into the majorgrooveof DNA, whereit contactsthe edgesofthe base pairs(seealsoFigure7-7).fhe N-terminala-helix(blue)functions primarilyasa structuralcomponentthat helpsto positionthe recognitionhelix.

cooH (A)

(B)

The group of helix-turn-helix proteins shorrynin Figure 7-l l demonstrates a common feature of many sequence-specificDNA-binding proteins. They bind as symmetric dimers to DNA sequencesthat are composed of two very similar "half-sites," which are also arranged symmetrically (Figure z-r2).This arrangement allows each protein monomer to make a nearly identical set of contacts and enormously increases the binding affinity: as a first approximation, doubling the number of contacts doubles the free energy of the interaction and thereby squares the affinity constant.

HomeodomainProteinsConstitutea SpecialClassof Helix-Turn-Helix Proteins Not long after the first gene regulatory proteins were discovered in bacteria, genetic analyses in the fruit fly Drosophila led to the characterization of an imoortant class of genes, the homeotic selector genes,that play a critical part in orchestrating fly development. As discussed in chapter 22, they have since proved to have a fundamental role in the development of higher animals as well. Mutations in these genescan cause one body part in the fly to be converted into another, showing that the proteins they encode control critical developmental decisions. \Mhen the nucleotide sequences of several homeotic serector genes were determined in the early 1980s, each proved to code for an almost identical stretch of 60 amino acids that defines this class of proteins and is termed the homeodomain. \A/hen the three-dimensional structure of the homeodomain was determined, it was seen to contain a helix-turn-helix motif related to that of

"t

tryptophan repressor

l a m b d aC r o

.JP',

I

l a m b d ar e p r e s s o r f ragment

CAPfragment

DNA

Figure7-1 1 Somehelix-turn-helixDNA-bindingproteins.All of the proteinsbind DNAasdimersin whichthe two copies of the recognitionhelix (redcylinder)are separatedby exactlyone turn of the DNA helix (3.4nm).The other helixof the helix-turn-helixmotif is coloredblue,as in Figure 7- 10.The lam bda repressor and Cro proteinscontrolbacteriophage lambdageneexpression, and the tryptophanrepressor and the cataboliteactivatorprotein(CAp)controlthe expression of setsof E.coli genes.

421

DNA-BINDING MOTIFSIN GENEREGULATORY PROTEINS FigureT-12 A specificDNA sequencerecognizedby the bacteriophage labeledin greenin this sequenceare lambdaCro protein.The nucleotides arrangedsymmetrically, allowingeachhalfof the DNAsiteto be recognized in the sameway by eachproteinmonomer,alsoshownin green.See Figure7-11 for the actualstructureof the protein.

s ' , rA A C A C

GCG TGTT

TTGTG

ACAA

I ttrll

ttrl

u5

I c5'

the bacterial gene regulatory proteins, providing one of the first indications that the principles of gene regulation established in bacteria are relevant to higher organisms as well. More than 60 homeodomain proteins have now been discovered in Drosophila alone, and homeodomain proteins have been identified in virtually all eucaryotic organisms that have been studied, from yeasts to plants to humans. The structure of a homeodomain bound to its specific DNA sequence is shor,rmin Figure 7-13. \Mhereas the helix-turn-helix motif of bacterial gene regulatory proteins is often embedded in different structural contexts, the helix-turn-helix motif of homeodomains is always surrounded by the same structure (which forms the rest of the homeodomain), suggestingthat the motif is always presented to DNA in the same way. Indeed, structural studies have shor,vnthat a yeast homeodomain protein and a Drosophilahomeodomain protein have very similar conformations and recognize DNA in almost exactly the same manner, although they are identical at only 17 of 60 amino acid positions (seeFigure3-13).

ZincFingerMotifs ThereAreSeveralTypes of DNA-Binding The helix-turn-helix motif is composed solely of amino acids.A second important group of DNA-binding motifs includes one or more zinc atoms as structural components. Although all such zinc-coordinated DNA-binding motifs are called zinc fingers, this description refers only to their appearancein schematic drawings dating from their initial discovery (Figure 7-l4A). Subsequent structural that they fall into several distinct structural groups' two of studies have sho',n"ryr which we consider here. The first type was initially discoveredin the protein that activates the transcription of a eucaryotic ribosomal RNA gene. It has a simple structure, in which the zinc holds an cr helix and a B sheet together (Figure 7-l4B). This tlpe of zinc finger is often found in tandem clusters so that the cr helix of each can contact the major groove of the DNA, forming a nearly continuous stretch of o helices along the groove. In this way, a strong and specific DNA-protein interaction is built up through a repeating basic structural unit (Figure 7-r5). Another type of zinc finger is found in the large family of intracellular receptor proteins (discussedin detail in Chapter 15). It forms a different type of

1

Arg

Figure7-13 A homeodomainbound to its specificDNA sequence.Two different viewsof the samestructureareshown' (A)Thehomeodomainisfoldedinto three whichare packedtightlY o,helices, The togetherby hydrophobicinteractions' part containinghelices2 and 3 closely the helix-turn-helixmotif. resembles (B)The recognitionhelix (helix3, red)forms importantcontactswith the majorgroove (Asn)of helix3, for of DNA.Theasparagine example,contactsan adenine,asshownin FigureT-9.A flexiblearm attachedto helix 1 formscontactswith nucleotidepairsin the minorgroove.Thehomeodomain shownhereisfrom a yeastgeneregulatory protein,but it closelYresembles from manyeucaryotic homeodomains organisms.(AdaPtedfrom C.Wolbergeret al.,Cell67:517 -528, 1991. from Elsevier.) With oermission

422

Chapter7:ControlofGeneExpression

25 HOOC---N\ K\ H

.D ./ \

V

1 ,,Y---NH2

c(*

/'

C I

o C

H I R I S I

I S

I I R I S I E

F 10

\./

KV E 12

(A)

Figure7-14 One type of zincfinger protein.This protein belongsto the Cys-Cys-His-His familyof zincfinger proteins,namedafterthe aminoacidsthat graspthe zinc.(A)Schematic drawingof the aminoacidsequenceof a zincfinger from a frog proteinof this class.(B)The three-dimensional structureof this same type of zinc finger is constructedfrom an antiparallel B sheet(aminoacidsi to 10) followedby an o helix(aminoacids12to 24).Thefour aminoacidsthat bind the zinc (Cys3, Cys6, His19,and His23)hold one end of the o helixfirmlyto one end of the B sheet.(Adaptedfrom M.S.Leeet al., Science245:635-637,1989.With permissionfrom AAAS.)

structure (similar in some respects to the helix-turn-helix motif) in which two s helices are packed together with zinc atoms (Figure 7-16). Like the helix-turnhelix proteins, these proteins usually form dimers that allow one of the two cr helices of each subunit to interact with the major groove of the DNA. Aithough the two types of zinc finger structures discussed in this section are structurally distinct, they share two important features: both use zinc as a structural element, and both use an cr helix to recognize the major groove of the DNA.

p sheetsCanAlsoRecognize DNA

sequencerecognized depends on the sequence of amino acids that make up the B sheet.

(B)

Figure7-15 DNA binding by a zincfinger protein. (A)The structureof a fragmentof a mousegeneregulatoryproteinboundto a specificDNA site.This protein recognizes DNAby usingthreezincfingersof the Cys-Cys-His-His type (seeFigure7-1 4) arrangedas direct repeats.(B)The threefingershavesimilaraminoacid sequences and contactthe DNAin similar ways.ln both (A)and (B)the zincatom in eachfinger is representedby a small sphere.(Adaptedfrom N. Pavletichand C. Pabo,Science252:8'10-817, 1991.With permissionfrom AAAS.)

PROTEINS MOTIFSIN GENEREGULATORY DNA-BINDING

SomeProteinsUseLoopsThatEnterthe Majorand Minor DNA Groovesto Recognize

423

Figure7-16 A dimer of the zincfinger domain of the intracellularreceptor family bound to its sPecificDNA sequence,Eachzincfingerdomain containstwo atomsof Zn (indicatedby one stabilizesthe the smallgrayspheres); DNArecognitionhelix(shownin brownin one subunitandredin the othed,and one stabilizesa loop (shownin purple) involvedin dimerformation.EachZn atom is coordinatedby four appropriately spacedcysteineresidues.Likethe helix-turn-helixproteinsshownin Figure 7-11,the two recognitionhelicesof the dimerare heldapartbYa distance to one turn of the DNA corresponding doublehelix.The specificexampleshown is a fragmentof the glucocorticoid Thisis the proteinthrough receptor. whichcellsdetectand respond to the glucocorticoid transcriptionally hormonesproducedin the adrenalgland in responseto stress.(Adaptedfrom B.F.Luisiet al.,Naturc 352:497-505' 1991from Macmillan With permission Ltd.) Publishers

many tumors, as we shall see in Chapter 20. Many of the p53 mutations obseived in cancer cells destroy or alter its DNA-binding properties; indeed, Arg 248, which contacts the minor groove of DNA (seeFigure 7-lB) is the most frequently mutated p53 residue in human cancers'

TheLeucineZipperMotif MediatesBothDNABindingand Protein Dimerization Many gene regulatory proteins recognize DNA as homodimers, probably because,as *" hau" seen, this is a simple way of achieving strong specific binding (seeFigure 7-12). Usually, the portion of the protein responsible for dimerizition is distinct from the portion that is responsible for DNA binding. One motil however, combines these two functions elegantly and economically. It is called the leucine zipper motif, so named because of the way the two o helices, one from each monomer, are joined together to form a short coiled-coil (seeFigure 3-9). The helices are held together by interactions between hydrophobic amino acid side chains (often on leucines) that extend from one side of each helix. Just beyond the dimerization interface the two s helices separate from each other to form aY-shaped structure, which allows their side chains to contact the major groove of bNR. The dimer thus grips the double helix like a clothespin on a clothesline (Figure 7-lS).

Figure7-17 The bacterialMet repressor protein.The bacterialMet repressor the genesencodingthe regulates methionine enzymesthat catalYze Whenthis aminoacidis synthesis. abundant,it bindsto the repressor, causinga changein the structureof the proteinthat enablesit to bind to DNA of the tightly,shuttingoff the synthesis enzyme.(A)In orderto bind to DNA mustbe tightly,the Met rePressor methionine, complexedwith 5-adenosyl outlinedin red.Onesubunitof the dimericprotein is shown in green,while the other is shown in b/ue'The twostrandedB sheetthat bindsto DNAis formedby one strandfrom eachsubunit and is shown in darkgreenand dorkblue. (B)Simplifieddiagramof the Met reoressorbound to DNA,showinghow the two-strandedB sheetof the repressor bindsto the majorgrooveof DNA' For clarity,the other regionsof the repressor havebeen omitted. (A,adaptedfrom 5. Phillips,Cun.Opin.Struct.Biol.1:89-98, from Elsevier; 1991,with permission B,adaptedfrom W. Somersand S. Phillips,Nature 359:387-393, 1992' from Macmillan with permission Ltd.) Publishers

424

Chapter7: Controlof GeneExpression

Figure7-18 DNA recognitionby the p53 protein,The most important DNA contactsaremadeby arginine248and lysine120,whichextendfrom the protrudingloopsenteringthe minorand major grooves.The folding of the p53 protein requiresa zinc atom (shownas a sphere), but the way in whichthe zincis graspedby the protein is completely differentfrom that of the zinc finger proteins,describedpreviously.

Heterodimerization Expandsthe Repertoireof DNAsequences ThatGeneRegulatoryproteinsCanRecognize

structureshown is of the yeastGcn4protein,which regulates transcription in response to the availability of aminoacidsin the environment.(Adapted from T.E.Ellenbergeret al.,CeII 7 j :1223_1237, 1992.With permissionfrom Elsevier.)

Figure7-20 Heterodimerizationof leucinezipper proteins can alter their DNA-bindingspecificity.Leucine zipperhomodimersbind to symmetricDNAsequences, as shown in the left-handand centerdrawings.Thesetwo proteinsrecognizedifferentDNA sequences, as indicated by the redand b/ueregionsin the DNA.The two different monomerscan combineto form a heterodimer,which now recognizesa hybrid DNAsequence,composedfrom one red and one 6/ueregion.

PROTEINS MOTIFSIN GENEREGULATORY DNA-BINDING

4

2

There are, however, Iimits to this promiscuity: for example, if all the many tlpes of leucine zipper proteins in a typical eucaryotic cell formed heterodimers, the amount of "cross-talk" between the gene regulatory circuits of a cell would presumably be so great as to cause chaos. lVhether or not a particular heterodimer can form depends on how well the hydrophobic surfaces of the two Ieucine zipper a helices mesh with each other, which in turn depends on the exact amino acid sequencesof the two zipper regions.Thus, each leucine zipper protein in the cell can form dimers with only a small set of other leucine zipper proteins. Heterodimerization is an example of combinatorial control, in which combinations of different proteins, rather than individual proteins, control a cell process. Heterodimerization as a mechanism for combinatorial control of gene expression occurs in many different rypes of gene regulatory proteins (Figure 7-21). Combinatorial control is a major theme that we shall encounter repeatedly in this chapter, and the formation of heterodimeric gene regulatory complexes is only one of manyways in which proteins work in combinations to control gene expression. Certain combinations of gene regulatory proteins have become "hardwired" in the cell; for example, two distinct DNA-binding domains can, through gene rearrangements occurring over evolutionary time scales,become joined into a single pollpeptide chain that displays a novel DNA-binding specificity (Figure 7-22).

425 FigureT-21 A heterodimercomposedof two homeodomain proteins bound to its DNA recognitionsite.Theyel/owhelix4 of the proteinon the right (Mato'2)is in the absenceofthe protein unstructured forminga helixonly on the left (Mata1), The DNA upon heterodimerization. sequenceis recognizedjointly by both proteins;someof the Protein-DNA contactsmade by Mato2 were shown in Figure7-13.Thesetwo proteinsarefrom buddingyeast,wherethe heterodimer a particularcelltype (seeFigure specifies 7-65).The helicesare numberedin with Figure7-13' (Adapted accordance from T. Li et al.,Science270:262-269,1995. With permissionfrom AAA5.)

and DNA MotifAlsoMediatesDimerization TheHelix-Loop-Helix Binding Another important DNA-binding motif, related to the leucine zipper, is the (HLH) motif, which differs from the helix-turn-helix motif helix-loop-helix discussed earlier. An HLH motif consists of a short cr helix connected by a loop to a second, longer crhelix. The flexibility of the loop allows one helix to fold back

Figure7 -22 fwo DNA-bindingdomains covalentlyjoined bY a flexible polypeptide.The structureshown(called of both a consists a Pou-domain) homeodomainand a helix-turn-helix structurejoined by a flexiblepolypeptide by the brokenlines. "leashi'indicated A singlegeneencodesthe entireproteln, asa continuous which is synthesized polypeptidechain.The covalentjoining of two structuresin this way resultsin a in the affinityof the largeincrease proteinfor its specificDNA sequence comparedwith the DNAaffinityof either separatestructure.The grouP of mammaliangeneregulatoryproteins exemplifiedby this structureregulatethe productionof growth factors, and other molecules immunoglobulins, The particular involvedin development. exampleshownisfrom the Octl protein.(Adaptedfrom J.D'Klemmet al', Cell77:21-32, 1994.With permission from Elsevier.)

426

Chapter7: Controlof GeneExpression Figure7 -23 A helix-loop-helix (HLH) dimer bound to DNA.The two monomersare held together in a fourhelixbundle:eachmonomercontributes two o helicesconnectedby a flexible loop of protein (redJ.A specificDNA sequenceis bound by the two s helices that projectfrom the four-helixbundle. (Adaptedfrom A.R.Ferre-DAmare et al., Nature363:38-45,1993.With permission from MacmillanPublishers Ltd.)

and pack against the other. As shown in Figure 7-23, this two-helix structure binds both to DNA and to the HLH motif of a second HLH protein. The second HLH protein can be the same (creating a homodimer) or different (creating a heterodimer). In either case, tvvo s helices that extend from the dimerizati,on interface make specific contacts with the DNA.

It ls NotYetPossible to Predictthe DNAsequences Recognized by All GeneRegulatory Proteins The various DNA-binding motifs that we have discussed provide structural frameworks from which specific amino acid side chains to conracr spe"*t"nd cific base pairs in the DNA. It is reasonable to ask, therefore, whether there is a

Having outlined the general features of gene regulatory proteins, we turn to some of the methods that are now used to studv them.

a c t i v eH L Hh o m o d i m e r

inactiveHLH heterodimer

Figure7 -24 lnhibitory regulation by truncatedHLHproteins.The HLHmotif is responsible for both dimerization and DNAbinding.On the /eft,an HLH homodimerrecognizes a symmetricDNA sequence.On the right,the binding of a full-lengthHLHprotein (blue)to a truncatedHLHprotein(green)that lacks the DNA-binding cr helixgenerates a heterodimer that is unableto bind DNA tightly. lf presentin excess,the truncated proteinmoleculeblocksthe homodimerization of the full-lengthHLH proteinand therebypreventsit from bindingto DNA.

427

PROTEINS DNA-BINDING MOTIFSIN GENEREGULATORY Figure7-25 One of the most common protein-DNA interactions' Becauseof its specificgeometryof hydrogen-bondacceptors(see guanine. recognizes Figure7-7),the sidechainof arginineunambiguously Figure7-9 showsanothercommonprotein-DNAinteraction.

a r g I nI n e

A Gel-MobilityShiftAssayReadilyDetectsSequence-Specific Proteins DNA-Binding Genetic analyses,which provided a route to the gene regulatory proteins of bacteria, yeast, and Drosophila, are much more difficult in vertebrates. Therefore, the isolation of vertebrate gene regulatory proteins had to await the development of different approaches.Many of these approachesrely on the detection in a cell extract of a DNA-binding protein that specifically recognizes a DNA sequence known to control the expressionof a particular gene. One of the most common ways to detect and study sequence-specificDNA-binding proteins is based on the effect of a bound protein on the migration of DNA molecules in an electric field.

major 9roove H

N-H

lllllllllllllllo

N illillillllllllH-

mrnor groove

6E 5 4 3 2&

TTKfinger 1

TTK finger 2

6l 5 4 3

Zif finger 1

Zif finger 2

G L If i n g e r 4

G L If i n g e r 5

FigureT-26 Summaryof sequencespecificinteractionsbetween six different zinc fingers and their DNA recognitionsequences.Eventhough all sixZn fingershavethe sameoverall structure(seeFigure7-14),eachbindsto numbered a differentDNAsequence.The aminoacidsform the q, helixthat recognizesDNA (Figures7-14 andT-15), and thosethat makesequence-specific DNAcontactsaregreen.Basescontacted by protein arc orunge.Although contactsarecommon arginine-guanine (seeFigure7-25),guaninecan alsobe recognizedby serine,histidine,and lysine,as shown.Moreover,the same in this example)can aminoacid(serine, recognizemore than one base.Two of the Zn fingersdepictedarefrom the TTK protein (a Drosophilaprotein that functionsin development);two arefrom the mouseprotein (Zif268)that was shownin Figure7-15;and two arefrom a humanprotein(GL1)whoseaberrant forms can causecertaintypes of cancers. (Adaptedfrom C. Brandenand J.Tooze, lntroductionto ProteinStructure,2nd ed. 1999') NewYork:GarlandPublishing,

428

Chapter7: Control ofGene Expression

A DNA molecule is highly negatively charged and will therefore move rapidly toward a positive electrode when it is subiected to an electric field. \fhen anaIyzed by polyacrylamide-gel electrophoresis (see p. 534), DNA molecules are separared according to their size because smaller molecules are able to penetrate the fine gel meshwork more easily than large ones. protein molecules bound to a DNA molecule will cause it to move more slowly through the gel; in general, the larger the bound protein, the greater the retardation of the DNA molecule. This phenomenon provides the basis for the gel-mobility shift assay, which allows even trace amounts of a sequence-specificDNA-binding p.otein io be readily detected. In this assay,a short DNA fragment of specific iength and sequence (produced either by DNA cloning or by chemical synthesis, as discussed in chapter B) is radioactively labeled and mlred with a cell extract; the mixture is then loaded onto a polyacrylamide gel and subjected to electrophoresis. If the DNA fragment corresponds to a chromosomal region where, for example, several sequence-specificproteins bind, autoradiography (seepp. 602-603) will reveal a series of DNA bands, each retarded to a different exrent and representing a distinct DNA-protein complex. The proteins responsible for each band on the gel can then be separated from one another byiubsequent fractionations of the cell extract (Figure z-27). once a sequence-specificDNA protein has been purified, the gel-mobility shift assaycan be used to study the strength and specificity of its interactions with different DNA sequences, the lifetime of DNA-protein complexes, and other properties critical to the functioning of the protein in the cell.

DNAAffinitychromatography Facilitates the purification of proteins Sequence-Specific DNA-Binding A particularly powerful protein-purification method called DNA affinity chromatography can be used once the DNA sequencethat a gene regulatory protein recognizeshas been determined.A double-strandedoligonucleotideof the correct sequence is synthesizedby chemical methods and linked to an insoluble porous matrix such as agarose;the matrix with the oligonucleotide attached is

radioactive

I

c

a c o c

c5 freeDNA

I

tr

a

I I

I

er -

-C4

-

-c2

-C5 -

...-C3

gel result

, 8 * r ,' - *

o

-

-

C 1-

-C1 c o

',

C2-k#q@ C3-

,,.,W

-C4

ri,'',"}*irs*

freeDNA

-C5

o

+

r:risr@N6r:1i:r

-

c6

-free DNA

@

_c6 (A)

c o o o

10

20

30

40

f r a c t i o nn u m b e rf r o m c h r o m o t o g r a p h y c o l u m ne l u t e dw i t h i n c r e a s i n g s a l tc o n c e n t r a t i o n

Figure7-27 A gel-mobility shift assay. The principleof the assayis shown schematically in (A).In this examplean extractof an antibody-producing cellline is mixedwith a radioactive DNAfragment containingabout 160nucleotides ofa regulatoryDNAsequence from a gene encodingthe light chainof the antibody madeby the cellline.Theeffectof the proteinsin the extracton the mobilityof the DNAfragmentis analyzedby polyacrylamide-gel electrophoresis followedby autoradiography. Thefree DNAfragmentsmigraterapidlyto the bottom of the gel,whilethosefragments boundto proteinsareretarded; the findingof six retardedbandssuggests that the extractcontainssixdifferent sequence-specific proteins DNA-binding (indicatedasC1-C6)that bind to this DNA (Forsimplicity, sequence. any DNA fragmentswith morethan one protein bound havebeenomittedfrom the figure.)In (B)a standardchromatographic technique(seepp. 512-513) wasusedto fractionatethe extract(top) and each fractionwasmixedwith the radioactive DNAfragment,appliedto one laneof a polyacrylamide gel,and analyzedas in (A). (8,modifiedfrom C.Scheidereit, A Heguy and R.G.Roeder,Cell51:783-793,1987. With permission from Elsevier.)

429

PROTEINS DNA-BINDING MOTIFSIN GENEREGULATORY

then used to construct a column that selectively binds proteins that recognize the particular DNA sequence (Figure 7-28). Purifications as great as 10,000-fold can be achieved by this means with relatively little effort. Although most gene regulatory proteins are present at very low levels in the cell, enough pure protein can usually be isolated by affinity chromatography to obtain a partial amino acid sequenceby mass spectrometry or other means (discussed in Chapter 8). If the complete genome sequence of the organism is known, the partial amino acid sequence can be used to identify the gene. The gene not only provides the complete amino acid sequenceof the protein; it also provides the means to produce the protein in unlimited amounts through genetic engineering techniques, also discussedin Chapter 8.

ProteinCan by a GeneRegulatory Recognized TheDNASequence Experimentally BeDetermined Gene regulatory proteins can be discovered before the DNA sequence they recognize is known. For example, many of the Drosophilahomeodomain proteins were discoveredthrough the isolation of mutations that altered fly development. This allowed the genes encoding the proteins to be identified, and the proteins could then be overexpressedin cultured cells and easily purified. DNA foot' printingis one method of determining the DNA sequencesrecognized by a gene regulatory protein once it has been purified. This strategy also requires a purified fragment of duplex DNA that contains somewhere within it a recognition site for the protein. Short recognition sequences can occur by chance on any long DNA fragment, although it is often necessaryto use DNA corresponding to a regulatory region for a gene kno',.tmto be controlled by the protein of interest. DNA footprinting is based on nucleasesor chemicals that randomly cleaveDNA at every phosphodiester bond. A bound gene regulatory protein blocks the phosphodiester bonds from attack, thereby revealing the protein's precise recognition site as a protected zone, or footprint (Figure 7-ZS). A second way of determining the DNA sequencesrecognized by a gene regulatory protein requires no prior knowledge of what genes the protein might

D N A - b i n d i n gp r o t e i n s from step 1

t o t a l c e l lp r o t e i n s aa aa o ao

STEP2 aoo at

t a

II

t c o l u m nw i t h m a t r i x c o n t a i n i n go n l y GCGCCC CCqGGG

m e d i u m - s a lwt a s h removesall proteins n o t s p e c i f i cf o r

L]

?3 a

1t . .i ' t!

h i g h - s a lw t ash e l u t e sr a r e p r o t e i n that specifically recoqnizes GGGCCC -

cccG6G

a o

oo

aat'

Figure7-28 DNA affinity chromatography. In the firststep,allthe proteinsthat can from the remainder bind DNAareseparated of the cellproteinson a columncontaining a hugenumberof differentDNAsequences. DNA-binding Mostsequence-specific proteinshavea weak(nonspecific) affinity for bulk DNAand arethereforeretainedon the column.Thisaffinityis due largelyto and the proteinscan be ionicattractions, washedoff the DNAbYa solutionthat of salt' containsa moderateconcentration In the secondstep,the mixtureof DNAbindingproteinsis passedthrougha columnthat containsonlYDNAof a all the DNAparticularsequence. Typically, bindingproteinswill stickto the column, the great majoritybY nonsPecific Theseareagainelutedby interactions. solutionsof moderatesaltconcentration, leavingon the columnonlythoseproteins (typicallyone or only a few)that bind and thereforeverytightlyto the specifically particularDNAsequence. Theseremaining proteinscan be elutedfrom the columnby solutionscontaininga verYhigh of salt. concentration

430

Chapter7:Controlof GeneExpression region of DNA protected b y D N A - b i n d i n gp r o t e i n

(A)

R A N D O MC L E A V A GB EY N U C L E A S E O R C H E M I C A LF,O L L O W E B DY REMOVAO L FT H EP R O T E IA NN D SEPARATION OF THEDNA STRANDS

f a m i l yo f s i n g l e - s t r a n d eDdN A m o l e c u l e sl a b e l e da t t h e 5 , e n d

,rro^o'o,., By cELELEcrRopHoRESrs |

ililill (B)

"footprint, " w h e r e n o c l e a v a g ei s observed

tliltl t top of gel

Figure7-29 DNA footprinting. (A)Schematic of the method.A DNA fragmentis labeledat one end with 328a proceduredescribedin Figure8-34; next, the DNAis cleavedwith a nuclease or chemicalthat makesrandom,singlestrandedcuts.Afterthe DNAmoleculeis denaturedto separateits two strands, the resultantfragmentsfrom the labeled strandareseparated on a gel and detectedby autoradiography(seeFigure 8-33).The patternof bandsfrom DNA cut in the presence of a DNA-bindingprotein is comparedwith that from DNAcut in its absence.When protein is present,it coversthe nucleotides at its bindingsite and protectstheir phosphodiester bonds from cleavage.As a result,those labeled fragmentsthat would otherwise terminatein the bindingsiteare missing, leavinga gap in the gel patterncalleda "footprint." In the exampleshown,the DNA-binding proteinprotectsseven phosphodiester bondsfrom the DNA cleavingagent.(B)An actualfootprint usedto determinethe bindingsitefor a generegulatoryproteinfrom humans. Thecleavingagentwasa small,ironcontainingorganicmoleculethat normallycutsat everyphosphodiester bond with nearlyequalfrequency. (8,courtesyof MicheleSawadogoand RobertRoeder.)

without protein with protein

f..tp"[,lt gene regulatory p r o t e i no f u n k n o w n D N A - b i n d i n gs p e c iifc i t y

sequencerecognizedby a gene regulatoryprotein is known, computerized genomesearchescan identifu candidategeneswhose transcriptionthe gene

Figure7-30 A methodfor determiningthe DNAsequencerecognizedby a gene regulatoryprotein.A purifiedgeneregulatoryproteinis mixed with millionsof differentshort DNAfragments,eachwith a different sequenceof nucleotides. A collectionof suchDNAfraqmentscan be

r,#xadrtq4l f*1!Pitcrs$

separation is throughgel-mobilityshifts,as illustrated in Figure7_27.After separationof the DNA-proteincomplexesfrom the free DNA,the DNA fragmentsare removedfrom the proteinand typicallyusedfor several additionalroundsof the sameselectionprocess(not shown).The nucleotidesequences of thoseDNAfragmentsthat remainthrough multipleroundsof bindingand release can be determined, and a consensus DNArecognitionsequencecanthus be generated.

I

I I I +

R E M O V EP R O T E TANN D D E T E R M I NSEE Q U E N C E OSF TIGHTLY B O U N DD N A FRAGMENT5

DNACONSENSU SS EQUENCE R E C O G N I Z EBDY G E N E REGULATORY PROTEIN

431

DNA.BINDINGMOTIFSIN GENEREGULATORY PROTEINS

D N As e q u e n c efsr o m f i v e c l o s e l yr e l a t e dy e a s ts p e c i e s - - -TGATGACAGTCTTAATATCATCTGCAAC---TCTTGAAATCTTGCTTTATAGTCAAAATTTACGTACGCTTTTCACTATATAATATGATTTGTCAAT ---CAACGGTAGTTTCGAGGTTGCATATAAT---CCGGTGGA-CTGGlcGTTAAAGTTAGEAGTCCACTTCACTTCT-TC--ATTG-TATTCTGTCTTATC TCTTGATGGCAGTCTTGATACCGTGTAAJAAC---CCACGTGGTCTAGTCTCATACTCAAAATT-ACGTCCACTTTCCCCTGTATATTATGTTTTGTCGAT ---CAAAACTGATCC-AGAAGCACTCCTGATTCACCTTTGATACCAATCGATTCATTCAAATCGGCCTGAACCTTG-AT---ATA-TGTATCTTGCCCCT ---TAATACTGATCCCATAGGTGCTCTTAA--CACCCGCAGT-CTGGCCTTATGATTGAAAGTTAATCGAACTTTT-ATTGTATA-TAACGCTATGTATT b i n d i n gs i t ef o r g e n e r e g u l a t o r yp r o t e i n

t1

- TTCATGTTGA GTGATGAGTGAATGTCTCCCTGTTACCCGGTT AACATCCGT GTC- - - CGTAGAGCACTCTCTGTTACCCGGATATTCCTGTTATCTTTTGTTTCAAGCTTTAA ATCGTATCAGAATTTATTGG-ATTACCCGGGCCGACCCTTTTTTGCGTGTTTCAAGCTTCAA-GGCACAACCAAATATATTTTCGTTACCCGAACCAGCTTTTAATTATCTGTTTCAGG

Ll

tiI

---TGTTTTGTGACAGCACCTGTCAATTT-TAGGATAGTAGCAATCGCAAAC---GTTCTCAATAATTCTAAGA----- -CTACTTAACAGCTCTAGCA- - - - CTCTGCTCTATAGTAGCACTTCTAACTTCATTGAGAAACAATAAAGACAGAA- - -TGTTTTGTGATAGCACTTCTCAGTTT-TGAAATAACAGCAACCGCAGAC---A- ---CAAAACCTCTAAA- - -- -- - -TGTTGTACGATAGCACCCTTGTGTTCGCTTGAAAACACCAAAGGAAGACAGCTAGCCCCATCCCCACGACTCCAGC ---TGTTTTGTGATAGCACTCTCAAGTTTACTTGAAAAGGACAAAAGAAGAA---CCGCCCGACGCCTCCAAT------

upstreamof the samegenefrom five footprinting.ThisexamplecomparesDNAsequences Figure7-31 Phylogenetic footprintingrevealsDNArecognition yellow. Phylogenetic in yeasts; highlighted nucleotides are identical closelyrelated Onlythe regionupstream than surroundingsequences. sitesfor regulatoryproteins,asthey aretypicallymoreconserved genomes. The gene entire to analyze used is typically particular the approach geneis shownin this example,but of a regulatoryproteinsthat bind to the siteoutlinedin red areshownin Figure7-21.Someof the shorterphylogenetic footprintsin this examplerepresentbindingsitesfor additionalgeneregulatoryproteins,not all of which havebeen Ltd.,and (FromM. Kelliset al.,Nature423:241-254,2003, from MacmillanPublishers with permission identified. from NationalAcademyof Sciences.) 01:18069-18074,2004,with permission D.J.Galgoczyet al.,Proc.NatlAcad.Sci.IJ.S.A.1 regulatory protein of interest might control. However, this strategy is not foolproof. For example, many organisms produce a set of closely related gene regulatory proteins that recognize very similar DNA sequences, and this approach cannot resolve them. In most cases, predictions of the sites of action of gene regulatory proteins obtained from searching genome sequences must, in the end, be tested experimentally.

Sequences FootprintingldentifiesDNARegulatory Phylogenetic Genomics ThroughComparative The widespread availability of complete genome sequencesprovides a surprisingly simple method for identi$ring important regulatory sites on DNA, even when the gene regulatory protein that binds them is unknown. In this approach, genomes from several closely related species are compared. If the species are chosen properly, the protein-coding portions of the genomes will be very similar, but the regions between sequencesthat encode protein or RNA molecules will have diverged considerably, as most of this sequence is functionally irrelevant and therefore not constrained in evolution. Among the exceptions are the regulatory sequences that control gene transcription. These stand out as conserved islands in a sea of nonconserved nucleotides (Figure 7-31 ) . Although the identity of the gene regulatory proteins that recognize the conserved DNA sequencesmust be determined by other means, phylogenetic footprinting is a powerful method for identifuing many of the DNA sequencesthat control gene expression.

ldentifiesManyof the SitesThat Chromatinlmmunoprecipitation ProteinsOccupyin LivingCells GeneRegulatory A gene regulatory protein will not occupy all of its potential DNA-binding sites in the genome at a particular time. Under some conditions, the protein may not be synthesized,and so will be absent from the cell; it may be present but lacking a heterodimer partner; or it may be excluded from the nucleus until an appropriate signal is received from the cell's environment. Even if the gene regulatory

432

Chapter7:Controlof GeneExpression Figure7-32 Chromatinimmunoprecipitation. Thismethodallowsthe identification of all the sitesin a genomethat a generegulatoryprotein occupiesin vivo For the amplificationof DNA by a polymerasechain reaction(PCR), seeFigure8-45.The identitiesof the precipitated, amplified DNAfragmentscan be determinedby hybridizingthe mixtureof fragmentsto DNAmicroarrays, asdescribedin ChapterB.

regulatoryproteinA :w

w: gene1 proteinB

regu

living cell

g e n e2

protein is present in the nucleus and is competent to bind DNA, components of chromatin or other gene regulatory proteins that can bind to the same or overlapping DNA sequences may occlude many of its potential binding sites on DNA. chromatin immunoprecipitation provides one way of empirically determining the sites on DNA that a given gene regulatory protein occupies under a particular set of conditions (Figure z-32).ln this approach, proteins are covalently cross-linked to DNA in living cells, the cells are broken open, and the DNA is mechanically sheared into small fragments. Antibodies directed against a given gene regulatory protein are then used to puriff DNA that became covalently cross-linked to that protein in the cell. If this DNA is hybridized to microarrays that contain the entire genome displayed as a seriesof discrete DNA fragments (see Figure 8-73), the precise genomic location of each precipitated DNA fragment can be determined. In this way, all the sites occupied by the gene regulatory protein in the original cells can be mapped on the cell's genome (Figure 7-33). chromatin immunoprecipitation is also routinely used to identify the positions along a genome that are packaged by the various types of modified histones (discussedin chapter 4). In this case,antibodies specific to the particular histone modification of interest are employed.

Summ a r y Gene regulatory proteins recognizeshort stretchesof double-helical DNA of defined sequenceand therebydetermine which of the thousandsof genesin a ceII will be transcribed.Thousandsof gene regulatory proteins haue been identified in a witJe uariety of organisms.Although each of theseproteins has unique features, most bind to DNA as homodimers or heterodimersand recognizeDNA through one of a small number of structural motifs. The common motifs include the helix-turn-helix, the homeodomain, the leucine zipper, the helix-loop-helix, and zinc fingers of seueral rypes.The preciseamino acid sequencethat isfolded into a motif determinesthe particular DNA sequencethat a gene regulatory protein recognizes.Heterodimerization increasesthe rangeof DNAsequencesthat can be recognized.Powerful techniquesare now auailable for identifying and isolating theseproteins, the genesthat encodethem, and the DNA sequencesthey recognize,and for mapping all of the genes that they regulate on a genome.

HOWGENETIC SWITCHES WORK In the previous section, we described the basic components of genetic switches: gene regulatory proteins and the specific DNA sequences that these proteins recognize.we shall now discusshow these components operate to turn geneson and off in response to a variety of signals. In the mid-twentieth century, the idea that genes could be switched on and wa9 revolutionary. This concept was a major advance, and it came originally 9ff from the study of how E coli bacteria adapt to changes in the composition of their growth medium. Parallel studies of the lambda bacteriophage lea to many of the same conclusions and helped to establish the underlying mechanism. Many of the same principles apply to eucaryotic cells. However, ihe enormous complexity of gene regulation in higher organisms,combined with the packaging

pRorEtNS To I cnoss-r-rrur +

D N A W I T HF O R M A L D E H Y D E

I I LYSECELLS I ; B R E A KD N A I N T OS M A L L | (- 300NUCLEOTDE) V FRAGMENTS m X mx

w,

*i*twssr

+ many other DNA fragments that comprisethe rest of the genome

DNAustNG I cnecrerrnre

SA T N SG TE N E I A N T T B O D TAEG R E G U L A T O RPYR O T E TAN + wXwx R E V E R SFEO R M A L D E H Y D E CROSS-LINKS; REMOVEPROTEIN

I I A M P L T FTYH EP R E C T P T T A T E D I DNABYPCR t D N A C O R R E S P O N D ITNOGT H O S E P O S I T I O NI N S T H EG E N O M ET H A TW E R E O C C U P I EB DY G E N ER E G U L A T O R Y P R O T E IA N I N T H EC E L L S

HOWGENETIC SWITCHES WORK

433

cnromosome numoer

C

1

FUS3

RDH54

to

.'

I

i

c

MATU1 & MATri2

HO

4

I C S 2& A M N l

I

STE 5

ol

I

Mt-,4.i

I

(_l

I

5

MFa-2

8ax

GPAl

ta)

9 10 11

RME,l

ot

tl

7

|

1201

FARl S A G1 | 5TE3 | YLR04OC C C W 1 2 & HOG1

tx o

5TE18

I NEJ1

|

13 't4

Io DDR2

1s|o to

5TE4

I

oI

I MFal

AXLl

of their DNA into chromatin, createsspecial challenges and some novel opportunities for control-as we shall see.We begin with the simplest example-an on-off switch in bacteria that responds to a single signal.

TheTryptophanRepressor !sa SimpleSwitchThatTurnsGenes On and Off in Bacteria The chromosome of the bacterium E. coli, a single-celled organism, is a single circular DNA molecule of about 4.6 x 106nucleotide pairs. This DNA encodes approximately 4300 proteins, although the cell makes only a fraction of these at any one time. The expression of many genes is regulated according to the available food in the environment. This is illustrated by the five E. coli genesthat code for enzymes that manufacture the amino acid try,ptophan. These genes are arranged as a single operon; that is, they are adjacent to one another on the chromosome and are transcribed from a single promoter as one long mRNA molecule (Figure 7-34). But when tryptophan is present in the growth medium and enters the cell (when the bacterium is in the gut of a mammal that has just eaten a meal of protein, for example), the cell no longer needs these enzyrnes and shuts off their production. The molecular basis for this switch is understood in considerable detail. As described in Chapter 6, a promoter is a specific DNA sequence that directs RNA polymerase to bind to DNA, to open the DNA double helix, and to begin

promoler

&.

E .c o / i c h r o m o s o m e

operator m R N Am o l e c u l e

r+ttt Of-a.o e n z y m e sf o r t r y p t o p h a nb i o s y n t h e s i s

Figure7-33 A gene regulatorycircuit:the complete set of genescontrolled by three key regulatory proteins in budding yeast, as deduced from the DNA siteswhere the regulatoryproteinsbind,The regulatory proteins-calledMata1,Mat()(1, and Mato2-specify the two differenthaploid matingtypes(analogous to maleand femalegamates)of this unicellular organism. The 16chromosomes in the yeast genomeare shown (gray),withcoloredbars indicatingsiteswherevariouscombinations of the threeregulatoryproteinsbind. Aboveeachbindingsiteis the nameof the proteinproductofthe regulatedtarget gene.Mats1,actingin a complexwith anotherprotein,Mcm1,activates expressionof the genesmarkedin red; Mat02,actingin a complexwith Mcm1, represses the genesmarkedin blue;and Matal in a complexwith Matcx,2 represses the genesmarkedin green(seeFigures 7 -21 and 7-65). Doublearrowheads genes, represent divergentlytranscribed whicharecontrolledby the indicatedgene regulatoryproteins. Thiscompletemap of bound regulatoryproteinswasdetermined usinga combinationof genome-wide (seeFigure chromatinimmunoprecipitation 7-32) and phylogeneticfootprinting (see Figure7-29).Suchdeterminationsof completetranscriptional circuitsshowthat transcriptional networksare not infinitely complex,althoughthey may appearthat way initially. Thistype of studyalsohelpsto revealthe overalllogicof the transcriptional circuitsusedby modern cells.(FromD.J.Galgoczyet al.,Proc.Natl Acad.Sci.U.5.4.101:18069-18074,2004. With permission from NationalAcademyof Sciences.)

Figure7-34 The clusteredgenesin E colithat code for enzymesthat manufacturethe amino acid tryptophan. Thesefive genesof the Irp operon-denoted as TrpA,B,C,D, and E-are transcribed asa singlemRNA molecule, whichallowstheirexpression to be controlledcoordinately. Clusters of genestranscribed asa singlemRNA moleculearecommonin bacteria. Each suchclusteris calledan ooeron.

434

Chapter7: ControlofGeneExpression promoter start of transcription 35

J

-10 operaror

+1

+20

inactiverepressor R N Ap o l y m e r a s e

€++

tryptoph

GENEs ARE JNI

active repressor

GENES AREOFF

synthesizing an RNA molecule. Within the promoter that directs transcription of the trlptophan biosynthetic genes lies a regulator element called an operator (see Figure 7-34). This is simply a short region of regulatory DNA of defined nucleotide sequence that is recognized by a repressor protein, in this case the tryptophan repressor, a member of the helix-turn-helix family (see Figure 7-ll). The promoter and operator are arranged so that when the tryptophan repressor occupies the operator, it blocks accessto the promoter by RNA polymerase, thereby preventing expression of the tryptophan-producing enzymes (Figure 7-35). The block to gene expression is regulated in an ingenious way: to bind to its operator DNA, the repressor protein has to have two molecules of the amino

Figure7-35 Switchingthe tryptophan geneson and off. lf the levelof tryptophaninsidethe cell is low,RNA polymerase bindsto the promoterand transcribes the fivegenesof the tryptophan (Irp) operon.lf the levelof tryptophanis high,however, the tryptophan repressoris activatedto bind to the operator,where it blocksthe bindingof RNApolymerase to the promoter.Wheneverthe levelof intracellular tryptophandrops,the repressor releases itstryptophanand becomesinactive, allowingthe polymerase to begintranscribing these genes.The promoterincludestwo key blocksof DNAsequenceinformation, the - 3 5 a n d - 1 0 r e g i o n sh i g h l i g h t e idn yellow(seeFigure6-12).

tryptophan

GENES A R EO N

GENES A R EO F F

Figure7-36 The binding of tryptophan to the tryptophan repressorprotein changesits conformation.Thisstructural changeenablesthis generegulatoryproteinto bind tightlyto a specificDNAsequence(theoperator), therebyblocking transcription of the genesencodingthe enzymesrequiredto producetryptophan(the Trpoperon).Thethree-dimensional structureof this bacterialhelix-turn-helixprotein,asdeterminedby x-raydiffractionwith and withouttryptophanbound, is illustrated' Tryptophanbindingincreases the distancebetweenthe two recognitionhelicesin the homodimer,allowing the repressorto fit snuglyon the operator.(Adaptedfrom R.Zhanget al.,Nature327:591-597 ,1 9g7.With permissionfrom MacmillanPublishers Ltd.)

HOW GENETIC SWITCHES WORK

Becausethe active, DNA-binding form of the protein serves to turn genes off, this mode of gene regulation is called negative control, and the gene regulatory proteins that function in this way are called transcriptional repressorsor gene repressorproteins.

Transcriptional ActivatorsTurnGenesOn We saw in Chapter 6 that purified E. coli RNA polymerase (including its o subunit) can bind to a promoter and initiate DNA transcription. Many bacterial promoters, however, are only marginally functional on their own, either because they are recognized poorly by RNA polymerase or because the polymerase has difficulty opening the DNA helix and beginning transcription. In either case these poorly functioning promoters can be rescued by gene regulatory proteins that bind to a nearby site on the DNA and contact the RNA polymerase in a way that dramatically increases the probability that a transcript will be initiated. Because the active, DNA-binding form of such a protein turns genes on, this mode of gene regulation is called positive control, and the gene regulatory proteins that function in this manner are known as transcriptional actiuators or geneactiuator proteins.In some cases,bacterial gene activator proteins aid RNA polymerase in binding to the promoter by providing an additional contact surface for the polymerase. In other cases,they contact RNA polymerase and facilitate its transition from the initial DNA-bound conformation of polymerase to the actively transcribing form by stabilizing a transition state of the enzyme. Like repressors,gene activator proteins must be bound to DNA to exert their effects. In this way, each regulatory protein acts selectively,controlling only those genes that bear a DNA sequence recognized by it. DNA-bound activator proteins can increase the rate of transcription initiation up to 1000-fold, a value consistent with a relatively weak and nonspecific interaction between the activator and RNA polymerase. For example, a 1000fold change in the affinity of RNA polymerase for its promoter corresponds to a change in AG of -4 kcal/mole, which could be accounted for by just a few weak, noncovalent bonds. Thus gene activator proteins can work simply by providing a few favorable interactions that help to attract RNA polymerase to the promoter. As in negative control by a transcriptional repressor,a transcriptional activator can operate as part of a simple on-off genetic switch. The bacterial activator protein CAP (catabolite actiuator protein), for example, activates genes that enable E. coli to use alternative carbon sourceswhen glucose, its preferred carbon source, is unavailable. Falling levels of glucose cause an increase in the intracellular signaling molecule cyclic AMII which binds to the CAP protein, enabling it to bind to its specific DNA sequence near target promoters and thereby turn on the appropriate genes. In this way the expression of a target gene is switched on or off, depending on whether cyclic AMP levels in the cell are high or low, respectively. Figure 7-37 summarizes the different ways that positive and negative control can be used to regulate genes. Transcriptional activators and transcriptional repressors are similar in design.The trlptophan repressorand the transcriptional activator CAB for example, both use a helix-turn-helix motif (see Figure 7-l l) and both require a small cofactor in order to bind DNA. In fact, some bacterial proteins (including CAP and the bacteriophage lambda repressor)can act as either activators or repressors, depending on the exact placement of the DNA sequencethey recognize in relation to the promoter: if the binding site for the protein overlaps the promoter, the poll.rnerase cannot bind and the protein acts as a repressor (Figure 7-38).

Repressor A Transcriptional Activatorand a Transcriptional Controlthe LocOperon More complicated types of genetic switches combine positive and negative controls. The Lac operonin E. coli, for example, unlike the Trp operon, is under both

435

436

Chapter7:Controlof GeneExpression

N E G A T I VR EE G U L A T I O N bound repressorprotein preventstranscription

(A)

P O S I T I VREE G U L A T I O N bound activator protein promotestranscription

(B)

R N Ap o l y m e r a s e

boundactivator protein,

oounorepressor protein\ fl

G E N EO F F

is

GENE ON 8i

I

I

mRNA

5',

I ADDITION O FL I G A N D

protein

S W I T C H EGSE N EO N B Y R E M O V I NR GE P R E S S O R PROTEIN

A D D I T I O NO F L I G A N D S W I T C H EGSE N EO F F BYREMOVING ACTIVATOR PROTEIN

G E N EO F F

GENE ON

,g

II

I

mRNA

''13' inA.tiva

B YR E M O V I N G R E P R E S SPORRO T E I N

rahra'8:eq)rzlaruPC "lela aluerl-leIayl'yuor;'):uPZ purlesou;o 'slla) 'arn}ln) (O) a^lau uorl6ueb prnbrl o))eqof u1 sllar leutlat ;o {seltnor'V) stse;qo^u uroJol 6ursn; aleal)nul]lnu ))lq) (8) terpauund(f) 'slla)al)snLu 'slsplqorqU rq6;'19-g eln6t3 asno6(V)'atntln)ul slla)Josqde,r6onlur

urri 0S

())

r.l.rrl 001

V N Up u e ' V N C

(s)

url OZ

' s u t a l o l 66 u 1 1 e 1 n d t u e: 6 31 reldeql

t0s

ISOLATING CELLSAND GROWINGTHEM IN CUIIURE

in many species, regenerate a whole new plant. Similar to animal cells, callus cultures can be mechanically dissociated into single cells, which will grow and divide as a suspension culture (seeFigure B-4D).

Eucaryotic CellLinesArea WidelyUsedSourceof Homogeneous Cells The cell cultures obtained by disrupting tissues tend to suffer from a problemeventually the cells die. Most vertebrate cells stop dividing after a finite number (discussed ofcell divisions in culture, a process called replicatiue cell senescence in Chapter 17). Normal human fibroblasts, for example, typically divide only 25-40 times in culture before they stop. In these cells, the limited proliferation capacity reflects a progressiveshortening and uncapping of the cell's telomeres, the repetitive DNA sequencesand associatedproteins that cap the ends of each chomosome (discussed in Chapter 5). Human somatic cells in the body have turned off production of the enzyrne, caIIed telomerase,Ihat normally maintains the telomeres, which is why their telomeres shorten with each cell division. Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase;in this case, they can be propagated as an "immortalized" cell line. Some human cells, however, cannot be immortalized by this trick. Although their telomeres remain long, they still stop dividing after a limited number of divisions because the culture conditions eventually activate cell-cycle checkpoint mechanlsms (discussedin Chapter 17) that arrest the cell cycle-a process sometimes called "culture shock." In order to immortalize these cells, one has to do more than introduce telomerase. One must also inactivate the checkpoint mechanisms. This can be done by introducing certain cancer-promoting oncogenes, such as those derived from tumor viruses (discussed in Chapter 20). Unlike human cells, most rodent cells do not turn off production of telomerase and therefore their telomeres do not shorten with each cell division. Therefore, if culture shock can be avoided, some rodent cell types will divide indefinitely in culture. In addition, rodent cells often undergo genetic changes in culture that inactivate their checkpoint mechanisms, thereby spontaneously producing immortalized cell lines. Cell lines can often be most easily generated from cancer cells,but these cultures differ from those prepared from normal cells in several ways, and are referred to as transformed cell llnes. Transformed cell lines often grow without attaching to a surface, for example, and they can proliferate to a much higher density in a culture dish. Similar properties can be induced experimentally in normal cells by transforming them with a tumor-inducing virus or chemical. The resulting transformed cell lines can usually cause tumors if injected into a susceptible animal (although it is usually only a small subpopulation, called cancer stem cells, that can do so-discussed in Chapter 20). Both transformed and nontransformed cell lines are extremely useful in cell research as sources of very large numbers of cells of a uniform type, especially since they can be stored in liquid nitrogen at -196'C for an indefinite period and retain their viability when thawed. It is important to keep in mind, however, that the cells in both types of cell lines nearly always differ in important ways from their normal progenitors in the tissues from which they were derived. Some widely used cell lines are listed in Table 8-1. Different lines have different advantages;for example, the PtK epithelial cell lines derived from the rat kangaroo, unlike many other cell lines which round up during mitosis, remain flat during mitosis, allowing the mitotic apparatus to be readily observed in action.

Medicine Embryonic StemCellsCouldRevolutionize Among the most promising cell lines to be developed-from a medical point of view-are embryonic stem (ES) cells. These remarkable cells, first harvested from the inner cell mass of the early mouse embryo, can proliferate indefinitely

505

506

Chapter8: ManipulatingProteins, DNA,and RNA

Table8-1 SomeCommonlyUsedCellLines

3T3 BHK21 MDCK HeLa PtKl L6 PCl2 5P2

cos 293

cHo DT4O R1 E',t4.1 H 1 ,H 9 5l

BY2

(mouse) fibroblast (Syrian fibroblast hamster) epithelial cell(dog) epithelial cell(human) epithelial cell(ratkangaroo) myoblast(rat) chromaffincell(rat) plasmacell(mouse) kidney(monkey) kidney(human); transformed with adenovirus ovary(Chinese hamster) lymphomacellfor efficienttargetedrecombination (chick) embryonic stemcell(mouse) embryonic stemcell(mouse) embryonic stemcell(human) macrophage-likecell(Drosophi la) undifferentiated meristematic cell(tobacco)

* M a n y o f t h e s elcr n ee l ls w e r e d e r i v e d f r o m t u m o r s A l lo f t h e m a r e c a o a bi lnedoef f r n r e r e p l i c a t l o n i n c u l t u r e a n d e xeparset s so amt e o f t h e s pcehcarraa c t e r i s t c s o f t h e i rocrei gl li' ns o f in culture and yet retain an unrestricted developmental potential. If the cells from the culture dish are put back into an early embryonic environment, they can give rise to all the cell types in the body, including germ cells (Figure g-5). Their descendants in the embryo are able to integrate perfectly into whatever site they come to occupy, adopting the character and behavior that normal cells would show at that site. cells with properties similar to those of mouse ES cells can now be derived from early human embryos, creating a potentially inexhaustible supply of cells that might be used to replace and repair damaged mature human tissue. Experiments in mice suggestthat it may be possible, in the future, to use ES cells to produce specialized cells for therapy-to replace the skeletal muscle fibers that degeneratein victims of muscular dystrophy, the nerve cells that die in patients with Parkinson'sdisease,the insulin-secreting cells that are destroyed in type I

fat cell

c e l l so f i n n e r c e l l m a s s neuron

macrophage e a r l ye m b r y o (blastocyst) s m o o t hm u s c l ec e l l

Figure8-5 Embryonicstem (ES)cells derived from an embryo.Thesecultured cellscan giveriseto all of the celltypes ofthe body.EScellsare harvestedfrom the innercell massof an earlyembryo and can be maintainedindefinitely as stemcells(discussed in Chapter23)in culture.lf they areput backinto an embryo,they will integrateperfectlyand differentiateto suit whatever environmentthey find themselves. The cellscanalsobe kept in cultureasan immortalcell line;they can then be suppliedwith differenthormonesor growth factorsto encouragethem to differentiateinto specificcell types. (Basedon E.Fuchsand J.A.Segr6,Cell 100:143-1 55,2000.With permission from Elsevier.)

s07

ISOLATING CELLSAND GROWING THEMIN CULTURE

diabetics, and the cardiac muscle cells that die during a heart attack. Perhaps one day it may even become possibleto grow entire organs from ES cells by a recapitulation of embryonic development. It is important not to transplant ES cells by themselves into adults, as they can produce tumors called teratomas. There is another major problem associatedwith the use of ES-cell-derived cells for tissue repair. If the transplanted cells differ genetically from the cells of the patient into whom they are grafted, the patient's immune system will reject and destroy those cells.This problem can be avoided, of course, if the cells used for repair are derived from the patient's own body. As discussed in Chapter 23, many adult tissues contain stem cells dedicated to continuous production of just one or a few specialized cell types, and a great deal of stem-cell research aims to manipulate the behavior of these adult stem cells for use in tissue repair. ES cell technology, however, in theory at least, also offers another way around the problem of immune rejection, using a strategy known as "therapeutic cloning," as we explain next.

MayProvidea Wayto SomaticCellNuclearTransplantation Personalized Generate StemCells The term "cloning" has been used in confusing ways as a shorthand term for several quite distinct types of procedures.It is important to understand the distinctions, particularly in the context of public debates about the ethics of stem cell research. As biologists define the term, a clone is simply a set of individuals that are genetically identical because they have descended from a single ancestor.The simplest type of cloning is the cloning of cells. Thus, one can take a single epidermal stem cell from the skin and let it grow and divide in culture to obtain a Iarge clone of genetically identical epidermal cells, which can, for example, be used to help reconstruct the skin of a badly burned patient. This kind of cloning is no more than an extension by artificial means of the processesof cell proliferation and repair that occur in a normal human body. The cloning of entire multicellular animals, called reproductiuecloning, is a very different enterprise, involving a far more radical departure from the ordinary course of nature. Normally, each individual animal has both a mother and a father, and is not genetically identical to either of them. In reproductive cloning, the need for two parents and sexual union is bypassed.For mammals, this difficult feat has been achieved in sheep and some other domestic animals by somatic ceII nuclear transplantation.The procedure begins with an unfertilized egg cell. The nucleus of this haploid cell is sucked out and replaced by a nucleus from a regular diploid somatic cell. The diploid donor cell is typically taken from a tissue of an adult individual. The hybrid cell, consisting of a diploid donor nucleus in a host egg cytoplasm, is allowed to develop for a short while in culture. In a small proportion of cases,this procedure can give rise to an early embryo, which is then put into the uterus of a foster mother (Figure 8-6). If the experimenter is lucky, development continues like that of a normal embryo,

Oct Oo)

Figure8-6 Reproductiveand therapeuticcloning,Cellsfrom adult tissuecan be usedfor reproductive cloningor for generatingpersonalized therapeuticcloning). EScells(so-called Kt

PRODUCTIVE CLONING

calf

/ e m b r y op l a c e di n foster mother

C E L LF U S I O N ORNUCLEAR INJECTION

c e l l sf r o m e a r l ye m b r y o t r a n s f e r r e dt o c u l t u r ed i s h

\ E Sc e l l s unfertilized egg from an adultfemale

r e m o v a lo f e g g c e l ln u c l e u s THERAPEUTIC CLONING

508

Chapter8: ManipulatingProteins, DNA,and RNA

giving rise, eventually, to a whole new animal. An individual produced in this way, by reproductive cloning, should be genetically identical to the adult individual that donated the diploid cell (except for the small amount of genetic information in mitochondria, which is inherited solely from the egg cyoplasm). Therapeutic cloning, which is very different from reproductive cloning, employs the technique of somatic cell nuclear transplantation to produce personalized ES cells (seeFigure 8-6). In this case,the very early embryo generated by nuclear transplantation, consisting of about 200 cells, is not transferred to the uterus of a foster mother. Instead, it is used as a source from which ES cells are derived in culture, with the aim of generating various cell tlpes that can be used for tissue repair. Cells obtained by this route are genetically nearly identical to the donor of the original nucleus, so they can be grafted back into the donor, without fear of immunological rejection. Somatic cell nuclear transfer has an additional potential benefit-for studying inherited human diseases.EScells that have received a somatic nucleus from an individual with an inherited disorder can be used to directly study the way in which the diseasedevelops as the ES cells are induced to differentiate into distinct cell types. "Disease-specific"ES cells and their differentiated progeny can also be used to study the progression of such diseasesand to test and develop new drugs to treat the disorders. These strategiesare still in their infancy, and some countries outlaw certain aspects of the research. It remains to be seen whether human ES cells can be produced by nuclear transfer and whether human ES cells will fulfill the great hopes that medical scientistshave for them.

HybridomaCellLinesAre Factories ThatProduceMonoclonal Antibodies As we see throughout this book, antibodies are particularly useful tools for cell biology. Their great specificity allows precise visualization of selected proteins among the many thousands that each cell typically produces. Antibodies are often produced by inoculating animals with the protein of interest and subsequently isolating the antibodies specific to that protein from the serum of the animal. However, only limited quantities of antibodies can be obtained from a single inoculated animal, and the antibodies produced will be a heterogeneous mixture of antibodies that recognize a variety of different antigenic sites on a macromolecule that differs from animal to animal. Moreover, antibodies specific for the antigen will constitute only a fraction of the antibodies found in the serum. An alternative technology, which allows the production of an infinite quantity of identical antibodies and greatly increasesthe specificity and convenience of antibody-based methods, is the production of monoclonal antibodies by hybridoma cell lines. This technology, developed in 1975, has revolutionized the production of antibodies for use as tools in cell biology, as well as for the diagnosis and treatment of certain diseases,including rheumatoid arthritis and cancer.The procedure requires hybrid cell technology (Figure B-7), and it involves propagiting a clone of cells from a single antibody-secreting B ly,rnphocyteto obtain a homogeneous preparation of antibodies in large quantities. B lymphocytes normally have a limited life-span in culture, but individual antibody-producing B lymphocytes from an immunized mouse or rat, when fused with cells derived fiom a transformed B lymphocyte cell line, can give rise to hybrids that have both the ability to make a particular anribody and the ability to multiply indefinitely in culture. These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody (Figure 8-8). Each type of monoclonal antibody recognizesa single type of antigenic site-for example, a particular cluster of five or six amino acid side chains on the surface of a protein. Their uniform specificity makes monoclonal antibodies much more useful than conventional antisera for most purposes. An important advantage of the hybridoma technique is that monoclonal antibodies can be made against molecules that constitute only a minor component of a complex mixture. In an ordinary antiserum made against such a mix-

'os op slla) eulopuq^q aq] ,(;uo'unno slr uo aleJaJtlold pue anrnlns ue) uorsnJletltutaql loJ pasnad{r lla> raqlrsuasne)ag'asnoul paztunuuilaql r.r.ror] paurplqoslla)lptulouaql u! l)elu! s! 'roLunt g aqr ulolJpa^uapautl lr lnq lla) lla) luetnu aql ur antt)alapsr{emqled srql'spt)e)tal)nuJaqt aztsaqlu^s o1{emqled ssed,{qe asn alolalaqt }snur slla)aql 'apeuraresaptloal)nuq)rqM^q s,{emqted )llaqlu^solqleurrouaql sl)olq lollqlLlulue sutetuo) teql (uuatdourure) oals uorsnJlla) aql ralle pasnLUntpauJ qu,ror6anr]>e;as aLll ,;x uaotlue,, se paleu6rsap srlseralur;ouabrlue aql'araH'ua611ue reln>|1lede 1su1e6e sa!poqrlueleuol)ououl olal)as leql seuoplrq^q1o uorleredal6g-g arn6r1

a

t

s

I

lpoqrluey-r1ue l o a ) J n o s6 u r n u r l u o :e eprnordsauo;: anrlrsod sarpoqrlue x - r l u er o l p e l s e ls l u e l e u r a d n s; e n p r n r p u r p u e ' { 1 d r 1 1 nou} p a m o l l es l l a ) t

ul

i'

t

l l a Mr a o l l a ) l - l e p a J n l l n )l l a M e n r l r s o du r o r ; s l l e l p u e ' ^ p o q r l u e x - l l u e r o ] p a l s a ll u e l e u r a o n s

dpoqrgueypala u r n r p e uer ^ r D e l a sa q ] u r e t e l a J r l o r dp u e e n r r u n s q l e re L u o p r : q i {rq{ ; u o

p e r n l l n )s 1 1 ae: u r o p u q { q6 u 1 1 ; n s a . r I NOrSnr

( u n r p a u ra ^ r l ) a l e su r a r p l n q u r n r p a u r l e u r o u u ! ^ l a l r u r + a p uMr o l 6 s l l a ) )

( a i n l l n ) u r s { e pm a l e g raue arp) say(:ot1dur^1

:1.

lo

{Poqrlue Y-;1ue 6ur1eu,r11at

o.9o lo-l

o

y ue6rlue ql;nn pazlunuur esnour

say{:oqdu{; I }o roun} e u l o r +p a ^ u e pe u r l l l a l ] u e l n u r

'europuqAq

'uorlf,unJpue aJnlcnJtsslr Lpnls ol uraloJd aqt lgund ol puE 'lueruenoru slr ^\olloJ ol 'sanssrlpuB slleJ ur uraloJd aq] azrlPJolol pasn aq UPJ]l 'epEtu uaaq seq,rtpoqpue ue ef,uo 'eldlues pcrSolorq e ur ureloJd LuE (aJoJaJaql'aldrcurrd uI 'sarlrluenb ]surE8uepeu aq ueJ,.(poquuP Ieuol3ouoru E palrlulun uI dpoqnue tBql aonpord ot se os dlalrugapur euoprrq,{q patJeles eq} ate8edoJd o] pue ,r(poqquelpuoloouoru Jo adrtt parrsap aqt seonpord 1eq1auo ]calas ol arnlxru a3re1aql uo4 sauolc pruoprJq^q pnpr^rpur uaerf,s o1 alqrssod seuoJaq lI (sEruopuq,{qolur aperu ale runJesr}ueslql Jo sluauodruof, snorJe^ eql acnpord teql seldJoqdru,{1g aq} Jr tng 'lnJasneq o} iletus oo} aq plnolv\tuau ,aJnl -oduroJ Jounu aq] ezruSooeJ]Eql salnJalour ^poqpup Jo uorpodord eql

prrq{qaq}'aurllla) roLun} e palle)sr 11ar e uor] sPMslla)lualeo oql Jo auoJl 'saurl puq{q lla) ;elrouLuro1aslranr6ue: s;;atpuq{q q)ns'snal)nua6.re; e;6urse ur raqla6o]]q6nolq aq ol sauosotj,lolq) aql lle 6ur^^olle'palqLUossestp uaoqa^eqsaoola^ua leal)nualptedos oMt aql qrqM ur ;;a: prrq{qe satnpold pue srsoll.rto1spaarold uo{le>oralaq e ^llenlua^l'asnJol [!aq] sa)nputteql piuseldaql {eane ur slle)}o sauelqulaLu clNo'l) NtHrluv H)rH^^'s'r'Ef srolleq)rqMJo q)ea 'lo)^16auallqla,(;od otuS H tt^o)38 qlrMro sasnJtA pale^tl)eut qltM utelta) tslHl'ltvusilroud paleal]st s;;attrouorsuedsns e ,(;;etrd,(1 cNV ln Inuns 'rel)nualeleoasoMl r]ltM of sNoAuv)ou3r_rH lla) paurqulo) A-rNOSAO]tV e 'uofuDoalarl p ulloJo] laqloue nntclN lnrDlrrs qtlM lla) euo asnjot alqtssodst ll .slla) p;rq{q;o uollrnpord aql l-8 arn6rJ s;;a: puqi{q }o seuol) ealq}

60s

lla)Jounl lla) leullou asnour peletluela+]tp

]UV H)IHM ,SNOAUV)OU]I]H lo NoU-VWUol oNV NOtSnl'l'llf

clccv 1Nr9v 9Ntsnlv HltM Cl9nJU-LNl)Sld^l_ 'l'll) oMr_ro NotsNsdsns

lunlrnf NtwlHI9Ntl ouDcNVs-nl) 9Ntlvtosl

'J.t ,rtq oLlluteluteulol ulals^s sllrJlPql uorlnlos lIEs elnlp e Jo dol uo puBq ulql e uI alBuaSoluoq aql Sulraz(e1 le aldLues uorlerabulalaql 6utMollepuplolol aLl] pa^erqcEeq upJ uorlEJedasJo eeJSapJauu v 'aZIsuI ,{l}PeJ3ragrp reqr slueuoduoJ 'suorleuol]oerJ 6urluanaid'uo!])UJ sa)npal Jo 6urleaLl uouu8ryIJtue3 eq] sl uI dals dyuo saleredes lsJg tsoru lr ]nq unn)en aql'luaurpas o1a;duteseq1ur 'seull IeJa^as sallrUedasnp)q)tqM'satroy1e6nlttua> eJnpa3oJduoDe8nJrJlueraql Suueedar pue lafied eq] Sutpuadsnsar^q pa^oueJ snourouasateraua6 rolor aql Jo uotlelol '(0I-8 aq ueJ slueurueluoJ aq] Jo .{ueu 1nq 'erndtut aJEsuol}Je4 eseqt Jo IIV prdeg'.ro.1o.r lplaLue ut saloq;e>rrpur;{r arn31g; palJalloo aq upJ seuosoqrJ eq] uaq] pue selJISaApasoll ileus aql ISJIJ Jo 6uu p otur palresurarplpql saqnlul poureluo)sra;duresaql'a6n1.r1ue>er1;n 'uorle8n;rrlueJ Jo sporJedJa8uol qly\^ pue speadsJaq8q uela le pue lpallsodap a^lleredardaql 6-g arn6rJ sr Errpuoqcotlu Jo ]e[ed e 'paads raq8rq fpq8us tP laqn] a8n;u1uac eq] Jo 'paads Iuouoq aq] ]e lelled E uroJ ol lualulpas Ialcnu se qcns slueuodluoJ a3re1 ro10ur ir,ro1Llanrlelar 1y ,{.1prder}solu aq} a^ou pu€ acro; p8qrrlual lseBrEI aql alua uorlera6u;ar -rradxa slrun 1sa3re1aql 'lpJaue8ut :l1tsuap pue azISzlq sluauodruoo IIaJ selerc -das ]uaruteer1srqJ'16-9 arnS;g) spaadsq8rq re sfiec ua{orqJo slrer]xa salelor qclqm 'aBntuluacatlln anuDJadaJdaq1se ulrroDl lueunrlsul ue Jo s9761,{Fea aqf ur luarudola^ap lerJra{uuoJ eql rage.{po alqrssod eluef,eq suot]euol}f,erJIIaJ qJnS 'pateredasaq ueql lsntu aleuaSouroq aql Jo slueuodruoc 1uara;;lp aql 'sauradord leorrueqJolq pu€tro raqlJo lsoru urclaJ -saruosorcru pallpJ 'runlnJllal ctuseldopua eql {uoq peAIJep salJISaAaq} Surpnlcur-sluauodruoJ snorJel eql '(a[euEBJoqJea JoJJoile pue IEIJ]z{q)uas -oqc rtln;areJ uaaq sEq lunlpalx uollezluaSoluoq eql ]eq] papl^oJd ,!1suap pue 'a8reqc 'azrsalrlcurlsp e qlrm qcee 'sellaueBropasolcua-aueJqruelu ,&arrene Jo surPluoJ Wrql (1JDJryato atauaSoruoq e pele) [-rrn1s4c1qt e o] pa3npeJ ,{qaraqt saruosrxoJedpue 'setuososdl'sn}ereddu sr sllal Jo uorsuadsnsaql ']celut ,{1a8re1 r31og aql 'elrpuoqf,olltu 'IelJnu se qJns sallaue8ro eluel saJnpacorduoqdnrstp 'salJrse^pJsolc aql 'JJ^eMoq'rno pJrJlEJ,{1;n3a.rer llztus LuroJo1 leasa.r,{1a1e Jl -rparurur leql sluaru8eq olul (runpcllar cnuseldopua pue eueJquaru eurseld . r a q u r e qpl a r o u r e l e u e l e u r6 u r l u a u r p a s aql Surpnlcur) IIaJ aql Jo seuErquau eql ;o Lueru leeJq saJnpacord asaql 'uorlerqn cluoseJlln 'Japualq 'aclJuo punor8 ro e ur dn IIEIus B qBnorqt patJoJ snoIJEAur dn ua4orq eq ueJ Jo {Joqs cr}oruso o1 palcafqns eq uEJ r{aqt :s.{ervr silal 'llao aql aprsu ruo{ palJeJ}xeeq }sJIJlsnru lI 'uta}old e d;und o} JepJouI

suolt)erl luauodulo) Jlaql olul palerede5ag ue) slla) ,i1tctycads Sutsearcur (lEIJalEureI{} Jo ,Qrxalduroc aqt ;o sdals uorlecr;rrnd ,{q paatolog uaq} sI pue aJnpal o] uorleuorlJe4 rBlnllaJqns qlIM sgels ^,(1ensnarnpacord uotlecr;trnd e (anssl] IErnlEuE Jo IIar paraaut8uaue sr uralord eIDJo eoJnoseq] Jaqlaqr\\'JeISea qcnu uorleJgund s1r3uqeu,{qaraqt 'uleloJd uan€ e;o sappuenb a8rel Surcnp -ord olur s11ac,,3uqcrr1,,.{q 1se1 slql dJIrduIS ,(lsnoruroua uet [3o7out1Ja1VNO 'oJ]lttu? uoI]JunJ utalord ,tpnls ruDutqluoJal taldeqc slql q ralel aes fleqs a,\/\sV ol JepJour euroJre^o aq ]snlu ]nq 'auo alqepluroJ B sI IIac e ut luasard suralord Je{DoJo spuesnoql eq} ruo4 ura}ord yo ad,rfia13utse 3u4e1ost;o a8ualeqc aq1

DNl^llund sNEroud 'sasnastp pua asou8atp iaau oi sDIIamsn'suratotd11ac f)uolrouout {tnd puo papp oi pasnaro rlJII4m'sarpoquua u.uoltun lo salllluanb palwqun acnpotd o1 pato1dtua,{Iapru arv sIIaJ autopuqtg 'astutotd prtpau ryaB p7ot1atopnqt [at11 'tpoq aqt lo sad[t lac ryatatltp aqt U?) arnl1nca ut ,fialru{apul atatalqotd otul arupuatatllpol r{l1tqaatq Suturutatalll1m'14sry sa [\anuyfapmpaulqlulqru aq uoJ uotlalndtuour uDJ sIIaJwals ctuorQqwfl'sauq71ac 'suols ulauaB Jo suouDlnut snoauaruodsqBnotql pazlf4Joruwr uaaq anDulLottLsUn -trtrppat lo nqunu a1ru1[anltn Sutputtpdols s17ac lowturt fuout qBnoqlly'sa1nca1ow puZrc alaudotdda pua srya14nuSutulaluor unlpaur arnllnJ alqvllns o l'lllm papnotd rua1dpua lnuttua [uDW'sarn] ata [.at1tltqstparnt]nJ a ut alataJ4otdpua antntnss11at -1nc17ac to luawLlsqqotsaaqt nl n ststlaua loJtwaqrolq rct pasn pua pa{und aq uor sadtl llac lDnprnrput tlnqm utott's77acryauodruor naqi olut palDnosstpaq uoJ sanssl.

freuurn5 vNu pup'vNo

'sulelor66ul1e;ndrue61 :gtaldeq)

0ts

511

PURIFYING PROTEINS Figure8-10Cellfractionation Repeated centrifugation by centrifugation, of cellsinto at progressively higherspeeds willfractionate homogenates the theircomponents. Ingeneral, thesubcellular component, thesmaller for greater values it.Typical isthecentrifugal forcerequired to sediment thevarious centrifugation stepsreferred to in thefigureare: Iowspeed: 1000timesgravityfor 10minutes mediumspeed:20,000 timesgravityfor 20 minutes highspeed:80,000 timesgravityfor t hour veryhighspeed: 150,000 timesgravityfor 3 hours

a centrifuge tube. \Mhen centrifuged, the various components in the mixture move as a series of distinct bands through the salt solution, each at a different rate, in a process called uelocity sedimentation (Figue 8-f f A) . For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles)finds itself on top of a lighter one (the salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device.The resulting density gradient-with the dense end at the bottom of the tube-keeps each region of the salt solution denser than any solution above it, and it thereby prevents convective mixing from distorting the separation. lVhen sedimented through such dilute sucrose gradients, different cell components separateinto distinct bands that can be collected individually. The relative rate at which each component sediments depends primarily on its size and shape-normally being described in terms of its sedimentation coefficient, or S value. Present-day ultracentrifuges rotate at speeds of up to 80,000 rpm and produce forces as high as 500,000 times gravity. These enormous forces drive even small macromolecules, such as IRNA molecules and simple enzymes, to sediment at an appreciable rate and allow them to be separated from one another by size. The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8-l lA, but it eventually reaches a position where the density of the solution is equal to its own density. At this point the component floats and can move no farther. A series of distinct bands is thereby produced in the centrifuge tube, with the bands closestto the bottom of the tube containing the components of highest buoyant density (Figure 8-11B). This method, called equilibrium sedimentation, is so sensitive that it can separate macromolecules that have incorporated hear,yisotopes, such as I3C or rsN, from the same macromolecules that contain the lighter, common isotopes (lzC or 14N).In fact, the cesium-chloride method was developed in 1957to separatethe labeled from the unlabeled DNA produced after exposure of a growing population of bacteria to nucleotide precursors containing l5N; this classic experiment provided direct evidence for the semiconservative replication of DNA (see Figure 5-5).

Systemsto StudyCellFunctions CellExtractsProvideAccessible Studies of organelles and other large subcellular components isolated in the ultracentrifuge have contributed enormously to our understanding of the functions of different cell components. Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy into forms that the cell can use. Similarly, resealedvesicles formed from fragments of rough and smooth endoplasmic reticulum (microsomes) have been separatedfrom each other and analyzed as functional models of these compartments of the intact cell. Similarly, highly concentrated cell extracts, especially extracts of Xenopus laeuis (African clawed frog) oocyes, have played a critical role in the study of

cell homogenate

L O W - S P E ECDE N T R I F U G A T I O N

+

-

pellet contains wholecells nuclei cytoskeletons

O SU SUPERNATAN T B J E C T ETD M E D I U M - S P E ECDE N T R I F U G A T I O N

p e l l e tc o n t a i n s mitochondria ly5osomeS peroxrsomes TD O SU SUPERNATAN T BJECTE H I G H - 5 P E ECDE N T R I F U G A T I O N

+

p e l l e tc o n t a i n s mrcroS0meS smallvesicles

V E R Y - H I G H - S P ECEEDN T R I F U G A T I O N

-

pellet contains ribosomes vlruSes large macromolecules

512

Chapter8: ManipulatingProteins,DNA,and RNA

(A)

(B)

VELOCITY SEDIMENTATION

EQUILIBRIUM SEDIMENTATION

sample steep sucrose gradient ( e . g, 2 O - 7 O o h )

I

CENTRIFUGATION

I

s l o w - s e idm e n t i n g component f a s t - s e idm e n t in g component

I

FRACTIONATION

II + low-buoyantdensity component high-buoyantdensity component

such complex and highly organized processesas the cell-division cycle, the separation of chromosomes on the mitotic spindle, and the vesicuiar-transport steps involved in the movement of proteins from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane. cell extracts also provide, in principle, the starting material for the complete separation of all of the individual macromolecular components of the cell. we now consider how this separation is achieved, focusing on proteins.

ProteinsCanBeSeparatedby Chromatography Proteins are most often fractionated by column chromatography, in which a mixture of proteins in solution is passed through a column containing a porous solid matrix. The different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separatelyas they flow out of the bottom of the column (Figure 8-12). Depending on the choice of matrix, proteins can be separated according to their charge (ion-exchangechromatography), their hydrophobicity (hydrophobic chromatography), their size (gel-fittration chromatography), or their ability to bind to particular small molecules or to other macromolecwles (affinity chromatography). Many tlpes of matrices are commercially available (Figure g-13). Ionexchange columns are packed with small beads that carry either a positive or a negative charge, so that proteins are fractionated according to the arrangement of charges on their surface. Hydrophobic columns are packed with beads from which hydrophobic side chains protrude, selectively retarding proteins with

Figure8-1 1 Comparisonof velocity sedimentationand equilibrium (A)In velocity sedimentation. sedimentation, subcellular components sedimentat differentspeedsaccordingto theirsizeand shapewhen layeredovera dilutesolutioncontainingsucrose. To stabilize the sedimentingbandsagainst convectivemixingcausedby small differencesin temperatureor solute concentration, the tube contatnsa continuousshallowgradientof sucrose, which increases in concentration toward the bottom of the tube (typicallyfrom 5olo to 20olo sucrose).After centrifugation,the differentcomponentscan be collected individually, mostsimplyby puncturing the plasticcentrifugetube and collecting dropsfrom the bottom,as illustrated here.(B)In equilibriumsedimentation, subcellular componentsmoveup or down when centrifugedin a gradient untilthey reacha positionwheretheir densitymatchestheirsurroundings. Althougha sucrosegradientis shown here,densergradients, whichare especially usefulfor proteinand nucleic acid separation,can be formed from cesiumchloride. Thefinalbands,at equilibrium, can be collectedas in (A).

513

PROTEINS PURIFYING

exposed hydrophobic regions. Gel-filtration columns, which separate proteins according to their size, are packed with tiny porous beads: molecules that are small enough to enter the pores linger inside successivebeads as they pass down the column, while larger molecules remain in the solution flowing between the beads and therefore move more rapidly, emerging from the column first. Besides providing a means of separating molecules, gel-filtration chromatography is a convenient way to determine their size. Inhomogeneities in the matrices (such as cellulose),which cause an uneven flow of solvent through the column, limit the resolution of conventional column chromatography. Special chromatography resins (usually silica-based) composed of tiny spheres (3-10 pm in diameter) can be packed with a special apparatus to form a uniform column bed. Such high-performance liquid chromatography (HPLC) columns attain a high degree of resolution. In HPLC, the solutes equilibrate very rapidly with the interior of the tiny spheres, and so solutes with different affinities for the matrix are efficiently separated from one another even at very fast flow rates. HPLC is therefore the method of choice for separating many proteins and small molecules.

BindingSiteson ExploitsSpecific AffinityChromatography Proteins If one starts with a complex mixture of proteins, the types of column chromatography just discussed do not produce very highly purified fractions: a single passagethrough the column generally increases the proportion of a given protein in the mixture no more than twenty{old. Becausemost individual proteins represent less than 1/ 1000of the total cell protein, it is usually necessaryto use several different types of columns in succession to attain sufficient purity (Figure 8-f 4). A more efficient procedure, known as affinity chromatography, takes advantageof the biologically important binding interactions that occur on protein surfaces.If a substrate molecule is covalently coupled to an inert matrix such as a polysaccharide bead, the enzyme that operates on that substrate will often be specifically retained by the matrix and can then be eluted (washed out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically

COLUMN CHROMATOGRAPHY s o l v e n tc o n t i n u o u s l y a p p l i e dt o t h e t o p o f s a m p l e c o l u m nf r o m a l a r g e

0

0

f r a c t i o n a t e dm o l e c u l e s e l u t e da n d c o l l e c t e d

Figure8-1 2 The separationof moleculesby columnchromatography. The sample,a solutioncontaininga is applied mixtureof differentmolecules, glassor plastic to the top of a cylindrical columnfilledwith a permeablesolid A largeamount matrix,suchascellulose. of solventis then PumPedslowlY throughthe columnand collectedin tubesas it emergesfrom the separate variouscomponentsof bottom.Because the sampletravelat differentrates throughthe column,they are fractionatedinto differenttubes'

514

Chapter8: ManipulatingProteins, DNA,and RNA solvent f low

s o l v e n tf l o w

s o l v e n tf l o w

;i

o.o '

a a a a

o. oO

(A)

I O N - E X C H A N G EC H R O M A T O G R A P H Y

'

p o r o u sb e a d

.lD. .' o .tO

a

.

a a .

o. molecule

o e\ a ,

aa

a

'

o

.-

retarded S m a l lm o l e c u l e

s

\f)-.

5-

t!)

a,

I

,nretardeo :l a' r"g"e'm- 'o"l e c u l e

( B ) G E L - F I L T R A T IC OH NR O M A T O G R A P H Y

\

ir.

b e a dw i t h c o v al e n t l y attached substrate bound enzyme motecute other proteins

/t (C) AFFINITY CHROMATOGRAPHY

Figure8-13 Threetypesof matricesusedfor chromatography. (A)In ion-exchange chromatography, the insolublematrix carriesionicchargesthat retardthe movementof molecules of oppositecharge.Matricesusedfor separatingproteins includediethylaminoethylcellulose (DEAE-cellulose), which is positivelycharged,and carboxymethylcellulose iCM-cellulose) and phosphocellulose, whichare negatively charged.Analogousmatricesbasedon agaroseor other polymersareatso frequentlyused.Thestrengthof the association betweenthe dissolvedmolecules and the ion-exchange matrixdependson both the ionicstrengthand the pH of the solutionthat is passingdown the column,which may thereforebe varied (asin Figure8-14)to achievean effectiveseparation. systematically (B)In gel-filtration chromatography, the matrixis inert but porous.Molecules that aresmallenoughto penetrateinto the matrixaretherebydelayedand travelmoreslowly throughthe columnthan largermolecules that cannotpenetrate. polysaccharide (dextran,agarose, Beadsofcross-linked or acrylamide) areavailablecommercially in a wide rangeof poresizes,makingthem suitablefor the fractionation of

ff:T:i::?iffi::::ffii.?Jil: *.;l':flff:il:Hff ffi:T;J:["Jil:il:;],',i.fi::;i:1fi:il't.,ecu an enzyme substrate, thatwillbinda specific protein. Enzyme molecules thatbindto immobilized substrates on such

columns canbeelutedwitha concentrated solution of thefreeformof thesubstrate molecule, whilemolecules thatbindto immobilized antibodies canbeelutedby dissociating theantibody-antigen complex withconcentrated saltsolutions or solutions of highor lowpH.Highdegrees of purification canbeachieved in a singlepassthroughanaffinitycolumn.

designed sequence can be immobilized in this way and used to purit/ DNAbinding proteins that normally recognize this sequence of nucleotides in chromosomes (seeFigure 7-28). Alternatively, specific antibodies can be coupled to a matrix to purify protein molecules recognized by the antibodies. Because of the great specificity of all such affiniry columns, 1000- to 10,000-fold purifications can sometimes be achieved in a single pass.

Genetically-Engineered TagsProvidean EasyWayto purify Proteins using the recombinant DNA methods discussedin subsequent sections,any gene can be modified to produce its protein with a special recognition tag attached to it, so as to make subsequent purification of the protein by affinity chromatography simple and rapid. often the recognition tag is itsell an antigenic determinant, or epitope, which can be recognized by a highly specific antibody. The antibody, can then be used both to localize the protein in cells and to purify it (Figure s-r5). other types of tags are specifically designed for protein purification. For example, the amino acid histidine binds to certain metal ions, including nickel and copper. If genetic engineering techniques are used to attach a short string of histidines to one end of a proiein, the slightly modified protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used t6 purify the modified protein from a complex molecular mixture. In other cases,an entire protein is used as the recognition tag.when cells are engineered to synthesize the small enzyme glutathione S-transferase (GST) attached to a protein of interest, the resulting fusion protein can be purified from the other contents of the cell with an affinity column containing glutathione, a

515

PURIFYING PROTEINS (A) ION-EXCHANG CEHR O M A T O G R A P H Y

I

I =

E

E 6

o a o

-L-----i' fraction number +

p o o l t h e s ef r a c t i o n sa n d a p p l yt h e m t o t h e n e x t c o l u m nb e l o w

( B ) G E L - F I L T R A T IC OHNR O M A T O G R A P H Y

E f

E o o

E

E

(D) 1 f )

fractionnumber+

I I p o o l t h e s ef r a c t i o n sa n d a p p l yt h e m t o t h e n e x t c o l u m nb e l o w

220-

(C) AFFINITC Y HROMATOGRAPHY

t

I

96-

w

E l

o

65-

E E o

f r a c t i o nn u m b e r p o o l t h e s ef r a c t i o n sw , h i c h n o w c o n t a i nt h e h i g h l yp u r i f i e dp r o t e i n

substrate molecule that binds specifically and tightly to GST.If the purification is carried out under conditions that do not disrupt protein-protein interactions, the fusion protein can be isolated in association with the proteins it interacts with inside the cell (Figure 8-f O). As a further refinement of purification methods using recognition tags, an amino acid sequence that forms a cleavagesite for a highly specific proteolltic enzyme can be engineered between the protein of choice and the recognition tag. Becausethe amino acid sequencesat the cleavagesite are very rarely found by chance in proteins, the tag can later be cleaved off without destroying the purified protein. This tlpe of specific cleavageis used in an especially powerful purification methodology known as tandem ffinity purification tagging (tap-tagging). Here, one end of a protein is engineered to contain two recognition tags that are separated by a protease cleavage site. The tag on the very end of the construct is chosen to bind irreversibly to an affinity column, allowing the column to be washed extensively to remove all contaminating proteins. Protease cleavage then releasesthe protein, which is then further purified using the second tag.

Figure8-14 Proteinpurificationby chromatography.Typicalresults obtainedwhen threedifferent stepsare usedin chromatographic to purifya protein.In this succession example,a homogenateof cellswasfirst by allowingit to percolate fractionated resinpacked throughan ion-exchange into a column(A).The columnwas washedto removeall unbound and the bound proteins contaminants, werethen elutedby passinga solution c o n t a i n i n ag g r a d u a l liyn c r e a s i n g of saltonto the top of the concentration with the lowestaffinity column.Proteins resinpassed for the ion-exchange directlythroughthe columnand were collectedin the earliestfractionseluted from the bottom of the column.The remainingproteinswereelutedin sequenceaccordingto their affinityfor the resin-those proteinsbindingmost tightlyto the resinrequiringthe highest of saltto removethem. concentration The oroteinof interestwaselutedin severalfractionsand was detectedby its Thefractionswith enzymaticactivity. activitywerepooledand then appliedto column(B).The a second,gel-filtration elutionpositionof the still-impure proteinwasagaindeterminedby its enzymaticactivity,and the active fractionswerepooledand purifiedto homogeneityon an affinitycolumn(C) that containedan immobilizedsubstrate of the enzyme.(D)Affinity purificationof cyclin-bindingproteinsfrom S.cerevisiae, asanalyzedby SDSpolyacrylamide-gel which is describedbelow electrophoresis, in FigureB-18.Lane1 is a total cell extract;lane2 showsthe proteinseluted from an affinitycolumncontainingcyclin 82;lane3 showsone majorProtein elutedfrom a cyclin83 affinitycolumn. Proteinsin lanes2 and 3 wereeluted from the affinitycolumnswith salt,and blue. the gel was stainedwith Coomassie Thescaleat the left showsthe molecular weightsof markerproteins,in (D,from D. Kellogget al., kilodaltons. With J. CellBiol.130:675-685,1995. fromThe Rockefeller oermisison Press.) University

x utsloJd q l r Mp e r a l u r l e q l s u r a l o l dq l l M l e q l a 6 o l u r a l o . r ou o r s n +s e l n l a u o t l n l o sa u o r q l e l n l 6

X u r a l o r do l p u r q s u r a l o r d6 u r p e . r a l u+r 1 ' p a p p Ps rp e r l x a lla) uaqM

I |

3

"

'(S L-8 arn6ll aas)6el adolrdero uralordlleus luarua^uo)raqlouero 159 lsutebe serpoqrlue ureluo)ol appu.r aq osleue) suurnlo)Ilru;gy'(17-g atnbtl asaql aas)Illauro.rlrodssseu Iq paururalapeq uer suralotdleuor]tppe qtlm patnlaaq uaLllue) 'tl ol Illq6rl punoq aLll'auorLllelnl6 ,o sarlrluapr ereleql lla) aql u! suralo.rd leqto qllM 6uo;e'utalotduorsnleql'Ienne qllM paleo) paqseMoJespeaqaql o] punoqlou sutalord'auorqletn;6 speaq6urureluo)uurnlol{1rur;;eue uo palnlderaq ue)'sanbtuqla}VNO 'suralo.rd uorsn1 uo;sn1 lueurquro)arqllM slla)ur pa>npord ISg'uga1o.td pa66e1-159e 6ulsn {q saxe;duor u;a1ord,o uolle)Ulrnd 91-g aln6r3 uunloJ [q pa{rmd aq UDJ stJor]xa alqnps u! sunlold aqJ'paqonu! Vuauod nac 'sassaiord -luoc raqrc puo sunrcid ar4| 77acxa\d nD to uoqoJ|Jtrnd ailsua$a SwJInbaJ -woc to sllvpp JalnJapra aqt Sulununiap nt papaau aJD swa$[s aa{-pac pa{und [trtBtu 'pado1anap aq o1 sualsts aa$-11ac puoucunt Sulmon?1 'siuaiuoJ nat4t Suuz) pua Luaril SwtdnJup [q [T1acnuaLlJoIqpazt1aun aq uoc syacto suotla1ndo4

.:ltiil-uolpuJt it:it:i

s p e a qp a l p o ) - a u o r q 1 e l n 1f 6 o l p u n o q u r a l o t du o l s n ; | y uralotd

l(.reuun5

15

( I S 9 ) a s e J a l s u e l l -a5u o r q l e l n ; 6 pue y uralo.rduaeMlaq uotsnl eleul ol p e s na r e s a n b r u q : ay1p q l u e u ! q u o ) e r

'sepqnlorclru Suole uodsueJl 'sllal ur JnJJo luq] sassacoJdJeqlo ,{upu pue elJUJed pue (uorlJeJluoJ elcsnu 'uoDPISuEJ]uleloJd '3utct1dsVNU 'uollducs -uEJl VNCI pue uorleJrldar yl11q Jo sllelap JelnJaloru aq] reqdrcap ol 'aldruexa JoJ 'pasn uaaq e^eq daq; 'srualsdsaa;;-1ac qons uI salpnls l(q paranocsrpuaaq sEq IIac aqt Jo .{Solorq relnf,alolu aql lnoqE depol ,vrou1a^ lEql!\ Jo pap lear8 e 'auop aq ol sureruarqcnru qBnoq][V 'uollJe luslueqceu astcardrgaq]lno {Jo.tr Jo o1 pannbar se (suorleJluaJuocJreql IoJluoJ puE ssacord aq] JoJpepeeu sluauod -r.uo3aql Jo lle aurJapauo uEJz(ernsrql uI [Iuo 'ua]s,{s ee4-llal palllJnd B ul sseJ -o.rdpcrSolorq Lrana Jo uollnlllsuooeJ aq] st slst8oyotqIIeJ JoJpo8 roferu y 'ssacord IIeJe^oeql (alqellele ur aloJ ]Jexe slr eurJapo1 flaleredas plaqql]M Jo pappe aq plnoJ qJEa aJeMslueuodruoc amd pnptntput acu6 Lraurqceru cqaqlur(s-uralord eq] alnlps -uoc pue 'svNul 'saurosoqlJeql uJnl ut pacnpord raqlaSo] sarurtzue snoIJPA leq] 'sulaloJd acnpord ol salnJaloru 'da1sLq dals 'aleuaSouroq slql Jo uoIlEuoIlJEJC vNu elPlsueJ] plnoJ leql aleuaSouoq ilac E qll1l^ ueSaq stsaqlu.,ts utalord 'aldruexa JoC 'lseJelul Jo sseJ Jo srusrueqJau aql raqdrcap ol sluelulJadxa aql -ord pcrSolorq e azdplec 01 papaau aJBleql salncalouoJJeru pnpIAIpuI eql Jo qcea Surrlrrnd;o rurc eqt qlllrt paleuoqce4 are saleuaSouroqIIef, 'alqtssod stql e{pu oI'srualsfs aa{-IIeJ pegpnd Sulsn dq 1ac Suvrr e uI JnJOoleql suollJear aprs xaldruoJ eql Jo IIB ruo4 ae{ sassaJordpctSolorq ,(pn1so1 luegodrut s111

suo!l)unlreln)elowJo uoll)assloas!)ardeql roJpel|nbaualv suals^s salJ-lla)polJund suraloJopaler)osse , { u ep u e u r a l o r d n:66o1n

prder uorle:r,rr.rnd

6el uralo.rd o 1 s a r p o q r l u e6 u r s n uo!]ezrle)olounurul

,,*k.pa66e1

'pagrrnd dprder aq o] ulalord '8e}-de} e sapocua lEqf lseart .{.ue,{,\olleot pet3nr}suoJ ueeq seq VNCI ol pesnJ aua8 e qlu qJee 'suleJlslsead 6969l.laleurrxorddeJo les e 'aldruexaro; luara;yp 'uoJJeel]lll ^la^l]Elar qll^ uollecgund 'snql'd8o1orq flac ur,tyanrsualxapesn sI ]l uralord ;o aar8ap q8rq ,(lercadsa u€ sepllord d8elerls dals-orrnlslql esneJeg

-adolrda

erelrd'ard seuo!]nros r:"J'"jil::;il?:JtX;i?""1H: ro]nouraqr

pa66eraql 6ututetuor aq} luralo.td aql lurl-ssor){;;errpadssatpoqtlue uorlnlose ol peppeare6el adotldaaql lsu;ebepal)orp satpoqrluP 'uorlelrdr>ardounurur (1rur;eto uotle}tdttardounuJtltl ul ',{qde.r6o1euo.rqr ,(ql! ,!lrnd olro slla)ut ulalo.tdaqtlo uotlP)olaql autulalap ol pasnaq ue) 'palaqpl{;qer;ns,{poqrlueaq1'{poqtluealqelre^e {;;eureur,uo> a}eudolddeue,(q palebtelaq ue) tl 'adoldaJo'}ueululalap rrue6rlueueJlaslrsr6et aqlJl'tsalalutJo utalotde ot pappeaq uel 6e1 'su1a1ord >tlauabplepuels6uts1'1 aprldeduoqs e'sanbruqral6ulraaul6ua ;o uolterglrnd ,o uollez!le)ol aql ro; 6u;66e1adolrdf g 1-g atnbtl

rrl: orr.rr I r)naourNr I evt:aolat f rcrrdrd9NrcofNlI vNo-LUlsNt I l s a l e l u r+ o u r e l o t dt o 1 a u a 6

VNUpue'VNC'suteloldbullelndtueyl:gtaldeqf 9ts

'uljoJ paztuotsll ur araquMoqssrscs aql'stsaloqoollfala la6-apturel^r)eIlod 565 rol suraloroazrlrqnlos ol pasnaJeslp)tuleq) oMl asaql'loueqlaoldp)Jau-f,rue6e 6uPnparaql pue (SOS)arelns |{>apop unlpos 1ua6ra1ep aql 11-g arn6r1 loueqlaolde)raur-d HS '.Hl : ) '_ Hll

I

HO

)u5 rnPN v rlO

"I

O:S:O I

o

,ui ,Hi ,Hi ,Hi .Hf ZHf l ._ H J

,Hi ,Hi ,Hi ,Hl ,Hf

'urelord E Jo uorlBJIJrJndeq] ur sa8elsa^Issacrnsaql Jo qJea az.{.I 'sureloJd -puE ot pesn uaaq seq ]pql Ia8 pJo qderSoloqd e stueserd 6I-g arn8lc Jo uorlrsoduoc lrunqns aql puE rq81amrelnJalolu 3q] lnoqP uorleruJoJursepr^ -ord tr 'ezrsdq sepqded^lod sateredespoqlau eql esneJeq puv 'saueJqruaruur sureloJd ^uplu eql se qJns-JalE^\ ur alqnlosur fileluJou eJBleq} esoq] Sulpnlcur 'suralord sl 1IDVd-SCS ;o sadfi IIE aleJedas ueo tr asnecaq pasn ,{.1aprrr,t 'pueq e ur palJelap aq ueJ uralord;o 3u 91 su aIDII se leqt os 'urels plo8 Jo JeAIrse qlllvr paleaJl sle8 ur uaas are suralord rourru ualg 'anlq arsseruooJ sE qcns al.p e qlpr 1aBaqr u1 suralord aql Sururels Iq palcalap [ppuar are sura]ord ro[eur aq;'(gl-g arn31g) relnf,eloru Jo JepJo ur peBuBJJuspueq uralord elerf,srpJo sarJese olur lq31a,,r,r peleuorlJeq sr suralord;o arnlxrur xaldruoJ u 'l1nseJu sV 'seuo IIBrusueql eJoru qJnu papJeleJ aJEsuralord a8rel'anars Jplnoeloru e sp slce qJIq \'1aB apnuel 'Jnq '}no 'uor}nlos -r{rcer{1odaql Jo qsaur aq} ur larueJ plnol slJaJJaoml arl} aaq uI '3erp ra8rel B ol osle pup seJJoJIEJrJlJelera8rel o1 palcafqns aJp 'eBJEqO aJoru qlr,4i\'suralord ra8rel 'a8reqc aAIlEBeuJo lunoue elues eq] e^eq eJoJeJeql pue scs Jo lunolue atups eql purq,taqt (z) pue 'alu€s eq] are sadeqs rraq] lEq] os 'SCS aqr -{q paploJun i{1ala1druocsr aJn}lnJls alr}eu Jraql (I) asneJeq spaads rulrurs qryn 1aBaql q8norqt aloru o] pua] azrs aues aql Jo suralord 'paqdde sr a8ellon e uaqm apoJlf,elaanrlrsod eql pJemo] aler8nu ot ll esneJ pue a8reqc JrsurJlur s,uralord aql {seru qJIqM 'salnoaloru lua8ralap pa8reqr dlanuu8au aq] Jo sraqunu a8rel spurq epcaloru uralord qceg ;1aBapnueldrce.{1odgo quls e q8norql unJ sr suralord pazrtqnlos-Sqs Jo eJnlxru p uaq^ suaddeq 1eq11 ,(laleredas pez&leue aq ueJ suralord lrun 'suratord aq} u1 sa8e4url -qnsq1ntu ur saprtdad,{yodluanlqsuoo aqt Jo IIe teqt os g-g ,{ue {perq o} pappe dlensn sl OI-8 arn8rg ees) Ioueqlaoldecraru-fl sE qrns 'uonlppp uI 'uorlnlos lua8u Surcnper B lua8ralap aql ul alqnlos dlaar; perapuer pup selnJaloru pldl Jo sure]oJd Jeq]o q]lm suorleroosspJraql ruo4 paseeleJeJB saplaloru uralord pnprlrpur aql 'supqc ap4daddlod papuaua olur ploJun ol ruaql SursneJ'selnJaloruuralord aql;o suo€ar crqoqdorp.,(r{ol spurq lua8ralap srql esneJag '(zI-g arnS;g) sos ro 'ateJlnsflcapop unrpos ']uaSratap peSrEqr dlanrle8au p;ra,,vrod p sapnlour euo ut lnq uonnlos snoenbu aldrurs e ur ler{} ']seJelur sapcalotu uralord aql go uoqerSrur ]ou ale sallasueql suralord aql Jo eq] pretar o1 q8noua sr leqt os palsnfpe eq uBOya8aqr Jo azrs arod aql rl Iprus :sreruouoruJouortezrJa(u,rtlod dq paredard sr 1aBaq; 'aler8ru suralord aq] qf,rqm q8norqt xrrleru uaur aqt se apnuel,{rce,,(1od3o la8 palull-ssoJJ dp8tq e sasn '(1ICVd-SOS)slseroqdorlcala U 1aE-ap;urufi.rcafiod SOS st rirradord srqtJo uop -ec11dderelndod tsotu aql'adeqs pue ezrsslr uo pue a8reqc leu slr uo spuadap ]Eql elEJe 1e aler8rur ol uralord eql sesneJ elnceloru uralord e Surureluoc uorl -nlos E o1paudde plerJJrJlceleuV 'uruluof, daq] sprce ourure pa8reqc go aJnlxrru aql uo Surpuadap 'a8reqc a,lpe8au ro anrlrsod lau E ssessoddlensn suraloJd

s!soloqdolt)alf

eg ue) sulalord sos ^q pa}eredas lag-aprue;fure{;o6 'ruaq1.{pnls o1 sanbruqcat apnlu1nru e go luarudola^ep aq} palelntuns Jo seq suorlJury pue sarnlJnrls uralord;o dlauel lear8 aq; 'lleJ er{l Jo s}uauala '{Jonr IernlcnJls roferu aq] sE enJespue IpJrueqJau op o1 srsdlorp.,(qapqoalcnu asn 'suorlJeal crloqelaru azftlept r(aq] :s11ac ur sassacordlsoru ruJoJJadsuralor4

'uouaclfrmd uaql 8uttlt\dturs [poaB fqataqt'sutalotd o1paqrquD aq oj sBpjuotttuSocatpttcadsmolp raruI paqrJJsap aq o1 'sanbruqlaj VNe tuoutqwoJav '$au aLU oj panddq aJ?tuunloJ auo tuoLt paulaiqo suo1lco.4{ pa?4Juuaal7-urru m suwnp) ryatatllp la)anastl¬qt passad st a\duos aql 'uottoclfuncl ytctcltl o uI 'salncapu raq]o rcl &ryyffn ro 'srtjslraJcD -ttn4caBtaqc 'tlnlqoqdotptt1 'lq8pm rulncapu 4aqt s6oq aqj uo pauntodasaq unc to suralotd artllcn[11acfio1otq'xuJDruutun1octo ad& aq] uo Sutpuadap:tqda8olaulou4r LLS

sNEtoud9NtzAtvNV

518

Chapter8: ManipulatingProteins, DNA,and RNA (A)

(B)

s a m p l el o a d e do n t o g e l by pipette cathode

e

p l a s t i cc a s i n g

protein with two s u b u n i t sA , and B, joinedby a disulfide bridge A

single-subunit protein

c

I

-s

I

I HEATE D W I T H S D SA N D M E RCAPTOETHANOL

I

I + )a n o o e

-B

^ |

I

POLYACRYLAM IDE.GEL ELECTROPHORESIS

tl

It Figure8-18 SDSpolyacrylamide-gel (SDS-PAGE). (A)An electrophoresis (B)Individualpolypeptidechainsform a electrophoresis apparatus. complexwith negativelychargedmolecules of sodiumdodecylsulfate (SDS) and thereforemigrateas a negatively chargedSDS-protein complex througha porousgel of polyacrylamide. Because the speedof migration undertheseconditionsis greaterthe smallerthe polypeptide, this techniquecan be usedto determinethe approximatemolecularweightof a polypeptidechainaswell asthe subunitcompositionof a protein.lf the proteincontainsa largeamountof carbohydrate, however,it will move anomalously on the gel and its apparentmolecularweightestimatedby SDS-PAGE will be misleadino.

"::it:it.l,ali:']]i:r:irl.:i:,'

'a::'::.:ra.iiari.r:::'

@ s l a b o f p o l y a c r y l a m i d eg e l

specificProteinscan Be Detectedby Blottingwith Antibodies

molecular weight (daltons) 100,000

of labeled antibody to reveal the protein of interest. This method of detecting proteins is called Western blotting, or immunoblotting (Figure g-2O).

Figure8-19 Analysisof proteinsamplesby SDSpolyacrylamide-gel electrophoresis. The photographshowsa Coomassie-stained gel that has beenusedto detectthe proteinspresentat successive stagesin the purification of an enzyme.The leftmostlane(lane1) containsthe complex mixtureof proteinsin the startingcellextract,and eachsucceeding lane analyzes the proteinsobtainedaftera chromatographic fractionation of the proteinsampleanalyzedin the previouslane(seeFigureg-14).Thesame total amountof protein(10 pg) was loadedonto the gel at the top of each lane.Individualproteinsnormallyappearas sharp,dye-stained bands;a band broadens,however,when it containstoo much protein.(From T.Formosaand B.M.Alberts,J.Biol.Chem.261:6't07-6i l B. 1986.)

519

PROTEINs ANALYZING

(B) Figure8-20 Westernblotting.All the proteinsfrom dividingtobaccocellsin culturearefirstseparatedby two-dimensional (described in FigureB-23).In (A),the positionsof the proteinsare revealedby a polyacrylamide-gel electrophoresis to a sheetof nitrocellulose proteinson an identicalgel werethen transferred proteinstain.In (B),the separated sensitive during residues on threonine phosphorylated are proteins that onlythose and exposedto an antibodythat recognizes by this antibodyarerevealedby an enzyme-linked The positionsof the dozenor io proteinsthat arerecognized mitosis. secondantibody.Thistechniqueis alsoknownas immunoblotting(orWesternblotting).(FromJ.A.Traaset al.,PlantJ Publishing') from Blackwell 1992.With permission 2:723-732,

Methodfor Provides a HighlySensitive MassSpectrometry ldentifyingUnknownProteins A frequent problem in cell biology and biochemistry is the identification of a protein or collection of proteins that has been obtained by one of the purification procedures discussed in the preceding pages (see, for example, Figure s f m o s t c o m m o n e x p e r i m e n l . aol r g a n B - 1 6 ) .B e c a u s et h e g e n o m e S e q u e n c e o isms are now known, cataloguesof all the proteins produced in those organisms are available. The task of identifiiing an unknown protein (or collection of unknown proteins) thus reduces to matching some of the amino acid sequences present in the unknown sample with known catalogued genes.This task is now performed almost exclusively by using mass spectrometry in conjunction with

then dried onto a metal or ceramic slide. A laser then blasts the sample, ejecting the peptides from the slide in the form of an ionized gas,in which each molecule cattl"t one or more positive charges.The ionized peptides are acceleratedin an electric field and fly toward a detector. Their mass and charge determines the time it takes them to reach the detector: Iarge peptides move more slowly, and more highly charged molecules move more quickly. By analyzing those ionized peptides that bear a single charge,the precise massesof peptides present in the original sample can be determined. MALDI-TOF can also be used to accurately -"ir.rre the mass of intact proteins as large as 200,000daltons. This information is then used to search genomic databases,in which the masses of all proteins and of all their predicted peptide fragments have been tabulated from the genomic sequencesof the organism (FigUreS-zf A). An unambiguous match to i particular open reading frame can often be made by knowing the mass of only a few peptides derived from a given protein' MALDI-TOF provides accurate molecular weight measurements for proteins and peptidei. Moreover, by employing two mass spectrometers in tandem 1an arrangement known as MS/MS), it is possible to directly determine the

520

Chapter8: ManipulatingProteins, DNA,and RNA

p r o t e i no f Interest

P E P T I D ERSE L E A S EBDY T R Y P T ID C I G E S T I OANN D T H E I RM A S S E M S EASURED U S I N GM A L D I - T O M F ASS SPECTROMETRY

II

j -

M A S S S P E C T R O M E T EGRI V E S PEPTIDE M A S S E SA , S IN (A)

o tr o c l

E A C HP E P T I DIEST H E h F U R T H EFRR A G M E N T E D A T P E P T I DBEO N D S

o

u

m a s stto m l z ((mass mlz o ccharge h a r g erratio) atio)

1600

II

M A S S EO S F F R A G M E N TMSE A S U R EO DN A c o u P L E DS E C O N D MRS / M 5 ) M A S SS P E C T R O M E T(E

t

P R O T E IS NE Q U E N CDEA T A B A S ESSE A R C H EFDO R M A T C H EW S I T HT H E O R E T I C A MLA S S E C SALCULATED F O RA L LT R Y P S I N - R E L E A P SE P DT I D E S

II

t

IDENTIFICATION O F C O R R E S P O N DG I NEGN E (A)

q C o E c

)

o

zvu 200

mlz (massto charqe ratio)

1500

T H EM A S SD I F F E R E N CBEEST W E E F NR A G M E N TCSA N B EU S E D T O C O N S T R U CATP A R T I AA L M I N OA C I DS E Q U E N CTEH ED A T A M A Y A L L O WG E N EI D E N T I F I C A T I O ONR,P R O V I DT EH EM E A N S F O RC L O N I N T GH EG E N E (B)

Figure8-21 use of massspectrometryto identify proteins and to sequencepeptides.An isolatedprotein is digested with trypsinand the peptidefragmentsarethen loadedinto the massspectrometer. Two differentapproaches cJn then be usedto identifythe protein.(A)In the firstmethod,peptidemasses aremeasuredprecisely usingMALDI-TOF mass spectrometry' Sequence databases arethen searched to find the genethat encodesa proteinwhosecalculated tryptic digestprofilematchesthesevalues.(B)Massspectrometry can alsobe usedto determlnedirectlythe aminoacidsequence of peptidefragments. In this example,trypticpeptidesarefirstseparatedbasedon masswithina massspectrometer. Each peptideis then furtherfragmented,primarilyby cleavingits peptidebonds.Thistreatmentgenerates a nestedsetof peptides,eachdifferingin sizeby one aminoacid Thesefragmentsarefed into a second coupledmassspectrometer, and their masses aredetermined.The differencein massesbetweentwo closelyrelatedpeptidescan be usedto deducetne "missing"amino acid.By repeatedapplications of this procedure, a partialaminoacidsequenceof the originalproteincan be determined. Forsimplicity, the analysis shownbeginswith a singlespeciesof purifiedprotein.In reality,mass spectrometry is usuallycarriedout on mixturesof proteins,suchasthoseobtainedfor affinitychromatogiaphy (seeFigure8-16),and can identifyall the proteinspresentin the mixtures. experiments As explainedin the text,mass spectrometry can alsodetectpost-translational modifications of oroteins. amino acid sequences of individual peptides in a complex mixture. As described above, the protein sample is first broken dor.tm into smaller peptides, which are separated from each other by mass spectrometry. each peptiae is then further fragmented through collisions with high-energy gas atoms. this method of fragmentation preferentially cleaves the peptide bonds, generating a ladder of fra!ments, each differing by a single amino acid. The second miss spectrometer then separates these fragments and displays their masses. The amino acid sequence of a peptide can then be deduced from these differences in mass (Fig-

ure 8-2lB). MS/MS is particularly useful for detecting and precisely mapping posttranslational modifications of proteins, such is phosphorylutiorn o. acetytations. Becausethese modifications impart a charicteristic mass increase to an amino acid, they are easily detected by mass spectrometry. As described in

521

PROTEINS ANALYZING

Chapter 3, proteomics, a general term that encompassesmany different experimental techniques, is the characterization of all proteins in the cell, including all protein-protein interactions and all post-translational modifications. In combination with the rapid purification techniques discussedin the last section, mass spectrometry has emerged as the most powerful method for mapping both the post-translational modifications of a given protein and the proteins that remain associatedwith it during purification.

Powerful MethodsareEspecially Separation Two-Dimensional Because different proteins can have similar sizes, shapes, masses, and overall charges, most separation techniques such as SDS polyacrylamide-gel electrophoresis or ion-exchange chromatography cannot typically display all the proteins in a cell or even in an organelle. In contrast, two-dimensional gel electrophoresis, which combines two different separation procedures, can resolve up to 2000 proteins-the total number of different proteins in a simple bacterium-in the form of a two-dimensional protein map. In the first step, the proteins are separated by their intrinsic charges.The sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, together with B-mercaptoethanol and the denaturing reagent urea. This solution solubilizes, denatures, and dissociates all the polypeptide chains but leaves their intrinsic charge unchanged. The pollpeptide chains are then separated in a pH gradient by a procedure called isoelectric protein focusing, which takes advantage of the variation in the net charge on a molecule with the pH of its surrounding solution. Every protein has a characteristic isoelectric point, the pH at which the protein has no net charge and therefore does not migrate in an electric field. In isoelectric focusing, proteins are separated electrophoretically in a narrow tube of polyacrylamide gel in which a gradient of pH is establishedby a mixture of special buffers. Each protein moves to a position in the gradient that corresponds to its isoelectric point and remains there (Figure 8-22). This is the first dimension of two-dimensional polyacrylamide- gel electrophoresis. In the second step, the narrow gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size,as in one-dimensional SDS-PAGE:the original narrow gel is soakedin SDSand then placed on one edge of an SDSpolyacrylamide-gel slab, through which each pollpeptide chain migrates to form a discrete spot. This is the second dimension of two-dimensional polyacrylamide-gel electrophoresis. The only proteins left unresolved are those that have both identical sizes and identical iioelectric points, a relatively rare situation. Even trace amounts of each pollpeptide chain can be detected on the gel by various staining procedures-or by autoradiography if the protein sample was initially labeled with a radioisotope (Figure 8-23). The technique has such great resolving power that it can distinguiih between two proteins that differ in only a single charged amino acid.

4 c o

at low pH, the protein is positively charged

Ho B' -7 o^ i6 o

10

at high pH, the protein is negatively charged

at the isoelectric point, the protein h a sn o n e t c h a r g e a n d t h e r e f o r en o l o n g e r m i g r a t e si n t h e e l e c t r i cf i e l d ; for the protein shown the isoelectricpH i s6 . 5

Figure8-22 Separationof Protein moleculesby isoelectricfocusing.At low the pH (highH+concentration), carboxylicacid groupsof proteinstend to be uncharged(-COOH)and their basicgroupsfully nitrogen-containing charged(forexample,-NH:+),giving most proteinsa net positivecharge'At acidgroupsare high pH,the carboxylic negativelycharged(-COO-)and the basic groupstend to be uncharged(for example,-NHz),givingmost proternsa pH' net negativecharge.At its isoelectric a proteinhasno net chargesincethe positiveand negativechargesbalance. Thus,when a tube containinga fixedpH gradientis subjectedto a strongelectric fieldin the appropriatedirection,each proteinspeciespresentmigratesuntil it pH, formsa sharpbandat its isoelectric as shown.

Chapter8: Manipulatingproteins,DNA,and RNA

522

basrc

G c

s t a b l ep H g r a d i e n t

1 0 0-

;

!

o

-;

c

E o

t'

:

s*

___-_

5o:

I

:-1.c1

.if'r !-

5 c

3:

-

-

J,

-^]---

-*J$,,.;1Fry,:.,.i,'"XffUa!cl,r,*r,ri:,:r*d -;r - :lti_ .-S-,:

s.-

ri:l'1,:i,.l.i. {

6 o E o

25-'*

-

1 &-

l**

A different, even more powerful, "two-dimensional" technique is now available when the aim is to determine all of the proteins present inan organelle or another complex mixture of proteins. Because the technique relies on mass spectroscopy, it requires that the proteins be from an organism with a completely sequenced genome. First, the mixture of proteins present is digested with trypsin to produce short peptides. Next, these peptideJ u." sepu.uted by a series of automated liquid chromatography steps. As the second dimension, each separated peptide is fed directly into a tandem mass spectrometer (MS/MS) that allows its amino acid sequence, as well as any post-translational modifications, to be determined. This arrangement, in wtrictr a tandem mass spectrometer (MS/MS) is attached to the output of an automated liquid chromatography (LC) system, is referred to as LC-MS/MS. It is now becoming routine to subject an entire organelle preparation to LC-MS/MS analysis una to ideltify hundreds of proteins and their modifications. of course, no organelle isolation procedure is perfect, and some of the proteins identified will be contaminating proteins. These can often be excluded by analyzing neighboring fractions from the organelle purification and "subtraiting" ihed out from the peak organelle fractions.

HydrodynamicMeasurementsRevealthe Sizeand Shapeof a ProteinComplex Most proteins in a cell act as part of larger complexes, and knowledge of the size and shape of these complexes often leads to insights regarding their function. This information can be obtained in severalimportant ways. Sometimes, a complex can be directly visualized using electron microscopy, as described in chapter 9. A complementary approach relies on the hydrodynamic properties of a complex, that is, its behavior as it moves through a liquid medium. 0sually, two separatemeasurements are made. one measure is the velocity of a complex as it moves under the influence of a centrifugal field produced byan ultracentrifuge (seeFigure 8-llA). The sedimentation constant (or S-valuej obtained depends on both the size and the shape of the complex and does not, by itself, convev especially useful information. However, once a second hydrodvnamic measure_ ment is performed-by charting the migration of a compiex thiough a gel-filtration chromatography column (seeFigure g-r3B)-botrr tne upp.*imite shape of a complex and its molecular weight can be calculated. Molecular weight can also be determined more directly by using an analytical ultracentrifuge, a complex device that allows protein absorbince measurements

Figure8-23 Two-dimensional polyacrylamide-gel electrophoresis, All the proteinsin an E.coli bacterialcell are separated in thisgel,in whicheachspot correspondsto a differentpolypeptide chain.The proteinswerefirst separated on the basisoftheir isoelectric pointsby isoelectricfocusingfrom left to right. Theywere then further fractionated accordingto their molecularweightsby electrophoresis from top to bottom in the presenceof SDS.Note that different proteinsare presentin very different amounts.The bacteriawerefed with a mixtureof radioisotope-labeled amino acidsso that all of their proretnswere radioactive and couldbe detectedby autoradiography(seepp. 602-603). (Courtesyof PatrickO'Farrell.)

ANALYZING PROTEINS

to be made on a sample while it is subjected to centrifugal forces. In this approach, the sample is centrifuged until it reachesequilibrium, where the centrifugal force on a protein complex exactly balances its tendency to diffuse away. Becausethis balancing point is dependent on a complex's molecular weight but not on its particular shape, the molecular weight can be directly calculated, as needed to determine the stoichiometry of each protein in a protein complex'

Setsof InteractingProteinsCanBeldentifiedby Biochemical Methods Because most proteins in the cell function as part of complexes with other proteins, an important way to begin to characterize the biological role of an unknor,tryr protein is to identiff all of the other proteins to which it specifically binds. One method for identifying proteins that bind to one another tightly is coimmunoprecipitation.In this case,an antibody recognizesa specific target protein; reagents that bind to the antibody and are coupled to a solid matrix then drag the complex out of solution to the bottom of a test tube. If the original target protein is associatedtightly enough with another protein when it is captured by the antibody, the partner precipitates as well. This method is useful for identifuing proteins that are part of a complex inside cells, including those that interact only transiently-for example, when extracellular signal molecules stimulate cells (discussed in Chapter 15). Another method frequently used to identify a protein's binding partners is protein affinity chromatography (seeFigure B-l3C)' To employ this technique to capture interacting proteins, a target protein is attached to polymer beads that are packed into a column. \A/henthe proteins in a cell extract are washed through this column, those proteins that interact with the target protein are retained by the affinity matrix. These proteins can then be eluted and their identity determined by mass spectrometry. In addition to capturing protein complexes on columns or in test tubes, researchers are developing high-density protein arrays to investigate protein interactions. These arrays, which contain thousands of different proteins or antibodies spotted onto glass slides or immobilized in tiny wells, allow one to examine the biochemical activities and binding profiles of a large number of proteins at once. For example, if one incubates a fluorescently labeled protein with arrays containing thousands of immobilized proteins, the spots that remain fluorescent after extensivewashing each contain a protein to which the labeled protein specifically binds.

Interactions CanAlsoBe ldentifiedby a Protein-Protein Two-HybridTechniquein Yeast Thus far, we have emphasized biochemical approaches to the study of protein-protein interactions. However, a particularly powerful strategy, called the two-hybrid system, relies on exploiting the cell's own mechanisms to reveal protein-protein interactions. The technique takes advantage of the modular nature of gene activator proteins (see Figure 7-45). These proteins both bind to specific DNA sequences and activate gene transcription, and these activities are often performed by two separate protein domains. Using recombinant DNA techniques, two such protein domains are used to create separate "bait" and "prey" fusion proteins. To create the "bait" fusion protein, the DNA sequence that codes for a target protein is fused with DNA that encodes the DNA-binding domain of a gene activator protein. lVhen this construct is introduced into yeast, the cells produce the fusion protein, with the target protein attached to this DNA-binding domain (Figure 8-24). This fusion protein binds to the regulatory region of a reporter gene, where it serves as "bait" to fish for proteins that interact with the target protein. To search for potential binding partners (potential prey for the bait), the candidate proteins also have to be constructed as fusion proteins: DNA encoding the activation domain of a gene activator protein is fused to a large

523

524

Chapter8: ManipulatingProteins, DNA,and RNA target D N A - b i n d i n gd o m a i n p r o t e i n

bindino partne; \

transcriptional a c T l v a t l oO n omaln

R E C O M B I N A NGTE N E S ENCODING B A I TA N D P R E YI N T R O D U C EI D NTO YEASTCELL

t r a n s c r i p t i o n aalc t i v a t o r b i n d i n gs i t e

ENE

number of different genes. Members of this collection of genes-encoding potential 'rprey"-are introduced individually into yeast cells containing the bait. If the yeast cell receives a DNA clone that expressesa prey partner for the bait protein, the two halves of a transcriptional activator ate ,-,.rited, switching on the reporter gene (seeFigure 8-24). This ingenious technique sounds complex, but the two-hybrid system is relatively simple to use in the laboratory.Although the protein-protein interactions occur in the yeast cell nucleus, proteins from every part of the cell and from any organism can be studied in this way. The two-hybrid system has been scaled up to map the interactions that occur among all of the proteins an organism produces. In this case,a set of bait and prey fusions is produced for every cell protein, and every bait/prey combination can be monitored. In this way proiein interaction maps have been generated for most of the proteins in yeast, c. elegans, and Drosophila.

produces combiningDataDerivedfrom DifferentTechniques ReliableProtein-lnteraction Maps As previously discussedin chapter 3, extensiveprotein-interaction maps can be very useful for identiSring the functions of proteins (seeFigure 3-g2) . For this reason, both the two-hybrid method and the biochemical technique discussedearlier knornmas tap-tagging (seepp. 515-516) have been automited to determine the interactions between thousands of proteins. Unfortunately, different results are found in different experiments, and many of the interactions detected in one laboratory are not detected in another. Therefore,the most useful protein-interaction maps are those that combine data from many experiments, requiring that each interaction in the map be confirmed by more than one technique.

opticalMethodscan MonitorproteinInteractions in RealTime once two proteins-or a protein and a small molecule-are knor.vnto associate, it becomes important to characterize their interaction in more detail. proteins can associate with each other more or less permanently (like the subunits of RNA polymerase or the proteosome), or engage in transient encounters that may last only a few milliseconds (like a protein kinase and its substrate). To understand how a protein functions inside a cell, we need to determine how tightly it binds to other proteins, how rapidly it dissociatesfrom them, and how covalent modifications, small molecules, or other proteins influence these interactions. such studies of protein dynamics often employ optical methods.

Figure8-24 The yeasttwo-hybrid systemfor detecting protein-protein interactions.The target protein is fused to a DNA-binding domainthat directsthe fusion proteinto the regulatoryregionof a reportergeneas"bait."When thistarget proteinbindsto anotherspecially designedproteinin the cell nucleus 1"prey"\,their interactionbringstogether two halvesof a transcriptionalactivator, whichthen switcheson the expression of the reportergene.

525

ANALYZINGPROTEINS

Certain amino acids (for example, tryptophan) exhibit weak fluorescence that can be detected with sensitive fluorimeters. In many cases,the fluorescence intensity, or the emission spectrum of fluorescent amino acids located in a protein-protein interface, will change when the proteins associate. \.{/hen this change can be detected by fluorimetry, it provides a sensitive and quantitative measure of protein binding. A particularly useful method for monitoring the dynamics of a protein's binding to other molecules is called surface plasmon resonance (SPR).The SPR method has been used to characterize a wide variety of molecular interactions, including antibody-antigen binding, ligand-receptor coupling, and the binding of proteins to DNA, carbohydrates,small molecules, and other proteins. SPRdetects binding interactions by monitoring the reflection of a beam of Iight off the interface between an aqueous solution of potential binding molecules and a biosensor surface carrying an immobilized bait protein. The bait protein is attached to a very thin layer of metal that coats one side of a glass prism (Figure 8-25). A light beam is passedthrough the prism; at a certain angle, called the resonanceangle, some of the energy from the light interacts with the cloud of electrons in the metal film, generating a plasmon-an oscillation of the electrons at right anglesto the plane of the film, bouncing up and down between its upper and lower surfaceslike a weight on a spring. The plasmon, in turn, generates an electrical field that extends a short distance-about the wavelength of the light-above and below the metal surface.Any change in the composition of (A)

p r i s m ,o r g r a t i n g

reflected-lig ht detector

s u r fa c e p l a s m o n se x c i t e d in gold film by light at a , / s p e c i f i cr e s o n a n c e angle g o r of l r m (-50 nm) L bait molecule attachedto gold

r i r m b v r r e x i b r e +g a

t

(

f

solution of prey molecules

( B ) T h e b i n d i n go f p r e y m o l e c u l e tso b a i t m o l e c u l e si n c r e a s etsh e r e f r a c t i v ei n d e xo { t h e s u r f a c el a y e r , h i c h c a n b e m e a s u r e db y a d e t e c t o r . T h i sa l t e r st h e r e s o n a n c e a n g l ef o r p l a s m o ni n d u c t i o n w

s o lu t l o n of prey molecules

t

Gr

(.t

association

+ liiT.::" dissociation

o E o o c 6 o

prey added

buffer wash

Figure8-25 Surfaceplasmon resonance. by (A)SPRcan detectbindinginteractions monitoringthe reflectionof a beam of light off the interfacebetweenan aqueous solutionof potentialbindingmolecules (green)and a biosensorsurfacecoated with an immobilizedbait protein (red,). (B)A solutionof prey proteinsis allowedto flow pastthe immobilizedbait protein. to the bait Bindingof preymolecules changein proteinproducesa measurable angle,asdoestheir the resonance when a buffersolutionwashes dissociation them off.Thesechanges,monitoredin real and time,reflectthe association of the molecularcomplexes. dissociation

526

Chapter8: ManipulatingProteins, DNA,and RNA

the environment within the range of the electrical field will cause a measurable change in the resonance angle. To measure binding, a solution containing proteins (or other molecules) that might interact with the immobilized bait protein is allowed to flow past the biosensor surface. Proteins binding to the bait change the composition of the molecular complexes on the metal surface, causing a change in the resonance angle (see Figure 8-25). The changes in the resonance angle are monitored in real time and reflect the kinetics of the association-or dissociation-of molecules with the bait protein. The association rate (korj is measured as the molecules interact, and the dissociation rate (kor) is determined as buffer washes the bound molecules from the sensor surface.A binding constant (.fiJis calculated by dividing komby kon.In addition to determining the kinetics, spR can be used to determine the number of molecules that are bound in each complex: the magnitude of the sPR signal change is proportional to the mass of the immobilized complex. The sPR method is particularly useful becauseit requires only small amounts of the protein, the protein does not have to be labeled in any way, and the interactions of the protein with other molecules can be monitored in real time. A third optical method for probing protein interactions usesgreenfluorescent protein (discussedin detail below) and its derivatives of different colors. In this application, two proteins of interest are each labeled with a different fluorochrome, such that the emission spectrum of one fluorochrome overlaps the absorption spectrum of the second fluorochrome. If the two proteins-and their attached fluorochromes-come very close to each other (within about l-10 nm), the energy of the absorbed light is transferred from one fluorochrome to the other. The energy transfer, called fluorescence resonance energy transfer (FRET), is determined by illuminating the first fluorochrome and measuring emission from the second (Figure 8-26). This technique is especially powerful because,when combined with fluorescencemicroscopy, it canbe used to characlerizeprotein-protein interactions at specific locations inside living cells.

SomeT e c h n i q u eCsa nMo n i to rS i n g l eMolecules The biochemical methods described so far in this chapter are used to study large populations of molecules, a limitation that reflects the small size of typical bio, logical molecules relative to the sensitivity of the methods to detect them. However, the recent development of highly sensitive and precise measurement methods has created a new branch of biophysics-the study of single molecules. Single-molecule studies are particularly important in cell biology becausemany processesrely on the activities of only a few critical molecules in the cell. bluefluorescent protetn blue

g r e e n f u o r e s c e nt protein

blue light e x c i t a toi n

:fii.";,'6!*l''""

9reen l i gh t e mr s S t o n

@

(A)

p r o t e i nX

blue

violet

tght)

protein Y

green violet

Wl'*

l;;fi]

,rr+ir'[0,!] ,,rtrii

@ ( B ) N O P R O T E I INN T E R A C T I O I \ N O E X C I T A T I OONF G R E E I " F L U O R E S C EPNRTO T E I N ; B L U EL I G H TD E T E C T E D

( c ) P R O T E TTNN T E R A C T T O N F L U O R E S C E NRCEES O N A N C E E N E R GT YR A N S F E R ; G R E E NL I G H TD E T E C T E D

Figure8-26 Fluorescence resonance energy transfer(FRET). To determine whether(andwhen)two proteins interactinsidea cell,the proteinsarefirst producedasfusionproteinsattachedto differentcolorvariantsof green protein(GFP). (A)In this fluorescent example,proteinX is coupledto a blue protein,which is excitedby fluorescent violetlight (370-440nm) and emitsblue light (440-480nm);proteiny is coupled protein,whichis to a greenfluorescent excitedby blue lightand emitsgreen light (510nm).(B)lf proreinX and y do not interact,illuminatingthe samplewith violetlight yieldsfluorescence from the bluefluorescent proteinonly.(C)When proteinX and proteinY interact,FRET can now occur.llluminatingthe samplewith violetlight excitesthe bluefluorescent protein,whoseemissionin turn excites the greenfluorescent protein,resultingin an emissionof greenlight.The fluorochromes mustbe quiteclose together-within about 1-10 nm of one another-for FRET to occur.Because not everymoleculeof proteinX and proteiny is boundat all times,someblue light may stillbe detected.Butasthe two proteins beginto interact,emissionfrom the donorGFPfallsasthe emissionfrom the acceDtor GFPrises.

527

ANALYZING PROTEINS

The first example of a technique for studying the function of single protein molecules was the use of a patch electrode to measure current flow through single ion channels (seeFigure ll-33). Another approach is to attach the protein to a larger structure, such as a polystyrene bead, which can then be observed by conventional microscopy. This strategy has been particularly useftrl in measuring the movements of motor proteins. For example, molecules of the motor protein kinesin (discussed in Chapter 16) can be attached to a bead, and by observing the kinesin-attached bead moving along a microtubule, the step size of the motor (that is, the distance moved for each ATP molecule hydrolyzed) can be measured. As we will see in Chapter 9, optical microscopes have a limited resolution due to the diffraction of light, but computational and optical methods can be used to determine the position of a bead to a much finer precision than the resolution limit of the microscope. Using such techniques, extremely small movements-on the order of nanometers-can easily be detected and quantified. Another advantage of attaching molecules to large beads is that these beads can serve as "handles" by which the molecules can be manipulated. This allows forces to be applied to the molecules, and their responseobserved.For example, the speed or step size of a motor can be measured as a function of the force it is pulling against. As discussed in the next chapter, a focused laser beam can be used as "optical tweezers" to generate a mechanical force on a bead, allowing motor proteins to be studied under an applied force (seeFigure 9-35). Beads can also be manipulated using magnetic fields, a technology known as "magnetic tweezers."If multiple beads are present in a magnetic field, they will all experience the same force, potentially allowing large numbers of beads to be manipulated in parallel in a single experiment. VVhilebeads can be used as markers to track protein movements, it is clearly preferable to be able to visualize the proteins themselves. In the next chapter, we shall see that recent refinements in microscopy have now made this possible.

HNI s4N/ (A)

O

CH:

cH-

H monastrol

ProteinFunctionCanBeSelectivelyDisruptedWith Small Molecules Chemical inhibitors have contributed to the development of cell biology. For example, the microtubule inhibitor colchicine is routinely used to test whether microtubules are required for a given biological process; it also led to the first purification of tubulin several decades ago. In the past, these small molecules were usually natural products; that is, they were symthesizedby living creatures. Although, as a whole, natural products have been extraordinarily useftrl in science and medicine (see,for example, Table 6-4, p.385), they acted on a limited number of biological processes.Howeve! the recent development of methods to slmthesize hundreds of thousands of small molecules and to carry out large-scale automated screens holds the promise of identifuing chemical inhibitors for virtually any biological process. In such approaches, Iarge collections of small chemical compounds are simultaneously tested, either on living cells or in cell-free assays. Once an inhibitor is identified, it can be used as a probe to identiff, through affrnity chromatography (see Figure 8-13C) or other means, the protein to which the inhibitor binds. This general strategy, often called chemical biology, has successfirlly identifled inhibitors of many proteins that carry out key processesin cell biology. The kinesin protein that functions in mitosis, for example, was identified by this method (Figure &-27). Chemical inhibitors give the cell biologist great control over the timing of inhibition, as drugs can be rapidly added to or removed from cells, allowing protein function to be switched on or offquickly.

ProteinStructureCanBe DeterminedUsingX-RayDiffraction The main technique that has been used to discover the three-dimensional structure of molecules, including proteins, at atomic resolution is x-ray crystallography. X-rays, like light, are a form of electromagnetic radiation, but they have a much shorter wavelength, typically around 0.1 nm (the diameter of a hydrogen

5pm

(c)

L_5,,,"---_l

Figure8-27 Small-moleculeinhibitors for manipulating living cells. (A)Chemicalstructureof monastrol,a kinesininhibitoridentifiedin a largethat scalescreenfor smallmolecules disruptmitosis.(B)Normalmitotic spindleseenin an untreatedcell.The microtubulesare stainedgreenand chromosomesblue.(C)MonoPolar soindlethat forms in cellstreatedwith monastrol.(Band C,from T.U'Mayeret al.,Science286:971 -974, 1999.With oermissionfrom AAA5.)

528

Chapter8: ManipulatingProteins, DNA,and RNA x-ray diff raction pattern obtained from the protein crystal

\

diffracted beams

beam of x-rays

x - r a ys o u r c e

(A)

Figure8-28 X-raycrystallography. (A)A narrowparallelbeamofx-raysis directedat a well-ordered crystal(B).shown hereis a proteincrystalof ribulose bisphosphate carboxylase, an enzymewith a centralrolein CO2fixationduring photosynthesis. Theatomsin the crystalscattersomeofthe beam,and the scattered wavesreinforce one anotherat certainpointsand appearasa pattern of diffractionspots(C).Thisdiffractionpattern,togetherwith the aminoacid sequenceof the protein,can be usedto producean atomicmodel(D).The completeatomicmodelis hardto interpret,but this simplifiedversion,derived from the x-raydiffractiondata,showsthe protein'sstructuralfeaturesclearly(a, green;p strands, helices, red).Thecomponentspicturedin A to D arenot shown to scale.(8,courtesyofC. Branden; C,courtesyofJ. Hajduand l. Andersson; D,adaptedfrom originalprovidedby B.Furugren.)

(C)

atom). If a narrow parallel beam of x-rays is directed at a sample of a pure protein, most of the x-rays pass straight through it. A small fraction, however, are scattered by the atoms in the sample. If the sample is a well-ordered crystal, the scatteredwaves reinforce one another at certain points and appear as diffraction spots when recorded by a suitable detector (Figure g-2g). The position and intensity of each spot in the x-ray diffraction pattern contain information about the locations of the atoms in the crystal that gave rise to it. Deducing the three-dimensional structure of a large molecule from the diffraction pattern of its crystal is a complex task and was not achieved for a protein molecule until 1960. But in recent years x-ray diffraction analysis has become increasingly automated, and now the slowest step is likely to be ihe generation of suitable protein crystals.This step requires large amounts of very pure protein and often involves years oftrial and error to discover the proper crystallization conditions; the pace has greatly acceleratedwith the use of recombinant DNA techniques to produce pure proteins and robotic techniques to test large numbers of crystallization conditions. Analysis of the resulting diffraction pattern produces a complex threedimensional electron-density map. Interpreting this map-translating its contours into a three-dimensional structure-is a complicated procedure that requires knowledge of the amino acid sequence of the protein. Largely by trial and error, the sequence and the electron-density map are correlated by computer to give the best possible fit. The reliability of the final atomic model depends on the resolution of the original crystallographic data: 0.5 nm resolution might produce a low-resolution map of the polypeptide backbone, whereas a resolution of 0.15 nm allows all of the non-hydrogen atoms in the molecule to be reliably positioned. A complete atomic model is often too complex to appreciate directly, but simplified versions that show a protein's essential structural features can be readily derived from it (see Panel 3-2, pp. 132-133). The three-dimensional

/n\ \vl

529

PROTEINS ANALYZING

structures of about 20,000different proteins have now been determined by x-ray crystallography or by NMR spectroscopy (see below)-enough to begin to see families of common structures emerging. These structures or protein folds often seem to be more conserved in evolution than are the amino acid sequencesthat form them (seeFigure3-13). X-ray crystallographic techniques can also be applied to the study of macromolecular complexes. In a recent triumph, the method was used to determine the structure of the ribosome, a large and complex machine made of several RNAs and more than 50 proteins (see Figure 6-64). The determination required the use of a synchrotron, a radiation source that generatesx-rays with the intensity needed to analyze the crystals of such large macromolecular complexes.

NMRCanBeUsedto DetermineProteinStructurein Solution Nuclear magnetic resonance (NMR) spectroscopyhas been widely used for many years to analyze the structure of small molecules. This technique is now also increasingly applied to the study of small proteins or protein domains. Unlike xray crystallography, NMR does not depend on having a crystalline sample. It simply requires a small volume of concentrated protein solution that is placed in a strong magnetic field; indeed, it is the main technique that yields detailed evidence about the three-dimensional structure of molecules in solution. Certain atomic nuclei, particularly hydrogen nuclei, have a magnetic moment or spin: that is, they have an intrinsic magnetization, like a bar magnet. The spin aligns along the strong magnetic field, but it can be changed to a misaligned, excited state in response to applied radiofrequency (RF)pulses of electromagnetic radiation. \.Vhenthe excited hydrogen nuclei return to their aligned state, they emit RF radiation, which can be measured and displayed as a spectrum. The nature of the emitted radiation depends on the environment of each hydrogen nucleus, and if one nucleus is excited, it influences the absorption and emission of radiation by other nuclei that lie close to it. It is consequently possible, by an ingenious elaboration of the basic NMR technique known as twodimensional NMR, to distinguish the signals from hydrogen nuclei in different amino acid residues,and to identify and measure the small shifts in these signals that occur when these hydrogen nuclei lie close enough together to interact. Becausethe size of such a shift revealsthe distance between the interacting pair of hydrogen atoms, NMR can provide information about the distances between the parts of the protein molecule. By combining this information with a knowledge of the amino acid sequence,it is possible in principle to compute the threedimensional structure of the protein (Figure 8-29).

(B)

Figure8-29 NMRspectroscopy.(A)An exampleof the datafrom an NMR NMR Thistwo-dimensional machine. soectrumis derivedfrom the C-terminal The domainof the enzymecellulase. spotsrepresentinteractionsbetween hydrogenatomsthat are nearneighbors in the orotein and hencereflectthe them.Complex distancethat separates computingmethods,in conjunctionwith enable the knownaminoacidsequence, possiblecompatiblestructures to be derived.(B)Tenstructuresof the enzyme, whichall satisfythe distanceconstraints on equallywell,areshownsuperimposed one another,givinga good indicationof the orobablethree-dimensional (Courtesy of P.Kraulis.) structure.

530

Chapter8: ManipulatingProteins,DNA,and RNA

For technical reasonsthe structure of small proteins of about 20,000daltons or less can be most readily determined by NMR spectroscopy. Resolution decreasesas the size of a macromolecule increases.But recent technical advances have now pushed the limit to about 100,000daltons, thereby making the majority of proteins accessiblefor structural analysis by NMR. BecauseNMR studies are performed in solution, this method also offers a convenient means of monitoring changes in protein structure, for example during protein folding or when the protein binds to another molecule. NMR is also used widely to investigate molecules other than proteins and is valuable, for example, as a method to determine the three-dimensional structures of RNA molecules and the complex carbohydrate side chains of glycoproteins. Some landmarks in the development of x-ray crystallography and NMR are Iisted in Table 8-2.

ProteinSequence and StructureProvideCluesAboutprotein Fu n c t i o n Having discussed methods for purifying and analyzing proteins, we now turn to a common situation in cell and molecular biology: an investigator has identified a geneimportant for a biologicalprocessbut has no direct knowledgeof the biochemical propertiesof its protein product. Thanks to the proliferation of protein and nucleic acid sequencesthat are catalogued in genome databases,the function of a gene-and its encoded protein-can often be predicted by simply comparing its sequence with those of previously characterized genes (seeFigure 3-14). Becauseamino acid sequence

Table8-2 Landmarksin the Developmentof X-rayCrystallography and NMRand TheirApplicationto BiologicalMolecules 1864 Hoppe-Seyler crystallizes, and names, the proteinhemoglobin. 1895 Rcintgen observes that a newformof penetrating radiation, whichhe namesx-rays, is producedwhencathoderays (electrons) hit a metaltarget. 1912 Von Laueobtainsthe firstx-raydiffractionpatternsby passingx-raysthrougha crystalof zincsulfide. W.L.Braggproposes a simplerelationship betweenan x-raydiffraction patternandthe arrangement of atomsin a crystalthat producethe pattern. 1926 5ummerobtainscrystals of the enzymeurease fromextracts of jackbeansanddemonstrates that proteinspossess catalyticactivity. 1931 Paulingpublishes hisfirstessays on 'TheNatureof the Chemical Bondidetailingthe rulesof covalentbonding. 1934 Bernaland Crowfootpresentthe firstdetailedx-raydiffractionpatternsof a proteinobtainedfrom crystalsof the enzymepepsin. 1935 Patterson develops an analytical methodfor determining interatomic spacings fromx-raydata. 1941 Astburyobtainsthe firstx-raydiffractionpatternof DNA. 1946 BlockandPurcelldescribe NMR. 19 5 1 Paulingand Coreyproposethe structure of a helicalconformation of a chainof L-amino acids-the crhelix-and the structure of the B sheet,bothof whichwerelaterfoundin manyproteins. 1953 Wallon and Crickproposethe double-helix modelof DNA,basedon x-raydiffraction patternsobtainedby Franklin and Wilkins. 1954 Perutzandcolleagues developheavy-atom methodsto solvethe phaseproblemin proteincrystallography. 1960 Kendrewdescribes the firstdetailedstructure of a protein(spermwhalemyoglobin) to a resolution of 0.2nm,and Perutzpresents a lower-resolution structure of the largerproteinhemoglobin. 1966 Phillipsdescribes the structure of lysozyme, the firstenzymeto haveitsstructure analyzed in detail. 1971 Jeenerproposes the useof two-dimensional NMR,andWuthrichandcolleagues firstusethe methodto solvea protein structure in the early1980s. 1976 Kimand Richand Klugandcolleagues describe the detailedthree-dimensional structure of IRNAdetermined by x-ray diffraction. 1977-'1978Holmesand Klugdetermine the structure of tobaccomosaicvirus(TMV), andHarrisonandRossman determine the structure of two smallspherical viruses. 1985 Michel,Deisenhofer andcolleagues determine the firststructure protein(a bacterial of a transmembrane reaction center)by.x-ray crystallography. Henderson andcolleagues obtainthe structure of bacteriorhodopsin, a transmembrane protein,by high-resolution electron-microscopy methodsbetween1975and'1990.

ANALYZING PROTEINS Score = 399 bits (1025), Expect = e-111 = L98/290 (58t), Positives = 24L/290 (82t), Identities

531

Figure8-30 Resultsof a BLASTsearch. can be searched to Sequence databases find similaraminoacidor nucleicacid I.IE}IFQKVEKIGEGTYGWYKARNKLTGEWAI,KKIRLDTETEGVPSTATREISI,I,KEI.NH 116 QUETy:57 Here,a searchfor proteins sequences. ME ++KVEXIGEGTYGWYKA +K T E +AI,KKIRI,+ E EGVPSTAIREISLI,RE+NH regulatory similarto the humancell-cycle Sbjct ! 1 MEOYEKVEKIGEGTYGWYKALDK.ATI{ETIAI,KKTRLEOEDEGVPSTAIREISLI,KEMNE 6O proteinCdc2(Query)locatesmaizeCdc2 OUETY! 117 PNIVKIIIDVIHTEIIKLYLVFEFLSODLKKFUDASTI,IKTPLPIIKSYLFQLLOGLAFCES 175 (Sbjcf)which is 680loidentical(and82olo NIV+L DV+E+E ++YTVFE+IJ DI'KKFUD+ LIKSYL+O+IJ G+A+CES similad to humanCdc2in its aminoacid SbJCt! 51'GNIVRIEDWI{SEERI,YLVFBYI,EITDLKKFMDSEWFNXSIflTLIKSYI,YQII,EGVAYCHS 120 Thealignmentbeginsat residue sequence. that 57 of the Queryprotein,suggesting OUETYt I 7 7 HRWERDLKPONI,I.INENKAIKLADFGLAXAFGVPVRTYTEEWTI,WYRAPEII,I,CdKE2 35 ERWHRDIJKPO$LLI+ A+KI'ADFGLARJAFG+PVRT+TEEV\ITLYIYRAPEII'I'G + the humanproteinhasan N-terminal Sbjct: 121 IIRVI.ERDLKPONLI,IDftM}IATKLADFGLARAFGIPVRTFTHEWTLWYRAPEILLGIIR&180 regionthat is absentfrom the maize protein.The greenblocksindicate 295 QUETY:235 YST*VDIIVSLGCIFAEUVIIRR&FPGDSEIDOLFRIFRI,T,GTPDE$VITPGUTSUPDYKSS differencesin sequence,and the yel/owbar YST VD+YIS+GCIFAEMV++ I,FPGDSEID+LF+IFR I,GTP+E I{PGV+ +PD+K + sbjctr 181 YSXWDVWSVGCIFAEWHQKSTFPGDSEIDELFKIFRXLGTPNEO6TVPGVSfi,PDFKIDA when the two 240 the similarities: summarizes the areidentical, aminoacidsequences QUETY! 2 9 5 FPKIfi&ODF.SS/VPST,DBOGN$LLSO}TTSYDPNKRISAKIAIAIIPFFQDV 3 4 5 conservative aminoacid residue is shown; FP+IV OD + vvP IJD G I'I'S+}TL Y+P+KRI+A+ A! E +F+D+ areindicatedby a plussign substitutions sb j ct : 2 4 1 FPRIIO*ODI.ATWPIEDS*61IALLSKIT{LRYEPSKRITAR0ATIHBYFKDL 2 9 0 (+).Onlyone smallgap hasbeen introduced-indicated by the redarrow at determines protein structure, and structure dictates biochemical function, proposition194in the Querysequence-to teins that share a similar amino acid sequence usually have the same structure maximally. The alignthe two sequences which is expressed and usually perform similar biochemical functions, even when they are found in alignment score(Score), in two differenttypes of units,takesinto distantly related organisms. In modern cell biology, the study of a newly discovand account oenaltiesfor substitutions protein proteins ered usually begins with a search for previously characterized gaps;the higherthe alignmentscore,the that are similar in their amino acid sequences. of the betterthe match.Thesignificance Searching a collection of known sequences for homologous genes or pro(E) alignmentis reflectedin the Expectation teins is typically done over the World Wide Web, and it simply involves selecting how oftena match value,which specifies a databaseand entering the desired sequence.A sequencealignment programthis good would be expectedto occurby chance. The lowerthe E value,the more the most popular are BI,AST and FASTA-scans the database for similar the match;the extremelylow significant sequences by sliding the submitted sequence along the archived sequences (e-111) indicatescertain value here until a cluster of residues falls into full or partial alignment (Figure 8-30). The E valuesmuch higherthan 0.1 significance. results of even a complex search-which can be performed on either a For areunlikelyto reflecttrue relatedness. nucleotide or an amino acid sequence-are returnedwithin minutes. Such comexample,an E valueof 0.1meansthereis a parisons can predict the functions of individual proteins, families of proteins, or 1 in 10 likelihoodthat sucha matchwould even most of the protein complement of a newly sequenced organism. arisesolelyby chance. €tpa -

L/290

As was explained in Chapter 3, many proteins that adopt the same conformation and have related functions are too distantly related to be identified as clearly homologous from a comparison of their amino acid sequencesalone (see Figure 3-13). Thus, an ability to reliably predict the three dimensional structure of a protein from its amino acid sequencewould improve our ability to infer protein function from the sequence information in genomic databases.In recent years,major progresshas been made in predicting the precise structure of a protein. These predictions are based,in part, on our knowledge of tens of thousands of protein structures that have already been determined by x-ray crystallography and NMR spectroscopy and, in part, on computations using our knowledge of the physical forces acting on the atoms. However, it remains a substantial and important challenge to predict the structures of proteins that are large or have multiple domains, or to predict structures at the very high levels of resolution needed to assist in computer-based drug discovery. \.A/hilefinding homologous sequencesand structures for a new protein will provide many clues about its function, it is usually necessary to test these insights through direct experimentation. However, the clues generated from sequence comparisons typically point the investigator in the correct experimental direction, and their use has therefore become one of the most important strategiesin modern cell biology.

Summary Most proteinsfunction in concert with other proteins,and many methodsexistfor identifying and studying protein-protein interactions.Small-molecule inhibitors allow thefunctions of proteins they act upon to be studied in liuing cells.Becauseproteinswith similar structuresoften hauesimilar functions, the biochemicalactiuifitof a

532

Chapter8: ManipulatingProteins,DNA,and RNA

protein can often be predicted by searchingdatabasesfor preuiouslycharacterizedproteins that are similar in their amino acid seauences.

ANALYZING AND MANIPULATING DNA Until the early 1970s,DNAwas the most difficult biological molecule for the biochemist to analyze.Enormously long and chemically monotonous, the string of nucleotides that forms the genetic material of an organism could be examined only indirectly, by protein or RNA sequencing or by genetic analysis.Today, the situation has changed entirely. From being the most difficult macromolecule of the cell to analyze, DNA has become the easiest.It is now possible to isolate a specific region of almost any genome, to produce a virtually unlimited number of copies of it, and to determine the sequence of its nucleotides in a few hours. At the height of the Human Genome Project, large facilities with automated machines were generating DNA sequences at the rate of 1000 nucleotides per second, around the clock. By related techniques, an isolated gene can be altered (engineered) at will and transferred back into the germ line of an animal or plant, so as to become a functional and heritable part of the organism'sgenome. These technical breakthroughs in genetic engineering-the ability to manipulate DNA with precision in a test tube or an organism-have had a dramatic impact on all aspects of cell biology by facilitating the study of cells and their macromolecules in previously unimagined ways. Recombinant DNA technology comprises a mixture of techniques, some newly developed and some borrowed from other fields such as microbial genetics (Table 8-3). Central to the technology are the following key techniques: l. Cleavage of DNA at specific sites by restriction nucleases,which greatly facilitates the isolation and manipulation of individual genes. 2. DNA ligation, which makes it possible to design and construct DNA molecules that are not found in nature. 3. DNA cloning through the use of either cloning vectors or the polymerase chain reaction, in which a portion of DNA is repeatedly copied to generate many billions of identical molecules. 4. Nucleic acid hybridization, which makes it possible to find a specific sequenceof DNA or RNA with great accuracy and sensitivity on the basis of its ability to selectivelybind a complementary nucleic acid sequence. 5. Rapid determination of the sequence of nucleotides of any DNA (even entire genomes), making it possible to identify genes and to deduce the amino acid sequence of the proteins they encode. 6. Simultaneous monitoring of the level of mRNA produced by every gene in a cell, using nucleic acid microarrays, in which tens of thousands of hybridization reactions take place simultaneously. In this section, we describe each of these basic techniques, which together have revolutionized the study of cell biology.

Restriction Nucleases Cut LargeDNAMolecules into Fragments unlike a protein, a gene does not exist as a discrete entity in cells, but rather as a small region of a much longer DNA molecule. Although the DNA molecules in a cell can be randomly broken into small pieces by mechanical force, a fragment containing a single gene in a mammalian genome would still be only one among a hundred thousand or more DNA fragments, indistinguishable in their average size. How could such a gene be purified? Becauseall DNA molecules consist of an approximately equal mixture of the same four nucleotides, they cannot be readily separated, as proteins can, on the basis of their different charges and binding properties. The solution to all of these problems began to emerge with the discovery of restriction nucleases.These enzymes,which can be purified from bacteria, cut the DNA double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long double-stranded DNA molecule into frasments of

ANALYZING ANDMANIPULATING DNA

533

Table8-3 SomeMajorStepsin the Development Technology of Recombinant DNAandTransgenic obtainedfroma nearby Miescher bandages firstisolates DNAfromwhitebloodcellsharvested frompus-soaked hospital. transformation. duringbacterial 1944 Averyprovides the geneticinformation evidence that DNA,ratherthanprotein,carries Franklin and Wilkins. propose x-ray results of 1953 Watsonand Crick basedon modelfor DNAstructure the double-helix probes. 1955 Kornbergdiscovers DNApolymerase, the enzymenow usedto producelabeledDNA of nucleicacidhydridization 1961 Marmurand Dotydiscover andfeasibility the specificity DNArenaturation, establishing reactions. andusein nucleases, leadingto theirpurification 1962 Arberprovides the firstevidence for the existence of DNArestriction DNAsequence characterization by Nathansand H.Smith. 1966 Nirenberg, Ochoa,andKhoranaelucidate the geneticcode. 1967 together. Gellertdiscovers DNAligase, the enzymeusedto join DNAfragments at Stanford 1972-1973 DNAcloningtechniques of Boyer,Cohen,Berg,andtheircolleagues aredeveloped by the laboratories University andthe University of California at SanFrancisco. gel-transfer DNAsequences. 1975 Southerndevelops hybridization for the detectionof specific methods. 1975-1977 Sangerand BarrellandMaxamand GilbertdeveloprapidDNA-sequencing fruitflies. 1981-1982 Palmiterand Brinsterproducetransgenic mice;Spradlingand Rubinproducetransgenic 1982 GenBank, at LosAlamosNationalLaboratory. NIH'spublicgeneticsequence database, isestablished 1985 Mullisandco-workers inventthe polymerase chainreaction(PCR). stemcells. in mouseembryonic 1987 Capecchi targetedgenereplacement andSmithiesintroducemethodsfor performing andstudyingproteininteractions. 1989 Fieldsand Songdevelopthe yeasttwo-hybridsystemfor identifying mapsof of DNAthat areusedto makephysical 1989 Olsonandcolleagues describe sequence-tagged sites,uniquestretches humanchromosomes. for homologybetweenDNAandproteinsequences. 1990 Lipmanandcolleagues release BLAST, an algorithmusedto search to carrylargepiecesof cloned BACs, chromosomes, 1990 artificial Simonandcolleagues studyhowto efficiently usebacterial humanDNAfor sequencing. technology. 1991 Hoodand Hunkapillar introducenewautomatedDNAsequence influenzae. 1995 Venterand colleagues sequence the firstcompletegenome,that of the bacteriumHaemophilus of a of the firstgenomesequence the completion 1996 Goffeauandan international announce consortium of researchers 1869

eucaryote, the yeast Saccharo mycescerevisioe. which allow the simultaneousmonitoring 1996-1997 Lockhart and colleaguesand Brown and DeRisiproduceDNA microarrays,

1998 2001 2004

of thousands of genes. the nematode organism, of a multicellular producethe firstcompletesequence SulstonandWaterston andcolleagues w orm Caenorhabditiselegans. Consortia of researchers announce the completion of the drafthumangenomesequence. Publication of the"finished" humangenomesequence.

strictly defined sizes. Different restriction nucleases have diff'erent sequence specificities, and it is relatively simple to find an enzyme that can create a DNA fragment that includes a particular gene.The size of the DNA fragment can then be used as a basis for partial purification of the gene from a mixture. Different species of bacteria make different restriction nucleases, which protect them from viruses by degrading incoming viral DNA. Each bacterial nuclease recognizes a specific sequence of four to eight nucleotides in DNA. These sequences,where they occur in the genome of the bacterium itself, are protected from cleavageby methylation at an A or a C nucleotide; the sequences in foreign DNA are generally not methylated and so are cleavedby the restriction nucleases.Large numbers of restriction nucleaseshave been purified from various species of bacteria; several hundred, most of which recognize different nucleotide sequences,are now available commercially. Some restriction nucleases produce staggered cuts, which leave short single-stranded tails at the two ends of each fragment (Figure 8-31). Ends of this type are known as cohesiueends,as each tail can form complementary base pairs with the tail at any other end produced by the same enzyme (Figure 8-32). The cohesive ends generated by restriction enzymes allow any two DNA fragments to be easily joined together, as long as the fragments were generated with the same restriction nuclease (or with another nuclease that produces the same cohesive ends). DNA molecules produced by splicing together two or more DNA fragments are called recombinant DNA molecules.

534

Chapter8: ManipulatingProteins, DNA,and RNA Figure8-31 The DNA nucleotidesequencesrecognizedby four widely usedrestrictionnucleases. As in the examplesshown,suchsequences are (thatis,the nucleotide often sixbasepairslong and"palindromic" sequenceis the sameif the helixis turnedby 180degreesaroundthe centerof the shortregionof helixthat is recognized). Theenzymescut the two strandsof DNAat or nearthe recognitionsequence. Forthe genes encodingsomeenzymes, suchas Hpal,the cleavageleavesblunt ends;for others,suchas EcoRl, Hindlll,and Pstl,the cleavageis staggeredand createscohesiveends.Restriction nucleases areobtainedfrom various parainfluenzae, speciesof bacteria:Hpal is from Haemophilus EcoRlis lrom Escherichia colt,Hindlll isfrom Haemophilus influenzae, and Pstlis from Providenciastuartii.

CUT EcoRl

s'fl

GelElectrophoresis Separates DNAMolecules of DifferentSizes The same types of gel electrophoresismethods that have proved so useful in the analysis of proteins can determine the length and purity of DNA molecules. The procedure is actually simpler than for proteins: because each nucleotide in a nucleic acid molecule already carries a single negative charge (on the phosphate group), there is no need to add the negatively charged detergent SDS that is required to make protein molecules move uniformly toward the positive electrode. For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels allow the separation of molecules that differ in length by as little as a single nucleotide (Figure 8-33A). The pores in polyacrylamide gels, however, are too small to permit very large DNA molecules to pass; to separate these by size, the much more porous gels formed by dilute solutions of agarose (a polysaccharide isolated from seaweed) are used (Figure 8-338). These DNA separation methods are widely used for both analytical and preparative purposes. A variation of agarose-gel electrophoresis, called pulsed-field gel electrophoresis,makes it possible to separate even extremely long DNA molecules. Ordinary gel electrophoresisfails to separatesuch molecules becausethe steady electric field stretches them out so that they travel end-first through the gel in snakelike configurations at a rate that is independent of their length. In pulsedfield gel electrophoresis, by contrast, the direction of the electric field changes periodically, which forces the molecules to reorient before continuing to move snakelike through the gel. This reorientation takes much more time for larger molecules, so that longer molecules move more slowly than shorter ones. As a consequence,even entire bacterial or yeast chromosomes separateinto discrete bands in pulsed-field gels and so can be sorted and identified on the basis of their size (Figure 8-33C). Although a typical mammalian chromosome of 108 base pairs is too large to be sorted even in this way, large segments of these chromosomes are readily separated and identified if the chromosomal DNA is first cut with a restriction nuclease selected to recognize sequencesthat occur only rarely (once every 10,000or more nucleotide pairs). The DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or stained in some way. one sensitivemethod of staining DNA is to expose it to the dye ethidium bromide, which fluoresces under ultraviolet light when it is bound to DNA (seeFigure 8-338,c). An even more sensitive detection method incorporates a radioisotope into the DNA molecules before electrophoresis;32Pis often used as it can be incorporated into DNA phosphates and emits an energetic B particle that is easily detected by autoradiography,as in Figure 8-33. (For a discussion ofradioisotopes, see p. 601).

s', 5

s',

3',

s', 5',

anneatino Figure8-32 The use of restrictionnucleasesto produce DNA fragments that can be easilyjoined together,Fragments with the samecohesive endscan readilyjoin by complementary base-pairing betweentheir cohesiveends,as illustrated. Thetwo DNAfragmentsthat join in this examplewere both producedby the EcoRlrestrictionnuclease,whereas the three other fragmentswere producedby differentrestrictionnucleases that generateddifferentcohesiveends(seeFigure8-31). Blunt-ended fragments, likethosegeneratedby Hpal(seeFigure8-31),can be spliced togetherwith more difficulty.

I 5',

5 l

535

ANALYZING AND MANIPULATING DNA Figure8-33 Gel electrophoresis techniquesfor separatingDNA isfrom moleculesby size.In the threeexamplesshown,electrophoresis

taneS 4 123

jfffi l",J""n'"i,1,1,1,,.,, llSl?"131'.[;lTii:lll"o:"',",.:-,i:,xiT; poreswas usedto fractionatesingle-stranded DNA.In the sizerange10to 500 nucleotides, DNAmolecules that differin sizeby only a single nucteotidecan be separated from eachother.In the example,the four lanes represent setsof DNAmoleculessynthesized in the courseof a DNAsequencingprocedure. The DNAto be sequenced hasbeenartificially replicated from a fixedstartsiteup to a variablestoppingpoint,producing a setof partialreplicas of differinglengths.(Figure8-50 explainshow such setsof partialreplicas aresynthesized.) Lane1 showsall the partialreplicas that terminatein a G,lane2 all thosethat terminatein an A, lane3 all those that terminatein a I and lane4 all thosethat terminatein a C.Sincethe DNAmoleculesusedin thesereactions their positions wereradiolabeled, can be determinedby autoradiography, as shown. (B)An agarosegel with medium-sized poreswasusedto separate doublestrandedDNAmolecules. Thismethodis mostusefulin the sizerange300 to 10,000nucleotidepairs.TheseDNAmolecules arefragmentsproduced and nuclease, by cleavingthe genomeof a bacterialviruswith a restriction they havebeendetectedby theirfluorescence when stainedwith the dye ethidiumbromide.(C)Thetechniqueof pulsed-field agarosegel electrophoresis was usedto separate16 differentyeast(Saccharomyces cerevisiae) chromosomes, which rangein sizefrom 220p00to 2.5 million as largeas nucleotidepairs.TheDNAwasstainedas in (B).DNAmolecules 107nucleotidepairscan be separated in this way.(A,courtesyof Leander Laufferand PeterWalter;B,courtesyof KenKreuzer;C,from D.Vollrathand R.W.Davis,NucleicAcidsRes.15:7865-7876, 1987.With permissionfrom OxfordUniversitvPress.)

& @

o

o

=

& PurifiedDNAMolecules Labeledwith CanBeSpecifically Radioisotopes or ChemicalMarkersin vitro

12

q&*

A

15 7

t&1

TWo procedures are widely used to label isolated DNA molecules. In the first method, a DNA polymerase copies the DNA in the presence of nucleotides that are either radioactive (usually labeled with 32P)or chemically tagged (Figure 8-34A). In this way, "DNA probes" containing many labeled nucleotides can be produced for nucleic acid hybridization reactions (discussedbelow). The second procedure uses the bacteriophage enzyme pol)'nucleotide kinase to transfer a single 32P-labeledphosphate from AIP to the 5' end of each DNA chain (Figure 8-348). Because only one 32Patom is incorporated by the kinase into each DNA strand, the DNA molecules labeled in this way are often not radioactive enough to be used as DNA probes; becausethey are labeled at only one end, however, they have been invaluable for other applications, including DNA footprinting, as discussedin Chapter 7. Radioactivelabeling methods are being replaced by Iabeling with molecules that can be detected chemically or through fluorescence.To produce such nonradioactive DNA molecules, specially modified nucleotide precursors are used (Figure B-34C).A DNA molecule made in this way is allowed to bind to its complementary DNA sequence by hybridization, as discussed in the next section, and is then detected with an antibody (or other ligand) that specifically recognizes its modified side chain (Figure 8-35).

Wayto NucleicAcidHybridization Reactions Providea Sensitive DetectSpecificNucleotideSequences \.A/henan aqueous solution of DNA is heated at 100'C or exposed to a very high pH (pH ' 13), the complementary base pairs that normally hold the two strands of the double helix together are disrupted and the double helix rapidly dissociates into two single strands. This process, called DNA denaturation, was for many years thought to be irreversible. In 1961,however, it was discovered that complementary single strands of DNA readily re-form double helices by a process called hybridization (also called Dl/A renaturation) if they are kept for a

w

950,000

a

wast '?e*

'!659' ... i,h**s| '&':

wt:

E f

c o

nucleotide parrs

Ero ,qkf.

*

5 10 , 0 0 0 v f

8 9$,

rry**, ,wl '*i$ :8Ai!i '{*#r,

(,{i

9 3 6 1

536

Chapter8: ManipulatingProteins, DNA,and RNA

(A)

(B)

53',

f

3',

s,

5

D N A l a b e l e da t 5 ' e n d s w i t h p-olynucleotidekinaseand "P-labeled ATP

d e n a t u r ea n d a n n e a lw i t h mixture of hexanucleotides

+

u restrictionnucleasecuts DNA helix into two differentsizedfragments

5

and I aOOorunpolymerase labeled nucleotides I separationby gel electrophoresis

35

+ 3',

5' DNA polymeraseincorporateslabeled n u c l e o t i d e sr,e s u l t i n gi n a p o p u l a t i o no f D N A m o l e c u l e tsh a t c o n t a i nl a b e l e de x a m o l e so f a l l sequenceson both strands

the desiredDNA fragment with a singlestrand labeled at one end

t h i s r e g i o ns t i l l

ooo ililil

o--P-O-P-O-P-O

ltl o-

o-

o-

prolonged period at 65"c. similar hybridization reactions can occur between any two single-stranded nucleic acid chains (DNA/DNA, RNA/RNA, or RNA/DNA), provided that they have complementary nucleotide sequences. These specific hybridization reactions are widely used to detect and characterize specific nucleotide sequencesin both RNA and DNA molecules. Single-stranded DNA molecules used to detect complementary sequences are known as probes; these molecules, which carry radioactive or chemical markers to facilitate their detection, can range from fifteen to thousands of nucleotides long. Hybridization reactions using DNA probes are so sensitive and selective that they can detect complementary sequences present at a concentration as low as one molecule per cell. It is thus possible to determine how many copies of any DNA sequence are present in a particular DNA sample. The same technique can be used to searchfor related but nonidentical genes.To find a gene of interest in an organism whose genome has not yet been sequenced,for example, a portion of a knor,rrngene can be used as a probe (Figure 8-36).

Figure8-34 Methods for labeling DNA mofeculesinvitro. (A)A purifiedDNA polymerase enzymelabelsall the nucleotides in a DNAmoleculeand can therebyproducehighlyradioactive DNA probes.(B)Polynucleotide kinaselabels only the 5'ends of DNAstrands; therefore, when labelingis followed by restriction nuclease cleavage, as shown,DNA molecules containinga single5'-endlabeledstrandcan be readilyobtained. (C)The method in (A)is alsousedto producenonradioactive DNAmolecules that carrya specificchemicalmarkerthat can be detectedwith an appropriate antibody.The modified nucleotideshown can be incorporatedinto DNA by DNA polymerase, allowingthe DNAmoleculeto serveas a probe that can be readily detected.The baseon the nucleoside triphosphate shownis an analogof thymine,in whichthe methylgroupon T hasbeen replacedby a spacerarm linked to the plantsteroiddigoxigenin. An antidigoxygeninantibodycoupledto a visible markersuchas a fluorescentdye is usedto visualize the probe.Otherchemicallabels suchas biotin can be attachedto nucleotides and usedin essentiallv the sameway.

ANALYZING AND MANIPULATING DNA

537

Figure8-35In situhybridization to locatespecificgeneson chromosomes. Here,sixdifferentDNAprobeshavebeenusedto markthe locations of theirrespective nucleotide seouences on humanchromosome 5 at metaphase. Theprobeshavebeenchemically labeled anddetected withfluorescent antibodies. Bothcopies of chromosome 5 areshown, aligned sideby side.Eachprobeproduces two dotson eachchromosome, sincea metaphase hasreplicated itsDNAandtherefore chromosome (Courtesy contains two identical DNAhelices. of DavidC.Ward.)

Alternatively, DNA probes can be used in hybridization reactions with RNA rather than DNA to find out whether a cell is expressinga given gene. In this case a DNA probe that contains part of the gene's sequence is hybridized with RNA purified from the cell in question to see whether the RNA includes nucleotide sequencesmatching the probe DNA and, if so, in what quantities. In somewhat more elaborate procedures, the DNA probe is treated with specific nucleases after the hybridization is complete, to determine the exact regions of the DNA probe that have paired with the RNA molecules. One can thereby determine the start and stop sites for RNA transcription, as well as the precise boundaries of the intron and exon sequencesin a gene (FigureS-37). Today, the positions of intron/exon boundaries are usually determined by sequencing t},:'e complementary DNA (cDNA) sequences that represent the mRNAs expressedin a cell and comparing them with the nucleotide sequenceof the genome.We describe later how cDNAs are prepared from mRNAs. The hybridization of DNA probes to RNAs allows one to determine whether or not a particular gene is being transcribed; moreover, when the expression of a gene changes,one can determine whether the change is due to transcriptional or post-transcriptional controls (seeFigure 7-92). These tests ofgene expression were initially performed with one DNA probe at a time. DNA microarrays now allow the simultaneous monitoring of hundreds or thousands of genes at a time, as we discuss later. Hybridization methods are in such wide use in cell biology today that it is difficult to imagine how we could study gene structure and expressionwithout them.

single-stranded

B Gl.__,f

/ mixture of sing le-stranded DNAmolecules

,l/Z

\

\

/a-'\**..., F

h y b r i d i z a t i o ni n 5 0 % formamide at 42"C

only A forms stable d o u b l eh e l i x

A, C, and E all form s t a b l ed o u b l e h e l i c e s

Figure8-36 Stringentversus nonstringent hybridizationconditions. To usea DNA probeto find an identical conditions match,stringenthybridization are used;the reactiontemperatureis kept just a few degreesbelow that at which a perfectDNA helixdenaturesin the solventused(itsmeltingtemperoture), so that all imperfecthelicesformed are Whena DNAprobeis being unstable. usedto find DNAswith related,as well as lessstringent sequences, identical, is conditionsareused;hybridization performedat a lower temperature,which allowseven imperfectlypaireddouble helicesto form. Only the lowerconditionscan temperaturehybridization be usedto searchfor genesthat are but relatedto geneA (Cand nonidentical E in this example).

538

Chapter8: ManipulatingProteins, DNA,and RNA exon1

intron

exon2

5', 3',

TREATMENT I sr ruucleasr

degraded DNA

I D E G R A D ESST N G L E - S T R A N D E D I N U C L E IA CC I D S 3' 5',

5' 3',

untreated o o c

control *-

eXOn 2

--

exon 1 a o

I a l k a l i n e a g a r o s eg e l

Northernand SouthernBlottingFacilitate Hybridization with Electrophoretically Separated NucleicAcidMolecules In a complex mixture of nucleic acids, DNA probes are often used to detect only those molecules with sequences that are complementary to all or part of the probe. Gel electrophoresiscan be used to fractionate the many different RNA or DNA molecules in a crude mixture according to their size before the hybridization reaction is performed; if the probe binds to molecules of only one or a few sizes, one can be certain that the hybridization was indeed specific. Moreover, the size information obtained can be invaluable in itself. An example illustrates this point. Suppose that one wishes to determine the nature of the defect in a mutant mouse that produces abnormally low amounts of albumin, a protein that liver cells normally secreteinto the blood in large amounts. First, one collects identical samples of liver tissue from mutant and normal mice (the latter serving as controls) and disrupts the cells in a strong detergent to inactivate nucleasesthat might otherwise degrade the nucleic acids. Next, one separates the RNA and DNA from all of the other cell components: the proteins present are completely denatured and removed by repeated extractions with phenol-a potent organic solvent that is partly miscible with water; the nucleic acids, which remain in the aqueous phase, are then precipitated with alcohol to separate them from the small molecules of the cell. Then, one separatesthe DNA from the RNA by their different solubilities in alcohols and degradesany contaminating nucleic acid of the unwanted type by treatment with a highly specific enzyme-either an RNase or a DNase.The mRNAs are typically separatedfrom bulk RNA by retention on a chromatography column that specifically binds the poly-A tails of mRNAs. To analyze the albumin-encoding mRNAs, a technique called Northern blotting is used. First, the intact mRNA molecules purified from mutant and control liver cells are fractionated on the basis oftheir sizesinto a seriesofbands by gel electrophoresis. Then, to make the RNA molecules accessible to DNA probes, a replica of the pattern of RNA bands on the gel is made by transferring ("blotting"l the fractionated RNA molecules onto a sheet of nitrocellulose or nylon paper. The paper is then incubated in a solution containing a labeled DNA probe, the sequence of which corresponds to part of the template strand that

Figure8-37 The useof nucleicacid hybridizationto determinethe region of a clonedDNAfragmentthat is presentin an mRNAmolecule.The methodshownreouiresa nuclease that cutsthe DNAchainonly whereit is not base-paired to a complementary RNA chain.The oositionsof the intronsin genesare mappedby the eucaryotic methodshown.Forthis type of analysis, the DNAis electrophoresed througha denaturingagarosegel,which causesit to migrateas single-stranded molecules. Thelocationof eachend of an RNA moleculecan be determinedusinq s i m i l am r ethods.

539

ANALYZING AND MANIPULATING DNA r e m o v en i t r o c e l l u l o s e p a p e rw i t h t i g h t l y b o u n d n u c l e i ca c i d s

stackof paper towels

(c)

(B)

n itrocelIulose paper

l a b e l e dR N Ao r D N Ao f k n o w n s i z e s s e r v i n ga s s i z em a r k e r s agarose gel spon9e a l k a l is o l u t i o n N U C L E IA CC I D SS E P A R A T E D A C C O R D I NTGO S I Z EB Y A G A R O S E G E LE L E C T R O P H O R E S I S

CC I D SB L O T T E D S E P A R A T ENDU C L E IA B YS U C T I O N O N T ON I T R O C E L L U L OPSAEP E R RH R O U G H G E LA N D P A P E R O F B U F F ET

Figure8-38 Detectionof specificRNAor DNA moleculesby gel-transfer hybridization.In this example,the DNAprobeis detectedby its radioactivity. methods DNAprobesdetectedby chemicalor fluorescence arealsowidelyused(seeFigure8-34).(A)A mixtureof eithersingleDNA strandedRNAmolecules(Northern blotting)orthe double-stranded fragmentscreatedby restrictionnucleasetreatment(Southernblotting)is (B)A sheetof separated accordingto lengthby electrophoresis. RNAor nitrocellulose or nylonpaperis laidoverthe gel,and the separated DNAfragmentsaretransferred to the sheetby blotting.(C)The nitrocellulose sheetis carefullypeeledoff the gel.(D)The sheetcontaining the bound nucleicacidsis placedin a sealedplasticbag togetherwith a bufferedsaltsolutioncontaininga radioactively labeledDNAprobe.The sheetis exposedto a labeledDNAprobefor a prolongedperiodunder (E)The sheetis removedfrom the bag and conditionsfavoringhybridization. washedthoroughly,so that only probemolecules that havehybridizedto the RNAor DNAimmobilizedon the paperremainattached.After autoradiography, the DNAthat hashybridizedto the labeledprobeshows up as bandson the autoradiograph DNAmolecules ForSouthernblotting,the strandsof the double-stranded process; this is on the papermust be separated beforethe hybridization done by exposingthe DNAto alkalinedenaturingconditionsafterthe gel hasbeenrun (not shown). produces albumin mRNA. The RNA molecules that hybridize to the labeled DNA probe on the paper (because they are complementary to part of the normal albumin gene sequence) are then located by detecting the bound probe by autoradiography or by chemical means (Figure 8-38). The sizes of the hybridized RNA molecules can be determined by reference to RNA standards of

known sizesthat are electrophoresedside by side with the experimental sample. In this way, one might discover that Iiver cells from the mutant mice make albumin mRNA in normal amounts and of normal size; alternatively,you might find that they make it in normal size but in greatly reduced amounts. Another possibility is that the mutant albumin nRNA molecules are abnormally short; in this casethe gel blot could be retestedwith a seriesof shorter DNA probes, each corresponding to small portions of the gene, to revealwhich part of the normal RNA ls mrsslng. The original gel-transfer hybridization method, called Southern blotting, analyzes DNA rather than RNA. (It was named after its inventor, and the Northern andWestern blotting techniques were named with referenceto it.) Here, isoIated DNA is flrst cut into readily separablefragments with restriction nucleases. The double-stranded fragments are then separated on the basis of size by gel electrophoresis, and those complementary to a DNA probe are identified by blotting and hybridization, as just described for RNA (seeFigure B-38). To characterize the structure of the albumin gene in the mutant mice, an albumin-specific DNA probe would be used to construct a detailed restriction map of the genome in the region of the albumin gene (such a map consists of the pattern of DNA fragments produced by various restriction nucleases).From this map one

TO HYBRIDIZED LABELED PROBE DNA BANDS COMPLEMENTARY BY AUTORADIOGRAPHY VISUALIZED (E)

positions of labeled marKers

labeled bands

540

Chapter8: ManipulatingProteins,DNA,and RNA

could determine if the albumin gene has been rearranged in the defective animals-for example, by the deletion or the insertion of a short DNA sequence; most single-basechanges,however, could not be detected in this way.

GenesCanBeClonedUsingDNALibraries Any DNA fragment can be cloned. In molecular biology, the term DNA cloning is used in two senses.In one sense,it literally refers to the act of making many identical copies of a DNA molecule-the amplification of a particular DNA sequence.However, the term also describes the isolation of a particular stretch of DNA (often a particular gene) from the rest of a cell's DNA, becausethis isolation is greatly facilitated by making many identical copies of the DNA of interest. As discussed earlier in this chapter, cloning, particularly when used in the context of developmental biology, can also refer to the generation of many genetically identical cells starting from a single cell or even to the generation of genetically identical organisms. In all cases,cloning refers to the act of making many genetically identical copies; in this section, we will use the term cloning (or DNA cloning or gene cloning) to refer to methods designed to generate many identical copies of a segment of nucleic acid. DNA cloning in its most general sensecan be accomplished in severalways. The simplest involves inserting a particular fragment of DNA into the purified DNA genome of a self-replicating genetic element-generally a virus or a plasmid. A DNA fragment containing a human gene, for example, can be joined in a test tube to the chromosome of a bacterial virus, and the new recombinant DNA molecule can then be introduced into a bacterial cell, where the inserted DNA fragment will be replicated along with the DNA of the virus. Starting with only one such recombinant DNA molecule that infects a single cell, the normal replication mechanisms of the virus can produce more than l012identical virus DNA molecules in less than a day, thereby amplifying the amount of the inserted human DNA fragment by the same factor. A virus or plasmid used in this way is knor.tmas a cloning uector,and the DNA propagated by insertion into it is said to have been cloned. To isolate a specific gene, one often begins by constructing a Dl/A librarya comprehensive collection of cloned DNA fragments from a cell, tissue, or organism. This library includes (one hopes) at least one fragment that contains the gene of interest. Libraries can be constructed with either a virus or a plasmid vector and are generally housed in a population of bacterial cells.The principles underlying the methods used for cloning genes are the same for either tlpe of cloning vector, although the details may differ. Today,most cloning is performed with plasmid vectors. The plasmid vectors most widely used for gene cloning are small circular molecules of double-stranded DNA derived from larger plasmids that occur naturally in bacterial cells. They generally account for only a minor fraction of the total host bacterial cell DNA, but they can easily be separated owing to their small size from chromosomal DNA molecules, which are large and precipitate as a pellet upon centrifugation. For use as cloning vectors, the purified plasmid DNA circles are first cut with a restriction nuclease to create linear DNA molecules. The genomic DNA to be used in constructing the library is cut with the same restriction nuclease,and the resulting restriction fragments (including those containing the gene to be cloned) are then added to the cut plasmids and annealed via their cohesive ends to form recombinant DNA circles. These recombinant molecules containing foreign DNA inserts are then covalently sealedwith the enzyme DNA ligase (Figure 8-39). In the next step in preparing the library, the recombinant DNA circles are introduced into bacterial cells that have been made transiently permeable to DNA. These bacterial cells are now said to be transfectedwith the plasmids. As the cells grow and divide, doubling in number every 30 minutes, the recombinant plasmids also replicate to produce an enormous number of copies of DNA circles containing the foreign DNA (Figure 8-40). Many bacterial plasmids carry genes for antibiotic resistance (discussedin chapter 24), a property that can be

,

ANALYZING AND MANIPULATING DNA c i r c ul ar dou ble-stranded p l a s m i dD N A ( c l o n i n gv e c t o r )

.. 1';..

.

DNAfragment to be cloned

v

CLEAVAGE WITH RESTRICTION NUCLEASE

r e c o m b i n a nD t NA

.:

COVALENT LINKAGE BYDNALIGASE

'

?'

541

Figure8-39 The insertionof a DNA fragment into a bacterialplasmid with the enzymeDNA ligase.The plasmidis nuclease(in cut open with a restriction this caseone that oroducescohesive ends)and is mixedwith the DNA fragmentto be cloned(whichhasbeen preparedwith the samerestriction nuclease). DNAligaseand ATPareadded. and DNA Thecohesiveendsbase-pair, ligasesealsthe nicksin the DNA backbone,producinga complete recombinantDNAmolecule. (Micrographs courtesyof Huntington Potterand DavidDressler.)

200nm

200nm

exploited to select those cells that have been successfullytransfected;if the bacteria are grown in the presence of the antibiotic, only cells containing plasmids will survive. Each original bacterial cell that was initially transfected contains, in general, a different foreign DNA insert; this insert is inherited by all of the progeny cells of that bacterium, which together form a small colony in a culture dish. For many years, plasmids were used to clone fragments of DNA of 1000 to 30,000nucleotide pairs. Larger DNA fragments are more difficult to handle and were harder to clone. Then researchersbegan t o vse yeastartificial chromosomes (IACs), which could accommodate very large pieces of DNA (Figure 8-4f). Today,new plasmid vectors based on the naturally occurring F plasmid of E. coli are used to clone DNA fragments of 300,000to I million nucleotide pairs. Unlike smaller bacterial plasmids, the F plasmid-and its derivative, the bacterial artificial chromosome (BAC)-is present in only one or two copies per E. coli celI. The fact that BACs are kept in such low numbers in bacterial cells may contribute to their ability to maintain large cloned DNA sequencesstably: with only a few BACs present, it is less Iikely that the cloned DNA fragments will become scrambled by recombination with sequencescarried on other copies of the plasmid. Because of their stability, ability to accept large DNA inserts, and ease of handling, BACs are now the preferred vector for building DNA libraries of complex organisms-including those representing the human and mouse genomes.

TwoTypesof DNALibrariesServeDifferentPurposes Cleaving the entire genome of a cell with a specific restriction nuclease and cloning each fragment as just described produces a very large number of DNA fragments-on the order of a million for a mammalian genome. The fragments are distributed among millions of different colonies of transfected bacterial cells. d o ub l e - s t r a n d e d recombrnant p l a s m i dD N A i n t r o d u c e di n t o b a c t e r i acl e l l

i,

;.."',

. , "' \"; \l

1* bacteriaI cell

""r /2-,

,"\

"t

: c e l lc u l t u r ep r o d u c e s h u n d r e d so f m i l l i o n so f new bacteria

\

)-

m a n y c o p i e so f p u r i f i e d r e c o m b i n a np t l a s m i di s o l a t e d f r o m l y s e db a c t e r i acl e l l s

Figure8-40 The amplification of the DNAfragmentsinsertedinto a plasmid. To producelargeamountsof the DNAof the recombinantplasmidDNAin interest, Figure8-39 is introducedinto a whereit will bacteriumby transfection, replicatemanymillionsof timesasthe bacteriummultiplies.

542

Chapter8: ManipulatingProteins, DNA,and RNA

Y E A S TA R T I F I C I ACLH R O M O S O MVEE C T O R

H U M A ND N A

B TEL BamHl B a m HA l NDEcoRl DIGESTION TEI

A ORI CEN

B

TEL

rlm+K left arm

rioht arm

l a r g ec h r o m o s o m aflr a g m e n t s

DNALIGATION A N D Y E A S TC E L LT R A N S F O R M A T I O N TEL

B

A ORI CEN

Figure8-41 The makingof a yeast artificial chromosome(YAC).A YAC vectorallowsthe cloningof very large DNAmolecules. TEL,CEN,and ORIarethe telomere,centromere, and originof replicationsequences, respectively, for the yeast Saccharomycescerevisiae;all of theseare requiredto propagatethe YAC. BamHland EcoRlaresiteswherethe corresponding restrictionnucleases cut the DNAdoublehelix.The seouences denotedA and B encodeenzymesthat serveas selectable markersto allowthe easyisolationofyeastcellsthat have takenup the artificialchromosome. Because bacteriadividemore rapidly than yeasts,most large-scale cloning projectsnow useE.colias the meansfor amplifyingDNA.(Adaptedfrom D.T.Burke,G.F.Carleand M.V.Olson, Science236:806-812,1987.With permission from AAAS.)

TEL

I

5 . 5x 10 3 n u c l e o t i d ep a i r s

up to

'106 n u c l e o t i d ep a i r s

3 9 x 1 0 3n u c l e o t i d ep a i r s

Y E A S TA R T I F I C I ACLH R O M O S O MW E I T H I N S E R T EHDU M A N D N A

\A/hen working with BACs rather than typical plasmids, larger fragments can be inserted, and so fewer transfected bacterial cells are required to cover the genome. In either case, each of the colonies is composed of a clone of cells derived from a single ancestor cell, and therefore harbors many copies of a particular stretch of the fragmented genome (Figure 8-42). Such a plasmid is said to contain a genomic DNA clone, and the entire collection of plasmids is called a genomic DNA library. But because the genomic DNA is cut into fragments at random, only some fragments contain genes.Many of the genomic DNA clones obtained from the DNA of a higher eucaryotic cell contain only noncoding DNA, which, as we discussedin Chapter 4, makes up most of the DNA in such genomes. An alternative strategyis to begin the cloning processby selecting only those DNA sequencesthat are transcribed into mRNA and thus are presumed to correspond to protein-encoding genes.This is done by extracting the mRNA from cells and then making a DNA copy of each mRNA molecule present-a so-called complementary DNA, or cDNA. The copying reaction is catalyzed by the reverse transcriptase enzyme of retroviruses,which synthesizesa complementary DNA chain on an RNA template. The single-stranded cDNA molecules synthesizedby the reverse transcriptase are converted into double-stranded cDNA molecules by DNA polymerase, and these molecules are inserted into a plasmid or virus vector and cloned (Figure 8-43). Each clone obtained in this way is called a cDNA clone, and the entire collection of clones derived from one mRNA preparation constitutes a cDNA library. Figure 8-44 illustrates some important differences between genomic DNA clones and cDNA clones. Genomic clones represent a random sample of all of the DNA sequencesin an organism and, with very rare exceptions, are the same regardlessof the cell type used to prepare them. By contrast, cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Becausethe cells of different tissuesproduce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library. Figure8-42 Constructionof a human genomicDNA library.A genomic libraryis usuallystoredasa set of bacteria, eachbacteriumcarryinga differentfragmentof humanDNA.Forsimplicity, cloningof just a few representative fragments(colored)is shown.In reality,all of the gray DNA fragmentswould alsobe cloned.

h u m a nd o u b l e - s t r a n d eD d NA E rrH I CLEAVw I RESTRTCTTON NUCLEASE I V

m i l l i o n so f g e n o m i c DNAfragments

I

DNAFRAGMENTS I N S E R T EID NTO PLASMIDS

J

h u m a n g e n o m i cD N A l i b r a r y

ANALYZING AND MANIPULATING DNA

Irssue ( e . 9 . ,b r a i n )

I '. :' ,543

LYSECELLS AND PURIFY mRNA

H Y B R I D I ZW EI T H POLY-TPRIMER AAAAAA l

mRNA I

TTTTTTT M A K EC O M P L E M E N T A R DY NA TRANSCRIPTASE 5 COPYWITH REVERSE 5 AAAAAAA TTTTTTT

CDNA

s',

D E G R A DR ENA WITH RNaseH

I

offtft TTTTTT

3, SYNTHESIZ A ES E C O N D c D N AS T R A N D U S I N GD N A P O L Y M E R A S E : R N AF R A G M E NATC T SA S P R I M E R

5

AAAAAAA ] L

TTTTTTT d o u b l e - s t r a n d ecdD N Ac o p yo f o r i g i n a lm R N A

Figure8-43 The synthesisof cDNA. TotalmRNAis extractedfrom a particular tissue,and the enzymereverse transcriptaseproducesDNAcopies (cDNA)of the mRNAmolecules(see p. 320).Forsimplicity, the copyingof just one of thesemRNAsinto cDNAis A shortoligonucleotide illustrated. to the poly-Atail at the complementary in Chapter 3' end of the mRNA(discussed 6) is first hybridizedto the RNAto act as a primerfor the reversetranscriptase, whichthen copiesthe RNAinto a DNAchain,thereby complementary forming a DNA/RNAhybrid helix.Treating the DNA/RNAhybrid with RNaseH (see Figure5-12) createsnicksand gapsin the RNAstrand.The enzymeDNA polymerase then copiesthe remainingsinglestrandedcDNAinto double-stranded cDNA.Thefragmentof the originalmRNA is the primerfor this synthesisreaction, the DNApolymerase asshown.Because usedto synthesizethe secondDNA throughthe bound strandcan synthesize the RNAfragmentthat is RNAmolecules, base-oairedto the 3' end of the first DNA strandusuallyactsasthe primerfor the finaloroductofthe secondstrand ThisRNAis eventually synthesis. cloning degradedduringsubsequent steps.As a result,the nucleotide at the extreme5'ends ofthe sequences areoften originalmRNAmolecules absentfrom cDNAlibraries.

c n r o m o s o m aDl N A

exon

InTron

nontranscri bed DNA

RESTRICTIO NN U C L E A SDEI G E S T I O N

II

rRANscRrPloN I II II

RNA I transcripts

rII II II

+

RNA SPLICING

ororr.nr"n.,

I

I

I

mRNAs

-

-

I

DNACLONING

I

I

I

R E V E R STER A N S C R I P T I A ON ND D N A C L O N I N G

I

ffi I

ffiiiin

I

- i : : : :

r i' .' 1' t

iriii'r' ta5;!

H H

rF H H H I

g e n o m i cD N Ac l o n e si n g e n o m i cD N A l i b r a r y

CDNAclonesin cDNAlibrary

Figure8-44 The differencesbetween cDNAclonesand genomicDNA clones derived from the same region of DNA. In this example,geneA is infrequently whereasgeneB is frequently transcribed, and both genescontain transcribed, invons (green).Inthe genomic DNA library,both the intronsand the nontranscribedDNA (pink)are included in the clones,and mostclonescontain,at most,only part of the codingsequence of a gene (red).In the cDNAclones,the intron sequences(yellow)havebeen removedby RNAsplicingduringthe formationof the mRNA(blue),and a continuouscodingsequenceis therefore geneB is presentin eachclone.Because morefrequentlythan geneA transcribed in the cellsfrom whichthe cDNAlibrary was made.it is representedmuch more frequentlythan A in the cDNAlibrary.In A and B arein principle contrast, equallyin the genomic represented DNAlibrary.

544

Chapter8: ManipulatingProteins, DNA,and RNA

cDNAClonesContainUninterrupted CodingSequences There are severaladvantagesin using a cDNA library for gene cloning. First, specialized cells produce large quantities of some proteins. In this case,the mRNA encoding the protein is likely to be produced in such large quantities that a cDNA library prepared from the cells is highly enriched for the cDNA molecules encoding the protein, greatly reducing the problem of identi$ring the desired clone in the library (seeFigure B-44). Hemoglobin, for example, is made in large amounts by developing erl,throcytes (red blood cells); for this reason the globin geneswere among the first to be cloned. By far the most important advantageof cDNA clones is that they contain the uninterrupted coding sequence of a gene. As we have seen, eucaryotic genes usually consist of short coding sequences of DNA (exons) separated by much Ionger noncoding sequences (introns); the production of mRNA entails the removal of the noncoding sequences from the initial RNA transcript and the splicing together of the coding sequences.Neither bacterial nor yeast cells will make these modifications to the RNA produced from a gene of a higher eucaryotic cell. Thus, when the aim of the cloning is either to deduce the amino acid sequence of the protein from the DNA sequence or to produce the protein in bulk by expressingthe cloned gene in a bacterial or yeast cell, it is much preferable to start with cDNA. cDNA libraries have an additional use: as described in Chapter 7, many mRNAs from humans and other complex organisms are alternatively spliced, and a cDNA library often representsmany, if not all, of the alternatively spliced mRNAs produced from a given cell line or tissue. Genomic and cDNA libraries are inexhaustible resources,which are widely shared among investigators.Today, many such libraries are also available from commercial sources.

GenesCanBeSelectively Amplifiedby PCR Now that so many genome sequencesare available,genescan be cloned directly without the need to first construct DNA libraries. A technique called the polymerase chain reaction (PCR)makes this rapid cloning possible. Starting with an entire genome, PCR allows the DNA from a selected region to be amplified several billionfold, effectively "purifying" this DNA away from the remainder of the genome. To begin, a pair of DNA oligonucleotides, chosen to flank the desired nucleotide sequence of the gene, are synthesizedby chemical methods. These oligonucleotides are then used to prime DNA synthesis on single strands generated by heating the DNA from the entire genome. The newly synthesizedDNA is produced in a reaction catalyzed in uitroby a purified DNA polymerase, and the primers remain at the 5' ends of the final DNA fragments that are made (Figure 8-454). Nothing special is produced in the first cycle of DNA synthesis;the power of the PCR method is revealed only after repeated rounds of DNA slmthesis.Every cycle doubles the amount of DNA synthesized in the previous cycle. Because each cycle requires a brief heat treatment to separatethe two strands of the template DNA double helix, the technique requires the use of a special DNA polymerase, isolated from a thermophilic bacterium, that is stable at much higher temperatures than normal so that it is not denatured by the repeated heat treatments. with each round of DNA synthesis,the newly generated fragments serve as templates in their turn, and within a few cycles the predominant product is a single species of DNA fragment whose length corresponds to the distance between the two original primers (seeFigure 8-458). In practice, effective DNA amplification requires 20-30 reaction cycles,with the products of each cycle serving as the DNA templates for the next-hence the term polymerase "chain reaction." A single cycle requires only about 5 minutes, and the entire procedure can be easily automated. pcR thereby makes possible the "cell-free molecular cloning" of a DNA fragment in a few hours, compared with the several days required for standard cloning procedures. This technique

545

ANALYZING AND MANIPULATING DNA

is now used routinely to clone DNA from genes of interest directly-starting either from genomic DNA or from mRNA isolated from cells (Figure 8-46). The PCR method is extremely sensitive;it can detect a single DNA molecule in a sample.Trace amounts of RNA can be analyzed in the same way by first transcribing them into DNA with reverse transcriptase.The PCR cloning technique has largely replaced Southern blotting for the diagnosis of genetic diseasesand for the detection of low levels of viral infection. It also has great promise in forensic medicine as a means of analyzing minute traces of blood or other tissues-

(A)

S T E P1

S T E P2

S T E P3

FIRSTCYCLE (B)

s e p a r a t et h e D N A s e p a r a t et h e D N A s t r a n d sa n d a n n e a l primers

DNA synthesis /

/

DNA

!ru\a

s e p a r a t et h e D N A s t r a n d sa n d a n n e a l primers

DNA synthesis

./

---I-_'-I

@+r@w

FIRSTCYCLE (producingtwo double-stranded DNA molecules)

SECOND CYCLE ( p r o d u c i n gf o u r d o u b l e - s t r a n d e d DNA molecules)

THIRDCYCLE (producingeight double-stranded DNA molecules)

Knowledgeof the DNAsequenceto be amplifiedis Figure8-45 Amplificationof DNA by the PCRtechnique. to the sequenceon one strand primerDNAoligonucleotides. One primeris complementary usedto designtwo synthetic, of the DNAdoublehelix,and one is complementary to the sequenceon the otherstrand,but at the oppositeend of the which is performedby a DNA regionto be amplified. Theseoligonucleotides serveas primersfor in vitroDNAsynthesis, DNA,and polymerase, and they determinethe segmentof the DNAto be amplified.(A)PCRstartswith a double-stranded the two strands(step1).Afterstrandseparation, eachcycleof the reactionbeginswith a briefheattreatmentto separate allowstheseprimersto coolingof the DNAin the presence of a largeexcessof the two primerDNAoligonucleotides hybridizeto complementary sequences in the two DNAstrands(step2).Thismixtureis then incubatedwith DNA polymerase DNA,startingfrom the two primers(step3).The to synthesize and the four deoxyribonucleoside triphosphates DNAstrands.(B)As the procedure the newlysynthesized entirecycleis then begunagainby a heattreatmentto separate fragmentsserveastemplatesin theirturn,and within a few cycles is performedoverand overagain,the newlysynthesized the predominantDNAis identicalto the sequencebracketedby and includingthe two primersin the originaltemplate.Of the DNAput into the originalreaction,onlythe sequencebracketedby the two primersis amplifiedbecausethereare no primersattachedanywhereelse.In the exampleillustrated in (B),threecyclesof reactionproduce16 DNAchains,8 of which(boxedin yellow)arethe samelength as and correspondexactlyto one or the other strandof the originalbracketed which is sequenceshownat the far left;the other strandscontainextraDNAdownstreamof the originalsequence, exactlyto the original replicatedin the firstfew cycles.Afterfour morecycles,240of the 256 DNAchainscorrespond all ofthe DNAstrandshavethis uniquelength. bracketedsequence, and afterseveralmorecycles,essentially

546

Chapter8: ManipulatingProteins, DNA,and RNA

i s o l a t eD N A

|

i s o l a t em R N A

|

l-r

# :==:x-:,r I ADD FIRST PRIMER, R E V E R STER A N S C R I P T A S E , AND DEOXYRIBONUCLEOSIDE TRIPHOSPHATES

SEPARATE STRANDS AND ADD SECOND PRIMER

I

i

I

I

P C RA M P L I F I C A T I O N

P C RA M P L I F I C A T I O N

II

+

t

@::a,!i

9enomrc clones (A)

cDNA crones (B)

evenaslittle asa singlecell-and identifyingthe personfrom whom the sample cameby his or her genetic"fingerprint" (Figure 847).

CellsCanBe UsedAs Factories to ProduceSpecificProteins The vast majority of the thousands of different proteins in a cell, including many with crucially important functions, are present in very small amounts. In the past, for most of them, it has been extremely difficult, if not impossible, to obtain more than a few micrograms of pure material. One of the most important contributions of DNA cloning and genetic engineering to cell biology is that they have made it possible to produce any of the cell's proteins in nearly unlimited amounts. Large amounts of a desired protein are produced in living cells by using expression vectors (Figure 8-48). These are generally plasmids that have been designed to produce a large amount of a stable nRNA that can be efficiently translated into protein in the transfected bacterial, yeast, insect, or mammalian cell. To prevent the high level of the foreign protein from interfering with the transfected cell's growth, the expression vector is often designed to delay the slnthesis of the foreign mRNA and protein until shortly before the cells are harvested and lysed (Figure 8-49). Because the desired protein made from an expression vector is produced inside a cell, it must be purified away from the host-cell proteins by chromatography after cell lysis; but because it is such a plentiful species in the cell lysate (often 1-10% of the total cell protein), the purification is usually easy to accomplish in only a few steps.As we saw above, manv expression vectors have been

Figure8-46 Useof PCRto obtain a genomicor cDNAclone.(A)To obtaina genomiccloneusingPCR,chromosomal DNA is first ourifiedfrom cells.PCR orimersthat flank the stretchof DNAto be clonedareadded,and manycyclesof the reactionare completed(seeFigure 8-45).Sinceonlythe DNAbetween(and including)the primersis amplified,PCR providesa way to obtain a short stretch of chromosomal DNAselectively in a virtuallypure form. (B)To use PCRto obtaina cDNAcloneof a gene,mRNAis first purifiedfrom cells.The first primer is then addedto the populationof mRNAs, and reversetranscriotase is usedto make a complementary DNAstrand.The secondprimeris then added,and the single-stranded cDNAmoleculeis amplifiedthroughmanycyclesof PCR,as shown in Figure8-45. For both types of cloning,the nucleotidesequenceof at leastpart of the regionto be clonedmust be known beforehand.

547

ANALYZING AND MANIPULATING DNA

(A)

S E P A R A TPEC RP R O D U C TOSN G E L

p r i m e r sf o r PCR a m p l i fi c a t i o n

I o t

o

E a o

I repeated sequences ofa V N T Rl o c u s

FY -b9 o m

't-

locus3ll_l_L

-]

ll30

--

gz5

--

I o

o

!

o20 o

-5

-:

---

E o

-

o a

tc

I

10

-

-

I

that createthe variabilityusedin this analysis Figure8-47 How PCRis usedin forensicscience.(A)The DNAsequences . . .,whicharefound in variouspositions(loci)in the human suchasCACACA containrunsof short,repeatedsequences, genome.The numberof repeatsin eachrun can be highlyvariablein the population,rangingfrom 4 to 40 in different sequencemicrosatel/lte individuafs. A run of repeatednucleotidesof this type is commonly referredto as a hypervariable variablenumberoftandemrepeat)sequence.Becauseofthevariabilityinthesesequencesateac a l s o k n o w n a s a V N T(R locus,individuals usuallyinherita differentvariantfrom their motherand from theirfather;two unrelatedindividuals usingprimersthat bracketthe locusproduces A PCRanalysis thereforedo not usuallycontainthe samepairof sequences. the maternalvariantand the other one band representing a pairof bandsof amplifiedDNAfrom eachindividual, representing the paternalvariant.The lengthof the amplifiedDNA,and thusthe positionof the band it producesafter exampleshownhere,the same electrophoresis, dependson the exactnumberof repeatsat the locus.(B)In the schematic primers)from three selectedoligonucleotide threeVNTRlociareanalyzed(requiringthreedifferentpairsof specially electrophoresis. suspects(individuals A, B,and C),producingsix DNAbandsfor eachpersonafterpolyacrylamide-gel Althoughsomeindividuals haveseveralbandsin common,the overallpatternis quitedistinctivefor each.The band fourth lane(F)containsthe patterncanthereforeserveasa "fingerprint"toidentifyan individualnearlyuniquely.The productsof the samereactions startingmaterialfor sucha PCRcan be a singlehairor carriedout on a forensicsample.The a tiny sampleof bloodthat was left at the crimescene.Whenexaminingthe variabilityat 5-10 differentVNTRloci,the odds 1 in 10 billion.In the would sharethe samegeneticpatternby chancecan be approximately that two randomindividuals whereasindividualB remainsa clear A and C can be eliminatedfrom furtherenquiries, caseshownhere,individuals suspectfor committingthe crime.A similarapproachis now routinelyusedfor paternitytesting.

548

Chapter8: ManipulatingProteins, DNA,and RNA Figure8-48 Production of largeamountsof a proteinfroma proteincodingDNAsequence clonedinto an expression vectorand introduced intocells,A plasmid vectorhasbeenengineered to contain a highlyactive promoter, whichcauses unusually largeamounts of mRNA to be produced fromanadjacent protein-coding geneinserted intotheplasmid vector. Depending on thecharacteristics of thecloning vector, theplasmid is introduced yeast, intobacterial, insect, or mammalian cells, wherethe geneisefficiently inserted transcribed andtranslated intoprotein.

d o u b le - s t r an d e d p l a s m i dD N A e x p r e s s i ovne c t o r

sequence

cur DNAwrrH RESTRICTION NUCLEASE

designed to add a molecular tag-a cluster of histidine residues or a small marker protein-to the expressedprotein to allow easy purification by affinity chromatography (seeFigure 8-16). A variety of expressionvectors are available, each engineered to function in the tlpe of cell in which the protein is to be made. In this way, cells can be induced to make vast quantities of medically useful proteins-such as human insulin and growth hormone, interferon, and viral antigens for vaccines. More generally, these methods make it possible to produce every protein-even those that may be present in only a few copies per cell-in large enough amounts to be used in the kinds of detailed structural and functional studies that we discussed earlier. DNA technology also can produce large amounts of any RNA molecule whose gene has been isolated. Studies of RNA splicing, protein synthesis, and RNA-based enzyrnes, for example, are greatly facilitated by the availability of pure RNA molecules. Most RNAs are present in only tiny quantities in cells, and they are very difficult to purify away from other cell components-especially from the many thousands of other RNAs present in the cell. But any RNA of interest can be synthesized efficiently in uitro by transcription of its DNA sequence (produced by one of the methods just described) with a highly efficient viral RNA polymerase. The single species of RNA produced is then easily purified away from the DNA template and the RNA polymerase.

I 3 99

+

e e

overexpressed mRNA

overexpresseo protein

Proteinsand NucleicAcidsCanBeSynthesized Directlyby ChemicalReactions chemical reactions have been devised to synthesize directly specific sequences of amino acids or nucleic acids. These methodologies provide direct sources of biological molecules and do not rely on any cells or enzyrnes.Chemical synthesis is the method of choice for obtaining nucleic acids in the range of 100 nucleotides or fewer, which are particularly useful in the PCR-basedapproaches discussed above. chemical slmthesis is also routinely used to produce specific peptides that, when chemically coupled to other proteins, are used to generate antibodies against the peptide.

time at 42"C 25"C

DNACanBeRapidlySequenced Methods that allow the nucleotide sequence of any DNA fragment to be determined simply and quickly have made it possible to determine the DNA sequences of tens of thousands of genes, and many complete genomes (see Table 1-1, p. l8). The volume of DNA sequence information is now so large (many tens of billions of nucleotides) that powerful computers must be used to store and analyze it.

o o o o c o

Figure8-49 Productionof largeamountsof a proteinby usinga plasmid expressionvector.In this example,bacterialcellshavebeentransfected with the codingsequencefor an enzyme,DNAhelicase,.transcription from this codingsequenceis underthe controlof a viralpromoterthat becomes activeonlyat temperatures of 37"Cor higher.Thetotal cellproteinhas beenanalyzedby SDSpolyacrylamide-gel electrophoresis, eitherfrom bacteriagrown at 25'C(no helicase proteinmade)or aftera shiftof the samebacteriato 42"Cforup to 2 hours(helicase proteinhasbecomethe mostabundantproteinspeciesin the lysate). (Courtesy of JackBarry.)

I

I

549

ANALYZING AND MANIPULATING DNA

Large-volume DNA sequencing was made possible through the development in the mid-1970s of the dideoxy method for sequencing DNA, which is based on in uitro DNA slnthesis performed in the presence of chain-terminating dideoxy'ribonucleosidetriphosphates (Figure 8-50).

(A)

d i d e o x y r i b o n u c l e o s i tdrei p h o s p h a t e

deoxyribonucleosidetriphosphate

P P P-o-fH,

P P P

o-(.1 l,

3, ( )ft

/

/

/

3',/s',

/

preventsstrand extensionat 3' end

a l l o w ss t r a n de x t e n s i o na t 3 ' e n d

CGATGGACG T A CC T C T G A A G C G

s i n g l e - s t r a n d eDdN A m o l e c u l e to be seouenced -l

(c)

5' GCATATGTCAGTCCAG 3' 3' CGTATACAGTCAGGTC 5' l a b e l e dp r i m e r

s' {iGAT: 3'

double-stranded 1DNA

I I

5' 3'CGTATACAGTCAGGTC

single-stranded DNA

l+dd f + DNApolymerase

GCAT

'tll-+

GCAT

ATCG DNA sequencereading directlyfrom the bottom of the gel upward, is ATGTCAGTCCAG 112

Figure8-50 The enzymatic-or dideoxymethod of sequencingDNA. (A)This method relieson the useof dideoxyribonucleoside of the normal derivatives triphosphates, that lack triphosphates deoxyribonucleoside the 3'hydroxylgroup.(B)PurifiedDNAis synthesizedin vitroin a mixturethat contains molecules of the DNAto be single-stranded sequenced(gray),theenzYmeDNA polymerase,a short primer DNA (oronge)to to startDNAsynthesis, enablethe polymerase and the four deoxyribonucleoside triphosphates(dATedcTB dGTP,dTTP:b/ueA, analog C,G,andT). lf a dideoxyribonucleotide (red)of one of thesenucleotidesis also presentin the nucleotidemixture,it can into a growingDNA becomeincorporated this chainnow lacksa 3'OH chain.Because group,the additionof the next nucleotideis blocked,and the DNAchainterminatesat that point.In the exampleillustrated, a small amount of dideoxyATP(ddATBsymbolized hereasa redA) hasbeenincludedin the nucleotidemixture.lt competeswith an blueA), excessofthe normaldeoxyATP(dATP, so that ddATPis occasionallyincorporated,at random,into a growingDNAstrand.This reactionmixturewill eventuallyproducea set of DNAsof differentlengthscomplementary to the templateDNAthat is beingsequenced and terminatingat eachof the differentAs. The exactlengthsof the DNA synthesis productscanthen be usedto determinethe positionof eachA in the growingchain.(C)To determinethe completesequenceof a DNA DNAis first fragment,the double-stranded into its singlestrandsand one of separated the strandsis usedasthe templatefor Fourdifferentchain-terminating sequencing. (ddATP, triphosphates dideoxyribonucleoside ddCTBddGTBddTTBagain shown in red)are reactions usedin four separateDNAsynthesis DNA on copiesof the samesingle-stranded template (gray).Eachreactionproducesa set of DNAcopiesthat terminateat different points in the sequence. The productsof these four reactionsare separatedby in four parallellanesof a electrophoresis gel (labeledhereA,L C,and polyacrylamide G).The newly synthesizedfragmentsare detectedby a label(eitherradioactiveor fluorescent)that has been incorporatedeither into the primeror into one of the usedto triphosphates deoxyribonucleoside extendthe DNAchain.In eachlane,the bands representfragmentsthat haveterminatedat a givennucleotide(e.9.,A in the leftmostlane) but at differentpositionsin the DNA.By readingoffthe bandsin order,startingat the bottom of the gel and working acrossall lanes,the DNA sequenceof the newlY strandcan be determined.The synthesized sequenceis given in the greenorrowtothe rightof the gel.Thissequenceis complementaryto the template strand(gray) DNA from the originaldouble-stranded molecule,and identicalto a portionof the green 5'-to-3' slfand.

550

Chapter8: ManipulatingProteins, DNA,and RNA L:

1il

ITTTT.

,f

T

|] -

Figure8-51 AutomatedDNA sequencing. Shownat the bottom is a tiny part of the raw datafrom an automatedDNA-sequencing run as it appearson the computerscreen.Each prominantcoloredpeakrepresents a nucleotidein the DNAsequence-a clear stretchof nucleotidesequencecan be readherebetweenpositions173and 194 from the startof the sequence. Thesmall peaksalongthe baselinerepresent "noise"and,as long asthey background aremuch lowerthan the "signal"peaks, they areignored.Thisparticularexample is takenfrom the internationalproject that determinedthe completenucleotide sequenceof the genomeof the plant (Courtesyof George Arabidopsis. Murphy.)

G'r

Although the same basic method is still used today, many improvements have been made. DNA sequencing is now completely automated: robotic devicesmlx the reagentsand then load, run, and read the order of the nucleotide bases from the gel. chain-terminating nucleotides that are each labeled with a different colored fluorescent dye facilitate these tasks; in this case, all four synthesis reactions can be performed in the same tube, and the products can be separated in a single lane of a gel. A detector positioned near the bottom of the gel reads and records the color ofthe fluorescent label on each band as it passes through a laser beam (Figure 8-51). A computer then reads and stores this nucleotide sequence. some modern systems dispense with the traditional gel entirely, separating nucleic acids by capillary electrophoresis, a method that facilitates rapid automation.

NucleotideSequencesAre Usedto Predictthe Amino Acid Sequencesof Proteins Now that DNA sequencing is so rapid and reliable, it has become the preferred method for determining, indirectly, the amino acid sequencesof most proteins. Given a nucleotide sequence that encodes a protein, the procedure is quite straightforward. Although in principle there are six different reading frames in which a DNA sequencecan be translated into protein (three on each strand), the correct one is generally recognizable as the only one lacking frequent stop codons (Figure 8-52). As we saw when we discussed the genetic code in Chap-

r e a d i n g d i r e c t i o n f o r s e q u e n c eo f t o p D N A s t r a n d -

ti

N

reaornq ?

t,u."i DNA

[i k,

N

leu

13', 1 reading frames

ile

N- tyr

phe

tyr

val ser

ile asn

glu

pro

arg ser

phe

asp

thr leu

thr

leu

asn lys

asn phe

arg ala

lys glu

arg

leu thr

thr his

ser

leu

-C

thr

asn -C

5'

lys ile

C- asn

lys

glu

ile asn

lys

leu

arg ser

glu

leu

thr thr

ile asn

val

arg

phe

lys

gly

seT arg

leu

phe

ala

val arg

arg ser

ser

phe val

lys

lys glu

ser

r e a d i n g d i r e c t i o n f o r s e q u e n c eo f b o t t o m D N A s t r a n d

r e a d i n gd i r e c t i o nf o r s e q u e n c eo f t o p D N A s t r a n d *

-3' DNAi, 55'

r e a d i n gd i r e c t i o nf o r s e q u e n c eo f b o t t o m D N A s t r a n d 5 0 0 b a s ep a i r s

-N

val

val

reading f rames

+

-C

arg leu

3'

-

(B)

arg ser

TTATTTTATTTCGAGTAATTCGACCTTAAACGCGAAACTTCACTTAAC

C-

3

phe

ile

AATAAAATAAAGCTCATTAAGCTGGAATTTGCGCTTTGAAGTGAATTG

C-

2

phe

leu phe

a(g leu

ser

N -N

Figure8-52 Findingthe regionsin a DNA sequencethat encode a protein. (A)Any regionof the DNAsequencecan,in principle, codefor sixdifferentaminoacid sequences, becauseany one ofthree differentreadingframescan be usedto interpretthe nucleotidesequenceon each strand.Notethat a nucleotidesequenceis alwaysreadin the 5'-to-3'direction and encodesa polypeptidefrom the N-terminus to the C-terminus. Fora randomnucleotidesequencereadin a particular frame,a stop signalfor protein synthesis is encounteredr on average, aboutonceevery20 aminoacids.In this samplesequenceof 48 basepairs,each suchsignal(stopcodon)is coloredb/ue, and only readingframe2 lacksa stop signal.(B)Searchof a 1700 base-pair DNA sequence for a possibleprotein-encoding sequence. The informationis displayedas in (A),with eachstop signalfor protein synthesisdenoted by a blueline.ln addition,all of the regionsbetween possiblestartand stop signalsfor protein (seep. 381) aredisplayedasred synthesis bars.Onlyreadingframe1 actually encodesa protein,which is475 amino acidresidueslono.

ANALYZING AND MANIPULATING DNA

ter 6, a random sequenceof nucleotides, read in frame, will encode a stop signal for protein synthesis about once every 20 amino acids. Nucleotide sequences that encode a stretch of amino acids much longer than this are candidates for presumptive exons, and they can be translated (by computer) into amino acid sequences and checked against databases for similarities to known proteins from other organisms. If necessary,a limited amount of amino acid sequence can then be determined from the purified protein to confirm the sequencepredicted from the DNA. The problem comes, however, in determining which nucleotide sequences-within a whole genome-represent genes that encode proteins. Identifying genes is easiest when the DNA sequence is from a bacterial or archaeal chromosome, which lacks introns, or from a cDNA clone. The location of genes in these nucleotide sequencescan be predicted by examining the DNA for certain distinctive features (discussedin Chapter 6). Briefly, these genesthat encode proteins are identified by searching the nucleotide sequence for open reading frames (ORFs)that begin with an initiation codon, usually ATG, and end with a termination codon, TAA, TAG, or TGA. To minimize errors, computers used to search for ORFs are often directed to count as genes only those sequencesthat are longer than, say, 100 codons in length. For more complex genomes, such as those of animals and plants, the presence of large introns embedded within the coding portion of genes complicates the process. In many multicellular organisms, including humans, the average exon is only 150 nucleotides long. Thus one must also search for other features that signal the presence of a gene, for example, sequences that signal an intron/exon boundary or distinctive upstream regulatory regions. Recent efforts to solve the exon prediction problem have turned to artificial intelligence algorithms, in which the computer learns, based on known examples, what sets of features are most indicative of an exon boundary. A second major approach to identiffing the coding regions in chromosomes is through the characterization of the nucleotide sequences of the detectable mRNAs (using the corresponding cDNAs). The mRNAs (and the cDNAs produced from them) Iack introns, regulatory DNA sequences, and the nonessential "spacer" DNA that lies between genes. It is therefore useful to sequence large numbers of cDNAs to produce a very large database of the coding seguencesof an organism. These sequencesare then readily used to distinguish the exons from the introns in the long chromosomal DNA sequencesthat correspond to genes.

TheGenomesof ManyOrganisms HaveBeenFullySequenced Owing in large part to the automation of DNA sequencing,the genomes of many organisms have been fully sequenced;these include plant chloroplasts and animal mitochondria, large numbers of bacteria, and archaea, and many of the model organisms that are studied routinely in the laboratory, including many yeasts, a nematode worm, the fruit fly Drosophila, tliremodel plant Arabidopsis, the mouse, dog, chimpanzee, and,last but not least, humans. Researchershave also deduced the complete DNA sequences for a wide variety of human pathogens.These include the bacteria that cause cholera, tuberculosis, syphilis, gonorrhea, L).ryne disease,and stomach ulcers, as well as hundreds of virusesincluding smallpox virus and Epstein-Barr virus (which causes infectious mononucleosis). Examination of the genomes of these pathogens provides clues about what makes them virulent and will also point the way to new and more effective treatments. Haemophilus influenzae (a bacterium that can cause ear infections and meningitis in children) was the first organism to have its complete genome sequence-all 1.8 million nucleotide pairs-determined by the shotgun sequencing method, the most common strategy used today. In the shotgun method, long sequences of DNA are broken apart randomly into many shorter fragments. Each fragment is then sequenced and a computer is used to order these pieces into a whole chromosome or genome, using sequence overlap to guide the assembly. The shotgun method is the technique of choice for

551

552

Chapter8: ManipulatingProteins, DNA,and RNA

sequencing small genomes.Although larger, more repetitive genome sequences are more challenging to assemble,the shotgun method-in combination with the analysis of large DNA fragments cloned in BACs-has played a key role in their sequencing as well. With new sequences appearing at a steadily accelerating pace in the scientific literature, comparison of the complete genome sequences of different organisms allows us to trace the evolutionary relationships among genes and organisms, and to discover genes and predict their functions (discussed in Chapters 3 and 4). Assigning functions to genes often involves comparing their sequences with related sequences from model organisms that have been well characterized in the laboratory, such as the bacterium E. coli, the yeasts S. cereuisiae and S.pombe, the nematode worm C. elegans,and the fruit fly Drosophila (discussedin Chapter I). Although the organisms whose genomes have been sequenced share many biochemical pathways and possessmany proteins that are homologous in their amino acid sequence or structure, the functions of a very large number of newly identified proteins remain unknoltn. Depending on the organism, some l5-40To of the proteins encoded by a sequenced genome do not resemble any protein that has been studied biochemically. This observation underscores a limitation of the emerging field of genomics: although comparative analysis of genomes reveals a great deal of information about the relationships between genes and organisms, it often does not provide immediate information about how these genes function, or what roles they have in the physiology of an organism. Comparison of the full gene complement of severalthermophilic bacteria, for example, does not reveal why these bacteria thrive at temperatures exceeding 70'c. And examination of the genome of the incredibly radioresistant bacterium Deinococcusradiodurans does not explain how this organism can survive a blast of radiation that can shatter glass.Further biochemical and genetic studies, like those described in the other sections of this chapter, are required to determine how genes, and the proteins they produce, function in the context of living organlsms.

Su m m a r y DNA cloning allows a copy of any specificpart of a DNA or RNAsequenceto be selected in a cell and producedin unlimited amounts in ftom the millions of other sequences pure form. DNA sequences can be amplified after cutting chromosomal DNA with a restriction nucleaseand inserting the resulting DNAfragments into the chromosome of a self-replicating geneticelement such as a uirus or a plasmid. Plasmid uectorsare generally used,and the resulting "genomic DNA library" is housed in millions of bacterial cells,each carrying a dffirent cloned DNA fragment. Indiuidual cellsfrom this library that are allowed to proliferate produce large amounts of a single cloned DNA fragment. The polymerasechain reaction (PCR)allows DNA cloning to be performed directly with a thermostable DNA polymerase-prouided that the DNA sequenceof interest is already known. The proceduresused to obtain DNA clones that correspondin sequenceto nRNA moleculesare the sameexceptthat a DNA copy of the nRNA sequence,called cDNA, is first made. rJnhke genomic DNA clones,cDNA clones lack intron sequences,making them the clonesof choicefor analyzing the protein product of a gene. Nucleic acid hybridization reactionsprouide a sensitiuemeans of detectinga gene or any other nucleotide sequenceof interest.Under stringent hybridization conditions (a combination of soluent and temperature at which euen a perfect double helix is barely stable), two strands can pair to form a "hybrid" helix only if their nucleotide sequencesare almost perfectly complementary. The enormous specificity of this hybridization reaction allows any single-stranded sequence of nucleotides to be labeled with a radioisotopeor chemical and usedas a probe to find a complementary partner strand,euenin a cell or cellextractthat containsmillions of dffirent DNAand RNA sequences.Probesof this type are widely used to detect the nucleic acids corresponding to specificgenes,both to facilitate their purification and characterization, and to localize them in cells, tissues,and organisms.

STUDYING GENEEXPRESSION AND FUNCTION

The nucleotide sequenceof DNA can be determined rapidly and simply by using highly automated techniquesbasedon the dideoxy method for sequencingDNA. This technique has made it possible to determine the complete DNA sequencesof the genomesof many organisms.Comparison of the genomesequencesof different organisms allows us to trace the euolutionary relationships among genesand organisms, and it has proued ualuablefor discoueringnew genesand predicting their functions. Takentogether,thesetechniquesfor analyzing and manipulating DNA hauemade it possible to identify, isolate, and sequencegenesfrom any organism of interest. Related technologiesqllow scientiststo produce the protein products ofthese genesin the large quantities neededfor detailed analysesof their structure and function, as well asfor medical purposes.

ANDFUNCTION EXPRESSION STUDYING GENE Ultimately, one wishes to determine how genes-and the proteins they encode-function in the intact organism. Although it may seem counterintuitive, one of the most direct ways to find out what a gene does is to seewhat happens to the organism when that gene is missing. Studying mutant organisms that have acquired changes or deletions in their nucleotide sequencesis a timehonored practice in biology and forms the basis of the important field of genetics. Becausemutations can disrupt cell processes,mutants often hold the key to understanding gene function. In the classical genetic approach, one begins by isolating mutants that have an interesting or unusual appearance:fruit flies with white eyes or curly wings, for example. Working backward from the phenotype-the appearance or behavior of the individual-one then determines the organism's genotype, the form of the gene responsible for that characteristic (Panel 8-t). Today,with numerous genome sequencesavailable,the exploration of gene function often begins with a DNA sequence.Here, the challenge is to translate sequence into function. One approach, discussed earlier in the chapter, is to search databases for well-characterized proteins that have similar amino acid sequencesto the protein encoded by a new gene, and from there employ some of the methods described in the previous section to explore the gene'sfunction further. But to determine directly a gene'sfunction in a cell or organism, the most effective approach involves studying mutants that either lack the gene or express an altered version of it. Determining which cell processeshave been disrupted or compromised in such mutants will usually shed light on a gene'sbiological role. In this section, we describe severalapproachesto determining a gene'sfunction, starting from a DNA sequence or an organism with an interesting phenotype. We begin with the classical genetic approach, which starts with a genetic screenfor isolating mutants of interest and then proceeds toward identification of the gene or genes responsible for the observed phenotype. We then describe the set of techniques that are collectively called reuersegenetics, in which one begins with a gene or gene sequence and attempts to determine its function' This approach often involves some intelligent guesswork-searching for homologous sequencesand determining when and where a gene is expressed-as well as generating mutant organisms and characterizing their phenotype.

by Random Genetics Beginsby Disruptinga CellProcess Classical Mutagenesis Before the advent of gene cloning technology, most geneswere identified by the abnormalities produced when the gene was mutated. This classical genetic approach-identifying the genes responsible for mutant phenotlpes-is most easily performed in organisms that reproduce rapidly and are amenable to genetic manipulation, such as bacteria, yeasts,nematode worms, and fruit flies. Although spontaneous mutants can sometimes be found by examining or tens of thousands of individual extremely large populations-thousands

s53

GENES A N DP H E N O T Y P E S Gene:

a functionalunit of inheritance,usuallycorresponding to the segment of DNA coding for a single protein. G e n o m e :a l l o f a n o r g a n i s m ' sD N A s e q u e n c e s . locus:the site of the gene in the genome Wild-type:the normal, naturallyoccurringtype homozygous A"/A

heterozygousa/A

GENOTYPE:the specific set of allelesforming the genome of an individual

Mutant:differingfrom the wild-type becauseof a genetic c h a n g e( a m u t a t i o n ) homozygousa/a

YY/lryl

PHENOWPE:the visible characterof the individual alleleA is dominant (relativeto a); allelea is recessive(relativeto A) In the exampleabove,the phenotypeof the heterozygoteis the same as that of one of the homozygotes;in caseswhere it is differentfrom both, the two allelesare said to be co-dominant.

a c h r o m o s o m ea t t h e b e g i n n i n go f t h e c e l l cycle,in G.,phase;the single long bar representsone long double helix of DNA

THEHAPLOID-DIPLOID CYCLE O FS E X U A L REPRODUCTION

l o n g" q " a r m a chromosomenear the end of the cell cycle,in metaphase;it is duplicatedand condensed,consistingof two identicalsisterchromatids(eachcontainingone DNA double helix)joined at the centromere. A normal diploid chromosomeset, as seen in a metaphasespread, preparedby burstingopen a cell at metaphaseand stainingthe scattered chromosomes.In the exampleshown schematicallyhere,there are three pairsof autosomes(chromosomes inheritedsymmetricallyfrom both parents,regardlessof sex) and two sex chromosomes-an X from the mother and a Y from the father. The numbersand types of sex chromosomesand their role in sex determinationare variablefrom one classof organismsto another,as is the number of pairsof autosomes. sexcnromosomes

S E X U A LF U S I O N( F E R T I L I Z A T I O N )

For simplicity,the cycle is shown for only one chromosome/chromosome pair.

M E I O S I SA N D G E N E T I C RECOMBINATION m a t e r n a lc h r o m o s o m e MEIOSIS AND RECOMBINATION

AB paternal chromosome

7// ab

2

diploid germ cell

senotype4p

h a p l o i dg a m e t e s( e g g so r s p e r m )

The greaterthe distance betweentwo loci on a single chromosome,the greateris the chancethat they will be separatedby crossingover occurringat a site between them. lf two genesare thus reassortedin xo/oof gametes, they are said to be separatedon a chromosomeby a geneticmap distanceof x map units (or x centimorgans).

TYPES OFMUTATIONS

deletesa segmentof a chromosome DELETION: P O I N TM U T A T I O Nm : a p st o a s i n g l es i t e i n t h e g e n o m e , correspondingto a single nucleotidepair or a very s m a l l p a r t o f a s i n g l eg e n e

invertsa segmentof a chromosome INVERSION: lethalmutation:causesthe developingorganismto die prematurely. conditionalmutation:producesits phenotypiceffectonly under certainconditions,calledthe restrictiveconditions, Under other conditions-the permissiveconditions-the effect is not seen. For a temperature-sensitivemutation, the restrictiveconditiontypicallyis high temperature,while the oermissiveconditionis low temoerature. loss-of-function mutation:either reducesor abolishesthe activityof the gene.Theseare the most common classof mutations.Loss-of-fu nction m utationsa re usually recessive-theorganismcan usuallyfunction normally as long as it retainsat leastone normal copy of the affected gene. mutationthat completely null mutation:a loss-of-function abolishesthe activityof the gene.

TRANSLOCATION:breaksoff a segmentfrom one chromosomeand attachesit to another gain-of-functionmutation:increasesthe activityof the gene these or makesit activein inappropriatecircumstances; mutationsare usuallYdominant. dominant-negativemutation:dominant-actingmutationthat phenotype blocksgene activity,causinga loss-of-function even in the presenceof a normal copy of the gene.This ohenomenonoccurswhen the mutant gene product interfereswith the function of the normal gene product. suppressormutation:suppressesthe phenotypiceffectof anothermutation,so that the double mutant seems normal. An intragenicsuppressormutation lies within the gene affected by the first mutation; an extragenicsuppressor mutation lies in a secondgene-often one whose product interactsdirectlywith the product of the first.

T W O G E N E SO R O N E ? G i v e nt w o m u t a t i o n st h a t p r o d u c et h e s a m e p h e n o t y p eh, o w c a n w e t e l l w h e t h e rt h e y a r e m u t a t i o n si n t h e s a m e g e n e ?l f t h e ( a st h e y m o s t o f t e na r e ) ,t h e a n s w e rc a n m u t a t i o n sa r e r e c e s s i v e b e f o u n d b y a c o m p l e r n e n t a t i ot n est

In the simplesttype of complementationtest, an individualwho is homozygousfor one mutation is mated with an individual who is homozygousfor the other.The phenotypeof the offspringgives the answerto the question'

COMPLEMENTATION: GENES MUTATIONSIN TWO DIFFERENT h o m o z y g o u sm u t a n t m o t h e r

homozygous mutant mother

i

'i,'.&:

, I

hybrid offspringshows normal phenotype: one normal copy of each gene is present

h o m o z y g o u sm u t a n tf a t h e r

;*ls'

a

hybrid offspringshows mutant phenotype: no normal copiesof the mutatedgene are present

556

Chapter8: ManipulatingProteins, DNA,and RNA

organisms-isolating mutant individuals is much more efficient if one generates mutations with chemicals or radiation that damage DNA. By treating organisms with such mutagens, very large numbers of mutant individuals can be created quickly and then screened for a particular defect of interest, as we discuss shortly. An alternative approach to chemical or radiation mutagenesis is called insertional mutagenesis.This method relies on the fact that exogenous DNA inserted randomly into the genome can produce mutations if the inserted fragment interrupts a gene or its regulatory sequences.The inserted DNA, whose sequence is knor,r'n,then serves as a molecular tag that aids in the subsequent identification and cloning of the disrupted gene (Figure B-59). rn Drosophila, the use of the transposable P element to inactivate geneshas revolutionized the study of gene function in the fly. Transposable elements (seeThble 5-3, p. 3lg) have also been used to generatemutations in bacteria, yeast,mice, and the flowering pfant Arabidopsis. such classical genetic studies are well suited for dissecting biological processes in experimental organisms, but how can we study gene function in humans? Unlike the genetically accessibleorganisms we have been discussing, humans do not reproduce rapidly, and they cannot be intentionally treated with mutagens. Moreover, any human with a serious defect in an essential process, such as DNA replication, would die long before birth. There are two main ways that we can study human genes. First, because genes and gene functions have been so highly conserved throughout evolution, the study of less complex model organisms reveals critical information about similar genes and processesin humans. The corresponding human genes can then be studied further in cultured human cells. Second, many mutations that are not lethal-tissue-specific defects in lysosomes or cell-surface receptors, for example-have arisen spontaneously in the human population. Analyses of the phenotypes of the affected individuals, together with studies of their cultured cells, have provided many unique insights into important human cell functions. Although such mutations are rare, they are very efficiently discovered because of a unique human property: the mutant individuals call attention to themselves by seeking special medical care.

GeneticscreensldentifyMutantswith specificAbnormalities once a collection of mutants in a model organism such as yeast or fly has been produced, one generally must examine thousands of individuals to find the altered phenotype of interest. Such a search is called a genetic screen, and the larger the genome, the less likely it is that any particular gene will be mutated. Therefore, the larger the genome of an organism, the bigger the screening task becomes. The phenotype being screened for can be simple or complex. Simple phenotypes are easiest to detect: one can screen many organismJ rapidly, ?or example, for mutations that make it impossible for the organism to survive in the absence of a particular amino acid or nutrient. More complex phenotypes, such as defects in learning or behavior, may require more elaborate screens (Figure B-54). But even genetic screensthat are used to dissect complex physiological systemsshould be as simple as possible in design, and, if possible, should permit the simultaneous examination of large numbers of mutants. As an example, one particularly elegant screen was designed to search for genesinvolved in visual processingin zebrafish.The basis of this screen, which monitors the fishes' response to motion, is a change in behavior. wild-type fish tend to swim in the direction of a perceived moiion, whereas mutants with defects in their visual processing systems swim in random directions-a behavior that is easily detected. one mutant discovered in this screenis called lakritz,which is missing B0%of the retinal ganglion cells that help to relay visual signals from the eye to the brain. As the cellllai organization of the zebrafish retina is similar to that of all vertebrates, the study of such mutants should also provide insights into visual processing in humans. Becausedefects in genes that are required for fundamental cell processesRNA syethesis and processing or cell-cycle control, for example-are usually

Figure8-53 Insertionalmutant of the snapdragon,Antirrhinum.A mutationin a singlegenecodingfor a regulatory protein causesleafyshootsto developin placeof flowers.The mutation allows cellsto adopt a character that would be appropriateto a differentpart of the normalplant.The mutantplantis on the left,the normal plant on the nght. (Courtesyof EnricoCoenand RosemaryCarpenter.)

557

STUDYING GENEEXPRESSION AND FUNCTION

Figure8-54 A behavioralphenotype detected in a genetic screen.(A)Wildtype C.elegansengagein socialfeeding. Thewormsmigratearounduntilthey encountertheir neighborsand commence (B)Mutantanimals feedingon bacteria. feed by themselves.(Courtesyof Cornelia Bargmann,Cell94: cover,1998.With permissionfrom Elsevier.)

.F

1mm

lethal, the functions of these genes are often studied in individuals with conditional mutations. The mutant individuals function normally as long as "permissive" conditions prevail, but demonstrate abnormal gene function when subjected to "nonpermissive" (restrictive) conditions. In organisms with temperature-sensitiuemutations, for example, the abnormality can be switched on and off experimentally simply by changing the temperature; thus, a cell containing a temperature-sensitive mutation in a gene essentialfor survival will die at a nonpermissive temperature but proliferate normally at the permissive temperature (Figure 8-55). The temperature-sensitive gene in such a mutant usually contains a point mutation that causesa subtle change in its protein product. Many temperature-sensitive mutations were found in the bacterial genes that encode the proteins required for DNA replication. The mutants were identified by screening populations of mutagen-treated bacteria for cells that stop making DNA when they are warmed from 30'C to 42'C. These mutants were later used to identify and characterize the corresponding DNA replication proteins (discussed in Chapter 5). Similarly, screens for temperature-sensitive mutations led to the identification of many proteins involved in regulating the cell cycle, as well as many proteins involved in moving proteins through the secretory pathway in yeast (see Panel 13-1). Related screening approaches demonstrated the function of enzymes involved in the principal metabolic pathways of bacteria and yeast (discussedin Chapter 2) and identified many of the gene products responsible for the orderly development of the Drosophila embryo (discussedin Chapter 22).

MutationsCanCauseLossor Gainof ProteinFunction Gene mutations are generally classedas "loss of function" or "gain of function." A loss of function mutation results in a gene product that either does not work or works too little; thus, it revealsthe normal function of the gene.A gain of function mutation results in a gene product that works too much, works at the wrong time or place, or works in a new way (Figure 8-56).

m u t a n t c e l l sp r o l i f e r a t e a n d f o r m a c o l o n ya t the permissive temperature

m u t a g e n i z e dc e l l s p r o l i f e r a t ea n d f o r m c o l o n i e sa t 2 3 o C

c o l o n i e sr e p l i c a t e d onto two identical p l a t e sa n d i n c u b a t e d at two different temperatures

\

m u t a n t c e l l sf a i l t o proliferate and form a c o l o n ya t t h e n o np e r m l s S l v e temperature

Figure8-55 Screeningfor temperaturesensitivebacterialor yeast mutants. cellsareplatedout at the Mutagenized Theydivideand permissive temperature. form colonies,which aretransferredto two identicalPetridishesby replica plating.One of theseplatesis incubated at the permissivetemperature,the other Cells temperature. at the nonpermissive containinga temperature-sensitive for mutationin a geneessential candivideat the normal, oroliferation permissive temperaturebut fail to divide at the elevated,nonPermissive temperature.

558

Chapter8: ManipulatingProteins, DNA,and RNA

wild type

loss-of-functionmutation

point mutation truncation

gai n-of-function mutation

conditionalossof-function mutation

370C

250C

An important early step in the genetic analysis of any mutant cell or organism is to determine whether the mutation causesa loss or a gain of function. A standard test is to determine whether the mutation is dominant or recessiue. A dominant mutation is one that still causes the mutant phenotype, in the presence of a single copy of the wild-type gene.A recessivemutation is one that is no longer able to cause the mutant phenotlpe in the presence of a single wild-type copy of the gene. Although caseshave been described in which a loss-of-function mutation is dominant or a gain-of-function mutation is recessive,in the vast majority of cases,recessivemutations are loss of function, and dominant mutations are gain of function. It is easy to determine if a mutation is dominant or recessive.one simply mates a mutant with a wild-type to obtain diploid cells or organisms. The progeny from the mating will be heterozygous for the mutation. If the mutant phenotype is no longer observed, one can conclude that the mutation is recessiveand is very likelv to be a loss-of-function mutation.

complementation TestsReveal whetherTwoMutationsAre in the SameGeneor DifferentGenes A large-scalegenetic screen can turn up many different mutations that show the same phenotype. These defects might lie in different genes that function in the same process, or they might represent different mutations in the same gene. Alternative forms of a gene are knolrm as alleles. The most common difference between alleles is a substitution of a single nucleotide pair, but different alleles can also bear deletions, substitutions, and duplications. How can we tell, then, whether two mutations that produce the same phenotlpe occur in the same gene or in different genes?If the mutations are recessive-if, for example, they represent a loss of function of a particular gene-a complementation test can be used to ascertain whether the mutations fall in the same or in different genes. To test complementation in a diploid organism, an individual that is homozygous for one mutation-that is, it possessestvvo identical alleles of the mutant gene in question-is mated with an individual that is homozygous for the other mutation. If the two mutations are in the same gene, the offspring show the mutant phenotype, because they still will have no normal copies of the gene in question (seePanel 8-l). If, in contrast, the mutations fall in different genes,the resulting offspring show a normal phenotype, because they retain one normal copy (and one mutant copy) of each gene; the mutations thereby complement one another and restore a normal phenotype. complementation testing of mutants identified during genetic screens has revealed, for example, that 5 genesare required for yeast to digest the sugar galactose,20 genesare needed for E. coli to build a functional flagellum, 48 genes are involved in assembling bacteriophage T4 viral particles, and hundreds of genes are involved in the development of an adult nematode worm from a fertilized egg.

GenesCanBeOrderedin Pathways by Epistasis Analysis

Figure8-56 Gene mutations that affect their protein product in different ways. In this example,the wild-typeproteinhas a specificcellfunctiondenotedby the redrays,Mutationsthat eliminatethis function,increase the function,or render the functionsensitive to higher temperatures areshown.The temperature-sensitive conditional mutantproteincarriesan aminoacid substitution(red)that preventsits proper foldingat 37oC,but allowsthe proteinto fold and functionnormallyat 25oC.Such conditionalmutationsareespecially usefulfor studyingessential genes;the organismcan be grown underthe permissive conditionand then movedto the nonpermissive conditionto studythe functionofthe gene.

s59

ANDFUNCTION S T U D Y I N G E N EE X P R E S S I O

activity of another protein, we would say that the former gene acts before the latter. Gene order can, in many cases,be determined purely by genetic analysis without any knowledge of the mechanism of action of the gene products involved. Suppose we have a biosynthetic process consisting of a sequence of steps, such that performance of a step B is conditional on completion of the preceding step A; and suppose gene Ais required for step A, and geneB is required for step B. Then a null mutation (a mutation that abolishesfunction) in gene Awill arrest the process at step A, regardlessof whether gene B is functional or not, whereas a null mutation in gene B will cause arrest at step B only if gene A is still active. In such a case,gene A is said to be epistatlc to gene B. By comparing the phenotypes of the different combinations of mutations, we can therefore discover the order in which the genes act. This type of analysis is called epistasis analysis. As an example, the pathway of protein secretion in yeast has been analyzedin this way. Different mutations in this pathway cause proteins to accumulate aberrantly in the endoplasmic reticulum (ER)or in the Golgi apparatus.\.A/hena yeast cell is engineered to carry both a mutation that blocks protein processing in the ER and a mutation that blocks processing in the Golgi apparatus, proteins accumulate in the ER. This indicates that proteins must pass through the ER before being sent to the Golgi before secretion (Figure 8-57). Strictly speaking, an epistasis analysis can only provide information about gene order in a pathway when both mutations are null alleles.\A4renthe mutations retain partial function, their epistasisinteractions can be difficult to interpret. Sometimes, a double mutant will show a new or more severe phenotlpe than either single mutant alone. This type of genetic interaction is called a syntheticphenotype, and if the phenoq,pe is death of the organism, it is called synthetic lethalify. In most cases,a synthetic phenotype indicates that the two genes act in two different parallel pathways, either of which is capable of mediating the same cell process. Thus, when both pathways are disrupted in the double mutant, the process fails altogether, and the synthetic phenotype is observed.

Genesldentifiedby MutationsCanBeCloned Once the mutant organisms are produced in a genetic screen, the next task is identifying the gene or genes responsible for the altered phenoq,pe. tf the phenotype has been produced by insertional mutagenesis, locating the disrupted gene is fairly simple. DNA fragments containing the insertion (a transposon or a retrovirus, for example) are collected and amplified by PCR, and the nucleotide sequence of the flanking DNA is determined. Genome databasescan then be searchedfor open reading frames containing this flanking sequence. If a DNA-damaging chemical was used to generate the mutations, identifying the inactivated gene is often more laborious, but it can be accomplished by several different approaches. In one, the first step is to experimentally determine the gene'slocation in the genome. To map a newly discovered gene, its rough chromosomal location is first determined by assessinghow far the gene lies from other known genes in the genome. Estimating the distance between genetic loci is usually done by linkage analysis, a technique that relies on the tendency for genes that lie near one another on a chromosome to be inherited together. Even closely linked genes, however, can be separated by ER

p r o l e rn

secretory Golgi apoaratus vesicles

n o r m a lc e l l

protern secreteo

s e c r e t o r vm u t a n t A

secretorymutant B

d o u b l em u t a n tA B

o r o t e i na c c u m u l a t e s in ER

p r o t e i na c c u m u l a t e s i n G o l g ia p p a r a t u s

p r o t e i na c c u m u l a t e s in ER

Figure8-57 Usinggeneticsto determinethe order of functionof genes.In normalcells,secretoryproteins whichfusewith areloadedinto vesicles, the olasmamembraneto secretetheir medium. contentsinto the extracellular Two mutants,A and B,fail to secrete proteins.ln mutant A, secretoryproteins accumulatein the ER.In mutantB, secretoryproteinsaccumulatein the Golgi.In the doublemutantAB,proteins that accumulatein the ER;this indicates the genedefectivein mutantA acts beforethe gene defectivein mutant B in the secretorypathway.

Chapter8: ManipulatingProteins, DNA,and RNA recombination during meiosis. The larger the distance between two genetic loci, the greater the chance that they will be separated by a crossover (seepanel B-l). By calculating the recombination frequency between two genes, the approximate distance between them can be determined. If the position of one gene in the genome is knor,tm,that of the second gene can thereby be estimated. Becausegenesare not always located close enough to one another to allow a precise pinpointing of their position, linkage analyses often rely on physical markers along the genome for estimating the location of an unknor,rmgene.These markers are generally short stretchesof nucleotides,with a knonm sequenceand genome location, that can exist in at least two allelic forms. The simplest markers are single-nucleotide polymorphivns (sl/Ps), short sequences that differ by one nucleotide pair among individuals in a population. SNps can be detected by hybridization techniques. Many such physical markers, distributed all along the length of chromosomes,have been collected for a variety of organisms.If the distribution of these markers is sufficiently dense, one can, through a linkage analysis that tests for the tight co-inheritance of one or more SNps with the mutant phenotype, narrow the potential location of a gene to a chromosomal region that may contain only a few gene sequences.These are then considered candidate genes,and their structure and function can be tested directly to determine which gene is responsible for the original mutant phenotype.

HumanGenetics Presents SpecialProblems and Opportunities Although genetic experimentation on humans is considered unethical and is legally banned, humans do suffer from a large variety of genetic disorders. The linkage analysis described above can be used to identifu the genes responsible for these heritable conditions. such studies require DNA samples from a large number of families affected by the disease.These samples are examined for the presence of physical markers such as SNPsthat seem to be closely linked to the diseasegene, in that they are always inherited by individuals who have the disease and not by their unaffected relatives. The disease gene is then located as described above (Figure 8-58). The genes for cystic fibrosis and Huntington's disease,for example, were discovered in this way. c h r o m o s o m ep a i r i n m o t h e rw i t h d i s e a s e

I

t egg -

disease SNPmarker

++++ +++ TESTS P E R F O R M EODN 7 C H I L D R E N c o N c L U S l o N : g e n e c a u s i n gd i s e a s ei s c o - i n h e r i t e dw i t h s N p m a r k e rf r o m d i s e a s e d mother i n 7 5 % o f t h e d i s e a s e dp r o g e n y -l f t h i s s a m ec o r r e l a t i o ni s o b s e r v e di n o t h e r f a m i l i e st h a t h a v eb e e n e x a m i n e dt,h e g e n e c a u s i n gd i s e a s ei s m a p p e dt o t h i s c h r o m o s o m ec l o s et o t h e s N P N o t e t h a t a n s N Pt h a t i s e i t h e rf a r a w a y f r o m t h e g e n e o n t h e s a m ec h r o m o s o m eo r l o c a t e do n a d i f f e r e n tc h r o m o s o m ef r o m t h e g e n e o f i n t e r e s tw i l l b e c o - i n h e r i t e do n l y 50% of the time.

Figure8-58 Geneticlinkageanalysis usingphysicalmarkerson DNAto find a human gene.In this example,the co-inheritance of a specifichuman phenotype(herea geneticdisease) with an 5NPmarker.lf individuals who inherit the diseasenearlyalwaysinherita particularSNPmarker,then the gene causing t h e d i s e a saen dt h e S N Pa r el i k e l y to be closetogetheron the chromosome, as shown here.To provethat an observed linkageis statistically significant, hundredsof individuals may needto be examined.Notethat the linkagewill not be absoluteunlessthe SNPmarkeris locatedin the geneitself.Thus, occasionally the SNPwill be separated from the diseasegeneby crossingover duringmeiosisin the formationof the egg or sperm:this hashappenedin the caseof the chromosomepairon the far right.Whenworkingwith a sequenced genome,this procedurewould be repeatedwith sNPslocatedon eitherside of the inirial5NP,untila 100o/o co-inheritance is found. Notethat the egg and spermwill each contributeonly one chromosomeof each pairfrom the parentto the child.

GENEEXPRESSION ANDFUNCTION STUDYING

WhichCanAid HumanGenesAreInheritedin HaplotypeBlocks, Disease in the Searchfor MutationsThatCause With the complete human genome sequence in hand, we can now study human genetics in a way that was impossible only a few years ago. For example, we can begin to identify those DNA differences that distinguish one individual from another. No two humans (with the exception of identical twins) have the same genome. Each of us carries a set of polymorphisms-differences in nucleotide sequence-that make us unique. These polymorphisms can be used as markers for building genetic maps and performing genetic analysesto Iink particular polymorphisms with specific diseasesor predispositions to disease. The problem is that any two humans typically differ by about 0.1% in their nucleotide sequences (approximately one nucleotide difference every 1000 nucleotides). This translates to about 3 million differences between one person and another. Theoretically, one would need to search through all 3 million of those polymorphisms to identify the one or two that are responsible for a particular heritable diseaseor diseasepredisposition. To reduce the number of polymorphisms we need to examine, researchers are taking advantage of the recent discovery that human genes tend to be inherited in blocks. The human species is relatively young, and it is thought that we are descended from a relatively small population of individuals who lived in Africa about 10,000 years ago. Because only a few hundred generations separate us from this ancestral population, large segments of human chromosomes have passedfrom parent to child unaltered by the recombination events that occur in meiosis. In fact, we observe that certain sets of alleles (including SNPs) are inherited in large blocks within chromosomes. These ancestral chromosome segments-sets of alleles that have been inherited in clusters with little genetic rearrangement acrossthe generations-are called haplotype blocks. Like genes, SNPs, and other genetic markers-which exist in different alleleic forms-haplotype blocks also come in a limited number of "flavors" that are common in the human population, each of which represents an allele combination passed dor,',nfrom a shared ancestor long ago. Researchersare now constructing a human genome map based on these haplotype blocks-called a haplot5rye map (hapmap). Geneticists hope that the human haplotype map will make the search for disease-causing and disease-susceptibility genes a much more manageable task. Instead of searching through each of the many millions of SNPsin the human population, one need only search through a considerably smaller set of selected SNPsto identify the haplotype block that appears to be inherited by individuals with the disease. (These searchesstill involve DNA samples from large numbers of people, and SNPs are now typically scored using robotic technologies.) If a specific haplotype block is more common among people with the diseasethan in unaffected individuals, the mutation linked to that disease will likely be located in that same segment of DNA (Figure 8-59) . Researcherscan then zero in on the specific regi,onwithin the block to search for the specific gene associated with the disease.This approach should, in principle, allow one to analyze the genetics of those common diseasesin which multiple genes confer susceptibility. A detailed examination of haplotype blocks can even tell us whether a particular allele has been favored by natural selection. As a rule, when a new allele of a gene arises that does not confer a selective advantage on the individual, it will take a long time for that allele to become common in the population. The more common-and therefore older-such an allele is, the smaller should be the haplotype block that surrounds it, because it will have had many chances of being separated from its neighboring variations by the recombination events that occur in meiosis generation after generation. A new allele may quickly spread in a population, however, if it confers some dramatic advantage on the organism. For example, mutations or variations that make an organism more resistant to an infection might be selected for because organisms with this variation would be more likely to survive and pass the

561

562

Chapter8: ManipulatingProteins, DNA,and RNA ' / 15 , 0 0 0 1 h o f t h e h u m a n g e n o m e( 2 0 0 , 0 0 0n u c l e o t i d ep a i r s )-gene

1

r

f-gene

haplotype block

2-

_-, l

MODERN DESCENDANT W I T HG E N E T I D CI S E A S E

C O N C L U S I OA N :M U T A T I O N l N G E N E1 C A U S ETSH ED T S E A S E

mutation on to their offspring. working with haplotype maps of individual genes, researchershave detected such positive selection for two human genes that confer resistance to malaria. The alleles that confer resistance are widespread in the population, but they are embedded in unusually large haplotype blocks, suggestingthat they rose to prominence recently in the human gene pool (Figure 8-60). In revealing the paths along which humans evolved, the human haplotype map provides a new window into our past; in helping us discover the genesthat make us susceptible or resistant to disease,the map may also provide a rough guide to our individual futures.

Figure8-59 Tracingthe inheritanceof SNPswithin haplotype blocksto reveal the locationof a disease-causing gene. An ancestorwho acquiresa diseasecausingmutationin gene 1 will passthat mutationalongto hisor her descendants. Partof this geneis embeddedwithin a haplotypeblock (redshadlng)-a clusterof variations(about30 SNPs) that havebeen passedalongfrom the ancestorin a continuouschunk.In the 400 generations that separate the ancestorfrom modern descendants with the disease, SNPs locatedover most of the ancesrral 200,000-nucleotide-pair regionshown havebeen shuffledby meiotic recombination in the descendant genome (b/ue). (Notethat the overlapof yellowand red is seenas orange,and the overlapof yellowand blue is seenasgreen.) The30 SNPswithin the haplotypeblock,however, havebeeninheritedasa group,as no crossovereventshaveyet separatedthem. To locatea genethat causesthe inherited disease, the SNPpatternsin a numberof peoplewho havethe diseaseneedto be analyzed. An individualwith the disease patternof SNps will retainthe ancestral locatedwithinthe haplotypeblockshown, revealingthat the disease-causing mutationis likelyto lie withinthat haplotypeblock-thus in gene 1.The beautyof usinghaplotypemapsfor this type of linkageanalysis is that onlya fractionof the total SNPsneed to be examined: one shouldbe ableto locate genesaftersearching throughonly about 10olo of the 3 millionusefulSNPspresentin the humanqenome.

( A ) s i z eo f h a p l o t y p eb l o c ks u r r o u n d i n ga t y p i c a la l l e l e a l l e l e so i g e n e1

ndvdua. Qfftl]il0l0illl{]|ul0ill0il0 ilt]10til0{]iltf i n d i v i d u aSl N p s ( B ) u n u s u a l l yl a r g eh a p l o t y p eb l o c ks u r r o u n d a s p a r t i c u l aar l l e l eo f g e n e2 gene 2

utt0iltI t0t00ill t00il001 h a p l o t y p eb l o c k

Figure8-60 ldentificationof allelesthat have been selectedfor in fairly recent human historyby the unusuallylarge haplotype blocks in which they are embedded.TheSNPsareindicatedin this diagramby verticalbars,whichareshown as whiteor black accordingto their DNA sequence. Haplotypeblocksareshadedin red,genesin yellow,and the restof the chromosomein b/ue.Thesedata suggest that this particularalleleof gene2 arose relatively recentlyin humanhistory.

GENE EXPRESSION ANDFUNCTION STUDYING

Are Influenced by MultipleGenes ComplexTraits A concert pianist might have an aunt who plays the violin. In another family, the parents and the children might all be fat. In a third family, the grandmother might be an alcoholic, and her grandson might abuse drugs. To what extent are such characteristics-musical abiliry obesity, and addiction-inherited genetically?This is a very difficult question to answer. Some traits or diseases"run in families" but appear in only a few relatives or with no easily discernible pattern. Characteristicsthat do not follow simple (sometimes called Mendelian) patterns of inheritance but have a genetically inherited component are termed complex traits. These traits are often polygenic; that is, they are influenced by multiple genes, each of which makes a small contribution to the phenotlpe in question. The effects of these genes are additive, which means that, together, they produce a continuum of varying features within the population. Individually, the genes that contribute to a polygenic trait are distributed to offspring in simple patterns, but because they all influence the phenotype, the pattern of traits inherited by offspring is often highly complex. A simple example of a polygenic trait is eye color, which is determined by enzyrnes that control the distribution and production of the pigment melanin: the more melanin produced, the darker the eye color. Becausenumerous genes contribute to the formation of melanin, eye color in humans shows enormous variation, from the palest gray to a dark chocolate brown. Although diseasesbased on mutations in single genes (for example, sicklecell anemia and hemophilia) were some of the earliest recognized human inherited phenotypes, only a small fraction of human traits are dictated by single genes. The most obvious human phenotlpes-from height, weight, eye color, and hair color to intelligence, temperament, sociability, and humor-arise from the interaction of many genes. Multiple genes also almost certainly underlie a propensity for the most common human diseases:diabetes, heart disease,high blood pressure,allergies,asthma, and various mental illnesses,including major depression and schizophrenia. Researchers are exploring new strategiesincluding the use of the haplotlpe maps discussed earlier-to understand the complex interplay between genes that act together to determine many of our most "human' traits.

Beginswith a KnownGeneand Determines Reverse Genetics Requirelts Function WhichCellProcesses As we have seen, classical genetics starts with a mutant phenotype (or, in the case of humans, a range of characteristics) and identifies the mutations (and consequently the genes) responsible for it. Recombinant DNA technology' in combination with genome sequencing, has made possible a different type of genetic approach. Instead of beginning with a mutant organism and using it to identify a gene and its protein, an investigator can start with a particular gene and proceed to make mutations in it, creating mutant cells or organisms so as to analyze the gene's function. Because this approach reverses the traditional direction of genetic discovery-proceeding from genes to mutations, rather than vice versa-it is commonly referred to as reverse genetics. Reverse genetics begins with a cloned gene, a protein with interesting properties that has been isolated from a cell, or simply a genome sequence' If the starting point is a protein, the gene encoding it is first identified and, if necessary,its nucleotide sequence is determined. The gene sequence can then be altered in uitro to create a mutant version. This engineered mutant gene, together with an appropriate regulatory region, is transferred into a cell where it can integrate into a chromosome, becoming a permanent part of the cell's genome. All of the descendants of the modified cell will now contain the mutant gene. If the original cell used for the gene transfer is a fertilized egg,whole multicellular organisms can be obtained that contain the mutant gene, provided that the mutation does not causelethalitY.In some of these animals, the altered gene

563

564

Chapter8: ManipulatingProteins, DNA,and RNA

will be incorporated into the germ cells-a germ-line mutation-allowing mutant gene to be passed on to their progeny.

the

GenesCanBe Engineered in SeveralWays we have seen that mutant organisms lacking a particular gene may quickly reveal the function of the protein it encodes. For this reason, a gene "knockout"-in which both copies of the gene in a diploid organism have been inactivated or deleted-is a particularly useful type of mutation. However, there are many more types of genetic alterations available to the experimenter. For example, by altering the regulatory region of a gene before it is reintegrated into the genome, one can create mutant organisms in which the gene product is expressedat abnormally high levels,in the wrong tissue, or at the wrong time in development (Figure 8-6r). By placing the gene under the control of an

genome than to replace the endogenous genes with it. The dominant-negative strategy exploits the fact that most proteins function as parts of larger protein complexes.The inclusion of just one nonfunctional component can often inactivate such complexes.Therefore, by designing a gene that produces large quantities of a mutant protein that is inactive but still able to assembleinto the tomplex, it is often possible to produce a cell in which all the complexes are inactivated despite the presence of the normal protein (Figure g-62). As noted in the earlier discussion of classicalgenetics,if a protein is required for the survival of the cell (or the organism), a dominant-negaiive mutant will be inviable, making it impossible to test the function of the protein. To avoid this problem in reverse genetics, one can couple the mutant gene to an inducible promoter in order to produce the faulty gene product only on command-for example, in response to an increase in temperature or to the presence of a specific signal molecule. In studying the action of a gene and the protein it encodes, one does not always wish to make drastic changes-flooding cells with huge quantities of the protein or eliminating a gene product entirely. It is sometimes useful to make

n o r m a lp r o t e i n in complex

ww' /,

\\

ACTIVE

mutant protein complex

c o m p l e xo f m u t a n t p r o t e i na n d n o r m a l protein

,itfl

Figure8-61 Ectopicmisexpression of Wnt, a signaling protein that affects developmentof the body axisin the earlyXenopusembryo. In this experiment, mRNAcodingfor Wnt was injectedinto the ventralvegetal blastomere, inducinga secondbody axis (discussed in Chapter22).(FromS.Sokol et al.,Cell67:741-752,1991.With permission from Elsevier.)

Figure8-62 A dominant-negative effect of a protein.Here,a geneis engineered to producea mutantproteinthat preventsthe normalcopiesof the same proteinfrom performingtheirfunction. I n t h i ss i m p l ee x a m p l et ,h e n o r m a l proteinmustform a multisubunit complexto be active,and the mutant proteinblocksfunctionby forminga mixedcomplexthat is inactive.In this way,a singlecopyof a mutantgene locatedanywherein the genomecan inactivate the normalproductsproduced by othergenecopies.

ANDFUNCTION S T U D Y I N G E N EE X P R E S S I O

56s

sequence into one of its two strands. After transfection, plasmids that carry the fully modified gene sequence are obtained. The modified DNA is then inserted into an expressionvector so that the redesigned protein can be produced in the appropriate type of cells for detailed studies of its function. By changing selected amino acids in a protein in this way-a technique called site-directed mutagenesis-one can determine exactly which parts of the pollpeptide chain are important for such processes as protein folding, interactions with other proteins, and enzymatic catalysis (Figure 8-63).

GenesCanBelnsertedinto the GermLineof Many Engineered Organisms Altered genes can be introduced into cells in a variety of ways. DNA can be microinjected into mammalian cells with a glassmicropipette or introduced by a virus that has been engineered to carry foreign genes.In plant cells, genes are frequently introduced by a technique called particle bombardment: DNA samples are painted onto tiny gold beads and then literally shot through the cell wall with a specially modified gun. Electroporation is the method of choice for introducing DNA into bacteria and some other cells.In this technique, a brief electric shock renders the cell membrane temporarily permeable, allowing foreign DNA to enter the cltoplasm.

(D)

Figure8-63 The use of a synthetic oligonucleotideto modify the proteincoding region of a gene by site-directed (A)A recombinantplasmid mutagenesis. into containinga geneinsertis separated itstwo DNAstrands.A synthetic p l a s m i dc l o n i n gv e c t o r i n s e r t e dn o r m a lg e n e t-: primercorresponding to oligonucleotide (A, .,N$rriltilil ililililtilt iltrilltilt,rililll I i I lll lll llllr/ part of the genesequencebut containing a singlealterednucleotideat a ,?,, point is addedto the predetermined " / i r l , , i l i l ti l l t i l l tl l l l l l l l l t r l l l r l llllrl l i l l l l l l llll l , r l l l l l ' 1 i l \ DNAunderconditions single-stranded ,-*oro that permitimperfectDNAhybridization SEPARATION (seeFigure8-36).(B)The Primer eotide to the DNA,forminga single hybridizes desired nucleotidepair.(C)The mismatched recombinantplasmidis madedoublestrandedby in vitro DNA sYnthesis (startingfrom the primer)followedby sealingby DNAligase.(D)The doublestrandedDNAis introducedinto a cell, using Replication whereit is replicated. coMPLETtoN I srnaruo one strandof the templateproducesa I sv orunPoLYMERASE normalDNAmolecule,but replication I AND DNA LIGASE V usingthe otherstrand(theone that containsthe primer)producesa DNA moleculecarryingthe desiredmutation. Onlyhalfof the progenycellswill end up rrririrrirririiririrrriiirrirriiirirrirrirrriiririirrriiriiiriirrr,rLirrrr. with a plasmidthat containsthe desired I mutant gene.However,a progenycell , oLLowED I l N T R o D U c r l o Nl N T o c E L L s F A N D S E G R E G A T I o N B Y R E P L I C A T I o N that containsthe mutatedgenecan be I RE L L S I I N T OD A U G H T E C from othercells,and identified,separated t\ culturedto producea pure populationof cells,all of whichcarrythe mutatedgene. u:;,i3,,. Onlyone of the manychangesthat can be engineeredin this way is shownhere. of the l l r i ; 1l l l l l l l l l l l l l illl l l l l l l l l i l l l l l l l l i l l l l l l l l l l l l l l l lll1i \ \ With an oligonucleotide morethan one appropriatesequence, I rnnruscnterton I rnnruscnrRrroru aminoacidsubstitutioncan be madeat a V V time,or one or moreaminoacidscan be < , f 3 ' 3' s'. insertedor deleted.Althoughnot shown IV rnarusrartoru I rnnr'rsrnrroru in thisfigure,it is alsopossibleto createa V mutationby usingthe site-directed Ala Asp and PCR appropriateoligonucleotides p r o t e i nw i t h t h e s i n g l ed e s i r e d n o r m a lp r o t e i n (insteadof plasmidreplication) to amplify c h a n g e a c i d amino the mutatedgene.

566

Chapter8: ManipulatingProteins, DNA,and RNA

N O R M A LG E N EX

GENE REPLACEMENT

GENE KNOCKOUT

GENE ADDITION

I I

I

V o n l y m u t a n t g e n e i sa c t i v e

n o a c t r v eg e n e r sp r e s e n t

ll VV b o t h n o r m a la n d m u t a n t g e n e sa r e a c t i v e

(A)

(B)

(c)

:

I

I :.:::.."'-'-'

Unlike higher eucaryores (which are multicellular and diploid), bacteria, yeasts,and the cellular slime mold Dictyostelium generally exist as haploid single cells. In these organisms, an artificially introduced DNA molecule carrying a mutant gene can, with a relatively high frequency, replace the single copy of the normal gene by homologous recombination; it is therefore easyto produce cells in which the mutant gene has replaced the normal gene (Figure g-6,1A).In this way, cells can be made in order to miss a particular protein or produce an altered form of it. The ability to perform direct gene replacements in lower eucaryores, combined with the power of standard genetic analysesin these haploid organisms, explains in large part why studies in these types of cells have been so important for working out the details of those cell processesthat are shared by all eucaryotes.

AnimalsCanBeGenetically Altered Gene additions and replacements are also possible, but more difficult to perform, in animals and plants. Animals and plants that have been genetically engineered by either gene insertion, gene deletion, or gene replacement are called transgenic organisms, and any foreign or modified genes that are added are called transgenes. we concentrate our discussion on transgenic mice, as enormous progressis being made in this area. If a DNA molecule carrying a mutated mouse gene is transferred into a mouse cell, it usually inserts into the chromosomes at random, but about once in a thousand times, it replacesone of the two copies of the normal gene by homologous recombination. By exploiting these rare "gene targeting" events, any specific gene can be altered or inactivated in a mouse cell by a direct gene replacement. In the special case in which both copies of the gene of interest is completely inactivated or deleted, the resulting animal is called a "knockout" mouse.

homologous recombination event is likely to have caused a gene replacement to occur are isolated. The correct colonies among these are identified by pcR or by Southern blotting: they contain recombinant DNA sequences in which the

Figure8-64 Gene replacement,gene knockout,and gene addition. A normalgenecan be alteredin several waysto producea transgenic organism. (A)The normal gene(green)can be completelyreplacedby a mutant copy of the gene (red).fhisprovidesinformation on the activityof the mutantgene without interference from the normal gene,and thus the effectsof smalland subtlemutationscan be determined. (B)Thenormalgenecan be inactivated completely, for example,by makinga largedeletionin it. (C)A mutantgenecan simplybe addedto the genome.In some organisms this is the easiesttype of geneticengineering to perform.This approachcan provideusefulinformation when the introducedmutantgene overrides the functionof the normal gene,aswith a dominant-negative mutation(seeFigure8-62).

567

STUDYING GENEEXPRESSION AND FUNCTION

mice are mated, one-fourth of their progeny will be homozygous for the altered gene. Studies of these homozygotes allow the function of the altered gene-or the effects of eliminating the gene's activity-to be examined in the absence of the corresponding normal gene. The ability to prepare transgenic mice lacking a knonm normal gene has been a major advance, and the technique is now being used to determine the functions of all mouse genes (Figure 8-66). A special technique is used to produce conditional mutants, in which a selectedgene becomes disrupted in a specific tissue at a certain time in development. The strategy takes advantage of a site-specific recombination system to excise-and thus disable-the target gene in a particular place or at a particular time. The most common of these recombination systems,called Cre/lox, is widely used to engineer gene replacements in mice and in plants (seeFigure 5-79). In this case,the target gene in ES cells is replaced by a fully functional version of the gene that is flanked by a pair of the

T E S TF O RT H ER A R E C O L O N YI N W H I C H T H ED N A F R A G M E N T H A SR E P L A C EODN E C O P YO FT H E N O R M A LG E N E

EMBRYo I HvenroEARLY FROM FORMED I PARTLY I EsCELLS

I N T R O D U CHEY B R I D EARLYEMERYOINTO PSEUDOPREGNANT MOUSE

E Sc e l l sw i t h o n e c o p yo f t a r g e t g e n e r e p l a c e db y m u t a n t g e n e

T R A N S G E NM I CO U S E WITH ONECOPYOF G E N ER E P L A C E D TARGET B Y A L T E R EG DENE I N G E R ML I N E

Figure8-65 Summaryof the procedures used for making gene replacementsin mice.In the first step (A),an altered versionof the geneis introducedinto culturedES(embryonicstem)cells.Only a few rareEScellswill havetheir normalgenesreplacedby corresponding the alteredgenethrougha homologous event.Althoughthe recombination procedureis often laborious,theserare cellscan be identifiedand culturedto eachof producemanYdescendants, whichcarriesan alteredgenein placeof one of its two normalcorresponding genes.In the next stepof the procedure (B),thesealteredEScellsare injectedinto a very earlymouseembryo;the cellsare into the growingembryo, incorporated and a mouseproducedbYsuchan embryowill containsomesomaticcells (indicatedby orange)that carrythe alteredgene.Someof thesemicewill alsocontaingerm-linecellsthat contain the alteredgene;when bred with a of normalmouse,someof the ProgenY thesemicewill containone copyof the alteredgenein all of theircells.lf two such mice are bred (not shown),someof the progenywill containtwo altered in all genes(oneon eachchromosome) of their cells. lf the originalgenealteration completelyinactivatesthe function of the gene,thesehomozygousmiceare knownas knockoutmice.Whensuch micearemissinggenesthat function they oftendie with duringdevelopment, specificdefectslong beforethey reach adulthood.Theselethaldefectsare carefullyanalyzedto help determinethe normalfunctionof the missinggene'

568

Chapter8: ManipulatingProteins, DNA,and RNA

(B)

short DNA sequences, called lox sites,that are recognized by the cre recombinase protein. The transgenic mice that result are phenotypically normal. They are then mated with transgenic mice that expressthe cre recombinase gene under the control of an inducible promoter. In the specific cells or tissues in which Cre is switched on, it catalyzesrecombination between the lox sequences-excising a target gene and eliminating its activity. similar recombination systems are used to generate conditional mutants in Drosophila (seeFigure 2249).

Transgenic PlantsAre lmportantfor BothCellBiologyand Agriculture A damaged plant can often repair itself by a process in which mature differentiated cells "dedifferentiate," proliferate, and then redifferentiate into other cell types. In some circumstances,the dedifferentiated cells can even form an apical meristem, which can then give rise to an entire new plant, including gamet"s. This remarkable developmental plasticity of plant celli can be exploited to generate transgenic plants from cells growing in culture.

culture (Figure S-67).

made it possible, for example, to modify the lipid, starch, and protein stored in seeds, to impart pest and virus resistance to plants, and to create modified plants that tolerate extreme habitats such as salt marshes or water-stressedsoil. Many of the major advances in understanding animal development have come from studies on the fruit fly Drosophilaand the nematode worm c. ele-

Figure8-66 Transgenicmice engineered to expressa mutant DNA helicaseshow prematureaging.The helicase, encoded by the Xpd gene,is involvedin both transcription and DNArepair.Compared with a wild-typemouseof the sameage (A),a transgenic mousethat expresses a defectiveversionof Xpd (B)exhibitsmany of the symptomsof prematureaging, includingosteoporosis, emaciation, early graying,infertility, and reducedlife-span. The mutationin Xpdusedhereimpairs the activityof the helicase and mimicsa mutationthat in humanscauses trichothiodystrophy, a disorder characterizedby brittle hair,skeletal abnormalities, and a very reducedlife expectancy. Theseresultsindicatethat an accumulation of DNAdamagecan contributeto the agingprocessin both humansand mice.(FromJ.de Boeret al., Science296:"1 276-1 279, 2002.With permissionfrom AAAS.)

569

AND FUNCTION STUDYING GENEEXPRESSION

l e a f d i s c si n c u b a t e dw i t h g e n e t i c a l l ye n g i n e e r e d Agrobacterium for 24 h

discsremoved from t o b a c c ol e a f

_:

proliferate

shoot shoot-inducing medium

transJer shoot to rootinducing medium

(A)

grow up rooted seedling adult plantcarrying t r a n s g e n et h a t w a s o r i g i n a l l yp r e s e n ti n the bacteria

b a c t e r i acl e l l

cytosol

recombinant p l a s m i di n Agrobacterium

plantchromosome

D,N A I SE X C I S EFDR O MP L A S M I D W I T H I NT H EB A C T E R I U M N T OT H E E IDR E C T LIY A S A L I N E A RM O L E C U LAEN D I ST H E NT R A N S F E R RD DT OT H EP L A N TC H R O M O S O M E P L A N TC E L LW , H E R EI T B E C O M EISN T E G R A T EI N

several major advantagesas a "model plant" (seeFigures 1-46 and 22-ll2).The relatively small Arabidopsis genome was the first plant genome to be completely sequenced, and the pace of research on this organism now rivals that of the model animals.

LargeCollectionsof TaggedKnockoutsProvidea Toolfor the Functionof EveryGenein an Organism Examining Extensive collaborative efforts are underway to assemble comprehensive libraries of mutations in a variety of model organisms, including S. cereuisiae,C. elegans,Drosophila,Arabidopsis, and the mouse. The ultimate aim in each case is to produce a collection of mutant strains in which every gene in the organism has been systematically deleted or altered in such a way that it can be conditionally disrupted. Collections of this type will provide an invaluable resource for investi-

Figure8-67 A procedureused to make a transgenicplant.(A)Outlineof the process. A discis cut out of a leafand incubatedin culturewith Agrobocterium cellsthat carrya recombinantplasmid marker that containsboth a selectable The geneand a desiredtransgene. woundedcellsat the edgeof the disc that attractthe release substances Agrobacteilumcellsand causethem to injectDNAinto thesecells.Onlythose plantcellsthat takeup the appropriate marker the selectable DNAand express gene surviveto proliferateand form a of growth The manipulation callus. and nutrientssuppliedto the regulators callusinducesit to form shoots,which root and grow into adult subsequently (B)The plantscanyingthe transgene. preparation of the recombinantplasmid and its transferto plantcells.An plasmidthat normally Agrobacterium carriesthe T-DNAsequenceis modified by markergene substitutinga selectable gene) (suchasthe kanamycin-resistance and a desiredtransgenebetweenthe When T-DNArepeats. 25-nucleotide-pair recognizesa plant cell, the Agrobacterium it efficientlypassesa DNA strandthat into the plantcell, carriesthesesequences usingthe specialmachinerythat normally T-DNAsequence. the plasmid's transfers

570

Chapter8: ManipulatingProteins, DNA,and RNA

sequence h o m o l o g o u st o yeasttarget g e n ex

selectable marKergene

u n i q u e" b a r c o d e " sequence

target gene

osome

Yeasttarget gene x ---

:1. +I

l

HOMOLOGOUS RECOMBINATION

primer PCR bas targetgene t a r g e t g e n e x r e p l a c e db y s e l e c t a b l e m a r k e rg e n e a n d a s s o c i a t e"db a r c o d e "s e q u e n c e (A) YEA5T

(B)

I t

PCRproduct detected on gel only if t r a n s p o s a b l e l e m e n th a s i n s e r t e di n target gene of interest

ARABIDOPSIS AND DROSOPHILA

Figure8-68 Makingcollections (A)A deletioncassette of mutantorganisms. for usein yeastcontains (red) DNAsequences homologous to eachendofa targetgenex,a selectable gene(btue), marker anda unique"barcode"sequence approximately 20 nucleotide pairsin length(green).This DNAis introduced intoyeastcells,whereit readilyreplaces the targetgeneby homologous recombination. Byusinga collection of suchcassettes, eachspecific foronegene,a library of yeastmutants canbeconstructed containing a mutantforeverygene.(B)A similar approach canbetakento prepare taggedknockoutmutantsin Arabidopsis andDrosophilo. In thiscase,mutations aregenerated by the accidental insertion of a transposable element intoa targetgene. ThetotalDNAfromthe resulting organism canbecollected andquicklyscreened fordisruption of a geneof interest by usingPCRprimers thatbindto thetransposable element andto thetargetgene. productisdetected A PCR on thegelonlyif thetransposable g-45). element hasinserted intothetargetgene(seeFigure gating gene function on a genomic scale.In some cases,each of the individual mutations within the collection will express a distinct molecular tag-in the form of a unique DNA sequence-designed to make identification of the altered gene rapid and routine. rn s. cereuisiae,the task of generating a complete set of 6000 mutants, each missing only one gene, is made simpler by yeast's propensity for homologous recombination. For each gene, a "deletion cassette" is prepared. The casiette consists of a special DNA molecule that contains 50 nucleotides identical in sequence to each end of the targeted gene, surrounding a selectablemarker. In addition, a special "barcode" sequencetag is embedded in this DNA molecule to facilitate the later rapid identification of each resulting mutant strain (Figure 8-68). A large mixture of such gene knockout mutants can then be grown r.rder various selective test conditions-such as nutritional deprivation, a temperature shift, or the presence of various drugs-and the cells that survive can be rapidly identified by their unique sequence tags. By assessinghow well each mutant in the mixture fares, one can begin to assesswhich genes are essential, useful, or irrelevant for growth under the various conditions. The challenge in deriving information from the study of such yeast mutants lies in deducing a gene'sactivity or biological role based on a mutant phenotype. Some defects-an inability to live without histidine, for example-point airecity to the function of the wild-type gene. other connections marnot be so obvious. \Mhat might a sudden sensitivity to cold indicate about the role of a particular gene in the yeast cell? such problems are even greater in organisms that are more complex than yeast.The loss of function of a single gene in the mouse, for example, may affect many different tissue ty?es at different stages of development-whereas the loss of other genes may have no obvious effect. Adequate$ characterizing mutant phenotypes in mice often requires a thorough examination, along with extensive knowledge of mouse anatomy, histology, pathology, physiology, and complex behavior. The insights generated by examination of mutant libraries, however, will be great. For example, studies of an extensive collection of mutants in Mycoplasma genitalium-the organism with the smallest known genome-have identified the minimum complement of genes essential for cellular life. Analysis of the mutant pool suggeststhat growth under laboratory conditions requires about three-quarters of the 480 protein-coding genesin M. genitalium. Approximately

urn0z

())

('p]-lsreqsllqnd uelltLu)PW arruoN"p 1aIz>uo9 ij urorJ'f pue 8) r!o4 uolssruiladq}|^ '0002 tgEE-''EE:BOI? 'alel6rr.u palleJ regrnuo.rd oMl aq1'IVNU^q pale^tl)eu!uaaqsequotst^tplla) ol aneq ur pa^lo^uraua6e q>rqrvr ur a6elsauresaql 1eo{rqua ulo6 (l) 'ofuqua aql }o }leq roualsodaql ur raqta6oleuro) pue pele.rbtuaneq(speaqmolepar)tal)nuoldLulads pue 66a aql'pazrlrllaj uaaq seq 66a aqt lage ,(;lloqso,tqr.uauloar ed,t-pllM (g) '1n6 ypgsp ^q (Z) lo ,(llrerrp 6ugtrefur eqt olul 11or 3 q];rt VNUspaql 6utssatdxa sruroMaql 6urpaal,(q (L)suDbap) olur pa)nporluraq ue) (VNUsp) VNUpapuerts -alqnoc (v) 'o)uoraJrolulvNU rtq PalPar)uollelnur enllebau-1ueuruo659-g arn611

(v)

\) @ ul.rom{q ua}ea 'VNU popuerls-alqnop 11o: g t 6urssa.rdxa

uor8al papuEJts-alqnopE aJnpord 'Surrredes?q q8norq] 'pue JIaslI uo {ceq ploJ uef, lEq] VNU pau8lsap.{llBlJadsE sl slq} uaUO 'reloluord elqlJnpul ue Jo loJ}uof, eql Jepun IVNu aq] ssaJdxalPql spluluE 3IueSsuEJla{EIu ol pesn eJBsanbluqJe} 'asEJslq] VNCIluPurqruoJer taqlBJ lesnour aql 01peJJo papaful ]ou sI IVNU eql uI 'acrru ot perlddp flap4 ueaq osp seq anbruqce] pelelal e ',{puaca.raro61 'seua8ruJolr,rJo luauraydruoc eJque aql ol suop -cun; Suru8rsseur dlaq ol pesn Suraq sr rVNU (paruanbas uaaq ser4sun&ala'JJo euoua8 eJrluaaql esneJag 'sadft anssp luaJaJrp ur aue8 1e8re1aqlgo uorssaJdxa sllqHul 1l aJeqM 'ruJo^ aql Jo ,{poq aql lnoqSnorql pelnqlrlslp sl vNu aqJ 'g '(69-9 arn81g) VNU aq] ernpord o1 pa;aaufue qoJ qtlm peJ aq ueo ruro^\ aql palcafur eq upo VNU eql :aydurs almb Jo 'lprurue eqlJo euqselur eql olur r(11carrp papuens-alqnop aqt Surcnporlur 'sruro.l,rqryv\ SuplJo^ uaqlgy'sua&a1a'3 sl VNU 'saua8 'epolerueu eql ul uollf,urg aua8 Lpnls o1 pasn dlaprruueeq oqe seq IVNU uprunq pue esnou 000'gZ eqr qllm srsr(pue 3o addl erups eql 1no ,rtrrec ol a1q -rssod aq IIIM ]l 'uoos 'slleJ pernlpc SursnpaJolruou eq UEJleql ssacordduB ur ^ olle (aua8 drala aua8 ,(g d-ra,taJo eloJ aql lsel ol (sqluou [BJeAesul (sJeqJJBasaJ roJ euo) selnJelou yygg nllqdosolc, 000'9I Jo slas 'peapul 'saull arnllno llec uprleurrueru pue npqdosotg ur saua8 alelrlJ-pur ol pasn .{puanbe4 sI IVNU

IoJroore^rlreJJe,{1e rcadsauervNusa{E ru

"":""#XiilH:: il':i::.:

":HJ*T" B otur Sur8e4cedslr 3ur 'ulletuoJqJ uJoJ passardar/.11€uorldrJcsupJl IEnp srqJ Jo -lJeJrp J1as1raua8 ta8rel aq] qllm tf,EJalurpue snalJnu aql Jelue uec losopb aql ur uoqeper8ap [q pacnpord s]ueru8e4 VNU 'saua8 elEArlJBuI d1qe1suec rg111g qJIqM q8norql usrueqoau puoces e sr ereq] '2 raldeq3 ur lttes a.tt sE ']ng 'uors -sardxaaue8 ur sa8ueqc elqelrJeqesneJ ueJ ry51g 's11ac LuaSord o1uo passedaq uec sluaur8e4 VNU iloqs asaql asneJeg 'VNUru 1a8re1aql;o uopeuruqe panup -uoJ aql stcerrp qclq ^'VNu pepuerls-elqnop aroru eJnpord or VNU paper8ap srqt Jo sluaru8erg Ilprus sasn ,(puanbasqns IIeJ eqJ 'uopuper8ap stl slcerrp pue aua8 la8re1aq] [q pacnpord VNUUr aqt q]lm sazrprrqdqtl 'passerord sl VNU eql reUV'pele^rloeur aq o1aua8 aqlgo uedgo leqt saqcleru acuanbas eppoelcnu asoqm elnJeloru VNU papuerls-alqnop e ursrue8ro Jo IIaJ e olut secnpoJlur anbruqcal eqJ '(gII-Z a;n3rg ees) slueruala alqesodsuen pue sasnJrl urpueJ '6un; 'sleturue 's1ue1dfueru ur pesn lsurc8e se^lasuaql ]calord ol eozolord puu ursrueqJeru leJnleu e slroldxa poqleur slql '(Uoqs JoJ 'I-VNU)eJuaJaJJeluI VNU palle:) 'para,rocsrpdpueJal ueeq seq saua8alelpcuul o1zterurarseeqJnru B 'aue8 eql Jo suorlcunJ aql Surpuelsrapun ro; qceordde 1ryraa,todlsoru aq] sdeqrad sr sacuanbasuof, aql SurLpnls pue ursrue8ro uE ur aua8 e 1no Suqeou>l qBnoqrlV uo!l)unl auag lsal ol {e4 p;deupuealdurs e sl o)uareJre}ul vNU 'paJa^oJsrpaq o11al.aneq aJrTerlrapun ]eq] susrueqJeu rulnoeloru Jrspq aqlJo Jaqrunu Sursrrd -Jns e leql slsaSSnsqclqm'uollJury u,{i\oDlunJoare saua8ppuesse eseqlJo 00I

LLS

NOIDNnJONVNOISSIUdXI lN:19DNIACnls

'o^lqua DI!ttdosor] e ut e)uanbas,{roteln6eraue6 aa3aq} 4o ,tlnltre aql rolruourol pasnsraua6 1o6tr1 aql Ll)rqMur aloulexoue sMoqsBss-l arn6!l '(92-6aln6rgaas)639 ro urelord lua)saronUuaal6pue (BSS-Zernbl1 aas)(to64) aseprsolte;e6-fl au{zua aql are suralo.rdlalrodal pasn{;uor.ur.uor oml'slla)>rlolletnaur sluauuadxalo1 '()) ut pozueululns aJes]lnsolaqI'slla) uetleulureu,o sadll lualaJJtpJo {leuen e olut uot})aJsueJ} raql rauPuorssatoxa loJ palsaluaql are saln)alorrrvNc lueutqulo)ol asaqf 1 aua6lal.rodal6urporuasaln)alou VNOlsal ernpordol suotleutquo) ur pappe aie sa>uanbas {lo1e1n6e.r aleplpue)6urureluory56 10s1uau6er1 snouen(g)'auPsaql ale l pue x ro; ureped uorssaldxaaql l uralord tetlodarro; eluanbas6urpotaq1,{q pare;darsry urelordto; atuanbas6urpor aql'aldurexastql ul (v) 'uorssardxe qaua6 e 1o ureped aql aulultalap og ugalordregrodare 6ursl g1-g arn6r3

C l l a ) u r X e u e 6 ; 4 os u ; n 1r ( ; ; e u . r o u; a r u a n b e sr f u o 1 e 1 n 6 a r 'l 'C slla) ur aue6 uo sulnl i(;;eul.rou a:uenbesrfuolelnberI pue X Z I lla) ur X eue6 uo su.rnlI;;eu.rou g aruenbasfuo1e1n6er- SNOIS6'I)NO) ())

A lN39 Ult_UodlU l o N U S X V dN O t S S I U d X I

teI tqitlglitif,$stffi;r tifn:ifr

uotssoloxa A aue6 reuodal;o u.ra11ed

,r

r

q

)

slla)

I

v

uotsseJoxe y a u e 6 l e u r r o u ; ou r e l l e d

rlcfSv

A UrAlordJaUOdOr ro,t a:uanbes6urpor srseqlu{s vNu Jo] alrs uels

}UeUlquro)aJ y aua6;o uopssa:dxe eql eututalep leql se)uanoasvNc itroleln6a.r -

/i\

tzt leuJou

x uraloJoJoJ a r u a n b a s6 u 1 p o r

slla) X ]N]9 JO NUSl_-LVd NOrSSrUdXl

s:nn)310nvNq gNrtuv$ (v)

'(02-g ernBIC)slleo olur se1nceloru VNO ]ueurqruocar eseql SuIJnpoJtuI pue 'pesseJdxa IoJluof, rleql Japun aueS JeuodeJ e Sulceld dq parpnls dpsea aq uE3 sr aue8 eql araq ^ pue ueq^\ ,(pcuxa eurruJalap qclq a 'sacuanbas drolep8ar eseqJ 'uorsserdxaaua8 loJluoc 'uor8el Sutpoc eqt Jo uEallsrmop Jo rueeJlsdn palecol 'sacuanbas VNC dJolElnaat 'L rcldeq3 uI llelap uI passnJslp sV '(LZ-6puv 9Z-6 sern8l{ eas) }onpord ulelord sllJo fltnt}Je f,I}Eurdzuaro ef,uecsaJonuaq] SuqceJ] /tq parolruoru ueq] sl auaS reuodeJ eql Jo uolssaJdxe aql 'seseolsoru ul 'eue8 JeuodeJ e qtrm.{pnls Jepun aua8 aq} Jo uoluod Surpoc aql Suroulder,{qpaqsrtdruoJoeaq UEJuolsseJdxeeue8Jo 3uru4} pue uJat}ed aq} Sururruralaq 'rusrue8Joaloqm eq] ur Jo IIac aqt ur pesserdxasI eua8 E aJaq ApuB uaq a Sururupxe dq peuretqo eq uago uer uol]runJ aue8 olul sfqBISuIluepodrul

passerdxlsl eueg e nrlsu, pue sauagleuodeu oraq^ pue uaq^ lee^euuorlezrpuqr(H 'o1 anlc SuoDs 'uorlsunJ eue8 purJou Jo JooJd E ^luessacau lou lnq E se pe^\er^ eq lsnlu lueurJadxa 1y59,{ue Jo s}Insal aql 'dlelulunln 'eue3 elues eql Jo suorSar luaraJJrp 01 paqJtEru salnrelolu vNu IIEIUSaldppur esn ol sI stual -qord qrns pro^E ot d8aterls auo 'aue8 pela8ret eql ol uop1ppe uI sauo8 pal€ler saf,npoJd setulleruos aJoJeJeqlr\/NU 'dlIJelIUIs SurlenDcuur 'stf,eJJa,,leBJEl-JJo,, aJuenbes llqlllxa qcrqMJo sJaqruarueql 'serllueJ aua8 a8Jululeluoo srusluE8Jo ,(ueru asneceq sesrJeurelqoJd Jeqlouv '(sapoleureu uI suoJneu 'alduexa ro;) r-vNu Jo uorlcu eqt ol luElslsal eq ^elu sanssll uleuec 'sutslueSro aloql!\ ulqll A teloeJoq 'seue8 IIB ele^rlJeul fpualcgga ]ou saop tvNU 'suoseal u^aou{un Joc 'slno{f,oDl f,r}aua8 enJl q}r a paJedruos suouellul ppuatod IBJeAesseq ll lnq'srusrue8Jo,{uelu ur }uarcuJepue alduns solleue8 asra^eJapBu sEq[-VNU 'uPlap elPJoqBIeur pazdpue eq ol seue8 la8re1 aql Jo suollJunJ aql Sul aolle 'luarudolaaap ul eurp Jelncrupd e 1e[1uo Jo anssl] pagrcads e ut dpo pacnpord aq uec r-VNUaq] 'pasn JeloruoJd alqrcnpur aqt uo Surpuadaq 'ecuenbas IVNU eqt qJtetu dpcexa ]eqt saua8aql dpo selB^rlJeursse3oJdaq1 ,traurqcetu IVNU aql ^q pezpSocar s1]eq]

V N Up u e ' V N O

' s u l a l o r d6 u l l e l n d ! u e 6 1 :glaldeql

zLs

573

GENEEXPRESSION AND FUNCTION STUDYING

(A) 1tt

It is also possible to directly observe the time and place that the mRNA product of a gene is expressed.Although this strategy often provides the same general information as the reporter gene approaches discussed above, there are instances where it provides additional information; for example, when the gene is transcribed but the mRNA is not immediately translated, or when the gene's final product is RNA rather than protein. This procedure, called in situ hybridization, relies on the principles of nucleic acid hybridization described earlier. \pically, tissues are gently fixed so that their RNA is retained in an exposed form that can hybridize with a labeled complementary DNA or RNA probe. In this way, the patterns of differential gene expression can be observed in tissues, and the location of specific RNAs in cells can be determined (Figure 8-71). In the Drosophilaembryo, for example, such patterns have provided new insights into the mechanisms that create distinctions between cells in different positions during development (described in Chapter 22). Using similar approaches, it is also possible to visualize specific DNA sequences in cells. In this case, tissue, cell, or even chromosome preparations are briefly exposed to high pH to disrupt their nucleotide pairs, and nucleic acid probes are added, allowed to hybridize with the cells' DNA, and then visualized (seeFigure B-35).

Using Expression of IndividualGenesCanBeMeasured QuantitativeRT-PCR Although reporter genes and in situhybridization reveal patterns of gene expression, it is often desirable to quantitate gene expression by directly measuring nRNA levels in cells.Although Northern blots (seeFigure 8-38) can be adapted to this purpose, a more accurate method is based on the principles of PCR (Figure 8-72). This method, called quantitative RT-PCR(reversetranscription-polymerase chain reaction), begins with the total population of nRNA molecules purified from a tissue or a cell culture. It is important that no DNA be present in the preparation; it must be purified away or enzyrnatically degraded' Two DNA primers that specifically match the gene of interest are added, along with reverse transcriptase, DNA polymerase, and the four deoxlmucleoside triphosphates needed for DNA synthesis.The first round of synthesis is the reversetranscription of the nRNA into DNA using one of the primers. Next, a series of heating and cooling cycles allows the amplification of that DNA strand by conventional PCR (seeFigure 8-45). The quantitative part of this method relies on a direct relationship between the rate at which the PCR product is generated and the original concentration of the mRNA species of interest. By adding chemical dyes to the PCR reaction that fluoresce only when bound to double-stranded DNA, a simple fluorescence measurement can be used to track the progressof the reaction and thereby accurately deduce the starting concentration of the mRNA that is amplified (seeFigure 8-72). Although it seems complicated, this quantitative RT-PCRtechnioue (sometimes called real time PCR)is relatively fast and simple

Figure8-71 ln situ hybridizationfor RNA patternof localization.(A)Expression DeltoCmRNAin the earlyzebrafish embryo.Thisgenecodesfor a ligandin the Notchsignalingpathway(discussed in Chapter15),and the patternshown here reflectsits role in the development of somites-the future segmentsof the vertebratetrunk and tail.(B)HighresolutionRNAin slfu localizationreveals the siteswithin the nucleolusof a pea cellwhereribosomalRNAis synthesized. pm in structures,0.5-1 The sausage-like diameter,correspondto the loopsof DNAthat containthe chromosomal genesencodingrRNA.Eachsmallwhite of a single transcription spot represents rRNAgene.(A,courtesyofYun-JinJiang; B,courtesyof PeterShaw.)

I o c q

E J

+ time (numberof PCRcYcles) Figure8-72 RNAlevelscan be The measuredby quantitative RT-PCR. measuredis generatedby a fluorescence onlywhen boundto dye that fluoresces DNAproductsof the double-stranded reaction(seeFigure8-468). the RT-PCR The red samplehasa higher of the mRNAbeing concentration measuredthan doesthe blue sample, sinceit requiresfewerPCRcyclesto reach of concentration the samehalf-maximal DNA.Basedon this double-stranded difference,the relativeamountsof the mRNAin the two samplescan be precisely determined.

574

Chapter8: ManipulatingProteins, DNA,and RNA Figure8-73 UsingDNA microarrays to monitorthe expressionof thousandsof genessimultaneously. To preparethe microarray, DNA fragments-eachcorresponding to a gene-are spottedonto a slideby a robot.Prepared arraysarealsowidelyavailablecommercially. In this example,mRNAis collectedfrom two differentcellsamolesfor a direct comparison of their relativelevelsof geneexpression; the two samples, for example,couldbe from cellstreatedwith a hormoneand untreatedcellsof the sametype.Thesesamplesareconvertedto CDNAand labeled,one with a redfluorochrome, the otherwith a greenfluorochrome. The labeled samplesare mixedand then allowedto hybridizeto the microarray. After incubation, the arrayis washedand the fluorescence scanned.In the portion of a microarray shown,which represents the expression of 110 yeastgenes, redspotsindicatethat the gene in sample1 is expressed at a higherlevel than the corresponding genein sample2; greenspotsindicatethat expression of the geneis higherin sample2 than in sample1. yellowspots revealgenesthat areexpressed at equallevelsin both cellsamples. Dark spotsindicatelittleor no expression in eithersampleof the genewhose fragmentis locatedat that positionin the array.(Microarray courtesyof J.L.DeRisiet al.,Science278:680-686,1997. With permissionfrom AAAS.)

to perform in the laboratory; it has displaced Northern blotting as the method of

choicefor quantifuingmRNAlevelsfrom any givengene.

Microarrays Monitorthe Expression of Thousands of Genesat Once

i cN A m o l e c u l e s c o l l e c t i o no f g e n e - s p e c i { D

+ + +

P C Ra m p l i f i c a t i o n r o b o t i c' p r i n t i n g 'o n t o g l a s ss l i d e

mRNA from s a m p l e1 l a b e l e d with red f luorochrome

m R N Af r o m s a m p l e2 l a b e l e d with green f luorochrome

HYBRIDIZE

+

WASH

I S C A NR E DA N D G R E E N SIGNALS A N D C O M B I N IEM A G E S

I

So far we have discussedtechniques that can be used to monitor the expression of only a single gene (or relatively few genes) at a time. Developed in the 1990s, DNA microarrays have revolutionized the analysis of gene expression by monitoring the RNA products of thousands of genes at once. By examining the expression of so many genes simultaneously, we can now begin to identify and study the gene expression patterns that underlie cell physiology: we can see which genes are switched on (or off) as cells grow, divide, differentiate, or respond to hormones or to toxins. DNA microarrays are little more than glass microscope slides studded with a large number of DNA fragments, each containing a nucleotide sequence that s m a l lr e g i o no f m i c r o a r r a yr e p r e s e n t i n g expressionof 110 genesfrom yeast

oligonucleotides that are synthesizedon the surface of the glasswafer with tech-

To use a DNA microarray to monitor gene expression, mRNA from the cells

Typically the fluorescent DNA from the experimental samples (labeled, for example, with a red fluorescent dye) are mixed with a reference sample of cDNA fragments labeled with a differently colored fluorescent dye (green, for example). Thus, if the amount of RNA expressedfrom a particular gene in the cells of interest is increased relative to that of the reference sample, the resulting spot is red. conversely, if the gene's expression is decreased relative to the referenCesample, the spot is green.If there is no change compared to the referencesample, the spot

575

STUDYING GENEEXPRESSION AND FUNCTION

time 0 1 5m i n 30min th 2h 3h 4h 8h 12h 1 6h 20h 24h

w o u n d h e a l i n gg e n e s

c e l lc y c l eg e n e s

cholesterolbiosynthesis genes

Genesthat belongto the regulated. Figure8-74 Usingclusteranalysis to identifysetsof genesthat arecoordinately dataareobtained microarray analysis, a cluster Toperform or processes. samecluster maybe involved in commonpathways in theirexpression changes andgenesthatshowcoordinate conditions, fromcellsamples to a variety of different exposed serumwasthen of serumfor48 hours; weredeprived patternaregrouped humanfibroblasts together. Inthisexperiment, Ofthe timepoints. at different analysis for microarray addedbackto thecultures at time0 andthecellswereharvested patterns in theirexpression variation justover3OO or greater threefold showed 8600genesanalyzed on theDNAmicroarra, Onthe greenis in expression. a decrease in expression; an increase In response redindicates to serumre-introduction.Here, on similar based the8600geneshavebeengroupedin clusters basis of theresults of manymicroarray experiments, to areturnedon in response in woundhealing patterns showthatgenesinvolved ofexpression.The results ofthisanalysis areshutdown.(From biosynthesis andcholesterol in regulating cellcycleprogression serum, whilegenesinvolved of Sciences.) Academy fromNational Withpermission 1998. M.B.Eisen et al.,Proc.NatlAcad.ici. LJ.S.A.95:14863-14868,

is yellow Using such an internal reference,gene expressionprofiles can be tabulated with great precision. So far, DNA microarrays have been used to examine everl,'thing from the changes in gene expressionthat make strawberries ripen to the gene expression "signatures" of different tlpes of human cancer cells (see Figure 7-3); or from changes that occur as cells progress through the cell cycle to those made in response to sudden shifts in temperature. Indeed, because microarrays allow the simultaneous monitoring of large numbers of genes,they can detect subtle changes in a cell, changes that might not be manifested in its outward appearance or behavior. Comprehensive studies of gene expression also provide an additional layer of information that is useful for predicting gene function. Earliel we discussed how identifying a protein's interaction partners can yield clues about that protein's function. A similar principle holds true for genes: information about a gene'sfunction can be deduced by identi$ring genes that share its expression pattern. Using a technique called cluster analysis,one can identify sets of genes that are coordinately regulated. Genes that are turned on or turned off together under different circumstances are likely to work in concert in the cell: they may encode proteins that are part of the same multiprotein machine, or proteins that are involved in a complex coordinated activity, such as DNA replication or RNA splicing. Characterizing a gene whose function is unknown by grouping it with knorrm genesthat share its transcriptional behavior is sometimes called "guilt by association." Cluster analyses have been used to analyze the gene expression profiles that underlie many interesting biological processes,including wound healing in humans (Figure 8-74). In addition to monitoring the level of mRNA corresponding to every gene in a genome, DNA microarrays have many other uses. For example, they can be used to monitor the progression of DNA replication in a cell (see Figure 5-32) and, when combined with immunoprecipitation, can pinpoint every position in the genome occupied by a given gene regulatory protein (see Figure 7-32). Microarrays can also be used to quickly identify disease-causingmicrobes by hybridizing DNA from infected tissues to an array containing genomic DNA sequencesfrom Iarge collections of pathogens.

"Noise" Biological AnalysisReveals Single-Cell GeneExpression The methods for monitoring mRNAs just described give averageexpressionlevels for each mRNA across a large population of cells. By using a fluorescent

576

Chapter8: ManipulatingProteins, DNA,and RNA

reporter protein whose expressionis under the control of a promoter of interest, it is also possible to accurately measure expression levels in individual cells. These new approaches have revealed a startling amount of variabiliry often calIed biological noise,between the individual cells in a homogeneous population ofcells. These studies have also revealed the presence ofdistinct subpopulations of cells whose existence would be masked if only the average across a whole population were considered. For example, a bimodal distribution of expression levels would indicate that the cells can exist in two distinct states (Figure 8-75), with the averageexpression level of the population being somewhere between them. The behavior of individual cells has important implications for understanding biology, for example, by revealing that some cells constantly and rapidly switch back and forth between tvvo states. Currently, there are two approaches for monitoring gene expressionin individual cells. In the imaging approach, live cells are mounted on a slide and viewed through a fluorescence microscope. This method has the advantagethat a given cell can be followed over time, allowing temporal changes in expression to be measured. The second approach, flow cytometry works by streaming a dilute suspension of cells past an illuminator and measuring the fluorescenceof individual cells as they flow pasr the detector (see Figure 8-2). Although it has the advantage that the expression levels of very large numbers of cells can be measured with precision, flow cytometry does not allow a given cell to be tracked over time; hence, it is complementary to the imaging methods.

Su m m a r y Geneticsand geneticengineeringprouide powerful toolsfor the study of genefunction in both cellsand organisms.In the classicalgeneticapproach, random mutagenesisis coupled with screeningto identfu mutants that are deficient in a particular biological process.Thesemutants are then usedto locateand study the genesresponsiblefor that process. Genefunction can also be ascertained by reuersegenetic techniques. DNA engineering methoclscan be used to alter genesand to re-insert them into a cell'schromosomesso that they becomea permanent part of the genome.If the cell usedfor this gene transfer is a fertilized egg (for an animal) or a totipotent plant cell in culture, transgenic organismsc(tn be produced that expressthe mutant geneand passit on to their progeny.Especiallyimportant for cell biology is the ability to alter cellsand organisms in highly specific ways-allowing one to discern the effecton the cell or the organism of a designedchange in a single protein or RNAmolecule. Many of these methods are being expanded to inuestigategene function on a genome-wide scale. The generation of mutant libraries in which euery gene in an organism has been systematicallydeleted or disrupted prouides inualuable toolsfor exploring the role of eachgene in the elaboratemolecular collaboration that giuesrise to life. Technologiessuch as DNA microarrays can monitor the expressionof thousands of genessimultaneously,prouiding detailed, comprehensiuesnapshotsof the dynamic pcttternsof geneexpressionthat underlie complex cell processes.

PROBLEMS Whichstatementsare true? Explainwhy or why not.

Figure8-75 Different levelsof gene expressionin individualcellswithin a popufation of E.coli bacteria.Forthis experiment,two differentreporter proteins(onefluorescing green,the other red)controlledby a copy of the same promoter,havebeenintroducedinto all of the bacteria. Whenilluminated, some cellsexpressonly one genecopy,and so appeareither red or green,while others expressboth genecopies,and so appear yellow.f his experimentalsoreveals variablelevelsof fluorescence, indicating variablelevelsof geneexpression within an apparentlyuniformpopulationof cells.(FromM.B.Elowitz,A.J.Levine, E.O.Siggiaand P.5.Swain,Science 297:1183-1186,2002.With permission from AAAS.)

real time, using small amounts of unlabeled molecules,but it does not give the information needed to determine the binding constant (^'J.

8-1 Becausea monoclonal antibody recognizesa specific antigenic site (epitope),ir binds only to the specificprotein againstwhich it was made.

8-4 If eachcycleof PCRdoublesthe amount of DNA synthesizedin the previouscycle,then 10 cycleswill give a 103fold amplification, 20 cycleswill give a 106-foldamplification, and 30 cycleswill give a 10e-foldamplification.

8-2 Given the inexorableprogressof technology,it seems inevitable that the sensitivity of detection of molecules will ultimately be pushed beyond the yoctomole level (10-2amole).

Discussthe following problems.

8-3 Surfaceplasmon resonance(SPR)measuresassociation (koJ and dissociation(kon)ratesbetween moleculesin

8-5 A common step in the isolation of cells from a sample of animal tissue is to treat it with trypsin, collagenase, and EDTA. \A/try is such a treatment necessary,and what

577

END-OF-CHAPTER PROBLEMS

doeseach component accomplish?And why doesthis treatment not kill the cells? Do you supposeit would be possibleto raisean anti8-6 body againstanother antibody?Explainyour answer. Distinguish between velocity sedimentation and 8-7 equilibrium sedimentation. For what general purpose is each technique used?\A/hichdo you supposemight be best suited for separatingtwo proteins of different size? 8-B Tropomyosin,at 93 kd, sedimentsat 2.6 S, whereas the 65-kd protein, hemoglobin,sedimentsat 4.3S. (The sedimentation coefficient S is a linear measure of the rate of sedimentation:both increaseor decreasein parallel.)These two proteins are shown as s-carbon backbone models in Figure Q8-1. How is it that the bigger protein sediments more slowlythan the smaller one?Can you think of an analogy from everyday experience that might help you with this problem? tn

hemoglobin

FigureQ8-1Backbone models of tropomyosin andhemoglobin (Problem 8-B). In the classicpaper that demonstratedthe semi-con8-9 servativereplication of DNA, Meselsonand Stahl began by showingthat DNA itself will form a band when subjectedto equilibrium sedimentation. They mixed randomly fragmented E coli DNA with a solution of CsClso that the final solution had a density of 1.71 g/ml. As shor.tnin Figure Q8-2, with increasing length of centrifugation at 70,000 times gravity, the DNA, which was initially dispersed throughout the centrifuge tube, became concentratedover time into a discreteband in the middle. A. Describewhat is happening with time and explain why the DNA forms a discreteband.

B. \A4ratis the buoyant density of the DNA? (The density of the solution at which DNA "floats" at equilibrium defines the "buoyant density" of the DNA.) C. Even if the DNA were centrifuged for twice as long-or even longer-the width of the band remains about what is shown at the bottom of Figure QB-2.\iVtrydoes the band not becomeevenmore compressed?Suggestsome possiblereasons to explain the thickness of the DNA band at equilibrlum. 8-10 Hybridoma technologyallows one to generatemonoclonal antibodies to virtually any protein. \lVhy is it then that tagging proteins with epitopes is such a commonly used technique, especially since an epitope tag has the potential to interfere with the function of the protein? 8-1 1 How many copiesof a protein need to be presentin a cell in order for it to be visible as a band on a gel?Assume that you can load 100 pg of cell extract onto a gel and that you can detect 10 ng in a singleband by silver staining.The concentrationof protein in cellsis about 200 mg/ml, and a typical mammalian cell has avolume of about 1000pm3 and a \pical bacterium a volume of about I pm3. Given these parameters,calculatethe number of copiesof a 120-kdprotein that would need to be presentin a mammalian cell and in a bacterium in order to give a detectableband on a gel. Youmight try an order-of-magnitudeguessbeforeyou make the calculations. 8-12 You want to amplify the DNA between the two stretchesof sequenceshown in Figure Q8-3. Of the listed primers choosethe pair that will allow you to amplify the DNA by PCR. D N At o b e a m p l i f i e d :

fUA-

J

pnmers (1)5'-GACCTGTCCAAGC 3 '- 3' (2) 5' _ CTGGACACCTTCG -LUSUUtsf

LLAU-r

- 3', (4) s',- GCTTCCACAGGTC

c e n t r i f u o a fl i e l d +

LAIAUUSSAI

uulsuL

5',

\5,' )

hours

_uAuuruf

(5) 5',-CATACGGGATTGA3' (5) s',-GTATGCCCTAACT3', (7) 5,_TGTTAGGGCATAC_ 3' /c\ q/-Taa

aTaaarfTATr:-

1'

PCRprimers andpotential FigureQ8-3DNAto beamplified ( P r o b l e8m- 1 2 ) .

0 2.1 4.3 6.4 8.5 10.7 128 149 1 71 19.2 21.3 235 36s 435

FigureQ8-2 Ultraviolet absorptionphotographs showingsuccessive stages in the bandingof E.coli DNA(Problem8*9).DNA, whichabsorbsUV light, showsup as darkregionsin The the photographs. bottom of the centrifuge tube is on the right.(From M. Meselson and F.W.Stahl, Proc.NatlAcad.Sci.U.S.A. 44:671-682,1958. With permission from National Academyof Sciences.)

8-13 In the very first round of PCR using genomic DNA' the DNA primers prime slnthesisthat terminatesonlywhen the cycle ends (or when a random end of DNA is encountered).Yet,by the end of 20 to 30 cycles-a typical amplification-the only visible product is defined preciselyby the ends of the DNA primers.In what cycleis a double-stranded fragment of the correct sizefirst generated? 8-14 Explain the difference between a gain-of-function mutation and a dominant-negative mutation. \A/hyare both thesetypes of mutation usually dominant? 8-15 Discussthe following statement:"We would have no idea today of the importance of insulin as a regulatoryhormone if its absencewere not associatedwith the devastating human diseasediabetes.It is the dramatic consequencesof its absencethat focusedearly effortson the identification of insulin and the study of its normal role in physiology."

578

Chapter8: ManipulatingProteins, DNA,and RNA

REFERENCES General AusubelFN4, BrentR,Kingston REet al (eds)(2002)ShortProrocols in Molecuar Biology, 5th ed NewYork.Wlley BrownTA (2002)Genomes 2, 2nd ed NewYork:Wiley-Liss SpectorDL,GoidmanRD& Leinwand tA (eds)(1998)Cel/sA Laboratory ManualColdSpringHarbor, NY:Co d SpringHarbor Laboratory Press WatsonJD,CaudyAA,MyersRM& Witkowski )A (2007)Recombinant DNA: GenesandGenomes A ShortCourse, 3rded NewYork.WH Freeman lsolating Cellsand Growing Them in Culture E m m e r t - B uM c kR ,B o n n eR r FS , m i t hP De t a l ( 1 9 9 6L) a s ecr a p t u r e microdissectron Science )74:998 1001 H a mR G( ' 1 9 6 5C)o n a lg r o w t ho f m a m m a l i acne l si n a c h e m i c a l l y defined,syntheticmedium ProcNatlAcadSciIISA53.288-293 Harow E& LaneD (1999)UstngAntibodies: A Laboratory Manual Cold SpringHarbor,NY:Co d SpringHarborLaboratory Press Herzenberg LA,SweetRG& Herzenberg LA(1976)Fluorescenceactivated cellsortingSciAm 234.108-116 Levi-Montalc ni R (1987) Thenervegrowthfactorthirty-five yearslater Science 237:1154-1162 LerouPH& DaleyGQ(2005) potentialof embryonic Therapeutic stem cellsBloodRev19.32131 Mllsten C (]980)Monoclonal antibodiesSciAm 243:6674 Purifying Proteins de DuveC & Beaufay H (1981)A shorthistoryoftissuefractionation J CellBtol91.293s-299s KroganN1,CagneyG,Yu H et al (2006)Globallandscape of protein complexesin the yeastSaccharomyces cerevrsrae Nature44a.637-43 LaemmlUK(1970)Cleavage proteinsduringthe assembly of structural of the headof bacteriophage14 Nature2)7:680-685 Nirenberg MW & Matthaei .lH(1961) Thedependence of cell-free proteinsynthesis in F collon naturally occurring or synthetic polyribonucleotides ProcNatI Acad SciIJSA47.1588-16a2 O'Farrell PH(l 975)High-resolution two-drmensional electrophoresis of proteinsJ BiolChem250.4A07-4021 Palade G (l 9/5) Intracellular aspects of the process of proteinsynthesis Science 189347-358 ScopesRK& CantorCR(1994)ProteinPurification: Principtes and Practice, 3rded NewYork:Springer Verlag Analyzing Proteins Sranden C &Toozei (1999)Introduction to ProteinStructure,2nd ed NewYork:GarlandScience FieldsS &SongO (1989)A novelgeneticsystemto detect protein-proteininteractionsNature340.245246 Giepmans BN,AdamsSRet al (2006) Thefluorescent toolboxfor proteinocationandfunctionScience312:217 assessing 24 Kendrew J C( 1 9 6 1T) h et h r e ed i m e n s i o nsatlr u c t u roef a p r o t e i n moieculeSciAm 205:96111 KnightZA & ShokatKM(2007)Chemicagenetics: Wheregeneticsand pharmacoogy meet Cell128:425-30 RigautG,Shevchenko A, RutzB et al (1999) A genericprotern purification methodfor proteincomplexcharacterization and proteomeexp oration NatureBtotechnol 17:10301032 Washburn MP,WoltersD andYatesJR(2001)Large-scale analysis of the yeastproteomeby multidimensional proteinidentification technoogy NatureBtotechnol 19:242-7 WuthrichK (,1989) Proteinstructure determination in solutionby nuclearmagnettcresonance spectroscopy Science 243.45-50 lsolating,Cloning,and SequencingDNA AdamsMD,Celniker SE,HoltRAet al (2000) Thegenomesequence of Drosophila melanogaster Science 287.2i85 2195 AiwineJC,KempDJ& StarkGR(19l/) Methodfor detectionof specific RNAsin agarose gelsby transfer to diabenzyioxymernyr

paperand hybridization with DNAprobes, ProcA/dtl AcadSciUSA 74:5350-5354 Blattner FR,Plunkett G,BlochCAet al (1997) Thecompletegenome qeclren.Faf Fsrhcrirhin .oli K-12Science 27/:1453-1474 CohenS,ChangA, BoyerH & HelllngR (,1973) Construction of plasmids biologically functional bacterial in vitroProcNatlAcadSci usA 70.32403244 (2000)Initial International HumanGenomeSequencing Consortium sequencing and analysis of the humangenome,A/ciure 409864-921 (2006) Internationai HumanGenomeSequencing Consortium The DNAsequence, annotation andanaysisof humanchromosome 3 Nature44A.11941l9B Jackson D,SymonsR& BergP (1972)Biochemica methodfor inserting new geneticinformation into DNAof simianvirus40: circular SV40DNAmolecules containing lambdaphagegenes and the .rala.loqeoneronof Escherichta ColiProcNatlAcad 'ci USA69.2904-2909 genesfrom libraries Maniatis T et al (1978) Theisolation of structural of eukaryoticDNA Cel/15:687701 MullisKB(1990)Theunusualoriginof the polymerase chainreaction SciAm 262:56-61 Nathans D & SmithHO (1975)Restriction endonucleases in the analysis and restructuring of dna molecules AnnuRevBiochem 44:273-93 SaikiRK,GelfandDH,StoffelS et al (1988)Primer-directed enzymatic amplification of DNAwith a thermostable DNApolymerase Science239.487 491 ) o l e c u l aCrl o n i n gA: L a b o r a t oM S a m b r o oJk,R u s s eDl l( 2 0 0 1M r ya n u a, 3rded ColdSpringHarbor, NY:ColdSpringHarborLaboratory Press SangerF,NicklenS & CoulsonAR(1977)DNAsequencing with chainterminatinginhibitorsProcNatlAcadScrUSA74.5463-5467 SmithM (1994)NobellectureSyniheiicDNAand biolog, BiosciRep14:5166 SouthernEM('1975) Detection of specific sequences amongDNA fragmentsseparatedby gel electrophoresis J Mol Biol98503 517 (2000)Analysis Ihe Arabidopsis GenomeInitiative of the genome sequenceof the f oweringplantArabidopsis thalianaNature 408.796Bt5 (1998)Genomesequence fie C.elegans Sequencing Consortium of the nematodeC.elegans: a platformfor investigating biology Science282:2012 2018 VenterJC,AdamsMA,MyersEWet al (2000) Thesequence ofthe human genome Scien ce291:1304-1351 Studying Gene Expressionand Function BooneC,Bussey H & AndrewsU QA07)Exploring geneticinteractions and networkswith yeast NatureRevGenet8:437-449 Botstein D,WhrteRL,Skolnick M & DavisRW(1980)Consrruction of a geneticlinkagemap in man usingrestriction fragmentlength polymorphisms Am J HumGenet32:314-331 DeRisi JL,iyerVR& BrownPO(1997)Exploring the metabolic and geneticcontrolof geneexpression on a genomicscaleScience 278:680-686 (2005)A haplotypemapof the International HapMapConsortium human genomeNature437:1)99-320 Lockhart DJ& Winzeler EA(2000)Genomics, geneexpression and DNA atraysNature405.U/ -836, MelloCC& ConteD (2004)Revealing the worldof RNAinterference Nature431:338-342 Nusslein VolhardC & Weischaus E (1980)Mutations affecting segment numberand polarityin DrosophilaNature287795 801 Palmiter RD& Brinster Rt (1985) Transgenic miceCell41:343-345 RubinGM& Sprading AC (1982) Genetictransformation of Drosophila with transposable elementvectorsSclence 218:348353 SabetiPC,Schaffner SF,FryB et al (2006)Positive naturalselection in the human ineage, Sclence 3 12:1614-1620 WeigelD & Glazebrook J (2001)Arabidopsls A Laboratory Manual ColdSpringHarbor, NY:ColdSpringHarborLaboratory Press

619 'edoJsoJJrr.u ]q3[ aq] qlyvr Suruur8aq 'uorleredard uarurcads pup s]uaunJ]sur q]oq Jeprsuoo eJoJeJaqlervr'suorssncsp Surivrollo;eql uI Jlasll edocso.rcrruaql Jo esueruro;rad eql uo se uarurcads eql SuuedeJd ro; sanbruqJel uo qonru se saqar.{docsorcrur fep slql o1 'a8raura o1 ur8aq plnoo aJnlonJls eurJ Ieuralur Jreq] Jo sapxaldruoc IInJ eql eJoJaqslac Surumls pue 3um-rasardroy sanbruqcal rlr.euJo luaurdolanep aql parmbar s9761,,(peaeq] ur paonporlur adocsoJcrruuoJlcela p;ramod aroru JeJaq] ',{.pegurrg'elqls1,rsaJnleal asoq} e{Eru ol lseJluoc }uarcrrgnspapnord leql surels Jo dlarrel e 3o '.{rnluac q}ueeleuru aq1 ;o pud Japel aq} u1 'luarudolelap eq] uo papuadap sernleeJ luuJelur urcru Jraql go fra,rocsrp aql ,(puanbesuo3 'luaonlsupJ] pue ssalJoloo oslp alp faqr rnq ,tup dpo lou eJp sllal IBruruV 'dSoyorq ilet Jo I{ulq [EuroJ eq] $lreru 'eulrlJop IIaJ aq] sEumoul 'ggg1 ur.{-raloJsrprreqJ'slleJ pnpwpurJo sale8arS3eeJe,t,rsanssrl prurue pue lueyd IIE leq] asodord uup^rqf,S puP uapraFIJS plp r{rn}uac qluee}euru aql 1o ged z(Fea aq} uI elqBlrp^e erupseq sedocsorcrurlq8q poo8 rarJedluo 'ada pa4eu eq] ol alqrsu alcqred lsellerus eql Jo azrs eql qUIJ-euo ]noqe sr qclqm teteuurp ur urrl 97-91 sr ilec lerurue IpJIdr{1V

tdo)sou)lwIH9lt tHI Nlsllt) IV DNuoot 'rueql ol uoucnpoJlur ue se 3ur lr -^ etn ueql reqter {ooq eql Jo sraldeqc JatEIeql q}IM uol}Junfuoc ur ]l peer pue eJueJaJere se raldeqc srql asn 'Surlsaralur ueq] se{Brx leql IIeo aql Jo eJnlJe} -rqf,JeIEJnlJnJls aq] ]noqE Je^oJsrp ol slspuerss palqEue aneq rtaql leq^\ sr lr ,spo -qlau lueuodrur are LdocsorcrruuoJlcela pue,,(docsorcnupcqdo q8noqlry 'suorsuerurp

rdo)sou)tw

ldofsouftwIH9|l IHI Ntsrt3) IV 9Nt)OOt

619

NOUT)3I] ]HI Nlsltn)ltot^ cNV stllf IV DNt)toot

v09

retdeq)slql ul

aaJq] ul pu€ 'uorlnloseJ orurol€ ]sorule lE sllec ulqlp{ saxaldruoc JEInJelou -oJJBruaql a8erur uec ,{.docsortrruuoJ}cale 'ppa}sur suoJlJele ureeq e Sursn Jo dg 'lq3ll alqlsp yo qfualanem aqr ,tq uorlnloseJ ur palru4l sr ,{docsorcrur pc -pdo 'sllao Suuru ur suorlopJelur pue 'scnueudp 'luerualolu JIeq] qclem snql ueJ am 'suralord lueJseJong dlecrsurrlur sp qcns 'saqord luecseJong qlFl sluauod -tuoc IIaJ cgrcads 3ur33er dg 'elllJnltsepuou Llanrlelar sr lq8q reqr sr d.docso.rrnu pcrldo ;o a8eluenpe luelrodrur uV 'eJnlJallqJJp luuorsuaurp-aeJq] Jreq] Jo uorlonJlsuoJeJ eql pup sluenlusuoc Jelnllal Ienptntput;o 3u€urur pue 3ur ,lueuodrur -1aqe1cgrcads eql roJ spoqtau;o luarudola^ep eql ol Suulo ,{1a3-re1 aJoru JaAeeruoseq seq,r(docsorcrurpcqdo srea,{}uaoar ul 'lool IBl}uasseup ru]s sl pue 'adocsorcrur qllm aqt ue8eq iSolorq esnucaq tl Suurets rno lqBI ]urod IIeo aq pa,rddocsorclu pcrldo 'uorpury s1ac,,uoq SuruJpal JoJ ppuessa alrsmbarard ue sr sllal Jo uotpzrue8ro prnpnrls aq1SurpuelsJapun 's11ac ,i.pn1sol pesn spo -qlaur ,tdocsorcrurpdrcuud aq1yo a{uos aqrJosap,{garrq a,u taldeqc srql uI 'spoqleu s1rgo Surqlauros ,trou>Io1dressacau sl 'eJoJ lr -araql 'd8olorq 1ac.{rerodrueluoJ puplsJapun og'r{3o1orqIIaJ ur seoup,rperoferu ur palFseJ dpuanbar; seq sanbruqoel lr,reuJo uoutnpoJlul aql pup ,slleo tnoqe uJeel ueJ ala leqm euruJelep psodsrp Jno lp slool aqJ 'uoncury sluauodruor snorrel Jreql lr,roqlno pulJ o] I[ls JepJEqpue 'uorlsoduoJ Jelnoaloru Jraql JaAoJ -s1po] pJEq raJruJnJlsJraq] aes ol pJpq s1]1 'xaldruoc pue llerus eJe sllef, esneteg

slla) 6ulzllenstn

'ado)soJ)rLu uorl)alaaqlro raModaql puo^eq^llensn are'slauedo^^llsel oql ut uMoqsse 'saln)alouor)eu jo slrelap)il.I|olv'Ipoq Jnour saln)aloururalord{ueu aql;o auo;o lred 6urr.u.rol suole Jololsnl) e ol 'aLuosoqu e ol'slla) urls q6norqt'qunql e uorl uorssarbord ,{reur6euur ue ut ual Jo rotre; e {q parlu6er,ua6eut ue sMotls q)el'sruole pue sgler6urnr; r"ue.r6erp uoamteqale)s ro asuosV l.-6 ern6rJ

ruTiZ'0 o] (]alor roJ) urr'lt'O tnoqe ruo{ sa8ueJqclqm'}q31 alqtsur.JoqlSuale^Em aql dq les eJoJareqlsI adoJsotclru IqBII E Jo uopnlosar aq] ot llurll etEurliF aql 'q]8uela^p^ u ^o slr ueq] Jell€us qJnu slelep IPJnpnJls aqord o1 pasn eq lou -uP3 uorlPlpuJ Jo ed.{l ue^rS € tBql sl sedoJsoJclur IIEJo uoIlBlIIuIT lelueruEpunJ v

lredv utd Z'Osllelaga^losauue) edolsorllp tt{6!1aql 'azrlEnsr^uE3 sedoJSoJsIIuJoseddl luaJaJJIpleql azISJosaSuEJaql pue 'lauPd sernlsnJls JelnlleJqns pu€ JBlnlles snoIJeAJo sezs aql sMoqs z-6 eJntlc '1aued glr{8ra pup q}ua^es aql }noqE ueeMleq o} adoJsoJJIIu uoJlsele eql puE qunoJ eql lnoqe ol puaue pFo^ edorsorclur ]q8ll eq] Jo uollnlosar aql 'sle -ued paleu eql'uoIleJIJIu8eu uI eseaJcul plnoJ ada salnleeJ aes or\ eql uI l ]sJrJ qJeg 'suole Jo Jelsnlf, e ol qunql E ploJuel E sluaseJdeJa8erur eAISSaJOnS ruo4 uorssaJSorddreur8erul ue aleJlsnlll I-6 arntlg uI se8elul Jo selles aqJ

s 1 1 a63u ; z 1 1 e n s:t61r a t d e q l

089

581

LOOKING AT CELLSIN THELIGHTMICROSCOPE

(for deep red). In practical terms, bacteria and mitochondria, which are about 500 nm (0.5 pm) wide, are generally the smallest objects whose shape we can clearly discern in the light microscope; smaller details than this are obscured by effects resulting from the wavelike nature of light. To understand why this occurs, we must follow the path of a beam of light waves as it passes through the lenses of a microscope (Figure 9-3). Because of its wave nature, Iight does not follow exactly the idealized straight ray paths that geometrical optics predict. Instead, light waves travel through an optical system by several slightly different routes, so that they interfere with one another and cause optical dffiaction effects.If two trains of waves reaching the same point by different paths are precisely in phase, with crest matching crest and trough matching trough, they will reinforce each other so as to increase brightness. In contrast, if the trains of waves are out of phase, they will interfere with each other in such a way as to cancel each other partly or entirely (Figure 9-4). The interaction of light with an object changes the phase relationships of the light waves in a way that produces complex interference effects. At high magnification, for example, the shadow of an edge that is evenly illuminated with light of uniform wavelength appears as a set of parallel lines (Figure 9-5), whereas that of a circular spot appears as a set of concentric rings. For the same reason, a single point seen through a microscope appears as a blurred disc, and two point objects close together give overlapping images and may merge into one. No amount of refinement of the lenses can overcome this Iimitation imposed by the wavelike nature of light. The limiting separation at which two objects appear distinct-the so-called limit of resolution-depends on both the wavelength of the light and the numerical apertureof the lens system used. The numerical aperture is a measure of the width of the entry pupil of the microscope, scaled according to its distance from the objec| the wider the microscope opens its eye, so to speak, the more sharply it can see (Figure 9-6). Under the best conditions, with violet light

lcm

10 0 L r m

:

plant cell animal cell

1 0r m

-

bacterium

vtrus ribosome

globular protein

small molecule relrna

arom

Figure9-2 Resolvingpower.Sizesof cellsand theircomponentsaredrawnon a logarithmicscale,indicatingthe range of objectsthat can be readilyresolvedby the nakedeyeand in the light and Thefollowingunits electronmicroscopes. of lengtharecommonlyemployedin mrcroscopy: = 10-om pm (micrometer) = 10-em nm (nanometer) m A (Angstromunit)= 1O-10

"0,".*,"" I specrmen conoenser

(A)Diagramshowingthe light path in a Figure9-3 A light microscope. compoundmicroscope. Lightisfocusedon the specimenby lensesin the condenser. A combinationof objectivelensesand eyepiecelensesare arrangedto focusan imageof the illuminatedspecimenin the eye. (B)A modern researchlight microscope.(8,courtesyof Andrew Davies.)

582

Chapter 9:Visualizing Cells T W O W A V E SI N P H A S E

TWO WAVESOUT OF PHASE

M (A)

B R IHGT Figure9-4 Interference betweenlightwaves.Whentwo lightwaves combine in phase, theamplitude of theresultant waveislarger andthe brightness isincreased. Twolightwaves thatareoutof phase cancel each otherpartlyandproduce a wavewhoseamplitude, andtherefore brightness, isdecreased. (wavelength = 0.4 pm) and a numerical aperture of 1.4,the light microscope can theoretically achieve a limit of resolution of just under 0.2 pm. Microscope makers at the end of the nineteenth century achieved this resolution and it is only rarely matched in contemporary, factory-produced microscopes. Although it is possible to enlargean image as much as we want-for example, by projecting it onto a screen-it is never possible to resolve two objects in the light microscope that are separated by less than about 0.2 pm; they will appear as a single object. Notice the difference between resolution, discussed above, and detection. If a small object, below the resolution limit, itself emits light, then we may still be able to see or detect it. Thus, we can see a single fluorescently labeled microtubule even though it is about ten times thinner than the resolution limit of the Iight microscope. Diffraction effects,however,will cause it to appear blurred and at least 0.2 pm thick (seeFigure 9-17). Becauseof the bright light they emit we can detect or see the stars in the night sky, even though they are far below the angular resolution of our unaided eyes.They all appear as similar points of light,

Figure9-5 lmagesofan edge and ofa point of light. (A)The interference effects,or fringes,seenat high magnification when light of a specific wavelengthpasses the edgeof a solid objectplacedbetweenthe light source (B)The imageof a and the observer. point sourceof light.Diffraction spreads this out into a complex,circularpattern, whosewidth deoendson the numerical apertureof the opticalsystem:the smallerthe aperturethe bigger(more blurred)the diffractedimage.Two point sourcescan be just resolvedwhen the centerof the imageof one lieson the firstdark ring in the imageof the other: this definesthe limit of resolution.

: t h e r e s o l v i n gp o w e r o f t h e m i c r o s c o pd e e p e n d so n t h e w i d t h o f t h e c o n eo f i l l u m i n a t i o na n d t h e r e f o r eo n b o t h t h e c o n d e n s ear n d t h e o b j e c t i v el e n s l t i s c a l c u l a t e du s i n gt h e f o r m u l a t h e o b j e c t i v el e n s c o l l e c t sa c o n eo f light raysto create an rmage

t h e c o n d e n s elre n s focusesa cone of l i g h t r a y so n t o e a c hp o i n t o f t h e specrmen

,:nsin0inthe e q u a t i o na b o v ei s c a l l e dt h e n u m e r i c aal p e r t u r e o f t h e l e n s( N A )a n d i s a f u n c t i o no f i t s l i g h t c o l l e c t i n ga b i l i t y F o r d r y l e n s e st h i s c a n n o tb e m o r e t h a n 1 , b u t f o r o i l - i m m e r s i o nl e n s e si t c a n b e a s h i g h a s 1 4 . T h e h i g h e rt h e n u m e r i c a l

r e s out t | o n

0 . 6 1i , nsin0

where: 0 = h a l f t h e a n g u l a rw i d t h o f t h e c o n eo f r a y sc o l l e c t e db y t h e o b j e c t i v el e n s f r o m a t y p i c a lp o i n t i n t h e s p e c i m e n ( s i n c et h e m a x i m u mw i d t h i s 1 8 0 o , s i n0 h a sa m a x i m u mv a l u eo f 1 ) n = t h e r e f r a c t i v ei n d e xo f t h e m e d i u m ( u s u a l l ya i r o r o i l ) s e p a r a t i n gt h e s p e c i m e nf r o m t h e o b j e c t i v ea n d c o n o e n s e tre n s e s l , = t h e w a v e l e n g t ho f l i g h t u s e d( f o r w h i t e l i g h t a f i g u r e o f 0 5 3 p m i sc o m m o n l y assumed) a p e r t u r e ,t h e g r e a t e rt h e r e s o l u t i o na n d t h e b r i g h t e rt h e i m a g e( b r i g h t n e s iss i m p o r t a n ti n f l u o r e s c e n cm e i c r o s c o p yH ) o w e v e r t, h i s a d v a n tage is obtained at the expenseof very short w o r k i n g d i s t a n c ea s n d a v e r ys m a l ld e p t h o f f i e l d

Figure9-6 Numericalaperture.The path of light rayspassingthrougha transparent specimenin a microscope illustrates the conceotof numerical apertureand its relationto the limit of resolution.

LOOKING AT CELLSIN THELIGHTMICROSCOPE

s83

differing only in their color or brightness. Using sensitivedetection methods, we can detect and follow the behavior of even a single fluorescent protein molecule with a light microscope. We see next how we can exploit interference and diffraction to study unstained cells in the living state.

LivingCellsAreSeenClearlyin a Phase-Contrast or a Different ia|-|nterference-Cont rast Microscope Microscopists have always been challenged by the possibility that some components of the cell may be lost or distorted during specimen preparation. The only certain way to avoid the problem is to examine cells while they are alive, without fifng or freezing. For this purpose, Iight microscopes with special optical systems are especially useful. \A/hen light passes through a living cell, the phase of the light wave is changed according to the cell'srefractive index a relatively thick or dense part of the cell, such as a nucleus, retards light passing through it. The phase of the light, consequently, is shifted relative to light that has passed through an adjacent thinner region of the c1'toplasm.The phase-contrast microscope and, in a more complex way, the differential-interference-contrast microscope exploit the interference effects produced when these two sets of waves recombine, thereby creating an image of the cell's structure (Figure 9-7). Both types of light microscopy are widely used to visualize living cells. A simpler way to see some of the features of a living cell is to observe the light that is scattered by its various components. In the dark-field microscope, the illuminating rays of light are directed from the side so that only scattered Iight enters the microscope lenses. Consequently, the cell appears as a bright object against a dark background. With a normal bright-field microscope, light passing through a cell in culture forms the image directly. Figure 9-8 compares images of the same cell obtained by four kinds of light microscopy. Phase-contrast, differential-interference-contrast, and dark-field microscopy make it possible to watch the movements involved in such processesas mitosis and cell migration. Since many cellular motions are too slow to be seen in real time, it is often helpful to make time-lapse movies. Here, the camera records successiveframes separatedby a short time delay,so that when the resulting picture seriesis played at normal speed, events appear greatly speededup.

lmagesCanBeEnhanced and Analyzedby DigitalTechniques In recent years electronic, or digital, imaging systems,and the associatedtechnology of image processing, have had a major impact on light microscopy. Certain practical limitations of microscopes,relating to imperfections in the optical (A)

i n c i d e n tl i g h t (white)

(B)

i n c i d e n tl i g h t (green)

Figure9-7 Two ways to obtain contrast in light microscopy.(A)The stained portionof the cellwill absorblightof whichdependon the somewavelengths, to stain,but will allowotherwavelengths passthroughit. A coloredimageof the cellistherebyobtainedthat is visiblein the normalbright-fieldlight microscope. (B)Lightpassingthroughthe unstained, very littlechange livingcellexperiences in amplitude,and the structuraldetails cannotbe seenevenif the imageis The phaseof the light, highlymagnified. however,is alteredby its passage througheitherthickeror denserpartsof can the cell,and smallphasedifferences be madevisibleby exploiting interferenceeffectsusinga phasecontrastor a differential-interferencecontrastmicroscope.

584

Chapter9:Visualizing Cells

Figure9-8 Fourtypes of light microscopy. Fourimagesareshownof the samefibroblastcellin culture.All imagescan be (A)Bright-field (B)Phaseobtainedwith mostmodernmicroscopes by interchanging opticalcomponents. microscopy. contrastmicroscopy.(C)Nomarskidifferential-interference-contrast microscopy.(D) Dark-fieldmicroscopy.

system have been largely overcome. Electronic imaging systems have also circumvented two fundamental limitations of the human eye: the eye cannot see well in extremely dim light, and it cannot perceive small differences in light intensity against a bright background. To increase our ability to observe cells in Iow light conditions, we can attach a sensitive digital camera to a microscope. These cameras contain a charge-coupled device (CCD), similar to those found in consumer digital cameras.Such CCD cameras are often cooled to reduce image noise. It is then possible to observe cells for long periods at very low light levels, thereby avoiding the damaging effects of prolonged bright light (and heat). Such low-light cameras are especially important for viewing fluorescent molecules in living cells, as explained below. Becauseimages produced by CCD cameras are in electronic form, they can be readily digitized, fed to a computer, and processedin various ways to extract latent information. Such image processing makes it possible to compensate for various optical faults in microscopes to attain the theoretical limit of resolution. Moreover, by digital image processing,contrast can be greatly enhanced to overcome the eye's limitations in detecting small differences in light intensity. Although this processing also enhances the effects ofrandom background irregularities in the optical system, digitally subtracting an image of a blank area of the field removes such defects.This procedure revealssmall transparent objects that were previously impossible to distinguish from the background. The high contrast attainable by computer-assisted differential-interferencecontrast microscopy makes it possible to seeeven very small objects such as single microtubules (Figure 9-9), which have a diameter of 0.025pm, lessthan onetenth the wavelength of light. Individual microtubules can also be seen in a fluorescencemicroscope if they are fluorescently labeled (seeFigure 9-15). In both cases,however, the unavoidable diffraction effects badly blur the image so that the microtubules appear at least 0.2 pm wide, making it impossible to distinguish a single microtubule from a bundle of several microtubules. Figure9-9 lmageprocessing. (A)Unstainedmicrotubules areshownhere in an unprocessed digitalimage,capturedusingdifferential-interference(B)The imagehasnow beenprocessed, contrastmicroscopy. firstby digitallysubtracting the unevenlyilluminatedbackground, and secondby digitallyenhancingthe contrast. The resultof this imageprocessing is a picturethat is easierto interpret.Notethat the microtubules aredynamic and somehavechangedlengthor positionbetweenthe before-and-after images.(Courtesy of VikiAllan.t

LOOKING ATCELLS INTHELIGHTMICROSCOPE

585 movement of . m i c r o t o m ea r m

IntactTissues Are UsuallyFixedand Sectioned beforeMicroscopy Becausemost tissue samples are too thick for their individual cells to be examined directly at high resolution, they must be cut into very thin transparent slices, or sections.To first immobilize, kill, and preserve the cells within the tissue they must be treated with a fixatiue. Common fixatives include formaldehyde and glutaraldehyde, which form covalent bonds with the free amino groups of proteins, cross-linking them so they are stabilized and locked into position. Becausetissues are generally soft and fragile, even after fixation, they need to be embedded in a supporting medium before sectioning. The usual embedding media are waxes or resins. In liquid form these media both permeate and surround the fixed tissue; they can then be hardened (by cooling or by polymerization) to form a solid block, which is readily sectioned with a microtome. This is a machine with a sharp blade that operates like a meat slicer (Figure 9-f 0). The sections (typically 1-10 pm thick) are then laid flat on the surface of a glass microscope slide. There is little in the contents of most cells (which are 70Towater by weight) to impede the passageof light rays. Thus, most cells in their natural state, even if fixed and sectioned, are almost invisible in an ordinary light microscope. There are three main approachesto working with thin tissue sections that reveal the cells themselves or specific components within them. First, and traditionally, sections can be stained with organic dyes that have some specific affinity for particular subcellular components. The dye hematoxylin, for example, has an affinity for negatively charged molecules and therefore reveals the distribution of DNA, RNA, and acidic proteins in a cell (Figure 9-f f). The chemical basis for the specificity of many dyes, however, is not known. Second,sectioned tissues can be used to visualize specific patterns of differential gene expression. In situ hybridization, discussed earlier (p. 573), reveals the cellular distribution and abundance of specific expressedRNA molecules in sectioned material or in whole mounts of small organisms or organs (Figure S-12). A third and very sensitive approach, generally and widely applicable for localizing proteins of interest, depends on using fluorescent probes and markers, as we exolain next.

50 Um

1 O Or m

Y L/

embedded .specimen t n w a xo r r e s r n

r i b b o n o f s e c t i o n so n g l a s ss l i d e ,s t a i n e d a n d m o u n t e du n d e r a g l a s sc o v e rs l i p

tmryIln Figure9-10 Makingtissuesections.This showshow an embedded illustration tissueis sectionedwith a microtomein preparation in the light for examination mlcroscope.

Figure9-1 1 Stainingof cellular components.(A)Thissectionof ducts cellsin the urine-collecting of the kidneywasstainedwith a combinationof dyes,hematoxylin and eosin,commonlyusedin histology. Eachduct is madeof closelypackedcells(with nuclei stainedred)that form a ring.The ring is surroundedby extracellular matrix,stainedpurple.(B)This sectionof a young plant root is stainedwith two dyes,safranin and fast green.The fast green cellwallswhile stainsthe cellulosic the safraninstainsthe lignified xylemcellwallsbrightred. (A,from P.R. Wheateret al., 2nd ed. Histology, Functional London:ChurchillLivingstone, 1987;8,courtesyof StephenGrace.)

586

Chapter9:Visualizing Cells

-.*,,--

SpecificMolecules CanBeLocatedin Cellsby Fluorescence Microscopy Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength. If we illuminate such a compound at its absorbing wavelength and then view it through a filter that allows only light of the emitted wavelength to pass, it will glow against a dark background. Becausethe background is dark, even a minute amount of the glowing fluorescent dye can be detected. The same number of molecules of an ordinary stain viewed conventionally would be practically invisible becausethe molecules would give only the faintest tinge of color to the light transmitted through this stained part of the specimen. The fluorescent dyes used for staining cells are visualized with a fluorescence microscope. This microscope is similar to an ordinary light microscope except that the illuminating light, from a very powerful source,is passedthrough tvvo sets of filters-one to filter the light before it reachesthe specimen and one to filter the light obtained from the specimen. The first filter passes only the wavelengths that excite the particular fluorescent dye, while the second filter blocks out this light and passes only those wavelengths emitted when the dye fluoresces (Figure 9-13). Fluorescence microscopy is most often used to detect specific proteins or other molecules in cells and tissues.A very powerful and widely used technique is to couple fluorescent dyes to antibody molecules, which then serve as highly specific and versatile staining reagents that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix. TWo fluorescent dyes that have been commonly used for this purpose are fluorescein, which emits an intense green fluorescence when excited with blue light, and

Figure9-12 RNArn situ hybridization. As describedin chapter8 (seeFigure 8-71),it is possibleto visualize the distributionof differentRNAsin tissues usingln sltuhybridization. Here,the transcriptionpatternof five different genesinvolvedin patterningthe earlyfly embryois revealedin a singleembryo. EachRNAprobehasbeenfluorescently labeledin a differentway,somedirectly and someindirectly, and the resulting imagesfalse-colored and combinedto seeeachindividualtranscriotmost clearly. Thegeneswhoseexpression pattern is revealedhere are wingless (yellow),engrailed(blue),short gastrulation (red), intermediate neuroblastsdefective(green),and muscle specifichomeobox(purple).(From D. Kosmanet al.,Scrence 305:846,2004. With permissionfrom AAA5.)

A eyeprece

LIGHT

3 s e c o n db a r r i c rf i l t e r :c u t so u t u n w a n t e df l u o r e s c e nst i g n a l s p a s s i n gt h e s p e c i f i cg r e e n {luoresceinemissionbetween )zu ano )bu nm

SOURCE

2 b e a r n - s p l r t t i nm g i r r o r :r e f l e c t s light below 510nm but t r a n s m i t sl i g h t a b o v e5 1 0 n m 1 t i r s t b a r r i e rf i l t e r : l e t st h r o u o h only bluelight with a wavelenjth between 450 and 490 nm

o b j e c t i v el e n s

Figure9-1 3 The optical systemof a fluorescencemicroscope.A filter set consistsof two barrierfilters(1 and 3) and a dichroic(beam-splitting) minor (2) Thisexampleshowsthe filtersetfor detectionof the fluorescent molecule fluorescein. High-numerical-apertu re objectivelensesareespecially important in this type of microscopybecause,for a givenmagnification, the brightness of the fluorescent imageis proportionalto the fourth powerof the numerical aperture(seealsoFigure9-6).

587

LOOKINGATCELLSIN THELIGHTMICROSCOPE

:l fl3;fi :;lf,lii:ffi:1:ffi'"T'iff"T?n#I"ft'xilt"J:?3,:ffi

relationto the correspondingcolorsof the spectrum.The photon emitted by a fluorescent moleculeis necessarily of lowerenergy(longer wavelength) than the photonabsorbedand this accountsfor the differencebetweenthe excitationand emissionpeaks.CFP,GFBYFPand RFPare cyan,green,yellow and red fluorescentproteinsrespectively.These arenot dyes,and arediscussed in detaillaterin the chapter.DAPIis widely usedasa generalfluorescent DNAprobe,whichabsorbsUV light and fluorescesbright blue.FITCis an abbreviationfor fluorescence isothiocyanate, a widely usedderivativeof fluorescein,which fluoresces label brightgreen.Theother probesareall commonlyusedto fluorescently antibodiesand other oroteins.

rhodamine, which emits a deep red fluorescence when excited with green-yellow light (Figure 9-14). By coupling one antibody to fluorescein and another to rhodamine, the distributions of different molecules can be compared in the same cell; the two molecules are visualized separately in the microscope by switching back and forth between tlvo sets of filters, each specific for one dye. As sholrm in Figure 9-15, three fluorescent dyes can be used in the same way to distinguish between three types of molecules in the same cell. Many newer fluorescent dyes, such as Cy3, Cy5, and the Alexa dyes, have been specifically devel-

oped for fluorescence microscopy (see Figure 9-14). These organic fluorochromes have some disadvantages.They are excited only by light of precise, but different, wavelengths, and additionally they fade fairly rapidly when continuously illuminated. More stable inorganic fluorochromes have recently been developed, however. Tiny crystals of semiconductor material, called nanoparticles, or quantum dots, can all be excited to fluoresce by a broad spectrum of blue light. Their emitted light has a color that depends on the exact size of the nanocrystal, between 2 and 10 nm in diameter, and additionally the fluorescence fades only slowlywith time (Figure 9-16). These nanoparticles, when coupled to other probes such as antibodies, are therefore ideal for tracking molecules over time. If introduced into a living cell, in an embryo for example, the progeny of that cell can be followed many days later by their fluorescence, allowing cell lineages to be tracked. Fluorescence microscopy methods, discussed later in the chapter, can be used to monitor changesin the concentration and location of specific molecules inside liuingcells (seep. 592).

10 pLm

Figure9-1 5 Multiple-fluorescent-probe microscopy.In this composite micrographof a cellin mitosis,three differentfluorescentprobeshavebeen usedto stainthree differentcellular The sPindle

components. microtubufesare revealedwith a green fluorescentantibody,centromereswith a redfluorescentantibodyand the DNAof with the the condensedchromosomes b/uefluorescentdye DAPI.(Courtesyof KevinF.Sullivan.)

588

Chapter9:Visualizing Cells semiconductor

(A)

5 nm

(B)

0 sec

20 sec

120 sec

AntibodiesCanBeUsedto DetectSpecific Molecules Antibodies are proteins produced by the vertebrate immune system as a defense against infection (discussed in Chapter 24).They are unique among proteins because they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule (or antigen). The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. \Mhen labeled with fluorescent dyes, antibodies are invaluable for locating specific molecules in cells by fluorescence microscopy (Figure g-17); labeled with electron-dense particles such as colloidal gold spheres,they are used for similar purposes in the electron microscope (discussedbelow). When we use antibodies as probes to detect and assayspecific molecules in cells we frequently amplify the fluorescent signal they produce by chemical methods. For example, although a marker molecule such as a fluorescent dye can be linked directly to an antibody used for specific recognition-the primary antibody-a stronger signal is achieved by using an unlabeled primary antibody and then detecting it with a group of labeled secondaryantibodies that bind to it (Figure 9-f 8). This process is called indirect immunocytochemistry. The most sensitive amplification methods use an enzyme as a marker molecule attached to the secondary antibody. The enzyrne alkaiine phosphatase, for example, in the presence of appropriate chemicals, produces inorganic phosphate that in turn leads to the local formation of a colored precipitate. This revealsthe location of the secondary antibody and hence the location of the antibody-antigen complex. Since each enzl.rne molecule acts catalytically to generatemany thousands of molecules of product, even tiny amounts of antigen can be detected.An enzyme-linked immunosorbent assay(ELISA)based on this principle is frequently used in medicine as a sensitive test-for pregnancy or for various types of infections, for example. Although the enzyme amplification makes enzyrne-linked methods very sensitive, diffusion of the colored precipitate away from the enzyme limits the spatial resolution of this method for

l0um

Figure9-16 Fluorescent nanoparticles or quantum dots.Quantumdots aretiny nanoparticles of cadmiumselenide, a semiconductor, with a coatingto make (A).Theycan be them water-soluble coupledto proteinprobessuchas antibodiesor streptavidin and,when introducedinto a cell,will bind to a protein of interest.Different-sized quantumdots emit lightof different colors-the largerthe dot the longerthe wavelength-but they areall excitedby the sameblue light.(B)Quantumdots can keepshiningfor weeks,unlikemost fluorescent organicdyes.In this cell,a nuclearprotein is labeled(green)withan organicfluorescent dye (Alexa488),while microtubulesare stained(red)with quantumdots boundto streptavidin. On continuousexposureto blue lightthe fluorescent dye fadesquicklywhilethe quantumdotscontinueto fluoresce. (8, from X. Wu et al.,Nat.Biotechnol. 21:41-46, 2003.With permission from MacmillanPublishers Ltd.)

Figure9-l 7 lmmunofluorescence. (A)A transmission electronmicrographof the peripheryof a culturedepithelialcell showingthe distributionof microtubules and otherfilaments.(B)The samearea stainedwith fluorescent antibodies againsttubulin,the proteinthat assembles to form microtubules, using the techniqueof indirect (seeFigure9-18). immunocytochemistry Redorrowsindicateindividual microtubules that arereadily recognizable in both images.Notethat, becauseof diffractioneffects,the microtubules in the light microscope appear0.2pm wide ratherthan theirtrue width of 0.025pm. (FromM. Osborn, R.Websterand K.Weber,J. CellBiol. 77:R27-R34, 1978.With permissionfrom The Rockefeller UniversityPress.)

589

LOOKINGATCELLSINTHELIGHTMICROSCOPE p r i m a r ya n t i b o d y r a b b i ta n t i b o d y d i r e c t e da g a i n s t a n t i g e nA

I

s e c o n d a r ay n t i b o d i e s : m a r k e r - c o u p l ea dn t i b o d i e s d i r e c t e da g a i n s tr a b b i t antibodies m ar K e r

_L_ microscopy, and fluorescent labels are usually used for the most precise optical localization. Antibodies are made most simply by injecting a sample of the antigen several times into an animal such as a rabbit or a goat and then collecting the antibody-rich serum. This antiserum conlains a heterogeneousmixture of antibodies, each produced by a different antibody-secreting cell (a B lymphocyte). The different antibodies recognize various parts of the antigen molecule (called an antigenic determinant, or epitope), as well as impurities in the antigen preparation. Removing the unwanted antibody molecules that bind to other molecules sharpens the specificity of an antiserum for a particular antigen; an antiserum produced against protein X, for example, when passed through an affinity column of antigens X, will bind to these antigens, allowing other antibodies to pass through the column. Purified anti-X antibody can subsequently be eluted from the column. Even so, the heterogeneity of such antisera sometimes limits their usefulness.The use of monoclonal antibodies largely overcomes this problem (seeFigure 8-8). However, monoclonal antibodies can also have problems. Since they are single-antibody protein species,they show almost perfect specificity for a single site or epitope on the antigen, but the accessibility of the epitope, and thus the usefulness of the antibody, may depend on the specimen preparation. For example, some monoclonal antibodies will react only with unfixed antigens, others only after the use of particular fixatives,and still others only with proteins denatured on SDSpolyacrylamide gels,and not with the proteins in their native conformation.

lmagingof ComplexThree-Dimensional with Objectsls Possible the OpticalMicroscope For ordinary light microscopy, as we have seen, a tissue has to be sliced into thin sections to be examined; the thinner the section, the crisper the image. The process of sectioning loses information about the third dimension. HoW then, can we get a picture of the three-dimensional architecture of a cell or tissue, and how can we view the microscopic structure of a specimen that, for one reason or another, cannot first be sliced into sections?Although an optical microscope is focused on a particular focal plane within complex three-dimensional specimens, all the other parts of the specimen, above and below the plane of focus, are also illuminated and the light originating from these regions contributes to the image as "out-of-focus" blur. This can make it very hard to interpret the image in detail and can lead to fine image structure being obscured by the outof-focus light. TWo distinct but complementary approaches solve this problem: one is computational, the other is optical. These three-dimensional microscopic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting the light that comes from out-of-focus regions above and below that plane. Thus one seesa crisp, thin optical section.From a seriesof such optical sections taken at different depths and stored in a computer, it is easy to reconstruct a three-dimensional image. The methods do for the microscopist what the CT scanner does (by different means) for the radiologist investigating a human body: both machines give detailed sectional views of the interior of an intact structure.

Figure9-18 Indirectimmunocytochemistry.Thisdetectionmethod is very becausemanymoleculesof the sensitive each antibodyrecognize secondary primaryantibody.Thesecondary antibody is covalentlycoupledto a marker moleculethat makesit readilydetectable. Commonlyusedmarkermolecules dyes(forfluorescence includefluorescent microscopy), the enzymehorseradish (foreitherconventional peroxidase light microscopyor electronmicroscopy), colloidalgold spheres(forelectron and the enzymesalkaline microscopy), (for phosphatase or peroxidase biochemical detection).

590

Chapter 9:Visualizing Cells

Figure9-1 9 lmage deconvolution. (A)A light micrographof the large polytenechromosomeslrom Drosopht stainedwith a fluorescentDNA-bindin dye.(B)The samefield of view after imagedeconvolutionclearlyrevealstf bandingpatternon the chromosomes Eachbandis about0.25pm thick, approaching the resolutionlimit of thr light microscope.(Courtesyof the Joh SedatLaboratory.)

5lrt

The computational approach is often called image deconuolution. To understand how it works, remember that the wavelike nature of Iight means that the microscope lens system produces a small blurred disc as the image of a point light source (see Figure 9-5), with increased blurring if the point source lies above or below the focal plane. This blurred image of a point source is called the point spreadfunction. An image of a complex object can then be thought of as being built up by replacing each point of the specimen by a corresponding blurred disc, resulting in an image that is blurred overall. For deconvolution, we first obtain a series of (blurred) images, usually with a cooled CCD camera, focusing the microscope in turn on a seriesof focal planes-in effect, a (blurred) three-dimensional image. The stack of digital images is then processedby computer to remove as much of the blur as possible. Essentially the computer program uses the microscope's point spread function to determine what the effect of the blurring would have been on the image, and then applies an equivalent "deblurring" (deconvolution), turning the blurred three-dimensional image into a series of clean optical sections. The computation required is quite complex, and used to be a serious limitation. However, with faster and cheaper computers, the image deconvolution method is gaining in power and popularity. Figure 9-19 shows an example.

TheConfocalMicroscopeProducesOpticalSectionsby Excluding Out-of-Focus Light The confocal microscope achieves a result similar to that of deconvolution, but does so by manipulating the light before it is measured; thus it is an analog technique rather than a digital one. The optical details of the confocal microscope are complex, but the basic idea is simple, as illustrated in Figure $-20, and the results are far superior to those obtained by conventional light microscopy (Figure

9-2r). The microscope is generally used with fluorescence optics (seeFigure 9-13), but instead of illuminating the whole specimen at once, in the usual way, the optical system at any instant focuses a spot oflight onto a single point at a specific depth in the specimen. It requires a very bright source of pinpoint illumination that is usually supplied by a laser whose light has been passed through a pinhole. The fluorescence emitted from the illuminated material is collected and brought to an image at a suitable light detector. A pinhole aperture is placed in front of the detector, at a position that is confocalvnth the illuminating pinhole-that is, precisely where the rays emitted from the illuminated point in the specimen come to a focus. Thus, the light from this point in the specimen converges on this aperture and enters the detector. By contrast, the light from regions out of the plane of focus of the spotlight is also out of focus at the pinhole aperture and is therefore largely excluded from the detector (seeFigure 9-20). To build up a two-dimensional image, data from

LOOKINGATCELLSINTHELIGHTMICROSCOPE

iri'.i., . .l:r.

(A)

objective 3 - Ds p e c i m e n pointof focus

emitted fluorescent lightfrom in-focus point is focusedat p i n h o l ea n d r e a c h e s detector

' I .' ', 59't

Figure9-20 The confocalfluorescence Thissimplifieddiagram microscope. of showsthat the basicarrangement opticalcomponentsis similarto that of the standardfluorescencemicroscope shownin Figure9-13,exceptthat a laser is usedto illuminatea smallpinhole whoseimageis focusedat a singlepoint in the specimen(A).Emittedfluorescence from thisfocalpoint in the specimenis focusedat a second(confocal)pinhole (B).Emittedlight from elsewherein the specimenis not focusedat the pinhole and thereforedoesnot contributeto the finalimage(C).By scanningthe beamof a verysharp lightacrossthe specimen, imageof the exact two-dimensional planeof focusis built up that is not significantlydegradedby light from other regionsof the specimen.

confocal pinholes

f l u o r e s c e nst o e c i m e ni s i l l u m i n a t e d with a focusedpoint of light from a oinhole

,

emitted light from outof-focuspoint is out of f o c u sa t p i n h o l ea n d i s l a r g e l ye x c l u d e d from detector

each point in the plane of focus are collected sequentiallyby scanning acrossthe field in a raster pattern (as on a television screen) and are displayed on a video screen. Although not shown in Figure 9-20, the scanning is usually done by deflecting the beam with an oscillating mirror placed between the dichroic mirror and the objective lens in such a way that the illuminating spotlight and the confocal pinhole at the detector remain strictly in register. The confocal microscope has been used to resolve the structure of numerous complex three-dimensional objects (Figure 9-22), including the networks of cytoskeletalfibers in the cytoplasm and the arrangements of chromosomes and genesin the nucleus. The relative merits of deconvolution methods and confocal microscopy for three-dimensional optical microscopy are still the subject of debate. Confocal microscopes are generally easier to use than deconvolution systems and the final optical sections can be seen quickly. In contrast, the cooled CCD (chargecoupled device) cameras used for deconvolution systems are extremely efficient at collecting small amounts of light, and they can be used to make detailed three-dimensional images from specimens that are too weakly stained or too easily damaged by the bright light used for confocal microscopy. Both methods, however, have another drawback; neither is good at coping with thick specimens. Deconvolution methods quickly become ineffective any deeper than about 40 pm into a specimen, while confocal microscopes can only

10tt

Figure9-21 Conventionaland confocal fluorescencemicroscopycompared. Thesetwo micrographsare of the same intact gastrula-stageDrosophiloembryo that has been stainedwith a fluorescent (A)The probefor actinfilaments. conventional,unprocessedimage is blurredby the presenceof fluorescent structuresaboveand below the planeof focus.(B)In the confocalimage,this outof-focusinformationis removed, resultingin a crispopticalsectionofthe cellsin the embryo.(Courtesyof Richard Warnand PeterShaw.)

592

Chapter9:Visualizing Cells

20

obtain images up to a depth of about 150 pm. Specialconfocal microscopes can now take advantage of the way in which fluorescent molecules are excited, to probe even deeper into a specimen. Fluorescent molecules are usually excited by a single high-energy photon, of shorter wavelength than the emitted light, but they can in addition be excited by the absorption of two (or more) photons of lower energy,as long as they both arrive within a femtosecond or so of each other. The use of this longer-wavelength excitation has some important advantages. In addition to reducing background noise, red or near infrared light can penetrate deeper within a specimen. Multiphoton confocal microscopes, constructed to take advantage of this "two-photon" effect, can tFpically obtain sharp images even at a depth of 0.5 mm within a specimen. This is particularly valuable for studies of living tissues, notably in imaging the dynamic activity of synapses and neurons just below the surface of living brains (Figure S-Zg).

"t

Figure9-22 Three-dimensional reconstructionfrom confocal microscopeimages.Pollengrains,in this casefrom a passionflower,havea complexsculpturedcellwallthat containsfluorescent compounds.lmages obtainedat differentdepthsthroughthe grain,usinga confocalmicroscope, can be recombinedto give a threedimensional view of the wholegrain, shownon the right.Threeselected individualopticalsectionsfrom the full set of 30,eachof which showslittle contributionfrom its neighbors, are shown on the left. (Courtesyof BradAmos.)

Fluorescent Proteins CanBeUsedto TagIndividualProteinsin LivingCellsand Organisms Even the most stable cellular structures must be assembled, disassembled,and reorganized during the cell's life cycle. other structures, often enormous on the molecular scale, rapidly change, move, and reorganize themselves as the cell conducts its internal affairs and responds to its environment. complex, highly organized pieces of molecular machinery move components around the cell, controlling traffic into and out of the nucleus, from one organelle to another, and into and out of the cell itself. Various techniques have been developed to make specific components of living cells visible in the microscope. Most of these methods use fluorescent proteins, and they require a trade-off between structural preservation and efficient labeling. All of the fluorescent molecules discussed so far are made outside the cell and then artificially introduced into it. Now new opportunities have been opened up by the discovery of genescoding for protein molecules that are themselvesinherently fluorescent. Genetic engineering then enables the creation of lines of cells or organisms that make their or,rmvisible tags and labels, without the introduction of foreign molecules. These cellular exhibitionists display their inner workings in glowing fluorescent color. Foremost among the fluorescent proteins used for these purposes by cell biologists is the green fluorescent protein (GFp), isolated from the jellyfish Aequoria uictoria. This protein is encoded in the normal way by a single gene that can be cloned and introduced into cells of other species.The freshly translated protein is not fluorescent, but within an hour or so (lessfor some allelesof

Figure9-23 Multi-photon imaging. Infraredlaserlightcauseslessdamageto livingcellsand canalsopenetratefurther, allowingmicroscopists to peerdeeper into living tissues.Thetwo-photon effect, in which a fluorochromecan be excited by two coincidentinfraredphotons insteadof a singlehigh-energy photon, allowsus to seenearly0.5mm insidethe cortexof a live mousebrain.A dye, whosefluorescence changeswith the calciumconcentration, revealsactive synapses(yellow)on the dendriticspines (red)thatchangeasa function of time. (Courtesyof KarelSvoboda.)

LOOKING INTHELIGHT ATCELLS MICROSCOPE protein(GFP). of GFBshown Figure9-24Greenfluorescent Thestructure p strands of a hereschematically, highlights thatformthestaves theeleven (darkgreen)thatis barrel. Buried withinthebarrelistheactivechromophore formedpost-translationally of threeamino fromtheprotruding sidechains (Adapted acidresidues. fromM.Ormoet al.,Science 273:1392-1395,1996. Withpermission fromAAAS.) the gene, more for others) it undergoes a self-catalyzedpost-translational modification to generate an efficient and bright fluorescent center, shielded within the interior of a barrel-like protein (Figure S-24). Extensive site-directed mutagenesisperformed on the original gene sequencehas resulted in useful fluorescence in organisms ranging from animals and plants to fungi and microbes. The fluorescence efficiency has also been improved, and variants have been generated with altered absorption and emission spectra in the blue-green-yellow range. Recently a family of related fluorescent proteins discoveredin corals, has extended the range into the red region ofthe spectrum (seeFigure 9-14). One of the simplest uses of GFP is as a reporter molecule, a fluorescent probe to monitor gene expression.A transgenic organism can be made with the GFP-coding sequence placed under the transcriptional control of the promoter belonging to a gene of interest, giving a directly visible readout of the gene's expressionpattern in the living organism (Figure S-25).In another application, a peptide location signal can be added to the GFP to direct it to a particular cellular compartment, such as the endoplasmic reticulum or a mitochondrion, lighting up these organelles so they can be observed in the living state (seeFigure 12-358). The GFP DNA-coding sequence can also be inserted at the beginning or end of the gene for another protein, yielding a chimeric product consisting of that protein with a GFP domain attached. In many cases,this GFP-fusion protein behaves in the same way as the original protein, directly revealing its location and activities by means of its genetically encoded contrast (Figure 9-26). It is often possible to prove that the GFP-fusion protein is functionally equivalent to the untagged protein, for example by using it to rescue a mutant lacking that protein. GFP tagging is the clearest and most unequivocal way of showing the distribution and dynamics of a protein in a living organism (Figure 9-27).

ProteinDynamics CanBeFollowedin LivingCells Fluorescent proteins are now exploited, not just to seewhere in a cell a particular protein is located, but also to uncover its kinetic properties and to find out whether it might interact with other proteins. We now describe three techniques in which GFP and its relatives are used in this way. The first is the monitoring of interactions between one protein and another by fluorescence resonance energy transfer (FRET). In this technique, whose principles have been described earlier (see Figure 8-26), the two molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome overlaps with the absorption spectrum of the other. If the two proteins bind so as to bring their fluorochromes into very close proximity (closerthan about 5 nm), one fluorochrome transfers the energy of the absorbed light directly to the other. Thus, when the complex is illuminated at the excitation wavelength of the first fluorochrome, fluorescent light is pro-

Figure9-25 Greenfluorescentprotein (GFP)as a reporter.Forthis experiment, carriedout in the fruit fly,the GFPgenewasjoined (using recombinantDNAtechniques) to a fly promoterthat is activeonly in a Thisimageof a livefly embryowascapturedby a specialized setof neurons, eachwith 20 neurons, fluorescence microscope and showsapproximately (axonsand dendrites) with other long projections that communicate (nonfluorescent) cells.Theseneuronsarelocatedjust underthe surfaceof the (FromW.B.Grueberet animaland allowit to senseits immediateenvironment. 2003.With permissionfrom Elsevier.) al.,Curr.Biol.13.618-626,

593

594

Chapter9:Visualizing Cells Figure 9 -26 GFP-taggedproteins. (A)The upper surfaceof the leavesof plantsare coveredwith huge Arabidopsis branchedsingle-cell hairsthat riseup from the surfaceof the eoidermis.These hairs,or trichomes, can be imagedin the (B)lf an scanningelectronmicroscope. plant is transformedwith a Arabidopsis DNAsequencecodingfor talin(anactinbindingprotein),fusedto a DNA sequencecoding for GFP,the fluorescent talinproteinproducedbindsto actin filamentsin all the livingcellsof the transgenicplant.Confocalmicroscopy can revealthe dynamicsof the entire actin cytoskeletonof the trichome (green).fhered fluorescencearisesfrom chlorophyllin cellswithinthe leafbelow (A,courtesyof Paul the epidermis. Linstead;B,courtesyof JaideepMathur.)

1 0 0p m

duced at the emission wavelength of the second. This method can be used with two different spectral variants of GFP as fluorochromes to monitor processes such as the interaction of signaling molecules with their receptors, or proteins in macromolecular complexes (Figure g-28). The complexity and rapidity of many intracellular processes,such as the actions of signaling molecules or the movements of cytoskeletalproteins, make them difficult to study at a single-cell level. Idealty, we would like to be able to introduce any molecule of interest into a living cell at a precise time and location and follow its subsequent behavior, as well as the response of the cell to that molecule. Microinjection is limited by the difficulty of controlling the place and time of delivery. A more powerful approach involves slmthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot of light on it. This process is referred to as photoactivation. Inactive photosensitive precursors of this type, often called caged molecules, have been made for many fluorescent molecules. A microscope can be used to focus a strong pulse of light from a laser on any tiny region of the cell, so that the experimenter can control exactly where and when the fluorescent molecule is photoactivated. one classof caged fluorescent proteins is made by attaching a photoactivatable fluorescent tag to a purified protein. It is important that the modified protein remain biologically active: labeling with a caged fluorescent dye adds a bulky group to the surface of a protein, which can easily change the protein's properties. A satisfactory labeling protocol is usually found by trial and error. once a biologically active labeled protein has been produced, it needs to be introduced into the living cell (see Figure 9-34), where its behavior can be followed. Tubulin, labeled with caged fluorescein for example, when injected into a dividing cell, can be incorporated into microtubules of the mitotic spindle.

5tt

Figure9-27 Dynamicsof GFPtagging. Thissequenceof micrographs showsa setof three-dimensional imagesof a livingnucleustakenoverthe courseof an hour.Tobacco cellshavebeenstably transformedwith GFPfusedto a spliceosomalproteinthat is concentrated in smallnuclearbodiescalledCajal bodies(seeFigure6-48).The fluorescent Cajalbodies,easilyvisiblein a livingcell with confocalmicroscopy,are dynamic structures that movearoundwithin the nucleus.(Courtesy of KurtBoudonck, LiamDolan,and PeterShaw.)

urrl 0 t

(a)

( s1sr6o;o19 ;o,{ueduol aql uolJ u o r s s r t u r aq dl l M ' 2 0 0 2 ' S l ,L l , - tOL L : SLL 'ps 'xaliol ila)'t "p ta uoileM l'cl Luorl) lla) aql ol tuaurq)Plleutl)eJo ssa)old aqr qllm ssalordleql sl)auuo)Illetrs{qd srql'r{laurqteLu >r1r(>opua luaLuuadxa aqt;o lred uro; ol urvroul,(pea.l;e semd tels aluts '(l) ebeul paltatrot pue parolo)-asleJ aql ut uMoqs'leuOrs 1393 e alereuebpue polet)ossp l;aso;t ate {aql areqmasoql pue '(g u poaqlAoilD) auolepuno;s; d ldqy elaLlmosoL1l '(V ul stAurD)auolp puno; srd 1e;5araqrr asoql 'slrosaarql Jo ale 'pasodr,uuadns are(f) pue'(g)'(V)uoqMuaas'(C) xauo) aql le sloosaql'(uru 5'0 u!qllM) patelrosse l1lq61larese;nta;ouomt aql uaqiu{;uo ln)lo llt^/\q)tql ijl^ aqr uoll pall!uroe)ualsalonlj aLlt^luo 6urplotar lnq djl aql 6urlr)xa,(qpaurelqo sl'()) par ur araqpa{e;dslp'(92-B elnbU oslpaas)leu6rs1351aq1'(dlf) dl9 Jo lupue^uelt e qlrnn(g)ulatorduorsnl e se passaldxa srd gdqyallqM,(dl1) e qtlM ulatord dlg Jo luPue^ano;;a,( uorsnJe sp (V)lla) lsea^aql ur passardxe srd t elS';1a> lsee{ 6urppnqe ;o e)eJrnsaql te luauqleDe ut]le le)tuo) ul pa^lonutst r1)l,lm'dldqy peller,utalold raqloueqlllr Illq6ll ])eralurue>d 1e15 pelle)uralolde leql smoqsluauruadxa srql'6ur6eur (IlUl) rsrsuerr(6leua a)ueuoso,a)uo)saJonll gg-6 eln6r1

s69

'VS6-lV6tZLl rallaJal)oueql uor; uorssruradql11111't66 t fsser6Ilrs.renrun 'lolg 'f1 'l') 'pa6ueqrun 'uosrq)lrW pue urMeS Luorl) {;a6re; sureua.r ilD T (a)ua)saronU pa.,eql ^q pozrlensrn) urlnqntpalaqel-aururepoqr e;purds aq1q6noql uanaprema;od6urnouu I;snonurluotst utlnqnl1eq1bullet;pul 'aloda;purds aql preMolsanou aql Ual ;eu6rsurlnqnl-uro)serong-peberun '(C 'J ur salnuru S'Zraue ur salnurL!S't raue)salnururMaj lxau aql ra^O 'a1e1d aseqdelaur aqt Jo aprsUal aql ol lsnfIlureu X;1ero1 urlnqnlpolaqel -ura)saronl;-pa6e) aql sa6e)unlq6ll An Jo ureoqV (g)'lq6tttalor^ellln ,{q patenrlrelllun }ua)saronguou srtr asne)aqelqrsrlurs! lnq selnqnlor)lr.u aql lleolur palerodlo)uroslesrq)rqM'urlnqnlpeleqel-ura)saronU -paber pue'saLuosoLuorq) aql sloqelfeql oIp 6ulpu;q-yp6anlq e'salnqnlor)rru aLllllelreur ot (par)urlnqnlpalaqel-auruepoqr :sra)reul lua)saJonUaaJqlpalerodro)urseqs66asndouay1ot)e;lxa ue wca!ultA u/ pauro, alpurdsaseqdelauV (V)'urlnqnlol palurl ugarsarong pa6er ql!^1alpulds)rlotlu aql u! xnB alnqnloDlur6u1u1ure1e6 67-6 arn6r1

'(It-6 arnSlc) surel -ord raqlo tuo4 selBJuorlBlJossrppuB Surpurq Jo 'seluJuodsuEJ] a^uJe '1uor.u sasn(V)ur umoqsguauuadxaaq1 'uor6arpaqrealqaql otul anoursaln)aloLu tua)solonU6urureuataql se'aul!l ranoi(rano)otloltuor.lluel lsrdotsot>lr.u peuUepe utq]!M oql 'lla) e;o uor6a.r saln)aloururalordpa66e1r(;lua:saron;1 Ilanrlra;es Jo las e 6urqreaqqotoqd ^B dlg lo a)uo)solonljaql 'q)ealq r o ' q s t n 6 u t l xlal l Ml q 6 l lr a s e1; oa s l n d y'(dVUl) 6urqrea;qoloqd pasn)oJ6uo.r1s re11e{renorer a)uo)saronll 16-5 arnErl

fsvw uror] qlm'€002' L6-Lg:008 uolsslLurao DUaDS '1'uosraued'H'g puezlreanqr5-porurddrl urorJ'B)'palolruouJ aq ue) surolord ;o s,{emqtedanrleperbappue ra^ourn} '6u11>rger1 aql'lla) aql ultlllm]ua)satonU ere surelo.rdpalenrlreoloqdaql ,{;uoaru15'polnseauaq uer'uor6arsrql lr se ijlg Jo luaula^our ,o lno sasnJJrp aql'Uail6) uor6alpalte;asaql ur ,{11q6;rq sarsaron;; I;prderlr /Luu€ [t 1eas;ndlaselnn e 6ursnlanarnoq339 aql Jo uorle^rl)elaUV'ulu 88t le lq6ll anlq Iq pall)xo uaqM (ap)Dpar) uotba) pol)alasaql u! pal)alap sra)ua)saronu '(0 aLurl)uorle^rlfearoJaB dl9 ou ro alll!l 'lla) leturuepornlln)e ur passaloxo s! dlg Jo lupue^alqele^ll)eoloqo e luauuaoxasrq]ul 'alelsa^rl)e ue ol aln)alou.r uaur ueJo uorlp^rl)e parnpur-tq6r1 aql sruorte^rl)poloqd 'uolle^!ltPolor,ld 0€-6 arn6rl

\/

u o r 6 a rp a p e ; e su r Arano:alo)uarseronlJ

paq)ealq u o r 6 a rp a p a ; e s

urfl 0t

ulur0z ulur 0z

ulur0g ulul 09

q)ealq-lsoo

ulur 0

'uollca Ira;eJ;JX?::ijt#Xfiff: srrar rurorcrru 'orpaeu aq]rnoqlr^ ,x?fi]#

p apr^oJd ol susrue8Jo Jaqlo pue sluBld uI dllef,IueBsu?JlpasseJdxa uaaq oslBseq urronbav'(Zt-6 alntld) pezIIUaJ sr 33a aq1uaqM srncJo leqt tusEldol-,{r aq] olrrr +7.eJeetlJo esBelar paz\eJol uappns eql 01 esuodsar ul lq3II Jo qsEIJe slrrua urJonbae 'aldruexaJoJ'33aue olul pelJefulorclruJI 'INrl 0I-9'0Jo aBUPJeql ur uorlEJluesuoJ +ze) ur saSuBqJol spuodseJpuu +zEJJo aJueseJdeq] ul ]q8I slrua 1r lqsr/.11afaurJpruE tuoq pelElosl ulaloJd lue3seululnl e s uuonbav '11q8uot arnsodxa uo ]q8q Suprpra) ]uarsaro -nlJ eJe sraqlo alrqa '(,{.lsnoaueluods tqBU 8ur}lrue) luaJsaulurnl eJEsJo}ellpul asoq.u eseql Jo eruos 'uor eql Jo uollPJlueluoc pcol eql slJeueJuolsslrue 1q311 'sJolBJIpuI aaplsuas-uol qty\^ pezltleue eq ueJ se8ueq3 qJnS 'qeu8ls JBInlleJ -BJUaol puodseJ ol slleJ 3uu,ro1puI aloJ lusuodul ue aABqleql +ze3clToso!{.3 Jo uorlerluaruoc aqt u sa8ueqc luelsueJl pue prder aq] pJoJaJol pellns,{1pap1 'snq; 'ctlerre lEq Aaluos pu? ^\ols aJEsesuodser lou eJBsepoJlJeleorsru aseql (llal P Jreql '+zPJ sP qrns 'uollEJluacuoJ ]!\ol Lran e le luasard uol ue JoJ pue ur lurod auo le Lluo uoqurluaruof, uol eq] IeaAeJsepoJlJalaoJcllueAIlISuas-uoI 're^e,4,\oH'+7El pue '-lJ '*) '*pN'*H se qons 'suot ctue8roul uoutuoJJo suolluJl -uacuoc Jelnllace4ur eql eJnsEaluol pesn sI anbluqcal sIqJ'aueJquaur eruseld aql qBnorqt Jorrelul [oc eql olut Lpcartp epoJlJaIeoJJFu aAI]ISues-uoI'ssel3 'aug u;o dp aqr aug uasul ol sI IIaJ SuIAIIapurs u;o.,fu1stuaqc eq] dpnls o1-,te.r,t

suollerlua)uo)uol relnlle)eJlul 6u;6ueq1{gp;degarnseewue) srote)rpul6ul11lurl-lq6!'l

)as 0

l+ uor6ar pape;as ur a)ua)saronl]

uorle^!peoloqo

slla) 6urz!lensrn:6 reldeL-|J

965

597

LOOKINGATCELLSINTHELIGHTMICROSCOPE protein protein. Figure9-32Aequorin, Theluminescent a luminescent aequorin emitslightin thepresence of freeCa2+. Here, aneggof the medaka fishhasbeeninjected withaequorin, whichhasdiffused witha sperm throughout thecytosol, andtheegghasthenbeenfertilized Thefour andexamined withthehelpof a verysensitive camera. photographs weretakenlookingdownon thesiteof spermentryat intervals of 10seconds andreveala waveof release offreeCa2*intothe justbeneath Thiswave cytosol frominternal stores theplasma membrane. in sweeps across the eggstartingfromthe siteof spermentry,asindicated the diagrams reproduced fromJ.C.Gilkey, on the /eft.(Photographs With 1978. L.F. Jaffe,E.B. Ridgway andG.T.Reynolds, J.CellBiol.76:448-466, permission fromTheRockefeller University Press.)

Bioluminescent molecules like aequorin emit tiny amounts of light-at best, a few photons per indicator molecule-that are difficult to measure.Fluorescent indicators produce orders of magnitude more photons per molecule; they are therefore easier to measure and can give better spatial resolution. Fluorescent CaZ*indicators have been synthesized that bind Ca2*tightly and are excited by or emit light at slightly different wavelengths when they are free of Caz+than when they are in their Ca2*-bound form. By measuring the ratio of fluorescence intensity at two excitation or emission wavelengths,we can determine the concentration ratio of the Ca2+-boundindicator to the Ca2*-freeindicator, thereby providing an accurate measurement of the free Ca2* concentration. Indicators of this tlpe are widely used for second-by-second monitoring of changes in intracellular Caz* concentrations in the different parts of a cell viewed in a fluorescence microscope (Figure 9-33). Similar fluorescent indicators measure other ions; some detect H+, for example, and hence measure intracellular pH. Some of these indicators can enter cells by diffusion and thus need not be microinjected; this makes it possible to monitor large numbers of individual cells simultaneously in a fluorescence microscope. New types of indicators, used in conjunction with modern image-processing methods, are leading to similarly rapid and precise methods for analyzing changes in the concentrations of many types of small molecules in cells.

10

zo

500 pm

Several Srategies AreAvailableby WhichMembrane-lmpermeant Substances CanBeIntroducedinto Cells It is often useful to introduce membrane-impermeant molecules into a Iiving cell, whether they are antibodies that recognize intracellular proteins, normal cell proteins tagged with a fluorescent label, or molecules that influence cell behavior. One approach is to microinject the molecules into the cell through a glassmicropipette. \fhen microinjected into a cell, antibodies can block the function of the molecule that they recognize.Anti-myosin-II antibodies injected into a fertilized sea urchin egg, for example, prevent the egg cell from dividing in two, even though nuclear division occurs normally. This observation demonstrates that this myosin has an essentialrole in the contractile process that divides the cltoplasm during cell division, but that it is not required for nuclear division.

by usinga Figure9-33 Visualizingintracellular Ca2+concentrations fluorescentindicator.The branchingtreeof dendritesof a Purkinjecellin from other neurons. morethan 100,000synapses the cerebellumreceives Theoutput from the cellis conveyedalongthe singleaxonseenleaving the cellbody at the bottom of the picture.Thisimageof the intracellular in a singlePurkinjecell(fromthe brainof a guineapig) Ca2+concentration fluorescent wastakenwith a low-lightcameraand the Ca2+-sensitive indictorfura-2.The concentrationof free Ca2+is representedby different colors,red being the highestand b/uethe lowest.The highestCa2+levels (Courtesy of arepresentin the thousandsof dendriticbranches. D.W.Tank,J.A.Connor,M. Sugimoriand R.R.Llinas.)

1 0 0p m

598

Chapter9:Visualizing Cells m e m b r a n e - e n c l o s ev d esicles /

I

gtass micropipette containing substanceX

microinjectioo nf substance into cell

c e l l p l a c e di n s u b s t a n c X e between two electrodesand subjectedto a very short electricshock

I t r a n s i e n tp o r e sm a d e i n t h e m e m b r a n ea l l o w s u b s t a n c e t o e n t e r t h e c e l l b e f o r er e s e a l i n g II t

I

I

I

i n d u c e dm e m b r a n ef u s i o n b e t w e e n v e s i c l eas n d p l a s m am e m b r a n e of target cell releasessubstance into the cytoplasm I v

DNA-coatedgold particles s h o t i n t o c e l la t h i g h v e l o c i t y a l l o w ss t a b l et r a n s f o r m a t i o n , or transient expressionof new genes

I oo

(A)

(B)

Microinjection, although widely used, demands that each cell be injected individually; therefore, it is possible to study at most only a few hundred cells at a time. other approaches allow large populations of cells to be permeabilized simultaneously. Partly disrupting the structure of the cell plasma membrane, for example, makes it more permeable; this is usually accomplished by using a powerful electric shock or a chemical such as a low concentration of detergent. The electrical technique has the advantage of creating large pores in the plasma membrane without damaging intracellular membranes. Depending on the cell type and the size of the electric shock, the pores allow even macromolecules to enter (and leave) the cytosol rapidly. This process of electroporarlonis valuable also in molecular genetics, as a way of introducing DNA molecules into cells. with a limited treatment, a large fraction of the cells repair their plasma membrane and survive. A third method for introducing large molecules into cells is to cause membrane-enclosed vesicles that contain these molecules to fuse with the cell's plasma membrane thus delivering their cargo.Thus method is used routinely to deliver nucleic acids into mammalian cells, either DNA for transfection studies or RNA for RNAi experiments (discussedin chapter 8). In the medical field it is also being explored as a method for the targeted delivering of new pharmaceuticals. Finally, DNA and RNA can also be physically introduced into cells by simpry blasting them in at high velocity, coated onto tiny gold particles. Living cells, shot with these nucleic-acid-coated gold particles (typically less than 1 pm in diameter) can successfullyincorporate the introduced RNA (used for transient expression studies or RNAi, for example) or DNA (for stable transfection). All four of these methods, illustrated in Figure g-34, are used widely in cell biology.

LightCanBeUsedto ManipulateMicroscopic ObjectsAsWellAs to lmageThem Photons carry a small amount of momentum. This means that an object that absorbs or deflects a beam of light experiencesa small force.with ordinary light sources,this radiation pressure is too small to be significant. But it is important on a cosmic scale (helping prevent gravitational collapse inside stars),and, more

(D)

Figure9-34 Methodsof introducinga membrane-impermeant substanceinto a cell.(A)The substance is injectedthrougha micropipette,either by applyingpressure or,if the substance is electrically charged, by applyinga voltagethat drivesthe substance into the cellasan ioniccurrent (a techniquecallediontophoresls). (B)The cellmembraneis madetransiently permeableto the substance by disrupting the membranestructurewith a briefbut intenseelectricshock(2000V/cm for 200 psec,for example).(C) Membraneenclosedvesicles areloadedwith the desiredsubstance and then inducedto fusewith the targetcells.(D)Goldparticles coatedwith DNAare usedto introducea novelgeneinto the nucleus.

: r'

LOOKINGATCELLSINTHELIGHTMICROSCOPE

0 sec

30 se1erus11ac'sdnor8 peeq pue sprce ,{pe; lualeJJrp IeJaAasSururq -tuoc .,(g 'dnor8 puaq sadrt 1ue.ra;;rp auo ol pelu[ sr uJnl ur qcrq ^ Jo IpJeAes Jo 'dnor8 aleqdsoqd p o] paqJe]le sr ruole uoqJEJ pJrrp aq] pue '1orac,{13 aqr 1o sruotp uoqreo luacefpe ot spuoq ralsa q8norqt pe{uu are sprceft1e; upqc-3uo1 o,t,t'J'(Z-0I arn8rg ees) euoq{Jeq1otact18uoqr€J-earql e aABqqtqrv\'sap;racdy8 -oqdsoqd eql eJe saueJquau IIaJ lerurue lsotu ur sprdrtoqdsoqd umu aql 'relpl ssnssrp a.\/\sp 'au€rqruau aql Jo ,tlplnu aq] 3ur1ca33e ,{qaraql taq}ouu auo }sure8e4ced salncayoruprduoqdsoqd ,^noqaJuenuur slre} plce fi1e; aqt Jo uorlernles pue {r8ua1 aq} ur saruaraJJlq '1l.eteqt ur {uq ilptus B salEeJJpuoq elqnop-st, qoee '7-91 arn8ggur u^ oqs sV'(parumru2ssr ]l 'sl leql) lou seop lel Jaqlo aql alq ^'(pai0Jnl0sun sr lr'sr ieql) spuoq alqnop-sp eJotuJo auo spq,{1pcrdft IrEt auo '(sruote uoqrec VZpue ?I uaamteq ureluoJ,{lpurou ^eqr) q]8ual ul raJJIpuuc,(aq] pue 'sproefi1e; dlpnsn ere slret eql 'sllar Ierralreq pue '1ueyd'purrue uI'sflat uoqtocotpttq crqoqdorpf.q o^alpue dnor8 peaq relod B eluq asaql'spgd;loqdsoqd eql arc sprdq auerquaru tuepunqe lsoru aqJ 'pue tu1oduou ro (,,3urrea;-Jatem,,) puu pue ru1od to (,,3urno1-ra1eir,r,,) c;qoqdorpr(q e c;gqdorpz(q e aneq r(aqt 'sr tpql-Jlllqd;qduru eJEseuuJqruau IIaJ ur salnJalou ptdl aqr Jo IIV 'ilac Ierurue IIEus e Jo aueJqueru eruseld aq] ut salnf,eloru ptdtt o0I ]noqe ro tadepq pldll Jo eare ulrl 1 x urd I E ur selnJelou pldq ng1 x g dlaleuxordde are aJeql 'urelord Suraq Jepurerual eql Jo IIe fpeau 'soueJq -tuaru IIeJ Iprurue lsoru Jo sseru aq] Jo 0609]noqe alnlusuoJ salnJelour prdrl

se u e r q u a wI le ) u! s pl dl l loferyaqt alv sloJalspup'sprdrlo6urqd5'sapg,rar(;boqdsoqd 'suorlrpuor lerJrJruealdurs rapun uanasra,{e1q olut dlsnoaueluodselquesseqJIqM 'salnJelou prdq aqr ;o sarlradordprcads

()) alnrelotlj uraloJd

-

a l n ) a l o | . xp r o r l

fia

arnl)ruls euerquew:g1raldeq3

8 t9

619

THELIPIDBILAYER

cH,-N*(CHlh

t-

CHOLINE

CH, I

o I

PHOSPHATE

O:

l'-O-

I GLYCEROL

tl ll tl CH., t'l' 9H,

12

ll CH,

t-l^

CH. I

o rl

nonpolar (hydrophobic) tails

v

o

CH. 9H, CH, CH,

t'l' 9t lH ,

9H,

CH,

CH,

t-l 9H, !,, \"'

9H, 1,,

z

c,s-double

"kbond

,z I cHr

molecule.Thisexampleis a phosphatidylcholine, Figure10-2 The partsof a phosphoglyceride model,and (D)asa symbol'Thekink (Bi by; iormula,(C)asa space-filling (A)schematicaily, represented emphasis. for is exaggerated bond resultingfrom the cis-double

CH:

@ NHr

\?l I CH C Hr

fn' fn' o

I

C QH Hz I

o: f:

il

o I

,P P-(, --

I o I

- C HH - CC HH z

CHr-CH-CHr tt l- l

I NH I I

oo tl

tl --6

\

ri\L\

I

l^ O:P-O I o I

CHr I CH:

4-6

o OH

tl

OH

HC-CH-CH] lln $H NHp ll'

CH

I

,l

.|' JJ

! J = =

z

-

nAr.\O

UU > > rLU

UUUv F F

FFFT_ [ d L L

phosphatidylethanolamine phosphatidylserine (A)

f

(B)

=' :.{':: .'? I'q '

i>.

:Ei l

.,$ ....

p h o s p h a t i d y l c hionle

s p h i n g o m y ei nl

sphingosine

(c)

(D)

(E)

by differentcolorsin the Figure10-3 Fourmajor phospholipidsin mammalianplasmamembranes.Differentheadgroupsarerepresented The moleculein (D)is sphingomyelin, whicharederivedfrom glycerol. shownin (A-C)arephosphoglycerides, Th. lipid molecules ,y,"nbolr. carriesa net negativecharge,the (E)and is thereforea sfhingolipid.Notethat only phosphatidylserine which is derivedfrom sphingosine pH,carryingone positiveand one negativecharge' neutralat physiological importanceof whichwe disiusslater;the otherthreeare'elecirically

620

Chapter10:MembraneStructure

p o l a r h e a dg r o u p

.H

rigid steroid nng structure

C H rC H r CH I

9H,

nonpolar hydrocarbon tail

9n, CH: I

CH CH: CH, (A)

(B)

In addition to phospholipids, the lipid bilayers in many cell membranes contain cholesterol and glycoliplds. Eucaryotic plasma membranes contain especially large amounts of cholesterol (Figure l0-4)-up to one molecule for every phospholipid molecule. cholesterol is a sterol. It contains a rigid ring structure, to which is attached a single polar hydroxyl group and a short nonpolar hydrocarbon chain. The cholesterol molecules oiienf themselves in the bilayer with their hydroxyl group close to the polar head groups of adjacent phospholipid molecules (Figure r0-5).

Figure 10-4 The structure of cholesterol. Cholesterolis represented(A)by a formula,(B)by a schematic drawing,and (C)asa space-filling model.

Phospholipids Spontaneously FormBilayers The shape and amphiphilic nature of the phospholipid molecules causethem to form bilayers spontaneously in aqueous environments. As discussedin chapter 2, hydrophilic molecules dissolve readily in water because they contain charged groups or uncharged polar groups that can form either favorable electrostitic interactions or hydrogen bonds with water molecules. Hydrophobic molecules, by contrast, are insoluble in water because all, or almosi all, of their atoms are

The hydrophobic and hydrophilic regions of lipid molecules behave in the

Being cylindrical, phospholipid molecules spontaneously form bilayers in aqueous environments. In this energetically most favorable arrangement, the hydrophilic heads face the water at each surface of the bilayer, and the hydrophobic tails are shielded from the water in the interior. The same forces that-drive phospholipids to form bilayers also provide a self-healing property. A small tear in the bilayer createsa free edge with water; because thii is energLtically unfavorable, the lipids tend to rearrange spontaneously to eliminate the free edge. (In eucaryotic plasma membranes, the fusion of lntracellular vesicles repairs larger tears.)The prohibition offree edgeshas a profound consequence: the only way for a bilayer to avoid having edgesis by closing in on itself and forming a sealedcompartment (Figure r0-8). This remarkable behavior, fundamental

E

c

il:It 4

Figure10-5 Cholesterolin a lipid bilayer.Schematic drawingof a cholesterol moleculeinteractingwith two phospholipidmoleculesin one monolayerof a lipid bilayer.

'alqeroAeJun ,(;1e>rlabraua aq plnoA q)rLlMlalem >rqoqdorp{qaql ol slre]uoqre)orpr(q JOarnsooxaaq] spro^elr asne)aqalqels sraln])nlls pasollaql'luoruueduor poleasp urol or rale;gqp;d;loqdsoqde to arnsol)snoaueluodseq1 g-g 1 etn6t1

'adeqsllaql uo 6ulpuadaplaleM ul alnl)nJlsJaqloaql lo saln)aloulpldll 'uoll)asssoJ)ul uaasJa^ellqpldll e pue auo uroj ^lsnoauetuods padeqs-taput;{l saln)alouprdr;oqdsoqd alle)lLupldfl y (g)'sraIelrqw)o! (lvloPq) pldt; padeqs-auol (v) 'luauluor!^uo searaqM'salla)rulw)o! (a^oqD)saln)aloLrl snoenbeue u! saln)aloulptdrl;o slueua6uerre6u;pe6 /-O t eln6l3

l'rsvuonvl A'l'lv)trt9u3Nl

-rai(e;rq p r d r ; o q d s o q^dq p e u r o + lusurueou_ro)paleas

rfo slla)ru p!o!l

O

o

leleM ol pasodxe sa6pe qlrnii r a i { e l r qp r d r l o q d s o q d : e u e l d

7\ 7\ 7\

/--

'a o1l saln)aloul p r d r l; o 6 u t p e d

l'lSvuonvlNn A'l'lv)trl 9u3Nl

3ln)elotU prdrl 1o adeqs

'saUrosodg Jeleuerp ur lud I o] tuu gz lnoqe ruoJJ azrs ur fuBA ueJ qllql\ palles 'salJISaAIEclraqds Jo luroJ aq] uI apBlu sJe.li.ellq(I) :salpnls qJns uI InJ -asn ,{ran uaaq a^uq suol}eJedaJdJo sad,q o/V\J'sJaLellq pldll 3l}aqluds Jo selpn}s 'sredellq pldll uqlllvl dlee4 asnJJIpol elqP uro4 arupr uorlErlsuotuap pUIq aqJ '026I punorv aJe salnJalou pldl pnpNpul leq] pazuSoJeJ ]sJU sJaqsJEaseJ

e slra^ell8p!d!] eql p!nll leuolsueurp-oMl 'suolpunJ fueru ol I€I3nJ:) euEJqrlau 'seuPJqluelu fiac JoJ sr qJrq a drlppu slr sI asaql Jo luuuodllll ]solu aql Jo euo arntonrls Ieapr ue tl e{eur leqt sf,ltslra}f,ereqo raq}o seq osp re^EIIq pldl V 'alnlelolu pldnoqdsoqd aqlSo arnlBu '1ac 3urn11e Jo uopeerJ aqt o] rrTrqdrqduB pue adBqs aq] luo4,(pcarp s^/\olloJ

alesuro}e puelolo) ut umotls atesdnot6te;oduou ,{o;6ut urvroqs aqf 'laleM -g 1oqu,(s pue'a6req: anrle6au e sate)tput*g anrlrsod relo6.a6reqr leltrede salellpul ;erlted abe)alllarl tl)ttlm'salnl)nlls aer1 aql saseat)ut srpunoduo:srql'(6raua ,(1lenurn aloraraqt utalqnlosut

'raleM q]!M suotl)elalul alqPlo^eJ [!JoJ ]ouue) ]l olur azrueo;oal ol saln)aloul .relervrluatefpe a)loj plnoM ]! ' r a r e M u t s a ^ l o s s l p , ( ; r p e aal u o l a l e ' s n q 1 a s n e ) a 8 ' t r q o q d o r p { q { l e r r 1 u as t a u e d o j d l ^ q } a u - Z ' l s e r t u o ) I B ( B ) ' t e ; o d o s l e a l e q ) t q M ' s a l n ) a l o u r a l e m q l r m s u o r l ) e r o l u ! ) ! ] e l s o J t ) a l a a l q e l o ^ e Ju l l o j u P ) l l l e l o d s l a u o l a l e asnp)a8 (v) lalervl qllM ^llua.raJJlp l)eralu! saln)oloru r;qoqdorplq pue >rlrqdorp{q mog 9-g 1 arn6t1

releM ur auola)P

raleM ur auedold l^qlaur-Z

lale^^ *9

uu "\^,'"

+g

U

_s euola)e .:tHf,

/'

so

I

urAVl|SCldll lHl-

tz9

'sauelquau tllaqluls Jo satuadold {1r;rqeatu.red aq} arnspauo} pasn aresauelquauJ))elB'sluauueduo) snoanbponnl6urleledesuollllled e ur aloq llpulse ssol)esulloJlt uaqM reue;dsrql .ra{epq 1relqsreadde.relelrq prdlJrrleqlu{s e ,euelqutaul)plq e y g 1-g 1 arn611 ,o rvro!^leuotl)as-ssoJ)

( a u e r q u r a u1r: e 1 q )r a f e l r q p r d . ,

p o] pmbrT alels E tuoU sa8uEqc prduoqdsoqdyo ad,{1 elSurs e ruo4 apEruJadBIrq Jlleqtuds tpeqlu^s sarprus paleJlsuouap ur d[peal sr se ,arn]BJad V'sJa^EIrq Jo -ua] slr puE uortrsodruor qloq str uo spuadep pldll E Jo &rpnu aql ra^Elrq 'la^al ploqsaJq] e puo^eq pasearJur fleluaruuadxa sr dlrsoJsr^ ra^p[q eql ueq A aseao'aldurexa JoJ (sar]rll]ce eru[zue pue sasseJord]rodsuerl auerqruau ureual 'paleln3eJLIasrJeJdaq ol seq sauBJqruaruIIaJJo dllpmu eqJ (.rat^asllruolJ uolssrrllaoqlm'5002' Ltt: L6t.low(zug 'qlaw']reqnH 'M pue 'd ,v) lrraparl urolJ 'sarpnls lpluauuadxaut Sauelqu.jalrj lapoLuse pasnl;uoturuot ale sauosodrl 'uorlfas ssor) ut uaas eujosodtl;eruaqds .arnlejadual lleus p Jo 6urrrnetp V (B) ua6o.rlruprnbrlo1 uazo.r;i(;prdetrelem ur-seuosodr;-salltsa^pldrloqdsoqd pautelsun'paxqun1o qdet6ortrr.u uorl)alauV (V)'sauosodrl 6-91 arn611

uolllsodruo)sll uo spuadacra/(eltgp!d!l e Jo/qtptnljaql '71 raldeq3 ur pessnJsrp sE taqlo eq] ol ral.elouoru auo uorg sprdsoqdsoqdSo dog-drg prder aql az^le,teJqllqm 'srorDrolsuauprdqoqd -soqd pa1eJ saul,tzuaaueJqurerusuerlJosselJpreads e,,(qpallos sr uralqord aq1 'epptu eq tou ppoc radelq prdrl .^,rautedelouoru crloso{ruou aq} o} [FdruoJd 'aueJquau z{yqeuoseara}er8rru pFor salnf,aloru eppu flarrau asaq] Jo auou Jl

U J US Z

-ol\^l E sr eueJqrueru pcr3010rq e;o luauodruoc prdl aq] tEq] alerlsuoruap ,taq; 'sra.{e1q c4aqlufs ur asoql o] JeFurs s]lnseJ anrSs11acSurnrl ur puE sauBJqrueru pcrSolorq palelosr ur salnlaloru prdu palaqel uo serpnls drilqoru lelrurs

JaleM

a8rel e qf8ual eqt sasruJrp I tnoqe ur (uri alnJaloru prdu a8e Z-) IIIJ lerralJeq Jo -JaAeuE lEq] suBau qslq^4 'Jas/zruJ uorsnJJrpp qllM s_0I lnoQe;o (o) ]uar3rJJaoJ 'uorsn3grp lera]el prder e o] asrr san€ srq;.(puocas rad satuq 10I-) Jafelouoru e ulLlrym sroqqSrau rraq] qlIM sareld aSueqcxa -,{lpear salnc-10ur prdl .tser} -uol uI dprder dog-drg uEr puB elnr slql ot uondalxe ue sr Ioratsaloqcq8noqtp 'alnJelolu pnpr^Ipu due ro; qluoru E atuo uBql ssal sJnf,Jo,,,dog-drg,,sE rr AODI 'ssecord slql.reqto eq] uo leqt ol eprsauo uo (la[oa\ E pelpc oqe) radelouo"qi uor; aler3[u,r(larer dra'r sradelq cpaqluds ur salnJalou prdroqdsoqd 1eq1-troqs

-euJally 'aueJqrueru E ur salnJalotx pnpNpur ue^a Jo uorsnJJrpaq].ry\olloJpuE dnorB peaq relod s1r paqJeue ol alcrped plo8 e ro artp e qlpn;a1d tuaosa.rong IIErus -ruexa roJ 'elnf,alourpld{ e }JnJlsuos uEJ auo 'sluauodtuoc rreql pue salncalolu pldll uorlou aql aJnsearu pasn uaaq aleq sanbruqcel snorren ol 1pnp4lpur.Io (0I-0I arnS;g; sluaurlredtuoc sno -anbe oml uaa-,\^laq uorlrlred e ur eloq e ssol:)e pauJoJ 'serrBJqr.uaur{rBIq palleJ 'sraitepq reueld (z) pue 1(6-0I arn8lc) pernpord are daqr ^'oq uo Burpuadap

arnlrn.tlseueJquary:0t JaldeqJ

zz9

623

THELIPIDBILAYER l a t e r a ld i f f u s i o n

\ L.. ., I flrp-Trop | (rarelYoccurs) t I

I {lexion

rotation

a c i dt a i l s l i p i d h e a dg r o u p s

water molecules

with > Starting lipidbila in an artificial molecules Figure10-11 Themobilityof phospholipid the lculated a c bilayer, regular in a arranged phosphatidylcholine molecules 100 a modelof (taking calculations time.Fromthesetheoretical of simulated position of everyatomafter300picoseconds of the all for almost that accounts emerges processor lipid bilayer the model of 1995), a time in weeksof per numberoflipidmolecules suchasitsthickness, properties lipidbilayer, of a synthetic measurable Notethatthetailsin one of thetwo surfaces. andunevenness depthof waterpenetration, membrane area, (B)Thedifferent if thetailsarelongenough. withthosein theothermonolayer, monolayer caninteract With 1995. J.69:1230-1245, Biophys. (A, et al., Chiu on 5.W. based in a bilayer. motionsof a lipidmolecule permission Society.) fromthe Biophysical

two-dimensional rigid crystalline (or gel) state at a characteristic freezing point. This change of state is called a phase transition, and the temperature at which it occurs is lower (that is, the membrane becomes more difficult to freeze) if the hydrocarbon chains are short or have double bonds. A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another, in both the same and opposite monolayer, and cls-double bonds produce kinks in the hydrocarbon chains that make them more difficult to pack together, so that the membrane remains fluid at lower temperatures (FigUre f0-f2). Bacteria, yeasts, and other organisms whose temperature fluctuates with that of their environment adjust the fatty acid composition of their membrane lipids to maintain a relatively constant fluidity. As the temperature falls, for instance, the cells of those organisms synthesizefatty acids with more cls-double bonds, and they avoid the decrease in bilayer fluidity that would otherwise result from the temperature drop. cholesterol modulates the properties of lipid bilayers. lvhen mixed with phospholipids, it enhances the permeability-barrier properties of the lipid bilayer. It inserts into the bilayer with its hydroryl group close to the polar head groups of the phospholipids, so that its rigid, platelike steroid rings interact with-and partly immobilize-those regions of the hydrocarbon chains closest to the polar head groups (seeFigure 10-5). By decreasingthe mobility of the first

unsaturated h y d r o c a r b o nc h a i n s with crs-doublebonds

saturated h y d r o c a r b o nc h a i n s

Figure 10-12 The influenceof crsdouble bonds in hydrocarbonchains. The double bonds make it more difficult to packthe chainstogether,thereby makingthe lipidbilayermoredifficultto freeze.In addition,becausethe hydrocarbonchainsof unsaturatedlipids aremorespreadapart,lipid bilayers containingthem arethinnerthan bilayers formed exclusivelyfrom saturatedlipids.

624

Chapter10:MembraneStructure

Table10-1 ApproximateLipid compositionsof Differentcell Membranes

Cholesterol Phosphatidylethanolami ne Phosphatidylserine Phosphatidylcholine Sphingomyelin Glycolipids Others

17 7 4 24 19 7 22

23 18 7 17 18 3 14

22 15 9 10 8 28 8

3 28 2 44 0 Irace 23

few cH2 groups of the hydrocarbon chains of the phospholipid molecules, cholesterol makes the lipid bilayer less deformable in thiJregion and thereby decreases the permeability of the bilayer to small water-soluble molecules. Although cholesterol tightens the packing of the lipids in a bilayer, it does not make membranes any less fluid. At the high concentrations found in most eucaryotic plasma membranes, cholesterol also prevents the hydrocarbon chains from coming together and crystallizing. Table l0-l compares the lipid compositions of several biorogical membranes. Note that bacterial plasma membranes are often composed of one main type of phospholipid and contain no cholesterol; their mechanical stability is enhanced by an overlying cell wall (seeFigure ll-lg). In archaea,lipids usually contain 20-25-carbon-long prenyl chains instead of fatty acids, prenyl and fatty acid chains are similarly hydrophobic and flexible (seeFigure ro-2op). rnus, lipid bilayers can be built from molecules with similar features but different molecular designs. The plasma membranes of most eucaryotic cells are more varied than those of procaryotes and archaea, not only in containing large amounts of cholesterol but also in containing a mixture of different phospholipids. Analysis of membrane lipids by mass spectrometry has revealed that the lipid composition of a typicai cell membrane is much more complex than originally thought. According to these studies, membranes are .omposed of a bewildering variery of 500-1000 different lipid species.\A/hile some of this complexity reflects the combinatorial variation in head groups, hydrocarbon chain lengths, and desaturation of the major phospholipid classes,membranes also contain many structurally distinct minor lipids, at least some of which have important functions. TJeeinositol phosphotipids, for example, are present in small quantities but have crucial functions in guiding membrane traffic and in cell signaling (discussed in chapters 13 and 15, respectively). Their local synthesis and destruction are regulated by a large number of enzymes,which createboth small intracellular signaling molecules and lipid docking sites on membranes that recruit specific proteins from the cytosol, as we discusslater.

DespiteTheirFluidity,LipidBilayers CanFormDomainsof DifferentCompositions Becausea lipid bilayer is a two-dimensional fluid, we might expect most types of lipid molecules in it to be randomly distributed in their o-wnmonolayer. The van derwaals attractive forces between neighboring hydrocarbon tails are not selective enough to hold groups of phospholipid molecules together. with certain lipid mixtures, however, different lipids can come together iransiently, creating a dynamic patchwork of different domains. In syntheiic lipid bilayerr composed of phosphatidylcholine, sphingomyelin, and cholesterol, van der waals forces between the long and saturated hydrocarbon chains of the sphingomyelin molecules can be just strong enough to hold the adjacent moleiuleslogether transiently (Figure f 0-f 3).

6 17 q

40 5 trace 27

0 70 trace 0 0 0 30

qllM'zo0z'0169z-99692:llz'waq) ('{6o;o;9reln)alo6 pue (llsruaqrorgro; ,(1aoo5ue)uauv aql LuorJuorssrr.utad 'uotltsodtuor 'p!B'f ''p ,(1sano;se5 'l'O LuorJ'V\'Pail6 1a ryDp)suralo.rdauelqurau rr;rradsalerlualuo) or lq6noql are suel pue'(9alduexe prdrlpue ssaul)lqtpasear)ur lraqt qloq Jo osne)ag(g)'sureuopUeraql olur uotltlted,{lleltuate;atd '61-91ernbr3ur palertsnllr) oq} ot paLl)elleale q)rqtvr'soln)aloul e,{q:a,{elrq roq)ue(ldg)lorlsoul-llplleqdsoqd;IsorI;6 pue ur1a,(uo6urqds uralordpele.rodJo)ur ureluo){;ueuud sueroql 'g 1-61aln6t1ut aresalrdsano;;aI'dreqsaq1';oralsaloq) a)roj )rtuolP{q pazlpeue'sryel se lraIe;rqaql Jo lsal aql ueql lal)tqt ate'abuotour umoqs'seaJeUeJaql leql aloN ',(dorso.trtur prdr;6urureluo:ra,(e;rq)ltaqlu^s e jo sinoluo) a)eJrnsaql (V)'sre{elq p;d;g;e;r;Itre ur sger prdr;lo sl)e#a oql t !-91 arn6t1 u.lu00s

(v)

+ P Jp r o r l

(s)

puP sprsP[DeJ ruoq pezsaq]u,{.seJEqslq^a'sJelse aql u1 sarurkua.{q loralsaloqJ 'spldl Ier]nau erols s]aldorp pldl'I Ioralsaloqs puP seprre3[181,bernse qJns 'IueeI]spoolq eq] qSnorql slles rer.{lo o} peuodxa pue pueuep uo paleraql aq uEf, sprsp^neJ qsrq a luo{ 's1a1dorpprd11a3re11osJaqrunulsen urcluor r(eq; 'ftq-Vl ern8rCaes) a8erots pldq roy pezrrelcadseJE'sailrodlpe pellpc osp 'silec 'eJJnos pooJ B sB Jo srsaqluds auurqurau JoJ $lJolq Suppnq sE poAaIJlaJ IBC aq uec daq] araq,r,ruror; 's1a1doJppldfl ur sprdq Jo ssecxa ue eJo]s slleJ lso6

Ia^elouou1p;d;loqdsoqde ^q pepunolrns arv slaldoJ6 p!d!l (ii;r"tiil; ur passnf,srp) sauorernterprlurolur spu'rs .,nFllererxatr"^"",

('allrMq)S ertadJo,(saltnot'6 ilat^asll ulolj uotsstuiladqtm't002 '69-LLiLVL'lolg 'Dnrls ";e e,(qe; 'p 1a 7 uJorJ'v)latauerp ur sralauoueuMaJe se lleus seeq {eu sgetalaqm'sauelquaur ur paltadxaleqt ueql re6.re; ;err6o;orq q)nui srsauosodr;let)Utue1uer6asaql ur pauro1sureuropaql Jo eztsabetane aql'saseqdaql Jo auo otursuorlrtred {;;erluaralatdleql a{p }ua)saJonU e Jo suorlerlua)uo)a)ell qllM paulels aresauosodrlaq1'saseqd alqt)stuull oml i.llrMsralelrquilo; lololsaloq)pue 'ut;a,(uo6urds'aur;oqt;Iprleqdsoqd yo alnlxrur !: [: t e Luol;parnpotd sauosodr; (B)sealaqM'sla,{e;rq utjo}tunuuoj pue eur;oq>1,(plteqdsoqd ur;aluro6urds 1o arnlxrur[: I e utot; parnpo.rdsauosodr; ]uer9 (V)'sra{e;rqprdl1lerr;ryeu1 g g-g 1 arn6r1 uolgeredeseseqd 1era1e1

se 'sarlqruasseuralord ur raqlaSot 3ugro,,rnJoJro (gI raldeq3 uI passncslp)self, -rsal aueJqruaru ur lrodsuerl JoJJaqlre ruaql SunerluaruoJ-sulalord auerquaur azrue8ro dlaq ppor sger prdt '[ear srql u1 'ssacord Surzflqe]s dgenlmu e aq 'aldrcurrd ur'plnom sururuopUeJolur sprdu;o pue suralord;o uorle8ar8asleJalel aql 'snql'(?I{I arn31g1sura}ord eueJqrueu ureileJ etepoturuoJre Jeneq pue (ZI-0I arn8rg aas)radelq aql;o sged raqto ueqt relclql ere suleluop gur 'sprdq aueJqruaru Jaqlo Jo asoq] ueql ratq8rcr1s pue ra8uol are sprdlo8uqds 1o suruqc uoqrecorpfq aq] asnecag 'suEr eseql azlrqels dlaq araql alquessB tEql sulelord cgtcads aql ]eql ]q8noql sl ll puE 'loralseloqf, pue sprdqoButqdsut paqrlrua arE '(tI raldeql ur pessnJsrp)srsol,bopua ur pallolur aoloanal eql sB qJns 'aueJq -ruaru eruseld aql yo suorBar pazqercads uruua3 'sger p1d;1pellef, 'sururuop pazr -prcads otur elquasse flluarsueD ueo sllec 1erurueJo eueJqueu euseld aql ur salnf,alotupldl aql Jaqlaq i\ slsrluercsSuoruu elpqep 3uo1e ueeq seq eJeql urns

(8)

ruti 0 t

szg

ul^VltSCrdtllHl

'sra^elouout Llloq ut Illenba Ilq6not palnqulsrpaq ot lr.l6noqlsr (uMor.ls tou) '(aryq sdnu6 peaqre;od loratseloq) qllm umelparesprdr;or{;6 ;euo6exaq /uorlrppeul'E-0t ernbr3ur pa)npollul esoqtarpsdnor6peaqprdrloqdsoqd eql roJpasnslolol aqf 'slls) poolq po, ueurnq4o.relellqpldll aqr u1sp;d;1or{16 pue sprdrgoqdsoqd ;o uorlnqrrlsrp eq1 91-91 arnbr3 ;e>grleuur{se

'losot_A)

l)vds uv'tn't'tlfvul_xl

,!1n11cuslr roJ prdloqdsoqd paBJBI{Jdla,rrle8au slql saJrnbalpue 'peleJlue3uor sr aurJasll.pqeqdsoqdaJaq A'auurqrualu puseld eqt Jo af,EJJllosolrtJ aql 01 spulq lI 'spuSrs JelnllaJeJlxa snorJEAo] esuodsaJ ur polEArlJBsr 'eldruexaroJ'ONA J asou!4 ularcJd aurfzua aql 'raf.epqprdl aqt Jo re^ulouour JrlosoilJ eqt q punoJ sdnor8 ppaq prdrl orJrcedsot pulq surelord '191Jaldeql ur pessnssrp)sauo JelnllesBJturolur slEuSrsJelnlleJ f,rTosol-{3,r(uery -eJlxa SurUeAuoJ ur,tlercadsa 'luelrodurr f1euorlcury sr ltr1ourur.{.sppldl'I AJIETUUIASE

prdq urc1urcrupue alpJaue8sroleJolsueJ]pldloqdsoqd punoq-eueJqueru ^aoq ZI Jaldpq3 ur ssnJsrpa A'Ja,{ulrqeqlJo sa^pq ol\i\laq] uaaMlaq a8Jeqcur acuaJaJ -JIp ]ueJrJru8rsE sr aJaq] tal.elouoru Jeuq eql uI peleJol sI aulJasl^plleqdsoqd pa8reqc .,(1a,r4e8auaql asnecag '(gI-0I ernS;g) ra,,(elouour Jauur aq] ur erp (aurrasldprleqdsoqd pue aurrueloueqlel.{pppqdsoqd) dnor8 oulue dreruud pu -Irure] e urcluoc luql IIe lsorup seaJaq^ tadelouour Jelno eql ur eJe (u1edruo8 -urqds pue euqoqrll.prreqdsoqd) dnor8 peaq Jraq] ur-HOzHOzHO*NE(sHJ) -eurloqJ aleq ]eq] salnraloru prdrtoqdsoqd aqr 1o I1e ]soru1e 'ald -ruexa ro; 'auerqurau poolq peJ uerunq aq] uI 'luaJaJJrp,(puryrr1seJeseupJq ilac -uraur dueru ur radelq prdg aqr sJa^Blouoruoml eql Jo suol]tsodruoc ptdu aql 1o

p!d!Taql Jo^rteuu{sy eql luelrodurlI;;euollrunj sl ror(elrg 'suralord pue sprdrloqdsoqdgo ;adelouour Surpunorrns Jlaqt aJlnbce pue ruroJ deru slaldorp pIdI irroq Jo Iepour euo smoqs 91-91 arntlC 'palerl -uacuoJ eJe ruslloqelaul prdq Jo sau^zua fueur araq,rn eueJqr.ueu runlnJl]aJ crurseldopua aq];o suor8al aleJosrpruo4 ruJoJLaq; 'sprce ,i1ey 3o suoneJlueJ -uoc q8rq o1 pasodxa eJe sllal uaqn,,(prdeJ rxJoJ slaldorp ptdtl 'uanou4un are lsoru Jo suor]Jury aql lnq 'rusrloquleur pldl ul pallolur seudzue are sutalord aql 'suralord;o dlarrel a8rel e sureluoJ qJIqA\ 'sptduoqdsoqd;o radelouour Jo auos al8urs e dq papunorrns are r(aq] asnecaq sallaue8ro enbrun are slaldorp prdrl 'sra.,(epqolur ueqt Jeqler slaldorp puolsuaulp-aeJql olul ale8arSSe qJIqM 'salncaloru crqoqdorp,,{q ,tlanrsnlcxa are .{aq} 'sdnor8 peaq cruqdorpdq ureluoJ ]ou op sprdq asaql asnereg 'euBJqueu unlnJrlal cnuseldopua

[pl] sleqsllqnduellru)ewuror] qll^'9002'I Lt -ELEiL'lolgila) uorssrr-urad 'pw'^aa'lpN /uoued'9'u pue ullrew 's uot; peldepy;'sulalo.rdpolet)osse pue sprdr;oqdsoqd ;o rele;ouotu al6urse Iq pepunor.rns'a;;eue6ro anbrune seauelqurotuunlnltlal )ruseloopuaaqt uolJJJosaq)uto pup spnqq)!qM'laldoJpleuotsuaultp -aarq]e olur elp6a.r66e r(aql'eraql 'auerqutauluinln)tlal )ilJlseldopuaaql ;o sra{e;ououromt aql uaam}aqpalrsodap aresprdrl;erlnag's1a;dorpprdrllo uolleuro, aql ro] lapotu y g g-g 1 arn611

sJelsa loJolsaloq) pue s1ola:I;614:eu1

aJnpnrls aueJquaw :0 t JaloeqJ

929

sluaLu6er1 6 ur ; e u 6 r s

'pele^r]rE suoll aq] uea,/\i\leq ol\/\l sr sJe^Elouolu 'Z -f,aJrpqloq ut dfiecrJrcadsuouspldlToqdsoqdsJaJSUEJ] ]eql ,,asulqlueJcs,, V 'palelrlceur sr ra.{elouoru cqosol,tc aql o} radelouoru ctloso$.cuou 'I aql ruo4 prdu qql strodsuer],(lpurou ]€ql rolerolsuel prdloqdsoqd aql :srusrueqraru oan1^q Jnrro o] ]qBnoql sI slleJ tltoldode ut aurrasldprleqdsoqd eqlJo uorleJolsuer] eqJ'll lsa8rp pue IIeJ peap aql asoldco8eqdo1'sa8eqdorceur sE qJns 's1ac Sutroqq8rau spu8rs eJPJrnsIIaJ aqt uo pasodxa aurrasldprluqd -soqd aql rafelouotu relnllaf,Er]xe aq] o] seluJolsuerl ,tlprder ta.,(epq ptdl aueJqruaru eruseld aq];o ra.{eyouourcqosoptc eq} o} peuguoc dlpurrou sl qJIqM 'auuas1,(pqeqdsoqd'191ralduq3 uI passnf,slp'Uleap ilac paurueJSord;o ruroJ e) srsoldode o8rapun qler Iprulue ueq ysllao peep pue e^II ueamloq qsrn8upslp o] seuerquaru euseld raqt Jo drlauudse prdrtoqdsoqd aql lroldxa spruruv '(fgZf-Ot arn8rg) runlnJllar cturseldopua eql ruor; +7eJJo eseeleraq} selplnurls pue losoldc aq] olul paseelersI Jaqlo aql allqm'3 aseurl uralord alen -pce sdlaq pue aueJqrueu aq] ur sulerueJqJIq aJo auo 'sluaurS€4 oml aleraua8 ol auerquau uruseydaql;o radelouoru rqosolb eql q prdloqdsoqd loltsout ue se^BelJ'aldurexa rc1 5 asadqoqdsotl4'sJolelpau JelnlleJer}ul pelll-uoqs se 'selnlaloru prduoqdsoqd cgrc lJe teqt se1ncaloruasaql Jo sluaru8er; Suueraua8 -ads azrealco1 spu8rs relnllacerua itq palunrlee eJPleql sasadqot4dsoqdsnouet sureluoJ euerquau eruseld aqJ'seuo relnlleoeJtul olut spu8ts JelnllaoeJlxaual -uoc o] r(eanJaqloue 1adur pasn arE aueJqruorueruseld aq] uI spldlloqdsoq4 'lrodsuert aueJqurauraprn8 leql suralord ]lnJcer o1 d1aq,{qeraqtpue saueJq -ruetu Jelnllacerlur ur sprdqoqdsoqd loltsout a1e1froqdsoqdsaseuq pldll TEIITUIS '(VZI-SI eruseld aq] arn31g1 auerqruaru Jo eJeJotloso{c eq} ol sulalord Surleu -8rs ol asuodsarut pue quu8ts JelnlleoeJlxe oi sdlaq replacerlur rr;rcads lInJJar s7aseur)lptdq e qcns pele^rtJe sr qJIq^^ '(asauq-g 14)asouly-€ aprlrsoulor4dsot1d ;o aldruexa lueuodrut uV'auerqueu aq] rl1 losoldc aqt uorJ sutalord cgtcads '3urr lrnJcal leql salrsSurputq Surlearc lolrsout eql uI suoDlsod ]ruustp le sdnor8 aleqdsoqd ppe uec saseuDl prdq snotren 'sauuJqrualu IIaJ Jo radelouotu ct1 -osolrb aql uI paleJlueJuoc aJeleql sprdloqdsoqd rourur aqlJo auo sI 'eJueJsul rc!'prlsourltryar4dsoq4'ace1d pue arul] rPInJIued e le salts Sutputq-utalord 'sesel Jaqlo uI eleeJf,o] parJrporuaq lsJrJlsnru sdnor8 peaq prdrl cgtcads

'(B)ur uMoqss,{ennq}ed (9't) sraleqdsoqdrp 6ut1eu6ts aql u! aleladol sasedr;oqdsoqd'umoqs anea;lsasedt;oqdsoqd arnl)nrlsaql'sprdrloqdsoqd lorlsoull,(p!reqdsoqd;o araqMsalrsaql (f)'(B€-st aln6rl osleaas) Jo sassel)luarajJrp lla) aqt olur ;eu6rsaq1,(elerdlaq ]eqr sluaubet;6utleraua6'sp1d;loqdsoqd alenrlresleu6lsle;n1;alerlxa a^eal)teqt sasedr;oqdsoqd lo]rsour lo, salrs6utllop relnlla)ellulsnolJeA auros(g)'sutalord6ur;eubts q)tqM'aseul)-€ld ale^!l)e 6urlear:'sprdr;oqdsoqd ;olrsoutsa1e1,(.toqdsoqd s;eu6rs .re;n;;arerlxa auos (V)'suerquau euse;d eql;o la;;ea;11;osoilr aqr u1spldllogdsoqd;o1|sou; ,o suorl)un,6ur;eu6tsom11 1-g1 arn6t1

(8)

u ralord loloa)al paleArlfe

I

prdrloqdsoqd l o l r s o uI palelrtoqdsoqd

jt_ leu6rs l

l"l1r""lr1 u3AVrSCldll lHl-

LZg

saprsorl8ue8xalduoJ Jraql uI luarf,Uap eJB lEql e3rtu ]uBlnru la^a^ oq Jo IIe '.{lSursrrdrng'(6I Jatdeql ur passnlsrp) uorseqpe [a]-llaoJo ssacord aq] ul sure] -ordor;tp pue sprdqoc,{13qtoq uo sdnor8 re8ns aq} o} pulq guuraD sura}ord 8ur -purq-aterp,r(qoqrEJpunoq-auBJqruau qclq^ ur 'sassacorduorlruSocar-llal ur uorlJunJ o1 lq8noqr osle ale sprdrtoc,,(19'aoeJJnseueJqruaru aq] ]e-+zp3 ,(llerf, -adsa-suor Jo suorleJluaJuoJ eql pue aueJqruau aql ssoJJEplag [BJrJlJalaaq] sralle aJuasard rraql :slJeJJe Jraql asnecaq aq .{pru 'saprs luelrodrur lp3rJlcala Jo -orpue8 se qrns 'sprdqoc,,tppa8req3 '(saru.,kuaa,rrqeper8ap suorleJluaJuoJ Jo q8rq pue gd rtol se qons) aJaql punoJ r(lluanbar; suorlrpuoJ rlsrcq eql lsure8e aupJqrueu aql lralord o1 dlaq Leu ,(aql ararlm 'aJeJJnspcrde pasodxa aq] ot paurJuoJ are sprdqoc,r(p'aldruexa JoJ 'sllal pqaqlrda Jo aueJqruau euseld aqt q 'uorlez\eJol Jraqt ruo4 auoc sprdqocr(p yo suorlJunJ aq] ol slulH -sE 'secl f [[aJJaqlo ur sarlrluenb Jalletus qcnru ur punoJ osle are faql lsseruprdq IE]o] eq] Jo %11'-g 'sllat elnlrtsuoc saprsoq8ue8 aJeq^ alrau eupJqruau eruseld aql ur eJppagt1 Jo -uapr uaaq a^eq leql saprsolpue8 tuaraJJrp97 ueel eroru eqt tuepunqe ]soru Jo aqJ'(8I-0I arn31g)a8reqc a,Lrte8auleu p saprsorpue8 anr8qtlqm'senprsar prJe Jrlers aroru Jo auo qly!\ seprJeqcceso8qoureluoJ 'sap1so118ue8 aqt 'sprdrtocdlS aql;o xaldruoJ lsotu aqJ 'saueJquaru JelnllaJerlur euros ur punoJ osp are daq; 'ra,r(elouoruralno aq} ur salncalotu prdrr eql Jo %g }noqe a}nlrlsuoJ ,{leraua8 ,{aql araq,r,t'seueJqruaueruseld IIef,Ieturup 1e ur rncco,{lqeqord sprdrtocdlg 'sSurpunorrnssll qlIM IIef, aq] Jo suor]f,eJaturur salor luelrodurr aneq rtaqt araqan'(gI-0I arn8rg aas) aJeJJnsIIac aql 1e pasodxa are sdnor8 re8ns aql 'aueJqruau eruseld eql ot paJanrlapare ,(aql sV '(ZI Jal -deql ur passnJsrp) aqlJo rorrelxe aqt ot d1ecr3o1odol parn]f,EJ sr ]uap^rnba IIaJ -nueru are .{aqt qJIqM ur luaurlreduoJ aql 'snqJ 'snleJedder31ogaql Jo uerunl aq] ur salnJaloruprdu aq] ot sdnor8 reSns;o uoqrppp aql ruo4 sllnsal ra,rtepqaqt ur sprdrloc,r(139o uoqnqulsrp crrlaurudse eqJ'sger prdq olur uorllued flequara -;ard ,ri.eru daql pue 'sureql uoqrecorpr(q ]qBIEJlspue 3uo1Jreq] uaamlaq seJJoJ slee1,1rapuea q8norq] -{pred pue sre8nsrraq} ueemteq spuoq uaSorpdq q8norqr ,(pred 'alercosse-JIas ol pual salncalou Surn8rrlur asaq;'u1a.{ruo8urqds a1l }sn[ 'aurso8urqdsuorJ eperu are r(aql sllal 'uoqnqrJlsrp auerqueru Jraq] ul Ier.urueuJ ,{rtaruu,r(seaureJ}xalsotu aq} aneq 'ra.{epqprdl aql;o radelouoru crlosoldcuou aq] ur ,{lanrsnlcxapuno; 'spgdgloJrtltpellef, selncaloru prdrl Surureluoc-re8ng

sauerqurow euseldllv Joa)eJrnsaql uo punoj arv sptdtlo)/(lg ( V N V N )p ! ) e ) r l P r s( f )

a P r s o r l 6 u er r69 ( 8 )

TE

tf >r\ ^T

HO

a p r s o r q a r a r o p e ; e(6y )

T

T

n z

--l

tZ

T

al

I

o:r

tl lll

HN NH I I

AOO

f

-

-l

UL 'pa6reqrunate ste6nsaa;q1 asoqllaururpsot>e;e6;r(1e>s-N = tVNleg ' e s o t n ; 6= ) l g : a s o l ) p l e 6 = le9 u '^ '(g-61arn613eas1ur;a{luo6urqds n roJase)aLllst se 'aurso6urqds uo paseqarespldr;o>i(16 llelsoulle d s l l a ll e u r u eu r ' s p r d l l o q d s o q lsou ere se';orarI;6uolJ pa^uapate sprdr;o>{;6 slueldpue eual)equt seajaqM llelsor,ule '(J) ur uMor]ssrarnl)nrlsasoqM,(VNVN ro 'pr)p)tutulplnaul^la)e-N pallel oslp) sanprsat pt)e )tletspabreqtI;aar1e6au arol.rJ ro auo sutpluo)s{em;eaprsor;6ue6 V (g) pe6req)unsrdnol6 peaq s]l sruroJleq] rebnsaql asnetaqp1d11oil16 (V) Iulnau e palle)st aptsorqarr)ot)pleD 'saln)alotuprdrlo>I;9g1-g 1 atn6r1

zfD-Hf-

Hf Hf Hf

tl

O:] Hf ttl HN Hf .Hl-Hf-

Hf I

oHo

'HlI

arnl)nrtsaueJquaw:0t raideq)

829

629

eql ra^Elouotu closol-{3 eq} olul suasul leq] uleqs plle &lEJ E Jo luauqsunE Jo 'auBrquau aq] lualB^os aqJ Jo epls Jaqlle uo Jelel ol pesodxe eJe suoISeJ rlrqdorp^q rraqJ 'relem luo{ [e^ae peJa]senbes are deq] areq^^ tedelq aql pue Jo rorrelur eql uI seFcalour prdl eq] Jo slle] crqoqdorpdq eql qlyv\ lJeralul 'suo€ar epqdorpdq eueJqueru aqt q8norqt ssed suor8ar crqoqdorpdq rtaq; 'sroq pue crqoqdorpr(q Suureq 'cqtqdtqdure are sula1o.rdarruJqruerusuuJl eseql '1 -q8rau epISJaqtle uo sseur arn8rg) pue'Z saldurexa'6I-0I prdu rraql e{l'I'(t 'auerqtuaru eql qllm ale rraqlJo ped qryn tafe1rq prdl aql q8norql pualxa rtue6l -IJossBuec suralord eupJqlueru qllqm ut sr(ea,rluaraJJlp eq] smoqs 51-91 arn8lg

s^e^ snolren auelqulaw e8 ue) sulalord utre^elt8prdrlaql qllMpale!)ossv 'suoqcury qclq/v\'ra,te1q prd;1aqt q]IM elelJosse rtaqt Jlaql slcageJ esJe^Ip d;el suralord aueJquel/{ 'sserudq uralord o699 ,{em aql uI pup aJn}cnJlsur dlaprrrn, arB ]Bq] sauerqruaru 1eJ uI elnJaloru utalord qcee JoJ salnoalotu prdu 99 lnoqu -saueJqueu IIaJ uI salnf,eloru uralord IIEqI salnoeloru prdl arour duuru srterr,rp aJe eJeql 'salncalour utalord qlpt pareduroJ lleurs eJE salnoeloru pldu asnecag 'SsptuslrJoJIBq tnoqe rogSullunoJJe ulalord q1r,rn'uaamlequI eJeqmaluosSIaueJq pue PIJpuot{J -ruaru ErusEIdpctddr V'utalord sr o692.{laleurxordde '(s1se1doro1qc -olrruJo seugrquau IpuJalul aql se qJns) uo4cnpord 4JVUI pe^lolul seuelqlueul 'suoxe aql q 'lseJluoc .,(g 'utalord st sseur aueJqueu eI+ Jo o/ngzueql ssal flac alrau roJ uouplnsul [EcIJ]Jelase r(lureru sa les qJIqM'eueJqrualu urla[ur aql u1 'alqerrenr{1q8lq sadIl pue s}unorue aqf ,tlSurproccy uI sutalord;o e aJp eugrquaru 'saqradord sll euErqlualu IIaf, 1o adif qcea a,r€ aro; puollJury sllslJalceJEqJ -aJaq] pue s,eueJqruau aqlJo lsotu uro;rad sureloJd arr?Jquau eql s4se1 cgrcads 'sauerqruaur pcrSolorq Jo aJn]onJts JISeq eqt sapnord ra,{epq prdl aqr qBnoqlry

SNI]IOUd]NVUBIAJ]W 'sa1nca1our ru7n77noJ1u! 3u17au31s 77owsalotauaBol sptd1oqdsotld cttlcads saalaqm 'sutalotd Sut1ouBtscr1osoltc ptlsoul ulvryraranaa1csasacll1oqdsoqd rc! sailsSur4cop tutot o1 sptd1\asaqlto sdnoB pqaq aql aru\fuotldsorldsasauryptdtl rDlnilaJouxa ol asuodsatur :Sut1ouBrs c$cads ,s1au?rc 77acu1 uad luauodutt ua [o\d 'sprdqoqdsorld nrbpq prdq auotquraw nuso1d at4llo nfaay u1osolr(caLUu! Lplqm to '17ac crlottacna a1$urso to sauuqwau snolJan swlc rounu a ata spqoqdsotld7o11sou1 aql w s0 lpm sD ,sadr(truatatllp lo q\aclo sauoJquaw alu u! punot atn sptdt\ lo satnl -xttu ryatalfiq 'auzrqlaaut 77aco to sacatoml at41to suollcunl ruatallrp aLPSuUra[aJ ,ryata!!1pata uatnlouow ratno puD Jauu! arpto suotltsodutocp1d17 a14J'sassolrroullu 'Iora$apqc 'sprdt1ot6dsot4d-sptdt1auorquau lo spatpuntl pua-sprdt1octt1? pua nfou aatqr arD anqJ 'satcadsp1d17ruanllrp 000t-009 ula]uor sIIaJ lo sassa1c siuautitadwoc paf)as utot q4qm'uatapq olur [lsnoaualuodsalqwassofaql nrum ut pacn\duaq16'ct\tt|dtlldwaar0 salncapu prdq auotqtuaw aw Latu1ouow umo 4aqi u!r4r!m[1p1dot asnlllp ot alqo salnralou prdq Tanpmtpu!LlUm'p!n[ q n,tapq pldll slI4J'paryaqua aro sunlord auotquaw lpnlm w salnralorltprd11!onrb\ alqnop snonuuuoc o lo lsrsuocsauarqwaw \ac8o\otg

[reuun5 'eurlsalur aq] olul JaIBlvrpue +eNJo xng;a a8rel e sasneJuJnl uI qclqv\'(9I Jalduq3 uI pessnJslp) pa8uolord u ol speel dInJVcrtcrb rglnllaceJlur Jo uollplluaf,uoc aq] uI eseaJcul'aleJJns 'sllao rtaql uo IWg Iellaqltda Puqsalut Sutpnlcut ilac e olut ,,fulua sU 'EJeloqf, a^eq ]eql slleJ asoql ,{po sralua pue ol spulq ulxol eleloqf, Jo BaqrrBlp e se slce Superllqap eql sesnef,lEq] uFot lErJatcuqaql toJ JoldeJal ef,EJJns-ilac '(gI-0I 'aldruexa eas) IWJ eptsollSue8 eqJ'suuo] IPIJatf,equleuac arn8tg JoJ 'uollcunJ ro3 slurod .,fu1uaapr,,r.ordsprdqocf.lS eluos IeruJou Jleql Ja^alel{ \ elrJelsrtlluanbasuos arE puB selsel aql ut.{lpurou euora} -solsal Uodsuel} ]ouuEc selElu aq] q8noqlp 'sal]Ileuuouqe snol^qo ou l!\oqs

SNI]lOUd]NVU8Y\J]W

'euprquraruB ssoJf,EsalnJelou JrlrqdoJp,{q oJ'aueJqruaru eurseld [erus JaJSuEJI aql Jo aprs atrsoddo eql uo spu8rs JelnllaceJlur ]ueJeJJrpaleJaue8 pue aceds JelnlleJeJtxa eq] ut salnJelou pu8rs purq ]eql suraloJd auerqruarusueJt ere 'aydruexaroJ (sJoldaJaleJEJJns-lle3'U ssoJJpselnJelou Jo Je,{plrqaq} UodsueJl 'urelold aql Jo saprsqloq uo uor]ounJuec sureloJd eupJqruausueJ] .{luo Jo uoq -cunJ eql slJauar Jefelrq prdl aqr qtl.\/\palerf,osspsr uraloJd auerquaru e ^\oH

su!alord6u;1eu6;5 auos Jo uotlezrle)olaueJqulewaql lotluo) sJorl)uvptdtl 'sugalord arruJqluaru prtalq pefiec ale sure]oJd asaq;'st(e,vreseq] ur pesealaJ eq louuEr redPlrq prdU aqf Jo arot Jrqoqdorp,{.qaql otq Uasur }eqt suor8aJ ap4daddlod rrqoqdorp^q ro sdnor8 pldll rtq radepq eql ur plaq surelord.{upru 'z ta]oeql ut passn)stp aJe I pue suratoJd eueJqtuerusuer; 'sula1ord erruJqrueu praqd;rad se o] peJJaJaJ ralelrqprdrlaql qllM palpt)osse aulo)aq eJe suraloJd eseql llJelur Ja^elrq prdU aqf a^eal lnq suorlJuralur ureloJd-urel sutaloldauelqutaulMorlJo sltelap aql'gs-zt oln6u ut palerlsnllt st paLuroJ -ord qrlm eJaJJalurqclqm 'gd aruarlxa Jo Jo r{}3ueJls cruor ,tlrol Jo q8rq dra,t sr(9)ur uMoqsroq)ue ldg aqt q)tqMul suorlnlos o1arnsodxa sp qJns 'seJnpecoJduorlJEJlxaaFuaSdla^ue1ar[q auerq Jo ^eMaq] allqM'02-0t arn6r3ur palprlsnlll -tueru aqt ruoq pesealar aq ueJ addl sqt go suretord aql ^u€W .(g pue 1 sald Jo srpauljo}st (g)ut alnl)nlls aLllq)tqM -ruexa '6I-0I arnBrg) suraloJd aueJqrrrau Jaqlo qllm suortf,EJelur lualeloJuou ur {eanaq1'suraloldauerquauu.raqlo dq auerqurau aqtJo eJeJJeqlre ol punoq peelsur are daqt l1e 1era,{epq pldll eql LllrMsuorl)elalutlualelofuou{q {1uo auerqLuauaql ol poq)eilealp sutaloJd Jo JorJelur crqoqdorpdq aq] olur pualxa tou op suraloJd aueJqrueu atuos 'auerqureru aql uro{ ruaql Surseayar { u e u ^ ; ; e u r (18 ' 1 )r o q ) u e l d 9 e p a l l e ) dqaraql ,sJoqcueJreq}ruo4 ae4 suratoJd -lar{e;ououttr;oso1^>uou eql ut asaq] slnc aut[zua sIqI '] esedqoqdsoqd cgrcads-1o]rsouqdpueqdsoqdpalpc lo}tsoul -llplteqdsoqdol'ralurl apueqr>eso6r;o aurrtzuaue Jo esn aq] ^q peqsn8urlsrp,r(lpeeJaq uef, JoqJue IdC E rtq aueJqtuau ue et^ (9)'to ia^elouoLu >r;osoy(t eruseld eqt ol punoq suretord '191raldeq3 ur pessnosrp)auerquau eursuld aql aql u!-(02-0 L ern6rgaas)dnor6 1,(uatd o] uralo.rd aqt re^qep ,{1en1uanasalJrsal uodsup{ '(71 raldeq3 ur pessncsrp) e ro ureq)ppe Igle; p raqlla-ulpq) pldll Joqlue sq1 ,(q ,t1a1os auerqueur aq] Jo ef,pJJnscrlosoldJuou aql ol punoq urel paq)etle,{;lua;enor e Iq {1e1os ielelrq -ord aqt 3uneal 'pappe sr roqrrre (IdC) 'xtlaq aql ol paq)eue E pue JJo are sraqto (s) oq] Iorlsoup$lruqdsoqdptsoc{8 '(Ug) pe^Eelcsr ulalord aql ;o luaruBaseueJqruarusuenaql ,Ug aql ur Ilrls apl 1oare; rqoqdorp^q aLlrqbnorqtra{e;rq prdrlaql Jota{e;ouor-u :r;oso1,{> aql unlnf,rleJ cnuseldopua eql ur sureloJd auBJqruarusuerl ssed-a13ursse apBru 'Jele^\oq 'g olur suortrlred 'dnor8 ]eql xllaqn >rlrqdlqdr-ue p aJe aldurexa ur suralord pldl aql Jo tueluqJelle ]ualploJ ue Iq arepnsrrlosoy{reql o} paloLl)ue aql ,{q eueJqruau eql 01 paJoqJup Lpuanbesqns aJe pue 1oso1,(c aql ur suralord aJeosaqlJo atuos (t) 'auelqulaul alqnlos sp aperu are 6I-0I aln8rC ur g aldurexa ur suralord pa1u11-prdqaql aqt Jo aprseuo {1uo1epasodxaate '(g '6I-0I aldurexa arn8rg) aueJqureru surelordauplqutotltlaqlo .(t) ra,(e;ouoru eruseld eqi Jo ral(elouotu prdrl ralno aqt ur 1o1rsou11r(ppeqdsoqd prdrltr;osoilt aql ut pallasututeqf o1 (apr.reqc -Jeso8qo cgrcads e ern) a8e4ull lueleloc e ,.(q,{po ra.{epq prdt aql ot par{Jp]}e pDe i{11e;paq)el}e,{1tua;enor e aneq sura1o.rd,,ssedr11nu,, pue,,ssed-a16urs,,Suraq 'ace;rns IIaJ lpuJalxa aq] lp pasodxa ,{1ar4ua are suralord auerquraru asaql oruos '(lalleq e; taaqs Jo fl fl Jaqlo ]a,1'(g aldruexa '6I-0I arn8rg) surpqc prdu paqceUe dpualeloJ aJoru Jo dn-pallole se (€)ro /sa)tlaqn a1dr1;nur euo,{q ro (7 aldruexe'6I-0I arn8rg) uralord eqtJo aJBJJnsaqt uo pasodxa xqaq s e( Z )' x r l a qn a 1 6 u res( [ ) s el a l p l r q n crpqdrqdrue ue dq raqlla tafepq prdil aqlgo re.(elouorucqosoptc eq] q]lm pe]e aql ssor)epualxaol rq6noqraresuralord -rJosse pue aJE auerqulausuetltsoyy'ra{e;rqprdr; losoldc aq] ur [leJrtua paleJol are suralord eupJqueu Jaq]O '(1 alduexa ,6I-0I arn8rg eas) sural aql ql!/vlale!)ossesula1o.rd auplquaul -ord auerquerusueJl eseqt er.uos fircrqoqdorp,{q aqt saseeJourradelq prdq t{)il,l/vlut sitervr snoueTl61-g 1 arn611 Jo Jo

@

o I

raAellq p!o!l I

*ffim-d

ernl)nlls euetquew :0[ ra]deqf

0€9

631

M E M B R A NP ER O T E I N S

a membrane transport protein must provide a path for the molecules to cross the hydrophobic permeability barrier of the lipid bilayer; the molecular architecture of multipass membrane proteins is ideally suited for this task, as we discussin Chapter 11. Proteins that function on only one side of the lipid bilayer, by contrast, are often associatedexclusivelywith either the lipid monolayer or a protein domain on that side. Some intracellular signaling proteins, for example, that are involved in converting extracellular signals into intracellular ones are bound to the cytosolic half of the plasma membrane by one or more covalently attached lipid groups, which can be fatty acid chains or prenyl groups (Figure 10-20). In some cases,myristic acid, a saturated l4-carbon fatty acid, is added to the N-terminal amino group of the protein during its synthesison the ribosome. All members of tl:'e Srcfamily of cytoplasmic protein ty'rosinekinases (discussedin Chapter l5) are mlristoylated in this way. Membrane attachment through a single lipid anchor is not very strong, however, and a second lipid group is often added to anchor proteins more firmly to a membrane. For most Src kinases, the second lipid modification is the attachment of palmitic acid, a saturated 16-carbon fatty acid, to a cysteine side chain of the protein. This modification occurs in response to an extracellular signal and helps recruit the kinases to the plasma membrane. lVhen the signaling pathway is turned ofl the palmitic acid is removed, allowing the kinase to return to the c)'tosol. Other intracellular signaling proteins, such as the Rasfamily small GTPases(discussedin Chapter 15),use a combination of prenyl group and palmitic acid attachment to recruit the proteins to the plasma membrane.

ChainCrosses In MostTransmembrane Proteins the Polypeptide the LipidBilayerin an cr-Helical Conformation A transmembrane protein always has a unique orientation in the membrane. This reflects both the asymmetric manner in which it is inserted into the lipid (A)

(B)

HO lll c -c -o-cHr I C F I, I t h i o e t h e rl i n k a g e . " ---- '--between cysteine a n d p r e n y lg r o u p \ n,

a m i d el i n k a g e

ripidt bilaver

=

I ( F ) f a r n e s y al n c h o r

( E ) p a l m i t o y la n c h o r

( D ) m y r i s t o yal n c h o r

o

o c-o"'

A/W\AA/\

Figure 10-20 Membraneprotein attachment by a fatty acid chain or a prenyl group. The covalentattachmentof either in the cytosol.(A)A fatty acidchain proteinto a membraneafterits synthesis type of lipidcan help localizea water-soluble (palmiticacid)is attachedvia (B) acid chain glycine. A fatty (myristicacid)is attachedvia an amidelinkageto an N-terminal group)is attachedvia a (C)A prenylgroup(eitherfarnesylor a longergeranylgeranyl a thioesterlinkageto a cysteine. Afterprenylation, C-terminus. from the protein's thioetherlinkageto a cysteineresiduethat is initiallylocatedfour residues is methylatedbeforeinsertionof the anchorinto the terminalthreeaminoacidsarecleavedofl and the new C-terminus of the lipidanchorsareshownbelow:(D)a myristoylanchor(a 14-carbon the membrane(not shown).Thestructures fatty acidchain),and (F)a farnesylanchor saturated saturated fatty acidchain),(E)a palmitoylanchor(a 16-carbon (a 15-carbonunsaturated chain). hydrocarbon

632

Chapter10:MembraneStructure Figure10-21A segment polypeptide of a transmembrane chaincrossing the lipidbilayerasan crhelix. Onlythecr-carbon backbone of the polypeptide chainisshown, withthehydrophobic aminoacidsin greenand yellow.The polypeptide segment photosynthetic shownispartof the bacterial reaction centerillustrated in Figure 10-34, thestructure of whichwas determined (Based by x-raydiffraction. on datafromJ.Deisenhofer et al.,Nature 318:618-624, 1985, andH.Michelet al.,EMBO J.5:1149-11 58,1986. Allwith permission fromMacmillan Publishers Ltd.)

bilayer in the ER during its biosynthesis (discussedin chapter 12) andthe different functions of its cytosolic and nonc],tosolic domains. These domains are separated by the membrane-spanning segments of the polypeptide chain, which contact the hydrophobic environment of the lipid bilayer and are composed largely of amino acids with nonpolar side chains. Because the peptide bonds themselvesare polar and becausewater is absent,ail peptide bonds in the bilayer are driven to form hydrogen bonds with one another. The hydrogen-bonding between peptide bonds is maximized if the polypeptide chain forms a regular a helix as it crosses the bilayer, and this is how most membrane-spanning segments of polypeptide chains traversethe bilayer (Figure f 0-Zl). In single-pass transmembrane proteins, the polypeptide chain crosses only once (seeFigure 10-lg, example l), whereas in multipass transmembrane proteins, the polypeptide chain crossesmultiple times (seeFigure l0-lg, example 2). An alternative way for the peptide bonds in the lipid bilayer to satist/ their hydrogen-bonding requirements is for multiple transmembrane strands of a polypeptide chain to be arranged as a B sheet that is rolled up into a closed barrel (a so-called B barrel; see Figure 10-19, example 3). This form of multipass transmembrane structure is seen in the porin proteins that we discuss later. Rapid progress in the x-ray crystallography of membrane proteins has enabled us to determine the three-dimensional structure of many of them. The structures confirm that it is often possible to predict from the protein's amino acid sequence which parts of the polypeptide chain extend across the lipid bilayer. segments containing about 20-30 amino acids with a high degree of hydrophobicity are long enough to span a lipid bilayer as an o, helix, and they can often be identified in hydropathy p/ors (Figure lo-zz). From such plots, it is estimated that about 20Toof the kind of an organism's proteins are transmembrane proteins, emphasizing their importance. Hydropathy plots cannot identify the membrane-spanning segments of a p barrel, as t0 amino acids or fewer are sufficient to traverse a lipid bilayer as an extended B strand and only every other amino acid side chain is hydrophobic. The strong drive to maximize hydrogen-bonding in the absence of water means that a polypeptide chain that enters the bilayer is likely to pass entirely through it before changing direction, since chain bending requires a loss of regular hydrogen-bonding interactions. But multipass membrane proteins can also contain regions that fold into the membrane from either side, squeezing into spacesbetween transmembrane cr helices without contacting the hydrophobic core of the lipid bilayer. Becausesuch regions of the polypeptide chain interact only with other polypeptide regions, they do not need to maximize hydrogenbonding; they can therefore have a variety of secondary structures, including helices that extend only part way across the lipid bilayer (Figure 10-23). Such regions are important for the function of some membrane proteins, including the K+ and water channels; the regions contribute to the walls of the pores traversing the membrane and confer substrate specificity on the channels, as we discuss in chapter 11. These regions cannot be identified in hydropathy plots and are only revealed by x-ray crystallography,electron diffraction (a technique similar to x-ray diffraction but performed on two-dimensional arrays of proteins), or NMR studies of the protein's three-dimensional structure.

Transmembrane crHelices OftenInteractwith OneAnother The transmembrane s helices of many single-passmembrane proteins do not contribute to the folding of the protein domains on either side of the membrane.

EXTRACELLULAR (200) sPACE GLY \ PHEI-

sER

o a

ILE

GLY ,, TYR GLY:

o

.cYs GLY

c

c

CYTOSOL

/^

633

MEMBRANE PROTEINS

(A) G L Y C O P H O R I N

(B) BACTERTORHODOPSTN

HzN

HzN

@

@

(c)

E. coli

COOH 12345tr7

S. cerevisiae

H. sapiens

c q o

!

o

E f c

p r o t e i n sw i t h a t l e a s to n e p r e d i c t e dt r a n s m e m b r a n o e .h e l i x

h y d r o p h o b i c i to yf the most h y d r o p h o b i c2 0 - a m i n o - a c i d s t r e t c hi n e a c hp r o t e i n

As a consequence,it is often possible to engineer cells to produce the cltosolic or extracellular domains of these proteins as water-soluble protein. This approach has been invaluable to study the structure and function of these domains, especially of those in transmembrane receptor proteins (discussedin Chapter l5). A transmembrane crhelix, even in a single-passmembrane protein, however, often does more than just anchor the protein to the lipid bilayer. Many single-passmembrane proteins form homodimers, which are held together by strong and highly specific interactions between the two transmembrane cr helices; the sequence of the hydrophobic amino acids of these helices contains the information that directs the protein-protein interaction. Similarly, the transmembrane s helices in multipass membrane proteins occupy specific positions in the folded protein structure that are determined by interactions between the neighboring helices.These interactions are crucial for the structure and function of the many channels and transporters that move molecules acrosslipid bilayers. In many cases,one can use proteasesto cut the loops of the polypeptide chain that link the transmembrane u helices on either side of the bilayer and the helices stay together and function normally. In some Figure10-23Two a helicesin the aquaporinwater channel,eachof the which spansonly halfwaythrough the lipid bilayer.In the membrane, suchthat the proteinformsa tetramerof four suchtwo-helixsegments, coloredsurfaceshownhereis buriedat an interfaceformedby protein-protein The mechanismby whichthe channelallows interactions. in more acrossthe lipid bilayeris discussed the passage of watermolecules d e t a i il n C h a o t e lr1

Figure|0-22 Usinghydropathyplotsto localizepotentiala-helicalmembranespanningsegmentsin a polypeptide chain.Thefreeenergyneededto transfer segmentsof a polypeptide successive chainfrom a nonpolarsolventto wateris from the aminoacid calculated compositionof eachsegmentusingdata obtainedfrom modelcompounds.This is madefor segmentsof a calculation fixedsize(usuallyaround10-20amino acids),beginningwith eachsuccessive aminoacidin the chain.The"hydropathy index"of the segmentis plottedon the Y axisasa functionof its locationin the that free chain.A positivevalueindicates energyis requiredfor transferto water (i.e.,the segmentis hydrophobic), and the valueassignedis an indexof the amountof energyneeded.Peaksin the hydropathyindexappearat the positions of hydrophobicsegmentsin the amino (A and B)Two examplesof acidsequence. laterin this membraneDroteinsdiscussed (A)hasa chapterareshown.Glycophorin crhelixand singlemembrane-spanning peakin the one corresponding (B) hydropathyplot.Bacteriorhodopsin o helices hassevenmembrane-spanning peaksin the and sevencorresponding hydropathyplot. (C)The proportionof predictedmembraneproteinsencoded by the genomesof E.coli,S.cerevisiae, and human.Theareashadedin green the fractionof proteinsthat indicates containat leastone predicted helix.Thedatafor transmembrane representthe E.coli and S.cerevisiae wholegenome;the datafor human represent only part of the genome;in eachcase,the areaunderthe curveis proportionalto the numberof genes analyzed.(A,adaptedfrom D. Eisenberg, Annu.Rev.Biochem.53:595-624,1984. from AnnualReviews; With permission C,adaptedfrom D. Boydet al.,ProteinSci. from 7:201-205, 1998.With permission The ProteinSociety.)

634

Chapter10:MembraneStructure

(A)

f r a g m e n t 1 f r a g m e n t2

Figure10-24 Convertinga single-chain multipassproteininto a two-chain multipassprotein. (A) Proteolytic cleavageof one loop to createtwo fragmentsthat staytogether and functionnormally.(B)Expression of the sametwo fragmentsfrom separategenes givesriseto a similarproteinthat functionsnormallv.

s i n g l e - c h a im n ultipass m e m b r a n ep r o t e i n

(B) g e n ef o r p r o t e i n fragment1

g e n ef o r p r o t e i n f r a g m e n t2

cooH t w o - c h a i nm u l t i p a s s m e m b r a n ep r o t e i n t h a t f u n c t i o n sn o r m a l l y

cases,one can even expressengineeredgenesencoding separatepieces ofa multipass protein in living cells, and one finds that the separate pieces assemble properly to form a functional transmembrane protein (Figure lV24), emphasizing the exquisite specificity with which transmembrane cr helices can interact. In multipass membrane proteins, neighboring transmembrane helices in the folded structure of the protein shield many of the transmembrane helices from the membrane lipids. \.\hy, then, are these shielded helices nevertheless composed primarily of hydrophobic amino acids?The answer lies in the way in which multipass proteins are integrated into the membrane during their biosynthesis. As we discuss in chapter 12, transmembrane s helices are inserted into the lipid bilayer sequentially by a protein translocator. After leaving the translocator, each helix is transiently surrounded by lipids in the bilayer, which requires that the helix be hydrophobic. It is only as the protein folds up into its final structure that contacts are made between adjacent helices and protein-protein contacts replace some of the protein-lipid contacts (Figure f0-25).

Somep BarrelsFormLargeTransmembrane Channels Multipass transmembrane proteins that have their transmembrane segments arranged as a B barrel rather than as an o helix are comparatively rigid and tend to crystallize readily. Thus, some of them were among the first multipass

n e w l ys y n t h e s i z e d m e m b r a n ep r o t e i n

folded m e m b r a n ep r o t e i n

Figure10-25 Stepsin the folding of a multipasstransmembraneprotein, Whenthe newlysynthesized transmembrane cxhelicesare released into the lipid bilayer, they areinitially surroundedby lipidmolecules. As the proteinfolds,contactsbetweenthe helicesdisplacesomeof the lipid molecules surrounding the helices.

635

MEMBRANE PROTEINS

membrane protein structures to be determined by x-ray crystallography. The number of B strands in a B barrel varies widely, from as few as B strands to as many as 22 (Figure f 0-26). p barrel proteins are abundant in the outer membrane of mitochondria, chloroplasts, and many bacteria. Some are pore-forming proteins, which create water-filled channels that allow selected small hydrophilic molecules to cross the lipid bilayer of the bacterial outer membrane. The porins are well-studied examples (example 3 in Figure 10-26). The porin barrel is formed from a 16strand, antiparallel B sheet, which is sufficiently large to roll up into a cylindrical structure. Polar amino acid side chains line the aqueous channel on the inside, while nonpolar side chains project from the outside of the barrel to interact with the hydrophobic core of the lipid bilayer. Loops of the pollpeptide chain often protrude into the lumen of the channel, narrowing it so that only certain solutes can pass. Some porins are therefore highly selective: maltoporin, for example, preferentially allows maltose and maltose oligomers to cross the outer membrane of E. coli. The FepA protein is a more complex example of a B barrel transport protein (example 4 in Figure f 0-26). It transports iron ions across the bacterial outer membrane. It is constructed from 22 p strands, and a large globular domain completely fills the inside of the barrel. Iron ions bind to this domain, which is thought to undergo a large conformational change to transfer the iron acrossthe membrane. Not all B barrel proteins are transport proteins. Some form smaller barrels that are completely filled by amino acid side chains that project into the center of the barrel. These proteins function as receptors or enzymes (examples 1 and 2 in Figure 10-26), and the barrel servesas a rigid anchor, which holds the protein in the membrane and orients the cytosolic loops that form binding sites for specific intracellular molecules. Although B barrel proteins have various functions, they are largely restricted to bacterial, mitochondrial, and chloroplast outer membranes. Most multipass transmembrane proteins in eucaryotic cells and in the bacterial plasma membrane are constructed from transmembrane cr helices. The helices can slide against each other, allowing conformational changes in the protein that can open and shut ion channels, transport solutes,or transduce extracellular signals into intracellular ones. In B barrel proteins, by contrast, hydrogen bonds bind each B strand rigidly to its neighbors, making conformational changes within the wall of the barrel unlikelv.

ManyMembraneProteinsAreGlycosylated Most transmembrane proteins in animal cells are glycosylated.As in glycolipids, the sugar residues are added in the lumen of the ER and the Golgi apparatus

o

8-stranded OmpA

\z)

I 2-stranded OMPLA

tt

15-stranded porln

Figure 10-26 p barrelsformed from different numbers of p strands. (1)TheE collOmpAproteinservesasa receptorfor a bacterialvirus.(2)The E coll OMPLAprotein is an enzyme(a lipid molecules. lipase)that hydrolyzes Theaminoacidsthat catalyzethe enzymaticreaction(shownin red,l protrudefrom the outsidesurfaceof the barrel.(3)A porinfrom the bacterium Rhodobactercapsulatusforms a waterfilledporeacrossthe outer membrane. Thediameterof the channelis restricted by loops (shownin blue)that protrude into the channel.(4)The E coli FepA proteintransportsiron ions.The insideof the barrelis completelyfilledby a globularproteindomain(shownin b/ue.) that containsan iron-bindingsite(not shown).Thisdomainisthoughtto to transportthe changeits conformation bound iron,but the moleculardetailsof the chanqesarenot known.

636

Chapter10:MembraneStructure Figure10-27A single-pass protein.Notethatthe transmembrane polypeptide chaintraverses thelipidbilayer asa right-handed s helixand thattheoligosaccharide chains anddisulfide bondsareallon the noncytosolic surface of themembrane. groupsin the Thesulfhydryl cytosolic domainof theproteindo notnormally formdisulfide bonds because thereducing environment in thecytosol maintains thesegroups (-SH)form. in theirreduced

(discussedin Chapters 12 and 13). For this reason, the oligosaccharide chains are always present on the noncytosolic side of the membrane. Another important difference between proteins (or parts of proteins) on the two sides of the membrane results from the reducing environment of the cytosol. This environment decreases the likelihood that intrachain or interchain disulfide (S-S) bonds will form between cysteines on the cltosolic side of membranes. These bonds form on the noncytosolic side, where they can help stabilize either the folded structure of the polypeptide chain or its association with other polypeptide chains (Figure 10-27). Becausemost plasma membrane proteins are glycosylated, carbohydrates extensivelycoat the surface of all eucaryotic cells.These carbohydrates occur as oligosaccharide chains covalently bound to membrane proteins (glycoproteins) and lipids (glycolipids).They also occur as the polysaccharide chains of integral membrane proteoglycan molecules. Proteoglycans, which consist of long polysaccharide chains linked covalently to a protein core, are found mainly outside the cell, as part of the extracellular matrix (discussedin Chapter l9). But, for some proteoglycans,the protein core either extends acrossthe lipid bilayer or is attached to the bilayer by a glycosylphosphatidylinositol (GpI) anchor. The terms cell coat or glycocalyx are sometimes used to describe the carbohydrate-rich zone on the cell surface.This carbohydrate layer can be visualized by various stains, such as ruthenium red (Figure f 0-28A), as well as by its affinity for carbohydrate-binding proteins called lectins, which can be labeled with a fluorescent dye or some other visible marker. Although most of the sugar groups are attached to intrinsic plasma membrane molecules, the carbohydrate layer also contains both glycoproteins and proteoglycans that have been secretedinto the extracellular space and then adsorbed onto the cell surface (Figure l0-288). Many of these adsorbed macromolecules are components of the extracellular matrix, so that the boundary between the plasma membrane and the extracellular matrix is often not sharply defined. one of the many functions of the carbohydrate layer is to protect cells against mechanical and chemical damage; it also keeps various other cells at a distance, preventing unwanted protein-protein interactions. The oligosaccharide side chains of glycoproteins and glycolipids are enormously diverse in their arrangement of sugars.Although they usually contain fewer than 15 sugars, they are often branched, and the sugars can be bonded together by various covalent linkages-unlike the amino acids in a pollpeptide chain, which are all linked by identical peptide bonds. Even three sugars can be put together to form hundreds of different trisaccharides.Both the diversity and the exposed position of the oligosaccharides on the cell surface make them especially well suited to function in specific cell-recognition processes.As we discuss in chapter 19, plasma membrane-bound lectins that recognize specific oligosaccharideson cell-surface glycolipids and glycoproteins mediate a variety of transient cell-cell adhesion processes, including those occurring in sperm-egg interactions, blood clotting, lymphoclte recirculation, and inflammatory responses.

MembraneProteinscan BeSolubilized and Purifiedin Detergents In general, only agents that disrupt hydrophobic associations and destroy the lipid bilayer can solubilize transmembrane proteins (and some other tightly bound membrane proteins). The most useful of these for the membrane biochemist are detergents, which are small amphiphilic molecules of variable

interchain d i s ul fi d e oono

o li g o s a c c h a r i d e s transmembrane a helix

CYTOSOL (reducing environment)

)tr

s ul f h v dr v l group

NH,

637

MEMBRANE PROTEINS (A)

Figure10-28Thecarbohydratelayeron the cell surface.Thiselectronmicrograph of the surfaceof a lymphocytestained the thick with rutheniumred emphasizes the layersurrounding carbohydrate-rich cell.(B)The carbohydratelayeris made sidechainsof up of the oligosaccharide glycolipids and integralmembrane glycoproteinsand the polysaccharide chainson integralmembrane proteoglycans. In addition,adsorbed glycoproteins, and adsorbed proteoglycans(not shown)contributeto the carbohydratelayerin many cells. is on Notethat all ofthe carbohydrate the noncytosolicsurfaceof the membrane.(A,courtesyof AudreYM. Cook.) Glauertand G.M.W. 200 nm

(B) transmemorane proteoglycan

carbohydrate rayer

lipid bilayer

CYTOSOL

structure. Detergents are much more soluble in water than lipids. Their polar (hydrophilic) ends can be either charged (ionic), as in sodium dodecyl sufute (SDS),or uncharged (nonionic), asin octylglucosideandTriton (Figure f 0-29,{). At low concentration, detergents are monomeric in solution, but when their concentration is increased above a threshold, called the critical micelle concentration or CMC, they aggregateto form micelles (Figure 10-29B-C). Detergent molecules rapidly diffuse in and out of micelles, keeping the concentration of monomer in the solution constant, no matter how many micelles are present. Both the CMC and the averagenumber of detergent molecules in a micelle are characteristic properties of each detergent, but they also depend on the temperature, pH, and salt concentration. Detergent solutions are therefore complex systems and are difficult to study. \Alhen mixed with membranes, the hydrophobic ends of detergents bind to the hydrophobic regions of the membrane proteins, where they displace lipid molecules with a collar of detergent molecules. Since the other end of the detergent molecule is polar, this binding tends to bring the membrane proteins into solution as detergent-protein complexes (Figure f0-30). Usually, some lipid molecules also remain attached to the protein. Strong ionic detergents, such as SDS, can solubilize even the most hydrophobic membrane proteins. This allows the proteins to be analyzed by SDSpolyacryIamide-gel electrophoresis(discussed in Chapter B), a procedure that has revolutionized the study of membrane proteins. Such strong detergents unfold (denature) proteins by binding to their internal "hydrophobic cores,"

638

Chapter10:MembraneStructure

(A)

(B)

oO Nu@

h.",on

O:S:O

ll Ho*.-rf I

I o I

CHr

tCH:

fn' fn' 1n' fn'

I

I

fH, I

CHr

I

Ct -H r I CHr

f*' f"'

I CHr I CH: I CHr I CHr I CHr I

rA

C^ PO

i'c

vc

detergent concentration(total)

(c)

fn'

CH:

hydrophilic h e a dg r o u p hydrophobic tail

CH:

s o d i u md o d e c y ls u l f a t e (SD5)

T r i t o nX - 1 0 0

p-octylgIucoside

Figure10-29Thestructure (A)Threecommonly andfunctionof detergent micelles.

useddetergents aresodium (SDS), dodecyl sulfate ananionic detergent, andTritonX-100andB-octylglucoside, two nonionic detergents. TritonX-100is a mixture of compounds in whichtheregionin brackets isrepeated between 9 and10times. Thehydrophobic portionof eachdetergentisshowninyellow, portionisshownin orange. andthe hydrophilic (B)At lowconcentration, detergent molecules aremonomeric in solution. Astheirconcentration isincreased (CMC), beyond thecritical micelle concentration someof thedetergent molecules formmicelles. Notethattheconcentration of detergent monomer staysconstant aDove (C)Because theCMC. theyhavebothpolarandnonpolar ends,detergent molecules areamphiphilic; andbecause theyare cone-shaped, theyformmicelles (seeFigure10-7).Detergent ratherthanbilayers micelles haveirregular shapes, and,due to packing constraints, thehydrophobic tailsarepartially exposed to water. Thespace-filling modelshowsthestructure of a micelle composed of 20p-octylglucoside predicted molecules, (B,adapted by molecular dynamics calculations from G.Gunnarsson, B.Jonsson andH.Wennerstrom, J.Phys. Chem.84:3114-3121 R.M. Venable and ,1980;C,fromS.Bogusz, R'W.Pastor, J.Phys. Chem. B.104:5462-5470,20OO. Withpermission fromtheAmerican Chemical Societv.) thereby rendering the proteins inactive and unusable for functional studies. Nonetheless,proteins can be readily separated and purified in their SDS-denatured form. In some cases,removal of the detergent allows the purified protein to renature, with recovery of functional activity. Many hydrophobic membrane proteins can be solubilized and then purified in an active form by the use of mild detergents.These detergents cover the hydrophobic regions on membrane-spanning segments that become exposed after lipid removal but do not unfold the protein. If the detergent concentration of a solution of solubilized membrane proteins is reduced (by dilution, for example), membrane proteins do not remain soluble. In the presence of an excessof phospholipid molecules in such a solution, membrane proteins incorporate into small liposomes that form spontaneously. In this way, functionally active membrane protein systems can be reconstituted from purified components, providing a powerful means of analyzing the activities of membrane transporters, ion channels, signaling receptors, and so on (Figure l0-31). Such

MEMBRANE PROTEINS

639

Figure| 0-30 Solubilizingmemblane proteinswith a mild nonionic detergent.Thedetergentdisruptsthe lipidbilayerand bringsthe proteinsinto solutionas protein-lipid-detergent in the The phospholipids complexes. bv the membranearealsosolubilized detergent.

hydrophobic tail hydrophilic head

----: sdetergent monomers detergent micelles

membrane protein in lipid bilayer

6."-,

water-solubleproteinl i p i d - d e t e r g e nct o m p l e x

'"?' ..?o

lipid-detergent micelles

lipid bilayer

f-s

*1{*

$r;"

1";T::*l'*"* ,

+

ft' t!l',*.* i'{a-.,..&

e -,'fi i-r

r's l$

'J

6*

l i p i d - d e t e r g e nm t icelles

PURIFICATION OF Na'-K* pump

REMOVALOF DETERGENT f

,.o

detergent m i c e l l e s+ monomerS

f u n c t i o n a lN a * - K ' p u m p i n c o r p o r a t e di n t o p h o s p h o l i p i dv e s i c l e

Figure10-31The useof mild nonionic detergentsfor solubilizing,purifying, and reconstitutingfunctional membrane protein systems.In this example,functionalNa*-K+PumP arepurifiedand incorporated molecules The Na+-K+ vesicles. into phospholipid pump is an ion pump that is presentin the olasmamembraneof mostanimal cells;it usesthe energyof ATPhydrolysis to pump Na+out of the celland K+in, as in Chapter11. discussed

640

Chapter10:MembraneStructure

functional reconstitution, for example, provided proof for the hypothesis that the transmembrane ATPasesuse H+ gradients in mitochondrial, chloroplast, and bacterial membranes to s1'nthesizeNlP. Detergents have also played a crucial part in the purification and crystallization of membrane proteins. The development of new detergents and new expression systems producing large quantities of membrane proteins from cDNA clones has led to a rapid increase in the number of structures of membrane proteins and protein complexes that are known.

Bacteriorhodopsin ls a Light-Driven ProtonPumpThatTraverses the LipidBilayerasSevena Helices In Chapter 11,we consider how multipass transmembrane proteins mediate the selectivetransport of small hydrophilic molecules across cell membranes. But a detailed understanding of how a membrane transport protein actually works requires precise information about its three-dimensional structure in the bilayer. Bacteriorhodopsinwasthe first membrane transport protein whose structure was determined. It has remained the prototy?e of many multipass membrane proteins with a similar structure, and it merits a brief digressionhere. The "purple membrane" of the archaean Halobacterium salinarum is a specialized patch in the plasma membrane that contains a single speciesof protein molecule, bacteriorhodopsin (Figure l0-32). Each bacteriorhodopsin molecule contains a single light-absorbing group, or chromophore (called retinal), which gives the protein its purple color. Retinal is vitamin A in its aldehyde form and is identical to the chromophore found in rhodopsin of the photoreceptor cells of the vertebrate eye (discussed in chapter 15). Retinal is covalently linked to a lysine side chain of the bacteriorhodopsin protein. \A/hen activated by a single photon of light, the excited chromophore changes its shape and causesa series of small conformational changes in the protein, resulting in the transfer of one H* from the inside to the outside of the cell (Figure f 0-39). In bright light, each bacteriorhodopsin molecule can pump several hundred protons per second. The light-driven proton transfer establishes an H+ gradient across the plasma membrane, which in turn drives the production of AIp by a second protein in the cell's plasma membrane. The energy stored in the H+ gradient also drives other energy-requiring processesin the cell. Thus, bacteriorhodopsin converts solar energy into a proton gradient, which provides energy to the archaeal cell.

u^t2 (A)

\ Patchof / bacteriorhodopsin molecules /

1 0n m

single b a c t e r i o r h o d o pns i molecule

2nm

Figure 10-32 Patchesof purple membrane,which contain bacteriorhodopsin in the archaean Holobacterium solin arum. (A)These archaealive in saltwaterpools,where they areexposedto sunlight.Theyhave evolveda varietyof light-activated proteins,includingbacteriorhodopsin, whichis a light-activated protonpump in the plasmamembrane.(B)The bacteriorhodopsin moleculesin the purplemembranepatchesaretightly packedinto two-dimensional crystalline (C)Detailsof the molecular arrays. surfacevisualized by atomicforce microscopy. With thistechnique individualbacteriorhodopsin molecules can be seen.(D)Outlineof the approximate locationsof three bacteriorhodopsin monomersand their individuala helicesin the imagesshown in (B).(B-D courtesyof Dieter Oesterhelt.)

suraloJdJoJlua^los puorsueurp-aeJq] B selnlrlsuoJ JaIB^^se lsnf'eueJqueu aql uI suleloJd eql JoJtuo^los puorsuaurp-o^q E Supn]11suocse sprdl auerqueu aql Jo {uq] UBJel 'eSreqJ puE 'edpqs 'azrsur reJJrpleql sdnorS ppeq q}u 'sprdll Jo dleuu^ B qJns ureluoJ saupJquaru Jrlo.ftEJna .{qm ureldxa sdlaq suoqceralur uretord-prdrl asaq]Jo &1cg1cadseql 'suounlos lue8relep ur surelord aq] ot {cEq pappe ale spIdII JIJIcedsJr Jo 'uorlJeJlxa ]ue8Jelep Suunp punoq ureuar sprdrl aqt Jo auos;r .,{1pea.r elou azrlfE]sfucpue lsaq IroM qJrqM 'suretordaueJqruau .ri.ueruazqrqels dleq o1 lq8noql ere sprdfl ogrJads q]y!r suoqcera]ul '(gtt-gl arnBIC)ef,eJrnsuratoJd aq] uo saceld Junads ur punoq ere ]Eq] salnJelour prd[ .{ueur spanar ursdopoqrorJe}f,EqJo eJn}JnJls IelsIJJ uorlnlosaJ-q8q aql (ssordf ttlape)v .{1pre3ure}ord }ueroup LuorJuolsslr.urad qllM .666t ' LL6_669:l6a uE Jo seqJuprq luelslp .{1rreuoqn1o,,reomt ot 3uo1aq .{yqeqord snq} pue .{tupl lolg'low'r'le ta a))enl H urorl'B -rrurs acuanbes ou Moqs daql 'repurs d13uopr1saJEsgld3 pue sursdopoqJorJel iSVVVLuo.rJ uorssrrurad qilM .666t -Jeq Jo sernlJnJls aqt q8noqfry '91 raldeq3 ur ssnJsrpe^ se '(sy,Jd.t) stoidacat '092-9szt98zDuaDS,I p ia al)anl H rlor] paldnoc-u1a1otd-g pa11etaroJaraqt arp pue IIer aql eprsu (uralord g) uralord paldepe'V)'uralordaql Jo a)eJlnsaql uo Surpurq-41g e Surlenrlce ,,(qpu8rs JelnlleceJlxauE 01 spuodseJ qJea :sreuod sa)pld)Ur)adso] punoq,{llqbrlere teql -supJl se ueql JeqleJ sJeJnpsupJlleu8rs sE uorlrunJ suralord eseql 'saJrlaq (sdnor6peaqpd qllM lAoila()saln)alour prdrlr{ueLu smoqsulsdopoqroua})pq n euerqueusueJl ualas uro4 lllnq osle eJe selnoaloru pu8rs replace4xa purq aqf (B) Jo aJnl)nrls;els{lt uorlnlosal-q6tLl leql sulaloJd Joldef,al aceJJns-ilac,(ueru pue eurlal alerqeue^ aq] Jo sllaf, por '(aryq)auufue'(pal ppe trlredse'(a,uuo) ur ursdopoqJ 'eldtuexa Jo{ 'suorlJunJ luereJJrplnq saJnlcnJlsJelrrursqtlm sure} poe rrue1nl6:apo)lolo)'alets 6urlrelsslr -ord auerquaru ;o i{pue;radns a8rel e Jo Jeqtuetu e sr ursdopoqJorJelf,Bg o] aLu{zuaaLl}uin}at pue a;r{r 6urdurnd 'a1c,{cSurdrund aq] alaldLuor-auelqLuauaqt q6norqi +H slr Surrnp suoqeruJoJuoc a}erpauJelur spralord qleo e aurl aqlJo aurosJo salnlJnrls ar{} JurruJa}apo1 alqrssodueaq oslEspq lr'sarn}Bled sureLl) pt)e apts outu.le ]Pql -ue] ^ ol Lra,l 1e ueq] 3uzee4 pue qplsLJc uraloJd peJepro rrpqdorplqaql 6utztlttnpue pale)tpul 1a,vrd;an 8uurc1qo 'sa18ue rapro aql ut-sjaJsupll le)uaLunu +H ,r{g }uereJJrp,tpq8rls1eraLelq prdrt eq] q8norql ssed qcrq,r '(sprle ourure laqlo,{lluanbasqn5'a.roqdor.uorqr gZ lnoqe Sururetuocqcea) satrlaq tr pelled,{1aso1cueles o}ur paploJ sr elnoaloru aq1{q uoloqd e;o uorldrosqe ursdopoqrorratrpq qceg dqderSolelsdrc der-x dq uorlnlosar q8rq dran o1 uodn s;nr:o leql (aloqdoLuolrl) aq] ol papuaua pue peluruuos Jelpl se.,urtqderSollelsdJtuoJlJele ^q peulPlqo aJnlJnJls ]xau pale)ol'paJ)SgpDe>rgredse;o ureqt aql 'ursdopoqJorJalJeqroC 'suorlnlos lua8Jelep tuoq azrllels,{rcoi lpcgylp eq o} oprsaLllot eloqoouolq) aql uolJ +H e;o 6urssed punoJ aJaMlerl] sureloJd eupJqruau fueru go s^ er^ [BJn]JnJ]slsJrJaql pappord eql srdals,(e1pue lslu aq1 'uMoqsalp a;t,(: 6urdrundpa1enrlre1q611 seq lI 'slsdpuE uoItcE4JIp.{er-xdq sula}oJdelqnlos Jo splsfrc lEuorsueurp-eaJq] aql 6urrnpsuolotdIq ua1e1leanqted go .{pn1s aq} ot snoSoleue sr 'fqderSol1els,i.rc uoJlJala se rrllroDl 'eJnpeJoJd a;qeqordaql pue (aldtnd)aroqdouorqt stql'srs.{1eueuorlJE4JrpuoJlJalapue Idocsorcrru uoJ}calaJouor}Burquoc p sasn 'sa)tlaLl n se leurlaloql Jo uottelolaqf luql qcuordde ue dq (y uolrnloseJatpJaporuol auprqueu eq] ut ursdopor{JorJ t) sauJr]usnasrar{e;rq prdrlaq} sassor)uteq) -e]f,eqJo uouptuarJo pue ernlJnJls aprldadi(1od aql (V) .aln)alotu Ieuorsue{urp-aaJq}eql euruJelap o1 alqrssod ursdopoqlolrel)eqe ]o oJnl)nrls ]I apeur seq Surxced reln8ar aql 'p1s,{rc puorsueurp-orr,q reueld e se pe8upJJe aq1 gg-g 1 arnbrS are eueJquraru aldrnd aql ur selnoaloru ursdopoqJorrelJeq snoJeunu aqJ leuo!suau.lp-aerq1

(v)

'rosor_A)

z +n

Ln9

SNI]IOUd]NVU8W]W

uelllul)ew Ipt] slaqs!lqnd rlorJ uolssll,j'Jjad qll^ 's86t 'vzg-glg:gLi alruDN^P la ra]oquostac'r urotj elep uo pesequospreq)lu'f ^q 6urme.rp e Luorlpaldepyl'astonbtnlut umoLls sr (tt ratcleqfur passn)stp) saln)alou ;;Iqdoro;q> 1o.rod looadsaql'sa)tlaq aql uaamlaqsaredsaql ur pabuerre are seu(zuao: aql leqr aloN ')rDlg ur uMotlsare saLu{zuoo> lauJe) uoJ})alo snoue^aql Jo suorle)olaq1'te,{e;rq prdrl aql ueosleql sa)rlaqn a^u suretuo)qlea pue lolue) uor])eal aql ro aio) aLlluJlo, slrunqnsyt1pue I aq1'auorqtoy(r e pue 'H 'W 'l slrunqnsrnoJ slstsuo)xa;duot Jo aql'xe;dutot ulalotdauelqueusuell slq],o slels^.r) uor})er#rp Jo srs,{;eue ,(er-x{q paurura}apsemaln})nl}s aqf p u tA sououlopnasdopo 's, qA unlral)eq aql ]o ralua) uo!l)ear rrlaqlu{soloqd aq} ro oJnl)nlls leuorsuaur!p-aorqloqI tg-O l. orn6rl

}unqns H

'losorA)

tI I

r e , { e l r qp r d r l ]o aro) :rqoqdorp{q

I I

l)vds uv]n't'll) -VUI-X]

I

l l u n q n sl A

atuorqrop{r

eql alqeeresurelordeueJquaudueru (uoqrppeul'(uotnlllp louo!rurct)re,,i.PIIq ;o aueldaq] ol relnlrpuedJadsrxEuE ]noqPelelor op.{aq}}nq te.{EIIqptdu aql ssoroe(do[-dtD elqun] ]ou op sule]ordeuErqrueu'sptdqauerquau ]sol'ueIIT

auerquew eql Jo aueldaql u! esn#tc suleloldeuerquew [ue6; '(gI raldeql uI pessnoslp)seuo relnlleJerlul olul spu8ts relnllef, -eDxe Sulf,npsuerl roJ osle ]nq 'r{3rauaJo sluJoJ snoIJBASuqsa,treq JoJ^luo lou 'saf,rleqeuerquau 'saxaldruoc suleloJd euerqua6 pa8uerre eJB uauo eBJBI uI 'eldurexe JoJ 'Eualseqoue[J -sueJ] 0g JaAo llallr pue sllunqns ulaloJd 6I suleluoc uroq xalduroc II uals^soloqd snouroue eql 'JalueouollJeeJ sllaqlu^soloqd aql 'stsaqlufso]oqd ut ueql JeBJeluene ale uodsueJl uoJlcele pue 'Suldurnd uolord pallonur saxaldruoc uraloJd euuJqueu eql Jo ^ueIN 'eueJqruelu aql ssolJe suol -ord drund ol lr asn pue ,iSraua 1q311arnldec ol uollcunJ saxalduoc Jllaqludsol -oqd qcns ^\oq ssnJslp aA\'tI raldeq3 uI '(?t{I alntlf,) eulqs€ru ulalord xeld -ruoc E ruJoJ ol euPJquau P uI alPlcossE sepDdeddlod aldqpur /t^oq elull ISJIJ aql roJ paMoqs [aq1 asnecaq,{8o1orqeuBrqluelu o] ecueuodlul IEJeueSJo ala^\ srsl.leue srql Jo slFsar aqJ 'uoItcErJJIp^eJ-x dq pez,r{.puepue pazqplsdrc aq o1 uolpaal Jlnqlutsol xaldruoc uralord aupJqruarusueJl ]sJIJeql sem qclqm 'JaJLtaJ -ot4d leualteq e sI auo .{qder8olersdrc Ler-x dq pelpnts ueeq eABqqrlq aJo IeJe -Aes 'sexeldruoc luauodurocppur Jo ]red se uol]f,urg sueloJd auerquraur [uu141

saxelduo) e6le'l se uoll)unJ uauo sulaloldeuerquew ,tlunlce JoJuol Jelnolued E aJInbeJ uoqnlos snoanbe ur saurr(zua,,i.ueruse ]sn[ 'sdnor8 peaq prdq cgtcads Jo eJue -sard aq1u1,t1uouorlJunJ ueo sulaloJd aueJqtualu e11ros'uonnlos snoanbe ue ur

arnpnrls auerquaw:0I laloeLl)

zv9

(slsl6010lB ;o lueduol aql uolJ uorssrrulad 'r'utptpl 'w qllM '016t '9EE-6t"8:t'l)S //af pue efu1'o'l uo paspg)'pal srautulepoql pue uaarbst uta)selonU asnelaq ado>so.nru.r a)ua)seronlJ e ur paqsrn6urlsrp aq ue) sutaloloaql aztlenst^ ol pasn sarpoqrlupoMl aqf 'autl tano xil.xlalul ^aql rnq 'auerquaur er"use;d uo,(re>orelaq paurrolI;mau aql jo sanlequMo ltaql ol pauuuo)r{;;erlrur aresuralo.rd upunq pue asnouraqf 'sllo)puqlq ueunq-asnoul Jo aue.tquauteurse;daqt u; surelord1o uolsnJJ!peq16uglerlsuouep lueugladxauy gg-91 arn6r1

pe{Jeu aq} JoJluarcrJJeosuoISnJJIpaq} e}elncpc sanlp^ eqJ'ureloJd esPJJns-lleJ uer a^ 'sluaruernsearudITc pue dvuc qJns urorc '(s9s-0l eJn3rfl selncalolu pelaqel ,(1]uacsaronu Jo euerqruaru Surpunorrns aql SuFeldap flenper8 ,{qaraqt '}l o}ur esnJJIpteq} salnJaloru luaoseJong eq} IIB qJealq o} ouprqlueru 'el. H'(1I'IA 8un1caa1q Jo eaJe IIErus B selerperJr.{lsnonurluoJ rueeq Jaspl E -o1ot1d u! ssol acuacsatonpfsr anbruqeal d-ruluaruaydruoc y '(Vgt-gl ernS;g) parnseerusr pare peqoeelq eql otur asnJJIpot dCC ro pue311paqcealqun Surl;rec suralord eueJqrueu luacefpe JoJualel eruq eqt pup 'urpeq Jesel€,,(qauerqruaru Jo eere llerus e ut peqJealq uaql sl dnor8 tuacsarong aqJ'(6 ralduq3 ur pessnc -slp) (dCC)uralo.rdtuacsarong uaar8 ol pesnJuralord aq] ssardxao1,i8o1ouqca1 VNCI ]ueurqruof,ar q4,,t,rro uralord eql ot spurq reqr dpoqpue paleqel-aroqdor -ong E sE qf,ns pue8l luacsaJonlJp qly\,\ reqlra euop eq UBJ srq; 'dnor8 luac -saJong f,grJadse q1ra,rlsaralurJo uratord eueJquaru aq1Suotreur sanlonur.{ypnsn poqtau aql'@Wfl Sutqcaa1qotor4dn{n [tanocat acuacsatonTf;o anbruqca] aq] Sursn dq parnseeu eq ueJ suralord aueJqrueru Jo selpJ uorsnJJlpleJelel aql '(9t-0I arn -t;g) rnoq up J1eqtnoqp ur aJpJrnsIIaJ eJllue eql relo paxlu pue pasnJJlpsulal -ordgo slas o^ ] aql 'uofrecoJa]eq peuJoJ zi.prauaqtJo se^pq u ^o Jreql o] peurJ -uoc ala^ suralord ueunq pue esnou aq] lsJUlE q8noqipsuralord aupJqrueu eurseld ueunq pue esnoru palf,alasqsm8u4srp ol pesn eJeMselpoqpue pelaqel ,(lluara;;p o^ I 'eueJqtuetu aql ;o aueld eq] ur alqou are suralord auerq -rrrerrrerrrseld auros leql aJueprle lJeJrp lsJrJeql papuord (suotLacotalaq) slleJ plrq,(q acnpord o] sflac u€unq ql!v\ pesnJ z(lprcryge eJe1!\sllef, esnoru qJIqM ur luarurradxa uy '(uotsntltp IDJaTD eupJqrueu aql ulqrlv\ ,,(1era1e1alour ol

salnulul 0t = auJll

)otg -LVNOtfVSn)Nt s e l n U l u J0 = a u l l l

(a) eurLuepoqrLllr^/\ p a l a q e l' u r e l o r d a u e J q t r a u ur e ( x n q I s u r e 6 e( Y )s o r p o q r l u e

(a) urarseronl]L{}r^^ p e l a q e l' u r e l o r d eueJqt!euresnou.r l s u ! e 6 e( \ r , ) s a r p o q r l u e

I.I

"o v o t

ii

NO UV)OU]I]H

u raloro auerq(xau

'l'llf NVANH

€v9

SNI]IOUd]NVUBW]W

rraqt SurTJBrtpue salcrued ploS duq Jo safp tuacseronu ot peldnoJ selpoqltuE qll^\ salnceloru eusrqrueu pnpNpur 3u11aqu1 .{q uelqord slql aruosra^o senblu -qJet sdeqrad-euerqlueu Sur4raq aIJuJDd-aFa?S'surelord lutele)so1{J,{q st Jo uor8eJ lleurs /tJeA B 01 luerue^oru slr ul palcrJlseJ lsnf Jo ellqolulultp4 elnsalou aql JeqleqM IIel louueJ euo 'eldruExa JoJ (PeJBpeqJEelq E olul alBrSIIu o1 slleJ uraloJd P JI 'selnJeloru ureloJd IPnpr^rpur 1\^olloJlouues auo laueJq -ruaru Jo eeJp aBJEILlalrlelar e ur selnoeloru Jo suoqelndod a8rel Jo luerue^oru pue eql 01 euo eql rolruou faqr sr sanbruqJel ruqr dI'Ic dvuc IJeqMEJp .lro eAIIo Jo leq] o] slqt uI pepedul elqeJPdruoJ rblsocslr e e^eq seuBJqr.ueluiler lEql alBOIpu zte,,vr dlerurulur eJe lpqt surelord Jo sluarueJnseet{ 'seeJ8ep3utften o} sueloJd eql 'a1qerrell.1q8rq Jo uorsryJrpeqt apadur suralord raqlo qll^\ suorlcurelul esnBJeq ere sllal tueJeJJIpq suretoJdauerqueu tueJeJJIpJoJsluelogJeoouoIsrUJIpaqtJo

I eere paq)ealq

t

,"r.

lueulaJnseour

-a(xt]

p a""* q)eolq

I

9;

== en

eole luaulelnseeu

ytlvts U:ls\fl | Hr.tAA H)v3'18 |

ol o o

'xuleutlelnlla)erlxa ro uolalelsol^loql ol paroq)ue {lluaueuled lou pup a;rqor.u ale Xaq} se 6uo1seipaq)ealqete sa;nte;ouuralord tuarsaronuaql,o lle [llenluera:urasnJJtp salnlalourpaq)ealqpue lno asnJjtpsutaloto luo)saronlJsp sasear)ap{;anrssa.r6ord earepuo)osaql ut a)ua)salonll 'ParealPlPoase ut palnseaust a)ua)salonB pue'{;snonurluo)paletpeJlt st auplqtrJauJ jo earelleu.rs e ,enbruq)aldlll ar.1l ul (g) '{ranoler oq} ratspJ aq}'ulatord auerquraur aq] Jo luar)U4ao)uorsnJJtp aql ralea.r6 aql:{ranorarJo alel aq}Joqder6e uor; paleln)le)srluar)ujao)uotsn]ltpaql'(sMot^ dot pue aprsur araqumor,ls) earepolplpelll aq] otu! asnJJrp saln)alourpaLl)ealqun pue{errne asnJJtp saln)aloupaq)ealq aLllse sja^o)ol ,{trsualura)uo)salonlJ aqf 'ureeqlasele 6ursnealelleus e ur paq)ealqate selnlaloullua)salonl, 'anbruqral 'lua)salonlj dVUl aqt ul (V) ,{;;errsur.rlur sr q)rLlM'(dlg)utalold lua)saronUuaar6qllm uralotduorsn; e se pessaldxaaq uel lsalalu!;o urelo.rd rr;oads V 'sanbruq>a1 6urq>ee;q -o1oqdIq urelo,rdaueJquaul e Jo uotsn#tp lpralel,o aler aql 6urrnseeyl9€-01 arn6rl

H)Vt'18

dr'H

(8)

*r^"-, I I aru!l P a J ep a q l e e l q

Ulno)3u

lltwgu:ls\n I

g=

H.r.lMH)vr18

|

OX

lr

dVUI

arnl)nrlseuerque4:0[ ra]deqf

(V)

Wg

64s

PROTEINS MEMBRANE p r o t e i nA

tight Ju n c t io n

a p i c a lp l a s m a / membrane

p r o t e i nB

l a t e r a lp l a s m a m e mo r a n e

b a s al a m i n a

movement by video microscopy. Using single-particle tracking, one can record the diffusion path of a single membrane protein molecule over time. Results from all of these techniques indicate that plasma membrane proteins differ widely in their diffusion characteristics,as we now discuss.

DomainsWithin CellsCanConfineProteinsand Lipidsto Specific a Membrane The recognition that biological membranes are two-dimensional fluids was a major advance in understanding membrane structure and function. It has become clear,however,that the picture of a membrane as a lipid sea in which all proteins float freely is greatly oversimplified. Many cells confine membrane proteins to specific regions in a continuous lipid bilayer.We have already discussed how bacteriorhodopsin molecules in the purple membrane of Halobacterium assemble into large two-dimensional crystals, in which individual protein molecules are relatively fixed in relationship to one another (seeFigure 10-32); large aggregates of this kind diffuse very slowly. In epithelial cells, such as those that line the gut or the tubules of the kidney, certain plasma membrane enzymes and transport proteins are confined to the apical surface of the cells, whereas others are confined to the basal and lateral surfaces (Figure f 0-37). This asymmetric distribution of membrane proteins is often essential for the function of the epithelium, as we discuss in Chapters 1l and 19.The lipid compositions of these two membrane domains are also different, demonstrating that epithelial cells can prevent the diffusion of lipid as well as protein molecules between the domains. Experiments with labeled lipids' however, suggestthat only lipid molecules in the outer monolayer of the membrane are confined in this way. The barriers set up by a specific type of intercellular junction (called a tight junction, discussedin Chapter l9) maintain the separation of both protein and lipid molecules. Clearly,the membrane proteins that form these intercellular junctions cannot be allowed to diffuse laterally in the interacting membranes. A cell can also create membrane domains without using intercellular junctions. The mammalian spermatozoon, for instance, is a single cell that consists of several structurally and functionally distinct parts covered by a continuous plasma membrane. \dhen a sperm cell is examined by immunofluorescence microscopy with a variety of antibodies, each of which react with a specific cellsurface molecule, the plasma membrane is found to consist of at least three distinct domains (Figure f 0-38). Some of the membrane molecules are able to diffuse freely within the confines of their own domain. The molecular nature of the "fence" that prevents the molecules from leaving their domain is not known. Many other cells have similar membrane fences that confine membrane protein diffusion to certain membrane domains. The plasma membrane of nerve cells, for example, contains a domain enclosing the cell body and dendrites, and another enclosing the axon. In this case, it is thought that a belt of actin filaments tightly associatedwith the plasma membrane at the cell-body-axon junction forms part of the barrier.

Figure10-37 How membranemolecules can be restrictedto a particular membranedomain.In this drawingof an epithelialcell,proteinA (in the apical membrane)and proteinB (in the basal candiffuse and lateralmembranes) laterallyin their own domainsbut are preventedfrom enteringthe other domain,at leastpartlyby the specialized celljunctioncalleda tight junction.Lipid in the outer(noncytosolic) molecules monolayerof the plasmamembraneare likewiseunableto diffusebetweenthe two domains;lipidsin the inner (cytosolic)monolayer,however,areable to do so (not shown).The basallaminais matrixthat a thin mat of extracellular epithelialsheetsfrom other separates in Chapter19). tissues(discussed

646

Chapter10:MembraneStructure

Figure10-38Threedomainsin the plasmamembraneof a guineapig sperm.(A)A drawingof a guineapig sperm.In the threepairsof micrographs, phase-contrast micrographs areon the Ieft,and the samecell is shown with cell-surface immunofluorescence staining onthe right.Differentmonoclonal antibodiesselectively labelcell-surface molecules on (B)the anteriorhead, (C)the posteriorhead,and (D)the tail. (Micrographs courtesyof SelenaCarroll and DianaMyles.)

a n t e r i o rh e a d

l

posteriorhead

#

Figure l0-39 shows four common ways of immobilizing specific membrane proteins through protein-protein interactions. (A)

Thecorticalcytoskeleton GivesMembranes Mechanical strength and Restricts MembraneProteinDiffusion As shown in Figure 10-398 and c, a common way in which a cell restricts the lateral mobility of specific membrane proteins is to tether them to macromolecular assemblies on either side of the membrane. The characteristic biconcave shape of a red blood cell (Figure 10-40), for example, results from interactions of its plasma membrane proteins with an underlying cytoskeleton,which consists mainly of a meshwork of the filamentous protein spectrin. Spectrin is a long, thin, flexible rod about 100 nm in length. Being the principal component of the red cell cytoskeleton, it maintains the structural integrity and shape of the plasma membrane, which is the red cell's only membrane, as the cell has no nucleus or other organelles.The spectrin cytoskeleton is riveted to the membrane through various membrane proteins. The final result is a deformable, netIike meshwork that covers the entire cltosolic surface of the red cell membrane (Figure r0-4r). This spectrin-based cytoskeleton enables the red cell to withstand the stresson its membrane as it is forced through narrow capillaries. Mice and humans with genetic abnormalities in spectrin are anemic and have red cells that are spherical (instead of concave) and fragile; the severity of the anemia increaseswith the degree of spectrin deficiency. An analogous but much more elaborate and complicated cltoskeletal network exists beneath the plasma membrane of most other cells in our body. This network, which constitutes the cortical region (or cortex) of the cytoplasm, is rich in actin filaments, which are attached to the plasma membrane in numerous ways' The cortex of nucleated cells contains proteins that are structurally homologous to spectrin and the other components of the red cell cytoskeleton. We discuss the cortical cytoskeleton in nucleated cells and its interactions with the plasma membrane in Chapter 16.

Figure 10-39 Fourways of restrictingthe lateralmobility of specificplasma membraneproteins.(A)The proteinscan self-assemble (as into largeaggregates seenfor bacteriorhodopsin in the purple membraneol Halobacterium); they can be tetheredby interactions with assemblies (B)outsideor (C)inside of macromolecules the cell;or they can interactwith proteins on the surfaceof anothercell(D).

647

MEMBRANE PROTEINS

Figure 10-40 A scanningelectron micrographof human red blood cells. shape The cellshavea biconcave

s n d o t h e ro r g a n e l l e s . a n d l a c ka n u c l e u a (Courtesy Chailley.) of Bernadette

5tt

The cortical cltoskeletal network underlying the plasma membrane restricts diffusion of not only the proteins that are directly anchored to it. Because the cytoskeletalfilaments are often closely apposed to the cltosolic membrane surface, they can form mechanical barriers that obstruct the free diffusion of membrane proteins.Thesebarriers partition the membrane into small domains, or corrals (Figure lO-42), which can be either permanent, as in the sperm (see Figure 10-38).or transient.The barriers can be detectedwhen the diffusion of individual membrane proteins is followed by high-speed, single-particle tracking. The proteins diffuse rapidly but are confined within an individual corral; occasionally,

j u n c t i o n aI .....-..-complex

!f

tropomyosrn

(A)

anKyrrn glycophorin 1 0 0n m

cytoskeletonon the cytosolicsideofthe human red Figure10-41The spectrin-based shownin the drawinghasbeen blood cell plasmamembrane,(A)Thearrangement of purifiedproteinsin yitro.Spectrin deducedmainlyfrom studieson the interactions dimers(enlargedin the box on the right)arelinkedtogetherinto a netlikemeshworkby 'Junctional in the box on the /eft).Eachspectrinheterodimer complexes"(enlarged flexiblepolypeptidechainscalledct and looselyintertwined, consistsof two antiparallel, to eachotherat multiplepoints,including B Thetwo chainsareattachednoncovalently The at both ends.Boththe crand p chainsarecomposedlargelyof repeatingdomains. junctionalcomplexes arecomposedof shortactinfilaments(containing13actin monomers),band4.1,adducin,anda tropomyoslnmoleculethat probablydetermines is linkedto the membranethrough Thecytoskeleton the lengthof the actinfilaments. proteins-a multipassproteincalledband3 anda single-pass two transmembrane proteincalledglycophorin.The spectrintetramersbind to someband 3 proteinsvia and to glycophorinand band 3 (not shown)via band 4.1 proteins. ankyrinmolecules, (B)Theelectronmicrographshowsthe cytoskeleton on the cytosolicsideof a red blood Thespectrinmeshworkhasbeen cellmembraneafterfixationand negativestaining. purposelystretchedout to allowthe detailsof its structureto be seen.In a normalcell, the meshworkshownwould be much morecrowdedand occupyonly aboutone-tenth of this area.(B,courtesyofT. Byersand D. Branton, Proc.NatlAcad.Sci.U S.A. from NationalAcademyof Sciences.) With permission 82:6153-6157,1985.

648

Chapter10:MembraneStructure

1 0 0n m (A)

lpm transmembrane

(B)

however, thermal motions cause a few cortical filaments to detach transiently from the membrane, allowing the protein to escapeinto an adjacent corral. The extent to which a transmembrane protein is confined within a corral

Figure10-42Corralingof membrane proteins by corticalcytoskeletal filaments.(A)How cytoskeletal filaments arethoughtto providediffusionbarriers that dividethe membraneinto small (B)High-speed, domains,or corrals. single-particle trackingwas usedto follow the pathsof a fluorescentlylabeledmembraneproteinovertime.The traceshowsthat membraneproteins diffusewithin a tightlydelimited membranedomain(shownby different colorsof the trace)and only infrequently escapeinto a neighboringdomain. (Adaptedfrom A. Kusumiet al.,Annu.Rev. Biophys. Biomol. Struct. 34:351-378, 2005. With permission from AnnualReviews.)

Su m m a r y Whereasthe lipid bilayer determinesthe basicstructure of biological membranes,proteins are responsiblefor most membrane functions, seruing as specific receptors, enzymes,transport proteins, and so on. Many membrane proteins extend acrossthe lipid bilayer.some of thesetransmembraneproteins are single-passproteins, in which the polypeptide chain crossesthe bilayer as a single a helix. others are multipass proteins, in which the polypeptide chain crossesthe bilayer multiple times-either as a seriesof a helicesor as a B sheetin theform of a closedbarrel. All proteins responsible for the transmembranetransport of ions and other small water-solublemoleculesare multipassproteins.some membrane-associated proteinsdo not span the bilayer but instead are attached to either side of the membrane.Many of theseare bound by noncoualentinteractionswith transmembraneproteins,but othersare bouncl uia coualently attached lipid groups. In rhe plasma membrane of all eucaryotic cells,most of the proteins exposedon the cell surfaceand some of the lipid moleculesin the outer lipid monolayer haue oligosaccharidechains coualently attached to them. Like the lipid moleculesin the bilayer, many membrane proteins are able to diffuse rapidly in the plane of the membrane.Howeuer,cells haue ways of immobilizing specific membrane proteins, as well as ways of confining both membrane proteiin and lipid moleculesto particular domains in a continuouslinid bilaver.

PROBLEMS Whichstatementsare true?Explainwhy or why not. 10-1 Although lipid molecules are free to diffuse in the plane of the bilayer,they cannot flip-flop acrossthe bilayer unless enzyme catalystscalled phospholipid translocators are presentin the membrane. 10-2 lVhereas all the carbohydrate in the plasma membrane facesoutward on the external surfaceofthe cell, all the carbohydrate on internal membranes faces toward the cltosol.

10-3 Although membrane domains with different protein compositionsare well known, there are at presentno examples of membrane domains that differ in lipid composition. Discussthe following problems. l0-4 \Ahen a lipid bilayeris torn, why doesit not sealitself by forming a "hemi-micelle" cap at the edges,as shornmin Figure Qr0-l? 10-5 Margarine is made from vegetable oil by a chemical process.Do you supposethis processconvertssaturatedfatty acids to unsaturatedones,or vice versa?Explain vour answer.

649

PROBLEMS END-OF-CHAPTER

do you supposemight accountfor the differencein extentof lossof ESRsignalin experimentswith red cell ghosts(Figure (Figure Q10-3C and D) versus those with normal red cells B). Q10-3Aand C. Were the spin-labeled phospholipids introduced equally into the two monolayersof the red cell membrane?

tear in bilayer lri U! lr I r b !! ! e g ! . )l:: I ;'

!!!sf,eLLbrt,t,66FI

,:,:: ' '1" , gre!!!atggb!i6

$lLtit9atSll ','l:' : :

't:'lt

5

a c G

] , ,

aLta6rG6!L{,tG

6

10-B Monomeric single-passtransmembrane proteins span a membrane with a single cxhelix that has characteristic chemicalpropertiesin the region of the bilayer.lVtrich of the three 20-amino acid sequenceslisted below is the most likely candidate for such a transmembrane segment? Explain the reasonsfor your choice. (Seeback of book for one-letter amino acid code; FAMILYVlV is a convenient mnemonic for hydrophobic amino acids')

witha hypothetical"hemisealed FigureQ10-l A tornlipidbilayer 10 4). cap(Problem micelle" 10-6 If a lipid raft is tlpically 70 nm in diameter and each lipid molecule has a diameter of 0.5 nm, about how many lipid molecules would there be in a lipid raft composed entirely of lipid? At a ratio of 50 lipid moleculesper protein molecule (50%protein by mass)how many proteins would be in a typical raft? (Neglectthe loss of lipid from the raft that would be required to accommodatethe protein.)

A.

]

TL

I

YFGVMAGVI

I

S

B,

I

TP

I

YFGPMAGVIGTPLL

I

S

TE

I

YFGRMAGVIGTDLL

I

S

C,

I

GT

I

LL

1o-7 A classicpaper studied the behavior of lipids in the t O-9 You are studying the binding of proteins to the c)'totwo monolayers of a membrane by labeling individual plasmic face of cultured neuroblastoma cells and have molecules with nitroxide groups, which are stable free radifound a method that givesa good yield of inside-outvesicles cals (Figure Q10-2). These spin-labeled lipids can be from the plasma membrane. Unfortunately,your preparadetectedby electron spin-resonance(ESR)spectroscopy,a tions are contaminatedwith variableamounts of right-sidetechniquethat doesnot harm living cells.Spin-labeledlipids out vesicles.Nothing you have tried avoids this problem. A are introducedinto small Iipid vesicles,which arethen fused friend suggeststhat you passyour vesiclesover an affinity with cells, thereby transferring the labeled lipids into the column made of lectin coupled to solid beads.\Mhat is the plasmamembrane. point of your friend'ssuggestion? The two spin-labeled phospholipids shown in Figure 10-10 Glycophorin, a protein in the plasma membrane of Q10-2 were incorporated into intact human red cell membranes in this way. To determine whether they were introthe red blood cell, normally exists as a homodimer that is duced equally into the two monolayersof the bilayer,ascorheld together entirely by interactions between its transbic acid (vitamin C),which is a water-solublereducingagent membrane domains. Since transmembrane domains are that does not crossmembranes,was added to the medium hydrophobic, how is it that they can associatewith one to destroyany nitroxide radicalsexposedon the outside of anotherso specifically? the cell.The ESRsignalwas followed as a function of time in _ R E DC E L L S - R E DC E L L S ( B ) P H O S P H O L I P2I D the presenceand absenceof ascorbic acid as indicated in ( A ) P H O S P H O L I P1I D Figure Ql0-3A and B. A. Ignoring for the moment the differencein extent of loss c 100 of ESRsignal, offer an explanationfor why phospholipid I (Figure Q10-3A) reacts faster with ascorbate than does phospholipid 2 (FigureQ10-3B).Note that phospholipid I c reachesa plateau in about 15 minutes, whereasphosphoc-" o Iipid 2 takesalmost an hour. 25 B. To investigate the difference in extentof loss of ESRsignalwith oo12 the two phospholipids,the exper%1020 ni t r o x i d e iments were repeated using red r a di c aI (D) PHOSPHOLIPID 2 - GHOSTS 1 -GHOsTs (c) PHOSPHOLIPID cell ghoststhat had been resealed to make them impermeable to - ascorbate ascorbate(FigureQI0-3C and D). I 100 red cell ghostsare missResealed ing all of their cltoplasm but have ,' a c an intact plasma membrane. In 6 q n- ' these experimentsthe loss of ESR c p h o s p h o l i p i d 1 phospholipids was signalfor both 1E 25 negligiblein the absenceof ascorbate and reacheda plateauat 50% 0123 oo 30 zo to in the presenceof ascorbate.\A4rat @

time (minutes)

E

ot two FigureQ10-2 Structures lipids(Problem nitroxide-labeled 10-7).The nitroxideradicalis shownat the top,and its positionof attachment is shownbelow. to the phospholipids

b ' A J

' J

p h o s p h o l i p i d2

time (hours)

in ESRsignalintensityas a functionof time FigureQl0-3 Decrease and absenceof inlntact redcellsand red cellghostsin the presence 1 and (Problem10-7).(A and B) Phospholipid ascorbate 1 and phospholipid2 in intactred cells'(Cand D) Phospholipid phospholipid 2 in red cellghosts.

650

Chapter10:MembraneStructure

REFERENCES General Bretscher MS(1973)Membrane structure:some generaiprinciples Sctence 181:622629 Edidn M (2003)Lipidson the frontier:a centuryof cellmemDrane bilayersNctRevlr.4ol CellBiol4:4i4-418 Jacobson K et a (1995)Revisiting the fluidmosaicmodelof membranes Science 268.14411442 Lipowsky R& Sackmann E (eds)(,1995) Thestructure anddynamics of membranes Amsterdam: Esevier SingerSJ& Nicolson Gl (1972) Thefluidmosaicmodelof the structure of cel membranes Scrence 175.12A n1 The Lipid Bilayer Bevers EM,Comfurius P & ZwaalRF(,1999) Lipidtranslocation across the pJasma membraneof mammalian cellsBtochim Biophys Acta 1439:317-330 DevauxPF(1993)tipid transmembrane asymmetry and flip-flopin biological membranes and in ltpidbilayers CurrOpinStruct Biol 3.489494 DowhanW (1997)Molecular basisfor membranephospholipid diversity:why arethereso manylipids? AnnuRevBiochem 6 6 1 9 92 3 2 Hakomori procNatl SiSl(2002)Inaugural Article:The glycosynapse Acad SciUSA99 225-232 HarderT & SimonsK (1997) Caveolae, DlGs, andthe dynamics of sphingolipid-cholesteroi microdomains CurrOpinCellBiol9:534-542 H a z el lR ( 1 9 9 5T) h e r m aal d a p t a t i oi n b i o l o g i c a ml e m b r a n e s : i s homeoviscous adaptation the explanation? AnnuRevphysiol 57:19-42 lchikawa S & Hirabayashi Y (,1998) Glucosyiceramide synthase and glycosphingolipid synrhesis Trends CellBiolB.1gB202 Kornberg RD& McConnell HM (t9Zt) Lateral diffusion of phospholipids in a vesiclemembraneProcNatlAcadSciUSA6g.25642568 Mansilla MC,Cybulski LE& de MendozaD (2004)Controlof membrane lipidfluidityby molecular thermosensors J Bacteriol 186:6681_6688 McConnell HM & Radhakrishnan A (2003)Condensed complexes of cholesterol and phospholipids Biochtm Biophys Actaj61O:j59_73

Rothman JE& LenardI (19l7)Membrane asymmetry Science 195.74353 SimonsK & VazWL(2004)Modelsystems, lipidrafts,and ceri membranesAnnuRevBrophys BtomolStruct33:269-95 TanfordC (1980) TheHydrophobic Effect: Formarion of Micelles and BiologicaMembranesNewyork:Wi ey van MeerG (2005)Cellular lipidomicsEMBO J 24:31593165 Membrane Proteins BennettV & Baines AJ(2001 and ankyrin-based pathways: ) Spectrin metazoaninventionsfor integratingcellsinto lssues physiolRev B1:1353-1392 Bqlmakers MJ& MarshlV (2003) Theon off storyof protein palmitoylationTrends CellBiol 13:32-42 Branden C &ToozeJ (1999)lntroduction to proteinSrructure,2nd ed NewYork:Garland Science Bretscher MS& RaffMC (19l5)Mammalian p asmamembranes /Vdrure 258.43-49 Buchanan SK(,1999) Beta-barrel proteinsfrom bacterial outer membranes:structure, functionand refoldingCurrOpinStruct Biol 9:455-461

ChenY,Lagerholm BC&Jacobson K (2006)Methodsto measure the lateraldiffusion of membranelipidsand proteinslt4ethods 39:147-153 CurranbAR& Engelman DM (2003)Sequence motifs,polarinteractions andconformational changesin helicalmembraneproteinsCurr OpinStructBiol13:412 Deisenhofer J & MichelH (1991) Structures photosynthetic of bacterial reactioncenters,4nnuRevCellBiol7:1-23 Drickamer K & TaylorME(1993)Biologyof animallectinsAnnuRevCell Biol9,23764 Drickamer K & TaylorME(1998)Evolving viewsof proteinglycosylation TrendsBrochemici 23:321-324 FryeLD& EdidinM (1970) Therapidintermixing of cellsurface antigens afterformationof mouse-human heterokaryons, J CellSci7:319-335 Helenius A and SimonsK (1975)Solubilization of membranes by detergentsBiochimBiophys Acta 415.29-79 H e n d e r s oRn& U n w i nP N( 1 9 7 5T) h r e ed i m e n s i o nm a lo d e o l f purple membrane obtainedby electronmicroscopy, Nature257:28-32 Kyte-1& DoolittleRF(1982)A simplemerhodfor displaying the hydropathic character of a proteinJ ltlolBiol157ia5_l32 le MaireM,Champeil P et al (2000)Interaction of membraneproteins and lipidswith solubilizing detergentsBiochim BiophysActa 1 5 0 8 : 8 61- 1 tee AG(2003)Lipid-protein interactions in biological membranes.a structuraiperspectiveBtochimBiophys Acta 1612:1-40 Marchesi W, Furthmayr H et al (1976) TheredcellmembraneAnnuRev Biochem45.667-698 Nakada C,Ritchie K & KusumiA (2003)Accumulation of anchored proteinsformsmembranediffusion barriers duringneuronal polarization NatCellBiol5.626-632 Oesterhelt D (1998) Thestructure and mechanism of the familyof retinalproteins from halophilic archaeaCurrapinStructBiol 8:489 500 ReigN &van derGootFG(2006)Aboutlipidsandtoxins FEBSLett 580:5572-5579 Reithmeier RAF(,l993) Theerythrocyte (band3) Curr aniontransporter OpinCellBiolT.7A7-7 14 Rodgers W & Glaser M (1993)Distriburions of proteinsand lipidsin the erythrocytememb rane Biochemistry 32.17591- i 2598 SharonN & tis H (200a)History of lectins:from hemagglutinins to biological recognition moiecules Glycobtology 14:53R-62R SheetzMP(200'1) Cellcontrolby membrane-cytoskeleton adhesion NatRevMolCellBiol2:392 396 Silvius JR(1992)Solubilization andfunctional reconstitution of bromembrane componentsAnnuRevBiophys BiomolStruct 21.323-348 SteckTL (1974lhe organization of proterns in the humanredblood cellmembraneA reviewJ CellBiol62:1-i9 Subramaniam S (1999) Thestructure of bacteriorhodopsin:an emerging consensusCurrOpinStructBiol9.462-468 VielA & BrantonD (1996)Spectrin:on the pathfrom structure ro function CurrOpinCellBiol8:49-55 WaliinE & von HeijneG (1998)Genomewideanalysis of inregral membraneproteins from eubacterial, archaean, and eukaryotic organrsmsProtetnSci7.1029-1038 WhiteSH& WimleyWC(1999)Membraneproteinfoldingand stability:physical principlesAnnuRevBiophys BiomolStruct 28319 36s

ts9

'pa^Io^ur sesseroJdpue sureloJd aql SurzrralcereqcJoJser8eleJlseruos pup uodsue4 aueJqueluJo sruJoJsnoIJeAeq] aqrJssepol pesn sruJel eql Jo eruos acnpoJlur uaql a A'sJe/tPIq prdq crlaqlu(s 'aar;-uralord seluedoJd ,fiqrqeaurad eql Sulqucsap ^q uoqces slql uFaq aM Jo

lO Slldl)Nlud ruodsNvulINVUBWlW

S]NVUBIA]W io sltluldoHd

I-UOdSNVUI rNVUSrlW l^tlfv CNVSU]IUOdSNVUI

v99

lVflUt)lll lHr CNVSI]NNVH)NOI

199

ts9

IUOdSNVUI ]NVU8Y!]IA lO Slldl)NlUo

ratdeq)s!r{Iul

'slEal ueunq eql IIB s,uIEJq Sulqsruotse 'uoqecpqqdos lno ftJec ol suoJneu Jo s{Jomtau 3u11qeua Jo la^al lsaq8rq rreql 'sllac aseql q '(slleJ a^reu) suoJnau uI sleuusqJ le ruJoJJed sureloJd IauuEqJ uorJo suorlJunJ 3ql uo l(Iulelu eleJluasuoc er\/\teldeqJ aqlJo ued tsEI eql uI 'sraldeqc JaIBIul passnJ -srp se '11aorrlofrecne aql Jo saueJqrueu Jaq]o eq] ssoJoealerado suslueqJalu JBIlurs lnq (euBJqrxaur eruseld aql ssoJJE ilodsuerl uo .{prcru uolssncslp Jno snJoJaM aJV s,llaoeqlJo lsotu e{eru (BUe}Jeqpue 'slseldoJolqJ 'Pupuoqc 'sassacotd -olrru ur) pue 'sllaf, alqelroxe,{1ecu1ra1aut spu8ts IeJIJ}3eler{.azruoc 'sluarper8 uodsueJl snorJel elrrp qf,rqm IeolrueqcoJlJeleJo urJoJeql ut d8raua ppualod erols ueo seueJqueu gac tadepq ptdq aqt ssoroe secueraJJlpuolleD -ueJuoJ cruor Surleraua8 Lg 'sallaue8ro pasoloue-eueJquau ulqllm plng eql ro (I-II aIqBD prnlJ relnllaceJue eqt reqlla Jo rEql qll/\^ paredruoc loso/.c aql a8rel salEaJJlrodsuerl elllce pue &ryqearu ;o uorlrsodruoc eql ur seoueJeJJrp 'lrodsueJl -rad pue all]ou az[1e1etol ,iSraua e arrssud elrlJeles uorleurquoo Jo 'suor crue8roul ilerus ;o ,i.purutrd Jo aJJnos e o1 paldnoc eq ueJ sralrodsuer; ']uarueloru aueJqrueusuerl arrrssud8ulr,ro1p 'arod cqtqdorpfq morreu E IUJoJ qJIqM 'slauuaqJ pue 'sauuJqruelu ssoJJPseFceloru cgtcads ]rodsuerl ol slred Surrrou e^Eq qJrq^^ 'staitodsuau:sradelrq ptdll ssoJf,equoJ pue IJeq selnJelou Jo JrJJe[ slql elelpetu lpq] sula]oJd auerquraur Jo sassPIOuleru oml eq] 'uJnl ur 'Japrsuocuaql eM'saueJquau IIas asJe^eJlselnJalou elqnlos-Jelel IIEus ,r,roq;o saldrcuud praua8 aruos Sutqrrcsap,'i.q.raldeqc sgqtur8aq all 'sassacord uodsuerl eueJqruau o1 uoqdrunsuoc d8raua f,rloqptau Ielot Jraql Jo spJrql-oml ol dn alonap slleo uelletuureur pazqetcads euros 's11ecIIB ur sure]ord auerquraur aql Jo %0S-gI dn a>1eruqJIWv\ 'sutalotd uodsuer] JoJ apoJ ]eql sursrue8JoIIp ur sauaS;o raqunu a3.re1aql uI pe]oegeJ sr ]rodsuer] eueJqruau ;o acuelrodul eqJ 'tI puu 71 sraldeq3 uI pessnf, -srp eJertaqt pue 'salncalour lerus SutrraJsueJlJoJpesn esoq] Ixo4 JeJJIpsesEJ eseqlJo lsoru ur pellolur srusrueqoar[ eq] lnq'saueJqtueur Jleql ssoJJBsalcqred a8rel uala pue selnJeloruoJJeru JaJsupJlosp uec s11a3'ra,(e1q prdrl aqr ssoJou selnJelou crue8ro elqnlos-Jelem IlEIxs pue suol ctuu8rout uodsuerl 01 sulel 'suolluJluecuoc uol JelnlleJeJlul eleln -ord pazqercads asn slle3 auerqruerusueJl 'slualJlnu -8ar puu 'slcnpord etsemorloqelaru aleJOXe lel]uessa]saButol JepJouI seuprqruaurJreql ssoJcesuol pue salnceloru elqnlos-JelEMct;tcads Surrra;suer1 go szlem.eAIoAeo] ppq e^pq sllac le^a^ oq 'JauJBq slq] uro4 llJauaq oI 'sluau -]redruoc pesoloue-aueJqueu Jelnllef,EJluraqtJo qoee ur pue plnlJ JEInlleJEJlxe eql uI esoql luo4 JaJJIpteql 1oso1,b sll q selnlos Jo suolleJluasuoJ ulPlulsru 01 fiec eq] smollu uorlJunJ Jarrreq srqJ 'salnoelour relod lsolu Jo a8essed aql sluanard seueJqrrreru1ac3o raLelq prdl aqt touelur crqoqdorprtq sllJo asneoag

seueJquawJoseluedord le)ul)all aqt puesaln)elow lleus Jolrodsuerleuerquaw

)o^eltqoql ssor)esasnJJtp aln)alou aql /(lptdelaloLu aql'raleMqltMsolet)osse lt ^l6uorts s s a al q ] ' { ; l u e l r o d u ar l o u ' p u e a l n l o l o u eql ralletrjsaql'saln)alou Jo sssspl) luara;Jrpo1ra{e;rqprdrll1eqlu{s e aq1 1- 11e.rn6r3 1oI1r;rqeeuladanl1e1ar raiieltq pto!l :r1aq1u{s

t)

*t 6 tltl ' -P-)

.,,.1'-,:

+) :UJH

sNol

*EN,*H

asor)ns asornl6

5lt n)tto|/\ UVIOd CI9UVH)Nn ]DUVI

1o-rer{16 Sllnfl1OlAl uvlod earn cl9uvH)Nn n zu ttvtAs sauou.]loq ptoJals ZN

zo) zO

slln)l'lot )tS0HdoucAH

's,4aupDlaql ur seuols aups^J uorleruroJ aq] o] speal aurrn eq] ul eulls^J Jo Jo uorlElnrunJJe Suqpsar aql :poolq aql otur aunsalq eqt Jo eurJn eql Jaqlra luo4 (autalsdr Jo reurp pe{url-aprJlnsp aql 'aur1s,{cSurpnlcur) sprJe ourrup ureuaJ UodsueJl louuet 'aldruexarc!'arJnurts[J esBasrppalrraqu aq] q]y!\ slBnpr^pul 'adft 'eurlsalur faupq aql q a]nlos crJrladsE 1ac Jeqlo Jo Jo uodsuer] eql Jap -ulq sesEasrppalrJequ snorre^ ruo4 JaJJnssuorleln{u relrurs qlp^ sueunq ]eq] 'suraloJd leql ^ ou) ^^ou aM uodsupJl eueJqrueu Jo .,{}rJrJroadsaql 3ur}BJls -uoruap,{qaraq} 'euBrqueu eluseld Jlaql ssoJJesJBBnsUodsuBJtol elqeun aJoM uollelnlu auaS-alSurse qlr^\ prretrEq lPql punoJ s096I aq] uI sarpnls 'ssplr aqlJo salcadsJBInJalou ureuac dpo uago pue (sprle ourrue Jo 'sJBBns'suor se qcns) alnoelotu Jo ssplc JelnJrued e suodsueJ] uraloJd qceg 'saueJqurarupcrSolorq Jo saddt IIe ur puB sruJoJl(uelu ur rnJco sura]ord eseqJ'seueJqrueu IIaJ ssoJJp salnlos qcns JaJSUEJIsulaloJd UodsueJl erruJqrueu IerJedS d1.,vro1s ,rfuan,r{po sre,r(epqprdl crlaqluds ssorc leql saqoqelaru IIaJ dueru pue 'saprloalJnu 'sprce oururE 'sre8ns 'suor sp qcns (salnJeloru Jplod snorJe^go a8essed aql ^\olle o] anpq osp tanamoq'sauerqruau IIaJ 'uorsnJJrpaldurrs^,(qaleaurrad ol salnceloru Jeloduou pue ralem ,lrolle sauerqrxau IIaJ 'sredelrq pldll f,Ileqlufs e{r'I

slauueq)pue sralJodsuerl :sulalorduodsuerlauerquawJosassel)urewoAI arv aroql '(z-II arnSlc) +) Jo +BNSEsuor Iletus qcns ua^a ()1uEql Jelel ol alqBeruJadaroru selull 60I are sra,{epqprdq crlaqluds 'snq1 'ra^,(e1q aq};o aseqd uoqrecorp.,{qaq} Surra}ua ruoq uaq] sluanard salnJelou qJns Jo uorlerp.,tqgo aar8ap q8q pue a8reqc aql :llerus ^ oq Jetleru ou '(suor) salnJalou pa8reqc ol alqeauJadur fp8rq aru s;adelq pIdI']sertuoc /tS'(I-II arn8tC),{1,r,ro1s eJor.uqJnu tlaqlE'redBlrq E ssorJe esnJJIposle 'eaJn Jo Jale^ se qlns 'salnJeloru JBIod pe8JeqJun IIeUS 'tueql ssorcedprder asnJJlparoJaraq]pup sra/tepqpldl uI anlossrpdppear '263 'ra,r(e1qpldl B ssoJce esnJJrp pue zO se qcns 'salncaloru Jeloduou leurg IIrM (crqoqdorp,{qaroru aqt) l1 fprder arou eqt '(s1tr 'reloduou ro Iro ur sr }l alqnlos eJoru aq] pue alnJalotu aqt Jallerus aql 'praua8 q 'Io ut &mqnlos alrlelal slr 'flsnoruJoue serJel uo ,r(1]soru tnq a1ncalotuaql Jo azrs aql uo -{1uedSurpuadap 'Jale,troq 'uorsnJJrp aleJ aqJ ']uerpEJBuorlerluacuoo sll ulrop radegq pldl Jo aar;-uralord e ssoJJe esnJJIpIlr,rr alnoalotu ,tue l.1pnlrrn 'aruq q8noue uelrC

suolol olqpouredurlIlq6!H arv sralel!8p!d!] oarl-ulalord

satrauebro p J r o t s ' + z p J . r o J 'spauleu e l s q n s l a qpl ou es u r a l o rodt p u n o q, { l i s o ua r eq t o q s n o u eu Ar q ] r M y1luOZlnoqprroleto]e sraraql suoroar1Jqlro; arpuanr6 lnq'sllal ur rzEJlAu.rZ I pup*761,\1 pue+.eJlo suorlerlurluolrq_L(rte'sdnot6;{xoqrerpueateqdsoqd 6utfuretsattloqptrur ,761,1 , O t ) P ) t J r 1 L ' ( J . o r o lrdo a ' O t H \ p O 6 , e U t , { o r , r r e 6 o u d , p q l U J " l l t I S U O ) l r J ) l s o t r ' l ) e l J : J l \ y L p J r c rl o l J ( L o . L pr J q l o^ J P J s L r P t L oL)d) J q t ' l ) o l u o r l r p pJer ' s . r q _{ e r l n a u , {e1) u l ) J l J 'sl puear,tltsod aq ]snLU a,rtle6au tl tpq])sa6-reqr 1osaltluenblenbauteluotlsnLUllJ) aql+

0t,

I r-9

-l)

*suoruv f t ' L H d ) oh l , r _ 0 t )s 0 [ x i Z_L z-L

s 9VL

ft'LHdroWzz_0t)s0LxL r-o I :U

obL 5t - 5

+H *zPJ

*zbw +) +eN

lla) ue!leuuew ;errd{1e apls}ng pup aprsulsuo!}erlua)uo)uol Jo uospeduo) V t- t t alqel

seuerqtuew ro sarlJedord le)!Jl)el:leql pue saln)alowlleus ro Uodsue{ auprquaw: t I Ja]deq)

zs9

653

PRINCIPLES OF MEMBRANE TRANSPORT Figure 11-2 Permeabilitycoefficientsfor the passageof various

aso'u'le ffj::ll,"jJiH::i;',,::iil113:1f;f li:,n"J?,:1."I:5nof

high permeability

this Multiplying concentration on thetwo sidesof themembrane. (inmol/cm3) coefficient bythepermeability concentration difference (incm/sec) persquare givestheflowof solutein molespersecond of of tryptophan A concentration difference centimeter of bilayer. (10-4110-3 a flowof wouldcause L = 0.1M),forexample, 10-amol/cm3 = 10-11mol/sec or x 10-7cm/sec through1 cm2of bilayer, 10-amol/cm3 through1 ;rm2of bilayer. 6 x 104molecules/sec

-- 10'

All membrane transport proteins that have been studied in detail have been found to be multipass transmembrane proteins-that is, their polypeptide chains traversethe lipid bilayer multiple times. By forming a continuous protein pathway across the membrane, these proteins enable specific hydrophilic solutes to cross the membrane without coming into direct contact with the hydrophobic interior of the lipid bilayer. Transporters and channels are the two major classesof membrane transport proteins (Figure 1l-3). Transporters (also called carriers,or permeases)bind the specific solute to be transported and undergo a series of conformational changes to transfer the bound solute across the membrane. Channels, in contrast, interact with the solute to be transported much more weakly. They form aqueous pores that extend acrossthe lipid bilayer; when open, these pores allow specific solutes (usually inorganic ions of appropriate size and charge) to pass through them and thereby cross the membrane. Not surprisingly, transport through channels occurs at a much faster rate than transport mediated by transporters. Although water can diffuse across synthetic lipid bilayers, all cells contain specific channel proteins (called water channels,or aquaporins) th'atgreatly increase the permeability of these membranes to water, as we discuss later'

tryptophan + g l u c o s e+

Ll

-

1 9a

_

t n-10

+

K++ Nar

t a-12 ru

+-

-

10 - 1 4

low permeability

Coupledto an ActiveTransportls Mediatedby Transporters EnergySource All channels and many transporters allow solutes to cross the membrane only passively ("downhill"), a process called passive transport, or facilitated diffusion. In the case of transport of a single uncharged molecule, the difference in the concentration on the two sides of the membrane-its concentration gradient-drives passivetransport and determines its direction (Figure f f -44). If the solute carries a net charge, however, both its concentration gradient and the electrical potential difference across the membrane, the membrane potential, influence its transport. The concentration gradient and the electrical gradient combine to form a net driving force, the electrochemical gradient, for each charged solute (Figure 11-4B). We discuss electrochemical gradients in more detail in Chapter 14. In fact, almost all plasma membranes have an electrical potential difference (voltage gradient) across them, with the inside usually negative with respect to the outside. This potential difference favors the entry of positively charged ions into the cell but opposes the entry of negatively chargedions.

and channel Figure11-3 Transporters proteins.(A)A transporteralternates betweentwo conformations,so that the siteis sequentially solute-binding on one sideof the bilayerand accessible a then on the other.(B)In contrast, pore channelproteinformsa water-filled acrossthe bilayerthroughwhichspecific solutescandiffuse.

solute

solute-binding site

aqueous pore (A) TRANSPORTER

(B) CHANNEP L ROTEIN

'l Chapter l: MembraneTransportof SmallMolecules and the Electrical Properties of Membranes

654 (A)

t r a n s p o r t e dm o l e c u l e

c h an n e l proletn

I

ripia I bilaver I

concentration qradient

\?^ simple diffusion

channelmediated

I

tra nsporterm e di a t e d

PASSIVE TRANSPORT

Aac,

I

I

ACTIVETRANSPORT

(B) ."+ .1 +t.t-tf'titr+ '+ ** ouTSlDE

i+ ].:!.

+ +

*rtr+ +++

+

+

+

+-

+

+

++

+

electrochemical electrochemical g r a d i e n tw i t h n o g r a d i e n tw i t h membranepotential membranepotential n e g a t i v ei n s i d e

electrochemical g r a d i e n tw i t h membranepotential p o s i t i v ei n s i d e

cells also require transport proteins that will actively pump certain solutes across the membrane against their electrochemical gradients ("uphill"); this process,known as active transport, is mediated by transporters, which are also called pumps. In active transport, the pumping activity of the transporter is directional because it is tightly coupled to a source of metabolic energy,such as ArP hydrolysis or an ion gradient, as discussed later. Thus, transmembrane movement of small molecules mediated by transporters can be either active or passive,whereas that mediated by channels is alwavs Dassive.

Su m m a r y Lipid bilayers are highly impermeable to most polar molecules.To transport small water-solublemoleculesinto or out of cellsor intracellular membrane-enclosed compartments, cell membranescontain uarious membrane transport proteins, each of which is responsiblefor transferring a particular solute or classof solutesacrossthe membrane. Thereare two classesof membrane transport proteins-transporters and channels. Both form continuous protein pathways acrossthe lipid bilayer. Wereas transmembranemouementmediatedby transporterscan be either actiueor passiue, soluteflow through channel proteins is always passiue.

TRANSPORTERS ANDACTIVE MEMBRANE TRANSPORT The process by which a transporter transfers a solute molecule across the lipid bilayer resemblesan enzyme-substrate reaction, and in manyways transporters behave like enzymes. In contrast to ordinary enzyme-substrate reactions, ho*ever,the transporter does not modify the transported solute but instead delivers it unchanged to the other side of the membrane. Each type of transporter has one or more specific binding sites for its solute (substrate). It transfers the solute across the lipid bilayer by undergoing

Figure11-4 Passive and active transport compared.(A) Passive transportdown an electrochemical gradientoccursspontaneously, eitherby simplediffusionthroughthe lipid bilayer or by facilitated diffusionthrough channelsand passive transporters. By contrast, activetransportrequiresan input of metabolicenergyand is always mediatedby transporters that harvest metabolicenergyto pump the solute againstits electrochemical gradient. (B)An electrochemical gradient combinesthe membranepotentialand gradient;they can the concentration work additivelyto increase the driving forceon an ion acrossthe membrane (middle)or can work againsteach other (right).

65s

TRANSPORT TRANSPORTERS AND ACTIVEMEMBRANE

Figure11-5 A model of how a conformationalchangein a transporter could mediate the passivemovement of a solute.Thetransportershowncan exist in two conformationalstates:in stateA, the bindingsitesfor soluteareexposedon the outsideof the lipid bilayer;in stateB, the samesitesareexposedon the other Thetransitionbetween sideof the bilayer. the two statescanoccurrandomly.lt is and doesnot completelyreversible dependon whetherthe solutebindingsite is occupied.Therefore,if the solute is higheron the outsideof concentration the bilayer,moresolutebindsto the than in transporterin the A conformation and thereis a net the B conformation, transportof solutedown its concentration gradient(or,if the soluteis an ion,down gradient). its electrochemical

s o lu t e stale A

Stale E

OUTSIDE

ripid I bilaver I

transportermediating oassrvetransDort

solute-bindingsite

reversible conformational changes that alternately expose the solute-binding site first on one side of the membrane and then on the other. Figure I l-5 shows a schematic model of how a transporter operates.lVhen the transporter is saturated (that is, when all solute-binding sites are occupied), the rate of transport is maximal. This rate, referred to as V,r'* (Vfor velocity), is characteristic of the specific carrier. I/-* measures the rate with which the carrier can flip between its two conformational states.In addition, each transporter has a characteristic affinity for its solute, reflected in the K- of the reaction, which is equal to the concentration of solute when the transport rate is half its maximum value (Figure I l-6). As with enzymes, the binding of solute can be blocked specifically by either competitive inhibitors (which compete for the same binding site and may or may not be transported) or noncompetitive inhibitors (which bind elsewhere and specifically alter the structure of the transporter). As we discuss below it requires only a relatively minor modification of the model shown in Figure 11-5 to link a transporter to a source of energy in order to pump a solute uphill against its electrochemical gradient. Cells carry out such active transport in three main ways (Figure I l-7): 1. Coupled transporterscouple the uphill transport of one solute across the membrane to the dornmhilltransport of another. 2. ATP-driuen pumps couple uphill transport to the hydrolysis of ATP 3. Light-driuen pumps, which are found mainly in bacteria and archaea,couple uphill transport to an input of energy from light, as with bacteriorhodopsin (discussedin Chapter l0). Amino acid sequence comparisons suggest that, in many cases,there are strong similarities in molecular design between transporters that mediate active transport and those that mediate passivetransport. Some bacterial transporters, for example that use the energy stored in the H+ gradient across the plasma membrane to drive the active uptake of various sugarsare structurally similar to the transporters that mediate passive glucose transport into most animal cells. This suggestsan evolutionary relationship between various transporters. Given the importance of small metabolites and sugars as energy sources,it is not surprising that the superfamily of transporters is an ancient one' We begin our discussion of active transport by considering transporters that are driven by ion gradients.These proteins have a crucial role in the transport of small metabolites across membranes in all cells. We then discuss ATP-driven pumps, including the Na+ pump that is found in the plasma membrane of almost all cells. r

Figure11-6 The kineticsof simplediffusionand transporter-mediated the rateof simplediffusionis alwaysproportionalto diffusion.Whereas diffusion the rateof transporter-mediated the soluteconcentration, Thesolute reaches a maximum(V."*)when the transporteris saturated. the when transportis at halfits maximalvalueapproximates concentration to for the soluteand is analogous bindingconstant(Kr) of the transporter Thegraphappliesto a transporter the Kr of an enzymefor its substrate. movinga singlesolute;the kineticsof coupledtransportof two or more solutesis morecomplex.

g

v'"'

c 6

g 1/2V^6, o

concentrationof + t r a n s p o r t e dm o l e c u l e

656

Chapter11:MembraneTransportof SmallMolecules and the Electrical Properties of Membranes

I

ll electrochemical qradient

-

Figure 1I -7 Threeways of driving active transport.Theactivelytransported moleculeis shownin yellow,and the energysourceis shown in red.

it

ADP COUPLED TRANSPORTER

ATP-DRIVEN PUMP

LIGHT-DRIVEN PUMP

ActiveTransportCanBeDrivenby lon Gradients

same direction, performed by symporters (also called co-transporters),or the transfer of a second solute in the opposite direction, performed by antiporters (also called exchangers)(Figure ll-8). The tight coupling between the transfer of two solutes allows these coupled

gradient of which provides a large driving force for the active transport of a second molecule. The Na+ that enters the cell during transport is subsequently pumped out by an ArP-driven Na+ pump in the plasma membrane (as we discuss later), which, by maintaining the Na+ gradient, indirectly drives the transport. (For this reason ion-driven carriers are said to mediate second.aryactiue transport, whereasArP-driven carriers are said to mediate primary actiue transport.) Intestinal and kidney epithelial cells, for example, contain a variety of symporters that are driven by the Na+ gradient across the plasma membrane. E'ach Na*-driven symporter is specific for importing a small group of related sugarsor amino acids into the cell, and the solute and Na* bind to different sites on the transporter. Becausethe Na+ tends to move into the cell down its electrochemical gradient, the sugar or amino acid is, in a sense,"dragged" into the cell with it. The greater the electrochemical gradient for Na+, the gieater the rate of solute

t r a n s p o r t e dm o l e c u l e

c o - t r a n s p o r t e ido n

,/\

-l ,,0,0 uitaver I \ UNIPORT

SYMPORT

I

ANTIPORT

c o u p l e dt r a n s p o r t

Figure 1I -8 Threetypes of transportermediated movement.This schematic diagramshowstransporters functioningas uniporters, symporters, and antiporters.

657

TRANSPORT TRANSPORTERS AND ACTIVEMEMBRANE

Na*

u*

+' + +

++

+

stateA a1 eq1 gg- 11 arn611 Frit*]

-,oulq'q

I

r89

lv)lul)lll lO Slll-UldOUd SINVU8IAIW

IHI CNVSl:INNVH)NOI

Jaqlra sr ]r :pe^orueJ,{lprder sr lr 'pa}aJJasuaaq suq Ja}lrusueJloJnau aqt Jauv 'uaq] Suruado pue (gt-II arn31g) spuuDLIJ uot pary?-taiqusuau o1 Surpurq r{q 1ac cr}deu,,(slsodaql ur a8ueqo leorJ}Jalaue sa4onordpue Uelc cqdeufs aql ssorcedlprdeJsasnJJrpJallnusuerlornau aqJ'slsoldcoxa Lq pasealal pue selorse^ cndeuds pasolcue-euerqueu ur peJols erp qorqm'sJelllusrruJloJneu sp u AoDI salncalotu pu8rs aseeler ol sraSSrn cr1deu,,(sard aql ur pr]ua]od lr leus flac '{alr uidauts Morrpu e [q Iecrrlcele ;o a8ueqc V 77ac4ldautslsod aq] uro4 paleredas Swaqyac utdautsatd eqt taqloue auo ruo4 palelosr l.1pcu1ca1aare slleJ eqJ 'lJeJrpur sr uorssnusueJlJo ursrueqcau Iensn aql 'sasdewb sE rr \oDI loeluoc Jo salrs pazrprcads ]B IIaJ ol IIaJ ruoq pailr{usu€Jl are spu8rs puorneN

sesdeu/(S le)rureq)le seuOle)ul)otf olur sleu6rsle)ruraq)ue^uo) sleuueq)uol paleg-rell!ursue4 ' ( s 1 gr o ' s u o r s -pnuoc) seJnzlescpdayda asnec suoJnau;o sdnor8 a8rel;o Surrg pazruorqcu.4.s alrsseJxa qJrqm ur asnp3 uec ureJq aql ur sleuueqJ +) Jo +ENloeJJElpql 'uorlJeJ}uol alJsnu pue uorlezrJelodap eueJqruau salerl "{sdapda suorlelnru .{pepurg -rurar,4.1pa1eadar pue seqsrugpuualod uorlJe ue JeUpslsrsrad.'fulua+eN ']lnseJe se l,{lerurou elEAr}JEuro} IruJslauueqJ IeruJouqe aq} esneJeq sJnf,cosrql'saseO eruos uI 'sruseds alf,snru p;ured Sursnec 'uollcurluoc drelunlon JeUe uoqp -xeler qclq alJsnu ur.{e1ap E sr aJaq} ^ ul uoplpuoc e'aruorctw asnec uec 'a1d -urexaJoJ'sllal alJsnu pe1e3-a3€]lolapocua leql seue8 IplaleTsur sleuueql *e111 ur suortetnl4l'suorlJunJflerurou eue8 luptnru aql dq papooua lauueqJ eql sllaJ 'saseasrp 'ureJq 'apsntu 'elJau ,r(larrene tuo4 JaJ qJIqM ur Surpuadap Jo uear{ Jo -Jns ueo suralord uor Surpocua seua8 luelnru llJequr oq \ sueunH Iauueqc 'sasuodsals1ralndruoc o] sleuueqJ uor luaJeJJrpJo Jaqrunu a8rel e sesn suoJnau 7gg ,r(po go dn apeu ruatsl.s snolJau aydrursp uene leql seleorpul ftr-xaldruoc srqJ 'slauueqJ +) patEIaJtnq tuaJaJJrpapocue ]eql saua8 gg surpluoc sunBa1a'3 ruJo^\ aq] Jo eruoue8 aql 'lauueqJ *; pa1e8-a8etlo^e JoJsepoo lpql aua8 a13urs p sureluoJ aacrralaJ'S ]sea,(pa11ac-a13urs eql spaJer{\'saydrcuud u8rsap aq};o .,(ueu areqs pue suralord peleleJ .{1ern1cnr1spue .{lueuorlnlo^e;o l.Irue;radns a8rel e o1 3uo1aq p ,{aqr 1eq1Surlerlsuouap 'sarlrrepurrsSuurr}s ^\oqs slau -ueqJ pue '+) '*e1.tr pa1e3-a8e11on prce U^,ltoDI eql saJuanbas ourup aq] +ze3 Jo 'sseleqleuoN 'aua8 aures pacnpord eqt tuo4 sldrrcsuer] Surcrlds alrleu VNU Jo -rallB eql ,tq pue saua8 aldrlpu .{q qfoq paleraua8 'sasselJeerql asaq} qJpa Jo ulqllm ,{lrsranrppuor}cunJ pue SursrrdrnsB sr alaqJ }unoure IeJnlcnJls Jo 'sleuueqf, +ENuo ueql JaqlEJslauuaqJ+pJ pawS-aBalpn uo puadap 'aldurexaJoJ'sllaf,eurJcopua pue '33a 'elJsnru aruos ur spuualod uorlte aql 'lerluelod uor]Jp ue aleJe -ua8 uec leql puol dpo aq] lou aJEslauueqJ +BN IauuEqJ uorleJ pa1e3-a8e11on;o

patelau{;;ern1rnl15 pue^l!teuorlnlo^lotv slauueq)uo!le) paleg-a6etlon '(IS-II arn8rg o1pua8al aas)puuetod auerqruaur aq] ur sa8ueqcdq paralp sr ]eq] Surddrg lsurp8p suorleruroJuoJpelplrtceur pue 'uado 'pesolJ aql Jo drgq -els anrlelal aql sl 1l pue 's8urpunorrns eql Jo sluatualoru leureql uopuer aql dq tlof luarclggnse ua,rr8;r uolleruJoJuooJeqloue ol ,,dU,,ueJ uorlpruJoJuoJqcuf, 'lauueqc aq1 uado leql seBuEqJ lpuorlpturo;uoc Suua8Brrl 'pre,,vqnoSurnoru .{q uoqezuelodap o1puodsar 1eq}s}uaur8esaueJqtueursueJlJraqlJo auo ur spr3e ourrue pa8reqc ,,{lanrlrsodcl}srJel3eJeqJaleq 'aldurexe JoJ 'slauueqc +zEJ pue '*) '*eN pa1e8-a8e11on'plarJ aqlJo qlSuerts aq] uo puadap serlr1rqetsesoq^ suorl -eurJoJuoca^rleuJalle ldope uec sleuueqc uor pa1e8-a8ellol ]nq ,luecgru8rsur dlqeqord ale plerJ IeJrJlcaleeueJqrueu aql ur sa8ueqJ Jo slJaJJaaql suralord aueJqr.ueu ,r(uerurog 'alnlsnJls Jplncalou eq] uo saJJoJsuexe eJoJaraq] plerJ pcrrlcele aqJ 'sruole snorJpl Jreql ueemleq spuoq pezrreyodse lla,tlrse ,sdnor8 pa8reqc ,r(ueruaneq 'sreqlo ilp a{I 'suralord esaqJ 'uorteruJoJuoJJraq} }JaJJE ,{lpunoyord ueJ leqt plarJ leJrr}Jela a8rel ,{ran e ol palcafqns snqt aJe aueJquraru

seueJquraw 1osalyador6le)lrpelf aql pue saln)alowlleulsJoyodsuerl euerqruaw:11laldeql

289

'auPJquau IIaJ ]a3JE1etuBseql ul ]uaseJd eJP]Pq1sleuueqc uollEs palES -e8ello^ fqreau Jo Jaqrunu luerouJns e uado ol q8noua sazlJelodep1el]uelod auerqrueu pcol aq] JI ^luo etrs slql uroq peJe88lJl eq uBf, lEllualod uol]ce uv'aJaql slsrsJadl! 3uo1 ^ oq puP esdEuds aql lE pasEaleJJeilnusuEJloJnau 'ppualod euBrqluau Jo sa8ueq3 Jo lunolue aq] ol Surprocce pepeJ8 aJPlEql aJueq puB 'se8upqJ ,'blllqeetuJad Iecol eJnpoJd z(aql 'pealsul 'uol]E]Icxe SuLiI -Idue-Jlas e ef,npord sallasueqt rtq louuec eJoJeJeq]pue puuetod auerqruaur aq] ol a^Illsuesut rtlannelal ere slauueqJ palES-JatllrusueJ] 'sprlualod uollJe JoJ ('aureU )upef lo ^sauno)'B)'lle) aql a{llun '(VSg-f f arnBICeas) auurqrueu alqrsuodsar sleuuEqr pa1e3-a8e11o^ )rtdeulslsode Jo elupuape uo sasdeuls eql ur e8ueqc ftlpqearurad Jauq e Surcnpord ,{qeraql 'salncalotu Jal}Ilusuer}oJ leurural a^rau oMl Jo qdelbol)il,u -nau Surpurq aql o] esuodsal ur dllualsu€J] uedo pue asdPu.{saqt Jo uolSal aq} uorl)alauorl)asurql v (€)'a^laupatt)xa Jo ur IIes srldpu^slsod eql Jo euerquau eluseld aql q paleDuaJuos eJEsleuueqJ aql uror1leu6rse 6urprusuetlIqaraql '1;ar 'sasdeufs auelqulaul 1a6releqt;o ;er1ue1od aq1 leJruaqc 1e spu8ts lEf,IJlcelaolut qeu8ts IscllueqJ rElnlla]eJlxe aql rollpsmolJuor6ur1;nsat eq1'asdeu{s pazler3eds slauueqJ uol pa1e3-replrusrru.{L ,(prder are Surlrarruoc JoJ aqt le llar la6.reltrldeulslsodaq1 'lualxe rlJnlu B o] lnq suoJnau r(q pasn oslP eJe qclq^l. JellElus Joauerqureueurselooq] ut palelluo)uo) '(61 raldeq3 ur pessnJsrp)sasdouts Tac4pala le suollJunf de8 ern Suldnor leclr] slauueq)uorpa1e6-te11rtusuet1 -Jala lcanp ueql alqeldepe pue e[]esJal eJoIu JBJslsasdnuts laJnaaLp qcns BI aq] suaoopue ol spurqrallrursuellolnau SuueuBrs'aes IIEqs arlt sV'llal cDduu.,klsodeql 01 palef,IunurruoJ.,(lalerncceaq uEJ sluela Sulpu8rs prder 'paleadar;o Sunup aq] leql os 'pasealeJsI JellllusueJ] -oJnau aslnd gau aq] eJoJeqgalc cudeuds aq] sJealJll pue 'slleJ Surroqq8rau Jo 'asdeuds acuanuur IIr^\ JelltusueJloJneu aql teql secueqc aql sesBerlaP ]I e te Surrcu8rsJo uorsnerd produral pue prleds qloq salnsua pnourar ptdeg 'asealar saler q31q qlpr dn daal ol slleJ 8umo1p 'palcdcar eJ€ sJaullusuerl Jo -orneu ,{em srql ur lsra}rodsueJl Ja}}IusueJloJneu luapuadap-*eg ;o ,tlauen e ,{q palerparu sr aleldneu 'sllaf, pq8 Supunorrns ztq Jo tI paseeler }eqt puru -Ja] alJau eq].{q dn ua1e1Jo UelJ cudeu,{s aql ut saurrtzue f,UIJeds,rtqpe,'(orlsap pasealaloqf'leuturrala^lau aqt Jo auprqrrau etuseldaql qllm asnJsal)tsan aql uoqMloualxalla) aql o] pasealal ale pue sal)rsan rrldeur(sur pauteluo) aresalnfelourraDrLusuellolnau aLll'rallil.llsuerlornau slt asealalol 1r';1er>r1deu{setd leururalaql salelnLUrls e ur leurulra]a^lau aql saq)eat uorl)pue uaqM(V) ler1ua1od 'asdeuls le)!uaq) y gg- 11 arn613

(8)

saprsanrrldeui{s

(v)

Sffi" 0(}

ISdVNAS'lV)ilAlH) lAlr)V

auelqulau eurselo uiti Z

lsdvNAs'1v)ilA3 Hf 9Nll-SlU .. 1 1 a1r a 6 r e 1 .....>r1deu[s1sod tauueu) *"oo'oo ,;;;;;'; N/ v'

-raulLusuerl

;rep:rldeuAs s a ; : r s arnr l d e u , { su t Jelltr!suerlojnau 11e:rldeufse.rd ] o l e u r t ! J a ]a ^ J a u

SlNVUgllW JO Slll-uldOud]V)lUrf:lll IHJCNVSIINNVHfNOI

€89

'peururJalep ueaq sEr{ aJnlJnJ}s 'peJuenbas ('sraqsrlqnd le lraqp leuorsueurp-aaJql slr pue frtlapP)v raMnl) uolj uotssil'uJad qllM puE pauolJ eq ol aueS IauuBqJ uor ]sJu eql oslE se^ euaS stl 'pepJoJeJ '186t'0t [ - [ 0 t : 0 t ' l o ] b u n a N T , e l e q a n^ se^ Isuueqf, uado a13urse Jo leuSrs leJrJlJele eql qJrq \ JoJ]sJUeql pue 'sJede[q pue rlesac 'r Luoll)'uMoqsst al)snuJ lla) prdll Jrlaqlufs ur pelnlnsuoJar fllpuoncury aq ol lsrg eql 'peururalep eJuanbas letalalse uo uoxeal6utselo uotleutiulal 'pagrrnd aq ol aql'60r, e u! uoll)un[reln)snu.tornau prcp ourrue alaldruoc str eAEr{ol }srg eqt lauueqJ uor tsJg eql selltU 'slauuer{J prJeds uor;o.,{.ro1srq aql u1 aceld e seq JoldeJal eulloqJlqaJu eql e Jo qdeJ6o.t)luluorl)ala 6uluuers 'tuels^s sno^Jeu y 99-11 ern613 uo11e>111ubeu-mo1 IuJluaJ eql ur sasdeuds aqt Jo tsotu aIIFn ,(pn1s pcrSolorsdqdorlcala o] elqrsseJce ,{lrpear sl tl asneJaq palu8llse^u ,(lanrsualur ueeq seq asdeurts s1ql '(gt-I I arn8lg 1ar alJsnur lelela{s e puu uoJneu Joloru e ueemleq asdeur(s pcruaqc pezuercads aql-uollJun[ repcsnuoJnau E ]u leurruJal e^Jau aq] ruoq pasEalal aurloqs 'sllal alJsnru p}ala{s go to1dacat -y&ace ,(q ,{.puarsuerl pauado sr lauueqr srql auryoqcg&aco eql sr lauueqc uor pa1e3-reltrusueJlEgo alduexa parpnls-tsaq aql

sleuuPr.l) uorle] pale9-raD!usupll elv uolpunr lelntsnutotnaNaql le sloldo)auoutloq)l^le)veql

u o x ep a l e u r ; a { u t

lla) opsnur

'sef,uenbesuosslr ur 8uqsu1raBuol pue 'xa1duof, eJor.upue Je ^ols JBJeq ol spual sroldacar paldnoc -aurfzua pue sroldacar paldnoc-uralord-g o1 Surpurq spue8u rtq parerpau 3ur 'aldurrs 'alerpaururr dlpraua8 $ sleuupqJ uor pa1e8-reglrusueJl -pu8rs ?euq pup o1 Surpurq sJallrusueJlornau ,irolrqrqur pue drotelrcxa ,(q palerpaur SuueuBrs sparar{ A'9I raldeq3 ur Irelep ulstoldacat pagdnoc-auttzua pue uoldacat pa1dnoc -u1a7oh-g pel1po-oseseql ssnosrp a1q ,(ltcarrpur dpo slauueqJ uor a1e1n3arleql sroldacar ol pulq 'sappdadornau;o dlauen a8rel e Surpnlcur 'sleunuJal alJeu dq palarcas aJe teql selnoelotu Sulpu8rs aqt;o r(ue6 'slauueqf, uor pa1e3-pue8rt q8norql saleredo uralsrk snolreu aql u1 Surpu8rs pcruaqc 11elou telellroH 'qleap 'suolsln^uos puE 'srusedselosnru sesnpJ 'aurcd18 uorlJe dJollqqul aqr 3ur-1co1q puu sroldacar Jo aurc.{13o1 Surpurq dq 'alduexa ro; 'auruqcfuls :sJenrusupJloJneu ,fuo}lqqul ;o acuelrodur eql aleJlsuouap uorloe Jreqt {Jolq leql suxot Jo slceJJe eqJ ']JeJJerelrrms B ser{ sleuueef, +) Jo Suruado eqJ 'lleJ eql elloxe ot aJueq pup euprqrueu eqt azuelodap ol tlnJrJJrperor.utl se{Eru sleuueqr _13;o Suruado eql 'snql 'uorJlezrJelodapaqr lJpJelunoo pue ilac eql Jelua suor _lJ pa8reqc dlanrle8au aJour 'ezuulodap o1 slJuls euuJqrueu eql se 1p4ua1od eueJqrueu eql JaJJnq01 spuat slauuuqc -13 Suruado 'uoseal srql JoC 'alrle8au aJoru uele Jo-lprlualod Surlsar eql ol asolo sl -1f, roJ Ippualod urnrrqlpnba eql 'suoJnau dueur rog'tJeJuI 'xngul slr sasoddoppuatod eueJqrueu aqt lnq'(Zgg'd'I-II elqeJ ees) eprsur uuqt eq] eprstno raq31q qcnru sr _lJ Jo uor]eJtueJuoJ eqJ flec 'smolloJ se sleuueqo -13 Suruado Jo lcaJJaar{l pue}sJapun uec eAA'eueJqueru B saztrelodap slauueqJ uorleJ go Suruado eql ^\oq pessnJsrpdpearp eAEqaM 'ureJq aleJqaual aq] ur SurTeu8rs,fuolellcxa aql 'aJuulsur Jo lsoru salerpau JoJ 'aleurelnlC 'sJe1rrusuerl drolrqrqur se pesn arc au1ct73pue (VW| ptco ctttl 'sJallrlusuert -nqoutrun-l, pue ,,fuotelrJxe pasn aJE uruololas pue 'a1.nua7n73 sp 'auqoqcl,Qace talamoq f.lpnsn 'ot spurq stoldacar au11oqc1rfiace;oad.{1eql ll uo Sutpuadap 'llqqul Jo elrf,xeraqlle upo 'aldurexarcg'auqoqcyQary'Jelunocue daqt reqr suoprpuoc Jruor eq] pue 'ol purq ,{aqt sro}dacar }eqm 'paseeleJ are ,taql eJeqm uo Surpuadap 'drolrqrqur ro ,{rolelrcxa Jar{}le eq uec sJe}}rru -suer1due141'aueJqruaru cqdeudslsod aql azlreyodap o] saJuangur drolelrcxa rog rapreq lg 3uorcur dq Suug sassarddns qqt pue 'slauueqc +) ro slauueqc _lJ raglra uado '1ser1uoc dq 'srallFusrru4oJneu ,fuof1q;qu1 'ppualod uorlJu ue Suug ro; puualod ploqseJqt eq] prulv\ol euprqrueu cpdeuklsod aql sazlrelodap leql 'slauueqJ uorleJ uado srelllursrruJloJneu ,fuo1u1gcxg *eN Jo xnlJur ue Sursnec 'asuodsar cqdeudslsod eq] Jo aJnleu eql seurruJelap sql :euerqrueur eurseld eqt ssoJJE ssed 1a1daqr reqr suor Jo ad.,! aql ur alrlJalas are ,taql 'slauueqJ se 'puof,as 'purruJel anrau ruduu.{sard aql ruo4 pasealeJ sr luql Ja}}tusup4 -oJneu eql JoJ alrs Surpurq enrlJeles [UBlq E eABqdaq] 'sroldacar se '1srrg 's.{.u,u. luelrodurr prelas ur Joqloue euo uorJ raJJrp slauueqr uor pa1e8-rallrursuer;

{.rolrqrqu;ro/fuolel!)x;lag ue) sasdeul5;elueq3 sauprquaw ;o saryedo.rSle)tJl)el:leql pue saln)elow lleus lo l.rodsuetl auerqulaw :1 1 raldeql

Vgg

I O NC H A N N E LA SN DT H EE L E C T R I C P AR L O P E R T IO EF SM E M B R A N E S

685

only moderate resolution. There were at least two reasonsfor the rapid progress in purifying and characterizing this receptor. First, an unusually rich source of the acetylcholine receptors exists in the electric organs of electric fish and rays (these organs are modified muscles designed to deliver a large electric shock to prey). Second, certain neurotoxins (such as a-bungarotoxln) in the venom of certain snakesbind with high affinity (Ku= 10sliters/mole) and specificity to the receptor and can therefore be used to purify it by affinity chromatography. FIuorescent or radiolabeled cr-bungarotoxin can also be used to localize and count acetylcholine receptors. In this way, researchershave shown that the receptors are densely packed in the muscle cell plasma membrane at a neuromuscular junction (about 20,000 such receptors per pm2), with relatively few receptors elsewherein the same membrane. The acetylcholine receptor of skeletalmuscle is composed of five transmembrane pollpeptides, two of one kind and three others, encoded by four separate genes. The four genes are strikingly similar in sequence, implying that they evolved from a single ancestral gene.The two identical polypeptides in the pentamer each contribute to one of two binding sites for acetylcholine that are nestled between adjoining subunits. \A/hen two acetylcholine molecules bind to the pentameric complex, they induce a conformational change: the helices that line the pore rotate to disrupt a ring of hydrophobic amino acids that blocks ion flow in the closed state.With ligand bound, the channel still flickers between open and closed states,but now it has a 90% probability of being open. This state continues until hydrolysis by a specific enzyme (acetylcholinesterase)locatedat the neuromuscular junction lowers the concentration of acetylcholine sufficiently. Once freed of its bound neurotransmitter, the acetylcholine receptor reverts to its initial resting state. If the presenceof acetylcholine persistsfor a prolonged time as a result of excessivenerve stimulation, the channel inactivates (Figure f f -37). The general shape of the acetylcholine receptor and the likely arrangement of its subunits have been determined by electron microscopy (Figure lf-38). The five subunits are arranged in a ring, forming a water-filled transmembrane channel that consists of a narrow pore through the lipid bilayer, which widens into vestibules at both ends. Clusters of negatively charged amino acids at either end of the pore help to exclude negative ions and encourage any positive ion of diameter less than 0.65 nm to pass through. The normal traffic consists chiefly of Na+ and K+,together with some Ca2*.Thus, unlike voltage-gated cation channels, such as the K+ channel discussed earlier, there is little selectivity among cations, and the relative contributions of the different cations to the current through the channel depend chiefly on their concentrations and on the electrochemical driving forces.\.Vhenthe muscle cell membrane is at its resting potential, the net driving force for K+is near zero, since the voltage gradient nearly balances the K+ concentration gradient across the membrane (see Panel l1-2, p. 670). For Na+,in contrast, the voltage gradient and the concentration gradient both act in the same direction to drive the ion into the cell. (The same is true for CaZ*,but the extracellular concentration of Ca2* is so much lower than that of Na+ that Caz* makes only a small contribution to the total inward current.)

unoccupied and closed

and closed ( in a c t i v a t e d )

\ \

occupied and open

Figure11-37 Threeconformationsof the acetylcholinereceptor.The binding opens molecules of two acetylcholine ion channel.lt thistransmitter-gated then maintainsa high probabilityof has beingopen untilthe acetylcholine been hydrolyzed.In the persistent presenceof acetylcholine,however,the (desensitizes). channelinactivates is rapidly the acetylcholine Normally, and the channelcloseswithin hydrolyzed well before about 1 millisecond, occurs. desensitization significant would occurafterabout Desensitization in the continued 20 milliseconds presence of acetylcholine.

686

Chapter11:MembraneTransportof SmallMolecules and the Electrical Properties of Membranes a c e t y l c h o l i n e - b i n d i ns g ite

a c e t y l c h o li n e -

binding SrIe

1 I

lipid bilayer

4nm

(A)

CYTOSOL (B)

gate

Therefore, the opening of the acetylcholine receptor channels leads to a large net influx of Na+ (a peak rate of about 30,000ions per channel each millisecond). This influx causes a membrane depolarization that signals the muscle to contract, as discussedbelow.

Transmitter-Gated lon Channels AreMajorTargetsfor Psychoactive Drugs The ion channels that open directly in responseto the neurotransmitters acetylcholine, serotonin, GABA, and glycine contain subunits that are structurally similar and probably form transmembrane pores in the same way, even though they have distinct neurotransmitter-binding specificities and ion selectivities. These channels are all built from homologous polypeptide subunits, which probably assemble as a pentamer resembling the acetylcholine receptor. Glutamate-gated ion channels are constructed from a distinct family of subunits and are thought to form tetramers resembling the K+ channels discussed earlier. For each classof transmitter-gated ion channel, there are alternative forms of each type ofsubunit, either encoded by distinct genesor generatedby alternative RNA splicing of the same gene product. The subunits assemblein different combinations to form an extremely diverse set of distinct channel subtypes,with different ligand affinities, different channel conductances,different rates of opening and closing, and different sensitivities to drugs and toxins. vertebrate neurons, for example,have acetylcholine-gatedion channels that differ from those of muscle cells in that they are usually formed from two subunits of one type and three of another; but there are at least nine genes coding for different versions of the first type of subunit and at least three coding for different versions of the second, with further diversity due to alternative RNA splicing. subsets of acetylcholinesensitive neurons performing different functions in the brain express different combinations of these subunits. This, in principle, and already to some extent in practice, makes it possible to design drugs targeted against narrowly defined groups of neurons or synapses,thereby specifically influencing particular brain functions. Indeed, transmitter-gated ion channels have for a long time been important targets for drugs. A surgeon, for example, can relax muscles for the duration of an operation by blocking the acetylcholine receptors on skeletal muscle cells with curare, a drug from a plant that was originally used by South American Indians to make poison arrows. Most drugs used to treat insomnia, anxiety, depression, and schizophrenia exert their effects at chemical synapses,and many of these act by binding to transmitter-gated channels. Both barbiturates and tranquilizers, such as valium and Librium, for example, bind to GABA receptors, potentiating the inhibitory action of GABA by allowing lower concentrations of this neurotransmitter to open cl- channels. The new molecular biology of ion

Figure| 1-38 A modelfor the structure of the acetylcholinereceptor.(A)Five homologoussubunits(cr,cr,F, y, 6) combineto form a transmembrane aqueouspore.Thepore is linedby a ring of fivetransmembrane crhelices, one contributedby eachsubunit.In its closed conformation, the poreis thoughtto be occludedby the hydrophobicsidechains of five leucines,one from eachcr helix, whichform a gate nearthe middleof the lipidbilayer. The negatively chargedside chainsat eitherend of the poreensure that only positivelychargedionspass throughthe channel.(B)Bothof the cxsubunitscontributeto an acetylcholine-binding sitenestled betweenadjoiningsubunits;when acetylcholine bindsto both sites,the channelundergoesa conformational changethat opensthe gate,possiblyby rotatingthe helicescontainingthe occludingleucinesto moveoutward.In the structuraldrawing (right),thepartsof the channelthat movein resoonse to AChRbindingto openthe poreare coloredin b/ue.(Adaptedfrom N. Unwin, Cel/72[5uppl.]:31-41, 1993.With permission from Elsevier.)

687

ION CHANNELS AND THEELECTRICAL PROPERTIES OF MEMERANES

channels, by revealing both their diversity and the details of their structure, holds out the hope of designing a new generation of psychoactive drugs that will act still more selectivelyto alleviate the miseries of mental illness. In addition to ion channels, many other components of the synaptic signaling machinery are potential targets for psychoactive drugs. As mentioned earlier, after releaseinto the synaptic cleft, many neurotransmitters are cleared by reuptake mechanisms mediated by Na*-driven transporters. The inhibition of such a transporter prolongs the effect of the transmitter and thereby strengthens synaptic transmission. Many antidepressant drugs, including Prozac,for example, inhibit the uptake of serotonin; others inhibit the uptake of both serotonin and norepinephrine. Ion channels are the basic molecular components from which neuronal devices for signaling and computation are built. To provide a glimpse of how sophisticated the functions of these devices can be, we consider several examples that demonstrate how groups of ion channels work together in slmaptic communication between electricallv excitable cells.

Activation Transmission lnvolvesthe Sequential Neuromuscular of FiveDifferentSetsof lon Channels The following process,in which a nerve impulse stimulates a muscle cell to contract, illustrates the importance of ion channels to electrically excitable cells. This apparently simple response requires the sequential activation of at least five different sets of ion channels, all within a few milliseconds (Figure I l-39). 1. The processis initiated when the nerve impulse reachesthe nerve terminal and depolarizes the plasma membrane of the terminal. The depolarization transiently opens voltage-gated CaZ* channels in this membrane. As the Ca2* concentration outside cells is more than 1000 times greater than the free Ca2* concentration inside, Caz* flows into the nerve terminal. The increase in Caz*concentration in the cltosol of the nerve terminal triggers the local releaseof acetylcholine into the synaptic cleft. 2. The released acetylcholine binds to acetylcholine receptors in the muscle cell plasma membrane, transiently opening the cation channels associated with them. The resulting influx of Na+ causes a local membrane depolarization. 3. The local depolarization of the muscle cell plasma membrane opens voltage-gated Na+ channels in this membrane, allowing more Na+ to enter, which further depolarizes the membrane. This, in turn, opens neighboring voltage-gated Na+ channels and results in a self-propagating depolarization (an action potential) that spreads to involve the entire plasma membrane (seeFigure f1-30). R E S T I NN GE U R O M U S C U LJAURN C T I O N

n e r v et e r m i n a l a c e t y l c h o li n e

A C E T Y L C H O LEI -N GATEDCATION CHANNEL

VOLTAGE-GATED c a 2 *C H A N N E L

VOLTAGE-GATEDNa'CHANNEL

sarcoplasmic reticulum

muscte plasma memorane

Figure 11-39 The systemof ion junction. channelsat a neuromuscular for Thesegatedion channelsareessential the stimulationof musclecontractionby a nerveimpulse.Thevariouschannelsare numberedin the sequencein whichthey areactivated,as describedin the text.

JUNCTION NEUROMUSCULAR ACTIVATED

688

Chapter11:MembraneTransportof SmallMoleculesand the Electrical Properties of Membranes

4. The generalized depolarization of the muscle cell plasma membrane activates voltage-gatedCa2*channels in specializedregions (the transverse ITl tubules-discussed in Chapter l6) of this membrane. 5. This, in turn, causes Caz+-gatedCd* releasechannels in an adjacent region of the sarcoplasmic reticulum (SR) membrane to open transiently and releasethe Ca2+stored in the SRinto the cltosol. The T-tubule and SRmembranes are closely apposed with the two t!?es of channels joined together in a specialized structure (see Figure 16-77).It is the sudden increase in the cltosolic Ca2*concentration that causesthe myofibrils in the muscle cell to contract. \A/hereasthe activation of muscle contraction by a motor neuron is complex, an even more sophisticated interplay of ion channels is required for a neuron to integrate a large number of input signals at synapsesand compute an appropriate output, as we now discuss.

SingleNeuronsAreComplexComputation Devices In the central nervous system, a single neuron can receive inputs from thousands of other neurons, and can in turn form slmapseswith many thousands of other cells. Severalthousand nerve terminals, for example, make synapseson an averagemotor neuron in the spinal cord; its cell body and dendrites are almost completely covered with them (Figure r r-40). some of these synapsestransmit signals from the brain or spinal cord; others bring sensory information from muscles or from the skin. The motor neuron must combine the information received from all these sources and react either by firing action potentials along its axon or by remaining quiet. of the many s)mapseson a neuron, some tend to excite it, others to inhibit it. Neurotransmitter releasedat an excitatory slmapsecausesa small depolarization in the postsynaptic membrane called an excitatory postsynaptic potential (excitatory PSP),while neurotransmitter released at an inhibitory slrlapse generally causesa small hlperpolarization called an inhibitory PSPThe membrane of the dendrites and cell body of most neurons contains a relatively low density of voltage-gated Na+ channels, and an individual excitatory pSp is generally too small to

I

01mm

I (A)

Figure 11-40 A motor neuron cell body in the spinalcord.(A)Manythousandsof nerveterminalssynapseon the cellbody and dendrites. Thesedeliversignalsfrom otherpartsof the organismto controlthe firingof actionpotentialsalongthe single axonof this largecell.(B)Micrograph showinga nervecellbody and itsdendrites stainedwith a fluorescent antibodythat recognizesa cytoskeletalVotein (green). Thousandsof axonterminals(red)from other nervecells(notvisible)make synapses on the cellbody and dendrites; they arestainedwith a fluorescent antibody that recognizes a proteinin synaptic (8,courtesyof OlafMundigland vesicles. Pietrode Camilli.)

aseqd SuUIeJaqt Suunp ltluo uado ^aq] srrlaupl ra^\olsrreqlJo asneraq tnq 'pale8 -e8ello^ pnuatod aqlJo uorlu8edord aqt ot uou ^,(eql'QZg'd aas) uouce eJe 'ISEI -elal passnJslp slql urrograd sleuuuqJ ur f.lsnornard sB *y pafelap aq1 'urc3e arg o] IIar aq] aredard o1 prlualod uortoe qJpe JaUeeuurqureru aql azlrelodeJ01 'aJoJeJeq]'papaau st ad.,ilIauupqc puourppe uV'pauretureru spm (sdsd ruo4) snlntut1sSurzrrelodap8uor1s aq1 se 3uo1 se Jnoco lou plnom qclq^A 'anpn a,rqe8au,{ran e o1 a8ellon aueJqrueu aql 'uouelrlf,pur Jreq] ruo4 ralocal ol eAEqplnom Jo urnler e arrnbar plnom leql lng slauueqJ esaql 'alg plnor puuelod uorlce Jar{}ou€aJoJeg'slauueqf, +eN eql Jo 'aJIJ uorlelrlcpur .{.q,{ea,r Ipnsn eq} ur peleulluJa} aq plnom prlualod uollce eqJ ppo^ ppualod uorloe uE pup 'uado ppo,vr slauueqo +eN aql 'pessoJf,aq ppo^a qll6'lepue}od uot}ce ue raSStrl ploqseJq] aq] 'uorlelnul]s Sursearcur,{.1pnper8 o] tuerJrJJnsureq plnom auerquau luaur8as Ipuur eql Jo uorlezlJelodap aqr 'slauueqJ 'uor1e1nur1s cpdeuds +pN aq] aJaM Jo Ia^al ploqseJql ureueJ e ^aoleg l.1uo aql 1 uaddeq plnom IIac a^Jeu eql ur luaserd slauueqc uot pa1e8-a8e11on 'sleuueqc go sadfi aldqpru roJ paau eqt puelsrapun oJ ]pq ^ tsJrJJeprsuoJ 'spuuaqc 'pafa1ap sp uaq] DI parunlpo- +pJ puu Supanpcout [1p1dat ol raJal lleqs alrrisauradord luaJaJJIpa^eq sleuueqo *)Jo sepalJel aarq] eeJ'*ze3 JoJ e^r]3elesauo puE +) roJ a^rlJalas aalq]-qauu€q3 uor Jo sesselsJaqlo JnoJ 'Surpocuagouorlf,ury lseal lp sureluoc osp luau8as pDrur eqlJo auerqruau eql p;4ua1d qtp\ uornau prcads s1r urro;rad ot tng 'sleuueqc *eg pa1u3-a8e11orr rlcea Jo uor8ar anbrun e 'lueur8as IEITIq eql le paleurur ale spqualod uollf,V 'sppuatod uorlJe;o .4ouanbar;aqt raq8rq eql 'uopeptulls aqt ra8uorls aq] :serlJ uoJnau aql ]eqt spqualod uorllp Jo ,iouanber; aql ,(q uorssrursueJ]acuelsrp-3uo1 JoJpepoJua sr salracal uoJneu e uoue1nrulls;o firsualur aq] t€ql uaas aAEqaM

slauueq)+)|Jospur) aarql lseal le,o uolleurquo) e serlnbauuollPlnduo) leuorneN '(gt-I arn8rg ees)yJolltll uoxv to 'luatu&as I Tnltrur alql sE rrltlroDl uor8ar e ur ',4.poqIIaJ aqt o1 luacefpe 'uoxe eql Jo esuq eq] le dlrsuap q8q re luaserd are '(If-I ]pqt sleuupqc uor pale8 ;o las ptcads e Lq pa,raqre st Sutpocua sIqJ I ern -3gg) spqualod uorlce;o Burtrl lo [cuanbad er4 otw 'papoJua ro 'pa]elsuun ueql sr dsd eqlJo apnlu8eru peurquoc aql 'uorssrtusueJ]eJuelsrp-3uo1rog ,{poq lac peards pooqroqq8rau qJee ruo4 sdsd eqJ'Ielol aq] uo a8ra.tuoc pue ^,(lenrssed aql ol uonnqtrluoc a'rtle8au e 3uorcu s454 rfuo1rqrqulQllm isdsd lenplr\Ipul eql 'eer} Jrtrrpuep aql Jo runs aqr.{lq8nor aq III.,ltpooqroqq8rau }eq} ur dsd IBto} aq} 'asdeuds ;o uor8ar arues aql ur sasdeu.{sIEJaAas1ei{ysnoauellnrurs aArrJEqeu8rs;1 eql Jo alrs aq] uro4 acuelsrp qlrm saseaJoapqJrqm 'apnlru8eru paper8 Jo dsd 'ppelsul 'ppuarod uortcp ue ra33rr1 IEJoI p ur patJagal sr pu8rs Sururocur qJEa 'drqsuorle;ar (l) alrqMijSdpaurqLuo) aql sazueururns aql urasear)ur ueqllM lerauab sosear)ur uoxeueJobuanberl6uuuaqlMorlsmotls (B)pue(V)lo uosuedruo> paurquorrabre;e a:npordo1auo V iCSd 6urpa>a.rd aqlol sppeparnpord aLUes aqlle a^ureslplluatod uorl)ea^rssa))ns uaq6's;e;1ua1od uo;1re dsdLl)ea'asdeu,(s paulquo)aql;o epnlru6eur s!(dsd);e;1ua1od trldeu(sgsod aql 19-11 arn611 ;o 6uu11 1obuanba,rlsql ul pal)alJar ( s p u o ) a s r l l r uaru) r l 002

00r

( s p u o ) a s r l l r uaru) i r l 002

00t

0/- oP QO 6rr n =

o

- J - q

n "

(i -6

oo ff

I oo,

6
pop ro;alqrsuodsar SIUVNS aql 'xa;druor IUVNS-sup,u a u e r q u r a uer u r s e l ol l a ) a ^ l a u

( S Z de u S ) ]UVNS-}

al)rsaA rrrdeur{s

E9L

IUOdSNVUJ ]NVU8W]IAI JO SWSINVHf]t^I UflNf]IOhJ ]HI

764

Chapter13:Intracellular Vesicular Traffic

Rab proteins, which can regulate the availability of SNAREproteins, exert an additional layer of control. I-SNAREsin target membranes are often associated with inhibitory proteins that must be releasedbefore the t-SNAREcan function. Rab proteins and their effectors trigger the release of such SNARE inhibitory proteins. In this way, sNARE proteins are concentrated and activated in the correct location on the membrane, where tethering proteins capture incoming vesicles. Rab proteins thus speed up the process by which appropriate sNARE proteins in two membranes find each other. For vesicular transport to operate normally, transport vesicles must incorporate the appropriate SNARE and Rab proteins. Not surprisingly, therefore, many transport vesicleswill form only if they incorporate the appropriate complement of SNAREand Rab proteins in their membrane. How this crucial control process operates during vesicle budding remains a mystery.

Interacting SNAREs Needto BePriedApartBeforeTheyCan Fu n c t i o n Again Most SNARE proteins in cells have already participated in multiple rounds of vesicular transport and are sometimes present in a membrane as stable complexes with partner SNAREs.The complexes have to disassemble before the SNAREscan mediate new rounds of transport. A crucial protein called NSF cycles between membranes and the cltosol and catalyzesthe disassemblyprocess.It is an ArPase that uses the energy of ATP hydrolysis to unravel the intimate interactions between the helical domains of paired SNAREproteins (Figure r3-rg). The requirement for NSF-mediated reactivation of SNAREsby sNARE complex disassembly helps prevent membranes from fusing indiscriminately: if the I-SNAREs in a target membrane were always active, any membrane containing an appropriate v-SNAREmight fuse whenever the two membranes made contact. It is not known how the activity of NSF is controlled so that the sNARE machinery is activated at the right time and place, but Rab effectors are likely candidates to play a part in this process.

ViralFusionProteins and SNAREs MayUseSimilarFusion Mechanisms Membrane fusion is important in other processesbeside vesicular transport. The plasma membranes of a sperm and an egg fuse during fertilization (discussedin chapter 21), and myoblasts fuse with one another during the development of multinucleate muscle fibers (discussedin chapter 22). Likewise, mitochondria fuse and fragment in a dlmamic way (discussedin chapter r4). A1lcell membrane fusions require special proteins and are tightly regulated to ensure that only appropriate membranes fuse. The controls are crucial for maintaining both the identity of cells and the individuality of each type of intracellular comparrmenr. The membrane fusions catalyzed by viral fusion proteins are the best understood. These proteins have a crucial role in permitting the entry of enveloped viruses (which have a lipid-bilayer-based membrane coat) into the cells that they infect (discussedin chapters 5 and24). For example, viruses such as the human immunodeficiency virus (HIV), which causesAIDS, bind to cell-surface

ttn SNARE dissociation

Figurel3-18 Dissociation of SNARE pairs by NSFafter a membranefusion cycle.After a v-SNARE and t-SNAREhave mediatedthe fusionof a transportvesicle on a targetmembrane,the NSFbindsto the SNARE complexand,with the help of two accessoryproteins,hydrolyzesATPto pry the SNAREs apart.

THEMOLECULAR MECHANISMS TRANSPORT OFMEMBRANE

765

m e mo r a n e n u c l e o cp as i d

H I Vf u s i o n protern oD120

-

-

CYTOSOL chemokine receplor CD4ATTACHMENT

C H E M O K I NR EECEPTOR BINDING

MEMBRANE INSERTION

E N T R YO FV I R A L NUCLEOCAPSID

(B)

showinghow HIVentersa cellby fusing Figure13-19The entry of envelopedvirusesinto cells.(A)Electronmicrographs HIVbindsfirstto the CD4protein its membranewith the plasmamembraneof the cell.(B)A modelfor the fusionprocess. protein,mediatesthis HIV fusion to the protein, is bound gp120 which on the surfaceof the targetcell.Theviral (discussed proteinon the targetcell,which normallyservesasa receptorfor chemokines interaction. A secondcell-surface the protein allowing from HIV fusion the releases 9p120, in Chapters24 and 25),now interactswith gp120.Thisinteraction prevrously fusionpeptideto insertinto the plasmamembraneof the targetcell.Thefusionprotein, buriedhydrophobic

HHH:JI:ff:i[,'"'l;ff]:inffi"ffi'"::ll;T#ffiff:"ffi:ff:J:,T;Til'J.1il together, copies of thefusionproteinpullsthetwo membranes in multiple energyreleased bythisrearrangement Thus,likea mousetrap,theHIV fusion. prevents membrane thatnormally energybarrier overcoming thehighactivation work.(A,from to do mechanical andharnessed whichisreleased energy, fusionproteincontains a reservoir of potential permission B,adaptedfroma drawingbyWayneHendrickson.) fromElsevier; 1987.With B.S. Steinet al.,Cell49:659-668, receptors and then fuse with the plasma membrane of the target cell (Figure f 3-f 9). This fusion event allows the viral nucleic acid inside the nucleocapsid to enter the cytosol, where it replicates.Other viruses, such as the influenza virus' first enter the cell by receptor-mediated endocytosis (discussedlater) and are delivered to endosomes; the low pH in endosomes activates a fusion protein in the viral envelope that catalyzes the fusion of the viral and endosomal membranes, releasing the viral nucleic acid into the cytosol. The three-dimensional structures of the fusion proteins of HIVand influenza virus provide valuable insights into the molecular mechanism of the membrane fusion catalyzed by these proteins. Exposure of the HIV fusion protein to receptors on the target cell membrane, or exposure of the influenza fusion protein to low pH, uncovers previously buried hydrophobic regions. These regions, called fusion peptides, then insert directly into the lipid bilayer of the target cell membrane. In this way, the fusion proteins transiently become integral membrane proteins in two separate lipid bilayers. Structural rearrangements in the fusion proteins then bring the two lipid bilayers into very close apposition and destabiIize them so that the bilayers fuse (seeFigure 13-19). Thus, viral fusion proteins and SNAREspromote lipid bilayer fusion in similar ways.

766

Chapter13:lntracellular Vesicular Traffic

Su m m a r y Directed and selectiuetransport of particular membrane componentsfrom one membrane-enclosedcompartment of a eucaryoticcell to another maintains the differences between those compartments. Transport uesicles,which can be spherical, tubular, or irregularly shaped,bud from specializedcoated regionsof the donor membrane. The assemblyof the coat helps to collectspecificmembraneand soluble cargo moleculesfor transport and to driue theformation of the uesicle. There are uarious types of coated uesicles.The best characterizedare clathrincoated uesicles,which mediate transport from the plasma membrane and the trans Golgi network, and COPI-and COPII-coateduesicles,which mediate transport between Golgi cisternaeand betweenthe ER and the Golgi apparatus, respectiuely. In clathrincoateduesicles, adaptor proteins link the clathrin to the uesiclemembraneand also trap specificcargo moleculesfor packaging into the uesicle.The coat is shed rapidty after budding, enabling the uesicleto fuse with its appropriate target membrane. Local synthesisof phosphoinositidescreatesbinding sitesthat trigger coat assembly and uesiclebudding. In addition, monomeric GTPases help regulateuariousstepsin uesiculartransport, includingboth uesiclebudding and docking. The coat-recruitment GTPases,including sarl and the Arf proteins, regulatecoat assemblyand disassembly. A largefamily of Rab proteinsfunctions as uesicletargeting GTPases. Rab proteins are recruited to transport uesiclesand target membranes.The assemblyand disassemblyof Rab proteins and their ffictors in specializedmembrane domains are dynamically controlled by GTP binding and hydrolysis.Actiue Rab proteins recruit Rab ffictors, such as motor proteins, which transport uesicleson actin filaments or microtubules, and filamentous tethering proteins, which help ensure that the uesiclesdeliuer their contentsonly to the appropriate membrane-enclosed compartment. complementary usN/fiE proteins on transport uesiclesand i-SNAREproteins on the target membrane form stable trans-SNAREcomplexes,whichforce the two membranesinto closeapposition so that their lipid bilayerscanfuse.

TRANSPORT FROMTHEERTHROUGH THEGOLGI APPARATUS As discussedin chapter 12, newly synthesizedproteins cross the ER membrane from the c)'tosol to enter the biosynthetic-secretory pathway. During their subsequent transport, from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface and elsewhere,these proteins are successivelymodified as they pass through a seriesof compartments. Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. Some transport vesicles select cargo molecules and move them to the next compartment in the pathway, while others retrieve escaped proteins and return them to a previous compartment where they normally function. Thus, the pathway from the ER to the cell surface consists of many sorting steps, which continuously select membrane and soluble lumenal proteins for packaging and transport-in vesicles or organelle fragments that bud from the ER and Golgi apparatus. In this section we focus mainly on the Golgi apparatus (also called the Golgi cpmplex). It is a major site of carbohydrate slmthesis, as well as a sorting anl dispatching station for products of the ER.The cell makes many polysaccharides in the Golgi apparatus, including the pectin and hemicellulose of the cell wall in plants and most of the glycosaminoglycansof the extracellular matrix in animals (discussedin chapter 19). The Golgi apparatus also lies on the exit route from the ER, and a large proportion of the carbohydrates that it makes are attached as oligosaccharideside chains to the many proteins and lipids that the ER sends to it. A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesiclesthat then transport them to lysosomes.But most proteins and lipids, once they have acquired their appropriate oligosaccharidesin the Golgi apparatus, are recognized in other ways for targeting into the transport vesicles going to other destinations.

C E L LE X T E R I O R

TRANSPORT FROM THEERTHROUGH APPARATUS THEGOLGI

767

ProteinsLeavethe ERin COPI|-Coated Transport Vesicles To initiate their journey along the biosynthetic-secretory pathway, proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into small COPII-coated transport vesicles.These vesicles bud from specialized regions of the ER called ER exit sites,whose membrane lacks bound ribosomes. Most animal cells have ER exit sites dispersed throughout the ER network. Originally, it was thought that all proteins that are not tethered in the ER enter transport vesiclesby default. It is now clear, however, that entry into vesicles that leave the ER is usually a selectiveprocess.Many membrane proteins are actively recruited into such vesicles, where they become concentrated. It is thought that these cargo proteins display exit (transport) signals on their cytosolic surface that components of the COPII coat recognize (Figure 13-20); these coat components act as cargo receptors and are recycled back to the ER after they have delivered their cargo to the Golgi apparatus. Soluble cargo proteins in the ER lumen, by contrast, have exit signals that attach them to transmembrane cargo receptors,which in turn bind through exit signals in their cl,toplasmic tails to components of the COPII coat. At a lower rate, proteins without exit signals can also enter transport vesicles,so that even proteins that normally function in theER(so-calledERresidentproteins)slowlyleakoutoftheERandaredelivered to the Golgi apparatus. Similarly, secretory proteins that are made in high concentrations may leave the ER without the help of exit signals or cargo receptors. The exit signals that direct soluble proteins out of the ER for transport to the Golgi apparatus and beyond are not well understood. Some transmembrane proteins that serve as cargo receptors for packaging some secretoryproteins into COPII-coated vesicles are lectins that bind to oligosaccharides.The ERGIC53 lectin, for example, binds to mannose and is thought to recognize this sugar on two secretedblood-clotting factors (FactorV and FactorVIII), thereby packaging the proteins into transport vesiclesin the ER.ERGIC53'srole in protein transport was identified becausehumans who lack it owing to an inherited mutation have Iowered serum levels of FactorsV and VIII, and they therefore bleed excessively.

CanLeave ThatAreProperlyFoldedand Assembled OnlyProteins theER To exit from the ER, proteins must be properly folded and, if they are subunits of multimeric protein complexes,they may need to be completely assembled.Those that are misfolded or incompletely assembledremain in the ER, where they are bound to chaperone proteins (discussedin Chapter 6), such as BiP or calnexin' The chaperones may cover up the exit signals or somehow anchor the proteins in f o r m i n g E Rt r a n s p o r tv e s i c l e 5 a r 1- G T P s u b u n i t so f C O P IcI o a t

e x i t s i g n a lo n membrane-bound c a r g op r o r e r n\

e x i t s i g n a lo n receptor

CYTOSOL e x i t s i g n a lo n cargo receptor e x i t s i g n a lo n s o l u b l ec a r g o proren r

r e s i d e n tE R p r o t e rn E RL U M E N

c h a p e r o n ep r o t e i n sb o u n d t o u n f o l d e do r m i s f o l d e dp r o t e i n s

Figure13-20The recruitmentof cargo By moleculesinto ERfiansportvesicles. bindingdirectlyor indirectlyto the COPII coat,membraneand solublecargo proteins,respectively, become as in the transportvesicles concentrated they leavethe ER.Membraneproteinsare packagedinto buddingtransportvesicles of exit signalson throughinteractions theircytosolictailswith the COPIIcoat. Someof the membraneproteinsthat the coat trapsfunction as cargo receptors, bindingsolubleproteinsin the lumen and helpingto packagethem into A typical50-nmtransportvesicle vesicles. containsabout200 membraneproteins, whichcan be of manydifferenttypes.As indicated,unfoldedor incompletely proteinsareboundto assembled and retainedin the ER chaoerones comoartment.

768

Chapter13: Intracellular Vesicular Traffic

CYTOSOL

a n t ib o d y h e a v yc h a i n

RETAINEDIN ER

Figure13-21 Retentionof incompletely assembledantibodymoleculesin the ER.Antibodiesaremadeup of two heavy and two light chains(discussed in Chapter25),whichassemblein the ER. ThechaperoneBiPisthoughtto bind to all incompletely assembled antibody molecules and to coverup an exit signal. Thus,only completelyassembled antibodiesleavethe ERand aresecreted.

antibody l i g h tc h a i n

SECRETED

the ER (Figure r3-2r). Such failed proteins are eventually transported back into the cltosol, where they are degradedby proteasomes(discussedin chapters 6 and 12).This quality-control step prevents the onward transport of misfolded or misassembled proteins that could potentially interfere with the functions of normal proteins. There is a surprising amount of corrective action. More than 90% of the newly sFrthesizedsubunits of the T cell receptor (discussedin chapter 25) and of the acetylcholine receptor (discussedin chapter ll), for example, are normally degraded without ever reaching the cell surface where they function. Thus, cells must make a large excessof many protein molecules to produce a select few that fold, assemble,and function properly. The process of continual degradation of a portion of ER proteins also provides an early warning system to alert the immune system when a virus infects cells. Using specialized ABC-type transporters, the ER imports peptide fragments of viral proteins produced by proteases in the proteasome. The foreign peptides are loaded onto class I MHC proteins in the ER lumen and then transported to the cell surface.T ly'rnphocytesthen recognizethe peptides as non-self antigens and kill the infected cells (discussedin Chapter 25). sometimes, however, there are drawbacks to the stringent quality-control mechanism. The predominant mutations that cause cystic fibrosis, a common inherited disease, result in the production of a slightly misfolded form of a plasma membrane protein important for cl- transport. Although the mutant protein would function perfectly normally if it reached the plasma membrane, it remains in the ER. This devastating diseasethus results not because the mutation inactivates the protein but because the active protein is discarded before it reachesthe plasma membrane.

vesicular TubularclustersMediateTransport from the ERto the G o l g i A p p a r a tu s After transport vesicleshave budded from ER exit sites and have shed their coat, they begin to fuse with one another. This fusion of membranes from the same compartment is called homotypic fusion, to distinguish it from heterotypic fusion, in which a membrane from one compartment fuses with the membrane of a different compartment. As with heterotypic fusion, homotypic fusion requires a set of matching SNAREs.In this case,however, the interaction is s).rynmetrical, with both membranes contributing v-SNAREsand t-sNAREs (Figure L3-22). The structures formed when ER-derived vesicles fuse with one another are called uesiculartubular clusters,because they have a convoluted appearance in

.

TRANSPORT FROMTHEERTHROUGH THEGOLGIAPPARATUS

S T E P1

STEP2

3 STEP

the electron microscope (Figure f3-23A). These clusters constitute a new compartment that is separatefrom the ER and lacks many of the proteins that function in the ER. They are generated continually and function as transport containers that bring material from the ER to the Golgi apparatus. The clusters are relatively short-lived because they move quickly along microtubules to the Golgi apparatus, with which they fuse to deliver their contents (Figure 13-238). As soon as vesicular tubular clusters form, they begin to bud off transport vesicles of their o'o,'n.Unlike the COPII-coated vesicles that bud from the ER, these vesiclesare COPI-coated.They carry back to the ER resident proteins that have escaped,as well as proteins such as cargo receptors that participated in the ER budding reaction and are being returned. This retrieval process demonstrates the exquisite control mechanisms that regulate coat assembly reactions. The COPI coat assembly begins only seconds after the COPII coats have been shed. It remains a mystery how this switch in coat assembly is controlled. The retrieual (or retrograde) transport continues as the vesicular tubular clusters move towards the Golgi apparatus. Thus, the clusters continuously mature, gradually changing their composition as selected proteins are returned to the ER. A similar retrieval process continues from the Golgi apparatus, after the vesicular tubular clusters have delivered their cargo.

Pathwayto the ERUsesSortingSignals TheRetrieval The retrieval pathway for returning escapedproteins back to the ER depends on ER retrieual signals.Resident ER membrane proteins, for example, contain signals that bind directly to COPI coats and are thus packaged into CoPl-coated v e s i c u l atru b u l a r c l u s t e r

769

Figure13-22 Homotypicmembrane fusion.In step1, NSFpriesapart and t-SNARES identicalpairsof v-SNARES in both membranes(seeFigure13-18).In matching steps2 and 3, the separated on adjacentidenticalmembranes SNAREs interact,whichleadsto membranefusion and the formationof one continuous tubular comoartmentcalleda vesicular the compartment cluster.Subsequently, growsby furtherhomotypicfusionwith vesicles from the samekind of membrane, Homotypic displayingmatchingSNARES. fusion is not restrictedto the formationof in a similar vesicular tubularclusters; process,endosomesfuseto generate Rabproteinshelp largerendosomes. regulatethe extentof homotypicfusion and hencethe sizeof the compartments in a cell(not shown).

Figure13-23Vesiculartubular clusters. (A)An electronmicrographsectionof formingfrom tubularclusters vesicular the ERmembrane.Manyof the vesicleseenin the micrographare likestructures crosssectionsof tubulesthat extend aboveand belowthe planeof thisthin sectionand areinterconnected. (B)Vesicular tubularclustersmovealong to carryproteinsfrom the microtubules COPIcoats ERto the Golgiapparatus. that mediatethe buddingof vesicles As returnto the ERfrom theseclusters. the coatsquicklydisassemble indicated, after the vesicleshaveformed. (A,courtesyof WilliamBalch.)

microtubule

770

Chapter13: Intracellular Vesicular Traffic

v e s i c u l atru b u l a r c l u s t e r

secrerory prorern

KDELreceptor protern

(A)

soluble E Rr e s i d e n t protein

(B)

ER

transport vesicles for retrograde delivery to the ER. The best-characterized retrieval signal of this type consists of two lysines, followed by any two other amino acids, at the extreme c-terminal end of the ER membrane protein. It is called a KKXX sequence,based on the single-letter amino acid code Soluble ER resident proteins, such as BiB also contain a short retrieval signal at their c-terminal end, but it is different: it consists of a Lys-Asp-Glu-Leu or a similar sequence.If this signal (called tlr.eKDEL sequence)is removed from Bip by genetic engineering, the protein is slowly secreted from the cell. If the signal is transferred to a protein that is normally secreted,the protein is now efficiently returned to the ER, where it accumulates. unlike the retrieval signals on ER membrane proteins, which can interact directly with the coPI coat, soluble ER resident proteins must bind to specialized receptor proteins such as rhe KDEL receptor-a multipass transmembrane protein that binds to the KDEL sequence and packagesany protein displaying it into coPl-coated retrograde transport vesicles (Figure lJ-24). To accomplish this task, the KDEL receptor itself must cycle between the ER and the Golgi apparatus, and its affinity for the KDEL sequence must differ in these two compartments. The receptor must have a high affinity for the KDEL sequence in vesicular tubular clusters and the Golgi apparatus, so as to capture escaped,soluble ER resident proteins that are present there at low concentration. It must have a low affinity for the KDEL sequence in the ER, however, to unload its cargo in spite of the very high concentration of KDEL-containing resident proteins in the ER. How does the affinity of the KDEL receptor change depending on the compartment in which it resides?The answer is not knovrm,but it mav be related to the different ionic conditions and pH in the different compartments, which are regulated by ion transporters in the compartment membrane. As we discuss later, pH-sensitive protein-protein interactions form the basis for many of the sorting steps in the cell. Most membrane proteins that function at the interface between the ER and Golgi apparatus, including v- and t-sNAREs and some cargo receptors, enter the retrieval pathway back to the ER. \fhereas the recycling of some of these proteins is mediated by signals,asjust described,for others no specific signal seems to be required. Thus, while retrieval signals increase the efficiency of the retrieval process, some proteins randomly enter budding vesicles destined for the ER and are returned to the ER at a slower rate. Many Golgi enzymes cycle constantly betvveenthe ER and the Golgi, but their rate of return to the ER is slow enough for most of the protein to be found in the Golgi apparatus.

cis Golgi network

Golgi stack

trans Golgi network

Figure l3-24 A model for the retrieval of solubleERresidentproteins.ER residentproteinsthat escapefrom the ER arereturnedby vesicular transport. (A)The KDELreceptorpresentin vesicular tubularclustersand the Golgiapparatus capturesthe solubleERresidentproteins and carriesthem in COP|-coated transportvesicles backto the ER.Upon bindingits ligandsin this environment, the KDELreceptormay change conformation, so asto facilitateits recruitmentinto buddingCOPI-coated vesicles.(B)The retrievalof ERproteins beginsin vesicular tubularclustersand continuesfrom all partsof the Golgi apparatus. In the environmentof the ER, the ERresidentproteinsdissociatefrom the KDELreceptor,which is then returned to the Golgiapparatus for reuse.

TRANSPORT FROMTHE ERTHROUGH THEGOLGI APPARATUS

771

ManyProteinsAreSelectively in Retained in the Compartments WhichTheyFunction The KDEL retrieval pathway only partly explains how ER resident proteins are maintained in the ER. As expected, cells that express genetically modified ER resident proteins, from which the KDEL sequence has been experimentally removed, secretethese proteins. But the rate of secretion is much slower than for a normal secretory protein. It seems that a mechanism that is independent of their KDEL signal anchors ER resident proteins and that only those proteins that escapethis retention mechanism are captured and returned via the KDEL receptor. A suggestedretention mechanism is that ER resident proteins bind to one another, thus forming complexes that are too big to enter transport vesiclesefficiently. BecauseER resident proteins are present in the ER at very high concentrations (estimated to be millimolar), relatively low-affinity interactions would suffice to tie up most of the proteins in such complexes. Aggregation of proteins that function in the same compartment-called kin recognition-is a general mechanism that compartments use to organize and retain their resident proteins. Golgi enzymes that function together,for example, also bind to each other and are thereby restrained from entering transport vesicles leaving the Golgi apparatus.

TheGolgiApparatusConsistsof an OrderedSeriesof Compartments Becauseof its large and regular structure, the Golgi apparatus was one of the first organelles described by early light microscopists. It consists of a collection of flattened, membrane-enclosed compartments, called cisternae,that somewhat resemble a stack of pita breads. Each Golgi stack usually consists of four to six cisternae (Figure 13-25), although some unicellular flagellates can have up to

cls FACE

G o l g iv e s i c l e

m e d i acl i s t e r n a trans cisterna

(A)

secretoryvesicle

trans FACE

Figure13-25 The Golgiapparatus. (A)Three-dimensional reconstruction of the from electronmicrographs animal Golgiapparatusin a secretory cell.The cisfaceof the Golgi stackis that closestto the ER.(B)A thinsectionelectronmicrograph zone the transitional emphasizing betweenthe ERand the Golgi apparatusin an animalcell.(C)An electronmicrographof a Golgi apparatusin a plantcell(thegreen seenin cross algaChlamydomoncs) section.In plantcells,the Golgi apparatusis generallymoredistinct from and moreclearlyseparated than in membranes other intracellular animalcells.(A,redrawnfrom A. Rambourgand Y.Clermont,Eur. J. CellBiol.51:189-200,1990.With permission fromWissenschaftliche B,courtesyof VerlagsgesellschafU BrijJ. Gupta;C,courtesyof George Palade.)

nuclear envelope roughER

vesicular tubular clusters cls Golgi network

*

(.SaMPH stJq)Jo {seunot 'B jue^tN)W ye6 pue {a1uaguqo;1o{salrno>,yy 's1>e1s r6;o9 arp (lolo) asleJ)slodsabuplo 1q6rrqaq1'uralotdluarsaron;;uaal6 ol pasnJatu{zua16;o9}ueprsare;o 6urlsrsuoturalotduorsnle 6urssaldxa sr reqt lla) lueld e ur snlpredde16109 aql (B) 'uollexgaroleq6ur;anel> semllo) aql q)rqMur uoll)allpaql 6upeJ'pezue;od srsnlereddet6109eql'1par)utatord tuaprsarr6;o9e sezru6o)al leql {poqllue lua)saronue qltM pauletslselqorqu parnllnl e ur snleredde161o9 aq1(y) 'sllerlueld pup leutue u; snleredde r6log orll Jo uollezrle)o'l9Z-€ I ain6rl

'}JEJ pJE^/\O] pelEJluaJuoJ aJBsdals 8ur SUDJI eql aPuJelsrs eql uI -ssacoJdJelel Burz.,tlplp3saurdzue sPeJaq^ '4ce1s€1og eq] eJeJst, eql pJE^\ol Jo eeuJelsrJ aq] ul pelEJlueJuos aJB sdels Surssacord [1tea Eutz[1e]eJ seuilzue :ef,uenbes e se IIal\/\sB [Br]BdsB ur sJnJOoSurssacord]Eql Jeel3 sl lI Ipcruaqf,orq 'ssaleqleuoN 'aru^,(zue Surpacardeql ^q pesserord ltlradord uaaq seq lI reup dluo et€Jtsqns e se ureloJdoc.{13e ldecae uec aru^zua Surssacord aprreqcceso8qo qrEa eJurs ',{ressacauunIueas lq3[u uor]ezrp]uauueduoJ srqJ ']run Surssac 'IJpls eql ssoJJpeuJalsrt o} euJalsr3 -oJd e8etsulnur p sruJoJ {f,Els aq} }Eq} os 'saludzue (uo4 peglpolu e^o(u.{aq} se sa8els e^rsseJ3ns ur aJp sure}oJd Sulssac '>1ce1s -oJd r31og Jo af,uepunqe JIlslJelJeJEqce Surupluoc euJalsro qJEa qllivr aq] uI aruanbas pazrue8ro uu ur Jnrco sdals SurssaoordeprJeqJOESoBqo aq; 'suorleuusep lrodsuer] IBUrJJreql 01 peqclsdsrp pue saSe>1ced luaJaJJrpolur pele8eJ8asare suraloJd araq ^ eJeld eql 'NCI eql qlr^ snonun -uoc eq o1 sr aeuJalsrJsuou er4l ueunl aq; 'palalduroc sr uorleldsoc lq8noqt lo -d13araqm 'apuJalsrJsztou er4lol dfleug pue (apura]src IBIpeur eql) lueuueduroo txau aq] o] alour uaql [aq1 '(aeuJa]srJrBIoO slo eql) slueu]reduror Surssac 'Iromleu r31og sre aql q8norql ssed -ord r31og eql Jo lsrrJ aq] Sulretua eJoJeq suleloJd 'NCJ eq] ur IBATJJE uodn 'suraloJd alnleru ur uees saJnlJnJlseprJeql -ceso8rlo snoauaSoJalaqeql seleJeue8qolqlv\ 'snlerudde r31og aqr ur suaddeq srqJ 'suortoury ^ eu JoJ seprreqJJeso8qoeql u8rsepar ot eerJ sr IIaJ eqt (pelllJ -lnJ uaeq aleq suorlJunJ Ug eseql eouo 'Ug aq] ruo4 SuDxa suralord;o drUenb aql Su1loJluoc ur eloJ ]upuodur ue.,(e1dr(aq1'snq1 'uorluperBaproJ losol.,{'caqt ot suraloJdpeploJsru uodsue4 dlaq o1pup ploJ suratoJddlaq o1 elres suorlJeeJ Surururrrt aql dq paleeJo selerperuJalur eprJEqJ3EsoBIIoeqJ 'Ug aq] ur ilDS sr uleloJd eql elrqM paruurrl ueql puB UA eql ur suraloJd,{uelJJolJolq ua patqJBlle sapuaqrrosoBt1o pa4uq-71;o sarcadsa18urse'71 raldeq3 ur paqrrosep sV 'lualuuPdruof, Jerpee ue o] pauJnleJ eq ueJ osJe.{.aq1'aJBJrnsIIaJ eql lo 'salJrsal ,fuotalcas 'sauososi{y :uor}eurlsap lxau Jraql o1 Sulproccu peuos eJp pue prEl uo e^oru 'Ug eql o] paurntar aq ro snluredde NOI aq] uro4 3u4xa suratord dlreprurg €1og aql ur pJe^ uo e^ou Jeqlre uec NDC aql Suualua suralord :Surlrosuralord rog tueuodurr are s{Jomleu qlog 'luaurlreduroo Jeqloup Jo eceJJnsIIaJ eql JoJ punoq {Jomlau rBlog suatl eqt ruoq tlxa pue lJon,rleu r31og srr aql ralua sprdl pue sureloJd 'Ug eqt ruor; Surnrrre sralsnlJ Jelnqn] JplnJrsal pesnJ Jo uor]tal -loJ e sr NCC aqJ flanrlcadsar '(NCI) {romleu 131ogsuo4 aq} pue (NCC) {ro,rr{ -1au srr :saJnlonJls eql ;31o9 leuJalsrf, pue Jelnqnl palJauuoJJelur Jo Uomlau e 'sluarulredruoc prcads qly!\ pelerJosser(1aso1c are seJvlsuuJl ;o pasodruoJ qJEe puB sl, qlog '(elEJ ]IXe Jo) aaulsurr4 e pue (ace; [J]ua Jo) aJBJslr e :seceJ]Jul]slp oml seq >1ce1s r31og qJpA 'suorlecutpotu luelenoo Jo sarJaspaJepJoue o8rapun se1ncelorupalrodsuerl 'snteredde r31og aqr q8norqt a8essedrlaql Surrng .(ggZ_et arn8rg) urseldo{c aql tnoq8norq} pasradsrp s{rpls r31og pnprnpul Jo sparpunq e^eq 's11ac luu1d lsoru Surpnycut'silec aruos 'selrs fr-xeUg o1 luacefpe ,urseldo{c aq} lnoq8norql punoJ eJelpql DIJels pnptnrput olur sazrueBroarsnleredde r31ogaqr 'pazrraur,rtlodap z(leluauuedxa are salnqnloJctu 'salnqnloJJrru uo spuadap JI uorlpzrleJol slqJ '(VgZ-t1 arnt;g) aruosoJlual eq] ol esolJ puu snolcnu IIaO aqt JBeu pelecol flensn sr qcrq ^ 'xaldruoc a13urse Sunuro; snql ,$lJels ^ueru ,sllaf, '0g Iurl aeuralsrc SurpuodsarJoJuae^\leq suorlcauuoJ JBInqn Ieunu€ uI

)!#eJIrpln)lsenrelnlla)ettul:€[ raldeq3

ZLL

TRANSPORT FROMTHEERTHROUGH THEGOLGIAPPARATUS

773

Figure13-27Molecular compartmentalization of theGolgiapparatus. (A)unstained, A series of electron micrographs showstheGolgiapparatus (B)stained withosmium, of whichispreferentially reduced bythecisternae theciscompartment, and(CandD)stained of to reveal thelocation specific enzymes. Nucleoside diphosphatase isfoundin thetransGolgi (C),whileacidphosphatase cisternae isfoundin thetransGolginetwork (D).Notethatusually are morethanonecisterna isstained. Theenzymes to a therefore thoughtto be highlyenriched localized ratherthanprecisely (Courtesy specific cisterna. of Daniel 5.Friend.) Investigators discovered the functional differences bet\iveen the cis, medial, and trans subdivisions of the Golgi apparatus by localizing the enzymes involved in processingl/-linked oligosaccharidesin distinct regions of the organelle,both by physical fractionation of the organelle and by labeling the enzymes in electron microscope sectionswith antibodies. The removal of mannose residuesand the addition of l/-acetylglucosamine, for example, were shown to occur in the medial compartment, while the addition of galactoseand sialic acid was found to occur in the trans compartment and the trans Golgi network (Figure 13-27). Figure 13-28 summarizes the functional compartmentalization of the Golgi apparatus. The Golgi apparatus is especially prominent in cells that are specialized for secretion of glycoproteins, such as the goblet cells of the intestinal epithelium, which secretelarge amounts of polysaccharide-rich mucus into the gut (Figure f 3-29). In such cells, unusually Iarge vesicles are found on the trans side of the Golgi apparatus, which faces the plasma membrane domain where secretion occurs.

Oligosaccharide ChainsAreProcessed in the GolgiApparatus \Mhereasthe ER lumen is full of soluble lumenal resident proteins and enzymes, the resident proteins in the Golgi apparatus are all membrane bound. The enzymatic reactions in the Golgi apparatus seem to be carried out entirely on its membrane surface. All of the Golgi glycosidasesand glycosyl transferasesare single-pass transmembrane proteins, many of which are organized in multienzyrne complexes. Thus, the two synthetic organelles in the biosyntheticsecretory pathway, the ER and the Golgi apparatus, are organized in fundamentallv different wavs.

I I SORTING of o phosp oligos on

c i sG o l g i nelworK

l Golgi apparatus

medial Golgi c r s t e r n a stack

. removal of Man o a d d i t i o no f G l c N A c

l osulfation of tyrosines and carbohydrates

/ lysosome

I

I plasma membrane

cls crsIerna

trans cisterna

trans Golgi network

Figure 13-28 Oligosaccharide processingin Golgi compartments.The localizationof eachprocessingstep shownwasdeterminedby a combination includingbiochemical of techniques, of the Golgiapparatus subfractionation and electronmicroscopy membranes afterstainingwith antibodiesspecificfor someof the processingenzymes. enzymesare not restrictedto Processing their instead, a particularcisterna; distributionis gradedacrossthe stackenzymesare suchthat early-acting presentmostlyin the cisGolgicisternae and lateractingenzymesaremostlyin the trdnsGolqi cisternae.

774

Chapter13:lntracellular Vesicular Traffic Figure13-29A goblet cellof the smallintestine.This cell is specialized for secretingmucus,a mixtureof glycoproteins and proteoglycans synthesized in the ERand Golgiapparatus. Likeall epithelialcells,gobletcellsarehighly polarized, with the apicaldomainof their plasmamembranefacingthe lumenof the gut and the basolateral domainfacingthe basallamina.The

secretionof mucusfrom ^^i-^t dprtdr

:#J:Bi'J:i[iil::.['iJ,L?runi*f fi'".;l"ffin"'ffi n:ffi :, R.VKrstic, lllustrated Encyclopedia of HumanHistology. NewYork: Springer-Verlag, 1984. Withpermission fromSpringer-Verlag.)

TWo broad classesof l/-linked oligosaccharides,the complex oligosaccharides and the high-mannose oligosaccharides, are attached to mammalian glycoproteins (Figure f3-30). Sometimes, both types are attached (in different places) to the same polypeptide chain. Complex oligosaccharidesare generatedwhen the original l/-linked oligosaccharide added in the ER is trimmed and further sugars are added. By contrast, high-mannose oligosaccharidesare trimmed but have no new sugars added to them in the Golgi apparatus. They contain just two l/-acetylglucosamines and many mannose residues,often approaching ihe number origin.attypresent in the Iipid-linked oligosaccharideprecursor added in the ER.Complex oligosaccharides can contain more than the original two l/-acetylglucosamines,aswell as avariable number of galactoseand sialic acids and, in some cases,fucose. Sialic acid is of special importance becauseit is the only sugar in glycoproteinsthat bears a negative charge.\A4rethera given oligosaccharideremains high-mannose or is processed depends largely on its position in the protein. If the oligosaccharide is

secretory vesicles c o n t ai n i n g mucus

G:Lql , apparatus

I I

o

NH I

I Asn I X I Seror Thr I I

co

/a\

CORE REGION

I

(B) COMPLEX OLIGOSACCHARIDE

KEY = N - a c e t y l g l u c o s a m i(nGel c N A c ) O 93"

-,@'

= m a n n o s e( M a n ) = gatactose(Gat) N - a c e t y l n e u r a m i nai c i d ( s i a l i ca c i d ,o r N A N A )

t

-q/ q p-r o

( C ) H I G H - M A N N O SOEL I G O S A C C H A R I D

Figure13-30 The two main classes (N-linked)oligosaccharides of asparagine-linked found in maturemammalianglycoproteins, (A)Bothcomplexoligosaccharides and high-mannose oligosaccharides sharea commoncoreregionderivedfrom the originalN-linkedoligosaccharide addedin the ER(seeFigurel2-50) and typicallycontainingtwo N-acetylglucosamines (GlcNAc) (Man).(B)Eachcomplexoligosaccharide and threemannoses consistsof a coreregiontogether with a terminalregionthat containsa variablenumber of copiesof a specialtrisaccharide unit (N-acetylglucosamine-galactose-siolic acid)linkedto the core mannoses.Frequently, the terminalregionis truncatedand containsonly GlcNAcand galactose (Gal)or just GlcNAc. In addition,a fucoseresiduemay be added,usuallyto the coreGlcNAcattachedto the asparagine (Asn). Thus,althoughthe stepsof processing and subsequent sugaradditionarerigidly ordered,complexoligosaccharides can be heterogeneous. Moreover, althoughthe complex oligosaccharide shownhasthreeterminalbranches, two and four branches arealsocommon, dependingon the glycoprotein and the cellin which it is made.(C)High-mannose oligosaccharides arenot trimmedbackallthe way to the coreregionand containadditional mannoseresidues. Hybridoligosaccharides with one Man branchand one GlcNAcand Gal brancharealsofound (not shown). Thethreeaminoacidsindicatedin (A)constitutethe sequencerecognized by the oligosaccharyl transferase enzymethat addsthe initialoligosaccharide to the protein. = threonine;X = anVaminoacid,exceptproline. Ser= serine;Thr

1 0p m

-,,-1^-^ )ur rdLg

775

TRANSPORT FROMTHEERTHROUGH THEGOLGIAPPARATUS

accessibleto the processingenzl'mes in the Golgi apparatus,it is likely to be converted to a complex form; if it is inaccessiblebecauseits sugarsare tightly held to the protein's surface,it is likely to remain in a high-mannose form. The processing that generates complex oligosaccharide chains follows the highly ordered pathway shown in Figure 13-31. Beyond these commonalities in oligosaccharide processing that are shared among most cells,the products of the carbohydrate modifications carried out in the Golgi apparatus are highly complex and have given rise to a new field called glycobiology. The human genome, for example, encodes hundreds of different Golgi glycosyl transferases,which are expresseddifferently from one cell type to another, resulting in a variety of glycosylatedforms of a given protein or lipid in different cell tlpes and at varying stages of differentiation, depending on the spectrum of enzymes expressedby the cell. The complexity of modifications is not limited to l/-linked oligosaccharidesbut also occurs on O-linked sugars,as we discuss next.

Proteoglycans AreAssembled in the GolgiApparatus In addition to the l/-linked oligosaccharide alterations made to proteins as they pass through the Golgi cisternae en route from the ER to their final destinations, many proteins are also modified in other ways. Some proteins have sugars added to the hydroxyl groups of selected serine or threonine side chains. This O-linked glycosylation (Figure 13-32), like the extension of ly'Iinked oligosaccharide chains, is catalyzed by a series of glycosyl transferase enzymes that use the sugar nucleotides in the lumen of the Golgi apparatus to glucosidase' I

, ]],t*Uil-

I ir'tfqfi-&

Golgi

nnosidase

-UDP

I

u

+lq_1tl

r314

1

\r'\

1

\rtr

r'rYI

ryry

I

/n ttl

next a d d e dh e r e !

, I ,

nigh-rr"^"* oligosaccharide E RL U M E N

KEY: = N-acetylglucosamine (GlcNAc) t

complex o l i g o s a c c hrai d e

G O L GLI U M E N

(Gal) = mannose (Man) I = glucose (Glc) \!i = galactose

acid,or NANA) acid(sialic ff-acetylneuraminic

pathwayis highlyordered,so that eachstep Figure13-31 Oligosaccharide processingin the ERand the Golgiapparatus.The processing initially from the oligosaccharide showndependson the previousone.Step1: Processing beginsin the ERwith the removalof the glucoses transferred to the protein.Then a mannosidase in the ERmembraneremovesa specificmannose.Step2:Theremainingstepsoccurin the I then addsan transferase Golgistack,whereGolgimannosidase I firstremovesthreemoremannoses. Step3: N-acetylglucosamine that is yieldsthe finalcoreof threemannoses N-acetylglucosamine. Step4: Mannosidase ll then removestwo additionalmannoses.This to attack in the corebecomesresistant presentin a complexoligosaccharide. At this stage,the bond betweenthe two N-acetylglucosamines treatmentwith this enzymeis by a highlyspecificendoglycosida in the pathwayarealsoEndoH-resistant, se (EndoH).Sinceall laterstructures as shownin Figure13-30,additional widelyusedto distinguish complexfrom high-mannose oligosaccharides. Step5: Finally, occurin the galactoses, of a complexoligosaccharide /V-acetylglucosamines, and sialicacidsareadded.These finalstepsin the synthesis that have usingsugarsubstrates enzymesact sequentially, cisternalcompartments ofthe Golgiapparatus.Three typesofglycosyltransferase containspecificcarrierproteinsthat alloweach beenactivatedby linkageto the indicatednucleotide.The membranes of the Golgicisternae phosphates afterthe sugaris attachedto the proteinon the lumenal sugarnucleotideto enterin exchangefor the nucleoside that are released face. insidethe lumenfrom from the ER:all sugarsareassembled Notethat asa biosynthetic differsfundamentally organelle, the Golgiapparatus partlyin the cytosoland partlyin the lumen,and is assembled sugarnucleotides. Bycontrast,in the ER,the N-linkedprecursor oligosaccharide (seeFiqure12-52). all lumenalreactionsusedolichol-linked suoarsastheirsubstrates

776

Chapter13:lntracellularVesicularTraffic

N-LINKEDGLYCOSYLATION protein backbone

O-LINKEDGLYCOSYLATION protein backbone _ --L------

-

--L------

I

I

-CH H -l C l |

CH,

t-

C:O

I

o

Asparagine

NH

Figure13-32/V-and O-linked glycosylation. In eachcase,only the singlesugargroupthat is directly attachedto the proteinchainis shown.

|nreontne

cH2oH

._o OHH

N - a c e t y l lgu c o s a m ine

N-acetylgalactosam ine

1H H r e m a i n d e ro f o li g o s a c c h a r i d e s i d ec h a i n

H NHCOCH3

r e m a i n d e ro f o l i g o s a c c h a r i dsei d ec h a i n

add sugar residues to a protein one at a time. Usually, N-acetylgalactosamineis added first, followed by a variable number of additional sugar residues,ranging from just a few to 10 or more. The Golgi apparatus confers the heaviest O-linked glycosylation of ail on mucins, the glycoproteinsin mucus secretions(seeFigure 13-29),and on proteoglycan core proteins, which it modifies to produce proteoglycans. As discussed in Chapter 19, this process involves the polyrnerization of one or more glycosaminoglycan chains (long unbranched polyrners composed of repeating disaccharide units; seeFigure 19-58)via a xyloselink onto serineson a core protein. Many proteoglycans are secreted and become components of the extracellular matrix, while others remain anchored to the extracellular face of the plasma membrane. Still others form a major component of slimy materials, such as the mucus that is secretedto form a protective coating on the surfaceof many epithelia. The sugarsincorporated into glycosaminoglycansare heavily sulfated in the Golgi apparatus immediately after these polyrners are made, thus adding a significant portion of their characteristically large negative charge. Some tlrosines in proteins also become sulfated shortly before they exit from the Golgi apparatus. In both cases,the sulfation depends on the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(Figure 13-33), which is transported from the cltosol into the lumen of I|rretrans Golgi network.

Whatls the Purposeof Glycosylation? There is an important difference between the construction of an oligosaccharide and the synthesis of other macromolecules such as DNA, RNA, and protein. \Mhereas nucleic acids and proteins are copied from a template in a repeated series of identical steps using the same enzyme or set of enzymes, complex carbohydrates require a different enzyme at each step, each product being recognized as the exclusive substrate for the next enzyme in the series. The vast abundance of glycoproteins and the complicated pathways that have evolved to synthesize them suggest that the oligosaccharides on glycoproteins and glycosphingolipids have very important functions. l/-linked glycosylation, for example, is prevalent in all eucaryotes,including yeasts. l/-linked oligosaccharidesalso occur in a very similar form in archaeal cell wall proteins, suggesting that the whole machinery required for their synthesis is evolutionarily ancient. l/-linked glycosylation promotes protein folding in two ways. First, it has a direct role in making folding intermediates more soluble, thereby preventing their aggregation.Second,the sequential modifications of the l/-linked oligosaccharide establish a "glyco-code" that marks the progression of protein folding and mediates the binding of the protein to chaperones (discussed in Chapter 12) and lectins-for example, in guiding ER-to-Golgi transport. As we discuss later, lectins also participate in protein sorting in the trans Golgi network.

HOO -O- I P:O

I

o3'-phosphoadenorln"-5'-phosphosulfate (PAPS) Fiqure 13-33 The structureof PAPS.

TRANSPORT FROMTHEERTHROUGH THEGOLGIAPPARATUS

\

Figure13-34 Thethree-dimensional structureof a smallN-linked Thestructurewas oligosaccharide. determinedby x-raycrystallographic This of a glycoprotein. analysis containsonly 6 sugars, oligosaccharide whereasthereare 14 sugarsin the that is initially N-linkedoligosaccharide to proteinsin the ER(see transferred F i g u r e 1s 2 - 5 0a n d 1 2 - 5 1 )(.A )A backbonemodelshowingall atoms (B)A space-filling excepthydrogens. indicatedby model,with the asparagine darkatoms.(8,courtesyof Richard Feldmann.)

Man

q

\-

Man

-/"

).'\\ ,t-

Man

,-1 l)-

\, \'1

\,'

\

GlcNAc

I \ GlcNAc

\ 1l

\

\ / t:'

.

/

z

GlcNAc

.,\ -,

A/\

\{.!.g f n Y

asparaoine

^_oa

---4 (A)

777

A

(B)

Because chains of sugars have limited flexibility, even a small l/-linked oligosaccharide protruding from the surface of a glycoprotein (Figure f3-34) can limit the approach of other macromolecules to the protein surface. In this way, for example, the presenceof oligosaccharidestends to make a glycoprotein more resistant to digestion by proteolyic enzymes. It may be that the oligosaccharides on cell-surfaceproteins originally provided an ancestralcell with a protective coat. Compared to the rigid bacterial cell wall, a mucus coat has the advantagethat it leavesthe cell with the freedom to change shape and move. The sugar chains have since become modified to serve other purposes as well. The mucus coat of lung and intestinal cells, for example, protects against many pathogens. The recognition of sugar chains by lectins in the extracellular space is important in many developmental processesand in cell-cell recognition: selectins,for example, are lectins that function in cell-cell adhesion during lymphocyte migration, as discussedin Chapter 19.The presenceof oligosaccharides may modify a protein's antigenic properties, making glycosylation an important factor in the production of proteins for pharmaceutical purposes. Glycosylation can also have important regulatory roles. Signaling through the cell-surface signaling receptor Notch, for example, determines the cell'sfate in development. Notch is a transmembrane protein that is O-glycosylatedby addition of a single fucose to some serines, threonines, and hydroxylysines. Some cell t],pes expressan additional glycosyl transferasethat adds an ly'-acetylglucosamine to each of these fucoses in the Golgi apparatus. This addition changes the specificity of the Notch receptor for the cell-surface signal proteins that activate the receptor.

Transport Throughthe GolgiApparatusMayOccurby Vesicular Transportor by Cisternal Maturation It is still uncertain how the Golgi apparatus achievesand maintains its polarized structure and how molecules move from one cisterna to another. Functional evidence from in uitrolransport assays,and the finding of abundant transport vesicles in the vicinity of Golgi cisternae, initially led to the view that these vesicles transport proteins between the cisternae,budding from one cisterna and fusing with the next. According to this vesicular transport model, the Golgi apparatus is a relatively static structure, with its enzymes held in place,while the molecules in transit move through the cisternae in sequence,carried by transport vesicles (Figure 13-35,{). A retrograde flow ofvesicles retrieves escaped ER and Golgi proteins and returns them to preceding compartments. Directional flow could be achieved because forward-moving cargo molecules are selectively allowed access to forward-moving vesicles.Although both forward-moving and retrograde types of vesicles are likely to be CoPl-coated, the coats may contain different adaptor proteins that confer selectivity on the packaging of cargo

778

Chapter13: Intracellular Vesicular Traffic

\ lJ+

( A ) V E S I C U L ATRR A N S P O RMTO D E L

(B) CISTERNAL MATURATION MODEL

molecules. An alternative possibility is that the transport vesicles shuttling between Golgi cisternae are not directional at all, instead transporting cargo material randomly back and forth; directional flow would then occur because of the continual input at the cis cisterna and output atllrre trans cisterna. A different hypothesis, called the cisternal maturation model, views the Golgi as a dynamic structure in which the cisternae themselvesmove. The vesicular tubular clusters that arrive from the ER fuse with one another to become a cls Golgi network. According to this model, this network then progressively matures to become a cis cisterna, then a medial cisterna, and so on. Thus, at the cls face of a Golgi stack, new cis cisternae would continually form and then migrate through the stack as they mature (Figure l3-35B). This model is supported by microscopic observations demonstrating that large structures such as collagen rods in fibroblasts and scales in certain algae-which are much too large to fit into classical transport vesicles-move progressively through the Golgi stack. In the cisternal maturation model, retrograde flow explains the characteristic distribution of Golgi enzymes. Everlthing moves continuously forward with the maturing cisterna, including the processingenz),Tnesthat belong in the early Golgi apparatus.But budding COPI-coatedvesiclescontinually collect the appropriate enzgnes, almost all of which are membrane proteins, and carrythem back to the earlier cisterna where they function. A newly formed cis cisterna would therefore receive its normal complement of resident enzynes primarily from the cisterna just ahead of it and would later pass those enzymes back to the next crs cisterna that forms. As we discusslater, when a cisterna finally moves forward to become part of Ihe trans Golgi network, various types of coated vesiclesbud off it until this network disappears,to be replaced by a maturing cisterna just behind. At the same time, other transport vesicles are continually retrieving membrane from postGolgi compartments and returning this membrane to the trans Golgi network. The vesicular transport and the cisternal maturation models are not mutually exclusive.Indeed, evidence suggeststhat transport may occur by a combination of the two mechanisms, in which some cargo is moved forward rapidly in transport vesicles, whereas other cargo is moved forward more slowly as the Golgi apparatus constantly renews itself through cisternal maturation.

GolgiMatrixProteinsHelpOrganize the Stack The unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton, as already mentioned, and cytoplasmic Golgi matrix proteins, which form a scaffold between adjacent cisternae and give the Golgi stack its structural integrity. Some of the matrix proteins form long, filamentous tethers, which are thought to help retain Golgi transport vesicles close to the organelle.lVhen the cell prepares to divide, mitotic protein kinases phosphorylate the Golgi matrix proteins, causing the Golgi apparatus to fragment and disperse throughout the cwosol. The Golei frasments are then distributed evenlv to

m a t r i xp r o t e i n s

Figure13-35Two possiblemodels explainingthe organizationof the Golgi apparatusand the transportof proteins from one cisternato the next.lt is likely that the transportthroughthe Golgi apparatusin the forwarddirection(red arrows)involveselementsof both models.(A)In the vesicular transport model,Golgicisternae arestatic organelles, whichcontaina characteristic complementof residentenzymes. The passingof moleculesfrom crslo trans throughthe Golgiis accomplished by forward-moving transportvesicles, which bud from one cisternaand fusewith the next in a cis-to-trans direction. (B)Accordingto the alternative cisternal maturationmodel,eachGolgicisterna maturesas it migratesoutwardthrough the stack.At eachstage,the Golgi residentproteinsthat arecarriedforward in a cisternaaremovedbackwardto an earliercompartmentin COPI-coated vesicles. Whena newlyformedcisterna movesto a medialposition,for example, "left-over"cls Golgienzymeswould be extractedand transportedretrogradely to a new clscisternabehind.Likewise, the medialenzymeswould be receivedby retrograde transportfrom the cisternae just ahead.In this way,a clscisterna would matureto a medialcisternaas it movesoutward.

TRANSPORT FROMTHETRANSGOLGINETWORKTO LYSOSOMES

the two daughter cells,where the matrix proteins are dephosphorylated, leading to the reassembly of the Golgi apparatus. Remarkably, the Golgi matrix proteins can assemble into appropriately localized stacks near the centrosome even when Golgi membrane proteins are experimentally prevented from leaving the ER. This observation suggeststhat the matrix proteins are largely responsible for both the structure and location of the Golgi apparatus.

Summary Correctlyfolded and assembledproteins in the ER are packaged into COPII-coqted transport uesiclesthat pinch offfrom the ER membrane.Shortly thereafter,the uesicles shed their coat and fuse with one another to form uesiculartubular clusters.The clusters then moueon microtubule tracks to the Golgi apparatus, where theyfuse with one another to form the cis-Golgi network. Any residentER proteins that escapefrom the ER are returned therefrom the uesiculartubular clustersand Golgi apparatus by retrogradetransport in COPl-coateduesicles. The Golgi appctratus,unlike the ER,contains many suger nucleotides,which glycosyl transferaseenzymesuse to perform glycosylation reactionson lipid and protein moleculesas they pass through the Golgi apparatus The mannoseson the N-linked oligosaccharidesthat are added to proteins in the ER are often initially remoued,and further sugarsare added.Moreouer,the Golgi apparatus is the site where O-linked glycosylation occursand where glycosaminoglycanchains are added to core proteins to form proteoglycans.Sulfation of the sugars in proteoglycansand of selectedtyrosines on proteins also occursin a late Golgi compartment. The Golgi apparatus modiftes the many proteins and lipids that it receiuesfrom the ER and then distributes them to the plasma membrane, Iysosomes,and secretory uesicles.The Golgi apparatus is a polarized organelle,consistingof one or more stacks of disc-shapedcisternae,each stack organized as a seriesof at least threefunctionally distinct compartments, termed cis, medial, and lrans cisternae.The cis and trans cisternaeare both connectedto specialsorting stations, called the cis Golgi network and thelrans Golgi network, respectiuely. Proteinsand lipids mouethrough the Golgi stack in the cis-to-trans direction. This mouementmay occur by uesiculartransport, by progressiuematuretion of the cis cisternae as they migrate continuously through the stack,on most likely, by a combination of thesetwo mechanisms.Continual retrograde uesiculartransport from more distal cisternaeis thought to keep the enzymesconcentrated in the cisternaewheretheyare needed.Thefinished new proteinsend up in the trans Golgr network, which packagesthem in transport uesiclesand dispatchesthem to their specificdestinations in the ceII.

TRANSPORT FROM THEIRAIV5 NETWORK GOLGI TOLYSOSOMES The trans Golgi network sorts all of the proteins that pass through the Golgi apparatus (except those that are retained there as permanent residents) according to their final destination. The sorting mechanism is especially well understood for those proteins destined for the lumen of lysosomes,and in this section we consider this selective transport process.We begin with a brief account of lysosome structure and function.

Lysosomes Arethe Principal Digestion Sitesof Intracellular Lysosomes are membrane-enclosed compartments filled with soluble hydrolytic enzymes that control intracellular digestion of macromolecules. Lysosomes contain about 40 types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases, and sulfatases.AII are acid hydrolases. For optimal activity, they need to be activated by

779

780

Chapter13:Intracellular Vesicular Traffic

proteoly'tic cleavageand require an acid environment, which the lysosome provides by maintaining a pH of about 4.5-5.0 in its interior. By this arrangement, the contents of the cytosol are doubly protected against attack by the cell's own digestive system: the membrane of the lysosome keeps the digestive enzymes out of the cltosol, but even if they leak out, they can do little damage at the cltosolic pH of about 7.2. Like all other intracellular organelles, the lysosome not only contains a unique collection of enz).rynes, but also has a unique surrounding membrane. Most of the lysosomal membrane proteins, for example, are unusually highly glycosylated,which helps to protect them from the lysosomal proteases in the lumen. Transport proteins in the lysosomal membrane carry the final products of the digestion of macromolecules-such as amino acids, sugars, and nucleotides-to the cltosol, where the cell can either reuse or excretethem. A uacuolar H+ ATPasein the lysosomal membrane uses the energy of ATP hydrolysis to pump H+ into the lysosome, thereby maintaining the lumen at its acidic pH (Figure f 3-36). The lysosomal H+ pump belongs ro the family of V-type ATPasesand has a similar architecture to the mitochondrial and chloroplast AIP synthases(F-type ATPases),which convert the energy stored in H+ gradients into ATP (seeFigure 1l-12). By contrast to these enzymes, however, the vacuolar H+ AIPase exclusively works in reverse,pumping H+ into the organelle. Similar or identical V-type AIPases acidify all endocytic and exocytic organelles,including lysosomes, endosomes, selected compartments of the Golgi apparatus, and many transport and secretory vesicles.In addition to providing a low-pH environment that is suitable for reactions occurring in the organelle lumen, the H+ gradient provides a source of energy that drives the transport of small metabolites acrossthe organelle membrane.

0.2-0 5

pH-7.2 illlif'

"rr'i pH-5.0

A C I DH Y D R O L A S E 5 : nucleases proteases glycosidases ltpases phosphatases sulfatases phospholipases

ADP

*

Pi

Figure| 3-36 Lysosomes. Theacid hydrolases are hydrolyticenzymesthat areactiveunderacidicconditions. A V-typeATPase in the membranepumps H+into the lysosome, maintainingits lumenat an acidicoH.

Lysosomes Are Heterogeneous Lysosomes were initially discovered by the biochemical fractionation of cell extracts; only later were they seen clearly in the electron microscope. Although extraordinarily diverse in shape and size,staining them with specific antibodies shows they are members of a single family of organelles.They can also be identified by histochemistry, using the precipitate produced by the action of an acid hydrolase on its substrate to indicate which organelles contain the hydrolase (Figure f 3-37). By this criterion, Iysosomesare found in all eucaryotic cells. The heterogeneity of lysosomal morphology contrasts with the relatively uniform structures of most other cell organelles.The diversity reflects the wide variety of digestive functions that acid hydrolases mediate, including the breakdown of intra- and extracellular debris, the destruction of phagocytosed microorganisms, and the production of nutrients for the cell. The diversity of lysosomal morphology, however, also reflects the way lysosomes form: late endosomes contain material received from both the plasma membrane by endocl.tosis and newly synthesized lysosomal hydrolases, and they therefore already bear a resemblance to lysosomes.Late endosomes fuse with preexisting lysosomes to form structures that are sometimes referred Lo as endolysosomes, which then fuse with one another (Figure f3-38). \Mhen the majoriry of the endocytosed material within an endolysosome has been digested so that only resistant or slowly digestable residues remain, these organellesbecome "classical" Iysosomes.These are relatively dense, round, and small, but they can enter Figure13-37 Histochemical visualization of lysosomes. Theseelectron micrographs showtwo sectionsof a cellstainedto revealthe locationof acidphosphatase, a markerenzymefor lysosomes. The largermembraneenclosedorganelles, containingdenseprecipitates of leadphosphate, are lysosomes. Theirdiversemorphologyreflectsvariationsin the amountand natureof the materialthey aredigesting. Theprecipitates areproduced (to fix the enzymein place)is when tissuefixedwith glutaraldehyde incubatedwith a phosphatase substratein the presence of leadions.Red arrowsin the top panelindicatetwo smallvesicles thoughtto be carrying (Courtesy acidhydrolases from the Golgiapparatus. of DanielS.Friend.)

200nm

(ssabrng'1;o,{sa1rno1) aq1 1se;douo1 pelle)sreuPrquauralon)eAaqf 'alon)eA snouloua aqt,(q 1|emlla) aql lsutp6e passerd'stse;doro;qr 6urureluorlaIe; un-{}e se ;osoy{reql smoqsJealo))eqol 6unoI e ur slla)Jo qdelborrruuoJl)ale srqf 'elon)e^;;ar1ue1deq1 69-9 1 ern6r3

LLtri 0 I

Jleql ul plnlJ aq] Jo ^llcluol aql uI se8ueqJ a8Jul elldsap aJnssaJdJoBJn]luels -uoJ lsolup up urelurBru slles luBld rtueru '.{Fu1u4s 'luElsuoJ losol-,b eq} ul Hd aq] daal ol spual qJIqM 'alonJe^ aql olul +H Jo UodsuEJ] pesPeJJuIue ^q 'Ued ur ]seel le '.peJuelBqsr losotdJ eq] olq +H Jo xnlJ aql 'eldtuexa JoJ 'sdoJp luau -uoJr^ua aql ur Hd eq] uaq \'lueruuoJr^ua Jleql ur suorlElJe^apl.ry\puelsqll t o] slleJ lueld SuIqPue 'ecl^ap sllBtsoaluoq e se ]uEuodul sI alonce^ eqJ 'a8eJolspue uoDSeBIpse qcns 'suollJunJ lsullslp qtl^\ selonJe^ luaraJJrp aAPq.{elu IIas arues eqJ'(SuDII^\ luo4 lueld eql sdaa{ puE II€,\4. IIaJ aq] uo pJP&qnosaqsnd ]eq] amsseJd Jlloruso erTl)alnssard ngJru lo JallorluoJ E se pue '(O?-tI aJn8lf,) ezls IIeJ Sursearcut Jo .{u^\ Ieclluouoce ue se ']uaurlredruoc e^Ileper8ap e se (slJnpoJdalsempuu slualJlnu qloq JoJallaue8Jo e8eJols p se lce upc alonlel ]ueld aqJ 'esJeAIp,(lqe4reruar aJe suollJurg Jlaql 'satudzua pue seruosos,{l yo dlauel B ule}uoc cr1-{1orp,,tq ]nq IIaJ IEluIue ol pe}elaJ 'alunlo^ aJBseloncE^'(6S-tI arn8ld) sedd] IIaJ eluos uI %06 sEqrnru sBpue IIaf, eq] Jo %0t upq] aroru ,{dncco ,tlpcldrir Leql 'selonce,r pellef, salclsa^ pefiu-pmu 'a8re1dran pre^es ro auo uIBluoJ (s1suafSurpnlcur) sfiac p8unJ pue 1ue1d1so141

alv salon)Pnlebunl pue lueld seuososll alrlesran(1qe>;leruau ']xeu ssnJSIpa^t su 'sllaJ 'saurfzua 1ue1dut sq] ueq] uoqlugap Ja,lroJJeue l.1dde ol pJEq fletcadsa q lI 'sallaue8Jo Jllr(lorydq Jo ]ualuoc qBIq E sI qf,IqM Jo ernteal uorutuoJ eql ]ruuslp Jo uollJallol snoeuesoJeleq e sP pe^AeIASeruualuos aJE setuosos1(l 'uoseeJ srql roc 'elJ,{J uorleJnlelu E;o sa8els lueJaJJIput are ,taql leq] ]dalxa pue seruosopua elel uaa^ueq uol}3ul}slp per ou erups aq] are faqt :saruosos,{.1 aql sr eJaql 'snqJ 'saruososrtlopua Jo saruosopua alBI qllm 3utsn; dq ure8e a1c^'tc

auososr{;

'araquMor.ls a;rb uorlernleuraql ut sa6e1s1ua.ra;;rp aql se llamse'e1;aue6ro aql ol paJanrlap sleualeLu aql arnleu aql luara#rp Jo '1r ed u r's1re;1ar,(6oloqdror.u ;er-uosos,{; oqI'uo!teJnlpur ;o {1raua6oralaq auosos{1ro, lapour V g€-€ t srnbrl

eu.rosos{;opua

euosopue elel

sluautyeduto: gd-nno1'enrper{1 Ieltp{1otp[q

ol-)uo^rlN l9lo9sNvullHr woull-uodsNVUr slv\Josos^l

L8L

'(uMoqs ]ou) sluaueluutl)eJo salpunq Iq pazr;rqe1s r.riseldoy{t ;o spuerls ,{q uor6a.r Jeellnuoq} ol pal)auuol sl qrrqmle{e; leraqduadutql e ol pauUuol {;;enluanasr ;oso1{raqf 'alon)p^ 6urpuedxaue olut talemjo aleldn aq] saruPdulo)>e 1eq1luaua6le;ua e sluauolleMlla) aq] 1;eruanup-to6.rn] ;o 6urueleampaztle)ol ;osoy(teqt;o aurnlo^aql 6urseat)ut ]noqltMalilnlo^ur asear)ura6rele anarqteuet;;at 1ue;dy 's;1a> gue;dro aztseql 6ur;1o.r1uor u! alon)el aqlJo alol aqlgy-61 ern611

Jo auerqueur alqnop e .{q alaueSJo uP Jo aJnsolcua aqr qll,\/\ ur8aq o} sureas sse3oJd aqJ 'sallaue8ro raqlo sE IIa^ se (BrrpuoqJolru (SuUSeBrp.{lqeruns -aJd puu) Suruteluoc saruosos^l IeeAaJslleJ IeruJouJo sa8erurcrdocsoJJnuuoJl 'eldruexe -Jele pue 's,{ep 0I }noqp Jo aupaJII E sEq uorJpuoqJolru aBeJeAeue roJ 'lleJ ralrl E uI 'f8Bqdolnu paller ssacord e-Jlastr IIac eqt Jo sued etelosqo Jo asodsrp ol satuosos,(lur,{e,,rnqleduopepeJ8ep puof,as e esn sed.&IIaJ IIV 'NCJ aql Jo saruosopue ol IJeq surelord aseql re^rlep ]eq] selorseluodsueJ] ^q aurosos^lSurdolarrapaql ruoJJpa^arJtalfla^rlcalas aJPsura]ord auPJqruarulBruosopue'eJnlBursaIUososl(l sV 'Hd lEurelul ur esEaJJapJaqUnJ e dq paruedruoJrp 'saruosopua alBI ruo4 ssesoJduorleJnleru E ^q uroJ satuosos,{laln]eru 'e^oqp passntsrp sy 'sur8aq sa1ncalorupasoldcopua eql Jo uouse81pcpr(lorpr(qeql eraqm etrs aq] sl tl pue '(gUd) JIpIf,e,(lp1nusr seuosopua atEI eqlJo JorJaturaql'sawosopua alal olur uo ssedsreqlo elrqm '{au€Jqtuarueuseld aq] ol palcl.cal pue panarrlar.{1a^r}Jelas ere selnJalou palsa8ur aq] eruos 'snleredde €1og aqr ruoq auosopua aql o] Jo paJa^rlapare qJrqM 'sasBlolp/iLl aql leeu lsJu slPrJalpupaso$oopua lPr.uosos^l enH'saruosopua [paa pallec saflaue8ro replacerlur padeqs d1;eln8au '1purs ol salorsel ur peJalrlap.{11ur1rur ere salnJelou paso1-bopuete}el [e]ap ur passnc -slp sV 'srsoltcopua f,q pnU JelnllaoeJlxe ruor; dn ua{el salncaloruorceu t(q pamolloJauo eql sr seruosos^lur sqtpd uoqeper8ap asaqtJo parpnts lseq eqJ 'uoqsa3rp roJ saruososf1olur sef,uBlsqnspeeJsa]Jnos tuaJaJJrpruor; sqlpd eaJql lseel lE alrqm 'seu^zua anqsa8rp lsoru srelrlap snteJeddp r31og aqt e1nUg aqt ruoq spre^ lno speel teq] etnor V'a8raluor trJ -JpJtJplnfiaceJ]ur sureeJ]s Jo IEJe^aseJaq a saculdSuqaarul.lpnsn aJesauososf'I

sautosos^l ol sleualewro^tlecsleMqledaldrllnw 'srolepard tsurp8p esuaJape apnord pa8eruep ro uelee sr lueyd e uaq ^ salonce^ ruo4 pasealer salncelou snouou elrq A 'slcasur 3ur1eur11odlcerlle ol sp os sJeMolJ.,(ueurgo sleled eql Joloc salonJpl ur palols sluaur8rd urue^J -oqluv'o^rq{ua Surdolanapaql roJ,{1ddnspooJ e apnord sprre ourrue Su4psar eql pue pazfyorpfq eJBsureloJd aseq] 'aleurruJa8spaas aql uer{M'supaq pue sead;o asoql sE qJns 'spaasfueru;o sllal eBBJo]seql Jo salonJpl aq] ur sJBe^ ro; panrasard eq uef, 'aydruexaroJ 'suralord 'uor]JunJ Jrloqelaru e aAEqslcnpord 'cqre8;o paJols,{uey41 Surroneg aql o1urnrdo ol JaqqnJruor; a8uer asaql 'sarcads 'selonoe^ luaJeJJrpuI lueld ur paJols saJuelsqns lse^pq ueuo sueunH 'saxng aseql IoJluoJ o] aueJqueu qJEe ur sralrodsueJl lcur1srpJo serlr -AIIJEaq] saleln8ar arnssardro8Jnl aqJ'auerquretu JBIonce^eql pu€ auerqureur eurseld aql ssorJe selrloqelaru Jaqlo pue 'sprJe ourure 'sJe8nsJo salpJlrodsue.rl aql Suualp ,tq rred ur pue 'aloncen eql ur areqdsoqd,rtyodse qcns sraur,{yod ;o stsaqtufser pup rrmop]Barq pellor]uoo aqt dq ped ur-alonoe^ pup losoldc eq] Jo alnssard crlourso aqt Sur8ueqc ,{q os op ,{aq1 '}ueruuorrlua alprparu{ur

I

snal)nu a u e : q u a t ue u s e l d

alon)e^

l l P ^ ^l l o ) urseldoy{r ]o spueJls

)ulP4.relnrlsen relnlla)etlul:€[ re]oeqf

z8L

TRANSPORT FROMTHEIRAN5 GOLGINETWORKTOLYSOSOMES

783

unknown origin, creating an outophagosome,whichthen fuses with a lysosome (or a late endosome). The process is highly regulated, and selected cell components can somehow be marked for lysosomal destruction during cell remodeling. For example, the smooth ER that proliferates in a liver cell in a detoxification responseto lipid-soluble drugs such as phenobarbital (discussedin Chapter 12) is selectively removed by autophagy after the drug is withdrawn. Similarly, other obsolete organelles, including senescent peroxisomes or mitochondria, can be selectively targeted for degradation by autophagy. Under starvation conditions, large portions of the cytosol are nonselectively captured into autophagosomes. Metabolites derived from the digestion of the captured material help the cell survive when external nutrients are limiting. In addition to maintaining basic cell functions in balance and helping to dispose of obsolete parts, autophagy also has a role in development and health. It helps restructure differentiating cells by disposing of no longer needed parts and helps defend against invading viruses and bacteria.Autophagy is uniquely suited as a mechanism that can remove whole organelles or large protein aggregates, which other mechanisms such as proteasomal degradation cannot handle. We still know very little about the events that lead to the formation of autophagosomes,or how the process of autophagy is controlled and targeted at specific organelles.More than 25 different proteins have been identified in yeast and animal cells that participate in the process.Autophagy can be divided into four general steps: (1) nucleation and extension of a delimiting membrane into a crescent-shapedstructure that engulfs a portion of the cytoplasm, (2) closure of the autophagosome into a sealeddouble-membrane-bounded compartment, (3) fusion of the new compartment with lysosomes, and (4) digestion of the inner membrane of the autophagosome and its contents (Figure 13-41). Many mysteries remain to be solved, including identifying the membrane system from which the vesicles that form the autophagosomal envelope derive, and how some target organellescan be enclosed so selectively. As we discuss later, the third pathway that brings materials to lysosomes for degradation is found mainly in cells specializedfor the phagocytosisof large particles and microorganisms. Such professional phagocyes (macrophages and neutrophils in vertebrates) engulf objects to form a phagosome,which is then converted to a lysosome in the manner described for the autophagosome. Figure 13-42 summarizes the three uathwavs.

A Mannose6-Phosphate Receptor Recognizes Proteins Lysosomal in the TransGolgiNetwork We now consider the pathway that delivers lysosomal hydrolases and membrane proteins to lysosomes. Both classes of proteins are co-translationally transported into the rough ER and then transported through the Golgi apparatus to the TGN. The transport vesiclesthat deliver these proteins to endosomes (from where the proteins are moved on to lysosomes) bud from the TGN. The vesicles incorporate the lysosomal proteins and exclude the many other proteins being packaged into different transport vesiclesfor delivery elsewhere.

lysosome

Figure13-41 A model of autophagy. After a nucleationevent in the a crescentof autophagosomal cytoplasm, of membranegrowsby fusionof vesicles unknownoriginthat extendits edges. Eventually, a membranefusionevent sequestering closesthe autophagosome, a portionof the cytoplasmof the cellin a Theautophagosome doublemembrane. containing then fuseswith lysosomes acidhydrolases that digestits content.

784

Chapter13:lntracellular Vesicular Traffic

(A)

Figure 13-42 Three pathwaysto (A)Materials degradationin lysosomes. in eachpathwayarederivedfrom a differentsource.Note that the hasa double autophagosome membrane.(B)An electronmicrographof containinga an autophagosome mitochondrion and a oeroxisome. (8,courtesyof DanielS.Friend,from D.W Fawcett,A Textbookof Histology, 12thed. NewYork:Chaomanand from Kluwer.) Hall,1994.With permission

LATE ENDOSOME

LYSOSOME f : ,

. mitochondrion

-' i"

:.:,, ltt

i .4N

3.

--__j_ 1^

How are lysosomal proteins recognized and selected in the TGN with the required accuracy?Weknow the answer for the lysosomal hydrolases.They carry a unique marker in the form of mannose }-phosphate (M6P) groups, which are added exclusively to the l/-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cls Golgi network (Figure f 3-43). Transmembrane M6P receptor proteins, which are present in the TGN, recognize the M6P groups. The receptor proteins bind to lysosomal hydrolases on the lumenal side of the membrane and to adaptor proteins in assembling clathrin coats on the cltosolic side. In this way, they help package the hydrolases into clathrin-coated vesiclesthat bud from the TGN. The vesiclesshed their coat and deliver their contents to earlv endosomes.

TheM6PReceptor ShuttlesBetweenSpecific Membranes The M6P receptor protein binds its specific oligosaccharide at pH 6.5-6.7 in the TGN and releasesit at pH 6, which is the pH in the interior of late endosomes. Thus, as the pH drops during endosomal maturation, the lysosomal hydrolases dissociate from the M6P receptor and eventually begin to digest the material delivered by endocytosis. An acid phosphatase removes the phosphate group from the mannose, thereby destroying the sorting signal and contributing to the releaseof the lysosomal hydrolasesfrom the M6P receptor. Having releasedtheir bound enzymes, the M6P receptors are retrieved into retromer-coated transport vesiclesthat bud from endosomes; the receptors are then returned to the membrane of the TGN for reuse (Figure 13-44). Transport in either direction requires signals in the cltoplasmic tail of the M6P receptor that direct this protein to the endosome or back to the Golgi apparatus. These signals are recognized by the retromer complex (seeFigure 13-9) that recruits M6P receptors into vesicles in endosomes. The recycling of the M6P receptor resembles the recycling of the KDEL receptor discussedearlier, although it differs in the type of coated vesicles that mediate the transport. Not all the hydrolase molecules that are tagged with M6P get to lysosomes. Some escape the normal packaging process inthe trans Golgi network and are transported "by default" to the cell surface, where they are secreted into the extracellular fluid. Some M6P receptors, however, also take a detour to the plasma membrane, where they recapture the escapedlysosomal hydrolases and return them by receptor-mediated endocytosis to lysosomes via early and late endosomes.As lysosomal hydrolases require an acidic milieu to work, they can do little harm in the extracellular fluid, which usuallv has a neutral pH of 7.4.

m a n n o s e5 - p h o s p h a t e (M6P)

N-linked

o li g o s a c c h a r i d e

rysosomal hydrol ase

Figure13-43 The structureof mannose 6-phosphateon a lysosomalhydrolase.

lolda)or aql qllM snleredder6logaql ol ulnlar aql lpqr 6uunsua lou op seselorp^q raqlrnJ'saselorp^q aqr o1paqrellesrebns asouueLueql u,rorlpo^oual srateqdsoqd aql'saurosopua aql ul 'tlodsuell Jo spunorraqlrnJro; snleledder61o9aqr palPo)-lauollal ut pa;>r{raj ol sal)lSaA are sroldo)ar^ldrua aqt pue's.ro1dare.l sase;orpi{q d9Waql uorj aler)ossrp aql 'aurosopuaaqt 1o pd ramolaql I;rea qtlrvr asnJpue'leo) lV'saurosopua Iaql paqs'N9I aqt u.lor]JJopnq sal)rsa^ paleo)-uuqlel)aq1'sase;orpIq ;er-uosos{; polJlporu aql pulq'urnt ur 'q)rqM 'srolda)er d9w aql purqleo) uuqlel) aqt ur suralo.rd roldepe)uaurouoLu asne)aqNDI aqt ur suralotd1o sed,(traqto lle uorJ a1e6e.r6as ueqr {aq1 '(gy-g arnbr3aas)saur{zua 1 ;er.uosos{; ;o srosrn>ardaqr or sdnot6 16914;; ateqdsoqd-9 asouueLu sppelloMlau t61ogsuotl pue s/, aql ur sau{zua oml Jo uorl)e;erluanbes aql 'sauososl; o1 sese;orpIqleuososl; pezlsaqluls {1meu;o l.rodsuerlaq1trtr-g 1 arnbr3

'sllac .sluarlEd er4lut suotsn\cut uI e}plnrunocese}PJlsqnspelse8pun a8rel ruro; lpuanbasuoJ qJIqM 'saruosos.{1 rraql puE 'slselqorqrJ Jo sauosos^l aq] Iuo4 Sulssnu aru seu^zue 3ll-{loJpl.q eql Jo IIE lsorrrle'uoltlpuoc srq] uI'@saaslp uax-I) asuaslp llaJ-uolsnljul pellPJ rapJosrp arEJ.{rarl e sr ta.ta.r,roq'aseeslp eSBJotsIPlxosos,{l Jo IUJ0Jere^as '8urss[u Jo a^rloeJep sI suIPqJ ueJ^lSouluresoc1tF saddl uIEUec lsour aql Jo Jo u^ op{eeJq eql JoJpeJrnbeJerul.zue eql qJIqM uI 'aldruexa rcJbsoaslp s,JaIJnHur pruosos,{l pnpt^Ipul ue JoJsepof,]Eql eueSleJn}f,nJlse uI sJnJcosrqJ'esBloJp,r(q uol]Plnru P sr eJeql 'sasPcJsoruuI 'Iualsl.s sno^Jau aql ul ueuo lsoru 'sesuenbes -uoJ IEJISoloqled eJe^esqll^\ 'seluosos.{luI selBqsqns pa}se8lpun Jo uoqelnlu -n3Ju uP ul ]psal slJaJap aql 'sasueqp atBJols lBtuososfl ueunq Jo Jeqlunu E asnEJ seseloJp,{qlEruosos^l eq} Jo aJoru Jo euo }leJJP}eq] slcaJap Jlleuec

eoeJols sueunHu! aseeslc )vN)lDeql u! sl)arac Ieuosos^le osne)aseralsuerroqdsoqd rotdarar dgIN eq] roJ IeuBIs rirpgJe 'sapueqccesoStloaldrlpu 'sanplser -qBIq arlnboe daql B Surpnord 496,{ueur e pulqeq urpluoJ saselorp,,{.qpurosos,{1 lsoru eJuIS 'JaIJeru dghtr pe}PaJc,,i1,rnau 8unea1 'enprsar JVNJIC aq] JJo selpalf, ueqr FIoC sunu erft uI eudzue puo -Jas V'(S?-Sf arn31g) urBqJ apIJeqJJesoBIIoqJPe uo senplseJesouuPu eqlJo omt ro auo o] aleqdsoqd-cygrlg sppe pue aseyorp,(qaqt spulq,{lecgtcads leqt r31ogsre aq] ur espraJsuerloqdsoqdcy51r1gP sI lsrlJ eql 'saselorpfq pruososf1 ot sdnor8 uop1ppe eq] azltleleJ o] [fienuanbas ]rP seudzua o,trJ dgIAI Jo 'eJeJJnss,uleloJd qf,Ea 'r,lJlod lryu&lse se rrrv\ou{ uo sprJe oultue SurroqqSrauJo JolsnlJ e st puSts uopluSoseJ aq] lEql pel€e^aJ e^Bq slueuuadxe Surraaur8ua JIlaueC 'aselorpdq qcee Jo ulEqo eplldaddlod eqt ur aJaq ^aruos eprser lsnu seplJeqscesoSllo o1 sllun dgl tr eql Suppe roy puSrs eq] 'surpqr aprreqcrPsoSllo pa>1ulr{ IPlPuepI qllM UA eql e^eel suralordoc,(p ile eJurs i1911Surppe roJ elqlsuodser setu/tzuar31o9 aqr,rtq sasul 'snleredde rB1o9aql ut suralord -orpfq aq] Jo uoqruSocar cgtcads sartnbar stql -oc[13 alerrdordde aql o1,{po pappu are sdnor8 dgIN asneoeqslro^ seuosopua puososdl salu8ar8asleql ua}srts SupJoseqJ ol ueqt saqcledsrppue saselorp,'{.q

uo!l!ppvd9w tol en) aqr ul q)led leublsv aql sapt^orduteq) oplrdad^lodeselolp,(H s n l e : e d d er 6 1 o 9

eurosopue[;rea

a p r s a n6 u r p p n q ur roloalar d9l^

9Nt'r)Aflu uol_d3)3u

rosJn)alo ase;o-rpr(q leulososr{;

NO

leo) rauJorlaJ

el)rse^ u o d s ue r l

]eo) urJqlell

)vN)19-d lo NotJ(lcv

ul Luorl

IUOdSNVUI r N 3 CN 3 dl C - U O I d 3 ) 3 U

!d + dqv

SSOUU eUl

losJn)aro aselorpi(q;euosos(;

lHr t/louJluodsNvul or-)|uoMIlNlDlogsNHU-L slr^rosos^r

s8L

aptteqrresobt;our asouueu o] paq)elle J'- rypr19 qlrm a s e l o r p , {e qu o s o s , 4 l

alts uotltuoo:el

iaql sautzua JuttoJp/tlt aqnlos to lgaunn aprm Dpuo sunlord auuqtuaut anbtun uru1 -uoc taql salncalowotJaw to uousaBrpJalnllaJalrut aql rc! pazuanadsata satuosos[1

{reuun5 ' (rusrurqp) uopeluauSldodr(q ;o surro; ol Surpeal'ssacordJaJsuEJt slq] {Jolq srsol-{coxa erxosoueleur ur sloeJep'sJepJo -srp cqaua8etuosuI 'uopeluaru8rduols IpruJouo1Surpeay'sa/oourlera1 dq dn ue{el uaql s luaru8rdaql'srsofroxa,(q snuraprdaaqlSoacedsreplacur}xe eq} olur luaur8rdJreq]eseeleJ sauosouapu Surureluoc-lueu8rdasaq; 'saurososf1 Jraqlul sluaru8rdeJolspue acnpord'aydurexa JoJ'UDIS eqlulsaubouopry'euetq -ruaur eruseldeql qlIM uorsry ro;,{raurqceru fuessacauaql parpbce e^eq leq} pazgercads saruosos,{1 ure}uof,telamoq 'sadd11ac eruos'pesseJls alp sllaf,eq} uaq,u.dpopasn'd.emqled rourure eq ot sueasslql 'sllaolsoruJo{ 'srJqepalqqsa8 -Ipun palsa8pun lo uoualJasTnutososf1 eluurrurTe o] sflac salqeua ]ualuof, [e deruqledeq] Jo pue aq] ,tlgressaceu lou sr satuososl.lol IprJalerugo Supa8rel

slsov()oxfobrapunsauosos{]eutos 'NCI aq] ol peuJnleJ eq ]ou peeu pu? sluap -IseJ lEuosos.{1 are suralord eueJqueru asoql-sroldecal dgIAI e{rlun-a3urs .,tletcadsa 'suralord purososrtl punoq-eueJqrueu pup elqnlos ro; alerado sruslueqraru q8noqlp 'saurososdl SulspdJns sdeqrad sr ]ualaJrp teql lou 1r JBelJun sl lI lJnJlsuoc o1 demqled Suuros euo upql aJou paau s11ac.,(qrvr. 'suralord roldepe lueJagrp esn pue saselorpdqpa33e1 -dglAtreql uodsueD leql esoql ruo{ lourlsrp aJe }Eq} self,rsel peleoc-urrqlelo ur 'aspes1p NCI aq] Iuo4 lIXe suralord aueJqruerueseqJ ilec-I ul Ipurou eJoJeJeql are suralord esoql pue 'saruosopua elEI ol Uodsuel JoJ NCI aqt ruoq seruos -os,'(1 suralo.rd aueJqueu eql suos slleo ;o IIe ur de,r,rqledluepuadapul-dgl4tr ue 'dlreprurg 'rrmoDlun sr r(enqled 'sJaqlo luapuadapur-dglN slql Jo eJnleu eqJ lou 1nq sadfi 1ac aruos dq pasn sr teqt saurosos.{1o} saselorpdq Supcerrp ro; deirnqled Jeqloue sI eJeql pruJou E urel Sultldrur 'sarudzua pruosos.,tl;o reqr luarualduroc -uoc 'selrtcoledaqse qcns 'sedIl eruos ur saurosos.,tl eql resuesrpflac-JuI IIaJ '.{unrqled }lneJap e ltq palarcas pue eJeJrns IIac eql ol perJJuJare saselorp,,(qpruososdl aq] 'pealsul 'NCI eql ur selclsal lrodsuerl aleudorddu aql olq ruaqt ate8ar8eslou op sroldacar dgl treqr '1romtau r31ogsrc aqt ur paley,(roqdsoqdlou eJpsaurfzua pruososrtl esneoeg 'espJaJsueJ]oqdsoqd -rVNclC pef,BJl Surssru Jo alrlJaJap E ol ueeq suq Suprosspu aql'saurososdl o1 'sn}uredde r31og aqt ug,t1ra Der peleJJes are saselorp.{.qeq} 'poolq eqt ut punoJ aJe saruososl.luror; Surssnu F asneoeg seesrp IIeJ-l qtll,r stuarted u1 'aua8 e^pceJep aq];o serdoc pul ul .ii.1uosrncco ll 'sl teql-e^rssesal s1 11 'sarcuarcr;ap u e)H 'pue loeJep aua8 a18ursE ot enp sr espesrpIIeo-I

alrs lrltr;e1e> /

\

ese:e;suelloqdsoqdlypll9 ann . ......:.

,

I

'pasodxa 'pasodxa 'ryJ1>;9 aql 6urnee; 6urnee; 'ryp>;9 aql aql sa^eal)au(zua au(zua d9w d9W aql Jrosa^eal) Jro puo)asv'sase;otp,(q leuosos,{; }o a)erlnsaqt uo /(;uo e ol sputqalrsuorlru6otareu tuasardsr1eq1qtled

l

01 d -)vN)19 Jo UtlsNVUt_ "j"t;;;tffi?

\ \

I ;eubrs 'rvN)rD-don puesapreq))eso6r;o pa1ur1-L1 asorueu-q6i[

;jitil".'|$l];3]3'":

qloq spurqelrs>r1{;e1et aqf 'sallsuorlru6olalpue rr1{;e1e: aleredasseqsnleledder6;o9aqt ur saselotplq;euuososr{; saztu6olalteqt er.ulzuaasera;sue.rloqdsoqd tyyrl9 aqt 'ase;orp{q aq1 gy-g I arn6r3 ;etuosos{;e lo uog11u6o>a.r

ase;o-rpr{q leu-rososi{;

rf#prlrqnrlsa^relnllarerlul;€tlBldEqf

ggl

lhlosooNS

'luvl

ur urerueJIIr ^ saruelsqns elqqsaSpur ,,(uv 'peperSep ueql sl IerJs]BrupalseSur aq] pue 'ller aql eprsur saruosos.{lrl}lm esnJ seruoso8eqd aql'(g?-tl arntlU) 'aycrlred palsa8ur Jlastr IIer cq,,bo8eqd aqt se a8rel se lsotup eq uer daql pue 'rurog aql pauruualep ezs eql dq ale sJeleuerp eAEq saruoso8eqd ]eq] Jo -run pue aJEsrsol-{courd pallolur ur salJrsal cpdcopua eq} seeJaq \ Ipus 'aldruexa ro; fep,fuana sn;o qJee ur sllal poolq peJ luecsaues rr0I ueq] arou asol-,(co8eqdsa8eqdorceu rno :luelrodul lsoru aqr rel dq sr qlec ppap pue luarsaues Jo aJupJEalJaq] 'sruJe]anpelpuenb uI '(gI raldeq3 ur pessnJsrp)srs -otdode dq pe1pe^eq leq] sllef,pue sllal luaosauas8u€ua,recs ur aloJ lueuodun ue elpq osp sa8eqdorJel4l 'uorloeJur lsure8e sn pueJap o] srusrue8rooJtrru Surpenur lsa8ur deqt pue '(g7 raldeq3 ur pessncsrp) sllal urals crlarodouraq uror; doyanep sllao esaqJ 'sgqdorlnau pue saSuqdoJJuru-satdoo8eqd puorssa;ord se IJE sllac poolq elrq a Jo sasselJoarq 'sleurureu u1 'saltcoBaqd ,uor1 Tauotssatotd palleJ-os-sllal pezrlerJads ,tq zt1urcu lno perrJeJ sr 1r pue -IJlnu ueql raqlo sasodrnd ro; slerurup lsoru ur tueuodrur sr srso{co8eq4 'slcnpord srs,{lorpdq aqt s[ec pup 'salcrped pooJ umop lrodrur ilerus 'aldruexe JoJ 'slelurue;o 1n3 aq} uI fllual3rJJa sels {eeJq sassaJordrelnlacerrya -pred a8rel qcns lsa8ur ol elqe are sursrueSroJulnlleorllnru ut slleo rlteJ taAaMoH 'pooJ pesn se aq o] Iosoldc aq] olul ssed sassacordanrlsaSrpluanbasqns aq] 'saruosos,{1 ur dn pua saruoso8eqd o}ur dn ua1e1salcqred ;o slcnpord aql pue a8rel :8upaa;Jo ruJoJe sr srso{co8eqd'eozolord uI 'sllet peep pup srusr -ue8roo.rcnu qJns se salrnred a8reylsa8ur ol saruosoteqd palpc salcrsan cr]r(c -opua a8rel sesn IIaJ e qorq ^ ur srsoldcopueJo ruJoJprcads e sr sgsoldco8uq4

sel)ruede6re1lsa6uluef slla) )!l/()o6eqdpaztler)ads 's1ac crl,bo8eqd pazrrcrcads,{qdltualcrga lsoru palsa8ur are salcrlred a8rel lsrsoldcourd,r(qsalnlos pup pmg Suqsa8urIlenu4uoc eJBsllal cglodrecna ' ]sohl (Jeleuerp ur ruu 00I ]noqe) sapsan 44tcoutd ilerus erl palsa8ur are selnlos pup pmg '(,,3uqurrp 11aq,1stsoltcoutd u1 '(relaruetp ur ruu gg7< .rtleraua8) sawoso&aqdpalleJ selJrsa,ra8rel un palsa8ul are salcrlred a8rey,(,,8upeailac,,) stso1tco&nqd uI 'parrrJoJ selrrsan opdcopua aqt Jo ezrs aq] ol Suprocce ragrp srsolrbopua;o saddl urEru o^al 'aycqred ro e3uplsqns palsaBur aq] Surureluoc ap6an c4tcopua uB ruJoJ ol JJo saqJurd ueql pue saleurBenul tsJU qJIqM ,auerq -ruaru eruseld aqr go uorlrod s palsa8ur aq o1 IIEus e dq pasolcua dyanrssarSord 'ssaJord 'slleJ 'sespJ ,pue ,secuels sql uI reqlo uala pazrprcads ur IerJalerueql -qns elepcqred 'salncalouroJoprudn a1e1sllec qtrqm ,,(q,srsolbopua Jo ssec -ord aql qlIM upls saurosos.,(1 ol aJBJrnsilec aq] ruo{ pJp^ ur peel leq} selnoJ eqJ

SISOU)OCN]:SNVUBW]W VWSVIdlHI WOUJ II]) ]HI NIIUOdSNVUJ '4JOmlaU t31og sueg aql uott sutalotd autuqtuaw Tar"uososty juaplsar ranqap ol sapsan paWoc -ulrqruIr sasnwalsts xtodsuau alotodas V'lououJarrprun sasnlotptq aq1to ltodsuotl aql SurYotu 'stotdacatasaql uott alazJosstpoj sasaprpltq pwosost\ aqt asnocdnot? dgry aqi uto{ alatldsot4daqt lo lanowar puv saLuosopuaur 11d mol aLlJ .sawosopua asaqlpua 18109sue[ aql uaampq rluot pua 4J0q auruLF stoldacatdgw aqJ TJompu 'sawosopuaol sjua7uoJJraqj.ranryap yodsutul Sulppnq orut uaqj aBa4cnd sa1usan ruqj sd1aqpua sasa1otptt1aqt sawBat&as lvqt yrompu fi1og suerl aqJ ur utalotd toldacat 4914ua fq panuSocatata sdnotB (49141alar4dsoqd-gasouuaw asaql .palzt7Uoqdsoqd aJDsanprsalasouuotanaql tuql os 4lomlau 87og so aql w [am anbtun o ur pa9ppout [1]ua1anocarv wr67sapuor4ccoso&1to pntull-Nulz1uor sasa1otptttlTatuososf1aq1 'saplsan lJoasuuJl palooJ-ulJt4lDIJ lo suaau [.q sawosopuaawl oj ryomtau 87og sue[, aql uto{ partJoo uaq] puD tsnl -atnddo t31ogaql q?notql paltodsuotl ,gg aW uawry pattalsunt1 aqt olut art) sulaj to '1qdmol sn4LsutojulDu auo)qutara -otd Tauosos[l pansazqluts[1,ma111 aq| Twuososr(1 ut dtund *g uanrrp-dJvuy'sauosos[1togd TnutalulaqJfl qJtqm,ggd 1o6aq a]otado

stsolA)ocNlrtNVUSwlw vwsvldlHt_WouJllt) tHl0t NtruodsNvul

L8L

('t16[ 'uolletodro))oo8 :IJo MaN'eseestc pue LlrleaH ut su_tstueq)aw le)rpawle]uaur]uo)ralul 'uoluregj ,(qtoro6;o{se1.rno>'y) 'auosobeqd6urr.uro; rry(>o6eq6 eql,o euerqureur aql u! saprlrsouroqdsoqd rg;>ds 1ouollelnurn))e pue uor]ezuer.ur(lod aqt ot puodsarq)r[1M'uor]ezrue6roer urlre {q uenupare uorleuro1atuosobeqdpue uorsua}xapodopnasd(g) '6u;p;nlp ssa>o.rd aql ul s! q)rqM/urnual)eqe 6ursol,(ro6eqd 1o uorl)ala uV (V)'slsoybo6eqd ;rqdorlnaue;o qder6or>rr-u 6ugrnpauerquraueurse;de 6ugdeqser ;lqdollnauy 19-91 ern69 (8) a s e u r ) - €l d euerqtxeur euselo

€a(s't'e)ra

zd(s'r)td

podopnasd

unuapeq

urnrJellPq

'llal ppap eqt srsolri.JoSeqd eql reSSlJlo1 sdleq ]l ereqM 'llal eq] Jo apls Jo -tno aql uo pesodxa ^^ou sI tafegq prdq aqf 5o leuEal Jlloso]tf, eql o] peu.IJuoJ 'acuanbasuoJ P sV flerurou sr qorq ^ 'autrasp{ptleqdsoqd pe8Jeqo ztlanpe8au 'aueJqruatu eruseld 11eq]q spldloqdsoqd;o uorlnqlrlstp ctrlaururziseaq] esol 'stusluE8 slleo srlotdodv'srsoldode ^q pelp a^eq ]eql slle3 azluSoJersreqlo ilFS -JOOJJrruurElJeCJO eJeJrns 3rD uo saplJeqJJesoSqo aztuSocar rtpoarp sJeqlo '(p7 raldeq3 uI passncslp)uollsnJlsep JoJsaqoJJllu Surla8ret uI selpoqnue qly!\ 'pazlJelceJBqJ aleJoqPllos qJIq \ 'sluauodurol ruawalduoJ ezruao}eJ aruos uaaq aAEq srsoldcoSeqd aloruoJd ]Bql sroldeceJ Jo sassels reqlo leJa^es 'uoltBruJoJauoso8eqd uI sdels lElluenbas sepm8 sepplsouloqdsoqd '(gzt-tl eJn f,rJrf,edsJo uoudunsuoo pue uorteraua8 paJepro eqt ,{ervrslql uI -3rg) euroso8eqd SuIuroJ aqlJo uoueur8enur eql allrp dlaq o1lroa4eu uIlf,E eql Surdeqsarol alnqrJluoc oqe .{EIu pup aluosoBeqd aqf Jo elnsolc JoJpeJlnbeJsI sd(9't'€)Id 'td(g't't)Id 01tr sua^uor qJIqM'esEuDI-€Id B ot pal3afqns st zd(9'7)Id se eseq s1r1e pazrrarudlodapsr ullJe ']ueluJp8ua s1talalduroc pug eulosolpqd eq] JJo [Pes oJ '(tt-st eJnSIc eas) euErqureu aql ul zd(g'7)Id Jo uol]elntunccE 'seseul) uE ol asuodseJur sJnJco uopezlJauil.lod uIIJP IEIlluI pue Id leJol Jo .,{]I -^IIJB eseuq aq] uo seqclu s asPdlg oqu eAItJeuv'spodopnasd aql sadPqs'(9I pue 9I sreldEql uI passnrslp) sCgC-oqU Supe,tpce reql pue sesBdIC l11ueJ -oqg rtq patEIlIuI 'uotlezrrarudlod ulloe pezllsso'I '(VZ7-SI ern81g) auosoBeqd P ruroJ ot sdq rtaql le esnJ puP elsltJEd eql JInBue leql spodopnesd pueue o] ilac 'slrqdorlnau pue se8€qdoJJEIII af,eJ cpdco8eqd aql secnpur Surpurq esoqM Jo -rns eql uo sJotdararrc Julceds rtq pazruSocarsl leoc,{.poqr1ueeqJ,'(92 reldeqS 'apcaloru z{poqlrueqoee ur pessncsrp)uot3ar rC eql pelleJ sI uolSer IIel slql Jo JorJelxe eql uo uoISeJ IIel aql sesodxa leql leoo E luJoJ ol susIuEBJooJcILusnoq -JeJurJoef,EJrnsaql ol fiurpu1q,{.qsn tca}ord qJIqM 'sarpoq4ue are qsoltcofieqd 'r(1sno 'ilec eqr Jo spaeu eql ;o ssalpru8ar ;o sra33rr1pezlJat3Ereqf,-lseqeql -nurluoJ sJncco lI 'ssacoJdeAIlnlIlSuoJ e st srsolrtcourd'1set1uoc,(g 'asuodsar eq] aleFlul pue JorJalurIIes eq] o1 spuSrs llususrl leqt sroldaceJJo uope^llJe 'llel eqtJo ,tJaulqcetu eql serpber ll 'sl leql'sseooJd para83rr1e sr stsotbo8eqd crl,bo8eqd eqt o] pe{uI ,tlpuorlcun; eJE }Pq} sJoldeoeJ eoeJJnspezqercads ;o 'e1l{c Ilarre,r e aneq sa/.co8eqd 'JeAa toq 'palse8ut eJeputq 1eq1salcrped ile loN -o8eqd aq] Jo eoeJrns eql ot pulq lsrlJ lsnru salcllJed 'pasolrtco8eqd aq o1 .eueJquau uruseld eq] o] peuJnleJ pue salJrse^]rodsuer] ur auosoSeqd aql ruoq pe^erJlar are,{aq} asnelaq 'aurososr(1aq} qf,PaJJeleu s}uauoduroc auerq -Iuelu eluseld pazqeuralu aql Jo aruos 'pessncstp .{lsnovrard aneq a^/\Se 'SISol -dcoxa ,{.qslac (uo4 palaJcxe aq uEJ qJIq { 'salpoq IanpKaJ SuturroJ 'saruososdl

fla^au lneduearJo {sa1rnol)'sllal pal aql l;nbuao} srello) se 6urpualxaareleql abeqdoneu aql urql 1osa6pa ;o (spodopnasd)sassaro.rd ol luloo s/t^u)Dpai aqf 'slla) poolq paJ paralleI;;e>ruaLl)oMl 6ursoy(ro6eqd abeqdor>etuasnouie Jo qderborrrr.u y'abeqdoneu uol]rale6uruue>s e {q srsoy(robeqS99-6 1 arn611 ufi S

)rteJl reln)lsen lPlnlle)ellul :t L laloellf

88/

TRANSPORT INTOTHECELLFROMTl.lEPLASMA MEMBRANE: ENDOCYT0S15

789

Remarkably,macrophageswill also phagocytosea variety of inanimate particles-such as glassor latexbeadsand asbestosfibers-yet they do not phagocltose live animal cells.Living animal cellsseemto display"dont-eat-me"signals in the form of cell-surfaceproteinsthat bind to inhibiting receptorson the surfaceof macrophages. The inhibitory receptorsrecruittyrosinephosphatases that antagonizethe intracellularsignalingeventsrequiredto initiatephagocytosis, therebylocally inhibiting the phagocyticprocess.Thus phagocltosis,like many other cell processes, dependson a balancebetweenpositivesignalsthat activatethe processand negativesignalsthat inhibit it. Apopototic cells are thought both to gain "eat-me"signals(such as extracellularlyexposedphosphatidylserine)and to Iosetheir "don't-eat-me"signals,causingthem to be very rapidlyphagocytosed by macrophages.

PinocyticVesiclesFormfrom CoatedPitsin the Plasma Membrane Virtually all eucaryotic cells continually ingest bits of their plasma membrane in the form of small pinocytic (endocltic) vesicles,which are later returned to the cell surface.The rate at which plasma membrane is internalized in this process of pinocytosis varies between cell types, but it is usually surprisingly large. A macrophage, for example, ingests 25Toof its own volume of fluid each hour. This means that it must ingest 3% of its plasma membrane each minute, or 100%in about half an hour. Fibroblasts endocytose at a somewhat lower rate (17oof their plasma membrane per minute), whereas some amoebae ingest their plasma membrane even more rapidly. Since a cell's surface area and volume remain unchanged during this process, it is clear that the same amount of membrane being removed by endocltosis is being added to the cell surface by the converse process of exocytosis.In this sense,endocltosis and exocltosis are linked processesthat can be considered to constitute an endocytic-exocytic cycle.The coupling between exocytosis and endocytosis is particularly strict in specialized structures characterized by high membrane turnover, such as the neuronal slmapse. The endocytic part of the cycle often begins at clathrin-coated pits. These specialized regions typically occupy about 2To of the total plasma membrane area.The lifetime of a clathrin-coated pit is short: within a minute or so of being formed, it invaginates into the cell and pinches off to form a clathrin-coated vesicle (Figure f3-48). It has been estimated that about 2500 clathrin-coated vesicles leave the plasma membrane of a cultured fibroblast every minute. The coated vesicles are even more transient than the coated pits: within seconds of being formed, they shed their coat and are able to fuse with early endosomes. Since extracellular fluid is trapped in clathrin-coated pits as they invaginate to

Figure13-48Theformationof clathrincoatedvesiclesfrom the plasma membrane,Theseelectronmicrographs illustrate the probablesequenceof eventsin the formationof a clathrincoatedvesiclefrom a clathrin-coatedpit. Theclathrin-coated oitsand vesicles shownarelargerthan thoseseenin normal-sized cells.Theytakeup lipoproteinparticlesinto a very largehen oocyteto form yolk.The lipoprotein particlesboundto their membranebound receptorsappearas a dense,fuzzy surfaceof the layeron the extracellular olasmamembrane-which isthe inside surfaceof the vesicle.(Courtesyof M.M.Perryand A.B.Gilbert,J. Cel/Scr. 39:257-272,1979.With permissionfrom The Companyof Biologists.)

.ri, ll O 1 ilm

790

Chapter13: Intracellular Vesicular Traffic Figure13-49 Caveolaein the plasma membraneof a fibroblast. (A)This electronmicrographshowsa plasma membranewith a very high densityof caveolae.Note that no cytosoliccoat is visible.(B)This rapid-freeze deep-etch imagedemonstrates the characteristic 'tauliflower"textureof the cytosolicface of the caveolaemembrane. The regular texture is thought to resultfrom aggregates of caveolinin the membrane. A clathrin-coated oit is alsoseenat the upper right. (Courtesyof R.G.WAnderson,from K.G.Rothberget al.,Cell68:673-682,1992.With permissionfrom Elsevier.)

.*

(B)

# form coated vesicles,any substance dissolved in the extracellular fluid is internalized-a proces s calIed fluid -ph ase endocyt osi s.

Not All Pinocytic Vesicles AreClathrin-Coated In addition to clathrin-coated pits and vesicles,there are other, less well-understood mechanisms by which cells can form pinocytic vesicles. One of these pathways initiates at caveolae (from the Latin for "little cavities"), originally recognized by their ability to transport molecules across endothelial cells, which form the inner lining of blood vessels.Caveolaeare present in the plasma membrane of most cell types, and in some of these they are seen in the electron microscope as deeply invaginated flasks (Figure f3-49). They are thought to form from membrane microdomains, or lipid rafts, which are patches of the plasma membrane that are especially rich in cholesterol, glycosphingolipids, and GPl-anchored membrane proteins (seeFigure 10-14). The major structural proteins in caveolae are caveolins, which are a family of unusual integral membrane proteins that each insert a hydrophobic loop into the membrane from the cytosolic side but do not extend acrossthe membrane. In contrast to clathrin-coated and COPI- or COPII-coated vesicles,caveolae are thought to invaginate and collect cargo proteins by virtue of the lipid composition of the calveolar membrane, rather than by the assembly of a cytosolic protein coat. Caveolins may stabilize these raft domains, into which certain plasma membrane proteins partition. Caveolaepinch off from the plasma membrane using dynamin, and they deliver their contents either to an endosomelike compartment (called a caueosome)or to the plasma membrane on the opposite side of a polarized cell (in a process called transcytosls,which we discuss later). Becausecaveolins are integral membrane proteins, they do not dissociate from the vesiclesafter endocltosis; instead they are delivered to the target compartments, where they are maintained as discrete membrane domains. Some animal viruses such as SV40 and papilloma virus (which causes warts) enter cells in vesicles derived from caveolae.The viruses are first delivered to caveosomes,and they move from there in specialized transport vesicles to the ER.The viral genome exits from the ER acrossthe ER membrane into the cytosol, from where it is imported into the nucleus to start the infection cycle. Endocytic vesicles can also bud from caveolin-free raft domains on the plasma membrane and deliver their cargo to caveosomes.Molecules that enter the cell through caveosomesavoid endosomes and lysosomes and are therefore shielded from exposure to low pH and lysosomal hydrolases;it is unknown how they move from caveosomesto other destinations in the cell.

791

TRANSPORT INTOTHE CELLFROMTHE PLASMA MEMBRANE; ENDOCYTOSIS

CellsUseReceptor-Mediated Endocytosis to lmport Selected Extracel Iular Macromolecu les In most animal cells, clathrin-coated pits and vesiclesprovide an efficient pathway for taking up specific macromolecules from the extracellular fluid. In this process, called receptor-mediated endocytosis, the macromolecules bind to complementary transmembrane receptor proteins, accumulate in coated pits, and then enter the cell as receptor-macromolecule complexesin clathrin-coated vesicles(seeFigure 13-48). Becauseligands are selectivelycaptured by receptors, receptor-mediated endocltosis provides a selective concentrating mechanism that increases the efficiency of internalization of particular ligands more than a hundredfold. In this way, even minor components of the extracellular fluid can be specifically taken up in large amounts without taking in a large volume of extracellular fluid. A particularly well-understood and physiologically important example is the processthat mammalian cells use to take up cholesterol. Many animals cells take up cholesterol through receptor-mediated endocytosis and, in this way, acquire most of the cholesterol they require to make new membrane. If the uptake is blocked, cholesterol accumulates in the blood and can contribute to the formation in blood vessel (artery) walls of atherosclerotic plaques, deposits of lipid and fibrous tissue that can cause strokes and heart attacks by blocking arterial blood flow In fact, it was a study of humans with a strong genetic predispositionfor atherosclerosisthat first revealed the mechanism of receptor-mediated endocytosis. Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid-protein particles known as low-density lipoproteins (LDLs) (Figure 13-50). \A/hen a cell needs cholesterol for membrane synthesis, it makes transmembrane receptor proteins for LDL and inserts them into its plasma membrane. Once in the plasma membrane, the LDL receptorsdiffuse until they associate with clathrin-coated pits that are in the process of forming (Figure f3-5fA). Since coated pits constantly pinch off to form coated vesicles, any LDL particles bound to LDL receptors in the coated pits are rapidly internalized in coated vesicles. After shedding their clathrin coats, the vesicles deliver their contents to ear$ endosomes,which are located near the cell periphery. Once the LDL and LDL receptors encounter the low pH in the endosomes,LDL is released from its receptor and is delivered via late endosomes to lysosomes.There, the cholesteryl esters in the LDL particles are hydrolyzed to free cholesterol, which is now available to the cell for new membrane slmthesis. If too much free cholesterol accumulates in a cell, the cell shuts off both its o',tm cholesterol synthesis and the synthesis of LDL receptors, so that it ceaseseither to make or to take up cholesterol. This regulated pathway for cholesterol uptake is disrupted in individuals who inherit defective genes encoding LDL receptors. The resulting high levels of blood cholesterol predispose these individuals to develop atherosclerosis

(A)

c l a t h r i n - c o a t epdi t

(B)

LDLreceptor protein with defectivecoated-pit-bindingsite

22 nm

phospholipid monorayer

cholesterylester molecule

surfaceprotrusion on proteinmolecule Figure 13-50 A low-densitylipoprotein (LDL)particle.Eachsphericalparticlehas a massof 3 x 106daltons.lt contatnsa molecules coreof about 1500cholesterol esterifiedto long-chainfatty acids.A lipid monolayercomposedof about 800 phospholipidand 500 unesterified molecules surroundsthe core cholesterol A singlemoleculeof esters. of cholesterol protein organizesthe a 500,000-dalton particleand mediatesthe specificbinding of LDLto cell-surfaceLDLreceptors.

Figure13-51 Normaland mutant LDL receptors.(A) LDLreceptorsbinding to a coatedpit in the plasmamembraneof a normalcell.The humanLDLreceptoris a glycoprotein transmembrane single-pass composedof about840aminoacids, only 50 of whichareon the cytoplasmic sideof the membrane.(B)A mutantcell areabnormal in whichthe LDLreceptors and lackthe sitein the cytoplasmic domainthat enablesthem to bind to adaptorproteinsin the clathrin-coated pits.Suchcellsbind LDLbut cannot 1 in ingestit. In mosthumanpopulations, 500individualsinheritsone defectiveLDL receptorgeneand,as a result,hasan increasedriskof a heart attackcausedby atheroscIerosis.

792

Chapter13: Intracellular Vesicular Traffic

prematurely, and many would die at an early age of heart attacks resulting from coronary artery diseaseif theywere not treated with drugs that lower the level of blood cholesterol. In some cases,the receptor is lacking altogether.In others, the receptors are defective-in either the extracellular binding site for LDL or the intracellular binding site that attaches the receptor to the coat of a clathrincoated pit (seeFigure 13-5f B). In the latter case,normal numbers of LDL receptors are present, but they fail to become localized in clathrin-coated pits. Although LDL binds to the surface of these mutant cells, it is not internalized, directly demonstrating the importance of clathrin-coated pits for the receptormediated endocytosis of cholesterol. More than 25 distinct receptors are known to participate in receptor-mediated endocytosis of different types of molecules. They all apparently use clathrin-dependent internalization routes and are guided into clathrin-coated pits by signals in their cytoplasmic tails that bind to adaptor proteins in the clathrin coat. Many of these receptors, like the LDL receptor, enter coated pits irrespective of whether they have bound their specific ligands. Others enter preferentially when bound to a specific ligand, suggesting that a ligandinduced conformational change is required for them to activate the signal sequence that guides them into the pits. Since most plasma membrane proteins fail to become concentrated in clathrin-coated pits, the pits serve as molecular filters, preferentially collecting certain plasma membrane proteins (receptors) over others. Electron-microscope studies of cultured cells exposed simultaneously to different labeled ligands demonstrate that many kinds of receptors can cluster in the same coated pit, whereas some other receptors cluster in different clathrin-coated pits. The plasma membrane of one clathrin-coated pit can probably accommodate up to 1000receptors of assortedvarieties.Although all of the receptor-ligand complexes that use this endocytic pathway are apparently delivered to the same endosomal compartment, the subsequent fates of the endoq,tosed molecules vary, as we discuss next.

Endocytosed Materials ThatAre Not Retrieved from Endosomes EndUp in Lysosomes The endosomal compartments of a cell can be complex. They can be made visible in the electron microscope by adding a readily detectable tracer molecule, such as the enz)..rneperoxidase, to the extracellular medium and leaving the cells for various lengths of time to take it up by endocytosis.The distribution of the molecule after its uptake reveals the endosomal compartments as a set of heterogeneous,membrane-enclosed tubes extending from the periphery of the cell to the perinuclear region, where it is often close to the Golgi apparatus. TWo sequential sets of endosomes can be readily distinguished in such labeling experiments. The tracer molecule appears within a minute or so in early endosomes, just beneath the plasma membrane. After 5-15 minutes, it has moved to late endosomes, close to the Golgi apparatus and near the nucleus. Early and late endosomes differ in their protein compositions. The transition from early to late endosomes is accompanied by the releaseof Rab5 and the binding of Rab7, for example. As mentioned earlier, a vacuolar H+ AIPase in the endosomal membrane, which pumps H+ into endosomes from the cytosol, keeps the lumen of the endosomal compartments acidic (pH -6). In general, later endosomes are more acidic than early endosomes.This gradient of acidic environments has a crucial role in the function of these organelles. We have already seen how endocytosed materials mix in early endosomes with newly syrthesized acid hydrolases and eventually end up being degraded in lysosomes. Many molecules, however, are specifically diverted from this journey to destruction. They are instead recycled from the early endosomes back to the plasma membrane via transport vesicles.Only molecules that are not retrieved from endosomes in this way are delivered to lysosomes for degradation.

793

TRANSPORT INTOTHECELLFROMTHEPLASMAMEMBRANE: ENDOCYTOSIS

Although mild digestion may start in early endosomes, many hydrolases are synthesized and delivered there as proenzymes, called zymogens,which contain extra inhibitory domains at their N-terminus that keep the hydrolase inactive until these domains are proteolltically removed. The hydrolases are activated when late endosomes become endolysosomes as the result of fusion with preexisting lysosomes,which contain a full complement of active hydrolases that digest off the inhibitory domains from the newly synthesized enzymes. Moreover, the pH in early endosomes is not low enough to activate lysosomal hydrolases optimally. By these means, cells can retrieve most membrane proteins from early endosomes and recycle them back to the plasma membrane.

ht junction

t

SpecificProteinsAre Retrievedfrom EarlyEndosomes and Returnedto the PlasmaMembrane Early endosomes form a compartment that acts as the main sorting station in the endocytic pathway, just as the cls and trans Golgi networks serve this function in the biosynthetic-secretory pathway. In the mildly acidic environment of the early endosome, many internalized receptor proteins change their conformation and release their ligand, as already discussed for the M6P receptors. Those endocltosed ligands that dissociate from their receptors in the early endosome are usually doomed to destruction in lysosomes,along with the other soluble contents of the endosome. Some other endocytosed ligands, however, remain bound to their receptors, and thereby share the fate of the receptors. The fates of receptors-and of any ligands remaining bound to them-vary according to the specific type of receptor. (1) Most receptors are recycled and return to the same plasma membrane domain from which they came; (2) some proceed to a different domain of the plasma membrane, thereby mediating transcytosis;and (3) some progressto lysosomes,where they are degraded (Figure 13-52). The LDL receptor follows the first pathway. It dissociates from its ligand, LDL, in the early endosome and is recycled back to the plasma membrane for reuse,leaving the discharged LDL to be carried to lysosomes (Figure 13-53). The recycling transport vesiclesbud from long, narrow tubules that extend from the

LDLreceptors

Figure 13-52 Possiblefates for transmembranereceptorproteinsthat have been endocytosed.Threepathways from the endosomalcompartmentin an eoithelialcellareshown.Retrieved are returned(1)to the same receptors olasmamembranedomainfrom which they came (recycling)or (2) to a different domainof the plasmamembrane (3) Receptorsthat are not (transcytosis). specificallyretrievedfrom endosomes followthe pathwayfrom the endosomal wherethey compartmentto lysosomes, are degraded (degradation).The in formationof oligomericaggregates the endosomalmembranemay be one of into the the signalsthat guidereceptors pathway.lf the ligandthat is degradative endocytosedwith its receptorstays boundto the receptorin the acidic environmentof the endosome,it follows the samepathwayas the receptor; otherwiseit is deliveredto lysosomes.

p l a s m am e m b r a n e CYTOSOL

coated pit

e a r l ye n d o s o m e

hydrolytic enzymes lysosome

Figure I 3-53 The receptor-mediated endocytosisof LDL.Notethat from its receptorsin the LDLdissociates the acidicenvironmentof the early Aftera numberof steps endosome. (shownin Figure13-55),the LDLendsup where it is degradedto in lysosomes, releasefree cholesterol.In contrast,the LDLreceptorsare returnedto the plasma transport membranevia clathrin-coated vesicles that bud offfrom the tubular as shown. regionof the earlyendosome, only one LDLreceptoris Forsimplicity, shownenteringthe celland returningto Whetherit is the olasmamembrane. occupiedor not, an LDLreceptortypically makesone roundtrip into the celland backto the plasmamembraneevery '10 minutes,makinga totalof several hundredtrips in its 20-hourlifespan.

794

Chapter13: Intracellular Vesicular Traffic

early endosome

late enoo50me

# early endosomes.It is likely that the geometry of these tubules helps the sorting process:becausetubules have a large membrane area enclosing a small volume, membrane proteins become enriched over soluble proteins. Transport vesicles that return material to the plasma membrane begin budding from the tubules, but tubular portions of the early endosome also pinch off and fuse with one another to form recycling endosomes,which serve as way-stations for the traffic between early endosomes and the plasma membrane. This recycling pathway operates continuously, compensating for the continuous endocltosis occurring at the plasma membrane. The transferrin receptor follows a similar recycling pathway as the LDL receptor, but unlike the LDL receptor it also recycles its ligand. Transferrin is a soluble protein that carries iron in the blood. Cell-surface transferrin receptors deliver transferrin with its bound iron to early endosomes by receptor-mediated endocy'tosis.The low pH in the endosome induces transferrin to release its bound iron, but the iron-free transferrin itself (called apotransferrin) remains bound to its receptor. The receptor-apotransferrin complex enters the tubular extensions of the early endosome and from there is recycled back to the plasma membrane (Figure f 3-54). \Mhen the apotransferrin returns to the neutral pH of the extracellular fluid, it dissociates from the receptor and is thereby freed to pick up more iron and begin the cycle again. Thus, transferrin shuttles back and forth between the extracellular fluid and the endosomal compartment, avoiding lysosomesand delivering iron to the cell interior, as needed for cells to grow and proliferate. The second pathway that endocltosed receptors can follow from endosomes is taken by many signaling receptors, including opioid receptors (see Figure 13-54) and the receptor that binds epidermal growthfactor (EGD. EGF is a small, extracellular signal protein that stimulates epidermal and various other cells to divide. Unlike LDL receptors, EGF receptors accumulate in clathrincoated pits only after binding EGB and most of them do not recycle but are degraded in lysosomes,alongwith the ingested EGE EGF binding therefore first activates intracellular signaling pathways and then leads to a decrease in the concentration of EGF receptors on the cell surface, a process called receptor down-regulationthat reduces the cell's subsequent sensitivity to EGF (seeFigure 15-29).

Figure13-54 Sortingof membrane proteins in the endocytic pathway, Transferrinreceptorsmediateiron uptake and constitutivelycyclebetween endosomes and the olasmamembrane. By contrast,activatedopioid receptors aredown-regulated by endocytosis followedby degradationin lysosomes; they areactivatedby opiatessuchas morphineand heroin,aswell as by endogenouspeptidescalledenkephalins and endorphins. Endocytosis of both typesof receptorsstartsin clathrincoatedpits.The receptorsarethen deliveredto earlyendosomes,wnere their pathwayspart:transferrinreceptors aresortedto the recyclingendosomes, whereasopioid receptorsare sortedto lateendosomes. The micrographshows both receptors-labeledwith different fluorescent dyes-30 min after endocytosis(transferrinreceptorsare labeledin red and opioid receptorsin green).At this time, someearly endosomes stillcontainboth receotors and are seenasyellow,due to the overlap of redand greenlightemittedfrom the fluorescentdyes.By contrast,recycling endosomes and lateendosomes are selectively enrichedin eithertransferrin or opioid receptors,respectively, and thereforeappearasdistinctred and (Courtesy greenstructures. of Markvon Zastrow.)

795

TRANSPORT INTOTHECELLFROMTHEPLASMAMEMBRANE: ENDOCYTOSIS

Clathrin-dependent receptor-mediated endocl'tosis is highly regulated. The receptors are first covalently modified with the small protein ubiquitin. But, unlike polyubiquitylation, which adds a chain of ubiquitins that typically targets a protein for degradation in proteasomes (discussed in Chapter 6), ubiquitin tagging for sorting into the clathrin-dependent endocytic pathway adds one or more single ubiquitin molecules to the protein-a process called monoubiquitylation or multiubiquitylation, respectively. Ubiquitin-binding proteins recognize the attached ubiquitin and help direct the modified receptors into clathrin-coated pits. After delivery to the endosome, other ubiquitinbinding proteins recognize the ubiquitin and help mediate sorting steps.

m u l t i v e s i c u l abro d i e s

Multivesicular BodiesFormon the Pathwayto LateEndosomes As previously stated, many of the endocltosed molecules move from the early to the late endosomal compartment. In this process, early endosomes migrate slowly along microtubules toward the cell interior, while shedding membrane tubules and vesiclesthat recycle material to the plasma membrane and TGN. At the same time, the membrane enclosing the migrating endosomes forms invaginating buds that pinch off and form internal vesicles;they are then called multivesicular bodies (Figure f 3-55). Multivesicular bodies eventually fuse with a late endosomal compartment or with each other to become late endosomes.At the end of this pathway, the late endosomes convert to endolysosomesand lysosomes as a result of both their fusion with preexisting lysosomesand progressive acidification (Figure l3-56). The multivesicular bodies carry those endocltosed membrane proteins that are to be degraded.As part ofthe protein-sorting process,receptors destined for degradation, such as occupied EGF receptors described previously, selectively partition into the invaginating membrane of the multivesicular bodies. In this way, both the receptors and any signaling proteins strongly bound to them are made fully accessibleto the digestive enzymes that will degrade them (Figure f 3-57). In addition to endocltosed membrane proteins, multivesicular bodies also contain the soluble content of early endosomes destined for late endosomes and digestion in lysosomes.

Golgistack Figurel3-55 Electronmicrographof a body in a plant cell.The multivesicular largeamountof internalmembranewill be deliveredto the vacuole,the plant for digestion. equivalentof the lysosome,

plasmamembrane

,--'--)a-r------tyL-

t

\tooo \l\ /

early

t

enoosome ?

MTCRoTUBULEf MEDIATED \ TRANSPORT

Cotgi network bule

Figure13-56 Detailsofthe endocytic pathway from the plasmamembraneto Maturationof early lysosomes. occurs to lateendosomes endosomes throughthe formationof multivesicular bodies,whichcontainlargeamountsof membraneand internal invaginated (hencetheir name).Multivesicular vesicles bodiesmoveinwardalongmicrotubules, continuallysheddingtransportvesicles that recyclecomponentsto the plasma Theygraduallyconvertinto membrane. eitherby fusingwith each lateendosomes, late otheror by fusingwith preexisting The lateendosomesno longer endosomes. to the plasmamembrane. sendvesicles

796

Chapter13:lntracellularVesicularTraffic

Figure13-57Thesequestration proteinsinto internal of endocytosed proteases membranes of multivesicular bodies. Eventually, in andlipases lysosomes digestallof theinternal membranes withinmultivesicular bodiesproduced processes bytheinvaginations. Theinvagination are essential to achieve complete digestion of endocytosed membrane proteins: because theoutermembrane of themultivesicular bodybecomes continuous withthelysosomal membrane, forexample, lysosomal hydrolases couldnotdigestthecytosolic domains of endocytosed proteins transmembrane suchastheEGFreceotor shownhere.if the proteinwerenotlocalized in internal vesicles.

Sorting into the internal vesicles of a multivesicular body requires one or multiple ubiquitin tags,which are added to the cltosolic domains of membrane proteins. These tags initially help guide the proteins into clathrin-coated vesicles. Once delivered to the endosomal membrane, the ubiquitin tags are recognized again, this time by a seriesof cytosolic protein complexes, called ESCR?-O -1, -II, and -111,which bind sequentially, handing the ubiquitylated cargo from one complex to the next, and ultimately mediate the sorting process into the internal vesicles of multivesicular bodies (Figure l3-S8). Membrane invagination into multivesicular bodies also depends on a lipid kinase that phosphorylates phosphatidylinositol to produce PI(3)B which servesas an additional docking site for the ESCRTcomplexes; these complexes require both PI(3)P and the presence of ubiquitylated cargo proteins to bind to the endosomal membrane. A second PI kinase adds another phosphate group to PI(3)B producing PI(3,5)Pz, which is required for ESCRT-III to form large multimeric assemblies on the membrane. It is not knolnm how the assembly of ESCRTcomplexes ultimatelv

e a r l ye n d o s o m e

OG FF I PTNCHTN +(SEQUESTRATION)

multivesicular body

rysosome

@ ESCRT-O

CYTOSOL

ESCRT-tl

ESCRT-il1

t

ESCRT-il1

\

Figure13-58 Sortingof endocytosedmembraneproteinsinto the internalvesiclesof a multivesicular body.A seriesof complexbindingeventspassesthe ubiquitylated cargoproteinssequentially from one ESCRT complexto the nexr, enventually concentrating them in membraneareasthat bud awayfrom the cytosolinto the lumenof the endosometo form the internalmembranevesicles of the multivesicular body.ESCRT complexes aresolublein the cytosoland are recruitedto the membraneas needed.First,ESCRT-O bindsboth the ubiquitinattachedto the cargoproteinand to pl(3)p headgroups.ESCRT-O dissociates from the membrane,handingthe ubiquitylated cargoproteinoverto the ESCRT-I complex;next ESCRT-I dissociates, handingthe cargoproteinoverto ESCRT-Il complex;and finallyESCRT-Il dissociates and -1,and -ll, ESCRT-Ill ESCRT-Ill complexes assemble on the membrane.By contrastto ESCRT-O, doesnot bind to the ubiquitylated cargodirectly.Insteaditsassemblyinto expansive multimericstructures is thoughtto confinetne cargo moleculesinto specialized membraneareasthat then invaginate, leavingthe ESCRT componentson the endosomesurface. (redcylinders) An AAA-ATPase then disassembles the ESCRT-Ill complexes so that they can be reused.

797

TRANSPORT INTOTHECELLFROMTHEPLASMAMEMBRANE: ENDOCYTOSIS

drives the invagination and pinching-off processesrequired to form the internal vesicles but the ESCRTcomplexes themselves are not part of the invaginating membranes. Mutant cells compromised in ESCRTfunction display signaling defects. In such cells, activated receptors cannot be down-regulated by endocytosis and packaged into multivesicular bodies and therefore mediate prolonged signaling, which can lead to uncontrolled cell proliferation and cancer. The same ESCRTmachinery that drives the internal budding from the endosomal membrane to form multivesicular bodies is also used by HIV ebola, and other enveloped viruses to bud from the plasma membrane into the extracellular space.The two processesare topologically equivalent, as they both involve budding away from the cltosolic surface of the membrane (Figure f 3-59).

TranscytosisTransfersMacromolecu lesAcrossEpitheliaI CelI Sheets Some receptors on the surface of polarized epithelial cells transfer specific macromolecules from one extracellular space to another by transcytosis (Figure 13-60). These receptors are endocltosed and then follow a pathway from endosomes to a different plasma membrane domain (seeFigure l3-52). A newborn rat, for example, obtains antibodies from its mother's milk (which help protect it against infection) by transporting them across the epithelium of its gut. The lumen of the gut is acidic, and, at this low pH, the antibodies in the milk bind to specific receptors on the apical (absorptive) surface of the gut epithelial cells. The receptor-antibody complexes are internalizedvia clathrincoated pits and vesicles and are delivered to early endosomes. The complexes remain intact and are retrieved in transport vesicles that bud from the early

vtrus particle

complexesin Figure 13-59 ESCRT multivesicularbody formation and virus budding.In the two topologically indicatedby the equivalentprocesses arrows,ESCRT complexesshape membranesinto budsthat bulgeaway from the cvtosol.

b a s o l a t e r adl o m a i n o f o l a s m am e m b r a n e E X T R A C E L L U LFALRU I D

t tra nscytosrs transPort vesicle I I

\ recycling endosome

\ \\ transport vesicle

i ng.trans; /,recyclvestcres

\

I

a p i c a ld o m a i no f p l a s m am e m b r a n e

\ i

Figure 13-60 The role of recycling endosomesin transcytosis.Recycling endosomesform a way-stationon the transcytoticpathway.In the example shownhere,an antibodyreceptoron a gut epithelialcellbindsantibodyand is endocytosed,eventuallycarryingthe plasma antibodyto the basolateral The receptoris calledan Fc membrane. receptorbecauseit binds the Fcpart of in Chapter25). the antibody(discussed

798

Chapterl3:lntracellularVesicularTraffic

i n s u l i n - s t i m u l a t ecde l l g Iu c o s e lransponer

insulin

@ \ s i g n a lc a u s e sr e l o c a l i z a t i o n of glucosereceptorsto p l a s m am e m b r a n et o b o o s t glucoseuptake into the cell

endosome and subsequently fuse with the basolateral domain of the plasma membrane. On exposure to the neutral pH of the extracellular fluid that bathes the basolateral surface ofthe cells, the antibodies dissociatefrom their receptors and eventually enter the newborn's bloodstream. The transcy-toticpathway from the early endosome to the plasma membrane is not direct. The receptors first move from the early endosome to an intermediate endosomal compartment, the recycling endosome described previously (see Figure 13-60). The variety of pathways that different receptors folIow from endosomes implies that, in addition to binding sites for their ligands and binding sites for coated pits, many receptors also possesssorting signals that guide them into the appropriate tlpe of transport vesicle leaving the endosome and moving to the appropriate target membrane in the cell. A unique property of recycling endosomes is that cells can regulate the exit of membrane proteins from the compartment. Thus, cells can adjust the flux of proteins through the transc).totic pathway according to need. Although the mechanism is uncertain, this regulation allows recycling endosomes to play an important part in adjusting the concentration of specific plasma membrane proteins. Fat cells and muscle cells,for example, contain large intracellular pools ofthe glucose transporters that are responsible for the uptake ofglucose across the plasma membrane. These membrane transport proteins are stored in specialized recycling endosomes until the hormone insulin stimulates the cell to increase its rate of glucose uptake. In response to the insulin signal, transport vesicles rapidly bud from the recycling endosome and deliver large numbers of glucose transporters to the plasma membrane, thereby greatly increasing the rate of glucose uptake into the cell (Figure 13-61).

Epithelial CellsHaveTwoDistinctEarlyEndosomal Compartments b u t a C o m m o nLa teE n d o so ma l C o mpar tm ent In polarized epithelial cells, endocytosis occurs from both the basolateral domain and the apical domain of the plasma membrane. Material endocy'tosed from either domain first enters an early endosomal compartment that is unique to that domain. This arrangement allows endocytosed receptors to be recycled back to their original membrane domain, unless they contain sorting signals that mark them for transcltosis to the other domain. Molecules endocltosed from either plasma membrane domain that are not retrieved from the early endosomes end up in a common late endosomal compartment near the cell center and are eventually degraded in lysosomes (Figure f 3-62). \A/trethercells contain a few connected or many unconnected endosomal compartments seems to depend on the cell type and the physiological state of the cell. Like many other membrane-enclosed organelles, endosomes of the same type can readily fuse with one another (an example of homotypic fusion, discussed earlier) to create large continuous endosomes.

Figure13-61 Storageof plasma membraneproteinsin recycling endosomes.Recycling endosomes can pool of serveasan intracellular plasmamembraneproteins specialized that can be mobilizedwhen needed.ln t h e e x a m p l es h o w n i,n s u l i nb i n d i n gt o the insulinreceptortriggersan intracellular signalingpathwaythat causesthe rapidinsertionof glucose transporters into the plasmamembrane of a fat or musclecell,greatlyincreasing olucoseintake.

UOIU:lIXl'l'll)

a^eel dlleluJou eu?Jquau erusEld aql JoJ peunsep selclse^ Uodsup{'Jorrelxe drolarcas eql auruExe puP snleJEdde r31og IIaJ aql 01 lno peel ]eq] s,r(e,rnqled aql ol uJnlar l ou e^\ 'sauosos,{1 uo aBJoAuoJleql JSJEJIeueJqureu Sururocur Jo sed& snorJel aqt pue uels,{s a^qse8rp [EuJelur sllal oq] paJeprsuoJSuneg

stsor^foxl:uolutlxtlltf lHt oI ITAOSOSA'l

sl'l) AUOI:

lcN3 lrvr

t9'lo9

Nn'rn)t1tu)tt lsv]dooNl

)UOAAIINl9lOD S/V

IHJ lAlOUlIUOdSNVUI

'papaauato [aql Urun paJo$ aq UDJsutaptd auotquaw Dlasaldpasoytcopua anqm 'sauosopua Suqc[tal sanpnu [amtgod stsoltcsuatl aq1 'srso1[tsuar]pallaJ ssacotda 'palauBuo I araLlmauDrqwau aql uoS uatlrp ruqt lpc ar41 to acatms a p pasualalaq o1puaBu at4l8u1snoc'utDuop auarquau Dwsald ruatall1po o1pauatsunty ata pua34 pua rcldacat LUoq'sasDJbrllo q 'salpoqJolnJwantllrua wtot o1 salrsan auurqruaraputosopua lo llo-8urycurd pua uotlau8anut aqt anup qcrqm 'saxa1duoc JAJS7 snolrDni.lnnal aotdacat paSSayu4mbtqn aqj'sasDcasaqtur luorla1n&at-umop nJdacat ut 3u47nsat 'satuosost1ut papatBapBunq dn pua puaBq aqj pua nldacat aLli tfloq'sasoJalLtosuI 'ruauLndutoc pwosopua aql utotl stamqlad nqlo mo11olunc saxa1dwocpua87-toldacal ing'asnal rct acatms 77araqi oj qJoq sa1l$anyodsua4. atrt palctcat ata aoldacat arlllo lsoru aqqm 'sautosostlut dn pua [11anyuana puv alaosopua aqlto luau.ruonlnuaJtpnD aLUut stoldacatuarll tuo{ alolcosslpspunBtyaqlto 1so141 'sawosopualUDa qirm asnJpuo slooJ urJrgDlcJDqi paqs ,t\p1dot salJ$anpaJDoJaq1 'stso1fcopuapawryala-toldacat pa\1ocssacotda ,salJlsan pawoc-urrqtLlJ w pazqDuraiut[puan$a ato spuo317 Jraqt pua uoldacat asaql ,jlnsal a sy's1td paiooJ-urnqLlJ otu! waLUsapmBquqm'ut1mblqn qrym pa33o1awoJaq salncapuolJour rDlnlpcnuxa cgfrcadspurq ruLU stoidacat aculms-11ac[uayg sap$an pua s11dpalaoc-uuq1a1c[q [7a3ru7parulpau s1a1c[t cr1tcoxa-ultcopua ayacs-aBnysrql 'stsoltcoxa [q acottns IIaJ aLU oj pauJnpr [11anur1.uocata pasol[topua ala wqi 6p1d4pua surarctdl sjuauodutocauarqwaw awsold aqt to lsotu asnoJaqaz$ ataasaqt utoual snal aqJ tno4 ftana paztpuJa]utst auatquau awsald aql lo uouca{ aBnT n ruqJ sIIaJ[uau ur anrcua]xaos sr srsoltcopug s1auBproInIIaJ -DrJxaoi asuodsatpataSSulo sa puo f1anpnlltsuoc LUoqsJnJJostso1tcopug'papaBap ato [aq1 araqm'sautosost1u1 dn pua t11on1.uana saluwd pua salncaplu pasoilbopua aqtlo [un74 'salctsanctu[topua wtot ol tlo qcutd pun alourSonutauotqwaw or.usa1d aqy 'stsoltcopua[q sa1ctltodpua 'salncaptu 'pm1l raBur sya7 to suo8at paztpcol qcrqm ut

{reuun5

'sauosos^lut palsabtp 6uraqaro;aq luaurlrpot!o)lptllosopua alel uouJt,tjo) e ut loau stsol^lsuplllo 6utl)Itol loJ sleu6tsuteluo)lou op leq] suteulop qloq urolJsaln)aloulpasol^)opua 'ranaMoHsluauluedLuo) eurosopua | {pea aleredasq}lMa}e)tunLuu.lo) auerquraueLuselo oq] Jo sutpLuop le)tde aql pue leraleloseq aql'lla) le!lsqllda ue ut sluaullJeduor ;euosopue {;rea 1ru11srp oml aql Z9-€L oJn6tJ

I eurosopua fupa

"' "roro/u" {;lee leieleloseq

stsol^)oxt :uotulrxl ttt) lHr ot-)uoMl_lNt9toDs/vyujlHl WoulIUodsNVUI

66t

'arnsserd )rloursoq6rqI;enrssarxa qbrq ue 6urleraua6 uorlerlua>uor lnoqlUvr le psrolsaq uer {aqt }pql os '(eutue}srq to1'suerr(;6oalo.rd) sa;nra;or.uorteu rr;rradso1paxa;duot ueuo aJe {aq} eraql'selrrsarfuolerlaspaulolard pet.rodsue.rl olur ;osoy(>aql LUorJ Iqanrlrearesalnlalouraseql:Iemqled relrurse r{qsrn>ro'sraDruJsueJlolnou pue aururelsrq se q)ns 'salnfelouj lleursjo uorlar)ospele;n6araql'uortar)as Jraqlsalelnurlsleu6rsrelnllerellxaue lllun parolspue palerlualuo)aresuraloJo r{rolaDasolur paua^rp eql alaqM'sal)rse^ areIJoMlau16;o9sua;;aql u; su;alord patralasq)!qM,(q Xennqled &ola.nas palelnbere aneqosles;1erftolanas pezrlerred5 suretoldpue sprdr;pazrsaqlu{s ^lMauqllm euerquraur euseldaql ser;ddns osleq)rqM {emqredsqql,(q 1;ar oql uor, palar)osI;;enurluoraresuralord 'slla) a;qn;os(uey11 lle ur saleredor(emqted {rolanes aArlnlrlsuo) aql'4om1au r61o9 suplt aql ur abranrps,{emqtedomt eqf 'slemqled (rolenas pe1e1n6er pue a^llnlllsuo>aql g9-g1 arn613

(eJBJJns 'xalduroJ aJoru eJesuoudo aq] luq] aes IIBqs a^a IIaJ aql Jo surPuop luereJJlp 01 pale^llap aq o] aAEq slJnpoJd luereJJlp eJeqM 'sllal pezuelod u1 'seurosos.{1o1 Jo uol}eJtas paleln8ar ol peel leql sr{emqled aq] JoJ pellalas Jo 'Jlaslr snleredde r31og aqr ut uralord lueplser e sP peuleleJ 'Ug aql 01 peurnlar ,{1ucr;rcads sl }l ssapn eceJJns IIao eql o1 de,uqled alpnl -llsuoJ aql u(qparJJeJ.{1ec4eruolne st sn}eredde r3log aql Jo ueunl eqt ul ulel -ord,{ue teq} suaas }l '}selqorqlJB ro IIaJ poolq etlqm E sPqons 1ar pazrrelodun up ur 'snql'furrrqlud lInBJap eq] palleo osle sI ll 'pu8ls repcqred e a.rrnbarlou saop femqled srql otul frlua asnecag 'aJeJJnsIIaJ aq] ol r(pcarlp sutalord raqlo 'salJrsezlzi.rol drolarcas elllnlrlsuoc e^llJelas-uou aq1, ]soru slrodsue4 ^,(ervrqled -alcas olur sutalotd tttoiatcasloeJlp ol rq8noqr are spu31s snoSopuu pue '(sasel -orpdq pruososzi.l;o eseJ aqt ut aleqdsoqd-g asouueru qt!v\) selclsenSurgedap crycads olur Sur8alced ro3 pa33ei ere setuosos,{1rog paupsap suralord 1eq1 palou fpearle a^Bq eM'(?g-tl arn81g; eJeJrnsIIeo eq] o1 drazrqapa]Elperurul JoJ paurlsap asoql puB 'sallrse^ ,trolaDas JoJ paullsep esoq] '(sauosopua eIA) saruososdlJoJ peuusap asoq]-{Jomlau rByogsuail aqt eneal daql eJoJaqsule} -ord;o sessplJeeJql ]seal ]e aluredas lsnu uolleJoaspalep8arSo alqeduc 1ac y

a)elrnsllo) aqt ol snlereddy;61o9eq1 ruor,^lle)rleuotnvpeure)ag ol uoas sp!d!]puesuletord^uew 'uollaJJas stuslueqoelu o,t,r]eql aredruoc Jo pue szi.errqted,fuolarcasasaql Jo qloq uI snlereddu t31o3 aqf Jo aloJ aql Jepls -uoJ a,t,r'uollcassql uI '(tg-tI ern81g)saurdzuaarrqsa8tpJo 'sJelllursueJloJneu 'sauoruJoqsE qcns-pueuap uo,(lprder slcnpord SupercasJoJpezqelsads s11ac 'esealal relel Jo! saIJ ur .{lururu puno; 'fum,qled f.rolarcas palup8ar aq} sI sIqI -tsan[tolatca,sur peJols,{.1pp1u1 are seJue]sqnsJeqlo pue sutalord elqnlos qJIqA\ ur Luarrqledrtrola.rcaspuocas e eleq telemoq 'q1ecfrolercas pazqetcadg dlsnon -ultuo3 salerado qJIq ^'furr,rqtuddroleroes elpnlpsuoJ slql armbar slleo IIV '61 reldeq3 ut pessnJsrpsr qf,rq i\ 'xlJryruJDInIIaJaJ$aeq] Jo sulelordocrtlSpue suecf.lSoalord 'aydurexaro; 'l.u.vrslq] uI'slsolboxa palpc aqlJo ]sour alaJcaspue arnpord s11ac 'aceds JelnllaJerue eq] sI aueJqlueu eurseld aql qllm selJISaAeql Jo uoISnJaq; o] peleJJaseJEselJISaAaql epISuIsuralord alqnlos aql ellqm'aueJqluatu eursuld s,lleJ aq] roy sluauoduro3 ^ eu apnord selJISaAesaq] ul sprdu aqt pue sulel -ord auurqureu eql'saFqn] paduqs dpep8arr se rueerls ^,(pea1s e uI NOI eql

5Uralolo

uorsnr

a rg

{to al)

s n l e . r e d d re6 1 o 9

" rpnarlo u . , " t

I al a

, i e m q l e d6 u r ; e u 6 t s r e ln Il a ) e r l uI

lallil.]l5 u eJlol nau lo auoulotl

f;-erz f r \

ca1uue Supcagar-tuqdnoJ JporusoFuaqJ sp rrMorDIssacorde dq saluradosasodrndpcrSolorqro; d8raua sseuJuqot d uouruoc aq; pue'slsuldorolqJ'pupuoqcolrur rtqpasndearrqle salor{record trodsuert-uorlcela AJVs,llaf,eql Jo lsoru ecnpordtuql sesseJord 'uIBluoO Jo ]es eleJoquleuE JoJlJorvrerue4eql sapurordeueJqlueu [BuJeluIsIqJ ,{aql auerqruaruleurelul Jo lunorue a8rul aql st slseldorolqJpue elJpuoqcolnu qroq Jo eJnleeJpcrSoloqdroru8uqu1s tsoru eql sqderSorcruuortoeleu1 'ae8p dlqelou }sou-spllsuld pue puu queld ur ,{po rncJo qclqm-s1se1do.ro1qc '(uozolord pue 'aetp 'que1d 'slelulue '1turg SurpnlJul) sruslue8rocpodrecna p ,t1pn1rp Jo sllao el{l q tuasard aru qJIqM 'BlJpuoqJollru ere sallaueSro Suptanuoc pesolcua-aueJqrueu eqJ aJV rreql Jo tsoru ecnpotd o1sa11auoBto -tBnua eplsurseuuJqruerupezrplcadseql esnpeelsul salod:ecng'11 raldeq3 ut peqrJosepsassacordilodsuerl aql JoJpelJeseJsl sllec cpod:ucnauI eueJqlualu eurseld eql tng 31y acnpord ol eueJqlualueruseld rlaql esn salodncor4 'selncalolxpooJ ruoq dIVJo slunoruua8re1acnpord ol ueSrixoesn ol sn salqeueleql uoperrdser olqoJaeeql ol sEllanlrsE 'slsaqludsotoqdut ,{8rauapuoq [EOIIueqJotuliSreua 'saJJnos ,fiauun apur,r lqBq 1o uorsreluoJ eql ot IBJluacsI lI 'eldurexeJoC 3o e urorgriSrauaarrnbceol slla3salqeuetl puE 'auEJqluelu€ sarlnbarssacordstq; 'aJII fuo1srqaql ur ,(1rua,i-rarrpareaddeuoperaua8,{8reua poqleu luelcgJe Jo Jo eroru qonru E lng '(Z raldeq3 ur pessnoslp)slleoJo dcuarrnc,iSrauapclueqc eqt AIV uJoJ 01 posn sr selnoelorua1erp,{qoqrecqctr-d8raueJo uouppFo lep -red aql uro{ pe^lJepr{3raua'1oso1beql ur rnoooleql suollJpaJJo}ese q8norq;

slseldorolq)pueelrpuoq)ol!w :uolsranuo)Aoteul

uo!lelor }lodsu erl r e l l a 6 P l l srsaqluls e u e J q w a u l eArpe leualleq

I (*H) uolold 1uarpe.r6 le)ruraq)olpela auerq|'xoulsuell

t I

I

I s u o l l ) a la r { 6 r a u a -6qr q

/\ /\

sJlnlspoojt lq6!luns

'slsprdororq)'erpuoq)o1ur t"^':ii:'utt1l":: ursuo')earburrnber-rbreu" p seseruasluarper6srql'auerquraue ssor)etuegper6 {6reuaa;tlesre^ uolord ;erueq)ortfala up alear) ol pesnlslU sr s#nlspooJro uorteprxo aql ro ]qbrlunsuorl ,{6rau3'6ur;dnor)rloulsoruaq) 7-j 1 arn611 esaql

(llal

'suralord rlaql lsoq P dq a>1etdnlErtrul Jleql a3urs Jo etuos roJ sepoJ

qclqm 'VNC u \o rlaqt uleluoc slspldorolqJ pue ErJpuoqJotru dqrr,nsuruldxa slseqlod.{q sHI'(ZI reldeq3 ur passncslp) ueql r{lpr dHsuopBlar rl}olquds € padoyarrappup sller cqo,,{-recnaanplurrrd dq paJln8ue erem }eqt setofJeooJd ruory paAIoAe selo.,fuecnaJo sallaue8Jo SulueluoJ-r{8raua eql lpql lq8noql sr U '(Ag-tt arn8rg ees)ueql serunsuoJ uorrpuoqJ -olru aq] seeJeqm'a1erp,{qoqreJpue zO saleraua8 lseldorolqc eql 'snqJ'el€Jp -.{.qoqrecazrsaqludso} (HOVN o} pe}Eler,t1aso1c punoduoc e 'HdCVN eI ) zOO ol peleuop are daql pue 'zg acnpord ol Jelem ruo4 ue{4 eJBsuoJloele :BrJpuoqJ -olru ur leql 01 alrsoddo uorloaJrp eql ul JeJsueJluoJlJela selrJp srualsdsoloqd lseldorolqc aqt rtq paleraua8 eJJoJalrloru-uoJlcele eqJ'lueJJnJ JrJlJeleue eAIJp ol lr esn pue d8raua ]qBII qrosqp qaued rulos ur qlacoloqd sp qonru 'suoJtJele Jo JeJsupn aq] a^rrp ol tr sesseuJeqpue dSraua lq8q sarnldec gdqdorolqc lueru -3rd uaar8 eql eJeqm sualstsolotqd aql arc esaql Suouru lsouerog 'eueJqrueru lelJpuot{co}rur aq} q punoJ }ou s}uauoduroc prcnJc eruos surcluoJ eueJqrueu lseldoroyqc aql 'uolrpuoqJolru aqt Jo esoq] ol Jelrurs are sluauodruoJ ururu pup 'srural rplluls ur paqrJosepeq ueo stlJo aql q3noqlly lseldorolqc Ieralas '(Ve-f arn31g ees) zg t o] suoJlcele aql Jo JaJsuerraql saz.{1e}pJ}pqt serJeseq} ur durnd +H pJIr{} eqt sl 'eupJqrueru ll pue lerJpuoqJotru rauur aql ur sdrund +H aaJr{}Jo serJese sanrJp atels,ri8raua-^aolp ol atets,r(3raua-q8q u uor; qled sHl uMop ./\/\olJ suoJlJele eql sEpaseelerlSraua eeq eqJ'rele^ uroJ ot (zO)se8 uaS,ixoSurcnparl.1pug 'srau -JeJ uoJlcale Jo urpqo e dq auerqruau eqt q8norqt peJJeJSuEJlerc-zOJ ot uop -eperBapstr esrnoo aql ur epceloru pooJ a1erp.{qoqreJB ruoq peseeleJueaq Jo eleq qJrqm-suoJlJale 'uorrpuoqcolgr eql u1 '1q31pns ruor; ,{8raua ueluoJ qJIqM 's1se1doro1qcur asoq] qtlm 'slanJ leJrrueqf, uror; l8reua iloluof, qJIqM 'errpuoqcolrru ur sasseJord uodsueJl-uonoele aql sareduoJ g-t,I arnt;g 'eueJqrueu lerJpuoqJotrru Jeuul aql ol paper8ep are selnoaloru pooJ eJaqM selrs eql luo4 suoJ]rala sarJJeJleql alnJelotu llBrus alqnlos-Jele^ B 'HCIVN aruoJeq ol (+H uE sn1dlsuorlcale oml dn salel qclqm'*CIVN sr sJerJJeJ uoJ]f,ele eseql Jo lsJU aql 'Eupuoq3ollru JoC 'JeqlouE ol rueql Je^llep puE uoll 'saru,r -EJol euo suoJ]f,ale Sullonp dn le lcrd uec tpql se1nceloruayqrsngrpdq lnq -uoc Lq lou Jeqlouu pup elrs euo ueemleq parJJpJeJe suoJlJale 'srualsz{.s pcr8o1 -olq ul tele,troH 'sdrund JrJlcelaJo 1ase qBnorql luarrnc e SurArJpIIaJ JrJlcele up o1 snoSopue sr lrodsuerl uoJlJelaJo rusrupqceu eqJ 'uIBqJ uodsuerl-uoJlJala ue pelleJ sr 'sJeJSuEJ] uoJlJale;o acuanbas dlrapro eql ul pallolur salnJelou 'aueJqureur eql ur suralord fletus eql qrlm raqraSo] Jo les eJltue eqJ (8) d-LV3)Vt\l Ol SSVHjNASdiv A8 oISS]NUVH

stl_Nltcvu9NoLoud:zt9vts

'e6e1sqtea le luauanou uolord pil a\I lo uorl)altp aql sMoqsn4o./Jo 'au{zue aseqlu{s 61y aqr Iq slsaqluIs dfv a^r;pol pasnaq ue) lpql elols {6leue ue se se^rasluarpetbuoford slql (g) 'aupJquauraLl]ssol)e luarpei6 uolo.rd le)rureq)olltalaue 6urlealt'suolotd dund ot (araquMor.ls tou sltelap)rarsuell uort)alaJo 16reueaql sassauleq dund aqf 'soln)alourralem [!olJ a;qe;rene {1aal1 areuMoqs(+H)suotoldaql'(_a)suoll)ala {6reue-q6rq;oe)rnose sn;d'aseqlu{s dfv ue pue ugalorddr.undp poppeqruaale q)rqMur-auejqulaut e ale stsoutsotulaq) .ro;sluauelrnbel letluasseaql (V) 'a1; rol (6.reua6ulsseure;1 1-y 1 e.rn6r3

(v) 3NVU8]EW SSOUfVSNOIOUd

sdnndrvHr dt ndStntuc ruodsNvurNourf3'll:t 39vls

o o o o o o

ooo oo

ooo o

{6raua niro; le uoipala

oo o

\

(suolord) suor+H

le uorpela

pup eupuoq)ol!W:uorsre^uo)Ibraul :y I tardeq3 slse;dorolq1

nLg

'lla) Dur^rlP ur paMollojsruoupuoq)olrur ue uaqMpa^lasqo lenpr^rpur uauo areadeqsJo sa6ueq)prdeu '{1>t1se;d leupuoq)ol!Wtr-9 1 arn6r1 -

s a l n u l u l0 Z -

'ulrpuoqso}Ilu 'suoseal }JEluI 3uI}EI lPcluqsal Joc IeJrrueqJorq asaql Jo .{ueu -osr JoJgt6I uI padole^ep salnpacoJd uo pepuadap tanaaloq 'uotlcun; rlaql 3uI -puElsJapunur ssalSordIEaHfJnlueJ qluaalaulu aq] Suunp palJlluapl ]SJIJaJe^\ ,(aqt pue 'adocsorcnu IqBII eq] uI uaas eq ol q8noua a8JelaJeeIJpuoqoolIIAI '19-71 arn31g1ru.radsp ur run11aBEU (llac alcsnu sBIpJBf,e uI slIJqIJo^u aqt punoJe fpq8lf padderarrJo 'aldruExaJoJ luacefpe uae^\]aq peloed-uopdunsuoo dJV q81q^llensnun Jo alts e o1,{1lcartp 'suleqo Jo 4;y apr,Lord.{aqt araq.u uolllsod auo ur pexrJ urerueJ ,,(aq1sraqlo uI stueuelrJ Sunour 3uo1ruro; sllal aruos uI Elrpuoqlollur aq] 'snqJ 'sllacJo sadd] lueJaJJrpur errpuoqJollru Jo uoDnqlJlslp pue uollelualJo anblun aq] aulruJalap uec qorqm '(g-tl aJnBId) sapqnloJorur qllu pelelJossp aq ol uees uago daql 'urc8e Suueredas uaq] pup Jaqloue auo 'ruseldogc aq] ul lnoqe anoru ,(aq] sy qtvrt Sursn; uale pue (p-71 arn31g1adeqs rtaqt 3u€ueq3.{llue}suoc 'sallaue8ro cqseld pue alrqour.{lqeryeruar arp prJpuoqJollu }eq} smoqs 'Jalalltoq 's1ac 8ut -nr1;o ,r(qderSoleruaurcorcruasdel-arull '€IJatJeqSutlqruasar 'lud I-9'0 Jo Jale -uerp e qlyrr srapur1.,{c pa1e8uo1a$lrs se palcrdap ztlensn erB elJpuoqJo}ll 'auop srs,t1oc,,(13,{q peJnp -ord st ueqt appru aq ot dJV eJotu salul] 9I sr olle slql 'OzH pue zol o1 zg ,{q pazrprxopue uorJpuoqcolru aql olul papodur sr atennrf.daql :pelelduroJ sI sJp -8ns;o rus[oqe]eu eq] 'ErJpuoqJotru uI 'uorleplxo asocny8uroq elqellenedlup -ualod aql Jo uoltf,pr; IIBLUse ,t1uosasealalll 'atennr,{.d sr .{8raua aa{ leql IElo} llqoraeue uel{r\\ aJV rloqt Jo II€ roJ stsr(1ocrt13 o1 asocnlS suanuor srs,{1oc.{13 uo puadap ol a^Bq plno^ slla3 Iprurue ,(ep-tuasard'errpuoqcolrur lnoqlr A'spur -rue xaldruoc Jo uortnlole eql JoJIelluassa uaaq aneq ,taql pue 's11ao cqorfuecna Jo erunlo^ crurseldoldc aql Jo uorlrod Ielluelsqns e ^,(dnccoEIJpUoTIJoUIAI

NO T HCNO H)OJl l ttH l _ 'sallaue8roasaql aprsurJnJOo leql suollJear cpaqludsotq pelJalas uo luapuadap aruoJaq osle eneq .{aql lluarualoru pue 'Surdurnd uor 'sasaqlur(sorqJoJpaau ^aqt dJV aql Jo qJnu ro; salaueBro asaq] uo luapuadap aruooeq e^eq sflec ]soq eq] 'flasranuo3 'allaue8ro eq] olul papodrut uaql pue '1oso1draql ul pazrsaqlu.{s'snaycnuaqt ur saua8dq papoJua ale leq} sutalord uo luapuadap,{lneaq eruoJaq pue saruoua8u,ntorraqt;o qlnru lsol aAEqsallauu8ro

'tse;doro;qr searaqM aqlJosl)nporda.re laql'uorrpuoq)olrur aq1ro;slnduralezOpuesaln)elour alerplqoqre> 'snqf'uor.rpuoq)olil.u P raJsuerl srsrr.{} ur uorl)ala alqero^eJ ,(1;e>r1a6raua aql ol allsoddo }eLl}pue'aterp,(qoqter Jouort)alrp ol OzHuror;ra;sup.rt oMlaql(q paterauab uort)ala anr.rp ol tse;doro;qr aqlsalqpua su.rals,(soloqd 1se;doto;qr a)roJanrlot!-uor])ala utalordaq1;o q)e1'sMoilo aq]teqlaloN'aueiqtlaue urpeppaqua sl(abuuo)sexa;du:or 'lq6r;uns 'uaatb pal pate>rpur Iq sr MolJ uorl)ala u.ror;,{6raue sue^uo) Joqtedaqtpue'anqares1>npord ry61ares1ndul aqj (g)'slanJ r.ror;{6rauasya^uo)uoupuoq)o}r.u aqf (V)'sassa)ord tse;dorolqr lrodsuerl-uoJl)al:l 5-y1arn6r1 lelruraq) s])npord

s]ln poro

ssln)olouJ alerp{qoqrer

to)

*

+

l u a r p e l 6* H

selnFlour alerp^qocuel pue qe+

l u a r p e l 6* H

rsvrdouorH)(s)

V) N O T U q N O H ) O r _ r r( A

918

NOIUONOH)OIlt J:lHt

'uMoqsetnl)nJlsaql aleel) ol roqtoueauo qllM asnJeupuoqfoltut aql pue lpaddesrpuaqt salnqnlor)rr! asaql:llelaql ut eupuoq)olil.u aqt az!le)old;aqor rq6noqlareIaql araqm'auJauoxe aql punoleI;lerrlaq '(8) pulMsalnqnlor)lLU l!el ruradsaq] Jo un1la6egaql;o lueudoqanapaq16uun6 '(t€0t'd aas)slrqr;o{tuall})elluo) aqt ot esol)pau6r;eetrpuoq)ol!r.u snordo>aql ulolJ 61y 1o{1ddns {peals e uo puedepol seq pue salols l6raua ur lllnq pa]!urlseq]l .Ilddns ,(6rauaalqerlere arrnbalsuotl)elluo) lenurluo)slr pue ,(poqaql u! al)snul pesnI;raeaqlsoul aql s! ueaq aql Jo lleMaql ur (v) al)snu )etple)';1e1ulads e pue al)snu )p!pJe)ut uollez!ltln dj.V q6lq ro sells leau eltpuorl)olrrrr Jo uollezlle)o'l9-tl eln6ll

(uaq) og ue11o,(se1tnol) 'salnqntol)r.!6uo;epau6r;eaq ot pual Pupuorl)o1!ur aqr teql eloN 'salnqnloJ)il.u ol pulq leql sarpoqltue lue)saronu qllM (uo!lex!,leue) paurelslla) aures eqt ;o qder6orlu alua)salonlJounuuJl uV (g)'slla)6urnr;ur eUpuoq)ol|tu s;aqe;{;lerrlrads}eql (€Zt autuepoqr) a{p luersaron;}e qilM paule}s seMlla) aqf 'alnlln) ut llo) uptleutuleu 6urnr; e ul eupuoq)ol!ul pale6uole jo sureqlJo qder6ortrr.u tqOllV (V) 'salnqnlor)tu,tpue eupuotl)ol!ul uao/\leq dlqsuollele.raq1 9-91 arn6l3

'lrvrr urds(8)

r])snn)vrouw(v) snle.reddealrllerluo) +o grrqr;o{tu

'selnJelou eseqt Jo ]as pelJeles [u3lq e suE]uoJ xrJleru eql 'sumluoJ lr salnselou Ipus eql 01 lsedseJ qlr { Ioso}d3 eql o} }uelB -Arnbe.(1pc[uaqc sr aceds eupJqrueruJe]uleq] seeJaqM'snql'auerqrueru Jeuur alqPeuJadrul eql ssBdlouuEs rueqtJo lsou lnq 'acedseuBJqr.ueruJalur eq] Jalue ueJ selnsalou qcns 'suretord Ipus SupnlJur 'ssel Jo suolpp 0009Jo selnsalolu [e ol elqpetuJed sl ]eql e^ars e salqueser snql euPJqrueu srqJ'(11 raldeq3 ur pessnJsrp)Je^elq prdil aql q8norqt slauueqc snoenbp a8rul surro; leqt ulelord uodsuuJl Jo adLl e 'selnJaloru utlod ^uelur sure]uof, arruJqrueu Jelno aqJ '(Zt ;aldeql ut passnrsrp) euEJqruaurlerJpuoqJollru (hiII) rauur pue (aueJqluetuJelno eqt Jo eseJolsuEJl 'IAtrOJ)retno eqlJo sespJolsuurluleloJd pezuelcads,,(qurseydot-,beql ruo4 uolJp -uoq3olru aql otul peuodru pue snelcnu eql q paporue eJesulaloJd IPIJpuoqc -olru lualeJJrpos Jo 000I eseqlJo lsoIAI'surelord;o uo11ca1ocenbrun E suleluoJ qJeg 'peuruJelap eq ueJ rueql '{q pasolcua seseds er{l Jo puP seueJqueu o^Al eqlJo qJEaJouorlrsoduoJ Iecrueqsorq eql'(z-tI alntld) sluauoduoc eleJedas olur pateuorlJe4 ueq] pue patdnrslp fllua8 are prJpuoqcotru pelJlrnd;1 'acuds erruJqruaurJelul Jal orJEu qsnru E pue xIJlBru purelq eql :slueuuEdtuoc 'suorlJurg lueJegrp ,rfuananeq IerJpuor.IJolr{ue}eJpdas oml eluaJJ ,,(aqrraqlaSol qclq^\ 'sauEJqr.uaurpezrTenads fUBIq o^1l ^q pesol3ue sI uolJpuoqf,ollru qf,eg

sluetuueduo) leuraluloMI pue 'oueJquaw Jelno ue suleluo)uoupuoq)ollweql ,reuulue 'oueJquraw 'elunlo^ IIaJ eql Jo qlJrJ-auolnoqP ddncco plol uI qJIqM 'EupuoqJollur 0002-000I suleluos IIaJ JeArTqf,Ee lre^rl ruoq parJrJnd elrpuoqcorlu qllm paruJoJJed eJe.rlrselpnls

ulfi 0[

slseldojolq) pup erJpuoq)ol!W:uolsro^uo) ^6leuf :tI ra]deLl)

918

817

THEMITOCHONDRION Figure14-7 Biochemical fractionationof purifiedmitochondriainto separatecomponents.Thesetechniqueshavemadeit possibleto study The method the differentoroteinsin eachmitochondrial comoartment.

i,ji!i*. ["J:;fl ::;:iil:1i;?:i::i'.';il:"":1il'"';:,Tiflil:T:ff yi;H:'."-ffi o,ds:l"lil"""f :::n'il'if :[T::,"JifJil:'#:r.'ffi and of onlytheinnermembrane a stucturecomposed

INTACT

matnx outer membrane i n n e rm e m b r a n e intermembrane space

srrength,waterflowsinto mitochondria and greatlyexpandsthe matrix

il:*:,:",f.t'.g As we explain in detail later, the major working part of the mitochondrion is the matrix and the inner membrane that surrounds it. The inner membrane is highly specialized. Its lipid bilayer contains a high proportion of the "double" phospholipid cardiolipin, which has four fatty acids rather than two and may help to make the membrane especially impermeable to ions (seeFigure 14-65). This membrane also contains a variety of transport proteins that make it selectively permeable to those small molecules that are metabolized or required by the many mitochondrial enzymes concentrated in the matrix. The matrix enzyrnes include those that metabolize pyruvate and fatty acids to produce acetyl CoA and those that oxidize acetyl CoA inthe citric acid cycle.The principal end products of this oxidation are COz, which is released from the cell as waste, and NADH, which is the main source of electrons for transport along the respiratory chain-the name given to the electron-transport chain in mitochondria. The enzymes of the respiratory chain are embedded in the inner mitochondrial membrane, and they are essential to the process of oxidatiue phosphorylation, which generatesmost of the animal cell'sATP As illustrated in Figure l4-8, the inner membrane is usually highly convoluted, forming a series of infoldings, known as cristae, that project into the matrix. These convolutions greatly increase the area of the inner membrane, so that in a liver cell, for example, it constitutes about one-third of the total cell membrane. The number of cristae is three times greater in the mitochondrion of a cardiac muscle cell than in the mitochondrion of a liver cell, presumably because of the greater demand for AIP in heart cells. There are also substantial differences in the mitochondrial enzyrnesof different cell types. In this chapter, we largely ignore these differences and focus instead on the enzymes and properties that are common to all mitochondria.

i n m e d i u mo f l o w o s m o l a r i t y t h e i n f l u x o f w a t e r c a u s e tsh e m i t o c h o n d r i o nt o s w e l la n d t h e o u t e r m e m b r a n et o r u p t u r e ,r e l e a s i n g the contentsof the intermembrane s p a c et;h e i n n e r m e m b r a n er e m a i n s intact

centrifugation leavesthe contents o f t h e i n t e r m e m b r a n es p a c ei n t h e n o n s e d i m e n t i n fgr a c t i o n

INTERMEMBRANE SPACE t r a n s f e rt o a m e d i u mo f h i g h o s m o l a r i t yc a u s e s h r i n k a g e

density-gradientcentrifugation s e o a r a t etsh e o u t e r m e m b r a n e f r o m t h e d e n s em a t r i xa n d i t s s u r r o u n d i n gi n n e r m e m b r a n e

Electrons High-Energy TheCitricAcidCycleGenerates Mitochondria can use both pyruvate and fatty acids as fuel. Pyruvate comes from glucose and other sugars,whereas fatty acids come from fats. Both of these fuel molecules are transported across the inner mitochondrial membrane and are then converted to the crucial metabolic intermediate acetyl CoAby enzymes Iocated in the mitochondrial matrix. The acetyl groups in acetyl CoA are then oxidized in the matrix via the citric acid cycle, described in Chapter 2. The cycle converts the carbon atoms in acetyl CoA to CO2, which the cell releasesas a waste product. Most importantly, this oxidation generates high-energy electrons, carried by the activated carrier molecules NADH and FADHz @igure l4-9). These high-energy electrons are then transferred to the inner mitochondrial membrane, where they enter the electron-transport chain; the loss of electrons from NADH and FADH2 also regenerates the NAD+ and FAD that is needed for continued oxidative metabolism. FigUre l4-10 presents the entire sequence of reactions schematicallv.

Process ConvertsOxidationEnergyinto ATP A Chemiosmotic Although the citric acid cycle is considered to be part of aerobic metabolism, it does not itself use the oxygen. Only in the final catabolic reactions that take place on the inner mitochondrial membrane is molecular oxygen (Oz) directly

d i s r u p t i o na n d c e n t r i fu g a t i o n s e p a r a t ei n n e r m e m b r a n ef r o m m a t r i xc o m D o n e n t s

IsS] OUTER MEMBRANE

818

Chapter14:EnergyConversion: Mitochondria and Chloroplasts

Matrix. Thislarge internalspacecontainsa highly concentrated mixtureof hundredsof enzymes,includingthose requiredfor the oxidationof pyruvateand fatty acidsand for the citricacid cycle.The matrix alsocontainsseveralidenticalcopiesof the mitochondrialDNA genome,specialmitochondrialribosomes, tRNAs,and variousenzymes requiredfor expressionof the mitochondrialgenes. Inner membrane.The inner membraneis folded into numerouscrrstae.

c h a r g e dm o l e c ul e s . Outer membrane.Becauseit containsa largechannel-formingprotein (a porin, VDAC),the outer membraneis permeableto all moleculesof 5 0 0 0d a l t o n so r l e s sO . t h e rp r o t e i n si n t f r i sm e m b r a n ei n c l u d ee n z y m e s i n v o l v e di n m i t o c h o n d r i al li p i ds y n t h e s iasn d e n z y m e st h a t c o n v e i t lipid substratesinto forms that are subsequentlymetabolizedin the matrix,import receptorsfor mitochondrialproteins,and enzymatic m a c h i n e r fyo r d i v i s i o na n d f u s i o no f t h e o r g a n e l l e . Intermembranespace.Thisspacecontainsseveralenzymesthat use the ATPpassingout of the matrix to phosphorylateofher nucleotides.

robnfr

300

Figure14-8 The structureof a mitochondrion. In the liver,an estimated670lo of the total proteinis locatedin the mitochondrial matrix,2lo/o is locatedin the lnner membrane,60lo in the outer membrane, and60/o in the intermembrane soace.As indicatedbelow,eachof thesefour regionscontainsa specialsetof proteins that mediatedistinctfunctions.(Large micrographcourtesyof DanielS.Friend; smallmicrographand three-dimensional reconstructionfrom T.G.Frey, C.W.Renkenand G.A.Perkins,Biochim. Biophys. Acta 1555:196-203, 2002.With permission from Elsevier.) "t

consumed. Nearly all the energy available from burning carbohydrates,fats, and other foodstuffs in the earlier stages of their oxidation is initially saved in the form of high-energy electrons removed from substratesby NAD+ and FAD.These electrons, carried by NADH and FADH2,then combine with 02 by means of the two high-energy e l e c t r o n sf r o m s u g a ro x i d a t i o n

u n s t a b l ei s o m e r

o

no

HO

-'-NH,

ELECTRON DONATION

-T* H

tc'c-NH, Itl

n-t-f-t-

I

+ h y d ri d e i o n

" l' lc

I

BOND REARRANGEMENT

n

I

H:

t H'

\

2e- +

two electronsto electronT r a n s p o rct h a t nI n m e m b r a n e

Figure14-9 How NADHdonateselectrons.In this diagram,the high-energy electronsareshownastwo reddotson a yellowhydrogen atom.A hydrideion (H-,a hydrogenatom with an extraelectron)is removedfrom NADHand is converted into a protonand two high-energy electrons: H- -+ H++ 2e-.Onlythe ringthat carriesthe electronsin a high-energy linkageis shown;for the completestructureand the conversion of NAD+backto NADH,seethe structureof the closely relatedNADPHin Figure2-60.Electrons arealsocarriedin a similarway by FADH2, whosestructureis shownin Figure2-83.

'dlv ut ^brauapuoq-aleqdsoqd olur uorteplxo(zHCVlpue)HOVN Jo lbraua aql Jo ued lofpu e 6urlranuor 'raqloueo1I6.rauapuoq le)tueLll Jo ruloJauo sabueqrleqt e)tnap e seso^lasauelqulau lpupuoq)olil.ll rauuraql'uorge;{roqdsoqd anrleplxo Jo ssa)ojdstqtul'uoupuoq)ollul aqt r(q pazllPle) uolsta^uo) I6reua gaurofeu aq1 11-tr1 atn6r3 L d+ d c v

o c u ot s t a n u o > - { 6 r a u a

( ozH+

*H+zoz/t* EN

'uluqc aql Suop ssed^aqt sp esol ^llenper8 pue ,{8rauaqBrq.{rair lsolu aqt Jo{ 1r qu^\ uEls suoJlcala aqJ 'urBq3 frolerrdsar eq] ur srarJJesuoJ]3ele lueJeJJrpgI uBql eJou aql Jo ]sJrJaql ol passPdare suorlJele o,{i\leql '(oz + *H e _H) suoJl -Jala o^ t pue uolord e olur peueluoJ sr pue (+CIVNaleJeueBeJot) HCVN uroq pe^orueJ sr uor aprrpfq eq] ueq ^ sufaq uodsueD uoJlcele yo ssacord aq; 'Gt-Vt arnBIU)epceloru uaBdxo eqt ol suorlJala eq] Jo uoutppp purJ aqt Lq palearc saSruqc alrle -3au aql ezrleJlnau01pesn are,{aql uaqm'dlluaueruJad peurnlar suolord eql eJp ureqc lrodsueJl-uoJlcele aql Jo pue eql ]e dpo lng 'peurquotar .,(FuersueJlaJe suoJlrale pue suolord ',{ea,r aq] Suop sdals IeJeAaslV'auerqueu IerJpuoqf,o]nu Jauur eql ur sJarrJpJuoJlf,ela Jo sarJese q8norql ssed suoJlJele er{J 'suoJlJele pue suolord olur paleredes lsJrJeJEsruole uaSorpr(qeql'lpeq sE luaruuoJrlua eql ol 1so1Suraq Jo peelsur paJols aq ueJ peseeleJ.{8rauaaql Jo }soru lpql os 'sdals leurs dueur ur rnclo o] eperu sr OzH

parols e r.lrro+ o] peua^uo) pue passaureq s t, ( 6 r a u a aql,o q)nui

az

+

A9U3Nl IV]H

ro rsvSllu ln tso'tdxl

+HZ

s u o r l : a 1 ap r e I *g olut aleredasI ,H NO|IVCrXO]V)tDO]Ol8 (8)

NOrlsnsnO) (V)

pue E!rpuoq)ol!W:uolslanuol (6reu3 :71 raldeql s1se1dorolq3

028

'xuleuraqt ur !d pue dov ujorj dfv azrsaqlu^sol anolj+H aqt Jo ^6leue aqt sasnleql xalduo) uralordauelquiaulsuell e'aseqlu^sdIV aq] qonolql l)e9 +H saAUpauejquau leuu! eql ssor)eluarpeJ6uoloJdle)!ulaq)oJl)ala 6u11;nsal aql'xuteul aql ro lno +H dund leql saxaldr"uor aulzua fuole.rrdsa.r aorqlaql sanrrppasea;el 16reua aq] ro oruos'ureq)Uodsuerl-uorl)ela aq] 6uolepassedsr uoJl)eleIbraua -qbrq e sy'uollel/fuoqdsoqdanrgeprxo Jo urslueq)au;ereuebaqlj1-yg arn613

oueiqurau relno

u r e q )u o o s u e r l - u o J p a l o

'llnseJ eql olul peue^uos ueeq sEq luelpEJS e lr/vroplv\ou uoloJd Jo ,{8leue eq} '(g9I-?I e sV 'pEeq dodglol eql aplsq dprdur urnl ol epelu dqareql sI qrlq \ 'ulds arn8rC u aryq) rotor eql o] peqceDB II€ls E surnl osIP Suputds sIqJ ol 8uu JoloJ eql sesneJ luetue^oru Jleql 'lJEluoJ JoloJ-Jolels eql lE peluroJ Ieu -ueq3 ualoJd eueJqruausuE[ Iecp sv'sllunqns qSnorql ssPd suoloJd /v\oJJeu e -uepl tI ol 0I Jo 8uu e;o pasodruoJ ,,JoloJ,, E qll \ lcEluoc uI sl Jolels sIqI 'auEJq -Iuelu eql ur ,,Jolels,,e ruJoJ ]Pql sqeloJd euEJqlualususJ] Jo dnorS e or ll 8ul]{l ,i.qaceld ur peeq qql sploq ruJBpelu8uole uV'aueJqluau lelJpuoqsolllu Jauul eql Jo eprs xrJteru aql uo s]3e(oJd'sllunqns 9 Jo 8uu e Jo pesodulot pue pPeq dodrqol u e)FI pedeqs 'uoruod Jppru^zua eBrel V'sls,tpleJ drelor fq slrom leql resEdIV suolpp 000'009 uPql eJolu Jo sselu E ql!v\ ulelord lFnqnspFtu e sl 1l ICo{ eq} peller osIV 'eseqtu^s dJV Jo erntf,n[s eql smoqs 9I-?I ern8lt 'Eeeqf,JEpuE erJalsPqJoeuuJqlualu Puseld eqt ul puE 'ae8p puu s1ue1d;o slseldorolqJ eqt 'silec pluluB Jo PlrpuoqJolllu eql uI sJn33oeu^zua eruEs eql iul8rro luersue;o sr aseqlurts dIV aqJ, 'Q7-7 anfu7 ees) dJV seIBIu leql Id pue dCV ueelrl\lequollseeJ elqelo1eJun l.lpcpeflraue aql eAIIp ol pesn aJe Laql 'aseqlufs 4gy aqt q8norqt.{ervr JIaq} peeIql suol eseql sV'luelpeJ8 IPJIrueqc -oJ]f,ele 4aql uMop ^\ou ol suoloJd so\ollB lEql auPrquau IElJpuoqJolIIu Jauul '^lsnoneJd pauoquau eql ssoJce Learrqlud JnlqdoJp^q e seleeJJ atJr/izue sql 'asuqlurfu 41y arudzue punoq-eueJqrueu eql ^q elqlssod epelu sI slqJ,'(?I-tI arn81fl uopBl^Joqdsoqd arrlfepgo Jo ssaJoJd IPJIIITOeq] ul qseqlurts 41y saltrp euEJqrueu IerJpuoqJollIu Jeuul aqt ssoJJsluelpEJSuoloJd leJlureqJoJlselaeqJ

slseqlu^sdIV se^llc luelpeJguolordeql '(nru Ogtnoqe Jo lepualod euerqrxelu e ol luelellnba 1ce;ga uE seq llun gd I Jo HdV qtee) strun Hd 9'0 ol t'0 tnoqe Jo luatper8 gd e puu nru 0ZI ol 0gI lnoqeJo leltualod auerqlueu e;o dn eperu sI ll pue'(e^pu8au aprsu1 Aur 06I ol 0gI lnoqp sl uolJpuolloolttu Suutdser eJo auerqluaur reuul aql ssoJce '(Atu) sllollllru aoroJ elllotx-uotord aql 'lleJ pcrdr! e uI Jo sllun ul paJnseau eq uEO r{Jlqllr 'eiloJ elpotu-uolord e suexe lualper8 uo}ord pclueqcoJlcale sl.Ho

+n

XJgtrVl\I:

+n

.xuleuraLllolu! +H a^up ot l)e sa)roJrllo8 '(Hdv) luerper6 +Haql ol anp a)roJralleuse pue 'slradxa (lxaislql ur71ypaleu6rsap lnq Iq hy paleubrsap {1;euortrperl) ler1ua1od auerqurau.r aql ol anp a)ro; a6rele lo s l s r s u oa) u e r q u e u l e u p u o q ) o l r ur e u u l aql ssor)ea)ro; anrlour-uotord;e1o1aq1 'tualper6uolord ;erluaqrorl)ala aql 1o slueuodruo) oml aql € [-t 1 arn611

+ H * H * H * H* H * H +H *g*H *;14H *H *H.u * *H*H U *H

;*fiHEffi{Fsffi illllid#d,f; #Fxlii,i

o

:r*ffi,lii#H!{ii AV tlv

lellualod aueJqttaul

ol anp a)lol eAtloltt-uOlold

llVdS INVUE:k1llr$EtINl

1Hl NOIUONOH)OJIW

LZ8

('Pll sraqsrlqnd uellru)ew uolJ uorsslullad qlll 't66I '829-LZgtjLEilntDN ''p sureqelqv la ij.r ulorJ'Jalle4 uLlofJo Isallnot ,g) .uotyod auejqulauJsueJl aql u_lolJ paq)elap uaqM'ld pue dCV ot dIV Jo srs,(;otpr(q aql I;aueu-uorlteal srsaqlu{s dIV aLlljo osie^alatll uo ,{ller ol ,(tlllqesll urorJaueu stt so^uapaseqluls dfv aql Jo rred srql',(qder6o;1e1sfut {er-x {q pautuJletap/ased1vtl aql Jo elnl)nlls lpuotsuaultp-aalql or]l (g) 'aleloj louue) ast^oltl qftqM 'ppaqaql suroJ tprjt slrunqnsflg pueng 1o 6urr e ol lolels aql soxtJ[!te stql 'ure pa1e6uo1a ue ateal)leql sl!unqns reqlo ol pelt'sllunqnse eueJqulaulsuetl uolJ pau.uojs paar6) )oPts aql 'auelquau aql ut sllunqns) t t-0 t Jo 6uu e Iq paurol @al )otd e ol paxl] ,sltunqns sr1;e1s6ur1e1ot V'palp)tputse aldltlnu ulorJpauuoJare0l pue Ll qlog '0Jpalle)laule) +HauelqulausueJlp pue'osed1vtl aql palle),uotuodpeaqp ;o pasodluot sratu{zue aql (V) 'eseqgu{s 61y 91-y1 ern613

,{8raua1e}oleql sI srql pue 'pazqoqp}aursr teq} esoJnlSJo elnoelour,,fuarra rog 'flsnornardpa]etssV dJVJo selnceloluZJoplad tau E sef,npoJdeuop srsdloJdlS

dlv s,lle)eql Jo lsow a)npordsluerper9uolotd 'Qt+t erntlg) Suop unuapeq eql qedord qrlqm'runlle8ell lerretreq aqlJo uoqeloJ prder aql selrJp osle luarpur8 slql 'elJetopq elloru u1 'sassacordJo sad,{l oml eseql elrJp ol passauJer.{sr aueJqr.ueu uuseld [erJa]Jeq eq] ssoJopluarpur8 JelrrursE 'euelcpq uI 'eueJqueur aql ssoJcpsalrToqeleruJo uodsueJl eql pue dJV Jo uouuruJoJ er{] qroq e^rJp ol pesn sr eueJqruau lerJpuor.IJolnu Jeuur eql ssoJJe luarper8 uoloJd lecruaqcoJlJela aql 'q1ac cpol:eone ur '^aoq ueas eABq aAA '(9I-?I erntlC) ragodsu?rl-or dIV-dCIV qql sa^rrp eueJqrueru eql ssoJse eJueJeJJrpe3pllo^ aql 'snql 'uolJpuor.lsollur aql Jo lno peloru Suraq a8reqc e^pe8eu auo Jo lelol p ur sllnseJ e8ueqcxa eppoelonu qJEe '4qy ue{l a8reqc enrle8eu aJoru auo seq elncalou dJV up eours 'ureloJd rauod -suer1 a13ulse ^q suorparrp alrsoddo ur peuodsuurl-oJ eJEdIV pue dOV 'xrJleru eq] olul seloru *H er{l se +H q}lm pJE^ ur peuodsuen-oo eJE(ld) aleqdsoqd JruEBJoulpue elulnr^d 'alduexa JoJ 'snqJ'xrnptu IBIJpuoqc -olFu eql olul +H Jo ^i\ou elqero^eJ ,tlpcpaSraua eql ol uodsueJ] rraql aldnoc ueJ selnselou essqt purq lEqt sJeuodsuprJ'uopJeJlp etlsoddo eql q pe olu eq ]snu AIV sp qJns 'sJeqlo elrqM 'losol,{J aql ruo{ xIJlBru eql olul pedund eJu 'ld pue AOV'alennild sBqJns 'selnJalourflerus peBJeqcduuur 'pupuoqJolr(u uI 'srs -eqtu^s dJV seprseqsesseJordraqlo sa^rJpluarper8 uolord lef,rrueqcoJtcelaeqJ

eueJquaw rauul or.ltssor)vuodsueJlpeldno) se^uo luetpetg uoloJdeql '(g pue uaaMleq € Jequnu leJ8etq-uou e dlerauaS q qclq a',,oper rea3,,slD dJVJo elnJaloru qcee e{eru ol aJrlap snole^Jeru srql q8norqt ssed ol peau luq} suo}ord Jo Jeqrunu aql saurrurelep leql Jeqrunu sq] q ll pue 'sesuqtu,(s aw luereJJrp ul lueJeJJrp sI roloJ aql ut strunqns Supucoysuerl-uolordgoJequnu eqJ'uounlonar rad 41y 'puoJes rad Jo salnJelou g Suqeraua8 61y1o sapceloru 661 uuel aroru ecnpord ol alqu u asuqluds dJV eqt 'deansrql uI 'sateeJJIlels Supulor aq] tpql uolleruroJ -uoc uralord ur sa8ueqcpeleadar q8norqr aql puoq d8Jeua olur paual IpJrrueqc 'aleqdsoqd orue8 -uo3 sr ,{3raua lEf,rupq3eru sp dIV ruJoJ o} ua^lJp eJu eser{J -Joul pup JoJsalrs Surpurq ureluoo peeq aql ur slrunqns x.rseql Jo eeJqJ dCIV 'suratord peeq 8uu dreuouels e 1surc8eSurqsnd suralord 1p1s Jo Suputor :Jeqlo qcee ]suru8e Surqqnr suralord Jo slas oml Jo ,{8raua lecrueqceu uruo[

(V)

ssedlv rl

le!

l)vds lNVUSrArWUllNl

J e r J r e )+ H auerquraurSuell

slse;doro;qlpueplrpuor{to}thl:uolslanuol{6lau3:g1ra1deq3 ZZg

'asPqlu^s eql Iq pesn asoql dlv t/!sv]dol^l alqurasar ^eu tpql sulstueq)auIq Qo1e1s oql) suralord;o6uu lalno ue q6norql +H suolo.rdJo MoUe Iq uanrrpst uottelol aql 'un1;abe11 dund uolord aq] qllm elelol pue sauelquroru rauutpueialno aql ut poppequla (ouerquJeu.l ele q)tqM '(a6uDJo) s6uu uratotd euseld) f aueJquraurL Jo sauasp ol poq)pllesrun;;a6egaql 'moll l e r J e D e qr a u u l +H {q uenup utnlle6epleual)pq aql Jo uollelor aq1 4 1-y 1 ernbr3

puo)esJed suollnlo^al 00|' ueql erou le 6urlelo.rrolou.r.re;;a6e11 suraloro roloJ

su!oloro rolels

I

rale; u e : { l 6 o p r 1 d a dr "* auetqueu f leilapeq ralno L

l)vds uv]n't1t)vuIXl un11a6e11

eqt uI srsdlorpdr{ dIV,{q pacnpord selnralou dCIV aqt ACV roJ dJV se8upqcxe leql euBJqruau lErJpuoqJolr(u Jeuul eql q uleloJd JarrJEf,eql Jo asneseg

slla) ur olleu dcv:dlv tl6tu e ulelutewetrpuorl)oltw '(Ig-z ernSrcosleees1gt-rt alqpl) sleJJouorlEprxoeql urorJ pe^rrepsr tEqt zgcvg eq] pu? HCIVNaql uorJ dJVJo lunoup a3re1e sacnpord oslE uorrpuoqJolru eql q uoppllroqdsoqd e^nepxo 'auetqluau pupuoqf, -olrlu aql ur susluEqserusrlorusorueqs^q pasnpoJdsr IIeslpruruEuBur asoJnlS Jo uoqeplxoeq] uro4 pasnpoJddIV eql Jo dluofErulsp^ aq] ,uorsnlJuoJuI 'sdIV qlpr 0t lnoqp Jo pler^ leu p se^rS-uollp!fuoqdsoqd anrlepr-xo Surpuapup srst(loc^18 qlyvr3ur]rB]s-asoJnlSJo alnJalou euo Jo uoneprxoeteld -uoJ eql alEInJIeJupJ e^ 'VI-trI elqul ul pazrJeturunsalcLcprcu clJllo eql teq] pue srs.,(lord13 yo spyardtrnpord eql rxorC 'losol-,baql ur srsdlocdlS dq parnpord seFcelotu aql ruoJJro 'zHov{ uor; rrcd uortcelerad sepcalou dIV g.I HCIVN sarnpordosp uonel,fuoqdsoqd e^rlEplxo.losot{Jeqr ol dIV srq}Buruodsuerl roJ pepeeuISraua eq] Supcerlqnsraue AJVJo salnJelorug'Z tnoqe Jo uoneru -roJ eql roJ.,iSJaue aprloJd ol sr BrrpuoqJollu paJnpoJd ur lq8noqt HOVNeql,,(q 'uoppl.ftoqdsoqd peleuop suorlJaleJo qcEe rrpd e^rlEplxo Suunc .(7raldeq3 ur pessncsp)zg go acuasqeeql ul Jncooleql sassaoord uollelueurraleql roJpleil 16

t t raloeqf ur passnlstpst surolordtettodsuetl {q sauetqular,u ssol)esaln)alotrlJo trodsuerla^tl)eaqf 'spunoduo>aseql]o ^laalJst auelqurau,l Jalno lle ol alqeaLulad oqI uMoqssp'aptsutaarle6aust q)tLlM 'leltualodauetqutaulaql qltM uosuedL!ol roj pale)!pu!st saln)aloutpallodsuprl aql q)pa uo e6req> aqt']no padr,und sr Jo aql otut pa^ourarpdov dlv ollq^^'xtrleur pue'(ld) a t e q d s o q rdr u e 6 l o u r , a 1 e n n r , ( 6 'auptquau Jauu!aql le!,tpuotl)ol!tu ssorleluarpel6uolord ;erruaq)oJt)alo oql ^q ua^!Jpsassarotdl.rodsuell e^!l)e aqlro auo5 91:y1 arn6r3

aueJquraul lolno

_rdIV

alerntld

_€dClV

€28

NOTUONOHfOItW lHr

srs,{lorp,{qdIV eqJ '(Lg-6L'dd eas) rncoo }ou eslmleqlo plnom }eq} suollJeeJ 'pasn sI srs.{lorprtqsll JoJ Iuorrueqo Jeqlo aAIJp ol'suouJual pa1dnoc q8norqr (gy anqe8au a8rel) a8ueqc d8raua-aarg elqero^eJ 'a8ru1aq1 'slleo uI ,.elnJelolu '7 laldeq3 u1 JerJJeJ.{8raua pa}eAIlJE,,roferu eql se peqIJJSapsum dIV '(0 = ul) pnba are g pup VJo suollEJluacuoc reloul eq] I ueqmgVJo anle^ aq] spnba ]Eql enp^ acuereJeJe Lluo eJoJereqlsI gtv'ruqllru -3o1prnleu aql sr ul pue 'lgpue VJo suolleJluaJuoJ eq] alouep [g] pue [y] araqm tvl =9v + lsi't-rY JV 'suolleJluec 'g e V uollf,ear aldruts eql JoC -uoc JIeq] uo spuadap puof,as eql :salnoelourSurlcear aqlJo sJelcercqccIsuIJluI 'lsJg aql :sued oml eql uo spuadap '"gy'aBuoqc rtBnua-aattptnpuots eql pelleo 'd Jo runs aq] se ueilrJv\ eq uec uol]Jeer uaruBe JoJtv eql teq] 92 uo pa^ oqs eM 'rV 'uopceeJ e JoJa8ueqc 'lleJ e uI JnJJo IIIM uollceeJ slql Jeqleq ^ saulruJelap '7 ,{8reua-aar; eqJ '(tl ,,{8raua aeq Jo }dacuoc aql pecnporlul ervr raldeq3 u1

lla) aql ol Inlasn dIV lor gv Jo enlena^!le6aNe6rel v dIV saIPWsrs/(lorp/(H 'scrureudpourraql ;o sald -rcutrd rfueluaruala eruos replsuof, lsnlu arr,r'rtqrr,r umldxa o1 '14pue dCVJo suop -BJluacuocaqf qlyv\ paredruoc qBH st feq] dJVJo uolluJluacuoc e Surureluteur qlec uo spuadap 11:SurpueruapaJorusI aJII'lceJuI 'lnq loJezseqceeJdIVJo uop -eJluaJuoo eql ueq,{ .{1uodols pFo^ sassacordJelnllac leq} Iuees rq8nu 11 'auerqtuaru ',{e,rt qleep sesneJ ,(pcexa ul stql IelJpuoqsolllu Jauul 'selp aql ur uodsupJt uortoele qcolq qclq/v\ 'apruudc uostod aql IIeo eq] pue 'ualup ra$uol ou eJe suollf,ear elqgJo1eJunr(lecna8raue 'r(lpnluana :u4op sunJ Lralleq s,ller eqt pue IIEJsle^al dJV 'pe{colq sI Elrpuoqsollu aqlJo drpllcu aqr11 'saur8ua lrJlcele elrJp o] pasn aq uuc dJeilBq E leql ,teru arues aql qonlu uI sasseJ -ord replac e^Irp o] pesn eroJereqts1lood dIV aql'169-7 arn8tg aes)dJVJo sls -.{.1orplq elqeJo^eJ.{.lpcqa8raua eq} ot ueq} Surtdnoc Lq suotlcuar elqeroleJun '7 .,(leoqa8raua elrJp uego saurrtzua crlaqlur(so1q raldeq3 uI pessncslp sV aCIVJouorlerluacuoc eq] ueq] raq8rq sau4l 0I ]noqe sI ]eql dIVJo uorleJlueJuoc e sulglulelu IIef, aql pue 'elnulul red acuo ueql eJotu Sut8reqcar roJ (dCV se) 1r o1u1{Jeq pue uolJpuoqcollu e Jo lno sel}lnqs Apoq uErunq aqt ur alnoeloru dIV pcrd.,i1V'pepeeu are Leqt araqm losoldc aql olul padulnd ,{lprder are uo4ey,fuoqdsoqd allteprxo ,{q xrrleu Ielrpuoqcollu aql ut peuroJ salnJeloru dJV aq] ayq^u 'Sut8reqceJ roJ elJpuoqcolru ralua ,{lprder losoldc

1 d-198 + ZHCVISt + HOVNt€ e 6uo;e ,{Jprder {ran a^oul suolold (V)'Jale^rr ut aleqoq suolord rvrogg7-y1 arnbrl

)i

l{{

slse;do.ro;q1pue eUpuoq)ollW :uotslenuol {6lau3 :y1 raldeqS

828

') Jeuie)ol uojl)oloslt saleuop ll uaqMauelqutauaql Jo aptslaqlo aqt ol uoloroaql sasealal lr :v laule) uolJ (_a) uorl)alaue slda))elt uaqMauelquraul aql ro aprsauo uol] (+H)uolold e dn sl)rd B laulel uoll)ale'uel6etp srqlul oals q)eale uolordp aseolal pue purque) lt iauelqLUau la^eltq -prdrle ur pappaqLua utpq)llodsuell -uor])alaue 6uolpsasseduoll)ala ue sv'sauerquraussol)epadund aq ue) suolold MoH tz-tt atn6rJ +H uorllala A6.raue-nnoI

']ueuodur dleuorlcun; ]ou sI sed^,{} olul uoI}eJgISSEIJ eql pup ad.,! qcea;o sauoJqf,oldJ I€JeAesuIEluoJ 01u^ ou{ ^\ou eJEsllec q8noqf ue^e 'pa^r^JnssEq alnlPlf,uatuou slqJ 'J pue 'q'22seluoJqsofc su ruaql paleuSr -sep pup erlJeds uortdJosqe e^rlruuslp rleql ^q seuorqJo]-,{JJo saddf earql peIJ 'adocsorlcads e qllm senssp pue slleo Sut.rasqo dg 'slcasut -puapr sJaqJJEesaJ pue 'slseart'eualJeq se aleredstp se susIuEBJo 3ur,t11ul uollf,npeJ pue uonep -xo prder o8rapun ]uq] spunoduroJ sE9Z6I ul peJe^oJSIpaJa^asauoJqcoldc aqt rsnqJ 'rr,rroDl aJal suorlJunJ lJexe JIeI{l aJoJaq3uo1 sluauodtuoc aseq} ,,!rrnd paleJl o1 alqrssod aJoJeJeqlse^AlI 'seJnlxllu apnJc uI uana "tlectdoJsoJlJads pue runrlcads uorl aq o] Jorleqaq slr ,l olle o1 q8noua ]JuIlsIp eJEleq] rtlnrlcear 'lpJaue8 'pacnpal -drosqe pazlpxo daql uaq ^ rolof, a8ueqc ro are qcpa u1 up s€q uoJlJela aql;o ,r{.ue61 pue lq8rr elqls4 qJosqe urcqc ,fuolertdsar aq] uI sJaITJPJ

u!eq) /(rolerldsaS uorl)alf (uery paulluaplspoqlaw)ldo)sorllads aql u! s"talrre)

1 t

'i',:.,t,.i,, v i::::::':'::''l::l;l:::'

'peseelal sr teql d8raua aq] srql'sda1s lprus fuuu ur Jo JIeq,{peau aJots o} IIeJ aq} s^ olp ssacord asmrrda}s sJnJJoOzH

SS

(A) \

SS tl Cyr \.,,^-J

Cyt

Figure 14-23 The structuresof two types of iron-sulfurcenters.(A)A centerof the 2Fe2Stype. (B)A centerof the 4Fe4Stype. Althoughthey containmultipleironatoms, eachiron-sulfurcentercan carryonly one electronat a time.Thereare morethan sevendifferentiron-sulfurcentersin the respiratory chain.

832

Chapter14:EnergyConversion: Mitochondriaand Chloroplasts

hydrophobic hydrocarbontail

-,'/

oxidized ubiquinone

ubisemiquinone ( f r e er a d i c a l )

reouceo ubiquinone

deoxycholate,can solubilize selectedcomponents of the inner mitochondrial membrane in their native form. This permitted the identification and purification of the three major membrane-bound respiratory enzyme complexes in the pathway from NADH to oxygen. Each of these purified complexes can be inserted into lipid bilayer vesicles and shown to pump protons across the bilayer as electronspassthrough it. In the mitochondrion, the three complexes are asymmetrically oriented in the inner membrane, and they are linked in series as electron-transport-drivenH+ pumps that pump protons out of the matrix (Figure L4-26):

Figure 14-24 Quinone electron carriers. Ubiquinonein the respiratory chainpicks up one H+from the aqueous environmentfor everyelectronit accepts, and it cancarryeitherone or two electronsas part of a hydrogenatom (yellow). Whenreducedubiquinone donatesits electronsto the nextcarrierin the chain,theseprotonsarereleased. A long hydrophobictail confines u b i o u i n o n teo t h e m e m b r a n e and consists of 6-10 five-carbon isoorene u n i t st,h e n u m b e rd e p e n d i n g on the organism.The corresponding electron carrierin the photosynthetic membranes is plastoquinone, of chloroplasts which is For almostidenticalin structure. simplicity, we referto both ubiquinone and plastoquinone in this chapteras quinone(abbreviated as Q)

The NADH dehydrogenase complex (generallyknown as complex I) is the largest of the respiratory enzyme complexes, containing more than 40 polypeptide chains. It accepts electrons from NADH and passes them through a flavin and at least seven iron-sulfur centers to ubiquinone. Ubiquinone then transfers its electrons to a second respiratory enzyme complex, the cltochrome b-c1complex. 2. The cytochrome b-q complex contains at least 11 different pollpeptide chains and functions as a dimer. Each monomer contains three hemes bound to cltochromes and an iron-sulfur protein. The complex accepts electrons from ubiquinone and passes them on to cl.tochrome c, which carries its electron to the c)'tochrome oxidase complex. 3. The cytochrome oxidase complex also functions as a dimer; each monomer contains l3 different polypeptide chains, including two c1'tochromesand two copper atoms. The complex accepts one electron at a time from c)'tochrome c and passesthem four at a time to oxygen. The c1'tochromes,iron-sulfur centers, and copper atoms can carry only one electron at a time. Yet each NADH donates two electrons, and each 02 molecule must receive four electrons to produce water. There are severalelectron-collecting and electron-dispersing points along the electron-transport chain that coordinate these changes in electron number. The most obvious of these is cytochrome oxidase. i.

An lron-CopperCenterin Cytochrome OxidaseCatalyzes Efficient 02 Reduction Because oxygen has a high affinity for electrons, it releases a large amount of free energy when it is reduced to form water. Thus, the evolution of cellular ( A ) N O R M A LC O N D I T I O N S e

( B ) A N A E R O B TCCO N D T T T O N S

+

*Oz

p a r t i a l l yo x i d i z e d

a

*.b

r e du c e d

\T

b*c

d

fully reduced

inhibitor e-+

control e+a+

s u d d e na d d i t i o no f o x y g e n Oz

oxidized w a v e o f i n c r e a s i n o x i d a t i o nw i t h t i m e

Figure14-25 The generalmethodsused to determinethe path of electronsalong an electron-transport chain.Theextentof oxidationof electroncarriers a, b, c, and d is continuously monitoredby following theirdistinctspectra,whichdifferin their oxidizedand reducedstates.In this diagram,darkerred indicatesan increased degreeof oxidation.(A)Undernormal conditions, whereoxygenis abundant,all carriers are in a partlyoxidizedstate.The additionof a specificinhibitorcausesthe downstreamcarriers to becomemore oxidized(red)and the upstreamcarriersto becomemore reduced.(B)In the absence of oxygen,all carriers arein theirfully reducedstate(gray).The suddenaddition of oxygenconvertseachcarrierto its partlyoxidizedform with a delaythat is greatestfor the mostupstreamcarriers.

'OZHZ1el,t1p1derpue aAItJEeJflsnoreSuep seruoJeq ll .(_zo) IesrpeJ apxoradns E ruJoJol uoJlcela auo dn palcrd seq zg;o alnJelou p acuo 1ng .srsrt -plec c4eudzua q8norql uortoeer IEIUur slr Iorluor ol slleJ pel\ollu seq tcBJslql :uoJlcale]sJUslr dn Suqcrd elqnoJl spq lr esnuceq eqleaJq e'tt JrEeq] ut zO eleJa -lol uEf, a7q'fu1s[uaqc palecqsrqdos d.rane seJrnbeJte^a/v\oq fe^\ slql uI ze esn o] sruals,ts pcrSolorq Jo ifilqe aq; 'arrdsar srusrue8Jo raq8rq p d.qa,rdlqeruns -ard sr srr.IJ'urqloqeleru JrqoJapupruoq peAIJapaq uEJ ueqllsJeue eJorx qcnrx sseuJeq ol srusrueBro pelqeua talem ol pepeluoJ s1 z6 qclqiu ur ,uoqeldser

xe;du.ro: aseprxo aurotqro1l{r

xalduJo) l)-g euioJr.l)oil)

xa;duro: aseua6orp{qap HCVN

uu 0L

zoz/r+ *Hz 'saxatduotau{zua Iiolerrdsel aqlJo qleo {q euelqr"ueu aql ssol)e padurndale suolotd 'pole)!pursV'lxeuaql ol xe;duor auo uolj suotl)ale r{tra;leqt slaule) altqou seolias, euiolq)oil) pue auournbtqn'6aq pat ua6,(xoo1p6y11 uroJ1suoll)ela Jo lalsuell aqt 6urrn6 'umoqsale xaldurotqree 1oadeqs pue oztsaleurxoldde aq1 .sexalduor eu{zue ftogelldsar aeJql aql q6no.rql suorl)ale Jo qred aq1 97-9 1 arn6r3

l)vds ]NVUEIA]IAU]1NI

EE8

SdWNdNOI-OUd UI] HI CNVSNIVH)IUOdSNVU.L.NOUI)]I]

'pale)rpurse'll pue sl!unqnsur palerol I are sreure)uo.rl)aleaql (8) 'auerqLuaur rouu!aql ur pauorlrsoo lerJpuoq)ot!r! 'uMoqssr uralordarlua aql (v) '(LZ-tL anfil aos)ouprqureu aql ssor)e suolorddund ol uralo.rd eq1esner ,(aql'agnralouzO punoq s1ro1Iervieql uo uralordsrqlqbnolql ssedsuoll)ela sy 'ar"u,(zue aql Jo ero) leuor]runjaLlt r,urol{aqt pue'auouab leupuoq)olrur aql ^q papo)uaareslrunqnsporolo) aarql aq1'(suollep000't0Zjo sseur rauouoru) slrunqnsuralo.rdlua.ra;yrp EI qlrM Jaurouor.!P urorJpauIo, JauJp e sr uralo.rdsrql'aseplxo aurorqroy(r Jo ornl)nrls reln)alour eq1 g7-9 1 ern6r1

'JeulJEdalElrdoJd -deut ue qll,vr suonceJalut 8u{euunt s}I lueleJd ol uleloJd e eplsul q8noue daap 'l?Uuelod xopar uorlJela ue SurfrJPO,i.q papr,r,ord eq ol sl'uees uollElnsul sIqJ qBH e qtlm JelJJEf,e qlyv\ seplTlos lellualod xopar llrol e qllm relJJBJ uorlsele '3ulssn3 ue uaq r Jn33o esr araqlo plno1v\leql sllnJJlc uoqs slua^aJd uopelnsul -srp eJE a.ry\sessef,ordaqt ro; IesIlIJc sI tEql dladord lesluPqceu-IunluPnb p ,,'3uqeuunl,, uorlJele z(q rncco sdurnf eql 'ruu Z se a8Jel se dEB 3 ssorce 8uI -durnt Lq oslp lnq 'elnoelour p uqrlv\ spuoq lueleloo Suop Sunoru ,,(q,!uo 1ou sluelsl.s IPsrSolorquI ueql fuJec lPql selnJeloul eq] uee ueq a^otu suoJlf,elg '92-7I arn8tg uI rrmoqs eJuanbes eql ul 'slInJJIJ uoqs ou qlyv\ ll ol luasefps JerJJeseqr qll a,'(Iuo lseJelul 01 elqe sI JaIJJeJuoJlcele qcee :uleqJ eql Jo stueuodruof, eql uaemlaq suollJeJelul puoucunJ eql Jo ^rlJglcads aql o] ,(larque anp sr urpqo .{rotertdsar eql SuoP suo4sele Jo reJsueJl perepJo eqJ '(spuocesqllur 0Z-g fua^e eJuo ]noqe uoJtJele uE selleoeJpue seleuop xalduoc r{f,ea)raJsupJluoJ}celeJosaleJpalJasqo eq} JoJlunocJe uuo saxalduroc arudzua Sursryyp fyno1s arou eq] pue SJaIJJPJallqour esaql uaamleq suols1loc ruopupJJo aler patcadxe eql'auerquaru lelJpuoqcolllu rauul eq]Jo auuld eqt u1 dprder esnJJrp-J aurorqcoptc pue auoumblqn-uleqo ,fuolurtdsar aql;o saxald -uroo eur.{zue Jofeur eerql eq] uaemlaq suoJlJela,&JPc ]Eql slueuodluoo olvu eql

uoll)el3{q pere;pa61 suorsrllo)tuopueu6u;rnp6u1;auun1 arv eueJquawlPlJpuotl)ol!wJauuleql u! sJsJsuelluoJl)alf 'aulqoerrl utalord paunl 'suleloJd prralceq pue ]sea[ [1aug srql;o srusrueq]atu pellelep aq] SuIea^aJ eJB aq] ;o Surraaur8ua cuaua8 .{.q aurdzua aql olq peJnpoJ}ul suollelnru peJolIE} 'seJnlcn4s uopnl ^,tlasrcardJo ]ceJJaaql Jo salpnls c11s1ueqoeruqrl,lt' peutqtuoJ peuru -oser Jrruole aqj-'g1-al arnt;g uI pale4snly se '.{.qdur3o1p1sri.-rc,,fur-x,ri.q -Jelep ueeq seq xaldruoc aurdzua a8rey slqt Jo eJnlf,nJls alaldruoc aql 'aurdzua aq] Jo eroJ aq1 rrrJoJ lEql stlunqns aerql aql ;o dlqruasse eqt ro d1ur.r1ceaq] ol rueas esaqlJo lsotu 'sllunqns ;aqlta alepflar o1Surdlaq 'a1orzfuetptsqnse aAEL{ uralord tuaJeJJrpgI surc]uoJ sleruluelu uI eseprxo auorqcolb aqr q8noqrry 'uoqcnpord 4;y Sutcnpar z(puar8 ,{qaraql 'ltodsuer} uoJlf,ale do}s ol saxaldruoc aseplxo aurorqcoldc s.llal aql o1 Lpq8p purq ,,(aqr esnetaq crxol .{laruarlxe eJB apIZP pue aprueri3 'aJIIJlqoree ilp roJ IpIcnrJ aJoJaJaqls xaldruoc utalord sql'slleJ lsoru uI aletdn uaSrixoplol eqlJo %06 lnoqe roJ slunoJJe uollceal asuprxo aurorqcolb aq;

(v)

XIU-LVIA

l)vds ] NVUSY\]IAU]INI

: euro:q:oy{> urorlursuoJpala

slseldolo;q3 pue erJpuotl)ollW:uolsJanuolI6lau3 :71 reldeql

?€8

'(Og-tf arn81g),r(qder3o1els.{rc .{.er-xzi.qpeuluralap xalduroc Ic-q aruorqcolrtc eql Jo aJnlsnJls alelduoJ aql ,{q paple '.la^alsIIuolE aq} lP lno paIJoM Suleq IIIIS erE elqrssod slql eletu lPql sraJsuPrluorlcele Jo selJespatPslldruoJ eqJ 'Ja^a^aoq'xaldruoc Ir-q pedund eJesuoloJd o^aJ Jed aqt uI uoJlJele auorqco/c 'peuodsueJt uorlJala ufua,raro; Jefellq aq] ssoJJE +H auo Surrra;suerl ,rtqaraql 'aJpJJnseprslno eql Jpeu xaldruoJ Ie-q aurorqco{c aql o} ueqt aluuop puu eueJqueu eql Jo eJuJJnsaplsul eq] Jeau suoJlJalalde3JP plnos ]I 're/tPIIqpIdII aqt q elqotrl.{1aar; sr auoulnblqn arurs'(VZ-VI arnBICaes)uor}cale eql sasEalar ll uaqm ll seteJaqrlpue saIJJeJll uoJtJele qcea qllm Suop runtpatu snoanbe aql uorJ +H E dn s)JId auoulnb e .{lsnonaJd peuoguau sV'aloJ E aAPq.{.Fea1c sauournb aq1 'xaldruoc Ir-q aruorqJoils eql uI 'saxalduroc elul1lzueluaJaJJIp aaJql aql JoJluaJaJJIpsI Suldund *g o1 paldnof, sI uodsueJ] uoJlcale qJq^,ltf.q ursrueqJarupalrelep aqJ'o^\l durnd sraqlo sealaq^\ 'aueJqurarulelJpuoqf,olllu Jauur aq] ssoJJeuoJ]f,elarad +H auo dund saxaldruoc eutf\zuazfuoluldsar aruoS

soxalduo)aulzul rofew aalql aql u! srustueq)aw l)ullsto {q srn>rg6utdutn6+Haql 'eueJqluau euosodq aql ssoJcepalEs -olsuerl ere suolord 'xaldruoc aqt q8norqt ssed UEJ suoJlcela ]eql os pappe eJe roldaJf,B pue Jouop uoJtJala aleudordde uE uaq ^ :sauosodll o1ut,(laleredas1t Surterodrocur pue xaldruoJ aru.{zua.{ro}ertdsarqJEe SuIdJIJnddq palzrlsuourap aq uec uorsJaluoc srql 'xa1druof,leq] q8norq] sszd suorlcela sB luoIpBJBuo] -ord pcnuaqf,orloala ue SuIleaJJfqaraql 'eueJquratuJeuul aq] ssorJe *g drund ol a8ueqc ,{8raua-aar; slql Jo etuos Sulssaureq [q aJIAep uoISJaAuoJ-dBJaua ue se slJE xelduor aru,{zua qJea 'luaq} uee^ }aq sJeJsueJluoJlcela ue ueql peseelal .{8raua earJ aq} ol puoluodord ,{.pcar1psI sreIJJeJuoJlJele o^U LUP uee^ laq lErlualod xopar ur aSueqc aq1 'xaldruoc ,{rolertdsar Jofelu qJee ssorcP 'uleqc ,{rolertdsal eql SuoP auo 'sdals a8rel aarql ur dorp sppuelod asaqJ parnsearuspqualodxoper aqlJo eulpno ue sluasald 6Z-?I aJnBIg'(92'd aas) sarlrurJJP uoJlJala slJeuar ppualod xoper aql ^ oq pessnJstp,{lsnoltard e^eq a

A 'HCVNrxoll pauoosuel] suor])alaoMt aql ueql 6urdLUnd +H 6uldun6 *g ssalosne)aroJoraql {aqt laseua6orp{qep g6y5 6urssed(q'auournbrqn loJ I6taul aql sapt^old saxoldtuo) aul(zul flolelldseg o] ^lt)arp pessedaresuorlfalaoM] aalql aql ro q)ef ssor)v leltualod xopau ul do,lo e6re1 y sll (Zg-Zarn611 aas)a;tIt prre)u]!) aql Iq pue (19-7a.rnbr3 aes)uo!]ep!xopl)e Ipel {q palereuaboslpst ZHCVIut^ep 'xrJleru aqf ureq)fuolerrdser aq] loJsuorllalaJo lerJpuoqJolrur aql Jo lno suolord dund ot passeunq aJElxeu aq] ol JelJ -Jer uoJlJala auo ruo4 IEpualod xopeJ uI saSuuqceq] l oq lxau ssnJslp aM arrnosIluo aql lou st HCVNreq] oloN 'auosdurndxalduor aseprxoautolqroy{> aql seaJaqm'uoJl)ala rad oanldund *;1 q)pa saxalduro)L)-qauorq)o1{t pue zor/, + *HZ ozH p,(qapHCVNaqf 'auerquraur aseua6or 008 rauuraql ssorre*g dLund leupuoq)o]rLu xaldu.tor o1xa;duor au,(zuaqreaIq passaureq 002 aseprxo srabueqr(6raue-aallalqelo^e; eurorq>o1Ar c- oi 009 aqlJo Ued 'pale)!pursV xaldLuo) o o q)ea ur sraure)uoll)alaeldltlnut 00s = o o aql q6norqleruanbasur 6urssed {q 00t ot,i xa;duroraur{zuar{ro}errdsar e q6norq} x 00t MolJsuorl)all '(Arilo,fv ro abueqr o o lellualooxopore ssor)eparJalsuerl ol 002 Sl o suorl)ala raqurnuaql s! u aleqM lo o 'o,lV 00r Gaj'du- = 9V) aleurpropueq-Ual f 3 paurelqoaq uP) aln)alou aql u.rorJ 0 OZ: HCVNue {q pareuopsuor})alaoln}aq} o 0 0[ Jo qlea Jora;suerlaql roJ:9V'a6ueqr 3 002{6raua-ear;prepue}saq1'ua6{xo sz9 ol ureq)r(rolerrdsar aql uMopMolJ 008peleu6rsap) suol])olose sasear)ur 10,3 00t xoparaqf 'ureqrlrodsuell ;er1ua1od -uorl)als leupuoq)olr.uaqt 6uo1e +n sa6ueq>1er1ue1od xopeg67-tr1arnbrl

9€8

UI]HI-CNVSNIVH)I-UOdSNVUI-NOUD]I3 SdWNdNOI-OUd

'eperu eq re8uol ou ueo pue flalaldruoc paledrssrp sr ef,JoJalrloru dIV 'aseqluds -uolord eqt 'Suplncrll-uoqs slqr dJV eqt sassuddq]eq] Jo tlnser p sy eupJqrueu IEIJpuoqooUIu Jauur eql ssoJce+HJo ^aolJeql JoJdemqted e apnord zlaql pue '(saroqdouor *g) raLepq prdrr eq] ur srerrrpo +H elqlsnJJlpsE lou leql sprJe {Barlt elqnlos-prdl are slua8e Suqdnocun :1ue8a1apue aldrurs qloq sr }JaJJa srql JoJ uoqeueldxa eqJ 'paleJaua8 sr luarperB *11 ou lnq 'eleJ prder e ]e anup -uoc Sutdrund *g pue lrodsue4 uoJloele'1ua8eSuqdnolun uBgo ecuasard aql u1 'ua8dxo;o a1e1dnrtaq] Suucoyq lnoqlp\ Elrpuoqcolru dq srsaqluds 41y sdols sllef, o] spunoduroc cruu8ro ]q8lam-Jelnoeloru-Molasaql Jo uop1ppe eql 'srseql -u.{s 41y uror; }rodsuerl uorpaya SuqdnoJun 'sluaBaSut1dnoJun sElJe ol rrmou{ ueeq eleq-louaqdorlluIp-V'Z se qtns-secuulsqns IpJeAas's0t6l eql ecurs

slseqlu^SdIV LuorJilodsuell uotl)alf eldno)un seroqdouol+H 'ilec eql o1 1ryasndlerupdo 1l e{eu ol paleln8ar st urcqc rfuolerrdsar aqt lvroqJeprsuoc ol dpeal eJeent 'Surdurnd uolord puu podsueJl uoJ]f,ale JoJsrseq crlsrupqoeu eqt passnf,srpe^pq e^ lpqlrvroN 'It-tI a.rntlg ur paluasard sr rusrueqf,aru praua8 e lutalord drund aqr JoJ suorleruJoJuoJ lJurlsrp eaJql lseel le sarrnbar Surdund *g;o ad,,i1slqJ 'eueJquaru Jeuur lerJpuoqoolru eql ssoJJe*g durnd ol utalo;d eql esnec uJn] ur sa8ueqc puou"ruJoJuoJ eseql 'sluauodruoJ eql Jo elels xopeJ aql Surralp dq uoperuJoJuoc uralord ur sa8ueqc orJalsolle ppuanbas saltrp uodsueJl uoJlcele luql f.ya4l sruaas ll 'xeldtuoc eseprxo aruorqco/o eql pup xaldruoc aseuaSorpdqap HCIVN eql qtoq roC 'asraleJ ur Suruunr aseq] -u,{s 41y aqr dq paz,{lorpdq sr dJV ueqm padrund sr +H se }sn[ '+H durnd osp uec let{l suoppurJoJuoJuta}ord ur sa8ueqc JrJe}sollesesneoilodsuerl uoJlcelg apb O at4lseumou)sr'loodauoulnbaql olull>eq pa;t{taralesuoJl)ale aqlJoaruosqltqMur'ssa)ord srql'{6rauaxoparJoe6eio}saq}sa)ueque uraloldeql leql xe;dr.uot qbnotqtsuotl)eai laJsueJt uolordpueuorl)ala auournbrqn Iq paleuop I1;erlrur Joqledxalduorp MolloJ suoJl)ale aql'suojl)ala .lojsalrslrxapuearuerlua eql qtlM6uo;e'sraurer uoll)elelleqt (B)'slauouoru o/vrlaql ssol)esutolordaarqlaseqlJouorl)ejalureql(V)'@F)nd) lalue)lnjlns-uolrue 6urureluol tl auorqroy(r'(uaat61 utalolda>1salg aq] pue'(aryqS q auo.rqrol{r:au{zuaaqlJoalo) leuotl)unJ aql uloj sulaloidpalololaarql aql 'sleuJ ureuruI saln)alouuraloidlueia;;!pt I jo pasod r.uorstleuououl uollep-oo0'otz aql'iaulp e sruratoldslql.l)-q eruorqrog(r;oernl)ntls)!u,toleaql O€-91arn6tg (8)

N)

{

-q au ( z H O )a u o u r n b r q n uror+ut suojpele

XIU-tVIA

l)vds ] NVUSAI]IAU]INI

slseldoro;q1pup eupuoq)oltW :uotsrenuol {6.rau3:y 1 laldeql

9€g

uoBpzlFln HCIVN Jo erer eqr o] uoucnpord ttcvtt Jo salpr aql lsnfpe ol lcp AJV pranas Jo uoqlqqul {JuqpeeJ Sutpnlcur 'sloJ}uoo JulIruIS dq saur,{.zuazi.a>1 'uJnl uI 'luatper8 lrodsuer] uoJlJala Jo eler eql seJuequa S,rIIeJ aq1 .{prder aJoru }ueIpErB uolord leolrueqooJtJela aql saledtsstp,{.qereqt pue xrJlpru eql olq MolJ *H eroru slel aur,{zua aq} 'dn speads }l sV 'asueJoul Id pup dCIV saleJlsqns slr Jo suolleJluecuoJ eq] se JelsBJslrortr 'aldruexe JoJ 'aueJq -Iualu IerJpuoqcol[u Jeuur aql ut aseq]ur{s dJV eqJ'll?J o} olleJ eq} sasnBcdIV 'olluJ dCIV:dJV eql o1 pelsnlpe Jo uouezlpn peseercur ue Jeleueq ^ Sursearcur are sassacordeseqlJo salpr aql'lrodsueD uoJlcala pue 'a1cdcpIJP cIJlIs eql IIEJo 'umoplperq prce 'srsr(1ocd13 seler eql eteulprooc ]Bql slorluoo fi1e; {JBqpeeJ Jo aleJoqele ue ;o ped auo lsnl sI loJtuoc ,fuo]ertdsag ;o rualsds 3u11co1ra1ur '(6I-?I arn8tg eas) eseql -u,(s41y aqt;o dltpuorlcarlp eql slraJJPll se lsnf 'uodsuer] uorlsele Jo uol]Jarlp eq] pue aler eql qloq slJeJJeluatper8 uotord Iuclluaqcorpele eql Jo apnlru8eu uoJloele ro; a8uuqc r{8raua-aa4 eq} pue Sutdrund uolord eq] 'sl lpql-lrodsue4 paluq-godsupJl-uoJlJela ro; a8uuqc r{3raua-aar;eql uaamlaq eoueleq alduns e s]ragal loJluor,{rolerrdsar ieql slsaSSnsuollu^Jesqo slqJ'uleqf,,{roleldsar aql Jo suorloeseulos uI pelJatep equet mo4[uoulala asJanale pue ,(lalalduroc sdols 'aupJqtuetu peleaJs r(leluauruadxa Jeuu eq] ssoJoe uodsuen uoJlcala IpuJou 'smols ssec sr luarper8 uolord Ief,IluaqcoJlcala a3re1dletcgqre uE JI SeAoeJoIN

'(ZZ-€ern6H 'ses€aJculluarper8 aql '.]lnorJJrp -ord uoJlsele pue seluof,aq eql aJoru uodsuer] aas)tuaLuelU e 6uo;euorlrarrpe;6urs 'luatper8 aql sV'elEJ lerur-xpuraq] lE pe{oaqJun unJ o} earJs1}rodsuerl uoJlJele e urllem upl leql uralolde eleai) ot sasdellocraldnocun uB uar{ \'uodsuerl uorloele Jo e}EJeql uo luarpurS uolord palrnDarare suorleuJoJuo)oalql lseal pcrueqcorlf,ola aq] Jo eouengul fuotlqlqul lJarlp e eI 1f,8o1lq8noqt sI Ionuoc le leql uosealouresaql JoJ'ssa)old 'IorluoJ frolu4dsar;o acuelsxa aq] slsegal leqr a>ptdn uaSr{xouI aseeJcul 6urdund leuol)a^ e alear) o1pa.rrnbe.r aql are suorleujoJuol aolql l)utlstp lseel le ue ur Su4psar ',{llenuelsqns podsuuJ} uoJlsele Jo alsJ JIaq} aseeJsul o} EIJp 'sase) u1'srsIlorp,(q ruor; pa^uap -uoqJolru sasneJ slleo ol louaqdorltulp se qcns raldnocun uB Jo uoulppe eqJ dIV lle srIblaua aql'sdund uolotd laqlo 1a,( rol '(€E-0t arn613aes)lq6q1,(q paprnold sr16rauasrqlursdopoqtoualteq ro] searaqM'Uodsuetl uoll)ala {q paprnords! f ro}r pue o f ,(1;errtabraua sr+Heql sasealar a (o leLlllr uorleuiroJuo)o] I uorleLuroluo) ( M o l+ q 6 r q ) urorjuorlrsueJl aql'auerquau {6raua + HU O I aql ro aprslnooql uo +H e aseolorol ll l_tNHlv 6ursner'*;1.ro1{1rur;ervrole sequrelord o Ntdouc aqt') uorteuroluo)ul 'auPrquraur aql Jo aprsuraql uo +H e dn >prdol lr 6ursner '*11 q61q to;,{tlulge e seq utatotd aq}'V q6lq) ( * H r o +^ l l u t + + e 3 uorleuroluo) u; 'sar6reue]uaroJJ!p aneq a V NOI1VIAUOJNO) suorleuro;uo)urelordesaql'6uoeds /V lp)rtro^raql {q palerrpursV'l pue'g :suorleuroJuo)oarLllJo alrb e q6norql uanupsruratordaq1'ursdopoqroualreq 'dund uolord rglo,{re>ord uanrrp-tqbr; eqt,{q se lla/1/\ se'aseprxo auorqroy(r I p6yp ,(q pue eseua6o.rp,(qap asealaluololo pasneq ol lq6noql aretpql sulsrupq)au "H uo paspqsrurelordauerquuausuerl e {q (*H ro+ ^]lulrle /'^ol) 6urdr"und +HroJlapow srql'6urdtund*g f Notivt^UolNo) ro, lapou ;eraua6y 1g-91arn6;1

u!eq)eqr ,(lleuloNlolluo) ftoter!dsau qOnorqlrv\olluotpelf su!etlsou

I

I

f

f

f

I

sdt/!ndNoroudullHl-cNVsNlvH)ruodsNVur-Noul-flll

r€8

{8raua alqesn aql Jo aruosJo ssol qtr^ tnq-d11carrp pue l.lprder aroru ureqc lrodsueJl uorlJele aql Jelue ol ureql Surlqeua 'aueJquraurrauur prrpuoqJ -o]ru passed eqt eceJrns ratno dllcarrp aqt o1 are aleqdsoqd-g ep,(qappra Jo -c[p ruor; palrrep suor]Jele dSraua-q8rqaqt araH 'srsdloc[18JrqoJeEdq alVJo 'alcsntu slunorue a3re1,r(lercadsa acnpord sp qJns 'sllac pazlerf, leql lq3lg lcasur -ads aruos ur passedl.q sr rualsds elllnqs pellpo-os srql 'Sur8ruqJal JoJ loso/.c eql 01 pauJnlal are ^,(aqlqclqm JaUE 'iHCVN lerJpuoqJolru ruJoJ ol +(IVN ol suoJ]3alarraql JeJsuBrlselnJalou Jallelus asaq] 'xrJleru eql ur esuo 'auEJqluel'u IerJpuoqf,olrruJeuur aql q8norql aloru uer leqt Iosoilc eql ur se1ncalotuJellerus ol HOVN eql ruo4 passedaroJeJaqleru suoJlJalaaqJ'HOVN o1 alqeauradun sr eupJquau Jauur lurrpuoqJolnu eql lng 'uorJpuoqJollru eq] eprsur uor1e1.,fuoqd -soqd a,rqepxo pasn elrJp dlaq o1 aq f1enluana llrzltsuoJloale HCIVN eqJ 'aJaqMeurossuoJ] -cale HCVN d8raua-q8rq aq] Surrra;sue4 [q paleraua8ar aq o] speeu +CVN ag] pue 'ssecordaqt ul ol peue^uoc sl slLll'QL-Z arn8rg aas)a1eracd13 HCVN +CIVN -oqdsoqdsrq-t'I ol aleqdsoqd-g ap,{qapprac,r(13 slrazruocteql srs[1oc.,(13 ur uo1] -JeaJIeJluaJ eql JoJ*qy111roldacJe uorlcele aql ,(1ddns]uelsuoJ B peau sllaJ lo 'loso1dc aql ur Ipuualod xopar aqt Surragnq JoJIBf,rtrJJosle eJEerJpuoqf,olrt\ 'sauerqrueu ^aauplnq leqt slorets pue sprre fi1eg aq1lo uopcnpord aq] Supera -laJf,E 'eupuorlJolrtu pagodxa q ruor; ateJlrJ yosoltc pacnpord are yo3 uror; aql 'qeu8rs qwor8 o] asuodsar s,llef, E 'aldruexa 1&ace yo stunorue a8rel ;o ged se roJ 'snql'sasaqtu,{.sorqroJ suotelels uoqrpo pue HdCIVN q}oq eJnpord o} pazlo -qeleu sl ]r alaqm'1oso{c eq} ol luerppr8 pcnuaqroJ}f,ele s}rtrmop papodsue4 sr a1c,{opne f,rJlrJ aql ,(q xrleur lprJpuoqco}ru eql ul pacnpord e}Er}rossacxa 'asod -rnd sqt rog 'qp,ror8 roJ papaau HdCIVNpup suolelals uoqrpr qloq ateraua8 IIaJ osle errpuoqrotru '{elqe1re^p sr dJVJo fiua1d pue 'tuepunqe are sJJn]spooJaraqm suorlrpuoJ Japun ]ng '(srs[1oc,(p o] anr]EuJe]leue 'te.uq]ed aleqdsoqd aso] -uad aqr) sre8ns;o u ^oplparq aq] roJ,{u,,nqiedaprse [q losog.c aq] ur pacnpord sI HdCIVN er{} sEaJaqM'sreSns Jo umopIeaJq aql uro4 ,{1tcar1peruoJ suo}ele{s uoqreJ pepaeu aq] teq] alels srseqlu,{sorq;oadfi srql;o suoqdrrosaptsoni 'suo} -ala{s uoqrEs puB HdcvN Jo Jo urroJ aq} ur ra,nod Surcnpar;o.{1ddnsluelsuoc e sarrnbarloso{c eq} ur srseqludsorqaql tarraivroqt1 ol uourppe uy '(7 raldeq3 ur pessnJsrp) ptemol JepJosrp Sursearlur Sunup s,{emp sl asJelrun B ur sap leql -r1uapazrue8rodlq8rq sB se^lasueqt urelureru o] peeu sllec tpql dJV eq] Surcnp -ord ur erJpuoqJolnu aloJ aql azrseqdruaa^vr'raldeqc slqt lnoq8norql Jo IBlrlrJJ 'sasaqlu^,(sorq.{q paceldal eq tsnru fecap ]uqt selnJeloru asoql '8uu,ror8 ]ou aJE]eqt srusrue8Jopue sllal roJ uang 'sa8eIIaJ aql sE luaueJeldar ro rredar aJpqJrqm 'salncelouroJJeu Jo pasodruoc ,{1a8re1 are slla3 Jo paeu ur ,{11ue}suoc

usrloqelawlla) u! saloule)rlu) (uery seHuoupuor,{)ollw eql .ploc aq] ruor; sarqeq ueunq uJoqmeu Jo spaJeallllsuas lca}ord ol pup spurue Suueu -Jaqlq aArAaJoJ Surdlaq ,,'sped3urleaq,, se alres IEJ ulAoJq Surureluoc sanssrl 6;y ue{t }Eaq eroru acnpord pue eteJ prder e }e seJolstpJ Jraq} azrpuo o} slleo aql Sursnec'parrnbar sr uorleraua8 leeq uarlm uo paqJlr ^s sr uralord Surldnocun srql 'aseqluds dJV q8norqt Surssed tnoqtp^ luarper8 leJrrueqoor]f,elerraq] umop eloru ol suolord smolle leql 'uraloJd 8u[dnocun ue se umou>1,uralord lrodsuer] prcads e ureluof, slleJ esaql ur ErrpuoqJollur a8rel eql Jo saueJqruau rauur eqJ aJV olul paualuoo Suraqueq] reqter ]paq se paledrssrpsr uorluprxoJo d8raua aqt Jo ]soru 'sllac ]eJ umoJq se u,vrou>I,sllac asaq] u1 .srseq1u.{sdIV ruoq paldnocun .,tlerutou sr uorlerrdsal IprJpuoqJollru ,sllal ]eJ pezlprJads aruos u1

seurq)ew 6ulleteueg-leoH olur lel u/v\olgut eupuotl)ollwaql lJa^uo)sJaldnolunlernleN 'lsal Jo porrad e Surrnp ueq] esrJJaxesnonuans Jo pouad e Surrnp dprdeJ aloru seu4l 0I-g sre8nspuu steJsazlpxo dpoq aq] 'srusru 'uo os pue ,ureqc r(rolerrdsaraql dq -BqJaru loJluoc aldqpu asaq] Jo llnsal e sV slse;dorolql pue eUpuot{)ol!W:uolsJanuol {6leu3 :y1 reldeql

8€8

aurloJo

sNo|ltcNof f t8ou]vNv (8)

auelquleur etriselo leuapeq a l e ur ) ) n 5

e ur s A l

htsv'ldot-,,('f

rd * dQv aseqlu^sdIv u t e q )f u o l e i t d s a J

sNorrcNo))r8oulv (v)

'(V)urureq)fuotertdsal uoloJd asoqlsa^upteqltualpPJO eql uo puadap sassa>o.td l.rodsuetl 1eq1 uaqlaseqtu^s ueqsrlqelsa ol dfv srqlJoauossez,{;orp,(q lplruaq)ort)ale rapun )lqolaeue tuot;dIVs}lsa^llap suolllpuo) 61yeql'srs,{;or(16 aq1(B)'dfvaleu ol puella)aqlotu!slualrlnu 6urmor6 ulnuol)eq aurps auost.todsuerl ot qloqpasnuaqlsrluatpel6s!qliauelquaueLuseld ureq> {ioleltdsa.t uesa>npotd aLllssoJ)p uolordlel!uaq)oll)ala luatpetb '+eN p'runrrel)eq pue aq] olul slualllnu (v) sladxa ul )rqoree uP lla) a)loja^lloul-uolold sdundauerqueuer,use;d aqlssol)epalelauab V 'eual)equrlrodsuerlua^lrp-+H,o aq1g5-tr1etn6t3 a>uellodtul '(tt-tl 'lsBrluoc,{q 's11ac ernBIC) reuodur^,(slIJIJeds e q8norq} suo} IEruIue uI -ord aJoruJo auo qllm Suop IIao eq] olul peSSeJpsl lualJlnu qcee :sJEBns,{ueru pue sprcBoururP se qcns /slualJlnu Jo uodsuBJl pJeMuI eAIlJeeql JoJpasn lsour osp sr luarper8 srql 'sllef,crlo^,fuP3ne Jo drund *y-*eI\I eq] Jo eJeld eql sa{el }eql Jeuodrtup +H-+ENp eIA runlJelJeq aql Jo ]no +PNdrund ol pesn sI ]l pue tololu Jelle8eg e elrJp ol passeuJuq aq ueJ ]I 'eueJquau Pruseld Jlaql ssoJf,Eluelp 'euel3eq lsolu 'snql 'saqoJaeue -er8 aq] Surpnlcut uolord e urpluEru ]JIJls '(7 raldeq3 uI passnoslp) sassaooJduoueluaural ,r{qpaleraua8 st ssacord slq] JoJ pesn dJV eql'aueJq -luetu etusEld aql ssorceluatper8 uolord E qsllqelse PuB *H durnd ol sIS,{loJdlS dq pacnpord dIV aql Sursn 'esreneruI s{Jo^ esel{}u^s dJV aqr 'elJe}ceqJeq}o uI aIV a{pru ol eseq}u^s dJV aq} selrJp }Eql eueJqluau Eurseld aql ssoJJPaoJoJ anrloru-uolord e saqsllqelsa .,(qaraq]pue ilac aql Jo lno +H sdund UIEI{Juod -sueD-uorlJala aql }saAJErlo} uleqJ-uodsueJl-uoJ}3aleuE asn lBq} BIJa} "{8raua -f,Pqq 'PrJpuoqsolrru AJaAsI ]eq] esEqlu^sdJV uE sulPluoJ ur auo eq] 01JBIIIUIS '^lIsJe^Ipslql elldsec erreloeqJo ^,(1rro[eru lse^ eq] Jo euerqueu eurseld aql 'sJoldeoJeasaql ol suoJlceleeql JaJsuEJ]suIPqJdJol -errdsal lErJpuoqJolru uI asoql ol elqEJEduroJere leql euEJqluarueruseld aqt ur sJerrJEJuoJ]f,alaJo sarJasv'aldurexa JoJ'(aleuoqJPJJOelEJelun;)punoduoc uoqJPJE ro '(elIJInsJo alPJIns)punoduoo JnJInsE '(elullu Jo elEJlIu) punoduoJ 'roldacce uoJlsele puIJ eql uaSouru p aq uec Joldef,JeuoJlJale aAIleuJaUEaq; se ua3.4xoueq] reqlo alnf,alolu e s.,(oldrualEqt uleqf, UodsuErl-uoJlcale uB Iuoq ruory raq]ta .{8raue JIaq} Sunuap 'saqo Jo (uorleluarurag l(q1 auop srsr(1oc[18 -JaeuelJrJls aJesJeqlo 'aueJqrualulPlrpuoqcolllu rauul eql uI auo eql o] JBIIIUIS 'all^c pIJe clnls eq} sr leql auerqruaru eurseld Jraql ul umqc.{rolerldsar e pue 'srsLloc,r(13 .{q OzH pue zOJ o] azlpxo daq] sreSnsluo4 dIV ezsaqlurts ^aqt lJlqo -JeEeJE'slleole(urue a{q 'etuos 'seJrnos,{8rauaasrantprtlsnolrlJouaesn elJa}3eg

(6raug )llol,usoluaq)lloldxl oslv ellat)e8 ssaureH ol sr,uslueq)aw ' Qt-Vt arnt;g) ruslloqelelu relnlleJ Suturuluteru 'suoFlpuof, ]ueJeJJIpJepun ur suorlcunJ Iecrlrrc ,(ueu seq uolrpuoqJollu aql suorlrear luaraJJrplno Sul1trrec,{.q'snql 'uopcnpord dIV roJ HCVN acnpord o1 'splJBoullue pazrpxo pue erJpuoqJollluolul palrodrut erBsplle oulure aql pue o] u,{/\opua{oJq aJBselpoq Jno uI sule}oJd 'uol}e^Jels Jo suonlpuoc Jepun

'uorlpl^roqdsoLld paa;ol spne anrleprxo ,(lreJpue alennrld sazrprxo leqt a)eurnJ lle) oqt se eupuoq)olr.uuo srseqdua .rno;o(:enbepeuraqt azrseqdua'alaq palerlsnllr asoqtse qfns'eupuoq)o1rtu {q ]no poure)suorllPar)rloqelaurlerluassa {uetu aq1 'uo;}rnpo.rddIV saplsoq us!loqPtau lla) u! Pupuoq)ot!ur Jo salor;err1u1gg-71 atnbrg

r a m o d6 u t : n p a l ssa)xa lelrpuoq)o]rr.lr 6 u r s ns r s a q l u [ s o rrqo 1 ( H d C V Ns e )r a m o d 6 u t : n p e r q l l M l o s o l ^ lI l d d n s NOTUCNOH)OIy! s l o r a l sp u e s p r : e4 1 1 e 1 ; o s t s a q l u [ sr o ] a ] e r ] r ) ssa)xaqtrnnlosoy(: ,ilddns

uotpnpo.td 61y len+ol spr)eou||.ueosn sNotltcNo) Nol-LV uvrs

ss3)xl Jo SNorlrcNo)

]osor)

ruodsNvui-Noul-fll3 sdwndNoloudullHrcNVSNlvH)

6€8

p apnlrur slrnpord eq]'stueld uI 'salnJaloru dlensn) reBns1q8ra,r,r-JulnJelou-Mol crue8ro ^,(ueuracnpord ol AIV pue HdCIVN 'srsaqlufso]oqd 1o slcnpord elErpeuur aql osn sfiec cr]eqlu,(so1oq4 'srnoq tqSlldEp aql SuFnp ssaq]udso] -oqd urro;rad slseldoroyq3 'lse1doro1qJ aqt-efieue8ro replacerlur pazqercads e ur sJnf,Josrsaqtu,{so}oqdta1e1qcnu padole^ap qcrqm'ae31epue s1ue1du1 'stuJoJaJrTcrqoJeB luepunqp ;o luarudola^ap aql alqrssod apeur [1en1uele erJelopq crlaq]u.{sol -oqd a,rrlrurrrd aroru tuo4 erJatJeqouefc;o uonnlole eql lpq] rq8noqr sr ]r ,uoll -ces srq] ur aes e.,lt, sy'uoqeldroqdsoqd elrleprxo ro; parmbar ua8dxo aql araqds -oulB eql olul alBJeqIIosp ,r(aql'lzou +'(OzHf,) \ffizoJu + gz11u uorl3par 3upl11ds;o asrnoc aql uI 'uo1ruxgfuoqJDJ palpc ssacord IIEraAoeqt url ra1e,r,r e-spunoduroc crue8ro olul zOC crraqdsourle ueluoJ ol lq8rTuns ;o ,iSraua eql pue JaIE,rrruorJ suoJloela asn Laql 'sluaurarrnbeJ luarJlnu Ipururru eleq qolq,t,r'eualJeqouefc aql aJe errelJeq cqaqlu.{soloqd pacuulpe lsoru aqJ 'errepPq cqaqlurtsoloqd yo sadfi Lueru Surpnlcur 'stusrua&to Jlpqru/tsotottd [q, pacnpord ueeq eAEq s11ac3uur11dq parrnber slerJaleu crue8ro aq] Jo IIEfpeeu 'eruu lpql acurg 'o8e sreadgo suollrq dn pasn ara,,vr spunoduroc purSrro eseqt Jo lsou ]eq] rEelc sr 1l lnq 'sessacordpcruraqc -oa3.{q pacnpord spunodruoc cruu8ro aq}Jo aJuepunqu u€ ol sselte peq queg arrrlnurrdeql uo srusruu8rolsJrJeql 1eq1L1a1uq lI 'sessecordrulnlac selrJp teql d8raua Jrloqpleu eql pup srsaq1u,,{.sorq roJ suolela{s uoqJec eql qloq apnord spunoduoc aseql ']ueruuoJrlua Jraq] ruoq spunodruoc crueBro Jo sluno{ue a8rel ;o a1e1dn pnuuuoJ eq] uo .,(yarsrusueSlooJJlru lsotu pue slpturue 1y

SIS]HI-NASOIOHd CNV SISVIdOHOI Hf 'snal m srasruoqJau JrjowsorwaLp acua1todrur lo IDJjuaJatq ol saLfilsalsa7.ottocotd puo 'slsn1dotolllJ'Drrpuotlooitla w asaqtu[s dJV to acuasatd,lvsranrunaqJ 'xruaa aql otut ycoq smoll +H qJlqm qBnotql 'asnqruts dJV ar.lt[q aw ayaw 01 passauJoLl 'luatpvt? s1 s1t41 uojotd uD satnan nr1Dw aql lo mo *g lo tuatput8 luxrwaqJoJjJala Sutdwnd aqr o1suotpala yodsuau a1qatonnt [11ncljaBtaua at4llo Suqdnoc aq1 to '6291ua&[xotqlncapw e;;ore srxa;duuot ralua) uorpeal euualueeqf .ualsIsoloqd e ur relua) uotl)eoJ ;er;utaq>o1oqd pue xe;duroteuuatueaq1 gg-gg a.rn6rg

auournb

r o l u a ) u o r p e o Jl e ) r u a q ) o l o q o u r s a l n ) e l o L1 u; { q d o : o 1 q r , t ol l e o l e r ) e o s

xelduror P U U e l UU er saln)alou-r

vtAoul_s euerquraur ptolel,iql

JJVO)

cto)vr H-r.

xe;du.roreuualue

v a l n ) a l o u ip a z r p r x o uorpale 16:aua-nno; 6 u r f u J eV ) aln)aloul

'anlgur uMoqs spuoqoLltle^o paztle)olep aresuort)ell '(77-y1atn61aas)auJaqut uolt sputqteql 6uu urr(qd.rod aql ol pelelarsrq>rqm'6urr uu{qdrode ut plaq sl uroleunrsaubeuy ';;{qdorolq> arnl)nrls aql Zt-t I orn6rl 1o €Hf

€Hl

\,/ HJ

zHl zH)

'ui ,s:- i'r .sl 'Hi u o r 6 a rt r e r rrqoqdorp,iq

- | "Hl I f _ .. H lH zHl ._Hl J

,l

"Hf

l f_l "" H

*i '"Hl l l t_ H l

lfHl ',H l - f . . ,?/? J\ /J\' , , \ \lr/ H_l

/ )-N \ ,/

'HJ-'Hl-f

l-N

\/, I

ll f

o

H N: l

//

N-l t\

tt/

J

I

tHf

f-ltH

\// -) I H]

H

'surpqJ uodsueJt uoJtJala olul peJaJEleqt suoJlcalalSraua-q8rq pateJeue8 -lq8u Jo uorlJnpoJd aqt ur ]1nsardaqt pue 'elaldruoc o] atup eJoru aJrnbal eseqJ 'suouJeal ]uanbasqns roJ peuolltsod ,{1qelrns,{qa.raqtst uoJ}Jalaaqf 'alqp}s alou qJnru sl 1l eJeqM lueruuoJr^ua uB ol lr sJaJsuEJlJalual uorltpal IEtrrueqJ -otoqd aql 's[^qdorolq] eqt tuo4,{eme z(prder uorlcela,riSraua-qBrqeql Surloru [5,'ftV-at arnS;g) xalduoc ura]oJd aql ur sroldaJJe uoJltala Jo ureqc Suuoq -q8rau B ol passed.{lalerparuurrsr uoJlJala pe}rcxeaql asneJaqe}uenb uoqe}rJxe ro; de4 alqrsJa^arrruB sEslJe Jalual uorl3EeJaql ur salnJalourldqdorolqoJo JrEd 'erJel3EqJrtaqludsoloqd aarlllurrd ur oBBsree^ uollrq IerJadsaqJ t ueqt aJoru paAIoAaeABq ol lq3noq] sl ll 'slseqlu[soloqd Jo ilpeq ag] ]B sell tpql xelduoJ ]uau8rd-uralord auerqruatusueJl p sr Jalual uollceeJ lecluaqJoloqd ar{t 'GV-il arnS;g) pesn aq uec lr ereqm ,tlanrlcaga 'leuunJ p se s}Je dqaraqt alIS f,rJrcadse ot lr SurlcaJrppue,{8raua }q8q 3ur1ca11oJ xaldruoc Buuatue qJBg 'relueJ uorlJeal lecrrueqJotoqd aqt ur salncalou ldqd -oJolqc yo rrcd proads B seqceal ]l Ipun JeJsueJtd8raua ecueuosal dq raqloue ol e1nreloru auo tuo4 paJraJsuer].{prder sr .{8Jeueaql 'xaldruoJ euuelue aq} ur alncalotu 1,{qdorolqc B setrJxe1q311 uaq11 'sqlSualalem raq}o Jo IqBII toalloc dlaq uec pue uorlpprxo ruo4 s11,,{qdorolqceql }catord qrlqa '.sprcuarcrar pelle) 'auerquau pro{el sluaur8rd .[rossacce sure]uoJ osp xalduoJ euue]ue aqJ -,{qt aqr ut Llasroardruaq} 3ur}ualJo 'Ja}ual uortceeJJed sa1ncalou ldqdorolqc parpunq lere^as pulq suralord asaql teqleSol 1(saxaldurocSuqsanreq-]q8ll sE umool) saxaldurocuraloJd aueJquraru]cuDSIpJo Jaqrunu E Jo slsrsuoJ 11siseld -oJolqo u1 'd8raua lq8q SurrrydeJ roJ lupuodrur sr xalduroc Buueluu aqJ 'd8raua leJruaqJ olur peuenuoc aq ol dSraua]qBII elqeua leql selnJaloru 1.{qdorolqJ pue sura}oJd ;o xaldruoc e Jo SurlsrsuoJ ,Jeluac uorlJeal IeJrueqJoloqd e pue :JetuJJ uortJeer eql ol ll paal pue d8raua 1q8r1 arnldec teql selnoaloru luau8rd;o 1asa8rel E o] punoq suralord;o Suusrsuoc 'xaldruoc Euuelup uE :slueuodruoo paTurl flasolc oml Jo slsrsuoJ uelsfsoloqd 'surroJ V InJasn ot selnralou flqdorolqc palrJxe ur parnldec d8reua ]qBI aql Jo uorsJanuocaqt az,,(p1eJsruels.[soloqd palpc saxalduroc uralordqlnru a8rel

uals^soloqd e urol xelduro) euualuv ue snld raluo] uoll)eau Iellutaq)oroqdv

f-H

'1.ronn.,taql ^\oq eqltJsap ^lou ileqs a6 .alqrssodqueS uo aJll Jql 'UaJuoJ ur SurlJe 'saxalduroc ureloJd Jo sed& o,t,r]aseql .Jalual Jo lsoru eletu uoltcuar e t\qraJsuerl uortrela .,i3raua-q8rqpue xa1dtuocTtuuatua lauuaqrorcqd ue ^q JaJSUBJ1 .,{8raua acupuosar :saxelduroc uralord tuaJaJJrp o.,r,q,r(q pa1e1 -q1cegdpear8 eJesusrueqJaru oml lsel eq] ,srsaqlufso]oqd;o ssacord aql uI

zui

slseldoro;q1pue eUpuoq)ol!W :uotsrenuo3l6lau3 :71 laldeql

8r8

, , t . : : ,,,.,':.,.:, f O U

CHLOROPLASTS AND PHOTOSYNTH ESIS

Figure 14-44The arrangementof the electron carriersin the photochemical reactioncenter of a purple bacterium. shown

The pigmentmolecules areheldin the interiorof a proteinand are transmembrane surroundedby the lipid bilayerof the bacterialplasmamembrane.An electron in the specialpairof chlorophyll moleculesis excitedby resonancefrom and the an antennacomplexchlorophyll, excitedelectronis then transferred stepwisefrom the specialpairto the quinone(seealsoFigure14-45).A similar arrangementof electroncarriersis presentin the reactioncentersof plants (seeFigure14-47).

o o

E o E a

E o o o

€ o o

CYTOSOL

In a ReactionCenter,Light EnergyCapturedby Chlorophyll Createsa StrongElectronDonorfrom a WeakOne The electron transfers involved in the photochemical reactions just described have been analyzed extensively by rapid spectroscopic methods. Figure l4-45 illustrates, in a general way, how light provides the energy needed to transfer an electron from a weak electron donor (a molecule with a strong affinity for electrons) to a molecule that is a strong electron donor in its reduced form (a chargeseparation chlorophyll oxidized

chlorophyll

acceptor reduced

electrondonor oxidized

acceptor reduced

reactioncenter c h l o r o p h y l(l s p e c i apl a i r ) in restingstate

low-energy electron

high-energy electron

o-:-

WATER

OF DONATION H I G H - E N E R GEYL E C T R O N TO ELECTRON. T R A N S P O RCTH A I N

(B)

Figure l4-45 How light energy is harvestedby a reactioncenter chlorophyll molecule.(A)The initialeventsin a reaction centercreatea chargeseparation.A pigment-proteincomplexholdsa chlorophyllmoleculeof the specialpair (blue) preciselypositionedso that both a potentiallow-energyelectrondonor (orange)and a potentialhigh-energyelectron When light energizesan electronin the chlorophyllmolecule(redelectron),the acceptor(green)areimmediatelyavailable. excitedelectronis immediatelypassedto the electronacceptorand is therebypartiallystabilized.The positivelycharged chlorophyllmoleculethen quicklyattractsthe low-energyelectronfrom the electrondonor and returnsto its restingstate, Thesereactionsrequirelessthan 10-6 electron. that furtherstabilizeithe high-energy creatinga largerchargeseparation (A),the photosyntheticreactioncenter in the steps (B) process, follows which ln final stage of this secondto complete. the the high-energy is restoredto its originalrestingstateby acquiringa new low-energyelectronand then transferring the subsequently, As will be discussed electronderivedfrom chlorophyllto an electrontransportchainin the membrane. ultimatesourceof low-energyelectronsfor photosystemll in the chloroplastis water;as a result,light produces high-energyelectronsin the thylakoidmembranefrom low-energyelectronsin water.

850

Chapter14:EnergyConversion: Mitochondriaand Chloroplasts

molecule with a weak affinity for electrons). The special pair of chlorophyll molecules in the reaction center is poised to pass each excited electron to a precisely positioned neighboring molecule in the same protein complex (an electron acceptor).The chlorophyll molecule that loses an electron becomes positively charged,but it rapidly regains an electron from an adjacent electron donor to return to its unexcited, uncharged state (Figure r4-4s\, orange electron). Then, in slower reactions, the electron donor has its missing electron replaced, and the high-energy electron that was generated by the excited chlorophyll is transferred to the electron-transport chain (Figure l4-45B). The excitation energy in chlorophyll that would normally be releasedas fluorescence or heat is thereby used instead to create a strong electron donor (a molecule carrying a high-energy electron) where none had been before. The photosystem of purple bacteria is somewhat simpler than the evolutionarily related photosystems in chloroplasts, and it has served as a good model for working out reaction details. The reaction center in this photosystem is a large protein-pigment complex that can be solubilized with detergent and purified in active form. In a major triumph of structure analysis,its complete threedimensional structure was determined by x-ray crystallography (see Figure 10-34). This structure, combined with kinetic data, provides the best picture we have of the initial electron-transfer reactions that occur during photosynthesis. Figure l4-46 shows the actual sequenceofelectron transfers that take place, for comparison with Figure 14-45A. In the purple bacterium, the electron used to fill the electron-deficient hole created by the light-induced charge separation comes from a cyclic flow of electrons transferred through a c1'tochrome (see orange box in Figure 14-45); the strong electron donor produced is a quinone. one of the two photosltems in the chloroplasts of higher plants likewise produces a quinone carrying high-energy electrons. However, as we discuss next, becausewater provides the electrons for this photosystem, photosynthesis in plants-unlike that in purple bacteriareleaseslarge quantities of oxygen gas.

NoncyclicPhotophosphorylationProducesBoth NADPHand ATp Photosynthesis in plants and cyanobacteria produces both ATp and NADpH directly by a two-step process called noncyclic photophosphorylation. Because two photosystems-called photosystems I and ll-work in seriesto energize an electron to a high-enough energy state, the electron can be transferred all the Figure14-46 The electrontransfersthat occurin the photochemicalreactioncenterof a purple bacterium. A similarsetof reactions occursin the evolutionarily related photosystem ll in plants.At the top left is an orientatingdiagramshowingthe molecules that carryefectrons, whicharethosein Figure14-45,plusan exchangeable quinone(es) and a freelymobilequinone(Q)dissolvedin the lipid bilayer.Electroncarriers1-5 areeach bound in a specificpositionon a 596-amino-acid proteinformedfrom transmembrane two separate subunits(seeFigure10-34).Afterexcitationby a photonof light,a highenergyelectronpasses from pigmentmoleculeto pigmentmolecule, very rapidlycreating a stablechargeseparation, as shownin the sequenceof stepsA-C, in whichthe pigmenr moleculecarryinga high-energy electronis coloredred.StepsD and E then occur progressively. Aftera secondphotonhasrepeatedthis sequencewith a secondelectron, the exchangeable quinoneis released into the bilayercarryingtwo high-energy electrons. Thisquinonequicklylosesits chargeby pickingup two protons(seeFigurel4-24). L I GH T

l o w - e n e r g ye l e c t r o nf r o m a n

3 picoseconds ( 3 x 1 0 1 2s e c o n d )

tesozrqt5

ffi

I lso)

6peu

M qo)

M

zxo)

M

lxo)

r

n tlsol

n

su)

-Em tur

5,1sdL K tdte ffi tdte,.

iio;

9a1e

.

e4ueq)Jew

seuowou!l)aa

IN Z ISdJ

;im zldr IM LPEU

t*ffi gpeu M €peu

K fpeu

ffi Eu+c

[svvv urorj

ffi

u o l s s l u u aqdl l ^ 6 6 6 t ' L g v L - 9 L v L i E S z patdepv) nuaDs "le ra Ierg ' A'w LUorJ 'f xo).)l!unqns aseprxoaurorq)oil) , e pue '(qQ) g auorrl)ol^) 'Iut pue su) svNUleuosoquapo)uaasaLll:sauouo6 leupuoq)olluruMoul lle u! tuesard sauabrnoj Iluo arearaql'seuouab JobrelIq papo)ua ore leupuoq)o]il.r.r leql svNUleuiosoqllpue sulaloloeqlJo slasqnsapo)uasauoua6leupuotl)olrul xaldLuo)ssel'sauouableUpuoq)ol!ur ;o uosFedurol 6s-tl ainbrl

Voil

n IZF)

--/

sctlsvld cNVvluclNoHlorlwlo swlls^s )l1lNl9lHl-

t98

'uopnlo^a alerurrd ur sdals eq] se qons 'sluele d:euo4nlona lueoeJ d1a,u1e1ar Jo selep eq] Surlerurlsa JoJ InJesndyprcadsa sacuanbasVNO lerJpuoqJolru 3o uosrredruoc ? sa{eu saua8prrpuoqcotru puru€Jo uopnlo^aJo alur q8rq [1anpe1araql flalenbape pelsel uaaq ]ad tou seq srqr q8noqtle ,eupuoqco4u ur srs -aqlufs uralordgo &llapg aq] arnpeJ o1papadxa eq plnom,aldruexa roJ ,(sVNUJ '!I aql llol Jo azrs aql sprlqt-om] ueqt ssel) sVNUr eqt Jo azts lurus flpnsnun eqt pue sVNUt 77 tlluo 1o acuasardeqJ 'slleJ ur eJaq aesleasodrnd eruesaql JoJpasn asoql qlIM pareduroc aldurs dlalrlelar eJB sessaf,oJdasaqt urro;rad ler{l susru -eq3eru aqi ,(q^| umldxa ppoc sql 'spnpord aua8,u,a; [1anr1e1araq] yo euo 3ur -Seureplnoqtlv\ q8q,{1an11e1eJ eq uet seuosoqrr lerJpuoqootrurdq uralord olur 'asurarudlod ,uoq pelelsueJ] ro paqrrcsuerl .,(q trcdar dq peureluruur VruU VNO -ecqdar VNCI ,tq pardoc appoelonu red aler JoJJaaql ,eupuoqJolrru IIaJ lerulup ur suralord pue SVNUse passa.rdxapue palecrydal eq ol peau sepnoalcnu VNC 'qloq ro trcdar .uorlecrldar yl11q 009'gI lnoqe dpo asnef,eg VNO tuerorJJaur lerJpuoqcol[u Jo ,ilueprJ paJnpeJ B 01 enp aq o1,(1a41sr ef,ueJaJJrpsrql .aurouaS JEalJnueql ul ueql aruouaS eql ul ralearS sau4t 0I uaaq spq uoD leupuor.lf,ollru '(sa^IesJnoSurpnlcur) sprurus -nlo^e Surrnp uorlnlrlsqns eppoalJnu Jo ateJ aql elpJqaue^ ul 'leqt IeeAeJsrusruu8rolualeJJrpur sacuanbasVNCIJo suosrredruo3

uMou) suels^s )lleueg lselduts oql u!eluo) etrpuotl)oltw teutuv '[a] eqt,,(orlsap ,{qaraqlpuesuralorddueru;ouorlouryeq}rallpplnomaruoua8

a8rel e ur a8uuqc e r{Jns spaleqm 'alqpJalo} uopoc arer e;o Surueeru aq} ur a8ueqc lpuorseJJo ue sa{Bru auroua8 lprJpuoqoolrur aqr dq pepoJue suralordgo Jeqrunu 'r(lqerunsar4 'ErJpuoqJolrru ur apoJ cpaua8 eqt ur JnJf,o lerus dlpnsnun eql UBJ Urrp ruopuer B ]pql slsaSSnsuoBprJBAqons '(g-tl alqeJ aes) o1ttldosotq Jo srJpuoqJotlu eql ur aurJes JoJ sepos pue slErxtusru Jo ErJpuoqJolru eql ul .salpJq dols tol sepot ]r 1nq 'aurur8re ro; sapoc f.leurou CCV uopoJ eqt ,{1re1gu1g -auelur pue 'r8ury 'slelulueru ,eJeqm Jo erJpuoqJollu ur ueqdoldl-r1 sp pBaJ sr -asleuopor qJIqA\'Vgn dots B sr eqtJo apoo crlaua8 prepuels aql te1'snelf,nu IIeo ,sauoruou\IJaYueozolord eql ,69-?I uror; pe8ueqcun sr epoc cpaua8 er4l Jo ]ErIl arn8rg ur saua8 go Jequnu eql qll a uorJpuoqJolru aq] u1 'srusrueS.ro1ua lsa8rel -reJJIpur ]uereJrp sr apoJ cqaua8 [BrJpuoqJo]ru eqt teq] Burpug eql ruoq seruof, luq V i€rJpuoqJolnu dueru ur epoc cpaua8 eq] ul seJuaJaJJIp^aeJaql ureldxa a,\/\op 'uaq] ^\oH 'JolseJuE uoruruoc E ruoJI pa^Io^e e^eq slles IIe ]Eql eluep -rna Suorts sapnord srusrue8ro ur apoJ clliue8 ,t1,in1i-rr'"ro1, IIB 1o "ql "q1 '(t-7I elqur) saruoua8 Jaqlo ur suopoJ arues aql Jo asoqt ruo.ry ,,s8uruperu,,luaJaJJrp eAEqsuopoo tg eql Jo 7 :]ueraJrp sr epoo c4aua8 eq] tuql eluJrpu suralord SurpuodsarroJ eqt;o sacuanbasprce ourue eql puu sacuanbasaua8 prrp -uoqJolru;o suosrredruoc ,Sursrrdrns]soru sdeqra4 .apocu4aua? rurymA .t 'sa1ncalotu VNUI Je^ eJ qlpa s1saq1u.{s uralord slrrurad pue suopoc JnoJJo auo due qlpr rlud o] VNU1euo smolle 8urrrcd ,,€Jo tno 2,, qJnS 'uoylrsod (elqqo^ ) pJrql aqt ut sapuoalJnu rnoJ

apol ,,lesrJ^run,, aqt ulol] sial]tpapo) aqt teql aleltputs]letlpau*

VNV

law ilJ_

vn)

nal

D9V V9V

na'l

)as

6rv all

dot_s

v9n

*sapo) )llaue9 lellpuotl)ol!W PUPePo),,lesJa^lun,,aql uee/v\lagse)uala#t0 euos E-t I alqel slseldorolql pue eupuoq)oltW :uolsJanuolI6.rau3 :71 laideq3

298

THEGENETIC SYSTEMS OFMITOCHONDRIA ANDPLASTIDS

GenesContainIntrons SomeOrganelle The processing of precursor RNAs has an important role in the two mitochondrial systems studied in most detail-human and yeast. In human cells, both strands of the mitochondrial DNA are transcribed at the same rate from a single promoter region on each strand, producing two different giant RNA molecules, each containing a full-length copy of one DNA strand. Transcription is therefore The transcripts made on one strand are extensivelyprocompletely s).rynmetric. cessed by nuclease cleavage to yield the two rRNAs, most of the tRNAs, and about 10 poly-A-containing RNAs. In contrast, the transcript of the other strand is processed to produce only 8 tRNAs and I small poly-A-containing RNA; the remaining 90% of this transcript apparently contains no useful information (being complementary to coding sequences symthesizedon the other strand) and is degraded. The poly-A-containing RNAs are the mitochondrial mRNAs: although they lack a cap structure at their 5' end, they carry a poly-A tail at their 3' end that is added posttranscriptionally by a mitochondrial poly-A polymerase. Unlike human mitochondrial genes, some plant and fungal (including yeast) mitochondrial genes contain introns, which must be removed by RNA splicing. Introns also occur in some in plant chloroplast genes. Many of the introns in organelle genes consist of a family of related nucleotide sequences that are capable of splicing themselvesout of the RNA transcripts by RNA-mediated catalysis (discussed in Chapter 6), although proteins generally aid these self-splicing reactions. The presence of introns in organelle genes is surprising' as introns are not common in the genes of the bacteria whose ancestors are thought to have given rise to mitochondria and plant chloroplasts. In yeasts,the same mitochondrial gene may have an intron in one strain but not in another. Such "optional introns" seem to be able to move in and out of genomes like transposable elements. In contrast, introns in other yeast mitochondrial genes have also been found in a corresponding position in the mitochondria of Aspergillu.sand .ly'earospora,implying that they were inherited from a common ancestor of these three fungi. It is possible that these intron sequencesare of ancient origin-tracing back to a bacterial ancestor-and that, although they have been lost from many bacteria, they have been preferentially retained in some organelle genomes where RNA splicing is regulated to help control gene expression.

About Genomeof HigherPlantsContains TheChloroplast 120 Genes More than 20 chloroplast genomes have now been sequenced.The genomes of even distantly related plants (such as tobacco and liverwort) are highly similar' and even those of green algae are closely related (Figure 14-6f). Chloroplast genes are involved in four main tlpes of processes:transcription, translation, photosynthesis, and the biosynthesis of small molecules such as amino acids, fatty acids, and pigments. Plant chloroplast genes also encode at least 40 proteins whose functions are as yet unknor.tm. Paradoxically, all of the known proteins encoded in the chloroplast are part of larger protein complexes that also contain one or more subunits encoded in the nucleus. We discuss possible reasons for this paradox later. The genomes of chloroplasts and bacteria have striking similarities. The basic regulatory sequences,such as transcription promoters and terminators, are virtually identical in the two cases.The amino acid sequencesof the proteins encoded in chloroplasts are clearly recognizable as bacterial, and several clusters of geneswith related functions (such as those encoding ribosomal proteins) are organized in the same way in the genomes of chloroplasts, E coli and cyanobacteria. Further comparisons of large numbers of homologous nucleotide sequencesshould help clarify the exact evolutionary pathway from bacteria to chloroplasts, but some conclusions can already be drawn: 1. Chloroplasts in higher plants arose from photosynthetic bacteria.

863

864

Chapter14:EnergyConversion: Mitochondriaand Chloroplasts

-/R N Ag e n e s

Figure14-61The organizationofthe liverwort chloroplastgenome.The genomeorganization chloroplast is similarin all higherplants,althoughthe sizevariesfrom speciesto speciesdependingon how muchofthe DNA surrounding the genesencodingthe chloroplast's 165and 23SribosomalRNAs is presentin two copies.

-1.-

?_

total length of genome = 121,024nucleotidepairs

o,* KEY: t R N Ag e n e s

/:

ribosomalprotein genes

v

r i b u l o s eb i s p h o s p h a t e c a r b o x y l a s(el a r g e subunit)

photosystemI genes photosystemll genes ATPsynthasegenes

// ,P/ , .' /

lli rel

rrr I

t

I \I ut i tt tI , j iiilt l[I 2.

Many of the genes of the original bacterium are now present in the nuclear genome, where they have been integrated and are stably maintained. In higher plants, for example, two-thirds of the 60 or so chloroplast ribosomal proteins are encoded in the cell nucleus; these geneshave a clear bacterial ancestry, and the chloroplast ribosomes retain their original bacterial properties.

MitochondrialGenesAre Inheritedby a Non-Mendelian Mechanism Many experiments on the mechanisms of mitochondrial biogenesis have been performed vrtth saccharomyces cereuisiae(baker's yeast). There are several reasons for this preference. First, when grown on glucose, this yeast has an ability to live by glycolysis alone and can therefore survive with defective mitochondria that cannot perform oxidative phosphorylation. This makes it possible to grow cells with mutations in mitochondrial or nuclear DNA that interfere with mitochondrial function; such mutations are lethal in manv other eucaryotes.Second.

The abiliry to control the alternation between asexual and sexual reproduction in the laboratory greatly facilitates genetic analyses. Mutations in mitochondrial genes are not inherited in accordance with the Mendelian rules that govern the inheritance of nuclear genes.Therefore, long before the mitochondrial genome could be sequenced, genetic studies revealed which of the genes involved in yeast mitochondrial function are located in the nucleus and which in the mitochondria. An example of non-Mendelian (cytoplasmic) inheritance of mitochondrial genes in a haploid yeast cell is shornmin Eigure l4-.gz.In this example, we trace the inheritance of a mutant gene that makes mitochondrial protein synthesis resistant to chloramphenicol. \Mhen a chloramphenicol-resistant haploid cell mates with a chlorampheni, col-sensitive wild-type haploid cell, the resulting diploid zygotecontains a mixture of mutant and wild-type genomes.The two mitochondrial networks fuse in the zygote, creating one continuous reticulum that contains genomes of both parental cells.lvhen the zygote undergoes mitosis, copies of both mutant and

g e n e sf o r b 6 - fc o m p l e x genes R N Ap o l y m e r a s e genesfor NADH d e h y d r o g e n a sceo m p l e x

THEGENETIc SYSTEMS oF MITocHoNDRIA ANDPLASTIDS

EOS

wild-type mitochondrial DNA are segregatedto the diploid daughter cell. In the case of nuclear DNA, each daughter cell receives exactly two copies of each chromosome, one from each parent. By contrast, in the case of mitochondrial DNA, the daughter cell may inherit either more copies of the mutant DNA or more copies of the wild-type DNA. Successivemitotic divisions can further enrich for either DNA, so that subsequently many cells will arise that contain mitochondrial DNA of only one genotype. This stochastic process is called mitotic segregation. \.Vhendiploid cells that have segregatedtheir mitochondrial genomes in this way undergo meiosis to form four haploid daughter cells, each of the four daughters receives the same mitochondrial genes. This type of inheritance is called non-Mendelian, or cytoplasmic inheritance, to contrast it with the Mendelian inheritance of nuclear genes (see Figure 74-62). When nonMendelian inheritance occurs, it demonstrates that the gene in question is located outside the nuclear chromosomes. Although clusters of mitochondrial DNA molecules (nucleoids) are relatively immobile in the mitochondrial reticulum because of their anchorage to the inner membrane, individual nucleoids occasionally come together.This is most Iikely to occur at siteswhere the two parental mitochondrial networks fuse during zygote formation. lVhen different DNAs are present in the same nucleoid, genetic recombination can occur. This recombination can result in mitochondrial genomes that contain DNA from both parent cells, which are stably inherited after their mitotic segregation.

c h l o r a m p h e inc o l - r e s i s t a n t m u t a n t h a p l o i dy e a s t

wild-type h a p l o i dy e a s t

'o ,.' i"'. I

A

l I

SBR;fRi'Ji\"' N nDrPLorDcELLs . tr..

a l l f o u r p r o g e n ya r e r e s i s t a ntto c h l o r a m p h e n i c o l

a l l f o u r p r o g e n ya r e sensitive to chloramphenicol

Figure 14-62 The differencein the patternsof inheritancebetween mitochondrialand nucleargenesof yeast cells.Fornucleargenes(Mendelian two of the four cellsthat result inheritance), from meiosisinheritthe genefrom one of the originalhaploidparentcells(green and the remainingtwo cells chromosomes), inheritthe gene from the other (b/ock for Bycontrast, chromosomes). genes(non-Mendelian mitochondrial it is possiblefor all four of the inheritance), cellsthat resultfrom meiosisto inherittheir genesfrom only one of the mitochondrial two originalhaploidcells.In this example, geneis one that,in its the mitochondrial DNAdenoted mutantform (mitochondrial by bluedots),makesprotein synthesisin the to mitochondrionresistant chloramphenicol-aproteinsynthesis on the inhibitorthat actsspecifically procaryotic-like in mitochondria ribosomes We can detect yeastcells and chloroplasts. that containthe mutantgeneby their of abilityto grow in the presence suchas on a substrate, chloramphenicol glycerol,that cannot be usedfor glycolysis' blocked,ATPmustbe With glycolysis and providedby functionalmitochondria, thereforethe cellsthat carrythe normal (wild-type)mitochondrialDNA (reddots,) cannotgrow.

866

Chapter14:EnergyConversion: Mitochondriaand Chloroplasts

Organelle GenesAreMaternallyInheritedin ManyOrganisms The consequences of cytoplasmic inheritance are more profound for some organisms, including ourselves, than they are for yeasts. In yeasts, when two haploid cells mate, they are equal in size and contribute equal amounts of mitochondrial DNA to the zygote (see Figure 14-62). Mitochondrial inheritance in yeasts is therefore biparental: both parents contribute equally to the mitochondrial gene pool ofthe progeny (although, as we havejust seen, after severalgenerations of vegetative growth, the indiuidual progeny often contain mitochondria from only one parent). In higher animals, by contrast, the egg cell always contributes much more cltoplasm to the zygote than does the sperm. One would therefore expect mitochondrial inheritance in higher animals to be nearly uniparental-o! more precisely,maternal. such maternal inheritance has been demonstrated in laboratory animals. A cross between animals carrying type A mitochondrial DNA and animals carrying type B results in progeny that contain only the maternal type of mitochondrial DNA. similarly, by following the disrribution of variant mitochondrial DNA sequencesin large families, we find that human mitochondrial DNA is maternally inherited. In about two-thirds of higher plants, the chloroplasts from the male parent (contained in pollen grains) do not enter the zygote,so that chloroplast as well as mitochondrial DNA is maternally inherited. In other plants, the pollen chloroplasts enter the zygote, making chloroplast inheritance biparental. In such plants, defective chloroplasts are a cause of uariegation.'a mixture of normal and defective chloroplasts in a zygote may sort out by mitotic segregation during plant growth and development, thereby producing alternating green and white patches in leaves.The green patches contain normal chloroplasts, while the white patches contain defective chloroplasts (Figure f 4-63). A fertilized human egg carries perhaps 2000 copies of the human mitochondrial genome, all but one or two inherited from the mother. A human in which all of these genomes carried a deleterious mutation would generally not survive. But some mothers carry a mixed population of both mutant and normal mitochondrial genomes.Their daughters and sons inherit this mixture of normal and mutant mitochondrial DNAs and are healthy unless the process of mitotic segregation by chance results in a majority of defective mitochondria in a particular tissue. Muscle and nervous tissues are most at risk, because of their need for particularly large amounts of ATP we can identify an inherited disease in humans caused by a mutation in mitochondrial DNA by its passagefrom affected mothers to both their daughters and their sons, with the daughters but not the sons producing grandchildren with the disease.As expectedfrom the random nature of mitotic segregation,the slrnptoms of these diseasesvary greatly between different family membersincluding not only the severity and age of onset, but also which tissue is affected. Consider, for example, the inherited disease myoclonic epilepsyand ragged redftber disease(MERRF),which can be caused by a mutation in one of the mitochondrial transfer RNA genes.This diseaseappears when, by chance, a particular tissue inherits a threshold amount of defective mitochondrial DNA genomes. Above this threshold, the pool of defective IRNA decreasesthe synthesis of the mitochondrial proteins required for electron transport and production of ATp The result may be muscle weakness or heart problems (from effects on heart muscle), forms of epilepsy or dementia (from effects on nerve cells), or other symptoms. Not surprisingly, a similar variability in phenotypes is found for many other mitochondrial diseases.

PetiteMutantsin YeastsDemonstrate the Overwhelming lmportanceof the CellNucleusfor Mitochondrial Biogenesis Genetic studies of yeasts have had a crucial role in the analysis of mitochondrial biogenesis.A striking example is provided by studies of yeast mutants that contain large deletions in their mitochondrial DNA, so that all mitochondrial protein synthesis is abolished. Not surprisingly, these mutants cannot make

Figure 14-63 A variegatedleaf. In the white patches, the plantcellshave inheriteda defectivechloroplast.(Courtesy of JohnInnesFoundation.)

('sua^ets erPqreB,o ^sauno))'suraloldpopo)ua -snal)nuuorj ^larlua pal)nJtsuo) s ra l l a u e b r ao q l p u e ' 6 u r s s raur e slrnpo.tdauab pepo)ua-uoupuoq)olr.u eql lle'sluPlnuralrlaoul'luelnu alrlod e ur Pupuor.l)olrw (8)'erJpuoq)orlul 'slle) lseel leulrouJo arnl)nrts aql (V) ;o sqderbor>guuorl)al3 t9-'l arn6rl

'pldll IElo] eqt]JJ-o/o0zlnoqe setnlllsuoo lI eJaqM'auPJqluetu Jauul IEIJpuoqc '(99-tI -olru sFPl prse [11e; rno; sulPluor punoJ erntlc) dplEtu sl eq] ur lI '(lorerdlSlfppeqdsoqdstq) utdqo rpqt prdnoqdsoqd (,elqnop,,e st utdyotpreC -rpJEJol sprdq palrodur Jo uorsJeluor eql sI senlasuraql elJpuoqJollur eql fq pazl.plec srsaqludsorq pldll Jo uollceeJ uIEIu aql 'eultueloueqray.{ppeqdsoqd 'Blrpuoqoollu o1 aurrasldprleqdsoqd peuodul Supeldxoqrecep ot uoulppe uI Jo aupJqruaru Jalno aq] ol uaql JeJsueJ]uaql pue lunlnJllar cruseldopua eql uI eurresl,tpleqdsoqd pue aulloqclrtprleqdsoqd sprdqoqdsoqd aql azlseql -urts slac IBruruV 'spld[ Jreql Jo ]soru Uodrul 'lseJluoo uI 'eupuoqsoll6 'ereqm ,t1pco1pazsaqlu.,ts oslu ere tseldorolqe aqt;o sprdqoedlS rofeur eql -asle srnsco sprce ,r!1e; eq] Jo uoneJnlesep qSnoqrp ']seldorolqc aq] ut aceld 'armbar salel srseqlufs proe fi1e; re1n11ac1e 'aldruexa JoJ 'seleel qceutds u1 ,{aq1 sprdrl aqt a{Eru 01 pua} gseldorolq3 'sure}ord pue splse Jlalf,nu ol uop -ppe q sptdl sarmbar slseldorolqo pue elJpuoqsollu rvreuJosrsaqtudsolqaql

sileqlJo lsow lleql Jotsow uodtr|l Prlpuor'l)ol!w a{ew s}seldorolq)jsp!d11 's1se1doro1qc u1 sutalord pepoJue-snelcnu aqt Suorue punoJ osle eJu seJuaJeJJIpcgrcads-anssll 'palcadxa aq plnorv\ sV'eArAJns ol spnptntpq eqr 8ur,,uo1p,{lpurou uoltJury 's[ec el3snur uueq rraql Surpnlcur 'sllao Jeqlo Jlaql 'slleJ alssnlu lelela{s o1 cgtoads sI llunqns eql eours :eseprxoauorqooytc Jo llunqns e^pcaJapP eAEqaseeslpelJsnu crlaua8 e qlyu sueurnq auos 'snql ']rodsuu4 uoJlcela;o srolep8ar se lce o1 lq8noql are 'cgtcads-anssp lpq] slpnqns papocua-snelJnu IeJaAesuleluoc osp s1elulueru

roau'Iqluau'euull'I'puoqsorlu"ut"i.'"";r"J,1Tr""';Tfi ;fi ,",Hor", [X^J..lfu palrodun pue pezsaqlurts are saurrkua parmbar aq] pue la^ll aq] se qons 'sens -sl] ,t,reJe dpo ur sJnJco srsaqlurtsearn 'a1crbaql ut sdals leJe^esurro;rad xtrleru 'eam se eulrn aql uI peteJJxa IerJpuoqcolru eql ul sarufzua papoJua-snalcnN are slcnpord asaql 'uaSorlp upluoJ leq] slcnpoJd uruop4earqreplac;o Sutsod -slp JoJ slerutuetu ur d.e.mqledJrloqelau IEJluac eql s1 'aldruuxa JoJ 'a1ctcnatn eqJ 'slleo;o sad,{r relnrqred uI suol}cunJ pazlptcads eABq uer elrpuoqf,o}I141

snel)nNlle) aqr ul papo)uf elv leql surelord)ul)ads-enssllu!etuo) spllseldpue eupuot{)orlw 'arp 'luesqe sl VNC lsuldorolqc esnecaq Jo ro elllf,eJap s1ue1d eq] 's1ue1draq8q uI pe{colq st slse1d {Jep aqt ut s1ueydaq} Sursrer-,tqraqlre tarrarrnoq -orolqf, ernleruJo luarudola,rap eqtJI 'seteJlsqnselqeztpxo qU,vrpeppord eru ^'taq] JI alqeF,(1tca;rad arE pue slseldorolqc uleluof, IIIIS sJncf,o qsaqluds utalord 1se1d -orolqf, ou qcrqm ur ae8p luelnl4 'nua1&ngse qf,ns ae8p relnllaclun Jo sluelnlu aJP sluelnru el11adpupuoqJotlu ]sear(ol ]uapnrnba lsareau aql'slseldorolqc rog '(tt-ZI arn8rg aas)de.u srql 'auoue8 rrmo sllJo eJuesqe ur alecqdar.{.1purou saruosxorad leql ayqrssodsl 1I alalduoc eqt ur uale 's11accr1o,{recnaSuqera;qord ;o rusuldolb aql u1 d1ar1u -lJepul alecqdar ueJ uoISSIJ.{qsaprzrrp}eql allaue8ro ue leql Moqs osle slue}nlu elrled 'srseuaSorq prrpuoqJollu uI snalJnu aql Jo eJuelrodurr Suturlaqrr,rrano 'sarudzuaa1c.{optce ou aql Sutlerlsuouap-Sulalord auerqua(u Jauu! lsotu pue -llc aqlJo 1p 'saseraurdlodVNU pue VNCISupnlcur-1osolb eql luo4 palrodrut pue saue8 Jeopnu ,(q par;rcads eJe leql suralord lelJpuoqsollur eql ile ,(1pn1rrn arntlg) eulslrr padolanap,(pood qllirt aueJqureurrauul uP ureluoJ daqt'(lWf puE eueJqrueruJalno puJou u e^eq PlJpuoqJolllu esaql'eupuoqcolllu ulEluos sselar{ualau,{aql [IJV acnpord }Eql elJpuoqsollu e{eu louuec eJoJaraq}pue prJpuoqoolru Jraq] ul suralord aztsaqlurfu louueJ sluulnlu a1r1adq8noqlly sluatnut alpad 4usn1do1[c payet eJeElrpuoqc -olrru elrlceJep qons qllm sluelnlu 1e 'asorn18;o uolleJlueJuoJ lvrole Sutupluoc 'raq}a8 unrperu ur umor8 ueq^A seluolof, llerus .,{.1pnsnunruro; ,(aql esnef,eg -olle VNC Ierrpuoqrollul {cul s}ue}nur asaql Jo auos 'elJpuoqco}Iu Surrtdsar

198

f II-]N]9 ]HI JO SW]ISAS SCIISVIdCNVVIUONOH)OI-IW

pue seruos e Sururelureruasnpcaq 'lplllrl lou sr uorlsenb aq;;lou op 'seurosos,{1 -rxorad se qcns 'useldo{c arues eql aJpqs lpqt selaue8ro Jeqlo uaq A 'surel -sds cDaua8 aleredas umo rreq] arrnbar slseldorolqc pup ErJpuoqcolrurop dq11

esuals^s pueeupuor.l)ol!w )rlouogu/r^oraql a^pHslseldoro;q1 oc ^qM

yo s11dsurddod,{ldrurs ,{q sanrlrno suolordot alqeaq IIEqs "^;ffitttf:i::; stuees]l 'suerunqo1pre8arqU^ ]no ilps srfrnf aql q8noqtry 1e[1a>plun

'sJounl 'uorlJunJ Jo uorlEruroJpeseeJJurue e^eq pue lerJpuoqf, 'a8eruep -olnu ur aurlJap arnleuard ^ prrpuoqJolnu e oqs alrleprxo Jo lunorue 'Lyarnleruard a8e o] readde lou IBnsnun ue alElnruncce ,taql q8noqf uana op pue af,ttu prurou se Suoyse a^rl COSUIAtr Jo ]unorue IEruJoueql Jo JIpq aJnp -ord ol paraaur8uaecru 'srsaqlod,{.qe1c,{csnorf,rl eq}Jo lsel loeJrpE sy'a8euep ro suorlelnru Jo uorlelnruncce ^,(uetuoq lno Surrea,rnupqt Jaqler 'op [aq1 uaq^r,r arp pue a8e o] paruuerSord uaaq aAEqsursrue8roeseql lpql slsaSSnsBtEp srqJ 'sa8ueqcc4aua3;o requnu e euo u(ueqBnorqt pale8uola ,{lecqeurerp aq ueJ Jo suedsa;r1'sarlJpue sruJola se qcns srusrue8ro Jelnllaorllnur Jemol JoJ 'reAoeJoIAI 'ssacord xalduoc -{ran e sr Sur8e 1eq}JeelJ sr lI ieepl srqt ot qtnr1 r(ue ereq] sl 'a8e p1oSurlelgqap E 01 esrJ 3unr3-s.{ecap rusrue8ro aJrlua aql }eqt slJnpoJd uoqepxo ,,(ueu os Surcnpord eJesuerunq plo ro aJru ploJo €rJpuoqcolru aq] 1pun sanuquoc dool 'senssq esaqt ur sJoJrauorleprxo elpJ Surleralacceue TJBqpeaJInJurJeqsrqJ Jo sasnBsuJn] ur qJIq { 'sanssrlJlle(uos Jo vNC lerJpuor{Jolru eq} uI a}elnrunJJe ol suorlplnu sasnel a8euep uorlpprxo 'l atn slq] u1 'a8e sursrue8rodqm ureldxa ol sldrua]le teql slsaqlodrtq ,,a1cLcsnorJrn,,e o] pal aneq 's8uraq ueunq Japlo ur erJpuoqJolru Suruorlcury dlrood;o suorlelJasqo qrryr paldnoo 'suorlplJesqo yo sadfi asaqJ 'VNCI reelcnu aqt ueql sapqoelonu Ierurouqe 'pezrprxoJo la^el raq8tq ploJ-ual B ureluoJ ol pelJasqo sl aq] 'slerurue pru VNCI lerJpuoqcolrur -Jou ur ua,rg 'au,{zua srq}Jo suorsrenJBInllaJEJuaaq} ro Jrlosoilc aql raq}ra 1ce1 teq] aorru aI1un'a;rl ur.{pea e1p(OOSuru)aselnusrp apxoradns IerJpuor.lJolru ur ]uarJuap acrru leql ro 'saurfzua asepxorad auorqlelnlS pue ase]nrus1papl -xoradns u^aoJreq] ureluof, erJpuoqJolru leql Sulqrdrns lou eJoJeJaq]sr lI 'erJp -uoqcolru eprsur peuJoJ sr sllal ur paleraua8 _zg aql yo o696,{laleuxorddy '(dnor8 1t(rp,{qps aura}sfc sll q]lm auorqrelnp app -dadrn aqt sluasarder HSC alaq^\ '9SSC + OzH Z 1so1,{c go sadr{t eeJql elpr{ sllac 1eruruplso6

slNtwvt tJtvlltl)sol^)

lo

]UNI)NUIS)IWVNAC CNVAIBW]SSV-JI]S 1Hl

sejl

uotAVHlS Il) Nolltl)sol^)

ONV tHI

0r.0r suol_ow uflnfilow

266

SIN]WVIII tvl_il1)sol_AluEHl_ trvtn9tu slll) MoH SIN]IAVIII

tvl_ltl)sol_A) lo lunl)nuts

s96

fIWVNAC CNV lt8wlssv-Jtls tHl_

roldeq) slql ul

'suorlef,ol rvreuolJleslr eloru ol-eleudordde uaq^\-pue ,Jeqlouu o1 aceld auo ruo4 Jauupru pelJeJrp e ur salaue8Jo eloru o1 ,ecu;rns str laporueJ pue adeqs o] tepJo lpuJelur urelureru pue qstlqelse o] IIaJ eql Sullqeue ,srualsztsluerxelg ureru eeJql eql qllm lceJalu suralord Jaqlo ,lroq eqrJcsep uaq] eM.sequelncad 'z{.yquassesppue rtlqruasserraql Sur.rtlrapunsaldrcuud crseq lenprlrpur Jreql pue eql 'sluarxplggo sed,ft ureru eeJql eql SulquJsep r(q ur8aq e,\,\teldeqc srq] uI 'e^oru ol ,irmqe str pup 'adeqs s1r'q13uar1sslr IIef, e an€ ol d1a,r,11ca11oc uoucunJ dlerurou lsmu srualsds lueurelg pteleTsol,{t aeJql IIe os :eqteSol {ro^a ot salJsnru puu 'sauoq 'sluarue8rl rno eJrnbal arvrsE 'aluradooc slueuela lsnf tueJeJJrpaqt rvroq pup s{rom uo1a1a4so{c eqt MoqJo Surpuelsrapun praua8 E JoJsrseq eql ep4ord saldnurrd aseql'seldroulrd pluaruepury ureuac eJeqs eerql IIE lnq ,selor IeJr 'scrureu.,(p'saqradord pcrueqcaru -3o1orq pue tJurlslp seq luaruelggo adft qceg 'sluauelg yo sadfi urcru eaJql ruJoJ ol alquesse qclqm ,selnJelour uralord;o sarlrrueJeerqlJo rorler1eq eq} uo puedap suollJury pelrel spolaya4so{c eql 'sadeqs 1ec cqorfuecna;o .,i1rs -ranrp .setrJp Surzeure eql sloJtuoo pue IIB,r,rIIec 1ue1deqlJo qlmor8 aql saprn8 tl -uep pu" uoxB ue pueua o1 IIaJ a^Jeu eql ur puE uorlf,BJluos JoJ IIaJ elssnru aql ur rfuaurqoprueql sapnord lI 'seJeJrnsssorJp I1v\eJJol .slleJ poolq etrqM pue slselqoJqg se qons 'sJar{lo puu rurms ol rurads se qJns sllec salqeue 11.sa8ueqc pue sgrqs lueruuoJrlue eqt se gude paddp Suraq lnoqlpr suruJls pue sasseJls reaq [ec aq] lal ]eql saSoprl leoruer{Jarrr aql sapprord pue eupJqrueru erusuld ay8er; aq] suoddns lI 'reqlouu ol IIec aql go uud auo ruo+ slerJalpru Burdrra; 'sallaueBro lrJJerl relnllacprlur aql saprn8 pue selrJp 1I .ort,r]olur ilec Bupptp Jo aqt sqds ueql pue srsolru ]e uedB seruosoruoJqc aql sgnd uo1a1a1so1baq1 '(1-91 arn81fl uolalarlsofc eqt palluc stue[ueg ura1s,,(selqDlrBruer e uo puadap daqt pue ,aarSap q8q ,,fuarr 3o E o] suorlJurg pue prleds eseql 11epadolanap e^eq s[ec c4orfuecng leorueqJeru 'secuslstunsrlf, 8u€ueqc o1ldepe pup 'apr^rp rr,rorSrtaqt se sluauodruoJ lpuJelur rreqt a8uerreal ol alqe aq o] eAEqsllet IIV.ace1d o1 aceld uor; arrou pue adeqs rraqt a8ueqc o1 aneq,,(ueyqLleura1u1 parnpnrls dlradord pue ,lsnqor.,typcrsdqd 'padeqs ,tllcarroc eq ot elpq ,taq1 .luaruuoJrlue Jleql qfl^a ,,i.lpcrueqcaru 1ce -ralur pue aceds ur sellesueql azrue8ro lsnu daql flradord uoncury ol sllec JoC

uolelalsov()aql

'lleJ aI{} ilac eql qcurd ol elJSnIu,rtup e e411slclJlsuoJ qclq/!\ Sutt aplcn4uot aql puB Jo elpprur eq] punoJP lleq P ruJoJuaql ulsor(ur utalord Jololu pe]€Icossesll 'Suttotu sdols upcy'adeqs pctraqds eJour P selunsse pue 'dn spunoJ 1ac aqt lBql os elquesseslp qslp aql Jo esPJrnseq] ssoJlE I AeJco1 lselqorqlJ aql alqeue 'aurp arues eql lV 'IalJnu ralq8nup aler leql seJntcnJls urlJe pazlptcads eql -edas oml olur eluosoluorqc paleclldeJ qJee Jo sardoc oml aq] Surlu8arfias.{1a1er -nJJp Jo uoDJunJ IerplrJ aq] senJesqJIqM 'a1putdsJ!\oL!u'trelodrq aql olul peJn -Squoca; -{erre ePqntoJJIIu aseqdralut sueds urseldo/c aqt sr 1uql lnoq8norql 'qslp eJnllnJ enssll e ut Suu,ror8 aql 'palecqdal aAEq sauosoruoJqf, aql JaUV (uoIsIAIp 1ac Surrnp sJncsouo1a1a4so1dr lselqoJqrJu JoJZ-gI arnt;g uI u^^oqsse eql Jo uorleztue8roer ptder ;o alduruxa slletueJp pue luelrodtul uV 'speura8ug pue 'suoxe }snqoJ puu 3uo1 rreq se qcns sa8epuaddeq8nol IuJoJo] sn lltolle.{aqt pue pueua ot slleo anrau dlaq ro raqtaSol slaaqs 1ac plaqttda ploq uec ]eq] selqeJ Suorts otur petslmt are daq] '1oso{c aq} uI :VNCI s,llar eql ro; a8ec anpcalord e Sunuro; 'adolanua Jeelcnu eql Jo ef,PJJeuul eql euq stueuPIIJ elelperuJalul 'uoqdrosqe ]uelJlnu eoueque ol aql slac plaqltda pupsatulJo aoBJJns EaJeaceJJns1ac pcrde eql asuaJcur,(1lsen 'punos o] asuodsaruI spoJp€tr se lllf feql uo tl7ltoJJrrupazrueflroz(1relunspue slualuplrJurlJe Jo salpunq alqels ulEluos JBeJauul aqt uI sllal JIEqJo esBJJnseql uoDllnoanls Jo sepunq rep8ar eqJ'lJerluoJ o] alosntu alqeua pue unleJlsqns Sur.Llrapun ue lsurefie se1lesuaql eteJq ot sllao ltlrollp srterre a1qe1saJou :oml ur sllac applp o1.{puatsue4 salqurasseBuuallPaJiuor pesuq-ullJe aql'punore se^lasueql 1nd pue ,rfuolrrrataroldxa ol asn sflac leqt mpodo1{ pue atpodty -pwal eql se qJns ,sernlcnJls crureudp aJe asaql Jo eluos 'suopcaford aoBJJns -llar Jo sad,& .{ueru ruroJ osle ,,taq1 ra,(egq prdq uqr s1l o1 aduqs pue qlSuarls

q."e ." " d's'ar IEruru'roauer surpr'r'or T:r"jir#l;lii #lr'J::Hi"tH fi1'J",,0

'*o* 's1iac puornau 3uo1 1ue1O.ti o1 dlaq sepqruorJlru;o s.{erre paztue8ro umop slerreteurgo podsuur] eql roJ $loeJ] se enJasleql sapunq pau81p fpq8p ro ,lleJ eqt Jo aoeJrns aql uo ayaBnll pue DqlJ palpc sdtqrr elllotu IUJoJ osle uec .{aq; 'uorsr1rp1ac Sutrnp aTputdsctlo4ta relodtq e IIJoJ o] sanlasuraqla8uur -ruar r(11cmbupJ 'lle3 aseqdrelut uP Jo Jaluec aql 1IIo4 Surleueura Lurre cruseld -o{c a1q-ruls e uI puno; r(1}uanbar;are qclq.t,\'selnqn}olJllN 'saJn}JnJ}saseq} 'srualsfs }uaruEIIJclseq aaJql laued uI sqderSorctur eql Jo aruos pe^er I{I eq] 11104saJnlJnlls Jo aluer snoruroua ue p1nq o] sgac rtlodrecne Sa1ollgSlueul -elrJ IPleleIsol,tc aqt;o l.lqruasse puB rol^eqaq c[ueu.{p aql 3o uorlepSaS .aSueqc elllI sarrnbar BJua dSraua uaqm suoplpuoc IIef, E ur luauraSuerreal 'snql 'xnu ,lreD lue up Jo alB]s ]uBlsuoc e uI aJe Jo uorlEJal[Baql aI[ IBrnlcnrls E saJnlJnJls asaql dn e)e111leql sluauodruoc lelnoeloluoJJeul pnpwpul eq] ]ng .aullaJrTs,lleJ aq] o] dn atnunu e ueq] ssal uo4 Sur8uer erullJo sqfual ro3 3upse1 ,paeu ol Surprocce ,lsrslad ro a8ueqc uEf, saJnlsnJts ptalalso]r{.c apcs-a8rel ,{em Jelrurs e uI .uorlenlrs ^ eu eq] qll { pep 01firprder Surqsruolse qly!\Jlasll saSuer -JeeJarnlJnJls cnueu^,tpeql'e^Eal pue dn uealc sralcrucrd eqlJI Jo 'pooJJoeoJnos 'cllels 1nq Surq{ue ere rallaq pue ,t,raup purJ stnoJs lup aql JI IIEJ} aql uqryv\ slue lenptlrtpul aq] tnq 'a]ts ctuctd alqelJelep E ol lsau lue eql uror; Sutpuaga 's,,tulv\q8rqe}elsJetul uBq} ,sJnoq dueru ro; lsrsrad .{eru s}ue Jo IreD a13ursy slreJ] lue aIII aroru pazruu8ro 'alqeldepe pue ctueu,{p are suralsds p1a1a1sof3

elqerdepvpue)lueu(6 ery sluauellJlelalelsot^) 'ace;,{eru }I leq} ^lpn}uarra,(ue o1.(lprder puodsar asnuJ }Pql suslueqJeru ol alqe sr IIeJ aq] 'crureu,{p aq ol s}uau€1; p1a1a>1so1rtJ alqelrerual eql Jo asnecaq ']Eql aes IIEqs aM'seJnlJnJls paztue8ro-Jlaspue pezl eql Jo sluarue -re1od ,(lecrsulJlul ruJoJ ol drIIqB Jlsql uo snJoJ eM 'uo1a1a>lsoytJ -ru aql dn aleur 1eq1suralord aq];o sarlredord aql ssnJSIpe^ 'uollsas slql uI 'sanlesrueql slueruelu aql elotu Jo slueuellJ aqt Suop sallaue8ro eloru Jeqlla uBJ ]Eql eJJoJIeOlueqcau olul srsr(lorpdqdJVJo d8raua 'sutalotd tolota eq} sapnlcul aql Ue^uoc leql saulqJeu JEInJalou alqe{reIual 'suopeool relnf,Iued ur sluaurellJ lelalalsolb aqr;o .{lqruassepellor}uoo tl pup aql:9 L raldeqf uogala>1soy{1

996

ue!lllM fue^rllnS 'sloqq6raust! qrlM6ulplllo) lo Iseuno)) Luo4snel)nuq)ee 6urdaalalrq^ 'srsolrur aql aleledas lp saurosorrjorq) ol parnbarare'o^rquraburnrg e ur eraq uaes'(uaat6)sapqnlor)rur pue (pal) sluaurelUu!l)e aLllJo slueua6uelrea.r prderaql'use;dolb uouruo>e ur os Jo selnurur61 ,tana Jn))o suorsr^rp real)nuIUea aq]'lle) reel)nurllnur tuer6s;ql ul 'oftqure {Fee olltldosotg e 1o guaurdo;anopaql 6uunp pa^rasqouo!lez!ueOro1e1e1a1soy(> u! se6ueq)prdegg-91 ern6r1

aseqdeleur u!r.us t

aseqdoJd u l u rs I

aseqolalul

u!ru0

urri 9

urfl t

urnz

urn0

e)eJJns lla) u.lol,|.a)uels!p

'(g-91arnt1g) saln -urru eAU ueq] ssel ulqtyv\ se^lasueq] eSueJJeeJzi.1a1a1duroc uE3 suolelelso].{3 alnqnloJcrru puB urtce eql 'o^Jqlua DITdOS1JOe uI suoIsIAIp JBalcnu ,{Fea aql '}sPlqoJqg sE qJns 'sesecaruos uI :Jnoq uE tnoqe se{e} slua^e Jo asuenbas slq} P uI 'lle3 Jeqlou 3u11,uerc'pauaileu eql Jo suolsJa^ rellelus olul slles JelqSnsp dn papunor oml el{l uanuoJ o} seJn}f,nJlsaseqdJe}u 4eqt olu1 elquasseer s}sEI -qorqlJ ralq8nep o^\l eql Jo suolale{so}(c aq} 'atelduroc sI uoIsIAIp ual{ \'o1v\l uI

'sr{ean areredes raqt lmpr)o] tlaql 6ur;qeua'llalraqlour eql u! luasordaramleql asoqlJo suorsra^ ralleusolur suole;e>1soy(r u!])e pue alnqnlor)rrrraql qloq azrueOroer s;;ar rolq6nepoMl aql'a1a;duo:sruorsr^rp aneqsaluosor!olq) lla) JaUV'paleredas aql raue oml ur lla) aq1saqrurdleqt lle) oql,o ralua) aql ]e 6uu olrl)eJluo) e ulroJsluaurel!,ult)e eqf'(urlolg) sauiosouorq)pole)rldnpaql 6ulleredas uaql pue 6uru6r;e lo, alqrsuodsej sr q)rqM'a;purds rrlolrrule;odrqe ur.ro1 ol sa6uelrea:,{ele a;nqnloorurpazuelod aql'sapr^rplla) eqr uaqM'snal)nu aLllJo luorJ ur pale)ol Jaluo) 6urzrue6ro alnqnlor)rrua;6urse uorJ aleueuJo lpql 6uo;;o 6urlsrsu o)' (uaalb salnqnlor)r,.u ur uMoqs)uo1ala1so1{r alnqnlor)rur aql r(q patsrsse sr uo1a1a1so1b urlre aq1'1q6uaql preMol aql Jo uorlezuelod e6pe6urpeels1rqsndol salqLuassp uolalalsoy{>utlre reql (pal ur uMor.ls) rrueu{p'pezuelode seqoraquMerp ]splqorqq6ur;merraql'uo!s!^!pllal qlr/vr pale!)osseuollezlue6ro le1a1a4soy(> ugsa6ueqrprdeg7-91 ern6r3

196

slNttiv]l lvllll)sol^)

lHllo lunl)nuls )lt lvN^ccNVAlStJlSSV-llls

(^r) rqav rlan i(ril)uroqso ^ren i(il) eqsrapa) I fuueN :(r)uelurnO^oU ,o Issunor sqder60rlrW

' u r n r l a q l t d ea l t l u o o q l 6 u r u a q l 6 u e j l s^ q a l e q l l a q l o u e o 1 u o r p u n l ue u1 ql6uatp l l a ) - l l e )a u o u l o t 1u s e l d o p { ta q } u e d s^ a q } ' e n s s t ;l e r ; a q l r d a ' u i s e l d o p {e: q } s s o r l ep u a y a s a d { 1l a q l o auelquraur l e ) r u e q ) a usr 1 1 a6:u l n r 6 J e e l ) n ur a u u ra q l q l e a u a q1 s n Ie u r u e l r e e l ) n ua q ] p a l l e )] l o M q s a u l e surroJluauJelr+alerpeuiJelur1o ed[1 au6 ,{1tu.re1 snoauabo.ralaqpue a6le1 e a l n l r l s u o )q ) r q ^ / \ ' s u t a l o rldu a u e l t ] a l e t p a u J J a l ul ot a p e u a l e ^ e q l : u l u 0 l p u n o r e] o J e l a u r e r pe L l l t Ms J e q t ,a 1 r ; a d o a t J es l u o L ! e l ra+l p r p a u r l ; l u l

urusz

uru00I

StNI lAVl ll l-LVlC:ltAUlIN I (^r) rpsac V i(ilr)eurqs pr^eo i(il) l)utl M U pue MorpooM 1 O j(r)apeM preq)U +o ^salnol sqdej6ol)ru\

'eu/oso4ua) e pelle) ( ) O I W ) r a l u a ) 6 u r z r u e 6 t o - e l n q n l o t r rau;t6 u l se o l p e q ) e l l e p u a e u o a ^ e q[ l l e r r d r i 1p u e 1 q 6 r e . r 1 p su e 6 u o l a l e s a l n q n ] o J ) t l 4sl] u e u t e l U u r D e u e q l p t 6 u a l o u q ) n u Ja J e, { a q 1' u u S Z} o l a } a u l e t pl a l n o u e q } ! A u r ; n q n 1u r a l o . r da q ) ] o e p e u ls l e p u l l [ : m o l l o q ' 6 u o 1a l e s a l n q n ] o l ) r ! 4

uru00!

srrn8n10u)ry\ (rrr)abprrnE q]!e) :(ll)sso6rng U c pup erepnslpfl Id i(^r pue r) 6rer) ra6ou +o Isalrno) sqder60r)y\j

a u e r q u r a ue u s e ; da q ] q l p a u a ql s n f ' x a y o : a q l u t p a l e J l u e ) u o ) ^ l q 6 r ql s o r xe r e i ( a q l ' ; ; a : a q 1l n o q 6 n o J q lp e s l e d s t pa l e s l u a u e l t l u 1 1 : eq b n o q l l y s ; a 6 o r s u a u l p - e e t qplu e , s l J o M l e ul e u o t s u a u l t p - o m ] ' s a l p u n qr e e u r l [ 1 a u e ne; eoul u r p a z r u e E l oe . r er { e q }p u e , u ] u6 - s } o . / a } e L u e t p 'Lo s lqrxal+ e qllm 'sernDnJla s e r e a d d e{ a q t ' u r p e u r a l o . r de q 11 o s l a u f l o d le)llaq pepuerls-oMlale (sluaile/ryoDtwse uMoul osle)sluauelt] utllv

uru00[

sl_Ntytvll NUJV

('sla6oupt^p6 Iq peplo)al oopt^e uotl) .spuolasMaJp utLlltM jo uorl)altp pup uotlpluauo luaLria^ouJ s1ra6ueqrot lt salqeualla) stqt ur uola;a1sop{r ut})eaql ro I;quessea.l pue {lquasseslppldpU.eual)eq oql Jo uorle)ol aLllpreMol qsnd ol (pal) a6pe6urpealaql le lJomlsu utl)p asuap aql salqLUasseat I;1trnb ;rqdorlnau aql'anout euat)eqaLllsV .llr.ldojlnau e {q palnlder aq ol lnoqe sl (lvtoilD attq/A)elepeq;o durnlr e ,poolq ueurnq 1o uorletedaldstql ul .eual)eq y tr-91 arn6r1 ;o glnsrndur ;pqdorgnau

696

prp/v\ol Suqurod pup pau8qp selqBJ urlJp eq] ql!v\ ,pnq eqt Jo dl] Suriuor8 aql ]e pelsrlueJuoJ dlqSq e{uoJeq seqJ}ed unce aq} 'ssacord sH} uI 'raSJBI,rtluro; .eJEJJns -run SuluorB dldurrs pesoddo ol sp IIac eql uo elrs el8uts e ruoq pnq e ,tlroJ8ot eql .{ pazrJulod ollp ot ^HBIq eq slleo Surppnq Surlera;1 tsntu IIeJ }seed ,xauoc -or4 '(srsol,bopue uelrJp-urlJe Jo salrs Suqreur IIef, eql ql!v\ peleloosse slueuelrJ Jo sarlquesse IIBrus) ssqclEd ultf,E pue (slueuelrJ ullJp Jo selpunq 3uo1) salqec uqce :sllec eseql ur serlquessp luauelrJ urlJe Jo sedil omt aJBeJer.{J 'uo1e1a>1so1,b urlJe slleo er{} Jo uorle}uerro relod eql uro4 selrJap drlauudse sILIJ 'llal Jeqlou a8reye pue IIeo ralq8nep IIErus e eleerJ 01 Surppnq l{q apyrlp daqt ,{em eql ur luaprla 'd-qaruurdsepe{Jeu aql sl sfiac eseq] Jo erntlnJ]s eql Jo eJnleeJ elqelou lsotu eqJ ,ir1relod eIpss-e8JEIelqpls paau aq$uralas sat{LaoJ -Dqnas lsea,rtSurppnq aqt se qons sllec aldurs dypcrSoloqdroru .llel.usua^g '19-91 arnt;g) pcrsdqd raureq elrlcaJJa ue sp alJes ol slleJ Jo ra.,fu1al8urs srql elqeue 01 Jeqtoue qllm auo slJuluoc alrsaqpe Suorls urclureru lsmu osp daq; 'urBeJlspoolq eql ol eupJquraru eurseld aq] qBnoJqt sluarJlnu raJsupJl slleJ eql arcg,n'racoltnsluJaruIosaq eql ol [q sessedpooJ aJeq ^ auuselul aq] Jo ueunl eql ruo.t; sluerJlnu sqJosqe lar4l aJatJns 7n4da eql uaemleq sef,uaJeJJrpleuor}cunJ (sluaruplu urpe ,seFqnl IecrlrJJ eql urElureu 01 sluetuplu alerpetuJalul pup 'eldurExe JoJ ,eurlselur aq] -oJcrru Jo sdeJJEpazue8ro esn Jo SuruU aql uI punoJ asoql sp qJns sllal pezrrplod 'llef, er{l Jo eul}eJII eqt relo paurelurpu Ieneqlrde aq ueuo osp ]snu uo1a1a1so1tc aq] Jo uorlEzruuSJoeql dq papoJue uorleuroJur drlreyod apcs-a8re1 eqJ )req pup ]uoq ro (ruopoq pue do1 uaemteq aJuereJ -JIp eqt ilet ot sllar Suqqeua ,ir1re1odJelnllar apcs-a8re1rog alqrsuodseJosle sr uo1a1a4so1dc 'suorsnJlord eql acey;ns pazrlerJeds a1qels Sururrog saprsag IIaJ 'sapef,apro3 lsrsrad leql saml 'sJnoq -JnJls ecPJJns IIec alqels eseql ulqlll\{ ue^e 97.{rana aSeraneuo paceldar pue palapouar dlsnonuquoc ere pue crureu(p dl8urnrrls urerual sluaruelg ur]38 lEnpr^rpur eql 'ssaleqleuoN 'JenouJnl lou op silac aseql ecurs .lBrurup eql Jo eurpaJll eJrlue eql JoJuorlpzruESro a1qe1sJraq] urplurpru lsnru Jea Jeuur eqlJo sllef,Jrpq eq] uo prlrJoeratsJo seroJ eql lp sepunq urlJu eqt 1ng .sdep.ue; e fpo sI slq] slleo Ierleqllda puqsalur uo rllrlorJrru Jo seroJ aqt le sepunq urpe eql Joc '[ec eq] Jo eurrleJrleJr]ueeq] JeAoJelauerp pue 'q13ua1'uor1eco1luu]suoc p prlrJ urElurBru ot elqe eJe pue rllr^orsrru Surpnlcur suorsnrlord aceJrns pespq IIa3 '3un1 -p1a1a>1so1,b pue aqr eurlselul eq] sB qcns senss! auI lpql s11ac prlaqlrda pazqercads uO 'uorlezrue8ro re1n11ac JoJ seJnlonJlsapcs-a8rel ,alqpls apnord oslE ]snru uo1ala4so{c aql Jo slueuala cnueu,{p eql 'sllaf, plaqtrda ro suornsu eJnlpu sE qcns .,{3o1oqd.rourpelpnueJeJJrp.elqpls e pelerr{Je a^eq }eq} sfiec uI

sernl)nlls alqels uroJ oslv ue) uolalalsot{) aql 'raldeqe slql Jo uol]f,es IeurJ aql ul Fetep aJoruur pessnJsrpaq IIIrltsluarua8uerreeJlelale{so1,b prdergo spury esaq] '17-91 arn81J) spuoces ulqll^t sernl3nrls arrrsnrlord pazrelod slr azru Jo qlog (uorlJeJrp -e8roar pasrod o1 sl pqdorlnau aql luaJeJJrpe ur seloru r.ard prrapeq a^rsnla eql uar{liUslueuplrJ urlce pazrraur,,(1od,r{prrau qly!\ palllJ a8pa Burpeel aql le aJnlJnJls aarsnrlord e Surpualxa dq acuunpe slqdorlnau ,s1ac SurlaneJc]soru arrJ 'UDISeq] q slnJ qSnorql se fpoq eq]30 sped alrrals flerurou eql 0l ssaJcu urc8 dleluaprcf,B tpqt s11acp8urg pup lprretf,Eq s;p8ua pue sespqo ,llal poolq allq \ Jo ad.{} e '71t1do4nauaql 'aldurBxa Joc 'lla1n\se aseqdralur Buunp Buruorl -JunJ Ierurou neq] roJ sluarua8uerreeJ lelele{so{c prder arrnbar q1ac,,(uuytr )os E

)esz

)os I

)as 0

aulll

slNtwvl|Jtvl-ltl)souf Jo lunr)nuts )twvN o cNVAtSwlssv-ltls3Ht

(ssar6{lrsrenrul JellaJal)ou aqf uorJ uorssrurao q l l M ' 8 6 6t ' L L S \ - L j S L | Z V' lLo ! 8i l D ' f "le la e^odle) 'Sf ruoll) 'solqe)palueuo Ipuopuera^eqslle)qloq q6noqlle 'elrsuorsr^rp aql rPaupaleJlua)uo) areq)rqM'soq)ledMau uJoJ s;;a>rarq6neppue raqlour'uorsr^tp llal (O)'pezgelodureuar raue ^lalerpauLul 'pnq pazrs-llnJ e lle) laqlouraql u! salqef Jo ale;insoql ralo palnqulsrp{1tu.to;tun 'rltntor6 lsor.!leare seq)led ()) lleMllal MauJo alrssrqlpremollurod ol anurtuol ']t llo) laqtoul aql u! salqef uqllM pnq lleus ureuarsaq)ledlsor-u'srvror6 aql sV (g)'a6raua lllMpnq aql araqM alrsaql sl q)rq^^'saq)led Joralsnl)aql premotlurod pue dn pourlaresalqe)aqf 'pua auo le peielsnl)auro)aqsoq)led eqt Jo lsor! 'pnq eql Jo uolleuro] aql arolaq'lla) Jaqtoure ul (v) '(saurl popualxa)salqe)urt)epue (slodsrq6Uq punor)saq>tedull)e apnl)u!'u1p;olgeqd 'lla) tsea^ tua)saronljLlllMareq polaqel aql u! sarnl)nllsu!l)e snoluaLue;|1'a;r{> lsea{ aql lnoq6norql solqP)pue 11a> saqrledull)elo Ilrre1o69-91 arn6t1

uri0t

']ouueJ s]ueIlEIU pelqulesse aql seaJeqm prdpJ o8Japun ueo sllal '[ert sq] uI 'ruseldopb dprder asnJJIp uef, ^eq] 'ilerus eJe sllunqns esaql eql ulqll^\ (sllunqns asnBseg '$lJIJq Jo lno radBJJSEs e Sulplmq a)II ilelus eql Jo sreq -unu a8relJo flqurasse e^l]padal eqi,(q saJn]3nJ]sa8rel aq1splnq ilec eqJ'azIS e .{po f.1prauafi eJp uolele{so1,b aql Jo selnf,elou uleloJd ur sJalaruoueu ,11aJ pnptirlput aq] ]a 'sraleuloJcllu Jo speJpunq uene Jo suel Suluueds taqto aqf o1 IIaJ aq] Jo pua auo luo4 ,(em aql IIE qJeer dlluanbar; seJn]onJls lulelelsol'(f,

slrunqnsulaloJd ralleus uolJ pal)nllsuo) sl lueuellJ lelelelsov()lo ed^I q)el 'ssaoordUoISIAIp IIec eql Jo pua aql l3 IIef, ralqSnep eql olq peJe^rTepeq uE3 saruosoruorqJ palEJrldeJJo tes elaldluoJ P ,alpurds sltolllu eql lpql os Jo uopetuerJo aql saJuenlJul uJn] uI salnlJnJls uI]lE aJntIC) Suppnq3o elISeql o} sleIJe}EIu aqlJo uoBezruesJopaztrelod eql'(9{I 'ueq] Jaqlo pue IIE^ IIec ^\au Jo uollaJJas eql slserlp uollEZIueSJoullJs slqJ

'suorle)ol rado.rdrraql ot sluauodr.uorpezrsaqlu{s I;mau lrarrpol lla) aql salqeuoleql ualsl(saleurproorleqo;6e aprno.rdpue 'uroDoqaql ol lla) aqr 1odot oqt Luolj Illerrlran unt (uaafi) salnqnlor)rW'6 [ aq lll/v\salnl)nlls ratoeLlfur passn)srp e^rsaqpe luelJodrurasoql:lla)aql lo aprslPseqaLlluo xuleuJrelnlla)Prlxa 6ur{;rapunaql ol uaq} q)e}}e pue taaqs,(prnts e oturslla)1e1laql1da eql l)auuo) leql saulosoulsapluaq pue sauJosotusap 6utpn;rutsornl)nlls a^rsaqpe Jo spurl raqlo ol poloq)ue de pnlq) sluaurelualelpaulralul 'Ipoq aql olu! burleal u.lol;uaulnl aql Jo sluatuolaql lua^ardleql leurtsalur ol satnquluo) suorlrunflla)-llo)6urr-uro; sluaureluurl)eJo pueq lerluaraJun)ll) eqt Molaqlsn|poo] urorJ e'rllr^orfil.l-r eaJe ro, alqepene sluaulnu6urqrosqe a)e]lnslla) aql osear)urleql !ll!Aor)!Lu w)ol (Pu) stuaueluurl)e palpunq 'uaulnl aql 6ulre;'a)eJrns leutlsalur paddn)lerrdeeql lV'aultsolu!lleuls a q l e u ! ll p q t s l l a rl e r l a q r ; daaq l 6 u r p n l ) u l pezrler:ads s;1e> 1osadeqs)!lsual)ereq) aqt a)npold ol aleladoor uololalsot^r aql;o sluauodtuo)aqt llV 'slla) pazl.re;odu1uolalelsoy{r 1e;leqr1de aqlJo uollezlue6rg5-91 a'rnEr3

sluAuellloJllul

utPe

salnqnlol)ltl.J

->s----

sluauelu alelpaulalul

e u r u l e ll e s e qeuosor.!saprureq

auosoulsep

uorpunl sueraqpe ulpe ]o qa^^ leururJal

snllrAor)rul

uola;a1soy(3aql :9I raideql)

oL6

(8)

ll_tsA tN v IV 'rsWSSSVIU Sl_NltAV]H ro

sltNnSns Jo N O t S n T J CC t d V U cNV SINil TV]H

ro A'rStAlssvsro

a)i nos l U a r J l nu e se q)ns ' ; e u6 r s

+ raLuAlod s n o l u a u r e l ra; 6 r e l

s l ru n q n s a l q n l o sl l e u r s

LL6

Ierales ur spuoq leurpnlr8uol Jo sles Suqearq sarrnbar a1ppgr eql ut luerup 'lseJluoJ 'spuoq -g alrsodruoc eqlJo a8e4earq ul IeJelBIJosles oml ro auo reqlle pup purpnlr8uol se>lerulueruelrJotoJdauo ;o pua aqt spuoq euo $lpaJq Jo les Jo 'e3pl?l ]B lrunqns E Jo ssol ro uourppe eqJ lPslleq P ur reqtoue euo punoJE ]slMl stueruelrJolord aql l.1pcrddl ,(1era1u1reqloup euo qllm alerJosse ]Eql-pue ol pua paurof slrunqns Jo s8uuls ruau113uo1-slueurulgolo.rd aldrlpur Jo lno llnq are.{aqt esneceq,ftqrqeldepe ql!v\ q}3ueJ}s eurqruoJ srarur(1odp1a1a>1so1,(3 'aJnlcnJls InJesnssel pue Jrlels B uolalalsot tc aqt Suqeu 'ayqurassesrpplnoo slueuelrJ eq] qrq.tr te elpJ aqt lgrll plnom Surpurq 'sroqq8rau o^at sU ol .,(FqBp.{laruarga punoq sp^ tq8p qcns lueupl.IJ eql ur lpnqns qJEa sselun 'd8raua purJaq] luerqup ,(q a8aparq plo^e o] qlSuarls aql ppo,,u.de^\ sq] q peruJoJlueuElrJ B ]nq 'lla3 crlol.recna ilulus e Jo JeleuPrp >1ce1 aql uuds plnom'pue ol pua dn pauq 'alduruxeJoJ 'sJeurouou u{nqn} puesnoql y'a1g lq8re4s apurs e ur raqtaSol slrunqns Sut8uuls,{ldurrs dq t[nq tou erp sllac SurnrTur slueuelrJ plalalsogr ]snqor aq] 'JaAe^oH 'purq UEOllunqns reqloue lad qcrq,u o] pua ,vrauE saleaJcraudlod eql Jo pue eql o] lrunqns qcpe Jo uo1} -lppe aqJ '1 + zr qfual e aleraua8 ol slrunqns u sureluoc lpqt ]uaru luaurelu Jo 'uouceeJ uorleroossealdruts E se -€lrJp ]ueu Jo pue eql 01 spurq lpnqns aa4 V -elrJE ruroJ o1 raqlaSol slrunqns uralord;o Suuul eql 1\^aI^ue3 el 'praua8 u1

se!uedotdsnoo6elue^pv eldlrlnwuol, paulol sluaulel!l e^eHstuorueluolold 'enoru ol 'sasecduetu uI 'pup aJnlJnJls IEuJeluI alqFeg 1nq pazrue8ro l.1q3rq e urelupru ol IIeo cportrecna e elqeue sutalord frossacce eq] TaqtaSot Surlcv'a1c[c 1ac qrea Suunp rnoco teql uo1a1a>1so1dc aql raSSrrl luql esoql Supnlrur 'spuBts repl eql suorleruJoJsupJl oIlBuEJp Jo -leceJlur pue JelnllaJeJtxa IoJluoJ aq] Japun aJntJnJls p1a1a1so1dcSuuq sutal Jo -ord rfuossacceaq] 'sassecordaseql q 'aueJqruaru eursuld eql pue sallaue8ro se qsns saJnlJnJls IIac raqlo ol ro reqloue auo ol sluauPlrJ {uI 01 puP 'eJroJ alereua8 ol ,{3raua sseuJeq o1 'l.lqurassesrp pue dlqruasse tueruelg Jo sorleuDl aq] a8ueqc ol 'sruJoJllunqns pue luatuelrJ ueemleq suralord raurdlod 3o 8utuo1] -4red aq] aleln8ar o1 'sluaurelg ^aauJo ,{lqruasseJo sells eql aulruJa}ap o} sllun -qns Jleql Jo sluaruplrJeql ol pulq sutalord,{rossacceeseqJ'uol}Je p1a1a1so1,b olur s.{.ervrqledSurpu8rs q8norqt pallacer uol}eruroJul Surl.rarruoc 's}uelu -elrJ aqt Jo ror^Bqeq crueu.{p aqt pup uopnqlJ}slp pqeds aqt atuln8ar suratord 'llao eql ulqll111 zfuossaccepelprJosse-uo1a1a1so$.c lueJeJJrpJo speJpunq 'ue{oJq Jo peuroJ bureq spuoq lueluloc lnoqlr^ Llprder Jncoo uec .{lqurassesrp pue ,ri.lqruasspJleql dpuanb -asuo] ',,sraruz(1od,, p1a1a1so$cgo sad,fi eeJql eql raqlaSol ploq suollsPrelul 'sula}ord pue 'VNU 'VNC Sutpnlcut-sraurdlod luelp^oouou {ea^a 'lseJ}uoc uI pcrSolorq Lueu raqtaSor ploq strunqns rraq] ueemleq sa8eluu tualplo3 'luatuelrJ addl qcea Jo go sa4radord pcrueqJaru pue ,bnqels aql ul se3ueJeJJlp IEJIIIJoacnpord ruaql ueamlaq saJroJ elrlcprpe aql 3o sq13ua4s eql pue sllunqns eql Jo salnlonJls 'slceluoc ulalord apIS-ol-eplspue pua-ol-pua aql ur seoueJaJJrO Jo uolleulqruoJ e Sursn 'alerf,osse-Jles lpq] (gz-t arn8t4 eas) sllunqns Jo sellquasse IDJIIeq sP go sadft eeJql IIV 'salnqnlorolru toJvrunqns unnqnl ruroJ sluetrregl p1a1a>1so{c 'sluaruelrJ pue eJe ]eql sllunqns U4la-ll-lnqo13 ullou JoJ sryunqns lcedruoc 'snoJqIJpue pale8uola Jo aperu ere salnqnloJcru pue sluaruelu ullce seaJaq^ sallesueql eJElpql slrunqns ra[eurs Jo dn apeur eJEslueuellJ elEIpaIuJa]uI '14-gy arnElg) ferte JEJalrs raq]oue 1e ruaql 3u11q -ruasspar pue elrs euo le slueuBIIJ Suqqruasseslp 'suollezlue8roar IeJnlonJls

'leu6rs ut lla)e ut uola;a1soy{> ueo] asuodsat leuralxa prdeg(B)'ulalordlleurs e ulo.U uolleuto1 aqlJouorlezrue6roel lueuelu(V) 'uo1a;a1so/r pale;nbal pue,{lqr,uasse aql adeqsa.r ol ,(;qr.uassesrp tuaulelU uraloldJouolleulloJ sMolle slrunqns uralojdralleusqrnururor]sluauelU aq1'edeqs;;a>u1se6ueqr6u;rnpuola;elsoybeq14-9 1 arn6t1

Sl-Nlt JVlll lvl-ll3)SOIA) lO :lUnIfnUlS )IWVNACCNV AlBl llSSV-JllS 3Hl-

'luaq ^lrsPaale ^aql qOnoqlle /sa)roJ 6urq)lorlsol tuelstsal(;;er:adsa ,{;}uanbasuor are pue (em s;ql ur pauro} aresluaurelualprpaulelul'sl)eluo) leJelel 6uor1sqlrrvrsl!unqns snorq!Jpale6uo;a rror; paruro, tuauellr 6uor1sy 5-91 ernbrl

slU_uldoud t)t-l-ldou

" tr.

a l e u r u r o ps 1 r e l u o )l e r e l e l: s l r u n q n s6 u o 1p a r a 6 6 e 1 s

'sluetu -elrJelerpeuJalur op uBql luaq are ,{aq} uaqa,r,(lsea eJolu qcnu {PeJq sepqnl -orJru 'uosEeJ srql JoC 2ea^\ ,{1ang}EJedruo3 ale JeqleSol sluetuBllJoloJd tI aqt Burploq spuoq Ierelel aq] pup 'spuoq purpnlr8uol [q,{lrerurrd raqtaSot pyaq slrunqns repqop luo4 llrnq erE 'tseJluoc.{q 'salnqntorsll tr'(6-9I arntlg) sarnl -JnJls alrl-ador Suo4s Sunuro; 'Surpuaq pue SulqJlaJls al€Jelol stueruellJ ale -rpeuJalur 'luaruelrJ aql ur paraSSetsare slrunqns 1enpl^lpul aq] asnecag ']Un -qns snorqrJpale8uola qceeJo er8ual aqlJo lso{u ralo puetxe qJIqA\'s1tocpaltoc 'alduexa .rog IEf,rIaq-Duaamleq slJeluoJ IBJaIBISuorls Sururo; Lq alqruasse 'sluatuelu 'slueruElu p1ala>1so1-ri: roJ reJJIp eq] slcPluor elBrperuJalul luereJJlp '(?-t arn8rg aas) raqlaSol tuetue trunqns-lrunqns Jo sadfi pue suorterol aqJ -lrJlEtela{solr{ce ur slrunqns aq} ploq spuoq }uale^oJuou {Ee^\ puE suorlJeJa}ul crqoqdorpLq fueru 'suo1]oeJaturulalord-u1a1ord cgtcads Jaq]o q]Irlt sV ^lproeJ Jncco uE3 slrunqns Jo ssol puE uorl -lpp€ qJIq \ t€ sernlf,nrts cnueuf.p se spue ]ueuelrJ aqt Surneal apqa,t'a8e4earq IeruJaqllsrseJol slueuelrJ p1a1a1so1blsoru s^ olp sassecordoml eseql ueemleq acuaraJJrp,{8raua a8rel aql'19-91 arn8gg) aurp erues aq} }€ IIe slueuegolord

l'ldt-L'tnW 3'18VtSA'l'lVtAUlHl :SlNltAV'lHOt_OUd

'/

pal ut uMoqsele sluaLuelu aql ur raqlabolslrunqnsaqr 6urp;oqspuoq aqf 'sluaueluololo a^u ulotj peulloJsl luauelUalqetsaqt'a;durexale)ltaqlodA.l srqlu1'a6e1earq leullaqiol luelstsalale sa^lasLuaqt siuaureluaql altq/\^'r[ueu,{p aq ol spuaaLllsMolleluaureluolold auo ueql aloul LxolJ]uauelu eJo uottPulol .sPua ;e1e1a1sol,{> rrueu{p ql!tvrs}uaurelll1e1e1e1so1fr 1o(1r;rqe1s leureql ar,llg-9 [ arn6rl

l \r-

r'1€vlsNn A]lvhrulHr.lN3wv'lBoloud 319Nrs

uola;a4soy{3aql:9l. raldeq3

ZL6

THESELF-ASSEMBLY AND DYNAMICSTRUCTURE FILAMENTS OFCYTOSKELETAL

nu c l e a t i o n (lagphase)

elongation (growth phase)

steadystate ( e q u i l i b r i u mp h a s e )

c o

!

elongation (growth phase)

E o

c o E o

c

c

c l f

c f .o f

c

c

o

o

t i m e a f t e r s a l ta d d i t i o n+

steadystate ( e q u i l i b r i u mp h a s e )

c o m i n go n a n d o f f

p r e f o r m e df i l a m e n ts e e d sa d d e d h e r e

0 (A)

973

(B)

Nucleation ls the Rate-Limiting Stepin the Formationof a Polymer Cytoskeletal There is an important additional consequence of the multiple-protofilament organization of cltoskeletal polymers. Short oligomers composed of a few subunits can assemble spontaneously,but they are unstable and disassemblereadily because each monomer is bonded only to a few other monomers. For a new large filament to form, subunits must assemble into an initial aggregate,or nucleus, that is stabilized by many subunit-subunit contacts and can then elongate rapidly by addition of more subunits. The initial process of nucleus assembly is called filament nucleation, and it can take quite a long time, depending on how many subunits must come together to form the nucleus. The instability of smaller aggregatescreates a kinetic barrier to nucleation, which is easily observed in a solution of pure actin or tubulin-the subunits of actin filaments and microtubules, respectively.When polymerization is initiated in a test tube containing a solution of pure individual subunits (by raising the temperature or raising the salt concentration), there is an initial lag phase, during which no filaments are observed.During this lag phase,however,a few of the small unstable aggregatessucceed in making the transition to the more stable filament form, so that the lag phase is followed by a phase of rapid filament elongation, during which subunits add quickly onto the ends of the nucleated filaments (Figure f 6-f 0A). Finally, the system approaches a steady state at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation from the ends. The concentration of free subunits left in solution at this point is called the critical concentration, C.. As explained in Panel f6-2 (pp. 978-979),the value ofthe critical concentration is equal to the rate constant for subunit loss divided by the rate constant for subunit additionthat is, Cc = koff I kon. The lag phase in filament growth is eliminated if preexisting seeds (such as filament fragments that have been chemically cross-linked) are added to the solution at the beginning of the polymerizalion reaction (Figure l6-108). The cell takes great advantageof this nucleation requirement: it uses special proteins to catalyze filament nucleation at specific sites, thereby determining the location at which new cytoskeletal filaments are assembled. Indeed, the regulation of filament nucleation is a primary way for cells to control their shape and their movement.

to Create The Tubulinand Actin SubunitsAssembleHead-to-Tail P o l a rF i l a m e n t s Microtubules areformedfromproteinsubunitsof tubulin.Thetubulinsubunit is itself a heterodimer formed from two closely related globular proteins called ct-tubulin and B-tubulin, tigll'tly bound together by noncovalent bonds (Figure

timeaftersaltaddition+ Figure16-10 The time courseof actin polymerizationin a test tube. (A)Polymerization is begunby raisingthe in a solutionof pure saltconcentration is actinsubunits.(B)Polymerization begunin the sameway,but with preformedfragmentsof actinfilaments presentto act as nucleifor filament growth.As indicated, the 0/ofreesubunits reflectsthe criticalconcentration(Cc),the point at whichthereis no net changein porymer.

974

Chapter16:TheCytoskeleton

P-tubulin R

f,,uo tubulinheterodimer ( = m i c r o t u b u l es u b u n i t )

protofilament

prus eno

1

5U nm

I

mtnus eno d-tubulin

microtubule

(A)

f 6-l f ). These two tubulin proteins are found only in this heterodimer. Each a or B monomer has a binding site for one molecule of GTP The GTP that is bound to the cr-tubulin monomer is physically trapped at the dimer interface and is never hydrolyzed or exchanged;it can therefore be considered to be an integral part of the tubulin heterodimer structure. The nucleotide on the p-tubulin, in contrast, may be in either the GTP or the GDP form, and it is exchangeable.As we shall see,the hydrolysis of GTP at this site to produce GDP has an important effect on microtubule dynamics. A microtubule is a hollow cylindrical structure built from l3 parallel protofilaments, each composed of alternating s-tubulin and B-tubulin molecules. \Mhen the tubulin heterodimers assemble to form the hollow cylindrical microtubule, they generate two new types of protein-protein contacts.Along the longitudinal axis of the microtubule, the "top" of one B-tubulin molecule forms an interface with the "bottom" of the a-tubulin molecule in the adjacent heterodimer. This interface is very similar to the interface holding the a and B monomers together in the dimer subunit, and the binding energy is strong. perpendicular to these interactions, neighboring protofilaments form lateral contacts. In this dimension, the main lateral contacts are between monomers of the same type (cr-cxand 0-0). Togetnel the longitudinal and lateral contacts are repeated in the regular helical lattice of the microtubule. Becausemultiple contacts within the lattice hold most of the subunits in a microtubule in place, the addition and loss of subunits occurs almost exclusivelyat the microtubule ends (seeFigure 16-8). These multiple contacts among subunits make microtubules stiff and difficult to bend. The stiffness of a filament can be characterized by its persistencelength, a property of the filament describing how long it must be before random thermal fluctuations are likely to cause it to bend. The persistence length of a microtubule is several millimeters, making microtubules the stiffest and straightest structural elements found in most animal cells. The subunits in each protofilament in a microtubule all point in the same direction, and the protofilaments themselves are aligned in parallel (in Figure 16-11, for example, the cr-tubulin is dornmand the B-tubulin up in each hetero-

Figure 16-1 1 The structureof a microtubuleand its subunit.(A)The subunitof eachprotofilament is a tubulin heterodimer,formed from a very tightly linkedpairof cr-and P-tubulinmonomers. TheGTPmoleculein the o-tubulin monomeris so tightlyboundthat it can be considered an integralpart ofthe protein.TheGTPmoleculein the B-tubulin monomer,however,is lesstightlybound and hasan importantrolein filament dynamics. Bothnucleotides areshownin red.(B)One tubulinsubunit (cr-p heterodimer)and one protofilament areshownschematically. Each protofilament consists of manyadjacent subunitswith the sameorientation. (C)The microtubuleis a stiffhollowtube formedfrom 13 protofilaments alignedin (D)A shortsegmentof a parallel. microtubuleviewedin an electron (E)Electronmicrographof a microscope. crosssectionof a microtubuleshowinga ring of 13 distinctprotofilaments. (D,courtesyof RichardWade;E,courtesy of RichardLinck.)

[6rer1ra6og url)e paurels 1o,(salrnor'))'sluaLuelu Ilanrle6auJo sqder6orrrr.u uort)all ()) 'uolleluauoeuiesaql a^eq luaLuelu aq] ulqtlMslrunqnseqr llv'ruu 19,(.rana 6ulleadellsrMle qtrM'xrlaqe Jo spueils lallereoo/!{lseraqlo q)ea punorepurM q)rLlM'st)eluo) lerarel,(qraqraborplaq 'sluaue;ryolord orvrl Jo 6urlsrsuo)seJo lq6noql oq osleue) ll 'siaurouour Jo xrlaq a;6urse se paqu)sapuauo srluauelr, aqt qbnoqlly'luauielue ur srououou.ljo 'alnraloLu aql,o lalua) luaua6uerry(B) aql ul ual) daap e ur punoq (dcv lo dlv aplloal)nue seqraruouou]urt)e raLll!a) aqf (v)'luauieluurl)P puPJaurouour u!l)e ue ,o sarnl)nJlsoql Z [-9 [ oJn6rJ uru0s

())

'pua snullu aql pellBc sI pue Jaqlo eql pue 'pua snld aql pelleJ sI 'lsuJ aJEa3e>1uuqspue qtMoJS qloq ereqM']uaruelJ '}selseJsezlJsru^lodep oslB pua SuIMorS e Jo spue o^N eql Jo sIIuBu,{p eJolu aqJ -lseJeql 'uollsJluaouoJ IBOIIIJJaq] Moleq sdoJp uol]eJlueJuof, llunqns eeq eql lPql os pefnlp dprder arBstuauleluJI'(tI-gI ernBIC)roqlo eql uEql ralsPJqf,nlu selp8uole luelu8uJJ qcEe Jo pua euo 'slueIuBIIJ peluJoJeJdJo s}ueu8e4 pe{Ielu '.snql 'Jeqlo eqt lE oluo elquesse o] pe^ ollP sr sllunqns paulJnd Jo ssasxe ue JI 'rrorypue u9l-uopelcosstp ueql pue euo ]e JaleaJBqcnu uauo eJe-^lelllf,ad."t puP uorlPrsossEJoJsluelsuos alEJlllaupl aql 'luauEIIJ JPIod[lleJn]JnJls B uI 'pue qJPe lE lueJeJJIprfua,raq z{erus}uels -uoc alEJ asaql Jo sanle^ alnlosqe aqr q8noql uele (spua oml aql lE lecnuapl 'asPf,slq] eq ]snu gJo4luoq'slueJsuoJ eleJ pJE^DIf,EqpuB pJEl!\loJeql Jo olleJ eq] '(uop u1 'raur[1od eql Jo pua raq]Ie ]e s]runqns Jo uolllppe roJ eruBseql eq lsnur -eJluaJuoJ pue 'eouaJeJJlp eql eJoJeJeql pue) runuqiltnba eql ]uelsuoJ lecrlrJc 'slpnqns d8raua eer; aq1 'srsfloJp^q dIC Jo dIVJo acuesqe eq} uI I + u Jo }uau -EIrJ e ur sllnseJ slrunqns il Jo ]ueluPlg P Jo pue reqlle ol lFnqns E Jo uoplppv 'saler q1,r,ror8 lueuelrJ uo ]JeJJapuno;ord E eABqleql s,(E,vrul ]uaJeJJIpraurf1od qcea Jo spua oml aql seTetu uolleluelro srql Lhrrelod leJnlsnJls sapqruoJf,Iu pue slueruelu ullce sanr8 slrunqns Jleql Jo uoneluelro lalered Sep8ar aql

seleuluereJJlole /v\or9 leql spul l)u!ls!c o^ I e^eHsluaulelllull)v pue selnqnlol)lw 'lueluellJ ullcE IenpIAIpuI uE ueql JeSuoJlsqsnlu seJnlcnJls esaql Suqeru 'raqtaSol ueq] alpunq pue luqssorJ (lrtolaqeas) upJp elpJs-eBre1 'sJe]sIuoJOIlu sue] ana;e dpo suralord ,trossacce '1ac Suurg € uI ]ng 3o t{}3ua1 Jo aouelsrsJad B qrl \ 'salnqnloJJltu pcrrpulrtc ^ olloq eql qlIA\ pareduroc luaq ,i.1seapue alqFeg '(lazrqelar ere stueruelg ultov'4laq papueq-rq31J e uI reqlo qcea punoJe ]suvq leql slueuellJolord lalprud orvrlJo lslsuoc ol peJeplsuooeq uBf, lueIuEIIJ ullJe aql firrelod leJnlcnJls ]JuIlsIp E qllm slueruelu aleraua8 ol Irel-ol-peeq elquesse sllunqns uIl3E aq]'uqnqnl roJ sV'(ZI{1 arntlg) (dCC io) 'epuoelcnu P JoJalls dlg uBqt JeqtBI (dCVro) dJV sI eplloelsnu aql uDce JoJ]nq (ullnqnl e{I'I 'Jarulp e ueql Jeq}EJJeluouo(u Surpurq E spq lpnqns ullJe qJee e snq] sr pue urcqc apqdaddlod re1nqo13 aputs e sI lrunqns ul]f,e aqJ 'pue Jeqlo aqt pasodxa surpqnl-fl pue pue auo le pasodxa suqnqnl-n te 'eJoJareq; '(rarup qlp\ ,quelod prnlcnrls ]Jullslp B seq JIeslI eFqnlorf,Ilu aql (8)

(v)

pua snu!ur PUA SnUrUr

( l u a u l e l ! ,u ! uaq/v\dcv) dfv

puasnlo puesnlo

a l n ) a l o u ru r p e

sINlwvlH lvllll)sou)

sL6

lo lunl)nuls )lwvNACcNVll8l llssv-llls 1Hl-

rxr I

tpql .{la{II sI tr uaqt ,{lprder Suu,ror8sr }uauEIrJ eq}JI sr rer{r 'q8rq sr uor}rppp }run -qns Jo eler aq] 'uorlrppe ]1unqns atpr aqt qllm paredruoc srsdlorp,{qsrq} JI Jo Jo alpr eqt uo spuadap uroJ C eqt ro I aqt ur sr turuelrJ e;o pua.{ran aqt te tgnqns aql raqIJqM 'epuoalJnu punoq rlaqt paz,{1orp,riq aneq o1 are ,{aqt ,{1a111 a.ror-u aq] 'alrllel raru.r{1odaqt ur ueaq a^Pq strunqns }pql atup aq} raSuol eqJ aoc pup ploJ-ua] pup ueql raq8rq sr qloqJo uorterluaJ ]Eq] lnoqe dCVJo dJC dJV 's11ac -uoJ aa4 aq] se 'uJoJ aql ur aJEslrunqns ae4 eq] 3unr1 u1 J Jo lsotu .,r,ror8sraur.,(1odruro;-; a1q^a{urJqs aloJalaq} ilr^ sraru.,{1od ruJoJ-C'strunqns aa4Jo suolterluaJuoJ ureual JoC'(J)rJ ueq} ratuar8 sl (C)'J 'snq; 'raru.{1oduro;-1 aq} roJ oper Supuodsarroc aql ueqt ra8rel sl 't(O)'Jl uolteJluaouoJ Iecrlrrc s1ro1 pnba ,r(lpcrraunu sr qcrqm 'raudlod ruro; 'raru,(1od 'ory -C rory aql JoJ urroy-1 eqt ruo4 }lunqns / Jo orler eqt -{ltuanbasuo3 e Jo uorlerJossrproJ a8ueqc .{8rauaaa4 aq} ueq} anr}e8auaJoruraru.,(1od uJoJ-O aqt tuo4 llunqns e Jo uortprcossrproJ a8ueqc .{3;aua aa4 aqt sa{Bru srqJ 'acp}el raur.,(1od aqt ur parols sr puoq aleqdsoqd-aleqdsoqd d8raua-q8rqaq] Jo e8e^eelc ,{.qpasualar d8raua qlnu 'pazd1orp,4q aa4 aq} sr aprloalJnu eqt uaql\ Jo '(urFqn] roJ 'urlre roJ 'ultrE roJ punoq aql qlylr euo pue '(uInqnl roJ dcc dcIV) c,, ,,luroJ dJc ']sua uef, sernttnrls dJV) punoq eprtoalcnu aql Jo ,,ruroJJ,, eql qtl^\ auo lueru -EIIJJo sad,ft luarag;lp omt 'snqJ ('ZI-91 arn8rg aes-trunqns oqt Jo raluar aql reeu Ualo e ur daap sr eprloalJnu aql 'urlJe ur sEeJaqM'11-g1 arn8rg aas-slrun -qns Surroqq8rau o,r,rl uaamlaq aceJreluraq] te sarl alrs Surpurq-epqoalJnu aql 'ullnqnl uO) 'eJntJnJls luauelrJ aql ur paddeJl surcurer aluqdsoqdrp aprsoalJnu aql lnq 'llunqns qJea uorJ peseelal sr dnor8 aleqdsoqd eaq aql lsrncco srsf -1orp,{qeprloalJnu 'tueruelrJe olur trunqns uqnqnt Jo urlJe ue Jo uoueJodrorur rage.{llroqg 'slueruelrJolur palerodrof,ur aJEs}runqns aq} ueqm pe}pJaleJtesr ll 'ra,ra,r,roq:,r{1alo1s t(ra,nspaarord srs.{1orp,r(q 's]unqns aa4 aq} rog dlanrlcadsar slql 'aleqdsoqdrrl aprsoalJnu e AJC ro 41y ;o srs[1orp,{qaqt az[1e1et uEJ teqt saru,{zuaqloq aJEs}runqns uqnqnl pue urlce aq} 'sraru.{1odlualenocuou uroJ ol dllflqe rraq] o] uourppe ul 'sapqnlorcrur pup s]uauelrJ ur]ce qloq o1 sarldde teql IJEJ IeJrlrJJ e parou8r seq scruuu,{p tuaruelrJJo uorssnJsrp Jno 'rEJsnqJ

ull)v pue utlnqnl{q srs{;orp/(H ept}oal)nN Jo safuenbasuo)erv fil;lqelsu;l!ueu^C pue 6urllrutpeatl luaueltl 'JalEIssnJsrpa^ sE 'slleJ alrloru go a8pa Surpealaql apnrlord dlaq sluaurepyu4ce Suqe8uola ,{lrepurg 'aseqdeue Surrnp sralsrsrraql ruor; de^apseurosoruorqJJrlolnu pd dlaq ueJ selnqnloJJrru Suqurrqs pue 'seueJqruaru lno qsnd dlaq uec sepqnlorcnu Suqe8uola ,a1d -ruexa JoC 'peol peqJeue ue 1nd to qsnd o1 tepcrged ur-{Jo^ pcrueqJaru op ol uorlezrraru,{1odapro uor}ezrraudlod luarueg snoaueluods Surrnp peseelaJ d8raua aa4 asn ueJ IIac aql 'snql lsassacordsnoaueluods asar{}ol ssacord alqe 'orez ueql ralear8 sr a8uuqc .{3raua -roneJun,{lpcqa8raua ue aldnoc uet IIaf, V ea4 slqt uaqa,rllsnoaueluods spaacord uorlezrraru,{1odap}uaruplrJ ,asu a{r.I 'uorleJluaJuoc IeJrlrJJ aql spaeJxa uorlnlos ur slrunqns Jo uorleJluaJuoJ eql ueq ^ aseJ aql sl srqJ 'oJazueq] ssal sr lrunqns elqnlos aq] Jo uoUrppB roJ (gV) aBueqod8raua ae4 aq] uaqm,{lsnoaue}uods spaacord uorle8uola tuauelrC ('luauelrJ aql Suop punoq uaq^ speaq urso,{rugo acuereadde peaqarrorreaql Jo asnef,eq ,spua ,,pa1urod,, se spua snurru pue 'spua ,,paqreq,,sE ol parreJer ,{lensn JJE s}ueuel.rJurlce 'suosear 'pua snunu eq] pJpmol slurod Jaruouour Jo spua snld aql IuJrJolsrqJoC) aqt uo galc Surpurq-dlv aq] 'slueuelrJ ul]Je uO 'pua snld aqt te pasodxa aJE slrunqns fl pue 'pua snurur aql ]e pasodxa are slrunqns n ,selnqnlorJrur uO 1{srrog(re9,roIsalrnof)'qder6ortrr.l srqlugdolaqllp puaeqr'alpunq alnqnlot)Lll aqlJopuasn;daql 'suotltpuo> urorJ nnor6 sa;nqnlol)tW 6ulzuer-uI;od rapun sltunqns ]salspJ urlnqnlqlrMaLutl seM(ralplpassn)stp) urntlt)pJo Uoqse JoJpalpqn)ut

salnqnlol)t rx paujlo+ ^l^^au

a r o ) a q ] u r o r Jp a u t e l q o s a l n q n l o t ) i l . I ]J o o l p u n q a l q p l s e , a s e ) s t q l u l l a L l l o aql u e q l p u a a u o l e l a l s e J m o i 6 s l u e r . u e ; lu; t ] ) e p u e s a l n q n l o l l u l q l o g l e 'pue sn;d aql tp solnqntortru 1o qynolb lpllusJalald aq1 g 1-9 1 arnbrg

uola;a1soy(1 srll_:gI reldeq]

916

uld OI

qllM'/66 t 'vEt-LLtt6El aqf uot, uolsstt-uled (ssar6ArsranlunrallaJa))oU 'C'l pue lalols-ueulraleM'W')Luoll)'I1t1tqe1sut 'lo!8 lP) T'uoules s{e|ds1p oslepue snldaq1'sluuqs(ryalaq} uo) pua snuluraq} pue rrr"ueu{p pd aqlAq pate)lpul snofi (lq6paqt uo) pua sn;daql elrqM'(peaqMoile e i(q umoqsse)^reuollelssuleural allltel alnqntol)turaqt ulqllm)reLupue; 'U6!t ot aq o1sleadde arlllel alnqnlor)ruaql '])ej ul ']nq Ualuol; 6urpr;s uMoqsalnqnlorllu aql'ejaule) )luoll)ala anlllsuase qllM pa^losqouaql aqf'lua)salonuseM0z ul seMsalnqnlol)lu lenpl^lpulJo o)ue)salonlJ 'au!ulepoqla{p lualsarongaql o} ]eqt os l!unqnsurlnqntI I;aler.urxotdde seMlla) y'11ar6u;n;1 palur;,{1}ua;eno) uoaqpPqleqr urlnqnlq}!Mpa1>efur p u! pa^Josqose 'alnqntol)llue Jo ,o!^eqaq6url1rutpear1 91-91 ern6t3

'(9r-9r

) a s0 a u r ]

' 6 u r ; ; r u p e aur lt 6 u t 1 ; n s e t ' s l u r l q s pua sn;daql pua snururaLllolrqMsrrnot6 'sanleAoMl asaql uaaMlaqaraqMatuos s! uorlerlualuo)lrunqnslenl)eaql ,l r.urojc aql u! pue luauelu e lo] ueq] raMolsruilo, I aLllur pua luauelu e uo roJuorlerlua)uo)lefr}tl) uorlezuar.u{1od aql'strunqnsaaJJJo suollerluo)uo) (g) alerpau,ualur le sJn)lo6ut;1tupeatl 'turo, c aLllul aJepue slql l p s l r u n q n lse u r u r a o l s p u e ' p u as n u l L u aql le uoltebuolaueql ratsejst sts{;olp{q 'ra^e^oH 'rlroJ aql ur s^eMleorojalaql I arepua s!qlle sl!unqnsleululol aql pue 'a;dr,uexa s!ql u! pua sn;daqr re s1s{lotpIq uelll ralsejsruotlebuo;a'srrno.r6 1uaue1t1 aql sV'a)ruelluaueltj aqr u1srs{lotplq aprloal)nuobrapunuaqt pue'luauJelU p Jo spua qloq le azuaur{;od 6urniror6 (sl!unqnsuuro;-1) aleqdsoqdtll e p t s o a l ) npuu n o qq l l Ms t r u n q n s ' s p u a snuru pue snldaql te ()) suotlellua)uo) oql loJ uorleueldxl(V) le)rlrr)luaroJJrp 'uotttppe l!unqns s/'^ollo,leql s;s{;orp^q aleqdsoqdrrlaplsoal)nuaql Iq olqlssod apeu 'alnqnlor)lurro luauellJ u!l)P aql 71-91 a.rn6r1 ue 1o 6ur1;rupeerl

eJntIC) selnqntoroltu lBnpl^ryul roJ slla3 a^ll q pe^Jasqo aq uPf, Sugprupearl (uIl3Broloqln ?l?pa^resqo ueeq seq slueluellJ a{ll s{ool }eq} uoueuouaqd P puP a18urs;oSulpupearl eq] 'uleUecun sI IIeo eq] apISuISuqprupea4 Jo lualxe eq] aleqdsoqdr4 aprsoalsnu Jo {rrroJ aq} ut -{8raua;o uopdunsuoJ aln11,1:srs.,i.1oip.,tq ]uplsuoJ B seJrnbal ,,8urruupearl alels-l.peats,,slqJ'peSueqJun suEIuaJ lueluP -llJ eql Jo rp8ual lPlol aql ellq^a 'salels snolueluellJ puu ee4 eql ueeMleq '{lprder 'pue eql snullu le a3E{uuqs luelu€llJ aql sasuElPq alcrtc slrunqns eql '^\oN 'uolleJluecuoJ llunqns alElpauJelul ,(1]cexapue snld aq] le q] ^or8 luaurug eq] repcqrud e lV'elqlssod SurqnupeaJl sa)Pu,rtqaraql pue eFqnloJOIIu ro ]uaru -EIrJurlce eq] Jo spua snullu pue snld aq] le suollJeeJ uollelcosslp/uollelsosse aqilo,{8laua aargaqt uI eouareJJlpaq} o} aslr sa,tr8dea,taqt Suop sJncso teql stsd -lorpdq dIC ro dIV eql'turoJ C aql ut pua snuru eql ruoq peqs pue urroJ I eq] '3uq*upearl Surrnq ,rt rjrrXloa Jo pua snld aqt tu petrnr3al arp strunqns '(Z-gt "qt dlra Iaued pue ?I-9I arn8lg) Bullprpuarl luarueg;;o -dord alqu4reual eql o] speal sIqJ 'pue snulru aql ruoq sllunqns sasoldlsnoauel 'sl -lnurs pue ,pue snld aqt ]e strunqns sppP lueIxBIIJeql-(pue snulul eql ]uql) turoJ G aqlJo uorlEJtuaJuoJ IecllIJJ aql ueql Ja^ ol ]nq'(pua snld eql'sI lBq]) urJoJ J aql Jo uorleJluaJuoJ IuJrlrJJ aql uPql raqSrq-a8uuJ elulpaluJelul ue '(Alquesse pre^\ol AIIpBaJ ur sr uounlos uI sllunqns aa4 Jo uoneJlueJuoc eql JI eJolu supel uJoJ J eq] allqm .{lqruassestp pJe^ o}.{1puar eJoru sueel IUJoJC eql ,spJo^\Jeqto uI) 'tuJoJ aql ueql uolleJluaJuoJ IeJIIIJOraq8q € seq uJoJ C eI{} J ,paureldxalsnf sy .uortutuJoJuot c eql sldope pua snulul aql elq ^ 'uolletuJoJ -uoJ J aql q sulpureJlua1llelg aql Jo pua snld aqt 'asEl slq] uI 'pue snulu aql 'pua snld aql 1e srs'r{lorpdqeppoelf,nu epuoalsnu ueq} re^/Iolslnq 1e srsr(1orp,{.q 'sllunqns ueql JalseJaq 01uopppe llunqns Jo alEJaql JoJalqlssod aloJeJaqlsl 1I '(Z-9I ae4 Jo uorleJluecuof, elelpalurelul uu tV leuPd aas) spue o^\l aql uaao\leq asuareJJrpIernlJnJls p Jo asnPJaqpue snulu aql JoJueql luaruBlr; u;o pua snld ''! ]uelsuoc eleJ aql pue uolleJluef,uoJ llunqns aql JoJJelsBJqf,nlu sl "oZ aqJ aaJJeql Jo lrnpord aq} sl lueluellJ e Jo pua aql ]e uoFIppE }Iunqns Jo e}EreqJ ruroJ c aql uI aq ueql ilI ^ ]ueIuEIIJ '/\,\olsI aqf 1o dp eql puE 'pappB sI ]Iunqns lxeu eql aroJaq Jncco ^EIu sls'{lorpdq 'urro; 'doc I uoDlppe lpnqns Jo aleJ aql JI tanarvroll 419 rc dac dJV ue Suturo; 'pazl.1orp,'i.q uaeq seq llunqns peppe eql ur sureuar raudlod eqtJo dll aql leql os ^,{.lsnonardaqt ul appoalcnu aq} eroJaq raru,{1odeq} o} uo pp€ IIIM llunqns lreu e s r n ) r o6 u r l I L u p e a J l (a)5 >f > (f))

(g)

rol

a 6 u e l6 u t 1 l t t l P e a r 1

(v) (a)5 > (I)) purqeq s6e1srsr{lorp{q - l s e l s l u o r l r p p ep u a - s n 1 d

uorlerlue)uo) l r u n qn s

dn saqrle>srsf;o.tp[q - r l t o l s s l u o l l l p p ep u e - s n u r u

TT

-_r

+

sls^l0uo H clMo]'lol N

a rl

( C ) u l l o +C p u e ( l ) t u r o l l l o a r n l x l u e a . r es r o u r ^ l o d ( I ) u l l o +f u l a l e s l l u n q n se l q n l o s

lHl slNlwvlll lvllll)soIA) lo lunl)nuls )lt^vN^cONVAISWlSSV-llls

LL6

']ure.rlsuo) stLll sa^oulaj uotlezuot!^lodutlnqnl pue utl)e lrunqnsror|'9v oq] 'alolaraql 'le)tluaptst uotlet)osstp Jo+e+tunqnsaq] 'pua raq]te saruedLUo))e ]eql stsllolp^qaleqdsoqdulaptsoal)nu aql ,o olels leur+aq] pue }e lsol st ltunqnse uoqM ualolq oJe ')uuqs suorperolut llunqnseulesaq] I;Dexa asne)oqsrsrql .(srsIlolpIq319 spuaqloq ') > ) rot 1nno.r6 spueqloq ,) < lo] ,snq1rolsp+ ro ou) uotpeor uorlezuatu{;od eldurs q}oq ) e Jo+ spua dIV }e aulesaq} saultllno+ )uuqs oslelsnLU}t ,puosnutLuor.llueql latse+saull eq 1snur-) erueq pue-uo/ory orlel treql lauAlod aql spuasnutLU lo lnortsnnotbpua snldaql+t :spuaqloq 1e le)tluaptst ,puo oq] qltlvr pue snld aq] JoI sanle^luola#tp a^eq uo) q6noq1uan3 lltlvr,lo)pue ,ssol uorler)osse 'spuooMl aql slt to+ utnlrqr;rnbe aql luelsuo) souturlalap q)tqlvr o] ppe slrunqnsq)tqM}e solel aq] spo#e a6ueLl)leuotleuJroluo)stql

tau,{;od aq} sla}ua}! se ]runqnsq)ea +o uotleulloluo) aql ur se6ueq>Iq e;qrssod apeursr spuoonnleql le qUnotb+osalel aql ut a)uala#tp aql puo snutu aql polle) st pue bulnnot6-anols ar-{}seaJoqM 'pua snld oq] polle) sr pue 6uranol6-lset aqt.saleJ luara#tp le azueur{;odalnqn}oJ)tLu Jo }uotuelt+utpe ue }o spuaoA } oql

s o N ls n N t nc N Vs n t d 'srollrouotuo1 ))eq I;quassesrpo1 anp teullod aql 1o e6eluuqs aql so)ueleqI;asl>atduotltppeJaLuououlo1 anp leutfi;od aqt 1o qynol6 oq] uoqM poq)eal st ,alelsIpeals.ro ,aseqdu.rnuqr;rnbe aq1 'uorlebuo;a Uu r n n o t 6 luoueltl 6ursner'luauiel6 oql lo spuapasodxaaq] ol ppe srauouorxse srnl)o aseqdqlnnot6aq1 auJ!] uorleol)nuro+ ua)el autl ol spuodsettoteseqd6e1eql

wntu€tltnol :asJno)eur] 6urnnol;orr eq] sMoqsI11e:rd{1 tunl1e6elrt let:e})eqe lo }uauelt+ e se q)ns.raur{;od1e>r;eq ;e1a1e1soy{: 6uo; e olur urelo.rde,to {;qLuesse eql

NOtlVZtUltAAtOd lo lsuno) tmtl 'sluauelu utl)e Jo solnqnloj)tut pezuau{;od {pea.r;ertos1uaut6e.r,r se q)ns ,tol)nuapeuratd6urppeIq {;el1ua poqstloqeJo pa)npoJeq ue: aseqd6e1eq1 .uotlezuautfi;od 6ur.rnpuees aseqd6e1eql sureldxaqltqM lvrols{;enr1e;a.r st snal)nue 1o I;qurasseaq_ ' a u r e so q l s re ; d r : u u d

'(Eh-€ernbrl oas:uotlrppe ltunqns io+ luelsuo) unUqtltnbeaql s!) olor.lM) L

*"\

_J

uo) 1lo1 = 3

. u l n u q t l t n b sat q l }v 'ssol +runqnslo aleJ eq+s;enbeuorllppe l!unqns+o aler oql uorlerluo)uo)s!ql lv .()) uorlerlua)uo)le)tllJ)oql palle) 'onleAluelsuo) e saq)eal1r;r1undo.rpol panlasqost) pue 'dn 'sanol6 pasn oie sltunqns letu{;od aq1sy ') uo puadap soop (so)) aler luelsuo) lou leql p le pua Jou^lod oql a^eal lltnns]tunqnsaql 'Ouo)) lnq ltunqnsaol+oq] +o uotlelluo)uo) oql o] leuotyodotd aq lltlvrpuo)os red (a;nqnlotlul ro utpe) loul^lod luaulelt+ aq] ol ppe leq) sraurouor.ll ,|'oJaquinu aqf

NOtlVUtNl)NO) tV)U_tU)I HI

( s l r u n q n s! + u q l l M ) r e r u ^ l o d

.',Il'",

sDe a.rolaraqlq)tqM leuu] stLlloluo e>e;da>1e1 ue) uotltpperaulouourJaLlunl (slrunqnsu qllM) Jeul^lod

'olqelsaJouldnot6 elrlua sa)eu loulll e uJlo+ol loulououl utpe pllql e lo uotltppe+nq loqlo q)eo o] _aql {;1eennI;enr1e;e.r putq soln)aloulut1reoM} ,utpe }o ase)oLl}ul .s}tunqnslua>efpe uaaMlaqspeluo) ardrlrnLu {q pezr;rqe1s srlau.rr;odre)rroqv NOllvll)nN

'(1_>as slrun)#o) {q uanr6sr 1o ssol1|'oalel aLlI't_)os 1_u\| jo sllun seq q)tqIV\ ''o) ]uelsuol aler oq] Iq uanrbst (slaulouoLu palle))sltunqnsasoql+o uotltppe+o olej eq1 'teur{;odaq} +o spueaq} }e s}tunqns+o le^ourarpue uor]rppeeq1Iq (sazuaufilodep) solquassestp pue (sazuou^;od)sa;qu.resse 'olnqnloJ)tule lo luot!elt+utpe ue se q)ns 'saln)oloruurelo.rd,roteur{;odJeautlV

S]IVU IJO CNV S].LVUNO

-

{1r;rqelsur rrueulp palle) uouauroueqde 'Ilquassesrpprder}o porJade pue qynorb Mols+o por.rade uoaMloqalpurolle aro+araqlue) salnqnlor)il,!lenpr^rpul

eNt)NtuHs

O

O

oe) dI9 'sansuauorlezuaurllodapuaq+'lsol st lt,t lnq 'qporb sro^el de) 'utlnqn+ dI9 V dl9 6urureluo)auo uol+ ueql ullnqnl 6q9 6urureluo)pua ue uloJl Jolse+soulrlO0l lnoqe azuetuI;odepsa;nqnlotrryl

Ar_nrsvlsNt )IAVNAO '6ur;;rr.upeerg se uMou) lauIlod aq1q6nolql slrunqnslo xnl,t]au e sr oraql qbnoql uana'q16ua1 luelsuo) e suteluteul laruI;od aql'aler le)rluoprue le pua snururoql 1e{;quassesrp1au e pue pua snld eq11eI;quesse1eue o6.rapunslrunqns'e1e1s fipee1s stql }v'pua snutulaql ro; ) nno;eqlnq puo sn;daqt lo,r)3 enoqesr leql anle^ e soq)eorrauouor.uaorl +o uorlerluo)uo)aql ltlun spaaro.rduorlezueu{;od 'pasodxeale leufi;od e lo spuoq}oq +t 'snqI (pua snld) ') < (pua snurtu))

t

:leq] os lauI;od aql ,ro spuo qloq ]e oules aql oq lou paau uor)Poq) orler raql 'suorl)eal luaJo#rp o] Ja+ar'or) pr" #oo.>1 aruls :rar.u{1od oql +o spuoonn+aql le uorlelluo)uo) le)rlu) eq1abueqr ol sr uotleullol rau{;od sarueduorre1eq1srs{lotp{qapt}oal)nuaq1go aruenbosuo)oug

' s l u a u i e l ru+t D e u l aleuruuopeld Ieu 6ur;;rrupeallsealaqM 'salnqnlor)rulut aleurulopatd panar;aq o1 sr I1r;rqelsur >rr.ueulq'srsI;orp{qeleqdsoqdul aprsool)nuqltM polet)osseeJe qlog 'sretur{;od ut pa^lasqo 1e1a;a1soy(> sror^eqaqoMl ore gNl'l'lllAcvlul pue Al-ntEvJ_sNt )ilAVNAC

'alnqnloJ)il.ue uo de) dfg e lo ]ueuelt+ url)e ue uo der 31y ue-eleqdsoqdul aprsoal)nuaql 6utuleluo)sltunqnslo 'suotltpuo)q)ns ,,Cle)ile seq pua aql repup paz{;o.rp'{q sl eprloel)nupunoq roql q)rq/V\ le aleJ aql ueq] lalse+oq ue) alnqnloJ)tLU Jo luaueltl urpe 6urnnorb e ol slrunqnsto uotltppe+o sleJ aql

'(

-ffi)

aar+ lrunqns

aql lo uorpeal ebueqrxaaprlool)nue Iq peqsrualdar aq lsnu pezl;orpIq sl ]eql dI9 Jo dIV or.llasneloq'unuqr;rnbeanrl e lou pue alels {pea1se srsrql uo,)

H;i

=5

ro

# o o 1= ) u o r )

uaqm sosea)qlnnot6teur{;od}eql os 'lleus fuen{1;ensnate Iaql o)u!ssor) pue uo6) arou6ruer enn'sasodtnd ontlellsnllt.ro1') =;r;r1un nno.rb oq},aro+aqsV 1;rnnraurr{;od

:A1uopua sn;daq1le sluana6uueprsuo3 'sa^eal leql urol ffi aql pue lueuelr+aql o1 sppeler{} r.uro+ a q l I l l e n s n s t] l ( t u s t u e q ) o r u a ; q r s s o d e l o + 9 t - 9 1 , a . r n b r 3 e a s ) l u a u e l t + oLll+opuo q)ee [!oJ] o]pr)osstp o1 I;e1r1aJoul]t so)eu pue sltunqns6urloqqbreu rol lrunqnsoLll+o Ilrur#e 6urpurqaql sa)nparoptlool)nu punoq oql;o srsllotp{g (dcD ro 6 6 y 6 u r r t r e rr a u r o u o r x= C ) (d19ro 61y 6ur{rrer raurouotu= l-)

r a u r { ; o du r } r u n q n s

____.;s1*__-____ __

rau{;od oq} olur salquossealn)aloutorl} la4e uoosoln)olotudOg punoq I;1q611 e ol paua^uol sl leql dI9 punoq I;1q611e satle) oln)olotuutlnqnl qree '{;re;rur5:rau{;od oq} o}ut ,{;qruesse s}tlal+e uoosaln)aloul6qy punoq I;1q611 e o1 pez{;otp{q st }eq} eln)elor! dlv punoq {11q611 e saure)aln)olorr.l urpe q)el

sdv) dt9 cNV sdv) d-LV

sts^touoAH lctlolt)nN

crueu,tp eprsoalonu Jo stunorup a8rel az,{1orpr(q lsnur ilac eql .{}g11qe1sur ro Suqprupear] Jaqlra ;o ssacord e Suto8rapun eJB qf,Iq ^ Jo lsoru 'saFqnl -oJJrrrr P uleluleru o1 'd8;aua pup uolleJluaouoJ sluetuplg urlce luulsuol Jo 'acue13 }sJIJ rV Jo alse^ e e{rl suaas slueuelrJ Jo Jor eqaq crueudp aql 'selnurru Jo spuof,es suel l eJ Jo e dpo ro; Suqsrsrad stuetuelu lenpl^rpur qlyrt'plder fUectd& sI ralourn] luelue -lIJ ul]f,e ]eq] laoqs upc auo tanartoq 'r(docsorclu af,uef,saJonguo peseq sanbru -qca1 alerrdordde qllM 's11acSuurq uHtl ^ stueruellJ ullce pnpIAIpuI Jo spue aql a^loseJ ol llnJIJJIp aloru qJnu sr 1r '3ut1ced Jesuep pue ezIS rellelus JIaq] '(ZI{I ctureur(p }lqqxe ol etup IEer uI uees aq ernt;g) fi111qe1sur Jo asneoeg aJoJaraq]uEJ selnqnloJsrruIBnpIAIpuIJospua eql'satnultu a,ra;drana aSelurrqs pue qluo.r8 uaemleq seqcllms 'spua Suquuqs pue SuunorBsll uaemlaq sacue -JaJJIpleJnlcnrls rofeur q]!vr 'alnqnloJJltu e flpcrddl;1ec 3uurl e apISuI s]ueu -elg ul]f,e pue seFqnloJJIIu eql arc crtueudp arrog '1oso/o eql pue sluaruellJ eql ueemlaq alcl.car r(puelsuoc sllunqns IEnpI tpul allqm 'lualuoc luauellJ IIeJaAo arues eql uIBluIBIu ol ilac e ,tlrolle Sutqnupea4 pue .{tlrqu}sur cnueu.{p qlog

qder6orrruuol})all)'Ualleddn aql uo uMoqsst sluaulelualetpauilalul uoll)ala uV'uotl)alp ;o qder6ol>tr-u q>eaur 6urlurodale sraulrpasaql,oJleH 'uorl)as-ssor) (slto) palo) Zt) sreLutp ut '^ellp 9 [ spqq]tqM le)tlaqe ut laq]a60] pel)ed areslaueltal'luoulelualtl-odol r.I,u-o t leuu aLllul (l) taulpllat lei.lloue qllm ater)ossp o1116uranol;e {qalaql laqloue auo ol l)odsal q]!M lasJJoaie sraurpoMl aql'iaruerla]q)ea ulr.lfn (C) 'sluaurplu aletpaurlalut Jo ltunqns alqnlosaql stleuretlalaqf'suteq) aprldadllodlnoJJoraurellellalleredrtue ue ulo, ol aptsIq aprsdn eur;uaql sraurp o/1/\t()) 'lto) polro) e otur toqta6o] punoMpue lalleledut pau6tleele sureuop poi lellual pa^losuo)aql q)!q^/\ u! '(g)rauJrpe uiloJol laurououl le)ttuapl uP q]!M srecl (v) ut un/\or{s leulouoru eql 'uo!l)nrlsuo)tuaupl!J olelpaurelu! ,o lapour V 6l-9 t arnFll

'aleqdsoqdu E ulsluoc tou op sllunqns e JoJ ells Sutputq eprsoelcnu '(6I{I eJntlc) Je(uouotu lueuBIU alErpauralul aq] 'uqnqnl Jo ur]rE eql e{Iun ul]ce eql 01 ro teurp ullnqnl-dD eql 01 snoSolEue sl leq] ]Fnqns elqnlos eqt sluesaJdarraruEJlal srqJ'JeluPJla] pala8SetsP tuJoJ ol uolqseJ IellEJEdIluE ue ur salercosseuaq] sJarurp IelleJEdJo Jred v 'Jaruouoru Jaqloue qlll!\ IIOJ pelroc lallEJed e sruroJlBq] ureuop lBsrleq-}] IeJ]uac pepueue ue q]l.4i\selncelou pale8uole erp sluaruBlu alBlpeuralul Jo seppded.{lod Ienpl1\Ipul aqJ 'slueuelrJ elprperuJelur f,rrusEldoltf, 'eII -edor eJnpord o1 panlona eAEqsaleJrldnp aqt pue 'palecqdnp dpuaredde aneq seue8 urtupl 'uoltnlo^a upozBlalu Surrnp selull pra^as '(ZI JaldPqJ uI pessnc -s1p sl uorsrlrp IIac Suunp Jorlpqaq crrupu,{p JIeq}) salod JpalJnu puu seuos -ouorqc JoJ selrs eBEJoqJueapr^ord daq] araqrvr'adolanua JeelJnu Jlloftecne eq] Jo euEJqrueu Jeuur aql 3uru11lromqsaru P urJoJ luql sureloJd lueIuBIIJ elPrperuJelu arp sulluel Jealcnu eql'sulluol JOaIJnuluele^erd aJoru qcnru aqt 'sJolsecue pelelal elelpeluJetul JlruseldoilJ dlasolo eJE s}uaruellJ Jrar{l ot 'sleruruE Jerqsmbs eq] roJ sanssll 01 qlSueJls lecluPqf,eru 8uI -uedurr ur aloJ ]uEtJodril uE,{eld sluauelrJ alerperuJelul teq} sueas }I 'sruJapo -urqJa puB spodoJque se qJns suolelalsoxa prSu e^Pq lPql sIPIuIue uI punoJ lou ,(lerauaB aJp pue 'ssaJlslecrupqJaru o] tcafqns eJBleql sllao Jo ruseldoldc aq1 u1 'slueluellJ alelpelurelul qJns luaurruoJd dlrepcqred aJBslueuellJ elelpalurelul ureluof, ]ou op 'elduexa JoJ 'ruels^s sno^Jeu leJlues ateJqeue^ aq] uI ula,{.Iu uolalelsoil) aql:9 [ raldeLlf

v86

s86

uroI uaql qJIqMJo o^a] 'sJarurpoJe]equJoJ eseq] lsuleqJ ulleJa{ (clseq/leJ}nau) II ed,{t pue (JrprJe) I ad.{1 Jo arnlxru pnba ue 1o dn apelu sI tueruellJ uqera4 zfuang'surleJa{ lJullslp 09 lnoqe aq -,teruereq} }eql palea^al seq acuanbas auroua8 ueunq eq] Jo srsdpue isleu pue Jrpq o] cgrcads aJB leql aloru 0I 'sllec pleqtrda uerunq;o sadfi tnoqe pue 1uara;;rpu punoJ 0Z rnoqe are araql :,,(pure;tueruelrJ alprparuJelur esJelrp lsotu eql eJe suIlBJaX '(I-gI aIqBI) saddl ilac tualaJJrp ur passardxa aJE sluetuelrJ alerperuJelul Jo seIlIuBJ ]ueJeJJIC -{1}ear8 .[ren uec sureuop repqolS purruJa]-J pue -N aql lnq 's{uJoJosr}uaJeJJrpaq} ur Jplrurrssr ureruop slqJ'(6-t arn8rg aes)aJnlonJlsIroo-pelloJpepuelxe uE ruJoJ 'uqnqn] Jo leq] sJllour leadar peldaq os Jo 0t seq ureruop pcrTeq-n IeJlueJ V ullJe Jo sruJoJosraql ur sJncf,oueql suJoJosl lrunqns aq] ur uorlerren acuanbas aroru ,{1eque}sqns q}yrr 'sad.,i.1;o,{.}arre,r. eplirr e ur aruoJ s}uetuellJ e}Brpaurelul

s l l o) leuluV or ^t!llqersle)!uPqlawllPdul sluauel!l ele!paulalul 'sllJJ eseqt ur sualsfs 1eta1a1so/.c p}uauepunJ e sr aJaql tpq} Suuerls eeJql aql go uorler8alur leJruer{cau -uouap 'seseJaruos ur s{Jo^ }eu }ueurelrJurlJe pue elnqnloJorru aqt .{lqruas 3o -spsrp aql acnpur osle ueJ uorlcalur Jepel eql 'd18uqsara1ul'{Jo^ ]au }ueuelrJ atprpaurelul eqt Jo flqruessesrp prder aqt seJnpur tpnqns aqt yo uo13erIEOrIeq panresuoJ p ruo4 palrJap saprldad;o uorlcafur ue alrq ^ 'selnurur l aJ e ulq]lm sluaruelrJalerparuJalulSupsxa aq] oluo sallasuaq] ppe,{lprder sllal arn}lnJ ens -sp otq palcalurorcrtu s]runqns peleqel telouJnl prder ro; eJueprne sV'@Z-ZI arn8rg ees) srsolru ur surupl J€alcnu go ,tyqruassesrpeqt salep8ar uor1e1,{roqd -soqd leql fem arues aq] qJnru ur '.{lqurassesrpJraql salep8ar,r(lqeqord uor1e1 'slselqoJqrJse qJns slleo ur saJnl -,{roqdsoqd uralord 'suoplpuoc IEruJouJepun -cnrts crrueuLp l.U8tq wuo1uuuarura Surpnlcur s]ueuelrJ e]ErpetuJelur;o sadiil aluos lnq 'selnqnloJcru puE slueruEllJ ullJE Jo ueq] stualuulrJ alerpauJelur ;o .r(lquassesrppue flquasse Jo rusrueqcau aql ]noqe poo]srap1;es;[lT'", -rgrp.{laruarlxa aJei{aqf fnq'(ur}Je JoJsraleruoJJrruue} }noqe pue sepqnloJorru JoJsreleuqlrur IeJaAaso1paredruoc) JalauoJrru auo ueq] ssalgoqlSual eJuetsrs -rad e qtyrt'1uaq,{lsea aq uec.{aq1 'JalreJeqf,a1q-ador e s}ueuelrJ elerperuJalur sant8 'suralord goc-papoc ;o pcrddl suor]reJelur crqoqdorp.,tq IeJalEI Suorls eq] q]!v\ 'raqtaSot dn paug 1e sap4daddlod;o raqunu a8rel srqJ 'slror Iec1aq -n uorlcas ssoJJ e sBq eroJaJeqllualuElg elerperuJelul pnpl^ zt lPnpl^Ipul Jo -rpur qJeg 'sJarueJlet dn apeu stuarueluolord 3o lalpred lq8la sapnl3ur qJrqm ']uaruellJ aql uroJ o1 [1era1e1raqlaSo] >1cedsraruer]el aqJ 'saFqnloJJnu pup sluauelrJ ullJe roJ lef,rtrJJ sr ]pq] [1rre1od IEJn]JnJ]s IIeJaAoaq] s{cEI eJoJaJaq} ]uauelrJ alerpeuJalur palquassE aqJ 'atups aq] aJE spua o^1l slr 'suoucarrp alrsoddo ur Suuurod sJarurpo^ l Jo dn apeu sl lpnqns JrJerueJlelaql aJurs

suoJnau (slleupuereq "6'e)sa^lle^uap rlaql pueslla)letlaqlldo suolneuaLuos (slleruueMq)S auos puesay(rorlse) s;;ar1e;16 etfsnur urbuo {ueu ;eu{qruasetu ;o s;1er (adolo^ualeal)nuJo 6 u r u rr1a u u re) u ! u l eJle a l ) n u

(H-lNpue'w-lN ''t-lN) suralordluauellrornau ()rseq)surlerel;1ed{} ()!pl)e)surleral1adll urreqdtted

leuoxv lerlaL{}tdl

uralordrrpne,(.re;1uqr; 1er;6 urusap

real)nN

'v f pue'€ suluel

elrl-urluaurn

ullueulA

slla) elerqauanur suralo.rd lueuelrl elerpeulrelul;o sad{1lofew t-91 alqel

sl_Nlv\Jvtl tvlltl)sol^) lo tuntfnuls )twvN^ccNVAtSyllssv-llls lHr

ud O?

sr(xreproaJ|'olla) leseq

'uo] fssal6ftrslanrul JO spue aql JetlP sesPasrpasaq] esneJ ]Eq] suollelnru slJlseds eqt Jo ltuBw rallaJal)oUaql uJolj uorssil.ulad -eleIsol-,tc Jo uoI}BZIuEBJosIpP puB PIuneJl [eO urlEJa{ eql Suldtunls lualuelg Jo q l l M ' t 6 6t ' v L 9 L -t " 9 9L : SLL 'l o l Bi l D ' , r -rupqJeru Iq peudr! eJEselpeplu aseql Jo IIV af,uenbesuoJ e sP eJnldnr ilec Jo "le la aqLuolno)'vijLuoll)'eutulelleseq 'senssp esoql ot f,rJrcedssr uorssardxe asoqM sulleJel lueJeJJIpeqt ul suollElnlu 6ur{pepunaqt o} slueulelUullera)eql pasnec ale 'e.{eeq] Jo EauJooeq] pue '8urur1pe8eqdose 'qlnour eql Jo sJepJo l)auuo) q)!qM'(6t raldeq) ut passn)srp) Lq -srp Surpnlf,ur 'seseesrpSuualsqq Jo sed.,irraqlo '(IZ{I arntlC) sllec lesuq eqt saurosouisaptulaq aq] pue snal)nu aql uaamlaqarnldnr slla) oql 'Moilepa) salnldnJ qolq \ 'sseJls Ief,rueqcau tq8Is r(ran uana ol esuodseJ uI sJalsllq UDIS eql ^q pole)rpur sy'Idorso.rrru uollfela pelleJ JapJoslp e ecnpoJd daql 'snu qorq eql ^ ur'xaldutts asonnq s8tlouJaptda {q penresqose'srr-ulaprda -raprda luelnul pesseJdxe e^DceJapueq ^ 'eld eJe sullsre{ eqt ul aqt JedBI Jo IIaJ Ipseq eql;o ra,(e;lpseqoql ut slla)aarql,o -ruExe asnBc saueS ul]eJel uI suollstnlN Joc'sespasrp JrleueS ueunq leJa^as q)la)s v (f) '(s^orP pal tioqs) srluroprda '6I Jaldeql ur lPlep eJolu uI seJnlf,nJls luetnuloLllJorale; ;eseqaqt u! slle),o ssnJSIpaM'(9-9I eJnSrc ees)sauoso|usaprulaq pelleJ e^rsaqpe eseq] ]uEuodr.ul 6uunldnraql uJorJsllnsal6uualstlqeql ']f,EluoJ xrJlpru-lleo ro'saruosoltlsap pal]:P-J'lsBluo3 llac-lleJ Jo salls lB slueluBlg leq] Moqsurls (8)luelnui pup (v) leullou uI senssF lellaqllde 01 qlSueJls lecluEqcalu elerparuralul eql Suuoqf,ue dq ued ,o suorl)asssol),o sqder6oorutq611 'ulls aql sller '(02-gI arnglC) Jo lespqaql ut lroMlau uedur stueruelg urleJe) {romleu elSuIS e olul azlJeudlodoJ 'surtPre{ luaurelUullpJalaql sldn.rsrp,(qareql puB eseqt Jo sed.& aldnFlu acnpord ,(eu 1ac [BIIaq]Ide elSuIS V pue surlera)leullou aLllqll/\/^ salquasse luerulEarl Jo eJroqJ aql apmS or dlaq uEf, snqt puP pel€u urelordanrl:a;apaql'asnourlrua6suetl -r8uo Jaruec aq] qrq ^ ur enssll pllaqllda eq] Jo uonuclpul uE saAISpasseJdxe e ur passaJoxo seM(suteuopleutulal-) surleJeI Jo les JBInJrped aql se '(seurourcJef,)sJasuec 1etteqllde;o stsouSetp pue -N aql qloq 6ull)pl) ulaloldu!]elal (sA\BIc'sIeu 'JIPquI aql ur Iuesn dlecruqc sl suIlPJe{ q dllsJa lp eqJ 'selEf,spuP pale)unrl e 6utpo)uaaua6 1ue1nr.u y 'eue6 ullera>1 e Iq posne) pue uDISJo Jedel Jalno aql ul sE 'slerurue JoJs8uualoJ q8nol SururroJ 'slleo rleql luelnu urls srlllo 6urrelsr;g1g-91arn6r3 qlPep eqt ua^e a^r^rns,{eur spuoq epulnslp ^q Jeq}e8o} plaq s{rolli\leu ul}eJel Jo pa{url-ssorJ '(6I-9I arnSrg eas) }runqns JIJaIuPrlel plueluBpury aql uroJ o} u r d0 t

('o6reuy aurla6uen3 pue uaaJg uaalqle)lo {selrno))'sauepunoq lla) aLllro uorle)olaql lea^aro] pautelsuaoq setl(aryq uralo.rdpuolas y '(69 raldeql ur possn)srp) saurosoursep {q sroqqbrau strJo asoql ol pal)auuo) {llrargpurele lla) q)ea ul sluaureluaql'alnlln) ut slla) stuauelU lerlaqtldajo taaqse ur (uaar6,) ulleral ,o lroMlau aql 1o qder6orrru .slla) a)uafsalonuounuiull 1e;laqr;da ur slueuPlg urlerayg7-91 arnbrl

uolalalsolr() aql:9 l. raideqf

986

FILAMENTS OFCYTOSKELETAL THESELF-ASSEMBLY AND DYNAMICSTRUCTURE

the central rod domain, demonstrating the importance of this particular part of the protein for correct filament assembly. A second family of intermediate filaments, called neurofilaments, is found in high concentrations along the axons of vertebrate neurons (Figure 16-22). Three types of neurofilament proteins (NF-L, NF-M, NF-H) coassemble in uiuo, forming heteropolymers that contain NF-L plus one of the others.The NF-H and NF-M proteins have lengthy C-terminal tail domains that bind to neighboring filaments, generating aligned arrays with a uniform interfilament spacing. During axonal growth, new neurofilament subunits are incorporated all along the axon in a dgramic process that involves the addition of subunits along the filament length, as well as the addition of subunits at the filament ends. After an axon has gro\,&'nand connected with its target cell, the diameter of the axon may increase as much as fivefold. The level of neurofilament gene expression seems to directly control axonal diameter, which in turn influences how fast electrical signals travel dor,tmthe axon. The neurodegenerative disease amyotrophic lateral sclerosis (ALS, or Lou Gehrig'sDisease)is associatedwith an accumulation and abnormal assembly of neurofilaments in motor neuron cell bodies and in the axon, which may interfere with normal axonal transport. The degeneration of the axons leads to muscle weakness and atrophy, which is usually fatal. The over-expressionof human NF-L or NF-H in mice results in mice that have an ALS-like disease. The vimentin-like filaments are a third family of intermediate filaments. Desmin, a member of this family, is expressed in skeletal, cardiac, and smooth muscle. Mice lacking desmin shownormal initial muscle development,but adults have various muscle cell abnormalities, including misaligned muscle fibers.

DrugsCanAlterFilamentPolymerization Becausethe survival of eucaryotic cells depends on a balanced assembly and disassembly of the highly conserved cytoskeletal filaments formed from actin and tubulin, the two types of filaments are frequent targets for natural toxins. These toxins are produced in self-defenseby plants, fungi, or sponges that do not wish to be eaten but cannot run away from predators, and they generally disrupt the filament polymerization reaction. The toxin binds tightly to either the filament form or the free subunit form of a polymer, driving the assembly reaction in the direction that favors the form to which the toxin binds. For example, the drug latrunculin, extracled from the sea sponge Latrunculia magnifica, binds to actin monomers and prevents their assembly into filaments; it thereby

987

Figure 16-22 Two types of intermediate filaments in cellsof the nervous system. (A)Freeze-etch electronmicroscopic in a nervecell imageof neurofilaments cross-linking axon,showingthe extensive throughproteincross-bridges-an believedto givethis long arrangement The cellprocessgreattensilestrength. areformedby the long, cross-bridges at the C-terminus extensions nonhelical protein of the largestneurofilament (B)Freeze-etch (NF-H). imageof glial filamentsin glialcells,showingthat these filamentsaresmoothand intermediate (C)Conventional havefew cross-bridges. electronmicrographof a crosssectionof an axonshowingthe regularside-to-side which spacingof the neurofilaments, greatlyoutnumberthe microtubules. (A and B,courtesyof NobutakaHirokawa; C,courtesyof JohnHopkins.)

988

Chapter16:TheCytoskeleton

Table16-2 DrugsThat AffectActin Filamentsand Microtubules

Phalloidin

Latrunculin

bindsandstabilizes filaments capsfilamentplusends seversfilaments bindssubunitsandprevents theirpolymerization

Taxol Colchicine, colcemid Vinblastine, vincristine Nocodazole

bindsandstabilizes microtubules bindssubunitsand prevents theirpolymerization bindssubunitsandprevents theirpolymerization bindssubunits and prevents theirpolymerization

Cytochalasin Swinholide

stabilizes free tubulin, causing microtubule depolymerization. In contrast, taxol, extractedfrom the bark of a rare speciesof yew tree, binds to and stabilizes microtubules, causing a net increasein tubulin polyrnerization. These and some other natural products that are commonly used by cell biologists to manipulate the cyoskeleton are listed in Table 16-2.

o H3C-C -O

o C -NH-CH-CH-C

9oH

H,C

o

CH

-O

c-o o

(A)

1 5p m

o-c ll o

CH:

Figure 16-23 Effectof the drug taxol on microtubuleorganization.(A)Molecular structureof taxol.Recently, organic chemistshavesucceeded in synthesizing this complexmolecule, which is widely usedfor cancertreatment. (B)|mmunofluorescence micrograph showingthe microtubuleorganization in a liverepithelialcellbeforethe addition of taxol.(C)Microtubuleorganization in the sametype of cellafter taxol treatment.Notethe thickcircumferential bundlesof microtubules aroundthe peripheryof the cell.(D)A Pacificyew tree,the naturalsourceof taxol. (8,C from N.A.Gloushankova et al.,Proc. NatlAcad.Sci.U.S.A. 91:8597-8601,1994. With permission from NationalAcademy of Sciences; D courtesyof A.K.Mitchell 2001.o HerMajestythe Queenin Right of Canada, CanadianForestService.)

989

FILAMENTS OFCYTOSKELETAL THESELF-ASSEMBLY AND DYNAMICSTRUCTURE Figure16-24ThebacterialFtsZprotein,a tubulin homolog in procaryotes.(A)A band of FtsZproteinforms a ring in a dividing bacterial cell.Thisring hasbeenlabeledby fusingthe FtsZproteinto the green protein(GFP), whichallowsit to be observedin livingE coli fluorescent microscope. Iop, sideview showsthe ring asa bar cellswith a fluorescence in the middleof the dividingcell.Bottom,rotatedview showingthe ring using (B)FtsZfilamentsand rings,formedin vitro,asvisualized structure. Comparethis imagewith that of the microtubule electronmicroscopy. shownon the rightin Figure16-16C.(A,from X. Ma,D.W.Ehrhardtand 1996;B,from W. Margolin,Proc.NatlAcad.Sci.IJ.S.A.93:12998-13003, (A) :i,:5:Tffi'-.'#l;illir'ifl ,':?y.',i.'"::i1e-s23'lee6Arrw*h

1 rm

and CellDivisionDependon CellOrganization Bacterial Cytoskeleton Homologsof the Eucaryotic \Mhile eucaryotic cells are typically large and morphologically complex, bacterial cells are usually only a few micrometers long and assume simple, modest shapes such as spheres or rods. Bacteria also lack the elaborate networks of intracellular membrane-enclosed organelles such as the endoplasmic reticulum and Golgi apparatus. For many years, biologists assumed that the lack of a bacterial cltoskeleton was one reason for these striking differences between cell organization in the eucaryotic and bacterial kingdoms. This assumption was challenged with the discovery in the early 1990s that nearly all bacteria and many archaea contain a homolog of tubulin, FtsZ, that can polymerize into filaments and assemble into a ring (called the Z-ring) at the site where the septum forms during cell division (Figure 16-24). The three-dimensional folded protein structure of FtsZ is remarkably simiIar to the structure of o or B tubulin and, like tubulin, hydrolysis of GTP is triggered by polymerization and causes a conformational change in the filament structure. Although the Z-ring itself persistsfor many minutes, the individual filaments within it are highly dynamic, with an average half-life of about thirty seconds. As the bacterium divides, ttre Z-ring becomes smaller until it has completely disassembled,and it is thought that the shrinkage of the Z-ring may contribute to the membrane invagination necessaryfor the completion of cell division. The Z-ringmay also serve as a site for localization of specialized cell wall slnthesis enzymes required for building the septum between the two daughter cells. The disassembled FtsZ subunits later reassemble at the new sites of septum formation in the daughter cells (Figure f 6-25). More recently, it has been found that many bacteria also contain homologs of actin. TWo of these, MreB and Mbl, are found primarily in rod-shaped or spiral-shaped cells, and mutations disrupting their expression cause extreme abnormalities in cell shape and defects in chromosome segregation (Figure f6-26).MreBandMblfilamentsassemble inuiuotoformlarge-scalespiralsthat

(B)

(B)

(A)

il; time 0 (min)

35

37

, trm Figure l6-25 Rapidrearrangementsof FtsZthrough the bacterialcell cycle.(A)After chromosomesegregationis complete, thl ringformedby FtsZat the middleof the cellbecomessmallerasthe cellpinchesin two, much likethe contractilering asthe cellshaveseparated cells.The FtsZfilamentsthat havedisassembled formedby actinand myosinfilamentsin eucaryotic (red\from a redalga (B) chloroplasts Dividing cells. daughter two of the middle rings at the to form two new then reassemble alsomake useof a protein ring madefrom FtsZ(yeltow)forcleavage.(A,from Q. Sunand W. Margolin,J. Bacteriol. 180:2050-2056,1998.With peimissionfrom AmericanSocietyfor Microbiology;B,from S.Miyagishimaet al.,PlantCell With permissionfrom AmericanSocietyof PlantBiologists') 13:2257-2268,2001.

990

Chapter16:TheCytoskeleton

r

(c)

L-----.1 (B)

5um

Figure16-26 Actin homologsin bacteriadetermine cell shape.(A)The common soil bacteriumBacillussubtilis normallyformscellswith a regularrodlikeshape.(B)B.subtiliscellslackingthe actinhomologMbl grow into irregular twistedtubesand eventuallydie.(C)The Mbl proteinformslong helicesmadeof up manyshortfilamentsthat run the lengthof the bacterialcelland helpto directthe sitesof cellwall synthesis. (FromL.J.Jones,R.Carbadillo-Lopez and J. Errington,Cell104:913-922,2001.With permission from Elsevier.)

5[m

span the length of the cell and apparently contribute to cell shape determination by serving as a scaffold to direct the synthesisof the peptidoglycan cell wall, in much the same way that microtubules help organize the synthesis of the cellulose cell wall in higher plant cells (see Figure 19-82). As with Ftsz, the filaments within the MreB and Mbl spirals are highly dynamic, with half-lives of a few minutes; as for actin, ATP hydrolysis accompanies the polymerization proCCSS.

Diverse relatives of MreB and Mbl have more specializedroles.A particularly intriguing bacterial actin homolog is parM, which is encoded on certain bacterial plasmids that also carry genes responsible for antibiotic resistanceand frequently cause the spread of multi-drug resistancein epidemics. Bacterial plasmids typically encode all the gene products that are necessaryfor their owrsegregation, presumably as a strategyto ensure their faithful inheritance and propagation in their bacterial hosts. rn uiuo, parM assembles into a filamentous structure that associatesat each end with a copy of the plasmid that encodes it, and growth of the ParM filament appears to push the replicated plasmid copies apart, rather like a mitotic spindle operating in reverse(Figure 16-2z).Altholgh ParM is a structural homolog of actin, its dynamic behavior differs significantiy. ParM filaments undergo dramatic dynamic instability in uitro, more closeiy resembling microtubules than actin filaments in the way that they grow and shrink. The spindle-like structure is apparently built by the selective stabilization of spontaneously nucleated filaments that bind to specialized proteins recruited to the origins of replication on the plasmids. The various bacterial actin homologs share similar molecular structures but their amino acid sequence similarity to each other is quite low (- l0-15% identical residues). They assemble into filaments with distinct helical packing patterns, which may also have very different dynamic behaviors. Rather than uiing the same well-conserved actin for many different purposes, as eucaryotic celli do, bacteria have apparently opted to proliferate and specialize their actin homologs for distinct purposes. It is now clear that the general principle of organizing cell structure by the self-associationof nucleotide-binding proteins into dynamic helical filaments is used in all cells, and that the two major families of actin and tubulin are verv

monomers (A)

replication

f ilaments proteins (B) 2ttm

Figure 16-27 Roleof the actin homolog ParMin plasmidsegregation.(A)Some plasmids bacterialdrug-resistance (yellow)encodean actin homolog,ParM, that will spontaneously nucleateto form small,dynamicfilaments(green) throughoutthe bacterialcytoplasm. A secondplasmid-encoded protein(b/ue) bindsto specificDNAsequences in the plasmid,and alsostabilizes the dynamic endsof the ParMfilaments. Whenthe plasmidhasduplicated, so that the ParM filamentscan be stabilized at both ends, the filamentsgrow and pushthe duplicatedplasmidsto oppositeendsof the cell.(B)In thesebacterialcells harboringa drug-resistance plasmid,the plasmidsarelabeledin redand the ParM protein in green.Left,a short ParM bundleconnectsthe two daughter plasmidsshortlyaftertheir duplication. Right,the fullyassembled ParMfilament haspushedthe duplicatedplasmidsto the cellpoles.(A,adaptedfrom E.C.Garner, C.S.Campbellano R.D.Mullins,Science 306:1021-1 025, 2004.With permissionfrom AAAS;B,from J. Moller-Jensen et al.,Mol.Cell 12:1477-1 487,2003.With permission from Elsevier.)

FILAMENTS OFCYTOSKELETAL AND DYNAMICSTRUCTURE THESELF-ASSEMBLY

(A)

t L__ zpm

(B)

I

2pm

ancient, probably predating the split between the eucaryotic and bacterial kingdoms. However, the usesto which bacteria put their cltoskeletons appear somewhat different from their eucaryotic homologs. For example, in bacteria it is the tubulin (FtsZ) that is involved in cytokinesis (the pinching apart of a dividing cell into two daughters), while actin drives this process in eucaryotic cells. Conversely, eucaryotic microtubules are responsible for chromosome segregation, while bacterial actins (ParM and possibly MreB) help to segregatereplicated DNA in bacteria. At least one bacterial specieswith an unusual crescent shape, Caulobacter crescentus,even appears to harbor a protein with significant structural similarity to the third major class of cytoskeletal filaments found in animal cells, the intermediate filaments. A protein called crescentin forms a filamentous structure that seems to influence the cell shape; when the gene encoding crescentin is deleted, the Caulobacter cells grow as straight rods (Figure f 6-28)' Since we now know that bacteria do in fact have sophisticated dynamic cltoskeletons, why then do they remain so small and morphologically simple? As yet there have been no motor proleins identified that walk along the bacterial filaments; perhaps the evolution of motor proteins was a critical step allowing morphological elaboration in the eucaryotes.

Su m m a r y The cytoplasm of eucaryotic cells is spatielly organized by a network of protein ftla' ments known as the cytoskeleton.This network contains three principal typesof filaments:microtubules,actin filaments, and intermediatefilaments. All three typesof filamentsform as helical assembliesof subunits that self-associateusing a combination of end-to-endand side-to-sideprotein contacts.Dffirences in the structureof the subunits and the manner of their self-assemblygiue the fitaments dffirent mechanical properties.Intermediatefilaments are rope-likeand easyto bend but hard to break. Microtubules are strong, rigid hollow tubes.Actin filaments are the thinnest of the three and are easyto break. In liuing cells,the assemblyand disassemblyof their subunits constantly remodels all threetypesof cytoskeletalfilaments. Microtubules and actin filaments add and lose subunitsonly at their ends,with one end (theplus end)growingfaster than the other. Tubulin and actin (the subunits of microtubulesand actin filaments, respectiuely) birul and hydrolyze nucleoside triphosphates (tubulin binds GTP and actin binds ATP).Nucleotide hydrolysisunderliesthe characteristicdynamic behauior of thesetwo where fiIaments.Actin filaments in cellsseemto predominantlyundergotreadmilling, end. the other at disassembling simultaneously end while assembles at one a filament Microtubules in cellspredominantly display dynamic instability, wherea microtubule end undergoesalternating bouts of growth and shrinkage. tnAlhereas tubulin and actin haue beenstrongly conseruedin euolution, thefamily of intermediatefilaments is uerydiuerse.Thereare many tissue-speciftc formsfound in the cytoplasmof animal cells,including keratinfilaments in epithelialcells,neurofilamentsin neruecells,and desminfilaments in musclecells.In all thesecells,the primary job of intermediateftlaments is to prouide mechanical strength. Bacterial cellsalsocontainhomologsof tubulin, actin and intermediatefilaments that form dynamicfilamentous structuresinuoluedin determiningcell shapeand in cell diuision.

991

and Figure16-28 Caulobacter bacterium Thesickle-shaped crescentin. a protein, expresses crescenfus Caulobacter with a seriesof coiled-coil crescentin, to domainssimilarin sizeand organization the domainsof eucaryoticintermediate protein filaments.In cells,the crescentin formsa fiberthat runsdown the innerside of the curvingbacterialcellwall.Whenthe geneis disrupted, the bacteriaareviable form. but grow in a straightrod-shaped (FromN.Ausmees, J.R.Kuhnand er,Cell115:705-713,2003' C.Jacobs-Wagn from Elsevier.) With oermission

992

Chapter16:TheCytoskeleton

FILAM ENTS Microtubules, actin filaments, and intermediate filaments are much more dynamic in cells than they are in the test tube. The cell regulatesthe length and stability of its cytoskeletal filaments, as well as their number and geometry. It does so largely by regulating their attachments to one another and to other components of the cell, so that the filaments can form a wide variety of higher-order structures. Direct covalent modification of the filament subunits regulatessome filament properties, but most of the regulation is performed by a large array of accessoryproteins that bind to either the filaments or their free subunits. Some of the most important accessory proteins associated with microtubules and actin filaments are outlined in Panel 16-3 (pp. gg4-gg5).This section describes how these accessoryproteins modify the dynamics and structure of cltoskeletal filaments. we begin with a discussion of the way that microtubules and actin filaments are nucleated in cells, becausethis plays a major part in determining the overall organization of the cell's interior.

A Proteincomplexcontainingy-Tubulin Nucleates Microtubules \.{/hilecr- and B-tubulins are the regular building blocks of microtubules, another type of tubulin, carled y-tubulin, ]nasa more specialized role. present in much smaller amounts than cr- and B-tubulin, this protein is involved in the nucleation of microtubule growth in organisms ranging from yeasts to humans. Microtubules are generally nucleated from a specific intracellular location knor,rmas a microtubule-organizing center (MToc). Antibodies against y-tubulin stain the MToc in virtually all speciesand cell types thus far examined.

Microtubules Emanate from the centrosomein Animalcells Most animal cells have a single, well-defined MToc called the centrosome, located near the nucleus. From this focal point, the cytoplasmic microtubules emanate in a star-like, "astral" conformation. Microtubules are nucleated at the centrosome at their minus ends, so the plus ends point outward and grow toward the cell periphery. Microtubules nucleated at ihe centrosome continuously grow and shrink by dFramic instabiliry probing the entire three-dimensional volume of the cell. A centrosome is composed of a fibrous centrosome

y-tubul in

accessory proteins in y - t u b u l i nr i n g c o m p l e x

Figure16-29 Polymerization of tubulin nucleatedby ytubulin ring complexes. (A)Structureof the y-tubulinring complex,reconstructedfrom averaging electronmicrographs of individual purifiedcomplexes. (B)Modelfor the nucleationof microtubulegrowthby the lTuRC.The red outlineindicatesa pairof proteinsbound to two moleculesof y-tubulin;this groupcan be isolatedasa separate subcomplex of the largerring. Notethe longitudinaldiscontinuity betweentwo protofilaments. Microtubules generallyhaveone such "seam"breakingthe otherwiseuniform helicalpackingof the protofilaments. (C)Electronmicrographof a single microtubulenucleatedfrom the purified y-tubulinring complex.(A and C,from M. Moritzet al.,Ndf.CellBiol.2:365-370, 2000.With permission from Macmillan Publishers Ltd.)

('rarAasllruorJuorssrtIlJad qlm'/66t 'EtL-LLLijZL 'lotB'Dnrls,'r'lela uall?rql 'c urorJ poldepe'g'suo5I ,(allMuqof uorj q]!M 0002'6 LV-60Vt6V uolsslLurad ' 2su)tw'reulreu'B'r LPal'saa pue no I')|rew 'r'D uroll'v) red aloulue)aql Jo alnl)nlls (8) '{qreauuaesareselnqn}oJ)tul ratq6nep snoror.rtnN'eloulua) aql Jo uott)as;eurPnttbuo; e pue aloutue) laqlou-leql Jo ^^al^ auosorlua) uo-puauP 6urrvroqs e Jo uorl)asurqte Jo qdet6ottttu uoll)ala uV (V)'auosoJlua) aql u! alolllusr y 1g-91arn6t3

('ssard Allslo^runralleJol)ouaqf u.ror,uolssluladql111n'€002' 99v- LSVitgL 'I'l tuolj ')) 'tolB ilD'r "le ]a aloolo 'euosorlua)aql Luot;6utleuetueuaas aq ue) salnqnlor)ur la))lLllasuap Jo e '1a>suo0ala) e uor, )OIW aql Jo 'useldol^) aber,urpalrnrlsuorer e ul (l) aq] u! aar,sl alnqnloJ)lruqlea Jo pua snldaql searaqm'xa;duot6utt utlnqnt-,t e ulor; unror66utneq'euosoltua) aqi ur pappaquesl alnqnlol)ltuqfea Jo pua paq)elleqllM snururaLlI'salnqnlorlrLu aurosortua) V (g)'lxal aql ul paqu)sapse 'seloulua) rrede r(qpaztue6losl xllleur Jo srql'poq)elteele qlmot6alnqnlol)ltrr aleal)nuleql saxalduot6ut.tutlnqnl-/" aLllq)lqMot sulaloldsnolquro xlrleul 'snal)nuaql snoqdrou:euejo slstsuo)]t 'slla) ol txau r,useldoiltaql u; pale)o1 tofetu aql st eulosorlual lpurue Jo 191y11 aql (V)'auosotluo) aql OE-91arn6t1

'Ialuaf, sll le aulosoJlues aql uop seJnsolcuaIprcrJrupur ue^a slql saop ]I pue -rsod 01pup ilel eqlJo suorSerSurrtpnoeqt ^a^Jns ot eJIAepe se slcB eluosoJlueJ 'snelJnu aqlfteu pelJallor spua aql tuo4 Superper salnqnlorclu Jo lualsl's aql snurru aiqers pue draqdirad IIao eqt prumo] pJe,tirlnoSupulod spue snld rlrueu^p 'sllao IeruIuP uI qll^\ ']snqoJ zirarrsr selnqnloJJlru Jo uonBJnBIJuoJIPJISPeql 'selnqnloJslru Jlaql aleelJnu o1lI 'secueJeJJlp aseq] eltdsaq 'sa1o asn o] ruaas pue urlnqn]-1,uteluoc slleJ aseqt ile 'ado1a'Lua Jealcnu eql punoJe -rJluao uleluoJ s11ac tuuld ]soru Jou r8un; raqllag 'tpoq o] luaes slleJ ]uEId-JaqBE1 IIE palnqlJlslp sells lB salnqnloJJllu elPelJnu a1oda1purdseq] peller enbeld IIEtus 3 sE edolalue ruelrnu eq] uI peppaqua 'surolerp pue rSun; u1 sr lEql coIN ue lB pelealJnu aJe selnqnloJcllu 'poolsJapun-ile^Alou SIUOI1 -ecqdnp slr JoJ srsEqJplncelolu eqJ .suraloJd.{rossacceJo Jeqlunu a3re1u snld 'GL11-ZL1I 'selnqnlorJlru palJlpotu Jo JepuII^o uoqs € Jo slslsuoc elolJluec V 'sulSeq 'dd 't-zt aql ruro; '{aqt pue Ieued aas) apurds qloilur aql;o salod o^u srsolrruueq A snalcnu--"qt1o Saplsallsoddo ol a4olu Saluosolluac ralqfinep orul 'aseqd:a1urSurrnp slred a'saq1:red alolJlue3 pafecqdnR e Suruteluoc Jpq qcBa '11 raldeq3 uI peqIJJSap pue saleclldnp euosoJluef, eql pnbe orntro1u1s111ds rV'(f g-gf arn8gg) alei{dnp sallesueql selolr}ueo aql se a1c'ic1ac qoea Sulrnp '(pualuu JelolJluaclJed aqt palec oslu) xulutu etuos uopecldnp slr Sutrnsua -orf,r", aziuefiro selorrlueJ aql'(Jalel pequJsep) sllao alporu ur ella8eg pue "qi qJIqM 'selolrlual aql aru esaql'(0t-91 arn8lg) ellro Jo serpoq prnq "qi"-of,eq uopern8guorpadeqs-1 ue uI Jeqlo qJea o] sa13uurqBIJ 1Bpa8uerre seJnlsnJls pcrrpulrtc snorralsl.ru lEq^\eluos;o rred e sI aruosoJlueJeql uI peppaqurg 'lunl-,| eql ale^pcE puP llnrseJ 'peJaAoJSIpaq o} UIEIIIaJ xlJlelu slql IIIJOJ ,taqt lv\oq u^/\oDI ]a,{ }ou sI }I puB .3gn1-1,1osardocd4q ueqr eJolu suleluos ]eq] x!&aut ]eq} suraloJd aqt Jo lsontr

auosortua) alll +o s a x a l d u o r6 u u u t l n q n l - l ' 6ur,uo:6salnqnlol)tul i Luo.r1

(8)

(v)

. salorJlua) IO l|eo

+"..--d

a xlJleuJ auosoJlua)

6ul.tut1nqnl-/"1 (saxaldLUo) s a l t s6 u t 1 e a ; l n u

srNtWVllllvrlll)soi^)

866

ullHr llvln9lu slll) MoH

ACTIN FILAMENTS

F

{ formin

ARPcomplex

n u c l e a t e sa s s e m b l a y nd remains associatedwith the growing p l u se n d

thymosin b i n d ss u b u n i t s , p r e v e n t sa s s e m b l y

nucleatea s ssembly to form a web and remains a s s o c i a t ew d i t h t h e m i n u se n d

ar rro

profilin b i n d ss u b u n i t s , s p e e d se l o n g a t i o n

oa o -V

a

O2or

oo.

actin subunits

'r t

a't C ./

-\

actin filament

o\t.

A

a

cofilin b i n d sA D P - a c t i nf i l a m e n t s , acceleratesdisassembly

./ gelsolin s e v e r sf i l a m e n t sa n d b i n d st o p l u se n d

c a p p i n gp r o t e i n p r e v e n t sa s s e m b l a y nd disassemba l yt p l u se n d

f i l a m e n t b u n d l i n g ,c r o s s - l i n k i n ga,n d a t t a c h m e n t o m e m b r a n e s

fimbrin

a-actinin

filamin

Someof the major accessoryproteins of the actin cytoskeleton.Exceptfor the myosrnmotor proteins,to be discussedin a later,section, an exampleoi each.major type is shown. Eachof theie is discussed in the text. HoweveLmost cellscontain more than a hundreddifferent actin-bindin!proteins,and it is rit"rvihaiih*e are importanttypesof actin-associated proteinsthat are not yet recognized.

'pazru6o)al la^ lou oJeleq+suralordpaler)osse-alnqnlor)rur +o sodl+lueuodur sre sraql leLll ^lolrl sr]r-suroloJd poler)osse-urpe aq] rol se-pue 'surelo.rd 6urpurq-elnqnloJ)rur luaJa#rppoJpunqe ueql aJourureluo) slla)+sour la^omoH '1xo+aql ur possn)srp sr osaql+o q)el 'umoqssr ed[1 .rofewq)eo ]o a;duexe ue 'uorposia]el e ur passn)srp aq o1 'suralo.rdrolouJ +o sassel)o,V\lJo+lda)xl 'uola;a1soy{ralnqn}oJ)il,1r aq1,rosuralord fuosse>>e roleur eq} +o aulos

sluaurPlrjalerpauljalur ol s)u., u r1re1d

pue6urlpunq 6ur1ur;-sso.o luauelU s a p r s6 u o 1 e6 u r p u r q { q s a l n q n la z r l r q e } s

s a l n q n l o r l r r . l rs J a ^ o s

sdvy{

u ruele)

p u a s n l d1 e{ l q L r a s s e s r p : r q d o r l s e l e :s a ) u e q u a

+-

i ( l q u a s s es a l e r a l a r l ep u e s p u as n l ds a z t l t q e l s

EI u r s a u r l

s t zdvhlx

\

t

i

t\|

o

elnqnloJ)rur

c

-1, \rr ,

{lqurassesluanard ' s l r u n q n ss p u r q utuiqlels

il-

-G

\

)llrl )\rl

E(5gr*" p u a s n u t u3 q ] r..llrM p a l e r ) o s s seu r e u a r p u e ^ l q L U a s ssea l e e l ) n u )UnJ.-,t

sllnSntou)t1A

talua) 6utztue610 -alnqntoj)tulMau e ul paunq'luaur6elJ salnqnlor)rrx pezrue6JoaJ aql ro raluar aql reaudn pua spuasnutu qlliV\ raqt leql os azruebtoer 1uau6er1;1ar t u a u 6 e r l 1 ; a r/ paq)elapeql u! salnqnlol)tuaq]'alpaau e qllM#o tn) srlla) lueubrd qsr;e 'lla) p,o lalua) aql pulJ ,o rlre aql rauv uel {ere alnqnlor)lulV €€-91 ern6!l

saloulua) 6urpel raluar 6urzrue6ro -alnqnloJ)rur/l/\eu

llo) oroLloouelour

1uau6e.r1 lla) peraAos

lteo oloulue) 6urureluor euJosolluo)

pazflelP-Jeq ue3 uor]palJnu srqJ'lueuuoJl^ua leuJalxa sll uI se8ueqJ o] asuodsaJ ur dlprder sser4Jrtspue adeqs str e8uuqc 01 IIaJ eq] 3umo1p 'auerqtuaur etuseld eql ]E sluatuelg urlJe Jo uorlEelsnu aq] elElnSar ^lluenba.{ spuSIS lBurexa 'se8eqdOJJEur ur sdnr cll,tco8eqd eql pup 'salertsqns pqos re^o slleJ eloru dlaq teqf Dlpodll -pwql palleJ slre^ e^rsnrlord pE'aryodol{ rc qunoJJlla sB qf,ns sapunq allds apnlJur eseqJ 'suoucaford aJEJJnsIIaJ Jo sadIl ]uaJeJJIp^FuDIu]s ,(ueur urroJ uBc seJnlsnJlsurlJe 'au€JqrueruErusBIdeql ol puE Jaqloue euo o] slualuqceuP Jraql uo Surpuadap 'aldurexa JoC 'aJeJJns IIec eql Jo luarua^olu puE edeqs eq] surruJalep ]l ul slualuelrJ uI]38 aql puP 'xeuoJ IIaJ eql pall"3 sI euEJqlueu euseld eq] qtBeueq rsnf rai(e1aql draqduad ilec eql ]e sl sllal lsotu uI slueul 'euEJqruerusrusEld eq] reeu ro -PIrJ fpuenbesuo] urlf,e eqt &rsuap tsaq8q Jo 'snalJnu eql rueu ursBldoldc ]E sJnrso lilluanbe4 lsol'u uol}Palsnu ]ueluellJ ullcs eqf ulqlp{ daap l.Irzruud sJnJJo qJIq \ 'uouPalf,nu eFqn}oJf,Ilu ol }s€Jluoc uI

euerqureweurseldaql le poteal)nNueuo erv slueulellJulpv

fsaluar)sJo {urapery lpuor}eNutol; uotsstullodrlllM ' 166L' Lt Zg-8229:V6',V',S', n',pS'pDV ltoN iud "p 1a{;og '31 urorl)'arnsol)uaaql Jo slleMlnoJlle uo 6urqsndsolnqnlolltur aqt Jo uorlnqulsrp aql Moqsol paurelspue pexu/auiosoJlua)paletua) -JlasV (€)'sle^ralur alnurul-aalqlle ualel araMsarnt)roasaqf'llo/1/\ aql,o latua) aql ol auosorluaf aql sa)roj uor]rsooeql azrlrqels ot suorl)arp;;eur 6urqsnd;enbe roJluauarnDar aqf 'llali\aqr Jo slleM oql lsule6eqsnd{aql'auosorlue) aql ,(q paleapnu'azrratul;od salnqnlot)rur o q l s v ' u o r t n l o us r s l r u n q n us r l n q n q l ]rM 6uo;e's;;amasaqlJo auo oturpereldsenn aurosorlua)al6ursy'aletlsqnsrrlse;d e olur paurq)euror)rur araMsllaMaJenos 'oruosorlua)e lor^eqaq lleLus(v) ro 6uglaas-reluaraq1 7g-9 1 aln611

'(9-91 arn8rg eas) eJeJrns Ipseq eql se reJ sB Surpuaga spua snld qlIM 'llac eql Jo srxe 3uo1 aql Suop sruroJ selnqn] -oJJrru lalleJPd f.Feau Jo ^EJJEue (uoueJol crJleruludsE slql luoJc 'eueJqluau Eluspld pcrde aql reau uor8ar e ol e^otu spua snulru eFqnloJJIIu eql 'pazuElod {UBU saruocaq pue suorlcunf 1ac-11acsIuJoJIIaJ Ielleqtlde ue uaq \ 'aldruexa JoJ 'snql'surelsds eleurpJoo3 IEuJsluI elEJoqelaaloru Jleql qslqetsa ol sruslu -eq3eu SurrnsearupuoplppE asn lsnlu slleJ Ielleqllda pue 'salcsntu 'suoJneu '[ac aql ulqrp\ sE qrns serSoloqdrou xaldruoc qllm slleo pelellueraJJlp [tq8Uf selleuB8Jo^,(ueu uoltlsod o1 pasn uaql sl qJIqM 'uralsLs elEulpJooo IeJeuaB B seqsqqelseIIes aqtJo ratuas aql pulJ o] uolele{sotdc alnqnlorJllu eq}Jo,irulqe slql '(tt{I arntgg) aruosoJtual pelBlofl eq} dq pesn usluErlcaru Sutqsnd a1d -urs eq1 lsnf ueql slueuodruoJ eJolu aAIoAul^Eur ssaf,oJdsrqf q8noqlp teluec eql ]e peJalsnlc spua snurru eFqnloJsllu eqr qll^\leJJe padeqs-re1se olul se^Ies -ueql aBuBJJusellauB8rosnoeuuJqruau qlIMBullcEJalul seFqnloJcllu cnuuufp '(ZS-9I arnS;g) 'aruosoJlueo eql SuDIJpl luaru8e4 IIaf, pelelosl ue uI uaAA

uirl 0 t

uolelelsoil) aql :9I raldeql)

966

997

FILAMENTS THEIRCYTOSKELETAL HOW CELLSREGULATE

by two different tlpes of regulated factors, the ARP complex and the formins (discussedbelow). The first of these is a complex of proteins that includes two actin-related proteins, or ARPs,each of which is about 45% identical to actin. Analogous to the function of the y-TuRC,the ARP complex (also known as the Arp 2/3 complex) nucleates actin filament growth from the minus end, allowing rapid elongation at the plus end (Figure 16-34A and B). The complex can also

T..-l

".,inll

]

nrpz [

arpr [

]

]

(A) activating factor

mrnus eno

n u c l e a t e da c t i nf i l a m e n t ARPcomplex (B)

activeARPcomplex

1 0 0n m

t.\ \!/

(D) 10 nm

Figure 16-34 Nucleationand actin web formation by the ARPcomple; actin.Afthoughthe faceof the moleculeequivalentto the plus end (toP. differenceson the sidesand minus end (bottom)preventtheseactin-reli into filamentswith actin.(B)A modelfor actinfilamentnucleationby th are held by their accessoryproteinsin an orientationthat preventsthen indicatedby the bluetrianglebinds the complex,Arp2 and Arp3 are bro onto this structure, an actinfilament.Actinsubunitscanthen assemble when i filamentsmostefficiently 16-10).(C)TheARPcomplexnucleates roundsof branchingnucleationresultin a treelikeweb of filamentbranchthat growsat a 70. anglerelativeto the originalfilament.Repeated with purifiedARP actin filaments.(D)Top,electronmicrographsof branchedu'.tinfilur"ntt formed by mixing purifiedactin subunits beenfitted to the have ARP complex the and actin oi structures crystal the where complexes.Bottom,reconstructedimageof a branch the right wherethe ARPcomplex electrondensity.The mother filament runsfrom top to bottom, and the daughterfilament branchesoff to 86, 1998'With permission bindsto three actin subunitsin the mother filament (D,from R.D.Mullinset al.,Proc.NatlAcad.Sci.IJ.S.A.95:6181-61 MacmillanPublishersLtd') from permission With 2001. and from N.Volkmannet al.,Science293:2456-2459, from NationalAcademyof Sciences,

998

Chapter 16:TheCytoskeleton

20um

rR\ 1 0 0p m

attach to the side of another actin filament while remaining bound to the minus end of the filament that it has nucleated, thereby building individual filaments into a treelike web (Figure l6-34C and D).

near the plasma membrane in yeast, where it is required to form cortical actin patches (see Figure 16-6), and in plant cells, where it directs the formation of actin bundles at the surface that are required for the growth of complex cell shapesin a variety ofdifferent tissues (Figure f6-3b).

TheMechanism of Nucleation Infruences Large-scale Filament Organization

or microtubule and prevent both subunit addition and subunit loss at this end.

Figure16-35 Functionof the ARP complexin plant cells.(A)Cellsin the maizeleafepidermisform small,actinrichlobesthat lockneighboringcells togetherlikepiecesof a jigsawpuzzle. (B)The regularpatternof interlocking cellscoversthe leafsurface. (C)Epidermal cellsin a mutantplantlackingthe ARp complexdo not form the interlocking lobes.The brick-shaped cellsare normal in sizeand spacing,but form leavesthat appeartoo shinyto the nakedeye.(From M.J.Frank,H.N.Cartwrightand L.G.Smith,Development130:753-762, 2003.With permission from the Company of Biologists.)

] + a l )6 u r P u r q - d I V ur aln)elourdfv

'a)p!ns auerquraur aql qleauaqAll)eJrp luaureluu!t)e aqt lo uorlezuaullod oql p!epue'auerqureur lPuorlrasur eurseldlla) aq] ot pal)auuo)Ill)arlpu! '11ar {ueyt1 aql ur sarnlfnrts ole sururroJ reln>l1ted o] ]l lull pue ^t!^!l)est!aleln6ar suorbarlaqlg'eraq umoqssraln)elour abre;aqt;o tred ^luO'alqLuasse ot urr..tuo; Iunqns Mau Ll)easMollPt! se pua snloaql le pasooxaslrunqnsur])eoMl aql jo auo o16urpurqsll surelureur uraloldurr-u.ro1 aq1 sa1e6uo1e lr sp pua sn;d6urmor6 -{lprder qllm poler)osse aqt ureuarpue (pal)]uaLUPllJ ullf e Mau e Jo uorlPuuo] aql aleel)nuue) leql xaloulo))llouJp Iq e w)o! (uad6)surato.rduru.ro3'sugtu,rol palelporuuollebuo;aulr)v 9€-9[ a.tn6!l

'pua ('unqr5'l a)uarel)puer>1>,{zog laeq)rW }o (saynol) u!1)euelo {1;elttals slll }nq'luoulel!J uoll paluanatd snuruaqlot 6utputq ue)laulpolelaqult)P-ullUoro pueol pulqarolaraql puasn;daqlale6uola ull)PJoa)eJaqlol spulqu!llJord aqlallsoddo srrll'Ual)6u;putq-61y 'uaatb slaln)alour ul uMoqs st dIV'parut utl)eeql pue'an/q u7umor.ls arll 'rauiouoiuu!l)e ue ol punoqu;1;1o16 uralordurlr;o.rd 1g-91atn611 qslJ-urlJP Jo uolsuaxe eql puB uollEzlJelu1(lodullJe IBsol a^Isoldxa esnpord o] ullrJord elelllcp uBJ sleu8rsJelnllaoeJlxa'uoltecolslql ]V'araql spldloqdsoqd auerqruaru 3rpl3e o] spulq 1l esnecaq euBJqueu Bluseld eql Jo aJPJslloso]]tc aql l€ pezllesol sI uIIIJoJd 'euEJqueu Pluseld aql tE dlqruasse luauPIIJ JoJ IEJIIIJJ sr sluetuellJ Jo spue Suntor8 eq] oluo sllunqns ullse palelsenbes aloul 's]te ulllJoJd eJaqmsalls aql euuep UBJ sluslu ol dr$qp spggord 'aldruexa JoC -BqJeu aseqJ'spldlloqdsoqd IolISouI ol Supulq uIIIJord pue uorlel,fuoqdsoqd uqgord Sutpnlcut drprpce ullgord eleln8ar stuslueqf,eru JelnllaceJlul IeJaAaS '(gt-gl arn8lg) u1goJd ot punoq aq ullJe rlraluouolu aqt leql eJlnbar ueJ uorlB8uola ]uaruEIIJultJB 'sasec eseql q :(e^oqe pessnf,slp sutalord 3ut -]eelJnu-ur13e;o dpuel eql) sultuJoJuleueJ qll1v\pelelf,ossesI pua snld asoqrr slueruelg asoql JoJ uoll€Allf,E ullgord uo ,i.puor1s aJolu uela spuedep ql,lroJ8 'spue snld ]ueIuEIIJ oluo Iood punoq-ursoudq] peralsenbes tueruelu ullf,V eql uroq sllunqns ullJ€ Jo ]uatue^olu e sI selnJalour u1p;ord Jo uolle^Ilf,e 'sJeluouoru ullcE IenpIAIpuI ol Supulq ur utsourr(ql qlyvr IBOOIp Jo ]lnseJ lau aqt saladruoJ ulluoJd asnpJag ra8uol llunqns euo luaIuBIIJ UIIJE eql Sulteal 3go sllpJurTgord eql os 'ulllJord rog trrluUJBslr saJnpeJ ]eqt ultJe eqt ur e8ueqJ leuoll -BruJoJuooe sacnpul uop1ppe sql'pue snld aar; E oluo ppe ,(llpear uec xald -ruoJ ur]f,e-ulgord oql'Og-9I arntg) pua snld aq] ol spulq lBql reruouotu aq] uo alrs aql pasodxa 3unea1 allqm rpua snulru luetuellJ aqr qUltlraletcossu .{.1pru -rou plno^ lEr{l Jaruouou aq} Jo epIS aql SuDlcolq 'rya1cSutputq-dlv aq} a}IS -oddo uITgoJd'u11{otd'uralord Surpurq ol spulq aceJ eql ullJe eqtJo raurouoru 'pEslsul 'eseJaq] aq ol punoJ uaaq -Jaruouoru uo spuadap reqlouE ]ualullnJser 'ursourdql alep8ar plnoJ 9I raldeq3 uI passn3slpesoql se qJns lou seq srql lnq uollJnpsueJl pu8ts JI sE ruees tq8pr lt ;uopeztrarudlod ro; ueql esn s,'i.ea,rqted pue lood a8erots pereJJnq qql ruoq sreruouoru ultoe llnJf,er sllao op ,vroH 'apuoalcnu punoq rgaq] a8ueqcxa rou azf.lorpr(q reqltau uec pue luauEIU ulloe Jo spue snulru ro snld aql Jeqlle qllm alelcosselouuec ,(aq1 aJeqm'alels pe{ool e ur aJpursourdql ol punoq sJeruouoruultf,V'sulalord asaql '(ullnJunJlel Snrp eql Jo luepunqp lsoru eq] st utsouttt4t palleJ ulaloJd leurs V uollJe uE) elqEJo^EJssel qJnu uolluzuaurrtlod Suqeru Lqalaql Jo teql ol TEIITuIS 'sJaruouoru ptcads suleluoJ pulq sutalord eql ol uuce leql lood lrunqns aql 'elqnlos uPIuaJ ullce leq] sr uoseal aqJ isluatuullJ olul Surzrrarul.lod uuql JeqleJ eql Jo qcn{u os seop ,r(qq'aqn1 }sal e uI ul}ce arnd JoJpa^-IasqoINrl I ueq} sselJo uorlerluacuoJ leor]rrr aqt uanr8 'q8rqdlSursrrdrnsst sqt :(lru/3ru 8-Z) INt1002-09 'spu8rs puJelxa ol asuodsar dlecrdIl sr uolleJluaJuoc Jatuouou elqnlos aq; sl %09 pue s}ueuEIIJuI sI uI}rP ur.{lprder a8ueqc uer ollur slq} q8noqrle 'a1qn1os 'sllunqns elqn aqf Jo %09 ,(laleurxordde 's11aceleJqaue^ elJsnluuou lsoul ul -los Jo uoplppE eqt r(q a1e3uo1a dlerauaS slueluelu p1ala1so{c 'palEelsnu eJuo

uotte6uoll lueurelll,llpow sllunqnsaarJaql ol pu!8leql sulelord pua snulu.l

puasnld

srNrwvl|ltvllrr)sou) ullHrlrilnDlu slll) A oH

666

1000

Chapter16;TheCytoskeleton Figure16-38 Profilinand formins.Somemembersof the forminprotein familyhaveunstructured domainsor"whiskers"that containseveralbinding sitesfor profilinor the profilin-actin complex.Theseflexibledomainsserveas a stagingareafor additionof actinto the growingprusend of the actin filamentwhen forminis bound.Undersomeconditions, this can enhancethe rateof actinfilamentelongationso that filamentgrowthis fasterthan that expectedfor a diffusion-controlled reaction,and fasterin the presenceof forminand profilinthan the ratefor pureactinalone(seealsoFigure3-g0C).

motile structures such as filopodia and lamellipodia (see below). Besides binding to actin and phospholipids, profilin also binds to various other intracellular proteins that have domains rich in proline; these proteins can also help to localize profilin to sites that require rapid actin asrembly. As it does with actin monomers, the cell sequesters unpolymerized tubulin subunits to maintain the subunit pool at a level substantially higher than the

critical concentration. one molecule of the small protein smnmtibinds to two tubulin heterodimers and prevents their additlon onto the ends of microtubules. Stathmin thus decreasesthe effective concentration of tubulin subunits that are available for polymerization (an action analogous to that of the drug colchicine). Furthermore, stathmin enhances the rk;hhood that a growing microtubule will undergo the catastrophic transition to the shrinkin"g statel Phosphorylation of stathmin inhibits iis binding ro tubulin, and signils that c.ausestathmin phosphorylation can increase the rate of microtubui=eelongation andsuppress dynamic instability. Cancer cells frequently overexpressstuihmin, and the increased rate of microtubule turnover that results is thought to contribute to the characteristic change in cell shape associatedwith mahlnant transformation.

severingProteinsRegulate the Lengthand KineticBehaviorof ActinFilaments and Microtubules In some situations, a cell may break an existing long filament into many smaller filaments. This generates a large number of new filament ends: one iong filament with just one plus end and one minus end might be broken into dozens of short filaments, each with its own minus end and plus end. under some intracellular conditions, these newly formed ends nucleite filament elongation, and in-this case severing acceleratesthe assembly of new filament structures. under other conditions, severing promotes the depolymerization of old filaments, speeding up the depolymerization rate by tenfbldor more. In addition, severing filaments changes the physical and mechanical properties of the cytoplasmi stiff, large bundles and gels become more fluid when the filaments are severed. To sever a microtubule, thirteen longitudinal bonds must be broken, one for each protofilament. The protein katanin, named after the Japaneseword for "sword," accomplishes-thisdemanding task (Figure 16-39). Kaianin is made up of two subunits, a smaller subunit thai hydrolyies Arp and performs the actual severing, and a larger one that directs katanin to the centrosome. Katanin releasesmicrotubules from their attachment to a microtubule organizing center, and it is thought to have an important role in the rapid microtub"ule dep"oll,rnerization observed at the poles of spindles during mei,osisand mitosis. It may also be involved in microtubule releaseand depolyrnerization in proliferating cells in interphase and in postmitotic cells such ai nerr.ons. In contrast to microtubule severing by katanin, which requires ArB the severing of actin filaments does not requiie an extra energy input. Most actin-severing proteins are members of the gekotin superfamity,'whbsesevering activity is activated by high levels of cytosolic ca2*. Gelsolin has subdomains that bind to two different sites on the actin subunit, one exposed on the surface of the filament and one that is normally hidden in the longitudinal bond to the next subunit in the protofilament. According to one model for gelsolin severing,gelsolin binds on the side of an actin filament and waits until a thermal fluctuation hanpens to create a small gap between neighboring subunits in the protofilament; gelsolin then insinuates its subdomain into the gap, breaking the filament.

't t l

wrrH I nreear

I R E L O A D T NOGF ACTTN ON WH|SKERS + c o n t i n u e dr a p i d g r o w t h o f a c t i n f i l a m e n ta t p l u se n d

Luri0 !

('rar^asllurorj q]!M'966t 'Lgz-Llzit6 ila) uolssll..urad ^le 'f'f ta upurueH Luorl)salnqnlor)il.x 6uo;aqt;o suorle)olsnornard aql le 'sare;d slueu6er;llpLus ro sauase 6urnea; ,(ueu ur paralasuaaqaneqsluaurelU aqf 'uruelel uorlrppPaq] ra]resalnurur € Jo plauaruesaql (€) uruelPl,o uorlrppe aq] raljP spuo)os0€ solnqnlor)il.! aql ur slearqMaJe arearaqf (V) dfv qllm 6uolepappeseMuruelel par;undpue 'aprlssse;6e;o a)eJrnsaqt uo paqrospeale^^salnqnlol)rurpalaqPl 'u!uelel -aulurepoqr'pazrl rqels-loxef Iq 6ulrenesalnqnlor)lw 69-91 arn6r1

(ralueB'y,{re9 PuellaPueW 'M souref {satlnol) 'sa}tlpuaput }uasatdoslest ne} po}el^loqdsoqd Jo lnel palel{roqdsoqdun o1I;uo spulqnel })alap o} araqpasn{poqtlueaq1 'salupuapstrpue{poq slql lla) aqt ot peuUuolst 6ututelsZdVW'(uolnau ur paq)uerqpue 6uo1)uoxPaql ol pauUuo)st 6ututelsnel sealoqM'alnlln) e u (a6uDro)bututelsZdVt pue (uaatb)'uwels ur uornau ;edr.ueroddrq a)ua)salonlJounuulslqf npt,o uorlnqulstpaqt smoqsqde.r6o.r:tu 'uornau e sallrpuap pue uoxe ut sdVW uollPzlle)o'lg9-9 1 arn6t1 ,o Jo

alntlc) dpqSn eroru aFlll e lslxu ol lueuEIIJ ssarts l€3ruEq3eu srqJ 'ev$l 'sluJoJ }Iunqns aql SurJJoJ'lueluelrJ ullce eql Jo l{}8uel eq} Suop spuq uITIJOS 'tolcntSut aeJJpuE ]uauelrJ eql qloq uI ullJe o] spulq ll ]eq] uI pnsnun sI UIIIJOJ -nrautlodap uura palIeJ osIV 'slue(uellJ ullce sazulqBlsep qclq^\ 'ullltoJ sI slla3 JIlo^JeJne IIB ul ]uasaJd uleloJd Surpurq lueurBlg-upce luBuodlul Jeqlouv '(gz-gI aJnBrCees)JaIBIssnlslp e^\ se 'uollJeJluoJ 'uoseal alJsnu ur dals ]uEuodrul ue sI SuIpuIq ulso^urodoJ] Jo uoqeln8al aql slql JoJ lsuralord raqlo qlIM SutlcEralul luorJ luauel.IJ eql lue^aJd uec luaruep; '}uaurellJoloJdauo uI s}Iunqns ullce ur]re ue Suop urso.{tuodorl;o Surpurq eqJ luecEfpe ua^as ol .{lsnoauelpruls spulq lBq] ula}oJd pele8uole ue'urcotutodoJl 'slueul ;o Surpurq aqt ^q pazlllqels eJe slla3 ]sotu uI stueuEIIJ UI1JBpalcalas -ulrJ urlse s]f,aJJeosp sepls Jleql Suop suleloJd drossacce;o Surpurq aql 'eseaslp palelJossP qrL-v\ uollB slq] -Jeue8apoJneueqlJo eJuanbasuoJe Jo esnE3e aJPnulJo sa13uu1 eseql Jaqlaq^\ ^,fuelprqgorneupellec 'stuauely nelJo sale8erSSea8rel reelo ta^ lou sl ]I 'sa13ue1 sureluoc eseaslp s,Jarulaqzlvqlp eldoed Jo suIBJqeql q useldol-,tJ IIaJ elJeu eqJ 'suorleJluaouoJ q8rq .4.pua1clJJns tB luesard ueqm sluaruulg IeJIIeq umo str suroJ ulaloJd ne] 'saFqnloJJIIu Jo sepls eql Suop Surpulq o] uoulppe uI '(7-91 arn8rg aas) uoqe8 -ar8asaruosoruoJqJro; uoqeredard ur aypulds Jllolltu eq] uJoJ ot uolalalsopb apqnloJf,rru slr se8ueJreal IIaJ eq] sE JnJJo ]Eql scnueufp apqnloJf,Ilx '(11 raldeq3 uI passnrslp) ur sa8uuqc aql aleln8ar sarU^Itre dVIN telncFred u1 ayc,{clac aql q8norqt ssarSord sllar se JJopup uo peurn} eJeteql esoql ale sdvtr^tr aleln8ar ueo ler{l seseuDluralord lueuodrur aq} Suoury'sllal aplsul uoIlEZIIBJol pue d1r,lr1cesll qloq Suqlorluoc uI eloJ d;eurtrd E eleq uP3 d\'trAle;o uoqelrfu 'saseur{ uralord -oqdsoqd pue Suqpsar aql IBJe^es;o sla8rel eq} aJEsdVIAtr 'JelBlssnJSIp 'sJolotu relnJalotu ,{.quantrp sallaue8ropasolJua III,r,tJM qJtq,,t,t -euerquau;o godsuu4 aql ateln8ar osp uef, sluaruelg o1 Surpurq neJ '(It-gI 'uIEIxop Su1}caf arn8lgl salnqnlorcrru pa>1ced[1aso1ceJoru Jo sapunq IuroJ -ord ralroqs qonru E qll^ dVI tre 'nal Eursserdxaranoslleo allqm 'paceds flapyut 'uleurop Supcaford 3uo1 1da1 ere lpql sepqruorJllu elqpts Jo sepunq tuJoJ e seq qorqm 'Z1VW Sutssardxara^o slla3 'sdVIAtr]uaJaJJIpeJnpoJdJalo o] peJeeu -r8ua qlac uI palertsuouap se 'raqtaSo]4ced sapqnlorolru pateof,-dvlN.{.1aso1c 'pJe^ r oq eurruJalap ueJ ureuop Suqcaford eq] Jo r{}3ua1eql lno slcaford ]€ql JaqlouB pue aceJJnsaFqnloJJIIu aql 01spulq leql ulelxop auo lsEel lP eABq sdVIN eseqJ'(07-9I arn81g;.{poq 1ec aq} Ixoq puega }Eqt sallrpuap pue suoxe aqt Jo aJoJ aql uroJ salpunq eFqnlorJlu paz[qe]s alaqM 'suoJneu ul ]ueu 'sluauodruoc -nuord sr srqJ lasqns 1ac Jaqlo qlIM selnqnloJJllu Jo uollJeJelul eql ele1patu os1euec sdVIAtr Jo lasqns y ,{lqurassesp lsup8e salnqnloJclru azIIIq -Els uEJ sdVIAl 'loxel Snrp aqt a{l'I 'sdVIAtrro 'su1e1ord pelulcossu-eFqnloJoIru palpc ,{.1anuf,alloc are salnqnlorrrru Jo sepls aqt Suop pulq }pqt sulatord _ AIeAr]f,eosaJ 'raur^,(1od aqt azlrqelsep Jo azlpqpls reqtla dqaraql u(aql pue 'a1e1srarul.lod aql;o d8raua aa{ aq} asrer Jo Ja^ ol Jaq}Ie o} ^8Jaua Surputq JIaq} asn suta}ord pelerlossE-tuaruplg lueJeJJIC'sarlradord IBJIueqJau pue dlurqels s,]uauellJ aql ralp,{.eru raru.{1odeqlJo sepls aq} Suole pulq }eql suralord;o 1ase '1ood}Fnqns eql tuo4 pale8uola pup uonealf,nu,tq pauroJ sl lualuelg p1a1a1so{J e af,uo

ro az!l!qels ueql azrlrqelsec raqlll ue) sluauel!l Josaplseql 6uolv pu!8reql sulalold t00r

slNl wvt II tvl-ltilsor-A) ull Hr lrfl n9lu slll) MoH

('ssord A]tsra^t un rallalal)ouaql l..UorJ uolsslLulad q]!M l66t 'Lg/__Lll:g€L 'lolB 'f ''lP q6no9)W'V la ilD Luoll)'xtlaLlaq] jo ]stMlq)ea^q pauusds a)uetsrpaql 6ut)npalpue ^llq6ll aloul lt 6utlstM]'a)tilpl luauleluutl)e oql uuoJepot sanrasburpulqutluo)Jo I6raua aq1'ra1:1qr,(;iq61;s r(1uo s.readde luauelUaql os pue '(suo]lepolr) €t) ult)e ol paleduro)(suo]lepolr) tl) uletold e sru!lgol'luaurel!;aqt 6uo;e lleu,rs l l es l r u n q n us r ] > eo l { r } a u o t q ) t o l s[ : ! e u l spurqq)tqM'utluo)qtlM poteo)]uaLuelu utl)p ueJo uott)nrlsuo)au(B).x!laq u!])e aqlJo slstM]oMl Jo uedsaql sMoqs lallerq aqf'utllp orndJo apeu stuauleltJ ut uorlta;a-o{lrulor; 1osqder6ortr uorl)nJlsuo)at (V) leuotsuaultp-aalqI 'utltro)^q pa)npu! luauelrJ u!l)e uP ro 6ullsrrvrl7tr-91atn61

uruLs

urlr+o)+ luau,relr]|' utpe

(g)

wu vL

urpe (v) luerrrelr+

,,fuanaro; urso,,(urodorl auo ]noqe 'aydruexaJoJ)seular.uorqcrols q8rq r(pre; le luas -ard aq ot aleq [aq] suearu slq] pue i(1a1a1duroc ]uauelrJ aq] ]poJ ol peeu uago sulalord esaql 'ra^eMoq 'lJaJJarunrurxEur JoC 'Jor^Eqaq Jrureu^p s,]uauelrJ eql aSueqcuec lueuelrJ e Jo aprs aq] Suop purq leql suraloJd 'uaas 1sn[aleq e^ sV

s)!ureu/(c luauelrJe6ueq3 {;;e>rleruerg ue) spul }uauel!l qr!/v1 })eratul}eql sure}otd 'Surprerc IIaJ Ipuor]JeJrprunJoJelqrsuodsal 'pazrJelod ar{} JoJ IJoMlau urtJe ar{}Jo qte\oJ8patJalrp IeJrluJ sr stueuplrJ ur}Je ^\au lou lnq plo Jo .,{lquassesrppa}erpau-urlrJoJ aql 'lJalelssncsrp IIr,/\,\ a^\ sv dprder JaAouJnl stuauulrJ ulloe IIe leql Suunsue 'llal aq] ur sluatuelrJJaplo eql salluerusrpdlluarcrg;aaJoJaJaq]ulllJo:) 'urlgoJ .{q uor}uzrrarurtlodapol tue}srser ere pue dJV.{Fsour ure}uoJ III}s 11e]eql ur s]ueruelrJurlJe lsamau aql flquresse 's]ueruelrJ 3ururc1uoc-41y ]uauBIrJ ueql remols f1ensn sr srsd1orp,,i.q dJV aJurs ol ueqt Jaqler stuauelrJ urlce SurureluoJ-dCV o1,{lp4uaragard spurq ulllJo:) 'Surpurq urso^uodoJl ,rtqurqoc ruor; palcalord aq uec sluarxelrJurlJv'aqnl lsal e ur urlJe aJnd ruo4 pauroJ slueure -lIJaJeuBq] pe^rl-JauoqsqJnru aJEsllac aprsursluauElrJ urlJE eqlJo ]solu'llnsal e sy fyquassesrp ]uetuElrJ ur]JB elEJeleJ3e,(pear8 serlr^rlcp aseqJ ']uaruBlrJ aq] Jo pua snu[u eq] ruo4 alerf,ossrpol lrunqns urlJe-dcJv ue JoJJarseaqJnu 1r sa)Erutl 'uollrppe ul 'suonor.u[Er.r]Jeq] dq paranas.{.1rsea arour pue allluq luetu -elrJaql Suqeur 'luaruelrJ aql ut slrunqns ur]re uaamlaq slceluof, eql sue{eal ruu00€ (a)

('prl sraqs!lqnd uellil.!)ew nrorjuolssluiladqtlM 266t 'LVg-VLg:jgt \ arnlDN''P la uaql'n;o,{selrno: ,c pup f) 'ule 6url>aford qnel lroqsI;anr1e;a.r ueqttaqtabol Jo asne)oq()) ut arp,{aq1 {;aso;rarou pa>edsaresalnqn}or)tut aql alaH'ne16urssatdxarano lla) e ut alpunqalnqnlol)tut e q6norqtuotl)asssol)reltults(C) 6urltafordaql ZdVWaql Jo sr.ure Jo rl]6uolluelsuo)aqt Luorjtlnsatalpunq srqfur (sfw)salnqnlol)turaqlJo 6ut)eds re;n6araq1 74yy1; 6urssardxareno l l a ) e u t a l p u n qa l n q n l o r t r ue q 6 n o r q l uotl)asssol) e 6urmoqsqder6or:rr.u uort)all (1) dool 6ult>afotdtroqs e qllM'!ururral-)pue -N slt qloq le e)tuel alnqnlol)il.!aq] ol spurqnef (B)'pua raqlo aq] ]e ureurop6urpurq-a1nqn1ot>rr.u puo)ase qtlm [!re 6ulttalord6uol e spualxepue spuas]tjo auo lp e)tilel alnqnlor)il.ll eql 6uolespulqzdvw (v) 'sdVW^q salpunqalnqntol)!r,u ;o uollezgue6lg1y-91arn613

(8)

.lnqnlortr-/

'

uoFlelsoil) aql :9[ ro]deql

200t

'pua snutul luauPlu aql Jo ]eql ol surqsuorlelndodaql loJ uotlerlua)uo) l e ) r t u )a q l ' u o r ] r p p ue l ' u o t l e r l u a ) u o l lelrlu) eql Molaqsuotlprlua)uo) raLuouour llele taMolsaq llrM a6eluuqs]uaurelUpue /uotlellua)uo) le)r]u)eql a^oqesuolle.tlua)uol rauououj llplP laMolsaq lltM L.ltMor6 1s]runqns luaupl!JXliuanbasuo) asolro ppe ol alqpsr pua snutLr.l aLl]^luo '(aul pil) pua snldaq] sdp) leql uralordp Jo a)uasardaql ul (autluail6) sJawouow aaJJalqelrPAe Jo uotlellua)uol or.lluo 6urpuadop'a6e)urrrls lo qtmorbprderur 6ultlnsa,'spuasnurur puPsnlosLlir.lloqte slrunqnssasolpue sppesluaurelU paddetun;o uorle;ndody 'srrueu{p luouel!} uo stlaJ}a s1rpue 6ugdde)luatupllJgtr-91a.rn6r1

aleJ aqdoDsBlpJ eql sasEaJJuI.{puecgru8rsslopat aqdoJlsDlal se U^\Ou)Isule} -oJd palplaJ-ursauDlJo ,{lrueJ E 'aldrupxe JoC '(sencsal Jo ^cuenbarJ aq}) atels eq}) a}els Sur4urrqs Suurror8E ol SuDluuqs e ruo4 Jo (saqdorlseleJJo ,,{.cuanbar; E o] Suu or8 e ruo4 saqclu s elnqnlorcltu e qJIqM lB eleJ aql aluanlJul uef, ,(aqt '(gi-g t arn8rg aas)sa1nqnlonru;o,rfiqrqe]sul JIIueu.{paq} uo slcaJJaJI}eu -EJp a^Eq uec r(aq] 'relnJlued uI 'ulelord Surddec aldruts e ro; palcadxa asoq] puo.,(aqsaloJIETJnJJa^Eq seFqnloJJrruJo spua aqt le IJE ]eq] sutalord aruog '{}3ua1 ur ruJoJrun puE alqels qloq aJE salnqnlorJlru araq A '(Jalel passnJslpJ BITI3 ur selnqnlorJrru aqt Jo spua eql le punoJ xaldruoc uralord ptcads aqt sI sapqnl -oJJrur ro; uralord Surddec anJl Jeqlouv 'spua snurur rlaql sdec pue JaluaJ Surzrue8ro up te salnqnlorJrur ;o qrruror8aql selealJnu qloq qJIqM '(lgnJ-,t) xalduoc Surr urlnqnl-,t aql :raddef,elnqnlorcltu lupuodrul ue passnoslp.,{pearp JoJ serlilqrssod aJoru zlueu qlpu ']ueulellJ aleq a A'uorlJe uralord .,fuossacce urlce ue pua aq] ueq] arnlJnrls xalduoc aloru pue ra3re1qcnru P sI '(I I-9I eJn Jo -3rg aas)8urr,r,ro11oq E ur stueurulgolord uaaulqt qll.t,trelnqnloJJlrue Jo pue aql

spul alnqnlor)!w6u1nnor9 {lprdegro sa!}radord aql la}lv suletordlo spu!)ltuata}JI6 'pazllrqels tPq^\aruos uaaq.,{pearp,{qaraqt aleq pue urso'(uodorl ^,{qpaleoc ueeq a^EI{leq} sluatupllJ u r t J pJ o p u J s n u r u a q r o r . { 1 u os p u r q u r l n p o r u o d o r l' p u a s n u r t u e q r w u t l n p o r u -odot1 f,qpup pue snld aqt rc 7de3,{.q-spua qtoq }E paddec ,{.1prcads ere s}ueur -EIrJaq] 'panr1-3uo1 .{lpuoqdacxe ere sluauEIIJ ullJe aJeqm 'sllal alcsnu uI 'paddecun aJepue xaldruoc dUV aq] Iuoq pasealal erp sllec pcrddl ur spua snurur luel'uBlrJurlJE aqtJo,ri.ueuleql alqrssodsl ll qSnoqlP 'uorlEelJnu slr JoJalqrsuodsarsea,rleqt xalduror dUV aql ol punoq Suturetuar,{q paddec aq .,(eu luauelg urlJe ue 'pua snunu aqi tV'(ulelor4 Surdde3 palleJ osle sl lr lmoleq aJS 'pueq Z alJsnur Jql ur uortpJol stl roJ palueu) 7da3 su qtns sutalord ,{q pua snld rraq] le paddec ere IIac p uI sluaurpllJultte eql Jo lsoru 'paapul '(Sf-gI arn8gg) alrJJeur pua snld aqt Suopru dq uorlezrraru,r{lodaplueruelrJ pue q}^r,ror8 TIJIqM 'pua snld sll ]e lualuellJ ullJe tuauelrJ qloq Jo salBr aq] s,r,rols,4.11ear3 ue sazrlrqelsulalotd Sutddac pua snld e;o Surpurq aqJ 'pua snld aqt lE rnJro la^a^ oq 'sa8ueqcprder lsoru aqJ'ruJoJ C aqt ot ile^uoo ol dJVJraql paz[1orp,{q aleq pua teql le saln3alou urlJe aql acuo 'pua snullu s1tro snld sll reqlle uorJ .{.1pcr;rcads slrunqns asol uEJ 11:r(lprderazrraruf1odapueJ IIaJ aql ,(q pazrTrqE]s 'pessnJslp,r(lsnornard sy lou sr pue uorle8uola saseal leq] luaruellJ ullJp uE 'Tro,\i\]au ]uauelrJ urlce ue Jo aJntcatrqJJeaq] ruJoJsueJlol q8noua aq ueJ (sJaruouoruulloe 009-002 lnoqu rad auo ,{Wcrd,{r)}uauelrJ ur}ce rad uralord e qJns Jo alnJalotu auo 'spua }uarup -g le .{prerurrd rncco ssol pue uourppe lrunqns aJuIS 'slalal ,r,to1 ,{ran }e luasard are .,(aq]ueqm uela scrureufp ]uauelrJ uo s]JaJJaJl]prueJp eleq upJ slueuelrJ 'tsBJluoJuI '(llunqns utlce frana Jo spua aql o1,(1er1uara;ardpurq ]Eqt suralord JoJurlrJoJauo Jo 'slrunqns urlnqnl rno;.{rarra roJ nB} auo 's}Iunqns ul}ce ualas UOIIeJIUa)UO) JOt!OUOU] e

'-: TO

r3

{ 1 u op u a s n u r u r} e qyiror6:sluaurelrl +ouorlelnooo padoe)

0

I

spua s n u r L up u e s n l dl e qp.rol6:sluaurelr; +ouorlelnood paooe)un

€00r

s I NlWVlH

@

d

3 d a

'lvl 'EvSOtI)

)IEH-LtlvtnDtu

Stttf MOH

('utMps uayJo^sauno),g llatlesll ulo.lJuotsstt.lJled l . l l f ^ ' 9 0 0 2 ' v sL- V : I L ' l o ! 8 p2'u1d6 zn1 'peelua6ooH.J.) pue e^oueuqlv'v ruotj'v) 'slla) padeqs-poraql lo sa;odoM] aLlt re (pal)tgj;o bolouoq eql qIM palef)ossparc (uaa$)salnqnlot)tut eq] jo spuesnldaql'aqwod sat(wot o tltlosozlqr5 lsea{ uolssr; pedeqs-poraqr ul (g) '@arl LAj urolorodlI+ aql qllM pelet)osse srq)rqMpua snld6uuvrol6 e seq (uaar6) olnqnlol)tLuq)ea'alnlln) anssrlur ur*or6;;a>;e;leq1rda ue ul (v) 'solnqnlor)lur aql le ,o spue sn;d 6ugrvro"r6 punol sulelord d11+St-91 ern6ll

ulrl s

'(gt{I ernt.rd {urrqs ol ur8aq sepqntorclru aql ueqm spue eql {uo4 SurlelJosslp 'salnqnloJf,Iru Surirnor8,{lprdur Jo spua eql le sJe8uassedse IIaJ aql punoJu taIJoJ ol JBaddBpuE 'spue a^rlJe aseql ]u elplnrunf,Je (sdIJ+) sunrcJd Sur4Jau pua-sryd pelleo sulelord peluroosse-alnqnt 'lsEJluoc u1 'spua snld -oJJrIN'eJeds IIeo eJrlua aql eqoJd puE eJoldxadpuarcr;;a eqJ'se4s uorlezrraurdlodap alnqntoJcrru sB e^Jasesle Jo 'aruosoJluacaql qllllr uorlproosse dq pazllrquls eJe salnqnloJorru Jo spue snu[x eql 'slleo dueur u1 '(gg-Zt ern8rCees) epurds JItolFu aqlJo uollonrlsuoc lualo 'srsolrru -rJJe pe^Jasqo Suunp saFqruoJJrru aql JoJ IBJTITJJsr ]Br{} uoprsusJl B go ,{1gqe1sur Jrrupudp aql ur esealf,ur ploJuel p ur sllnsar UIqs eqJ '(?t{I eJn -tgg) srolceg aqdorlselec qlpr uoppedruoJ str Jo acueluq aql sUHs puu dlvrnce slr slrqrqu srsotnuSurrnp gIZdVI [XJo uor1efl-roqdsoqdaqJ'e]els Suquuqs u o1 Suurror8E ruo4 qJllrv\sJleqr ]lqg{ul pue spue aFqntoJJIIu aaq azlpquls ol Ir111qp prcads e seq ueloJd slqt'(suolppoFn ur ssBruJEInJelou sll o] sJaJeJJeqlunu eql pue 'uralord palerf,ossE-elnqnloJJlul sndouay roJ spuels dVIAIX) suElunq ol ]sead uo4 aBuer leq1 surque8Jo ur sSolouroq esolo seq leql gyT1yy1y snol -mbrqn aq] sp qons sdVIAtr ere suoqcu 4eql SulsoddO '(Cgt-gf arn8rg ees) alels Suopuqs eql Jo orlsrJeloeJeqo]uaruelrJolord pal-rnc eql olul pude 8ul8urrds uro4 elnqnloJorru E sluanard ler{l JerJJeqr{3raua uorlellloe leluJou aq] Suuemol 'pede slueurelgolord drd o1 uees pue spue eFqntorJrru ol ,{lpcgtcads putq ,taqt '(gg-gt arn8rg aas ldgure; tl-ulsauq eqt Jo sJeqlueru erp sutalord esaql)

paseerlu!

'(ratelpassn)s!p) (;rue#adns urelororolour utseutlaqllo lequ.taul e'€ t-ursaurlse q)ns slo])eleqdotlselet ale uotl)e s1r6ursoddg'eraq1 6u;pulq lelluaralerds1trq a;nqnlor>rtu 6urmol6e Jo pua oqt sazlltqels StZdV6X se q)ns dVWy'suralo.rdleoads{q s;1erur pelloJluo)sr 6urluuqsalnqnlol)tuipue qlrvror6e;nqnloJ)il,!uaoMlaquotltsuell oqf'sPua alnqnloJ)!utor pu!q reql sulalord 1o sl)e#a aql tt-91 arnbr3

(€[ ursaur{) rope] aqooJlselel

alnqnloj)il.r.,|+o pua snlouo oe) dI9

:f'lnstu

pa)ueque eleJ qy'iror6 roTpuepassorddns saqdo.rlsele:+o r{:uenbarl

.1'lnslu dVIA

uola;a1soy(1aql:9 [ laideq)

too t

THEIRCYTOSKELETAL FILAMENTS HOW CELLSREGULATE

Some of the +TIPs, such as the kinesin-related catastrophe factors and XMAP215 mentioned above, modulate the growth and shrinkage of the microtubule end to which they are attached. Others control microtubule positioning by helping to capture and stabilize the growing microtubule end at the location of specific target proteins in the cell cortex. EBl, a +TIP present in both yeasts and humans, for example, is essential for yeast mitotic spindle positioning, directing the growing plus ends of yeast spindle microtubules to a specific docking region in the yeast bud and then helping to anchor them there.

in Cells Structures into Higher-Order AreOrganized Filaments So far, we have described how cells use accessoryproteins to regulate the location and dynamic behavior of cltoskeletal filaments. These proteins can nucleate filament assembly,bind to the ends or sides of the filaments, or bind to the free subunits of filaments. But in order for the cytoskeletal filaments to form a useful intracellular scaffold that gives the cell mechanical integrity and determines its shape,the individual filaments must be organized and attached to one another in larger-scale structures. The centrosome is one example of such a cytoskeletal organizer; in addition to nucleating the growth of microtubules, it holds them together in a defined geometry, with all of the minus ends buried in the centrosome and the plus ends pointing outward. In this way, the centrosome createsthe astral array of microtubules that is able to find the center of each cell (seeFigure 16-32). Another mechanism that cells use to organize filaments into large structures is filament cross-linking. As described earlier, some MAPs can bundle microtubules together: they have two domains-one that binds along the microtubule side (and thereby stabilizes the filament) and another that projects outward to contact other MAP-coated microtubules. In the actin cytoskeleton, the stabilizing and cross-linking functions are separated. Tropomyosin binds along the sides of actin filaments, but it does not have an outward projecting domain. As we shall see shortly, filament cross-linking is instead mediated by a second group of actin-binding proteins that have only this function. Intermediate filaments are different yet again; they are organized both by a lateral self-association of the filaments themselves and by the cross-linking activity of accessory proteins, as we describe next.

and BundledInto Filaments AreCross-Linked lntermediate StrongArrays Each individual intermediate filament forms as a long bundle of tetrameric subunits (see Figure 16-19). Many intermediate filaments further bundle themselves by self-association; for example, the neurofilament proteins NF-M and NF-H (seeTable 16-I, p. 985) contain a C-terminal domain that extends outward from the surface of the assembled intermediate filament and binds to a neighboring filament. Thus groups of neurofilaments form robust parallel arrays that are held together by multiple lateral contacts, giving strength and stability to the long cell processesofneurons (seeFigure 16-22). Other tlpes of intermediate filament bundles are held together by accessory proteins, such as filaggrin, which bundles keratin filaments in differentiating cells of the epidermis to give the outermost layers of the skin their special toughness.Plectinis a particularly interesting cross-linking protein. Besidesbundling intermediate filaments, it also links the intermediate filaments to microtubules, actin filament bundles, and filaments of the motor protein myosin II (discussed below), as well as helping to attach intermediate filament bundles to adhesive structures at the plasma membrane (Figure 16-46). Mutations in the gene for plectin cause a devastating human disease that combines epidermolysis bullosa (caused by disruption of skin keratin filaments), muscular dystrophy (caused by disruption of desmin filaments), and neurodegeneration (caused by disruption of neurofilaments). Mice lacking a

1005

1006

Chapter16:TheCytoskeleton

u . 5u m

Figure16-46 Plectincross-linking of diversecytoskeletalelements.Plectin (green)is seenhere makingcross-links from intermediatefilaments(blue)to microtubules(red).ln this electron micrograph,the dots (yellow)are gold particleslinkedto anti-plectin antibodies. Theentireactinfilamentnetworkwas removedto revealtheseproteins.(From T.M.Svitkinaand G.G.Borisy, J. CellBiol. 135:991-1 007,1996.With permission from The Rockefeller UniversityPress.)

functional plectin gene die within a few days of birth, with blistered skin and abnormal skeletal and heart muscles.Thus, although plectin may not be necessary for the initial formation and assembly of intermediate filaments, its crosslinking action is required to provide cells with the strength they need to withstand the mechanical stressesinherent to vertebrate life.

cross-linkingProteinswith DistinctpropertiesorganizeDifferent Assemblies of ActinFilaments Actin filaments in animal cells are organized into two types of arrays: bundles and weblike (gel-like) networks (Figure lHz). As described earlier, these different structures are initiated by the action of distinct nucleating proteins: the long straight filaments produced by formins make bundles and the ARp complex makes webs. The actin filament cross-linking proteins that help to stabilize and maintain these distinct structures are divided into tvvo classes:bundling proteins and gel-forming proteins. Bundling proteins cross-link actin filaments into a parallel array, while gel-forming proteins hold two actin filaments together at a large angle to each other, thereby creating a looser meshwork. Both

Each type of bundling protein also determines which other molecules can interact with an actin filament. Myosin II (discussedlater) is the motor protein in stressfibers and other contractile arrays that enables them to contract. The

c o n t r a c t i l eb u n d l e

g e l - l i k en e t w o r k

1 0 0n m

Figure 16-47 Actin arraysin a cell. A fibroblastcrawlingin a tissueculturedish is shownwith threeareasenlargedto show the arrangement of actinfilaments. The actin fifamentsare shown in red,with pointingtowardthe minus arrowheads end. Stressfibersare contractileand exert tension.Filopodiaarespike-like projections of the plasmamembranethat allowa cell to exploreits environment. The cortex underliesthe plasmamembrane.

1007

THEIRCYTOSKELETAL FILAMENTS HOWCELLSREGULATE spectrin(tetramer)

fimbrin (monomer)

0-actinin (dimer)

5U nm

very closepacking of actin filaments causedby the small monomeric bundling protein fimbrin apparentlyexcludesmyosin, and thus the parallel actin filamentsheld togetherby fimbrin arenot contractile;on the otherhand,the looser packing causedby the larger dimeric bundling protein a-actinin allows myosin moleculesto enter,making stressfiberscontractile(Figuref6-49). Becauseof the very different spacing between the actin filaments, bundling by fimbrin automaticallydiscouragesbundling by o-actinin, and vice-versa,so that the two types of bundling protein are themselvesmutually exclusive. Villinis anotherbundling protein that, like fimbrin, has two actin-filamentbinding sites very closetogether in a single pollpeptide chain.Villin (together with fimbrin) helps cross-linkthe 20 to 30 tightly bundled actin filaments found in microvilli, the finger-like extensionsof the plasmamembraneon the surface of many epithelial cells (Figure l$-50). A singleabsorptiveepithelial cell in the human small intestine,for example,has severalthousandmicrovilli on its apical surface.Eachis about 0.08pm wide and I pm long, making the cell'sabsorptive surfacearea about 20 times greaterthan it would be without microvilli. \'Vhen villin is introduced into cultured fibroblasts,which do not normally contain villin and haveonly a few small microvilli, the existingmicrovilli becomegreatly elongatedand stabilized,and new onesareinduced.The actin filamentcoreof the microvillus is attachedto the plasma membrane along its sidesby lateral sidearmsmade of myosinI (discussedlater),which has a binding site for filamentous actin on one end and a domain that binds lipids on the other end' one binding actin filamentsto eachother and Thesetwo typesof cross-linkers, the other binding these filaments to the membrane, seem to be sufficient to form microvilli on cells.Interestingly,when the genefor villin is disrupted in a mouse,the intestinal microvilli form with apparentlynormal morphology,indicating that other bundling proteins provide sufficient redundant function for this purpose.However,the remodelingof intestinal microvilli in responseto certain kinds of stressor starvationis impaired.

Figurel6-48 The modular structuresof four actin-cross-linkingproteins.Eachof the proteinsshown hastwo actinbinding sites(red)that are relatedin Fimbrinhastwo directly sequence. sites,so that it adjacentactin-binding holds its two actin filamentsvery close together(14nm apart),alignedwith the samepolarity(seeFigure16-494).The two actin-bindingsitesin cr-actininare separatedby a spaceraround 30 nm long,so that it forms more looselY packedactinbundles(seeFigure 16-49A).Filaminhastwo actin-binding siteswith a V-shapedlinkagebetween actinfilaments them,so that it cross-links into a networkwith the filaments orientedalmostat rightanglesto one another(seeFigure16-51).Spectrinis a tetramerof two o and two 0 subunits, and the tetramerhastwo actin-binding sitesspacedabout 200-nmapart (see F i g u r e1 0 - 4 1 ) .

Figure16-49 The formation of two types of actin filament bundles. (A)cr-actinin, whichis a homodimet actin filamentsinto loose cross-links bundles,whichallowthe motor protein myosinll (not shown)to participatein actin Fimbrincross-links the assembly. filamentsinto tight bundles,which excludemyosin.Fimbrinand s-actinin tend to excludeone anotherbecauseof the very differentspacingof the actin filamentbundlesthat theYform. (B)Electronmicrographof purified c[-actininmolecules.(8,courtesyof JohnHeuser.)

a c t i nf i l a m e n t sa n d fimbrin

a c t i nf i l a m e n t sa n d d-actinin

50 nm contractilebundle l o o s ep a c k i n ga l l o w sm y o s i n - l l (A) to enterbundle

p a r a l l e lb u n d l e t i g h t p a c k i n gp r e v e n t sm y o s i n - l l f r o m e n t e r i n gb u n d l e

(B)

1 0 0n m

1008

Chapter16:The Cytoskeleton

amorpnous, densely s t a i n i n gr e g i o n p l u se n d o f a c t i nf i l a m e n t microvillus

acItn f ilament bundle

l a t e r a ls i d e a r m (myosin-1, calmodulin)

(A)

(B)

(c)

|

| 1rt

Figure16-50A microvillus.(A)A bundleof parallelactinfilamentscross-linked by the actin-bundling proteinsvillinand fimbrinformsthe coreof a microvillus. Lateralsidearms(composedof myosinI and the Ca2+-binding proteincalmodulin) connectthe sidesof the actinfilamentbundleto the overlyingplasmamembrane.All the plusendsof the actinfilaments areat the tip of the microvillus, wherethey areembeddedin an amorphous, denselystainingsubstance of unKnown (B)Freeze-fracture composition. electronmicrographof the apicalsurfaceof an intestinalepithelialcell,showingmicrovilli. Actinbundlesfrom the microvilliextenddown into the celland arerootedin the terminalweb,wherethey are linked togetherby a complexsetof proteinsthat includesspectrinand myosinll. Belowthe terminalweb is a layerof intermediate (C)Thinsectionelectronmicrographof microvilli. filaments. (8,courtesyof JohnHeuser; C,from P.T.Matsudairaand D.R.Burgess,ColdSpringHarb.Symp.Quont.Biol.46:845-854,1 985.With permissionfrom Cold Spring HarborLaboratoryPress.)

Filaminand SpectrinFormActinFilamentWebs The various bundling proteins that we have discussed so far have straight, stiff connections between their two actin-filament-binding domains, and they tend to align filaments in parallel bundles. In contrast, those actin cross-linking proteins that have either a flexible or a stiff, bent connection between their two binding domains form actin filament webs or gels,rather than actin bundles.

projections called lamellipodiathathelp them to crawl acrosssolid surfaces.Fil-

A very different well-studied web-forming protein i s spectrin,which was first identified in red blood cells. spectrin is a long, flexible protein made out of four elongated pollpeptide chains (two cr subunits and two subunits), arranged so B that the two actin-filament-binding sites are about 200 nm apart (compared

r.lr110 I

(8)

qlm '/66t 'Lsq-Svg:gtl eql uorsstutad (ssar6 {lrsranrun lolloJal)ou 'Il!lllor.rl 'f 'to!B '1' pue{6o;oqdtoulle)uo ;e1ar-ueq6uluunl tuorl) ilD aqlleqll)a;Ja lo a)uasaJd eJoa)uasqe a;6urs aneque)uralord le.rnl>nr1s /(lq6lLl s!Lll')llelselau Iueu jo ouosla;druexe puno;ord aql 6urlPllsuotuep uaaq I;1etlt;llte alPtuMousllafaql'palolsal arepueerpodr;;aue1 leiuJou aulesaLll(8)'azlselselaul q)tqMutslle)euouPlaul sequorssaldxa urutplt, auelquaul o] ]ou pualpue{uood;nnetlr{aq}'}lnsore sv ,;sqalq,, pueerpodt;;euel qllMpora^o) olepealsul leulroualeulol alqelou uea^eqleq}slla)eurouelaur jo la^alMol{;;eutouqe areslla)asaql'ulJrelU '/ttltrou sasne)ulurellr 1ossol75-91ern6r3 lla)lerulouqe ;o dnol6V (V) 'uollseJelul JulnlalorueJlul uP ^q aq uec uorleturoJuoc e^rlcE eq] o] SulqJlr1v\S 'uollEluJoJuoc peploJ a^Il3eul JeqlaSol plaq eJe IuIIuJel-3 puB -N eql qllq^\ uI ue pup 'utalord aueJqueusuBJl E puB uIIJP qloq ol spulq pue sazIJaIuoBIIo 'suollgluJoJuoc 06l ul ls1xe ugl sulelold lpql uoqeuloJuoc pepuelxa e1llce uE 'slEuBISrBlnlleJPJlxapuB mlnllacPrlul qtoq,(q pelup8ar erc sulelord ntrUg INUS ,r(qpalerparu eupJqueu Eruseld eqt pue ullse uaamlaq slueuqcune eql 'uEuoJnlEAq 'v?cll su qJns 'sulal ]uauoduos xrJlBur JelnllaJEJUa aql JoJ Joldacal eql -oJdoJ^18aueJqrueursueJ]aJotu Jo auo Jo af,EJJlluseldoldl aql ot sputq uletuop 'sluaruBllJullJe Jo sapls eq] o] fllJarlp spulq ulelord I trug ue IPurrurel-N eqJ .srsol{Jopue pue srsol-{3oxeur pe^Io^ur pue IlrJelod Jo urEruop IpuruJa]-J aqJ '(ulseoul pup II3OJo eJupualureru aql JoJ pennbeJ eJe teql sJaqlueu Suleluos 'sulaloJd 'urrnpeJ 'uuza 'srequtaru y'1gg aql Jel aaJql lsrlJ sll JoJ pelueu) tt11't;l-1 -nlleJeJtul patelar.{1aso1c Jo t{llrueJpeardsaprluV'poolsrapun dlprued lluo are eupJqueur eruseld aql ol uolela>1so$oullJe Ieoluoc eql Jo suollJeuuoo aql 'auurqueu EursBIdaql;o sprdll Jo sulelord puE {Joo\ -lau luauelrJ urlJg aq] ueegleq Slue111llJelleJIJIcedSSaJInbaJsaJnlsnJls esaql Jo sseue^IlcaJJaaql'sllaf, lullaqllda;o eare aceJJnse^IldJosqe eql eBJEIualeql rllr^oJcrruuI selpunq uI]3E-uIIIF eql pue seueJqrueu eruseld aql sallJepun lEql qe^a uIIJE-uIJlJads aqt :selduExa o^i\l passncsrp,{pearp e^eq eM'eueJqrxelu eruseld aqt Jo adpqs eql a8ueqJ pue ueJJIlsqloq sernpnrls Ielale{sol,{c ullsv

sauerqulewol sluauq)ellv {uery elew slueuall lelelalso}/() 'euerquau aJeJrns aql ueJJIls pue adBr{so1 dlaq oqe -,(aqlalaq^r'sadd111acelPJqaua^raqlo lsoluJo xauoJ eq} 'rfuepder e q8norql Sutzaanbsrage adeqs ur punoJ aJeurrlceds Jo sellt€lal aso13 'auuJqtualu eurseld 3utd1 purSrro slr ol {JEq Suuds o} IIaJ poolq pal aql Surruolp -JeAoeq] ro; ]roddns IeJIueqJaIu saplloJd ]Pql xagoo IIal JJIIse seleaJc{Iomleu Suqpsar aqJ '(It-gl arnBICaas)sutalord euerqrueu IBJBaIuIfq Ja,{e1qptdl aqr 'suralold euEJqluelu praqduad IoJ salls JBau peuoplsod se^lesruaq]aJe qJIqM Surpurq elpJedesseq lI esnp3eqaugIqlueu eusgld aql ol qe^ slql $lug ulJlceds :slualuellJ ultce uoqs ,i.q raqtaSot plaq qe^ puolsuaulp-oMl B sluroJ ll araqM 'aueJqureru euseld eq] qleeuaq lsnf pa]er]uecuoc sI UIJ]ceds '{lleJpoolq peJ eql uI .(87-gI eJnSrcaas 'ururlJe-D JoJruu 0t lnoqP pue UIJqIUIJJoJIuu 7I qu^a

'la6ro qam buorls(;ge:rueq>au e su-uo1 urulelU (q pa1ur1-ssorr sluaureluurl)ejo las V (B) 'sluaulelU urlre luarelpe oml uaaMlaq 'alqrxage sLuro;pue 1ur1al6ue-q61q papualxaI;;n; ueqan6uo; uuu09l lnoqe srraurpor.uoq ururelU tl)el 1y;'1ebe ;errs{qd aql qllm ryoMtau ;o segl.radord e olul sluauelu leuolsuour!p-aorql u!l)e s{u!l-ssor)ulrrelll 19-91arn6t1

srNrwvrl lvl-lll)sol^) ull Ht l-Lfln9lu sll3f MoH

600t

,(seFqnloJJrru pu€ 'lualuelg aqf Suop qJlqm anour,,bq] ur uorloalrp eql Jo urlJE raqlta) of pulq ,taqt eddf eqt ur raJJrp,{aq1 '1ac rr}o.,fueone Lra.l.a luarueg ur 1sr Jo -xeoJ suraloJd rolour luereJrpJo suezoq 'lf Suop,rtppeals eloru ol srs^loJp^q dJV Jo salJ,,bpelBeder ruo4 pe^rrap zt8rauaeqt esn puE tueuEIU Ielala{sotft pezrrElod E o] purq suraloJd alqDlJerueJ eseqJ 'suleloJd Jolour palleJ sJololu JBlnJelolu aqt eJPuolalelsoil.c aql q]!v\ alercossEleql sulaloJd Sulleurcse; tsolu eql Suoury

SUOIOW UilNf]IOW 'auurqruaw auso1d aqt o1 sluaua1g[Su1qcaLp [q s11acto sa4tadotdanlsaqpapuo adnqs pA's[am aqt auruuapp sularctd ftossacco Jaqjo pau{ap [11au4auoaB ur JaqjouD auo guauapfar4] o1waql But>1ut1-ssotc fq parapto saJnpnJ$ nBnT olur salquasw sulapJd [tossaccv ssDIJraLflouv'sluauBatt nllaus olut s7uaua77[ aql Suuanas r(q n sruaru lo -aN lo spua aqj o13u1pu1q[q sauoutp ryatualg[ n77a sulaprd asaql awos .sa(4as lo -uaqt sruatual{ aqt rc slrunqns aa{ arll raqtla oj pwq wt4t sutalotd [tossaccafq pala -rapcJv Jo pamolsJaqtn aq uac [Tquassnnppua [lquassD ]ualuuly[to sulaut4 aq1 'auoJqraau autso1d aql pa7aalrnu Jaau aJo sl.uauollf upca rout ,]wJtuoc q.Ipc aql .awosorluaxaqJ pawapnu aJDsalnqru. to nluac aql toau palacol [Tqraassaxalduoc a w -orrrw '.sqacputuo jsota uI s1uaua1y[mau uorlaa1cnuaql aruulu! wq] sassacotd to aql to uotrulnBatav.lls2saJnpnus pppqsou[xto sa11s aqllo tunutuuapp tQotuttd y 'sutalotd ftossaccaqcns to Tasqnspawc -pap aBnT a soq (sruauo1{ upca puD rstuaual{ arulpawtalm ,salnqnrcrrna) sassoll ruaua14frcfnw aarqt aql to tqcog'sutalotd [tossaccolo auol.tadataplaaan a uo puadap s77ac nlofuacna ul saJnpnr$ ruawallt ln1a\at1so1rb lo suotlcunt pua suto! pauan ar,11

fteurun5 '61 raldeq3 ur ssnf,srp e^,lt suorlJunf xrr]eur lpqt IIu -llao pue suorlcunf ,suor]JeuuoJ esaql Ilao-llal pazqercads aql armbar qJlr{lrt JoJ Ief,nrJJ are sluaruElrJ alerpauralu pue slueluelrJ uDJe qlog .eueJqrueu ]ueuasEq JElnllaf,EJlxeup o] Jo slles Jeqlo ol IIef,e Jo eJnlsnJlsleuJelur aql lJau -uoc o] SurdleqJo uorlJury .(966-t66 leuoqrppe aqt a^eq suralord esaqlJo euros 'dd) g-gt lauud ur pemarlal aJE selnqnloJJrru pue sluetuplu urtre Jo uorl 'uorlJes srql ur pessnJsrp,suralord -tsod pue ,,(lqruasse eql aq1 IoJluoJ leql '(21 raldeq3 ees) rll aoJB IIarJo loJtuoo aq] ol stuaruele lern]JnJts IIar slcauuoo leql ruels.{s)leqpaal p Jo suorleorpurdueur;o auo sr srqJ .uels^s snol -reu eqlJo slred raqlo ureueJ pue sanraudJolrpne aql ur doyarrapsJorunl uBruaq aldrlpur qJIqM ur'stsolvutotqglotnau pellec aseesrpcqaua8 uerunq eq] Jo ruJoJ e ur slFSeJ 'urlJerupalpc ,(prue; WUg eql Jo sJaqrueu aq] Jo euo Jo ssol aqJ '(t9-9I arn -tgg) 1ac eql ^q pelratal spu8rs;o dleuen e ol elrlrsuas auooeq xeUoc [e] eql yo saqradord eql 'tte.,tlr srql q '(sesEuDIeursordl JoldaceJIeJaAasJoslaBrel lcarrp are suralord ^gg ar{}) spu8rs JEInlleJeJ1xeo1 asuodsar ur ,aldurexa JoJ tncao uEf, q3rqm Jo raqtra 'zdld ol Surpurq ,(q ro uorle/.roqdsoqd uralord dq para88rrl

'sleuots lelnlla)eJlxa ol asuodseiut LUIoJ leql suotsnllold a)e]lns-llo)aztltqets pup aleraua6 ^qaralll ue) suratold{1;ue1-61g3 JOuorle^tl)v'utaloldauejqurausuell e loJ auo pue luauelu utt)e ue JoJ auo'salrs6urpurqoirll sasodxa,26;6o1 (q pasnet 6urpurq,(qto uortel,(roqdsoqd 'uralold{1;ue1-6153 ue106urp;o;un .aueJquauteuseld aql paleln6aU ol slueulellJuglre6ugqre11e u; surelord {lrue;-6193;o alor eq1 gg-91 arn613

uole;a4soy(3 eql:9 [ raldeql

0 t0 t

r-uu00|'

('uoloqs pr^e6Jo {salrnor'g)'urnurleldqtlm pamopeqs sa;nre;ouurso{r-u uorl)alaur uaas,{;leaqr aq ue) lte}aq} pue speaqtelnqo;6 ;o sqder6or>rr-u oMr aql (B)'lreloql pelle)sraln)alou aql,o lteo s!r.llpue 'uotlnlos ul por pepualxaup saleurluauabuelreltol-peltolaq1 (6-9 arn613 aas)spr)eourue rrqoqdorplqparedsIlre;nbaljo uotlet)osse aql Iq ualup 'lror-papo)p uuoJol .raqloq)ea punorederrnsureqr,{reaqaqt;o sa>r;aq D OMIOql UaqMSin))ouorlezuaurc'peaqursor{ru qlea uo luasaldsr ad,{1q>ee;o ,{do>auo pue 'sad,(1 l)utlstpo^i\}Jo ale sureq> }qbrlaql'@ryq) sureqr1q6r;rnol pue (uaafi)6uo1spr)eoururp0002lnoqe qtea)sureq) (neaq o,rn1 sralnlalour ;; ursolu.rV (V)'ll ulsortwgg-91 alnbr3 1o pasodr.uo> uru0sI u o r 6 a ra 6 u r q: o l r a u

snururral-)

I uru

\ \

- sureq) lqDrl

s a ) r l a qn o M l + o l r o ) - p a l r o )

snuIullal-N

IPurrural-N aq] ol asolJ purq surEqc]q8ll o^\t eql'(?g-gl ernSID uollezrrarurp urcqc fueaq selprpau ]Eql IloJ-pelroc papuaxa up sruJoJ]eql ecuanbas prcE ourure 3uo1,tran e dq pamollog 'draurqceur SuperauaS-a3JoJeql surEluof, ]eql snu -rruJe]-Nslr ]E ureruop peaq JelnqolSe seq urpqJ dleaq qJeg 'sureqJ1r{3I o^n1gs qree Jo serdoJ oml pup sureqo .,i,,reaqom1ruo4 peruJoJ sl leq] uraloJd pelp8uola ue sr (l\^olaq ees)il u8o/tru palle3 'urso,'ilu slqJ 'uorlJeJluoJ elJsnu JoJ acJoJeql saleraue8 qJIq { 'ulsoriru al3snru Iptala{s sE^aperJr}ueprureloJd roloru lsJg aql

(;lureltedn5 urso^waql ro sroquew alv sutalotdrolow paseg-utl)v 'sJor^eqeq 1ac xalduroc saleJauaS,{lsnonardpeqrJJsaps}uaruelu lelele{sol,{J cnueu.{p aql pue suraloJd Jo}oru ueeMlaq uorleJoqElloc e n/t.oqeuruexa ilu^ aantaldeqc slqlJo uoqcas puu eql uI 'stuaurelule]a1a4soilcuro{ }lrnq seJnlonJ}s ;o adeqs aql a8ueqc o] Jo sVNUtu pue sallaue8Jopesoltua-aueJqruerupodsuerl o] {JoM uec.,taq1,rnoqaqrJrsap uaq} el 'sulaud.ppue 'sursauDl'surso.{ru :suralo.rd Jotoru p1a1a1so1b;o sdnor8 aarql aql Surqrrcsap,,i.q u€aq a,rn'uorlJas srql uI '(uralord rolour pnpp -eur aqtJo uortoury pcrSolorq eq] eroJaraqlpue) o8reJ eqlJo.,{1quapleqt seuru ']1 -Jatap uralord rotoru aql Jo IIBI eql sealaqm Suop luerueloru Jo uor]oeJrpaql pue {JEr} eqlJo &euepr aql seurruJa}ap(peaq) uretuop Jo}oru aqJ'(sJelauouuu ,^i\eJe Jo aJuelsrp e ,{1eerdfi) }ueurEIU aqt Suo1e eurr} e le dals euo aloru o8rec palercosseslr pue uralord Joloru aq] 'Surputqal luaruplu pue ,uorlexelaJ puorleuroJuoc raseeler]uaruelrJ 'e8ueqc IeuorleuJoJuoJ ,Surpurq ]uauelrJ Jo a1c.,bpcruaqJoupqJau e q8norq;'punoqun are daqt qlrqm ur salpls pup s{JEr} ]uaruelrJ Jreql o] ,{13uor1spunoq a.re[aq] qJIq \ ur sale]s ueemleq alcdc sural -ord aq] 'sa8ueqc puorlpruroJuoc acnpord lpq] srs^loJp,{qaprloalcnu Jo selodc Lq ua,rrrq 61y saz(1orp.r{.qpuu spurq luq} 'umluop Joloru Jo 'uor8eJ ,,peeq,, e qSnorql s{ceJl luaruelrJJraq}q}I,,t,r eler3ossesureloJdJolou playalsolrb aq1 '(12-g g arn8rg aas) raldeq3 ur paurcydxase 'ura1o.rd e ur a8ueqc puorleuroJuoc apcs-a8rel e ot srs,{1orpz(q aleqdsoqdrrl aprs 'IJuJ} aq13o,firrelod prnlcnrls -oelJnu Surldnoc ,{q uoqoru aleraua8 uaql Jo IIV aq] uo luapuadap SurprtsJo uortcaJrpaql qtF '{oeJl JeaurTe Suop sallasueql 'seuosoqrr pue ladord o] dSraua lulrueqo asn ol drruqe aqt a^eq esaqt Jo IIV 'seseorlaq'saseraru,{1od '{ooq sn{} ul eraqmaslapassncsp VNU pue VNCIsp qcns saxaldruoc uralord pue suralord Jaqlo aruos Jo luacsrurruer eJe {oeJ} rarudlod peluerJo ue Suop ,{ypuo4carrprun aloru teql suralord ro}otu p1a1a>1so{3 'uorsrnrp '3ur1eaqdrerlrJ 'uorlJerluoJ alcsnru se eueurouaqd qcns sanrrp IIac pue leqt acJoJaqt Suueraua8 taqlo qcea 1surc3eapus ol Jo uorsua] uexa o] slueu -eru plala{so},tc asnec suralord Jo}oru Jaq}O 'ilec aq} ur suouecol alerrdordde Jlaql ol-salcrsan drolarcas Jo 'sTcels €1og 'eupuoqJolru sp qcns-sal1aue3;o pesopua-eupJqruau drrec suralord rotoru .,(ue141frrec .{aql ,,o8rec,, aq}

I t0t

suorowuiln)ltot^t

('q)tpndSsauef 'v) ']uaLua^our luaulplu Jo Isalrno) u!l)e jo uorl)arlpeql ale)!pu!sMoilepaJ a6relaqt'luauuadxa aql Jo r.xprOetC (B) ')as/uJrl t tnoqe Jo aler e le suotl)alp a]rsoddour 6ur^oura.law(uaafi auo pue pa)auo)uMor]ssluauJplu ut])e oMl aq] iuedepuo)asg'0 tnoqp paplo)arareM a)uanbassrqlur uMoqssauerJoapr^aq1 'luauelu q)Paol punoqspeaqurso^u Jo suozopaql Jo q)ee {q ua)el sdats (uer.uaql ol 6urmo'a)eJrns lpnpr^rpur aqi 6uoleaprl6o1ue6aqsluaLue;r; url)e aqt'pappeseMd-LVuaqM(V) 'speaq purq ursoA-u aql ol ol pamollppup poppparaMurpro;1eqd luarsaronl,q]rm p a l a q esl t u a u e l uu r l ) Pu a q tp u e ' e p t l s sse;6e o1paq)eilp ararvtspeaqursoftu 15parpnd'tuauuadxasrqtul 'peaq ulsolur oql ^l!^!l)e rolou.l Jo aql ro] o)uap!^al)orlq a9-9 1 arnEr3

(8)

a p r l ss s e l 6. - . - - -

tull S

's0z6l osle sB^ uraloJd uISo^IU pepeaq-oml TEIIIUISe leq] punoJ sraqcJeesal aql ur lnq 'alJsntu ur ,r(po luaseJd se^ urso^tu ]eql lq8noq] ^ileplul se^aU '(99{I arn8ld) pueq eqt uqll Adla}alduoo peureluoJ sr dlrntlJe Jolour aql teql Sut.ord 'oJlln ut Surpls luaruellJ aleJaua8 upJ auop luaru8er; IS eqJ'(IS pallpJ) lueur8e4 lcelul ue sPpeseelar sI uletuop pPaq aql 'ulPded pue ulsd^Dour,r(qc.{qpalse8lp sI ulso,{Iu elJSnIu E ueq \ 'uaql apooue seua8 'pa8uerre fyreyurs aJEleqt salnJaloru II ulso/tru ule]uoc el3 ]uereJJrpq8noqlp -snlx qlootus pue Jerpre] '(ralel passncsrp)uorlseJluos elJsnu uI stlnsal sluaur -elrJ urlre ;o Surprts ua^Irp-dJv aq] 'stuaruellJ {3lql ulso,{ru eq} Sulpunorrns ulq],, ut pau81e eJBsluaIuEIIJ ullce pa8ueJre.'{.1p;areeqclqm uI sderre ,,]uauBIIJ 'Jeqlo qcea 'allsnru lsed slueuelrJ ur}38 pa}uelro dlalrsoddo Jo sJled 1elala{suI Surprts 1e ]uerJIJJaluauEIU aqt se{eru luaruBIIJ {olql aq} uI speeq aql Jo uope} -uauo Sulsoddo aq1 'luaruellJ ullJe uP Jo pua snld aq] plemol {lem o} sls,{loJp-{q dJV Jo ,48raua aqt Sursn i11y saz,r(1orp.{qpue spulq peeq utso,{ru qceg '(99-9I arn8;g) luatuellJ {rlq} eql Jo spua oml aql }e suorloeJrpalrsoddo ur patuerro 'speaq urso^u paJpunq IEJaAese^eq leql ,,slualu -elrJ IJIql,, relodrq aBrel uroy suorlJpJelur Wl-lle_] asaqJ 'salnJelolu ulsortlu 'uleruop peaq raqlo Jo sllel eql qllmJIaslI sepunq IIE] Ilos-pa1oc Euol aq] ellqm 'V'Uuo paseq 'f '1-'6tet1'U pueuolped'UleqtMor:) ( ssard qil^ sg6t '6€v-6zv:vgL'tolB )ruape)Vuor] uorssil.urad tow 'ralletus 'V)'(ZL-gL (;re;rr-urs ul s}uauelU q)nurarPslla)al)snLu-uou qbnoqlle :treMals ,{elrn61 pazruebio arn6rg aas) 1o{sauno> uol})elauloljpal)nr}suo)al uy'sqdet6ot)rul trLuse;do1{: aq1'uaatb urpalq6rlq6rq staln)aloLu urso{Lu ;; urso,{r,u ;enprnrpur aqlJolo}ua)aql ul auozaleq s}srsuo) setuaulelt, e }o uotl)aslleulsV ()) 's;re1 }uaurelU ;1urso{ru 1;urso{tu ;o ,{;arr}ua saln)alou1;utso{tu oq1luaLu el!JaqlJoaprslno aqtot 6ulltafordspeaqraql q]!M'suor6ar lretrlaqljo sueau ,(qale6ar66e arPqleltua)aql atoN'al)snul6or;r.uot1 peaqlo aarJsrr..l)rr1m'auoz aql ale)sol umprp1ou'r,uerberp (B)'sureulop )lteuraq)S palelosr )i)rql;; urso,{r,u u! luauel!J))rq} lelodlq11ulsoltuaq195-91atn6t1 e 1oqder6or>rr.u uor})all(v)'al)sntrt luaurelu

; r e 1u r s o I r . u

uolalelsol^) aq] :9 I raldPql)

ZLOL

'uoll)al!put aoueLltstrllloJ alqrsuodsar {lqeqotdst 'sutsoftulaq}o ul punoj lou 'uteLtoppeaLlloloul sl! u!r'lll/v\ uoruasutlleulseql 'luauleluutlle upJo (puasnldeqlJo peatsur) pua snutu aql premol6urnou ur anbrunst 'slaqulaLu I;rue1.raqloo1{1ue;rutslelnl)nlls llela^o slralldsap'11ulso{y1'uteutoplolout auo lsnf qlrm'slaulouou_l se uotl)unJ ot uaas (Alx pue'xl 'l sp qlns)/!{aJe lnq 'oln)aloullad suteLuoplolor.u oMt qltM 'sleultpuJloJsurso{lu,(uey1'sraquau ^lrueJasaqlloJ ainl)nlls leln)aloul oql Jo suort)rdaparetqbrraql uO 'asla^tp Iran ere (aryqry6fl suotsualxaleututJal-N pue (uaarbtrl6tl)sllel leulutal-f lraqt lnq '(uaafi 4top ur uMoqs)sutpuloploloru repursareqssurso{r.u 1;y'sad(1urso,(ru auJosJo sureq>Ineaq arllJoaln])nJls .sloqtueu ureuropaq] Jo uosuedLUo) {;rue;rednsu;so(yy4g-91 arn6;g

(aopJJns '(09-gI aJnBrCaes) Ilrlor3rruJo uorlonJlsuoc ar11JoJJarlJpapassnJsrpsE IIeJ eqi lB seJnlJnJlsqcrJ-ur]rp Jo uorsnJloJd aqr SupnlJur-uorlezrue8ro re1n1 -lesPrlur ur pan1o,rur,,(1eraua3are,,(aq]pue 's[pl Jreql ul alrs Surpurq-aueJqrueu E Jo elrs Surpurq-uqcp puocas p ureluor uauo suralord 1 ursor(ruaq;'uoqer8rur '(sJel 1ac Surrnp ilac e Jo ,{poq aqf Jo uorleoolsupJl pJpMJoJaql JoJ se IIe^ se -q8nep oml olur 1ac Surprnrpe;o gede Surqcurd aq]) slsauHofc .rogparrnbar 'sllaf, alcsnruuou pue elf,snru qloq ut ,ir4lrce alrlJeJluoJ qtl \ ,(leraua8 sr 1I '}rodsue.u alaue8ro pup elcrsal ur pellolur sr ursort141 pelproosse sr 1yurso,{141 n 'pauruJalap aq ol urEruer sursor(ur aql Jo lsoru JoJ suorlsunJ tJexe aqJ 'pua snulu eql pJP.^ao]sa^oru qJIq ^ '1r1ursodru sr uortdacxa aq; 'spaads lueJeJJrp]e os op ,{aq1 q8noqrp ']uetuelrJ ullJe u€ Jo pua snld aql pJemol anotu auo ]dacxa sursortrueql Jo IIV '(tZ ratdeql ur pessnrsrp) spu8rs leJrJlJela olur sale^a punos ueluoJ pue punos ol asuodsar ul lU] eseql 1(99-6arn8rg aas) sllal aseql Jo eJEJrnsperde aql uo punoJ eJEleql prlrroeJels qJrJ-urlop Jo sepunq InJllneeq pue xaldruoo eql Jo uoqcury pup uorlJnJlsuoo aql JoJluelrodrut are surso,{rupazqercads,{yaurargaasaql'ssauJeap drelrparaq asnEc ol rr aou)I erE rueql Jo eArJur suorlelntu pup tea Jauur eql Jo sllac JrEq aqt u1 dlarrrsnloxaJo ,(preruud passardxa are sursortu upunq eql Jo aurN 'saua8 utsodru 0? ]noqe sepnlf,ur aruoua8 upunq eql :sasselJIeJn]f,nJls uales lspal 'saua8ursorturgI 'aldruuxeJoJ'sunBa1a apolpueu 1uSuquasardar lspal 1e seq J aq;'sursrue8Jo JplnllaJulnru ur r(1;epcrlred 'suorlcunJpazrleroadsaroru ru.ro;rad sutsodtu Jaqlo ]Eql pue eArAJnso1 1ac c4ol.r€cne e ro; dressarau are sursof.ur;o sadft aarql aseql teql alelncads o] Suudrua] sl ]I 'sA ulso.{ru oml pue 'II ursodtu auo 's1 ursortru oa,q :sursodru aAIJ suleluoo aursrnaJal sactruotaqccrg lseari. eql 'uonnlona crlodrecna ur f.pea asore sursofru leql 3uusa83ns 'sa]o^,fuecna 'JaAa^oq ']sontr '(XD seleJqepel ur [1uo punoJ ueeq elpq aruos [p uI punoJ eJp pue 's1ue1dur dpo punoJ uaeq a^pq (1y pue IIIA se qJns) surso,{.ruaurog 'sao8rec ]uaraJJrpqlyvl ]oeJalur ol pup slrunqns Jeqlo pulq ot suralord aqt llurad ol uorlnlole Surrnp pegrsrelrp,tpuaredde aneq 1,(1p -raua8 suralord Joloru slre] eql pue) slrpl ursor(uraq; dprue;radns aqt ur sell Jo -rue; ursodrulJurlqp Zg lspal ]e eJEaJaq]leq] aleJrpu salof.recnaasJe^rpSuoure suosrredruoc acuanbag 'gInX ursodu lspel le qBnorql 111urso,,tru),,fuanoc -slp Jo JapJo alerurxordde rlaql sloegal lrlrou arnlelJueurou aql pup '11ursortur pue 1urso,{ru o} palelal r(1enba,r(lalerurxorddeaJE}pq} sar}erJe^pepeeq-o^q puB papEeq-euoJo Jaqunu E epnlf,ur sursoziru;o sadzilpar;rluapr ,(prau aqJ '(zg-gl arn8gg) surEr.uop lpl lpururel-O Jo dlarrezr p qlu dlapVtt a8ranrp ueql pue 'snururJal-N eql ]B ureuop Jotoru ursodur alqezruSocarB q1.rmpels dleraua8 sureqJ dleaq aq1 'paJaloJsrp ararr saddl ursortur raqlo dueru .{puanbesqng '(pappeq-oml to1) utsotru perueueJ serr,rursoz(u puorl 11 -uenuoc aq] 1(papeeq-euoJoJ)I ursotru porueu sellt pup Jeruouoru e se uoqcury o] peuees alnf,aloru sIqJ 'llel tuaraJJrpLlalaldruoc e 1nq urso.{uralJsnruJo peaq aql ol Jelrrurs ureruop Jolotu e Suneq ur puonueluof,un sem lertrl qualp$aJ oqaouDqruaJy eqaouE Jate^ qserJ eql uI ursor(ru e punoJ sJeqJJeaseJJeqlo 'aurll aures aql lnoqe lV'slleo ueozolord Surpnlcur 's11acaycsmuuou ur luasard s p r ) eo u r u r e0 0 0 1 Atx IX XI iltn ilA

ill tl I ad{t

u r e u r o pr o l o u

urso{u.r

€t 0 t

suol_ow ufln)ttow

('rasnoH uqof Jo^sauno)'g) ual eql uo sureuoppeeqaql qll/!{aln)eloul ursaurleJo qder6orrrr-u uoll)ala q)la -azaarl(B)'elnqnlor)rrue,o pue snldaql Jo ppalsurpua snurureql pleMol'sutsaut),1 lo Iryofeur aq] urorJuor])alrpa]lsoddo aql ur lanerl11;eraua6 sursaut)osaql 'greyuralord tseal aqt pue prp uralotd oltLloosqoaqt sapnl)urleql ulsaur) 'd leurural-) e srt[-utsaut)'(€00t aas) srol)ej aqoorlsele)palle) arojalaql ale Ieql 1sa;nqnto.r>rru rrueulp ;o,(lrlrqelsur aseaJ)ur ol spuaalnqntolllr,.u ol putq peolsurpup {1rnr1re rololu le:rdft lsol a^eqleql sursaurl1o,{;ruele Joraquau p srll 'ureq)Ineaq aqt;o alppruraql u! pale)olureuop rolourslrseLl€ [-ursaur) '1;urso{r.u {q paurol s}uaurelU ))rql re;odrqaqt 1o(tln!t>eaqt o1sno6o;eue 'raqlo q)Patseo salnqnlor)il.uoM] eprls ol alqesrrauerlal S-ursour) relodtqeql 's;re1 rraqtIq aler)ossp sraulp oMt araqM srauerlalsuilo, s-ursaur)'salnqnlol)rul pasol)uo-auelquraur 6uo;esa;lauebro a^ou pue sror.lrouoru se uotl)unj ol uaas leql sulsaullJo ssel)lensnunue pasol)ua slueserder €-urseur)'a;;aue6ro -auerqureur e se q)ns'o6rerol saq)elte reqr lle] e suroj ureuop leur[!Jal-]aql 'uortpzuauJrp 6u!te!paulror-papor6uo; e suuoJureuropalpprJraql'ureq: Ineaq aqt Jo snuruual-Naql le ureuJop rolour aql seq [-ursaur)'pa^Jasuo) aresureuop rolou aq1{;uo'{;rureyadnsurso{r.u aql u! sv'slequralu,{;rule;redns ursaurl a^UJo sarnt)nrts(V)'sulalord palelar -ulsaull pue u!sau!)gg-91 arn6r3

'speeq ,taqt pue 's11arcqodrcJne IIB uI punoJ.{.1qeqordare suleudp clurseldol,li.:) sP suEruop rololu a8rel ortl qlF 'sJeulpouoq uluqs-,,ileaq f.1pcldfi eJe qJIqM 'sutaut[p JllusaldoltJ eq] sureluo3 qJueJq lueIJuE lsolu eql '(69{I eJntld) seq3uerq rofEtu o^\] seq,{.lrureJuleu.{p eqJ'suIEqJ lqBI pue suIPqJ elElpeluJe}ul pelprsosseJo requnu elqerJE^puB eSJele pu? (ureluop rolou eql epnlJul leql) surBqJd^eaq eeJql Jo o^^lJo pesoduo3 erP^eqJ dgure;radns ulseupl aql ol pelPl -eJun ^lIuPJ B aJE sulau^p eql patseJlp-pue-snul[u sJolour eFqruoJJrru Jo 'uoueluJoJ elpulds 'uorsr^rp 1ac Sur.rnpuorlPJPdesaluosoruoJqc uI pue cporalu puP 3r1o1[u ur saloJ srJrJadseAPq sreqtuetu .{FueJredns utsauol aql 'eFqnloJJrru Jaqloue ro a[eue8Jo pesolJue-euEJqluau P Jeqlle JoJ IIel ;o,{ue11 eql uI elrs Surpurq B i{Jrec sursauDllsow 'sluelllElu uDce JoJseop }usurellJ {3lql 'reqlouE seFqn}oJJlru peluelJo,(lalrsoddo sap11s euo ]s?d II ulso/(lu e se qJn{u 'urPruop leql Jolour JPIodrq P SuruJoJ IIEI eql qSnorqr elPlsosse-Jlasuec LilrxEJ g-ursauDlaqlJo sJaqurelfisJelulporeleq aJesJaqlo 1adpue 'sretulpoluoq aru sJe -qlo eluos 'I urso^ru o1snoSopue'sJeurouolu sPuollJunJ ol luees pue acuenbas 'eFqn}oJJlru eql Jo pua snulu IroJ-paFoc e {Jel sureqs .4leaq utsauDl euos eql pJPo\ol 'uorlseJlp ellsoddo eql ul qpA\ puE snulruJel-3 eql ]P uleluop Jololu aqt seq dgue;8uqsara1ur,(lrepcruud V'alnqruor3llu eqlJo pua snld aq] prPr!\ol IIe^\ puB ureqJ d^eeq aql Jo snuluJel-N eq] lE ulPluop Jololu eql e^EI{ ureql Jo 1so61fprue;Jadns urseurl eq] ul sarlrueJ ]oullqp ueaunoJ lseel lE eJPeJaqJ 'sulsaul{ '9t tnoqe e^eq suElunq puE 9I sEqsuDSaIa lsEa^ eqJ J epolPueu JqI'sursaull ]Jullstp xls sPq aDrcInilaJsaJlkaoJOt4Jsrs '(gg-gl arn81g) lueuala uorutuoJ ^luo eqt sl uleruop Jololu eql qJIqM JoJ L11ure; 'utsortur aIIT 'uoIlPZIJeruIp uleqc -Jedns uraloJd a8rel e Jo Jaqrueru P sI uIseuIT ,{,reaqro; alqrsuodserIteI IroJ-palloc pe}E8uoleuE pue suleruop Jololu peeq JEIn -qo13olu IuJoJ esaql lJolotu ell]f,e Jed suleqJ 1q311 omr pue suIBqJ Ileaq o^r13uI -AEqur II urso^u o1 ^,(lernlcnrls relIuIS sI uISauI) 'selnqnloJJrru Jo pue snld eql prE,\i\olSuUprt rtq pururral uoxB eql pJe,4,\o]fpoq 1ac Puorneu eq] luo4 Le]!\P selleuE8ropasol3ue-auEJqrueu seIrJPJll eJaqM 'plnbs eql Jo uoxP 1ue€ aql ut palJlruepl IsJIJsB^ ]I 'sapqntoJcru Suop saloru leql ulalord Jolotu E sI ulseury

su!au^c puesu!seu!)| :sulatord ,|olowalnqnlor)lw,o sad^IoMI oJVereql

(v) spr)eourue00s

? l - u r s a ur l a

)N

)

N N

)N

I

fN

€-urseurl r . u u0 l

fN

I

[ -ursaurl

)N uretrop rolour

uolelalsol^) aql:9[ ra]deq)

tt0t

(tat^asllulotJuotsstuled q t l M ' / 6 6t ' 9 V Z - 6 Ela' :l o t S D ' nlts 'utdo 'v'u pue souv 'v"l "iln)'ssol) 'ureuroplotor.llorlllo uro4 paldepv) suoruoduorl)npsueil-a)loJ pue 6ulpugq -raLu^lod u! se)uara#rprofer.uo1anp aqt ere sJolor.ll oMl aqlJo suotl)unJpue sezts luaraJ,rp{ren aql'r e1rLurs(len {l Iern}rnr}s arc (uo11at( ul papeqs)ursautlpue ursolur;o sureuop6urpurq-aprloaltnu lerlua)aLlI'speaqulsaull pue urso{ur ,o sarnl)nrls 1e1sfn{er-;1g9-91 ern6;1

sa]ls n l o r : ru r 6 ur p ur q - e ; n q

u l s a ur )

'aSuEqJ eql esnEc eJoJ IeJlueJ eql tuo{ pJe^ }no Sutpuaga sdool a3re1 Ieuoq -PruJoJuor crralsollP ue olur srs^loJpdl{ dIV elEIsuEJ} o} dJesseseu dJaulqceu aql pue Surpurq dJV Jo elIS eq] sepnlf,ul uoluruoJ uI a^eq suleloJd Joloru arn8lf,) seJoJ Ierlueo eql'(09{I Jo sed& omt eql leq] tuauala SuuerauaS-aJJoJ IPsrluepr fFeau punoJE tllnq aJ€ sulEluop Jololu o^\l eseql lBq] palEe^eJ seq urseuDlpue urso^ru Jo sureuop Joloru eql Jo eJnlcnrls IBuolsuaulp-eeJq] eql Jo uorleurruJalap ta^a^^oH 'selllJelruls ef,uanbes pIJe oulluP elqelJltuepl ou eAPq daql pue 'sattradord rllaul{ luaraJJlp e^Eq puu sluetuellJ }uereJJlp Suop >1cer1 suleloJd Joloru Jo sessEIJo1!\leqJ '09t lnoqB ql!v\ peJeduroc splcP oulluE 098 (sursauDl ^llepuetsqns sI susodru Jo ulEluop rolou eqJ Jnoqe Jo leq] upql JaBTPI

u!6UOfueuol1n;o^f uol,utuo) e sale)rpulursaul)pue ulso^w,o/buelluls lelnl)nrlsaql ^ oleq lro.nnr(aqt ^ oq ssnJslp fieqs aAA'casTurrlt-z ]noqe lP sapquoJsl[u Jleql e^our up3 surseuDllsalseJeqt 'uoslJ?druoJuI 'f,es/uli tI Jo eluJ elqPIJEureJeql te aqru lse] e uI sepqnloJJllu e^o{u uec suleu,{p lPluauoxB :}sa}sPJeql Suolue osle arE ,(aqt pue 'sJoloru rElncelolu rr^\ou)I el{l Jo }se8Jel aql eJe sulau^C 'ellrcJo Suqeaq 3ql ur pa^Io^ul aq 01slueaslnq sulau^p plueuoxe qtyruEql slru -se1do{c r{llrrr drpellunrs acuenbas relpeJ8 saleqs qJuEJq 'Joultu 'pJIq} V '(Ja1PI pessnJsrp)e11a3eg pue EIIIJJoSuqeaq aql e^Irp ]eql sa{nqnloJclruJosluaua^oru 'speeq Surprts lueIJUJapue plder eqt roJ pazllelJads r(tq8q are ri.aql ,tla,rpcadsar ureuop-Jolou eeJql Jo o^/tl qll^\ 'sJelulJloralaq pue sJarulpoJelaq apnlsul 'lleJ 'qcuerq aql raqlo aql Sutaufp a8rel Jo Je]uao eql JEau snlsJ lazuauoxv -edde r31og eql Jo uoFezIIBJol JoJsB IIa^ se 'SuDIJIJJEJ] elclsel JoJluulJodlul ale

flasnoH ur.lof 'ursaurl.rourso{u raqlra ;o Isetrnol) Jo peaqoql qllm pareduor a6re;fuan sr peaquraulp aql leql eloN'speaqoMl seqsleurrue uorl urau,(p^lerlr):speaq aarql seq pue ueozoloroe LUoJJ sr uMoqs urau{p{rer1oaql'aln)alourpapeaq -oml e sr urau{prrr-use;doy(r'ursaury pue p;urso{tua111'urau,{p(leuouoxe)Iletltl Jo aln)aloLue pue urau{prrr.use;do1,{r uoll)ala Jo aln)aloure;o sqder6o.rtrur q)la-azearl'sulau{q69-9 1 arn6r1

uraui(p[.retp:

st0t

u r a u ( pr r u s e ; d o p b

UflN]]IOIA SUOIOW

s/l\ollBSurycopun pue Suplcop ra{urTJo a1c[c slqt ]Bq] 0s 'pa]eurprooJ zi.laso1c aru speaq o^ l aq1 ur salrdc srsdlorpdq eppoelcnu eqJ 'peeq ]sJU aqt JoJ etrs Surpurq aql ueql pua snld eFqruoJonu eql 01JesolJ ruu I 'lueruelgolord eql uo elrs ]uaru -qJene ,t,reup purq o] alqe eq ily\^ ll ereqm uoltlsod e ot pJEMroJpeeq puooas eql s^\orq] srq] :peaq aqt Jo eprs aql Suop slcop uo€ar re{u[ slr 'peeq punoq srq] ol 'peJnlcnJlsun [1azr4u1ar sr uor8ar ra4ull slr 'e{oJ]s ra,,vr.od aql 41y;o Sutpulq eql uO aroJaqepqruorcrru p 01 punoq $ peeq ursauDl (Surpeaptuo{ aq} uarlq'(Ig-gI arn8rg ees) pua Jeqlo aq] ]e urpruop uorlezrJarurp1oc-pa1or aq] o] pue euo tB ppeq rotoru slqt slf,euuoc 1eq1uo6ar ra1u113uo1 B ot urcrxop peaq rolour aql go Suqcopun pue Suotcop aq1alepBar alrs Surpurq-appoalcnu aqt ]e sdool qcl,vrs 'ruJe 'urseupl p ppalsur Jelel Suqcor eql uI Jo sluarualoru llerus eql ;o Jo '(fg-gf arn8gg) luau -e^oruJo dals ay8urs e sacnpord (,,a{oJ}srarrod,, eq} sesneJqJlr{llr) asealareleqd 'srs,{1orp,{q -soqd pue epnoalonu'Supurq epuoelonuJo aycrtcpcnuaqooueqcaru 'raqtoue ]e ure8e ]l Jo ploq qcteus pue lurod euo tE lueuelrJ url3p IInJ aql aql uo drr8 slr asealeJol peeq ursodru aql 8umo1e 'urlJe JoJrtfpgJe Surpurq s1r ur sa8ueqc o1paldnoc are ursoLu aq] uoueuJoJuoJ aql ur saSueql eseqJ Jo 'ruu 0'9 lnoqe dq a^our o] x1leqeqt Jo pua reJ aql Sursnec talal 3uo1 p a)rl xueq aql 3ur.us uec lurod srql le a8ueqc IIErusV '(09-9I arn8rg eas)ureuop rapeluoc pellpo-os aqtJo suo4ptor llerus ol peeq eql ur Uap Supurq-41y aq] lE sluarualoru slcauuoJ ]eq] xtleq e{u-uolsrd e sr araql 'peeq 'sureqc aqt o] txau rure relel srqt Jo eseq eqt lV tq8q 1o Surpurq aqt dq pazrl -tqe1s,{lernlonJls sr qJIq^{ 'raJUJanalJo 'xtleq n Suoy-ruu-g'gup Sur8uu,rsaqt Jo ,i.qpalerauaS sr urlce Suop luaruanotu aq];o dals qcea 'ursodru JoC 'elnJelotu utalord eq] Jo lred ur ]uarualoru a8rel e eJJoJ ol srsdlorp,,(q pue Surpurq 'uouour dJV ruo{ pa^rJap,,{3rauaeql esn lsnru urelord ro}oru p IBuorlJeJrprun qrns JoC'luaruulrJeql uo alrs Surpurq ^ au p ol lueuelrJ e Suop uoqcarrp a18urs e uI prpMJoJruaql ladord lsntu aseeleJpue Surpurq 3o a1c.{cqope :luauarrnbar Jequrg E alpr{ sura}ord ro}oru eq} 'ulaloJd raulred e q}lm suog}ceralurcrycr{c acnpord o] selrs Surpurq-aleqdsoqdrrl-aprsoalJnu neql q sa8ueqc IeJnlcnJls asn qloq suralord Surpurq-41g pue suralord Jolotu plala>1so{c aqr q8noqrry

sa6ueq) euolteuiloruo) | ot srs^lorp{H a}eJauaD sureloldro}o4 aw 6u;1dno1{qa>.ro1 '$lrerl tuaurelg Jraqt o] Surpurq,{q palenrlce ^,(13uor1s sr suralord Joloru;o ,!nr1ce asedJv aql eJuls 'suorloeJrpqloq ur {Joilr ol srueas alrs srsflorpdq apl]oapnu aqt puu alrs Supurq-raurr(1odaql uaamleq sa8ueqc prntJnrts ;o .,{.e1araqJ 'ef,eJJetur Surpurq-raru.4.1odaql o} pa,{e1ar aq o1 alcitc srs,{lorpdq dJV eql Lq pasnec suoursuEJl lBrntJnrts aq} 3uu,ro11u ,{1a,r4cadsar'Surpurq ur}oe pue apqn}orJrru ur pellolur uralord aq};o suo€ar asoqt qll,lr ,{Janrsualxas}cerelur dool qcllvrs e 'ursol.ur pue ulseurl ul 'uralord ped yo aql e Jo uot1elor ]uaJaJJrp E asnpJ o1 pagqdruu putural ,ri.peprurs sr-aleqdsoqd E aouasqero acuasard Jo aql-alls e^rlJp aq] ur a8ueqc IBJntJnJls lerus .,t1a,r,qe1ar e 'pazdlorp.{q s1 413 ueql JaqlEJ dJV puP 'suralord rolo(u oe\l eql JoJ JeJJIpluarue^oru eql Jo slrEiep aqr q8noqrly'pasealar s1lareqdsoqd purural eq]) eleqdsoqd-/"paz,{1orp,{qaqt uaqm lno Suurs sdool aseql tnq 'elets punoq-dJg eql ur aleqdsoqd-/" aqr qrprr alrqou :suroJ ]reluoc esolJ ur aJe alrs Supurq-appoalf,nu aql ur sdool ,,qctIMS,, (a^rlJpur) punoq-dcg puE (a^Ucp)punoq-dJg Jraql uI suorlBruJoJuof,l3urlsrp tlq1qxe sutalord eseqr '(ZZ-t arn8rg aas) g JaldEqOur passnrsrp sy ,{pue;radns seg aq] Jo sesedJg llerus eql Jo elrs Surpurq appoelcnu eqt ol acuelqueser IEJnlsnrls aruos sreaq oslp aJoc Joloru aql leql uorlp^Jasqo eq] ruo4 aruoJ seq uorleJeua8 acro; ur pe^lolur sr aJoc IeJlueJ eql ,ttoq o] anlc luepodrur uy 'aroJ leJlueo aq] uro4 ]no Sunuoc sdool aqt;o uorlnlo^e q8norql uorleJrJrporu pue uorleorldnp aua8 erl esoJe suorlcunJ pazrTerJadssnorJel Jreql leq] pue losJncard uralord Joloru [pJlseJue uoruruoJ e ruoq papuecsep a.re surso,{ru eq} pue sursauDl eq} qtoq leqt rq8noqt sI lI ,(larrpcadsar ,ursauDl pue ursodur eql ur salrs Surpurq-elnqntoJcrru pue Surpurq-uuJe aq] epnlour sdool asaql'{JeDJo ecroqJ aql JoJalqrsuodsa; eJepue azrsureruop ur ecualaJJrp

uo1a1a1so{1 eql:9 t reldeql

9 tOt

fsvvv LlorJ uorssf qllM'866t lgs-}s:LgzDuaDS LrJrad ''lp la tuauleu 'l uo pase8)luaulplu url)p ol6utsp a^ou ol JeqteoolltoM ol su rsolur,{ueu 6 urrrno ;;e'a r.ur1 a;rr(>al rlua aq] Joo/o5 lnoqe I;uo lo; luaulelUutt)e aq] ol punoq surpr,.ual peeqaq1'a;t{: 11 'tuaulel4 u!t)e ue urso{utaql ul 6uo;e1;emo11gulso(tuIq pesnsa6upr{) lern))ntls,o ol)^) aql [9-9 t o/n6tl

.{puaredde tlp uI acuaraJJlp slql dlluara;yrp patuarro are speaq paqre}teun 'puoJes eql 'ayqeqsrn8urlsrput,{ller}uassaaJE alnqnloJJnu eq} o} paqcel}e lnq aJB]eql speeq eql 'saFqnlorJrru o1 punoq flruesradns urseuDl3q] Jo sJeqrueu 'peloau 3ur4p.,r,r-premIJpq pup SuDIIBllr-prp^/r4.JoJ Jo sa8prur uoqnlosar-q8q ,rI -uoJ eJEspeaq aq] qJIqA\ ur ,tem eql ur arl ol sruaesraMsuEaqJ isuouJeJrp elrs 'pcrluapr -oddo ur anoru Laql ,lrroq uEJ f1er]uasse eJe sursaur)Jo saddl oml asaq] 'pua snuru aql pJEl ol a^oru pue snurruJal-J aql le JOSurEruoprolour eql af,urs sureuop Jolour Jraql aleq sreqruerudprue;radns ,lvralE 'elnqnloJJrru eql Jo pua snld aql pJeA\ola^our 'snurruJet-Naq] ]e surctuop Jolotu Jreql qlrA\'.{puegradns ursauDlaqlJo sJeqruarulsoru searaq^ teql IIEsau'luauanoru;o dlqeuor]3aJrpslr auruJalap ol pue JarurpursauDlaql Jo (surptuopJoloru) speaq oml aql Jo salJ^J IeJluaqsoueqJaru eql elEurpJooc o] qloq srueas uEruop Foc-palros eql 'da1sruu-g eleJosrpe 8up4etaurl qtee '(Zgrgl aJngIC)Jeuueru asl,vrdals(ppaq-Ja^o-peaqJo) pueq-Jalo-pueq e ur aloru ot Jolour papeeq-omt aq]

F

l u e u j e l t +u t l ) e a q l u o u o t l t s o d n,raue ol pa^ourseq peaq aql leql aloN uollern6rluor . r o 6 ue u r l u a u . r p l tul t D e a q l o 1 , i ; 1 q 6 rp1a 1 t o 1u r e 6 e sr peaq urso(tuaq] ,al)b aqt +o pua aq] rV OlHlVltV

pua s n1 d

dav -1

ru

pua Snutul

I

r)oulsurmod

I a ; t { >a a a ue + o! e } s a q } o } 6 u r u . r n l a t ^qaraql 'dcv punoq slr sasolpeaq aq1 'a1or1s.rer'aod a q l ' o o s i n o )e q l u l u o r l e u l l o ; u o r1 e u r 6 r . sr o1 rs u l e 6 a l p e a q a q l q r r q m6 u u n p a d e q su r a 6 u e q t 6 u r l e t a u a 6 - a : . r o ; a q l - a ) o r l s i a M o d a q l s t a 6 6 u 1a s e a l a sJ t q t u r ] ) e o l p e a q a q l ] o 6 u t p u t ql q 6 ! f a q f q y n n{ 1 1 u e 1 r u . r o > u o : 'srs{lorpAq {q parnpotd e l e q d s o q dl u e 6 l o u r a q 1 6ty l o a s e a l a sl a s n e )l u a u l e l t ]u t D e a q l u o a l t sM a u e o l p e a q urso{uraq11o 6urpurqleam y gNlIvUlN!19-l)UOl

u r e l o r da q 1o 1 p u n o q { 1 g q 6 r 1 u r e t u a rp a r n p o t d( d ) a l e q d s o r l dt r u e 6 t o u rp u e d C V a q ] l n q ' s r n ) ) o6 t y 1 o s r s f l o r p { Hu ] u S } n o q p } o a ) u e } s t p e , { q 1 u a u . r e lar ;q } 6 u o l e p a t e l d s r pe q o } p e o q a q } s a s n e ) 1 e q 1a 6 u e q ra d e q sa 6 l e 1e 6 u u a 6 6 u 1 ' a l n ) a l o udl f v a q ] p u n o r e l l a q su r e l )e e ) r l s a s o l )] + a l )a q f a l ) l f , o f

t

srs^roua^H I ( u r p e a L l lo 1 a s o l :i { l a ns u r e w a tr { l q e q o t d p e a q a q l A l r l e e tu l q 6 n o q l l e' a 6 u e q )s l . { ls a z l s e q d u t a u r l r e p u e p e a q a q l u a a M l a qa l a q u M e l p a l e d sa q l ) ' t u a u r e l ra j q l 6 u o 1 ee ^ o u ol lr snnollp eue utpe lol peaq a q l ; o , { 1 r u r ; ; ea q } s a ) n p a ls t L l l a } t s6 u r p u r q - u r l >aeq } d n a ) e u l e q l s u t e u l o pa q l ] o u o t l p u t J o l u o a ) ql ur a6ueqr ] q 6 1 1es s a s n e :( ; a l e r p a t u L lpr u e ( ] u a u e l t l u t 1 r ea q l u r o J ]t s a q u n ] a p t sa q l u o ' s tl p q ] ) p e a q e q l + o , , ) ) e q , ,a q l u o g a p a 6 r e 1a q l o t s p u r qd 1 V ] o a l n ) a l o u rV O l S V l l : l U

luauJelr] )ilrq] u IsoAur

t _)

eq],{qpa1eurura1Aprder u.,t:d"'

t ii:#'#i:

I r""'ii"ff s r q t' a p s n u 6 u r p e . r l u o t^ l a ^ t ] ) eu e u l ( q l e a p+ o A l t p i 6 u aq1'sr1towto6rLto1 a;qlsuodsatst lr asne)aqpoujeu o s ) u o l l e t n b t l u o tt o 6 u e u t ] u a u l e l t ]u r l t e u e o l u o , { ; 1 q 6 1 1 p u a s nl d p a ) ) o l s ra p r l o e l ) n up u n o q e 6 u r 1 : e ;p e a q u r s o { u re 'aln6r; srqt ut uMor]sap{r aq1;o ue}s aq} }V O:ll{iVIfV

I

p e a qu r s o ^ u pua snu tLlt urlle luaure||+

LLOL

suolovuuflnfttoy\l

Ipr] sreqs!lqnd uPllru)ewuorJ uorssruJao qllM'866t '9 r8-€ [8:56€ atnloN''P ]a ullqPs'l ruoU) 'uorleluauoailsoddoaq] qlr/v\sraurlpsuloj'snururjal-Jaql le ureuop iolot! aql ql!/vlroloul pal)arrp-pua-snutwe' \DItrloosoto u! p)N palle))€ [-ursaur)(B)'dals 'pua snld ]xeu eql e)el ol pesrod alnqntor)r.ueql prpmol6urlurod sr peaqpunoqun'puo)os aql'(ar!s6urpurqaptloal)nu aqr u! dlv qtlM)alels alorls-lsod e ur alnqnlor)r[!aql ol punoq srrarurpaql Jo peeqauo uaq^ 'olnqnlor)rLU aql,o pua snloaql preMolsa^ou pue snuruilal-N s/uraloroaql le ureuop rolor.l_r slrseq(ursaur)leuoltua^uol) [-ursour)(y)'({do>sor>rru uorlre;aoft> ^q peurLuralap) salnqnlor)rutol peq)ellP sraurpaql,o a6eur uorln;osa.r ra/vlole oluo (,{qder6oq1e1s{rr {er -x {q peururalap)sraLurp uralord -Jolouraarj aqt Jo sarnl)nlls aql 6ulug,{q palerauabararrn sa6eur asaLll'salnqnlor)!ur ol punoq sulalordI;ruepadns u!sou!I pue -prervrJo, 6ur1;em-prem1)eq Jo uollelualrg 69-9 1 eln6;1

(ureurop rolour) peoq

(8)

l!er

llel

(ureuroprolour) (V) peaq

)N

)N

rolou pepalp-pua snld l-ursaull

rolor! paperp-pue snurur ( p ) N ) €[ - u r s a u r l

c

c

o luaurelrloloro - alnqnlor)rtri p e a qu r s a u r l punoqun

-{ooq V '(16-9 arn8r4 aas) 9 Ja}dPqO uI pessncslp esPdIV slJe(uExeq eq} Jo e^qeleJ xeldruoJ aJoru e eJoJaleql sl ]I luleluop pulluJal-J uIEI{J-,{^eeq aql snld surBruopwvXIs :suleruop ue^as luo4 peruloJ 3uu ruueld e Jo slslsuos pBeq aql 'ppeq pedBqs-8uu 'eleJoqeleue uJoJ ot pasn sI uIPq3.{,reaqaqtSo uonJod Jo[Eur aq] alrqM'alnseloru uleu,{p eq} uI suIEqJ lrreaq raqlo eq} o} speuuof, pu€ suleq:) 'luerue^olu aqt selEeJJ lq311o ]as E spuq teql IIB] e sruJoJuoluod pupural-g sll lPql ernlsnDs JrsEq eq] surroJ suolpp 000'009 uPql alolu Jo uleqs I^eaq 1uer3y 'eBuEqJ IEuollBIUJoJuooSulluJauaS-ac;o;e ol se ila^ sESurpurqun pue SuIpuIq apqnlorsrru 01 srs,ltlorp^q eppoelsnu eql SuIdnos Jo alnr [EJaueSeq] s,i\/\olloJ 'sursauDl rololu uleudp eql sI pelEleJun dlprnpnrls pue ol sulso^lu ilps ]nq ' (tg-gl aJntIC) luelualoru Joloru Jo dllpuorlcarp aq] saurruJelap,,(qaraqtpuE 'peeq puoces eql JoJ alts Sutputq ]xeu aql saselq 'dalslxauaql loJ 'urvroqs ssa>ord eql;o teadarl)exoue,{qspaalotdqrtqnn puestsfilotp{q (!d)aleqdsoqd aql'(esPalal pasrod ursaurl rvrou sraln)alour

dcv

tr a)eJJnsalnqnlor)rur

dfv sal|nballuaLuq)elap)punoq dCV tll!/vlalnqnlolllu, eql ulolJ peq)elapseqlt a)uo 'preMlo,uteirioploloLuleal aqr sllndUlqsslql'(llo) palro)poutmualutaLllpue uteutoprolou aql uaamlaqautl6utlleuuol e se aroq umelp st lalutl lrau oql) uolleui.lo;uor6urlutod-plervuo; e uol; Urqsol,,le)utl))au,,aql pa1;eraptldad;1eus e o16urlurod-premrear slql ul E pue z e sasne)ureuroprolor.ilsrqlol dIV,o 6urpu;qaql'(6utarretp ureuop rolot! luolJ aql u! dCV loj dIV lo abueqrxaue ,{q s;eueduaerr,rlaq) prennrol aql'al!s 6u1pu;q uanup srureuop rolou,rearaql,o luaula)eldsrp sr peeq6utpeello ]uol] sr!u! dov tlll/!{alnqnlol)ll.!aql ol punoq,(1asoo; '@aatb aql allqMijIV ol pue alnqnlor)lul aql ol punoq ,(llqbtl sl 4top) peaq 6ur66e1 ro rearaql'speoqutsautloml aqlJo auo'dalsq)ea,o uels aql lV .l)el] sl! jo {;a1alduor }noql!/v\alnqntot)ttuaLlluo sa)ue}srp6uo; to; anou o6 6ur11a1 'a1ts6u;putq sltll 6urs61 ue) raurp ursautlaql 'uoltotu,,pueq-la^o-pueq,, ullnqnlelqelle^elxau aql ol spulqaluaql puP'uleuloplolol'ulaulleo aqt sassed'e1rs6utpurqutlnqnlsll ulolJsaq)elappeaqJealaq],'dals,, ursour)e 6uunplrauueu paleutptoo)e ul llom suleuloplolou, ulseull oml aql'(8S-91arnbrleas)lre]llo)-pallo)6uo; e q6nolql pal)auuo) oMl Jolauilp e lolotu 6u1pu1q-ap;toel)nu ale leql (speoq)suleLuop sr [-ursaur)'ulsout>1o a;r(r ;erluaqrouetl)au aql 79-91 etn613

uolalalsolr() eql :9 L raldeqf

8 t0 t

MOLECULAR MOTORS

I019 AAA domains

(B)

AAA domains

power stroKe, 8nm

c

ADP + Pr

AAA domains in motorhead major ATPase

+

C - t e r m i n adl o m a i n

attachmento cargo o r a n o t h e rm i c r o t u b u l e

(c)

1 5n m

shaped linker region connects the hear,y-chain tail to the AAA domain that is most active as an ATPase.Between the fourth and fifth AAA domains is a heavy chain domain that forms a long anti-parallel coiled-coil stalk. This stalk extends from the top of the ring, with an ATP hydrolysis-regulated microtubule-binding site at its tip. Dlmein's "power stroke" is driven by the release of ADP and inorganic phosphate, and it causesthe ring to rotate relative to the tail (Figure f 6-64). Although kinesin, myosin, and dynein all undergo analogous mechanochemical cycles, the exact nature of the coupling between the mechanical and chemical cycles differs in the three cases. For example, myosin without any nucleotide is tightly bound to its actin track, in a so-called "rigor" state, and it is released from this track by the association of ATP In contrast, kinesin forms a rigor-like tight associationwith a microtubule when ATP is bound to the kinesin, and it is hydrolysis of AIP that promotes releaseof the motor from its track. The mechanochemical cycle of dlmein is more similar to myosin than to kinesin, in that nucleotide-free dynein is tightly bound to the microtubule and it is released by binding AIP However, for dynein the inorganic phosphate and ADP appear to be releasedat the same time, causing the conformational change driving the power stroke, while for myosin the phosphate is released first and the power stroke does not occur until the ADP subsequently dissociates from the motor head. Thus, cytoskeletal motor proteins work in a manner highly analogous to GTP-binding proteins, except that in motor proteins the small protein conformational changes (a few tenths of a nanometer) associated with nucleotide hydrolysis are amplified by special protein domains-the lever arm in the case of myosin, the linker in the case of kinesin, and the ring and stalk in the case of dgrein-to generate large-scale (several nanometers) conformational changes that move the motor proteins stepwise along their filament tracks. The analogy

Figure 16-64 The power stroke of dynein. (A)Theorganization of the domainsin each dyneinheavychain.Thisis a huge molecule, containingnearly5000amino acids.The numberof heavychainsin a dyneinis equalto its numberof motor heads.(B)Dyneinc is a monomericflagella greenalga dyneinfound in unicellular ChIomydom onas reinhardtii.rhe large dyneinmotor headis a planarring domain(gray)and containinga C-terminal sixAAAdomains,four of which retainATPbut only one of which bindingsequences, (darkred)hasthe majorATPaseactivity. Extendingfrom the headarea long,coiledcoil stalkwith the microtubulebindingsite at the tip, and a tail with a cargoattachmentsite.In the ATP-boundstate, the stalkis detachedfrom the microtubule, causesstalk-microtubule but ATPhydrolysis releaseof ADP Subsequent attachment. and Pithen leadsto a largeconformational "power stroke"involvingrotationof the headand stalkrelativeto the tail.Each a stepof about8 nm along cyclegenerates the microtubuletowardsits minusend. (C)Electronmicrographsof purified dyneinsin two differentconformations representingdifferentstepsin the cycle.(B,from mechanochemical S.A.Burgesset al.,Noture421:715-718, from Macmillan 2003.With permission Ltd.) Publishers

1020

Chapter16:TheCytoskeleton

between the GTPasesand the cytoskeletal motor proteins has recently been extended by the observation that one of the GTP-binding proteins-the bacterial elongation factor G-translates the chemical energy of GTP hydrolysis into directional movement of the mRNA molecule on the ribosome.

MotorProteinKinetics AreAdaptedto CellFunctions The motor proteins in the myosin and kinesin superfamilies exhibit a remarkable diversity of motile properties, well beyond their choice of different polymer tracks. Most strikingly, a single dimer of kinesin-1 moves in a highly processiue fashion, traveling for hundreds of ATPasecycles along a microtubule without dissociating. Skeietal muscle myosin II, in contrast, cannot move processively and makes just one or a few steps along an actin filament before letting go.These differences are critical for the motors' various biological roles. A small number of kinesin- I molecules must be able to transport an organelle all the way down a nerve cell axon, and therefore require a high level of processivity.Skeletalmuscle myosin, in contrast, never operates as a single molecule but rather as part of a huge array of myosin II molecules in a thick filament. Here processivity would actually inhibit biological function, since efficient muscle contraction requires that each myosin head perform its power stroke and then quickly get out of the way-in order to avoid interfering with the actions of the other heads attached to the same actin filament. There are two reasonsfor the high degree of processivity of kinesin-1 movement. The first is that the mechanochemical cycles of the two motor heads in a kinesin- I dimer are coordinated with each other, so that one kinesin head does not let go until the other is poised to bind. This coordination allows the motor protein to operate in a hand-over-hand fashion, never allowing the organelle cargo to diffuse away from the microtubule track. In contrast, there is no apparent coordination between the myosin heads in a myosin II dimer. The second reason for the high processivity of kinesin- I movement is that kinesin- 1 spends a relatively large fraction of its ATPasecycle tightly bound to the microtubule. For both kinesin- I and myosin II, the conformational change that produces the force-generating working stroke must occur while the motor protein is tightly bound to its polymer, and the recovery stroke in preparation for the next step must occur while the motor is unbound. But myosin II spends only about 5% of its AIPase cycle in the tightly bound state, and it is unbound the rest of the time. \.Vhat myosin loses in processivity it gains in speed; in an array in which many motor heads are interacting with the same actin filament, a set of linked myosins can move its filament a total distance equivalent to 20 steps during a single cycle time, while kinesins can move only two. Thus, myosin II can typically drive filament sliding much more rapidly than kinesin-1, even though the two different motor proteins hydrolyze NIP at comparable rates and take molecular steps of comparable length. This property is particularly important in the rapid contraction of skeletal muscle, as we will discuss later. Within each motor protein class,movement speedsvary widely, from about 0.2 to 60 pm/sec for myosins, and from about 0.02 to 2 pm/sec for kinesins. These differences arise from a fine-tuning of the mechanochemical cycle. The number of steps that an individual motor molecule can take in a given time, and thereby the velocity, can be decreasedby either decreasing the motor protein's intrinsic ATPaserate or by increasing the proportion of cycle time spent bound to the filament track. For example, myosin v (which acts as a processivevesicle motor) spends up to 907oof its nucleotide cycle tightly bound to the actin filament, in contrast to 5% for myosin IL Moreover, a motor protein can evolve to change the size of each step by either changing the length of the lever arm (for example, the lever arm of myosin V is about three times longer than the lever arm of myosin II) or the angle through which the helix swings (Figure f 6-65). Each of these parameters varies slightly among different members of the myosin and kinesin families, corresponding to slightly different protein sequencesand structures.

M y o s i nl l 5 to 10nm swing of lever arm

MyosinV 30to 40 nm swing o f l e v e ra r m

neao---

mrnuS end

p lu s end

Figure 16-65 The effect of lever arm length on the step sizefor a motor protein.The leverarm of myosinll is muchshorterthan the leverarm of myosinV.The power strokein the head swingstheir leverarmsthroughthe same angle,so myosinV is ableto takea bigger stepthan myosinll.

('eurqSpr^e6lo ^sallnof) 'urseldol^) sr 1;araqt tnoq6norqlpas.redsrp pue s1uau6e.r; snleredde1610g aqi ,(7-g1 alqel aas)azuau,{;odapo} salnqn}ol)il.u sasne)q)tqM'alozepo)ou o1atnsodxa rel;V (g)'snal)nueql,o saprsalrsoddo ol pe^oura^eq saulosoltua)pale)tldnp aql pue'aseqdordur sttlal aql uo lla) aql 'aurosorlua) a16urs e qlran'aseqdrelul u; s1 tqb7 a\t uo lla) aqf 'lla) aql jo lalua) aLll lP snal)nuaql ol asol)'aulosollua)aql rpaupaztle)olsrr6;o9eql,l)plut suteulal salnqnlor)l.ll 1ouals{s aqt se 6uo1 sy'1ura1o.rd r6;o9e lsurebetrpoqltre,e 6u6n1uaatbur palaqelsrsnleredder6;o9 aql pue pal ut palaqelale salnqnlor)trrj a q l ' l l a )l e r l a q r o p usar q ru l ( V ) 'snleredde r6go9 aqr uo salnqnloJ)tul 6u1z;raur{;odap Jo l)a#l 99-91 arn611

urn0[

alqpls 'aBJEISurcnpord,{q aseesrp s,Jeruraqzlv o} esrr sa^r8uralord srq}Jo Surssac -ord prurouqp aql 'suoxB ur JoldaJal Jolotu eupJquorusuEJlp eq o] IIaJ-aAJau p ol .,tl}JeJrpspurq r{Jrq^\ AdV pasodord sr pup -ursauD{ aqt uo ureqc lq8q I Jo IrEl 'ureloJd JosJnJaJdpro1,{ureaq} aq o} sureessJoldaJer eseql Jo aug .sraqruaru[1 -rupJurseuDl aleudorddu aqt slrel eql qluv\ dl]JeJrpur Jo dpcalp ]ceJelur slueu Jo -uedruoo pasoloue-aueJqrueruJgrcedsol pepos eJpteql sroldeJeJJolour palerc -osse-euEJqruehl'o3JpJallaueSJoeleudoJddp Jreql o] qJEue 01sJolotu eql lv\ollB suraloJdJoloru cgrcadsuo surpqcIqBII pelercosseJreql pue slrp]]ueJegrpar{J 'eruosoJlueJ aql lE spua snurru pJe.\i\o]$lJpJ] aFqnloJsrru Suop salcrsan r31og Sur,rou taluao eql JEau snleJedde r31og eql Suruoursod perrnbaJ JoJ IIaJ 'q}llroJ8 elnqnloJJnu q}IA\ pe}ercosse sr draqdued aJB surau^p 'r{yasra^rruo3 IIaJ eqt premol saFqn] UgJo luaue^ou pJemlno eql sllal Surnrlur ,asrmalrT'sllal ur Ug aqt a41 qcnu dral qa^ snouprquau e Sururro; pup suorsnlord repqnl olur lno seueJqrueurUA aqt 3ur33erp 'spua snld apqruoJJrru aq] pJemo] Ip^ pue 's{JeJ} elnqn}ortru paruro;erd ol saueJqruau palrJep-Ug Jaq}a} uEJ surseur) 'oJiln uI '(SS-gt arnS;g) useldo{c aq} }noq8norq} sasredsrp pue slueru8er; srueredde r31og aql e1tq^ 'llal eql Jo Ja]ual aq] o1 sasdellocUg aql ,elozupotou Jo aurJrq3loo sE qJns 'salnqnloJ3[u sazuaur.4.yodap lpq] Snrp e qlyv\ paleaJ] aJE sller uar{ ,1'aruosor}ual aq} Jpau pelpcol sr snluredde €1og aqr seeJeqm 'lleo aql Jo a8pa eql ol ]sorule spualxa pue selnqnloJf,rruqlrr!\ su8qu salnqnl euerquau aql'snteredde pup r31o5 (Ug) aqt urnpcltar trurseld Ug Jo {ro^a}eu -opue aql SurzrueBrour eloJ Jraq] sl sauerqurau relnllaJeJlur rorleqaq eql uo Jo sJolour alnqnlorJrru pup saFqntoJJtu Jo ]JaJJaarll Jo aldurexa lseJealJeqJ 'sursauDlse qJns sJolour palceJrp-pue-sn1d armbar draqdrrad aq] pJe^ao]sluaru -alour p8n;uluac searaq A'urau,(p curseydolrb se qJns suralo.rdJolotu palcaJrp -pue-snurru uorlf,e aql armbar JelueJ Jo IIaJ eql pJe^ ol selJrsel ro salaueBro;o sluaruanorupladrrluac 'snq;'l;aqdrrad lac aq] ol Sueuexa spue snydaq1pue 'eruosoJlueJ aql le IIaf, aq] Jo ralueJ aql JEeu spua snurur aql qllM paluerJo sr .fuesseoau,,(1pnba sr uodsupJ] 1ac aseqdratur uB ur,{erre alnqnlorcru pcrdfl V paztrelod Jleql (saJuBlsrp3uo1 qcns ra^of, lou paau sllal lsolu ur sallaue8ro q8noqry 's1eurruJetalJau ]uplsrp aqt ot uoxe aql Jo s.{emq8rq apqnlorJrru aql u^l,op slueuodruoc asdeu.{ssnorJe^pue (sJosJnJeJdalJrsa^,,fuolercas,erJpuor.lJ -olnuJo luatueloru prder aqt 'uodsuerl Ipuoxp lspJJoJalqrsuodsaruralord aq1se peguuepr dleu€rro se^ urseur) 'sallaue8ropesolJue-aueJqrueruJo Buruorlrsod pue lrodsue[ eq] sl s11ac aseqdralur ur sJo]our p1a1a>1so1dc Jo uorlJunJ rofeu y SAllAUe6ro pASOI)Ul

-auerquaw,o ltodsuell lelnlla)erluleql alerpewsulolotdtolow 'llef, eql Jo spaau 3uua;;rp o1 asuodsar ur parJrpou aq uEf, uraudp cluseldolrtc ;o saqradord leJrueqJaru aq] Jeqleqm ro l\^oq realc 1a.,(lou sl 'uraui{p cnuseldol,b Jo uJoJ auo -,(po ,{1ensn sr eJeql ]I '1ac cqodrecna pcrddl B ur punoJ sJaq{uau,{1pregurseuDlpue ursodu }ueJeJJrp dueru are alaq] seaJaq yJaqtuaru dgruey relnc4red lpq] Jo uorlounJ aq] roJ IIeJ aqt Jo spaau cr;rcadsaql o] Surprocce,{lrnrssacordpue paads ro3 ,ureurop Uel s}I q8norql peqJelle o8rec aql;o zlquapr aqr rtq peuruJalap sr uor]JunJ esoq ^ ,ural -ord rolour qJBa ror eqaq aql paun]-aurJ seq uounlo^a leq] paunsse sr ]I Jo LZOL

suol_ow uflnfltow

'aldtuExeJoc 'suoll (sls;6o1org JoJIEJrlrJcJolcPJuolldlJJsuEJl E 'ptoctg SutpoJua vNulu eq] ;o,(uedu:o1aql '6sg-Lvg:}lL urorjuolssluladqll^ '1.66L -nqrJlsrpvNuru pezlerreds asaql qsllqelse 01 tolJalsod eql reeu spua snld pue 'PS ''P la nM X uroll) uralord ilD'f flac aqlJo ued JoIJe]uPaq] uI peJe]snlceJespua snullu alnqnloJclru ]soIU areq { roloursrqlJo sardoryorequrnua6re1e qlrm 'uolele{so]Jb pazlrPlod s1r aFqn}orclu ;o eBB}ueApBsa{Pl eldcoo aqJ paler)osse sreuosouelau{ran3l urso{lu 'e],,tJooaql Jo uoISeJ e qllm palaqel tsure6e{poqrlue}ua)salonlJ 'uluelaur JorJalue aq] q pazrTPJolsr or^qua eql ul seJnlJnrls JoIJaluPJo uoIlPJUI3eds luaubrd urls aql lla) aues aql (g) 'al,{ooo eql ul qllM pall1;sa;;aue6lo pasol)ua-auPlquiaur JoJ,,fu€sseJeu sureloJd Sutpocua sVNUur Jo dnor8 tJullslp P pue 'oIJqLua aql are q)rq/1/\'saurosouplaur arp slods)i)elq ,{porralsod pazlTef,olsI 'sllef,tura8 aq} Jo luaudole^ep Surpnlcur Jo aql'asnour e uroiJpalelosre1,{>ouelaur uor8al JorJalsod aql Jo luaudole^ap JadoJdJoJdJesseJeusuleloJd apocue leql e;o uorl.rode;o a6eur lselluo)-aseqd (V) '(77 nldeqa uI passnrslp) srsauaSo,{rqure ^lrEa uI slua^a uoD sVNUru Jo dnor8 y 'sauosouelauuo ugso(61 B9-9t ernbrJ 1 -€JrJr3edsIIeJ pldeJ aqlJo uoUPdIJIluu uI ilec aql uqlF salls clJlsadso] sVNUur utti Ol papocua 1tllEuJaleru raqunu aBrPIB sazllPsol altlcoo apr4dosoJQ]uBI8 eqJ Jo (E) (v) 'suollunlls Jo 53fld1asaql uI selnJelolu VNUru uorlrsod puP Uodsuerl sulaloJd Jololu Plala{so}.{3 puB uolala{sotdc aq] 'rtlsulsudrns toN ,irlcpseld rlldeul's pue ^rorueru rure]-3uol Supep8ar ur selor ]ueuodurl .{eld sa}ts ct}dPuds aql le uoFeln8al uoIlPISu"Jl puE uollezllEf,ol 'suoJnau Aueur uI sesdEu^so] asolJ pazITPJol vNutu leql acuapl^e sI aJaql pue ,(lecgrcads eJe uollcunJ asdeu,{s uI pe^lonul sule}oJd Sutpocua sVNUur IeJaAes 'aldtuexa Jaqloup sV'saleJ toullsp qtllt sralq8nep o t1 eleJaua8 ol sapIAIpIIec ]ueJed e ueq ^ ]ueuodlul LFelncrped sl slqJ'salJlelulu^s? Jelnlle3 seqsqqelse 'selncelolu 1eq1ssacordE VNUtu s1r3utzleco1 Lq utalord relncured e;o slsaqlu^s eql lJulsel uauo slleJ 'uopcunJ Jo ells rtaql ]E suleloJd eleJluacuoc ol JepJouI

saln)olowvNU)u!)od5sazl;eroluolalelsol^)aql 'sa11aue3ro Jaqlo Jo dlarrerre pue sauosopua ' ( t t - 0 t a r n b r la a s ) '-{Fuatcg;a saltcouqe llal qlllvr pelercosse aJe 'I ursol.u Sutpnput 'sutsoLtu Jeqlo poolq paraq] uolalalsoli{> paler>osse Jo -Je{ aql ot peJe^llap }ou aJB seuosouEleru eql esnEf,aq pepPJ $lool JoloJ -auprqurauaq] o] rplrlrs 'urrIlue pue (aclru 'adflouaqd urrlreds1o)romtaue qbnorqtsa1;auebro rnJ qcrqM ur ,,alnllp,,P ul ]FsaJ auaSnutsori.rueq] ul suopelnlu q 'aqnl ]sel e ur ]uarue^oru peseq-ullJe Jleql alBlperu ol alqP fl puu (89-9I pasol)ua-ouerquauol xa;duo>abre; ' srr-l] arnElg) saruosoueleurJo aJpJJnsaql qll^\ pelerJossesr n utsoLl41(allul uI InJ eql Jo luaLuq)ellealerpaurIeu luaue;r; ldry aqr reqt tq6noqi sr11 1d.ryuralord aql o} pare^[ap eru ztaql eJaq^ Surl.lJe^o pup) seldtoulleJa{ uDIs aq] uJoJ lpql patPlar-ur])P aq] Jo apP|.U ,sa{couelaru eq} uI sassaJold Jl}upuap ]uaurPlualrl Jo spua aq} ol lno enolu seluosouelaru -url)P e uJoJleq] sluauoouro)pue lleurs esaqJ 'eJeJrns UDIS aql qleaueq salLJouapu pellec sllac uI pazlsaq] ;;aslrureulp ol purqteql sluauodruor -ur(s are ,sautosouDlautpallpJ (salnueJfilueufiId pesolsua-aueJquau 'SuEIunq 'salnqnloJ)rt! purqiPqt o1,(;leaarr pue eorru uI '(g9-gI aJnBrCees) ezIS dals a8rel e qlp\ ulso.'tru papeaq-oml sluauoduol sapnl)ur]€ql (par)xoldruo) 'slueruellJ pasol)ua abre;e srurlreu,(6'sa;;auebro e ^ ulso[ur se,rtrfiqtlour alleue8Jo e]elpeu 01 u ^oqs ulsolur lsJU aqJ -auerquraur q]rMaler)osse ol suralord P a^eq oslBsulaloJd Jolohtr urlJe SuolEuodsuEJt allaue8ro uI eloJ IuPOIJIuBIS 'llal fiossarre raqu,rnu e6re;e;o aruasard 1o poJ ulsdopoqJ er{} uI Jo SuDIJgJEnIeIuJou ro; parmber sI uoIl3BJa}uI 'ada eq] slleJ por eq] ul eqt salrnbarurau{6 'a;;auebropasol)ua sq] pue 'suleqr tq8l uraufp eql Jo euo ol fllcerlp spulq Jo -euerqulau e ol ulauIp lustuq)eup 1o punoJ uretord Surlcatap-]q3q aqr 'ulsdopoqr Jo IIet f,Iruseldotb aq;'o8rec naq] aql roJlapotuy 49-9 1 arn611 'seseJJeqlo uI '(29-9I aln qll-ry\^llJaJIp ]JpJaluI l.etu sJolotu utau.,(pcrurseldoldc rxusz -31fl allaue8ro aqt ot ulau,{p;o ruaruqJe}}e peluF8er eql alulperu,{eur }l-}uarue -llJ IdrV eql Prn-pue 'auerqruetu r31o3 eqr oi &nqets IeJnlcnrls sanr8dlqeqord derre utrlcads eqJ'(I?-01 arn8rg aes) uolala{so/.c auerqurau aptcorqldra aql alnqnloJ)ruj fl q luaru t Jo lue3srurluar IEJJp le]ala{sol-,(J ruueld B IuJoJ o1xalduroc ul}Jeu^p eql -eg ldry aq] qlp\ alelJosseol pasodord uaaq e^eq qclq ^ 'utllcads pue utrllue '(slueuEIIJ ullJe suraloJdaql qlrM paleoJ eJesnleJEdder31ogaqlJo sauEJquelN IPuorlue^uof, Jo uollBelf,nu aql uI paAIoAuIxelduoc duv eql Jo sluauoduoc aqt 'tdrv pue Tdry ruor; ]cunslp) l dry uralord petelar-ulloe aq] uroq sturoJlPq] ]ualupllJ a{[-ul]Je uoqs e sepnlcul xalduroc upceu,{p aq; z(larrrlcagasalleuE8Jo alpoolsuerl ol uuJzu[p palpc xaldruo] ue]oJd e8Jel puoJas e qlllvr uolle -IJossEseJlnber'xaldruoc uleloJd efinq e JIeslI 'uraufp crurseldof3 'SeueJqulatu 'utau.{p ro4 o] salelpatu uago r(lqruesserelnlaloluoJJBur a3rul e lualuqJBl]e '8u1pu8rs IIaJ pue uodsuerl ueamlaq Tull 'SuIPuBts e apnord deru sroldacal uIseuDI eseqJ 1ac qllrr,r petelJosse sulaloJd 'u0I}EZIIBJOI rleq} roJ Uodsuer} ploJJeJSere ,(sureloJdSunceralul-)Nl) sdl{ eqJ paseq-aFqnloJJrru uo ^lar leq] sallaueSJopunoq-auEJqrueu Jaqlo snoIJuAuo sB ,unlnsrlal crurseldopua aq] uo palJpuepl uaeq aAPqsurseuDlcgtcads ro; IIa^i\se uralord sJoldaJeJJeqlo '(96-9 arnflrg aas) uleJq aql Jo slleo a1Jeu uI selEBaJSSe

&

uolelelsol^) aql:9l. ieldeq)

zzol

fale^ uou pue eMezrlpf laladJo Isellnor ,B).lla)rarq6nep aql ol pauuuo)oslpst 'vNUutpaztlp)ol srqlLuor]paqu)supl]'utaloJd lqsv.(pnq 'araq) tatq6nepeqt;o a6te1e ;;r1s lla) dll re, aql o] pauUUo)sl VNUr!oql'lle) 6urprarpsrql ur (pal)VNUruI gsy aLlr 1sea,{ azrle)olol pesnseM(HSll)uollezlplrq,(q nJlsu/ ]ua)selonll (B)'^llstLuaLl)otq pue srlaua6 {q paururralep se ,uotlpztle)ol leln)aloLu vNUr.uI qsy lo u.tstupq)arrJ aqt (V)'dtr pnq lsea{ eql u! uo!}ezlle)ol VNUUIpazgelod69-9t arn6rl

'llEJa^o lno SuruurM ursauDlJaSuoJlseql qlrl\/\'suralord Joloru elnqnlorJrru o^\l aql uee^ JeM-Jo-8n] e ruoq IlluaJedde sluaua^oru pJemlno dryaf aql taq llnseJ 'sa1nuel3 ]uaru8rd aql qlll\i\ pelprcossE eJe n urso,r(ruJoloru urlJe aq] sE IIa^ase ursaur{ pue urau.,(psJoloru apqruorJnu aql qlog 'sda1spreaqcuq luanbar; q}yrt {1ra[ sr Juaruanour pJe^ ]no aql alq ^ 'qlootus pue prdur sr luarualoru premur aql teqt slpanar(IZ-9I arn31g1sapuer8 luaur8rd lenprlrpur Jo 8uqcu4 aq; ,{raqdrrad IIar aq} punore patnqrr}srp ere spua snld aqi a1rq^ ,llar aqt Jo retuaJ eq] ur spua snur{u rraql Surzrpcol 'salnqn}orf,rru eseq} salealtnu euosor}uel aql 'salnqnloJf,rruJo {romleu alrsualxa ue Suop Surnourr(q asradsrpro a1e8ar88esaln -uer8 luaru8rd aqt'(OZ-91 arn81g;uorlelnrut1spuoruJoq Jo IpuoJnauo1asuodsar ur uorlpool Jraqt Jallp UEJ leq] salnuer8 luaur8rd a8rel urc1uoc ,qsg go sarcads ,s11ac leJalas ur uorleJoloc UDISur saSueqc prdur roy alqrsuodsar aJe qJrq A lue€ asaql 'saldruexa Jrleruerp lsotu aql Jo auo apvrord saptcouularu qsrC 'sluarueloru IlaJ-aloq ^ ro salauu8ro pasolcua-auerquau s1rgo Suruorlrsod aql ur a8ueqc e Jarllle asnec dqaraqt pue surelord roloru 3o firnrlce aq1 alep8ar ueo IIaf, aql

uo!t)unJutetotdtolow eleln6auslle) '(69-9I arn31g1 uralord 4 ursofur aqt pue VNUru tqsv aql ur acuanbas crycads e uaamlaq {uq ]Jarrp E ruJoJ ]eql suralord olrl Jo xalduroc e se llarlr se Tleslr urltu pue ,url3ord 'urso.,(ruodorl'sunuJoJ aql Jo auo apnlJur aseql i&lJelod purrou ro; permbar aJe uolala{so{c aql qtIM pa}ErcosseeJe teq} s}f,npoJd auaB Jaq}o xts lseel lE ralq8nepTJaqlotu aqt ]dnJsrp lpql suorlulnru Jaqlo lpql pelea^al suq aouaJaJJrp JoJ uaarJs crlauaB V 'VNUru tr4sv Jo uonnqrJlsrp crrlaruurr{se sq} roJ parrnbar sr 'dyo[141']sea[ ur puno; 'llar surso^,(ru omt aq]Jo auo ralq8nep aq] ur,(po nadfi dn pua aJoJareqlpue pnq Suunor8 aq] ^le^rsnlcxa pezrleJol ol are uralord pue 'tllsv pe1eJ uralord ,trolep8ar aua8 e ,{q pasner aJp sacueJeJ VNUIU f rysy qlog -Jrpasar{} 'tue61 'alrs pnq uau reql Jo Jo af,rorlJeql uI pue (2 raldeq3 ur pessnl -srp) Surqclrrr,rs addt-3upeu o8rapun o] drl[qe luanbasqns Jraql ur seouaJaJJrp rofeur dq pelealal sE 'rsarlrluapl]Jurlstp urulal sllal ralqBnup pue raqloru lsead aq1 'luapuadap-urloe sr Surroqcue se IIaM se lrodsuerl VNUur ,sllal euros uI dpueg WUg aql Jo Jaqtuau e 'ursaotu palleJ ureloJd Surpurq-urlJe uE o] dpcarrp spurq 'aldurexa JoJ te>Iso SurpoouaVNUtu aql .uolala{so1,{ourlce 1ecr1 -Joc aql aAIoAurol sreadde selnqnloJJnu er,r,{ra,u1ap raUBsuor}pJo1alerrdordde Jlaql 01 sVNUru aq];o SurroqJue aqJ 'spua snld elnqnloJ3rru aql ol Uodsuerl slr JoJ I-urseuu sarrnbar'odrqura eqlJo JorJalsodaql u1 ]uarudoya,rap1ac urra8 ro; .,tressacauuralord e tE{sO Surpocua VNUru Jo }rodsuer} aql dlasranuo3 .JorJ -alue IIer aql lE spua snurru alnqnloJJnu aq] ol uodsuer] slr 8u[qeua dlqeruns -ard 'ureqc rq311uraufp cruseldopb e ol spurq urn] ur qclqm ,^aollelrtspallpo utalord P spulq lEql UIn ,t eql uHllllr eJnltnJls e spq ']uerudolanap JorJeluE

a)uanbas uotleztle)ol VNU

,{

tzot

suolowuvtnftlow

uiri 0 !

puasnurur

alnqnlor)il,!

puesnlo

('sa)uar)s sJaqrueu Jeqlo Jo uoqelnSaH 'sllac alJsnu qloolus uI uollJPJluoc Jo IoJluoJ eql 1or{uapery leuOrleNuo.r; uorssrr.urad Jo ]uauoduoc tueuodrul uB osls sI uoneldJoqdsoqd utsor(ru ^ oleq ssnf,slpilu qrl^ '166r '9ZLt-0ZLtit6'V'S'n'pS olul e^ sV'omt olut ilar cpollu aql saqcurd leq] 3u1ralpcertuoc paseq-urlce eql 'pDV ''p 1asla6og'l'S Luorl) '@L-91 'slsolltu Surrnp pele lloN iud alquasse o1 FlP osp sI )3'IIAI 'pua snlo aql preMol pua snurLrr 1yursodru Sursnec aql urorj pue luauEIIJ relodrq e olur dlqurassesll seloru sa^ouJpue uort)arp saq)lrMs,{lldnrqe arnS;g) uollJeJluoo IIaJ ol speel -ord qcrqrt 'elels papuelxa eq] arunsse ,tlpquara;ard ol II ursodru eql sasnuc Ir 'aruanbesosprnaql qbnorql Ierryleg 'pua snururaql pre^ol pua snldaql UJIW) asaury uruqt-ry?11utsotru luapuadap-runlf,lec eql Lq ureqc tq31&ote1 'peaq Jololu eq} q}yv\slf,eJa}ultltuaredde uletuop -n8ar aq1;o uor1e1,{.roqdsoqd LuorJ'aprls sse;6e uo alnqnlor)rure 6uo;e 'slueurelS relodtq ruJoJueo ]eql aluls papualxa sa^ow (/aoila()aurosouelaurpalelosl IrEt aql qf,rqm uI alels tueq e pue uv'alnqnlor)l[u e uo ouosouelaule 'sllal qJns uI saluts uE leuoperuJoJuoJ luaJaJJIp oml uI lsrxa ueJ 11 utsodur 'suleqr tq311pue ,o luauralour leuorl)aJrprg11-91 arn613 aq1 .{lqruasse }uetuelg {olq} pue &1nltce rolotu q}oq SulrreJJe aq uuc 11utsodur 'slleJ elJsnru dleaq qloq uo salts;o ,,!atJel e uo pa1e1,{roqdsoqd -uou alep8ar o1 uor1u1.{roqdsoqdasn osp ueJ IIac eql ursortu uI &lrrpce dlntlce pue luauqJe]le utalord Jolotu qloq areln8al leql sleu -8rs SurladuroJ Jo af,upleqxaldtuoc e t(q palonuoJ sI sula]oJd rolotu relnc[red 'JoloJ qlyv\ pelpoJ salaueflro aueJquau Jeqlo Jo ]uaruanolu aql fent relnuls P uI sqsrJaqt Sur8ueqc 'ralual IIaf, eqt pru^ o],(prder sapuerB luau8rd aql Serp o1 'a8uuqr Jolos uDIs qeu8ts luql uopel aar; urauLp 8unea1 'palenqceut sI uISauII -nurrls lBuoruroq P JeuE parelrfuoqdsoqdaruocaq suleqs 1q3u ulsauq eql uaqM tUfi OS

('oureH qeal ;o {salrno)'B)'AL\6U) lla) aqrJolalua) aq] ur patebar66e ro (ryal)useldov(r aqt tnoqbnorql pasraosrp raq]rasaurosouelaur prpllr)ue)uJv s1r6urrvroqs'qsq ueJo ale)se ur lla) a;6urse;o sa6eur plag-lq6;rg(8)'salnqnlor)rtu 6uole rn))o sauJosouelaui Jo suorlnqulsrpar qlog',(len$:adsar'(dwv)) 6yyytr;tIt Jelnlla)erlur ur asPar)ap ro asear)uruPol asuodsarur sauJosouelaur ;o uor1e6a.r66e pue ;esradsrp aql 6ulrtoqs';1arluaubrd e 'snlnurrls leuornau ,o Mar^)lleuaq)s (v) ro leuouiloqe ol asuoosalur lla) aq] u! uorle)olr;aql a6ueq>ue) saurosoueleur .ro'sa;nuerb aql' PMuq) saurosouelaur a6re1ureluor'qsr;;osaoads luar.u6rd ur uorlerolo)ur>1s ur sa6ueqr leranas rol a;qrsuodsa.r areLl)rqM's11ar tuerb asaqf 'slla) luau6rd qsg u! sluouanour ourosouplaurpa1eln6egO/-9 I arn6tl

c3suldslc

clIv91u99V

(v)

dIAV) aseoJ)ul dWV) aseal)ep

uola;a>1soy(1aql:9 L laldeLl]

vzoL

1025

THECYTOSKELETON AND CELLBEHAVIOR a c t i n - b i n d i n gs i t e m y o s i nl i g h t c h a i n s

@ b i p o l a rf i l a m e n t of 15-20 molecules m y o s i nt a i l r e l e a s e d

I N A C T I VS ET A T E : ( l i g h t c h a i n sn o t p h o s p h o r y l a t e d )

ACTIVESTATE: ( l i g h t c h a i n sp h o s p h o r y l a t e d )

(A)

Figure16-72 Light-chainphosphorylationand the regulationof the assemblyof myosinll into thick filaments.(A)The of one of the two light chains(theso-called controlledphosphorylation by the enzymemyosinlight-chainkinase(MLCK) r e g u l a t o r y l i g h t c h a i n , s h o wlni gi nh t b l u e ) o n n o n m u s c l e m y o siinnal lt e s t t u b e h a s a t l e a s t t w o e f f e cittsc:a u s e s a c h a n g e the myosintail from a "stickypatch" site,and it releases in the conformation of the myosinhead,exposingitsactin-binding to assembleinto short,bipolar,thickfilaments.(B)Electron on the myosinhead,therebyallowingthe myosinmolecules micrographof negatively stainedshortfilamentsof myosinll that havebeeninducedto assemblein a testtube by phosphorylation of their lightchains. Thesemyosinll filamentsare muchsmallerthan thosefound in skeletalmusclecells (seeFigure16-55).(8,courtesyof JohnKendrick-Jones.)

of the myosin superfamily is not as well understood, but the control of these myosins is likewise thought to involve site-specific phosphorylations.

Su m m a r y Motor proteins use the energyof ATP hydrolysis to moue along microtubules or actin filaments. Theymediate the sliding of ftlaments relatiue to one another and the transport of cargoalongfilament tracks.All known motor proteinsthat moueon actin filaments are members of the myosin superfamily. The motor proteins that moue on microtubules are either membersof the kinesin superfamily or the dynein family. The myosin and kinesin superfamiliesare diuerse,with about 40 genesencodingeachtypeof protein in humans. The only structural element shared among all membersof each superfamily is the motor "head"domain. Theseheadsarefused to a wide uarieryof different "tails,"which attach to different typesof cargoand enablethe uariousfamily membersto perform dffirent functions in the cell. Thesefunctions include the transportation and localization of specificproteins,membrane-enclosed organelles,and mRNAs. Although myosin and kinesin walk along different tracks and usedifferent mech' anisms to produceforce and mouementby ATP hydrolysis,they sharea common structural core,suggestingthat they are deriuedfrom a common ancestor.Thedynein motor protein has independentlyeuolued,and it has a distinct structureand mechanismof action.

THECYTOSKELETON ANDCELLBEH IOR A central challenge in all areasof cell biology is to understand how the functions of many individual molecular components combine to produce complex cell behaviors. The cell behaviors that we describe in this final section all rely on a coordinated deployment of the components and processes that we have explored in the first three sections of the chapter: the dynamic assembly and disassembly of cltoskeletal polymers, the regulation and modification of their structure by polymer-associated proteins, and the actions of motor proteins moving along the polymers. How does the cell coordinate all these activities to define its shape, to enable it to crawl, or to divide it neatly into two at mitosis? These problems of cltoskeletal coordination will challenge scientists for many years to come.

1026

Chapter 16:The Cytoskeleton

To provide a sense of our present understanding, we first discuss examples where specialized cells build stable arrays of filaments and use highly ordered arrays of motor proteins sliding them relative to each other to generate the large-scale movements of muscle, cilia, and eucaryotic flagella. Next, we consider two important instances where filament dynamics collude with motor protein activity to generate complex, self-organized dynamic structures: the microtubule-based mitotic spindle and the actin arrays involved in cell crawling. Finally, we consider the extraordinary organization and behavior of the neuronal c)'toskeleton.

Slidingof Myosinll and ActinFilaments Causes Muscles to Contract Muscle contraction is the most familiar and the best understood form of movement in animals. In vertebrates, running, walking, swimming, and flying all depend on the rapid contraction of skeletal muscle on its scaffolding of bone, while involuntary movements such as heart pumping and gut peristalsis depend on the contraction of cardiac muscle and smooth muscle, respectively.All these forms of muscle contraction depend on the ATP-driven sliding of highly organized arrays of actin filaments against arrays of myosin II filaments. Skeletalmuscle was a relatively late evolutionary development, and muscle cells are highly specialized for rapid and efficient contracrion. The long thin muscle fibers of skeletal muscle are actually huge single cells that form during development by the fusion of many separate cells, as discussed in Chapter 22. The large muscle cell retains the many nuclei of the contributing cells. These nuclei lie just beneath the plasma membrane (Figure f6-23). The bulk of the cltoplasm inside is made up of myofibrils, which is the name given to the basic contractile elements of the muscle cell. A myofibril is a cylindrical structure l-2 pm in diameter that is often as long as the giant muscle cell itself. It consists of a long repeated chain of tiny contractile units-called snrcomeres,each about 2.2 pm long, which give the vertebrate myofibril its striated appearance (Figure

ro-7$. Each sarcomere is formed from a miniature, precisely ordered array of parallel and partly overlapping thin and thick filaments. Tlne thin fiIaments are composed of actin and associatedproteins, and they are attached at their plus ends to a Z disc at each end of the sarcomere.The capped minus ends of the actin filaments extend in toward the middle of the sarcomere, where they overlap with thick fiIamenfs, the bipolar assembliesformed from specific muscle isoforms of myosin II (see Figure 16-55). \Mhen this region of overlap is examined in cross section by electron microscopy, the myosin filaments are seen to be arranged in a regular hexagonal lattice, with the actin filaments evenly spaced between them (Figure 16-75). cardiac muscle and smooth muscle also contain sarcomeres, although the organization is not as regular as that in skeletal muscle. Sarcomere shortening is caused by the myosin filaments sliding past the actin thin filaments, with no change in the length of either type of filament (Figure 16-74 c and D). Bipolar thick filaments walk toward the plus ends of two sets of thin filaments of opposite orientations, driven by dozens of independent myosin heads that are positioned to interact with each thin filament. Because there is no coordination among the movements of the myosin heads, it is critical

Figure16-73 Skeletalmusclecells(also calledmusclefibers).(A)Thesehuge multinucleated cellsform by the fusionof manymusclecellprecursors, called myoblasts. In an adult human,a muscle cellis typically50 pm in diameterand can be up to severalcentimeterslong. (B)Fluorescence micrographof rat muscle, showingthe peripherally locatednuclei (blue)in thesegiant cells.Myofibrilsare stainedred;seealsoFigure23-468. (B,courtesyof NancyL. Kedersha.)

myofibril 50pm

1027

THECYTOSKELETON AND CELLBEHAVIOR Z disc

dark band light band

_d E --l

) (B)

II I

one sarcomere

t h i c k f i l a m e n t( m y o s i n ) t h i n f i l a m e n t( a c t i n l light band dark band light band

2pm

electron Figure 16-74 Skeletalmusclemyofibrils,(A)Low-magnification micrographof a longitudinalsectionthrougha skeletalmusclecellof a rabbit,showingthe regularpatternof cross-striations. Thecellcontains manymyofibrilsalignedin parallel(seeFigure16-73).(B)Detailof the skeletalmuscleshownin (A),showingportionsof two adjacentmyofibrils and the definitionof a sarcomere(blackarrow).(C)Schematicdiagramof a singlesarcomere, showingthe originof the darkand light bandsseenin are the electronmicrographs. TheZ discs,at eachend of the sarcomere, the attachmentsitesfor the plusendsof actinfilaments(thinfilaments); M line,or midline,isthe locationof proteinsthat linkadjacentmyosinll filaments(thickfilaments) to one another.Thedarkbands,which markthe they locationof the thickfilaments, aresometimes calledA bandsbecause indexchanges appearanisotropic in polarizedlight (thatis,their refractive with the planeof polarization). The light bands,whichcontainonly thin filamentsand thereforehavea lowerdensityof protein,arerelatively isotropicin polarizedlightand aresometimescalledI bands.(D)Whenthe sarcomere contracts, the actinand myosinfilamentsslidepastone another (A and B,courtesyof RogerCraig.) without shortening.

that they operate with a low processivity, remaining tightly bound filament for only a small fraction of each AIPase cycle so that they one another back. Each myosin thick filament has about 300 heads muscle), and each head cycles about five times per second in the

e;'

1um

' -.

to the actin do not hold (294 in frog course of a

Figure16-75 Electronmicrographsof an insectflight muscleviewed in cross section.The myosinand actinfilaments arepackedtogetherwith almostcrystalline Unliketheirvertebrate regularity. counterparts,thesemyosinfilamentshave a hollow center,as seenin the on the right.Thegeometryof enlargement the hexagonallatticeis slightlydifferentin vertebratemuscle.(FromJ. Auber,J. de Microsc.8:197-232, 1969.With permission from Societ6frangaisede microscopie 6lectronique.)

1028

Chapter16:TheCytoskeleton Z disc Cap Z

m y o s i n( t h i c kf i l a m e n t ) tropomodulin

4!g!'llLlD

'

.:t

.ra!i!rrtl,:)

b l u se n d of actin f ilament

m t n u se n o

a c t i n( t h i n f i l a m e n t )

rapid contraction-sliding the myosin and actin filaments past one another at rates of up to 15 pm/sec and enabling the sarcomere to shorten by l0% of its length in less than 1/50th ofa second. The rapid synchronized shortening ofthe thousands of sarcomeres lying end-to-end in each myofibril enables skeletal muscle to contract rapidly enough for running and flying, or for playing the piano. Accessory proteins produce the remarkable uniformity in filament organizalion,length, and spacing in the sarcomere (Figure f 6-76). The actin filament plus ends are anchored in the Z disc, which is built fromcapzand o-actinin; the Z disc caps the filaments (preventing depolymerization), while holding them together in a regularly spaced bundle. The preciselength of each thin filament is determined by a template protein of enormous size, called nebulin, which consists almost entirely of a repeating 35-amino-acid actin-binding motif. Nebulin stretches from the Z disc to the minus end of each thin filament and acts as a "molecular ruler" to dictate the length of the filament. The minus ends of the thin filaments are capped and stabilized by tropomodulin. Although there is some slow exchange of actin subunits at both ends of the muscle thin filament, such that the components of the thin filament turn over with a half-life of several days, the actin filaments in sarcomeresare remarkably stable compared to the dynamic actin filaments characteristic of most other cell t!?es that turn over with half-lives of a few minutes or less. Opposing pairs of an even longer template protein, called titin, position the thick filaments midway between the Z discs. Titin acts as a molecular spring, with a long series of immunoglobulin-like domains that can unfold one by one as stressis applied to the protein. A springlike unfolding and refolding of these domains keeps the thick filaments poised in the middle of the sarcomere and allows the muscle fiber to recover after being overstretched.In c. elegans,whose sarcomeres are longer than those in vertebrates, titin is also longer, suggesting that it too servesas a molecular ruler, determining in this casethe overall length of each sarcomere (seeFigure 3-33).

A SuddenRisein Cytosolic Ca2+ Concentration InitiatesMuscle Contraction The force-generatingmolecular interaction between myosin thick filaments and actin thin filaments takes place only when a signal passesto the skeletal muscle from its motor nerve. Immediately upon arrival of the signal, the muscle cell

act in rapid successionon the same thin filament without interfering with one another. second, a specialized membrane system relays the incoming signal rapidly throughout the entire cell. The signal from the nerve triggers an action potential in the muscle cell plasma membrane (discussedin Chapter ll), and

FigureI 6-76 Organizationof accessory proteins in a sarcomere.Each gianttitin moleculeextendsfrom the Z discto the M line-a distanceof over 1 pm. Partof eachtitin moleculeis closelyassociated with a myosinthick filament(whichswitchespolarityat the M line);the restof the titin moleculeis elasticand changeslengthasthe sarcomere contractsand relaxes. Each nebulinmoleculeis exactlythe lengthof a thin filament.Theactinfilamentsare alsocoatedwith tropomyosinand troponin(not shown;seeFigurei6-78) and are cappedat both ends. Tropomodulin capsthe minusend of the actinfilaments,and CapZanchorsthe plusend at the Z disc,whichalso containso(-actinin.

1029

THECYTOSKELETON AND CELLBEHAVIOR

o l a s m am e m b r a n e

Ca2*-release c h an n e l s transverse(T) t u b u l e sf o r m e d from invaginations o f p l a s m am e m b r a n e ic s ar c o p l a s m reticulum 05pm

d e p o l a r i z e dT - t u b u l em e m b r a n e a a

l. :a

polarized T-tubule t membrane

a a

(A)

1 :iji: I

CYTOSOL

35nm

a t. t 'i:.:llf a

rl':rral

ai

ri

l.!

!liir.:

i't'r..t:,

arllili:! l

I

this electrical excitation spreadsrapidly into a seriesof membraneous folds, the transverse tubules, or T tubules, that extend inward from the plasma membrane around each myofibril. The signal is then relayed across a small gap to tlre sarcoplasmic reticulum, an adjacentweb-like sheath of modified endoplasmic reticulum that surrounds each myofibril like a net stocking (Figure lg-77{and B). rWhen the incoming action potential activates a Ca2+channel in the T-tubule membrane, a Ca2+influx triggers the opening of Ca2+-releasechannels in the sarcoplasmic reticulum (Figure l6-77C). CaZ*flooding into the cytosol then initiates the contraction of each myofibril. Becausethe signal from the muscle-cell plasma membrane is passed within milliseconds (via the T tubules and sarcoplasmic reticulum) to every sarcomere in the cell, all of the myofibrils in the cell contract at once. The increasein Ca2*concentration is transient becausethe Ca2* is rapidly pumped back into the sarcoplasmic reticulum by an abundant, AlP-dependent Ca2+-pump (also called a Caz*-ATPase)in its membrane (see Figure 1l-I3). Typically, the cytoplasmic Ca2* concentration is restored to resting levels within 30 msec, allowing the myofibrils to relax. Thus, muscle contraction depends on two processesthat consume enormous amounts ofATP: filament sliding, driven by the ATPase of the myosin motor domain, and Ca2* pumping, driven by the Caz*-pump. The Ca2* dependence of vertebrate skeletal muscle contraction, and hence its dependence on motor commands transmitted via nerves, is due entirely to a set of specialized accessoryproteins that are closely associatedwith the actin thin filaments. One of these accessoryproteins is a muscle form of tropomyosin, an elongated molecule that binds along the groove of the actin helix. The other is troponin, a complex of three polypeptides, troponins T I, and C (named for their tropomyosin-binding, inhibitory, and Ca2*-binding activities, respectively). Troponin I binds to actin as well as to troponin T. In a resting muscle, the troponin I-T complex pulls the tropomyosin out of its normal binding groove into a position along the actin filament that interferes with the binding of

Figure16-77T tubulesand the sarcoplasmicreticulum.(A)Drawingof the two membranesystemsthat relaythe signalto contractfrom the musclecell plasmamembraneto all of the myofibrils in the cell.(B)Electronmicrograph showingtwo T tubules.Notethe position channelsin the of the largeCa2+-release reticulummembrane;they sarcoplasmic "feet"that look likesquare-shaped connectto the adjacentT-tubule diagram membrane.(C)Schematic channelin showinghow a Ca2+-release reticulummembraneis the sarcoolasmic thought to be opened by the activation of a voltage-gatedCa2+channel. (8,courtesyof ClaraFranzini-Armstrong.)

1030

Chapter16:TheCytoskeleton

t r o p o ni n comPlex

rc

myosin-bindingsite exposed by Ca'*-mediatedtropomyosin movement

tropomvosin

T + ca2*

ca2* ,".

10 "t

Figure16-78Thecontrolof skeletal muscle contraction by troponin.(A)A skeletal muscle cellthinfilament, showing the positions of tropomyosin andtroponin alongtheactinfilament. Eachtropomyosin molecule hasseven evenlyspaced regions withsimilar aminoacidsequences, eachof whichisthoughtto bindto anactinsubunitin thefilament. (B)A thinfilament (binding shownend-on, illustrating howCa2+ to troponin) isthoughtto relieve thetropomyosin blockage of theinteraction between actinandthemyosin head.(A,adapted fromG.N.Phillips, J.P. Fillers andC.Cohen, J.Mol.Biol.192:111-131,1986. Withpermission fromAcademic Press.)

myosin heads, thereby preventing any force-generating interaction. \Mhen the level of Ca2*is raised, troponin C-which binds up to four molecules of Caz+causes troponin I to release its hold on actin. This allows the tropomyosin molecules to slip back into their normal position so that the myosin heads can walk along the actin filaments (Figure f 6-78). Troponin C is closely related to the ubiquitous Caz*-binding protein calmodulin (see Figure lS=44); it can be thought of as a specialized form of calmodulin that has acquired binding sites for troponin I and troponin ! thereby ensuring that the myofibril responds extremely rapidly to an increase in Ca2+concentration. In smooth muscle cells, so-called because they lack the regular striations of skeletalmuscle, contraction is also triggered by an influx of calcium ions, but the regulatory mechanism is different. Smooth muscle forms the contractile portion of the stomach, intestine, and uterus, the walls of arteries,and many other structures requiring slow and sustained contractions. smooth muscle is composed of sheets of highly elongated spindle-shaped cells, each with a single nucleus. Smooth muscle cells do not expressthe troponins. Instead, Caz* influx into the cell regulates contraction by two mechanisms that depend on the ubiquitous calcium binding protein calmodulin. First, Ca2+-boundcalmodulin binds to an actin-binding protein, caldesmon, which blocks the actin sites where the myosin motor heads would normally bind. This causesthe caldesmon to fall off of the actin filaments, preparing the filaments for contraction. Second,smooth muscle myosin is phosphorylated on one of its two light chains by myosin light chain kinase (MLCK.),as described previously for regulation of nonmuscle myosin II (see Figure 16-72).lVhen the light chain is phosphorylated, the myosin head can interact with actin filaments and cause contraction; when it is dephosphorylated, the myosin head tends to dissociatefrom actin and becomes inactive (in contrast to nonmuscle myosin II, light chain dephosphorylation does not cause thick filament disassembly in smooth muscle cells). MLCK requires bound ca2*/calmodulin to be fully active. External signaling molecules such as adrenaline (epinephrine) can also regulate the contractile activity of smooth muscle. Adrenaline binding to its G-protein-coupled cell surface receptor causesan increase in the intracellular level of cyclic AMf; which in turn activates cyclic-AMP-dependent protein kinase (pKA) (seeFigure l5-35). PKA phosphorylates and inactivates MLCK, thereby causing the smooth muscle cell to relax. The phosphorylation events that regulate contraction in smooth muscle cells occur realtively slowly, so that maximum contraction often requires nearly a second (compared with the few milliseconds required for contraction of a skeletal muscle cell). But rapid activation of contraction is not important in smooth muscle: its myosin II hydrolyzes ArP about l0 times more slowly than skeletal muscle myosin, producing a slow cycle of myosin conformational changes that results in slow contraction.

THECYTOSKELETON ANDCELLBEHAVIOR

1031

HeartMusclels a Precisely Engineered Machine The heart is the most heavilyworked muscle in the body, contracting about 3 billion (3 x 10s)times during the course of a human lifetime. This number is about the same as the average number of revolutions in the lifetime of an automobile's internal combustion engine. Heart cells expressseveralspecific isoforms of cardiac muscle myosin and cardiac muscle actin. Even subtle changesin these contractile proteins expressed in the heart-changes that would not cause any noticeable consequencesin other tissues-can cause serious heart disease(Figure 16-79). The normal cardiac contractile apparatus is such a highly tuned machine that a tiny abnormality anywhere in the works can be enough to gradually wear it down over years of repetitive motion. Familial hypertrophic cardiomyopathy is a frequent cause of sudden death in young athletes. It is a genetically dominant inherited condition that affects about two out of every thousand people, and it is associated with heart enlargement, abnormally small coronary vessels,and disturbances in heart rhythm (cardiac arrhythmias). The cause of this condition is either any one of over 40 subtle point mutations in the genesencoding cardiac B myosin heavy chain (almost all causing changesin or near the motor domain), or one of about a dozen mutations in other genes encoding contractile proteins-including myosin light chains, cardiac troponin, and tropomyosin. Minor missensemutations in the cardiac actin gene cause another type of heart condition, called dilated cardiomyopathy, that also frequently results in early heart failure.

Ciliaand Flagella AreMotileStructures Builtfrom Microtubules a n dD y n e i n s Just as myofibrils are highly specialized and efficient motility machines built from actin and myosin filaments, cilia and flagella are highly specialized and efficient motility structures built from microtubules and dynein. Both cilia and flagella are hair-like cell appendagesthat have a bundle of microtubules at their core. Flagella are found on sperm and many protozoa. By their undulating motion, they enable the cells to which they are attached to swim through liquid media (Figure f 6-80A). Cilia tend to be shorter than flagella and are organized in a similar fashion, but they beat with a whip-like motion that resembles the breast stroke in swimming (Figure f6-808). The cycles of adjacent cilia are almost but not quite in synchrony, creating the wave-like patterns that can be seen in fields of beating cilia under the microscope. Ciliary beating can either propel single cells through a fluid (asin the swimming of the protozoan Paramecium) or can move fluid over the surface of a group of cells in a tissue. In the human body, huge numbers of cilia (10s/cmzor more) line our respiratory tract, sweeping layers of mucus, trapped particles of dust, and bacteria up to the mouth where they are swallowed and ultimately eliminated. Likewise, cilia along the oviduct help to sweep eggs toward the uterus. The movement of a cilium or a flagellum is produced by the bending of its core, which is called the axoneme. The axoneme is composed of microtubules and their associatedproteins, arranged in a distinctive and regular pattern. Nine special doublet microtubules (comprising one complete and one partial microtubule fused together so that they share a common tubule wall) are arranged in

Figure 16-80 The contrastingmotions of flagella and cilia. (A)The wave-like motion of the flagellumof a spermcellfrom a tunicate.The cell was photographed illuminationat 400 flashesper second.Notethat with stroboscopic from the baseto the tip of the wavesof constantamplitudemovecontinuously the breaststrokein flagellum.(B)The beatof a cilium,which resembles swimming.A fast power stroke(redarrows),in which fluid is driven over the surfaceof the cell,is followed by a slow recoverystroke.Eachcycletypically to the axisof the requires0.1-0.2 secand generates a forceperpendicular axoneme(the ciliarycore).(A,courtesyof C.J.Brokaw.)

Figure 16-79 Effect on the heart of a subtle mutation in cardiacmyosin.left, normal heartfrom a 6-dayold mouse pup.Right,heart from a pup with a point mutation in both copiesof its cardiac myosingene,changingArg 403to Gln. Thearrowsindicatethe atria.In the heart from the pup with the cardiacmyosin mutation,both atriaare greatlyenlarged (hypertrophic), and the micedie withina few weeksof birth. (FromD. Fatkinet al., '103:1471999.With J. CIin.lnvest. , permissionfrom The Rockefeller UniversityPress.)

r032 Chapter16:TheCytoskeleton o u t e r d y n e i na r m r a d i a ls p o k e i n n e rs h e a t h c e n t r a ls i n g l e t microtubule

p l a s m am e m b r a n e i n n e r d y n e i na r m 1 0 0n m

(B) A microtubule B microtubule o u t e r d o u b l e tm i c r o t u b u l e

a ring around a pair of single microtubules (Figure r6-sr). Almost all forms of eucaryotic flagella and cilia (from protozoans to humans) have this characteristic arrangement. The microtubules extend continuously for the length of the axoneme, which can be f 0-200 pm. At regular positions along the length of the microtubules, accessoryproteins cross-link the microtubules together. Molecules of ciliary dynein form bridges between the neighboring doublet microtubules around the circumference of the axoneme (Figure f 6-S2). \fhen the motor domain of this dynein is activated, the dynein molecules attached to one microtubule doublet (seeFigure 16-64) attempt to walk along the adjacent microtubule doublet, tending to force the adjacent doublets to slide relative to one another, much as actin thin filaments slide during muscle contraction. However, the presence of other links between the microtubule doublets prevents this sliding, and the dynein force is instead converted into a bending motion (Figure 16-83). The length of flagella is carefully regulared. If one of the two flagella on a chlamydomonas cell is amputated, the remaining one will transiently shrink as the stump regrows until they reach the same length, and then the two shortened flagella will continue to elongate until both are as long as they were on the unperturbed cell. New flagellar components including tubulin and dynein are incorporated into the growing flagella at the distal tips. Thus, even in these

(A)

50 nm

Figure16-81The arrangementof microtubulesin a flagellumor cilium. (A)Electronmicrographof the flagellum of a green-algacell (Chlamydomonas) shownin crosssection,illustrating the "9 + 2" arrangement distinctive of (B)Diagramof the partsof microtubules. a flagellumor cilium.Thevarious projections from the microtubules link the microtubules togetherand occurat regularintervalsalongthe lengthof the axoneme.(A,courtesyof LewisTilney.)

1 0 0n m

Figure16-82 Ciliarydynein.Ciliary(axonemal) dyneinis a largeproteinassembly(nearly2 milliondaltons)composedof 9-12 polypeptidechains,the largestof which is the heavychainof morethan 500,000daltons.(A)The heavychainsform the majorportionof the globularheadand stemdomains,and manyof the smallerchainsareclusteredaroundthe baseof the stem.Thereare two headsin the outer dynein in metazoans,but three headsin protozoa,eachformed from their own heavychain(seeFigure16-598 for a view of an isolatedmolecule). Thetail of the moleculebindstightlyto an A microtubulein an ATP-|ndependent manner,whilethe largeglobularheadshavean ATP-dependent bindingsitefor a B microtubule(seeFigure16-81).Whenthe headshydrolyzetheir boundATP, they movetowardthe minusend of the B microtubule, therebyproducinga slidingforcebetweenthe adjacentmicrotubuledoubletsin a ciliumor flagellum.For details,seeFigure16-64.(B)Freeze-etch electronmicrographof a cilium showingthe dynein armsprojectingat regutar intervalsfrom the doubletmicrotubules. (8,courtesyof JohnHeuser.)

tuu001 (8)

'(IS-ZI eJnBrCees)usrueqJaru snorJels.^.ru e ^q eJnl ruls -cnr1spur8rro aql o] relncrpuedrad paurrogsr ralq8nep Jellelus p qJIq ^ uI 'ssaJ -ord uoqecqdar snorrnc e ,tq asue sarpoqIESBqpup salorJtuacrvrall'sa1odalputds aql;o ged aruof,aqpue JorralurIIaJ eql olul eloru selpoq IESeqaqt pue 'paqJosaJ ('I)u!l'M'u pue Morpoo^ l'c are ella8eg aq] 'aldurexa to!\auoraopfunpp e31e replaclun eqt uI slso]tru ; o { s a l r n o t ' V ) ' ( t € - 9 te l n 6 u aas)aLuesaql ,(;;erluessa st aloulua) qcea Surrnp :alqrualuoJJalur d1puo1tf,uryaJe salorJluef,pue sepoq Ipseq 'sursr p ur salnqnlor)rlu;olueua6uellpeq1 -ue8ro aruos ur 'paapul '(t8{I arntlg; IaaqMIeJ e ur pa8uerre salnqnloJJrru 'raqlabolsalnqnlor)rur 1o,{erelerupur;I> pasnJJo sdnor8 auru qllm 'saurosoJluaolprurueJo Je]ual eq] lB pappequa 1a1drr1 eqt ploq lPq] s)iurluilo] (€ urpal punoJ aJeleql salorJluar eql se urroJ eluEseq] e^Eq sarpoq IesBqeqJ'43€JJnsIIac ur uMoqs)suralordraqlo'(selnqnlol)tru aql te ela8eg pue prln crtortrecna loor l.1urg salpoq psoq pelll.l; seJntf,nJls alalduo)ul ) pup B aLlt)salnqntoj)rLu oMl ol pasnj(alnqnlor)rur ]uaprJlE IeJrJolsrqaleunuoJun ue V oq]) alnqnlor)rnrelaldtuotauo 6urureluot sr snleredde Surunumrs 5o sadfi lueJaJJrp.{ran o,rnleseql elouep ol arueu arups galdr.r1 laldu] q)ea'salnqnlorrrlu aq] Jo asn aqJ '(IZ-9I arn8rg aas) IIe^i\IIaf, IerrelJpq aql ur pappequa roloru Stpup aLuauoxe JOSlaSAU|U JOpaSOOUIO) ,fuelor prcads e dq uanrrp 'sralladord a{q a}etor ela8eg aq; 'urqa8eg ura}ord fterp>e;o uorltodramo;aql sultoJ aqt Jo strunqns Surleadar ;o dn aperu 'sluauelrJ IBcIIaq p€rr '3uo1 an a77aBa7l {poq ;eseqq)el'apts aq} LuoiJpaMor^ 'leeq ro aAeAA lou op pup urauLp Jo sepqruorJrur ureluoo lou Ipoq ;eseqe;o r"uel6er6 Iauapaq'pealsul (g)'ueozoloid 'ela8eg pallpJ seJnlJnJlsecEJJns op eseql Sursn tur^ s osp EIJelf,Eg P xauo) aql ut satpoq 1nq aelql 1ac Jo leseq q6norqtuolt)asssor)e;o qde.r6o.r>rr.u Q7 :e1deq1 uorl)all (V)'salpoqlesegtrg-91arn6r1 ur pessnJsrp)luaurdolanap cruo^,fuquadlrea Suunp dpoq aqtJo srxelq8u-ga1 aqr ur slJaJeppup 'puelJeq puE srJqeplualJ ol IreJlpql ]ce.ufuole.r Jo uoDEulr.uralep -rdsar paz.{.pred aql ur erlrc o} 3uu,ro suorlcaJur o1,fillqpdacsns q8rq e aql Suny 'rurads pazrreppJeqc elqorurur ot anp ,{}mrals apru,{q sr aurorpuf.s eql'eruoJp -u,r(s s,raua8eue) fre1gc ur ,fuelrparaq 'suerunq uI esneJ uraur{p sloaJap 'acuarradxa,{aq1sa8ueqc aql o1 ,{1pcrueu,{.p pue ,r(prder puodsar o} uo}ala{so}b aq} yo ftqrqeldepe pue ,Qr -11qFaUJrsurJ]uraq] asn s11ac's.{erreJotoru-tuaruelrJalqpts pue palapro ,(UBU 9NrCN3€

l'rn8n10u)rn srsnv) N E N A C: t / ! n t ] l 9 V ' l J .]VIAUON NI

(8)

9NtCtls3'lnSntou)r1 sl)ncoud NENAC:S3'tnSnrOU)ttA (v) rl]gnoc c3rv]ost Nt

t 'gg-91ern613 ur uaas sp'suorloru6urleaqJosaneM6urleatt 'uollotu6urpuaqe sasne)atoJataql uort)eloloru aql'ralqnopaqrJo 6utp1;s aql luanardslur;uralordalqrxap '(ulradse ur se Ll)ns)auauoxp l)elut up u1(g)'rredreqlo aqt lsurp6esa;nqnlorrru ]alqnopJo rredauo eprlsol speaq urau{peqt;o uot})eJoloulaql smollp d l v r o u o r ] r p p ae q l ' a s p )s t q lu l ' u a l o l q areraqla6olsalnqnlol)tru]elqnop 6 u u o q q 6 r e6uu r p l o qs a 6 e 1 uer 1q l ' u r s d f t t aLu^zuotr1{;oelordeq} o} pasodxa are sauauoxe uaLlM(v) 'atueuoxP ue 1o 6urpuaqaq1 gg-9 1 arn6r3

rrrf cNVNorrrrxsol^)IHI uorAVHrS

E€OI

'(98{I erntlg) a1purdsrltol1ru relodrq parueleq rtpuu8alaeq} sl tFsar IEUIJ aql'spua snulx epqnloJJnu asaql asrue8Jopup atealrnu o1dlaq leql seurosoJl -ueo rred e ^,(qpaprnB eJesasseooJdeseql 'sllaf,IEurIuElsoru uI 'sa1odalpurds Jo eqt ruroJ o1 raqlaSo] spua snunu eFqnloJorru eqt Jaqtp8 leql suralord ploJJpcs qlyvr saxalduroc crrauro8qo urJoJ tl-ulseuq pue utaudp cturseldoldc sJoloru pelJeJrp pue-snuru aql ,(1pu1g 'ssEru a(uosoruoJqc eq] ruo4 deme spua snul{u rtaql saqsnd pue salnqnloJJru palproosse-eruosot[orqf,Jo spua snld eq] pJelv\o] S)IEI\/\'t-ursauDl 'suup eruosouroJq3 ol punoq sr ]Bql urseuDl raqloue 'lxag 'Jaqlo qJpe uror; de,,vresuorlJerrp alrsoddo ur poluarJo erc l€ql sepqnloJclu seprTspue Lern lalpred e olur saFqnlorouu eql sepunq (gg-gl arn8tg aas) g-urseuDlrelodrq eql ']sJIC'sseru elqnloJcllu pezlue8rostp aq] uroq alpurds re1 'suorl -odrq luaraqoJ e plmq surelord Joloru IBJeAes Jo suottJe peluurprooc eql -oalrp ruopuBr ur Surlurod uqeurorqf, pasuepuoo uo elquesse selnqnloJf,Ilu sV'sauosouoJqo pasuapuoo aql punoJp suor8ar aql ul paJuequa aJu.,(yqurasse pue uoueelonu alnqntorcrl4l 'ctueudp aJou qcnru pue railoqs eJB salnqnl -oJJnu aq] srsolru Suunp lnq 'seqdoqseler eJer o8rapun pue 3uo1 ,!1ecgd,1 eJesepqnlorcnu aq].{erre aseqdralur eq} uI 'sepqn}orJrru aqrlo qfual a8erane pue Jorleqaq clueufp aq1 ug sa8ueqJ Jr]euerp eJe eJeql 'sIsoJILu,{1reau1 'Suporlpred euosoruoJqc alqBIIaJeJnsue o1 aceld ur srusrueqJau dnlceq aJe aJaq] 'uospal Lue ro; slleJ sluslueqf,elu 'dcuepunpar;o aar8ap snopuerueJlE eJnleal alputds Jllot$r Jo ]es euo Jr leql os aql yo SuruollJunJ pue uoucnJlsuoc aql 'lelJelpur cpaua8 aql Jo uolsslusueJl ur ,!1gqe11ar;o ecuegodur lErluar eql Jo esnEJeg 'sllar ralq8nep o^ l eql ol ,fi[apg 1ca;radqlp\ seuosoruorqc paleclldar aqt Sulre8ar8asro3 alqtsuodsar st teq] ernlcnrts alpurds relodlq eqt uroJ o1pazrue8roerpue pelquesseslp L1a1a1d -ruoc sr sepqnloJJlu;o ,(erru aseqdralur eqt 's[ec IPuruP ul Jnoq uB uel{} ssel se1e1d1ecld.,! ]eql slue^e Jo acuanbas prdur e uI 'sa(uosoruoJqo eql Jo uoped1c11 -red anrlce eqt sarnteeJ osle 1I 'sluauplrJ playa>1so1.{c cnueurtp qll&\ SullJeJelul suralord Joloru Jo srueel dq uoqezrue8ro-;1asyo raarrodaql;o aldruexa Surluurc -seJ pue luelrodrur l.1re1nc4rede sr alpurds ollollu aq] Jo uollJnJlsuoo eqJ alpurds Jllolru eql Jo uorlf,unJ IeJrupqoelu pue uollJnJls -uoJ aql ol elnquluoJ teql suslupqJar.u1e1a1a1soilc aqlJo ^ aJe ssnlslp fgauq a,vr'eJeH 'uorsrlrp IIac Jo slua^a aql go 3unur1 eqt seulurelap lpql ualsd.s loJluoc alcri.c aq] puE srsolrru;o ssacord eq] qloq lelap uI eqlJf,sepily!\ a^\ 'Ll rcl 1ac -duq3 uI 'uorsrlrp 1ac Surrnp uroJ leql 3uu aplcerluoo eql pue ayputdsJIlolIu aq] aJEeseql Jo JerlnupJlsoru aqJ 'sqof rraql alaldruoc ,taql acuo readdesrp uaql pue spu8ls luuJelxa ol asuodsar ur ro alcdc IIaJ al{l 3o sa8els cgtcads le readde 'luerualoru a,ulrladar leql seJruJnrls elrqel uo puadap slueruenoru IIaf, ]soru ]ng acnpord o1 pazqercads seJnlJnJls ]uauerurad [1anpe1araJEBIIIo pue sprqgor(7,q

suleloldrolow ^ue6lJo suoll)PJatulaql pue s)lueu/(c elnqnlor)!wselrnbauelpulds)!lol!w aql ro uoll)nrlsuo) 'U7-ZZ arn8rg aas) ofrqrua 3urdo1a,r.apaql u1 drtaruur,{se lq8rr-ga1 8ur -qs[qetse rog parmbar sr leeq o] alqu Sutaq ut pnsnun sI leql tunlllf, dreurud;o puDI pazrTerf,ads raqlouy'arnle;,taupo1 ur ztlunluala pue sue8ro aqt lnoq8norql s1s,b palg-prng a8rel Jo uolteluroJ aql u1 Suppsar-slleo pqaqlrda daupr4 aql 'eseeslpdaupq cp Jo uorleJaJllordranosasneJ leql JapJoslpcpaua8 uoluruol e -s,{c[1od sesneos11ac.{aupq aql q runlllf, ,{-rerupd eqt Jo s}ueuoduroc prntcnr}s Jeqlo Jo IeuuEqJ runrJlpo eql Jo sso'I 'uoqeraylord puu qu,rorS lac ,(aupq 8ur -lep8ar 'erlc Lrerurrd aql spueq ^ oU pmg eql ueqm pauado eJEslauueqc runlc -leJ enrlrsuesoueqralN'sa1nqn1faupH eq] q8norqt plngJo tuetuelolu eqt ]Jetep 'rualsrts snorr '.slleJ l€ql sJosuas molJ se ]Je erlrc dmutrd lellaqlrda ,i.aupq ug -Jau ler]uao eql ur suoJnau uo sJelllrusueJ]oJneuro; sroldacal pue '(096 'd aasl ,{e,u.qled Sulpu8rs Soqa8pag eql uI pa lonug suralord Sutpnlcut 'runrpc dletu '(s1ac -ud r(ue6 a8eggec) saldcorp pelpJluacuof, aJe sutalord Suueu8ts aq] ur 'slselqorqg Surpnlcur sa&! 'slleJ 'sllal -uoqo pup auoq'suoJnau 1ac Iellaqllda 'urau.{p luareJJrpdueru;o acpJrns eqt uo punoJ eJEdaql {Jel pue r{r8ua1uI sreta -ruorcru,ua; e;tpo dlensn eJEerlroLrerutrd 'unryn fnruud e peflec uoucalord eceJrns qJrr-eFqnloJlrur 'elrlour-uou e yo qu,ror8 aql eleelJnu ,tlluanbaq salo -rrtual 'e11a8egro ErlrcSurluaq padola,lep rtlnl 1ce1letll slleJ IeIuIue uI uelg uole;a1soy(1eql:9 L raldeql

t€0 t

(rar^asll r.uolJ uolsslLurao qllM'0002'g0su:0t 'ptg'iln) 'resa6'V rrrorJ') lssald{lrslenrul rollalallou aqf uot, uorssruladq1111 '096t'S9€-S9E:99'tolg ilD'1'11eba5'g'g pue uourles'c'3Luolr'B)'sauosoulolr{) aql ol salnqntol)tur orll t)auuo) leql /saloq)olautl aql soint)nrls pezr;er>ads lJeLuslodspal aqL'aryqut sarrosoutolq) aqt pue uaat6uga;purdseql Jo salnqnlol)r.uaql smoqsqde.r6otrtr,u a)ua)saronU slql ()) tofenbae;purds aql le paubr;esaulosoruolq) aqt qllM 'eseqdelau elputds)tloltul palelosl le ue 1o qderbot>trlllseltuo)-aseqdV (g) 'se;oda;purdsaql pJemolxng snonutluo) o6rapunqloq solnqnlol:rurle;odtalur pue salnqnlot)tulaloq)olaut)aqt apqm'spuasn;dtraqlle 6urluuqspue 6uranoi6'I1r;rqelsur :rueu,{po6lapun salnqnlolfturlellseaqt'smolreaql ,{q palp)rputsy',(enne6ulDa[ordspuasn;d raql pue'aprsarsauosollua)aql aleqM sa;oda;purdsaqt le spuasnutulltoql qllM polualroaJesalnqnlol)ilJloql lo llv'xauo) lla) aql qllM ])elatutue) leqt (uaaJ6) salnqnlol)tutletlsepue'raqta6ot alpurdsaqt Jo sa^leqoMl aql ploq fpqf (pal)salnqnlol)trule;odtalur'a;od elpurdsaql ol aulosoulolq)q)ea q)eue teql (anlg)solnqnlor)ruraroq)olaurl :aseqdptau1ea;putdstr1o1rr"u aql u! salnqnlor)rru )tueu^p Jo sassel) oerql are araqf (v) .slla) leul|ue ur e;purds)llorlu aql Sg-91arnbrl

'slueuoduoc SurzrueBroJeqlo pue sJoloru JElncelolu Jo suezop Jo suor]rE lEnpr^rpur aq] ql.rM selnqnloJsrru Jo saF -radord crueur(p aq] Sururquroc uoJJa eArlBJoqBIIoJe sluasardar alpurds JIlolIu eq] IIeJa O 'er8ual slr seurruJalap osp alpurds eql uI senrlrlce uralord Joloru 'zI reldeq3 ur passnssrp aq 30 saffi o1\^lesaql ueervuaqasuPlBq elEf,llep eq] IIIM sV'(98-gI arn8rg aas)pede ruaqt Surqsnd,tpuetsuoc ere teqt sapqnlorclru relodralur eql uo sJoloru g-urseuDlpatcalrp-pua snyd reyodrq aql pue 'selnqnl -oJorru aql q Suqaar ,(ltuelsuoc ere leql alod alpurds aql lE suralord roloru palcaJrp-pua snurru Jo uorlse aqr rtq ue^rJp sl xnu pJe^ elod 'a1od alpurds aql Jeau spue snurru Jreql lE ssol leu e Surcuepq 'spua snld rraql lp slpnqns uqnqnl 'xnp[ ptnmalod palec Jor eqaq e tlqlqxa sapqruoJJrrx Jo uonrppp tau e qlrm alpurds aql Jo ,(ueur 'aldurexa JoJ 'asuqdeuu u1 alerudas o1 u€aq ,i.luappns sauosoruoJqJ eq] ueq a ur8aq p^u. leql uorlf,p JoJ pasuel 'eJnlJnJls cnueufp ,tlaruarlxa ue ,tlenpe sr alpurds eq] esneJaq 'a,rpdacap sr acueruadde .{pea1s qql '(IZ ralduq3 aas) a1c[c IIaJ eql Jo uorssarSordaq] sraS8rr]uoqezlrueJ Irtun sqluoru ro sr(ep roJ slreM alpurds eql pue 'aseqdelaru cllorelu ur a1e,{.cIIaJ str slseJJu33a pazqrga;un aql 'slprurue Lueur u1 'eurl Jo sporrad 3uo1 rog luacsarnb pue alqets ruadde upJ lr 'pelquesse seq alpurds JIlolFu relodrq eq] JaUV

atrJosollue)

uri 0t

s a l n q n l o r l r uar J o q ) o l a u r ) s a l n q n l o J ) r ul e i rlse

+_)

ourosolluel

990r

UOIAVHIS lll)

CNVNol:lll)Sol-ll lHI

'pa) uMoqsst utl)e ur le)tuo) paztjalri^lod Ilnnaueq1'Ilqloorls premloJlla) aql 6utnou 'peteulproo),{lfq6lf aq uet sda}s esrrndals e ur lle (lenrleurallv'uotLlseJ 'paleadaiaq ue> preMroJ11araql 6urnou.r ol)I) eues aqf 'plemloj slMet)lla) aql sel)eq aqt le pelqurassestp ale souoplo pue'tuor] aqt te opeurare sl)eluo) le)oj MAN'(uorl)ejl)uorsuelaqlJo outosxelal ol ())pq lp Morreuaa;6)premro;;;at aql 1o,(poqeql s;adordlla) oqr Jo lear aql le uorl)erluo]'xauo) utl)e aql saq)lalls pue (luorJle sttotrc uaatdlpleMtoJ abpaaql sa^ou llar aqrro a6pe6urpea; aql le unlpodlllaLuel e,o luauiq)elle uuU pue uorsnllo.rdguapuadap -uollezuoullod-urt)e oql'pJeMroJlla) e elour xauo) r,l)!r-ull)paqt u! poleJeuab so)roJMoq ,o lapout y 9g-9 1 arn6r1

11urso{tu urpe pezr:eu{;odun ;o luatuanout

NOtSnUtOUdffi urnlpoorllauJel puasnld sapnJtoJd 1e uorlezuaur{1odurpe

uorsualrepun xeuol

l l

urnlerlsqns u r n r p o o r l l o u r e l

xeuo) urpe

sE qJns 's1ac SuqareJ3eruos uI '(98{I aJntIJ) pJElv\JoJrrx\eJp sI urseldoli.c 8uI 'uolpaq pu? llurueJ}sqns eql 01eueJqlualu PIusBId -lleJ] eqlJo {lnq aql qcrqM ur aql ssorJe s]3euuo3 uolala{solb ur}Je eql qslq ^ ul 'luau1q30110'.lleJ eql Jo 'uolsnJrcJd :pa,rlonut luo4 eq1 1B]no paqsnd are sarnlrnJls qJIJ-uIt3e qJlqo\ uI aJe sarlr^r]rE ]Jurlslp aeJqJ 'euEJqruau Pluseld aql qleauaq xailoJ tIcIJ-uIlJE eql uo luepuedep 'sseJotd peleJ8alul xaldruo3 {UBU e sI 8u!l ^eJOIIaD 'sasElsPle|.u ruJoJot,{poq aql ur selrs Jo slesse^ poolq olul IMEJJpue senssp Jeqlo le a8rerua uaql pu€ slesse^ qdur,ri.1 Surroqq8rau apelur Jounl ,{rerutrd e uI slleJ uaqm 'sJeJuEJ,{ueru uI eloJ E seq osp SurTMeJclac ',{laleunuoJun 'snlll eql go dr1 aql 1" lsol slle3 e^ltdrosqe 8ur -Jeldar 'rTIrA Ipuusatul eqt Jo saprs eq] dn 1aner1eunsalul aqt Jo Sulull lBlleqllda eql ur sllal aql 'uorssaJord parapro ue uI 'ftnful Jo sells lE seJn]JnJlspeSBluBp (senssll allpeuuoc plrnqal o1 Surdlaq pue LJessef,euaJeqo\ ueq] SulaporueJ q8norql elEJBru uec slselqoJqrgKlrepurrS'1e^eueJpue SuqepoueJ euoqJo ssaJ -ord snonurluor e ur 'ureq] ragp ,vrolloJteqt s]$lqoelso eql ,tq ut pa1pl ale teqt slauuBqJ Sulurro; 'euoq olur Ieuunl slsBlJoelso 'esuodseJaunlurul el?uul eql Jo UBd leorlrJ3e se sJepe^uru8raro;;p8ua puP uollJeJulJo sells ol p\EJf,slqdorl -neu pue se8eqdoJcelN 'q1ac Sugrr,reJJqlIM seqlaes osp PIuluB llnpe aqJ 'ri.u,uaql Suop xuleru JelnllaouJxe pue seJBJ -rns IIeo ot punoq spu8rs pup slEuBIselqnlosJo suolteulqruoc dq paprnB 'sla8rel Jrldeu^s Ipnluale Jreql ol IeAeJlsuoxe Surdolanap 3o sdp 8ulouu^pe eql l3 sauoo q] \oJBq3rJ-url3E aql ]Bq] /(elv\slql q sI 1I :Iualsds sno^Jeu eJnua aqlJo uollJnns -uoJ aql ol plueuepun; st Suptero e3uPtslp-8uo'J'ecBJ eqlJo seJnpnJls snoIJEA puu dJosuas 's1ac u11s Suturocaq pue 'eq8 pue Jlteqledurds suorneu luaur8rd 'saleJ esJelrp a^Er{ sllal eseqJ 'orfuqrua aq} }noq8noJql sa}IS ;o .Qeuel e o1 aqnl luJneu aql ur u13uo Jo alls JIaq] uro4 suoper8nu acuelstp-3uol Jlaql roJ alqe -{JurueJ ere s11ai$aJJ IUJnau 'seleJqegal uI '(tZ Je}deq3 uI pessnf,slp) sleeqs pI -aqllda aloqmJo slua(ualour paleulpJooJ aqt.{q puu suol}EJol IaBJE}JIJIsadsol (slseu sflac lpnprlrpur Jo suor]eJ8rru aq] r{q palearc sI Ieruluu uE Jo eJnlonJls eq} -a3o,{rqura Surrng 'urrads Suruula,rs;o uotldacxa elqetou aq} qllrlt '3uq,r,rercfq sJnJf,ouoporuoJol IIaJ IIe lsotup 'sletulue uI 'ral€,!\puodSo dorp e ut salella8eg pue selerlrJJallBr.rrs Jno^ap pue {JEllu ol pa^Jasqoaq ,tlsea uec ,t[aqlpue 'pooJ 'Ixu s ol elaBeg Jo PIIIs Jo qJJees ur z(lsnonulluoc I ^BJJ aeqeoluu drolepar4 'su1a1ordJo}ou 3ursn,{q uuql Jeqlpr saJEJJnsJaAoSulllvreJcr(q arrour slac duelX pue 'sJoleln8ar luaruelg 'sluaurpllJ lelala{so}^c 3o uo4er8elur crueu{.p aql alercardde uEJ al alaq \ eouelsur Jeqloue saprnordSupterc 1ac 3o ssacordaq1

urnlertsqnsp!lose ssol)v l/v\er)ue) slle) {ue6; uola;a1soy(1eql:9 [ raldeL]f

9€0 t

('ael la!lnrlo {seunol 'B pue 'snal)nuoql ot asol)suor6er V) aql ol pat)ulse) de (anlq)sluaurelu alerpauJalurpue (uail6) salnqnlol)tW 'luaura^our proer Ellar aql roj alqrsuodsor arepue tunrpodrllarue; a6relaqt lll, (pa4sluaueluu!l)v'lla) slql u! sluouelu lelololsoil) Jo uotlnqulsto ()) lear aql le unlerlsqns aql anoqedn peu.rer 'snel)nu aql 6u;pnpul [poq 1;erlleus pue unrpodr;;aLuel teg'peolqs1r6urrnoqs 'Ido>sooru uorl)ala 6uruuelsIq uees ay(rolerey(g)')as/uli SI lnoqe le 6urnou sr llo) srql'ueoe)as g I lnoqe ualel 'aJnlln)ur a1r{ro1eta1 e;o sqdeloolrru lq6!l (V) 'srureprde qsrl e uro,r;selI>o1ere1{ro1er6r1111 2g-9 1 arn6r1

sIeeAeJslql 'elEJ su Sururuexe pue urlsP Jo q3led IlEurs B SuolrBur ,{.qerpodq -leurpl eil.Jol?re{ ur parpnls eq uBJ s}ueuBIU urlJeJo roFeqaq JIIueuIp eqJ 'saptcoleral ,tuq a41 3uryoo1 'salaue8ro pasolcua-eueJqluau pue salnqnt 'r{.1purou IAerJ ol enur}uoJ r(aq} -oJrrru 4cu1,i.lpraua8 sluaur8ur; eqf q8noql[V 'elladrdorcFu e qfl1v\JJo peJIIS eq ueJ unrpodqlauel slqf Jo sluau8erg '(Z8-9I arntlg) Iunlerlsqns aql o] peqJBltE 'llElus e pue unrpodileuEl a8rel drarr e qlpvr adeqs lou sr lEql ,i.poq 1ac Sumer] a^rlJurlslp 3 arunsss sa1,{co1eral 'sllal lenpr^rpur s3 peJnllnc ual{]vUuluTuri gg o1 dn saleJ tE Surnour ,(1p1derdran spunon esoll ol pazrprcads are daql pue (leeqs lelleqlrda ue Sunurog dq purruu eql reloJ,tlprurou sllaf, esaql'slueIuBIIJ up€Je{ luepunqe JraqlJo asnecaqsaT/-cownq sErrmou{ eJEqJIqM's8or; pue qs.r; ;o srurraprda eqt Jo slleo pllaqlrda aqt q papnls larvr dlercadsa uaaq aleq daql ,ir111lourIIaJ roJ parmbar sr leql d:aurqreur eql Jo IIe uruluoJ utpodqlaurul 'erpodopnasd ro erpodolrJ Jer{}le JoJaABqe,rrrueql erpodllauelJo rusrueqcaru uorsnrlord pue uorlezrue8ro cnueudp eql lnoqe uon -Er.uJoJuraJou e^eq etrlr'adoJsoJcrul 1q311aql qlyv\ uorleurluexe JoJ luerue^uoc '1aB luauug ]soru sr drlauroa8 puorsuaurp-oml rlaql esnuJeq sduqra4 -ultJe ue qllm pellg suopcaford leuorsuaurp-aarq1 ,tqqnls are 'qtqdonneu pue epqaorup,(q paurrol 'ugpodopnasd 'runleJtsqns pilos aql 01 lallEr?d aueld e ur ell qJrq ^ Jo tsoru 'slueruelu ullop Jo qseur pa{uI-ssorc dleuoSoquo uE ululuoo 'leuorsueurp-oml aJu (suoJneu eruos ,tq sE se ,{aq; 'sarnlcnJ}s e{rl-leeqs IIel 'slselqorqg pue slleJ plaqrlda dq paurro; 'ugpod;1arue1 'cnueu,(p eroru sp lla,r,r sE teuulql pue ra8uol ]nq illr oJJrru ur asoql Jo luacsrur{ueJ eru r{Jrq ^ 'slua{u -BIUurlJe pepunq '3uo13oeJoJE ureluoc r(aq1 'leuorsuaurp-auo rtlprluassa eJe 'sl$lqoJquJo pue qyroJ8 sadrb Surlur8rur ri.qperuJoJ 'u;podogg auos sauoo 'suralord pelErJosse-urlJB tueJeJJrp;o acuasard eql uorJ sllnser slql moq passnJsrp ,(puarp eleq er\ pue -rtlarrrpadsal 'suorsuaurp eaJq] Jo 'oml 'euo ur-pezlue8ro sr urlJp eql qolqm ur ^,teivraqt u1 ,tlguurrrd ragrp saJnlonJls eeJql eqJ 'sallaue8ro pesoloue-aueJq 'elp -Iuelu sapnlsxa qsrqm 'ullJu snoluauelg Jo aloc asuep E qlyv\ pellrJ eJu IIV -odopnasd pue 'elpodq1aue1'(salldsoJJlrx sB rr ^ou{ osp) epodolg Supnlcur 'saJnlcnJls anrsnrlord go sad,fi 'prumlno lueJeJJrpaleraua8 sadr! 1ac lueJeJJIC auuJqueu eruseld aql Suqsnd uorlezrraurdlod uure ltq paleraua8 sacro; uo dp -rerurrd r(1aro1 sruees 'a8pa Surpeal E Jo uorsnr1ord 'uorlourorol ur dals IsJIJeql

uolsnrlord euerquaweurseld se^ucuoltezueu{1o6 u;py 'reln8arr pue 61ra[ sruorloruocolaql pue 'luapuadapulaJou ere sep.rllce eseql 'slselqorqrJsBqJns 'sllecJeqlo u1 'adeqs3u€ueqc lnoqlnt r(lqtooruspJeMJoJ apr13ol ueas sllacaql pue 'peteurproocrtlasolceJusarlrlrlJeasaq]'slurJaprdaqsg eql tuoq saldcolera>l LEOL

uorAVHtS rtl) cNVNollllxsolD tHI

(ssald^ltsla^!unralloJal)oU aql uor} uorssil,lladqlm.666 t '9201-600LtSnL'p!8 'r Xsuo€.D pue ilD eurll!^s f r.uoll)'urluo) qllM paler)osse lou ^llerauabalp a)tDeltuauelu aql ul (url)edCV ueql raqlei)utl)e dfv utpluo) ol ^la)rl]sot! ale leql pup,(;luatarlsotu patilioJa^eqleqt sauoaqt osleaJeq)tqM 'a6pa 6urpee; aqt ol tsasol)sluauelU ult)e eql'v ur a;bueltal a1lq^4 aqt qll/!^ polreu uor6a.r aql;o Mar^dn-asol)(B) 'a6pe6urpeal&an aql punoj te lou sr urlUo)'unrpodr;;aue;aqt q6norql,(em aql lle saq)ear)|lo/v\qsau utl)e asuapaql qbnoqrlv'no11a(rcaddede;lanooml aql araqivrsuor6araql',(poqrrue]ua)saJonlJ e q4uruaatbur palaqelurlqor pue ulplolleqdlua)sotonlJ {q pa; ur pa;aqe1 stuaureluull)e qllM ailrorera>lV (V) 'erpodrl;eure1 u! utltro) 69-9[ ern6rJ

urri0Z urlrlo)pueurpe

'(16-9I erntl0 preMioJ sa^oru ]l sE IIac aq] Jo saprseql ur raqlBS o1Surdlaq 'unrpodllaurEl SuuoruoJol aqlJo seprsaq] ur saqJurd tr puu uorsnJloJd slua^aJd uorlceJluos sIqJ'a3pa 3ur -pEel eq] o] IellEJedlsorulE uolleluelro ue ol aSpeSurpeal eqt ol rPlnJlpuedred dlreau uo4-uorleluerJo ^ au E olur ueqt ilnd pup qem aql uI sluaruellJ ullJe eqr qlF aterJossEot uaas sluaruslrJ II ulsodlx JEIodIq ,(1pu14'(06{I arntlgl urnrpodrllarupl eql ur ssecord Surlgurpearl puollJeJlplun 'luerorJJeuE surel -uleru lBr{1 rusrueqJaru p JoJ slsBq aq} apnord o1 lqSnoql sr ullJe sno}ueIuEIIJ dq srsu(lorpdq41ypa,(e1apeqt 'snql'stuaruelg replo eqt alqruasspslpr{pualcgga upr urlrJof, 'spaacord srs,{loJpdqdIV pue e8p slueuelg eq} sV '(68-9I aJnBIC) urlrJocIq uollEzlreurflodap o] luutsrseJeq pFoqs a8pa Surpualeql ]e petEJeue8 sluauelrJ ruJoJ-J^aeu eql '(urro; c eql) ullcB-dclv Surureluoc slueuPlrJ urlJB o1 ,(lequaragard pue ,{lanperadoof, spurq (ZV-qI ern8rC ees) urqtor asnecag 'a8pa Surpeal aqr purqeq IlalvrpeluJol selrs te srnrro uouezlrerudlodap luaru -PIrJ]sow 'pJB^\JoJeueJquau eurseld eql qsnd ol uoltEf,ol rEql ul zi.preurtrd Surrrncco ewor8 ]uauEIrJ urtoe ^ eu qlyv\'e8pa Surpeal eql lE pazlpJol sI uollu -elonu luauelrc 'sJolJEJ IeJaAesJo uorleJ8elur lef,rueqoarupuu uoqeradooo aqt eJInbaJol ]q8noqt s1erpodrqaurul.{q uorlou lPuol]f,erlplunJo aJuPueluIeIN 'surruJoJ ,(q paleurprooc f.yqrssod ,{lqruassesrp pue .,{yqruasse}uaIuEIIJ ullJp peleu 'xaydruoc -rpJoocsarrnbar dUV aql uo puadap lou seop ]eql uslueqJalu ilps ]nq p asn ot readde slselqorqrJ Surpnlcur slac Sunoru-.{.pro1saurog 'a8pa Surpual eql Jo uorsnrlo;d aq] alrJp ol sluaIuEIU ullJe Jltupur{.p asn UEJ slleJ leqt sderr IEJaAasJo euo dpo sr xalduoc dUV eqr f,q l11nqqem cltlrpuep B Jo SuHIIIupEe{ '(tf-gt pue passncsp stuauPIIJ ullf,E seFqnloJJIIu arn8rg aas) flsnonard 'IJEq eql le Suqquras Ienpr^pu ur sJncJo leql SuUIrupBaD eql Jo lueJslurueJ -seslp puB luo4 aql 1e Suqqurasse'Suqpupeal Suro8rapun aq ol sueas eloq ^ B sp qam aql'(gg-9l arn8gg; qa^aleuorsueurp-o^ l aq] IUJoJo1 Surdlaq '(7t-gl arn8rg aas) saxalduoJ dUV ,(q sluauep; urtre reqlo Jo sepls eql ot peqf,elle ,(puanba4 aru spue snurru aqJ 'prumloJ 3utce; spua snyd rleqr q]1lvrpatualro ,(psoru eJp {Jo^aqsau aql ut s}uatuplg ulloe aqJ 'aleJ}sqns aql ol lcadsar qlyvr freuor]e]s urerual s]uaruPlrJur]Je eqt'pJe/vlroJ yrterc etpodqleul?l aql eIq { 'leqt (ssar6r{lgsranrul qrll '666t '9201-600LiSVL'lolg rallaJal)ou aqf urorluotssil.lllact ila)'f '9 pueeur)ll^S tuo4)'(t€-9t alnbHaas)tueLuelg 6utlstxaald Xsrrog f xaldulotdUVaql uaqm e aprsaq] luauelUurl)eMaue soleal)nu Jo JJo paq)uelq paurola;6ue.0l)rlsual)ejeq) aq ue)sluaulellJ aqrqllm'uaas 'g qaM utlte aql 6utpea; aqt snoraunN ur uA oqs a6pa le Josuot6al alouapslaqel eqI paueuraqlJosMar^ seare dn-aso;)()) ') ul pa6le;ue 'lromqsaur 'eilroleral e;o abpe6u;peel 6ulnoqs url)p asuap eql luaurelq uoll)al] (8)'anltletsou.t e ;o qdel6o.t>tLu aqllo er;;darpaMoppr.{s-ulnulteld aqlro luolj aqlleaupaleJluo)uo) sruorleel)nu u!l)earaqM'unrpodrllauel omlaql alaqm Ilq6rqsrxa;dtror6gy aq1'rolalleaddedel.leno lpoqlgueue suor6a.r aql'suralo.rd luauodtuorsl!Joauolsutebepester t4tltrt {q pal uaafiurpalaqplxalduorduv aqrpue'urprol;eqd lua>saron;; u! paloqelsluauielU u!l)eqllmau(toletalV (V)'elPodlllauelu; xe;duro> qa/npueuolleal)nuluoutellJulpy gg-91atnbtl dUVeqr(q uorgeu.ro; uolalalsoy(1 aql:9 t teldeql

8€o t

1039

THECYTOSKELETON AND CELLBEHAVIOR

ts

9q

net filament assembla yt l e a d i n ge d g e

?

&u

$

d

!p d.

rd

$ ql

capprng 0 proTern

d q

t' d

lr

& 6

o

€ a

tnC po

q

The pushing force created by the polymerization of a branched web of actin filaments plays an important role in many cell processes.The polymerization at the plus end can push the plasma membrane outward, as in the example just discussed (see Figure 16-90), or it can propel vesicles or particles through the cell cltoplasm, as in the example of the bacterium Listeria monocytogenesdiscussed in Chapter 24 (seeFigure 24-37 ). Moreover, when anchored in a more complex way to the membrane, the same t)?e of force drives plasma membrane invaginations, as it does during the endocytotic and phagocl'totic processesdiscussedin Chapter 13. It is interesting to compare the organization of the actin-rich lamellipodium to the organization of the microtubule-rich mitotic spindle. In both cases,the cell harnessesand amplifies the intrinsic dynamic behavior of the cltoskeletal filament systems to generate large-scalestructures that determine the behavior of the whole cell. Both structures feature rapid turnover of their constituent cytoskeletal filaments, even though the structures themselves may remain intact at steady state for long periods of time. The leading edge plasma membrane in the lamellipodium fulfills an organizational role analogous to the condensed chromosomes in organizing and stimulating the dynamics of the mitotic spindle. In both cases,molecular motor proteins help to enhance cltoskeletal filament flux and turnover in the large-scale arrays.

Figure 16-91 Contribution of myosin ll to polarizedcell motility' (A)Myosinll bipolarfilamentsbind to actinfilamentsin the dendritic The myosin-driven meshworkand causenetworkcontraction. lamellipodial reorientation of the actinfilamentsin the dendriticmeshworkformsan to generatingthe actinbundlethat recruitsmoremyosinll and contributes forcesrequiredfor retractionof the trailingedgeof the moving contractile can be of a keratocyte cell.(B)A fragmentof the largelamellipodium or from the maincellbody eitherby surgerywith a micropipette separated by treatingthe cellwith certaindrugs.Manyof thesefragmentscontinue asthe organization to moverapidly,with the sameoverallcytoskeletal intact keratocytes. Actin (btue)formsa protrusivemeshworkat the front of the fragment.Myosinll (pink)is gatheredinto a band at the rear'(From A. Verkovskyet al.,Curr.Biol.9:11-20,1999.With permissionfrom Elsevier,)

Figure16-90 A model for protrusion of the actin meshworkat the leadingedge, Two time pointsduringadvanceof the with newly areillustrated, lamellipodium at the latertime structures assembled point shownin a lightercolor.Nucleation is mediatedby the ARPcomplexat the front.Newlynucleatedactinfilamentsare attachedto the sidesof preexisting filaments,primarilyat a 70'angle. elongate,pushingthe plasma Filaments membraneforward becauseof somesort of anchorageof the arraybehind.At a steadyrate,actinfilamentplusends becomecapped.After newly polymerized actinsubunitshydrolyzetheir boundATP in the filamentlattice,the filaments to depolymerization becomesusceptible by cofilin.Thiscyclecausesa spatial separationbetween net filament at the front and net filament assembly at the reat so that the actin disassembly filamentnetworkas a wholecan move forward,eventhoughthe individual with filamentswithin it remainstationary resoectto the substratum.

1040

Chapter16:TheCytoskeleton

Hrc-cH'-cg, /\ HrC-CH CHOH Hzc

CH

il CH

HC

HO

leading edge of cell

(c)

Hzc

20pm

au

CellAdhesionandTractionAllowCellsto PullThemselves Forward Lamellipodia of all cells seem to share a basic, simple type of dynamic organization where actin filament assembly occurs preferentially at the leading edge and actin filament disassembly occurs preferentially at the rear. However, the interactions between the cell and its normal physical environment usually make the situation considerably more complex than for fish keratocFes crawling on a culture dish. Particularly important in locomotion is the intimate crosstalkbetween

tightly to move forward. In these lamellipodia, the same cycle of localized nucleation of new actin filaments, depolymerization of old filaments, and myosindependent contraction continues to operate. But because the leading edge is prevented physically from moving forward, the entire actin mesh moves backward toward the cell body instead, pulled by myosins (Figure 16-92). The adhesion of most cells lies somewhere between these two extremes,and most lamellipodia exhibit some combination of forward actin filament protrusion (like keratocJ,tes)and rearward actin flux (like Ihe Aplysia neurons). As a lamellipodium, filopodium, or pseudopodium extends forward over a substratum, it can form new attachment sites at the cell front that remain stationary as the cell moves forward over them, persisting until the rear of the cell catches up with them. \.A/henan individual lamellipodium fails to adhere to the

(D)

cytochalasin B

Figure 16-92 Rearwardmovement of the actin network in a growth-cone lamellipodium.(A)A growthconefrom a neuronof the seaslugAplysiaiscultured on a highlyadhesivesubstratumand viewed by differential-interferencecontrastmicroscopy. Microtubules and membrane-enclosed organelles are confinedto the bright,rearareaofthe growth cone (to the /eft),while a meshworkof actinfilamentsfillsthe (onthe right).(B)After lamellipodium brieftreatmentwith the drug cytochalasin, whichcapsthe plusendsof actinfilaments(seeTable16-2,p. 988), the actinmeshworkhasdetachedfrom the front edgeof the lamellipodium and (C)At the hasbeenpulledbackward. time point shownin B,the cellwasfixed phalloidin and labeledwith fluorescent to showthe distributionof the actin filaments. Someactinfilamentspersistat the leadingedge,but the regionbehind the leadingedgeis devoidof filaments. Notethe sharpboundaryof the rearward-moving actinmeshwork. (D)Thecomplexcyclicstructureof cytochalasin B.(A-C,courtesyof Paul Forscher.)

Figure16-93 Lamellipodiaand rufflesat the leadingedge of a human fibroblast migratingin culture.Thearow in this scanningelectronmicrographshowsthe directionof cell movement.As the cell movesforward,lamellipodia that failto attachto the substratum aresweot backwardoverthe dorsalsurfaceof the cell,a movementknownas rufflinq. (Courtesy of JulianHeath.)

1041

THECYTOSKELETON AND CELLBEHAVIOR

substratum, it is usually lifted up onto the dorsal surface of the cell and rapidly carried backward as a "ruffle" (Figure 16-93). The attachment sites established at the leading edge serve as anchorage points, which allow the cell to generate traction on the substratum and pull its body forward. Myosin motor proteins, especially myosin II, seem to generate traction forces.In many locomoting cells, myosin II is highly concentrated at the posterior of the cell where it may help to push the cell body forward like toothpaste being squeezed out of a tube from the rear (Figure 16-94; see also Figure 16-91). Dictyostelium amoebae that are deficient in myosin II are able to protrude pseudopodia at normal speeds,but the translocationof their cell body is much slower than that of wild-type amoebae, indicating the importance of myosin II contraction in this part of the cell locomotion cycle. In addition to helping to push the cell body forward, contraction of the actin-rich cortex at the rear of the cell may selectivelyweaken the older adhesive interactions that tend to hold the cell back. Myosin II may also transport cell body components forward over a polarized array of actin filaments. The traction forces generated by locomoting cells exert a significant pull on the substratum (Figure 16-95). In a living animal, most crawling cells move across a semiflexible substratum made of extracellular matrix, which can be deformed and rearranged by these cell forces. In culture, movement of fibroblasts through a gel of collagen fibrils aligns the collagen, generating an organized extracellular matrix that in turn affectsthe shape and direction of locomotion of the fibroblasts within it (Figure 16-96). Conversely, mechanical tension or stretching applied externally to a cell will cause it to assemble stressfibers and focal adhesions, and become more contractile. Although poorly understood, this two-way mechanical interaction between cells and their physical environment is thought to be a primary way that vertebrate tissuesorganize themselves.

Membersof the Rho ProteinFamilyCauseMajor Rearrangements of the Actin Cytoskeleton

5[m Figure16-94The localizationof myosin land myosinll in a normalcrawling Dictyosteliumamoeba.Thiscell was crawlingtowardthe upperright at the time that it wasfixedand labeledwith antibodiesspecificfor two myosin isoforms.Myosin| (green)is mainly to the leadingedgeof restricted pseudopodia at the front of the cell. Myosinll (red)is highestin the posterior, of the actin-richcortex.Contraction cortexat the posteriorof the cellby myosinll may helpto pushthe cellbody of YoshioFukui.) forward.(Courtesy

Cell migration is one example of a process that requires long-distance communication and coordination between one end of a cell and the other. During directed migration, it is important that the front end of the cell remain structurally and functionally distinct from the back end. In addition to driving local mechanical processessuch as protrusion at the front and retraction at the rear, the cltoskeleton is responsible for coordinating cell shape, organization, and mechanical properties from one end of the cell to the other, a distance which is typically several tens of micrometers for animal cells. In many cases,including but not limited to cell migration, large-scalecyoskeletal coordination takes the form of the establishment of cell polarity, where a cell builds different structures with distinct molecular components at the front vs. the back, or at the top vs. the

1 0 0$ m

Figure16-95Adhesivecellsexert tractionforceson the substratum.These havebeenculturedon a very fibroblasts thin sheetof siliconrubber.Attachmentof the cells,followedby contractionof their hascausedthe rubber cytoskeleton, to wrinkle.(FromA.K'Harris, substratum P.Wild and D. Stopak,Science208:177-179' from AAAS.) 1980.With permission

1042

Chapter16:TheCytoskeleton Figure16-96Shaping of theextracellular matrixby cellpulling.This micrograph showsa regionbetween two pieces of embryonic chickheart (tissue explants richin fibroblasts andheartmuscle cells) thatweregrown in culture on a collagen gelfor4 days. A densetractof aligned collagen fibershasformedbetween thetwoexplants, apparently asa resultof fibroblasts (FromD.Stopak tuggingon thecollagen. andA.K.Harris, Dey. press.) Blol.90:383-398, 1982. Withpermission fromAcademic

bottom. Cell locomotion requires an initial polarization of the cell to set it off in a particular direction. Carefully controlled cell polarization processesare also required for oriented cell divisions in tissues and for formation of a coherent, organized multicellular structure. Genetic studies in yeast,flies, and worms have provided most of our current understanding of the molecular basis of cell polarity. The mechanisms that generate cell polarity in vertebrates are only beginning to be explored. In all known cases,however, the c],toskeletonhas a central role, and many of the molecular components have been evolutionarily conserved. For the actin cytoskeleton, diverse cell-surface receptors trigger global structural rearrangementsin responseto external signals.But all of these signals seem to converge inside the cell on a group of closely related monomeric GTPasesthat are members of the Rho protein family-cdc42, Rac, andRho. The same Rho family proteins are also involved in the establishment of many kinds of cell polarity. Like other members of the Rassuperfamily, these Rho proteins act as molecular switches to control cell processesby cycling between an active, GTp-bound state and an inactive, GDP-bound state (seeFigure 3-71). Activation of cdc42 on the plasma membrane triggers actin polymerization and bundling to form either filopodia or shorter cell protrusions called microspikes. Activation of Rac promotes actin polyrnerization at the cell periphery leading to the formation of sheet-like lamellipodial extensions and membrane ruffles, which are actin-rich protrusions on the cell'sdorsal surface (seeFigure l6-93). Activation of Rho promotes both the bundling of actin filaments with myosin II filaments into stress fibers and the clustering of integrins and associatedproteins to form focal contacts (Figure 16-97). These dramatic and complex structural changes occur because each of these three molecular switches has numerous downsueam rarget proteins that affect actin organization and dynamics. actinstaining

a c t i ns t a i n i n g

( A ) Q U I E 5 C E NCTE L L S

(B) Rho ACTIVATION

(C) RacACTIVATION

(D) Cdc42ACTIVATION 20 pm

Figure 16-97 The dramatic effectsof Rac,Rho,and Cdc42on actin organizationin fibroblasts.In eachcase, the actinfilamentshavebeenlabeled with fluorescent phalloidin. (A)Serum-starved fibroblastshaveactin filamentsprimarilyin the cortex,and relativelyfew stressfibers. (B)Microinjectionof a constitutively activatedform of Rhocausesthe raoid assembly of manyprominentstress fibers.(C)Microinjection of a constitutivelyactivatedform of Rac,a closelyrelatedmonomericGTPase, causesthe formationof an enormous lamellipodium that extendsfrom the entirecircumference of the cell. (D)Microinjection of a constitutively activatedform of Cdc42,anotherRho familymember,causesthe protrusionof manylong filopodiaat the cellperiphery. The distinctglobal effectsof thesethree GTPases on the organization of the actin cytoskeletonare mediatedby the actions of dozensof other protein moleculesthat are regulatedby the GTPases. These targetproteinsincludesomeof the variousactin-associated proteinsthat we havediscussedin this chapter.(From A. Hall,Science279:509-514,1998.With permissionfrom AAAS.)

1043

THECYTOSKELETON AND CELLBEHAVIOR

Some key targets of activated Cdc42 are members of the WASp protein family. Human patients deficient in WASp suffer fromWiskott-Aldrich Syndrome, a severe form of immunodeficiency where immune system cells have abnormal actin-based motility and platelets do not form normally. AlthoughWASp itself is expressedonly in blood cells and immune system cells, other family members are expressed ubiquitously that enable activated Cdc42 to enhance actin polymerization. WASp proteins can exist in an inactive folded conformation and an activated open conformation. Association with Cdc42-GTP stabilizes the open form of WASp, enabling it to bind to the ARP complex and strongly enhancing this complex's actin-nucleating activity (seeFigure 16-34). In this way, activation of Cdc42 increasesactin nucleation. Rac-GTP also activates WASp family members, as well as activating the crosslinking activity of the gel-forming protein filamin, and inhibiting the contractile activity of the motor protein myosin II, stabilizing the lamellipodia and inhibiting the formation of contractile stressfibers (Figure l6-98A). Rho-GTP has a very different set of targets. Instead of activating the ARP complex to build actin networks, Rho-GTP turns on formin proteins to construct parallel actin bundles. At the same time, Rho-GTP activatesa protein kinase that indirectly inhibits the activity of cofilin, leading to actin filament stabilization. The same protein kinase inhibits a phosphatase acting on myosin light chains (seeFigure 16-72). The consequent increase in the net amount of myosin light chain phosphorylation increasesthe amount of contractile myosin motor protein activity in the cell, enhancing the formation of tension-dependent structures such as stressfibers (Figure 16-988). In some cell types, Rac-GTPactivatesRho, usuallywith kinetics that are slow compared to Rac'sactivation of the ARP complex. This enables cells to use the Rac pathway to build a new actin structure while subsequently activating the Rho pathway to induce a contractility that builds up tension in this structure. This occurs, for example, during the formation and maturation of cell-cell contacts. As we will explore in more detail below the communication between the Rac and Rho pathways also facilitates maintenance of the large-scaledifferences between the cell front and the cell rear during migration.

Extracellular SignalsCanActivatethe ThreeRhoProteinFamily Members The activation of the monomeric GTPasesRho, Rac, and Cdc42 occurs through an exchange of GTP for a tightly bound GDP molecule, catalyzed by guanine nucleotide exchangefactors (GEFs).Of the 85 GEFsthat have been identified in

e zt>p) {qalaqm dool lreqpaa; anrlrsode aleal) qv"ror6luaue;U pue uorleal)nuurl)e ;ero;eql'6urpurqrol)e16ulteu Jo alls aql le uolleal)nuurl)e le)olot 6urpeal 'xelduor dUVaql sale^tl)e1eq1uralotd ,(1;ue1 d5yg e sete^tl)eZt)p)'slla) uerleuureuJ ur sV'Zt)pl'utalold 6u;pulq -619,(1;ueJoqu aql sate^ll)eurnl ul slr.ll'(Stroldeqf ur passnf,stp) uralo.rd-g )rJauJuloleleqe uoi] ]tunqnsDg punoq-dfg aql Jo uotle!tossrps.re66u1 roloa)er aq] jo uolle^lt)v'auerquiaul eurse;deqt ur loldatel pa;dnor-urelord-g e ol spulq rolre; 6ug1eurelnlla)eltxa aq1'asuodsarto1rel Eulleu lsea{ aql u; Iernqled 6ur;eu6rsaq1 96 1-9 1 arn6r3

'lg-sLiL f prl slorlsllqnduellrur)en urorJuorssrurad'lllM'666 [ 'tDN '(t-9 ernbrl osleaas)]! Ior]sep pue 'tolB "le le rauro A'c'o ruorj) [ il4 urnuat)eqaqr Jlnbuaplnoivr;rqdo.rlnau aql 'altadld Erole6llsa^utup ueql raqler 'aprldedaqt Jo a)rnosaql aroMurnual)eq led e I'(wollog) eplldad s!ql Jo a)rnosaql premolslmer)lla) aql (lleurl '@tpp!w)untpodt;;eue; aql pate)ol st ;1ursolurellDerluol Jo uorlrsodaqt allsoddolear aqt te ^lr.reuJud pue tuntpodt;1euelsltll spualxauaql ]Pq] os uolalalsov(r slr sazr.re;od '(dot) apltdad ll luet)eiueouraq) aql Jo a)rnosaql premol urntpodtllaulPl Mau e spualxar(|)|);nb|;r.ldortnou aql'(tz latdeq) ur passn)slp)ropP^ul u6raro;e;o trnpord aql seeprldedslql sazru6o)arl!qdorlneuueunq eql os 'sanprsarauruo!qlaurpale;Iu.ro3eneqsutelo.td leUal)eq{1u6'eq4-na1-1a61 -;{uro; aprgdadaq} Jo lunoure lleus e 6urleal sr1q6uaq} }e drl eDadldeql 'slxelouraq)pue uollezyelod llqdorgnap 1g1-91 arn6;1 'ursluuqraur rrMoDlun up qSnoJqJ'(96-9I ern8rJ ees) sllnsal uorsnJloJd lerpodl -leluel puB xelduoJ duv aql sale^ll3E ueql f,Bu 'esPdJc J?u eql selu^rl3? urnl uI teql (ed(9'7'g)Id) elnrelolu Suleu8rs paseq-prdl p selpraue8 qrlqM'(s)gld) sesBur)l ,t applsouroqdsoqd salBAIlJE Joldaf,eJ paldnoJ uralord-c sll ol pue '1eu8ls eqt pJemol uols -3q aqlJo Sulpurq '.slleJ JllJeloruaqo JoC ]uelcerDuoruaqJ '(66-9I arn8rg eas) -nrlord e spuelxe lseef Butooruqs aq1 u1 IIaJ SurpuodseJ eq]

sv .rerlJEapassnssrp sesBdlc,i.guel-oqg slJeuouoru eqt uo spuedap esuodser uopuzrretuf.lod urlJp srql 'spue8rl Jreqt purq sroldeceJ eril uaq ^ peteFurlls sl sJotdef,eJaql leeu upf,e Jo uorluzFaurdlod [EJol e [16Jy c11cfcgo acrnos E preo\ot euqeotrru unna$otlJlg Jo srxelotuaqr relrurs eql q pu€ espJ slqt uI qtog '(I0I{I arnS;g) req}o eql snsrel IIer eql Jo epls auo uo saprldad elqlsnJJlpeseqlJo uorlPrlueruor eql q 961dpolo esuaraJJrps lcelep 01 'ir4qe Jreql ^q sta8Jet IErJelJeq o1 paprn8 eJp slrqdoJtneu 'sJoldeJer esaql Sursn '(auruorqlaurlrturrog-1"rqly!\ srsaqtuds ureloJd ur8aq salo,{-recordfpo) suraloJd lerJalJBq ruo4 pa^rJep aJeleqt seppdad pale/ruro;-1ur aqlJo suopeJluaJuos ,/v\ol ,i-rarraql lJelep ol uaql alqpue spqdonnau Jo ef,eJJnsaql uo sulalord roldacag 'uorlJeJur 's\tt1do4nau pallm '.sllaf, poolq elrqM lprra]Jeq Jo eJJnos p prBl!\ol 'e^oqP Jo ssEIJ E Jo luarua^olu srlJuloruaqs aql s1 aldruexa parpnls-llaM euo peqlrosep ,ifIlloru [aJ roJ peJrnbeJ sn]eJeddB eq] Jo uopuzlus8ro eql SurJue -ngu1 ,tq firrelod IIaJ eIBJS-eBJeldn tas ol sulalord ,!nue; oqg eqt Je88ul sleu -3rs puraga araqm es€J SullseJalur dlJelnJrupd u sl slqJ 'leJlrueqo elqrsnJJrpe Jo luarper8 e ,{q pallorluoJ uorlceJrp e ur }uerualou IIec sp peuuep q suulowaqJ

uollerotwlle) ro uoll)arloaql eleplo ue) sleubtsleurelxf ,irpelod IIaJJo luauqslqelse eql ur pa^lo^ur esrma{rl ueryoeJEdaql araqir,r'srusrue8loJar{}our sSolouroqaleq ol punoJ uaaq eAEq suraloJdesaqlJofueur 'lseef ur uortuogltueprJIeq]ol ]uanbasqng'sluaruuadxa uorlezr:au{;od u!pe

II I

, 6 u r o o u l q sp, u e s r s o l ^ ) o x ap a z u e ; o d

t

uorlPuro] a l q e )u r p e

t

urulJo,

I

ureloro 9 pale^rpe

\

rope,r6urleur

5?0t

O

uor^VHrs trl) cNVNoIIT)solAf lHr

't-g arn6rl t aas'uJatsls6ur;eu6rsstql Jo ssaualtt)a#a aqt Jo alourexaaJtl-leale lol 'luauranolrlpsl)altp 6urlsrsse pue leal 'ller lla) aql Jo uor])ertuo)6ultelnLulls aql,o rearaLl]le Iluo palentl)est oqu 'llo) aql Jo luor1aql le alrl)e sr uorsnllo.rd paseq-)eU arur5'trlsruobelue {;;entnu are s{ervrqled oml aql'oqu Jo uotlenll)p aq1sra66111 leqt' (EL/Z L9 palouap) € [g pue 719 suralord9 )uotrtp] eqt ern'{ervrqted puoras 6u11eu6rs e salelnurlsosletoloa)al aureseqf 'luelnurtsaql ot eqt 1o uorbar lsasol)11at aql ol palrurl sr uorsnrloJdos 'pa^ll-uoqs are,{earqled srqlu!q}!Msla6uasseu puo)as'slJoMlauu!lte anrsnllold '!9 ;o quirol6o1 uralold g fuaulu] aql er^'speal{ealqred)eU aql Jo uollelnults ';eu6rs leuat>eqaql Jo a)lnosoql ol esol) 'uoltezueloos,lle) 'lla) aqr Jo luor, aqt lv aql ol alnquluor s{ervrqled6ur;eubrs l)urlsrp olvrl'tuorJaql le puebr;leuol)eq aq] o] punoq aq o1l;a1r;arourale rnq 'lla) aqtJo a)eJrnsaq] ro^o lle punoj are slolda:er asaql'fi r;l1orupat)altp alplnurls sltqoollnauoql uo s.loloa)al pa;dnor-ura1o.rd-9 ot se;ntaloulleus])eq e q t ; o 6 u r p u r g ' s ; r q d o r l n6auur p n ; t u r 'uels,{sounuult aql;o slleraql ,(q ubrarolse pazru6orelaJeleql saln)alou elarras{poq uetunqaql papelut oleLl tei.lteuatreg'uollezgre;od;;qdorlneu 6urrnp6ur;eu6!5ZO[-9t aln6!l

'spu8rs TBa/\/\o] asuodsal a^rlrsuos E Sur aolle 'p1ro,vreprslno eql ruo4 salracal uolala{soilJ ullJe eql leql uolleru 'ursruE -roJur dlrrelod aq] acro;urar ol sruees eruosoJlueJ eq] Jo uoueluarJo eql 'porJed paSuoloJd e roJ uorlcerrp a(uus aql ul -q3eru lrExe eql lsls Jo ssalpreSeu -rad o1z!ry1oru alrsnrloJd selqeua dool a^qrsod e 3ut1uarc 'uorsnJ] leql {Jeqpaal -ord raqury e8uJnoJua [qeJer{l pFo^a salnqn}oJJnu Jo uorlEJluef,uof, paseeJJur aqJ 'spua alnqnlorcltu Suur,ror8uo Surlelert sdII+ aq] o] pulq teql sCHD-oEU Suuanuap rtq uor8ar elrsnJloJd aql q uoDezueur,(lod urlJe eseaJJur Jequry ol esedJD ceu aql alp^rlse oslE pue uorsaqpe IeJol atpFpour dllcarlpul ,{eur spua snld apqnloJf,ru Jrtueudp eql'ilac aqlJo uor8al elrsnJloJd aqt olul eruosoJlueJ aq] ruor; Surpualxa spua snydJraql alpq seFqnloJJrru eseqlJo dueru lpql sueeur Suruorlrsodar slr pue 'salnqnlorctu crueur(p Jo Jaqunu a8rel e salealJnu eruos -oJluar eqJ'sluaua8uerruar urlJe eJuengur sJtueudp alnqnloJOrru 'uJnl uI 'salnqnloJJrru azrTrqels o] sreadde uope^Ipe oqg pue 'salnqnlorcrur Suzqrqelsap '(966-t66 'dd 't-gl laued aas) uruqtpls ulalord Surpurq ulnqnl aqt (tlqqul eJoJeJaqlpue) elelftoqdsoqd uec ceg dq pelelrlop esBuDIulalord u 'elduexa JoC ,tpcarrp scrueu^,tp alnqntoJcrur elulnporu oqg pue JEUJo ruealtsrrmop suraloJd JopaJJa IeJa^aS'sapqnloJJlru slr uo Surlpd r(q auosorlual aql a^otu leq] sulal -oJd Jolour alrT-ureudpalelrlJp,{.1pco1osle lnq (uorsnr}ord IEJoI eJoJaJaqlpue) aJaq] uorlezrraru,{1od ullle elelnrups .{1uo lou rq8lu IIaf, p Jo e8pe euo uo sJol -dacar;o uorlelrtre aq] ]eq] ]q8noql sI ]I 'pe^lo ur aq Leur ZwpJ ulalord d.11ue3 oqu eql ]eq] eJuapna sr aJaql qSnoqlp 'Jeelclou sl uollBluelJoal aluosoJlual 'uollezuelu 'snelJnu eq] Jo rusrueqJeru eqJ Jo aprs pJE^ JoJ aql uo puno; Sutaq -,{yodunce elrsnJ]oJdJo uorluool eq] dq peruengur sr aruosonuec eqlJo uoplsod aql 'sllaf, Suqourocol ,tueru u1 'sluaruElg uIlf,B JIeq] r{fyvr Suop saFqnloJJlru Jraql asn sllac uorlJaJrp JBInJrued e ur luarueloru luelslsJed ezrueSrc dlaq o1

uorloruo)olpue uollezuelodlla)-aloLl^seleu!proo) suolelelsorr()ulrrv pueelnqntor)lryaql uaa/nleguolle)lunuuo) '(Zg-9I arn8rg aas) suoneupsap radord rraql ot sauoo qlMoJBro s11ac Suqoruocol eq] raets o1 spu8rs alqrsnsp-uou pue elqlsryJlp Jo uoueuqruoo B uo puadap 'sauor qrrtor8 puoJneuJo slaAEJ]eql pue uollEJBIIuIIaJ-lsalJ-leJnau Surpnlcur 'slerurue ur suorler8nu IIaJ acuptsrp-3uo1 lsolz{ 'uopeztrarur{1od uqce p3]f,eJrp puu uorseqpe IIas peseaJJur asnBJ uec ,(aq] 'sroldacar elP^rlse slPu -3ts asaq] uar111'uoqur8rur IIaJ Jo uopcaJlp aqt eJuangu osle uEf, sllaf,Jo aoPJJns elqlsnJJlpuoN aql o1 Jo xrJluru JelnllaseJlxe aql ol peqsBllE sanJ lecluaqJ )Jeq aq] l3 uorlJPJl -uoJ pue a8pa Surpeal aql tp uorsnrlord qlyv\ &IJpIod [EuoI]JurU sll UIE]uIpru arn81g) JEaJaqt uI sel?ulruop uol}E^ltJe oqg ot IIaf, eqt selqeue slqJ'(Z0I{1 puE IIef, eql Jo luoJJ aql u! saleuruop uorlE^IlJp JEU 'llnsar e sE puB Teqlo qf,Ee llqlqul ,tpcarrp sassacord o^(1 aqJ dl1ltcer1uoc paseq-utsofru seJuuqua puP oqu uo surnl }eq1,(eMqlEd 3u1pu3rs JeqlouE salEArlJEroldacer s1to1 pue3l ]uel -cpJlleouaqJ eqlJo 3urpurq 'aruu eures aql tV'esuluqdsoqd ptdq arrqcer(larrpnl -llsuoJ e ,tq 2614olur If,Eq paua^uoc .{lprder sI }I eJuls 'qsaqluds 3o alls sll ruo4 JeJ asnJJrplouuec JEu sale^rlJE teql td(9'7't)Id aqJ 'uorsrulord;o uollJnpul aq1 SuruaqfueJls 'dool {cpqpeeJ anr}rsod e ul II1,rI}JB )tld Jo }uarueoueque Ierol raqury sasneoa8pa Surpealaql le qa ^ ullte pazlreyodeqlJo uollelnrunJce

,rraro-......'.......-/,uo,,n,ro,oi': -: O d

eu-l

uorperluo) urso{ur-urpe 'saleururop ol-l

uole;alsoy(3 aql:9 ! raldeql

9VOL

1047

THECYTOSKELETON AND CELLEEHAVIOR

t (A)

localized signal

Figure 16-1 03 The polarizationof a cytotoxicT cell after target-cell recognition.(A)Changesin the cytoskeletonof a cytotoxicT cell after it eventresultsin hasmadecontactwith a targetcell.Theinitialrecognition in both cellsat the siteof contact. signalsthat causeactinpolymerization betweenthe actin-richcontactzoneand In theT cell,interactions microtubulesemanatingfrom the centrosomeresultin reorientationof the centrosome,so that the associatedGolgiapparatusis directlyapposedto micrographin whichboth the the targetcell.(B)lmmuno-fluorescence T cell(top)and its target cell(bottom)havebeen stainedwith an antibody radiatingfrom and the microtubules Thecentrosome againstmicrotubules. it in the T cell are orientedtoward the point of cell-cellcontact.In contrast, the microtubulearrayin the target cell is not polarized.(B'from B.Geiger, With permissionfrom D. Rosenand G. Berke,J. CettBiol.95137-143,1982. The Rockefeller UniversityPress.) A similar cooperative feedback loop seems to operate in many other instances of cell polarization. A particularly interesting example is the killing of specific target cells byT lymphocytes. These cells are a critical component of the vertebrate's adaptive immune response to infection by viruses. T cells, like neu-

trophils, use actin-based motility to crawl through the body's tissue and find infected target cells. \Mhen a T cell comes into contact with a virus-infected cell and its receptors recognize foreign viral antigens on the surface of the target cell, the same polarization machinery is engaged in a very different way to facilitate kilting of the target cell. Rac is activated at the point of cell-cell contact and causesactin polymerization at this site, creating a specialized region of the cortex. This specialized site causes the centrosome to reorient, moving with its microtubules to the zone of T-cell-target contact (Figure f6-f03). The microtubules, in turn, position the Golgi apparatus right under the contact zone' focusing the killing machinery onto the target cell. The mechanism of killing is discussedin Chapter 25 (seeFigure 25-47).

of NeuronsDepends Specialization TheComplexMorphological on the Cytoskeleton For our final case study of the ways that the intrinsic properties of the eucaryotic cytoskeleton enable specific and enormously complicated large-scale cell behaviors, we examine the neuron. Neurons begin life in the embryo as unremarkable cells, which use actin-based motility to migrate to specific locations. Once there, however, they send out a series of long specialized processes that will either receive electrical signals (dendrites) or transmit electrical signals (axons) to their The beautiful and elaborate branching morphology of axons target cells. les neurons to form tremendously complex signaling netand dendri works, interacting with many other cells simultaneously and making possible the complicated and often unpredictable behavior of the higher animals. Both axons and dendrites (collectively called.neurites) are filled with bundles of microtubules that are critical to both their structure and their function. In axons, all the microtubules are oriented in the same direction, with their minus end pointing back toward the cell body and their plus end pointing forward toward the axon terminals (Figure f 6-104). The microtubules do not reach

(8) 10pm

1048

Chapter16:TheCytoskeleton

o v e s i c l ew i t h b o u n d d y n e i n o v e s i c l ew i t h b o u n d k i n e s i n microtubule r (A)

FIBRoBLAST

(B)

NEURoN

from the cell body all the way to the axon terminals; each is typically only a few micrometers in length, but large numbers are staggered in an overlapping array. This set of aligned microtubule tracks acts as a highway to transport many specific proteins, protein-containing vesicles, and mRNAs to the axon terminals, where synapsesmust be constructed and maintained. The longest axon in the human body reachesfrom the base of the spinal cord to the foot, being up to a meter in length. Mitochondria, large numbers of specific proteins in transport vesicles,and synaptic vesicle precursors make the long journey in the forward (anterograde) direction. They are carried there by plus-end-directed kinesin-family moror proteins that can move them a meter in as little as two or three days, which is a great improvement over diffrrsion, which would take approximately several decades to move a mitochondrion this distance. Many members of the kinesin superfamily contribute to this anterograde axonal transport, most carrying sp"iific subsets of membrane-enclosed organelles along the microtubuler. ih" g."ut diversity of the kinesin family motor proteins used in axonal transport suggests that they are involved in targeting their cargo to specific structures near the terminus or along the way, as well as in cargo movement. old components from the axon terminals are carried back to the cell body for degradation and recycling by a retrograde axonal transport. This transport occurs along the same set of oriented microtubules, but it relies on cytoplasmic dynein, t-tri.tr is a minus-enddirected motor protein. Retrogradetransport is also critical for communicating the presence of growth and survival signals received by the nerve terminus back to the nucleus, in order to influence gene expression. one form of human peripheral neuropathy, charcot-Marie-Tooth disease,is caused by a point mutation in a particular kinesin family member that transports slmaptic vesicle precursors dovrrnthe axon. other kinds of neurodegenerative diseases such as Alzheimer's disease may also be caused in part by dlsruptions in neuronal trafficking; as pointed out previously, the amyloid pr".r.r^o, protein APP is part of a protein complex that serves as a receptor for kinesin-l binding to other axonal transport vesicles. Axonal structure depends on the axonal microtubules, as well as on the contributions of the other two major cytoskeletal systems-actin filaments and intermediate filaments. Actin filaments line the cortex of the axon, just beneath the plasma membrane, and actin-based motor proteins such as myosin v are also abundant in the rxon, presumably to help move materials. Neurofilaments, the specialized intermediate filaments of nerve cells, provide the most important structural support in the axon. A disruption in neurofilament structure, or in the cross-linking proteins that attach the neurofilaments to the microtubules and actin filaments distributed along the ixon, can result in axonal disorganization and eventually axonal degeneration. The construction ofthe elaborate branching architecture ofthe neuron during embryonic development requires actin-based motility. As mentioned earlier, the-tips of growing axons and dendrites extend by means of a growth cone, a specialized motile structure rich in actin (Figure 16-105). Mosi neuronal growth cones produce filopodia, and some make lamellipodia as well. The protrusion

Figurel6-104 Microtubuleorganization in fibroblastsand neurons.(A)In a fibroblast,microtubulesemanate outwardfrom the centrosomein the middleof the cell.Vesicles with Dlus-enddirectedkinesinattachedmove outward, and vesicles with minus-end-directed dyneinattachedmove inward.(B)In a neuron,microtubuleorganization is more complex.In the axon,all microtubules sharethe samepolarity,with the plus ends pointing outward toward the axon terminus.No one microtubulestretches the entirelengthofthe axon;instead, short overlappingsegmentsof parallel microtubulesmakethe tracksfor fast axonaltransport.In dendrites,the microtubulesare of mixed polarity,with someplusendspointingoutwardand somepointinginward.

104!l

ANDCELLBEHAVIOR THECYTOSKELETON

tt(B) 1 0p m

1 0p m

Figure 16-1 05 Neuronalgrowth cones. (A)Scanningelectronmicrograph of two growth conesat the end of a neurite,put out by a chicksympathetic single neuronin culture.Here,a previously growth cone hasrecentlysplit into two' Notethe manyfilopodiaand the large of the Thetaut appearance lamellipodia. neuriteis due to tensiongeneratedby the forward movementof the growth cones, which are often the only firm points of attachmentof the axonto the substratum. (B)Scanningelectronmicrographof the growth cone of a sensoryneuroncrawling overthe innersurfaceof the epidermisof a Xenopustadpole.(A,from D. Bray,in Cell A. Curtisand Behaviour [R.Bellairs, UK:Cambridge G.Dunn,eds.l.Cambridge, UniversityPress,1982;B,from A. Roberts, BrainRes.118:526-530,1976.With permissionfrom Elsevier.)

and stabilization of growth-cone filopodia are exquisitely sensitive to environmental cues.Some cells secretesoluble proteins such as netrin to attract or repel growth cones. These modulate the structure and motility of the growth cone cytoskeleton by altering the balance between Rac activity and Rho activity at the leading edge (see Figure 15-62).In addition, there are fixed guidance markers along the way, attached to the extracellular matrix or to the surfaces of cells. \Mhen a filopodium encounters such a "guidepost" in its exploration, it quickly forms adhesive contacts. It is thought that a myosin-dependent collapse of the actin meshwork in the unstabilized part of the growth cone then causes the developing ixon to turn toward the guidepost. Thus, a complex combination of positive and negative signals,both soluble and insoluble, accurately guide the growth cone to its final destination. Microtubules then reinforce the directional decisions made by the actin-rich protrusive structures at the leading edge of the growth cone. Microtubules from the axonal parallel array just behind the growth cone are constantly growing into the growth cone and shrinking back by dynamic instability. Adhesive guidance signals are somehow relayed to the dynamic microtubule ends, so that microtubules growing in the correct direction are stabilized against disassembly. In this way, a microtubule-rich axon is left behind, marking the path that the growth cone has traveled. Dendrites are generally much shorter projections than axons' and they receive synaptic inputs rather than being specialized for sending signals like axons. The microtubules in dendrites all lie parallel to one another but their polarities are mixed, with some pointing their plus ends toward the dendrite tip, while others point back toward the cell body. Nevertheless,dendrites also form as the result of growth-cone activity. Therefore, it is the growth cones at the tips of axons and dendrites that create the intricate, highly individual morphology of each mature neuronal cell (Figure f 6-f 06).

a x o n ( l e s st h a n 1 m m t o m o r et h a n 1 m i n l e n g t h )

dendritesreceive s y n a p t i ci n p u t s c e l lb o d y

zlrm

t e r m i n a lb r a n c h e so f a x o n m a k es y n a p s eosn target cells

Figure16-106 The complexarchitecture of a vertebrate neuron.The neuron shown is from the retinaof a monkey. The arrowsindicatethe directionof travel signalalongthe axon. of the electrical The longestand largestneuronsin the human body extendfor a distanceof about 1 m (1 millionUm),from the base of the spinalcord to the tip of the big toe,and havean axondiameterof 15 pm. on (Adaptedfrom B.B.Boycott,in Essays and the NervousSystem[R.Bellairs E.G.Gray,eds.l.Oxford,UK:Clarendon Press,1974.)

1050

Chapter16:TheCytoskeleton

(B)

20 40 timein mins

60 |

lolr#

80

Although the neurons of the central nervous system are long-lived cells,they are by no means static. synapses are constantly being created, strengthened, weakened, and eliminated as the brain learns, evaluates,and forgets. uigh-r"rolution imaging of the structure of neurons in the brains of adult mice has revealed that neuronal morphology is undergoing constant rearrangement as synapsesare forged and broken (Figure l6-10z). These actin-dependent rearrangements are rhought to be critical in learning and long-term memory. In this way, the cltoskeleton provides the engine for construction of the entire nervous system, as well as producing the supporting structures that strengthen, stabilize, and maintain its parts.

Figure16-107Rapidchangesin dendrite structurewithin a living mousebrain. (A)lmageof corticalneuronsin a transgenic mousethat hasbeenengineered to express greenfluorescentprotein in a smallfraction of its braincells.Changesin thesebrain neuronsand their projections can be followedfor monthsusinghighlysensitive fluorescencemicroscopy. To makethis possible, the mouseis subjectedto an operationthat introduces a small transparent windowthroughits skull,and it is anesthetized eachtime that an imageis (B)A singledendrite,imagedover recorded. the period of 80 minutes,demonstratesthat dendritesareconstantlysendingout and retractingtiny actin-dependent protrusions to createthe dendriticspinesthat receive the vast majorityof excitatorysynapses from axonsin the brain.Thosespinesthat becomestabilized and persistfor months arethoughtto be importantfor brain function,and may be involvedin long-term memory.(Courtesyof KarelSvoboda.)

Summary Two distinct typesof specializedstructures in eucaryotic cells are formed from highty ordered arrays of motor proteins that moue on stabilized filament tracks. The

systemfunction in the adult animal-is another prime example of such complex, coordinated cytoskeletalaction. For a cell to crawl, it must generateand maintain an ouerall structural polarity, which is influenced by external cues.In addition, the cell must coordinate protrusion at the leading edge(by assemblyof new actin filaments), adhesion of the newly protruded part of the cell to the substratum, forcesgeneratedby molecular motors to bring the cell bodyforward. Complexcells,such as neurons,require the coordinatedassemblyof microtubules, neurofilaments (neuronal intermediatefilaments), and actin ftlaments, as well as the actions of dozensof highly specialized molecular motors that transport subcellular componentsto their appropriate destinations.

PROBLEMS Whichstatementsare true? Explainwhy or why not. 16-1 The role of ATP hydrolysisin actin polymerization is similar to the role of GTP hydrolysis in tubulin polyrneriza_ tion: both serve to weaken the bonds in the polymer and therebypromote depolymerization.

16-2 In most animal cells, minus end-directed micro_ tubule motors delivertheir cargoto the periphery of the cell, whereasplus end-directedmicrotubule motors delivertheir cargoto the interior ofthe cell. 16-3 Motor neurons trigger action potentials in muscle cell membranes that open voltage-sensitiveCa2*channels in T-tubules, allowing extracellular CaZ*to enter the cytosol, bind to troponin C, and initiate rapid muscle contraction.

1051

PROBLEMS END.OF-CHAPTER ( B ) P O S I T I OO NF K I N E S I N

( A ) E X P E R I M E N TSAELT U P

Discussthe following problems. 16-4 At 1.4 mg/ml pure tubulin, microtubules grow at a rate of about 2 pm/min. At this growth rate how many oBtubulin dimers (B nm in length) are added to the ends of a microtubule each second?

Euo a

16*5 A solution of pure aB-tubulin dimers is thought to nucleate microtubules by forming a linear protofilament about sevendimers in length.At that point, the probabilities that the next ap-dimer will bind laterallyor to the end of the protofilament are about equal.The critical event for microtubule formation is thought to be the first lateralassociation (Figure Qf6-f). How does lateral associationpromote the subsequentrapid formation of a microtubule? LATERAL ASSOCIATION

L I N E A RG R O W T H

o

od

o.cd

o -,

I I

J

e a

od "tJ

e

J

e .t

..ddd 'lj,t'l oofo

dddd dddd .t.s,lJ

,f

by purecrB-tubulin nucleation FigureQ16-1Modelfor microtubule d i m e r(sP r o b l e1m6 5 ) . 16-6 How does a centrosome"know" when it has found the center of the cell? 16-7 The concentration of actin in cells is 50-100 times greater than the critical concentration observed for pure preventsthe actin in a test tube. How is this possible?'v\rhat actin subunits in cells from polymerizing into filaments? \A4ryis it advantageousto the cell to maintain such a large pool of actin subunits? 16*8 The movements of single motor-protein molecules can be analyzeddirectly.Using polarizedlaserlight, it is possible to create interferencepatterns that exert a centrally directed force, ranging from zero aI the center to a few piconewtonsat the periphery (about 200 nm from the center). Individual molecules that enter the interferencepattern are rapidly pushed to the center,allowing them to be capturedand moved at the experimenter'sdiscretion. Using such "optical tweezers,"single kinesin molecules can be positioned on a microtubule that is fixed to a coverslip. Although a single kinesin molecule cannot be seen optically,it can be taggedwith a silicabead and trackedindirectly by following the bead (Figure Qf 6-2,{). In the absence of ATB the kinesin molecule remains at the center of the interferencepattern, but with AIP it movestoward the plus end of the microtubule.As kinesin moves along the microtubule, it encountersthe force of the interferencepattern, which simulates the load kinesin carries during its actual function in the cell. Moreover,the pressureagainstthe silica bead countersthe effectsof Brownian (thermal)motion, so that the position of the bead more accuratelyreflects the position of the kinesin molecule on the microtubule. Tracesof the movements of a kinesin molecule along a in FigureQ16-28. microtubule are sho'nr,.n

Fqo 20

8 .,.1 o".onlu (Problent alonga microtubule of kinesin FigureQ16-2Movement linkedto a silicabead, setupwithkinesin 16 s).(A)Experimental (asvisualized by (B)Position of kinesin movingalonga microtubule. pattern, as a of interference center relative to bead) of silica Dosition Thejagged alonqthemicrotubule' of timeof movement function motionof thebead' fromBrownian natureof thetraceresults o

m icrotubule microtubule

z

A. As shown in Figure Ql6-2B, all movement of kinesin is in one direction (toward the plus end of the microtubule). \A4ratsuppliesthe free energyneededto ensurea unidirectional movement along the microtubule? B. \Altratis the averagerate of movement of kinesin along the microtubule? c. \A/hatis the length of each step that a kinesin takes as it movesalong a microtubule? D. From other studies it is knor,rmthat kinesin has two globular domains that each can bind to B-tubulin, and that kinesin moves along a single protofilament in a microtubule. In each protofilament the B-tubulin subunit repeats at B-nm intervals. Given the step length and the interval between B-tubulin subunits,how do you supposea kinesin moleculemovesalong a microtubule? E. Is there anything in the data in FigureQl6-28 that tells you how many AIP molecules are hydrolyzed per step? 16-9 How is the unidirectional motion of a lamellipodium maintained? 16* 10 Detailedmeasurementsof sarcomerelength and tension during isometric contraction in striated muscle provided crucial early support for the sliding filament model of muscle contraction. Based on your understanding of the sliding filament model and the structure of a sarcomere, proposea molecular explanationfor the relationshipof tension to sarcomerelength in the portions of Figure Qf 6-3 marked L II, III, and IV (In this muscle, the length of the myosin filament is 1.6pm and the lengths of the actin thin filaments that project from the Z discsare 1.0pm.) FigureQl6-3 Tensionasa function of sarcomerelength duringisometriccontraction (Problem 16-10).

E l

E i 100 o E 'b -50 c az) q

0

23 sarcomerelength (Pm)

1052

Chapter16:TheCytoskeleton

REFERENCES General BrayD (2001)CellMovements: FromMolecules to Motility, 2nd ed NewYork:Garland Science Howard.J(2001)Mechanics of Motorproterns andthe Cytoskeleton S u n d e r l a nM dA , :S i n a u e r The Self-Assemblyand Dynamic Structure of Cytoskeletal Filaments DogteromM & YurkeB (1997)Measurement of the force-velocity relationfor growingmicrotubuies Science 218856 860 GarnerEC,Campbell CS& MullinsRD(2004)Dynamicinstability in a DNA-segregating prokaryotic actinhomologScience 306.1021 1O)5 HelfandBT,ChangL & GoidmanRD(2003) Thedynamicand motile propertiesof intermediate filamentsAnnuRevCellDevBiol 19.445,467 HillTL& Kirschner MW (1982)Bioenergetics and kinetics of microtubule and actinfilamentassembly-disassembly lntRevCytol7B:1 125 HotaniH & HorioT ('l9BB)Dynamics of microtubules visualized by darkfield microscopy: treadmilling anddynamicinstability CellMotil Cytoskeleton 10:229-236 JonesLJ,Carballido-Lopez R& Errington J (2001)Conrrolofcellshape in bacteria: helical, actinlikefilaments in Bacillus subtilis Cell 104913-922

LubyPhelpsK (2000)Cytoarchitecture and physical properties of cytoplasm: volume,viscosity, diffusion, intracellular surface area/nt RevCytol192189-221 Mitchison T & Kirschner M (1984)Dynamicinstability of microtubule growth A/rture312:237242 MitchisonI I (1995)Evolutionof a dynamiccytoskeletonphilosTransR SocLondBBiol Sci349:299-304 Mukherjee A & Lutkenhaus J (1994)Guaninenucleotide oepenoent assembly of FtsZintofilamentsJ Bacteriol 116:2754 2/58 OosawaF & Asakura S (1975) Thermodynamics of the polymerization of press,pp 41 55,pp 9O_1OB proteinNewYork:Academic PaulingL (1953)Aggregation of GlobularproteinsDiscuss Faraday Soc 13.170-176 Rodionov Vl & BorsyGG(,1997) Microtubule treadmilling ln vlvo S c i e n c2e/ 5 . 2 1 5 - 2 l B ShihYt & Rothfreldt (2006)The bacterialcytoskeletonl,licrobiolMol BiolRev7a.729-/54 TheriotlA (2000)The polymerization mator Traffic1:lg 28 How CellsRegulateTheir CytoskeletalFilaments AldazH,RiceLM,Stearns T & AgardDA (2005)Insighrs into microtubul nucleation fromthe crystalstructure of hurnangamma_ tubulin.A/d re 435523 52/ Bretscher A,Chambers D,NguyenR& Reczek D (2000)ERM-Merlin and proteinfamilies EBP50 in plasmamembraneorganization and function AnnuRevCellDevBiol i6.113 143 DoxseyS,McCollumD & Theurkauf W (2005)Centrosomes in cellular regulationAnnuRevCellDevBiol21:411-434 GarciaML& Cleveland DW(2OOl) Goingnew placesusingan old MAp: tau,microtubules and humanneurodegenerative diseaseCurrapin CellBiol13:41-48. HolyTE,DogteromM,YurkeB & LeiblerS (1997)Assembry anc positioning of microtubule astersin microfabricated chambersproc NarlAcad SciUSA94:6228-6231 MullinsRD,Heuser JA& Pollard TD (1998) Theinteraction of Arp2/3

usA 95.618t 6186 Robinson RC,Turbedsky K,Kaiser DA et al (2001)Crystal structure of Arp2l3complexScience 294:1 679-1684 QuarmbyL (2000)Cellular Samurai: kataninand the severing of microtubules.JCellSci113:2821-2827

Stearns T & Kirschner M (1994)/n /ltro reconstitution of centrosome assembly andfunction: the centralroleof gamma-tubulin Cel/ 76:623-637 WieseC & ZhengY (2006)Microtubule gammatubulinand nucleation: beyondJ Cel/5ct119.41 43-4153 ZhengY,WongML,AlbertsB & Mitchison T (1995)Nucleation of microtubuie assembly by a gamma-tubulin-containing ring complexNature 378.578-583 Zigmond5H (2004)Formin-induced nucleation of actinfilamentsCurr OpinCellBiol 16.99-105 MolecularMotors Burgess SA,WalkerML,Sakakibara H et al (2003)Dyneinstructure and powerstrokeNature421:715718 Hirokawa N (1998)Kinesin proteinsandthe anddyneinsuperfamily mechanism of organelle transportScience 219.519526 HowardJ,Hudspeth AJ& ValeRD('1989) Movementof microtubules by singlekinesinmoleculesNature342154-158 HowardJ (1997)Molecular motors:structural adaptations to cellular functions,Nature389:561567 Rayment l, Rypniewski WR,SchmidtBaseK et al (1993) Threedimensional structure of myosinsubfragment-1: a molecular motor Science261:50 58 Reck-Peterson SL,YildizA,CarrerAPet al (2006)Single-molecule analysis of dyneinprocessivity and steppingbehaviorCel/ 126:335348 RiceS,LinAW,SaferD et al (1999)A structural changein the kinesin motor proteinthat drivesmotility /Vdfure402:178-784 Richards TA & Cavalier SmithT (2005)Myosindomainevolutionand the primarydivergence of eukaryotes Nature436:11 13-1j j B SvobodaK,SchmidtCF,SchnappBJ& BlockSM(1993)Drrect observation of kinesin steppingby opticaltrappinginterferometry Nature365,721727 Vikstrom KL& LeinwandLA (,l996) proteinmutations Contractile and heartdiseaseCurrOpinCellBiolB:97-105 WellsAL,LinAW,ChentQ et al (1999)MyosinVl isan actin-based motorthat movesbackwards lVorure 401:505-5OB YildizA, ForkeyJN,McKinney SAet al (2003)MyosinV walkshandoverhand:singlefluorophore imagingwith 1 5-nmlocalization sclence 300.2061-2065 YildizA & SelvinPR(2005)Kinesin: walking, crawlingor slidingalong? TrendsCellBiol 15:112-120 The Cytoskeleton and Cell Behavior Abercrombie M (1980) Thecrawlingmovementof metazoan cellsproc RoySocB 207.129-147 CookeR (2004)The slidingfilamentmodel.1972-2004 1Genphysiol 123.643-656 DentEW& GertlerFB(2003)Cytoskeletal dynamics andtransportin growthconemotilityand axonguidanceNeuron 40:209227 Lauffenburger DA & HorwitzAF(1996)Cellmigration: a phystcally integratedmolecularprocessCel/84:359-369 Lo CM,WangHB,DemboM & WangYL(2000)Cellmovementrs guidedby the rigidityof the substrate Biophys J 79:144152 MaddenK & SnyderM (1998)Cellpolarityand morphogenesis in buddingyea:rAnnuRevMicrobiol52:687.744 RidleyAJ,Schwartz MA,Burridge K et al (2003)Cellmigration: integrating signals fromfrontto backScience 302J104-1709 Rafelski SM& Theriot.JA(2004) Crawling rowarda unifiedmodelof cell motility: spatialandtemporalregulation of actindynamicsAnnuRev BiochemT3:209 239 ParentCA& DevreotesPN(1999)A cell'ssenseof directionSclence 284:765-770 Pollard TD & Borisy cc (2003)Cellular motilitydrivenby assembly and disassembly of actinfilamenrsCelli12:453465 Purcell EM(1977)ttfe at low Reynolds' numberAmJ phys 45:3-11 WittmannI HymanA & DesaiA (200,l) Thespindle: a dynamic assemblyof microtubules and motors NatureCellBiol3:E2B-E34

gsor

l0tt Z60L

HIMOUD II]f CNVNOISIAIC rtl) Jo toulNol stslNl)ol^)

]SVHdS

LgOL

SISO1IW

LLOL

090r

?s0r

wlls^stourNof ltf^)-Itf

IHI

llf^) Il) lHl ro MTAU]AO

raldeq) slql ul

eql sreSSlrl ll ^|oq upldxa pup urals^s loJluo3 elc.{J-lleJ aqt eqlJ3sepueql eM 'elJltJ IIaJ aqt Jo ^ ar^re^o Jalrq B ql.Irv\u€aq eM'paleulpJooJ puu pallorluoJ eru.{.eql^\oq puE alf,l{JIIoc eqlJo slue^e snoIJEAaql saqlrcsep reldBqJ sIqJ 'sl 'uorsr^rp naql qll1!\ (ss€ru ]BI{}) q}Mor8 IIef, ur esuercul JIeq] Jraql eleulpJooJ lsnru silac Surpr^rp 'azrs Jreql urPluIEIu oJ'uolsplp qcPe qlyvl JellErus le8 plnoM sllef, ratqSnsp 'esMJeqlo lselnJelotuoJletu pue selleueSJo Jaqto rreqt aluJlldnp oqe sllaf, lsoru 'euloue8 rlaqt Supucrtdnp o1uoyllppe uI 'Jecuucq llnseJ ueJ suorsr^rp IIac e^rssesxe 'suorlJuruluu uralsrts eql uel{lv\ dpoq aqr 1o senssp aql ul sJeqrunu [ec Surlup8ar ul eloJ IeJlueJ p suq eJoJeJeqluralsds lortuoc alcdc -lla3 eql 'lou aJE,{aql ueq^| ll 3u14co1qpuu pepeeu aJE slleJ eroru ueq a uols -FIp IIaJ Supeprups 'slleJ reqlo uo4 spu8ts ot arusuodsar,(1q8q st uralsds aql 'leurrue JplnllacrlFru p uI 'ilec eql eplslno suolllpuoJ srolluoru osp rualsrts 1or1 -uoo eqJ 'a1a1duocs1uorlecqdal VNC lpun 'aydurexeJoJ 'saluosoruoJqt paleo -gdnp 3o uone8ar8as eql JoJ suoqerudard lnuad tou saop 11'palalduoJ ueeq eAEq sluala JerlJpaIpun sluele ralel sdulep pue alcrb IIaf, eql q8norql uotsser8 -ord srolruoru rualsrts IoJluoJ aql 'llal eql eplsul 'lleJ eql eplstno pue eplsu qloq uro4 spu31s snolJpl o1 puodsar ol rualsds IoJluoJ eql ,lrollu pue uoIsIAIp IIaJ puolllppe 'sllef, }sotu uI 'uoue8ar8as yo '{rqapg eq} acupqua uoqeln8ar;o srar{.e1 pup uorlporTdnp aurosoruorqc Surpnlcur 'alcztc eql Jo stua^a u1eru eql elelllul 'a1cdc lEqt saqJtrMs lBf,ruaq]orq Jo selJasperapJo ue s1rualsrfu slq] Jo aroc aql aq] sE u ^ou{ IIaJ eql q8norqt uorssarSord surano8 ferTt'utalsts ToJluoJayctc-11ac 'suralord ,fuo1ep8ar Jo {Jomlau xayduroc e peAIoAe e^eq slleo cqorfuucng '(1-2,1 arntlgl auroua8 erque eql go,tdor E selreJal qf,Eelpql os 'sllal rarq8nep oMl aql o1 6palnBa8ag petnqulslp dlalerncce eq uaql lsnrx seruosoruoJqc paleclldar eql pue 'sardoc alaldruoc orrnl acnpord o1paleclldar IIIryqrleJ eq lsJg tsnur eruosotuorqo qcea ul VNC eqt 'sllao ralq8nep leJpuepr ,(lpcpaua8 oa4 acnpord oI 'slleo Jo uopureue8 xau eql ol uoppruroJul cpeua8 slrJo uo Surssedeqt :{sul lplueuepury tsoru sll qsqdurocce ol lr ^\olle lpql esoql are uro;rad o] seq ilec e lpql sassaoordJo las IunIuIuIu eqJ'lesre^run eru talemoq 'sJIlslJe]JBJEqJuIPuaC 'eJII slrrspe8ro ue uI setup tuaraJJrplE pue ursrue8ro ot rusrue8ro uro4 fuen a1c,tc1ac eqlJo sllutep aqJ 's[ep rrag E ulqllm erp plnom arvr-aldruexe ro3 'sz{er-xgoasop a8re1 I-razre ol arnsodxa fq-paddols aJa^ uorsrllp IIaJ IIe JI :aAIAIns o1 dldurrs puo -cas drala sllao Jo suollnu ,(ueur arnlce;nueru lsnru sn Jo qcea 'IJBJ uI 'alp leq] s11ac aceldar ol pepeeu,'tlpnsn sr uorsrnrp1ac 'r(poql1npe eql uI uelg 'rustue8ro 3u1uo11cungu ecnpord o1 parmbar eJe suolsrnlp IIaJ Jo sacuanbas xaldruoc pue Suoy 'sarcads JulnlleJplnru u1 'rusrue8ro a,raualalduroo p seJnpoJd uotsvrtp 'stseadpue BrJatf,uqse qJns 'salcadsJEInlleJIun u1 'acnpordar s8uqt IIac qJBa 3ut rl 11eqclqm rQ usrueqf,eru prluasse aql s1'a1cfc ilac eqt sE trmou{ 'uotstrttp pue uorluJrldnp 1o alcdc srql 'or\^l q sepplp ueql pue slueluor qt salecqdnp ll qJIq a ur sluene go acuanbas ,{lrapro ue Sunuro;rad dq sacnpordar 1ac y 'o8e srear(uoIIIq eeJql JeAoquug uo aJrI ;o s8uruu€aq eqt o] etuu ul lceq Eulpua[e uols1lrlp pue qunor8 IIer Jo spunor paleadar;o slcnpord aJp 'leurru€ur Jelnllacr]Fru eql o] runlJeloeq JulnllaJrun aqt uroq 'srusrue8ro 3upil 11V'aJIIJo ,fignupuoc eqt JoJ a8essaur puno;ord e 1t qllm sarJJeJ'r{-rntuacqluaa}eup aqlJo alppnu aql ul paqslqelse }sJrJ'1cegaldurs sr{I'stsxa.{pearp teq} IIeo u elecgdnp ol $ IIaJ rvraue e{Eru o},,(e,rndpo aql

el)/() Ile) eql

'(srsaur)ol^)) oMl olut sapr^rp Jlaslrlla) eql q)tqM roue '(srsollulut) rol)nulalq6nepJo lred e olul paleoejbasale saulosoulolrl) petP)rldnpaql uaqM,aseLld t^l pue 'pale)tldnpalp sauosotuolLl) aql uaqM'aseqds ut rn))o al)I) lla) aql .al)^) Jo slua^aleuosoLuorq)Joreulaql lla) aql ]o slua^o rolpu atll z-l I arn6tl

slslNt)o1A) o 6

pate8efas aq] pue 'palquassesrp ueq] sr alpurds eql 'epurds aql Jo salod alrsoddo ol palpd aJeqcrqm 'sppeurorqc Jalsrseql saleredasasaqdnun 1o yels eql lp uorseqoo prleruoJqr-JalsrsJo uorlonJlsep aql 'asaqdaput pelleJ a8els e ur rolenba alpurds aql lu u8rp srelsrs 1e 'rtlenluale 'pue 'alpurds aql;o salod altsoddo ol peqJBpE aJEsprteruoJqJ Jelsrs '(gt raldeql ur pessnJsrp) saFqn] 'a1purdsJqollut aql ol peqop11eaurocaq srrcd -oJf,IIu go Lerre relodrq luer8 e 'srso}nu ur Ja}el selquasspsrp adolazruaJ€el3nu aql uer{M pDp{uoJqc Jelsrs aql 'uo&aqn p!ruu,toJqc-Ja7s1s ,tq raqlaSo] pa{ull ureruar qJIq&\ 'spllBruoJqJ Jel -sls paller spor lcedruoc pue pr8rr;o srmd otur pesuepuoopue papueluasrp z(1pn -per8 are selnrelolu VNC o1\^leq] 'asatldotd palpc a8els e lp srsolrru ur ,(Feg 'sa8e -1uq uralord pazqercads ,(q raqtaSor fpq8p pleq pue peurm]relur erp seruosoru -oJqJ palerrldnp yo rrcd qcea ur selncelotu eqt 'aseqd S Jo pue eqt ]V VNCI '17-21 arnt;g) omt ur sapwp 'srs Jleslr IIeJ eql ueq.^ -auryo1tcro 'uorsnrp cnuseldo/.c pue lrelJnu ralq8nep;o rmd e olur petnqrnsp aJBseruosoruorqcpardoc eql qJrqm Suunp 'srsolrutJo 'uorsrlrp JeelJnu :sluale rofeur om] sasrrdruocaseqd 6 '(llef,uBIIBururEruB ur Jnoq uu ueql ssel)eruq ssel qcnur sarrnbeJqJrqm '(srsoln JoJW) asuqdy1 ur JnJJo uorsrlrp IIaJ pue uone8 -ar8as aurosoruoJqJ'aseqd S JaUV'lleo uerprurueu pcrddl e ur arurt a1c.,b-1ac aqtJoJIeq lnoqe sardnccopue srnoq 71-91 se.rrnberqcrq a'(srsaqtuis VNCIroJ S) asoqd g Surrnp srnrro uo4eclldnp euosouoJq3 'a1c,{cfiac eq] Jo saseqdroferu oml eql aurJap sassacord eseqJ 'sllal rarq8nup IpJrluepr ,tlucllaua8 o^ l olur .,{lasrcardsardoc aql a}e8arSasueq} pue saruosoruoJqcaqr ul VNC Jo lunorue lsEA aq] ,{lalerncce alecrldnp o1 s alcd: IIaJ eq} Jo uorlf,unJ f,rseq }soru eql

saseqdrnol olu! pap!^!csl all^) lle) )rlo^re)nl eql o o E

I

u o r l e ) r l dn p a|.xosourorq)

'a1c.,b 1ac aql dprys o1 pasn suralsdsIIaJ Iapour puu spoqlau aq] Jo eruos JaprsuoJuer{l aM 'a1c[c 1ac cqo,,fuecnaarlt p sasaqdrnoJ aqt Jo uoqdrrcsap ;arJq E qlrm uorloas slql urBaq all

tt)A) ttr) lHl_Jo ltAUtAo 'peluurpJooJ eJBsasseoordor4 asaql ,rroq pue uorsrnlp puu qlr,ror8 IIaJ Jo seleJ aq] uralo8 spu8rs JEInllacEJue^aoqJaprsuoca,u.'dleurg 's1ac relq8npp oml eql olur pale8ar8as uaqt pue palecrldnp eJe seuosoruoJqJ eqt qJrqm ur 'a1c[c 11ac aql;o sa8els roleu eql 1elep ur Japrsuol xeu aM'ayc[c aqt go s]uele ]ueJeJJIp

NOrrvglu9ls S W O S O IO U H f

\

\ 'sa;>{t q6norqr6urobIq ;;a: leuorlrppe apr^rpot enuttuo)uauo lltMs;;atralqbnep aqrJo q)el 'a;r,(rq>eaur parnpotd ate s;;arralqbneple)rluapt{lletrleue6oan} MOqalerlsnllrol uMoqsst sot.uosorloJtlf omt Ll]rM;;et rrlol.rernalerlteqrod,(q e Jo uolsr^rp eq1 'a;r{r lla) aql 1-11 arn6r1

NOr-LV)r'rd3U ]tAOSOIAOUHf CNV Ht_lou9'l'lt) L

a1:(11;a1eqt:/ t rardeq3 t5g t

1055

OVERVIEW OFTHECELLCYCLE cytokinesis mitosis transition metaphase-to-anaphase

INTERPHASE DNAreplic

chromosomes are packaged into separate nuclei at telophase. Cltokinesis then cleaves the cell in two, so that each daughter cell inherits one of the two nuclei (Figure l7-3). Most cells require much more time to grow and double their mass of proteins and organelles than they require to duplicate their chromosomes and divide. Partly to allow more time for growth, most cell cycles have exrra gap phases-a G1 phase between M phase and S phase and a Gz phase between S phase and mitosis. Thus, the eucaryotic cell cycle is traditionally divided into four sequential phases:Gr, S, G2,and M. Gr, S, and G2together are called interphase (Figure l7-4, and seeFigure 17-3). In a typical human cell proliferating in culture, interphase might occupy 23 hours of a 24-hour cycle, with I hour for M phase. Cell growth occurs throughout the cell cycle, except during mitosis. The two gap phases are more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations are complete before the cell commits itself to the major upheavals of S phase and mitosis. The G1 phase is especially important in this respect. Its length can vary greatly depending on external conditions and extracellular signals from other cells. If extracellular conditions are unfavorable, for example, cells delay progress through G1 and may even enter a specializedresting state known as Go(G zero), in which they can remain for days, weeks, or even years before resuming proliferation. Indeed, many cells remain permanently in Geuntil they or the organism dies. If extracellular conditions are favorable and signals to grow and divide are present, cells in early G1 or G0 progress through a commitment point near the end of G1 knorvn as Start (in yeasts) or the restriction point (in mammalian cells).We will use the term Start for both yeast and animal cells. After passing this point, cells are committed to DNA replication, even if the extracellular signals that stimulate cell growth and division are removed.

Figure 17-3 The eventsofeucaryotic cell divisionas seenunder a microscope,The easilyvisibleprocesses and cell of nucleardivision(mitosis) called collectively division(cytokinesis), M phase,typicallyoccupyonly a small fractionof the cellcycle.The other,much longer,part of the cycleis known as which includesS phaseand interphase, the gap phases(discussedin text).The five stagesof mitosisare shown:an state abruptchangein the biochemical of the cell occursat the transitionfrom A cellcan pause to anaphase. metaphase in metaphasebeforethis transitionpoint, this point,the cell but onceit passes carrieson to the end of mitosisand through cytokinesisinto interphase.

M PHASE

mitosis (nuclear division) G 2P H A S E

5 PHASE ( D N Ar e p l i c a t i o n )

cytokinesis (cytoplasmic

G 1P H A S E

Figure 17-4 The four phasesof the cell cycle.In most cells,gap phasesseparate the major eventsof 5 phaseand M phase' Gr is the gap betweenM Phaseand 5 phase,while G2is the gaP between S phaseand M phase.

1056

Chapter17:TheCellCycle

Cell-Cycle Controlls Similarin All Eucaryotes Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism. The basic organization of the cycle, however, is essentially the same in all eucaryotic cells, and all eucaryotes appear to use similar machinery and control mechanisms to drive and regulate cell-cycle events. The proteins of the cellcycle control system, for example, first appeared over a billion years ago. Remarkably, they have been so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. we can therefore study the cell cycle and its regulation in a variety of organisms and use the findings from all of them to assemble a unified picture of how eucaryotic cells divide. In the rest of this section, we briefly review the three eucaryotic systems most commonly used to study cell-cycle organization and control: yeasts,animal embryos, and cultured mammalian cells.

Cell-Cycle ControlCanBe Dissected Geneticallyby Analysisof YeastMutants Yeasts are tiny, single-celled fungi, with a cell-cycle control system remarkably similar to our own. TWospecies are generally used in studies of the cell cycle. The fission yeast schizosaccharomyces pombe is named after the African beer it is used to produce. It is a rod-shaped cell that grows by elongation at its ends. Division occurs when a septum, or cell plate, forms midway along the rod (Figure r7-5A). The budding yeast Saccharomyces cereuisiaeis used by both brewers and bakers. It is an oval cell that divides by forming a bud, which first appears during G1 and grows steadily until it separatesfrom the mother cell aftei mitosis (Figure l7-58).

gene, becausewe avoid the complication of having a second copy of the gene in the cell. Many important discoveries about cell-cycle control have come from systematic searchesfor mutations in yeasts that inactivate genes encoding essential components of the cell-cycle control system.The genes affected by iome of

YEAST(Schhosaccharo mycespo m be)

Gr

I START

YEAST(5accharomyces cerevisiae)

@ Gr

Figure 17-5 A comparisonof the cell cyclesof fissionyeastsand budding yeasts.(A)The fissionyeasthasa typical eucaryotic cellcyclewith Gr,5, G2,and M phases. The nuclearenvelopeof the yeastcell,unlikethat of a higher eucaryoticcell,does not breakdown duringM phase.The microtubules of the mitotic spindle(lightgreen)forminside the nucleusand areattachedto spindle pole bodies(darkgreen)at its periphery. The cell dividesby forming a partition (knownasthe cellplate)and splittingin two. (B)ThebuddingyeasthasnormalG1 and S phasesbut doesnot havea normal G2phase.Instead,a microtubule-based spindlebeginsto form late in S phase;as in fissionyeasts,the nuclearenvelope remainsintactduringmitosis,and the spindleformswithinthe nucleus.ln contrastwith a fissionyeastcell,the cell dividesby budding.

('sllrw^uof Jo ^sauno)) uorlernleu 66a;o ssarordaql 6uunp uruop ua)iorqseqaoola^uareal)nuaqt'alnllto aqt ur uaasaq louue) slql q6noqtly 'use;doy(r66a aqt ra{e;ate;rnsaq1ur 1o lueubrd u/Aotqaqt pareldsrpspqq)rqM 'snal)nu aql Jo alrsaql smoqsdol aql reau]odsa;edaq1 'uo11ezrp1.ra;.ro; Ipear '66asndouayarnleur g-11 arn611 V

( xesraqnga;;o,(sa1lnol)'aseqd W alelJo )llsllal)ereq)aJeq)lq^^'spnq ulol} }lxaaq} alalduro) a6relqllm {;uro;tun lsa.ue{aq}'}lnsale sy'srsautloy(lpue slsolltLl aql le ua,rotb a^rllrJlsar luPrnLu9t)pf e ul (B) alelduor s;;a>'ernleredua] ]ouue)]nq aseqdeue 'a6e1s uorle;ndod;eulou a;r(r-;14)aql ol 6urptotteaztsut Ilen spnq'slla)lseaI 6utleta;r1otd;o e ul (V)'uoltetnw )p) e Iq palsarres;1allseel 6ulppnqlo I6oloqdrou aq1 ;-1 1 atn6t1 r.uri0Z (8)

ururs 0

'sepIAIp Surureluoc o^Jqua ue aleJaue8 ot 'Sut,r,roJ8 lnoqlu IIaJ luEIS elSuIS aql qcrqm ur'suorstt'trpaSanaalJpallel 'suoISIAIpIIeJ Jo asuenbas prder ,r{1$urqsr -uolsE ue sraSSlrtBBasndouay aql Jo uoUPZIpraC '(8-ZI arnBIJ) ,r(poqueutnq aql ul IIaJ a8pJa^euE ueq] urspldoldJ eJour sarull 000'00I suleluoJ pue Jala -ruerp ur ruru I JaAosr 'aldruexa ro!'sndouax 3or; aqrlo 33a aq1 'uoISIAIpIIaJ roJ papaau suraloJd aqt Jo salld{Jo}s a8Jel.{rrer qcrqm 'sletulue ^'(ueru;o s33apazr -llueJ luBI8 aql uI paz.{IeuBLllpeer aJealJ.{f,IIaJ eql Jo saJn}EaJ leJlruaqcolq aqJ

soltqul leuruv ur ,(lle)lutoq)o!8paz^leuv a8 ue) lotluo) al)^)-lla) '(z-zI arnSlc)palsarre

'(uMor,|s lou) lno unJsa^rasar r a q t u a q Mu o 6 u r p u a d a p lp)ruaq)orq -a;rl{r eq1;oe6e1sIue le ,{lprezeqdeq ]lpq'(uortlnpord61y se qrns)qlrvror6 pue srsaqlu{sorq ro; apl(r aq} }noq6nolq} {ressarausrleql ssaro.rd e ur }uar)Uap Jr'siuelnur)pJ-uou'lseltuo){g a6re; {;;erurouqe aLuolaq(aql anor6o} anur}uo) '(aldurpxa llllssluPlnurrp) aql asnp)ag srqiur 'aseqds Jo uorler]rur) alaldLuo) o] alqeunare{aq} 1eq1dalsrrlreds eq} ol aiuor {aql ;r1una;rb aql q6norqlsser6ord ol anurluolslla)luelnLuaqi'Illeurouqe suorl)unJlrnpord aua6luelnr.ueql q)!qMle 'arnleradura] (q6!q)arrlplrrsar aql ol 6urLurerrir uO (B)'(rolo)s1r,(q pale>rpur srlla) aqlJo eseqdaqt)apl{l eqllo saseqd apr^rpslla) lle ur puno}arepue,{1;eu.rrou aql lV (V) aql'arnleradural(Mol)anrssrurad 'luelnul rp) aArl!suos-srnlereduel e Jo ,ror^eqsq eq19-11 arn6r1

'ezISpnq pue 'pnq e sr tuptntu aql qtrq^a ]e ap,{t aq} ul lulod aql aleJlpul Jo aJuasqe ro asuasald eql :s[a] aql lE SuDIool]sn[ ^q ad,{1srql;o lseJJealJ-'{J-lleJ '(9-zI arnSlt) lpq -{aq] lulod slql ruroJrun e trelep uer a^\ 's1sea.{Surppnq uI 'ssaJSoJdraqunJ JoJpaJlnbar sI eua8 luelnlu aql tE pue Jo uollJunJ aql aJaq^ 'ern}eJadrue} lutod aql qoeal ^eql l!]un alc,{c IIaJ aql q8norql anulluoJ sllao raq8rq aql lV 'aua8 tuelntu aql Jo uorlJunJ er{l JJoqJ}I ^s ol (uourpuoJ anuJulsa.t aqt) aJnteJeduralraq8rq E ot pasIPJuaql pue (uoutpuoJ ansslruJacleql) aJnl -eradual l ol e lP pelEBEdordaq uec luelnut JpJ aAIlISuas-arnleJedlualV 'seJnl -eJadual llrol ]E uorsrnrpIIas .rrrolleo1 qSnoua IIe^ suorlJunJ lnq saJnlpJedrual 'suoltDinLuanqrcuas-aJnl q8rq fe uollrunJ ot sllEJuralord ]uplnlu aql qcrqM uI -DJadraalere suollelntu all^{J-llal PuolllpuoJ lsolN 'suoplpuol llJlsads UIEUaJ 'sI ur .,{1tro uortJunJ ol slleJ}JnpoJd aua8 lue}nu aql JI wql-IDuolllpuoc s ad^l '.snqJ 'pa}EB -oueqd paurclulelu puB pellalas aq uBc s}uE}ntu ,pJ rlaq] .{1uo JI -edord la^amoq 'alJ^3 IIaJ aql elelduo) louuel leql luelnru V aq louuec '1urod s1q] tsed IIac aq] la8 ol pallnber sI lJnpoJd aue8 leurJou aql lBI{13ul -tsa38ns'elct(t IIaJ aqt q lulod JrJrcedse te lsaJJeol sllao asneJ suollPlnlu aseql 'saua3 cp3 Jo 'saua8 alcrb-uolspqp-Ilac se u^AoDI erE suollelnlu aseql ;o .rfueyq

L9

IAZ9S

l^lr)lul-slu (8) tunrvu3dtAtl(H9rH)

run-LvutdFlsr(Mo'I) 1^l55ll/!U3d(v)

l1)A) lll) lHllo l llAUlAo

LSOL

'l)ejlxa )tuseldol^) aaJJ-lla) stqtul 6ulleradosr ulalslslolluo) al)l)-lla) aql 'stsoltLU pue uorlerrlder leql 6urle>rpur y1161osa;:{t paleadalqbnorql o6 uaql pue asuapuo)opre;tnur.urads aql dlv qll/vlraqta6ot'trol poppeale ra;>nuutiadspue'pal)allolsrr-use;do{r palnlrpunaqf 'sluauoduto)lla) raqlo ruor;urseldoy(> aql etetedaspue s66e 6ol;1o q>reqe6re;e uadoleelq ol pasn sr uorle6nplua) altuag .urelsls aa.tJ-lle) e u1a1>{>lla) aql 6ur{pn15g 1-1 1 ern6r3

ulur 09-0t :apib rrlolrur aer+-lla)

'suoprpuor alqellorluoJ puE pa$ldl.uls dlqSlq rapun slua^a alJ.,tr-llal alElndruErupue a^JasqouEJ a^\'slJEJlxe IIaJ qJns uI '(0I-ZI eJngIC) eqru lsal P ur alr^J IIaJ eql Jo slua^a dueu alruUsuoceJ puE sBBasndouay ]UJ04 ruseldol,b eJnd lsorup aredeJd ol alqrssod osle sr ]I 'uorsseJSordalcdc-1ac uo ]caJJaJreql aurruJalep ol 33a ue olur seJuBlsqns ]sa] lcefur ol ,,(seadlanrlelal sl ]I 'azrse8JplJraql sl srs^lpup a1c,,b-1acro; solJqua dlJea eseqtJo aBEluB^ppJaqlouv 's11acralq8nep oml otur uorle8eJ8esslr pue etuoue8 aql Jo uorlecqdnp aq1 -sluarueJrnbar pluaurepury lsoru aq] a^arqf,Eol papeeu unurrurru eql ol perJrld -rurs pue urrop paddrrls uals.{.s alcdc-1ac eql Jo sBuDIro ^ eql IBaAeJeJoJ IoJluoJ 'selJ.,(c -e;eqt saIJ[J 71accruofuquta fpaa asaqJ IIaJ xaldruoc eJoru ur a]EJedoleql (JalEI pessnf,srp) surstueqlaru IoJluoJ eql Jo ^ueur Jo qllr,roJ8Jaqlre Jo aJuesqe aqt rn uorsrlrp prder ,{lSurpaarxe Jo alqpdec snq} erB 'apqdosotg,{g 1pr3 aqr puealnsrdg ruplJ eqtJo asoql se [e^a se 'sndouay 1o s11ac cuod;qura i{yreaaql 'saseqdzg gI ro Ig alqelcarap salnurlu pue saseqd lnoqllm'qcea ]noqeJo ntr S olur papwp ,slplra}ur a}nur{u st a1c-,bqJEg 'sJnoq ulr{tlm sllao (zrZ)g60t Suronpord }noqp 2 -69 1e 'dlsnouorqcu,{sssalJo aJoru tnJf,o suorsrlrp eql ,salnur{u 06 }noqe lxeu I I se{et tpq} uorsrlrp }srrJE raUV '16-21 arn81g)s11ac raleurs Jo spupsnoq}

'(lou stitito;aq 6orJ aql ale)s aures aq] uo are,rnor dol aql ur lnq) s6u;anerp aql'6urpea; sur6aq a;odpel lle aql uaqM{;uo s1re1s qlMorg'aztsaues aq} sureurer oftqLuaaLl}pue'uotst^tp q)ea q}!Mre1;eus,(;anrssat6ord ta6 s;;at aqf 'oMl to {ep e ulqtlmalodpelJelnlla)rllnue 6urr.uro1-salnurur 0€ {ranea;>I>uotst^tpauo }o atel e te ,(1;erlrur-I;plder Ilan sanea;> 66e eql'uo!tez!llue1 uodn'(lZ ratdeql ur passn)srp) 66a ue olur salnleu I;;eur;pue 6o.11 taqlou aq];o,{reno aql ul sqluoul ,(ueu.r .ro;6ulprnrplnoqllM sanorba/roo aq1'sndouayur e6eaea;>66e pue qlMoJ6ey(rog 6-4 1 arn6l1

r

I 6 o J Jl l n p e u e seuo)aqpue'stsoqdlouJelaut sao6rapun'snnot6'spea;e;odpe1 uruj I

'. -

:,r,r:.+i.:iiirrii;

////////

(srnoq) 6urnirol6lnoqltM saprnrp66a paztltpa+

NOIIVZt'lU_Ull

(sqluour) 6urprnrplnoqlrnl saaol6ep(roo

epl3 11a1 aqJ_:1.I raldeq)

BS0t

('rarusor) olr)g)JoIseUno))'aIp lua)salonU pare qllMpeurets areslla)aql lv 'pail6) serpoqrlue npi€-lluelualselonu qllM6ulleqello, patedaldpuepaxuseMansslleql q)!qMlaue 'nprg peleqel-npl8 aqrJoslle)le!laqtlda ol pesodxe seMqsuaql'ln6 ilsuelqaz Jo qdeJ6oJ)lru 6u;1aqe1 uV'slla)eseqd-S a)ue)seronUounLuur ZI-LL etn69

'S 'IC Jo sqlSuel aql eulruJelep o] dJleruoltc rlog asn osp uec eM'(8I-ZI aln -t;g) s1aclo srequnu e8rplJo srs,(1euuJlleruotnu pue pldeJ eql smolle lqlrqr.,'taj -auoilb mo[ B pup se^p Surpurq-ygq ]uarsarongJo esn el{lfq palelllrcugdpearB sr qceordde sql 'eseqd S Suunp selqnop qJIq ^ 'lueluoJ OtO sll SuFnseaur

dqsralcdc s'qrrer*uijj"?;:uj,:lTff;1j"#i"'ul,l1X""o ileceq]"' peqceer " pue 'IC olur aseqd ry q8norqr 'aseqd htr olul z9 q8norqr ssarSordo1 lac aseqd -S uE JoJselel ]r 3uo1moq eurruJalepuBJ e a'elullJo sqfuel peJnseeu ro3 alcri.c eqt punor€ enurluoo ol slleJ aql 8u!vro[e puu npJg go aqnd e 3un€ Lq 'uo1] -lppe uI 'aseqd htr uorlernp eq] aleu4lse uet at't''(xapu! Jlioryu eql) slsotlru ul Jo sllao Jo uoprodord eql uorJ ,(yrupurrg'uoll€rnp alrdc-1ac eloqm aqt Jo uollcerJ u se aseqd S Jo uorlernp aq] ateurltsa uEf, ar'l''(xapu!Suqaqay eql) pelaqel aJE 'npJg teqt uopepdod e qcns ur slleJ Jo uoqrodord aq] ruorc Jo aslnd;auq e ,tq 'dlsnouorqc paleqel eruooeq pue luelsul due le aseqd g ul eq ill \ o/o1V-OE lnoqe -u,,(se1nq fprder Supera;rtord ile arp leq] s[a];o uorlulndod e ur ,{1pctd,,(; 'Qt-Lt a.rnt;g)sar -poqrlue palerodrocut anuq pazqunsll ueql ale r{.q r{tyn Sururels llpJg npJg-rlue '(nprg) aurprrndxoap-oruorq Sopue aupnudqt teqt Ielrnu IIaO [elsgluu eq] sE qJns 'VNC pazrsaqtur(sfpu.au olut pelerodrocul eJe ]eql sepcelotu alqEZIlBnsI qtr^arueq] Surdlddns,{.qadorsorcl{u eqt uI palJlluepl eq uec slac aseqd-5 dpep -urrg '(alpurds Jrlolnu aq] Sulearral) sepqruoJJlru aql su qJns sluauodruoc repl -1accgrcads azruSocar]eq] salpoquue qllm Jo (slsollru uI seluosoruoJqcJo uou -Esuepuoc eql lealar qcqnlr) sari.pluarsarong Surpurq-VNq tlllm s11acSutumls Lq uoqrsod alcdc-1aclnoqu senlo Ipuop1ppputu8 uec aM'slseuDloltc 3o ssacord aqt ur pelJesqo aq upc sJeqlo '(II-ZI arn8rg ees)slsolltu uI eJEpue dn papunor a^eq slleJ eql Jo uollJu4 E leql slealer eJnllnJ ut Suqura;qord sgac uEIIBtutuEru 'adocsorcrru p qlp s11ac3urn1 ]e {ool o1 ,tldruts st ;o uorlelndod e 1e acue13y ,i.urrn au6 ;a1crb 1ac aqt ur peqJEeJsuq IIaJ IeruIuP ue a8els leq ^ ile] ao\ uec ,rroH

u! palpnlseg ue) uolssal6oldel)I)-lle) s{ery1snolren '(97 raldeq3 uI pessnJslp)JaJueouI sloJluoJ asel{l ssol Jo aq1 SurpuelsJepun JoJ osle lnq sensslt uI sJeqlunu IIaJ Jo sloJluoo IEIuJou eql ('ralqang-lq)arqlV JoJLluo 1ou lueuodrut aJe salpnls qJnS 'suspe8ro replacrlpu Surpuelsrapun ratuanDJo ^satrno)) 'slselqorqu ur uoqereJrTord 1ac Jo loJluoc aq] Suurano8 srustueqcalu Julncalou aql Surur ler areqder6ot)ruruol])ala6uruuets -ruexe JoJ ^,(lercadsauaeq alpq sllal uelprurueru pemlpJ Jo salpnls InJesn sltltu! slla)aqf 'ernlln) ur 6urlere;r;o,rd 'loJluof, alcri.c-11ac uI peAIoAuI suralord aq] Jo sls slle) uelleu.rureyg 11-41 arnOrl -d1euepcruaqf,orq ol alqeuerue aru ,(aql pue 'sluela a1c[c-1acJo suopelJesqo uiri 0 I 's[ac pcrSolo$.c pelrelep ^ olle 01 a8rel ,tlluagelJJnsare sllaf, eseql 'uolllppu uI snoaua3oruoq ,(lecrlaua8 Jo eoJnos pellru[un ue apnord ,bql asnecaq-rtne -raua8 pue-selpnts alcrb-1ac ro3 pasn dlapu ere saull IIaJ qsns dSolorq roJ [ac 'sauq 'leturou lou are daql q8noqtff IIaJ ,.pazqeuouul,, sP eJnJIncur ,{laltugapur aleragqord uraql dlaq reqr suollelntu o8rapundleuolsef,Jo slleJ u€IIEIUTUPIN 'ralpl ssncsrp eM se 'aJuaJsauas an!ruJlldal pelleo sseloJd e 'suo1s IIaJ -1rr1p ra4e Sutpnrp aseec dlluaueurrad 'aldruuxa JoJ 'slsEIqoJqIJueunH 0?-gZ 'suo1]1puoc 'salczb uorsr^rp Jo Jaqrunu palltuI E JaUP 8upplp dols uago daq] pJepuets ur paJnllnc aJBsanssll uelPruruBur leruJou ruo4 slles uel{ \'Je^all/Ioq 'uorlerrldurol B sr eJeqJ '(II-ZI a.rn8;g) sJopeJ Jeqlo pue sluelJlnu lelluesse ;o acuesard eql ut saqsp eJnllnc cpsuld ur uvrorS pue sJorun] Jo senssu IEruJou uro4 palelosr ueeq a^eq lpql sllef, esn eJoJaJaqlIoJluoJ alcdr-11ar uelprurueru uo serpnls lsohtr 'lPruruPrulf,Blul ue uI sllal lPnpmlpul a^Jasqo ol r{.sualou sl }I

slla) uerletxurewpelnllnJ ul palpnlso8 ue) lorluo] ol)^)-lle) 650[

llf^)

Ilf

lHl- lo M3lAUlAo

tuels,{sIoJluoJ eql 'tua^e alJ^J-lla3 qf,EeJo uoFaldtuoJ aql JoJaturl Jo }unourp paxrJ e saprloJd ]eql Jarurt pauueJSoJd ,{lpr8rr p a{ll sr ruals.{s loJ}uoc aql -rarlrea passnJsrp sa1c,b 1ac cruodrqura umop-padduls aql ur uaas se-ruJoJ '(1I-ZI arn31g) acuanbas lsaldrurs stl uI las E ur a1c-,b1ac eql Jo sluela aqt sra8 -3rl teql JotellrJsoJo rarurl e a{rl qJnru salerado rualsrb loJluoc alcfc-11acaq1

al)I) lla) aql ro stua^l tofew ar{}sta66!rlurals{slolluo) al)^)-lla) aql 'ap,{r 11araql Jo slua^a aq] eleurpJooJpup eurl ol raqlaSol 4roan.,{aqlmoq pue rua}sLsIoJ}uoJ aq] Jo slueuodruoc uratord aqt ssnJsrpuaq] aM'salerado rualsds lorluoc alodc 'uol}Jes sq} uI -[ec qJIqM eql uodn saldrcurrd crseq ar{} JaprsuoJ }sJrJellt 'uo os pue 'uope8ar8asaruosoru -oJqJ 'uorleJrldar y111q;o sassacord aql uroyrad lBql suralord aq] ruo4 lJurlslp are .{aql }eq} uotezrleal aq} qryrrrSuop 'rua}s,{.s IoJluof, aql Jo suralord La1 aql yo uortecrJrtuapraql qlu!\ s086I elpl aq] ur eruef,q8norqtlearq roferu V'sallasruaql palloJluoJ llroqatuos srsauqo{c pup 'srsoutu 'srsaqluf.sVNCI Jo sassacordaql Jaqlaqm ro 'ure1s,{s aleredas e sem aJaql Jaq}aqm JealJ uale }ou spl }I IoJluoJ 'lleJ eql aprsu xoq uals,,(sIoJluof, alc,rtc-1ac eql 'stuala asaql )Je1q e,(1durs se^,r,t SuryyorluocureunJ aqt purqaq .{e1leq.u;o papr ou peq lnq srsauqo{c pue 'srs -olrlu 'srseqlu,r(s ygq;o noqs laddnd aql paqcle^ slsrSolorq1ac srea.{,tuelu Jo{

wltSAStoutNo) ltf^f-tt])

IHt_

'IoouoJ a7c[t-71ac ulofuacna lo mampatltun a oi pal anoq-atnqnJ ur slp) uvtlalutuDrapuo'sofuqua lvlaruD'slsoaf.Sutpnput-stualststo aBuataptm o ut satpnlspuD 'uounlonaSuunp panrasuoJ[7r.13tt1 uaaqanaq lor]uor puv uo4aztua8to a1c,(t-17a3'uorssatSotd a\ctc-yat ala\nBat qauBp r\lnnar2r#a puD ralnl -Iarortul snouon ua14m'z) puD It pa1pcsasaqddoB[q paryndas [11ansnatDasotldyg pua asaqdg'(usaut4o1[c1 oml ut s1t1ds uaqi llal aqi puv'(srcoilut)npnu pnprnrput ojul pala&at&as an sataosotuottlrpa1o41datatll 'awt1d Suunq 'a1c[caql moqBnotql [1sno 779 -tluquor palauldnp atu siuauodutoJIIar raLlp lsola soaraqm'a1c[.c 77acaql to asaqd g Suunp sJnJJououuJr1dnpawosowoJqJ'snalnlqBnop oml olur slualuoJasotpto uorl -nqu1s1p,tq pamollol 's1ua1uor sJIaJaqllo uo4act\dnp qum su8aq [1\onsn uosrnlp IpJ

{reuun5 ( s t r u n^ r e i l r q r e ) l l a ) r a o v N o + o l u n o u r ee ^ t l e l a J L '

J C

3 5

q o

'a1c.rtc aql q8norql sserSordslleJ aql ^ oq IeaAaJsllaf,Jo uorlelndod pazruorqcufs p qJns uo slueuraJnspeur]ua]uoc y111q:ap.,{cIIaJ aq};o aseqd relncrlred auo ur aq o} pa}Jalesarduaaq a^eq }eq} sllal palaqel-VNq Jo uorlelndod e arup rano 8ul,r,ro1o;,r(q'sasuqd IN + zC pue

'uo!lelndod s r q ]u ! W 1 z 9 u e { l r a b u o s; r L 9 1 e q61u r m o q'se s e q dl A + z g u t u p q }L g ur slla)jo sraqunu;aleer6arearaq]]eql sate)tputpolellsnlltasp)aqt ul slla)Jo uorlnqulsrp oqf 'aseqos ur arepue vNc Jo ]unoue aletpaulalutue a^eq]eql asoqlpup'aspqdW ro z9 u! arepup (lualuo)VNO LDeql o)tMl) I11n;e a^eqleq] asoql'lD ut atoJaraql alp VNCJo luauraldulotpale>r;dar pup VNC;o iuaua;duo: paletr;datunup a^pq]pq] osoq]:sarrobalet aalql otur llpjslla)aql lla) q)pa ul VNCJo lunourpaq] o] ;euolt.rodotd I;l>atrp sr3)ua)saronlJJo lunouJeaq] ]pql os'vNc ol sputqlt uaqMlua)salonlJ sau,ro)aq leqr a,{pe q}!Mpau!e}saraMaloLlpazl;eueslla)aql'(z-g arnbr3aes-erua)salonlJltaql ol 6urpro>>e slla)tlos ol pasnaq osleup) 'SfVl 'rauos ro pallp) e osle'lataLuol^) MolJV) lla) pale^tl)e-a)ua)salonU 'Jalauol^) pautuilalap MolJ e ut st slla)lenpt^tput sltJotualuo) VNC aql uaqMuorle;ndod;;at 6urlera;r;o.rd e ro; paurelqostlnsor;errdIl smoqs qderbsrql'.ra1eruo1fu ltog p qllm tualuot VNO,o srs(;euyg 1-2 1 aln6r1

a1r{311a1 aql:/ [ raldeLl) 090]

1061

THECELL-CYCLE CONTROL SYSTEM

lsall DNAreplicated? l s e n v i r o n m e n ft a v o r a b l e ? G 2 l MC H E C K P O I N T

Are all chromosomes attached to the spindle? METAPHASE-TO-ANAPHASE TRANSITION END T R I G G EARN A P H A S A P R O C E ETDO C Y T O K I N E S I S

ENTER C E L LC Y C L E A N D P R O C E ETDO S P H A S E START CHECKPOINT l s e n v i r o n m e n ft a v o r a b l e ?

in these cells is independent of the events it controls, so that its timing mechanisms continue to operate even if those events fail. In most cells, however, the control system does respond to information received back from the processesit controls. Sensors,for example, detect the completion of DNA synthesis, and if some malfunction prevents the successful completion of this process, signals are sent to the control system to delay progression to M phase. Such delays provide time for the machinery to be repaired and also prevent the disaster that might result if the cycle progressed prematurely to the next stage-and segregated incompletely replicated chromosomes, for example. The cell-cycle control system is based on a connected seriesof biochemical switches, each of which initiates a specific cell-cycle event. This system of switches possessesmany important engineering features that increasethe accuracy and reliability of cell-cycle progression. First, the switches are generally binary (on/off) and launch events in a complete, irreversible fashion. It would clearly be disastrous, for example, if events like chromosome condensation or nuclear envelope breakdor.trnwere only partially initiated or started but not completed. Second, the cell-cycle control system is remarkably robust and reliable, partly because backup mechanisms and other features allow the system to operate effectively under a variety of conditions and even if some components fail. Finally, the control system is highly adaptable and can be modified to suit specific cell types or to respond to specific intracellular or extracellular signals. In most eucaryotic cells, the cell-cycle control system triggers cell-cycle progression at three major regulatory transitions, or checkpoints (see Figure I7-I4).The first checkpoint is Start (or the restriction point) in late Gr, where the cell commits to cell-cycle entry and chromosome duplication, as mentioned earlier. The second is the GzlM checkpoint, where the control system triggers the early mitotic events that lead to chromosome alignment on the spindle in metaphase.The third is the metaphase-to-anaphase transition, where the control system stimulates sister-chromatid separation, leading to the completion of mitosis and cytokinesis.The control system blocks progression through each of these checkpoints if it detects problems inside or outside the cell. If the control system sensesproblems in the completion of DNA replication, for example, it will hold the cell at the G2iM checkpoint until those problems are solved. Similarly, if extracellular conditions are not appropriate for cell proliferation, the control system blocks progression through Start, thereby preventing cell division until conditions become favorable.

Figure'17-14Thecontrol of the cell cycle.A cell-cyclecontrolsystemtriggers processes of the cellcyclethe essential mitosis,and suchas DNAreplication, controlsystemis cytokinesis.The representedhereas a centralarm-the controller-that rotatesclockwise, processes when it triggeringessential on the outer reachesspecificcheckpoints dial.Informationaboutthe completionof events,aswell as signalsfrom cell-cycle cancausethe control the environment, systemto arrestthe cycleat these The mostProminent checkpoints. occurat locationsmarked checkDoints with vellow boxes.

1062

Chapter17:TheCellCycle

TheCell-Cycle ControlSystemDependson Cyclically Activated (Cdks) Cyclin-Dependent ProteinKinases Central components of the cell-cycle control system are members of a family of protein kinases knor,rm as cyclin-dependent kinases (Cdks). The activities of these kinases rise and fall as the cell progressesthrough the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle. An increase in Cdk activity at the G2lM checkpoint, for example, increasesthe phosphorylation of proteins that control chromosome condensation, nuclear envelope breakdown, spindle assembly,and other events that occur at the onset of mitosis. Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins that regulate these kinases.The most important of these Cdk regulators are proteins known as cyclins. Cdks, as their name implies, are dependent on cyclins for their activity: unless they are tightly bound to a cyclin, they have no protein kinase activity (Figure f7-f 5). Cyclins were originally named becausethey undergo a cycle of syrthesis and degradation in each cell cycle.The levels of the cdk proteins, by contrast, are constant, at least in the simplest cell cycles. Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of the cyclin-cdk complexes; this activation in turn triggers cell-cycle events. There are four classesofcyclins, each defined by the stageofthe cell cycle at which they bind cdks and function. All eucaryotic cells require three of these classes(Figure l7-f 6): l. G1/S-cyclins activate Cdks in late Gr and thereby help trigger progression through Start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase. 2. S-cyclins bind Cdks soon after progression through Start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events. 3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2lM checkpoint. Mechanisms that we discuss later destroy M-cyclins in midmitosis. In most cells, a fourth class of cyclins, the Gl-cyclins, helps govern the activities of the Gr/S cyclins, which control progression through Start in late G1. In yeast cells, a single cdk protein binds all classesof cyclins and triggers different cell-cycle events by changing cyclin partners at different stages of the cycle. In vertebrate cells, by contrast, there are four cdks. TWointeract with Grcyclins, one with G1/S-and S-cyclins,and onewith M-cyclins. In this chapter, we simply refer to the different cyclin-Cdk complexes as G1-Cdk, Gr/S-Cdk, S-Cdk, and M-Cdk. Thble l7-l lists the names of the individual Cdks and cyclins. How do different cyclin-cdk complexes trigger different cell-cycle events? The answer, at least in part, seems to be that the cyclin protein does not simply activate its cdk partner but also directs it to specific target proteins. As a result,

G,iS-cycl in

',,GrtM

i _ -m e t a p h a s e - a n a p h a s e iM

APC/C

Gr/s-Cdk

S-Cdk

G1

cyclin-dependent (Cdk) kinase Figure17-15Two key componentsof the cell-cyclecontrol system.When cyclinformsa complexwith Cdk,the protein kinaseis activatedto trigger specificcell-cycleevents.Without cyclin, Cdkis inactive.

Figure 17-16 Cyclin-Cdkcomplexesof the cell-cyclecontrol system.The concentrationsof the three major cyclin typesoscillateduringthe cellcycle,while the concentrationsof Cdks(not shown) do not changeand exceedthe amounts of cyclins.In lateG1,risingG1lS-cyclin levelslead to the formationof G1lS-Cdk complexes that triggerprogression throughthe Startcheckpoint. S-Cdk complexesform at the start of S phase and triggerDNAreplication, aswell as someearlymitotic events.M-Cdk complexes form duringG2but areheld in an inactivestateby mechanismswe describelater.Thesecomplexesare activatedat the end of G2and triggerthe earlyeventsof mitosis.A separate regulatoryprotein,the APC/C,which we discusslater,initiatesthe metaphase-toanaphase transition.

THECELL.CYCLE CONTROL SYSTEM

1063

Table17-1TheMajorCyclins andCdksof Vertebrates andBuddingYeast

G1-Cdk GrlS-Cdk 5-Cdk M-Cdk

cyclinD* cyclinE cyclinA cyclinB

Cdk4 Cdk6 Cdkz Cdk2,Cdkl** Cdkl

Cln3 Cln1,2 c l b s ,6 clb1,2,3,4

cdkl "* cdkl cdkl cdkl

D1,D2,and D3) " TherearethreeD cyclinsin mammals(cyclins **The originalnameof Cdkl wasCdc2in bothvertebrates andflssionyeast, and Cdc2Bin buddingyeast

each cyclin-Cdk complex phosphorylates a different set of substrate proteins. The same cyclin-Cdk complex can also induce different effects at different times in the cycle, probably because the accessibility of some Cdk substrateschanges during the cell cycle. Certain proteins that function in mitosis, for example, may become available for phosphorylation only in G2. Studies of the three-dimensional structures of Cdk and cyclin proteins have revealed that, in the absence of cyclin, the active site in the Cdk protein is partly obscured by a slab of protein, like a stone blocking the entrance to a cave (Figure l7-L7A). Cyclin binding causesthe slab to move away from the active site, resulting in partial activation of the Cdk enz)ryne(Figure l7-l7B). Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site. This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events (Figure I7-I7C).

Can InhibitoryPhosphorylation and CdkInhibitoryProteins(CKls) SuppressCdkActivity The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. Several additional mechanisms, however, fine-tune Cdk activity at specific stagesofthe cycle. Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin-Cdk complex. Phosphorylation of these sites by a protein kinase knor,vn as Weel inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase knor,rm as Cdc25 increases Cdk activity (Figure 17-18). We will see later that this regulatory mechanism is particularly important in the control of M-Cdk activity at the onset of mitosis. Binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk complexes. The three-dimensional structure of a cyclin-Cdk-CKl complex reveals

Cdk-activatingkinase(CAK)

cyclin

activesite (A)

TNACTTVE

(B) PARTLY ACTIVE

activating phosphate

(c)

Figure 17-17 The structuralbasisof Cdk activation.Thesedrawingsare basedon of human structures three-dimensional Cdk2,as determinedby x-ray locationof the bound crystallography.The enzymeis shownin ATPis indicated.The three states.(A) In the inactivestate,without cyclinbound,the activesite is blockedby a regionof the proteincalledthe T-loop(red). (B)The bindingof cyclincausesthe T-loopto move out of the activesite,resultingin partialactivationof the Cdk2.(C) Phosphorylationof Cdk2(by CAK)at a threonineresiduein the T-loopfurther the enzymeby changingthe shape activates of the T-loop,improvingthe abilityof the enzymeto bind its protein substrates.

1064

Chapter17:TheCell Cycle

that CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive (Figure f 7-fg). Cells use CKIs primarily to help govern the activities of Gr/S- and S-Cdks early in the cell cycle.

TheCell-Cycle ControlSystemDependson CyclicalProteolysis \fhereas activation of specific cyclin-Cdk complexes drives progression through the Start and G2lM checkpoints (see Figure 17-16), progression through the metaphase-to-anaphasetransition is triggered not by protein phosphorylation but by protein destruction, leading to the final stagesof cell division. The key regulator of the metaphase-to-anaphasetransition is the anaphasepromoting complex, or cyclosome (APC/C), a member of the ubiquitin ligase family of enzymes.As discussed in Chapter 3, many of these enzyrnes are used in numerous cell processesto stimulate the proteolytic destruction of specific regulatory proteins. They transfer multiple copies of the small protein ubiquitin to specific target proteins, resulting in their proteolytic destruction by the proteasomes. Other ubiquitin ligases mark proteins for purposes other than destruction. The APC/C catalyzesthe ubiquitylation and destruction of two major proteins. The first is securln,which normally protects the protein linkages that hold sister chromatid pairs together in early mitosis. Destruction of securin at the metaphase-to-anaphasetransition activatesa protease that separatesthe sisters and unleashes anaphase.The S- and M-cyclins are the second major targets of the APCi c. Destroying these cyclins inactivates most cdks in the cell (seeFigure 17-16). As a result, the many proteins phosphorylated by Cdks from S phase to early mitosis are dephosphorylated by various phosphatasesthat are present in the anaphase cell. This dephosphorylation of Cdk targets is required for the completion of M phase, including the final steps in mitosis and the process of cytokinesis.Following its activation in mid-mitosis, the APC/c remains active in G1,thereby providing a stable period of Cdk inactivity. \Mhen G1/S-Cdksare activated in late Gr, the APC/G is turned off, thereby allowing cyclin accumulation to start the next cell cycle. The cell-cycle control system also uses another ubiquitin ligase called SCF (after the names of its three subunits). It ubiquitylates certain cKI proteins in late G1 and thereby helps control the activation of S-cdks and DNA replication. The APC/C and SCF are both large, multisubunit complexes with some related components, but they are regulated differently. APC/C activity changes during the cell cycle, primarily as a result of changes in its association with an activating subunit-either cdc20 during anaphase or cdhl from late mitosis through early G1. These subunits help the APC/G recognize its target proteins (Figure l7-2oL). SCF activity also depends on subunits called F-box proteins, which help the complex recognize its target proteins. unlike Apc/c activity, however, scF activity is constant during the cell cycle. ubiquitylation by scF is controlled instead by changes in the phosphorylation state of its target proteins, as F-box subunits recognize only specifically phosphorylated proteins (Figure I7-20H.

actrve cyclin-Cdk complex

inactive p27-cyclin-Cdk comprex

Cdk

activating phosphate INACTIVE

Figure17-18The regulationof Cdk activity by inhibitory phosphorylation. Theactivecyclin-Cdkcomplexisturned off when the kinaseWeel phosphorylates two closelyspacedsitesabovethe active site.Removalof thesephosphatesby the phosphatase Cdc25activates the cyclin-Cdkcomplex.Forsimplicity, only one inhibitoryphosphateis shown.CAK addsthe activatingphosphate, as shown i n F i g u r e1 7 - 1 7 .

Figure17-19Theinhibitionof a cyclin-Cdkcomplex by a CKl.This drawingis basedon the threedimensional structureof the human cyclinA-Cdk2complexboundto the CKIp27,as determinedby x-ray p27 bindsto both crystallography.The the cyclinand Cdkin the complex, distortingthe activesite of the Cdk.lt alsoinsertsinto the ATP-binding site, furtherinhibitingthe enzymeactivity.

']ua^e elc^o-lleJ JUrJadsE sJaSSulqclq^\Jo qcpe (seqUrMs Ipcr -ureqJorq Jo selJasP elPArlJEo1dlsnoluouolnE ^llp4uessa saleJedoqcrq ^ ({JoM -lau e ruroJ ol JeqlaSol pe{ul ,r(leuorlcurg are suralord asaq; 'rualsrts lsnqoJ IoJluoJ alcdc-gaceqlJo slueuoduroc roferu eqlJo aruos sezrJer.uruns Z-Llelqe;

seq)lrMsle)!ueq)otg ,o lro/vuaNe sp suolpunl u,lels/(slolluoJ al){)-lle) eql 'rrl\/\ou)lun 'suon aJE sJeqlo ,tueur;o suorlJury aql lnq -rury a1cl.c-1leJrrlr,roDlqlyv\ sureloJd apocua saua8 asaql;o eruos 'a1r",b 1ac aql Surrnp elellrJso slelel esoqm sVNUur epoJua saua8 aql Jo %0I lnoqe ,aldruexa ro; ']sear( Suppnq u1 'Sursrrdrns aJp serpnls esar{l Jo s}FseJ aql 'a1cdcilac eql q8norqt sassarSord IIaJ aql se auoua8 aqt u1 saua8 eql Jo IIe go uorssardxa aq] ur sa8ueqc azf1eue ot (B Ja]deql ut pessncsrp) sderreorcrur VNC esn ueJ aAA 'slleJ lsoru ut sle^el uqcdc yorluoc dlaq 'aldruexa ro; 'uorldrrcsuerl aua8 uilcrb ur sa8ueq3 'uorlep8ar;o Ialal leuorlrppp ue sapnord IoJluo3 Ipuou 'suratord -drrcsuerl :alamoq 'sadft 1ac tsoru Jo salcrb 1ac xaldruoc aJotu eql u1 la8rer 4aql puu sase8q uppblqn pue qp3 3o uopeln8ar eqt eAIoAul teql sursru -EqJeru puorldrrcsuerl-lsod uo f1arusn1cxaspuadap loJluoc alcdc-1a3 rncco lou saop uopdlrcsuerl auaB terlJea pessnosrpa1c[c lac cruortrqura 3o4 aql uI

uorteln6auleuotldulsuell uo spuadacoslv lotluo) al)/()-lle) seuAzua uorle;r$rnbrqn

(

1v{osvtJ.oud trtttX: lO F NOt-LVCVU9tC

m

nbrqn

+

(r)))

*CJ u t a l o l d r o l r q r q u1 r P1

utlrnbrqn{;od

315 Aq srs{;oalord1o lot1uo: 191

seu{zua uorle;Ipnbrqn

.t

'ltunqnsxoq-; rr1>ads e Iq pazruboralaq o11a6le1aql smolle iumoqs ,ura1o.rd ly) aqt se q)ns 1a6le1 e ;o uor1e1{roqdsoqd aql.sad,{1luaragrp {ueu ate ataql q)tr.lMJo ,sutalold xoq-l palle)sltunqns6urpurq-elellsqns uo spuadep115ase611 urlrnbrqn aqr 1o,(r;n;lreaql (g) 'eulosealoid e ur paper6appue pazrubotat uaql s! tabrel perelr(lrnbrqn{;od aq1'uralotd1a6le1 aq1oluo saln)alour urlrnbrqnaldlrlnu sle;suerlf/)dv aql'zl pue I j palle)suralotdleuorltppe omtJo dlaq oqr qll6'sulalord1ebre1 raqlo pue ur;r{t-y1uo sa:uanbasprre ourue lrpads sezru6olalq)tqrvr,gz)p) lrunqns6utlenrlreaql qllM uotlet)osse Iq srsoilu ut pale^r])esl l/)dv aqJ_(V) 'ep{r ;1eraql 6urrnp J)S pue )/)dV r(q sgs(;oalord1o lorluo) aq1 g7-4 1 arn611

1f; urlrnbrqn

t )/)dv a^rpe 1\l/1

tu,)^r-^

\

)l)d

lleul

)-'{

/t (02)p))lrunqns 6urlenrpe

)/)dV ^q srs{;oalord+o loJ}uot (V)

s90t

wlr_s^S tourNo) rt)^)-ttl) lHl_

alouexa lo]'sall^) lla) )tuoi(lqulaIuea ul 6urssrr.u ere,{ueu :sad{t;;ar;;eur luasald lou aresusrueq)aluasaqf'fdo, slua^a apAr-;;a> alaldurorurpue'a6euep Jplnlla)ellxa aqt tnoqp lla)'luauruorAUa uorteuroJur aprnordq)rqM'sursrupq)aLu ^rollqlqulsnorren Iq pa)uanllul os;esrxa;duor qtea;o ,{1rnr1:e aql latPl lrelapalourur passn)srp sv '(/Aoila() saxa;duor 1p1-urp,(>1o seuas e Jo slsrsuo)uals{s ;oiluot a;r,{r-;;at aql jo aro) aql 'ua1s{s ;o,rluora;r{>-;;a> aqt Jo rvra!^ralouy 17-11 arnbrg

lp)-ll-

lP)-S -

rg

lp)-slrD

)p)-t9

TT \\ a6euep VNC pale:rlda:un VNC

luauuoJrAua relnlla)erlxa e l qe r o ^ e ]

'llelep arotu uI se8elsa1c,{c-1ac 'aseqd S qllr,r Surlrels 'polrad Ic elqBls E uI 8uI esaql ssnJsrpol ,{peal ,{i\ouare a A'Ja}EIssnJSIpa^ se -llnsar 'srsol[u .,!r,,r.r1ce rage 1p3 ssarddns o] aleJoqelloJ slusluBqcaru alduptu 'a1a1duocsr srsolnu uaqr\\ ' ( I Z-Z 1 arnS;g) slsolltu Jo uopalduroc aql pue uoue8 -aJBasprlpruoJqJ-JalsrsSutqsealun,{qaraqt 'uolJlsuerl aseqdeue-01-aseqdelau '\zJpJ JolPAIlsB aql le surlJ^c puE urJnf,as Jo uollcnJlsep aql sJeSSul str qtr^ raqlaSol '3734y aqr -{1eurg'alpurds rl}o}Iu aqt;o rolenba aq} }e splleru -oJqc Jalsrs;o luaruuSrle aql o] Surpeal 'slsolllu .{Fua ;o sluala aql pue lutod -{3eqJ htr/zCaql q8norqt uorssarSordsraSStr}uaq} uol}PAIlcE{pl-N 'slso}IluJo slua^a .{Fea aruos o} salnqlJ}uoo osle pue aseqd 5 uI uol}eJlldnp eluosoluoJq3 'Ja}el ssnJSIpallr salerlrul qrlq \ ',{tnr}re )pl-S Jo aAP^ B qseapn qpl-S/i9 srusrueqJeur.,(g'lurodlcaqJ uels eqt q8norqt uolsserSordseAIJpuaqt {pl-S/15) 'sur1c,r{c-S pue -S/I9 Sutpocua sauaS;o uolsseJdxe Jo uorlelrlce Suqpsar aq; aql selBlntups uJnl ur qJrq ^ 'Ipl-I3 Jo uoIleAIlJB aql alBlntulls spuSts pu -Jalur pue Ieurelxa snoIJeA'lqBIJ ere uouera;t1ord IIeJ JoJsuoplpuoo ueq \

,{}!nllre i(sleurueuu LZd'$ee(Eulppnq slqlro; palrnber {1;ensn ura}ord 1a6let;ouor}el^ror.ldsoqd 'lojluor Lgur pa^lo^ur saz^lele) lfs u! t)lS)sl)) oLuos 6urpn1>ur suralord {ro1e;nbar Jouoltep0lnblqn rernrlre lp) ^q pallqlqu! pueeseqdeue :LglnoLl6norql tr-lp) rayp^lr^rl)eJ/)dV surelureu leqt l!unqns6utle^tl)e-)/)dv luol}lsueJ} {}tnr}relp)-ti! ,(q palelnurl}s 0z)p) aseqdeue-ol-aseqdplau te )/lldv jo uolle^ll)elelllu!sra66u]lsllallleut ltunqns6ulle^lpe-f/)dv pue-Spueulln)as lsurll^l-y\1 s]runqns 6ullenllreqllMuollellosse {q pa1e1n6et sazllele) uorlel,tlnblqn )DdV 6ulpn;rur'srsolrLu urorjlrxaur{;ueurudpanlonur sura}ord ,(roteln6ar;o utl1nb1q1 srolP^!l)erraqlpue sese611 rafue) ur pele^rl)eurr{;}uenbet;:t9ul ^}l^l})e )pf-tD sassalddns serlrnrl)e)pf-S pue )pf-S/tD sassarddns obeuep y56 6urano;;o; jo}etluarejJtp I;;eululal 315 {q uorle;ftrnblqns}l sre66rr}Z)pf Iq uoltellloqdsot{d (aqt pue Ip)-S/Lg sassarddns glar :Lg uaqnn ep{r uorJ MerpqllM s1;ar sd;aq ur sar}r^r})e lp)-S :19 ut ,{ltnl}le 11p1sesselddns uorl)nrlsapstt sta66rrtLD,o pua aqi te [)p]) ,{q uorlel,(roqdsoqd

( s l e L U u e u9) [ d (sleuueul) tZd (sleuLueul)1Zd (rsea^6urPPnq)[)lS (sDl)} suFlord roUqlqul Ip)

stsolttrrJo tesuo otlt lP uotle^tl)e tlpf 6ur;;orluolul pa^lo^ulI;tteuttd isleLuueurul () 'g 'VSZrp)) srequaLU{qruie;ealql 1qp) LUorJ sateqdsoqd,{toltqtqu;sa^ouar slsol!Lll ero;aq,{lrnr1re11p) 6ursserddnsur po^lo^urI;ueuud lslpf ur satrsfuollqlqursalel{loqdsotld

aseleqdsoqdg7rpl aseur) leaM

()V)) aseul) 6u tlentlre-1P1 $lp) ul alts 6urlentl:eue sa1e1&oqdsoqd s1p1 {1potu leq} sase}eqdsoqd utelord Pue saseu!l ula}old

ro[e61aq11o{leuutn5 Z-l I alqel suralordltuolelnEegaP{3-11a3 al)^) lla) aql:/ [ ra]deq)

990 t

5 PHASE

Summary The cell-cyclecontrol systemtriggersthe euentsof the cell cycleand ensuresthat these euentsare properly timed and occur in the correct order The control systemresponds to uarious intracellular and extracellular signals and arrests the cycle when the cell either fails to completean essentialcell-cycleprocessor encountersunfauorableenuironmental or intracellular conditions. Central componentsof the cell-cyclecontrol systemare cyclin-dependentprotein kinases(Cdks),which depend on cyclin subunits for their actiuity. Oscillations in the actiuities of uarious cyclin-Cdk complexescontrol uariouscell-cycleeuents.Thus,actiuation of S-phasecyclin-Cdk complexes(S-Cdk)initiates S phase,while actiuation of M-phase cyclin-Cdk complexes(M-Cdk) triggers mitosis. The mechanisms that control the actiuities of cyclin-Cdk complexesinclude phosphorylation of the Cdk subunit, binding of Cdk inhibitor proteins (CIQs),proteolysisof cyclins,and changesin the transcription of genes encoding Cdk regulators. The cell-cycle control system also dependscrucially on two additional enzymecomplexes,the APC|Cand SCFubiquitin ligases,which catalyzethe ubiquitylation and consequentdestruction of specificregulatory proteins that control critical euentsin the cVcle.

S PHASE The linear chromosomes of eucaryotic cells are vast and dyramic assembliesof DNA and protein, and their duplication is a complex process that takes up a major fraction of the cell cycle. Not only must the long DNA molecule of each chromosome be duplicated accurately-a remarkable feat in itself-but the protein packaging surrounding each region of that DNA must also be reproduced, ensuring that the daughter cells inherit all features of chromosome structure. The central event of chromosome duplication is replication of the DNA. A cell must solve two problems when initiating and completing DNA replication. First, replication must occur with extreme accuracy to minimize the risk of mutations in the next cell generation. Second, every nucleotide in the genome must be copied once, and only once, to prevent the damaging effects of gene amplification. In Chapter 5, we discussthe sophisticated protein machinery that performs DNA replication with astonishing speed and accuracy.In this section, we consider the elegant mechanisms by which the cell-cycle control system initiates the replication process and, at the same time, prevents it from happening more than once per cycle.

S-CdkInitiatesDNAReplication OncePerCycle DNA replication begins at origins of replication, which are scattered at numerous locations in every chromosome. During S phase, the initiation of DNA replication occurs at these origins when specialized protein machines (sometimes called initiator proteins) unwind the double helix at the origin and load DNA replication enzymes onto the two single-stranded templates. This leads to the elongationphase of replication, when the replication machinery moves outward from the origin at tuvoreplicationforks (discussedin Chapter 5). To ensure that chromosome duplication occurs only once per cell cycle, the initiation phase of DNA replication is divided into two distinct steps that occur at different times in the cell cycle. The first step occurs in late mitosis and early Gr, when a large complex of initiator proteins, called the prereplicative complex, or pre-RC, assemblesat origins of replication. This step is sometimes called licensing of replication origins because initiation of DNA synthesis is permitted only at origins containing a pre-RC. The second step occurs at the onset of S phase, when components of the pre-RC nucleate the formation of a larger protein complex called the preinitiation complex. This complex then unwinds the DNA helix and loads DNA polymerases and other replication enzymes onto the DNA strands, thereby initiating DNA synthesis, as described in Chapter 5. Once the replication origin has been activated in this way, the

1067

1068

Chapter17:TheCellCycle p r e r e p l i c a t i vceo m p l e x e sa t r e p l i c a t i o no r i g i n s

I

replication elongation forks

M-Cdk activation

|

.hrotoro."

segregation

,.4^

Gl

pre-Rc is dismantled and cannot be reassembledat that origin until the following G1.As a result, origins can be activated only once per cell cycle. The cell-cycle control system governs both assembly of the pre-RC and assembly of the pre-initiation complex (Figure lZ-22). Assembly of the pre-RC is inhibited by Cdk activiry and, in most cells, is stimulated by the ApC/C. preRC assembly therefore occurs only in late mitosis and early G1,when cdk activity is low and APC/C activity is high. At the onset of S phase, activation of S-Cdk then triggers the formation of a preinitiation complex, which initiates DNA synthesis. In addition, the pre-RC is partly dismantled. BecauseS-Cdk and M-Cdk activities remain high (and APC/C activity remains low) until late mitosis, new pre-RCs cannot be assembled at fired origins until the cell cycle is complete. Figure 17-23 illustrates some of the proteins involved in the initiation of DNA replication. A key player is a large, multiprotein complex called the origin recognition complex (oRc), which binds to replication origins throughout the cell cycle. In late mitosis and early Gr, the proteins cdc6 and cdtl bind to the ORC at origins and help load a group of six related proteins called the Mcm proteins. The resulting large complex is the pre-RC, and the origin is now licensed for replication. The six Mcm proteins of the pre-RC form a ring around the DNA that is thought to serve as the major DNA helicase that unwinds the origin DNA when DNA synthesis begins and as the replication forks move out from the origin. Thus, the central purpose of the pre-RC is to load the helicase that will play a central part in the subsequent DNA replication process. Once the pre-RC has assembled in Gr, the replication origin is ready to fire. The activation of s-cdk in late G1triggers the assembly of severaladditional protein complexes at the origin, leading to the formation of a giant preinitiation complex that unwinds the helix and begins DNA synthesis. At the same time as it initiates DNA replication, S-Cdk triggers the disassembly of some pre-RC components at the origin. cdks phosphorylate both the oRC and cdc6, resulting in their inhibition by various mechanisms. Furthermore, inactivation of the APC/G in late Gr also helps turn off pre-RC assembly. In late mitosis and early G1, the APC/C triggers the destruction of a protein,

Figure 17-22 Control of chromosome duplication.Preparations for DNA replication beginin Gr with the assembly of prereplicativecomplexes(pre-RCs) at replication origins.S-Cdkactivationleads to the formationof multiprotein preinitiation complexes that unwindthe DNAat originsand beginthe processof DNA replication.Two replicationforks moveout from eachoriginuntilthe entirechromosomeis duplicated. Duplicatedchromosomesare then segregatedin M phase.The activationof replication originsin S phasealsocauses disassembly of the prereplicative complex,which does not reformat the originuntilthe followingG1-thereby ensuringthat eachoriginis activated only oncein eachcellcycle.

1Mg

S PHASE

O R C( o r i g i nr e c o g n i t i o nc o m p l e x )

u1

p r e r e p l i c a t i vceo m p l e x( p r e - R C )

COMPLETION OF DNA REPLICATION

t

61M

geminin, that binds and inhibits the pre-RC component Cdtl. Thus, when the APC/C is turned off in late G1,geminin accumulates and inhibits Cdtl.In these various ways, S- and M-Cdk activities, combined with lowAPC/C activity, block pre-RC formation during S phase and thereafter.How then, is the cell-cycle control system reset to allow replication to occur in the next cell cycle? The answer is simple. At the end of mitosis, APC/C activation leads to the inactivation of Cdks and the destruction of geminin. Pre-RC components are dephosphorylated and Cdtl is activated, allowing pre-RC assembly to prepare the cell for the next S phase.

of Chromatin Duplication Requires Duplication Chromosome Structure The DNA of the chromosomes is extensively packaged in a variety of protein components, including histones and various regulatory proteins involved in the control ofgene expression (discussedin Chapter 4). Thus, duplication ofa

Figure17-23 Control of the initiation of DNA replication.The ORCremains associatedwith a replicationorigin throughoutthe cellcycle.In earlyG1, Cdc6and Cdtl associatewith the ORC. The resultingproteincomplexthen on the Mcm ring complexes assembles adjacentDNA,resultingin the formation of the prereplicativecomplex(pre-RC). from another S-Cdk(with assistance protein kinase,not shown)then of several the assembly stimulates additionalproteinsat the originto form complex.DNA the preinitiation polymeraseand other replication proteinsare recruitedto the origin,the Mcm protein ringsare activatedas DNA helicases, and DNAunwindingallows to begin.S-Cdkalso DNAreplication blocksrereplicationby triggeringthe destructionof Cdc6and the inactivation ofthe ORC.Cdtl is inactivatedby the proteingeminin.Gemininis an APC/C target and its levelsthereforeincreasein 5 and M phases,when APC/Cis inactive. Thus,the componentsofthe pre-RC (Cdc6,Cdt1,Mcm) cannotform a new pre-RCat the originsuntil M-Cdkis inactivatedand the APC/Cis activatedat the end of mitosis(seetext).

1070

Chapter17:TheCell Cycle

chromosome is not simply a matter of duplicating the DNA at its core but also requires the duplication of these chromatin proteins and their proper assembly on the DNA. The production of chromatin proteins increasesduring S phase to provide the raw materials needed to package the newly synthesized DNA. Most importantly, S-Cdksstimulate a large increase in the synthesis of the four histone subunits that form the histone octamers at the core of each nucleosome. These subunits are assembledinto nucleosomes on the DNA by nucleosome assemblyfactors, which typically associatewith the replication fork and distribute nucleosomes on both strands of the DNA as they emerge from the DNA synthesis machinery. Chromatin packaging helps to control gene expression.In some parts of the chromosome, the chromatin is highly condensed and is called heterochromatin, whereas in other regions it has a more open structure and is called euchromatin. These differences in chromatin structure depend on a variety of mechanisms, including modification of histone tails and the presenceof non-histone proteins (discussedin Chapter 4). Becausethese differences are important in gene regulation, it is crucial that chromatin structure, like the DNA within, is reproduced accurately during S phase. How chromatin structure is duplicated is not well understood, however. During DNA synthesis, histone-modifying enz).rynes and various non-histone proteins are probably deposited onto the two new DNA strands as they emerge from the replication fork, and these proteins are thought to help reproduce the local chromatin structure of the parent chromosome.

Cohesins HelpHold5isterChromatids Together At the end of S phase, each replicated chromosome consists of a pair of identical sister chromatids glued together along their length. This sister-chromatid cohesion sets the stage for a successfulmitosis because it greatly facilitates the attachment of the two sister chromatids in a pair to opposite poles of the mitotic spindle. Imagine how difficult it would be to achieve this bipolar attachment if sister chromatids were allowed to drift apart after S phase. Indeed, defectsin sister-chromatid cohesion-in yeast mutants, for example-lead inevitably to major errors in chromosome segregation. Sister-chromatid cohesion depends on a large protein complex called cohesin, which is deposited at many locations along the length of each sister chromatid as the DNA is replicated in s phase.TWoof the subunits of cohesin are members of a large family of proteins called sMC proteins (for Structural Maintenance of chromosomes). cohesin forms giant ring-like structures, and it has been proposed that these might form rings that surround the two sister chromatids (Figure 17-24).

5 m cm o l e c u l e

y'--'-} r

hinge

CN

.

ATPase domain

(A)

(c)

20

"t

Figure 17-24 Cohesin,Cohesinis a proteincomplexwith four subunits. Two subunits,Smcl and Smc3,arecoiled-coil proteinswith an ATPase domainat one end; together,they form a large V-shapedstructureas shown.Two additionalsubunits,Sccl and Scc3, connectthe ATPaseheaddomains forminga ring structurethat mayencircle the sisterchromatidsasshown.

LLOL

Jo slueueSuBJJeeJpue edola^ue rEelf,nu aql Jo u^\op{Balq aq} sa}oruoJd oslE 'saJn}3nJls e{ll-poJ '}JEdrrros olul spllPruoJqs Jelsrs 'slla3 {pl-W IEruruE uI paurMualur eql Jo uorlBZruBSJoeJelef,s-e8JEla\l 'luo!rysuapuoJawosoutoJqJ sre88u] osp tI 'alpuds aqt Jo alod elrsoddo eq] ol peqopnp sr rrcd p ur prleru -oJqr qcpe pue go Jelsrs ,tlqurasse ernsue alpurds Jllollur aqt aq] eJnpur luql 'srsolnu 'urnrurunu go [1rea rnoco e sa8els aql ur ]snur Ipf,-W lV ]eql slueru '{pO-I 'eseur{ ura} -a8ue.rrear tr 1ac xalduroc pue asJelrp aql Jo IIE lnoqe s8urrq -ord ay8ursp leq] sl IoJluoJ alcdc-gac Jo saJnleal alqe{Jeual tsotu eql Jo euo

srsolrW olutr(rluf se^UOIp)-W 'uaq] etErtseqrro c/ldv aql pue {pl-W ,lt.or{pue srsolrru Jo sluale IuJrueqoaru ,{a1 aql equosap a,rr 'uorlJes sq} uI 'srsauqog,b^,(q aq]Jo uorsrllp aq] pup 'alpurds cpotlx aqlyo l.1q 1ac -Iuessuslp aql 'aseqdeue uorlalduroo eql Surpnlcur 'aseqd t{ elBI sluela 3o Jo IIB ro; parmbar sr qf,rr{,t,r's1a8re11p3 go uoqeldroqdsoqdap eql pue uorlelrlcpur {pJ ot speal qJIq ^ 'sur1c,bJo uorlJnrtsap aq1 sra33rrl oslp 3/ldv eqJ 'spueluoJqc JalsrseqlJo uoperedas sal€qrur ^qaJeql pue urseqoJ seleelc ]eql asealord e 8ur -leJeqII'uunJas uorlJnJlsap aql sraSSrr]l/JdV aql uaqm 'uorlrsue4 asuqdeue Jo -o1-aseqdelau eql lB su€aq srsolru;o ged roleu puocas eq; 'srrcd pl]EruoJr{J ralsrs eq] ot tueruqoptlp slr pup alpurds ollot1ru aqr go .{lqurasse eqt ot Surpeal 'suralord;o rilarJel E alef i-roqdsoqdsaseuq uralord crlollu reqlo pue {pO [BJeAas -y41'porrad s1q]Surrng '(asuqdelaurpuu 'aseqdeleruord 'eseqdord) srsolrurdlrea Jo sluane aq] sraSSrrllurod4caqc W/zC aqr le.,br.qce {pl-hl ur eseeJJurldnrqe ue 'lsJIC'ruals.{sIoJluor a1c[c-1acaq] Jo s]ueuodtuoc ]curlsrp dq paurano8 qcea 'suud roferu o.tt] o]ur pep4lp eq UBJsrsolnu 'r,rar,r.go 1urod,,{rolep8ar e ruorg 'aseqd 'snelf,nu W stuale rofeur aq] sezrJeru(unsI-ZI larred Jo 'sellpq oml olur Ief,rluapr ue qtlm qoea 61 IIeo aql saprlrp-srseuqo{c-aseqd 'pa]aldruoc sr srsolrrusV 'adocsorcrur Jo luala rofeu puocas aq] e uI uees sE Jorleqaq aruosoruoJql Jo srseq eql uo ,{preurrd peugep---?su2t4do7a1 pue'asnqdnua 'asnqdnlatu 'asoqdnlauotd'asnqdotd-saBels ang olur paprnrp ,(1e -uonrpeJ] sr srsolrhl 'aruoua8 aql;o .{doc umo s}r q}lm qJEe 'ralJnu ralqBnup pcll -uepr rrcd u ot {pary&a8as)pa}nqrJtsrp pue paleredes erp sprtpruoJr.IJJelsrseql Jo qclqm Suunp 'srsolrur qlyrt sur8aq srq; 'aseqd yq;o pneaqdn cBprueJp aql sao8 -Jepun IIaf, aq] '.zg q8noJql uonrsupJl pue aseqd g;o uorlaldurof, aql 3ura,ro11og

srsolrw 'a1c[c ryau aqi Urun pasnar 77ac aq rcuuoJ puo asnr"ldS qJaa u! aJuo [1uo pua acuo patyf st utBuo qcaa 'snq1 'uru&u uotpct\datvNcl aruuru! oJ u!8tro ruqj monD o1 patlnbat arq jotlj sutajotd vlqlqu! osID 4pJ-S 'asoqd g Suunp pawnlpo sr utBrto uo4an1dat a a?uo 'sur&tto uotl.ac -tPaJ panoc aqt ut puo purmun salls uouDJrldat st! alDlltut aLIt iD ruLHsulaj VNe VNe -otd saJnnuca uotlantlca pata33u1 aql tq st uotlau\dnp awosotuoJ.qJ ryrum'4p)-Sto 'uotl.cuntatuosotaotqc pun y111q stcadso snounn utanoB aW ajonosst)!.oq| sunj lo rylm -otd uryawottlcaql uoryattldnpa4j sD qcoaw alnralow VNe lo lpm sD'alaosoraoJqJ at\ua aq1to Surtdoc aruJnJOo aql sanlollurasnt4dg ur sawosoluolt\caql lo uollvcl\dnq

{reuun5 'Jel€l aqrJJsepalr,rse 'saxalduroc aseql ;o uoqdnrsrp uo dpreurud spuadap eJoJalaqluorlrsueJl aseqdeue-o1-aseqduleru aqt ]e uorseqooJelsrsJo ssol aqJ 'saxaldruocurseqoJ uo dyreruud spuadep uors 'pa^oruerueeq seq uoueualec eql ecuo '(tz-g aln8r{ ees) -eqo3 prlEruoJrlJ-Jelsrs '{perq eq} q8norqt reqlo eql Surssed,ayncaloru VNO ]nc aq] Suqeasaruaql pue pue asuqd S ueemlaq sVNCIJelsrspelpueluJ eql VNC euo 8uq1nc dq srsolrur,r(1rua salSueluesrpdlpnper8 II asBJaurosrodolaruufuueaq;.srsaqluds y1qq Suunp laaur $lJoJ uorlecrydar om1ueq a sJnJco leql selnJalour VNC JelsrsJo Suruunlralur aql 'uorlaualal y 'lred ur lseel le 'sllnseJosle uorsaqoopqeuorqo-Jalsrs IO ruo4

stsoltt

alpulds aqi ]o salod alrsoddo o l s p r l e | . r ] o r L l s. l a t s r sq f ,e ] ] P s o l n q n l o r J r L oUi o q l o l a u r ) a q l s a l o a a l p u r 0 sa q ] uaaMlaq AeMplrr iilnurds aql ]o rolenba aql ]e p a u 6 r l Pa l e s a r r o s o L L r o r q 3

aql

]v

o ln q nlorc il..Ir o r o L l c o l a ur )

a;oda;purds ]e aLUosolluac

]SVHdVT]IA

uorloir .l^r])P u auosouloJq)

T

alnqnlorlru aroq)olaur)

lUdLIlaAOrL/ JAll)P

obrapun pue saroq)olaur) r a q ] e r As a ln q n l o r ) t t t . l a l p u l d so l q ) P l l P/ ^ o u U P ) s a u r o s o u r o r q JJ d o l a ^ u a reapnu aq] +o irMop)eojq aqt qlrM AllonrqP su e]s

,' a d o l a ^ u ar e a l l n u +o sluau6Pr+

alod a l p u l d sl e au osotlu al

]SVHdVl]IAOUd Z -1

u a a t6 a ) e s a l n q n l o l ) l u l p u e a 6 u e ) o p J U r P l sa r e s a L U o s o L U o r q ) ' qPdr 6 0 r ) i l - u

q 1 6 u a 1r r a q t 6 u o 1 e r a q l a 6 o ] p l a q s p r l p u r o r q ) l a l s l s o M ] 1 o 6 u r l s r s u o r ' a L ! o s o u o r q )p a l e t t l d a r 6 u t s u a p u o l

oloqo aql ul ]uasalo aLl-ros - o L U O r q )q ) P a + o s a r d o )o A ] aq plnoM aiaql'slla)proldrp ul uMoqs ale saLUosoLl.lolql a a r q ] ^ l u o A | r r l d L u t sr o 3 Uede po^our pue patertldar aAeqq)rqM'sat]-]osorlua) o^^l aq] uaaMlaq solqu.lassP a l p u r d sl r l o ] r u l J q l ' s n a l r n u rds ,l a aql aprslno asuapuol l p u ')rlollu 'sprleuorq) .t p a l e D o s s e r o l s r s ,,:, burrurol ./ , 4 1 a s o 1orn n l + o 6 u r l s r s u o l ",.,,,,..,......... ./ q)ea'saL!osoLUorq) au.losollua) p e l e r r l d e re q ] ' ]V

aroLllolaurl /

.,t"'

adola^ue leallnu ]lelul

]SVHdOUd

; !..

L

ANAPHASE daLlqntef CnrOmOSOmeS

n r

"

hL

'

N -s) \)

faces The krletochore microtubules get shorter, a n d t h e s p i n d l ep o l e s a l s o move apart; both p r o c e s s e s c o n t f l b u t et o c h r o m o s o m es e l l r e g a t t o n

r-.----:\ q.-\,

\C\\-

.

C\

rr'

\a/

' snorlen ng kinetochore

the srster

c h r o m a t i d ss v n c h r o n o u s l Y s e P a r a t et o t o r n l t w o daughter chronrosomes, a n d e a c h i s t r r u l l e sd l o w l Y toward the sptndle Pole it

,:..,., ::

:1

s p i n d l ep o l e m o v r n go u t w a r o

m i c r o t ub L rl e

-1..,.:

TELOPHASE

5

D ur i n g . the two sets of daughler chromosomes arrive at the poles of t h e s p i n d l ea n d d e c o n d e n s e A n e w n u c l e a re n v e l o p e r e a s s e m b l e sa r o u n d e a c h s e t , c o m p l e t i ' r , lJl r e [ o r m a t i o n o f t w o n u c l e ra n d r n a r k i n g t h e e n d o f n r i t o s i sT h e d i v i s i o no f t h e c y t o p l a s m b e g i n s w i t h ( o i l l r a c t i o no r t h e c o n t r a c t il e r i n g

sel n' daUghte'ultr,,tnosomes at spindle pole c o n t r a c t r l en n g startrng to

R .r}J

CO NITA CI

GiS -S) -i C2

ot

,::.:t:::. '. '::;, ...

:..1

centrosorne

overlap

'

ntIcrorLtDLtIes

n u c l e a re n v e l o p e r e a s s e m b l i n g a r o u n d i n d i v i c i u a lc h r o m o s o m e s

ta:,;:

r.

,-t,:' "

6

CYTOKINESIS c o m p l e t e d n u c l e a re n v e l o p e surroundsdecondensing chrontosomes

During , the cytoplasm is divided in two b y a c o n t r a c t r l er i r r g o f a c t r na n d m y o s r n f i l a m e n t s ,w h r c h p i n c h e s the cell in two to create two daughtcrs.each with one nucleus

coftracill€] ilng

r e - f o r m a t i o Do i n l e r p h a s e

c r e a t l n gc l e a v a g e

a r r a y o f m r c r o t r l, ) u l e s t l L t c l e a t e c l

f rrr r o w

llv the centrosonre

( M i c r o g r a p h sc o u r t e s yo f J u l i e C a n m a n a n d T e d S a l m o n )

rl.l:

'IaaM lrqtqutol lpf_w Jo IIlrqe aq] Iq pe)uequastlleqpeaJ srql'))eqpaaja^rlrsodut 6utllnsai 'lp)-W e^tt)e Iq pele;nutls raqunl s! SZ)p) '97rp1 asereqdsoqd aql ^q zD Jo pua aql le pelenrt)euaql srxa;dtuor 'aseutl lpf-W a^lr)Pu!6ulllnsaraqf aql {q salrsr(rolrqrqur 14e11 ;o lred e uo pue ()v)) aseull6ullp^ll)e-Ip) aql ,(q alrs6ur1en;1)e ue uo pale;fioqdsoqd srxa;duot )pf-W 6ultlnser aqf 'asuI;;enpet6urp{t-y11 1os;ena; aql se urp{>-y1qllm sa}e!)osse tlpf 'Ip)-W Jo uolle^ll)e aq1 97-11 arn6r3

)p)-l l aArpeul

oseurl ^rolrqrqur-lpf

lp)-lA a^rpeul

Dp)

eseurl 6urlenrpe-1p;r ur;:{:-61

eseleqdsoqd e A r l ) e uI

{pl-W Jo uor}Blndodqns B Jo uor}?Arlre lel}red eq} ot spPel OIpf,-Sdq sdeqrad) gzJpJ Jo uortE^rlJE lellJed eql 'lepolu e^rlcerltP srql o1 Suproccy '(92-LI em -3r4 aasl sdool {ceqpaal el4rsod sallolur srsolllu uI uollplllcp {pC-hl tuqt s}se8 -3ns puE (gzcpf,) rote^IlJe u^ o sll ele^Ilse ol rotrqrqur slr u^ao llqlqul 11aa111 'IeaM eseuDlfrolrqrqur aql llqlqq osle deur {p]-N )pC Ip3-ht Jo ^rIIqE eql rpelelllJe aq osp ueo gzopl 'd18uqsara1u1 -yq '1a3ru1 ^q 'ued sll ul lseal ]e 'gzJpJ alBlnruus esEqdoJd dlrua puE zc uI aAIlsE are teq] s{pl-S aqt }eq} sI ItilqISSod euo 'poo}srepun IIa^\ tou are stsolnu d.lJeauI (IeeA sseJddns pue) Ilr^ucp gZJpJ qseaFn lpql susFeqJeru eqJ 'sespeJ3ul&yrlfce {pC-hl lEqt Suunsua JeqUnJ'passarddnssI IeeMesBurneqlJo III^IIJE drollqqul aql 'erlrp eluPs eql lM'Gz-Ll arn81g;{p3-hl ulerlsar lBql selEqdsoqd,r{Joltqqut eql selorueJ r{Jrq^A 'gZJpJ ese}Eqdsoqd uta}ord aql Jo uoIlEAI}Je aq} sI 'ueql 'tBtI71\ tuale IETJnJJaql;elrdlcots {pl-^ eq}Jo uorlplrlop aql sra38u} 'esPuD[eql Jo a]rs a^rl3e aq] Itolq lPql selEqdsoqd,(q passarddns sI lnq ]JP ol Lpear pue parurJd sr ]eq] {pO-IAIJo a161co1stuepunqe uP supluoc tI 'zt Jo pua eql seqJeerIIec eql etuu eql ,{q 'snql '(8I-zI arnStgees)se}ISSurroqqSrauo,\/\l}u uopel,fuoqdsoqd.,fuorrqrqur.,tqa1e1se^llJEul uE ul ll sploq Iae A esEuDIulalord eql terllPe pessncsrp sp '$Vl) aseuDl Supenpce->1pf,eql ^q elIS SuIlPAIlce uP 1e pa1e1froqdsoqdsr sexaldruo3eseqt uI {p] eql q8noqflV'slsollru saqceordde ilet eqt sP (urr3l.r-t{ puB IIpl Jo xaldluof, aql) Ipc-N Jo uollBlnrunsce SurpuodsarroJ e o] speal uralord uITJIJ-I I uI espeJJulaq1'uoqduJsueJl eue8 u17ctc-1t11uI esperJul ue o1,tpreupd Suulo 'hl pue z5 Surrnp seseaJlul stsaqlu.{s urlcdc-y41 le^emoq 'sadd11ac tsoru uI 'aseqdralut ut utalord eqt Jo Irllqels qBH pue 'e1czbIIaJ eql lnoq8norqt luelsuoJ aql uorJ sllnser uorlelnun3oe uqc^,b-141 sr uqcrb-6;o srsaqlurtsaql 'sa1c,b1ac cruo.{rqruauI '(I-ZI elqEl ees :slle3elEJq -euel ur g unc^J) u11c,b-61Jo uopelnurncop eql qlpn sur8aq uoltelllJe {pl-N

srsotrwJotasuo aql le lp)-w sale^ll)v uollel/(loqdsoqdec 'JeelJ lou eru srusrueqceu uollelllJu asIJaJdeql lnq ,trplrce >lp3-4 uo spuadap seseuDlerornv pue sasEuDIel1-olod Jo uoIlEAIlcV relul ssnf,slp ertl sP 'elpulds aq] ol spleuorqJ relsls Jo tuauqJellp sloJluoo g-EJornV seaJeq^\'alpurds aql ;o,fi1rqe1s pue ,tlqurasseaqt ura,ro8 luql sulalord Ionuoo sdlaq osp V-EJoJnV aseuDlBJoJnVaqJ 'srsotltu ut f.pea salod aypurdsaq] Jo uoperedas uI pellonul suralord salul,fuoqdsoqd1rasnecaqtred ut 'ayputdsJItotIu reyodtqe;o dlquesse 'aldurexe JoJ '{ld eseuDle)I-olod eqJ'stuala cltol1ru Ieturou eql JoJparmbar sr ,(1rea ;o loJluoc aq] 01 suollnqlJluoJ ]ueuodrut eIeru osp 'sasou!4 aJoJnV 'sISolIu aql pue sasau!4aql-olod aql 'sesBupluralordgo serlrruBJlEuolllppP o^ J .,tpeaur pe^lo^ul surelord,{a4 ate$roqdsoqd ol auop IJP tou saop {pf,-IAI 'ssacoJdeql uI 'pegpuepl uaaq ]art lou a^eq surelord asaql Jo lsoru qSnoqlp pallolur suralord cryrcadssaleldroqdsoqd ryC-lt ueq.r,rparaSSln eq 01 rq8noql sr sassacord esaql Jo qceg 'snleJeddu r31og eql puu uolele{soldJ uI}Je aq}

af)^) lle) aql:/ [ raldeq3 vLOL

1075

MITOStS

i:ffl :flH' :'fl ;;".'"';'*'#:il:?:il[::ffi ffi :?ili:,T::''.",:fr (Courtesy alongtheirlength. Theconstricted regions arethecentromeres. of TerryD.Allen.) complexes, which then phosphorylate more Cdc25 and Weel molecules. This leads to more M-Cdk activation, and so on. Such a mechanism would quickly promote the complete activation of all the M-Cdk complexes in the cell. As mentioned earlier, similar molecular switches operate at various points in the cell cycle to promote the abrupt and complete transition from one cell-cycle state to the next.

for Condensin HelpsConfigureDuplicated Chromosomes Separation At the end of S phase, the immensely long DNA molecules of the sister chromatids are tangled in a mass of partially catenated DNA and proteins. Any attempt to pull the sisters apart in this state would undoubtedly lead to breaks in the chromosomes.To avoid this disaster,the cell devotes a great deal of energy in early mitosis to gradually reorganizing the sister chromatids into relatively short, distinct structures that can be pulled apart more easily in anaphase.These chromosomal changes involve two processes: chromosome condensation, in which the chromatids are dramatically compacted; and sister-chromatid resolution, whereby the two sisters are resolved into distinct, separable units (Figure 17-26). Resolution results from the decatenation of the sister DNAs, accompanied by the partial removal of cohesin molecules along the chromosome arms. As a result, when the cell reachesmetaphase,the sister chromatids appear in the microscope as compact, rod-like structures that are joined tightly at their centromeric regions and only loosely along their arms. The condensation and resolution of sister chromatids depends, at least in part, on a five-subunit protein complex called condensin. Condensin structure is related to that of the cohesin complex that holds sister chromatids together (seeFigure 17-24).It contains two SMC subunits like those of cohesin, plus three non-SMC subunits (Figure 17-27). Condensin may form a ring-like structure that somehow uses the energy provided byATP hydrolysis to promote the compaction and resolution of sister chromatids. Condensin is able to change the coiling of DNA molecules in a test tube, and this coiling activity is thought to be important for chromosome condensation during mitosis. Interestingly, phosphorylation of condensin subunits by M-Cdk stimulates this coiling activity, providing one mechanism by which M-Cdk may promote chromosome restructuring in early mitosis.

1*

ATPasedomain

Machine TheMitoticSpindlels a Microtubule-Based The central event of mitosis-chromosome segregation-depends in all eucaryotes on a complex and beautiful machine called the mitotic spindle. The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase,thereby segregatingthe two sets of chromosomes to opposite ends of the cell, where they are packaged into daughter nuclei. M-Cdk triSgers the assembly of the spindle early in mitosis, in parallel with the chromosome restructuring just described. Before we consider how the spindle assemblesand how its microtubules attach to sister chromatids, we briefly review the basic features of spindle structure. As discussed in Chapter 16, the core of the mitotic spindle is a bipolar array of microtubules, the minus ends of which are focused at the two spindle poles, and the plus ends of which radiate outward from the poles (Figure 17-28). The plus ends of some microtubules-called the interpolar microtubules-interact with the plus ends of microtubules from the other pole, resulting in an antiparallel array in the spindle midzone. The plus ends of other

DNA Figure 17-27 Condensin.Condensinis a five-subunitproteincomplexthat cohesin(seeFigure17-24). resembles The head domainsof its two major subunits,5mc2and Smc4,areheld togetherby threeadditionalsubunits.lt the is not clearhow condensincatalyzes and comPactionof restructuring chromosomeDNA,but it mayform a ring loopsof DNAas structurethat encircles shown;it can hydrolyzeATPand coil DNA moleculesin a testtube.

1076

Chapter17:TheCellCycle spindlepole replicated chromosome (sisterchromatids) centro50me

Figure17-28 The three classesof microtubulesof the mitotic spindlein an animalcell.The plusendsof the microtubulesprojectawayfrom the centrosomes, whilethe minusendsare anchoredat the spindlepoles,whichin this exampleareorganizedby centrosomes. Kinetochore microtubules connectthe spindlepoleswith the kinetochores of sisterchromatids, while interpolarmicrotubules from the two polesinterdigitate at the spindleequator. Astralmicrotubules radiateout from the polesinto the cytoplasmand usually interactwith the cellcortex,helpingto positionthe spindlein the cell.

kinetochore ,/

motor + protern

astral microtubules kinetochoremicrotubules

interoolar microtubules

microtubules-the kinetochore microtubules-are attached to sister chromatid pairs at large protein structures called kinetochores,which are located at the centromere of each sister chromatid. Finally, many spindles also contain astral microtubules that radiate outward from the poles and contact the cell cortex, helping to position the spindle in the cell. In most somatic animal cells, each spindle pole is focused at a protein organelle called the centrosome (discussed in chapter l6). Each centrosome consists of a cloud of amorphous material (called the pericentriolar matrix) that surrounds a pair of centrioles (Figure lz-29). The pericentriolar matrix nucleates a radial array of microtubules, with their fast-growing plus ends projecting outward and their minus ends associatedwith the centrosome. The matrix contains a variety of proteins, including microtubule-dependent motor proteins,

lpm

microtubule

p e r i c e n t r i o l am r atrix pairof centrioles

Figure| 7-29 The centrosome.(A)Electronmicrographof an S-phase mammaliancellin culture, showinga duplicatedcentrosome. Eachcentrosome containsa pairof centrioles; althoughthe centrioleshaveduplicated, they remaintogetherin a singlecomplex,as shownin the drawingof the micrographin (B).Onecentrioleof eachcentriolepairhasbeencut in crosssection,whilethe other is cut in longitudinalsection,indicatingthat the two membersof eachpairarealignedat right anglesto eachother.Thetwo halvesof the replicated centrosome, eachconsisting of a centriolepair surroundedby pericentriolar matrix,will splitand migrateapartto initiatethe formationof the two polesof the mitoticspindlewhen the cellentersM phase.(C)Electronmicrographof a centriolepair that hasbeenisolatedfrom a cell.Thetwo centrioleshavepartlyseparated duringthe isolation procedurebut remaintetheredtogetherby fine fibers,whichkeepthe centriolepairtogetheruntil it is time for them to separate. Bothcentrioles arecut longitudinally, and it can now be seenthat the two havedifferentstructures: the mothercentrioleis largerand morecomplexthan the daughter centriole, and onlythe mothercentrioleis associated with pericentriolar matrixthat nucleates microtubules. Eachdaughtercentriolewill matureduringthe next cellcycle,when it will replicate to giveriseto its own daughtercentriole.(A,from M. McGill,D.p.Highfield, T.M.Monahanand B.R.Brinkley,J. ultrastruct.Res.57:43-53,1976. with permissionfrom Academicpress;C,from M. Paintrandet al.,J. Struct.Biol.108107-128,1992.with permissionfrom Elsevier.)

200nm

1477

MlroSlS coiled-coilproteinsthat link the motorsto the centrosome,structuralproteins, and componentsof the cell-cyclecontrol system.Most important, it contains the y-tubulin ring complex,which is the component mainly responsiblefor in Chapter16). nucleatingmicrotubules(discussed Somecells-notably the cellsof higherplantsand the ooc)'tesof many vertebrates-do not have centrosomes,and microtubule-dependentmotor prowith microtubuleminus endsorganizeand teins and other proteinsassociated focusthe spindlepoles.

Microtubule-Dependent MotorProteinsGovernSpindle Assemblyand Function The assembly and function of the mitotic spindle depend on numerous microtubule-dependent motor proteins. As discussed in Chapter 16, these proteins belong to two families-the kinesin-related proteins, which usually move toward the plus end of microtubules, and dyneins, which move toward the minus end. In the mitotic spindle, these motor proteins generally operate at or near the ends of the microtubules. Four major types of motor proteins-kinesin5, kinesin-14, kinesins-4and 10, and dynein-are particularly important in spindle assembly and function (Figure f 7-30). Kinesin-S proteins contain two motor domains that interact with the plus ends of antiparallel microtubules in the spindle midzone. Because the two motor domains move toward the plus ends of the microtubules, they slide the two antiparallel microtubules past each other toward the spindle poles, forcing the poles apart. Kinesin- l4 proteins, by contrast, are minus-end directed motors with a single motor domain and other domains that can interact with a different microtubule. They can cross-link antiparallel interpolar microtubules at the spindle midzone and tend to pull the poles together. Kinesin-4 and kinesin-10 proteins, also called chromokinesins, are plus-end directed motors that associate with chromosome arms and push the attached chromosome away from the pole (or the pole away from the chromosome). Finally, dyneins are minus-end directed motors that, together with associatedproteins, organize microtubules at various cellular locations. They link the plus ends of astral microtubules to components of the actin cytoskeleton at the cell cortex, for example; by moving toward the minus end of the microtubules, the dgrein motors pull the spindle poles toward the cell cortex and away from each other.

of a BipolarMitotic in the Assembly TwoMechanisms Collaborate Spindle The mitotic spindle must have two poles if it is to pull the two sets of sister chromatids to opposite ends of the cell in anaphase. In animal cells, the primary focus of this chapter, two mechanisms collaborate to ensure the bipolarity of the spindle. One depends on the ability of mitotic chromosomes to nucleate and stabilize microtubules and on the ability of the various motor proteins just described to organize microtubules into a bipolar array, with minus ends kinesin-14 s p i n d l em i c r o t u b u l e

k i n e sni - 5

centrosome s i s t e rc h r o m a t i d s k i n e s i n - 4 ,01/

Figure17-30 Major motor proteinsof of the spindle.Fourmajorclasses motor proteins microtubule-dependent (yel/owboxes)contributeto spindle assemblyand function (seetext).The coloredarrowsindicatethe directionof motor movementalonga microtubuleb/uetoward the minusend, and red towardthe plusend.

'ralseue palle)salnqnlol)tt! Jo ^eJe lPrpPluMoslrsaleal)nuMou alllosoJluaf q)el ateredasol ut6aqsa^leqo/V\l aql pue oM] u! sl!ldsxalduo) oql uaLlM 'aseqd ogl lllun xa;durot W lo 6uruurbaq leurosorluola;6urse ul taqla6o1aso;t ureLuar sred oloulua)onrlaq1 z9 i(q palaldtuo){;;ensnsrolou}ua)ralq6nep a q ] ; o u o r l e 6 u o laaq l ' t l o 1e ; 6 u e ]q6u p te pue alotJluo)raqtou q)pa Jo aseqaql reauanot6o1sur6aqoloulua) relq6nepe'eseqd5 6urrn6 slalaLuol)tr.u ana;e Iq aleledasrredeqi;o salouluo) oMl aq]'Lg u! tulod utetla)p lv'uaar6) xuleurrelouluatrradpaleoossepue rPo aloulua)e Jo Slstsuo) aLuosollua) aq1 'uo11e>1;dar alotrlua] 1g-21 arn613

iir'. 1ti{iliiiixii lririiir!t,:',

,g

JealJnu eql Suop uede a^otu seurosoJlual o^ l aql '{sllal IEturue u1 z(lqruasse alpurds saterlrur-,tu,rtce)pl-W ur asrJuappns aq] ,srsolnuJo SuruurBaqeqt lV

aseqdordur ^lqurassvelpuldssaletltul{p)-W 'a1c,{c 1at Jad aJuo o} uor}eorldnp euosoJ}uac }1urll sllao qcns ^\oq umouT lou sr lI 'palJolq sr uorleJrldnp auosotuoJqJ Jr sanupuoJ uorlecrldnp aruosorluac puE rusruPqJaure qcns urEluol lou op 's3or; pue 'surqJJnEes ,saruJo so^Jqrua ,{pea aqt ur asoq} Surpnlcur lalamoq 'sed^l IIaJ raq}O 'IJaqJ ur lda{ sr Jeq -runu aruosorluac qcrq,,rn,{q usrueqJeru auo Surpr^oJd,uorlecrldnp auosoJlua3 sTJolq srsaqlu.{sVNC Jo uoqrqrqur p}ueurJadxa 'sedd} IIaJ ^ueru uI 'ureueJun are a1c,{clac.rad aJuo ot uoqecrldnp aruosoJlueJlltull leql susrueqcau eqJ 'uoup8eJ8asauosoruoJqc ur sJoJJasnq] pue ,tlquasse alpurds ur slf,aJapol peel plnoJ saruosoJluacJo Jaqunu ltalroJur ue :sardoc orq dpo q4nr srsollu sJalua IIaf, aq1 ]Eql arnsue ol ,alJ.{J1ac rad acuo ,{1uopuB acuo a}ecrldar }snru 'saurosoruoJqcelrl .sauosoJlue3 'JIeq^\eu e uorlJnJlsuo3 roJ sa1e1dualse elJas pue aleredas sa^pq oml aq] qorq A ur Jo 'uouecrldnp .uopecqdnp eruosotu Jo tusrueqlaru anrlelJesuoo-rurese asn qlog -orqJ pue uorlecqdnp eruosoJlual uee^Naq slalered Surlsaralur are areqJ 'srsolllu sra]ue IIaJ aq] Flun snalJnu aql Jo aprsauo uo reqtaSol sureruarrred aurosoJlual slqJ'(Ig-ZI arn8lfl xrJletu JBIorJl -uacrrad pa8relua up urqlrl srred alorJluac oA\l ur Surlpsar ,elorJluaf,^\au a18urs e Jo uorlerxJoJ eql saleelJnu qJea pue 'aleredas etuosoJlual aql ur salorJluaJ o1\{laqJ'uonectldnp aluosorlual salelllul osle ^Jlua alrdc 1ac sraSSrrlteqt (I-ZI alqeJ aes :sllar Ieurrue ul alp3 pue g uqc^,tc;oxaldruoc E) {pl-S/IC aq1 'aseqd S sralua flac aq] sE erur] arups eq] ]noqe ]e sur8aq uorlecqdnp aruosor]ua3 'saruosorlual o,\/\laJpaJaql srsol[u saqceerIIaJ aq] auE aq1.{q leql os ,a1c.{c11ac aq] srelua [ac eq] uaqm saleJrldnp aurosor]uao eqJ 'salnqnlolJrru crurseldoi-,b s,lleJaql Jo lsotu satealcnu leq] euosoJluac a13urse ureluoJ sllal Ierurue lsolN

al)^) lla) oqf ur {ltel sln))O uorle)tldnc auosol}ua) 'umop{eeJq adolanua Jealcnu JaUelup} -rodrur,r(lrepcrlred saruocaqpue saruosoJlual arrnbar lou saop leqt rusrueqcau uorlezrue8ro-Jlasaql reprsuoJ uaqt a A'srsolrur z(pea ur dlquasse luapuadap 'dlqruassealpurds;o sdals aqt aqrrtsep llrou -aurosoJlual qtl,tlr Suruu€aq aM 'aruosoJtuac po,rtorlsep aqt seq ueeq Jasele qclq ,t,ur sllaf,palnllnf, ur pue sauosoJluac ,,(leurrou sllal ur sruro3 aypurdspuorlJunJ E aturs lala leql 1ce1 -moq 'alpurds relodrq e go ^,{yqruasse aql JoJlertuasse}ou aJesaruosorlua3 .sa1od alpurds peleorrqeJardgo rred e Surpmord ,r{q,{lqruassealpurds relodrq a}p}rIrJEJ satuosoJlual o^Al aqJ 'salnqnloJs[u ,r(erreprpeJ e sa]ealcnu qJIqM Jo qsEe 3o 'seurosorluecaorred e qtr.{\srsolru sratua IIar leurue pcrddl y'sa1od alpurds aq} uro; dlaq oJ seruosonuac;o rftryqe aq] uo spuadap raqto aqJ .auozprru alpurds eq] uI Jaqlo qcea qll,tr Surlceratur spua snld pue salod alpurds oml le pasnJoJ

al),() lle) aql:/ [ ra]deq)

9LOL

1079

MITOSIS (D)

(c)

(B)

2pm

n o r m a ls p i n d l e s

overexpressionof Ka13p

overexpressionof CinSp

on spindlelengthin buddingyeast.(A)A differentialof opposing motorproteins Figure17-32Theinfluence of thespindle andtheposition in green, ishighlighted spindle micrograph of a mitoticyeastcell.The interference-contrast forms in yeasts, andthespindle polesisindicat Thenuclear envelope doesnotbreakdownduringmitosis ed by redarrows. (B)Normal antibodies. anti-tubulin withfluorescent havebeenstained inside thenucleus. In (B-D), themitoticspindles protein) leadsto abnormally (C)Overexpression yeasrcells. motorproteinKar3(akinesin-14 of theminus-end-directed protein) leadsto abnormally (D) plus-end-directed motorproteinCinS(akinesin-5 spindles. Overexpression of the short lengthin thesecells. spindle determines motorproteins opposing thata balance between longspindles. Thus,it seems 1997. With CellS:1025-1033, (A,courtesy andM.A.Hoyt,Mol.Biol. V.Lengyel B-D,fromW.Saunders, of KerryBloom; permission forCellBiology.) fromAmerican Society envelope, and the plus ends of the microtubules between them interdigitate to form the interpolar microtubules of the developing spindle. At the same time, the amount of y-tubulin ring complexes in each centrosome increases greatly, increasing the ability of the centrosomes to nucleate new microtubules, a processcalled centrosomematuration. Multiple motor proteins drive the separation of centrosomes in early mitosis. In prophase, minus-end directed dlnein motor proteins at the plus ends of astral microtubules provide the major force. These motors are anchored at the cell cortex or on the nuclear envelope, and their movement toward the microtubule minus end pulls the centrosomes apart (see Figure 17-30). Following nuclear envelope breakdown at the end of prophase, interactions between the centrosomal microtubules and the cell cortex allow actin-myosin bundles in the cortex to pull the centrosomes further apart. Finally, kinesin-5 motors cross-link the overlapping, antiparallel ends of interpolar microtubules and push the poles apart (seeFigure 17-30). The balance of opposing forces generated by different types of motor proteins determines the final length of the spindle. Dynein and kinesin-s motors generally promote centrosome separation and increase spindle length. Kinesin14 proteins do the opposite: they are minus-end directed motors and interact with a microtubule from one pole while traveling toward the minus end of an antiparallel microtubule from the other pole; as a result, they tend to pull the poles together. It is not clear how the cell regulates the balance of opposing forces to generate the appropriate spindle length (Figure 17-32). M-Cdk and other mitotic protein kinases are required for centrosome separation and maturation. M-Cdk and aurora-A phosphorylate kinesin-S motors and stimulate them to drive centrosome separation.Aurora-A and PIk also phosphorylate components of the centrosome and thereby promote its maturation.

TheCompletionof SpindleAssemblyin AnimalCellsRequires NuclearEnvelopeBreakdown The centrosomes and microtubules of animal cells are located in the cytoplasm, separated from the chromosomes by the double membrane barrier of the nuclear envelope (discussed in Chapter 12). Clearly, the attachment of sister chromatids to the spindle requires the removal of this barrier. In addition, many of the motor proteins and microtubule regulators that promote spindle assembly are associatedwith the chromosomes inside the nucleus. Nuclear envelope breakdor.rmallows these proteins to carry out their important functions in spindle assemblv.

1080

Chapter17:TheCell Cycle

Nuclear envelope breakdown is a complex, multi-step process that is thought to begin when M-cdk phosphorylates several subunits of the giant nuclear pore complexes in the nuclear envelope. This initiates the disassembly of nuclear pore complexes and their dissociation from the envelope. M-Cdk also phosphorylates components of the nuclear lamina, the structural framework that lies beneath the envelope. The phosphorylation of these lamina components and of several inner nuclear envelope proteins leads to disassembly of the nuclear lamina and the breakdown of the envelope membranes into small vesicles.

MicrotubuleInstabilitylncreases Greatlyin Mitosis Most animal cells in interphase contain a cytoplasmic array of microtubules radiating out from the single centrosome.As discussedin chapter 16, the microtubules of this interphase array are in a state of dynamic instability, in which individual microtubules are either growing or shrinking and stochastically switch between the two states. The switch from growth to shrinkage is called a catastrophe, and the switch from shrinkage to growth is called a rescue(see Figure l6-16). New microtubules are continually being created to balance the loss of those that disappear completely by depolymerization. Entry into mitosis signals an abrupt change in the cell's microtubules. The interphase array of few, long microtubules is converted to a larger number of shorter and more dynamic microtubules surrounding each centrosome. During prophase, and particularly in prometaphase and metaphase (seepanel l7-l), the half-life of microtubules decreases dramatically. This increase in microtubule instability, coupled with the increased ability of centrosomes to nucleate microtubules as mentioned earlier, results in remarkably dense and dlmamic arrays of spindle microtubules that are ideally suited for capturing sister chromatids. M-Cdk initiates these changes in microtubule behavior, at least in part, by phosphorylating two classesof proteins that control microtubule dynamics (discussed in chapter 16). These include microtubule-dependent motor proteins and microtubule-associated proteins (MAPs). Experiments using cell-free Xenopus egg extracts,which reproduce many of the changes that occur in intact cells during the cell cycle, have revealed the roles of these regulators in controlling microtubule dyrramics.If centrosomes and fluorescent tubulin are added to these extracts,fluorescent microtubules nucleate from the centrosomes,and we can observe the behavior of individual microtubules by time-lapse fluorescence video microscopy. The microtubules in mitotic extracts differ from those in interphase extracts primarily by the increased rate of catastrophes,in which the microtubules switch abruptly from slow growth to rapid shortening. TWo classes of proteins govern microtubule dlrramics in mitosis. proteins called catastrophe factors destabilize microtubule arrays by increasing the frequency of catastrophes (see Figure 16-16). one of these proteins is a kinesinrelated protein that does not function as a motor. MAps, by contrast, have the opposite effect, stabilizing microtubules in various ways: they can increase the frequency of rescues,in which microtubules switch from shrinkage to growth, or they can either increase the growth rate or decreasethe shrinkage rate of microtubules. Thus, in principle, changes in catastrophe factors and MAps can make microtubules much more dFramic in M phase by increasing total microtubule depolymerization rates, decreasing total microtubule polymerization rates, or both.

constant throughout the cell cycle, the balance between the two opposing activities of the MAP and catastrophe factor would shift, increasing the dynamic instability of the microtubules.

'seluosoJlual ululuoc ,{lerurou }Eql sllaJ eql azrue8Jodlaq saurosouloJqJ aql ur ua1g '(gt-ZI arnglg) saluosoJlue3lnoqllv\ dola,rap so^rqlua clueSouaqUgd aseq] ur selpurds JItolIu eqt '(IZ Jalduq3 uI passnJslp) 33a ue sezITIUeJ tl ueq^ errrosoJlual eql sapl^oJdri.lprurou ulJeds etqlsc-:(/ttlDJ!rcuaSouaquad'sr teqt) uopezlllpeJ lnor{tlm s33auro4 dolanap ol pacnpu ueeq aAPq}eql sodJque 'sapulds IUJOJ ]Jasur ureuec ur selpurds alquasse ol pasn ssacoJdeql oslE s-r1I 'sal.{cooplulue i(ueru ol ssaooJduorlgzruefiJo-JIaspeseq-euosoluolqo slql esn puB slueld raq8lq Jo esoql sE qJns 'setuosorlual IJBI [llPluJou ]uq] slle3 '(?t-ZI arn8lf,) selod apulds olrt] eql luroJ 01 sepqnloJtrru aql Jo spua snul1u eql sncoJpue Iuq-ssoJf, uel{} sule}old Surputq 'saruosoruoJqJaq} uorJ Le^AEspue pue-snuru snorJpl pup ,7I -ursauDl,uleu,{c 'lJede snuru eql qsnd dlaq osp surre aluosoruoJqJ eql uo 0I pue ?-suISeuI) spua snulu Jreql qsnd puB uolleluelJo IalluJEdUuPue uI saFqnloJJIIu IUII-ssoJJ ueql '(gt-ZI aJnBrCees) /i.lilueJg-urseuDlaqlJo srequaur dlrclnolued'sulaloJd Jotontr'seruosoluoJqceq] punoJe sepqnlorclu Jo uopez[qe]s puE uopEalsnu 'JeIIJeepessns aql qtl^/\ urSeq o] lqSnoql sr flqulasse epurds puosorlueJv -srp sE ,seurosorlueJJo aJuasqe eql q salpulds JBIodIq IUJoJo1 sllec salqeue ]eql saFqnloJJru azruesJopue ez$qels o] seruosotuoJqf,Jo 'iruqu sHl sI 1I 'saluosotuoJqcpunoJe seFqnloJJIIU '1oso1dcaql ul Jo uol}ezllqBls puE uopEalcnu Iusol aq1 Supuprups fqaraqt '(71 raldeq3 saxalduoc ulaloJd luo4 sulaloJd 8urz1qe1s-aFqnloJf,Ilu seseeleJ ur pessnssrp) ]rodsuerl JeelJnu uI peAIoAuI oslE sl qlHlv\ alD-ueu pelE^lpe eql aCIOJo ereld ul dJD pulq ol uua pe1cr Iosot{r eql uI asEdIC lptus E se]EI -nrurls Cgg aqt :ullBtuorqc ol punoq sI ]eq] (CgD) JolsPJe8ueqcxa eppoallnu -auruEn8 e uo 'UEd uI lseal lP 'puedap ol sruaesseruosouroJqceql Jo ,'bradord sql .ezrJau^lodap uorlrsod raruroJ s,aluosoruoJqtaq1 ]E selnqnloJJlu elpulds eql alrqM ,aurosoruorqJ peuousod dpvrauaq] punoJE sJBeddPflptder saFqnl -oJlrru a1p111ds l au Jo SSpIue 'luaurufirp Jo lno pe33n] sI eluosoluoJqc alSuISP 'aseqdplelu uI sllaf,etuos JoC 'peuJoJ sBq alpulds aql JaUBuraql uoplsodeJ o] Jr elpeau ssEISeurJu 3ursn.{q palerlsuouap eq up3 saruosoruoJqsaq}Jo aruanuul 'uoFezIIIqElsepqnlorcllu pue eqJ'uoueuro; alpurds ur ued aAIlcEue /{eld.{aqt uopealJnu alnqn]orrrtu qloq sro^BJl€q] lueluuoJl^ue lesol e SullEaJJ,(S ,(tq -tuesspepulds;o ssacord aql ut sra8uassedellssed lsnf ]ou eJBSaIuoSoIuoJqO fplt sraqs!lqnd qllM'0002 urorJuorssrrj.uoo uellrLu)ew '61-EL:Z'lo!8 ?pN"le ]a azrqournol'u {1qruassyalpu!dSr elod!g e}oulordseuosotllolt{) )llorlUl ila) ruorl) 'salnqnlor)ruaqt ratloqsaql 'sa1ereqdo.rlseleraqt raq6rqaqt teql alou lslaueddo1aql ur uMoqsaresuotttpuo) leluauilaoxa luarajJrpaql u! pauroJ afua)saJonll sralspaql ro sqde.r6o.trtt-u ro1:e1 eqdorlsele: pue ]o])ej aqoorlsele)aql pue dvn dvw ]ferlxa l)erlxa +opalaldap 1opalaldap dvt^ aq] uoaMlaqa)ueleqaql uo spuaoapelel '])eJua )llolll.ll 'peJlxa )rlolrur aseqdralut )llolltx aqdortsele> aql 'snqf'sl)erlxelrlolrul ul T st ro; aqqtsuodsal saqdorlseler6ur1e;nut1s rol)ej srql]eql 6urlertput'sl)pllxa)ttoltul s'0 palaloap-dvt^aql ur aler aqoollsele) aql sa)npar{1teat6(1eryyutalotd o palelar-ursaur) aql) Jol)eJeqdorlsPle) n'r 3 )uDaose Jo uorllqrqul'sl)erlxe)llolltu 5 stlq!Llu! ut saqoorlsele) dvw srq]lpql o s't . 6urlerrpur'a1eraqdotlsele)aql saspal)ul a sl)prlxa)rlo]ruraql urolJ(S!Zdeux pallP)) 3 rrpads e;o uorla;dapaql'sl)erlxa 6yy11 0- -7 a c aseqdralur ur ueql frlo]rurut taqbtqst a1e.t o aqdorlseleraql'palredxasy'{do:sot>ttu sz oapr^a)uo)sorong{q paano1lo1 seMsat!osoJlua)aql LUor]paleal)nu r.uri0 t salnqnlol)rrulPnpr^rpur ro ror^eqaqaLll pue sauosorlua) pue'urlnqntlua)saronlJ qllm paleqn)u! oraMsl)eJlxe66a sndouay alnqnlor)!ul )llollur ro aseqdralu;'r,ll6ual pue seqdorlsele)olnqnlor)!trt ;o {ruanberl sql se)uanlru!sdVWpuP eqdorgsele)uaa/nlsq a)ueleq aql s.ro1>e; leql a)uopr^a;e1uaulradx3gg-41 arnbtl

t80[

stsollt

uri O!

s a l o da l p u t d s

(ssar6{lrsrenrun rallala))ouaql rrrorluorssrr.ulad q}lM.966t ,t6E[-€g€I: tt t 'lolg 'ue^rllnS 'M pueolleqdlulesap .BLuoll).saulosourorr.,l) ila) f palerr;dar oql ale6al6as o] olqeaipsa;purds qlog.16u 1osed^1 ,a;purds saopsaurosorlua) elputds aqlseaJeqM ]noqltMpeLuloJ aqr;oa;od q)ea]e Jalspuespqsauosollua) qlt/vl alputds aqlleql eloN.uotleztltuoJ paletltutleLllollqula ue ut saurosollua) lnoqtlMluaurdo;anap lnoqltm pauro;a;purds e sMoqsqde.r6olrru woyoqa'41'ofuqwa paztltuaj DJnDS, {;leurrou e ursaurosol}ua) qltmpeulJo1 a;purds qder6olrrr"u ;euloue smoqs a)ua)seionU clolaql'parsauJosoulolrl) ar4l'uaatb pautelsalpsalnqnlol)tru aq1'(1eu6snbunlro)D,rDIrS: >lleuabouaqlred l)asulaql 1osol(lqura u! seuosorluo) gg-41arn6;1 lnoqllM^lquessea1pulds.relod;g ruo4 SurMoJBsalnqnlorc[u 'eurll erues aql lV.luaur{Jupe uo-pue ue o] pagal .eruosoJluec aql -uoc dlprde.r sr eruosoruoJqJ eq] o] lualuqculle leJelpl aqJ prelv\ol 1l Suop z(prder epIIS ot ueqt pup eFqntorsrru aql Jo eprs eqt ol purq ol lsJrJuaas sr eJoqtolaupl aql 'edoJsoJorrue ur luala eJnldeo Ierlrq eq] elJesqo ue3 a^ eJaq ^ '.slleJSunl lmeu uI 'pppuroJq3 Jelsrs auo Jo eJoqcoleuDl aql ernldpc dlenluala pup sauosoJluec aq] uorJ pJeMlno elerpEJ selnqnloJf,rru 'elpurds eql ot seuosotuoJql Jrlolru Jreql qf,elle Jo spua snld cnueu.,i.paql ol rusruBqJaru,,eJnldp3puB qcJEas,, u doydruasaruosoJlueJ8ururc1uocs11a3 'relel ssnJsrpal sE 'alpurds eq] uo sr eJoqooleuDl ]uetualoru euosoruoJqJ Jo IoJluoJ eql JoJIBJTITJO eq] ]e uo4ezuaur,(yodap pue uorlezrrarudlod pua snld Jo uoue1n8ag .et-Ll arnS;g) pua slq] ]B slrunqns u{nqnt Jo IB^oruer Jo uortrppE eq} 8ur^aolle ilrls elrq^aaroqoolauDl eql o1 dpq8q aFqntoror{u eqt Suploq fqaraql ,pua s]r rpau epqnloJcrru aql spunoJJnsleql Jelloc uraloJd e surpluoJ elrs ]uetuqtelle qceg 'auo ]sn[ ure]uoc saJoqooleuDl]spe^ seaJaqM,sa]rs]ueruqcE]]e esaql Jo 0t-0I urBluoc saJoqJolauDl IIac puruv 'aJoqsolauDl aql ulqlll\ir selrs lueruqJellp -apqnloJorru pazquroadsut uo-peeq peppeqrue ale sepqruoJorru eJoqooleuDl Jo spua snld aql'(gS-ZI arnt;g) euosoruorr{J eq}Jo uor8er crJeruor}ue3eq} le stuJoJleql urleruoJqJoJeleq aql uo llmq sr leq] aJnlJnJls uralord paradellpru 'luer8 e 'aloqiolauDl aql ]e ppeuoJqt Jalsrsqlee ol paqcellu eJe selnqnloJorru alpurdg 'seruosoruoJr{Jaq} ol ,r(eupeql Jo luatuqcp11eeq} sr uorleruJoJalpurds ur dals Jofeur puooes aq] ,(eJJpalnqnloJctu JBIodrqEJo dlquasse eq] Buu oiloC

elputdsaql ol sptletuolq)lalsrs q)ellv satoq)otou!) 'srsauqoilc ur sarllpruJouqE ur Suqpsar 'pauoqlsodsltu ueuo st alpurds eql 'llnseJ e sp isllec purue ur alpurds aql Suruoursod roJ alqrsuodsar aJp qJrqm ,selnqnlorJrr.uIeDSe $lf,el ll fptu -Jou satuosoluoJqJ ale8ar8asuuc alpurds leruosoJlueceSuqpsar eql q8noql1v 'pa^olueJ aJB saruosoJlual eql y alputds Jllolru JBIodrq e 3o .,(lqruasseeql aloruord uEJ 'suralold Jolour snoIJeAJodlaq aqf qllm'pue salnqnloJJrrualpurds jaqlosnoraurnu llede olutspuasnutulasoqlsn)oJ'suralord ql;mleqtabol'sroloru tl-ursourlor, ,,."nj-"'l:1:5il::i: aqluot; {eane spuosnuluJ qsndpue sture aurosoLuorq) ol salnqntor)rul pue aq} pol)arp pua-snlo 0[ t-sursau!l lurl .soulosotuorq) allqM'salpunq olutsalnqntol)tt,r't asaqleztue6lo (0E-1[arnbrlaos)sutalold lalleledllue JotoruS-utseut) aq] aql ul salnqntot)rr.li uorleuro; aql pue alouroro 1oIlrut>tn aleal)nu suralord selelrl)e q)rqM,(umoqs Jo lpq] rou)dfgueU )rlolrll'suralordrolouriq uoglezrue6ro-1;es Jouoll)npoldle)olaqlalelnullssaurosor.rrorq) alpurdg t€-ll aln6rl

tt-ursaut)pueurau{p {q salod1obulsntol

g 1 ' 7 - u r s a u r{lq qsnd preMlno

g - u r s a u r{l q 6 u r 4 u r ; - s s ol.eol l e l e d t l u e

el)r() lle) aql:lt

uotleel)nu

raldeq)

ZgOt

'aloqlotaurlaql o] paq)Pllesureuar olnqnlor)lulaql allqMpua snldpasodxo aqt le rn))o ol uollezuau.l^lodap pue uorlezuau,(;od 6urmolle 'pua sn;dalnqnlor)ru,aqt spunorns lq-t4l(t^oilat)arnl)nrlsrello) e uteluot ol tq6noql sralls q)el'aroq)olaull e u! alls alnqnlor)!urV /€-lI atn6ll luauiq)e11e

alnqnlorltrll aroqrolaurl + op u a s n u r u r

a J o q ) o l e u r )r a u u l

'ueql loeJJo3 ol peAIoAeeAEqsluslueqcaur drol -e1n3arluefiala pup tnJJo op sluaruqJelle ]JeJJof,uI'ssalequaleN 'alod alputds aluusaqleJeJuusSeJoqso}auDlqloqlsqlpooqna{IleqtSeJnpeJleqluopuluelJo eql Jo uEd sI Ja1v\suB {3uq-o}-)ceq B ur palsnrlsuoJ alp seJoqcoleuDlJe}slslEqt alputds arues ;sa1odapurds qloq ol aroqsoleuDl euo Jo lueuqsullE aql ro alod 'uollulua aql ol seJoqsoleuDl qloq Jo lualuqceile aql slualard IEI{ \ ipa^elqlu ,lueurqJeue -IJo-Iq ^ oH 'aseqdeue ur aleredas daql uaqru sl slql epou pellef, Jo 'alpurds cpotgr eq} Jo salod elIS alrsoddo ot elorrr ,{aqr reqr os 1"r "ql-;o.pua -oddo o] qcEllp rred e ur spDprrroJqJJalsrsteq] spuBuep sISolIIuJo sseJ3nseqJ

rorlf pue lelrl ^q pe^arq)vsl uollelueuo-!8 'fty-Ll arn8tg aas) alod alpurds eql 1Bsulelord -rolour Lq pasnco; pu€ spua snulur Jeqlo ol pe{uq-ssoJf, eluooeq AIIEn}ue^eppo^ ,salpurds eerJ-atuosorluecur spue snuru Jeqlo aIII 'selnqnlorlllu eJoqsoleupl sepq$oJolru aqt;o qyvror8 asaqlJo spue snurru eqJ'aroqooleuDl eql uroq ^,i.ea,re 'aloqso]auDl eql Jo SJPI ur llnsal uaql ppo.ry\ spua snld esaql le uopezgraruflod -1ocSurpurq-pua-sn1deq] q pappaque euoceq pu? saloqsoleupl qlIM ]rurelul go ,tprcn aql III sepqnloJcr{u Uoqs tuq} sr dlqrqlssod aug seuosoruoJqi "qt seop lAoH iseluosoJlueJ Jo acuesqBeql ur JnJlo lualuqcellE aruosoruoJqf,'eluosotuoJqo '(8t-ZI arnt1$ luaruqoeile relodrq u Suturro; aqlJo eprs alrsoddo eqt uo eroqootaur{ aql o} qoet}e alod alpurds alrsoddo aql qllM'0/6t ('99151 ruorluorssrutao 'y) 'sta'{e1 'g'g;o,(salrno> '1 :r(a11uug 'O'f q}!M 'f 'isny'a)Mol'f'l '26-LLiEZ'pS'lo!8 uor; leuolrlppe pue sdeag-11apr6 e a^eqsaloq)olaull }sotu JeululellJl auoaq}'arn})nlls e Luorr) araquMorls {1;ensnun ueseq(e6leuae.r6 xa;dr,rjo: alnl)nJls uorl)alf(f) 'slla)uelleululeul ue;oqde.rbolrlLu qtlMplleuloJLl) aseqdeue slrol paq)euesalnqnlor)r,u alrqM'aroLl)oleurl e ol punoqsalnqnlol)lul eseqdelar-u aloq)olaurl Jolaqunuaql'aleuollua) ut t ruolJsatje^ autosut0' ol lsee^6urppnq aroq)olautl aqtuo enbeldp suloJaloqrolautlqrel 'selnqnlol)ru Jospuasnldeqlo] paq)ellesplleuolq) aqlro a)eJJns q)eaqllMpalelfosse alP'plleuioJq) e;o 6utmetp aseqdelau aulosotuolq) lalstso11ls1r6urmoqs V G)'paipaulels ueulnqqtlMpuea^plua)salonu qllMl)perleqtsatpoqtlueolne rr;Deds aioq)otautl oMlaq1'surato.rd auo,saloq)olaut) 'aroq)olau!1 aq19g-41eln613 a)uarsaronlj e;o qder6otrtu aseqdelatu V (V) r qrlr*p"rlrtt arrrosotuolq) 6urpurq-yy6 (8)

(v)

prleuo.Ll)

f

aJoq)olaurlI ur peppequlaI salnqnlor)rLU L

auosol.!olql

+o

luaura^our prleuiolql +o uorl)ejrp

aurosoulo.lq) pale)rloeJ

aloq)olau r)

SISOIIW

€80t

'a)eldur pel)ol ^qalaqt srtuauq)eup t)ailo) stql.^lturte 6urpurqelnqntol)tulut aseal)utue sra66u1q)tqM/solts6urpurq-a;nqn1o.orr.u roql ssol)euotsuolasuasol rq6noqr aresaroq)oleut)lalstsaql /atoq)olaut) puo)asaql o] sputqa;odalrsoddo aqt urolj alnqnlor)trrJpuo)as e uaqM alet)osstp ol spuelsalnqnlol)turoMl aql Jo auo ]eql os la^aMoq,olqetsun ete sluauJq)ellp l)alro)ut asaql.aloq)olautl auo ol rl)ellp ue>sa;odalpuldsqroq uorj salnqnlol)tutlo ,aloq)olautllalsts reqlo aqt ol q)ellp ue: e;oda;purdsaules aql ulol; alnqnlol)tuly'slem snouen ur auosoLuolq)oql o] putq uaql ue) salnqnlorlil,.U leuotltppVI tedptleuorq) Jalsrse ut atoq)olaut)auo ol sputq alod e;purdse LUoIJ alnqnlortru a;6urs e'{;;ertrul'luaruq)eltp auosouroJtl) Jo sruro]a^lleural;y 69-1 1 atn6r1

ltSvl_s

lrsvl_sNn

l'l8vr-sNn

& rrSvisNn

'alrs lueuqJellp aql Jo [1ru4;e eql SursparJur pue uor]B1d-roqd 'sJnJJouorlBluarJo-rqueq ^ petE^rlJEur -soqd aroqcolaur4 SurcnpeJ dqaJaql sr g -EJornv'pua snld epqnloJJrru e JoJ &rugJu s,elrsaql SurspaJf,ep,alrsluaruqcE]lB alnqnloJf,ru aq] Jo slueuodluos IeJe^essaleldroqdsoqd 11'uorsual Jo eJuasqe aql ur alnqnloJcrru qlSuans lualuqJenB aql seJnpar d-rolrqrqur ]eq] Jo IEuSrs eql aleJeua8 o] sr g-eJoJnV 'aroq3oleur) aql qly!\ pelerf,ossE lq8noql qorqlv\ sr 'g-proJne aspuDluralord aq] uo spuadap rusrueqteur Bursuas-uorsua] eql 'salnqnlororru a1d41nu;o pesodruoJ p Jo uor}eruroJ Jaq{ aJoqropqry {JIq} aq] ur slFSeJ srqJ 'aJoqJoleuDl aq] 01 saFqnloJJrru puollrppe Jo ]ueluqcBne aq] o] spBel oslP ]nq elrs ]ueluqJene aq] Jo ^lrurJJp aq] sesEaJf,ur,{luo tou uors ']uaruqf,EDe alnqnloJsrru -uel 'slleJ SuruaqlSuarls ,lpuSrs .,trolrqrqur IEruruE uI eqt JJo stnqs aloqf,oleur{ aq} lE uorsual q8rq aqf ,sJnoJo uor}e}uerJo_rq

'outptlsauosouolq) Jo uott)altpaql sale)tput(l) ul uouo trotbaql oltqM,qlmol6elnqntol)rul Jo uoll)altpaql salp)tput(V)ut MdrD par aqI 'soJoq)olau!I {q sa;nqn1o.rtlu euosorlua),o arnlder aq1 gg-4 1 etn6r3

luaut qlelle lelodtq ol ielodtun lia^uo) ol alod alpurds alrsoddo txol+ salnqnlol)tul salnlde) aloq)oleutl eaJ+ G)

-auDI eq] saop /v\oq '1ng 'ace1dur pa)Jol arp sluaruqf,Ellp lJaJJoJ alrr{lv\r}sel }ou op pue elqelsun duStq aJEslueruqJeltp lJeJJoJur:aldrcurrd aldurrs e uo paseq sI leql rolJa pue IBrJ]Jo rua1s,{se .,(qpalcarroJ aJe slueuqceup IJaJJoJUI

elnqnlo])tuj oloq)oleut) pelle) MOU alnqnloJ)trrj :luaulqleDp lelodtun 'uo-pua o] sueAUo) ]ueLuq)elle lerotel (c)

a ; o d a ; p u r d sp l e a n o l seprlsaulosorrJolqr())

aInq nlol)t ul l e r l s eu e o 1{ 1 1 e t a } e 1 s p u r qa r o L l l o l a u t )a u o ( 8 )

aseqdelatlo.rd le eurosouloJq) peq)el]eun (v)

a ; o da ; p u r d s

\

el)^) lle) aql:/ [ roldeqf

t8OI

'alodalpurdsaql pua snurul le alnqnlor)ilJraql preMolaloq)otautl aql a^ourllrMslq]'eldr)uudul 'pua snld alnqnlol)rruaql spunollnsleLllaJnl)nlls rello)aqt lsurebeqsndpue (gt-g t arn6rl oas)preMtnoUnr alnqnlor)il.u aqt Jo sluaureluolordaql 'srn))o uollezuaulllodapuoqM'alodelpurds eql PleMol aioq)olau!{ oq} llnd r(eur uoltpzlraurllodap,vloHot-l t arn6rJ

eroq)oleurl salnqnloJ)ru.r +opuesnlo]o uorlezuaur{1 odap

eroq)olaur)

'lzv-Ll ern8ld) alEld esEqdEleu ar{} tB srlud ppeuoJqc Je}srs peluelJo-Iq eql UBIIE sdleq ll ueqm 'eseqdpletu pue eseqdelauord uI lupuodlul dlJelnJlued sI eJJoJ srql'sayod alpurds aql uor; derrnesauosoruoJqc aql lrodsuuJl pup selnqnlorolrrr relodralur qll,lr lf,eJelut srure eruosouloJqf, uo srololu 0I puu t-usauDl pelJeJlp -pue-snld 'auol uotlca[a n1od et4 sr seruosoruoJqo uo Supce acroJ prlql V 'aseqdeuu ur aleredas .{aqt rage splle{uoJt{JJe}sIS }uau Jo -e^oru prerr,ralodeq] puE aloqcoleupl eql le uorsualJo uorlsreuaS eql ol salnqlJl -uoo qJlqa 'ecJoJpJemalod e sacuarradxexng qcns Suto8rapun eFqnloJclru e o] paqrepp sr leql aJoqooleuDl duy 'salnqnlorcrtu relodretu pue eJoqJolaul{ qloq pJemo]Ie Erl lpql saplcads]ueoseJong,(ur13oacuureaddu aql uo salod aql ur stlnseJ slql'(I1-ZI arnt;g) s11ac3urn1 olut palcalul aJe uIFqru ]uacsarong Jo slunoue 1eurs.{ral qJIqM ul poq}eu snorua8ur ue dq uaas eq uEf, salputds aseqdelaru ul xng e1nqnloroll l 'a1odalpurds aql premol sepqntoJ3lru Jo tueu -aloru aql atldsap ]uelsuoJ surprueJ r{13ua1epqnloJf,Itu }Eql os 'pue snulur aql ]e u1nqnl Jo ssol aqt roJ salesuadruooelnqnloJJrru e Jo pua snld eql tu ulFqnl ,,lreuJouorlrppp aql 'aseqdeuuJolasuo eq] Ulun 'spua snuru Jreql ]E palluerusp pue salod alpurds eql pJumol peloru eJp sellesueq] saFqnloJJlru aql ,ri.qararylt 'xnfl Lq alnqnlo.rc;ur sadril 1ac auos ul papr,rord sI ecJoJprerralod puoces V '(gt ;aldeql ur passnJsrp)eFqnlororru e pua eql o] sppe lrun Jo -qns urnqn] e reue srnf,Jo teqt dJC;o srs,(1orp,tqeqt (uoq saruoJ it1aleu4p 11 lsazrarudlodep apqntorJrru eqt ueqm paspalersl puu epqntoJJllu aqt ul perols sr eql selrJp leql .{3raua eql 'eloru ,{qaraql pue sepqnlorcrur 8ur luerueloru -zrraur.{.1odap o} paqf,E}leurprueJuec 'luasard dJV ou q}lm'aqn} }sa} E ur seJoqJ -oleuDl pagrrnd lpql rrmoqsueeq spq tl tnq'lsJg ]e alqrsneldurrruaesrq81rusrq; ',{.13uqsara1u1 6;y armbar ]ou saop acJoJpJp^\elod palerauaS-aroqcolaupl sql 'Jalel ssnf,srpaal se 'asuqdeue ur alurudas daql rage salod eql pJEMotsppeluoJqc ratsrs Surnotu ro; luuuodrur ,i.lreprqred sl pu€ aseqdularu pue aseqdelaruord Surrnp sauosoruoJqc uo slnd ecJoJslql''p'V-Zt arn8gg) pre,nelod eJoqcotaupl aql slpd teql eJroJ e sateraua8 ,lroqeruos eFqnlorcrru eqlJo pue snld aql te uol] -ezuaru^,t1odep'usrueqJeru ureuef,un ue dg Jlestr aJoqcoteupl eql lB suratord ,(q pecnpord q iI 'alod alpurds aq] pre^ ol aFqn]ororru eroqcoleupl aql Suop euosoruoJqJ paleroosseslr pue eJoqcoleuDlaqt slpd acro; roleru ]sJrJer{I 'tueilodrur dpulncqred aq o1 lq8noql eJe seJJoJroferu aarq; 'alpurds JI]olIu aql Jo seFqnloJJrru eql uo saruos -oruoJqo aloru lpql secroJeql aleraua8 srusrueqceruJeqlo pue suralord Jolontr

alpuldseqt uo sourosoruoil4) a^ow salrol oldlrlnw 'Jelel ssnJ 'aurq 8EI -slp ellt sB 'seurosoruoJqc aql Jo lspl eql Jo luaruqcu1p ruyodrq eqt JeUe alqercpard p qllm 'pacnpord sr pu8rs aq1 'aleredas o] spupuorqJ ralsrs eq] JoJ pu8rs aqt Suqpare 'a1e1daseqdetau aq] le ,tltua8 alelllf,so ueql saruosoruorqJ aql 'sIIeJ alerqeuel u1 'e1eydasuqduleru eqr pellec uolllsod e 'se1odelpulds oanl eql ueemleq luelspmba uorlrsod e Sununssertlpnlue,ra 'quog pue 4cuq pa33n1 eJe saruosoruoJqr aq] 'sa1odalpurds o^N aql ol lueruqJelp Jleql 3urarro11o4

srsolrw

980r

'aloo eqt preMolsaulosoulolr.l) llnd ol lqbnoql ale'xnUolnqnlol)rul ql!Mraqla6ol'aloq)olautlaLll le uortezuaullodapIq paleloue6so)ro, premelod'(O€-/ t aln6rl aas).rolenba alpuldseql pJemolsaulosoulolL.l) saqsndq>rqnn'ariol uorlrafare;odeq1 elereua6ol salnqnlol)l.u qltm l)elalul ol lqOnoqlele sulleauosoruolq)aql uo (0 t-ulsaull pup t-utsoutl) suralo.rd.rolor.u pal)arp-pua-sn;6'a1e1d aseqdeleuaq1 ol saurosomolLlfanoul ol ateledoo>{elu sarro;6ursoddoolvrlMoq Jo lapou V (g) 'uotslndarpespoJ)op e 6urlraget 'elod plemol aql sonoul oJoq)olautl aql qllM lleo oql seoreqM'alod aql uolJ ,{emer(;prderpaqsndst eloLl)olaut)ie paJa^asaql Jo ued lnoqllM aurosoutoJLlf aqf 'salnqnlol)rularoq)oleurl {q e10d a;6urse o] paq)eupstleql euiosotuorrl) aseqdelauo.rde slanost!eoq Jasel e'lueuuadxa s!Lllul .Jolenbe elpurds eql pleMolselodalpurdsaql ulor, (earresauosoluolqt saqsndlpql o)roJ uoll)afarel0de to; e)uapr^](v) .aleld eseqdegauraql ol sauosoutolq) a^Up (eur sarrol Eursoddomog 79-41 e.rn6r3

sutelororolor.! 0 ['t-ursaur) pepalrp-pue-sn lo

a;od u.rol1,{elae se^oureroq)oleu!l lnoqllM trjle

el nqnlor)rur l e r l s el o J e l o o J a l Iu

eql lseJeau eJoqJoleupl aql ur uorsual selPJeueS ecJoJ uopJefe Jelod SurseeJsur ue 'elod elpurds aq] pJpMot a^oru seruosoruoJqf, se lpq] 'alduruxa roJ 'pesod -otd ueeq seq ]I 'eJoqooteupl aql ur uolsuel Jo eeJ8ep eq] uo puadap deur satels o^tl eql ueeMlaq q3ll.\/\s eqJ'elod aq] ruo4 I?^aE ser.uosoruoJqJ eq] seqsnd eJJoJ uorlJefe Jelod eqt puE JJo peuJnl erE seJJoJ pJe^aelod aql uaq,\/\ 'elPls 'lsJlneu Jo '(dV) alod-eql-uo4-itu^^p ue pup 'elod aql pJe^\ot pellnd eJe saluosoruoJqJ eq] ueql\i\ 'elels (d) pJB^\elod e-salels o^ 1 uaeMleq qsllr\ s 01 uaes eJE sluelu 'slleJ -a^our eql ur ^doosoJJllx oepl^ dq parpnls uel{ A 'esEqdEleru 3un1 luau puP ,{roleltcso eql Jo luelxe^oru sno aseqdeleruoJd ur saruosoruoJqc -nurluos eql sr sllal alerqeue^ ul srsolrru Jo slcedse 3uoF1s lsou eql Jo euo

('Pl'lslaqs!lqnd uelllul)ew 'o'l pue urolj uotssilIladqllM ' looz 'Lz-ll1:E'tolgilD ?DN'uor.ules uosrq)llw'r1 uor,') pue g) 'puo snuru aql uioJ]pe^ouereJeslrunqns urlnqnlseateraurpsaql ]e pua snloolnqnloj)ruraq] le pappeereslrunqns urlnqn]^ au asne)aq,(1lue>;;1u6;s e6ueq)tou saopalpuldseseqdeleu aql u! qlbualelnqnlol)lW(O) preMalod6utnou alPsalnqnlor)rr,! aql leql 6ulterlpul'uruTruriS/'0 lnoqelo aler e le salodaql ple^ ol 'aurllJo^ouor6araql;o abetuotu onourol uaasaq ue) sall)odslenprnrpul p oleur ol aprs(q eprspalsedpue seurl ;erluanbes le ualel aJaM(g) '{do>sot>ttu ul (/Au)D)uor6arpaxoqlelnOuel)ar'urql'6uo;eqlJo saoeLul oaprnesde;-aur;1 r(qpalro;1o1 eq uer sa;1radslenpt^tpulJo luaula^oul aql (f) par are sal))ads urlnqnlaqt pue'ulAorqparolo) ale saulosoulolq) aql'lla) 1e;laql;da 6un;vvrau6urnr;e ur alpurdslrlolru e Jo sqdel60l)lr.ll a)uo)saronll (g) ',(do>sor>ru.r aruarsaron;;r{q peMetAuoqmarueieadde pallradse e^eqsalnqnlor)il.u q)nS'utlnqnlluo)salonUjo uotuodotd lleuis^le^ e qllM uiloJsalnqnlor)tr!lenpl^!pullPql os slla)6ul^ll olur palrafursr urlnqnllua)saronpJolunoue lleulsfuaAe'xnUalnqnloJ)rtu a^lasqoo1 (y) 'alpu1dsaseqdelauraql u! xnu elnqnlor)lw 1y-4 1 aln6g

auJ] Idpred dpo sr ramsue aqJ ill3dv aqt sate^rlf,e1eq,u 'aseqdeue sraSStrl l/JdV aqt JI '(ratel passnJsrp) srsauqoldc pue srsolru yo uoqalduoJ aq] ro; parmbar se 'lleJ eq] o1 saseleqdsoqdsrr,rolpuotl ur saleJlsqns1a8re11p3 fueru aq] a1e1,{roqdsoqdap 'uollJnr}sep -elrlJeur 1p3 'aseqdeue ur ,,i.lr,Lrlce {pJ lsour Jo ssol aq} ot Surpeal ro; surlcu{c-1xpuB -s aql slaSret osp :)/3dv eq} 'uunras o} uoulppP ul

'ftv-Lr

pue ,(pdnrqe splleluorqJ Je}sISeql pue ernt;91 aleredas .r(1snouorqcu.,{s 'ursaqoJ "{eme Jo slrunqns aql Jo auo a^Eelf, o1 aa4 uaql sI tlJlq^\ IIPJsursaqoJ aqJ 'asBJ 'aseredas sasealal aseqdelaru Jo pue eq] lB uI-InJasJo uollJnJlsap aql -edas palpc asealord e yo flvrqce aq] s]lqqul pue o] spulq uIJnJas 'aseqdeuu eJoJeg 'uor]lnrlsep roJ ulrnJas uratord drolrqtqur aql Surla8rel [q ssac -ord aqt salBIlIuI :)/JdV eqJ'alpurds aqt;o salod attsoddo o] aloru pue aleredas ot sJatsrsaqt smolle qrlq \ 'uorseqof, plleuorqc-Jalsls Jo uopdnrstp uappns E qlp,t sur8aq aseqdeuy'pede spqeruorqr ratsls aqr gnd teq] saf,roJprea'tayodaql 'aseqdelaru Suunq lsrsar raqlaSol spupruoJqJ Jalsrs eqt Surploq sulsaqoJ 'N1Z-LI arn8rg aes) uortJnrlsap rraq] 3urra33r4 ^,(qaraqlpue sutalord ,{rolep8ar cpo}Iu IBJe^esSurlelrfirnbrqn ,{q uoqeredas pueruoJqJ-Ja}srsse}ellrul }eq} qc}rms aql s^\orq} JerlJeepassnJ -slp (J/ldv) xaldruoc Sunourord-aseqdeue eq] 'tua^a srqt roJ a8els aql slas dlt -^rlre {pl-I q8noqlry'(t7-21 arn8gg)uortrsuerl aseqdeue-o1-aseqdelaruaqt te sp[euorqJ ralsrs ar{l Jo uorleredas aq} q}lm xerurlc s}r saqJeera1c.{c1ac aq} 'sIS -o1ru,{pea ur JnJf,oteqt sluaua8uerrear xalduoJ aq} para83rr1seq IpO-IN Ja}JV

srsolrwJo uorloldruo) aql pue uoltpledosptleruorq)-talslssle66ul )Ddv aql 'alpurds aqt;o rolenbe eql lB ser.uosoruoJqlJo uorlelnruncop aql ul 3ur11nsar.{lpnper8pue alels alod-aq1-urorg-rteme eql ol qJ}IMSe 3uua33tr1'a1od ('rafeg marpuy;o,(sa1rno11 'ullnqn] palaqel-plob sarpoqrlup qt;rur paulets r.uredsopu (All) aram a snLltuowaog ]sute6e ]eqls;1a> 1osqder6o.rrrur asaqluruMoqs preMola^ourpuealeredas,{;ueppns sprleuorq) ralsrs se-alpurds )rlolrrrt aqlJoselodalrsoddo rq61; '(B)aspLldeue geuogle,redas gy-21atn6;3 plleuorq)-ralsr5 ol (V)aseLldelaur ulorJuortrsuert aql ul 'aseqdeue tud 0Z (8)

(v)

I

/80r

stsoltw

fssar6{lrstenrunlallajal)ou aqf uorl u o r s s r u l eLdl t l M ' 9 6t6, | , 6t [ - [ BLt : [ t I 'lo!8 ila)'f "le la slalPM)'l worl)'(/AoilD allLllvAq pelplrpur'1opa1od1a;od elpurds aqt ol paq)eDPs! leql plteuorq) Jolsts aqlJo aroq)oleutlaql qllM polet)ossp sl ZpeWJo lunoule lews V '(/Aut0 pa; {q palertput 'Joppat)ptlpuloJr.l) lelsrs paq)eDeunaqlJo etoq)olaut)aql ol punoq srzppw reqt sale)rputsatpoqrlue zpew-llueLlllM6ululels'a;purds eLlt,oalod auo Iluo o1peqrellesrtred plleuorq) relsrsauo'an/gut sptleulolrl) rolsfseql pue uaafi ur a;purds)tloltui eql r.lllm'espqdelouo.rd ur lle) uetleuJuleul e smoqsqder6otrlr.ua)ue)salonU srql'saroq)oleull peq)eDeun uo ulelold 7pe4 99-41 ern6;3

'sllec eseql q es"qdPuE;o Surup aqt eulruJalep lsnu 'uv\ou{un }ed sE 'rusrueqserrr Jer[o euos 'sofJque ^u pue 8oq dlree go sllac eql puu slsee.{ sE qJns 'sllec aruos ur lulod{Jeqc dlqruasse apurds eql uo puadep lou seop eseqdEue Jo 3uII'4] IEIuJou aqf flSugsrrdJns 'esEqdeue pue uorleJEdas ppeurorqc-Jelsrs aJnl?ueJd sesnef, rusruBqJeru ]urodlseqc eqlJo uonrqlqul ptueulJedxg'ral?l salnuFu 0z ]noqP surSeqesuqdeue puE 'elp -urds aql uo paluerJo-lq saruo3eqJIBd prleruoJqJ Jelsrsls?l eql JaUp sluaruoru surBeqslleJ eseql ur urrnces Jo uortf,nrlsep aql 'asuqdEuEJo Sullup lelurou eql seurruralep ]urod{JeqJ dlqruasse elpuds eql 'slleJ Jl}eluos uErleluluelu uI 'zpeIN 'llf,dv-Ozrpf, ltqtqut puu pulq uec zpBIN tBqt os Jo uonPruJoJuoJ eql uI aSueqJ e sazfplsc leql au^zue uB eII slJe eJoqcoteupl peqJeueun aq] lBqt sr 'zpew Jo ses.[lBuelBJnlf,nJ]s pelrelep uo fluEruud peseq zbllqrssod Suluadde auo '(gt-ZI eJntIJ) uorlrurg ol tulodlraqc ,{lqruasse alputds eql JoJparmbar aJp pue seJoqJoleupl paqJEllPun ol pellnroeJ ere 'ZqDW Surpnlour 'suralord IpJeAesq8noqrp teelJ lou sr IpuBIs slql Jo slseq Julnf,aloru eqJ'llar eql lnoqSnorqr &I^llce 3/3dv-Ozrpf, srlqHul lsql IeuSls elqlsnJJlpe etereua8 lv\oqeluos seJoqJoleupl paqopDu ,tlalerrdorddBur ]eqt lq8noqt s1t1 'JnJJo ol uorleJedes plleruoJqo-Jelsrs Surmolle 'pelorueJ {oolq qql $ peqoeue .{padord sr aJoqooteupllsel aqt uaqrvr,(yug'uo11lsue4eseqdeup-ot-aseqdelaru qcolq leql IEuSIs arrpeSau u }no aq] s{rolq snql puP uor}?Arl3E 3/ldv-Ozrpc spuas alpurds eql ot paqJeDEdlradord lou sI teqt eJoqJoleupl duy'aroqcolaug eql le 'uorsuel ^lqrssod puE 'lualuqcEils elnqnloJ3lIu Jo qlSueJls aql sJolluou lEqt usruBqsaru Josues P uo spuedep luod{3eqJ ,{.lqluessEalputds aql 'elpurds JIlolFr eql uo palualJo-Iq dllcarroc aJEseruoso(uoJqo IIe glun aseqdeuu Jelue lou op sllal leql seJnsueruslueqJelu lurod{JeqJ aqJ'uoqrsupJ} aseqdeue-ol-eseqdeteu eq} q8norqt uolsserSord qcolq pue lueluleaJt Snrp aql [q palEAI]rB sl tEr{} rusrueqsaru lulod{raqr /qq -ruessualpugdspJo uoupcglluapr aqt ol pel uolle^Jesqo sIqI's.{ep uela ro srnoq JoJsrsolru ur sllac ]sarJe '(gI Jaldeq3 ur passnf,slp)eupsulqutrrJo auIcIqJIoJ se qsns 'saFqnloJJrrrrezlrqElssp lPql sSnJC'alEJedasol spueruoJqcJelsls sacnpul '1noqe 8u1pso['aleld aseqdE]elu aql uo pau8rle leq] IBuBISO/OdV eql SulllE1!\E saruosoruoJqJ aql qll,\,\ 'aseqdulaur ur srsolru Jo Jpq lnoqB puads dlensn s11a3

lu!odl)eq)(lquassvelpuldseql q) peqle$eun :uolleredesplleuorq)-Jalslsl)ol g seurosoulol eseqoeue

eseqoele[rJ

'uoltel^roqdsoqdop aseJeoes e^!pe

st! 6urMolleIq uorlenrlreaseledas selouiordosle(uot])nllsopur;tIt ruor; 6ur1;nsar) aseqdeueut uorte^ll)eu! lp) 'snqI'(uMoqs sltqtqutosle lou) aseJedes qpf ^q uollellroqdsoqd's;;a>leugue ul 'ueoesplleLuorq) ralsrseqt llnd uaql a;purdsrr1o1rlu oqt Jo sa)roJ6u;lgndaq1 'ft7-4 an6/'7aes)reqla6ot splleuolr..l) ratsrsaqt 6u1p1oq xe;duot utsaqo)aql Jo s,l/\olle lrunqnse '[)fS a^eal)ol asetedas uun)asJo uorl)nrlsepeqI'elels e^tDeul ue ur eseredas sp;oq{;leutou q:rqm 'uun)os uo!])nrlsappue uoqte;I1;nb;qn ro aqt ot spealgz)pf ^q )/)dV Jo uorte^tl)p aql')/)dV aql ^q uolletpdesplleuorqr -ralsls,o uotletltureql 99-41 etn6l3

J/JdV o^rlleul

0z)pl

ap{1 1;a1eqt:/ [ ra]deLl) 980t

'a)eJrns eql prPMot'Jaqto q)ea lla) ujorj ^eMesalodaqt llnd ot salnqntor)rru lellseuo ])e alod alpurdsq)ea reaueuerqureuieulseloaqt o] peq)eue suraloJd Jolourpue'uPdesalodoMl oql qsndalpurdslerlua)aql u! raqloue lelodraluraql ouo lsedsalnqnlor)rur ,o 6urprlspup uorlp6uolaaql:g aspqdeue roJolqrsuodsor eq or rq6noqreje sa)JoJ aleredasoMI'tJede a^oLl-i salodalpurds oMl oql'B eseqdeueul 'xnualnqnlor)rnl prPl aloo pue salnqnlor)il.!aroq)olaur)i aql uo spuadapv Jo uorlezuaur^lodep aseLldeue ur salodaql preMol]uauJa^our aurosourorq)'slle) ue!leuureuiul eseqdeuele splleuorq) Jals!saleJedes leql sa)ro, roleur aql gt-l I ain6rl

'leluJouqe aJPesEqdPuu JoIAPqeq Jo eFqnloJsrru puP sluaua^orrl etuosoruoJqJ eql ]nq sJncJo dlerauaS uoIlEJ -edas pupruoJqc-ralsls-eldtuexe JoJ'll3dV aql ^q pazluSoJerlou sl leql urroJ 'sullJIJ sI uollsnJlsep ullf,l.J-I^I JI tuutnru P Jo uorlsnpord eql fq-peluanard 'saleJlsqns Jo uor]f,nrtsap luepuedap-3/f,dv aql tuo4 sllnser sllac lsolu uI qslqM Ipl Jo uopellroqdsoqdap eql uo spuedap aseqdeu? le(uJou E Jo uouelduoJ 'g pue epulds uI uolte8uole aseqdeue aqt ^lluulJodrur 1so141 V eseqdeue ul 'y aseqdeue sluarue^oru euosoruoJqs lJeJJoJ aJnSUaOSp StUSIuBqJeIUJeqlo 3o sluerue^oru auosoruoJqc eql selElllul uoIlBJPdesppPlxoJqc-JelsISqSnoqllv 'uBdE selod eq] ilnd xel -Jo3IIac aql o] spua snld eFqnloJsllu IeJlseJoqJuBlEq] sJololu uleufp 'uoglppe uI 'ilpde salod eql qsnd 'sepqnlorJllu rulodJelu eql Jo spue snld Sqdd€lJe^o '(Og-Zf ornBIC aqt {uq-ssoJJ qclq a 'sutatord Jo}otu g-uISauDIpelJerlp pue-snld ees) srsolru f.Fea ut seluosoJlual oMl eql aleredas lEql esoqt ol IeIIIurs suISIue -qJaru ue^rJp-Joloruuo spuadep g aseqdeue8u1rnp uollPJEdasalod apulds 'aJollf,olauDl slpsar slles 3I}EIuos peteJeueS sesJoJ uro4.{.preurrd eql le 'aldurexa JoJ 'x[lu eFqnloJJllu uo elEJqaue^ pue lsee.{ uI luelue^orrr seeJeq A (sllecJluo^Jqrue uI:sedfl f.lureru spuadap luerueloru euosoruoJqJ IIeJ iueJeJJIp 'sJnof,o ur serJel aseqdeue Surrnp secJoJoml eseql Jo eoueuodrr ellleleJ eql uolluzuaurrtlodap pua snul(x aJewv\ 'alod elpulds eql pJB^aol sepqnloJ3lru eq] Jo luellle^olu pru^aelod aql sI qJIqM 'xnu eFqnlorctur Lq paprzrordsl puoJas aql'a1od eql prEMol sa^olu aroqJoleupl eql sE pue snld aql le sllunqns ulnqnt 'eJoqJolaupl eql lP uouezrraur(1odap eFqntoJclru Jo ssol aq] q slFSaJ qJrqm ,,i.qpaleraua8 eoroJeqt sl lsrlJ eql'reIIJEe paqlrJsep secroJpJemalodrofeu o,vr1 eql Jo uopuulqluof, u uo spuadap y asuqdeue uI luelueloru eluosoluoJqJ '@V-Ztarn8gg) tJEd?eouelslp e{uos palolu eAEqseuosoluoJqJ relq8nep eql pue paleredas e^eq sppeurorqc Jelsls eql JaUPsur8aq qolqm'sellaslueql 'puoces eqJ salod apurds eq] Jo uorleredas aql sl 'g asuqduuu se ol paJJaJeJ 'selnqnloJJrru eJoqooleupl aql;o Sulualroqs dq patuedruocce sI qJIqM 'seluos -oruoJqo eql Jo luaruelotu prea,ralod pplul aql s1 'y aseqduuu sE ol paJreJeJ ']sJg eql 'sassacordSulddepano pue luapuadepul o^al .{q anou seuosoluoJqc aurcsouorq, pelleJ-lleJ aIDJo salod alrsoddo o] sJalslsaqt 1nd ar47'uotrySatBas 'uotle.redasplleruoJq3-Jalsls o1 alpurds Jpollu aq] Jo seJJoJeqt s^ ollu qJlqm ol speel aseqdeueJo tasuo aql tB uolseqoc ppeluoJqf,-JelslsJo ssol ueppns eql

g pue v aseqdeuvut ele6ar6esseuosourolq)

salnqnloJ)il.rJ relod+opuasnld

ol saloo ueoe uiaql a^ou_r o q l u o ^ l p a r p s l ) p e ) r o +6 u r 1 1 ned ( 7 ) :elebuola osle salnqnloJ)r.! r e ; o d r a l u ra q l : U e d es a l o da q 1q s n do g se;odalrsoddo uroJ+salnqnlor)rulre;odtalur u a a M l a qp a l e : a u e 6s ra ) r o +6 u r p r l se ( ! )

8 ]SVHdVNV

s a i o q ) o l e u r l1 eI ; u r e u pelerauaEsa)ro] :salodol seuosourorq)ralq6nep ;o luaua^ou :solnqnloJlrl..lr e J o q ) o l a u r ); o 6 u t u e y o q s

V ]SVHdVNV

stsoIW

680r

uI uorlBSeJ8asprleuorqc-Jalsrs a)r.I 'seuosoruoJqo snoSolouoq eqt elEBaJ -3as ol llroq :srsoraru;o ualqord IBJlueoaql sallos uorsrnlp f,lloleru lsJrJaql '(ZV-Zy a.rnt;g) Soyouroqqcee splleruorqr Jo JalsISeql sale8ar8as'srsolr{u IeuorlualuoJ e{[ 'II srsorel4l'(sprlpruoJqJ Je]srs 3o rrcd pelulldpq8u ego pasodruoc qrpo) sSolouroq aq1 sale8ar8asI srsorahtrT1 pue I stsorau pallpJ 'uorle8ar8aseuosoruoJqt Jo spunoJ omf z(qpamolloJ'asnt4d 'uorlecudnp auosoruoJqJ S irrcnru palleo Jo punoJ e qlp su€aq srsorel4 '17 raldeq3 uI pelep aJotu ur passnJsrpsr srsoral4 'srsolrurJo asoq] qlyvr aredruoc daql .uoq uo srseqdurauE qllm'srsoreurJo uoqepBar pue susrueqJeu Jrspq aql Jeprsuoo e^ 'areH 'lenpr rpur ^ eu p uJoJ ol ppualod aql qlp\ aloBfz ploldp B ruroJ o] asq 33a pue urads E uaq ^ spua aycdcanrlcnpordar aql 'sagcadsaseq] uI 'sarcads ]soru ur urrads pue s3Ba-satawaB palleo slleJ anqcnpordar pazqercads olur 'srusrue8ro.{uuu uI 'atuosoruoJr1Jqcpe go ddoc alpuueJaJJrp proldeq slac aq] a18urse dpo Sulrfurucs11ac pto1darl sacnpord qJIq { 'srsonru pellpo ssaJord uors -IAIp Jeelcnu pazqercadse uo spuadap uorlonpoJdar pnxag 'luared qJBe ruo4 auo 'aruosoruoJqJqcea 1o 's&o1owot1ro 'sardoc luaJaJJrpdpq311so^ ] uruluoo daq] 's1wrql pto1dtp.{leraua8 are sursruu8roeseq} Jo sllar aqJ '(IZ raldeq3 ur pessncsrp)luared reqtra ruor; }cuuslp dlecrlaua8 eJe leq} Surrds;;o a}uraua8 ol xur sluared omlJo saruoua8aq] :dgenxasacnpordar sursrueBrocqodrecna 1so141

uo!lrnpordau lenxasu! pa^lo^uluotsr^toteal)nNJo LuloJlet)edse sl stsotaw 'aseqdolat pue aseqdeue ur pello^ur selerlsqns {pl Jo tasqns e saleldroqd -soqdap qJIqM 'rtrpJ pelleJ aseteqdsoqde;o uortelrlce aq] uo spuadap srsotrru 'aldruexaro; 'lseartSuppnq u1 'saseleqdsoqdyo uorlelrlJe uo 3o uoqalduroc aql ,{1arosp silac euos 'sllal lsotu ur alqrsuodsarr(1umrusr-uoncnJlsap uqcdc uor; fyreurrrd Suppsar-uolle^r]Jeur 1p3 q3noqtiv 'q]oq ro 'sasuleqdsoqd;o uorl -eAIllB eql 's{p3 Jo uorlelr}Jeur aqt dq para38rr1eq plnoo srsollu Jo uoqaldruoc eql pup suoqel,{roqdsoqdap asaqt 'aldrcurrd u1 'aseqdola} ur raltnu ralqSnep Jo uorlEruJoJ-alaql pue rtlqruassesrpalpurds ro3 parmbar sr suralord eruEsesaql 'slsolpu dpua ur u,u.op ;o uo4e1,{.roqdsoqdap aq} teq} Sursrrdrns tou snq} sl }I -Ipalq adolanua Jealcnu pue 'uorlesuepuoo euosotuoJqc .{yquasse apurds salourord 1pO-IAI,(q suralo.rdsnorJEAJouor1u1,{roqdsoqd leqr JarlJEa^ es elA 'olv\l Olur uorsr^rp slr alalduroc ol ilac aq] JoJsr surerueJluql IIV'alaldruoc sr srsolrrupue 'paleeJcueeq seq snalf,nu laau V'erunseJ ol uorldrrcsuerl aua8 Surrvrolp'alels aseqdrelur JIaq] olut panue8roeJ eJpsaruosoruoJqJcrlolrru pasuapuoo eql pue 'spuedxa sneltnu aql 'su1alord JEalJnu ur drund saxalduroc arod aq] 'pauro;-ar seq adolanua JEalJnu aql eJuO 'unlnJrleJ cnuseldopua aql qllllr snonurluoo sauocaq ure8e acuo adolalue aql pue 'sruJoJ-alpurruel reelJnu eqt 'edolelua eql olur peluJ -odrocur are saxalduroc arod JEelonN'adolanua JealJnu alalduroc eql uJoJ-eJ 01 aJselPof, pu€ ueql saruosoruoJqc sJalsnlc asolJue ,,tpred ol asn; sluaurSer; Jo euPlqureru eseqJ 'sauosoruorqc pnpr^rpur qlIM elersossBsluaru ef,EJJns eqr Jo -3er; auerqrueu JpalJnu ,{1pp1u1'adolarruaJBelJnu eql Jo uorle{urog-ar aql dq pe^\olloJ 'alpurds JIlolFu aqr3o.{lqruasspsrpeql sr asuqdolal;o tuala roferu lsrg aql 'IelJnu ralqSnep;o rrcd e olur peBelred are seuosotuoJqJ Jo sles oml aql 'slsolrurJo a8uls pur; aq] 'asuqdolel uI 'lleo eqlJo spua alrsoddo sdnor8 pnba 1e oml olur pale8er8aselpq seuosoruoJqc ralq8nep aq] 'aseqdeueJo pua eql dg

le ral)nNrelqbne6urpa6el)edervseuosoruorq, O3|Joqir?':* 'ssacordaql u1 qfual aseqdelaru aql sarup 9I ot dn o1 ale8uola salpurds rraql pue 'aseqdeue lE saruosoruoJr{f,aq} a}eredas o1 g aseqdeue esn Llrerurrd uoz -olord ureueJ pue slsear(gosalpurds aql ']seJluoJ ur lqfua1 aseqdelau slr eolml lnoqe sr alpurds eql uaq \ sdols pue y aseqdeuurage.,ilpoqs sur8aq g aseqdeue 's1ac uerpurueu uI 'adr! lac eql uo Supuadap .{pear8 dren uoue8ar8as aluosoluoJqc o1 g aseqdpue pue y aseqdeue Jo suorlnqrJluoc aluelal aqJ

a1>(11;e1aqI:/ [ raldeqS 060t

sSolouoq eqt fqeraqm '&uurudSolouoq qtyvrsur8aqssacordslqJ 7 asnqdotd rc asaqdotdcu.onlilpalpc porrade ut 'aseqdS JIloIeu JaUEsJnJsoteql ssacord e Lq sSolouroqueamleq uro; saBelurl ,tqfual pue xalduoc ,{1qe4.reruer 'llar aqr pd ol s8olouroq spua alrsoddo aqtJo Solourog o1alpurdsaql Suuvrolp'1aseqdeueJo tesuoaqt le peloruars a8e1u11 'sa1odalrsoddoo1paqcel]errede ut sSolotuoqaql qlIM'alputdssllolerulsJU3q] ueemleq asaq;'s3o1o{uoq uo paluerro-lqeq o] srrcdSolouroqeqt ^ olle sa8u4u11 Soloruoq'slsollur uolletuJoJaql uo spuadapI sISoIauut uone8ar8as sa8e1u11;o

s 1 1 er :a l q 6 n e pp r o l d r p

pro;deq sa1eu.re6

m

3SVHdVNVIV

ulrsts sor-LVrAouH) lo Notrv9tu93s

II ]SVHdVNV.LV

u3tsrs sor-LVt/\louH) lo Not-tv9lu9ls

l'lcNtds lHl_No A]tvnotntcNt dn lNl'l sllAosot/\rouH) c3_LV)t'tdnc

'uorsrnrp a;6urse ur a1e6ar6as splleurorq)ratsrseql pue'dn rred lou op s6o;otuoq'lserluo),(q 'a1>,(r lalsrs lla) )rlolil.ue ul (8)'sprleutoJq) 'll slsoreLu 'punor aql sale6ar6as puo)asaql seoreqM's6o;or.uoq 'punorlsrU eql sele6ar6es'l srsoraur q) ,o aq1 'uor1e6ar6as aurosor.lJoi spunoroMl {q pamol1o1 sr(aseqd5 )rlorau) uolter!ldnpauosouolq) Jo punora;6urse q)rqMur uorsrnrp reel)nuJo lrlroJP srsrsoraw(v) 'a;rI> ;;er )llollur aql pue slsolarrr 1o uoslredurol Lt-LL an6lJ

m

scoroulouI

crrv)rldnc lo 9NturvdI

a)

6o;ou.roq leuJaleuJ

m

No[vrtra;uvrucI

6olouroq Ieuraled

stsofl l

(8)

fr

ii{iliitii

(v)

r60t

slsoJtJ

'patrJroJ aq o] sla^ossol)aldtllnul lo, lensn srlr pue '6o;or-uoq laqlo aql Jo plteulolq) reLlllaqllM ra^ossol)p urlo1ue) 6o;ouoq p Jo sptteurorr.l) oMl aqt Jo raqtle lnq 'e;duexas!qtul splleuolq) lolstsuou oml uaaMtaqpalln))o seqlalossol) a;6ursy 'sqr6ualarlua ltaql 6uo;epe1>auuot ,(;tq6rrare 6o;ouoq q)ea ut sptleuJoJq) ralsrsaql'stsoltLu ur sy.s6o;ouroq uaamlaqtalossol) V gt-ll aln6rl JOAOSSOI)

s6o1ou-roq

'aseqdolal ur srsollu Jo uo4aldruor eq] JaUE,(proqs spua puu espqdpue ur sur8eq srseuploldJ 'slleJ Ieurue lsoru uI 'ralJnu eldqlnu oJInbJp,,(qeJeqlpue srseuDlol-{J}noq}ll\/\ slso}Iur o8Jepun 'sllal alJsnr.uUeeq pup sa$coledaq uerlerurupru etuos pue (Jelel pessnosrp)sodrque qtqdosotq [pea sPqJns 'sllal eruos q8noqfp 'srsolrurdrarrasaruedruoJcesrseuDlol,{o'llat IEJrddt B uI 'tuseldol,b aqryo uorsrlrp eql 'slsauplolb sr a1c,bIIaJ eq] ul dats IpurJeql

srsr Nr)

f

'Dpnu proldaqlo uollautot aq| w Suulnsar'uolla&atBasataosow -ott4c spunor pamo1lot ory [q s1uopacl1dnp auosoruoJtloto punot a1&utsa qur4m to ur uotsrnlp na1cnu pazl1anadsa y srcorcW'adopnua Joalcnu aqLto uoryau tutot lo -tol-at aqt pua alputds ary /qqwassastpaqi Sutpnlcur '|slsotlwalalduoc 1ot41sluana to aqt rct pannbat v s1aBn14pJ lo uorln1ttor4dsoqdap8ut11nsataWI'qpJ-W lo uo!run!1 -Jam aqi sru4ipuo uouJnltsap u17c[csatowotd os1a2p4y nqnSot sp!]DworqJ Ja$$ aqi ppq ryq1 suraptd ar4ito uournr$ap arq sawlnuns Llcllm 'J1J1V at47tq paraS8uj v asaqdauy [Tquassaa1putdsa1o71pcat o7sautosouual asn osIDs77ac tunyl 'tatta n7 -odtq a oiw salnqntotJtu anuDBJooi sunrctd rolout [.11pqaaq] uo sa sn '&qtq lo Tgam -rys pua uouuapnu alnqruoJJlw IOJII aruruwlls 01sauosolaotqc u1o11ut lo ,ttutqa aql uo fiaBn1 spuadapslp) Iawrua w uotluturot a1puldg'a1putdsar4lto sa1nqntoJllutot alad ptloutotq) ra$$ aqi to tuawt4callo n1odrq pua 'alpurdsctlollw aqi to lqquassD 'uolLosuapuocawosowotqc Surpn1cur 'srsolrw [pna lo sruana aqt staSStu 4p)-h[

fteurun5 'srsolrru ur seop U sB qJnu-uolleJedas ppeuroJqc -Jelsrs pue a8e,realc urseqoo f,rJatuorlueJ sraSSul eJoJaJeql uorlplrlJe :)/ldv '11aseqdeue 'JI srsoreu ur pe^orueJale srsoraruur eJeruoJlue3 I Jo lesuo aql lV aql ]e a8elealo ursaqoJ {Jolq leq} srusrueqcaruaqJ 'II srsoreruur alpurds aql uo uo[e]ualro-rq ]JarJoc rraq] 3uu,ro1e'I srsoraurlnoq8norql seJetuoJlueJJraql tB palu1 urpruar eJoJaraqtsrmd pqeurorqo-relsrs '(17 raldeq3 ur pessnJsrp) aseredasruo4 pelJelord sr uor8ar ]uq] ur urseqof,asneJaqI srsoraruur pe^Bel3un urcrueJ seJaruoJlueJeql JBeu saxaldruoc urseqoo 'rana.uoq 'srsolnu o] lsEJ] -uoJ 'ftV-LI pue 'uollelllJe aspr€ ul arn8rg aas) sruJe aql a8enealo Suop ursaqoc -das 'uotlcnrlsap urrnJes o] speal qlql 'uorlelrlce O/f,dV uo spuadap I srsoraru ur uorseqoc tuJE Jo ssol aql 'sarcadslsoru uI '1 aseqdeue Jo lesuo eql lE uoll -eJedesSolouoq sraSSularoJaleql sruJeprleuoJqr-Jalsrs ruo4 uorseqoJ ssol Jo aqJ'(8t-ZI arn8rg aas)uorsaqoJ prleruoJqJ-ralsrsLq palJauuof, eJe spneuoJqc JelsISeql Jo srure eql esneoeq.,tpo raqlaSol s;rcd Soloruoq ploq sJelossoJ3 'srsolrruur are ,(aql se alpurds aql uo palueuo-rq eq uPc srrcd pqeuorqc Jelsls aq] II srsorau ur ]eq] os 'I srsoraruJeUe pasJa^al aJe srusrupqJaruesaql 'WZVrc ern8rg aas) alod auo ]sn[ ot seqJp]te ]eqt l1un Surpurq-alnqnlorf,rru a13urse olur pesnJl oqaruos aJp seJoqooleuDlJelsrso,\/\laq] ta^al oq 'I srsoreur uI '(6t-ZI arn8rg aes) srsolnu Surrnp paprolp flpurrou sl lueruqJe]le Jo addl sql'a1od aruus eql ol qcpuu Soloruoq e ur saJoqJoleuDlJelsrsqloq lpql sarrnbar alod apurds e o1Soloruoq eJo ]uaruqJepe aq] snq] pue 'sppeurorqJ relsrs pa{urT dpq8p o,,t,r]suretuoc Soloruoq qceg 'alpurds f,poletu lsJrJ eqt o] peqceilB erp srrcd Solouroq eql ueq^\ pe^los aq uralqord Jrloleu dlanbrun Jeqlouv ]snu 'uorsr^rpJllolalu ]SJIJaq] sJatuaIIaJ aq] ueq^ pa]f,auuoc,(1pcrs.,(qdare rrcd rirana ur sSoloruoq eql lur{l Surrnsua tred SoyoruoqqJpe ur srnJco sJalossoJJeseqlJo euo lseel lV'(97-ZI arn81g) prleru -orqc sno3olouoq B Jo VNCI eqr qllm snonurluoJ aruoJeq ol JeAosessoJlprleu -oJqJ B VNCI eql ereqm 'stanossoJlpalpc sa8ueqcxa VNC pcordrcar ol peel Jo sluane eseqlJo aurog 'sSoloruoqaq] uaamlaq sluala uorleurqruocal VNCJo sJaq -runu a8reyur Suqpsar 'ppetuoJqJ JalsrsqJea ur suorlerol IeJaAaslE paruJoJeJB sTearqpueJts-elqnop VNq :rred Soloruoq qJEe ul splleruoJqc Jalsrsuouueemleq uorleurqruoJar snoSolouroq ,(q aculd ur pelJol ueql eJe sa8e1u11Soloruog 'sSolouroq oml aq] ur saJuenbes VNCI dreluarualdruoJ uee^aleq suorlcp -Jalur Jo llnsal e se ,(Ireturrd 'snalJnu aq] ur Jeqlo qJPe ol esols e^ou ,{1unper8

alrI1 11a3 aql:/ LraldeqS Z60L

('rnlnl olqso^Jo 'M'H uroJJ'B)'s;;elralq6nep lua)seu aql,o xauo) (salrno>'1 1rXer-u615 urorJuorssl.lladqlm'9/6 t 'O6Z-6LZ:tgpS 'ury'lassa) 'D'Upue suree8 seataqtrtr'(uaa.r6) alqlslnaqlro 11e seq;1ursor(tu aql ut suteuarlsal aql:os auop sequtl)e aqllo auos {;uo'6uu alll)er}uo)aq} ol patnqu}srper e 6ulplntpe Jo Molln, a6eneal> e)uo)saronll()) 'lla) leLulue e ;o sqderborrrul ll ulsolul pue (pal ult)e loJ paulelseqaoulpploul aurls 6urprnrp '6u!, olll)elluo) aql aql 1o qder6orlul uoll)ala uV (g) 'llar 6urp;ngpe ur Morrn;abeneelraql ;o burmerpV (V) OS-ZI orn6ll ;o a6pa 6urrvror6ur un s'0 urn0t

())

(s)

(v) anor:n1abeneagrur sluaurelt;utsoi{u.t pue urpe lo 6ut: eltlre:luo:

alpuroslerlue) uroJ, s e l n q n l o r l r nrre l o d r a l u t6 u t u t e u a . t

apr^oJdtEql suletoJd Jaqto snorelunu sulPluoJ oslEqJIqM'(09-ZI eJntlf) tuIJ eIIlJBrluoJ Sutlqruasse l.lprder aql ul elelnlunoJE ol ulSeq II ulsoltlu puE ulllB 'aseqdeueur elpJedassprluruoJr{f,ralsrs eql sV'paseeleJeJEs}ueuEIIJ II ulsol(ru pue 'sazruu8JoeJullf,e aql Jo qonu :elqulassEslp ulsoful puB ullJe Jo S^EJJE aseql (srsolrruralue slleJ sv '(9I raldE[{l uI pessnsslp) snq{ ssatlspelIBJ selp -unq orruseldo{c a8rel ruJoJoslEr(aql's11acauos uI'euBJqlualu Prusuldeq} 8ul -f.papun >lromlau IeJruoJ e uJoJ slueruelg ulsofiu pue ullJe 'q1ac aseqdrelul uI

s!seu!lov() Jo,e)tol eqt aleJauag6utu elll)etluo) aql u! ll ulso/(wpue ull)v

(';y eu615r.uot;uorssrluladqtl1l ' ' V ' p s s a ) ' 9 ' Up u p 9L 6L ' 0 6 2 - 6L Z : V qD' S w stleag'M'Hlrtor;') pue g) 'uor1err1u6eu raqbrqte MornJ e Jo a)e;rns aqf ()) 'll qteaurepun6utl a;tlrerluol aql Jo ^lr^rl)p aql ^q pasne)sr auerqLuaur aql'abre;,(;;ensnun lla) aLllJo 6urrnorrn; sr lla) aqt sP'pauuapllaMpue snor^qo I;;erradsesrMornj a6eneepaqr'66a 6or; 6urneelre;o qder6orrrr.u uort)ele 6uruuersuorlerr;ru6eu-nlrol srqlul (g) 'pJeMur auelquau aql sllnouorl)erluo) ltaql leql os'uMoqsse potuauo ore 6uu alll)erluo)aqt;o segpunq ursor(ru -ulpe aql (v) 'srsaurl0y(169-21 an6t7 urrl 9Z

())

'uopaldluoJ pue 'uoluasul eueJquelu 'uopoeJluoc 'uopep -rur-se8uls JnoJur JnrJo 01peJeplsuoceq ueJ slseuDlolb 'snql'sgac ralq8nep aql uee^\leq de3 aqr pas uorsnJpue uoluesul auerqueu'palalduroc sI uollssJl -uo3 uI asEaJ3ul EeJu aJeJJns seluEdIrloJJe Jrruseldolr(c 3uu uaq leql |uorsr^rp '3uu aql ol aqt JoJ salusueduoJ eueJqrueru Jo uoulppu sIqI luef,Efpe eueJq -(uaru l eu suesur eueJqruaru eurseld aqr qtIM selclsa^ rPlnlleseJtul Jo uolsnJ 'aru11arues eql lE 'pue 'slJeJluos,,tlpnper8 Surr eq1 '(I-ZI lauPd osle ees :6V-Zt 'eseqd?ue 8ur eJngIC) eueJqrueu eruseld eql qtuauaq lsnf selquasse 8uu eql -JnCI 'suralord,'t-rolep8ar pue IeJnlonJls l(ueru pue tslualuelg II ulsodru 'slueur -plrJ urlse 3o pasodruoc ,{lqtuassE Jllu?udp e--8utt anpDJluoJ aq] s ssacord slq] 8ul{lJapun eJnlcnJls aq} 'sato^JeJne rElnlleJlun ^ueur puu silec plulue uI 'o^\l ur ilac eql sepFlp dlalalduloc 1I Ipun IIas aql punoJE speards puu suedeep ,(prder r oJJnJ eql 'aJBJJnsIIeJ aql uo 'moJJ( aBnnaa1cJo ta)cnd P Jo ecue -rEeddpueppns eq] sl [e] plulup up uI slseuploil.sJoeBuEqJalqlsnlsru eqJ urn002 (8)

srstNr)ol^f

€60r

lB pele^rlJE sl voqu 'a8u^BelJJo ells aql l3 3uu alllJeJluoJ eq] Jo uopcuru puE dlq '.Vol4q -Iuassp aql slorluoJ '(g-gI elqeJ eas)fgure;radns spg aqlJo esEdJC 1euuse

6u;ga;;perluo)aql Jo uoll)erluo) pue {;quessy sta66ul vor{uJo uotle^lpv le)o'l

aqrur alpurds eql]uarroorsdreqrpqlxelroreqluotr#Tt;1t:i5: luanbasqns

,teur ,(aqt eJeq ^ 'lleJ qopa Jo eueJqrxeru uuseyd er{} Jo aprsq eqt uo urerueJ uago Lpoqpru IpnprseJeqt Jo slueuodr.uoJ eqt Jo euros dlaralduoc aterpdas slac ralq8nup aq] JaUV'(I9-ZI a.rntlg) Ierrelpu xrrleru esuep e ulqll a raqtaSol ,(pq8p palred 'euozpnu alpurds aqt Jo saFqruoJorru reyodralur lalpredrlue aql ruor; pelrJep eJnlonJls uleloJd a3ru1u 'alpurds IeJlueJ aqt Jo suletual aq] suleluoo pue slleJ ralq8nep omt uaalqeq aqt Jeqlet e se slsrsred z(poqpnu eq1 'dpoqppu eql ruJoJ ot sr oJJBu raoJJnJ aBerrealceql Jo aueJqruaru euseld aql se 'spua a8enealcuaqr!\ qlpvr pasuadsrp JeqlaBolp dluur; sr Surr alllceJluoJ eql 's1saup1o1bSurrnp [1enu4uoc sa8ueqc luaru -a8uurre reql pue 'crureufp [UBq aJe8uu eql ul slueuel.rJ urloe eql 'elosnru ur u1lcpeIIFn'JaAoaJoIN fppeals eseeJJapsuletuoc ll slueu€lrJJo Jaqrunu eql puu erunlol Iulo] slr leql 3u4se83ns 'ssaDlJrql erues eq] suplureu ll 'slJlrlsuoc 3uu aqt sV'qled str ur pauasur $ tuql elpaeu sse13aug e pueq o1 q8noua a8rel acro; e suexe 3up aqt 'sur8aq uoqJeJluoJ eJuO 'oml u1 ruseldoltJ aq] sepwp leql eoJoJeql aleraua8 ol tJeJluoo slueuplrJ 11ursodur pue urlJe go sr(erre Surddul 'aseqdeuereUV'(gI raldeq3 ur passnJsrp)sruauelg uqce peqouprqun -JeAo aqt 'reautl 3o sr(erre lalpred ;o dlqruasse aql eleelcnu lpql suraloJd uttutol uo spuadap qc1q \ 'sluaureyrgurlop llteu Jo uon€ruroJ leJol eqr ruo4 lred ur sllnseJ 8uu allcerluoo eql yo dlqurassy .{lqurasseSurr ur }srssuJo }roddns IeJnpnJls urrl I

aueJqtreureurselo

lerJeleurx!ilet! esuep

turi 0l

('su!llnw'w'r '8 Jo IsaUno) talqang-lq)alqlVraluangJo {salrnor 'V)'alputdslpJtue)aql Jo suteulal aqt 6urureluottuse;do/r;o puelts urqtsrql^q paq)eueutpurolslla)telqbnep aqt lnq 'alaldulo)lsotulesra6enee;1 'lle) e;o Ipoqprru lpultue6u;prnrp aql ;o qder6onru uoJl)aleleuotluenuo) 'slle)relq6nepoml eqt sutoflltls V (g) ,(poqpruaqt j6utptntp jo ssato.rd aqt ul lla) leuruepornllnre;o qderbontuluoJl)ola buruuersV (V)'Ipoqplur aq1 19-4g arn6r1

a;:I1 ;1a3aql:/ t raldeql

V6OL

'bulJaql uoll)PJluo) Jo 6ultoruord{qeraqr Ilrnrlre pue uorlpuro,luauelU;1utso{tusa}elnuir}s lr'l)ou se q)ns'saseurluralordpale^tl)e -oqg 6urlenrlreIg '6urrepllertuo)aql u! aqt salou.rold sluaueluu!l)e Jo(;qLuasse 6ulputq VoqUpale^tire'suttuloJ ,(g 'al!sabeneallarnlnJaq] ]p pasn)oJ punoq-df9a^rl)e srvoqu lo LuroJ aql'uralorddVDoqUe {q palenrlreu; pue uralordllgoqU e,{q pelentlre oqg raqlo alrl sl VoqU'sasedlg{1gtue1 'VoqUasedlD aql {q 6u1ralll)erluo) aql;o uotle;n6aggg-4 1 atn6t1

uorlel^roqdsoqd ureq:-1q6r; ,fuo1e;n6ar urso{u.r

(l)ou 6urpnl)ur) seseurlpole^rlle-oqu

\ \

\

voqu a^rpeul

{pO Jo uouellJoqdsoqdep esneceq eurl} llarJoJ aq} 1BsJnJf,ooslE slseuDlol-{J 'srsolrru etEI ur srsauDlol/tc Sunup lseJJoc eql ol salnqlrluoc oslB Iuslue 3o -qJaru slql 'elpulds asBqdeue aql uI eleuISIJosleu8ls aseq] asnE3eg'satuosolu -oJqJ palEJEdas;o slas o/\ 1 eql uee^ leq sJnJco uoISIAIplEq] Suunsua dqaraq] 'sa1od E uolleruJoJ MoJJnJelBIlIq ]Pql uoplsod ^8,1^plu eql uea^ lP alpurds laq 'elpulds JIlolIu eql uo puedep slEu8rssalereue8 apurds eql 'eseqdBueSuun0 ]Eqt slusrueqf,alu lueSale ,i.qpanatqce eJu sllal IeIuIuB uI slseuDloldJJo Suluoq -rsod pup 8unult ]JeJJoJaql']es alalduoc P seAIeJeJIIaJ Jatq8nep qcea 1eq13ut -Jnsuefqaraqf 'seruosourorqcralq8nep Jo s]as o^A1aq] uaaM]aq paceld aq lsnlu uorsrlrp Jo alIS eql pue taqlo qcea uror; pale8ar8asd1ry eJe seruosoluoJqcJo 'ace1d sles o^^l aqt Jaue.{1uorncco lsnlu sISeuDIo{3 }q8lr eql uI pue au41}q8IJ eql le sJnJco uoISWp leq] ernsua o1 ^aoq sI slsauDloldl uI uelqoJd IeJluaJ eqJ

uorsrnrc Ila) leuruv eql )!lol!waqlJosalnqnlor)lw Joaupldaql au!uralacalpurds ']xeu 'paAIoAuIeq ol rueas apulds aseqdPueaql el\/\ sE ssnJsrp Jo saFqn] -oJsrrueql qSnoqlp 'alls UOISIAIp aql tE pelE^IlcEro pezlpcol sI ca3oqu eq] A\oq 'voqu ol lnoqP epll ^ ou)I el dJc Jo Sutputq puB doc Jo esEeleraql selPlnunls pue elrs uorsr^rp ernlnJ aql lP xeuol IIes aql le punoJ sl qJIqM '(Jacoqu) Jo]lEJ a8ueqcxa appoelJnu auluen8 oq5 e uo puedep o1 lqBnoql sLry\oJJnJe8enealc eqt le voquJo uolle^Ilf,P IBcol aqJ,'(gt rardeql uI pessncslp) dlc ol punoq 'sasEdlc Jeqto e{I'I uaql\/\ e^rl3E pue dclc ol punoq uaq^ e^Ilceul sI voqu '3urr ursol.ru-ullJe aql Jo uollJeJluof, pue dlqruasse eql Suporu -ord dqaraql ,{lplrce rotolu pup uoItBIuJoJluaurBIIJ II ulso^Iu JEIodIq seteplulls l'Ihlu eqlJo uollBl^roqdsoqd'II ulsol.urJo sllunqns aqlJo euo sl qrlq ^'(3Tnlu) ureqc lq8q ursodu fuolep8ar eqt elel.{roqdsoqd saseur) aseql'(Zg-21 arn8g) 'seseulr uta}ord aldppur Supe,rpce.{q {JoU aseup{pele^r}Je-oqg eql SupnlJul suolloBrluoc pue flqurasse 1yutsodur salotuord ]l pue 'sultuJo; Eurlenrlce ^,(quolr -euroJ ]uauBIIJ ullce salotuord U'uorlcerluoc Surr pue 'z(lqurasse 11ursodtu'uor1 -eruJoJlueruelg uure saloruord 1raraqm 'ells UoISIAIpaJnlru eq] le xouoc IIaf, aql

stslNt)or^)

s60t

'ulaLlluaaMlaquotlelujoJ MollnJ a)nputol xallo) lle)Jo uot6aj]ua)p[pp aql JelleMoqaulosslalsPaq] leqt 6u!lsa66ns'sralse oml uaamlaq{errnplu uor6ataql ut sulloJs{em;es;1a> asaqt ur MollnJebeneapaql satnpordlpql 6uU aflt)eJluo) eql'^lruoreddV.(poaq/lu)D pa;)use;dot{t aues aql aJpqs lle) leuilouqeslql u! lnq-alpurds )ttolrLu e Iq pa1ur1 lou ale ]eqt slalseluatefpe oM] aql uaeMlaqoslelnq ,(spDaLlMu)D no11al)sapuds )tloltul luenbesqns oMl oql Jo q)ea jo auozpil.!aLll 1eI;uo 1ourn>to saberreelt luenbasqn5 'lla) aql aprs ellsoddoaq1uo Jo rn))o ol 6ur;re;'alalduotur sr 6urnnoln; auerqueuraql ,ppaqssel6p qtlM lla) aql Jo aprsauo ol peqsndI;lerrueq:ar,u st alputds)rloltu aqt,l .;1e> 66a a6.re;e u1a6enea;r1oaue;dluanbasqns aql uo slalse alnqnlol)!ut ]o uol11sod aql Jo a)uanUu!aql 6ullerlsuourap luaulradxa uy y9-11 arn611

p e u r o l a r e s 1 1 et a : l q b n e pt n o l pue '1ua:efpe{1durs ate leq} soulosol}ua) o M l e q t u a a m l e qp u e s a l p u r d ts; t o l ; t u { q p a 1 u r 1 saurosoJlua)eql ueeMleq qloq slntlo e6enea;t

srsolil.! lelua ral)nu qloq

^., a l e a l ) n u r qe 6 u r r n p o t d ' l l a ) a p r sa u o u o +o {Juo sur.ro;mortnrL

a l p u t o sa q l s a ) e l o s t pl l a ) a q l o l u l p e q s n dp e a q s s e l 6e

;;a:66e 6urprnrp

p e a qs s e ; 6

'alrs leql lE uorlJeJ}uo3lecluoJ Sunourotd snqt tolpnbe Jo so,{Jqua dlrea aql uI epurds aq] ]E Iurururur sr uorl€xBleJ IecrUoJ er4t'Iapow uorl2xalal IDUSDs!r4lol Sutproccy 'xeuoJ IIaJ aql le salpunq urso^u-urloe Jo uorlEXEIeJlpJol aq] elotu 'sadil 'leql sesodoJd -oJd sepqnloJJrru IIac eruos ur IpJlsBaql Iapour pJlql V 'srsauDIo]43 'stuelnru Dlrqdosot1 uD sureloJd eseql Jo eJnlreJur IFSaJ (alduexa JoJ Jo uoq '(99-ZI -cury eqt ur stf,eJeq aJnBIfl VoqU ale1nrupsderu teql suratord Surpnlcur 'sutalord Surpu8rs snoJeurnu qlIM aluroosseaypurds leJlueo aqt Jo saFqnloJJrru JElodJalur Surddelra,ro eqJ 'xeuo3 aql uorlBruJoJ ^ oJJry Jo etrs eq] saUI ]E IIec (euozp[u elpurds -oeds leqt pu8rs Surcnpur-MoJJrgB seleJeue8'alpurds [BJlueJJo aq] lPql s 'pporu uouDlnuus alputds eql pellBc ,{lilqrssod puoses v lDil.ua7 'fug-Zt arn81g)elpurds rrlotrur B ^q reqlo qcea o] petJeuuoJ tou ere sJelseaql SurlPalcnu satuosoJlual o^al eqt uaq^^ ue^e (sJelseo^u uee Aleq de,r,rprursuJoJ ^aoJJnJa8e^ealc E leql eleJlsuouap qf,rq^ 'slleJ cruofJqura e8Jpl uI slueurJedxa snorue8ul luoJJ se{uoc Iapou srql roJ eouepr^g 'selod elpurds eql ueaMtaq dea,gpq Surr e olur pasnJoJ^\oqaluos aJedeql eJaq ^ 'xauoc ilec aql o1 qeu8rs Surcnpur-,r,roJrnJ ,ftJec salnqnloJcnu IeJlsE eql ]pql sale1nlsod qJrq ^ 'ppout uoUaInuUS lDr$a aql paura] sr ]srrJ aqJ'(tg-ZI arntgg) asaqtJo uop -eulquo3 e,r{o1duao} JEaddesllac lsoru pue 'pesodord uaaq eABqsusrueqJetu IPraueB earqJ iuorsr^lp Jo elrs eql ,,i;rcadsepurds JIlotIu eql seop ^toH 'uorlPruJoJMorJnJ IEsol asnpur epurds aql dq petEJeueSspuSrs ]Eq] Eepreql 8ur -uoddns-alls epurds ^ eu eql qlyvt pJoJJp ur sdolelap euo llrau B pup 'sJEeddE -slp ^aoJJnJ a8enealc]uardrJur aq] 'aseqdeue,{1reaur elpeau sselSeurJe qlrm uol} -ISod^\eu e olut pa83nl sr alpurds eql 'edoJsoJJrrue qflm atup JI IpeJur palJesqo aq ueJ elpurds aql 'Jealc sr urspldol-{J aql asneJeg 's11ecJellptus pue Jellerus olur dn pepplp ,{lalrssarSord sr 33a pur8rro eq} t(e^a srql ul 'qtmor8 Jo sporrad Suruanralur ^lprder anealcsoftqura eseql (uonpz1lual JaUV'3urr aplcerl ]noqlrm -uoo aq] aql SuuruJelep ur sapqnloJJru epurds Jo eJuetJodrur tuarueJeld Jo eql peleelar selpJqaualur aurJeru ;o s33a pazllpJeJ eql Jo serpnls ]sJrJ 'sllal leruruEur srseuDlol,{cuo srseqdrueuB qll^\'llElap aloru ur sursrueqJeurdrolep8ar esaql eqrJosaplaou aM 'srseuDlo/.c saleqrul 'aseqdeue pue aseqdeleru ur uorlJnrlsap uqc,{c uo spuadap qJqrlr ,seleJlsqns

'slusrueblo ]ualaJ,tpur 6urftensulstueq)aul lualaJjtpaql Jo o)ueuodult aLll qlrM'sr.r,isrueq)au aseqlJo uotleutqu,o) e Iq pauru.ralap sr6uruorlrsod morrnlteq] r(;altlst lt pue'suotle^tasqo aqt ;1esureldxa ;apoLue;6urso5 '6u1re;r1rer1uo) aql to 6uruolgrsod eq1 a)uanUu!leql s;eu61s aleleua6a;purds aseqdeueaqlro selnqnlor)lu,t oql Motl Jo slapourluarin) earql gg-4 1 arn6r3

lepour uorlexelaJlellse (f)

lepou.ruorlelnur!lsalpurdslerluer (g)

lapour uorlelnurls lellse (V)

el)l() lle) eql:/ [ raldeq)

960 t

(rategMarpuvlo ^sairno)) 'slle) ur sau:osorlua) ou are tueld-raq6rq araqlasnP)oq'salnqntor)rLu lerlseou aie araqtteq] oloN'o^plua)salonue qllM paurelssrsaurosouroJq) ralqbnep;ostas o M l a q l u r v N o a q l p u e ' u l l n q nlls u r e 6 e pelaqe;-plob qllM pau!els sarpoqrlup aje alpurosaqlJo salnqnlol)lulaqf 'a6edaql aue;daqt ol re;nrtpuadred 1o auPldP ur paurloj seq (tpDaq/Au)D oml aql uaamloq)a1e;d1;a>,{;tea aql'qdel6or)r.urq6ll slqt u1'aseqdolalug 1ue;de ugs;sau11o1I3 99-2 1 arn6r3 11ar

url 0S

srlouru aq] ruo4 pa^lrap selnqnloJsllu suleluoc qclq^\'lsuldoruSurqd eq] pallEc arnlJnJls e .{q paprn8 sI pue asBqdEu€e}el uI sulSaq alEld IIeo aql Jo ^lquesse 'llP^\ 'elBld eqJ '(g9-ZI arnBIC)IelJnu Jalq8nBp o^\] eql uea^ leq IIaJ eql pallE3 IIas ^\eu e Jo uorlJnJlsuoc eq] r(q tno aplsu aql uro4 pauouluBd sI IIes lueld eqt Jo rusEldo{c aqt 'uI epls}no eql luo4 lusBldo!{.f,aql SuIpI^Ip Sutr aplrerl -uoJ B uEq] raqleu 'lpm IIas plSlrures E dq pasolsue are slles esaql 'slle3 luBld -raq8q uI sISauDIol-(JJoJ lueuodurl ^lrc1nclued sI uolllsodap auerquraql MoJJnJeql Ol SnleJ -edde r31o9 aq] ruo4 selnqntoJollu uo pauodsueJl ale leq] selslsel aueJqrueu lerus rtq pepr^ord ^lpreue8 sI pue MorrnJ e8enealcaqlJo a8pa rauul eql ]e pappP sr auPrqureru 1v\aN'auEJqlualu eusEld aql Jo eeJe aJeJJns aql uI eseeJcul uE 'silec plulue lsolu uI uo spuadap MoJJnJa8e,tea1caql Jo luelua^oru pJBMUIaql

slueldraq6lHur slsaulloy()saplnglseldou6elqdaql

(razlolgleeq)rWpue,{u1ar6 esr;y 'lla) aq} aueld ;errolenba 1o{salrno3) }o eqt tnoq6norqlperalle)ssalpunq alnqnlorlrur rlllM pate!)osses (uail6) y1I1 uralorda;purds;e.r1ua: aql apqM 'a)eJrns aql qlpouaq6uu e suro; lla) 'oraq uMoqs se 6urr a;r1>erluor @at)Votl[ srlla) aqlJo aueloaql ur pauor])as-ssol) aql uaq1 (B)'(B€S-/t pue 7g-1 1 sern6rg aes)xelJo) lla) oqt te ,(flnlfreVoqU aleraua6ol lorluo)dlaq leql s;eu6rs rq6noqlaresuralordasaql'salnqnlor)rLu relodlalurJospuasnld6urdde;rano aql ]p sexa;duoruro, lpql suretord,{role;n6ar '(uaatb)p4A1 lere^osJo auo sr q)rqM pa;;e>uralorde pue (pa4VoqUasedlg aqt Jo suolle)olaql slea^arstsautlol,{l1o aql te lla) ueuinqparntln)p Jo 6uruur6aq qder6orrrr.u a)ue)saronll(V)'lla)upunq aql lo alpurdslerlua) aql le srole;n6al sgsaul>1oyb 1o uollpz!lp)ol gg-2 1 arnbgg

'ssnf,slp A\ou e^ se 'o^/ll uI IIaJ aq] apFlp pue alquessP ilI^ [P^a IIes 'prruq asuqdoJdaJd eq] aJaqr elrs eql s{retu pue slso}Iu eJoJeq1sn[salqruasse 'slleJ lueld aql pallPf, 'slueurEllJullcP pue salnqnlorsllu Jo puPq pezlueSJouE q 'alqruasse '11ursodtu 3urpnlcur '8utr allrerluoJ aq] Jo sluauodtuoc raqlo qclq^\ oluo ploJJecsB IuJoJ o1 1q3noq1eJe sulldas eql'alIS uolsl^Ip alnlnJ eq] 'aldtuexe ro; 's1sea,{ Sutp tp ID elEI ur salquassu suqdas pelleJ sulalord;o Surr e -pnq uI 'srsolrrueroJaquasoqc st.{lquasse Sutr;o e}ISaql'sadIl 1ac aruos uI 'lepolu sII{l qll^\ ]ualsrsuo3 'xeuoc IIaJ aql lnoqSnorql &1npce alpsBJluoJ paseeJsul01 pual 'suaga\a'3 seFqnloJJru [BJlsEJo ssol aql uI ]Insal lBql s]uau]eaJl 'eldluExan1

sts3Nt}lol^l

L6OL

'aseqdoletq FIoC eq] ]cnJlsuoJar ol pepaeu slerJalerueql sllrequr IIaJ Jatq8npp qJEe teql Surrnsua 'alpurds eqt Jo spua alrsoddo ot pelnqulsrp ,{.qaraq1are pue sayodalpurds eql qtp{ elprcossesluaur8e4 €yog 'srsolnu Surrnp paluaru8er; pue pazrue8JoeJ sr srueredde r31og aq; 'srsaurloldc Surrnp o,l,r] ur ]nJ sr pue suIEIxeJ aqt 'slleJ uI 'UA eql sesealeJadolanua Jeelonueql 1ce1ur^,t1a8rel lsoru Ug 'aseqd Jo u^aop{BeJq pue seFqnloJf,ru aq} Jo uouezrue8roar eql IN olur drlua uodn 'uo1a1a>1so1,b aFqnloJJnu aql .{q pazue8ro sr pue eueJqrueru JpelJnu eql qlIM snonurluoo sr s11acaseqd;arq q Ug aql'a1c,{c qJea ecuo elqnop rtp8no.r sJeqrunuJIaq] 'a8ereneuo peluequr dlagesaq ol sJaqunu q8noua a8rel ur luas F -ard rtlensn are slseldorolqJ puu prrpuoqrolrru sp qons salaue8rg isepwp IIaJ E uel{ A alu8ar8assallauu8ropasolJua-euEJqrueu snorJe^aql op 'ueql 'AroH 'luaserd ,,(pearp sgll go ped auos ssalun 'l.lreyurrg 'sallaue8ro 3u4sr (gg) runpc4ar cnuseydopua lvrau qlac B a{eru louuec -xaard eqtJo uoIsIAIp pue ql toJB aql .{.qd.yuoasrre uec r{aql lsluauoduroJ Ienpn -IpuI Jleql ruo4 olou ap palqruassu aq louueJ slseldorolqc pue prJpuoqJotru sE qJns sallaueBro'71 raldeq3 ul pessnJsrpsy'sallaue8ro pesolcua-eueJqrueu aql Surpnlcur 'sluauodurof, IIeo lelluessa reqlo aqt Jo IIe tlJaqur oqe tsnu IIaJ ralq8nep qcee :ela^ oq 'sapplp 1ao rqoftecne B uer{ A'saruosoruoJqoJo tueru -a1duloc IInJ e salre3eJIIeJ ralq8nep qcee tpql seJnsueslsolru 3o ssacord aq;

stseutlol^)6uunc slla) rolq6necol palnqrrtslceg lsnw sellaue6ropasol)uf-euerqruew '(Lg-Ll arnS;g) IlelrlrIIar ^ au eq1 Jo uollcnJtsuoc aq] alayduroc o1 aluyd IIeJ aqt Jo xrJleru eq1 ulqtlm rrmop prpl ere slrrqrJorJrru eso1nllar tale.I 'omt ur IIaJ eqt sapplp pup llellr 1ac pufrro aql pue auuJquelu eurseld aql seqJpeJ1l Ipun uorsnJ elf,rsel Jequry ztqprervrlno spuedxa a1eld aql 'a1zt1d77actpaa aql paflec aJn]JnJ]s pesoloue-eueJqrueu ,a1r1csrp e uJoJ ol asrg '1parr11ac^\au ar4 Jo srsaqlufs aql JoJ parnbar surelordocflS pue aplreqcces,{1odqtr.n pe[g 'selorsel esaqJ 'relueJ IIe] eq] ol snteredde r31og aqr Iuo4 selnqnlorJllu asaql Suop selorsal leurs podsuerl suralord Jolo4 'alpurds 's;;arralq6nep omlaql 6urleredas 'osnJ {1e1a1duol euerqrlaul aq}pueauelquaueruseld pueaueJqtueut aq1'1;ean lleMlla)Mauaq}6urpunorns ;1at;eurbuo eulseld aql q)ealol ptPMlno qrrqan'lleM sanoi6 osoql6uo;epallodsuell lla)Mauoql uloJol asnJpuesalnqnlol)tul are'lerlaleullem-llel qllmpallU'sal)rsa^ panuap-r6;o9'rolenba la6uo;ou solnqnlol)lul lla)aqlle pualnq de;lano asaqllospuesn;daq1'1se;dou6erqd aql r-uro; aseqdo;et a;purds tr1o1rr.u aqllo salnqnlo.r:ru.re;od.relur aq1 1e6urureuret 'elputds plo aqlJo lolenbe aq] alquasse ol suelslleMlle)anau le lla)aqlaprsur e'pa1e6ar6as aneqseuosoulolLll aqllaue 'aseqdo;at1o 6uruur6aq eLll lV'xatlo)llal aqtle (pueqaseqdordatd aql)sluaujelU pueqe {q ult)epuesalnqnlol)lm;o aseqdWaroJaq paLlsrlqelsa srauelduotst^!p aql.llo)gueldraq6rqe ursrsauglol() ro seJnlpal ;elradseq119-41ern613 '9

srsourloy{r selnqnlojlruj

aseqclolel

tg

sel)rsaA pa^rrep -!6t09

, :1 s e ; d o u 6 e t q d

.\

llel/\ llo) Mau

salnqnloJ)tu.t oseqdlalul 1o {et.re ;errpor

selnqnlor)il.!alpurds t e l o d r a l u r] o s u r e u r a J

s l u a u r e l ru] r p e p u e solnqnlor)il.r.r ]o pueq oseqooJoaJo

op^) lle) eqt:/ [ reldeq3

860t

('auorts uesnsJo (saunol) 'r"urads pue s66eol asuanr6leql slla)aql ug,(1uo dn pua salnuer6-d eql leql os'suorsrnrp u! paleadal ;1arluanbasqnsleJa^as srsse:orduolleberbasaues eq1 '1q6y.l aLlluo umoqssp'sap!^!p66a aql uaqm se;nuer6-6 eq] sa^ra)ar;;arrelq6nep roualsodaql Iluo lpql aJnsuoot poluauo sraueldabeneepaql'#a/ oqt uo umoqs se'66epezrlrlra; eql;o alod .rolralsod aqt ol pate6or6asauo)aq lnq (uMor.ls aqlJo useldotl) lou) 66a pazrlryarun aql tnoLl6norqlIlr.uopuerpatnqUlsrp arp {aq1's;;e>ura6 aqt ouo)aq slla)q)lq^ aururalappue suraloropue vNU,o epeur ere sa;nuerb;;eus aseql'Ido)sor)r.rt paMar^pue sa;nuel6-6 arua>saronp,(q tsulebe^poqrlupue qlrmpaurelsslla) auresaql de Molaqs;1eraq1'{dorso.nrr.u a)ua)saronupue lserluo)-a)uaraJralul -leltua.lajrlptlloq ^q perualnale {aq} l(serpoq re;odpue)snal)nuaql Moqs ol olp lua)sarong'6u;pu;q-y56 ?nlg e qllM paulelsueeq e^Pqa ogp slla) aqf 'suobala apoleurouaql,o 660 pazllluoJ ) e Jo uorsrnrp tsrUaql 6urrnp;1arralq6nep auo ot stuauodtuorrlr-useldo1{r trlrads >ulauulse palloJluo) Jo uolle6ar6as aLllalPrlsnllr sqder6o:rrr,u tq6;1esaql ';;a> ralq6nep auo I;uo ol slueuodr!o) >1urse;dolfu6u11e6ar6as uorsrArp ;;ar >lr1aurur{seuy gg-41 arnbr3

660t

'sllac 'aldurexe ',{.ea,r pue se1lb elcsnu JoJ paleelonult1nru eruoJaq slqt ur ueaq 's11ac -oledeq etuos pue 'sleleluld poolq ecnpoJd qrlq a'sepboIre1e8a111 u€Ileru -rueru Jo sadq euros uI sJn33o osp slsauplot{c lnoqlpt UOISIAIpmelcnN '(69-2I erntlf,) 'pue snelcnu qJee asolcue ol JJo saqcurd '3utr ulsol.Iu-ul]f,8 uP Jo dlaq aql q]!v\ premul spuaxe euerqrueu eruspld eq1-'uoltozqulryIac palluc slseuDloltJ paleu -lpJoos Jo punoJ auo uI snelJnu qJBe punoJB palPeJceJeseuPJqrueu 'suoISIAIp reelJnu prder asaql Jauv 'uorsl^Ip qJEa roJ slsauPlolb Jo sdels eql ile qSnorqr oB ol aup eql e{E} ot elpq lou op slloo eql s€ 'luaudolenep ^lJea dn spaads ,(pear8 tuerue8uerp srql 'urn;1buds e pelpc sr ruseldo$o eIrES aql aruqs 'IalJnu relJnu aldrlpru qf,rqm ur IIaJ V'erBJJns eq] Jeau Jaf.Blouoru P ut peBueJJE puesnoq] IPJeAesSurureluoc ilac aSJPIal8uts e Jo uoneluJoJ al{l q Suppsar 'uorsrlrp crurseldoldc lnoqllm JnoJo uoIsIAIp JEelJnu Jo spunoJ €I lsJg eql 'aldruexe JoJ 'ofJqua DlnldosoJe fpee aqt uI 'uoIsIAIp cturseldo{c Suruarrra}ut 'suopdacxe lnoqllM uorsr rp Jeelcnu Jo spunoJ eldpFur oSJapun silac etxos ere aJeql 'uolst^Ip crurseldolb,(q parr,rollo;,{1pnsn sI UOISIAIpJPelJnu q8noqlry

stsou!Ioy()lnoql!/u\ln))o ue) slsollw 'salurqeua^ ul eloJ Jelrurs E e^eq ol uees suleloJd asaql eluos puu '(zz nl Jo -deqf, ur passnJsrp) suorsrlrp clrlaruurri.seqcns JoJpeJlnbeJ sutalord eqlJo eruos pegBuapr atteqn\tqdosotq puesunBala 3 ur sas,(puE clleua0 'uofar aleudorddu aql ol 'seFqnloJJrru lErls€ sll eIA 'selod apurds el{l Jo euo [nd aleql penTBsol suralord rolou teql pue sluatuelou alpurds qcns tf,aJlp xeuoo IIeJ aq];o suor8ar 'ller Su1pptp eql ulq]$ rauupru pellorluoo lecol ur sa8ueqc lpql ^laIII sruaas lI e ur peloru eq o] seq elpurds eqt ffipculatuudse uoIsIAIp;o aueld eqt uoplsod oI'(g9-ZI amt;g) slueuoduor esaql sllrel{ur 1ac ralq8nep alerrdordde eql lBql os uorsrlrp ;o aueld eql uoplsod uaql pue IIaJ aql Jo epls euo ol (srunulutlapp a1al77acpelIBJ) slueuodruoc uleuac ale8ar8as tsrg lsnru IIaJ Jeqlou aql 'Leansrql ur salpJtueJeJJrpqllm sllao ralq8nep ateerc oI's,,(ervrqledlueJeJJIpSuop dola,rap 'qloq ut Jo rllJequl ol pauusep erp sllec ralq8nep iueJeJJIp oml eql ,{1pnsn ,taql slualuoc ctuseydo/J aqt ul 'ezISul raJJIpleql sllec om1acnpord ol dlluoulaur -uLse aprnrp slles ueqM te^al oq 'luatudolarrap uI secuelsur,{uuru eJEeJeqJ 'ssacordSurraluac eql ol elnqlJluoJ seFqnlorJlru eseql uo 1nd ro qsnd raqtla leql suleloJd Joloru pue seFqruoJOIIu 'a1pu1ds 'rusuldordc aqr ur Jlaslr JelueJ ol spual seseJ lsoru uI qclqm IeJlsV 'sluauodruoc aures aql Jllol$u eql Jo lueueceld aql luoq sllnseJ drleruurrfu slql qllj\/\ pue azrs pnba Jo slleJ ralqSnep o,r4 Sutcnpord '11acluared aql;o rolenba eql punom srurog 3uu allppJluoo aql :r(lpcrrleunurts ap1lr1psilec IPurIuP lsolN

{;;errrleuur{sy ep;n;got elpuldstlaql uotltsodeuslla) auos

ststNt)ot_Af

oseqdLg e loJ poJtnbar se,stsolil.! laue ^ll^ll)e )pf ,o uorssarddns penurluol e solnsuastqf '(uMoqslou lsurelo.rd rollqlLlu!lpf Jo uotlelnunt)paql ot se llaMse))/fdv-tqpJJo uotle^tl)e aql ol spealstsoltualel u! ,(lrnrtte l p ) - t u ! d o r pa q l ' a s p q dt 9 e 6 u r u r e l u o r slle)ul (g)'ure6e6ur1e;nutnt>e ur6aq ol surP,{>-y1 sMolleq)tqM,stso}tLU laue uorle^rt)eut l/)dv ol spealurl)^)-wJo ssolaql xtln!t>e)/)dv-oz)p) sotelnulrls ,{ll^ll)elp)-t4 asne)aB'uotllnJ}sop ur1:It-y11 6urre66rr1,aseqde1ar,u ]o pua aqt le sesrr{}rnrt>e)/)dV-OZ)p) 'sa;rb ;1atrruo,{lqu:aIllee ul (V) 'slsolrulJatleuorltqtqur 1p1 alqegs{q oseqdt9 p,o uollea.t)aq199-21 a.rn613 , &4Q9,,)l)iv:€$f]':.ltt.:],,t]

:t,:::tt:::,r:,:t:ttt..,:lt:,tt,tl,t,t:,

oseqd rD ou qll/!^slla) lluofuqure (V)

ulrl 0 I (8)

'(V09-ZI arntgg) elrdr ueu ar{lroJ ullr,{c-IN.r au 3ur -lelnrunJ3p ur8aq,ltpl3rnbo1 aql s^\ollp }r sp 'salJdr 1ac cruodrqrua prdBJur 1ac uor}e^rlceur f,/JdV srr{J 'lleo Jruo InJesn.,tlercadsasr srsolrruraue .{.lalErparurur -.,fuqruauE ur .{lrlr}Je l/ldv IIe Jo uortelrlJeul aq} ol speel uoos slsol1u atel ur uIIr.{r-N Jo uor}rnr}sap eqt 'snqJ .fipr11cef,/f,dV-OZOpOsetelnruns {pl-N }eqt 'ralel\oq 'llEJeU'JellJeepassncsrp'JlJdV-SZJpJ Jo uorlce aq] o] dlaJrluelsorup enp sI srsolrru elel uI {pl-hl Jo uor}E^rlJBureql 'soz{JquralBrurup .,(Feau1 'uorsr^rp ^tau E ol Suplpfiuoc eJoJeqlueu 'aseqd de8 IC e saluraua8 -uoJIAueslr sJolruoru pue s^ or8 IIaf,aqt qJtlm SurJnp d.tntlceur {pl Jo elels srql 'slleJ }sotu uI 'elo^3 IIec ^ eu u Jelue o1 saredard 1ac aql se .{t1,r,pleur{pl Jo alels B ot ruets.{s Iorluoc elcfr-llec aqt 8up}asar roJ rusru 'sur8rJouorlecrldar -eqcau B seprnoJdosp saxalduroc e pEOIIdaJeJd lI ]e VNCI (srseuplolb selouroJd 'slsolrur alpl Jo srseqluds eq1 salqeua pup Jo sluale eql sreSSulll :suorlcunJdueur seq aseqd 61 el€l ur qpl Jo uope pJeul eq] teldeqr slql ul rerlrea paqrrJsep sV 'uorlcnrlsep urTc.{r}uepuedep-f,/Odv ,,(q,tpreurrrd uallrp sl qJlq^a 's{pl Jo uorlulrtopul aql s1aseqd ntralBI ul tuela .{rolpp8ar da4 y

(1rnr1reu; lp) Joelels alqelse sl eseqdtg aql 'aseqd I Jo pua eqt lE palarqop sr elpls slql ^\oq Surssncsrp,ri.quoncas slq] pue 'a^Ilceul u(psour eJE s)p] qclqM ur (ID Jatua sllec 'srseuoloi-,b ragy lsoru

el

('ue^rllnS ue!lllM pup nAeurlsu)jo Isauno)'B) 'aseqdelar-u a1:{r aql qbnolq} ur lle arp,{aql'araq1I;snouorqruls o6 ra;>nuaql lleleql aloN pal sourosoJluaf aql pue uaa;6paurelsare salnqntor)r.u aqf uorlezuelnllo) aroleqo{rquraolrqdosotg e ur sa;purds rrlolru a;dr1;nu-r;o qder6o.r:ru.r pallel a)ua)saronll(B)'uorlpzuelnlla) ssa)oroP ur slla)lenpr^rpur Lurojol snal)nuq)ea punorjnsolJJosaq)uro pup preMurspualxaauerquraur euse;daqt pue'xelrol aql ot alerbrr-u uaql ral)nuaqi Jo lso6 'unrll(ru,{sabre;e alear>o} uorsrnrp rrluseldoy(r rn>rosuorsr^rp rpal)nu€ [ ]srUaqf (V) lnoqlrMpue{;snouorqru,{s 'o{rqura opqdosotqI;rea aql ugslsau;1oy(r lnoqllm slsollw 6s-l I aln6lJ aue./quaur

tunlv{:u,{se u r r e l l n uI u e u . r

(v) 66e pezrlrpal

tc sNotstn uvt'tfnN

cll_l'ldtAo) NOtIVZrUV'tn't'll)

el)^) lle) eql :/ [ ra]deqf

00t t

OF CELLDIVISION AND CELLGROWTH CONTROL

Rapid cyclin accumulation immediately after mitosis is not useful, however, for cells with cell cyclescontaining a G1phase.These cells employ severalmechanisms to prevent Cdk reactivation after mitosis. One mechanism uses another APC/C-activating protein called Cdhl, a close relative of Cdc20. Although both Cdhl and Cdc20 bind to and activate the APC/C, they differ in one important respect. \ArhereasM-Cdk activates the Cdc20-APC/C complex, it inhibits the Cdhl-APC/C complex by directly phosphorylating Cdhl. As a result of this relationship, Cdhl-APC/C activity increasesin late mitosis after the Cdc20-APC/C complex has initiated the destruction of M-cyclin. M-cyclin destruction therefore continues after mitosis: although Cdc20-APC/C activity has declined, Cdhl-APC/C activity is high (Figure l7-608) A second mechanism that suppresses Cdk activity in G1 depends on the increased production of CKIs, the Cdk inhibitory proteins discussed earlier. Budding yeast cells, in which this mechanism is best understood, contain a CKI protein called Sicl, which binds to and inactivates M-Cdk in late mitosis and G1. Like Cdhl, Sicl is inhibited by M-Cdk, which phosphorylates Sicl during mitosis and thereby promotes its ubiquitylation by SCF.Thus, Sicl and M-Cdk, like Cdhl and M-Cdk, inhibit each other. As a result, the decline in M-Cdk activity that occurs in late mitosis causesthe Sicl protein to accumulate, and this CKI helps keep M-Cdk activity low after mitosis. A CKI protein called p27 (seeFigure l7-I9l17-19) may serve similar functions in animal cells. In most cells, decreasedtranscription of M-cyclin genes also inactivates MCdks in late mitosis. In budding yeast,for example, M-Cdk promotes the expression of these genes,resulting in a positive feedback loop. This loop is turned off as cells exit from mitosis: the inactivation of M-Cdk by Cdhl and Sicl leads to decreasedM-cyclin gene transcription and thus decreasedM-cyclin synthesis. Gene regulatoryproteins that promote the expressionof G1/S- and S-cyclins are also inhibited during G1. Thus, Cdhl-APC/C activation, CKI accumulation, and decreasedcyclin gene expression act together to ensure that the early G1phase is a time when essentially all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the use of multiple regulatory mechanisms makes the suppression system robust, so that it still operates with reasonable efficiency even if one mechanism fails. So how does the cell escape from this stable Gr state to initiate a new cell cycle? The answer is that Gl/S-Cdk actMty, which rises in late Gr, releasesall the braking mechanisms that suppress Cdk activity, as we describe in the next section'

Summary After mitosiscompletestheformation of a pair of daughter nuclei, cytokinesisfinishes the cell cycle by diuiding the cell itself. Cytokinesisdepends on a ring of actin and myosin that contractsin late mitosis at a site midway betweenthe segregatedchromosomes.In animal cells,the positioning of the contractile ring is determined by signals emanatingfrom the microtubules of the anaphasespindle. Dephosphorylation of Cdk targets,which resultsfrom Cdk inactiuation in anaphase, triggerscytokinesisat the correcttime after anaphase.After cytokinesis,the cell entersa stableGt stateof low Cdk actiuity, where it awaits signals to enter a new cell cycle.

ANDCELLGRO TH OFCELLDIVISION CONTROL A fertilized mouse egg and a fertilized human egg are similar in size, yet they produce animals of very different sizes.\Alhatfactors in the control of cell behavior in humans and mice are responsible for these size differences?The same fundamental question can be asked for each organ and tissue in an animal's body. \Alhat factors in the control of cell behavior explain the length of an elephant's trunk or the size of its brain or its liver? These questions are largely unanswered, at least in part because they have received relatively little attention compared with other questions in cell and developmental biology. It is neverthelesspossible to say what the ingredients of an answer must be.

1101

1102

Chapter17:TheCell Cycle

The size of an organ or organism depends mainly on its total cell mass, which depends on both the total number of cells and the size of the cells. Cell number, in turn, depends on the amounts of cell division and cell death. organ and body size are therefore determined by three fundamental processes:cell growth, cell division, and cell death. Each is tightly regulated-both by intracellular programs and by extracellular signal molecules that control these programs. The extracellular signal molecules that regulate cell size and cell number are generally soluble secreted proteins, proteins bound to the surface of cells, or components of the extracellular matrix. They can be divided operationally into three major classes: 1. Mitogens, which stimulate cell division, primarily by triggering a wave of Gr/S-Cdk activity that relieves intracellular negative controls that otherwise block progress through the cell cycle. 2. Growth factors, which stimulate cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules and by inhibiting their degradation. 3. suruiual facfors, which promote cell survival by suppressing the form of programmed cell death known as apoptosis. Many extracellular signal molecules promote all of these processes,while others promote one or two of them. Indeed, the term growth faAoris often used inappropriately to describe a factor that has any of these activities. Even worse, the term cell growthis often used to mean an increasein cell numb er,or cell nroliferation. In addition to these three classesof stimulating signals,there are extracellular signal molecules that suppresscell proliferation, cell growth, or both; in general, Iessis known about them. There are also extracellular signal molecules that activate apoptosis. In this section, we focus primarily on how mitogens and other factors, such as DNA damage, control the rate of cell division. we then turn to the important but poorly understood problem of how a proliferating cell coordinates its growth with cell division so as to maintain its appropriate size.We discussthe control of cell survival and cell death by apoptosis in Chapter lg. microtubule

MitogensStimulateCellDivision u-nicellularorganismstend to growand divideasfastasthey can,and their rate of proliferationdependslargelyon the availabilityof nutrientsin the environ-

1[m

PDGFis only one of over50proteinsthat areknornmto act asmitogens.Most of theseproteins have a broad specificity.pDGII for example,can stimulate many types of cells to divide, including fibroblasts,smooth musclecells,and

Figure17-61A platelet.Platelets are miniaturecellswithouta nucleus. They circulatein the bloodand helpstimulate bloodclottingat sitesof tissuedamage, therebypreventingexcessive bleeding. Theyalsorelease variousfactorsthat stimulatehealing. The plateletshown herehasbeencut in halfto show its secretory vesicles, someof whichcontain platelet-derivedqrowth factor (PDGF).

OF CELLDIVISION AND CELLGROWTH CONTROL

neuroglial cells. Similarly, epidermal growth factor (EGD acts not only on epidermal cells but also on many other cell types, including both epithelial and nonepithelial cells. Some mitogens, however, have a narrow specificity; erythro' poietin, for example, only induces the proliferation of red blood cell precursors. Many mitogens, including PDGE also have other actions beside the stimulation of cell division: they can stimulate cell growth, survival, differentiation, or migration, depending on the circumstances and the cell type. In some tissues,inhibitory extracellular signal proteins oppose the positive regulators and thereby inhibit organ growth. The best-understood inhibitory signal proteins are TGFp and its relatives.TGFBinhibits the proliferation of several cell t1pes, either by blocking cell-cycle progression in G1 or by stimulating apoptosis.

Nondividing CellsCanDelayDivisionby Enteringa Specialized State In the absence of a mitogenic signal to proliferate, Cdk inhibition in Gt is maintained by the multiple mechanisms discussedearlier,and progressioninto a new cell cycle is blocked. In some cases,cells partly disassembletheir cell-cycle control system and exit from the cycle to a specialized, nondividing state called G6. Most cells in our body are in Gs, but the molecular basis and reversibility of this state vary in different cell types. Most of our neurons and skeletal muscle cells, for example, are in a terminally dffirentinted Ge state, inwhich their cell-cycle control system is completely dismantled: the expression of the genes encoding various Cdks and cyclins are permanently turned off, and cell dMsion rarely occurs. Other cell types withdraw from the cell cycle only transiently and retain the ability to reassemble the cell-cycle control system quickly and reenter the cycle. Most liver cells, for example, are in Gs, but they can be stimulated to dMde if the liver is damaged. Still other t!?es of cells, including fibroblasts and some Iymphocytes, withdraw from and re-enter the cell cycle repeatedly throughout their lifetime. Almost all the variation in cell-cycle length in the adult body occurs during the time the cell spends in G1or Go.By contrast, the time a cell takes to progress from the beginning of S phase through mitosis is usually brief (typically 12-24 hours in mammals) and relatively constant, regardlessof the interval from one division to the next.

Activities MitogensStimulateGr-Cdkand GrlS-Cdk For the vast majority of animal cells, mitogens control the rate of cell division by acting in the Gr phase of the cell cycle.As discussedearlier,multiple mechanisms act during G1to suppressCdk activity and therebyblock entry into S phase.Mitogens release these brakes on Cdk activity, thereby allowing S phase to begin. As we discussin Chapter 15, mitogens interact with cell-surfacereceptors to trigger multiple intracellular signaling pathways. One major pathway acts through the small GTPaseRas,which leads to the activation of a MAP kinase cascade.This leads to an increase in the production of gene regulatory proteins, including Myc. Myc is thought to promote cell-cycle entry by several mechanisms, one of which is to increase the expression of genes encoding G1 cyclins (D cyclins), thereby increasing Gr-Cdk (cyclin D-Cdk4) activity. As we discuss later, Myc also has a major role in stimulating the transcription of genes that increase cell growth. The key function of Gr-Cdk complexes in animal cells is to activate a group of gene regulatory factors called the E2F proteins, which bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins involved in DNA synthesis and chromosome duplication. In the absence of mitogenic stimulation, E2F-dependent Seneexpressionis inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family.

1103

1104

Chapter17:TheCellCycle mrtogen

Figure 17 -62 Mechanismscontrolling cell-cycleentry and S-phaseinitiationin animalcells.As discussed in Chapter15, mitogensbind to cell-surface receptors to initiateintracellular signaling pathways.One of the major pathways involvesactivationof the smallGTPase Ras,whichactivates a MAPkinase cascade, leadingto increased expression of numerousimmediateearly genes, includingthe geneencodingthe gene regulatoryproteinMyc.Myc increases the expressionof many delayed-response genes,includingsomethat leadto increased G1-Cdk activity(cyclinD-Cdk4), whichtriggersthe phosphorylation of membersof the Rbfamilyof proteins. Thisinactivates the Rb proteins, freeing the generegulatoryproteinE2Fto activatethe transcription of G115genes, includingthe genesfor a G1lS-cyclin (cyclinE)and S-cyclin(cyclinA).The resultingGrlS-Cdkand S-Cdkactivities furtherenhanceRb protein phosphorylation, forminga positive feedbackloop.E2Fproteinsalso stimulatethe transcription of theirown genes,forminganotherpositive feedbacklooo.

I nas

I

+ I

a c t i v a t i o no f g e n e r e g u l a t o r yp r o t e i n

cYrosol

I

rfl

NUCLEUS I

x

n"*11,"*on

I '

gene

regulatory gluyc.*f protein

I delayed-response gene expresston

)

positivefeedback

G 1/S-cyclin (cyciln rl S-cyclin ( c y c l i nA )

active + S.CdK

DNA SYNTHESIS

i nactivated E 2 Fp r o t e i n

inactivated Rb protein

F.?ffi" \A/hencells are stimulated to divide by mitogens, active Gr-cdk accumulates and

in turn increase Rb protein phosphorylation and promote further E2F release (seeFigure 17-62). The central member of the Rb family, the Rb protein itself, was identified originally through studies of an inherited form of eye cancer in children, knonm as retinoblastoma (discussedin chapter 20). The loss of both copies of the Rb gene leads to excessivecell proliferation in the developing retina, suggestingthat the Rb protein is particularly important for restraining ceUaivision in this tissue. The complete loss of Rb does not immediately cause increased proliferation of

AND CELLGROWTH CONTROL OF CELLDIVISION

retinal or other cell types, in part because Cdhl and CKIs also help inhibit progressionthrough Gr and in part becauseother cell tlpes contain Rb-related proteins that provide backup support in the absenceof Rb. It is also likely that other proteins, unrelated to Rb, help to regulate the activity of E2E Additional layers of control promote an overwhelming increase in S-Cdk activity at the beginning of S phase.We mentioned earlier that the APC/C activator Cdhl suppressescyclin levels after mitosis. In animal cells, however, Grand G1/S-cyclinsare resistant to Cdhl and can therefore act unopposed by the APC/C to promote Rb protein phosphorylation and E2F-dependent gene expression.S-cyclin, by contrast, is not resistant to Cdhl, and its level is initially restrained by Cdhl-APC/C activity. However, Gr/S-Cdk also phosphorylates and inactivates Cdhl-APC/C, thereby allowing the accumulation of S-cyclin, further promoting S-Cdk activation. Gr/S-Cdk also inactivates CKI proteins that suppress S-Cdk activity. The overall effect of all these interactions is the rapid and complete activation of the S-Cdk complexes required for S-phaseinitiation.

TheDNADamageResponse DNADamageBlocksCellDivision: Progressionthrough the cell cycle, and thus the rate of cell proliferation, is controlled not only by extracellular mitogens but also by other extracellular and intracellular mechanisms. One of the most important influences is DNA damage, which can occur as a result of spontaneous chemical reactions in DNA' errors in DNA replication, or exposure to radiation or certain chemicals. It is essentialthat the cell repair damaged chromosomes before attempting to duplicate or segregatethem. The cell-cycle control system can readily detect DNA damage and arrest the cycle at either of two checkpoints-one at Start in late Gt, which prevents entry into the cell cycle and into S phase, and one at the GzlM checkpoint, which prevents entry into mitosis (seeFigure 17-21). DNA damage initiates a signaling pathway by activating one of a pair of related protein kinases called ATM and ATR, which associate with the site of damage and phosphorylate various target proteins, including two other protein kinases called Chkl and Chk2. Together these various kinases phosphorylate other target proteins that lead to cell-cycle arrest.A major target is the gene regulatory protein p53, which stimulates transcription of the gene encoding a CKI protein called p21;this protein binds to Gr/S-Cdk and S-Cdk complexes and inhibits their activities, thereby helping to block entry into the cell cycle (Figure 17-63). DNA damage activates p53 by an indirect mechanism. In undamaged cells, p53 is highly unstable and is present at very low concentrations. This is largely because it interacts with another protein, Mdm2, which acts as a ubiquitin ligase that targets p53 for destruction by proteasomes. Phosphorylation of p53 after DNA damage reduces its binding to Mdm2. This decreasesp53 degradation, which results in a marked increasein p53 concentration in the cell. In addition, the decreasedbinding to Mdm2 enhances the ability of p53 to stimulate gene transcription (seeFigure 17-63). The protein kinases Chkl and Chk2 also block cell cycle progression by phosphorylating members of the Cdc25family of protein phosphatases,thereby inhibiting their function. As described earlier, these kinases are particularly important in the activation of M-Cdk at the beginning of mitosis (see Figure 17-25).Thus, the inhibition of cdc25 activity by DNA damage helps block entry into mitosis (seeFigure 17-21). The DNA-damage response also detects problems that arise when a replication fork fails during DNA replication. \{hen nucleotides are depleted, for example, replication forks stall during the elongation phase of DNA synthesis.To prevent the cell from attempting to segregatepartially replicated chromosomes, the same mechanisms that respond to DNA damage detect the stalled replication forks and block entry into mitosis until the problems at the replication fork are resolved. The DNA damage response is not essential for normal cell dMsion if environmental conditions are ideal. Conditions are rarely ideal, however: a low level

11 0 5

1 106

Chapter17: The Cell Cycle

(discussed in chapter 20). This loss of p53 function allows the cancer cell to accumulate mutations more readily. Similarly, a rare genetic disease known as ataxia telangiectasiais causedby a defect in ArM, one of the protein kinasesthat is activated in response to x-ray-induced DNA damage; patients with this diseaseare very sensitive to x-rays and suffer from increased rates of cancer. \Mhat happens if DNA damage is so severe that repair is not possible?The

individual cell. Cells that divide with severeDNA damage threaten the life of the organism, since genetic damage can often lead to cancer and other diseases. Thus, animal cells with severeDNA damage do not attempt to continue division, but instead commit suicide by undergoing apoptosis. Thus, unless the DNA damage is repaired, the DNA damage response can lead to either cell-cycle

DNA DNA damage

I I ATM/ATRkinaseactivation

+

C h k l/ C h k 2k i n a s ea c t i v a t i o n

II +

Mdm2

PHOSPHORYLATION OF p53

stable, a c t i v ep 5 3

p53 UBIQUITYLATION AND DEGRADATION IN PROTEASOMES

A C T I V Ep 5 3 B I N D ST O R E G U L A T O RRYE G I O N O Fp 2 7 G E N E

p21 gene

TRANscRrproN I V

p2, mRNA

rnarusmloru{ p21(cdk i n h i b i t o rp r o t e i n )

ACTIVE G1/S-Cdk and 5-Ldk

INACTIVE G r l S - C da knd S-Cdk c o m p l e x e dw i t h p 2 1

Figure17-63 How DNAdamagearrests the cellcyclein G1.WhenDNAis damaged,variousproteinkrnases are recruitedto the siteof damageand initiatea signalingpathwaythat causes cell-cycle arrest.Thefirstkinaseat the damagesite is eitherATMor ATR, dependingon the type of damage. Additionalproteinkinases, calledChkl and Chk2,arethen recruitedand activated, resultingin the phosphorylation of the generegulatory proteinp53.Mdm2 normallybindsto p53 and promotesits ubiquitylation and destructionin proteasomes. Phosphorylation of p53 blocksits binding to Mdm2;asa result,p53 accumulates to high levelsand stimulates transcription of the genethat encodesthe CKIprotein p21.The p21 bindsand inactivates G1lS-Cdk and S-Cdkcomplexes, arresting t h e c e l li n G 1 . I ns o m ec a s e sD, N A damagealsoinduceseitherthe phosphorylation of Mdm2 or a decrease in Mdm2 production,whichcausesa furtherincrease in p53 (not shown).

CONTROL OF CELLDIVISION AND CELLGROWTH

arrest or cell death. As we discuss in the next chapter, DNA damage-induced apoptosis often depends on the activation of p53. Indeed, it is this apoptosispromoting function of p53 that is apparently most important in protecting us against cancer.

ManyHumanCellsHavea Built-lnLimitationon the Numberof TimesTheyCanDivide Many human cells divide a limited number of times before they stop and undergo a permanent cell-cyclearrest.Fibroblaststaken from normal human tissue, for example, go through only about 25-50 population doublings when cultured in a standard mitogenic medium. Toward the end of this time, proliferation slows dolrm and finally halts, and the cells enter a nondividing state from which they never recover. This phenomenon is called replicative cell senescence, although it is unlikely to be responsible for the senescence(aging) of the organism. Organism senescenceis thought to depend, in part, on progressiveoxidative damage to long-lived macromolecules, as strategies that reduce metabolism (such as reduced food intake), and thereby reduce the production ofreactive oxygen species,can extend the lifespan of experimental animals. Replicative cell senescence in human fibroblasts seems to be caused by changes in the structure of the telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. As discussed in Chapter 5, when a cell divides, telomeric DNA sequences are not replicated in the same manner as the rest of the genome but instead are synthesizedby the enzyme telomerase. Telomerasealso promotes the formation of protein cap structures that protect the chromosome ends. Becausehuman fibroblasts, and many other human somatic cells, are deficient in telomerase, their telomeres become shorter with every cell division, and their protective protein caps progressively deteriorate. Eventually,the exposed chromosome ends are sensedas DNA damage,which activates a pS3-dependent cell-cycle arrest that resemblesthe arrest caused by other types of DNA damage (seeFigure l7-63). Rodent cells, by contrast, maintain telomerase activitywhen they proliferate in culture and therefore do not have such a telomere-dependent mechanism for limiting proliferation. The forced expressionof telomerase in normal human fibroblasts, using genetic engineering techniques, blocks this form of senescence.Unfortunately, most cancer cells have regained the ability to produce telomerase and therefore maintain telomere function as they proliferate; as a result, they do not undergo replicative cell senescence.

Arrestor AbnormalProliferation SignalsCauseCell-Cycle Apoptosis,Exceptin CancerCells Many of the components of mitogenic signaling pathways are encoded by genes that were originally identified as cancer-promoting genes,or oncogenes,because mutations in them contribute to the development of cancer.The mutation of a single amino acid in the small GTPaseRas, for example, causes the protein to become permanently overactive,leading to constant stimulation of Ras-dependent signaling pathways, even in the absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc stimulate excessive cell growth and proliferation and thereby promote the development of cancer. Surprisingly, however, when a hyperactivated form of Ras or Myc is experimentally overproduced in most normal cells, the result is not excessiveproliferation but the opposite: the cells undergo either cell-cycle arrest or apoptosis. The normal cell seems able to detect abnormal mitogenic stimulation, and it responds by preventing further division. Such responseshelp prevent the survival and proliferation of cells with various cancer-promoting mutations. Although it is not knolrm how a cell detects excessivemitogenic stimulation, such stimulation often leads to the production of a cell-cycle inhibitor protein calledArf which binds and inhibits Mdm2. As discussedearlier, Mdm2 normally

1"t07

1108

Chapter17:TheCellCycle

Figure 17-64 Cell-cyclearrestor apoptosisinduced by excessive stimulationof mitogenicpathways. Abnormallyhigh levelsof Myccausethe activationof Arf,whichbindsand inhibits p53 levels Mdm2 and therebyincreases (seeFigure17-60).Dependingon the cell p53 type and extracellular conditions, then causeseithercell-cycle arrestor apoptosis.

excessiveMyc production

a c t i v eM d m 2

stable, active p53

promotes p53 degradation. Activation of Arf therefore causes p53 levels to increase,inducing either cell-cycle arrest or apoptosis (Figure lZ-64). How do cancer cells ever arise if these mechanisms block the division or survival of mutant cells with overactive proliferation signals?The answer is that the protective system is often inactivated in cancer cells by mutations in the genes that encode essential components of the checkpoint responses,such as Arf or p53 or the proteins that help activate them.

Organismand OrganGrowthDependon CellGrowth For an organism or organ to grow cell division is not enough. If cells proliferated without growing, theywould get progressivelysmaller and there would be no net increase in total cell mass. In most proliferating cell populations, therefore, cell growth accompaniescell division. In single-celledorganisms such as yeasts,both cell growth and cell division require only nutrients. In animals, by contrast, both cell growth and cell proliferation depend on extracellular signal molecules, produced by other cells, which we call growth factors and mitogens, respectively. Like mitogens, the extracellular growth factors that stimulate animal cell growth bind to receptors on the cell surface and activate intracellular signaling pathways. These pathways stimulate the accumulation of proteins and other macromolecules, and they do so by both increasing their rate of synthesis and decreasing their rate of degradation. They also trigger increased uptake of nutrients and production of the ArP required to fuel increased protein slmthesis. one of the most important intracellular signaling pathways activated by growth factor receptors involves the en4rme PI 3-kinase, which adds a phosphate from AIp to the 3 position of inositol phospholipids in the plasma membrane. As discussedin Chapter 15, the activation of PI 3-kinase leads to the activation of a kinase called ToR, which lies at the heart of groMh regulatory pathways in all eucaryotes. ToR activates many targets in the cell that stimulate metabolic processesand increase protein slmthesis. one target is a protein kinase called s6 kinase (s6&, which phosphorylatesribosomal protein 56, increasingthe ability of ribosomes to translate a subset of mRNAs that mostly encode ribosomal components. ToR also indirectly activates a translation initiation factor called eIF4E and directly activates gene regulatory proteins that promote the increased expression of genes encoding ribosomal subunits (Figure f7-6S).

ProliferatingcellsUsuallycoordinateTheirGrowthand Division For proliferating cells to maintain a constant size, they must coordinate their growth with cell division to ensure that cell size doubles with each division: if cells grow too slowly, they will get smaller with each division, and if they grow

1109

CONTROL OFCELLDIVISIONAND CELLGROWTH

growth factor

aminoacids{

Figure 17-65 Stimulationof cell growth by extracellulargrowth factors and in Chapter15, the nutrients.As discussed occupationof cell-surfacereceptorsby growth factorsleadsto the activationof whichpromotesprotein Pl 3-kinase, througha complexsignaling synthesis pathwaythat leadsto the activationof the proteinkinaseTOR;extracellular nutrientssuchasaminoacidsalsohelp activateTORby an unknown pathway. to TORemploysmultiplemechanisms as shown;it stimulateproteinsynthesis, alsoinhibitsproteindegradation(not shown).Growthfactorsalsostimulate productionof the gene increased regulatoryprotein Myc (not shown), of the transcription whichactivates variousgenesthat promotecell is an metabolismand growth.4E-BP initiation inhibitorof the translation factor elF4E.

"i,

I ,/

activatedgrowth factor receptor

gene regulatory factors

I

V ribosome synthesis

I

I 55

+l

I

too fast, they will get larger with each division. It is not clear how cells achieve this coordination, but it is likely to involve multiple mechanisms that vary in different organisms and even in different cell types of the same organism (Figure

r7-66). Animal cell groMh and division are not always coordinated, however. In many cases,they are completely uncoupled to allow growth without division or division without growth. Muscle cells and nerve cells, for example, can grow dramatically after they have permanently withdrawrl from the cell cycle. Similarly, the eggs of many animals grow to an extremely large size without dividing; after fertilization, however, this relationship is reversed,and many rounds of division occur without growth (seeFigure 17-9). Compared to cell division, there has been surprisingly little study of how cell size is controlled in animals. As a result, it remains a mystery how cell size is determined and why different cell qpes in the same animal grow to be so different in size (Figure 17-67). One of the best-understood casesin mammals is the adult sympathetic neuron, which has permanently withdrarnrn from the cell cycle. Its size depends on the amount of neruegrowth factor (NGF) secretedby the target cells it innervates; the greater the amount of NGF the neuron has accessto, the larger it becomes. It seemslikely that the genesa cell expressesset limits on the size it can be, while extracellular signal molecules and nutrients GROWTHFACTOR

MITOGEN

C E L LG R O W T H

C E L LD I V I S I O N

CELLGROWTH

II

t

CELLDIVISION (A)

(B)

(c)

cells,cellsizeis for coordinatingcellgrowth and division.ln proliferating Figure| 7-66 Potentialmechanisms coupling maintainedby mechanisms that coordinateratesof celldivisionand cellgrowth.Numerousalternative of these mechanisms arethoughtto exist,and differentcelltypesappearto employdifferentcombinations (A)In manycelltypes-particularlyyeast-the rateof celldivisionis governedby the rateof cell mechanisms. someminimalthreshold;in yeasts,it is mainlythe growth,so that divisionoccursonlywhen growthrateachieves nutrientsthat regulatethe rateof cellgrowthand therebythe rateof celldivision'(B)In levelsof extracellular factors(growth extracellular someanimalcelltypes,growthand divisioncaneachbe controlledby separate and cell sizedependson the relativelevelsof the two types of factors. factorsand mitogens,respectively), activating (C)Someextracellular factorscan stimulateboth cellgrowthand celldivisionby simultaneously progression. signalingpathwaysthat promotegrowthand other pathwaysthat promotecell-cycle

1110

Chapter17:TheCellCycle Figure 17-67 The sizedifferencebetween a neuron (from the retina)and a lymphocytein a mammal.Bothcellscontainthe sameamountof DNA. A neurongrowsprogressively largerafterit haspermanently withdrawn from the cellcycle.Duringthis time,the ratioof cytoplasmto DNA increases enormously(by a factorof morethan 10sfor someneurons). (Neuronfrom B.B.Boycott,in Essays on the NervousSystem[R.Bellairs and E.G.Gray,edsl.Oxford,UK:ClarendonPress,1974.)

regulate the size within these limits. The challenge is to identify the relevant genes and signal molecules for each cell type.

Neighboring CellsCompetefor Extracellular SignalProteins \Arhenmost types of mammalian cells are cultured in a dish in the presence of serum, they adhere to the bottom of the dish, spread out, and divide until they form a confluent monolayer. Each cell is attached to the dish and contacts its neighbors on all sides.At this point, normal cells,unlike cancer cells, stop proliferating-a phenomenon known as density-dependentinhibition of cell diuision. This phenomenon was originally described in terms of "contact inhibition" of cell division, but it is unlikely that cell-cell contact interactions are solely responsible. The cell population density at which cell proliferation ceasesin the confluent monolayer increases as the concentration of serum in the medium increases. Moreover, if a stream of fresh culture medium is passedover a confluent layer of fibroblasts to increase the supply of mitogens, the cells under the stream are induced to divide (Figure r 7-68). Thus, density-dependent inhibition of cell proliferation seems to reflect, in part at least, the ability of a cell to deplete the medium around itself of extracellular mitogens, thereby depriving its neighbors. This type of competition could be important for cells in tissues as well as in culture, becauseit prevents them from proliferating beyond a certain population density, determined by the available amounts of mitogens, growth factors, and survival factors. The amounts of these factors in tissues are usually limiting, in that increasing their amounts results in an increase in cell number, cell size, or both. Thus, the amounts of these factors in tissueshave important roles in determining cell size and number, and possibly the final size of the organ or tissue. The overall size of a tissue may also be governed in some casesby extracellular inhibitory factors. Myostatin, for example, is a TGFB family member that normally inhibits the proliferation of myoblasts that fuse to form skeletal muscle cells.\A/henthe gene that encodes myostatin is deleted in mice, muscles grow to be several times larger than normal. Remarkably, two breeds of cattle that were bred for large muscles have both turned out to have mutations in the gene encoding myostatin (Figure f 7-69).

cells proliferate

confluent monolayer: cells no longer proliferate

s t r e a mo f f r e s hm e d i u m p u m p e da c r o s sc e l l s

neuron

a lymphocyte

f l o w o f m e d i u ms t i m u l a t e s c e l l p r o l i f e r a t i o nu n d e rt h e s t r e a m

Figure17-68The effectof freshmediumon a confluentcell monolayer.Cellsin a confluentmonolayer do not divide(grafl.fhecellsresumedividing(green)whenexposeddirectlyto freshculturemedium. Apparently, in the undisturbedconfluentmonolayer, proliferation hashaltedbecausethe mediumcloseto the cellsis depletedof mitogens,for whichthe cellscompete.

1111

CONTROL OFCELLDIVISIONANDCELLGROWTH

Figure17-69 The effectsof a myostatin mutationon musclesize,The mutation in the massof leadsto a greatincrease in this Belgian muscletissue,as illustrated Bluebull.The BelgianBluewas produced by cattlebreedersand wasonly recently foundto havea mutationin the Myostatingene.(FromH.L.Sweeney, Sci.Am. 291:62,2004.With permission American.) from Scientific

AnimalsControlTotal CellMassby UnknownMechanisms The size of an animal or one of its organs depends largely on the number and size of the cells it contains-that is, on total cell mass. Remarkably,animals can somehow assessthe total cell mass in a tissue or organ and regulate it: in many circumstances,for example, if cell size is experimentally increased or decreased in an organ, cell numbers adjust to maintain a normal organ size.This has been most dramatically illustrated by experiments in salamanders,in which cell size was manipulated by altering cell ploidy (in all organisms, the size of a cell is proportional to its ploidy, or genome content). Salamandersof different ploidies are the same size but have different numbers of cells. Individual cells in a pentaploid salamander are about five times the volume of those in a haploid salamander, and in each organ the pentaploids have only one-fifth as many cells as their haploid cousins, so that the organs are about the same size in the two animals (Figure L7-7O andFigure 17-71). Evidently, in this case (and in many others) the size of organs and organisms depends on mechanisms that can somehow measure total cell mass. How animals measure and adjust total mass remains a mystery, however. The development of limbs and organs of specific size and shape depends on complex positional controls, as well as on local concentrations of extracellular signal proteins that stimulate or inhibit cell growth, division, and survival. As we discuss in Chapter 22, we now know many of the genes that help pattern these processesin the embryo. A great deal remains to be learned, however, about how these genes regulate cell growth, division, survival, and differentiation to generate a complex organism. The controls that govern these processesin an adult body are also poorly understood. \A/hen a skin wound heals in a vertebrate, for example, about a dozen cell types, ranging from fibroblasts to Schwann cells, must be regenerated in appropriate numbers, sizes, and positions to reconstruct the lost tissue. The

HAPLOID

DIPLOID

PENTAPLOID

11chromosomes

22 chromosomes

55chromosomes

Figure17-70 Sectionsof kidneytubules from salamanderlarvaeof different ploidies,In all organisms, from bacteriato humans,cellsizeis proportionalto ploidy. for example,have Pentaploidsalamanders, cellsthat aremuch largerthan thoseof Theanimalsand haoloidsalamanders. their individualorgans,however,arethe samesizebecauseeachtissuein the pentaploidanimalcontainsfewercells. Thisindicatesthat the sizeof an organism or organis not controlledsimplyby countingcelldivisionsor cell numbers; totalcell massmustsomehowbe (Adaptedfrom G. Fankhauser, in regulated. Analysis of Development[8.H.Willier, P.A.Weiss,and V. Hamburger,eds.l, 1955.) pp. 126-150.Philadelphia: Saunders,

1'112 Chapter 17:TheCellCycle Figure17-7'lThe hindbrainin a haploidand in a tetraploidsalamander. (A)Thislight micrographshowsa crosssectionof the hindbrainof a (B)A corresponding haploidsalamander. crosssectionof the hindbrainof a tetraploidsalamander, revealinghow reducedcellnumberscompensate for increased cellsize,so that the overallsizeof the hindbrainisthe same in the two animals.(FromG. Fankhauser, lnt.Rev.Cytol.1:165-193, 1952. With permission from Elsevier.)

mechanisms that control cell growth and proliferation in tissues are likewise central to understanding cancer, a disease in which the controls go wrong, as discussedin Chapter 20.

Su m m a r y In multicellular animals, cell size,cell diuision,and cell death are carefullycontrolled to ensurethat the organismand its organsachieueand maintain an appropriatesize. Mitogens stimulate the rate of cell diuision by remouing intracellular molecular brakes that restrain cell-cycleprogressionin Gy Growth factors promote cell growth (an increasein cell mass)by stimulating the synthesisand inhibiting the degradation of macromolecules. For proliferating cells to maintain a constant cell size, they employ multiple mechanismsto ensurethat cell growth is coordinatedwith cetldiuision.Animals maintain the normal sizeof their tissuesand organsby adjusting cell size to compensatefor changesin cell number, or uice uersa.The mechanismsthat make this possibleare not known.

PROBLEMS Whichstatementsare true?Explainwhy or why not. '17-'l

Since there are about 1013cells in an adult human, and about 1010cells die and are replaced each day, we become new people everythree years.

(B) 1 0 0p m

like to isolate the wild-type gene that correspondsto the defective gene in your Cdc mutant. How might you isolate the wild-type gene using a plasmid-basedDNA library prepared from wild-t1pe yeastcells?

17-7 Many cell-cycle genes from human cells function perfectly well when expressedin yeast cells. \A/try do you suppose that is considered remarkable?After all, many human genes encoding enzymes for metabolic reactions also function in yeast,and no one thinks that is remarkable.

17-9 You have isolateda temperature-sensitivemutant of budding yeast.It proliferateswell at25"C, but at 35'C all the cells develop a large bud and then halt their progression through the cell cycle.The characteristicmorphology of the cells at the time they stop cycling is knor,tn as the landmark morphology. It is very difficult to obtain s1'nchronouscultures of this yeast, but you would like to know exactly where in the cell cycle the temperature-sensitivegene product must function-its executionpoint, in the terminology of the field-in order for the cell to complete the cycle.A cleverfriend, who has a good microscopewith a heatedstageand a video camera, suggeststhat you take movies of a field of cells as they experiencethe temperature increase,and follow the morphology of the cells as they stop cycling. Sincethe cells do not move much, it is relatively simple to study individual cells.To make senseof what you see,you arrangea circle of pictures of cellsat the start of the experimentin order of the sizeof their daughterbuds.You then find the corresponding pictures of those same cells6 hours later,when growth and division has completely stopped. The results with your mutant are shor.trnin Figure Qf 7-f . A. Indicate on the diagramin FigureQ17-l where the execution point for your mutant lies. B" Doesthe executionpoint correspondto the time at which the cell cycle is arrestedin your mutant? How can you tell?

17-8 You have isolated anew Cdcmutant of buddingyeast that forms coloniesat 25"Cbut not at 37.C.You would now

17- 10 The yeastcohesinsubunit Scc1,which is essentialfor sister-chromatid pairine, can be artificiallv reeulated for

17-2 The regulation of cyclin-Cdk complexes depends entirely on phosphorylationand dephosphorylation. 17-3 In order for proliferatingcellsto maintain a relatively constant size, the length of the cell cycle must match the time it takesfor the cell to double in size. '17-4 \A4rileother proteins come and go during the cell cycle,the proteins of the origin recognitioncomplex remain bound to the DNA throughout. 17-S Chromosomesare positioned on the metaphase plate by equal and oppositeforcesthat pull them toward the two poles of the spindle. 17-6 If we could turn on telomeraseactivity in all our cells, we could prevent aging. Discussthe following problems.

1113

END-OF-CHAPTER PROBLEMS

FigureQ17-1 Time-lapse photographyof a temperaturesensitivemutant of yeast (Problem17-9).Cellson the innerring are arrangedin order of their bud size,which corresponds to their positionin the cellcycle.After6 hoursat 37"C,they havegivenriseto the cellsshownon the outer rlng. No further growth or divisionoccurs.

t f

bud s i z ea t t i m e o f temperature shift

/

+

\

expression at any point in the cell cycle. If expression is turned on at the beginning of S phase,all the cells divide satisfactorily and survive. By contrast, if Sccl expression is turned on only after S phase is completed, the cells fail to divide and they die, even though Sccl accumulates in the nucleus and interacts efficiently with chromosomes.'Why do you supposethat cohesin must be present during S phasefor cells to divide normally? 17-1'l If cohesins join sister chromatids all along their length, how is it possible for condensins to generatemitotic chromosomes such as that shornmin Figure Ql7-2, which clearly showsthe two sister chromatids as separatedomains? FigureQ17-2 A scanningelectron micrographof a fullycondensed mitotic from chromosome vertebratecells (Problem17-11). (CourtesyofTerry D.Allen.) 1*.

17-12 High dosesof caffeineinterfere with the DNA replication checkpoint mechanism in mammalian cells. \A/hy then do you supposethe SurgeonGeneralhas not yet issued an appropriate warning to heavy coffee and cola drinkers?A typical cup of coffee (150 mL) contains 100 mg of caffeine (196gi mole). How many cups of coffeewould you have to drink to reach the dose (10 mM) required to interfere with the DNA replication checkpoint mechanism? (A tlpical adult contains about 40 liters of water.)

stages of of a singlecellat different FigureQ17-3Lightmicrographs (Courtesy of ConlyL.Rieder.) 17-13). M phase(Problem Centrosomeswere used to initiate microtubule growth, and then chromosomeswere added. The chromosomesbound to the free ends of the microtubules, as illustrated in Figure Ql7-4.The complexeswere then diluted to verylowtubulin concentration (well below the critical concentration for microtubule assembly)and examined again (Figure Ql7-4). As is evident, only the kinetochore microtubules were stable to dilution. A. Why do you think kinetochore microtubules are stable? B. How would you explain the disappearanceof the astral microtubules after dilution? Do they detach from the centrosome, depoll'rnerize from an end, or disintegrate along their length at random? C. How would a time course after dilution help to distinguish among thesepossiblemechanismsfor disappearance of the astral microtubules? 17-16 'v\hat are the two distinct cytoskeletalmachinesthat are assembledto carry out the mechanical processesof mitosis and cytokinesis in animal cells? 17-17 How do mitogens, growth factors, and survival factors differ from one another?

17-13 A living cell from the lung epithelium of a newt is shown at different stagesin M phase in Figure Ql7-3. Order theselight micrographsinto the correctsequenceand identify the stagein M phasethat each represents. 17-14 How many kinetochoresare there in a human cell at mitosis? 17-15 Aclassicpaper clearlydistinguishedthe propertiesof astral microtubules from those of kinetochore microtubules.

REFERENCES General TheCellCyc{e: Principles of ControlLondon:New MorganDO (2007) Press Science NewYork: TheCellCycle: An Introduction MurrayAW& HuntT (1993) WH Freeman and Co

b e f o r ed i l u t i o n

after dilution

chromosomes,and FigureQ17-4 Arrangementsof centrosomes, beforeand afterdilutionto low tubulinconcentration microtubules (Problem 17-15). Overview of the Cell Cycle ln the yeasts Forsburg 5L& NurseP (1991)Cellcycleregulation pombe Annu Rev andSchizosaccharomyces cerevisrae Saccharomyces CellBiol7:227-256 JRet al (1974)Geneticcontrolof the cell HartwellLH,CulottiJ,Pringle 183:46-51 divisioncyclein yeastSclence J (1985)Thetimingof early M, NewportJ & Gerhart Kirschner 1:41-47 eventsin XenopusTrendsGenet developmental

1114 Chapter17:TheCellCycle NurseP,Thuriaux P & NasmythK(1976)Geneticcontrolof the cell divisroncyclein the fissionyeastSchizosaccharomyces pombe.l\lol GenGenet146:167-178. The Cell-CycleControl System EvansT, Rosenthal ET,YoungblomJ et al (1983)Cyclin:a protein specified by maternal mRNAin seaurchineggsthat isdestroyed at eachcleavage dtvisionCel/33.389-396 LohkaMJ,HayesMK& MallerJt (t9BB)Purification of maturarion promotingfactor,an intracellular regulator of earlymitoticevents ProcNatlAcadSclU5A85.3OO9-30'l 3 M a s uYi a n dM a r k e rCt L( 1 9 7 1C) y t o p l a s mci oc n t r ool f n u ce a rb e h a v i o r duringmeioticmaturation of frogoocytesJ ErpZaol177:129-146 MorganDO (1997)Cyclin-dependent kinases: engines, clocks, and microprocessors AnnuRevCellDevBiol13)61-291 MurrayAW& Kirschner IVW(1989)Cycin synthesis drivesthe early embryonic cellcycleNature339.275-2BA P a ve t i c hN P( , 1 9 9 9 M)e c h a n i s m o sf c y c l i nd e p e n d e nkti n a s ree g u l a t i o n structures of Cdks,theircyclinactivators, andCIPand Ink4inhibitors I l\,/olBiol287.821-B2B PeterslM (2006) promotingcomplex/cyc Theanaphase osomera machinedesgnedto destroyNatureRevMolCellBiolL644-656 (2005)Function Petroski MD & Deshaies R.J and regulation of cullinRINGubiquitinligasesA/cture RevMolCellBiol6:9-2A Wittenberg C & ReedSl(2005)Cellcycle-dependent transcription in yeast:promoters, transcription factors, andtranscriptomes Oncogene 24:2746-2755 S Phase AriasEE& WaterlC (2007) preventing Strength in numbers: rereplication viamultiplemechanisms rneukaryotic ce ls Genes Dev21,497-518 BellSP& DuttaA (2002)DNAreplication in eukaryotic cellsAnnuRev Biochem71.333-374 BellSP& StillmanB (1992)ATPdependentrecognition of eukaryotrc originsof DNAreplication by a multiprotein complex,^/drure 357:128134 DiffleyJF(2004)Regulation of eary eventsin chromosome replication CurrBiol 14.R778-R786 GrothA, RochaW,Verreault A et a (2002)Chromatin challenges during DNAreplication and repairCell128.721-733 Tanaka S,UmemoriI HiralK et al (2007)CDKdependent phosphorylation of Sld2and Sld3initlates DNAreplication in buddrngyeast A/cture445:328-332 Zegerman P & Diffley)F (2007)Phosphory arionof S d2 andSld3by cyclin-depende s N Ar e p l i c a t i oi n b u d d i n g k innt a s epsr o m o t eD yeaslNaturc445:28- 285 Mitosis Cheeseman lM,ChappieJS,WiisonKubalek EMet al (2006) The conserved KMNnetworkconstitutes the coremicrotubule binding siteof the k netochoreCell127983-99t DongY,VandenBeldtKJ,MengX et al (2002) Theouterplatein vertebrate kinetochores s a flexiblenetworkwith mu tip e microtubule interactions NatureCellBiol9:516-522 HealdR,Tournebize R,BlankT et al (1996)Self-organization of m i c r o t u beusi n t ob i p o l asr p i n d l easr o u n da r t r f i c icahl r o m o s o m ei ns xenopusegg extractsNature382.420-425 HiranoT (2005)Condensins: organizing andsegregating the genome CurrBiol15.R265R215 KapoorTM,LampsonMA,HergertP et al (2006)Chromosomes can congress to the metaphase p atebeforebiorientation Science 3 11: 3 B B - 3 9 1 Mitchison T & Kirschner M (1984)Dynamicinstability of microtubule growth,Nature312237 242, Mitchison TJ(1989)Polewards microtubule fluxin the mltoticspindle: evidencefrom photoactivation of fluorescence J CellBiol 109:637652 Mitchison TJ& SalmonED(2001)Mitosis: a historyofdivisionNature CellBiol3:817-E21 Musacchio A & SalmonED(2007) Thespindle-assembly checkpoint in spaceand lme NatureRevllol CellBiol8319-393

NasmythK (2002)Segregating sistergenomes: the molecular biology of chromosome separation Science297:559 565 NiggEA(2007)Centrosome duplication: of rulesand licenses Irends CellBiol17.215221 NurseP (1990)Universal controlmechanism regulating onsetof M-phaseNature344.503-508 PageSL& HawleyRS(2003)Chromosome choreography: the meiotic ballet Sclence 301.785-789 Petronczki M,SiomosMF& NasmythK (2003)Un menage; quatre:the molecular biologyof chromosome segregation in meiosisCel/ 112:4T 444 SalmonED(2005)Microtubules: a ringfor the depolymerization motor CurrBiol15:R299-R302 Tanaka TU,StarkMJ& Tanaka K (2005)Kinetochore captureand biorientationon the mitoticspindle,\arureRevltlolCellBiol6:929-942 UhlmannF,Lottspeich F & NasmythK (1999)Sister-chromatid separation at anaphase onsetis promotedby cleavage ofthe cohesinsubunitSccl,Nature40a37-42 Wadsworth P & Khodjakov A (2004) E pluribus unum:towardsa universal mechanism for spindleassemblyTrends CellBiol14:413-419 Cytokinesis Albertson R,RiggsB & Sullivan W (2005)Membrane traffic: a driving forcein cytokinesisTrends CellBiol15:92-101 Burgess DR& ChangF (2005)Siteselection for the cleavage furrowat cytokinesisTrends CellBtol 15.156-162 DechantR& GlotzerM (2003)Centrosome separation and central spindleassembly act in redundantpathways that regulate microtubule densityandtriggercleavage furrowformatioa. DevCell 4.333 344 EggertUS,Mitchison TJ& FieldCM (2006)Animalcytokinesis: from partslistto mechanisms AnnuRevBiochem /5:543-566 GlotzerM (2005) Themolecular requirements for cytokinesis Science 307:1735-1739 GrillSW,GonczyP,Stelzer EHet al (200'1) Polarity conrrols forces governingasymmetric spinde positioning in the Caenorhabditis eIegans embryo, Nature 4a9.630-633 JurgensG (2005)Plantcytokinesis: fissionby fusionTrends CellBiol 15:277283 Rappaport R (1986)Establishment of the mechanism of cytokinesis in animalce ls,lnt RevCytol105:245-281 Control of Cell Divisionand Cell Growth Adhikary S & Eilers M (2005) Transcriptional regulation and transformation by Myc proteinsNatureRevl,,4ol CellBiol6.635 645 Campisi J (2005)Senescent cells,tumorsuppression, and organismal aglng:goodc;tize1s, bad neighbo.sCell120:51352) Conlon1& RaffM (1999)Sizecontrolin animaldevelopmentCel/ 96.235-244 FrolovMV,HuenDS,Stevaux O et al (2001 antagonism ) Functional betweenE2FfamilymembersGenes Dev1521462160 H a r r i s oJnC& H a b e r J (E2 0 0 6S) u r v i v i nt g h e b r e a k u pt h: e D N Ad a m a g e checkpointAnnuRevGenet40:209235 Jorgensen P & TyersM (2004)Howcellscoordinate growthand divisionCurrBtol14.R1014,R102/ LevineAJ(1997)p53,the cellular gatekeeper for growthanddivision Cell 88:323-331 RaffMC (1992)Social controlson cellsurvival andcelldeathlVoture 356.397-4AA Sh-.rr Cl & DePinhoRA(2000)Cellular senescence: mitoticclockor cultureshock?Cell102:407-410 SherrCJ& Roberts JM (1999)CDKinhibitors: positive and negative r e g u l a t oor sf G 1 - p h a sper o g r e s s i oGne n eDs e v1 3 : 1 5 0 1 - 1 5 , 1 2 Trimarchi JM & Lees)A Q)aD Siblingrivalrytn the E2Ffamily,Nature RevAlolCellBiol3:,l1-20, VousdenKH& Lu X (2002)Liveor letdie:the cell'sresponse to p53 NatureRevCancer2594-604 Zetterberg A & Larsson O (1985)Kineticanalysis of regulatory eventsin G1 leadingto proliferation or quiescence of Swiss3T3cells,ProcNatl Acad SciUSA82.5365-5369

stlt

JoJ'ruels^ssno^JeuelpJqeue^Surdole^epaql uI 'SuHsluolseeq upc senssrl[Bru -rue llnpu pue Sudola^ep ul srncto lpqr qlpep IIecpauruerSord;o lunoue aqJ

slle) pelue/nunseleulutlllqreao lla) peuuerootd 'eseesrp uEtunqol elnqrJluoJ uec srsoldodeluelJgJnsu ro alrsseoxe,vroqpue ,uoqepSer str puu srsoldodeJo rusrueqJaruJplncalou eql 'slutulupul qleep IIecpeurrueJSord go suouJuruaql ssnsslparrntaldeqc sHl uI 'ueql JeprsuoJtou [eqs e1\^ pue ,sllac Ipurue ur srsoldodealelpatuleql asoqlruo4 toullsrpareseseceseqlq pellonur srusruuqoaruJplnJeJorueqJ 'plJeloeqpue slsuadBulpnlcur ,srusruetrore1n1 -leJrunur sJnJOo qleep peuruuJSor4'uorlca;urpue r{:n[uro1esuodse.r ue^e ilec eq] ur se su 'senuelpue sJervrogJo ecueJsaues q pue luaurdolanap8ur aql ile^ -rnp sJn33011's]ue1duI 'sprurueol peuuuoJlou sr qteep lec paruuerS0rd ' (f, I-8I arn8rg)esuodsar.{:oleunueguruu 3uqrc11e pue sroqqBreurraqt JaAoslueluoJ Jreql 8u1pds 'lsJnq pue sllac JItoJceN 'slsoJJau pelIEJssacorde dq os op Llunsn f1ddns poolqIIeMs e Jo €runeJl IIaJ Jo {cEI se qcns 'llnsur elnoe ue ol esuodsaru1dleluaplccp erp leql sllec Ipurue ,(uur8 -ord qluap Jelnllaf,EJtur ue Jo uoueJedoaql saqdunqJrqm)qleep IIaJ paruuurB -ord;o sr.uJoJ pezrJeloprpqc llalr,rsselJeqlo pue srsoldodeol lserluoc ,tg 'luelxe slr elerxrlserapun fyqeqord pue srear( dueur ro; srsol 11t1s 'srsoldode -dode pa>1oo1rano slsr3o1o1q dryr,,(lqeqord perp s srql ,i.q e^Eq sllac Jo sJeqrunua8reluaqirnuele 'uaeseq ol slleJpeepfl\al f1ensn eJBereql ,rt14cmb os palsa8rppuu ueleeale slleJaql esneceg'asuodsar droleuruugurSufeurep e 8ur -snec]noql!v\',{.uir,re parealcrtlprdersrpue rtlleausetpIIaJaq1demsrq}uI .(SI-8I arn8rg)stueluoJlaql pds uec,teql eroJaq'uaql s;p8ua,{prder (gZJe}deqf, ur passncsrp'llac cu,tco8eqdpazrprcadsu) aSuqdorceru e Jo IIaJSuuoqq8rau e leql os 'peJelledlecruaqc seuocaq serpoqclloldode Jo IIec eql Jo eouJrns aql fllueuodrur 1soy,1'sa?poq cllo\dodo pelleo slueu8er; pasolcue-eueJqruetu o1u1dn $leerq uago 'a8re1sr ilec eqt JI ,pu€ sqelq uego ereJrnsIIer eqJ .(VI{I arn8gfl sluau8er; o1u1 dn pu€ $leeJq sesuepuoo ulleruoJqc JEelJnu eq] pue 'selqruessesrp 'sasdelloc ,asuepuocpue adolanua JEalJnu aq1 uola1a1sol.,io eql o8rapunsrsoldode {urJqsdaqt .IOOC>'sa8uuqcpcrSoloqdrou cusrJalceJeqc dq 8u$p sga3 flqua8uuqJrelu srsoldodepue qteep IIeo perxuerSord srural eql asn uago slsrSolorq,(lSursrguoc'pue 'poolsJepunlseq pu€ uoruruof,lsoru eqt rBJ[q sl rl 'U]eap peuruerSord;o uroJ euo dpo s1srsoldodeqtnoqrry ilec '(aeJl e ruo4 selpal se ,,TJo3u11e;,,Suruearuprom IeeJC eql uro4) slsoldodu dq srncco 'dlarrrsnlcxa lou lnq l.lpnsn slerurueur qluap 1ac paruurerSor4 'loJluocsll pue qleap IIecpaunuerSordol peleJrpepsaua8lsrg eql pegnuepr rcr4tsuaBa1a 3 epolerueueql ur sepnls cpaua8uo papuadappue sruar{97reqtoue ,s0Z6I pasodord sem,urerSordqleep ur eql ur 1oo1ecueldacceprauaB sll tnq -lllnq EanpqslleJlerurueleql eepl aqJ 'qluep IIaJ paurursJ8o.rdseurvrou4ssacord e-derrrrpelJorluoJe ur sallasrueqlilpl pue ruerSordqleep Jelnllef,pJlurue elBA -llJu sllecaq] qclqv\ uI 'saprcFsere sqlsep eser0]eql Motr{ 1v\oue a IIaJ ,([pruJou,, 'qleep u,rnoulundq [Ie] aql seJuelBqrlpexa uorsr^lp[eJ 'srusruEqcau.,i-rolep8er 'asneJeq {urJqslou op senssprno rnoq z{ranaaunsalurpuBMoJJuruauoq eql ul etp sllar Jo suoq[q 'uerunq]InpEfqlleeq E uI 'pooqrppe o]q senu4uocdgensn1r puu 'luarudolalap luuld prrplerurrreur ued tuelrodur,tgercruc e s,{e1dqleep [eC

s!sotdodv

fsls;6o1o19 1oIuedLuoSoql urol1 ql6'0002'ZSZ1-SVZ;i uorssrrurad LZL luawdoP^ao "le le pooM'M ruorl) 'sller rrtotdode ana;,(renare araql ueqM 'rare;{ep auo uoasse'slrbrp6urdo;enep aql uaaMlaqanssrlaql peleuruJle seqqteop1|etlet;6;prelul evl (B) 'st;6rp6u;do;enapaqt uaamlaqslop uaatby.16uqse Jeeddeslla) )rloldode eqI'srsoldodeauo6rapuna^eqleql slla) s;aqe;{;;e>rltads1eq1aIp e qtlm peu!els uaaqseqsnlajesnourslqt u! Medeql (V) 'slsoldode {q med esnou 6ugdolanep aqr ul srl6lpaq16u;1d1nr5 7-91 arn6t1

'aJuuIBq ul ^pJEXa arP ,{.aq}lEql ernsue ol paleF8al fpq8p aq lsnu uoIsIAIp JI 'Surlurrqs Jou SuIMorSreqllau erP lBql senssp lFpu uI IIac puE qlEap IIes '(gZ reldeqf, uI pessnJslp)seqor3ltu elqlsuodsal el{l ^oJlsap pedleq a^eq .{aql rage 'uoltca;ut uu ^q pa}E^IpB se}boqdu.{1 aql Jo }sotu sa}uurulla osp ll lsnoJe8uupdlepuatod silec aql alulu leqt sloldaoeJ eAIlcPeI-JIesacnpord ro sJotdaoerJrJrJeds-ueBIluB Iryesn,{lepuetod acnpord ol IIeJreqlla }Bql seltJoqd -..rdt S pue l Surdolelep seleulluqe slsoldode araqru 'rualsfs eunrulul e lldep€ 'lElulus eql o] snoJeSuep ,tlppualod elEJqeue^ eql ur rnsJo saldlxExe 3uq1r15 'lueludo Jo 'IeuollJunJuou 'paceldsnu 'PurJouqe eJB leql sllaf, SuIleuIuIIe -le^ep ul ssesord loJluos-^lqPnb e se suollrury oslE qlEep IIaJ peruluerSord 'JalEI ssnJsrp a^\ sE 'ol lceuuoc sllef, a^reu eql leql sllaJ 'ald ]e8re] Jo Jaqrunu eql qolpru ol slleo alJeu Jo Jaqrunu aql slsnfpe qleep IIao 'sJequnu

(.aurnSerlnrJo -ruExe JoJ 'urals^s sno^reu Suldolanap eql uI flac elep8ar sdlaq qreap Isalrnol) 'srsoldode1oornlealalqeue^ 'seseJreqlo .{uBru q '(t-8I arnS;g) sJeaddeslp '3or; aq} ul pepaeu lou sI IIef, e arp (V)ur lla) oqt Jo r"use;doilreqt qJIqM rllBl eql puE 'alp IIEI aql uI slleJ eq] 'slsoqdJoruelelu lP 3or; e olut sa8ueq3 ur alqrsr^selon)e^a6re;aql'papoldxa sI ruJoJ daql arnlcnr}s aql uaqo\ elp sllaJ a^eqol suaas ()) ur lla) aqt sPaJeLlM elodpEl E uer{ \'papeau raBuol ou 'Z-gI 'seseJ '1re1url;anr1e1eJ peluJlsnlll se 'alp uaql ueemleq JoJ E ^ 3d asnour aJnBIC uI uI Jeqlo uiaaslnq pesuapuo) 'seJnlcnJls eTl-apBds slleJ eqt se ,{1uo elPJBdas s}l8lp pnpwpul eql pue o^eq(g)pue (v) u! slle)aLlrreql atoN'lla) rry(ro6eqde,{q pe;1n6ua ueaq se lno Uets .{eqt :luarudolanap Jluo^Jque Suunp leeJ puE spuEq ldpcs sdlaq 'slsoldode ,tq,(1pnsn 'sllaf,pelue^ un seleuruqe qleep 'aldruexe JoJ 'qleep seqpue enssrl6urdolenep e u! palp (g) IIaJ ur lle) eql seaieqM'qsrp arnlln) e ur parp 'luaurdolelap eq] 'seseceuos ul uJ 'JEelJsI Ja,lrSUP PruIUE IIao peruueJSord (f) pue (V)ur slla)aqI'()) srsor)aupallp) slql seop sesodJnd ie^Jes qleep IIaJ aAISSETu qreaplla) leluapr))ero adfi e {q ro (g ter{ \'sellesueqr ilH faql arup eql le ^q]Peq -{1rra;rad are ,!uo[eru lsPA aql sE pue srsoldode Iq pa;pa^eqlpq] slle) v) 'peurJoJ are rtaql ,,tletcadsa 'atp o1 qlac dupur os ro; p;a1survrl.1qu{JEIuaJsluaes 1I moqssqder6or)rur uorl)alaasaql'qleap 'eldlxexo JaUP uoos atp ,(leurrou slleJ elJau ;o sad.ril.,tueru3o Jpq ueql eJolu lle) Jo suuo, l)urlsrp orvrl 1-g 1 arn6r1

())

;;er:rybo6eqd

l l a ) p e e pp a ; ; n 6 u a

urn0l

raldeql srsoldody:g1,

9[ t t

APOPTOSIS

11"t7 Figure18-3 Apoptosisduring the metamorphosisof a tadpoleinto a frog. As a tadpolechangesinto a frog,the cells in the tadpoletail areinducedto undergo the tail is apoptosis; as a consequence, in thyroidhormonein lost.An increase all the changesthat the bloodstimulates including occurduringmetamorphosis, a p o p t o s iisn t h e t a i l .

part of the liver is removed in an adult rat, for example, liver cell proliferation increasesto make up the loss. Conversely,if a rat is treated with the drug phenobarbital-which stimulates liver cell division (and thereby liver enlargement)-and then the phenobarbital treatment is stopped, apoptosis in the liver greatly increasesuntil the liver has returned to its original size, usually within a week or so. Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate, although the control mechanisms responsible for such regulation are largely unknor,rm. Apoptosis occurs at a staggeringlyhigh rate in the adult human bone marrow where most blood cells are produced. Here, for example, neutrophils (a type of white blood cell discussed in Chapter 23) are produced continuously in very large numbers, but the vast majority die by apoptosis in the bone marrow within a few days without ever functioning. This apparently futile cycle of production and destruction serves to maintain a ready supply of short-lived neutrophils that can be rapidly mobilized to fight infection wherever it occurs in the body. Compared with the life of the organism, cells are evidently cheap. Animal cells can recognize damage in their various organelles and, if the damage is great enough, they can kill themselves by undergoing apoptosis. An important example is DNA damage, which can produce cancer-promoting mutations if not repaired. Cells have various ways of detecting DNA damage, and, if they cannot repair it, they often kill themselves by undergoing apoptosis.

ApoptoticCellsAre Biochemically Recognizable Cells undergoing apoptosis not only have a characteristic morphology but also display characteristic biochemical changes,which can be used to identify apoptotic cells. During apoptosis, for example, an endonucleasecleavesthe chromosomal DNA into fragments of distinctive sizes;because the cleavagesoccur in the linker regions between nucleosomes, the fragments separateinto a characteristic ladder pattern when analyzed by gel electrophoresis (Figure 18-44). Moreover, the cleavage of DNA generates many new DNA ends, which can be marked in apoptotic nuclei by using a labeled nucleotide in the so-calledTUNEL technique (Figure l8-4B). An especially important change occurs in the plasma membrane of apoptotic cells.The negatively charged phospholipid phosphatidylserineis normally exclusively located in the inner leaflet of the lipid bilayer of the plasma membrane (seeFigures 10-3 and 10-16), but it flips to the outer leaflet in apoptotic cells,where it can serve as a marker of these cells.The phosphatidylserine on the surface of apoptotic cells can be visualized with a labeled form of the Annexin V protein, which specifically binds to this phospholipid. The cell-surface phosphatidylserine is more than a convenient marker of apoptosis for biologists; it helps signal to neighboring cells and macrophages to phagocytose the dying cell. In addition to serving as an "eat me" signal, it also blocks the inflammation often associatedwith phagocltosis: the phosphatidylserine-dependent engulfment of apoptotic cells inhibits the production of inflammation-inducing signal proteins (cltokines) by the phagocytic cell. Macrophages will phagoc)'tose most types of small particles, including oil droplets and glass beads, but they do not phagocytose any healthy cells in the animal, presumably because healthy cells express"don't eat me" signal molecules on their surface.Thus, in addition to expressingcell-surface"eat me" signalssuch as phosphatidylserine that stimulate phagocltosis, apoptotic cells must lose or inactivate their "dont eat me" signals in order for macrophagesto ingest them.

1118

Chapter18:Apoptosis Figure18-4 Markersof apoptosis.(A)Cleavage of nuclearDNAinto a characteristic ladderpatternof fragments. Mousethymuslymphocytes weretreatedwith an antibodyagainstthe cell-surface deathreceptorFas (discussed later),inducingthe cellsto undergoapoptosis. Aftervarious times(indicatedin hoursat the top of the figure),DNAwasextracted, and the fragmentswere separatedby sizeby electrophoresis in an agarosegel and stainedwith ethidiumbromide.(B)TheTUNELtechniquewasusedto labelthe cut endsof DNAfragmentsin the nucleiof apoptoticcellsin a tissuesectionofa developingchickleg bud;this crosssectionthroughthe skinand underlyingtissueis from a regionbetweentwo developingdigits, as indicatedin the underlyingdrawing.The procedureis calledthe TUNEL dUTPnickend labeling)techniquebecausethe enzyme fidT-mediated (TdT)addschainsof labeled terminaldeoxynucleotidyl transferase (dUTP) deoxynucleotide to the 3aOHendsof DNAfragments.(A,from D. Mcllroyet al.,GenesDev.14:549-558,2000.With permisisonfrom Cold SpringHarborLaboratoryPress; B,from V.Zuzarte-Luis and J.M.Hu116, lnt.J. Dev.Biol.46:871-876, 2002.With permissionfrom UBCPress.)

time (hr) 0

Cells undergoing apoptosis often lose the electrical potential that normally exists across the inner membrane of their mitochondria (discussed in chapter l4). This membrane potential can be measured by the use of positively charged fluorescent dyes that accumulate in mitochondria, driven by the negative charge on the inside of the inner membrane. A decrease in the labeling of mitochondria with these dyes helps to identify cells that are undergoing apoptosis. As we discuss later, proteins such as cytochrome c are usually released from the space between the inner and outer membrane (the intermembrane space) of mitochondria during apoptosis, and the relocation of cytochrome c from mitochondria to the cytosol can be used as another marker of apoptosis (see Figure I8-7).

ApoptosisDependson an Intracellular Proteolytic Cascade Thatls Mediatedby Caspases The intracellular machinery responsible for apoptosis is similar in all animal cells. It depends on a family of proteasesthat have a cysteine at their active site and cleave their target proteins at specific aspartic acids. They are therefore called caspases (c for cysteine and asp for aspartic acid). caspases are synthesized in the cell as inactive precursors, or procaspases,which are typically activated by proteolytic cleavage.Procaspasecleavageoccurs at one or two specific aspartic acids and is catalyzedby other (alreadyactive) caspases;the procaspase is split into a large and a small subunit that form a heterodimer, and two such dimers assemble to form the active tetramer (Figure l8-5A). once activated, caspasescleave, and thereby activate, other procaspases,resulting in an amplifying proteolytic cascade (Figure l8-58). Not all caspasesmediate apoptosis. Indeed, the first caspaseidentified was a human protein calred interleukin-l-conuerting enzyme (ICE), which is concerned with inflammatory responses rather than with cell death; ICE cuts out the inflammation-inducing cltokine interleukin-I eLl) from a larger precursor

As shor,rmin Figure 18-58 and rable l8-1, some of the procaspases that operate in apoptosis act at the start of the proteolytic cascadeand are called initiator procaspases; when activated, they cleave and activate dor,tmstreamexecutioner procaspases, which, then cleave and activate other executioner procaspases' as well as specific target proteins in the cell. Among the many target proteins cleaved by executioner caspasesare the nuclear lamins (see Figure 18-58), the cleavageof which causes the irreversible breakdown of the nuclear lamina (discussedin chapter 16).Another target is a protein that normally holds

(8)

APOPTOSIs

1119

(A) procaspaseactivation D yc l e a v a g e

active caspase

( B ) c a s p a s ce a s c a d e o n e m o l e c u l eo f active initiator caspase

inactive procaspases

actrve caspase prodomains

/lt ttt\

/// |\\\

/ti |\r\

gf executiener'caglsef lnahymolecules

/\

n

n

/\/\

I'

.a

cleavage of cytosolicprotein

the DNA-degrading enzyme mentioned earlier (an endonuclease)in an inactive form; its cleavagefrees the endonuclease to cut up the DNA in the cell nucleus. Other target proteins include components of the cltoskeleton and cell-cell adhesion proteins that attach cells to their neighbors; the cleavageof these proteins helps the apoptotic cell to round up and detach from its neighbors, making it easierfor a healthy neighboring cell to engulf it, or, in the caseof an epithelial cell, for the neighbors to extrude the apoptotic cell from the cell sheet. The caspasecascadeis not only destructive and self-amplifying but also irreversible, so that once a cell reaches a critical point along the path to destruction, it cannot turn back. The caspasesrequired for apoptosis vary depending on the cell type and stimulus. Inactivation of the mouse gene encoding caspase-3,an executioner caspase,for example, reduces normal apoptosis in the developing brain. As a result, the mouse often dies around birth with a deformed brain that contains too many cells. Apoptosis occurs normally, however, in many other organs of such mice. From the earliest stagesof an animal's development, healthy cells continuously make the procaspasesand other proteins required for apoptosis.Thus, the apoptosis machinery is always in place; all that is needed is a trigger to activate it. How then, is a caspasecascadeinitiated? In particular, how is the first procaspasein the cascadeactivated?Initiator procaspaseshave a long prodomain, which contains a caspase recruitment domain (CARD) that enables them to assemblewith adaptor proteins into actiuation complexeswhen the cell receives a signal to undergo apoptosis. Once incorporated into such a complex, the initiator procaspasesare brought into close proximity, which is sufficient to activate them; they then cleave each other to make the processirreversible.The activated initiator caspases then cleave and activate executioner procaspases, thereby initiating a proteolytic caspasecascade,which amplifies the death signal and spreads it throughout the cell. The two best understood signaling pathways that can activate a caspasecascade leading to apoptosis in mammalian cells are called the extrinsic pathway and the intrinsic pathway. Each uses its own initiator procaspasesand activation complex, as we now discuss. Table18-1 SomeHumanCaspases Caspases involvedin inflammation Caspases involvedin apoptosis Initiatorcaspases Executioner caspases

1 (lCE),4, 5 caspases c a s p a s 2e ,s8 , 9 , 1 0 caspases 3,6, 7

cleavage of nuclear lamin

Figure 18-5 Procaspaseactivation is during apoptosis.(A)Eachcaspase initiallymadeas an inactiveproenzyme (procaspase). are Someprocaspases activatedby proteolyticcleavageby an two cleavedfragments activatedcaspase: molecules from eachof two procaspase associate to form an activecaspase, whichis a tetramerof two smalland two largesubunits;the prodomainsare as indicated.(B)The usuallydiscarded, firstprocaspases activatedarecalled which then cleave initiatorprocaspases, and activate many executionerprocospose producingan amplifying molecules, chainreaction(a proteolyticcaspase caspases then cascade). Theexecutioner cleavea varietyof key proteinsin the cell, includingspecificcytosolicproteinsand nuclearlamins,as shownhere,leadingto the controlleddeathof the cell.Although are not shown,the initiatorprocaspases activatedby adaptorproteinsthat bring togetherin close the procaspases proximitywithin an activationcomplex; cleave althoughthe initiatorprocaspases eachotherwithinthe complex,the cleavageservesonlyto stabilizethe activeprotease.

| 120

Chapter 18:Apoptosis

k i l l e rl y m p h o c y t e Fasligand

Fasdeath receptor

d e a t hd o m a i n death effector

activated c a s p a s e -o8r 1 0

//l l\\

+ ACTIVATION AND CLEAVAGE OF -10, PROCASPASE-8, OR BOTH

Cell-Surface DeathReceptors Activatethe ExtrinsicPathwayof Apoptosis Extracellular signal proteins binding to cell-surface death receptors trigger the extrinsic pathway of apoptosis. Death receptors are transmembrane proteins containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular death domaln, which is required for the receptors to activate the apoptotic program. The receptors are homotrimers and belong to the tumor necrosisfactor (TNF) receptor family, which includes a receptor for TNF itself (discussedin Chapter 15) and the Fasdeath receptor.The ligands that activate the death receptors are also homotrimers; they are structurally related to one another and belong to the TNFfamily of signal proteins. A well-understood example of how death receptors trigger the extrinsic pathway of apoptosis is the activation of Fas on the surface of a target cell by Fas ligand on the surface of a killer (c],totoxic) lymphocyte (discussedin Chapter 25). \.Mhenactivated by the binding of Fas ligand, the death domains on the cltosolic tails of the Fas death receptors recruit intracellular adaptor proteins, which in turn recruit initiator procaspases (procaspase-8,procaspase-7}, or both), forming a death-inducing signaling complex (DISC). Once activated in the DISC, the initiator caspasesactivate do'vrmstreamexecutioner procaspasesto induce apoptosis (Figure 18-6). As we discuss later, in some cells the extrinsic pathway must recruit the intrinsic apoptotic pathway to amplify the caspase cascadein order to kill the cell. Many cells produce inhibitory proteins that act either extracellularly or intracellularly to restrain the extrinsic pathway. For example, some produce cellsurface decoy receptors, which have a ligand-binding domain but not a death domain; becausethey can bind a death ligand but cannot activate apoptosis,the decoys competitively inhibit the death receptors. Cells can also produce intracellular blocking proteins such as Fl14 which resembles an initiator procaspase but lacks the proteolltic domain; it competes with procaspase-8 and procaspase-10for binding sites in the DISC and thereby inhibits the activation of these initiator procaspases.Such inhibitory mechanisms help prevent the inappropriate activation of the extrinsic pathway of apoptosis. In some circumstances, death receptors activate other intracellular signaling pathways that do not lead to apoptosis.TNF receptors, for example, can also activate the NFrB pathway (discussedin Chapter 15), which can promote cell

-

a p o p t o t i ct a r g e t c e l l

Figure 18-6 The extrinsicpathway of apoptosisactivatedthrough Fasdeath receptors.Fasligandon the surfaceof a killerlymphocyteactivatesFasdeath receptorson the surfaceofthe target cell.Boththe ligandand receptorare homotrimers.The cytosolictail of Fas then recruitsthe adaptorproteinFADD via the deathdomainon eachprotein (FADDstandsfor Fas-associated death domain).EachFADDproteinthen recruitsan initiatorprocaspase (procaspase-8, procaspase-1 0, or both) via a death effectordomain on both FADDand the procaspase, forminga death-inducing signalingcomplex (DISC). Withinthe DISC, the initiator procaspase molecules are broughtinto closeproximity,whichactivates them; the activatedprocaspases then cleave one anotherto stabilize the activated protease, which is now a caspase. Activatedcaspase-8 and caspase-1 0 then cleaveand activateexecutioner procaspases, producinga caspase cascade, which leadsto apoptosis.

APOPTOS|S

1121

survivaland activategenesinvolvedin inflammatoryresponses. \.A/hich responses dominatedependson the type of cell and the othersignalsactingon it.

ThelntrinsicPathwayof ApoptosisDependson Mitochondria Cells can also activate their apoptosis program from inside the cell, usually in response to injury or other stresses,such as DNA damage or lack of oxygen, nutrients, or extracellular survival signals (discussedlater). In vertebrate cells, such intracellular activation of the apoptotic death program occurs via the intrinsic pathway of apoptosis,which depends on the releaseinto the cytosol of mitochondrial proteins that normally reside in the intermembrane space of these organelles (see Figure I2-2lA). Some of the released proteins activate a caspaseproteolytic cascadein the cltoplasm, leading to apoptosis. A crucial protein released from mitochondria in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron-transport chain. \iVhen released into the cytosol (Figure l8-7), it has an entirely different function: it binds to a procaspase-activatingadaptor protein called Apafl (apoptotic proteaseactiuating factor-l), causing the Apafl to oligomerize into a wheel-like heptamer called an apoptosome. The Apafl proteins in the apoptosome then recruit initiator procaspase proteins (procaspase-9),which are activated by proximity in the apoptosome, just as procaspase-8and -10 proteins are activated in the DISC. The activated caspase-9molecules then activate downstream executioner procaspasesto induce apoptosis (Figure l8-8). As mentioned earlier, in some cells, the extrinsic pathway must recruit the intrinsic pathway to amplify the apoptotic signal to kill the cell. It does so by activating a member of tll'e BcI2 family of proteins, which we now discuss.

Bcl2ProteinsRegulate the lntrinsicPathwayof Apoptosis The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill themselves only when it is appropriate. A major class of intracellular regulators of apoptosis is the Bcl2 family of proteins, which, like the caspasefamily, has been conserved in evolution from worms to humans; a human Bcl2 protein, for example, can suppress apoptosis when expressedin C. elegans.

(A) CONTROL cytochrome-c-G FP

(B) UV TREATED cytochrome-c-G FP

m i t o c h o n d r i adl y e

1 0p . anti-cytochromec

25um

Figure 18-7 Releaseof cytochromec from mitochondria during apoptosis. of human micrographs Fluorescence cancercellsin culture.(A)Thecontrol with a gene cellsweretransfected of encodinga fusionproteinconsisting cytochromec linkedto greenfluorescent protein (cytochrome-c-GFP); they were alsotreatedwith a positivelychargedred in mitochondria. dye that accumulates distributionofthe green Theoverfapping andred indicatethat the cytochrome-c(B)Cells GFPis locatedin mitochondria. were expressingcytochrome-c-GFP irradiated with ultravioletlightto induce apoptosis,and after 5 hoursthey were stainedwith antibodies(in red)against is cytochromec; the cytochrome-c-GFP Thesixcellsin the alsoshown(ingreen). in B have bottom halfof the micrographs releasedtheir cytochromec from mitochondriainto the cytosol,whereas the cellsin the upperhalfof the havenot yet done so.(From micrographs J.C.Goldsteinet al.,Ndf.CellBiol. 2:156-162, 2000.With permissionfrom Ltd.) MacmillanPublishers

1122

Chapter18:Apoptosis

(A) Apafl

Apafl by cytochromec a n d h y d r o l y s i os f b o u n d d A T Pt o d A D P

r e c r u i t m e na t nd activationof procaspase-9

apoptosometriggered b y r e l e a s eo f d A D P i n e x c h a n g ef o r dATP (or ATP) APOPTOSOME

I I

I c a s p a s e -c9l e a v e sa n d t h e r e b ya c t i v a t e s e x e c u t i o n e pr r o c a s p a s e s

I

i

CASPASE CASCADE LEADINGTO APOPTOSIS 10

"rn

mitochondrion

Mammalian Bcl2 proteins regulate the intrinsic pathway of apoptosis mainly by controlling the release of cltochrome c and other intermembrane mitochondrial proteins into the cytosol. Some Bcl2 proteins are pro-apoptotic and promote apoptosis by enhancing the release,whereas others are anti-apoptotic and inhibit apoptosis by blocking the release.The pro-apoptotic and antiapoptotic Bcl2 proteins can bind to each other in various combinations to form heterodimers, in which the two proteins inhibit each other's function. The balance between the activities of these two functional classes of Bcl2 proteins largely determines whether a mammalian cell lives or dies by the intrinsic pathway of apoptosis. As illustrated in Figure l8-9, the anti-apoptotic Bcl2 proteins, including Bcl2 itself (the founding member of the Bcl2 family) and Bcl-Xr, share four distinctive Bcl2 homology (BH) domains (BH1-4). The pro-apoptotic Bcl2 proteins consist of two subfamilies-the 8H123 proteins and the BH3-onlyproteins. The main BH123 proteins are Baxand Bak,wh'ich are structurally similar to Bcl2 but lack the BH4 domain. The BH3-only proteins share sequence homology with Bcl2 in only the BH3 domain (seeFigure l8-9). \fhen an apoptotic stimulus triggers the intrinsic pathway, the pro-apoptotic BHl23 proteins become activated and aggregateto form oligomers in the mitochondrial outer membrane, inducing the releaseof cytochrome c and other intermembrane proteins by an unknor,vn mechanism (Figure fS-f0). In mammalian cells, Bax and Bak are the main BH123 proteins, and at least one of them is required for the intrinsic pathway of apoptosis to operate: mutant mouse cells that lack both proteins are resistant to all pro-apoptotic signals that normally activate this pathway. \MhereasBak is tightly bound to the mitochondrial outer membrane even in the absence of an apoptotic signal, Bax is mainly Iocated in the cltosol and translocates to the mitochondria only after an apoptotic signal activates it. As we discuss below the activation of Bax and Bak usually depends on activated pro-apoptotic BH3-only proteins. Both Bax and Bak also operate on the surface of the endoplasmic reticulum (ER) and nuclear membranes; when activated in response to ER stress, they are thought to release Ca2* into the cytosol, which helps activate the mitochondrial-dependent intrinsic pathway of apoptosis by a poorly understood mechanism. The anti-apoptotic Bcl2 proteins such as Bcl2 itself and Bcl-X1 are also mainly located on the cltosolic surface of the outer mitochondrial membrane, the ER, and the nuclear envelope, where they help preserve the integrity of the

Figure18-8 The intrinsicpathwayof apoptosis.(A)A schematicdrawingof how cytochromec releasedfrom mitochondria activates Apaf1.The bindingof cytochromec causesthe Apafl to hydrolyzeits bound dATPto dADP(not shown).The replacement of the dADPwith dATPor ATP(not shown)then inducesthe complexof Apafl and cytochromec to aggregateto form a large,heptameric apoptosome, whichthen recruits procaspase-9 througha caspase recruitmentdomain(CARD) in each protein.The procaspase-9 molecules are activatedwithin the apoptosomeand are now able to cleaveand activate procaspases, downstreamexecutioner which leadsto the cleavageand activation of thesemoleculesin a caspase cascade. Otheroroteinsreleased from the mitochondrial intermembrane spaceare not shown.(B)A modelofthe threedimensional structureof an apoptosome. Notethat somescientists usethe term "apoptosome"toreferto the complex (B,from containingprocaspase-9. D. Aceham et al.,Mol. Cell9:423-432,2002. With permissionfrom Elsevier.)

'r.r,rstueLl)aul uMoulun ue ^q losol^) aql olu! a)eosauelqLuaullalut aql u.rorJ sutatotdlaqlo pup-)aulolq)ol^) aseolorpue auplqtrjotrlleupuor])o]tul ratno aqt uo aleber66P sutatold 'snlnLurls )lloldodPue Iq EZt HBaql pate^tl)euaqM's!sotdodeJo ^e/ilqted )rsulrluraql u! sulaloldauplquaulalu! leltpuoq)ot!ruro asealalaql u! (),legpue xeg ^lureur)sutalotdZl)g )tloldode-oJd €z I H8 Jo alot aql o t-8 [ alnbrl

r auro;qrop{:

suraloJo €zlH8a^tpe pa1e6er66e

a)eos a u e r qu J a u l a l uI

-

,.. sn]nntls ' f il_ol_dodv

a 'snoJaJuEs eruoseq asr ^Jeqlo pFot leql IIas snoJeS -uep ^llerluelod E Surleurur1a ,{qaleql de.uqled crsurJlur aq} Ja88rD ueql sural -ord dluo-tHg aseql :axoN pue olund surel'ordl.1uo-g11geq1apoJue lpqt seue8 Jo uortdrJJsuErl aql sele^rtrE puB (02 pue ZI sJeldEql uI pessnJsrp) satpFru -n3ce tgd uraloJd JossaJddnsJorunl eql ,peJredaleq louueJ lBq] e8purBp VNC ol esuodsal ur ^lJEIrunS ',{e,r,rqtedJrsurJ}ur aq} sJa83u} uaq} qorq^ ,rulg ulel -ord ^luo-tHg aq] Sulporua aue8 aqt Jo uoudrrJsuerl aqt satelrtre)A/ aseupl dVIAtraql uo spuadep 1eq],{E^\q}ed Sulleu8rs relnllaceDur up ,aldruexe roJ (slBu .sureloJd,r(po-g11g -8rs pnrnrns pFIIeJeJua Jo pa^rJdap erc sllaf, aruos ueq \ luaJeJJrpSurlenrtce rlnurls luaJaJJrp qllm ,slsoldode ;o demqlpd Jrsuulur eql pu€ qnuus tpoldodp uaamleq {urT IETJnJJeq} apr^oJd sureloJd,{1uo-ggg 'suralord aueJqruatuJeluraql esealeJ dyaq,{qaraqrpue ErrpuoqJollu uo gZtHg sure}ord rl}o}dodp-ord esaq}Jo uoqe8 -ar33e pue uorlpnltce aqt raSSrrl pue dleq ot xeg d1}carrp purq.,teur o} sural {Eg -ord,{1uo-g119etuos '(II-gI arnS;g) srsoldode eJnpur suretord teq} lerrpuoqJ ,BupuoqJolrru aJBJJns -olru eueJquaruJalur eql Jo aseelaraql sraSSrrlqclq ^ Jo eqt uo {eg pue xeg Jo uoqe8arSSeaq} selqeua uourqrqu pue Surpurq slql ,usru -eqceu poolsrepun,{Food e ,r{g flrnrlce Jraql SurzrTeJ}neu'sura}ord 71cg cr1ol -dode-que uo aAooJBcrqoqdorp.{q Suoye ot spurq ureuop .suralord tHg JIaqJ 71cgcrloldode-que Su4rqlqq r(q,r(prcur srsoldode eloruord o1 lq8noql ere ^eq] pue 'snlnlulls f,lloldodp ue ol esuodseJur ruaql selE^rlceJo seJnpoJdJaqlra IIaO aq; 'suralord fpuel ZIJg Jo sself,qns]se8rel eq] ere sulalord zfiuo-tHg eqJ 'uoDrqrqur eql elurpeu surel -ord,r{po-g119 aql :srso}dodeacnpur o1 ,(e,r,rq1ed orsurr}ur eql roJ pe}lq1lul eq ]snrx sureloJd asaql;o Jaqrunu p ta^oeJol,\l .a^r^rns ol auo lsEal le seJrnbalIIaJ uerpluluelu Lrana pue 'suralord 71cgcrloldode-rlue uErlErxtuprueArJlsEel le aJE araqJ 'suralord auerquaruJalur rar{}o pue, aruoJqcop{c;o asealeJaq} Buplqqur ,{qaraql 'Surzrraruo8rloruo4 ll }uanard pue {€g o} pulq.{aq} ,aldurexeJoJ,aueJq 'losofc aql ur Jo seuBJquaru -tuaru esaql uo Jaqlre lerJpuoqJolru Jalno eq] ug -suralord 71cg cqoldode-ord Suqrqrqur pue o] Surpurq dq ,r(preru srsoldode tlqlqul suralord asaqJ 'Ug aq] ruoq +7BJJo pue erJpuoqf,olru uorJ suralord eupJqrueruJalurJo aseeleraleudorddeur ,aldruexaro; ,Surluanard-auerquau

u r a l o r oz l ) 8

'slaqulau,{llueJ tllotdode_rtue pue rrloldode-orduaannlaq suotl)eralul llarp aql salerpour1r:staquau ,{;lr-ue; Zl)€ lle^q pareqsureurop119i(1uoeqi sr ureuJop €Hgaqr ieql atoN.sula1ord Zl)g Jo sossel)eerql eq1 6-9 1 ern611

( e x o N ' e u r n d' p r g ' u r r g' p e g " 6 ' a 1 u r a l o r d^ l u o - t H g )loldode-ord (f)

,r,,..it,

zH8

LHg

€H8

()eg 'xeg' 6'a) u ralo.ro€z LHg )loldode-oid (g)

'7pg ''6 a) r:rr::'ll:rllrrl (ry-pg u r a l o r d3 p g ) r l o l d o d e - r l u e( V )

?H8

EZLI

srsoldodv

'a)eJlns leupuorl)o]il.uoql ol punoqarpsuralo.rd 7;rg rrloldode-rlueaql 'umoqs]ou qOnoqrlv'suralo.rd €ZtHBaql pue ol 6urpurq,(q,{;}rarrparou 6urtenrl>e alelnuills aseoloruralorolerJpuoq)ol!ur patpnll)eauros ,{etusuralo.rd{1uo-91-19 '1osov(roql olu! suralo.td lerJpuoq)otlur auerqueuilolurjo asealaraql alouloro pue auerquraur ralno leupuoq)olrur pue pslp^rl)eauo)aq aq1ut a1e6ar66e Mou Ll)rqM'surotold€z t Hg aql llqlqu! ra6uo;ou ue>Iaql teqt os sulalold71rg aqr ol pulq pue pale^rl)e rrloldode-rlue rrloldode {1uo-ggg'snlnLUrls eresuralo.rd ue Jo a)uesardoql ul (g)'(uMor.ls lou-losou{r aqt ur pue) auelquraul ralno leupuoq)otrur aql uo suralo.td €z IHBeql llqlqurpue ol purqsulalojo ltloldode Zl)B)!roldode-!tue'snlnulls uero aruesqeeql ul (V)'srsoldode;o {emqled )!sulrlur aq} a}e;n6ersugalord zl)8 )rloldode-t1uepue {;uo-ggg mo1111-g 1 ern6t3 >11o1dode-o.rd

'sulaloJd dvl eTBur osp silel IEruIue lsolu leql uMou{ lv\ou sI tI ('slleo Jeqlo SuIloaJuI pue Supecldar ruo4 snrll aql luanard ol se^lesuaql ilpl ,(ltuanbar; sllao Iprurue patoeJul-snrl1) 'slsoldode ,tq laslr S.qtlpl uorJ snJIAeq] .{.qpalcagursI leql IIaJ }soq e }uenard ol sutalord d!'I epocue qJIqA\'(sesnJlloln -Jpq) sasnrrl loesur uleuao uI pagquepl lsrlJ eJeir (s4y1)s;soldodu;o srollqlqul

sosedse)tlq!qulsdvl 'aln4dosotg ut r(letcadsa'srsoldode Surssarddns ug ped tuetrodrur ue ,te1d osp sutalord (stsoldode Jo Jollqlqul) 'srsoldode;o srolep8ar relnllaoertur dpo aql lou are suralord 71cg dVI aqJ 'suralord 71rg ,{1uo-g11gaq} ut stso}dode;o sJolPAI}le}ualod lsoru eql aJPBrund ;o ,,tpuregqns pue 'rurg 'plg 'snqJ 'sutalord rqoldode-pue aql Jo lesqns Ilsrus e ,{1uo}lqlqul uec suralord ,{1uo-g11gJeq}o eql sealeq ^ 'sutalotd 71cg cpotdode-pue aql Jo 'tulg 'prg sutalord dpo-ggg aql IIE tlqlqul uer (6-8I arn8tg eas) Eund pue 'pu8rs qteap aql 8ur,fiqdure,(qaraql'sutalord euurqrueluJelul reqto pue r aurorqcoldc aseelero1 suratord tzIHg c1]oldode-ord;o uorlelar38e 'EupuoqJolltu aq1 sra33r4 pue sutalord 71cg cpoldode-puu sllqlqq ll ereq ^ 'plg seleels ol selpcolsueJlplgt 'plfl pel1lalplg Jo ruroJ paleJunJ] e Sutcnpord 'g-asedsec 'asedsec rol€ulq eq] 'sllal eseql ul Leruqled cISuIJlxaeql elenllcu sroldecar qleep ueq ys,,(emqted omt eql ueemleq {uI eq} sI p18rulalord ,{1uo -gHs eqJ 'llar aqt ilDI o] epecsBc asedsec aq] rqIIduP ol .{ea,tqled lISuIrluI eql slrnJJeJrtua,rqledcpotdode slsulJlxe aql sllec eruos uI tallIPe pauouueu sV

r aurorqroy{r'

sutalord , € zI H 8 a ^ r p epa1e6a.r66e

euerquleurJolu!pasealal

uralo.td,{1uo-Egg pale^rpe

u r e l o r d7 ; : g ]|loldode-rlue pale^rl)euI AVAAHIVd)ISNIUINIJO NOIIVAIDV (S)

a )e o s aueJqurauilalul ur suralo-rdreqlo

: atuo.tr.l:olr{:

)8 )tlotdode-!lue a^!pe AVAAHIVd)ISNIUINI ]AIDVNI

s l s o l d o d v : g1 t a l d e q l

(V)

tZLl

'srsoldode ol 6ulppal'epe)sp)asedse) p ale^tl)eMou uet q)tqM,saulosoldode lo Ilqurasspallt slo66tJlI auolt.ltoll) pasealat aq]'eu]|taues oLlllv .sdvl aql lo ^ll^!l)e {rolrqrquraql l)olq pue ol purqq)tqM'sutatolddvl-llueale afeds auerqueuJlalut aql uloll leupuoqtoltLu pasealalsuralotdaql 6uoue ,(e,rnq1ed )rsuuluraql salentltesnlnutls)lloldode ue uaqA (B)'(uMoqs 1ou)saurosealord ur uorleper6ap loJ uaql 6urlteu 'ot purq ,{eql sesedse> aq} atellltnbtqn urlrnbrqnosleale sdvl teql sase6r; auos'sasedse)paleAtl)er(;snoaueluods ^ue llqlqul pue ot purqpue ;oso/> aql u! pate)olale sdvl eql sesedserord aq1{q ,o uotlentl)esnoaueluods pasne)srsoloodpleluapt)felua^eldsdvl 'snlnurlsllloldodp ue a)uasqe aql Jo ul (V)'slla)uelleulueu u1srsoldode1o loJluo)aql ut sdvl-tluepue sdvl ,o salor aql ,ro;lepoul pesodold y 71-g 1 arn6r1

sdvl-rlue s u r a l o r o€ z I H 8 p a ] e ^ r ] l e

srsoldodv

i i sn-rnnrlslll01dodv

VAAH.LVd )ISNIUINIJO NOITVAIDV (8)

suralo./o tz lH8 a^tl)eul asedse) pal)olq

sdvl suIeulopolo NOIlVAIJJV snolNVrNods

,eel

; aLuo:qro$r sasedse:otd AVMH-LVd)tSNtULNtlA[)VNt

(v)

'sleluruBru ul luBurruop suraloJd zlJg pu€ seru uI lupuruop sdvl-rluB pup sdvl qtl^^ 'llntuqs Surcnpur-srsoldode ol flec purue ue Jo ^,{lrnrsuas aq] auruJe]ep sdvl-Due pup (sdvl'suralord zltg eqlJo sarlr^rlJe paurqruoJ eql ,IJeruuns uI 'srsoldode Surleln8ar ur saloJ leruJou Jreqt ]noqe fsranorluoc eq] aJueq-palJaJ ,sllal -;eun dpuaredde st srsoldode patelrlJeu! esnoru ur ara lLuO pua (O1SW7 pe[e] oslB) pel]€) Jaus sdv'l-Due uBrlBruueru U^\Ou)Io^\l Surposue seuoS eql talamoq 'uaq1ysrsoldode Suuouord dqaraqr pue Iosotdr aql uI sdyl Buqcolq 'palp^rlcp sr srsoldode go ,{ervrqledcrsurJ}ur aq} ueq a acpds euEJqrueurJalur IerJpuoq3olru eql tuo4 pespalar aJE sdvl-rlup ,zI-gI aJnSld ur paleJlsnllr sv 'lersJaloJluoJaJorusr srsoldode ur suralord upr1eturueruJoaloJ eqJ dVI-lluE ',{g aqr ur srso]dode Suqlorluoc JoJIerJnJc sr pue paleln8er dllq8n sI sd!'l-rlue pup sdVI uaamteq aJuEIeq eql flreeD 'srsoldode o8rapun ol o,,fuquedg Surdoya,rapaqt ul sllal eqlJo IIE sesneJnpqdosotq ul sdVI apof,ua 1eq1sauaS o1vllaq] Jo auo Jo uorlp^rlceur 'dlasranuol 'seu ur srsoldodP .asedsuc $lJolq plH pue 'urrrg tadeag Surpocua saua8 aarql aql Jo uouelaq ,s4tr1go e o1Supurq ruo4 ureuop aqt Suuualerd ureuop UIg aql ot spurq qolq ^ 'punural-51 '.uoqsrreql sl dlueltrurs Jqoru Surpulq-dvl [BJn]JnJ]s,(po rraqt pue 'prH pue'rurtg 'tadaay Surpnlcur ,serg ur sdvl-que eArJaJE areqJ .rlnurls Jltol -dode snorrel ol asuodseJur peonpoJd are qcrq,rn,suraloJd dyl-pue ,(q pazrprl -nau ,tseel aq UEJ sdVI ,{q papnord rarrreq ,{rolrqrqul slq} 1e npqdosotq u1 'srsotdodeta33rr1ol eruof,Jalo lsnu sesedseJpele^rtce ]Eql ploqseJql ^Jollqqur uE les s4tr1eq1 ,{em slql uI 'seruosealorddq uorlcnrlsep roJ sesedseceql 3ur)IJeur,sasedsecale1r$rn -brqn{1od osle sdVJatuos 'sasedseo pa}e^rlre pup pulq o} ot uaq} elqpua tlqqul qJIq ,\ 'sureruop (teadar d\al snJr^oFOEq) UIg aroru Jo auo elpq sdVI I1y gZLL

SISOIdOdV

'paleurLrJrla Alle)rleurolne areslla)a^lau ellxe aql leql pue slla)a^rau^q pal)eluo) arPslla)la6JelllPleq] sarnsua6urlln) {q pannollo;uorllnpord.rano ;o {6e1erts pro^eol srol)eJle^r^lnsJo srql'srsoldode ue a^ra)arslla)auros lunouieluar)uJnsur 'aro;araLll'slla) aq1{q posealar 1e6re1 srol)e]le^r^rns lo lunotuepallur!l aql ^q pauoddnsaq ue) ueqt parnpord ereslla)enrauaro4 'anssgl 1a6re1;o lunoue aLll ol slla) anrau 6ugdo;anep1o roqurnuaql 6urlsnfpeu! qlpep lle) pue srol)e, le^!^rnsJo alor oql € [-8 ! arn6rl

s l l a )a ^ r a u )rloldode

'slsotdodP.{q ruol}e^rP}sluo4 elp .{.llun}ua^esllac eql '(g1 raideq3 lou }nq 'saurosoSeqdolnuulq].Im ursgld ur passnJsrp)saurososf1qlpv\ esnJ ueq] qcq,iv\ -oltJ sll Jo sllq pue seileu€3JosrelsenbesIIaJ aql qJIqM vr'/t&Dtldomr q8norql 'sluelJlnu uodlul(IluarcrJ;a ]ouuP3 slleJ spaau crloqelau JIaq] IenJ sllec qcns eq] ,Je^el oq ,spu8rs IeArAJns]noqllM 1sro1ee3 IeAIAJnSJo acuesqP aql ul alnlln3 ur s{ee^ JoJ eArTaJoJaJeq}uuo pue ,{e,uqlud sISuIJluI eql a}PAI1oEo1 elqeun aJE 'aAIIesllac aql daa{ o} parlnber erE leq} IEg pue x?g qloq TJel ]Pql slles esnolN sulelold ZIIg Jlloldode-IluB aql Sutptrranortq stsoldode3or(ualqledcIsuIJluI eql alplrlJe qJIWv\ 'suralold ,tpo-g11g Jlloldode-ord Surlenpce pue SulJnpord Iq sa^lasueql ilpl ,taql 'sJolJeJIB^IAJnSJo pe^IJdap eJEsllas uellEluruelu ueII \ '(37I-8I 'sutalord 4y1 arn8rg) srsoldode ssarddns 01 suleloJd 451 Suuqeua u(qaraq] -Uue Surlg^llJeur pue Surfe{roqdsoqd ^q }lE sJo}cEJIeAIAJnseurcs'Dllt4dosot1 uI '(g7i-gl arn8rC) pDg se l{rns sulelord 71cg cllo}dode-ord dluo-g11g '(Wf-ef arnS;g) lx-lcg ro JIaslI ZIrg Jo uorpunJ eqt Suplqlqul ^q ]re sraqlo 'a1d sB qtns surelord 71cg crloldode-que Jo uollJnpoJd pasearcur ue alelnurqs -ruexe roJ 'sJolcpJIEAIAJnsetuos 'sutelord;o ,t1rue; ZIcg aql;o sraqruaru 3uqe1 -n8ar,(q uego ,ruprSordcrloldodu aqt ssarddns]Eql sdemqled Surrcu8rsrelnllar -erlul alellloe qJIq \ 'sroldeceJ aoeJJns-llacor pulq,{1ensn sJo}cPJIEAIATnS 'pooqtlnpe ur pue luaurdolalap Surrnp qloq 'senssll Jaqlo ul sJeqlunu IIeJ IoJluoJ o1 lqSnoql sr slla3 SuuoqqSrau z(qpacnpord sJolf,pJIeArAJnsJo slunolue patFull ro; uorlqadruo3 TPIIuTISV '(SI-8I arntlg) qlp peuuoc -{aql slleJ 1a3re13o Jaqunu aq] JoJalerrdordde sr tr lEq] os palsnfpe .{lpcqeruolne sI suoJ -nau Sur,rnrns Jo Jequnu aqr de,r,rslql uI 'elp sJeqlo eql e1q^a 'anq puBts pnrn -JnsJo ad,{l a}eudordde aql;o q8noua eAIaJeJ}Eql sllec eAJeN'ol lcauuoc dqeur -rou ,(aqt 13q1silet 1a3re1aq] dq palarcas aft leq] sJolceJ IBAr^JnsJo slunouE pal1ull ro; aladuroc ueql pue urals.{.ssno1JeuSurdolarrapeq] ur ssacxeuI pasnp -ord are 'aldruexa JoJ 'slleJ a1leN 'papaeu are ,taql a;aqru pug uaq ^ ,(po ant'r -Jns slleJ ]EI{} elnsua sdyaq.{puaredde luaruafluerre SursrrdJnssIqI 'stso}dode pIoAE ol slleJ Jeqlo ruor; Sulpu8ts snonurluoJ eJInbaJsllac Prulu€ lso4 'srolJuJ IBAI Jns pelpl dla,ttlcellor are qJIqM ,srsoldodellqHul leql selnJelourpu8ts Jelnllas€Jlxauo sncoJa^^te^aMoq'eIaH '(7-g1 ern8rg aas) se^lesuaq] ilpl ol seo] pue sra8ug Surdola.tapueemlaq sllaJ 'sd6g) sutalord ctuaSoqdrour auoq eteplulls (ZZpue g1 sraldeq3 uI pessnJslp pacnpord d1eco1allq^\,(t-gl arn8rg aas) stsoqdroluelaul le stsoldode o8rapun

'lou eJe ,(aql uaq/v\ sa^lasuaqt alelnurls salnselou puSrs replaseJlxe eluos 3ur1g4pue papaeu are faql uaq,u,Surrrnrns ^q'esef, slql ul-aloq]tv\e se ursrue8ro aql Jo pooS aqr JoJ a^eqeq sllef, IenpIAIpuI lBq] aJnsua leql sloJluoc ,,lelf,os" 'srsoldodu Surpnlcul 'slleJ IeuI IBruJouaq13oged aru spu8rs JBInlleJBIUeasaq; -Iue Jo salllllloe lsou alep8ar spu8ts Jelnllef,relu '91 raldeq3 uI pessnJslp sV

s^e1 snopenul stsoldodvllq!qulsrol)el le^!^rnsrelnlla)ellxl s r s o l d o d y: g1 t a l d e q 3

gzlL

'srsotdode 1to;q pue a^rt)eauo)aq q)tqM,sdvlsllq!qu! 'patel,Qoqdsoqd re6uo; p111 ou arug 's6y1 6urlrqrqur ,{q qieap ;1etsalor,uo.rd p;g 'pare;,toqdsoqd lou uaqM.plH uralorddVl-tlueaql lo uorle;,{toqdsoqd aqt 6ur1e;nr"ur1s Iq slsoldodelrqrqur srot)eJfe^l^lnsawos'DltLlclosuoul ()) '(uMoqs lou) stsoldodealoutold1eq} suralo.rd 6urpotuasaua6;ouorldrrlsuell aql alelnurtsleqr,{1;tuel peaqlrol aq}Jo suralo.rd {ro1e;n6at pue aua66urlenrlreul 6u11e;fi oqdsoqd,(qsrsoldode sassa.rddns osle]IV srsoldodessatddnsoi ZltB 6uraarl'saleoosst p peB'perelXloqdsoqd a)uo lzl)€ 6urlrqrqul pue ot 6urpurq,{q srsoldode salourotdpeg,parel,(roqdsoqd rou uaqM'(t9-g 1 arn6r3aes; pe6 uralordZl)Btlrotdode-ord{1uo-9119 aq] sale^rlleutpue salel,(torldsoqd 'sla6.relreqlo r(uer.u 6uor"ue,q>rqa,r 'llv aseurluraloldauruoalql/auUas aqt ate^rl)esreqlo^ueW(g)'1X-l)Bro Jlastl Zl)g se q)ns sutalotdZl)g )lloldode-tlue apo)ualeqt saue6;ouorldrrtsuerl aql 6ur1e1nutr1s ,(qsrsoldodessarddns sro])e; le^tnlnsauJos(V).slsoldode lrqrqu!ue) srol)eJle^!^lnsrelnlla)etlxa leql slervrealql tr 1-g 1 arnbrl

'suorlf,unl IIef, puP xIJlPru Jelnllef, -BJUa eql Jo suorlcunJ pue seJnlJnJls elqDIJPruaJaql ssnJsrp e^ teldeqs lxau eq] q 'suopJunf lles-llac pez{urcads ]P Jaq}oue auo o} eJeqpBol pup xrJ}Eu Jel -nllsceJlxe pasBaJsepe Surpnlsur tonEqeq Jraq] ur sepl eql 01 eJeqpe o],lilrlrqP -leruJouqedueur.,{.e1dsrp sfiec JaJUBJqJnS 'elplsord pue ']spaJq'lJeJl Ipunselur '8unl qJns pqaqlrda eq] ur asoq] sE senssp ur esrJe sJaJuEO uerunq lsontr .s8nrp eseqt o] eAIt -rsuesssel sllal reoupo sa{Eru osle uortourg tgdJo ssol eqt,(02 pue 21 sraldeq3 ur passncsrp)ursrueqJer.uluapuadap-ggd e l(q (]saJJea1c,{c-1acpue) srsoldode aJnpur s8nrp racuecrlue ,(ueur sV '(02 raldeq3 ur pessncsrp)lueu8qeru arour JeJueJ eql e)Eur qrlqm Jo euros 'suopelnru eJoru alelnrunJce sllal eql .{e,t. sgf ur lpa8uurep sl VNCI Jleql ueqm uele elpJaJrlord pue aArAJnso] slleJ Jeouec aql selqeua aroJeJeqtuorlounJ tgd Jo {Jpl aqJ 'a8eurep VNCI or asuodsar ur tseJJe a1c.,b-1acro srsoldode saloruord ra8uol ou ]r luql os sJeoupJuerunq Jo %09 uI paletnu sr ggd uralord rossarddnsJorunl aql Surpocua aua8 aql ,(lrepurrg 'srsoldode o8rapun ol sllal Jec -uec Sursnec dq qclql 's8nrp Jef,upJrlue ol dlnqlsuas ,sllal eq] 1ro,,rn.,t1uoruuroJ saseaJJaposp ]r lsJequmu Sursearcurpue IBATAJns 1ac 1ac Sur8uolord dqaraql 'srsoldode Sunrqrqur .,(q racuec ;o luaudolalap eql salourord uollecolsupJl aqt.{rrec 1eql sa{coqduzi.l aql ur uralord Z;p1;1olanal q8rq ar4y'aruor4drut177ac g slql uorJ arueu str sla8 Tycg'peopur juralord ZIJg eql Jo uoqcnpord elrsseJxe sesnec uorlecolsueJ] eruosoruoJqo p eJeq a 'suerunq ur JeJu€J a1-,tcoqduf1 'aldurexa ro3 'aua8 Jo ruJoJ uoruruoJ e ur perJrtuapl fsrg se-m ZIJq aLl;- fleurrouqe urerSord cpoldode aq] a]eln8ar uego sllef, raouet sp 'slournl rtueru ol uorlnqrJluoc uE se{pu osp srsoldode pesuarcaq luelrodurr 'senssp ul\o s.lenprlrpur eq] lsum8B lcuar salrtc -oqdru,{yeqr qJIq ^ ur 'aseasrpaunrururo}ne o} speal srql 'sespJ,(ueruu1 'spuep qdudl pue uaalds aq] ur sJaqrxnu elrsseJxaur alelnrunJce ol slleo asaq] Sursnuc 'sal,{coqdur[1etuos qtpap prurou aq] tuanard puu8rl seCaq] Jo roldacar qleap Jo sECaql epoJue leq] saua8 eql elp^pceq leql 'aldurexeJoJ'suprunq pue aJrru ur suorlelnl l 'srsoldode fq a1psllal ^\aJ oot araq^asuorlrpuoJ Jeqlo are eJaqJ 'suorlrpuoJ asaq] ul slac Sunes ur InJesnanord pm srsoldode {Jolq reql srolrqrqur asedsec se qJns s8nrp 'arnln; eqt ul '1pqt padoq sl tI 'slsotdode,(q erp slleo peloaJJessel '(,,(1ddns poolq alenbepeur) aq] eruos erueqcsr e se srsorcau,{q 1nq tlnsal Jo Jo 'suor}rpuoJ alnJe esaql uI 'se)oJ}spue $lcpDe arp s11ac,{.ueur ueaq eJBseldurexa lrlerueJp lsotu aql Suoury 'a8eruup anssrl ol alnqrJluoc .,(qaraqlpue srsoldode o8rapun slle3 Jo sraqtunu enrssecxeqclqm ur sJepJosrpupunq dueu ere araql

aseas!c ol alnqrrluo) up) stsoldodvlue!)lJJnsul lo eA!sse)xl,eql!l

irffi.ffi,i

T

ry,

*85.

I

ql))o'18 stsol_dodv

,,"roro a,r, T

I

I sdvl

VNU p r Ha ^ r l ) e u r p r He ^ t l ) e CP8 a^!l)eu I

asPut)il)v aArlfP

+ I

I

snl't)nN

pale^rpe

:\l /-

roDe] le^r^rns

sdvl-rlue+o uotle^tpeut ())

Joper|. leAr^rns

u!eloJo 7 1 : g{ 1 u o - g gg : ; t o t d o d e -ord +o uolle^tpeut (g)

Jopet lPAr^rns uralo.rd71:g:rlotdode-rlue ]o uorpnpord poseeJ)ut(V)

LZLL

SISOIdOdV

qll^ '966t '0sL-6ELiv6ilD,'lele eplqso 'H fral^asl]uor] uorsstuirad ruorl) '(g-g t Lualqord)a)lur uloqMou leuiJouo] a^llelal a)tu_t uroqMau1bdso) pue _/_tJDdV ur sMedJo a)ueleeddv t-g tO atnbll 6dse)

Ildld

-t-

Uedv

)ltl L-/tL^l

;srsoldodeilac-qa.\\ul raJJIpol eJnu cpoldode luarJgep esalp roJ alqrssodll sr ,troq 'r(errnqled atues aI{} ur uor}cunJ6-asedsecpue lpdy g1 '(141} a.rn -EId)sllSp pauroJ Llleurrou aleq acrrutuerrrJep-6-esedsec sBaraq^'srlSlpSuldole^apJlaql uaaMleqsqeMeql etpururrla ol IreJ acrur luarJgep-IJedy 'srviedrreqt ur seulpurouqB alrlcurlsrp aneq 5-asedsecro 1;edy ur luarf,rJapaorruuJoq -^\eu 'sarlrrcruJouqB ureJq Julrrurs rraql o1 uI g-g I lserluoJ 'Suruosear rnod ureydxgisrsotdode o8rapun ol IIer Jo ad! raqlrau ro 'qtoq 'auo ppo14 no,i. lcadxa )eg pup xpg roJ a,trlca;ap.,(1q -nop ara^\teqt sllacJo pue sllaoad,fi-pp.,r,r;o IosotLcaqt olul r aurorqcoldc 1cafurorcnuppoc nod ]er{} aufeurl s-gt 2suaBa1a I ul lno perJrecuaeq a^eq leql saua8,,qleap,,roJ suaalJs ctlaua8.,{ueru aql ur punoJlou se.vrsrsoldodeur alncaloruro1 -oeJJe aruorqco1lo Ierluace qrns leql asoddnsno,t op.,ir114'c

ro; aua8 aq] ul punoJ uaaq ra^a a^Bq auou 'apotpruauaql ur srsoldodeSupca;;usaua8luelmu ,{ueruaql;g 'srsoldode ur penlolur saua8 lsrg aql peupuapl teqt sluelnur petpl 'qteap -osrsuaBaga ? ur sluerurradxecrleuaSpcrsselJ IIaJ paurruerSorddq paleururrlaJalelaJEleqt s[a] I€I leuorlrppe ue sacnpordosp lr :silacJlleruos696-,(1}cexa saleraua8sur8 -ap sulpqDqJouaoJ epoteuau aql ;o luaudola,raq f-B[ 'Suluosear rno.{ ureldxg isllao rorunl eseql Jo IeAIArnseql ol alnqFl -uoc lq8nu uralord slql Jo uorssardxara^oleq] asoddns nort op ,vrog 'pue31 sECol spurq leql ulatord pelerJes e pup pagrldrueeAEqo] punoJ ala^ rogaua8e passardxarano 'srolunl uolof, pue 3un1.{reurrrdg6;o dpnls sJournlaqt JIeq e u1 'sa1dcoqdru,,(1 ra1p1,{q slac Journt Jo uorteurulla a1e -rpau ol sr pue8q sec pue secJo aloJtuelrodur auo €-B t 'sua;qord 6u;rvroglo; aql ssnfsrO 'lqBII An rq pecnpur srsoldodeo] ]uelsrseraq ppoqs c aurorqco{c elpq }ou op leq} silec uerleturuel4l Z-g t uorsr^rpIIaJ qleap IIac 'sansslllppp IBruJouuI t-gt sacupleq.,(1ensn '1ou(qrn.ro {qrtnu;eldq aan.ttare stuaurale$ q)!qn

SW]IBOUd

'uoqapat&ap pua pawnuJv t1at1l alotuotd sawdsnc ryqlqur sulalotd 49,1apqm'sutalotd auoJqluauJalur [q ,[umqlad Jrsurliu! aql a]ol lvtrpuot4coiu.u lo asaa1ataql 3ut77ot1uoc -nBatsutalotd cpoldodo-otdpuo cltotdoda-tluor4Jog'f)IaruDaql vltauaq4 uaqm 4cg [1uo san1asutary nq ltltDuuou snacruqi aJnsuao] wotSotd crtoldodo aql alnlnBat[pqBp sutalotd 4y7 pua sutaiotd elcg rvryilacDgut puv suralotd TnuBtsrulnlpcouxa Lflog '6-asadsacotdsatunrpapuv glnrxat pun awosol.dodaua olw salqwassvqlnlm '1tndy sapnllra aupuoqJoittu to acodsauoJquauJatul aql utott pasaa1atc auon4coltc '[amqlad J'tsurr]uraqt u! :JSI1 aql uttol o1sutaiotd toidupv r2tn puv g-sasndsocotd 0L ilnJJaJstoidacattpuap ar4i'[amt4lodJrsuluxa aqi uI'[Ianticadsat'autosotdodoaql pua uouanrpo puusrp ur palDnrpvaJDquqm'sasadwcotd nruU JSIA ary pa11nc'saxalduoJ -lur umo sl! sasnfomqlod qcog'passar].s arDsllal uaqm pawJauaBs\au8tsto\nllarquu! [q patonllco st [amq]nd ctsuluur aLUlstoldacatqtoap acntms-11ac o18urpu1qspunByn7 -n\\acat1xa[q pawnqca v [amLlwd JrsutJ$aatll :srsoldodaol Butpaagapacsacasodsncn n33yl puo sasodsocotdto7otltur aruatJo ol s[amqwd nu!$!p omj $DaI ru asnsIIaJ 'apDJSDJ t tltpatotd alq$r an '(IpJ -aut '8utt{r1dtltv uD Sunnpotd aLU ur sutalotd pBtol nqlo snouan pua) sasad 'sawdsocotdJauorlnJaxawaausumop arun -sucotd qcnlm Jauollncaxa Jaqlo alonuJa -UJopua anoa1c [aql'parualpa acuo:saxa1dutoc uouanrycout [trtutxotd olul lqBnotq uaqm patDnliJo alo sasodsacotdroiuq!ry 'sasodsocotdpallu) sJosrncatd antlcaur sa sIIaJlDurlua pataalJnu 770u1ruasatdata sasDdso)'nal aLuIM dpq o1sutal0td to\ -nlpJDrlu! cpfrcadsanoap qJrqm'sasodsncpa11acsatu[zuaul[loaiotd uo spuadap stsol -dodv stuatuoc ctwsa1dolfclo aBnyoayfua st ataql atotaqsluaw&a{ ro slp) aqt asolfc -oBaqd[1ptdu saBaqdotcnutn s77ac 8uuoqq8rcu puo 'luawBa{ [Tluanbatt puo 'asuap -uoJ'yutrLISsl1acaqL:stsoldoda[q tncco sqJDapasaqj'sasal$out u1 't1loaupua [74unb palaurutqa aq uoJ tustuaBtoaqt oi rualqi o atDJo 'papaaunBuoy ou 'paBawop[Tqtuan -aut pwrurz'tam atv sllar uql q'qruap 77acpawwatSotd pa11acssacotdo-tum toqi palloJiuoc a ur sanlasLuaqi IIl4 puo u,totSotdr4loapJvlnnacDuu! uD atanqro uoc sIIaJ

{reurun5 slsotdodv:g1reldeql

gZLL

1129

END-OF-CHAPTER PROBLEMS '18-7

\A/hen human cancer (HeLa) cells are exposed to Wlight at 90 mJ/cm2, most of the cells undergo apoptosis within 24 hours. Releaseof cltochrome c from mitochondria can be detected as early as 6 hours after exposure of a population of cells to UV light, and it continues to increase for more than 10 hours thereafter. Does this mean that individual cells slowly release their cltochrome c over this time period? Or, alternatively, do individual cells release their cytochrome c rapidly but with different cells being triggered over the longer time period? To answerthis fundamental question,you have fused the gene for green fluorescent protein (GFP) to the gene for c1'tochromec, so that you can observethe behavior of individual cells by confocal fluorescencemicroscopy.In cells that are expressingthe cytochrome c-GFP fusion, fluorescence shows the punctate pattern typical of mitochondrial proteins. You then irradiate these cells with W light and observeindividual cellsfor changesin the punctate pattern. TWo such cells (outlined in white) are shovrn in Figure QfB-2A and B. Releaseof cytochrome c-GFPis detectedas a change from a punctate to a diffuse pattern of fluorescence.Times after W exposureare indicated as hours:minutes below the individual panels. Which model for cytochrome c releasedo these observationssupport?Explainyour reasoning.

REFERENCES AdamsJM,HuangDC,Strasser A et al (2005)Subversion ofthe Bcl2 life/death switchin cancerdevelopment andtherapyColdSpring HarbSympQuantBiol7A.46977 KM& Salvesen Boatright GS(2003)Mechanisms of caspase activation, CurrOpinCellBiol15:725731 DanialNN& Korsmeyer SJ(2004)Celldeath:criticalcontrolpointsCel/ 116:205-219 ) echanism E l l i sR EY, u a nJ Y& H o r v i tR z A( 1 9 9 1M a sn df u n c t i o nosf c e l l death AnnuRevCellBiol7:663-698 givingphosphatidylserine FadokVA& HensonPM(2003)Apoptosis: recognition an assist-witharwisrCurrBial13:R655-R657 Galonek HL& Hardwick lM (2006)Upgrading the BCL-2network NatureCellBiolB:131/- 1319 ten minutesto dead Cel/ GreenDR(2005)Apoptoticpathways: 1 2 1. 6 7 1 , 6 7 4 HorvitzHR(2003) Worms,life,and death(Nobellecture)Chembiochem -7 11 4'697 Hyun-Eui K,FengheD,FangM & WangX (2005)Formation of is initiatedby cytochrome c-induced dATPhydrolysis apoptosome and subsequent nucleotide exchange on Apaf-1ProcNatlAcadSci USA102:175451755a MD,WeilM & RaffMC (1997)Programmed celldeathin Jacobson animalde',elnnme^raoll88347-354, apoptosis AnnuRev JiangX & WangX (2004)Cytochrome C-mediated Biochem/3.87-106 KerrJF,WyllieAH & CurrieAR(1972)Apoptosis: a basicblological phenomenon in tissuekinetics with wide-ranglng lmplications BJ Cancer26.239-257

10:09

l 0 : 15

17il0

17:18

analysis microscopic video,fluorescence FigureQ18-2 Time-lapse of individualcells from mitochondria of cytochromec-GFPrelease (Problem18-7).(A)Cellsobservedfor 8 minutes,10 hoursafter (B)Cellsobservedfor 6 minutes,17 hoursafter UV irradiation. UV irradiation.One cell in (A)and one in (B),eachoutlinedin white, theircytochromec-GFPduringthe time frameof the havereleased beloweachpanel. which is shownas hours:minutes observation, (FromJ.C.Goldsteinet al.,Ndt CellBiol.2:156-'162,2000. Ltd.) from MacmillanPublishers With permission

celldeath CellDeath functionin programmed Caspase KumarS (2007) Differ14:3243 J Cel/ PH(2005)Deathreceptorsignaling Lavrikl,GolksA & Krammer Sci118,265-267 tumoursuppression LoweSW,CeperoE & EvanG (2004)Intrinsic Nature432307 315. of LumJJ,BauerDE,KongM et al (2005)Growthfactorregulation Cel/ ofapoptosis, in the absence andcellsurvival autophagy 120,23748 of McCallK & Stellerl, (1997)Facingdeathin the fly:geneticanalysis Trends Genet13222-226 apoptosisin Drosophila. AnnuRevGenet apoptosis NagataS (1999)Fasligand-induced 33.2955 and programmed in development NagataS (2005)DNAdegradation celldeath,AnnuRevlmmunol23.853-875 Theapoptosome GS(2006) S & Salvesen PopC,TimmerJ,Sperandio MolCell22:269-275 by dimerization caspase-9 activates RaffMC (1999)Cellsuicidefor beginnersNature396:119-122 in of apoptosis Rathmell .JC& ThompsonCB(2002)Pathways anddiseaseCel/ homeostasis, development, lymphocyte 109.s97-107 celldeath of programmed TittelJN& StellerH (2000)A comparison between speciesGenomeBiol 1 AM,CoulsonEJ& VauxDL (2001)Inhibitorof apoptosis Verhagen Biol Genome proteinsandtheirrelatives: lAPsand otherBIRPs -300910 2:3009.1 VousdenKH(2005)Apoptosisp53and PUMA:a deadlyduo Science 685-1686 309:1 prot'oins how BH3-only WillisSN& AdamsJM (2005)Lifein the balance: induceapoptosisCurrapin CellBiol17:617-625

CELLSIN THEIRSOCIAL CONTEXT

[€tt

E6rr

tlvMttS)INVtd tHl

slnssu_ r^rlf tNNo) sLLL

IVWINVIO XIUIVW UilNII]fVUIX] ]HI NOISSHCV

6 9 rt V9LL

85t I,

xtulvy\j-tt3f CNVSNIU9]I.NI VNIY\JV-I IVSVB]HI VlVI^IS]COt/\JSVId CNVSNOIIfNNT dv9 :ttl) or tttf WOUISAVM]DVSSVd

vnlHltdl IO NOIIVZINV9UO ]HI 09r r cNV sNotlf Nnf 1H9U_ €€II

NOtStHCV Il)-ttl) CNVSNIU]HCV)

laldeq) sFlI ul

'elrs uorsaqpP ol alrs uorseqpPruo{ 'lle3 qJBe Jorra}ureq} ssoJJEsessaJls Jo SuplpusueD 'paJoqJup eJe slueruelg 1erala>lsol,(JeJeqm 'suorsaqpe llef,-lleo ,{q ,{.1lcar1preqlo qrea ol peqoupu ere sllec eql 'urnlaqtrda aqt ul{tl6'leeqs aql Jo aJeJ auo Surr(lrapun'(auatqutara luautasaq rc) aurruDl IDSaq aql peIleJ 'dluecs sr xrJlPru JElnllef,eJlxe aq1 'u11aqr1da leur ulql e ;o ,tlureur Supslsuoc pelleJ steeqs olur JeqtaSol punoq [1aso1caJe slleJ 'uDIs eqt yo Suuazrocpru -.raprdaeql ro aql go 3uru11eql sE qJns 'senssll p[eqrlde u1 'tserluoc ,{g ln8 '1rr{.qpelpd eq ol puu uo tl 1nd ot ueq] 8ur.nno1p'xuleur eql ot slueuqce1u luepodun a^pq slleJ eql lnq 'aJEJ dlarrpelar eJBJaqloue pue IIaJ euo ueemleq slueruqceue IJaJIC 'pa1cefqns sr ans -sp aqt qJIqM o] sseJlslBsrueqceu eql Jo tsolu sJeeq ]eql-slleJ eqt uBql JeqlPJ -xrJleru eql sI lI pue 'uaBa12oc,tlprcedsa 'sraudlod snoJqrJ ur qcrr sr xrJleru eql '11urqlpr pelnqrrlsrp ,(lasreds arB slles puu 'lryqueld sr 4r1eu rulnllaJEJlxe aql'uopue] Jo auoq se qJns 'sanssp eallJeuuoJ uI '(I-6I arntlgl sarueJlxeIBJnI -Jalrq3Je orr,q3uDuasardar 'sarro8ateJ peoJq oml Jo Jaqlo Jo auo olul IIBJrsou 'tZ raldeq3 ur eas 'peuel f.yreurproerge eJBsenssrl pru1uv lnq ilpqs elr,rsE 'senssp ]ueJeJJrpur sluelxe ]ueJeJJIpol lnq 'pesn eJp sa€alerls 'sprurue uI 'lleo qJBe punoJJns leql slle^ lpJnpelrqcJp qloq ilec eql ol qlSuarls Jrar{} amo sanssq 1ue1d:luegodurr-1e sr xrJleru JBInllecEJue aqt 'slueld u1 'raqlaSol slac SurroqqBrau Jo suolala>lsoptc eq] ap terft suolsatlpo IIaJ-IIar uo pue sllec eq] eprsur uo1a1a>1so{caqr 1o qr8ualls eqt uo spuadap d8ate.ttsJer{lo eql'eleroes slleJ eql 1uql surcqc aprruqccesf.lodpue suratord 'x4tola JDInIIaJoJ$a eqr go qr8uaJls eql uo spuadap I8a Jo {romtau xaldruor e -leJls euo 'aJnlsnJlsJelnilacrlpu B ssoJJPpeDrrususrl eq UPJsassarlsqsrqM ur sdea,roanl o1 Surpuodsarroc 'sar8alerls Surpgnq crseq oml ur serl JaMsuEaql 'eeJte Jo esroq e sE elqels sE ;alqrssod slq] sl ,vroH pue '3uor1s sp 'eAISSEru sB eJnlonJls e uroJ 01 suorTllruJraql ur eurqruoJ uec r(aql 1a.,(lauerqruaur euseld dsurg E ur pesoloue pue runrperu snoanbe uE qlrm pellrJ 'slcafqo e1lotu uego pue l(qsrnbs 'llerus eJEslleC 'lpeJ Sulsrrdrns lsoru E ]ues 'SuFaeur8ua -ardar susruu8ro ruplacppur a8rey leJntf,nJ]s;o saldruexa sy 'sesEasrp AleuBA snour Jo -Joue uB arTJepunsnluredde srqt ul slJeJeCI'saJnlJnJls JelnlleJrllnu;o scruruudp pue 'uorlcurg 'uorlezrue8ro aql;o lcadse ,{rana ro; IBJIIIJosl xrJlpru m1nlleceJxe pup 'sursrueqJaru uorseqpe IIac 'suorlcun[ IIaJ Jo snlerudde eql 'snql Jleslr srredar pue 'sdolanep 's,nirorBfpoq eqt se uaql Surprn8 'rusrue8lo eqr ulqllm eloru sllec fual aq] urarro8 xrJ]eru eq] Jo 3u11aporueql pue sluaruqJeilE eq] 'eJn]f,nJls ;o Surxearq pue Suqeu eqJ lpuJelur s,lleo qcee Jo uorlpluerJo eql IoJl -uoJ xrJleru JelnlleJeJue ol pue slleo Jeqlo ol slueruqJpltv 'uorssardxaauaS;o suraned rraql aleln8al pue Jor^eqeq Jreql eleurpJooJ leql spu8rs aq] a8ueqcxe 01 sllec aql Burvrolle 'uorlecrunruuroo ro; srtealqled alearc sllec ueemleq suorlcunf aql'sadr! IIeo luereJJrps1r;o luaura8uerft aqt pue 'q]Suarls s1r'adeqs s}1-ri.poq aql Jo eJnlJelrqJJB aq] urano8 uorsaqo3 Jo srusruuqJeu aqJ 'eJnlonJls Jelnllecnpru pazlue8ro uE uJoJ o1 are ,(aql g eraqoc 1snur,{aql 'Jaq}oueJo sueeur auo r{q lnq :alarcasrteql }uq} slerretE(uJElnlleoeJ}xa dq raqraSol punoq aq.(eru daqt ro 'suorlcunf fiac-llal lcarrp qSnorql Jaq]oue euo o] 3u11c,(eur q1a3 'raqleSol sller eqt ploq esoql aJE ]eq] lelueuepunJ 'rusruu8ro reFllef,rllnru e ur slleJ ueemleq suorlJpJelur prtos eql lsou eql IIp JO

xltlew lelnlle)elxll eqt pue 'uolsaqpv lle)'suollrunr Ile)

'uotllnpsuell (1;errdfi'sarnt)nrls suralordaprsbuole xa;druo:aresuorl)unf surolorda6eloq)ue6urnlonur leu6ts6utletpor.ll 6ur{e1er-;eu615 (C)'lla) o} lla) uor; ssedo1suorpue saln)alou.llleLusJoJsr(ervra6essed Lulo;(sura}ordwxauw )o uxauuo) suorllun[6ururo;-lauueq](l) .slla)1e;leqtgde suorl>unf uaaM]oqsdebleas(suralotdwpnDp6ul^lo^ur) 1o pasodr.uot) 6urpn;>tg(8)'(suralord wtbalut auerqurausuelletn^lle)tdIl) uUar4pnauerquraursuerlern xtJleulol;;a>to(suralord 'sanssrl I11e>tdI1) llo) ol lla) 1ur1suorlrunf6uuoqruy (V) ler!!up u! suorlrunf;;ar10ssssel)lpuoll)un,rno3 7-6 |, arn6tg

sNorDNnr 9NrA\nlu-'1vN9rs (a)

sNott)Nnr 9NI luol-'lSNNVH)

())

9Ntcn]f)o sNorDNnr

(8)

S N O I I J N N fD N I U O H ] N V

(v)

'uorsslusueJl IPuSrsur saloJ lueUodrur a^pq ueJ IIu 'sde,ru1uaraJ;IpuI 'suollJunf SuluJoJ-lauuPqc pue 'sJo]daJaJ 'JeAoeJoIN 'suorlrunI SurpnlcJo 'suor]cunI SulJoqcupluql ees IIEqsallr eql elE^llJe ot spuBSlleql JoJJaqloue euo qll^i\ lceluoJ ul plaq aq lsnlu seuEJq -luelu IIeo aqt :SupPeq sFIl rapun IIBJ'9I raldeql uI passnrslp se 'sroldeoerqdg pue surJqda Jo (qJloN puP ellec se qons sJled JoldaceJ-pue8I aueJqrueusueJl PrAuorleJrunruuroJ lles-lleJ Jo selrs 'seuo fluo eq] lou eJe Leql ]nq 'suoDcunf Surrtelar-pu8rsJo seldruexesnorlqo ]soru aql erc'(gZ Jeldeq3 uI pessnJslp)sllef, Suquasard-ue8rlue qU,t,rlJeJelur sal,boqdudl I eJeq \ 'sasdeu(s pctSoloumuurt pue (II raldeq3 ur passnJslp) urals[s snolrau aql uI sesdeu[s IPJItueqO ']Jeluos IIaJ-ol-lleJ Jo salls le seueJquau eluseld rlaql ssoJce 'V flac o] IIeJ luo4 pa^,(e1araq o1 spu8ts ^tolle suollrunl Sqfular-put;5 'slle3 lueJefpe 'S go surseldol,(f, aq] SuDIurI s.{emaBessedaleeJf, suollJun( tulruJoJ-Iauupq3 'JerJJBq(elqpaurJed,{lanpcalasro) elqeeluJadrurue olq leeqs IIar aql 'Z a{eru 01 se os eqaqtrde ur sllac ueeMteq sde8 aql pas suollJun[8upn1ccA 'llar aql aprsur sluauelrJ lelele{soilr ol pereq}al aJB pue sassalls l1rusuer1 'suowaLlpo 'I xlqaru-lpJ pue suorcaqpo ilac-Ilar qloq Sulpnlf,ul 'suollJunf tuFoqcuy

pue z-6I arntlg) srseqrerncero.,, e qryv\qr'a 'peqslndi:**Ht::* ruaraJJrp

suor]3unJ ureru Jnoc 'saJJoJlEcrs^qd ]IusuuJl 1sn[ ueq] eJoru op puB eJnlcnJ]s ur asJe^rpeJe xrJleru pue slleJ uaaMleq Jo IIac pu? IIaJ uea^\leq suollJunf ]nq 'senssr]p{eqtldauou uI pup eleqllda ut qloq'lell}uc sI luatuqf,E}le IUcIS^qd

| :::rr uabellor

onssrl aAtl)euuo)

uorSsolo uro) pue uorsuallo sessarls lelrueq)aursreeqi{;lrarrp

c

'll qleauaqanssl] xuleu relnlla)erlxe a^rl)auuo)aql ol anssrllerlaqltoa puoq slueuq)ellexuleu-lla) 'suor1>unf 6urroqrue {q 1;at o] lla) urorjpelurl'sa^lasuaqlslla) s e l r su o r s a q p e aql lo suolala)sot^>atll srlr 'anssrl l l e ) - l l a )p u e x u l e u j - l l a ) ol peroqrue slueurelr+ ul 'xuleu relnlla)eJlxa aql ;er;aqlrde {q lla) o} lla) sl tuauodruo)6urreaq-ssalls ureLu 1e1a1a1so1{> uror+pallrrusuerlere aql'anssrlanr])euuo)u1'raq1a6o1 sassarlS lP)!ueq)aur punoq sie sllo) le|'Urue q)!qM ur s(ervruleu or'nl 1-61 ern6r3

xfrlew,elnlla)ellxleqt pue'uolsaqpvlla) 'suoll)unrlla) :61raldeq3 zELl

'sllec q8norql eql ueemleq sdeB ulnrleqllda Ilao-llaoJo sedll oml eru eseql^ olag aql ssoJJe3q4ea1ruo4 selnJelourtupuanard '1sa1erqa1le^uISuolLJun[ 7q34se Ulrlrou>I) suollcunf tulpnlcco a1 xade aql ol lsasol3 rer{}oue euo qllm suopcunf pJawl Jo 'seplsJraql uo 'tunrpelu JelnlleJEJ$eeql ol e{Br.usllac eql 'seceJJns 'xadn pesodxapue qfyv\'pulurelleseqp uo pue]s eeq to'eceJJns lsourraddn 4eql 'araH 'etPrqeUa^B euFsetul aql IIBsllec1ergora,te1epuls e Jo IIEurserDJo 3u1u11 sBqcns runqeqlrdaJDuranlocagdrutse u1tueua8uerre pcrd,Q eql s,rnoqsurerS -e1peql'Jatel pessnosrp eq ol suollcunf;o sadfi raqlo ol uopeleJur polnqr4srp ere uoltres sl{l uI sn ureJuooIIIMlBql (suopcunftulroqcuu) suolseqpElleo-llec eql Aroq smoqs pue unqeqllda ernleru Jo uortJes e ur slEaAeJadocsorcrur uorlcela eqt leqr suollcunfgo sed! aql dlpcpeueqcs sa]Er]snllrg-51 arnftg 'alqrsurL1urc1d os sdertp lou eJe suonezrlerJedspuopcunf eq1areq,usenssprer{}oq osle lnq egaqtlda llnpu ur ,!uo 1ou1ue1rodu4'salnceloruJo srualsdsrJullsp ol etBIeJedocsorcruruonoele eql ul uees sernlonls tuaraJJlpeql leql rrmoqsaculs anuq,{to1o1qrc1nceloru pue ,{:lspueqcorg 'suopcunf 1ac Jo uorluogrsselcpraue8 lsJg eq] paprnord ddocsorcnuuoJlcele[q sansspeseqtJo dpnlg '1ac ol IIaJ;o ateroqcueperrp Suorls,{qraqratol plaq eJB}Bql 'elcsntuueeq sBqcns 'sansspJeqlo auos ul pue e11aq11da ernleru ur uees,'t1rea1c lsou eJEsuolsaqpBIIeJ-IIeJJo seJnlJnJlseqJ

NOTSTHCV llt)-]tl)

CNVSNtutHOV)

'uopcnJlsuoJJIerDur IIBrvr 1ec 1ue1daq1;o eloJleJlueceq] pue s1ue1dol uml IIBqservr'lse'I'suolseqpexuteu-l1ac qtnorql ll qtl \ lssJelq sllec eql qclqM ur s,te,neql puE slsrulrrEul xlJlsru JelnlleseJile eql ssnosrpueqt fleqseM'sleeqsplaqllda pazuelod otq sllecezrue8roraqleSol suorlcunf spuq eql Moq ees lleqs e1,4dlquassp pue srusruBqJeru lueJaJJrp Jo uolseqpu rvrergo eql ruo{ sesdeudspuoJnau aupuexe,(garJqllpqs lurod 4aqlJo a,vr'suopJunf Surr{e1ar-putrs ;o salduruxesy ,{llcar1pIIaJ ol IIoJ {u11leq] suop -cun[ SururroJ-leuueqcpue 'suorlcunf Surpnlcco'suorseqpeilec-ilac eql qr!v\ Suruu€aq 'sanssnpue slleoletulup uo snloJ p,r,rraldeqc s5{rJoped lsrg aql

suolt)unr lle) lo uo!le)!l!ssel)leuolpunl v 1-61 alqel

€€tI

lalel passn)s!p aq o] 'surolord loq)ue relnlla)ellut et^'l)oltpul 11;eraua6 sruola;a1so1{> aql o1a6e1ur; leureluraql'(surr6alul{q 11lerrdifi paterpau,r'a6etoqrue xuteul-lla))xuleul relnllo)erlxo ot ro (suuaqpetIq 11;e:td,{1 palerpaui'a6eroqrue lla)-lla))slla) raqloJo stredol leq]laaq {eru a6e1ur; leuralxaaqI'sarnl)nrls lelnlla)ellxo o1 uoga;a1so1I)aqr lull sulelord uolsaqpeauerqureusuell tr-6 1 ern611

slueurelrl_ Ielelalsol^)

raloeq) aql u! Jalelpassn)srp pezrlerred5 aresuorlrunf6ur(e1ar-;eubrs 'aurlsalu! llptusaqlJo sl1arlerlaqtlde uo paseqsr6urare.rp aql'paztue6ro {1re;n6ar ssolarpsaurosoursap leuortrppe pue suorlrunfde9 'xa1dr-uor ;euorltunf e palle)arn])nrtse uro; esaqllaqta6ot :saurosouJsap Jo mor 1a;;eled ;eoads e Iq uaql pue (llaq uorsaqpe) uorlrunf suaraqpeeqt (q pamo1lo4'uorlrsod le)rdelsotuaql sardn))ouolllunI lq6!t aql'ellaqltdaele.rqalran ;1e,(;teeu ur aues aql aresuorl)unfaql;o suorlrsod a^rlelaraqt /lla)aq]lo uoluod le)!de {reulrd rleql lsouraql u1'suor1>un; o16ugprorreporrrssel)'11ar 1e;laql1da alpiqolra^e u! punoJsuollrun[ 11ar snol.reA aql Jo fteuurns y 9-61 arn6r1

s u r a l o l dr o q r u e ielnlla)erlul

seuelquiaueulseld

'asrmeTrljsuorlcunf euosousep uJoJ pue sluauPIIJ elelpeuJa]ul ol {uI sJaqto allq.4i\'suorlsunf sueJeqpe IUJOJpue uuce ot luu suIJeL{pBJaluos :UOIIEZIIEIJ -eds sr aJaql ',{FtupJr{oEaulql.r A'xrJlEru ol slleo Jo }uauqoel}E elElpeu ,tgarqc ^,(1rue;radnsulr8elq eql Jo sqelord 'llao o] IIac Jo luaulqJe]1e elulpatu ,{ga1qc ,(luregradns ulraqpuJ eql Jo suleloJd '(Z-6I alqu.1)lualuqoepu leuralxe Jo spuDl crseq o^\l aql ol Surpuodsarroc (seIIIuPJJadns ol^1 olul .{ll?eu IIeJ salncalolu euerqruerusuPrl pe{u[-uolale{sotdc eseql'(?-6I eJntlt) }l eplslno seJnlJnJ}s reqlo ot Suopq pue raqlo aq] pue IIaJ aql epISuI uolale{soldJ aqt ol Suuun pua auo qll^\ 'auEJqureu aq] uPds leq] sulaloJd uolsaqpB errBJqruausrrBJl .{q padeld sr alor lertueJ aql 'suol]Junf 3uuoqcue Jo seddt JnoJaql Jo qcee ]V 'll 01 slueluelu elslpeluJelul JOqsuPSautosorusaprulaqauuL/fl'xlleru aql ol sluauEIIJ ullce JoqsuB (eJeqMesle 'auuselur luaurruord ]nq lletus aql ul ]sullsrpur) suorcaqpo xlJruu-Ilal paTuIl -ullJ0 isuorlsauuoJ lB]ela{soil3 Jleql 01 Sutprocce 'suolseqpPIIeJ-lleJ eq} aII 'suorseqpexrJleru-llar eseq],{JIssPIJeM'uollses Jelel e uI pessnlslp eq ilI \ pue eurluel IBseq aql 01 slleJ lelTeqllda aql qJe]le suolsaqpe Jo slas puoplppv 'suotpunf dDB pelle?, 'suorlcun[ Sururo;-lauueqJ aII 'suot]cunf euosousap Ieuonppe q]Ilrr pelSuFu ueuo Ja1!\olIIpS 'sluatuellJ elelpaluJelul JoJ sells e8EJol{f,ueeJEsuopcunl aruos -ousep lstueuelrJ ullJe roJ salts aBeroq3ueaJBsuollJunl sueJeqpv'suolseqpP

x r J l e [ !J e l n l l a ) e r ] x o a] l l e r e u r s ] u a u r e l r ] elerpat!JalursroLl)ueauiosoursapruaq

x r J l e wr e l n l l e ) e l l x oo l l ll)e sJoqlue lla) ur sluauielru u o r s a q p ex r J l e t u - l l a p) a l u l l - u l l l e IVSVS

lla) ol lla) u.roJl s a l n l a l o u .ai l q n l o s - r a l e M lleurs,o a 6 e s s e da q l s m o ; ; eu o r p u n I d e 6

sNolI)Nnr 9Ntv[uol-'l]NNVH)

lle)lxeu eql u r a s o q l0 1 l l a ) a u o u r s l u a u r e l r r t alerpauralur sDauuo) aulosoulsap

\

sNotr)Nnr gLttuoH)Nv I trJ-

lla) lxau oql u! leql q l l / ! ^l l a r a u o u r e l p u n ql u e u r e l r l u r l ) e s p e u u o ) u o g D u nsl u a : e q p e

s11a:1er1aq)1da u o a m l o qd e 6 s l e a su o r l r u n [] q 6 r l

| taJ

NOrr)Nnr 9Nrcn'l))o

aql pue'uolseqpvlle) 'suolpunrlla) :6t raldPq) xulew relnlle)PJlxf

VEIL

1135

CADHERINS AND CELL-CELL ADHESION Table19-2 AnchoringJunctions

junction adherens

c a dh e r i n (classica l dherin) ca

c a d h e r i ni n n e i g h b o r i n gc e l l

actin filaments

qesmoS0me

c a d h e r i n( d e s m o g l e i n , desmocollin)

d e s m o g l e i na n d in desmocollin n e i g h b o r i n gc e l l

intermediate filaments

actinlinkedcellmatrixadhesion

integrin

matrix extracellular proteins

actin filaments

t a l i n ,v i n c u l i na, - a c t i n i n , filamin,paxillin,focal adhesionkinase(FAK)

matrix extracellular proteins

intermediate filaments

plectin,dystonin(8P230)

hemidesmosome integrino6p4,type XVll collagen(8P180)

some integrins link to actin and form actin-linked cell-matrix adhesions,while others link to intermediate filaments and form hemidesmosomes. There are some exceptions to these rules. Some integrins, for example, mediate cell-cell rather than cell-matrix attachment. Moreover, there are other types of cell adhesion molecules that can provide attachments more flimsy than anchoring junctions, but sufficient to stick cells together in special circumstances.Cell-cell adhesions based on cadherins. however, seem to be the most fundamentally important class,and we begin our account of cell-cell adhesion with them.

Cadherins MediateCa2*-Dependent Cell-CellAdhesionin All An i m a l s Cadherins are present in all multicellular animals whose genomes have been analyzed, and in one other knor,rrngroup, the choanoflagellates.These creatures can exist either as free-living unicellular organisms or as multicellular colonies and are thought to be representativesof the group of protists from which all animals evolved. Other eucaryotes,including fungi and plants, Iack cadherins, and they are absent from bacteria and archaea also. Cadherins therefore seem to be part of the essenceof what it is to be an animal. The cadherins take their name from their dependence on Ca2*ions: removing Ca2* from the extracellular medium causes adhesions mediated by cadherins to come adrift. Sometimes, especially for embryonic tissues, this is enough to let the cells be easily separated. In other cases,a more severetreatment is required, combining Ca2*removal with exposure to a protease such as trypsin. The protease loosens additional connections mediated by extracellular matrix and by other cell-cell adhesion molecules that do not depend on Ca2*.In either case, when the dissociated cells are put back into a normal culture medium, they will generally stick together again by reconstructing their adhesions. This tlpe of cell-cell associationprovided one of the first assaysthat allowed cell-cell adhesion molecules to be identified. In these experiments, monoclonal antibodies were raised against the cells of interest, and each antibodywas tested for its ability to prevent the cells from sticking together again after they had been dissociated. Rare antibodies that bound to the cell-cell adhesion molecules showed this blocking effect. These antibodies then were used to isolate the adhesion molecule that they recognized. Virtually all cells in vertebrates, and probably in other animals too, seem to expressone or more proteins of the cadherin family, according to the cell type.

c[-catenin,B-catenin, plakoglobin (lcatenin;, p 12 0 - c a t e n i nv,i n c u l i n , c[-actinin plakoglobin (y-catenin), plakophilin, desmoplakin

1136

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix

'1.5 days

3 . 5d a y s

2 cells

32 cells

Severallines of evidence indicate that they are the main adhesion molecules holding cells together in early embryonic tissues. For example, embryonic tissues in culture disintegrate when treated with anti-cadherin antibodies, and if cadherinmediated adhesion is Ieft intact, antibodies against other adhesion molecules have little effect. Studies of the early mouse embryo illustrate the role of cadherins in development. Up to the eight-cell stage, the mouse embryo cells are only very loosely held togetheS remaining individually more or less spherical; then, rather suddenly, in a process called compaction, they become tightly packed together and joined by cell-cell junctions, so that the outer surface of the embryo becomes smoother (Figure l$-5). Antibodies against a specific cadherin, caTledE-cadherin, block compaction, whereas antibodies that react with various other cell-surface molecules on these cells do not. Mutations that inactivate E-cadherin cause the embryos to fall apart and die early in development.

1oPm

Figure19-5 Compactionof an early mouseembryo.Thecellsof the early embryoat firststicktogetheronly weakly.At about the eight-cellstage, they beginto expressE-cadherin and as a resultbecomestronglyand closely adherentto one another.(Scanning electronmicrographs courtesyof Patricia Calarco; 16-32-cellstageisfrom P.Calarcoand C.J.Epstein,Dev.Biol. 32:208-213,1973.With permissionfrom AcademicPress.)

TheCadherinSuperfamily in Vertebrates IncludesHundredsof DifferentProteins, IncludingManywith SignalingFunctions The first three cadherins that were discovered were named according to the main tissues in which they were found: E-cadherin is present on many types of epithelial cells; N-cadherin on nerve, muscle, and lens cells; and p-cadherin on cells in the placenta and epidermis. All are also found in various other tissues;Ncadherin, for example, is expressedin fibroblasts, and E-cadherin is expressedin parts ofthe brain (Figure 19-6). These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains. vtrhile all of them have well-defined adhesive functions, they are also important in signaling. Through their intracellular domains, as we shall see later, they relay information into the cell interior, enabling the cell to adapt its behavior according to whether it is attached or detached from other cells. There are also a large number of nonclassical cadherins more distantly related in sequence,with more than 50 expressedin the brain alone. The nonclassical cadherins include proteins with known adhesive function, such as the diverse protocadherlrzsfound in the brain, and the desmocollinsand, desmogleins that form desmosome junctions. They also include proteins that appear to be primarily involved in signaling, such as T-cadherin, which lacks a transmembrane domain and is attached to the plasma membrane of nerve and muscle E-cadheri n

R-cadherin

cadherin-6

Figure19-6 Cadherindiversityin the central nervoussystem,The diagram showsthe expressionpatternsofthree differentclassical cadherinsin the embryonicmousebrain.Morethan 70 othercadherins, both classical and nonclassical, arealsoexpressed in the brain,in complexpatternsthat are thoughtto reflecttheir rolesin guiding and maintainingthe organization of this intricateoroan.

satrs 01 purq suor +zBO'(96-6I ernBIC)e8uq e ,r(qururuop ulraqpss ueu eqt ol paurof 'lpn p€rr ssalJo eJoruE sruJoJuletuop uIJaqpEJqJeg '0t ueqt eJou eleq surJaqpef,IporssplJuouetuos lnq'g Jo t eJBeJeql suqloJoruseppue sutalSorusap ur pue 'sleadar eseql Jo g eJe eJaql saleJqauel Jo sulJeqpec IucIssPIJeql uI 'urDtuop uuaqpul eql pelleJ Jlloru e;o satdoc pralas Jo Supslsuoc uoprod repl -laJeJtxa ue eleq 'uop1ugep ,(q flruregradns eq] Jo sraqruelu eql 11V'selnJeloru uuaqppr Suqedrcured eqt Jo ernlcnrls eql uo spuadap pue paugap dlasrc -ard sr uorlcunf Surroqcue uu le seueJqruau IIeo eql ueem]aq Surceds aq1 '(V6-6I arn81g)Jeqlo aql Jo 1a1codeq] ur euo qJee Jo qoDI eql Jo uoluasul ,iq pulq seuuJqrueu lac alrsoddo ruo4 Sutpnrlord salnoeloru ulJeqpeJ eql pue 'lalcod rtqreau e pue qoDI IeuIIuJa] € sruJoJeJeq uleqJ utalord eqJ 'auuJq -tueu eql tuoq tsequrg aITteq] spue eql-selnf,alou ulreqpeJ aql;o sdp pu -ruJal-N aqt tE sJnJJoSurpurq aql 'laporu lueJJnJ e o1Sutproccy's1ac luacefpe uo addlqns palelar flasolc Jo arues eI{}Jo sapceloru ulJeqpeJ ol pulq IIaJ auo uo adfiqns cgrcadse Jo se1ncalou urreqpef, :(g-51 arnS1g'a4u-o1-a4l) cg;qdouoq ,{leraua8 sr surJaqpeJueemlaq Surpurq eql 'IJEJuI 'osle apls Jeqlo aql uo IIeJ eqt ul ul]oe ol aq IIIM lr 'uorlcunf aq]Jo apISauo uo IIao eq] ur 'aldruexaroJ 'uI]Je o1 sr a8e1u11eql JI :lecrJleururrts,{ypnsn eJE slleJ uee \laq suotpunf Sutroqcuy

uorseqpv)!l!qdouoHalerpewsulreqpe) atuaql Jo ,{ueru os eJeq} eJedq,,r,tpue 'suor}JunJ Jreql ol e}eleJ sula}ord asaql So seJnlonJls aql op ^loH 'suprunq ur sreqrxeur 0BI ueql eroru qlyv\ '(s-6I aIqBI puB 2-6I arn81g) flgrrugradns ulJaqpuJ eql e]n]psuoc sura]ord urJeqpeJ IpJISselJuou pue 'l.lantpadsar 'a1e1n8ar 'raqta3ol .{lgeyod IIeJ pue qlr'tor8 pllaqrrda IucrsselJ aql 'sutetord ler4laptldosot6' ur saua8go slcnpord aql se palJlluepl ]sJIJaJa^ qolqm oBulwa1g pue rul aql pue 'roqrue (IdC) Iolrsoullrtppuqdsoqdflsocd13 e t(q s11ac

uv'rn'r'r3)vulx3

u\fln1'll)vurNl uuaqpe)-l

6 qprd) suuaqpe)oloJo

(€zqpl) €z uuoqpe) (urllo)otusep) uuaqpe) leuJosoursap (lau) s u u a q p e )e s e u r lu r a l o . r d

'satltuJeJ utalololaLllo ur punoj osleale leql suteLuop pa^Jasuol /lpl ut sjtloul luasardarlau pup'o6urue11 parolo){;tuaragrpaql'uota;a1sov(raql ol uuaqpp)aql Jor.{lue }eql stuauodr-uot se llamsesaln)alout6ur;eu6rs 6urpnpur'spuebr; lplnlla)ellut;o,(larrerr aprme qllM suor])eJalut 6ultregat'pe!rea arouJeJPsuoruoolelnlla)ellutJtaqllnq '(slD^ouail6) ]ttoul utpulopuuaqpe) aql ;o sardoreldlrlnu 6urureluotsuorlrod Jelnlle)erlxa a^pqllesuralordasaql 'sraqureu{pr-ue;tadns uuaqpe>6uoue aq1 firsrenrpaql Jo atuossmoqsute.l6erp 'Ipue;redns ulraqpe)aql 4-61 arn6r3

LELL

(o6ururell) s u u a q p e )a u e J q u e u r s u e J l SSeO-UaAOS

(leJ) s u u a q p e la l t l - l e l ( uu a q p e ) - t ) u r J a q p Pl)e ) r s s e l )

-ilr)-]']lf NOTStHC]V

CNVSNTUtHCVf

'I;;etrlrqdotalaq purq /saln)olour letel passn)stp uotsaqpe lla) roqlo etuosl{11e:r;rqdoLuoq purq;erauab u r s u u a q p e )' 6 u r p u r q) t l t q d o t a l a q snslantrpqdouo;19-6 9 arn611

9 N r C N r s) | l r H d O U ] l _ l H

9 N t C N t S) n r H d O r O H

Jaqlo ^q pauJoJ suorsaqpexrJleu-llef, pup IIeJ-lleJ ]B salBrado osp ,,aydlJurrd orJIaA,,relrurs V 'uede peleed .{psealad pue orcla1 dq paurof ,,tpuor1seq uer JTJqEJ saoard o^ l sE lsnf '.{flenuanbassapcalotu aq] Suneredas .,{qpalquas Jo -sesrp,{1rsea aq UEJtl tJ^ pue 'puoq relnJeloruJalurlunpr^rpur,{uego }Eqt ueq} raleer8 reJ sr uorlrun[ srq];o q13uartsaqJ '(16-6I arn8rC)uol]Junf Sutroqcue uB tuJoJ ot aleloqPllor ,{ennsrqt ur aprs ,{q aprs paIJEd selnJelou ulleqpPJ duelX '1ac etues aqt uo salnJalou urJaqpeJreqlo ^ueru qtlm aprs-ol-aprspaJal -snlJ uauo aJEsalnf,alotuurJaqpeJ 'llal JeqtouPuo sJJuuEd peluarJo,r(lalrsoddo o1Surpurq uaq ylalleJed ur spuoq1Be^ qJns,{ueruJo uor}euJoJ aq} {uo4 llnsar q}r^ sJauued Jraq} 01 purq ,{1ecrdd1 sluaruqcellu 8uor1g .{lrur;;e ,r,ro1.{1a,Lr1e1al (suralord uorsaqpe llac-llar raqto tsoru pue) surreqpeJ '^lrurile qBIq qlF,r pue 'salncaloru -3q pu8rs cgrcads rreqt purq qrlqm alqnlos JoJ sJoldeJeJ ellun 'saufzua rr/.1o -a1ord,{qpaper8ap ,{.prderaJe+zEJJo ssol .{q r(errn slql q paz[rqe]sap salncalour urJeqpp3 '1ac alrsoddo aql uo alnraloru urJaqppo Surqcleur aql JoJ ,,(trurg;e Surpurq aq] Surue{Ee-tr.{1lq8r1s a8ueqc o1 lq8noql sr snururJe}N eql }e uolleur -JoJuooaql 'erult aruus aqt ly dddog saruocaqaJnlJnJtsaql puu '{xeguef, sa8urq aql 'pe^otuar sr +zE3 uarl\\ 'poJ 'pe^Jn3 ,(1tq3us'pr8rr e se sa^Pqaq surPruop urJaqpel Jo 3uuls a1oq.r,,r eql ]Bq] os '8uxag tuo4 ll ]uanard pue a8uq qf,ee JBeu

erlaqlloa sl)eJap aqnl lerneulrQue;od laqlo eLuospue {rosues suorlrunf;;ar-;;ar llel leueldpotdnrstp sauelquaurrrldeur(suou uotleloua6ep puesasdeu,(s suoJnau leuoJnau le)rujaq) suor]>unI e^epueutelqaJoj uels{s snonreu lle)raLllopuesnlnrar.uo;6 :ur6elqdetp snouen Iauprl ur r.u6elqdelp lllsJossol r;1s lerlue)pueerlorllloa JouotteuroJleu {1ue1od uals{s (ftue1od lla)leueldpa}dnrstp ;;arteue;d) l s r o u n lp u ps l s t pl e u ! 6 e upt e b l e l u e uorlrun[6urr{e;er-;eubrssnonreulerlue)pueer;aqlda slle)leLl{rosues ssauleep er;eqllda .leqlo'lealauul ur ellrfoarals uae^laq s)url uMoulun auou ueaLl'al)snu'suojnau uorsaqpe lla)-lla)ap{rourleta>| u!Is u!)s6uualsrlq saurosousap ,o ssolol anpaseastp u r l s ; o6 u u e 1 s r 1 q ulls saurosorrsap

o6urr-ue13 pe)olold suuaLl -Lpue'fl'n

(sleulLueLu u!) nel (Dlttldosot]ui rcJ €z uuaLlpe) uuaqpel-f ura;6ousag urllo)oursoc suuaqpDIDtssDl)uoN

(slla)lerlaqropua Jostsoldode) I eln)seAIeullouqe luauroola^ap pue;6{leu.rueuleulJouqe ]uauidolonap sl)aJeplJeequrorjerpso{lqua uollredtuoroblopuno] lteJ sofuqua:a6egs 1s(rolselqle qteep

uuaqpP)-ln

slla)lerlaqlopua suorl>un su f areqpe unrlaqllde s u o r t r u ns(u a r e q p e lsearq'srLurep!da'elua)Pld sesdeur(s le)ru;eq) s l s e l q o r qpuu e ' s u e l ' e l ) s n u r p u es u o r l r u nsfu e r e q p e lelelals'uPaq'suornou s u o r l r u ns(u a r e q p e

uuaqpe)-d uuaqpe)-N uuoqpe)-l

elleqllda,(ueu

sut)aqpDlDlssol)

{;rure;radn5ulraqpe) aq} }o srequta4 auos €-6 I alqel

xulew lelnllolerlxl eql pue 'uolsaqpylla) /suolpunrlla) :61raldeq3

8€ t t

11 3 9

CADHERINS AND CELL-CELL ADHESION 3 8 . 5n m

prasma memorane o f c e l l2

h i n g er e g i o n

< 0.05 mM Ca2+

c a d h e r i nr e p e a t

(A)

domain Figure19-9 Cadherinstructureand function.(A)Theextracellular how two such is shownhere,illustrating of a classical cadherin(C-cadherin) end-tomolecules on oppositecellsarethoughtto bind homophilically, end.The structurewas determinedby x-raydiffractionof the crystallized part of each C-cadherin extracellular domain.(B)Theextracellular polypeptideconsistsof a seriesof compactdomainscalledcadherin joined by flexiblehingeregions.Ca2+bindsin the neighborhood repeats, of eachhinge,preventingit from flexing.In the absenceof Ca2+,the moleculebecomesfloppyand adhesionfails.(C)At a typicaljunction, functioninglikeVelcroto manycadherinmolecules arearrayedin parallel, hold cellstogether.Cadherins on the samecellarethoughtto be coupled by side-to-side interactions betweentheir N-terminalheadregions,and via the attachments of their intracellular tailsto a mat of other proteins(not shown here).(Basedon T.J.Boggonet al.,Science296:1308-1313,2002. With permissionfrom AAAS.)

types of transmembrane adhesion proteins. The making and breaking of anchoring junctions plays a vital part in development and in the constant turnover of tissues in many parts of the mature body.

(c)

VertebrateCells SelectiveCell-CellAdhesionEnablesDissociated into OrganizedTissues to Reassemble Cadherins form specific homophilic attachments, and this explains why there are so many different family members. Cadherins are not like glue, making cell surfaces generally sticky. Rather, they mediate highly selective recognition, enabling cells of a similar ty?e to stick together and to stay segregatedfrom other types of cells. This selectivity in the way that animal cells consort with one another was demonstrated more than 50 years ago, Iong before the discovery of cadherins, in experiments in which amphibian embryos were dissociated into single cells. These cells were then mixed up and allowed to reassociate. Remarkably, the dissociated cells often reassembled in uitro into structures resembling those of the original embryo (Figure f9-10). The same phenomenon occurs when dissociated cells from two embryonic vertebrate organs, such as the liver and the retina, are mixed together and artificially formed into a pellet: the mixed aggregates

Figure19-10 Sortingout. Cellsfrom differentpartsof an earlyamphibian embryowill sortout accordingto their experimentshown origins.In the classical here,mesodermcells(green),neuralplate cells(b/ue),and epidermalcells(red)have and then beendisaggregated in a randommixture.They reaggregated reminiscent sortout into an arrangement of a normalembryo,with a "neuraltube" internally,epidermisexternally,and mesodermin between.(Modifiedfrom P.L.Townesand J. Holtfreter,J. Exp.Zool. 128'53-120,1955.With permissionfrom Wiley-Liss.)

1140

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix Figure19-11 Selectivedispersaland reassembly of cellsto form tissues in a vertebrateembryo.Somecellsthat areinitiallypart of the epithelial neuraltube altertheiradhesivepropertiesand disengage from the epitheliumto form the neuralcreston the uppersurfaceofthe neural tube.Thecellsthen migrateawayand form a varietyof celltypesand tissuesthroughoutthe embryo.Herethey areshownassembling and differentiating to form two clustersof nervecells,calledganglia,in the peripheralnervoussystem. Whilesomeof the neuralcrestcells differentiate in the ganglionto becomethe neurons, othersbecome

c e l l st h a t w i l l

::ffi:tti"l ;;,;[iL1d,#h',,:'#]ii#'.*']:*l'ii:fl

gradually sort out according to their organ of origin. More generally, disaggregated cells are found to adhere more readily to aggregatesof their own organ than to aggregatesofother organs. Evidently there are cell-cell recognition systems that make cells of the same differentiated tissue preferentiallv adhere to one another Such findings suggestthat tissue architecture in animals is not just a product of history but is actively organized and maintained by the system of affinities that cells have for one another and for the extracellular matrix. In the developing embryo, we can indeed watch the cells as they differentiate, and see how they move and regroup to form new structures, guided by selective adhesion. Some of these movements are subtle, others more far-reaching, involving longrange migrations, as we shall describe in chapter 22.rnvertebrate embryos, for example, cells from rhe neural crestbreakaway from the epithelial neural tube, of which they are initially a part, and migrate along specific paths to many other regions. There they reaggregatewith other cells and with one another to form a variety of tissues, including those of the peripheral nervous system (Figure r9-l t). To find their way, the cells depend on guidance from the embryonic tissues along the path. This may involve chemotaxis or chemorepulsion, that is, movement under the influence of soluble chemicals that attract or repel migrating cells. It may also involve contact guidance,inwhich the migrant cell touches other cells or extracellular matrix components, making transient adhesions that govern the track taken. Then, once the migrating cell has reached its destination, it must recognize and join other cells of the appropriate type to assembleinto a tissue. In all these processesof sorting out, contact guidance, and tissue assembly, cadherins play a crucial part.

I

I

r,o*o'o, oEe, ooq; 6

I

ooo^roo'on,

p e r i p h e r agl a n g l i a

Cadherins Controlthe 5elective Assortment of Cells The appearance and disappearanceof specific cadherins correlate with steps in embryonic development where cells regroup and change their contacts to create new tissue structures. As the neural tube forms and pinches off from the overlying ectoderm, for example, neural tube cells lose E-cadherin and acquire other cadherins, including N-cadherin, while the cells in the overlying ectoderm continue to expressE-cadherin (Figure ls-lzL, B). Then, when the neural crest cells migrate away from the neural tube, these cadherins become scarcely detectable, and another cadherin (cadherin-7) appears that helps hold the

studies with cultured cells support the suggestionthat the homophilic binding of cadherins controls these processesof tissue segregation.In a line of cultured fibroblasts called L cells,for example, cadherins are not expressedand the cells do not adhere to one another. \Mhen these cells are transfected with DNA encoding E-cadherin, however, they become adherent to one another, and the adhesion is inhibited by anti-E-cadherin antibodies. Since the transfected cells do not stick to untransfected L cells, we can conclude that the attachment

1141

CADHERINS AND CELL-CELL ADHESION

depends on E-cadherin on one cell binding to E-cadherin on another. If L cells expressing different cadherins are mixed together, they sort out and aggregate separately,indicating that different cadherins preferentially bind to their own type (Figure l9-l3A), mimicking what happens when cells derived from tissues that expressdifferent cadherins are mixed together.A similar segregationof cells occurs if L cells expressing different amounts of the same cadherin are mixed together (Figure l9-l3B). It therefore seems likely that both qualitative and quantitative differences in the expressionof cadherins have a role in organizing tissues.

TwistRegulatesEpithelial-Mesenchymal Transitions The assembly of cells into an epithelium is a reversible process.By switching on expression of adhesion molecules, dispersed unattached cells-often called mesenchymal cells-can come together to form an epithelium. Conversely, epithelial cells can change their character, disassemble, and migrate away from their parent epithelium as separate individuals. Such epithelial-mesenchymal transitionsplay an important part in normal embryonic development; the origin of the neural crest is one example (seeFigure 19-11).A control system involving a set of gene regulatory components called Slug, Snail, and TWist,with E-cadherin as a downstream component, seems to be critical for such transitions: in severaltissues,both in flies and vertebrates,switching on expressionof TWist,for example, converts epithelial cells to a mesenchymal character, and switching it off does the opposite. Epithelial-mesenchymal transitions also occur as pathological events during adult life, in cancer.Most cancers originate in epithelia, but become dangerously prone to spread-that is, malignant-only when the cancer cells escape from the epithelium of origin and invade other tissues.Experiments with malignant breast cancer cells in culture show that blocking expression of TWist can convert them back toward a nonmalignant character. Conversely, by forcing Twist expression, one can make normal epithelial cells undergo an epithelial-mesenchymal transition and behave like malignant cells. TWist exerts its effects, in part at least, by inhibiting expression of the cadherins that hold epithelial cells together. E-cadherin, in particular, is a target. Mutations that disrupt the production or function of E-cadherin are in fact often found in cancer cells and are thought to help make them malignant, as we shall discussin Chapter 2O.

Figure19-1 2 Changingpatternsof cadherinexpressionduring of constructionof the nervoussystem.The figure showscross-sections the earlychickembryo,asthe neuraltube detachesfrom the ectoderm and then as neuralcrestcellsdetachfrom the neuraltube. (A,B)lmmunofluorescence showingthe developingneural micrographs and (B)N-cadherin. tube labeledwith antibodiesagainst(A)E-cadherin (C)As the patternsof geneexpression change,the differentgroupsof cells they express. segregate from one anotheraccordingto the cadherins (Micrographs courtesyof KoheiHattaand MasatoshiTakeichi.)

r em : -

(c)

c e l l se x p r e s s i n E g -cadherin c e l l se x p r e s s i n cga d h e r i n6 8 c e l l se x p r e s s i n N g -cadherin c e l l se x p r e s s i ncga d h e r i n7

n e u r a lt u b e

1 0 0p m

'1142

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix

Catenins LinkClassical Cadherins to the ActinCytoskeleton The extracellular domains of cadherins mediate homophilic binding. The intracellular domains of typical cadherins, including all classicaland some nonclassical ones, provide anchorage for filaments of the cytoskeleton: anchorage to actin at adherens junctions, and to intermediate filaments at desmosome junctions, as mentioned earlier (seeFigure 19-3). The linkage to the c],toskeleton is indirect and depends on a cluster of accessoryintracellular anchor proteins that assembleon the tail of the cadherin. This linkage, connecting the cadherin family member to actin or intermediate filaments, includes several different components (Figure f9-f4). These components vary somewhat according to the type of anchorage-but in general a central part is played by B-cateninandlor its close relative y-catenin (plakoglobin). At adherens junctions, a remote relative of this pair of proteins, p120catenin, is also present and helps to regulate assembly of the whole complex. \Arhen pl2O-catenin is artificially depleted, cadherin proteins are rapidly degraded,and cell-cell adhesion is lost. An artificial increasein the level of p120catenin has an opposite effect. It is possible that cells use changes in the level of pl20-catenin or in its phosphorylation state as one way to regulate their strength of adhesion. In any case,it seems that the linkage to actin is essential for efficient cell-cell adhesion, as classicalcadherins that lack their cvtoolasmic domain cannot hold cells strongly together.

Adherens JunctionsCoordinate the Actin-Based Motilityof AdjacentCells Adherens junctions are an essential part of the machinery for modeling the shapes of multicellular structures in the animal body. By indirectly linking the actin filaments in one cell to those in its neighbors, they enable the cells in the tissue to use their actin cltoskeletons in a coordinated way. Adherens junctions occur in various forms. In many nonepithelial tissues, they appear as small punctate or streaklike attachments that indirectly connect the cortical actin filaments beneath the plasma membranes of two interacting cells. In heart muscle (discussedin chapter 23), they anchor the actin bundles of the contractile apparatus and act in parallel with desmosomejunctions to link the contractile cells end-to-end. (The cell-cell interfaces in the muscle where these adhesions occur are so substantial that they show up clearly in stained light-microscope sections as so-called intercalated discs.)But the prototypical examples of adherensjunctions occur in epithelia, where they often form a continuous adhesion belt (or zonula adherens)close beneath the apical face of the epithelium, encircling each of the interacting cells in the sheet (Figure r9-r5). within each cell, a contractile bundle of actin filaments lies adjacent to the adhesion belt, oriented parallel to the plasma membrane and tethered to it by the cadherins and their associatedintracellular anchor proteins. The actin bundles are thus linked, via the cadherins and anchor proteins, into an extensive transcellular network. This network can contract with the help of myosin motor proteins (discussedin chapter l6), providing the motile force for a fundamental process in animal morphogenesis-the folding of epithelial cell sheets into tubes, vesicles,and other related structures (Figure fg-f6).

Figure19-14 The linkageof classical cadherinsto actinfilaments.The cadherins arecoupledindirectlyto actinfilamentsvia B-catenin and other anchorproteins.s-Catenin, vinculin,and plakoglobin(a relativeof alsocalledlcatenin) areprobablyalsopresentin the linkageor B-catenin, involvedin controlof its assembly, but the detailsof the anchoragearenot well understood. Anotherintracellular protein,calledp120-catenin, also bindsto the cadherincytoplasmic tail and regulates cadherinfunction. hasa second,and very important,functionin intracellular B-Catenin signaling, aswe discussin Chapter15 (seeFigure15-77).

c e lI e x p r e s s i n g

c e l le x p r e s s i n g h i g h l e v e lo f E - c a d h e r i n

l e v e lo f E - c a d h e r i n (B) F i g u r e 19 - 1 3 C a d h e r i n - d e p e n d e n t c e l l s o r t i n g . C e l l si n c u l t u r e c a n s o r t t h e m s e l v e so u t a c c o r d i n g t o t h e t y p e a n d l e v e l o f c a d h e r i n st h e y e x p r e s s T . his can b e v i s u a l i z e db y l a b e l i n g d i f f e r e n t populations of cells with dyes of different c o l o r s .( A ) C e l l se x p r e s s i n gN - c a d h e r i n s o r t o u t f r o m c e l l s e x p r e s s i n gE - c a d h e r i n . ( B ) C e l l se x p r e s s i n gh i g h l e v e l so f E - c a d h e r i ns o r t o u t f r o m c e l l s e x p r e s s i n g l o w l e v e l so f E - c a d h e r i n .

other proterns

'ssaroroaq] ul alorluelroour ue a^eqol tq6noql osleorelaoqslellaqlldo aql ulql!/vlslla)aqt Jo sluaula6uelleel 'alequMoqslou q6noqllv '(77raldeql pue Z !-6 [ atnbt3aes) ut aqnl luaudo;anepalerqalra^,{pea lPrnouaqlJo uorleuJo,aql sl aloulexa uV'aqnl e olur dn llol ol laaqslellaqtlda aql sd;eqpue xaderaql lP MorPu ol aLl]sosne)sllaquorsaqpe slla)lerlaqlroa 6uo;e6uruunrsluauiel4ul])eJo solpunq aql Jo uoll)elluo) palualloaql 'aqnl up ulo, ol loaqs 1e11aqr;da 1e11eqr;da ue 1o 6u;p;o1aq1 91-61 atn6t3

rlrHs'l'llf J O S N O l g 3 UO i l J l ' l l s N l s 1 ' l l 8 N o l s l H c v NV A8 r o 9 N l N 3 l - H 9 l lC - SZINV9UO lo NolrvNl9v Nl ctsnv) tllHs'lvlllHlldl

I I |

s 1 1 al > e r l e q l r d a+ ol a a q s

'llec aql apISuI '(lzI-6I erB leql sluaruElu elPlpelurelul a{lledor Jo sepunq eq} arn8rg) rer{te3ot sllac aqt Suqanp 'uolsaqpe lelnllarra}ul;o slods alquo}}nq se 'lI reedde .{.1pc1dfisaruosoluseq luroJ }eql sulaloJd aql Jo eruos s^ oqs SZI-6I arn8rg pue 'aurosoursap E Jo eJnlJnJls IeJaueBaql sMoqs VZI-6I eJnSIC 'sezeJ8 IPuIue eql 'sx\o3 lnous eql snuraptda aql sBSuualleq luelsuoJ puelsqll.ry\o] seq qclq ^ Jo Jo sr serpnls lef,rruaqJolq JoJacJnos alIJoAuJe :uDISeql Jo Ja^BIJalno aql sIuJoJleql eJe pue 'eIaq}lda a}BJqel unrlaqlrda aq] 'slruJeplda aqt ul 1ryr1ua1d.{1auar1xa 'alrydosoJ1 'eldulexe JoJ'punoJ lou eJe -JeA uI ^eqJ eI€ uI eJnlelu lsoul ]uesald 'qfuarls lef,IueqJau lnq salgJqepa1 ul ]uegodrur are suollsunf e1uoso11rseq 'ullJe apnord ol sr uoncunJ urEur Jraql Jo ppelsur slueuellJ alulpaluJelul o1IuI ]nq suorlcunf suaraqpe ol Jelrurs ,(1prn1cnr1seJEsuol]cunf aurosoursaq

ql6uaJlSIellueq)aWe!laqtldleAIgsuo!l)unfauosousec a ) e } l n sl e s e q

N O I S l H C VI I ] ) _ 1 I ] )

€tt t

s n l l r ^ o i ) r ua p r s u l sluaurellu 1 rLl ) e

'laqte6olparlale s1;attuatefpe ul salpunqluauPlUullle eql',{ean slql ul lla) lualelpeaql uo suuaqpe)aq] pulq surPtuop Jo asoqlot,{1;err;lqdouoq raql pue'eueiquaul relnlla)erlxa euse;daql uedssuttaqpeteql 'surJaqpe) roq)uerelnlla)ellul ol suraloJo ^q paraqlalaresalpunqtuauielUutl)e aql'auelqualu euseld;euorlrunfaq1 :ruseldoy{reql 6uo1e6utuun.t Jo a)e1rns sluaueluu!l)e,o alpunqeF)erluo) e sl sll 's1;ar6ul1>etalul aJnlealsnor^qolsor.r.r aql Jo q)ea 6utplt>ua'laq uosaqPD uero uroJ oql salel uolt>un[suelaqpe oql'(eale a)eJrnsa^rlorosqeaql asPaJ)ul t1;tnol:ttu {ueu ol alras leql suorsnJtoJd) aql 6upe; aneq,(aql'1nbaqr;o uar-un| 'xedelraLlllP islueulnu uolldlosqe Jo areslle)asaqf'aullsalu! loJ pazrler)ads lleus aql ut slla) le!leqllde uaer'n1aq suogl>unf suareqpV9 !-6 [ etn6tl

s l l a )l P r l a q l r o a r r r:rafno

]toseuejquroLr.r euselole./alel

slueueIl urpe Jo alpunq u o r D u n fl q 6 ! ]

a)elJns le)rde ulol, 6urpualxe rllrnor)rtrt

CNVSNIU]HCVf

sLlrosor.!saptuiaq

eurrrreteseqaqlolpue'saurosousapqu.rurtr""lr"jlyi:H"tliiJ?Jl

,a;duexaslql ul-slla) {;lrerrpurare-out}se}urlleus aql Jo s;;at;er;aq}rda luerefpe;os)loMlauluouelUaletpoullalur ullelol aql .IroMlau luauelt, alp!paurolu!aql pue ,sauosoulsap!uaq,sauosoutsagg 1-61 arn613

'unrleqlrda pauesool aq] olul spmu,{poqJo aBeIBel q]!v\ 'uqs eql Jo Suuelsrlq alelas p ur sllnser srqJ .raqlaSot (sal-,bourte -re{) slleJ purraprda reql ploq }eql souosousap eql ldnrsp pue ol purq sarpoquup asaqJ 'surelord uuaqpeJ Ieruosousap u^ao Jrag] Jo auo ]sure8e sar -poqpue a{eru slenprlrpur petJeJJVsn8tqduad eseesrpuqs p1e; dlprlualod aql Jo sruJoJaruos ,(q paleJlsuouap sr suorltunf aurosorusap;o acueuodrur aql 'sllec alJsnu ur sluauto1{ utusap ueeq pue 'aldruexe JoJ 'sllal lerTaqlrda ]soru ur sluaunp[ u!ruJaY are [aq] :adr! 1ac eql uo spuadap sauosorusep aq] ol peqJplle sluatuelrJalerperuJalur;o addl rel -nc4red aqJ '(gI-6I arnS;g) enssu eqt lnoq8norql spuelxa leql Uomleu e Bune -arr 'slleo luacefpe ur salpunq Jelrurs o1 a8e4url qllm ,((IZI-61 arn8rg) qt8ueJ]s elrsual lear8 ;o {Jo^ eruer; prnpnJls B ruJoJ serxosousap aq} ol paJoqcue aulosotuSap

s l u a u e l t +u t l e J a l

/aH'M ulolJ ('svvv tuorj uolssrrlradqtlM E00z'€ t L-60t:20€afualrs iselols "l'o pue ur/1/\o] iJ 'C pue 'l! ol paq)elle sluaurelU oletpetrlJalur qllm'ou.rosoulsep )) a16urs e 6urmoqs'uolte>U!u6eu ieqolq le ensslleuresaqlJo Ued (C)'asnou Iqeq PJourls aql u! slla);er.uraprda uaaMlaqsuorltunfeuosousap uolt)alauV (l) 'stueurelU esaqlot aurosousapaqt 6ur,f ,{qaraq}'sluauelU ;o qdel6o.rrrr.u alerpau-11e}ur 'ut4oldowsap jo sapfsaql ol spurqurle;doLusa6 ol pu!q urnl ut q)tqM,(urueler-oztd Jo a^tlelarluplstpe) urltqdo4old pue (uruete>-1,) urqo16o4o1d putq slrelrruseldollt laql .suralo.rd uotsaqpeJo {;rr.ue;ulraqper aqlJo siaqLuauale ulllo)oulsappue utal6otusac'auosoulsap e;o sluauodruo)lpln)alou.t aLllJoauos (g) 'uslueq)aul e Iq taqtabolsauerquraur tuapuedap-+zP1 luarefpeaql ploq ol suteuoplelnlla)ettxa tteqt qbnorqlt)eJalulpue sanbeldaql ot purqIlrLue;urraqperaq] suralolduorsaqpe auplquJetusuetl Jo 'enbe;d q:ee;o a)ellns aql ol paq)euesrsluatrJelU elprpaurJalur ulleJalJo alpunqV .suralord lol'l)ue lelnllo)ellul elnlxtul e pasoduro) anbe;d asuap e st auelqurau eLuse;d Jo 6urltetalut rl)ea Jo .saulosousa6 e Jo s}uauodLuo> JOa)eJlns>ruse;do1{raql uo 'aLuosolusap lelnl)nl}s aqf (v) 2 1-61 a.rn613 ulu 00 I

(a)

ulll S 0

aueJqtrlau eulseld -\

())

s l u a u t eul alerpeLlllaluI

s u r e l o l ot o q ) u e , L oe n b e ; da s u a p

u rI q d o ) e l d

x f r l e w r e l n l l e ) e l t x f a q l p u e , u o t s e q p vl l e ) , s u o t l ) u n f l l o ) : 61 r a l d e q l

bVLL

CADHERINS ANDCELL-CELL ADHESION

Cell-Cell JunctionsSendSignalsinto the CellInterior The making and breaking of attachments are important events in the lives of cells and provoke large changes in their internal affairs. Conversely, changes in the internal state of a cell must be able to trigger the making or breaking of attachments. Thus there is a complex cross-talk between the adhesion machinery and chemical signaling pathways. We have described, for example, how changes in pl2O-catenin may regulate the formation of adherensjunctions, and several intracellular signaling pathways can control junction formation by phosphorylating this and other junctional proteins. Later, we shall discuss how the making and breaking of adhesions can send signals into the cell interior through mechanisms involving scaffold proteins on the intracellular side of the junction. Another of the central players in the two-way interaction between adhesion and signaling is thought to be B-catenin. In this chapter, we have mentioned it as an essential intracellular anchor protein at adherens junctions, Iinking cadherins to actin filaments. In Chapter 15,we encountered it in another guise, as a component of the Wnt cell-cell signaling pathway, moving from the cytoplasm to the nucleus to activate the transcription oftarget genes.Separateparts ofthe molecule are responsible for the adhesiveand gene-regulatory functions, but an individual molecule cannot do both things at once. Disintegration of an adherensjunction can set P-catenin molecules free to move from the cell surface into the nucleus as signaling molecules, and, conversely,the activities of components of theWnt signaling pathway (which regulate the phosphorylation and degradation of B-catenin) may control the availability of B-catenin to form adherens junctions. Some nonclassical cadherins transmit signals into the cell interior in yet other ways. Members of the Flamingo subfamily, for example, have a seven-pass transmembrane domain suggestingthat they might function as G-protein-coupled receptors. Vascular endothelial cadherin (VE-cadherin) provides another example.This protein not only mediates adhesion between endothelial cells but also is required for endothelial cell survival. Although endothelial cells that do not expressVE-cadherin still adhere to one another via N-cadherin, they fail to survive, because they are unable to respond to an extracellular protein called uascular endothelial gowth factor (WGF) that acts as a survival signal. VEGF binds to a receptor tyrosine kinase (discussedin Chapter 15) that requires VEcadherin as a co-receptor.

in the Cell-CellAdhesions MediateTransient Selectins Bloodstream The cadherin superfamily is central to cell-cell adhesion in animals, but at least three other superfamilies of cell-cell adhesion proteins are also important: the integrins,the selectins,and the adhesive immunoglobulin(l)-superfamilymembers. We shall discuss integrins in more detail later: their main function is in cell-matrix adhesion, but a few of them mediate cell-cell adhesion in specialized circumstances.Ca2*dependence provides one simple way to distinguish among these classesof proteins experimentally. Selectins,like cadherins and integrins, require Caz* for their adhesive function; Ig-superfamily members do not. Selectins are cell-surface carbohydrate-binding proteins AeUins)thatmediate a variety of transient, cell-cell adhesion interactions in the bloodstream. Their main role, in vertebrates at least, is in inflammatory responsesand in governing the traffic of white blood cells.'Whiteblood cells lead a nomadic life, roving between the bloodstream and the tissues,and this necessitatesspecial adhesive behavior. The selectins control the binding of white blood cells to the endothelial cells lining blood vessels,thereby enabling the blood cells to migrate out of the bloodstream into a tissue. Each selectin is a transmembrane protein with a conserved lectin domain that binds to a specific oligosaccharideon another cell (Figure l9-l9A). There are at least three types: L-selectinon white blood cells, P-selectinon blood platelets and on endothelial cells that have been locally activated by an inflammatory

1145

1146

chapter19:cell Junctions, cell Adhesion,and the Extracellurar Matrix -=-/

lectin domain E G F - l i kd eo m a i n

Dasat ..--l am i n a

P-selecti n

W E A KA D H E S I O N AND ROLLING (selectin-dependent)

STRONG ADHESION AND EMIGRATION (integrin-dependent)

blood vessel

white bloodcell

CYTOSOL

V

endothelial cell

(B)

ussue

aclrn f ilament

response, and E-selectln on activated endothelial cells. In a lymphoid organ, such as a lymph node or a tonsil, the endothelial cells expressoligosaccharides that are recognized by L-selectin on lymphocytes, causing the lymphocytes to loiter and become trapped. At sites of inflammation, the roles are reversed:the endothelial cells switch on expression of selectins that recognize the oligosaccharides on white blood cells and platelets, flagging the cells dornrrto help deal with the local emergency.Selectinsdo not act alone, however; they collaborate with integrins, which strengthen the binding of the blood cells to the endothelium. The cell-cell adhesions mediated by both selectins and integrin s are heterophilic-that is, the binding is to a molecule of a different type: selectinsbind to specific oligosaccharides on glycoproteins and glycolipids, while integrins bind to other specific proteins. Selectins and integrins act in sequence to let white blood cells leave the bloodstream and enter tissues (Figure l9-lgB). The selectins mediate a weak adhesion because the binding of the lectin domain of the selectin to its carbohydrate ligand is of low affinity. This allows the white blood cell to adhere weakly and reversibly to the endothelium, rolling along the surface of the blood ,r"rsei, propelled by the flow of blood. The rolling continues until the blood cell activates its integrins. As we discuss later, these transmembrane molecules can be switched into an adhesive conformation that enables them to latch onto other molecules external to the cell-in the present case,proteins on the surfaces of the endothelial cells. once it has attached in this way, the white blood cell escapesfrom the blood stream into the tissue by crawling out of the blood vessel between adjacent endothelial cells.

Membersof the lmmunoglobulin Superfamily of proteins MediateCa2+-lndependent Cell-CellAdhesion The chief endothelial cell proteins that are recognized by the white blood cell integrins are called ICAMs (intercellular cell adhesion moleculeg or vcAMs fuas-

Figure I 9-1 9 The structureand function of selectins.(A)The structureof P-selectin. The selectinattachesto the actincytoskeleton throughanchorproteinsthat arestillpoorly (B)How selectins characterized. and integrinsmediatethe cell-celladhesions requiredfor a white bloodcellto migrate out of the bloodstreaminto a tissue.First, selectins on endothelialcellsbind to oligosaccharides on the white bloodcell,so that it becomeslooselyattachedto the vesselwall.Thenthe white bloodcell activates an integrin(usuallyone called LFAl)in its plasmamembrane,enablingthis integrinto bind to a proteincalledlCAM1, belongingto the immunoglobulin superfamily, in the membraneof the endothelialcell.Thiscreatesa stronger attachmentthat allowsthe white bloodcell to crawl out of the vessel.

1147

CADHERINS AND CELL_CELL ADHESION Figure19-20Two membersof the lg superfamilyof cell-celladhesion molecules.NCAMis expressed on neuronsand manyothercelltypes,and

:;:#ffon[5 ffixlxT:[:il:i:lff illffi"".1']l!:iii.',",:::,Tffi adhesion. ICAMisexpressed covalently attached to it,hindering to an endothelial cellsandsomeothercelltypesandbindsheterophilically integrin on whitebloodcells. lg-like domains

acid units). By virtue of their negative charge,the long polysialic acid chains can interfere with cell adhesion (because like charges repel one another); NCAM heavily loaded with sialic acid may even serve to inhibit adhesion, rather than cause it. A cell of a given type generallyuses an assortment of different adhesion proteins to interact with other cells,just as each cell uses an assortment of different receptors to respond to the many soluble extracellular signal molecules, such as hormones and growth factors, in its environment. Although cadherins and Ig family members are frequently expressedon the same cells,the adhesionsmediated by cadherins are much stronger,and they are largely responsiblefor holding cells together, segregatingcell collectives into discrete tissues,and maintaining tissue integrity. Molecules such as NCAM seem to contribute more to the finetuning of these adhesive interactions during development and regeneration, playing a part in various specializedadhesivephenomena, such as that discussed for blood and endothelial cells.Thus, while mutant mice that lack N-cadherin die early in development, those that lack NCAM develop relatively normally but show some mild abnormalities in the development of certain specific tissues, including parts of the nervous system.

to Createa Act in Parallel ManyTypesof CellAdhesionMolecules Synapse Cells of the nervous system, especially, rely on complex systems of adhesion molecules, as well as chemotaxis and soluble signal factors, to guide axon outgrowth along precise pathways and to direct the formation of specific nerve connections (discussed in Chapter 22). Adhesion proteins of the Ig superfamily, along with many other classesof adhesion and signaling molecules, have important roles in these processes.Thus, for example, in flies with a mutation of Fasciclin2, related to NCAM, some €xons follow aberrant pathways and fail to reach their proper targets. Another member of the Ig superfamily, Fasciclin3, enables the neuronal growth cones to recognize their proper targets when they meet them. This protein is expressedtransiently on some motor neurons in Drosophila, as well as on the muscle cells they normally innervate. If Fasciclin3 is genetically removed from these motor neurons, they fail to recognize their muscle targets and do not make slmapseswith them. Conversely,if motor neurons that normally do not expressFasciclin3 are made to expressthis protein, they will synapsewith Fasciclin3-expressing muscle cells to which they normally do not connect' It seems that Fasciclin3 mediates these synaptic connections by a homophilic "matchmaking" mechanism. Ig superfamily proteins have similar roles in vertebrates. Proteins of the Sidekickssubfamily, for example, mediate homophilic adhesion, and different Sidekicks proteins are expressedin different layers of the retina, with synapsesforming between sets of retinal neurons that share expression of the same family member. \Alhenthe pattern of expression of the proteins is artificially altered, the pattern of synaptic connections changes accordingly. These Ig superfamily members are by no means the only adhesion molecules involved in initiating synapse formation. Misexpression of certain other synaptic adhesion proteins, unrelated to any of the types we have mentioned so far, can even trick growth cones into synapsing on non-neuronal cells that would never normally be innervated. Thus, if non-neuronal cells are forced to express neuroligin, a transmembrane protein evolutionarily related to the

fibronectin type lll domains

NCAM

1148

Chapter19:cell Junctions, cell Adhesion,and the Extracellular Matrix

enz]frJ].e acetylcholinesterase,neurons will synapse on them, as a consequence of binding of neuroligin to a protein called neurexin in the membrane of the presynaptic neuron.

ScaffoldProteinsOrganizeJunctional Complexes To make a synapse,the pre- and postsynaptic cells have to do more than recognize one another and adhere: they have to assemble a complex system of signal receptors, ion channels, slmaptic vesicles,docking proteins, and other components, as described in chapter 11. This apparatus for synaptic signaling could not exist without cell adhesion molecules to join the pre- and postsynaptic membranes firmly together and to help hold all the components of the signaling machinery in their proper positions. Thus, cadherins are generally present, concentrated at spots around the periphery of the synapse and within it, as well as Ig superfamily members and various other types of adhesion molecules. In fact, about 20 different classical cadherins are expressedin the vertebrate nervous system, in different combinations in different subsetsof neurons, and it is likely that selectivebinding of these molecules also plays a part in ensuring that neurons s).napsewith their correct partners. But how does the array of adhesion molecules recruit the other components of the synapse and hold them in place?scaffold proteins are thought to have a central role here. These intracellular molecules consist of strings of proteinbinding domains, typically including severalpDZ domains-segments about 70 amino acids long that can recognize and bind the C-terminal intracellular tails of specific transmembrane molecules (Figure rg-21). one domain of a scaffold protein may attach to a cell-cell adhesion protein, for example, while another latches onto a ligand-gated ion channel, and yet another binds a protein that regulates exocytosisor endocy'tosisor provides attachment to the cytoskeleton. Moreover, one molecule of scaffold protein can bind to another. In this way, the cell can assemble a mat of proteins, with all the components that are needed at the synapse woven into its fabric (Figure ls-22). Several hundred different types of proteins participate in this complex structure. Mutations in synaptic scaffbld proteins alter the size and structure of synapses and can have severe consequences for the function of the nervous system.Among other things, such mutations can damage the molecular machinery underlying learning and memory, which depend on the ability of electrical activity to leave a long-lasting trace in the form of alterations of synaptic architecture. The scaffold proteins, with their many potential binding partners, are involved in organizing other structures and functions beside synapses and synaptic signaling.The Dlscs large (Dlg) protein of Drosophilais an example (see Figure 19-2r). Dlg is needed for the construction of normal synapses;but we shall see that it, along with a set of other related scaffold proteins, ilso plays an

neuroligin g l u t a m a t er e c e p t o r s ( N M D Ar e c e p t o r s ) posrsyna

membrane

domains o t h e r s c a f f o l da n d scaffold-associated proteins

Dlg4 (PSD95)scaffoldproteins

Figurel9-21 A scaffoldprotein.The diagramshowsthe domainstructureof Dlg4,a mammalianhomologof the Drosophilaprotein Discs-large, along with someof its bindingpartners.Dlg4is concentrated beneaththe postsynaptic membraneat synapses, and is also knownas postsynaptic densityprotein 95,or PSD95. With its multipleproteinbindingdomains,it can linktogether differentcomponentsof the synapse. One moleculeof Dlg4can alsobind to anotheror to scaffolding molecules of othertypes,therebycreatingan extensive frameworkthat holdstogether all the componentsof the synapse. Scaffoldproteinsalsohaveimportant rolesat othertypesof celljunctions.

1149

CADHERINS AND CELL-CELL ADHESION

,Ut

v e s i c l e - d o cnkgi protern synaptrc vesrcles t.\

/ voltage-gated C a 2 *c h a n n e l

presynaptrc membrane poslsynapilc membrane \

ion channel

(c) essential part in almost every aspect of the organization of epithelia, including the formation of occluding junctions between the cells, the control of cell polarity, and even the control of cell proliferation. All these processeshave a shared dependence on the same machinery, not only in flies, but also in vertebrates.

Summary In epithelia, as well as in some other typesof tissue,cells are directly attached to one another through strong cell-cell adhesions,mediated by transmembraneproteins that are anchored intracellularly to the cytoskeleton.At adherensjunctions, the anchorage is to actin filaments; at desmosomejunctions, it is to intermediatefllaments. In both thesestructures,and in many lessconspicuouscell-cell junctions, the adhesiuetransmembrane proteins are members of the cadherin superfamily. Cadherins generally bind to one another homophilically: the head of one cadherin molecule binds to the head of a similar cadherin on an oppositecell. This selectiui4tenablesmixed populations of cells of dffirent types to sort out from one another according to the specific cadherins they express,and it helps to control cell rearrangementsduring deuelopment, where many dffirent cadherins are expressedin complex, changing patterns. Changesin cadherin expressioncan causecellsto undergo transitions between& cohesiueepithelialstateand a detachedmesenchymalstate-a phenomenonimportant in canceras well as in embryonic deuelopment. The "classical"cadherins are linked to the actin cytoskeletonby intracellular proteins called catenins Theseform an enchoring complex on the intracellular tail of the

Figure 19-22 Organizationof a synapse. (A)Electronmicrographand (B)line drawingof a crosssectionof two nerve on a dendritein the terminalssynapsing mammalianbrain.Notethe synaptic in the two nerveterminalsand vesicles materialassociated the dark-staining with the pre-and postsynaptic (C)Schematic diagram membranes. showingsomeof the synaptic at these comoonentsthat areassembled sites.Cell-celladhesionmolecules, c a d h e r i nasn d n e u r o l i g i nasn d including hold the pre-and postsynaptic neurexins, together.Scaffolding membranes proteinshelpto form a mat (corresponding to the dark-staining materialseenin (A))that linksthe adhesionmoleculesby their intracellular tailsto the componentsof the synaptic suchas machinery, signal-transmission ion channelsand neurotransmitter receptors.The structureof this large, complexmultiproteinassemblyis not yet knownin detail.lt includesanchorage sitesfor hundredsof additional not shownhere,including components, and various molecules cytoskeletal regulatorykinasesand phosphatases. (A,courtesyof CedricRaine.)

1 150

Chapter19:CellJunctions,Cell Adhesion,and the ExtracellularMatrix

cadherin molecule, and are inuolued not only in physical anchorage but also in the genesisof intracellular signals. Conuersely,intracellular signals can regulate the formation of cadherin-mediatedadhesions.B-Catenin,for example,is alsoa keycomponent of the Wnt cell signaling pathway. In addition to cadherins,at least three other classesof transmembranemolecules are also important mediators of cell-cell adhesion: selectins,immunoglobutin (Ig superfamily members, and integrins. selectins are expressedon white blood cells, blood platelets,and endothelial cells,and they bind heterophilically to carbohydrate groups on cell surfaces.They help to trap circulating white blood cells at sitesof inflammation. Ig-superfamily Ttroteinsalso play a part in this trapping, as well as in many other adhesiueprocesses; some of them bind homophilically, some heterophilically.Integrins, though they mainly serueto attach cellsto the extracellular matrix, can also mediate cell-cell adhesion by binding to the lg-superfumily members. Many different lg-superfamily members,cadherins,and other cell-cell adhesion moleculesguide the formation of nerueconnectionsand hotd neuronal membranes together at synapses.In these complicated structures, as well as at other types of cell-cell junctions, intracellulqr scaffold proteins containing multiple pDZ proteinbinding domains hauean important role in holding the many dffirent adhesiueand signaling moleculesin their proper arrangements.

TIGHT JUNCTIONS ANDTHEORGANIZATION OF EPITH ELIA An epithelial sheet,with its cellsjoined side by side and standing on a basal lamina, may seem a specializedtype of structure, but it is central to the construction of multicellular animals. In fact, more than 60% of the cell types in the vertebrate body are epithelial. Just as cell membranes enclose and partition the interior of the eucaryotic cell, so epithelia enclose and partition the animal body, lining all its surfacesand cavities, and creating internal compartments where specialized processesoccur. The epithelial sheet seems to be one of the inventions that lie at the origin of animal evolution, diversifying in a huge variety of ways (aswe see in chapter 23), but retaining an organization based on a set of conserved molecular mechanisms that practically all epithelia have in common. Essentially all epithelia are anchored to other tissue on one side-the basal side-and free of such attachment on their opposite side-the apical side. A basal lamina lies at the interface with the underlying tissue, mediating the attachment, while the apical surface of the epithelium is generally bathed by extracellular fluid (but sometimes covered by material that the cells have secretedat their apices).Thus all epithelia are structurally polarized, and so are their individual cells: the basal end of a cell, adherent to the basal lamina below differs from the apical end, exposed to the medium above. correspondingly, all epithelia have at least one function in common: they serve as selective permeability barriers, separating the fluid that permeates the tissue on their basal side from fluid with a different chemical composition on their apical side. This barrier function requires that the adjacent cells be sealed together by occluding junctions, so that molecules cannot leak freely acrossthe cell sheet. In this section we consider how the occluding junctions are formed, and how the polarized architecture of the epithelium is maintained. These two fundamental aspectsof epithelia are closely linked: the junctions play a key part in organizing and maintaining the polarity of the cells in the sheet.

TightJunctionsForma SealBetweenCellsand a FenceBetween Membrane D o ma i n s The occluding junctions found in vertebrate epithelia are called tight junctions. The epithelium of the small intestine provides a good illustration of their structure and function (see Figure rg-3). This epithelium has a simple columnar structure; that is, it consists of a single layer of tall (columnar) cells.These are of

T1GHT JUNCTIONS AND THE',ORGAN|ZAT|ON OF EPITHELIA

.,.:.'i.:,lr',

,'''.i'1''5.!

several differentiated types, but the majority are absorptive cells, specialized for uptake of nutrients from the internal cavity, or lumen, of the gut. The absorptive cells have to transport selected nutrients across the epithelium from the lumen into the extracellular fluid that permeates the connective tissue on the other side. From there, these nutrients diffuse into small blood vessels to provide nourishment to the organism. This transcellular transport depends on two sets of transport proteins in the plasma membrane of the absorptive cell. One set is confined to the apical surface of the cell (facing the lumen) and actively transports selectedmolecules into the cell from the gut. The other set is confined to the basolateral (basal and lateral) surfaces of the cell, and it allows the same molecules to leave the cell by facilitated diffusion into the extracellular fluid on the other side of the epithelium. For this transport activity to be effective,the spacesbetween the epithelial cells must be tightly sealed,so that the transported molecules cannot leak back into the gut lumen through these spaces (Figure f9-23). Moreover, the proteins that form the pumps and channels must be correctly distributed in the cell membranes: the apical set of active transport proteins must be delivered to the cell apex (as discussed in Chapter 13) and must not be allowed to drift to the basolateral surface, and the basolateral set of channel proteins must be delivered to the basolateral surface and must not be allowed to drift to the apical surface. The tight junctions between epithelial cells, besides sealing the gaps between the cells, may also function as "fences" helping to separatedomains within the plasma membrane of each cell, so as to hinder apical proteins (and lipids) from diffusing into the basal region, and vice versa (seeFigure 19-23). The sealing function of tight junctions is easyto demonstrate experimentally: a low-molecular-weight tracer added to one side of an epithelium will generally not passbeyond the tight junction (Figure lS-24).This seal is not absolute,however.Although all tight junctions are impermeable to macromolecules,their permeability to small molecules varies. Tight junctions in the epithelium lining the small intestine, for example, are 10,000times more permeable to inorganic ions, Na ' - d r i v e n glucose symport

t i gh t JUnCITOn plasma memoranes o f a d j a c e n tc e l l s i n t e r c eIlu l a r space p a s s r v ge r u c o s e carrer protern basolateraI surface

basal amina-i EXTRACELLULAR F L U I D / C O N N E C T-I V E TISSUE BLOOD

Figure 19-23 The role of tight junctions in transcellulartransport.Transport proteinsare confinedto differentregions of the plasmamembranein epithelialcells Thissegregation of the smallintestine. oermitsa vectorialtransferof nutrients acrossthe epitheliumfrom the gut lumen to the blood.In the examPleshown, glucoseis activelytransportedinto the cell by Na+-drivenglucosesymportsat its apicalsurface,and it diffusesout of the cell by facilitateddiffusionmediatedby glucosecarriersin its basolateral Tightjunctionsarethoughtto membrane. confinethe transportproteinsto their appropriatemembranedomainsby acting the asdiffusionbarriersor"fences"within lipid bilayerof the plasmamembrane; thesejunctionsalsoblockthe backflowof glucosefrom the basalsideofthe epitheliuminto the gut lumen.

1152

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix Figure19-24Therole of tight junctions in allowing epithelia to serveas barriers to solute diffusion. (A)The drawing shows how a smallextracellular tracermolecule addedon one sideof an epitheliumis preventedfrom crossingthe epitheliumby the tightjunctionsthat sealadjacentcells together.(B)Electronmicrographs of cells inanepithelium i n w h i c ha s m a l l , extracellular, electron-dense tracer moleculehasbeenaddedto eitherthe apicalside(on the /eft)or the basolateral side(on the right).Inboth cases, the tight junctionblockspassage of the tracer. (8,courtesyof DanielFriend.) (A)

(B)

fl 5*

such as Na+,than the tight junctions in the epithelium lining the urinary bladder. These differencesreflect differencesin the proteins that form the junctions. Epithelial cells can also alter their tight junctions transiently to permit an increased flow of solutes and water through breachesin the junctional barriers. Such paracellular transporf is especially important in the absorption of amino acids and monosaccharides from the lumen of the intestine, where the concentration of these nutrients can increase enough after a meal to drive passive transport in the proper direction. \t\4ren tight junctions are visualized by freeze-fracture electron microscopy, they seem to consist of a branching network of sealing strands that completely encirclesthe apical end of each cell in the epithelial sheet (Figure r9-25A and B). In conventional electron micrographs, the outer leaflets of the two interacting

microvilli

(A)

i n t e s t i n al u m e n

r i d g e so f t r a n s m e m b r a n e l a t e r a lp l a s m a p a r t i c l e sf o r m i n g s e a l i n g m e m b r a n e s t r a n d s( Pf a c e )

u ) [m

50 nm

Figure19-25The structureof a tight junction betweenepithelialcellsof the smallintestine.Thejunctionsareshown(A)schematicalry, (B)in a freeze-fracture electronmicrograph, and (C)in a conventional electronmicrograph. In (B),the planeof the micrographis parallelto the planeof the membrane,and the tight junctionappearsasa bandof branchingsealingstrandsthat encircleeachcellin the epithelium.The sealingstrandsareseenas ridgesof intramembrane particleson the cytoplasmic fracturefaceof the membrane(thep face)or as complementary grooveson the externalfaceof the membrane(theE face)(seeFigure19-26A).ln(C),the junctionis seenin crosssectionasa seriesof focalconnections betweenthe outer leafletsof the two interactingplasmamembranes, eachconnectioncorresponding to a sealing strandin crosssection.(Band C,from N.B.Gilula,in CellCommunication Cox,ed.],pp.1-29.Newyork:Wiley,1974.) [R.P.

11 5 3

TIGHTJUNCTIONS ANDTHEORGANIZATION OFEPITHELIA i n t e r a c tni g p l a s m am e m b r a n e s

cell 1

A i ntercelIu lar s p a c e(

I I

l o .6pm claudin

sealing strandsof tightj unction proteins \

occludin

proteins tight-junction (B)

ic cytoplasm half of lipid bilayer cell 1 (A)

plasma membranes are seen to be tightly apposed where sealing strands are present (Figure I9-25C). Each tight junction sealing strand is composed of a long row of transmembrane adhesion proteins embedded in each of the two interacting plasma membranes. The extracellular domains of these proteins adhere directly to one another to occlude the intercellular space (Figure 19-26). The main transmembrane proteins forming these strands are the claud.ins, which are essentialfor tight junction formation and function. Mice that lack the claudin-1gene, for example, fail to make tight junctions between the cells in the epidermal layer of the skin; as a result, the baby mice lose water rapidly by evaporation through the skin and die within a day after birth. Conversely, if nonepithelial cells such as fibroblasts are artificially caused to express claudin genes, they will form tight-junctional connections with one another. Normal tight junctions also contain a second major transmembrane protein called occludin, but the function of this protein is uncertain, and it does not seem to be as essential as the claudins. A third transmembrane protein, tricellulin (related to occludin), is required to seal cell membranes together and prevent transepithelial leakage at the points where three cells meet. The claudin protein family has many members (24 in humans), and these are expressedin different combinations in different epithelia to confer particular permeability properties on the epithelial sheet. They are thought to form paracellular pores-selective channels allowing specific ions to cross the tightjunctional barrier, from one extracellular space to another. A specific claudin found in kidney epithelial cells, for example, is needed to let MgZ* pass between the cells of the sheet so that this ion can be resorbed from the urine into the blood. A mutation in the gene encoding this claudin results in excessiveloss of MgZ* in the urine.

Playa KeyPartin the ScaffoldProteinsin JunctionalComplexes Controlof CellProliferation The claudins and occludins have to be held in the right position in the cell, so as to form the tight-junctional network of sealing strands.This network usually lies just apical to the adherens and desmosome junctions that bond the cells together mechanically, and the whole assembly is called a iunctional complex (Figure 19-27). The parts of this junctional complex depend on each other for

Figure 19-26 A model of a tight junction. (A)Thesealingstrandshold adjacent plasmamembranes together.Thestrands proteins arecomposedof transmembrane that makecontactacrossthe intercellular spaceand createa seal.(B)The molecular compositionof a sealingstrand.The claudinsarethe mainfunctional the roleof the occludins components; is uncertain.

1154

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix

a c t i ni n m i c r o v i l l i

Figure19-27 A junctionalcomplex betweentwo epithelialcellsin the lining of the gut. Mostapically, thereis a tightjunction;beneaththis,an adherens junction;and beneaththe adherens junction,a desmosomal junction.This exampleis from a vertebrate;in insects, the arrangement is different.(Courtesy of DanielS.Friend.)

tight junction (claudins) a d h e r e n sj u n c t i o n (cadherins)

d e s m o s o m aj ul n c t i o n (cadherins)

k e r a t i nf i l a m e n t s

their formation. For example, anti-cadherin antibodies that block the formation of adherens junctions also block the formation of tight junctions. The positioning and organization of tight junctions in relation to these other structures is thought to depend on association with intracellular scaffold proteins of the Tjp (Tight junction protein) family, also called ZO proteins (a tight junction is also known as a zonula occludens).The vertebrate Tjp proteins belong to the same family as the Discs-large proteins that we mentioned earlier for their role at synapses, and they anchor the tight-junctional strands to other components including the actin cytoskeleton. In invertebrates such as insects and mollusks, occluding junctions have a different appearance and are called septate junctions. Like tight junctions, these form a continuous band around each epithelial cell, but the structure is more regular, and the interacting plasma membranes are joined by proteins that are arranged in parallel rows with a regular periodicity (Figure lg-28). septate junctions are nevertheless based on proteins homologous to the vertebrate claudins, and they depend on scaffold proteins in a similar way, including in particular the same Discs-large protein that is present at synapses.Mutant flies that are deficient in Discs-largehave defective septatejunctions. Strikingly, these mutants also develop epithelial tumors, in the form of large overgrowths of the imaginal discs-the structures in the fly larva from which most of the adult body derives (as described in chapter 22). The gene takes its name from this remarkable effect, which depends on the presence of binding sites for growth regulators on the Discs-largeprotein. But why should the apparatus of cell-cell adhesion be linked in this way with the control of cell proliferation? The relationship seems to be fundamental: in vertebrates also, genes

_].",,,

Figure19-28A septatejunction. A conventional electronmicrographof a septatejunctionbetweentwo epithelial cellsin a mollusk.The interactingplasma membranes, seenin crosssection,are connectedby parallelrowsofjunctional proteins. The rows,which havea regular periodicity,are seenas densebars,or septa.(FromN.B.Gilula,in Cell Communication Cox,ed.],pp. 1-29. [R.P. NewYork:Wilev,1974.)

l-,

TIGHTJUNCTIONS AND THEORGANIZATION OF EPITHELIA

1155

homologous to Discs largehave this dual involvement. One possibility is that it reflects a basic mechanism for repair and maintenance of epithelia. If an epithelial cell is deprived of adhesive contacts with neighbors, its program of growth and proliferation is activated, thereby creating new cells to reconstruct a continuous multicellular sheet. In fact, a large body of evidence indicates that junctional complexes are important sites of cell-cell signaling not only via Discs-large but also through other components of these structures, including cadherins as we have seen.

Cell-Cell Junctionsand the BasalLaminaGovernApico-Basal Polarityin Epithelia Most cells in animal tissues are strongly polarized: they have a front that differs from the back, or a top that differs from the bottom. Examples include virtually all epithelial cells, as we have discussed, as well as neurons with their dendrite-axon polarity, migrating fibroblasts and white blood cells, with their locomotor Ieading edge and trailing rear end, and many other cells in embryos as they prepare to divide asymmetrically to create daughter cells that are different. A core set of components is critical for cell polarity in all these cases,throughout the animal kingdom, from worms and flies to mammals. In the case of epithelial cells, these fundamental generators of cell polarity have to establishthe difference between the apical and basal poles, and they have to do so in a properly oriented way, in accordance with the cell's surroundings. The basic phenomenon is nicely illustrated by experiments with a cultured line of epithelial cells, called MDCK cells (Figure f 9-29A). These can be separated from one another and cultured in suspension in a collagen gel. A single isolated cell in these circumstancesdoes not show any obvious polarity, but if it is allowed to divide to form a small colony of cells,these cells will organize themselvesinto a hollow epithelial vesiclewhere the polarity of each cell is clearly apparent. The vesiclebecomes surrounded by a basal lamina, and all the cells orient themselves in the same way, with apex-specific marker molecules facing the lumen. Evidently, the MDCK cells have a spontaneous tendency to become polarized, but the mechanism is cooperative and depends on contacts with neighbors. To discover how the underlying molecular mechanism works, the first step is to identify its components. Studies in the worm C. elegansand in Drosophila have been most informative here. In the worm, a screen for mutations upsetting the organization of the early embryo has revealeda set of genesessentialfor normal cell polarity and asymmetry of cell division (as discussed in Chapter 22). There are at least six ofthese genes, called Par (partitioning defectiue)genes.In all animal speciesstudied, they and their homologs (along with other genes discovered through studies in Drosophila and vertebrates)have a fundamental role not just in as),rynmetric cell division in the early embryo, but in many other processesof cell polarization, including the polarization of epithelial cells.The Par4 gene of C. elegans,for example, is homologous to a gene calledLkbl in mammals and Drosophila, coding for a serine/threonine kinase. In the fly, mutations of APICAL-BASAL POLARITY

APICAL-BASAL POLARITY

APICAL-BASAL POLARIW

Golgi

aminin nucleus tight junction ( A ) N O R M A LC E L LC L U S T E R

( B ) R A CF U N C T I O B NL O C K E D

( C ) R A CF U N C T I O B NL O C K E D P L U SE X O G E N O ULSA M I N I N

Figure19-29 Cooperativepolarizationof a clusterof epithelialcellsin cultureand its dependenceon Racand laminin.Cells of the MDCKline,derivedfrom dog kidney embedded epithelium,weredissociated, in a collagenmatrix,and allowedto proliferate, creatingsmallisolated in shownhereschematically colonies, crosssection.(A)Thecellsin sucha colony will normallyorganizethemselves into an epithelium spontaneously surrounding a centralcavity.Stainingfor ZO1 actin(whichmarksapicalmicrovilli), protein(a tight-junctionprotein),Golgi and laminin(a basallamina apparatus, component)showsthat the cellshaveall with becomepolarized, cooperatively apicalcomponentsfacingthe lumenof the cavityand basalcomponentsfacing the surroundingcollagengel.(B)When of a Racfunctionis blockedby expression form of the protein, dominant-negative the cellsshow invertedpolarity,failto form a cystwith a centralcavity,and cease to depositlamininin the normalmanner aroundthe peripheryof the cellcluster. (C)Whenthe cystis embeddedin a matrix richin exogenouslaminin,near-normal polarityis restoredeventhough Rac functionis stillblocked.(Basedon L.E.O'Brienet al.,Not CellBiol.3:831-838, from Macmillan 2001.With oermission Ltd.) Publishers

1156

Chapter19:CellJunctions, Matrix CellAdhesion,and the Extracellular

(A) NON-POLARIZE CD ELL

(B)

P O L A R I Z ECDE L L a c t i n - ifl l e d m i c r o v i l l i

L K B l1i n a c t i v e

L K B 1a c t i v e

ir

_;

this gene disrupt the polarity of the egg cell and of cells in epithelia. In humans, such mutations give rise to Peutz-Jegherssyndrome, involving disorderly abnormal growths of the lining of the gut and a predisposition to certain rare types of cancer. \A/hen cultured human colon epithelial cells are prevented from expressing LKBl, they fail to polarize normally. Moreover, when such cells in culture are artificially driven to express abnormally high levels of LKBI activity, they can become individually polarized, even when isolated from other cells, and surrounded on all sides by a uniform medium (Figure f9-30). This suggeststhat normal epithelial polarity depends on tvvo interlocking mechanisms: one that endows individual cells with a tendency to become polarized cellautonomously, and another that orients their polarity axis in relation to their neighbors and the basal lamina. The latter mechanism would be peculiar to epithelia; the former could be much more general, operating also in other polarized cell types. The molecules knor,rryrto be needed for epithelial polarity can be classified in relation to these two mechanisms. Central to the polarity of individual animal cells in general is a set of three membrane-associatedproteins: Par3, Par6, and atypical protein kinase C (aPKC). Par3 and Par6 are both scaffold proteins containing PDZ domains, and they bind to one another and to aPKC.The complex of these three components also has binding sites for various other molecules, including the small GTPasesRac and Cdc42.Theselatter molecules play a crucial part. Thus, for example,when Rac function is blocked in a cluster of MDCK cells, the cells develop with inverted polarity (see Figure 19-298). Rac and Cdc42 are key regulators of actin assembly, as explained in Chapter 16; through them, it seems,assemblyof a Par3-Par6-aPKCcomplex in a specific region of the cell cortex is associated with polarization of the cltoskeleton towards that region. The assembly process is evidently cooperative and involves some positive feedback and spatial signaling, so that a small initial cluster of these components is able to recruit more of them and to inhibit the development of clusters of the same t],pe elsewherein the cell. One source of positive feedback may lie in the behavior of Cdc42 and Rac: a high activity of these molecules at a particular site, by organizing the cytoskeleton, may direct intracellular transport so as to bring still more Cdc42 or Rac,or more of their activators,to the same site.This is suspectedto be an essentialpart of the polarization mechanism in budding yeast cells,and it may be the way in which cells such as migrating fibroblasts establish the difference between their leading edge and the rest of their periphery. It could be the core of the eucaryotic cell polarization machinery at least in evolutionary terms. The Par3-Par6-aPKCcomplex, combined with Cdc42 or Rac, seems to control the organization of other protein complexes associated with the internal face of the cell membrane. In particular, in epithelial cells, it causes the Crumbs complex,held together by the PDZ-domain scaffold proteins Discs-lost and Stardust, to become localized toward the apex of the cell, while a third such complex, called lhe Scribblecomplex,held together by the scaffold proteins Scribble and Discs-large (the same protein that we encountered previously) is localized more basally (Figure 19-3f ). These various protein assembliesinteract with one another and with other cell components in ways that are only beginning to be understood. But how is this whole elaborate system oriented correctly in relation to neighboring cells?In an epithelium, the Par3-Par6-aPKCcomplex assemblesat

Figure 19-30 Developmentof polarity in singleisolatedepithelialcells.Cellsof a linederivedfrom intestinaleoithelium were transfectedwith DNAconstructs codingfor regulatorycomponents throughwhichthe activityof the LKBI protein could be switchedon or off by a changein the compositionof the culture medium.WhenLKBlactivityis low,the cellsappearunpolarized; when it is high, polarized. they becomeindividually Their polarityis manifestin the distributionof tight-junctionproteins(ZO1) and proteins(p120adherens-junction whichaccumulate catenin), on one side of the cell,arounda cap of actin-filled microvilli, eventhough the cellsare isolatedfrom one anotherand makeno Thiscell-autonomous cell-celljunctions. oolarization occursevenwhen the cells areculturedin suspension, without contactwith any substratum that could tell them whichway wasup. (From A.F.Baaset al.,Cell 116:457-466, 2004. With permission from Elsevier.)

TIGHTJUNCTIONS ANDTHEORGANIZATION OF EPITHELIA

1157

Figure19-31The coordinated arrangementof three membraneassociatedprotein complexesthought to be criticalfor epithelialpolarity. A Drosophila epithelialcellis shown schematically on the left,and a vertebrate epithelialcellon the right.All three complex, complexes-thePa13-Pa16-aPkc the Crumbscomplex,and the Scribble complex-are organizedaroundscaffold proteinscontainingPDZdomains. The detaileddistributionof the complexes variessomewhataccordingto celltype.

/ C r u m b sc o m p l e x a d h e r e n sj u n c t i o n

tight junction

Par3-Par6-aPKC comprex

_ \

\ Cdc42lRac

septatejunction

S c r i b b l ec o m p l e x

G o l g i a p p a r a t u s/

_ D r o s o p h i l ae p i t h e l i a lc e l l

b a s a l a m i n a_ v e r t e b r a t ee p i t h e l i a cl e l l

cell-cell junctions-tight junctions in vertebrates, adherens junctions in Drosophila-because the scaffold proteins in the complex bind to the tails of certain of the junctional transmembrane adhesion proteins. Meanwhile, the cltoskeleton, under the influence of Rac or its relatives, directs the delivery of basal lamina components to the opposite end of the cell. These extracellular matrix molecules then act back on the cell to give that region a basal character (seeFigure 19-29C).In this way, the polarity of the cell is coupled to its orientation in the epithelial sheet and its relation to the basal lamina.

Figure19-32 Planarcell polarity.(A)Wing hairson the wing of a fly.Eachcellin the wing epitheliumformsone of theselittle or"hairs"atits apex,and spikyprotrusions all the hairspointthe samewa, toward the tip of the wing.Thisreflectsa planar polarityin the structureof eachcell. (B)Sensoryhaircellsin the innerearof a planar mousesimilarlyhavea well-defined polarity,manifestin the orientedpattern (actin-filled protrusions) on of stereocilia their surface.Thedetectionof sound deoendson the correct,coordinated orientationof the haircells.(C)A mutation in the geneFlamingoin the fly,codingfor cadherin, disruptsthe a non-classical patternof planarcellpolarityin the wing. (D)A mutationin a homologousFlomingo genein the mouserandomizes the orientationof the planarcellpolarity vectorof the haircellsin the ear.The mutantmicearedeaf.(A and C,from J. Chaeet al.,Development126:5421-5429, from TheCompany 1999.With permission Curtinet B and D,from .J.A. of Biologists; With al.,Curr.Biol.13:1129-1133,2003. from Elsevier.) oermission

A Separate SignalingSystemControlsPlanarCellPolarity Apico-basal polarity is a universal feature of epithelia, but the cells of some epithelia show an additional polarity at right anglesto this axis:it is as if they had an arrow written on them, pointing in a specific direction in the plane of the epithelium. This tlpe of polarity is called planar cell polarity (Figure l9-32A and B). In the wing of a fly, for example, each epithelial cell has a tiny asymmetrical projection, called a wing-hair, on its surface, and the hairs all point toward the tip of the wing. Similarly, in the inner ear of a vertebrate, each mechanosensory hair cell has an asymmetric bundle of stereocilia (actin-filled rod-like protrusions) sticking up from its apical surface: tilting the bundle in one direction causesion channels to open, stimulating the cell electrically; tilting in the opposite direction has the contrary effect. For the ear to function correctly, the hair cells must be correctly oriented. Planar cell polarity is important also in the respiratory tract, for example, where every ciliated cell must orient its beating so as to sweep mucus up out of the lungs, and not down into them (seeChapter 23). Screensfor mutants with disorderly wing hairs in Drosophilahave identified a set of genes that are critical for planar cell polarity in the fly. Some of these, e p i d e r m acl e l l si n f l y w i n g

s e n s o r yh a i r c e l l si n m o u s ee a r

I

e p i d e r m a cl e l l si n f l y w i n g

s e n s o r yh a i r c e l l si n m o u s ee a r

r(B) 5pm W I L DT Y P E

10 pLm

FlamingoMUTANT

1 158

Chapter19:Cell Junctions,Cell Adhesion,and the ExtracellularMatrix

such as Frizzled, for example, and Disheuelled, code for proteins that have since been shown to be components of the Wnt signaling pathway (discussed in Chapter 15). Two others, Flamingo (seeFigure l9-32C) and Dachsous,code for members of the cadherin superfamily. Still others are less easily classified functionally, but it is clear that planar cell polarity is organized by machinery formed from these components and assembled at cell-cell junctions in such a way that a polarizing influence can propagate from cell to cell. Essentiallythe same system of proteins controls planar cell polarity in vertebrates.Mice with mutations in a Flamingo homolog, for example, have incorrectly oriented hair cells in their ears (among other defects) and thus are deaf (seeFigure l9-32D).

Su m m a r y Occluding junctions-tight junctions in uertebrates,septatejunctions in insectsand molluscs-seal the gaps betweencellsin epithelia, creating a barrier to the diffusion of moleculesacrossthe cell sheet.Theyalsoform a bar to the diffusion of proteins in the plane of the membrane,and so help to maintain a dffirence betweenthe populations of proteins in the apical and basolateralmembrane domains of the epithelial cell. The major transmembraneproteinsforming occludingjunctions are calledclaudins;difin dffirent tissues, conferringdffirent perferent membersof thefamily are expressed meability properties on the uarious epithelial sheets. Intracellular scaffold proteins bind to the transmembranecomponentsat occluding junctions and coordinatethesejunctions with cadherin-basedanchoringjunctions, so as to createjunctional complexes.The junctional scaffold proteins haue at leasttwo other crucialfunctions. Theyplay a part in the control of epithelial cell proliferation; and, in conjunction with other regulatory moleculessuch as Racand Cdc42, they gouerncell polarity. Epithelial cellshauean intrinsic tendencyto deuelopa polarized apico-basal axis. The orientation ofthis axis in relation to the cell'sneighbors in an epithelial sheet depends on protein complexesinuoluing scaffold proteins that assembleat cell-cell junctions, as well as on cytoskeletalpolarization controlled by Rac/Cdc42and on influencesfrom the basal lamina. Thecellsof someepitheliahaueanadditionalpolarity intheplane of theepithelium, at right anglesto the apico-basalaxis.A separatesetofconseruedproteins,operatingin a similar way in uertebrates and in insects,gouernsthis planar cell polarity through poorly understoodsignaling processes that are likewisebasedon cell-celljunctions.

PASSAGEWAYS FROMCELLTO CELL:GAP JUNCTIONS AND PLASMODESMATA Tight junctions block the passagewaysthrough the gaps between cells, preventing extracellular molecules from leaking from one side of an epithelium to the other. Another type of junctional structure has a radically different function: it bridges gaps between adjacent cells so as to create direct passagewaysfrom the cltoplasm of one into that of the other. These passagewaystake quite different forms in animal tissues, where they are called gap junctions, and in plants, where they are called plasmodesmata (singular plasmodesma). In both cases, however, the function is similar: the connections allow neighboring cells to exchange small molecules but not macromolecules (with some exceptions for plasmodesmata). Many of the implications of this cell coupling are only beginning to be understood.

Gap JunctionsCoupleCellsBoth Electricallyand Metabolically Gap junctions are present in most animal tissues,including connective tissues as well as epithelia, allowing the cells to communicate with their neighbors. Each gap junction appears in conventional electron micrographs as a patch where the membranes of two adjacent cells are separated by a uniform narrow

r 159

PASSAGEWAYS FROMCELLTOCELL:GAPJUNCTIONS AND PLASMODESMATA

gap of about 2-4 nm. The gap is spanned by channel-forming proteins, of which there are two distinct families, called the connexins and the innexins. These are unrelated in sequence but similar in shape and function: in vertebrates, both families are present, but connexins predominate, with 2l members in humans. ln Drosophilaand C. elegans,only innexins are present, with 15 family members in the fly and 25 in the worm. The channels formed by the gap-junction proteins allow inorganic ions and other small water-soluble molecules to pass directly from the cltoplasm of one cell to the cltoplasm of the other, thereby coupling the cells both electrically and metabolically. Thus, when a suitable dye is injected into one cell, it diffuses readily into the other, without escaping into the extracellular space. Similarly, an electric current injected into one cell through a microelectrode causesan almost instantaneous electrical disturbance in the neighboring cell, due to the flow of ions carrying electric charge through gap junctions. With microelectrodes inserted into both cells, one can easily monitor this effect and measure properties of the gap junctions, such as their electrical resistanceand the ways in which the coupling changes as conditions change.In fact, some of the earliest evidence of gap-junctional communication came from electrophysiological studies that demonstrated this type of rapid, direct electrical coupling between some types of neurons. Similar methods were used to identify connexins as the proteins that mediate the gap-junctional communication: when connexin mRNA is injected into either frog oocytes or gap-junction-deficient cultured cells, channels with the properties expected of gap-junction channels can be demonstrated electrophysiologically where pairs of injected cells make contact. From experiments with injected dye molecules of different sizes, it seems that the largest functional pore size for gap-junctional channels is about 1.5nm. Thus, the coupled cells share their small molecules (such as inorganic ions, sugars, amino acids, nucleotides, vitamins, and the intracellular mediators cyclic AMP and inositol trisphosphate) but not their macromolecules (proteins, nucleic acids, and polysaccharides) (Figure f9-33).

A Gap-Junction Connexonls MadeUp of SixTransmembrane ConnexinSubunits Connexins are four-pass transmembrane proteins, six of which assemble to form a hemichannel, or connexon. \dhen the connexons in the plasma membranes of two cells in contact are aligned, they form a continuous aqueous channel that connects the two cell interiors (Figure l9-34A and Figure f 9-35). A gap junction consists of many such connexon pairs in parallel, forming a sort of molecular sieve.The connexons hold the interacting plasma membranes a fixed distance apart-hence the gap. Gap junctions in different tissues can have different properties becausethey are formed from different combinations of connexins, creating channels that differ in permeability. Most cell q,pes expressmore than one type of connexin, and two different connexin proteins can assembleinto a heteromeric connexon, with its own distinct properties. Moreover, adjacent cells expressing different

n

i nteracting p l a S m am e m D r a n e s

100,' 1000 i..l

soooa-;

Figure19-33 Determiningthe sizeof a gap-junctionchannel.Whenfluorescent molecules of varioussizesareinjectedinto one of two cellscoupledby gapjunctions, molecules with a massof lessthan about 1000daltonscan passinto the othercell, cannot. but largermolecules

Figure19-34 Gapjunctions.(A)A threedrawingshowingthe dimensional of two interactingplasmamembranes adjacentcellsconnectedby gap junctions.Eachlipid bilayeris shownasa oair of redsheets.Proteinassemblies calledconnexons(green),eachof which isformedby sixconnexinsubunits, penetratethe apposedlipid bilayers(red). join acrossthe Two connexons gap to form a continuous intercellular aqueouschannelconnectingthe two of connexins cells.(B)Theorganization into connexonsand connexonsinto can Theconnexons channels. intercellular and the be homomericor heteromeric, intercellular channelscan be homotypic or heterotypic.

homomenc

&4K

sf Er

I W OC O n n e X OtnnS r e g i s t e rf o r m i n g o p e n c h a n n e lb e t w e e n a d j a c e n tc e l l s (A)

connexon composedof s i xs u b u n i t s

!E

heteromeric c o n n e xnr s (B)

connexons

homotypic c

heterotypic

i n t e r c e l l u l acr h a n n e l s

I 160

Chapter19:CellJunctions, CellAdhesion, and the Extracellular Matrix

r ar g e gap Ju nctron

Figure19-35Gapjunctionsas seenin the electron microscope.(A)Thin-section and (B)freeze-fracture electron micrographs of a largeand a smallgap junctionbetweenfibroblasts in culture.In (B),eachgapjunctionis seenasa cluster of homogeneous intramembrane particles. particle Eachintramembrane corresponds to a connexon.(From N.B.Gilula,in CellCommunication lR.P.Cox,ed.l,pp. 1-29. New York:Wiley, 1974.\

memoranes

connexins can form intercellular channels in which the two aligned half-channels are different (Figure l9-348). Each gap-junctional plaque is a dyramic structure that can readily assemble, disassemble,or be remodelled, and it can contain a cluster of a few to many thousands of connexons (seeFigure l9-358). Studieswith fluorescently labeled connexins in living cells show that new connexons are continually added around the periphery of an existing junctional plaque, while old connexons are removed from the middle of it and destroyed (Figure f 9-36). This turnover is rapid: the connexin molecules have a half-life of a few hours. The mechanism of removal of old connexons from the middle of the plaque is not known, but the route of delivery of new connexons to its periphery seems clear: they are inserted into the plasma membrane by exocytosis,like other integral membrane proteins, and then diffuse in the plane of the membrane until they bump into the periphery of a plaque and become trapped. This has a corolIary: the plasma membrane away from the gap junction should contain connexons-hemichannels-that have not yet paired with their counterparts on another cell. It is thought that these unpaired hemichannels are normally held cRoSs SECTtONS

Figure19-36 Connexinturnoverat a gap junction.Cellsweretransfected with a slightlymodifiedconnexingene,codingfor a connexinwith a short amino-acidtag containingfour cysteines in the sequence...Cys-Cys-X-X(whereX denotesan arbitraryamino acid).Thistetracysteine Cys-Cys tog can bind strongly,and in effectirreversibly, to certainsmallfluorescentdye molecules that can be addedto the culturemediumand will readilyenter cellsby diffusingacrossthe plasmamembrane.In the experimentshown,a greendye wasaddedfirst,and the cellswerethen washedand incubated for 4 or t hours.At the end of thistime,a red dye wasaddedto the mediumand the cellswerewashedagainand fixed.Connexinmolecules alreadypresentat the beginningof the experimentare labeledgreen(and take up no red dye because theirtetracysteine tagsarealreadysaturated with greendye),whileconnexinssynthesized subsequently, duringthe 4- or 8-hourincubation, arelabeledred.Thefluorescence imagesshow opticalsectionsof gap junctionsbetweenpairsof cellspreparedin this way.Thecentralpart of the gap-junctionplaqueis green,indicatingthat it consists of old connexinmolecules, whilethe peripheryis red indicating that it consists of connexinssynthesized duringthe past4 or 8 hours.The longerthe time of incubation, the smallerthe greencentralpatchof old molecules, and the largerthe peripheralring of new molecules that have been recruitedto replacethem. (FromG. Gaiettaet al.,Sclence 296:503-507,2002.With permissionfrom AAAS.)

4 h incubation

8 h incubation E N - F A CVEI E W

8 h incubation 2pm

PASSAGEWAYS FROMCELLTO CELL: GAPJUNCTIONS ANDPLASMODESMATA in a closedconformation,preventingthe cell from losingits smallmoleculesby leakagethroughthem. But thereis alsoevidencethat in somephysiologicalcircumstancesthey can open and serve as channelsfor the releaseof small molecules,such as the neurotransmitterglutamate,to the exteriol or for the entry of smallmoleculesinto the cell.

GapJunctionsHaveDiverseFunctions In tissues containing electrically excitable cells, cell-cell coupling via gap junctions serves an obvious purpose. Some nerve cells, for example, are electrically coupled, allowing action potentials to spread rapidly from cell to cell, without the delay that occurs at chemical synapses.This is advantageous when speed and reliability are crucial, as in certain escaperesponsesin fish and insects, or where a set of neurons need to act in synchrony. Similarly, in vertebrates, electrical coupling through gap junctions synchronizes the contractions of heart muscle cells as well as those of the smooth muscle cells responsible for the peristaltic movements of the intestine. Gap junctions also occur in many tissues whose cells are not electrically excitable. In principle, the sharing of small metabolites and ions provides a mechanism for coordinating the activities of individual cells in such tissues and for smoothing out random fluctuations in small-molecule concentrations in different cells. Gap junctions are required in the liver, for example, to coordinate the response of the liver cells to signals from nerve terminals that contact only a part of the cell population (see Figure f 5-7). The normal development of ovarian follicles also depends on gap-junction-mediated communication-in this case,between the oocyte and the surrounding granulosa cells.A mutation in the gene that encodes the connexin that normally couples these two cell types causes infertility. Mutations in connexins, especially connexin-26, are the commonest of all genetic causes of congenital deafness: they result in the death of cells in the organ of Corti, probably because they disrupt functionally important pathways for the flow of ions from cell to cell in this electrically active sensory epithelium. Connexin mutations are responsible for many other disorders besides deafness, ranging from cataracts in the lens of the eye to a form of demyelinating disease in peripheral nerves. Cell coupling via gap junctions also seems to play a part in embryogenesis. In early vertebrate embryos (beginning with the late eight-cell stage in mouse embryos), most cells are electrically coupled to one another. As specific groups of cells in the embryo develop their distinct identities and begin to differentiate, they commonly uncouple from surrounding tissue. As the neural plate folds up and pinches off to form the neural tube, for instance (see Figure 19-16), its cells uncouple from the overlying ectoderm. Meanwhile, the cells within each group remain coupled with one another and therefore tend to behave as a cooperative assembly, all following a similar developmental pathway in a coordinated fashion.

CellsCanRegulate the Permeability of TheirGapJunctions Like conventional ion channels (discussedin Chapter 11), individual gap-junction channels do not remain continuously open; instead, they flip between open and closed states.Moreover, the permeability of gap junctions is rapidly (within seconds) and reversibly reduced by experimental manipulations that decrease the cytosolic pH or increase the cytosolic concentration of free Caz*to very high levels. The purpose of the pH regulation of gap-junction permeability is unknor.vn. In one case, however, the purpose of Caz* control seems clear. lVhen a cell is damaged, its plasma membrane can become leaky.Ions present at high concentration in the extracellular fluid, such as Ca2* and Na*, then move into the cell, and valuable metabolites leak out. If the cell were to remain coupled to its

't16"1

1162

Chapter19:CellJunctions, Matrix CellAdhesion,and the Extracellular Figure19-37The regulationof gapjunction coupling by a neurotransmitter. (A)A neuron in a rabbit retinawas injected with the dye Luciferyellow,which passes readilythroughgapjunctionsand labels other neuronsof the sametype that are connectedto the injectedcellby gap junctions.(B)The retinawasfirsttreated with the neurotransmitter dopamine, beforethe neuronwas injectedwith dye. As can be seen,the dopaminetreatment greatlydecreased the permeability of the gapjunctions.Dopamineactsby increasing intracellular cyclicAMPlevels. (Courtesyof DavidVaney.)

healthy neighbors, these too would suffer a dangerous disturbance of their internal chemistry. But the large influx of Ca2*into the damaged cell causesits gap-junction channels to close immediately, effectively isolating the cell and preventing the damage from spreading to other cells. Gap-junction communication can also be regulated by extracellular signals. The neurotransmitter dopamine, for example, reduces gap-junction communication between a classof neurons in the retina in responseto an increasein light intensity (Figure f 9-37). This reduction in gap-junction permeability helps the retina switch from using rod photoreceptors, which are good detectors of low light, to cone photoreceptors, which detect color and fine detail in bright light.

In Plants,Plasmodesmata PerformManyof the SameFunctions a sG a pJ u n c t io n s The tissues of a plant are organized on different principles from those of an animal. This is becauseplant cells are imprisoned within tough cell walls composed of an extracellular matrix rich in cellulose and other polysacharides, as we discuss later. The cell walls of adjacent cells are firmly cemented to those of their neighbors, which eliminates the need for anchoring junctions to hold the cells in place. But a need for direct cell-cell communication remains. Thus, plant cells have only one class of intercellular junctions, plasmodesmata. Like gap junctions, they directly connect the cytoplasms of adjacent cells. In plants, the cell wall between a tlpical pair of adjacent cells is at least 0.1 pm thick, and so a structure very different from a gap junction is required to mediate communication across it. Plasmodesmata solve the problem. With a few specialized exceptions, every living cell in a higher plant is connected to its living neighbors by these structures, which form fine cytoplasmic channels through the intervening cell walls. As shown in Figure l9-38A, the plasma membrane of one cell is continuous with that of its neighbor at each plasmodesma, which connects the cltoplasms of the two cells by a roughly cylindrical channel with a diameter of 20-40 nm. Running through the center of the channel in most plasmodesmata is a narrower cylindrical structure, the desmotubule,which is continuous with elements of the smooth endoplasmic reticulum in each of the connected cells (Figure l9-38B-D). Between the outside of the desmotubule and the inner face of the cylindrical channel formed by plasma membrane is an annulus of cytosol through which small molecules can pass from cell to cell. As each new cell wall is assembled during the cytokinesis phase of cell division, plasmodesmata are created within it. They form around elements of smooth ER that become trapped acrossthe developing cell plate (discussedin Chapter 17).They can also be inserted de nouo through preexisting cell walls, where they are commonly found in dense clusters called pit ftelds. \Alhen no longer required, plasmodesmata can be readilv removed.

a u e r q u a u Je u s e l o

rxusz

arDsuoxauuoJmau :saJnJJn4srllaqutp alo suolpunf doD 'suolpun[ asaq]to lgntqoala -rad aqi ataruSalual sIpJ aqJ 'txauatilto ap$w at4ioi IpJ auoto apcm aqt uto{ fpcanp ssadot suotf)p 000t noqq uqqj JaIIawssalnralow mo71a[\\onsn ]Dql suoxauuoJpalal -sn1ctosanba1dtorutol ar4layat asar.lJ'suortcunfdaBpallar suoltJuntBurtutol-1auuar4c [q pa1dnot an 'p1aqildauou puD pqaquda q]oq'sanssu louarua [uaw lo sIIaJaLIJ

[reuun5 'll q8norql ssed ol sB os leuueqf, aq] Jo saruadord uodsueJ] aql aleln8ar salnJaloruoJJeruIUJIA ro snouaSopua ^aoq,rfuatsfure ill]s sl ll pue 'u^ oulun eJe lJeJJeslq] a]elpau leq] Bleusepourseld;o sluauoduroJ leuollcunJ aq1 ,tlpctlelueJp Iauueqo eql ur Elerusepouseld aql;o sluauod Jo azrs arod anqca;;e aql aseaJJurleq] s.,(e,nn -ruoc ot purq ]eqt suratord acnpord sesnJIAasaql Lert sql ul IIaJ ol IIaJ ruoq ssed uec 'salcrlred snJrl ]Jelur uala Jo 'ygg pryr snollJaJul :elnoJ slq] lloldxa osle sesnrrl 1ue1dauog 'q3norqr ssed osp uec uralord aq] sepoJua ]pqt VNutu eq] 'saspo aruos uI 'a8essed Jlaql lue^eJd asu,traqlo plnolv\ ]Bq] rusluPqceu uorsnloxa ezrs aq] eprrralo pue eletusaporuseydeq] Jo sluauodruoc ol pqq daql 'elerusaporuseld q8norqt ilac ot IIaJ ruo4 ssed uorleuluJetap alEJIIat Jo ssacord slq] uI pe^lo^ur suralord rfuolep8ar aua8 auog '(77 rc1deq3 uI pessnJ -srp) seleJ ernlnJ rraql Surugap yo ssacord aq] uI Jeqloue euo o1 pu8ts sruals -rJaru]ooJ pue looqs aql ulqtl,|/I sllal Jo sdnorS 'luaurdolanap tueld Sulrnq 'poolsJepun lou aJesesuJasaq] uI uollEslunlutuoJ lclJlseJ lEql sl'uslu -eqJeu eqt letpusapourseld .'tpuaredde ,(q palcauuoJ are leq] 'slleo IEuJou 'slleJ uala Jo salnceloru ureual ueemlaq ]q31arr,r-relnceloru-Mol ;o sdnor8 ro 'eldurexe roJ 'sluarulradxa uorl ]uaruenoru eql ol sJerJJBqaq ueo ereq] ]eq] lrtoqs -cafur-ar(q 'paleln8ar sr elurusaporuseld q8norqt ]rodsue;l 'suotlcunf de8 qlpn sV 'suorlJunt de8 rol gornc lq8ram-repcalou aql ol JelIuIS sI qolq ^ '008 ]noqe ueq] ssal ;o lq8raa,rrelnJaloru e qllm salnJeloru ;o aBessedeql ,vrolleelerusap -oruseld sazts ]ueJeJJIpJo selnoaloru racer] Sutlcafut .,(qpaurelqo slsaSSns leql af,uapng 's,{e,,r,r repurs .{lqeryeural uI uoI}JunJ 01 ulaes daq} 'suollcunf de8 pue eleusaporuseld uaa,t{leq arnlcnJts uI eJueraJJlpIPoIpBJaq};o alrds u1

'Ot pueGl 'dd'6 ('Z86 :6raq;aprag I L'6elra-ra6uud5 'lleie^O'U LuollC pueJ) 'uorl)as euseldolor6 ur'6uruun9'S l'B pupa;loM'f 'uaasaq osleue)alnqnloulsap le;rurrs ssoJ)uruaaseursopor.lrseld V (O)

'lxau aql ol s]l pue Lunln)llal)lLuseloopuf llal lerlua)aql qllM uolleDosse auo uror]snonurluofsrpue arod aql seu!lauerqulauleulseloaql'ulal raleM uot1ral3(1) e 1oqde.r6ortttu e urorJeulsapor.use;d e;o uorl)asleurpnlrbuol 'Lunln)rlar aqt'aln])nlls qlooLUs Lrjor;panuap'alnqnlousap rrurse;dopua Jelnqn]auUe sureluo)osle^llensnll 'slla)pal)auuofoMl ol uol'.tlL.uol q)el (8) 'raq1a6o1 qrlm paull st eulsapoLrrsPld srleql auerquraureu.rseld rueldeql a>latdeleu.rsapouse;d 1ue;de ur sllal llel)auuo) pue lleM1;a> jo slauueqlrrr.use;doy{r aqf (V)'eteusapouseld95-61 atn6t1

(v)

(8) sllar lua)efpe o^^] 6ullrauuo:'eutsapo|.Useld 6 u r u r ;a u e t q u r e uer u r s e ; d

eleursapourselo

s l l e )l u e l o luerelpe 1o s l l e Ml l a ) losoll)

u n l n )r l a l >ruseldopua

€9rr

dV9:'II]f OIII]f VIVWS]COhISVId CNVSNOIIfNNT

IAIOUJ SAVM]9VSSVd

'taaqs a u e r q u J a u r e u J s e l ol l a ) a l ) s n u e u r u r e ll e s e q a n s s ral A r l ) a u u o ) llel lerlaqlroe letlaqltoa e u r u r Plle s e q aql ur sde6relnllafralul | .,. \' ."' s aql uedsvgl'sLu6uLldolpllls * !- " palle)'sarnl)nrls paseq-utalold raqlo uo spuaoaposleuorlPrllu aql'poolq aqt uorj auun aql olu! ssedllrMsaln)alou sn']nuty\lo-r9 lNcr) q)qM aururolapol 6utdlaq 'uor]lunl l1)snr! nnt'lSHLtdl a^rlroddns e se llaMse 6urra1lr; e sPqeuruielleseqaql pue'uraqlur sdebaneqslaaqs sureloJd eql azruB8Jo'r.usrloqBleurIIeJ eJuanlJur'd1rre1od ilac aurruJalapol alqe l l a l q l o q ' s n ; n r a u o ;{6a u p r 1 aJe^eqJ'Je^a,\,\oq'saloJSurJallu pup eldurs ueql erou eAEqeBurruBl aql ur leql oloN '(snlnraruol6 IeJruJnrls '(6t-6I aJnSlc) JallrJa^rlcalasE se suorlsunJ pue slaaqs ^auprl aqt ur se)slaaqs IesEg IIeJ o^\l ueaMleq lla) oMl uaaMlaqpasoolalul sarl purluel pseq e 'sn1nrauro13daupu aql sp qcns 'suorl€3ol Jeqto uJ 'uraql a J ep u e ' e r ; a q l l daer U a p u n uaal laq uorlJauuol lEJrueqreu eql sruJoJpue anss! a^llJeuuoc Surpunorrns '(slla)al)snur lelalalsse q)ns) pue slleJ aseql sa}eJedessnql eurruel IBsBqeql Jo 3ur,{lJepun aql ruor; erTaq}rda slla)uretralpunolrns(Moila() '(u11adruuJoJ ol suoxe IIaJ alJau leJaqdrJad punoJp der,u qcrqa,r)sllal uue^aqcs a P u r u elle s e g ' p a z l u e 6 r o puE 'sllec tpJ 'sllal elf,snru pnpr^rpw spunoJJnsosp lnq eleqlrde ile serlJapun sle aeururPlleseqq)tq/vl ,tpo 1ou euruel leseq Jo ]aaqs V )crql uru 0u-0v r(pcrdr! aJe aeurruel Ieseg ut sIBrvraarql 69-6 1 arn6t3

sad^l lla) lP!laqrrdauoN auos punoltnspue etlaqlldlllv alllepuneeultupllespg 'ralEl lrBlap ur pessncsrp aq ilu xrrlEru JElnllef,EJlxeJo stuJoJ raqlo asaqJ 'senss0e^llJauuoJ ur punoJXlJteurlEln[laJel]xaJo sed,q peuP^ aql qll.ry\suorlJeJelur Jraq] Surlerparu 'sad,,i11ac Jaqlo ur luaseJd oslP aJp surJSalur (saueJqruau ]pql ees IIEqsalrrpue IIaJ Iellaqlrda aqlulsularcJd utt&alut q8norql 'JaqJouE auo qlr^a lJeralur Buruel Ieseq pue slleJ Ielleqllda moq Jeprsuoc fleqs a1!tuorlf,eslxau aql ul Jlas]l eurupl pseq aq] ssnJsrpa1l\'uortlas srq] uI ,Qpuenb pue uoItIS -odluoJ qloq ur taqloue or run1,{qdpurue auo ruo4 alqerJel aloru aJExrJleru JelnllaJEJlxaJo sruroJ Jaqlo 'slErurue rcInllerrllnru Ip ol uourruoJ seJnlPeJ Surur;ap aql Jo euo eq o] suaas ]l 'surraqpeJ eql e{lT .{poq aq1Jo arntJa}rqlre aq] ul alor IEcrlrrJe seq 1I 'arunlon ur sl u se ilelus 'elaqlrda 1e;o Suruurdrapun ppuassa uE sr selnJalour xrJleru Jo taeqs elqFeg 'q8no1 'ulq] ^lSurpaacxasrqJ '(euBJqrueu lueuasEq aql se ol parJaJal osle) BuIurBI IEsBq aql-ernlonJls snonqo ssal qlntu e suJoJ leql xrJleu relnllaJerlxa aqt o] saoS aceld;o aprrd 'Mer^Jo 'uDIsaq}Jo lurod,{Jpuorlnlo^a ue ruo4 'Ja^a^\oq'praua8 ur slerurueroC radel purrap aq] pue 'uopue] 'euoq sE qlns sanssrl e^rlJauuoJ.{)lpq u1 punoJ ere xrJleru JplnllacpJga Jo sruJoJ pyquayd ]soru aq] 'sarpoq u^ o Jno uI 'ueq] pecnpord leql sllaf, aq] Jo saceJrnseql qlIM uorlerJosseasolc ur {Joli\qsau pazrue8ro uB olur palqtuassepue,{1eco1pe}aloas eJ€teql saprJeqJces.{1od pue suralord snorJplJo pasodruoc sr xrJleru svqlxulaw JoInIIaJoJWaaql 8ur]rupsuoJ selnoaloruoJceru 'acads ta1n11acoJlxa sr-ltud a8rel Jo {Jomlau a}EJrrlur ue .{q pardncco sr qJrq A ,{rane sarur}aruos-arunlo^ JraqlJo ged y 's1acgo ,{.1a1os dn apeur lou are senssrJ

VNIWVIIVSVB3H1 salnJalow aBnTrow lo a8nssndaqr 3un1co7qafqm Uaco177acutott ssodol salnralou Surllttutad ut suotlcunfdaBaq17 [TqaTtoutatuo4cunl faql'saln)apu 17aws -oJJow Tauotlautolu! uodsuau sawllauos uoc faqt puo'suou?un[ daB wql utott lo ruata[tp [\att]ua s! alnpnus naql q&noqtlv'aruwsapousa\d pa\\ncsuotjcun[ Suuacru 'vua1d 'namso s77ac sdnotBnr1lo ur a1otSu4nutptooc -ntuutoc payurl [q an syac uI lo D anaq ,bt1l pun 's77ac anrlco[77aut1ca7a lo saUmUrDaqj Surjuutptooc u1]ua\todutt atn suoqcunf dag'pa1dnoc [11ocuna1apuo [T1anutaqcatolataql arDpuo salnralow lfnus Jaqio puo suot ctuoBtour naql lo tuout anqs suotlcun[ doB [q papauuor sIIaC 'ralual st! wo4 panoluarqp -nuuuoJ aJDsuoxauuorplo af?4m'anba1d aqlto,Laqduad aqt ot patlnJJat[11anu4uoc xulew ,elnlle)eJlxf aql pue 'uolseqpv lle) 'suoll)unf lla) :61 reldeq3

Vgll

T H EB A S A LL A M I N A

1155

in adjacent plasma membranes, promote cell survival, proliferation, or differentiation, and serve as highways for cell migration. The mechanical role is neverthelessessential. In the skin, for example, the epithelial outer layer-the epidermis-depends on the strength of the basal lamina to keep it attached to the underlying connective tissue-the dermis. In people with genetic defects in certain basal lamina proteins or in a special type of collagen that anchors the basal lamina to the underlying connective tissue, the epidermis becomes detached from the dermis. This causes a blistering diseasecalled junctional epidermolysisbullosa, a severeand sometimes lethal condition.

Lamininls a PrimaryComponentof the BasalLamina The basal lamina is slmthesized by the cells on each side of it: the epithelial cells contribute one set of basal lamina components, while cells of the under$ing bed of connective tissue (called the stroma, Greek for "bedding") contribute another set (Figure f 9-40). Like other extracellular matrices in animal tissues, the basal lamina consists of two main classesof extracellular macromolecules: (l) fibrous proteins (usually glycoproteins, which have short oligosaccharide side chains attached) and (2) polysaccharide chains of the type called glycosaminoglycans (GAGI),which are usually found covalently linked to specific coreproteins to form proteoglycans (Figure f 9-4f ). In a later section, we shall discuss these two large and varied classesof matrix molecules in greater detail. We introduce them here through the special subset that are found in basal laminae. Although the precise composition of the mature basal lamina varies from tissue to tissue and even from region to region in the same lamina, it typically contains the glycoproteins laminin, type IV collagen, and, nidogen, along with the proteoglycan perlecan Together with these key components, present in the basal laminae of virtually all animals from jellfish to mammals, it holds in its meshes, or is closely associated with, various other molecules. These include collagen XWII (an atypical member of the collagen family, forming the core protein of a proteoglycan) and fibronectin, afibrous protein important in the adhesion of connective-tissue cells to matrix. The laminin is thought to be the primary organizer of the sheet structure, and early in development, basal laminae consist mainly of laminin molecules. Laminin-l (classicallaminin) is a large, flexible protein composed of three very

e p i t h e l i a lc e l l s

basal amina

c o rIa g e n

1 0l r .

Figure19-40The basallaminain the corneaof a chickembryo.ln this someof scanningelectronmicrograph, the eoithelialcellshavebeenremovedto exposethe uppersurfaceof the matlike basallamina.A networkof collagenfibrils in the underlyingconnectivetissue interactswith the lower face of the of RobertTrelstad.) lamina.(Courtesy

1166

Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix

perlecan type lV collagen

-------------=

-=-'----

------a

Figure19-41The comparativeshapes and sizesof some of the major extracellularmatrix macromolecules. Proteinis shown in green,and glycosaminoglycan in red.

aar nidogen f i b r i l l a cr o l l a g e n

iio.on"ain

{

aggrecan 1 0 0n m

Iong polypeptide chains (a, B, and 1) held together by disulfide bonds and arranged in the shape of an asymmetric bouquet, like a bunch of three flowers whose stems are twisted together at the foot but whose heads remain separate (Figure 19-42). These heterotrimers can self-assemble in uitro into a network, largely through interactions between their heads, although interaction with cells is needed to organize the the network into an orderly sheet. Since there are several isoforms of each type of chain, and these can associatein different combinations, many different laminins can be produced, creating basal laminae with distinctive properties. The laminin y-1 chain is, however, a component of most laminin heterotrimers; mice lacking it die during embryogenesis because they are unable to make basal lamina.

TypelV CollagenGivesthe BasalLaminaTensileStrength Tlrpe IV collagen is a second essentialcomponent of mature basal laminae, and it, too, exists in several isoforms. Like the fibrillar collagensthat constitute the bulk of the protein in connective tissues such as bone or tendon (discussed later), type IV collagen molecules consist of three separately syrrthesizedlong protein chains that twist together to form a ropelike superhelix; but they differ from the fibrillar collagens in that the triple-stranded helical structure is interrupted in more than 20 regions, allowing multiple bends. The tlpe IV collagen molecules interact via their terminal domains to assemble extracellularly into a flexible, felt-like network. In this way, type IV collagen gives the basal lamina tensile strength. But how do the networks of laminin and type IV collagen bond to one another and to the surfacesof the cells that sit on the basal lamina? Why do they form a two-dimensional sheet, rather than a three-dimensional gel? The molecules of laminin have severalfunctional domains, including one that binds to the perlecan proteoglycan, one that binds to the nidogen protein, and tvvo or more that bind to laminin receptor proteins on the surface of cells. Type IV collagen also has domains that bind nidogen and perlecan. It is thought, therefore,

i ntegrins

dystroglycan

penecan

(A)

Figure19-42The structureof laminin. (A)Thesubunitsof a laminin-1molecule, and someof their bindingsitesfor other molecules(yellowboxes).Lamininis a multidomainglycoprotein composedof three polypeptides(s, 0, and y) that are disulfide-bonded into an asymmetric crosslike structure. Eachof the polypeptide chainsis morethan 1500aminoacidslong. Fivetypes of o chains,three types of B chains,and threetypesof "ychainsare known;in principle, they canassemble to form 45 (5 x 3 x 3) lamininisoforms. Severalsuch isoformshavebeenfound, eachwith a characteristic tissue distribution. Throughtheir bindingsitesfor other proteins,lamininmoleculesplaya centralpart in organizingthe assembly of basallaminasand anchoringthem to cells. (B)Electronmicrographs of laminin moleculesshadowedwith platinum. (8,from J. Engelet al.,J. Mol.Biol. -1 20,1981.With permission 150:97 from AcademicPress.)

THEBASALLAMINA

1167

(A)

nidogen

n

n collagen Z\

\

\

I

nt(

=r

',

\

\r'

type lV collagen

i ntegrin

c*"!.*--*+

(' \

\

that nidogen and perlecan serve as Iinkers to connect the laminin and type IV collagen networks once the laminin is in place (Figure f 9-43). The laminin molecules that generate the initial sheet structure first join to each other while bound to receptors on the surface of the cells that produce them. The cell-surfacereceptors are of severalsorts. Many of them are members of the integrin family; another important type of laminin receptor is dystroglycan, aproleoglycan with a core protein that spans the cell membrane, dangling its glycosaminoglycan polysaccharide chains in the extracellular space. Together,these receptors organize basal lamina assembly:theyhold the laminin molecules by their feet, leaving the laminin heads positioned to interact so as to form a two-dimensional network. This laminin network then presumably coordinates the assembly of the other basal lamina components.

BasalLaminaeHaveDiverseFunctions As we have mentioned, in the kidney glomerulus, an unusually thick basal lamina acts as one of the layers of a molecular filter, helping to prevent the passage of macromolecules from the blood into the urine as urine is formed (seeFigure 19-39).The proteoglycan in the basal lamina seemsto be important for this function: when its GAG chains are removed by specific enz)rynes,the filtering properties of the lamina are destroyed. Type IV collagen also has a role: in a human hereditary kidney disorder (Alport syndrome), mutations in type IV collagen genes result in an irregularly thickened and dysfunctional glomerular filter. Laminin mutations, too, can disrupt the function of the kidney filter, but in a different way-by interfering with the differentiation of the cells that contact it and support it.

Figure 19-43 A model of the molecular structureof a basallamina.(A)The basal laminaisformedby specificinteractions (B)betweenthe proteinslaminin,type lV collagen, and nidogen,and the proteoglycanperlecan.Arows in (B) that can bind directly connectmolecules to eachother.Therearevariousisoforms of type lV collagenand laminin,eachwith tissuedistribution. a distinctive lamininreceptors Transmembrane (integrins in the plasma and dystroglycan) membranearethoughtto organizethe of the basallamina;onlythe assembly integrinsareshown.(Basedon H. Colognatoand P.D.Yurchenco,Dev.Dyn. 218:213-234,2000.With permissionfrom Wiley-Liss.)

1 168

Chapter19:Cell Junctions,Cell Adhesion,and the ExtracellularMatrix

The basal lamina can act as a selective barrier to the movement of cells, as well as a filter for molecules. The lamina beneath an epithelium, for example, usually prevents fibroblasts in the underlying connective tissue from making contact with the epithelial cells. It does not, however, stop macrophages, l1'rnphocytes, or nerve processesfrom passing through it, using specializedprotease enzyrnesto cut a hole for their transit. The basal lamina is also important in tissue regeneration after injury. When cells in tissues such as muscles, nerves, and epithelia are damaged or killed, the basal lamina often survives and provides a scaffold along which regenerating cells can migrate. In this way, the original tissue architecture is readily reconstructed. A particularly striking example of the role of the basal lamina in regeneration comes from studies on the neuromuscular junction, the site where the nerve terminals of a motor neuron form a chemical synapsewith a skeletalmuscle cell (discussedin Chapter I l). In vertebrates,the basal lamina that surrounds the muscle cell separatesthe nerve and muscle cell plasma membranes at the slmapse,and the synaptic region of the lamina has a distinctive chemical character, with special isoforms of type IV collagen and laminin and a proteoglycan called agrin. This basal lamina at the synapse has a central role in reconstructing the synapse after nerve or muscle injury. If a frog muscle and its motor nerve are destroyed,the basal lamina around each muscle cell remains intact and the sites of the old neuromuscular junctions are still recognizable.If the motor nerve, but not the muscle, is allowed to regenerate,the nerve axons seek out the original synaptic sites on the empty basal lamina and differentiate there to form normallooking nerve terminals. Thus, the junctional basal lamina by itself can guide the regeneration of motor nerve terminals. Similar experiments show that the basal lamina also controls the localization of the acetylcholine receptors that cluster in the muscle cell plasma membrane at a neuromuscular junction. If the muscle and nerve are both destroyed, but now the muscle is allowed to regeneratewhile the nerve is prevented from doing so, the acetylcholine receptors synthesized by the regenerated muscle Iocalize predominantly in the region of the old junctions, even though the nerve is absent (Figure f 9-44). Thus, the junctional basal lamina apparently coordinates the local spatial organization of the components in each of the two cells that form a neuromuscular junction. Some of the molecules responsible for these effects have been identified. Motor neuron €xons, for example, deposit agrin in the junctional basal lamina, where it regulates the assembly of acetyl-

s h e l lo f r e s i du a basal a

m u s c l ec e l l c u t s ot h a t i t degenerates

REGENERATED NERVE FIBER

t tr junctional i'basallamina

:

(\, DEGENERATE MD USCLE AND NERVE

REGENERATED MUSCLE FIBER

Figure 19-44 Regenerationexperiments demonstratingthe specialcharacterof the junctionalbasallaminaat a rratednerve junction.Whenthe nerve, neuromuscular rcturrrstositeo{ but not the muscle,is allowedto original junction regenerateafter both the nerveand musclehavebeendamaged(upperpart of figure),the junctionalbasallaminadirects nerveto the original the regenerating synapticsite.Whenthe muscle,but not the nerve,is allowedto regenerate(/ower part of figure),the junctional basallamina n e w a c e t y l c h o l i n e causes newlymadeacetylcholine receptorsbecome receptors(blue)to accumulateat the concentratedat originalsynapticsite.The muscle s i t eo f o r i g i n a l regenerates from satellitecells(discussed junction in Chapter23)locatedbetweenthe basal laminaand the originalmusclecell(not shown).Theseexperiments showthat the junctionalbasallaminacontrolsthe localization of synapticcomponentson both sidesof the lamina.

INTEGRINS AND CELL*MATRIX ADHESION

choline receptors and other proteins in the junctional plasma membrane of the muscle cell. Reciprocally,muscle cells deposit a particular isoform of laminin in the junctional basal lamina, and some evidence suggeststhat this binds directly to the extracellular domain of voltage-gated Caz* channels in the presynaptic membrane of the nerve cell, helping to hold them at the synapsewhere they are needed. Both agrin and the synaptic isoform of laminin are essentialfor the formation of normal neuromuscular junctions. Defects in components of the basal lamina or in proteins that tether muscle cell components to it at the synapseare responsible for many of the forms of muscular dystrophy, in which muscles at first develop normally but then degeneratein later years of life.

Summary The basal lamina is a thin tough sheetof extracellular matrix that closely underlies epithelia in all multicellular animals. It also wraps around certain other cell rypes, such as muscle cells.AII basal laminae are organized on a framework of laminin molecules,linked togetherby their side-armsand held closebeneath the basal endsof the epithelial cellsby attachment to integrins and other receptorsin the basal plasma membrane. TypeN collagen moleculesare recruited into this structure, assembling into a sheetlikemesh that is an essentialcomponent of all mature basal laminae. The collagen and laminin networks in mature basal laminae are bridged by the protein nidogen and the large heparan suffate proteoglycanperlecan. Basal laminae prouide mechanicel support for epithelia; theyform the interface and the attachment betweenepithelia and connectiuetissue;they serueasfilters in the kidney; they act as barriers to keepcells in their proper compartments; they influence cell polarity and cell dffirentiation; theyguide cell migrations;and moleculesembedded in them help to organize elaborate structures such as neuromuscular synapses. When cellsare damaged or killed, basal laminae often suruiueand can help guide tissue regeneration.

INTEGRINS ANDCELL_M RIXADHESION Cells make extracellular matrix, organize it, and degrade it. The matrix in its turn exerts powerful influences on the cells. The influences are exerted chiefly through transmembrane cell adhesion proteins that act as matrix receptors. These tie the matrix outside the cell to the cltoskeleton inside it, but their role goesfar beyond simple passivemechanical attachment. Through them, components of the matrix can affect almost any aspect of a cell's behavior. The matrix receptors have a crucial role in epithelial cells, mediating their interactions with the basal lamina beneath them; and they are no less important in connectivetissue cells, for their interactions with the matrix that surrounds them. Severaltypes of molecules can function as matrix receptors or co-receptors, including the transmembrane proteoglycans.But the principal receptors on animal cells for binding most extracellular matrix proteins are the integrins. Like the cadherins and the key components of the basal lamina, integrins are part of the fundamental architectural toolkit that is characteristic of multicellular animals. The members of this large family of homologous transmembrane adhesion molecules have a remarkable ability to transmit signals in both directions acrossthe cell membrane. The binding of a matrix component to an integrin can send a messageinto the interior of the cell, and conditions in the cell interior can send a signal outward to control binding of the integrin to matrix (or, in some cases,to a cell-surface molecule on another cell, as we saw in the case of white blood cells binding to endothelial cells). Tension applied to an integrin can cause it to tighten its grip on intracellular and extracellular structures, and loss of tension can loosen its hold, so that molecular signaling complexes fall apart on either side of the membrane. In this way, integrins can also serve not only to transmit mechanical and molecular signals,but also to convert the one type of signal into the other. Studies of the structure of integrin molecules have begun to reveal how they perform these tasks.

1169

'1170 Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix e x t r a c e l l u l am r a t r i xo r o t e i n

lntegrinsAreTransmembrane Heterodimers ThatLinkto the Cytoskeleton There are many varieties of integrins-at Ieast 24 in humans-but they all conform to a common plan. An integrin molecule is composed of two noncovalently associated glycoprotein subunits called u, and B. Both subunits span the cell membrane, with short intracellular C-terminal tails and large N-terminal extracellular domains. The extracellular portion of the integrin dimer binds to specific amino acid sequencesin extracellular matrix proteins such as laminin or fibronectin or, in some cases,to ligands on the surfacesof other cells.The intracellular portion binds to a complex of proteins that form a linkage to the cltoskeleton. For all but one of the 24 varieties of human integrins, this intracellular linkage is to actin filaments, via talin and a set of other intracellular anchorage proteins (Figure 19-45); talin, as we shall see later, seems to be the key component of the linkage. Like the actin-linked cell-cell junctions formed by cadherins, the actin-linked cell-matrix junctions formed by integrins may be small, inconspicuous and transient, or large, prominent, and durable. Examples of the latter are the focal adhesions that form when fibroblasts have sufficient time to form strong attachments to the rigid surface of a culture dish, and the myotendinous junctions that attach muscle cells to their tendons. In epithelia, the most prominent cell-matrix attachment sites are the hemidesmosomes, where a specific type of integrin (cr6p4)anchors the cells to laminin in the basal lamina. Here, uniquely, the intracellular attachment is to keratin filaments, via the intracellular anchor proteins plectin and dystonin (Figure 19-46).

IntegrinsCanSwitchBetweenan Activeand an Inactive Conformation A cell crawling through a tissue-a fibroblast or a macrophage, for example, or an epithelial cell migrating along a basal lamina-has to be able both to make and to break attachments to the matrix, and to do so rapidly if it is to travel quickly. Similarly, a circulating white blood cell has to be able to switch on or off its tendency to bind to endothelial cells in order to crawl out of a blood vessel at a site of inflammation under the appropriate circumstances. Furthermore, if

Figure19-45Thesubunitstructureof an activeintegrinmolecule,linking extracellularmatrix to the actin cytoskeleton.The headof the integrin moleculeattachesdirectlyto an proteinsuchasfibronectin; extracellular the intracellular tail of the integrinbinds to talin,which in turn bindsto filamentous actin.A setof other intracellular anchorproteins,including o-actinin,filamin,and vinculin,helpto reinforcethe linkage.

'urls jo aql aseostp Duualstlq e ol asrJanr6uet sluauodruo> esau] ;o {ue ur sl)a;a6'uorlrodsnoua6e;;or relnlla)eltxas]tol paq)elteuteuiop p seLlstql lllnx adll 6uruueds-auelquJatl ua6e;1o> laquetu {;rrue;ua6e;;otlpnsnun ue'urrbaluraql qllm ;a;;etedur /suteluo) oslexalduJo)e^tsaqpeeq].,(;te;n;;a:er1xa u r u r u r eoll p u e ' u l u o l s I pp u e u r l > a l d palle)surolordloq)ue etA{pe;n;1ater1ur slueLuelU utlplal ol 6urqre11e 'auerquau aq] sueds (uuOaluttdgn) urr6elurpazrlerreds V'auosousapstuaLl e jo sluauoor.!o)lelnlalow (B).ll aprsursluaueluu!]elal ol llat aLllaptslno u r u r u e 6; u 1 1 u're; u t t u pl le s e qa q l o l s l l a ) saLuosousapttueH (V) lerleqr;deplorvr-tods 'satuosousaptuag 9tr-6 1 ern6;1

'duO a q ] s u a 1 q 661u1r ; ; n dp u e 'paldno:orealn)alouraqt jo spuaatrsoddo sa6ueqr 1e leuorlpruloJuo) :relrursaresa;douudle)tupLl)aur aql lnq 'luala#rperee6e1ur;aql uubalurue ul 'spuaqloq le dll6 aq] lalqbl] aq] Jo slrelapaLll'aln)olour 'sbuo1aql uo taqlabol otuo) o] salpupq ;1ndaql relearbaql'tanoero141 aql sasne)6o; aql uo smel)aqt 6ursoppue :60laql du6 o1sme;r aqt sasnp)reqtabolsolpueqoql 6ulploH's6uollaqull/y-61 a.rn6rg elrlJeur 'paploJ slr q ielnJalou urJ8atul aqr Jo pua JelnllaJeJlur aql lp sluala ot atBIaJ urr8alur aqt Jo uor8al Jelnllacerlxe eql ut se8ueqJ eseql op .e\oq lng

' (S87-6I ern8rC)pup8rl ro;.firurge raq8rq E qtl^l, 'alrs Surpurq elqEJo^BJaJoru p selEaJc uoueruJoJuoo papualxa eq] Jo uoqdope 'dlasranuoc luorlpturoJuoJ .aAIlJe,, 'pepuelxa aqt Jo uoq -dope ro,Le;o] sBos uor8er srqt suotsrp pue3l eqlJo Supurg 'puBBIIrc1nllacerlxe aq] roJ alrs Surpurq aql suleluoJ 'laaru faql eJeq ^ 'uor8ar ppaq eqJ 'sureqJ $ pue n urr8alur eq] ol puodsarroc s8al o^u eql leql sleelar qJIq .r [qder3o1p1 -s-,fuc.,(er-x pelelep ruorJ etep qlr paredruoc aloru A aq ueJ seJnlcnJlsJo rred srql '(Vgt-6I arn8rg; uaq] aloqp qSrq uor8a.rpeaq E Suqroddns 'raqlo qJEa tuo4 paleredas pue paualq8rerls ^ ou eJelnq 'tueq ^pq8ll ra8uol ou are ]Bqt s8al o^ l qlp\'edeqs luaJaJJrpp olur puelxe pue elnoelotu sql putq surJ8eluraql'unrpaur eql o] peppp sr uralo.rd xrJleru JplnlleJeJlxa B urpuop Surpurq leJnleu Jo -urrSalur aqt stpunu leqt acuanbase Surureluoc ap4dad cpaqluzk llprus B JI lng 'spafqo padeqs-npaplog,{pq8u se readde ,(aql ,tdocsorf,rruJoJparedard dprder ueq] pue 'pueBII JelnllaoeJxe .,tue lnoqlplt lnq 'p1ng JulnlleJeJlxa prurou 01 relrrurs runrparu qf,rJ-unrolpc e ur 1da1are surr8alur aqt;1 .{docsoJtnu uoJ}cala Lq uo4nlosar qBH te uaq] Surunuexa pue salnf,aloru urr8alur yo uo4eredard paryrrnd paleJlsuouep e 3urne1 ,{q aq uet surr8alur ur sa8ueqc eqJ IpJnlJnJ}s 'QV6l arntlg) ,{8opue Ipcrueqreu alduns e apnord poo^^Jo s3o1;o ploq qer8 o1 asn slcelraqunl 1eq1s8uo] raqrup eqJ'aueJqruau IIeJ aq] ssoJJeuorlsaJrpJeqlre ur paurusuer] eq UBJsecuanuur leqt os taqlo aq] 1ea8ueqc prnlJnJls o1paydnocsr pue euo 1ea8ueqc IEJnlcnJlS 'alnralol'u eql Jo spua rBlnlleserlxa aql pue relnllaJEJlur aql qloq tJeJp tpql sa8ueqcpuorleruJoJuoc sao8rapun 1r 'spue8rl slr tuoq seqcelap ro o1 spurq uu8 -elur up se :uorleln8al f,rJalsolle sr eueruoueqd crureu^,{p asaq} JoJsrseqaqJ .aprsalrsoddo aq1 uo spue8rl;o les lualaJJrpe o] purq o1flrsuadord Jraql relle ]snu aupJqrueu aql ,aJeJS anrlceur Jo eprs auo uo spue8q Jraql Jo Surpurq aql pue :]ou op ,taql araqm up pue 'sluaurqoenp uro; dlpear.(aql araqar 'alpls eArlJBuE uaalAlaq qolrms ol elqp eq 1snru,{aq1'spua olrr}Jraql }e saqclud dlcr}s q}lm slcafqo pr8rr 'anrssedaq .{ldrurslouueJ sluaruqJe}le aql elerpeu pue eueJqrueu aq} ueds }eq} salnJalou utr8alur eql 'llal eql eprsu slueruqoeue p1a1a1so{c ;o .{lqruassusrp pue .{.lqruasse ldruord aqt ot peldnoo aq o} seq saseoasoq} [p ut s]uerxqJene Jelnllaf, -Erlxa eql Jo Suqearq pue Suqeu eql 'pepaau sr ll erer{rvrpalldde aq o} sr eJJoJ (8)

uabello)

ululuel -

1 ; 1 yu e 6 e 1 1 o t

(rden)

(v)

euruel lPseq aurosouJsepr uraq s l u a u e l r ,u r l e l a l lla) lerlor_l]roa

LLLI

NOIS]HCV XIU.LVW-II3f CNVSNIU9]TNI

'spue8rt s]I o] uIrBe]uI uP Surpurq eql Jo lrlllJlcads aql pue {IIUIJJB Jo eql qloq ecuenuur uP3 suollec luelE^lp 3qI'sllunqns d pue n eql uI sumluop Surpurq-uorleJ-luelplrp Jo acueseJd aql SuplegaJ'Iunlpatu JPInlleJEJlxa pue +zBJJo uollErlueJuot eqt ^q palcaJJBoslEsI spuPBIIJlaql ol surr8 aql ul +ZBIN -alur Jo Surpurq eqJ 'dtr rlce Surpurq Jleql alelnpolu ol sulJ8elul qll A lsBrelul (seuIJIJIcedsSurputq-pue uef, sJolJEJJrJrJads-ad&-lleJIeuorllppe reql stueestl -3q luara;;rp aAEqupc sed& ilec luaJaJJIpuI elnJalotu ulJ8elul arues eq] asnef,eq 'suolleulqruoJ teAoeJoIN'suoqcury pue seluedoJd elllcullslp seq uIJBeluIqouA 'seua8 uIEqJ-?oluaJeJJIp puE seue8 UIEqJ-$ tueragp lualaJJrp uI pezrJeurp 8I g Jo slJnpoJd aq] ruor; peruJoJ eJe upunq B uI punoJ suIJBeluI;o sadft vz eq;-

saseas!c ary sl)aJaoulJ6e}ul {ue6 roJalqlsuodseS )llauaDtuaroJJlo 'luetuuorl^ua IPcrueqc pu? IPcrs^qd slr puu IIaJ aql ueeMleq suollJBralul xeldluoJ eruos JoJ 's[eru. s^ ollp 'eueJqr.ueurflac eql ssoJJBsuopcaJlp q]oq ut slBuBISSupllusuerl -qled uouecrunruurof, asaql ile uaa^ leq IIEI-ssoJc aql 'srte,uqted raqlo .{q spu -3rs;o uorldacar aq] eouenlJul uer xIJlEru o] lueuqcu]1e dq suu8atul Jo uone^ -pJB aql {lasranuo3 '(6?-6I arntlg) uoI}PAIlJe uu8alul IoJ}uoJ snql ueJ qJIq ^ 'sesuuDlaursorr! JoldeJal puu sroldacar paldnoc-ulalord-g sB qJns 'sroldacal aJeJJns-llef,Jo sad^,ilJaqlo eIA IIaJ eqt aplslno Iuo{ pallef,ar spu8ts o} asuodser ur pacnpord sellasuaql eJE zdld sB qJns salnleloru puBrs relnlaf,EJlul 'spue8u qJeer ol salncaloru utr8 qer8 pue ploq rleqlJo rplnlleJerxe lno -elur slr raSSrrl ueo IIaJ eql eprsu paleraua8 pu8ts e 'z(earr sql u1 l.13uor1suruqc fl urr8alur eqt ot spurq tl ]eqt os urpl 3u11e4lce;o alqedec eq ol lq8noqr sl qrlqm '(gI raldeql ur pessnJslp) zdld epnlsouroqdsoqd aq] apnlsul esaql'seFceloru Lroleln8ar JplnllaoeJlur dq paraSStrl st ,.uoIlEAIlJE tno-eplsul,, slqJ 'uorleruJoJuoJa^Ilce'pspuaxe sll olur urr8alur eqlJo uoluod relnlacerye aql se^lJpsrql'pede Surrds ol elnf,eloru urrSalur aql Jo s3a1 orvrl aql Sul1v\olle 'a3e1uq $-n replaceJlul eql seopun 'uIPq3 ll ulEqc fl aqr or spulq ulel ueq1v\snql fl aqr 1o Ie] eq] uo alIS Surpurq 'eplsut o] aplslno slr JoJureqJ n uu8elul eql qlp saladruoc ulleJ Jo pealsul epls -lno ol eprsur ruo4 'asJaAeJuI alEJedo osl? UEJ]f,aJJepue esnec Jo urcqc eqJ ,.uoll?All3e ul-aplslno,, se o] peJJaJarsI sIqI 'elnJalou utrBalut aq1ol ']uaruqcelle;o lurod aqr re paldde eq uel acJoJleqt os uolayalsolb s1r8ur,$.{q s}rEarIIac aql 'lleJ aql epls}no puuSu s}IJo ploq seqf,}Bc sql uI '(gt-6l arn8tg eas)elnJeloru urr8alur eqrJo pue JEI urr8alur up ueqm de,nn -nllef,BJtur aql o] peroqf,ue slueluelg ullJe Jo ,(lquasse 01 speel uaql ulet;o 3u1 Ip]l slaqs!lqnd (llnsar E sV uellrul)ewurolJuotsstullao qltM -pqq eqJ'pasodxa sr ureqr fl aqtgo IIul aq] uo ullet JoJalls Sutputq e 'v002'19-6s:zwarruqN"le oerx 'uede anoru sureqJ asaqlJo suoruod (aueJqluausueJ] puu) JEInllef,PJluIeql pue le 1 uo paseq'8 :rar^oslfulolJ uorssil.llad ueIoJq sl l331uo3 slql 'sploJun uIPuIop JelnlleJEJlxa eql uel{ \ raqlous euo ol qilM'2002'tL9-66s:0tt ilD "P 'V) 'sluau.rberj eJeqpupuu raqlaSot asoloeII suleql $ pue n sllJo suoluod replacerlul eql'alels reln)alour lo rOplel'f LuoU pauUapJo {qder6o;1e1sfut Ie.r-xr,uo.r1 a)uapuuo) alqeuoseorqll/\/^paJleJu!aq 6 u r p u r qu r l e 1 ue) ernlfnJlsa1a;dr,uor aqt'punoq pue6r; 6uo.r1s lnoqllM pue LlllM'suorleLuro]uo) lernleu sll ul alnfaloull)elul oql azrl;e1s,(tr ol lln)UJrpsrrr qbnoql;y'Iqder6o11e1s,(lr {er-x r.uor;e}ppuo paseq'aln)alouJ uubaturue Jo sarnl)nrls(paplol)a^rl)eul pue (popualxa)enlrrv (g) 'sballrurlsrp oMl qlrn/\arnl)nlls popualxaue olul c9up;o;unsur.r6a1u! eql'aprldadO9Uue qlllvr palpqnluruaqpl'pap1o; {llLl6ttpue lleLus reeddesalnra;ouurrbaluraql'puebr; relnlla)ertxa,oafuasqeoLllul 'saln)alour urr6alurlenprnrpur Jo sqdel6o.r>rru uorllelepau6rleI;re;rurs,(ueLu 6urOerane Iq paxpord aramsebeu; (V)'pue6!l slr spurqll uaqmaln)alou.r ulr6elu;ue;o uolleulroluo) ur a6ueql Bt-6 ! ornbrl x fJ l e w l e l n l l a ) e J l x fa q l p u e ' u o l s a q P Vl l a ) ' s u o l D u n r l l a ) : 61 r a l d e q 3

zLLl

'patsrl arespueOrl 1op* 11e

oslPellaqllde Jaqto osleetlaqllde laqlo elleql!oau! u! sllajap 16uua5;;q ulxsere^as u! slro;epl6urrals;;q utls arales sauosor.usaplu.Jaq tdgn slsorladoalso pFLul(aseestp s,uueuzuelg) (aseasrp tuueuzuelg) uorle6ar66e ou l6urpealq slaloleld uaOougrqr; edqtrc ]aleleldou 16urpaa1q uo;teOar66e 1a;a1e1d suorDoJur 6urualeerqt-a;!l (wvlt) lua;ln)ol isasuodsar rfuoleuuregurpaJtedull sey(rornal srolda)ailaluno) (tvll) (6y1){ruarrgapuotsaqpeafiro>na1 slla)poolqalrqM ,{1tue;radns 61 Zdln Jolualrtlrn]larperredur (uoltelueldu! suorl>unfsnourpualo{u.r a^rllalap1{qdorls{prelnf,snur le) o^rqureJo qleap{pea al)snu ururulel dtn (uollelueldul os;ee;;aq1de reqlo ge)o{rqua;o qleap{1rea ur sllalap l6uualsrlqulls era^as snolrnbrqn uruil.Ilel [Bgp (uorlelueldur lseJ)leJnau'salluos'slasseA poolq ul spalapioliqure Jo qleep le) o^rquleJo qleap,!rea snolrnorqn urlrauorq$ tggn

sugr6alu;1o sad{1 auos t-6 t elqel '(uu8alur 1$1n aql rog pue8n ururruel aq] a{eu }ouueJ teql ecru op se) ,{qdoJlsdp repcsmu dolanap tnq e^r rns (elf,snur ur 1$ ro; rauped aqt; tlunqns Zn aql eleru ol alqeun l.1uo are '(luaurdolanap cruo.{rqrua dlrea ,{ran) uonelueldurr 1e arp surrSalur ur ]eql aoIIAI 'slleo sad,fi .,(ueruuo roldacar ururuel 1fl fuu aTeru louuec leql eJlru ]ue]nntr Jo B Idgn pue roldacar urtceuorqg e sr 1f,9n :sllal eterqeuel IIe lsourle uo punoJ are pup slrunqns ?o qtlm sraurp tuJoJ slrunqns 1fl aql tJulisp tspal le ZI 'e^IlJaJep aJe sureqc ro n urr8alur fl lenpr rpur uaqm llnsal ]eql sruel -qord aqr pue suu8alurJo sarlarJelJo auos slsl ?-6I alqul'alqelJetueJ eJoru sr ]eql suollJury urr8alur;o dllsranrp aql s1t1-alduexe roJ 'urunuel purq a^U lseel ]e-surr8atul tuareJJrpeqtJo seur llce aqt ur delrano eruos sr areql q8noqtry

'suu6alur qle) pue aqr alenrlfe Iqaraqr urlelJouotteuloJuol aql ialle ue)'saseutl autso.tll .roldarar pues.roldara.r pe;dnor-ura1o.rd-9 seqrns'stoldatalate;.tns lla),o sad{1raqloern 's{emqled6u;;eu61s s;eu615 ,aqlo uro.rJ lla)aql aprslnourorjpa^re)ar llel-sso.p{q su1r6e1u1 lo uo!le^!})v69-6g a.rn6r3 u r l e la ^ r p e u l

r+ luourel u!De

u r l e la ^ r p e

rolde)ar peldnol -ura1old-9anrpe

suu6alur enrpe

ELLI

NOIS]HCV XIUIVW_II]fONVSNIUD]INI

'ool aJeq saleJedo teIIJBa pauop Jo Suualsnlo uo spuadap uorsarlpe 3uor15 -uau 'eldrJuud oJJIeAeqJ 'aJEJrnsIIaJ aql uo uolleJluaJuoc JaqBIq ploJ-O0I ot -0I e ]e luasard ,r{1pnsnarB puE .{1rutJ;era^ ol qlu^ puP8n rleql pulq,{1ensn daql leql ur salnJelotu IEUBISelqnlos JelnlleJerua Jaqlo JoJpue sauoruJoq roJ sroldaJal aJEJrns-llacluo{ raJJIp'salnf,eloruuolsaqpP IIaJ Jeqlo e{II 'suu8alul

6uor15ulol ot rolsnl) sulroelul suorsaqpv 'elels aAIlJeuIue ol uJnlal ueql .{etu suu8 'a8esuasrpol IIaJ I aqt Sulmolle -etur aql f1ry patulnunrlsauoJeq o1 q8noua 3uo1tcetuoc uI surcrueJ1l leq] os IIac Surluasard-ua8rlueaqt o] 1{lSuoJtseraqpE ot IIaJ I aql alqeue uaq] suIJBaluI pelelrtre aql'(09-6I arn81g)sulr8atul 7f, s1talenqce leql flac J aq] uI s-,(e^,r,tqled Sureu8rs JelnllaoeJturuo saqJtlms (g7 raldeq3 uI passnJslp) 1ac Surluasard -ua8rlup up Jo af,EJJnsaq] uo uaBIluP cgrcads sll ol eldcoqdru,(11e 3o Surpurq eq] -{lrplnurs 'papaau sr tl ereq ^ atISeq} lsnf }e Surpaalq aql do}s ol sdlaq qrlqm '3n1d u ;o uorteruroJ ol spuel uaSouuqlJ qtlm slalaleld Jo uotlceralut 1a1a1e1d aq] uI ulr8alur g$ aqt Jo uollelllce 3utra83ul Suqpsar aqJ 'auerquaur 1a1a1e1d 'salnJaloru 3utleu8rs alqnlos snoIJeAo] pue Iesse^poolq pa8eruep E Jo Ile.treq] 'prder aq uec esuodsal uolsaqpe qllm lJuluoJ o1 puodsar 'aldurexaro; 's1a1a1e14 'snp papu8rs eql'otlou ap paz$aqlu.,{saq o1 paau lou op suIJBaluIaq} esne3ag -rurts alerrdordde ue -,i.qpalentlee are.{aqt p}un papadrulun a}EInJJIJo} sllao aql st Sulpu8ts }no-aplsul el^ Ill s^aolleuorsaqpe paleln8ag 'lueuodul{lrepc4red -nrlce aqr 'slalateld pue sllac poolq a}q^ qloq uI o1 ^,{1qrqe urrBatur aleln8ar paalq pue 8ur}}olc a^lpeJap uorJ JaJJns'sutr8a}ut ,{.1a.ttssacxa g$ ur luarcr;ap ,{lpcrlaua8 aJBoqm 'asaasrps,uuDwzualt q}IM suelunq pue '3ur1 -]olc qllm ol aleq slalaleld 'uaSoutt uaSoutrqr; poolq o1 alerpau tJeJalul IEruJou 'suralord xlrleur -rpt poolq aq] Sutpnlcut tonel Suutolc IEraAesputq,{aqr pue '(sllac Jaqlo snoIJeAsp poolq uo punoJ are surr8alur td aqJ IIe^ se) s1a1a1e1d

aIlrua eqrtJifi:X"i::\?Tffifflt:1iil:"qJ*:: pue'sroldacar .,raqr 7g1o.{'ueJ

-uoJ e sV 'slrunqns 7fl puorlcun; aztsaqluz(so1ye1ftuan{ap uorcaqpaaltcotnay paller aseaslpcrlaua8 eq] qll A a1doa4'(S6I-6I arn8tg aas) enssll palJeJul aq] 'pue uoIlJaJuI olur uearlspoolq aqt Jo ]no aler8tru ol 'tueruqJepe slq] qBnorql eq] o] ,(1urg qce]1e o] Jo satrs tE sllar Iurlaqlopua uo WVII utatord 't11ure1-31 '7fl1n; sselc srq];o up8 uaq] salqeua sllar poolq atlq^ uo (IVC'I sP u^touI osp -alur uE :raldeqc aq] uI JaIIreaaldurexaue paqlJcsapdpearp aleq eAA'sa1nJeloru 'sJoldaJalnlunoJ se uorsaqpe IIaJ-lla3 3o ,,(prue;radns 31 aqt Jo sJeqtualu eJe 'llal Jeqloue uo ol paJJaJaJ sarurtauros'spue31 aqJ 'lleJ Iellaqlopue ue se qons spue8rTcr;rcadsot Surpurq 'suollJeJalul xlJleru-lleJ ueql Jaqler IIaJ-lleJ alelparu .,(preru surr8atur Zd aqJ 'uollraJul tq8g o1 sllec asaql 3u11qeuauI alor Ielluessa ue aleq .{aqt araq.tr 'sllal poolq atlqm Jo acPJJnsaq} uo flantsnlcxa passardxa are pue llunqns n;o sadfi JnoJ lseal lE l{}Illt sJaIuIp llIJoJ s}Iunqns Zg eqJ

( ' 8 0 0 2 ' a ) u a r )psu e l r e 9 :lro^ MaN'pa ql/ 'IbololqounurLrrl s,i(eMouef "1e1aIqdrn6i y nror;petdepy)'asuodsar aunurrul >r;oadse lor;e {qeleqi pue lla) f aql alpAr])eo] aurt lla) 6urluasard-uabrlue lAVfI 01 A'r9NOU.LS l u o t A s c N t SN t u 9 l l - N t t v l ' l aql santbslql'WV)l ol {llua}srsrad pue r(16uor1s olorrlspurqlVl'l'llnsar e sV uubelur tVl-l or.llalp^rlre o] rolda>ar lla) I aql uor; patereua6sr ;eubrs r e l n l l a l p r l uure ' 1 ; a6>u r l u a s a r d - u a 6 r t u e aql uo aln)aloLu IHW aql Iq rl or paluasard'uabrluerrlr>ads sll sazlu6otar aurl aues eqt te roldatal lla)-1oq] rl ';;ar6urluasa.rd-ua6rlue aq] Jo auerquraul aql ul WVfl aln)aloulr{1tLue;tadns-61 a q t o ] l l a )f a q t u r u u 6 e l u r! V l l a q l 1o6urpurqqbnorqt{;1eamaraqpe}srU lp slla)oM] aLlf'lla) 6ugluase,rd-ua6r1ue ue ql!/!rey(roqdull f e ro ralunorua aql ur uorle^ll>eu1r6a1u; 65-6 1 arn611

Nru9rtNl lvll llv^ll-)v o-L ] V N 9 l S S C N I SU O l d l ) l U l l l ) - I 8 N O l l - 1 N 9 0 ) l UN l 9 l l N V +

iolda)ar

lla)-l

tAV)ror- Nlu9llNl t v j t l o 9 N t c N t S) v l ^ 1 1 a6: u r l u a s e l d - u a b t l u e

'''i"tti *u'* il

ff--%#"*

lla) J-

'suoll)unf xplew relnlle)erlxfeql pue'uotseqpV lla) :61taldeq3 VLLL lla)

1175

INTEGRINS AND CELL*MATRIX ADHESION

integrins, creating a plaque in which many cytoskeletal filaments are anchored, as at a hemidesmosome in the epidermis or at a focal adhesion made by a fibroblast on a culture dish. At focal adhesions, and probably also in the Iess prominent actin-linked cell-matrix adhesions that cells mainly make in normal tissues,activation of the small GTPaseRho plays a part in the maturation of the adhesive complex, by promoting recruitment of actin filaments and integrins to the contact site. Artificially mutated integrins that lack an intracellular tail fail to connect with cytoskeletal filaments, fail to cluster, and are unable to form strong adhesions.

Extracellular MatrixAttachments ActThroughIntegrinsto ControlCellProliferation and Survival Like other transmembrane cell adhesion proteins, integrins do more than just create attachments. They also activate intracellular signaling pathways and thereby allow control of almost any aspect of the cell'sbehavior according to the nature of the surrounding matrix and the state of the cell's attachments to it. Studies in culture show that many cells will not grow or proliferate unless they are attached to extracellular matrix; nutrients and soluble growth factors in the culture medium are not enough. For some cell t1pes, including epithelial, endothelial, and muscle cells, even cell survival depends on such attachments. lVhen these cells lose contact with the extracellular matrix, they undergo programmed cell death, or apoptosis.This dependence of cell growth, proliferation, and survival on attachment to a substratum is known as anchorage dependence, and it is mediated mainly by integrins and the intracellular signals they generate.Anchorage dependence is thought to help ensure that each type of cell survives and proliferates only when it is in an appropriate situation. Mutations that disrupt or override this form of control, allowing cells to escape from anchorage dependence, occur in cancer cells and play a major part in their invasive behavior. The physical spreading of a cell on the matrix also has a strong influence on intracellular events. Cells that are forced to spread over a large surface area by the formation of multiple adhesions at widely separate sites survive better and proliferate faster than cells that are not so spread out (Figure f 9-5f ). The stimulatory effect of cell spreading presumably helps tissues to regenerate after injury. If cells are lost from an epithelium, for example, the spreading of the remaining cells into the vacated spacewill help stimulate these survivors to proliferate until they fill the gap. It is uncertain how a cell sensesits extent of spreading so as to adjust its behavior accordingly,but the ability to spread depends on integrins, and signals generated by integrins at the sites of adhesion must play a part in providing the spread cells with stimulation. Our understanding of anchorage dependence and of the effects of cell spreading has come mainly from studies of cells living on the surface of matrixcoated culture dishes. For connective-tissue cells that are normally surrounded

a defined amountof f i b r o n e c t i ni n s i n g l ep a t c h

C E L LD I E S BY APOPTOSIS

tl the same a m o u n to f fibronectin d i s t r i b u t e di n s m a l ls p o t s

fall

C E L LS P R E A D S , SURVIVES, AND GROWS

aJat tc 50 pm

Figure19-51The importanceof cell spreading.In this experiment,cell growth and survivalareshownto dependon the extentof cellspreadingon a substratum, ratherthan the merefact of attachmentor the cell the numberof matrixmolecules contacts.(Basedon C.S.Chenet al., 1997.With Science276:1425-1428, permissionfrom AAAS.)

1176

Chapter19:Cell Junctions,Cell Ad:hesion,and the ExtracellularMatrix

by matrix on all sides, this is a far cry from the natural environment. Walking over a plain is very different from clambering through a jungle. The ty?es of contacts that cells make with a rigid substratum are not the same as those, much less well studied, that they make with the deformable web of fibers of the extracellular matrix, and there are substantial differences of cell behavior between the two contexts. Nevertheless,it is likely that the same basic principles apply. Both in uitro and in uiuo, intracellular signals generated at cell-matrix adhesion sites, by molecular complexes organized around integrins, are crucial for cell proliferation and survival.

IntegrinsRecruitIntracellular SignalingProteinsat Sitesof Cell-Substratu m Adhesion The mechanisms by which integrins signal into the cell interior are complex, involving several different pathways, and integrins and conventional signaling receptors often influence one another and work together to regulate cell behavior, as we have already emphasized. The Rasi MAP kinase pathway (see Figure 15-61), for example, can be activated both by conventional signaling receptors and by integrins, but cells often need both kinds of stimulation of this pathway at the same time to give sufficient activation to induce cell proliferation. Integrins and conventional signaling receptors also cooperate in activating similar pathways to promote cell survival (discussedin Chapters 15 and 17). One of the best-studied modes of integrin signaling depends on a cltoplasmic protein tyrosine kinase called focal adhesion kinase (FAK). In studies of cells cultured in the normal way on rigid substrata, focal adhesions are often prominent sites of tyrosine phosphorylation (Figure f g-5ZA), and FAK is one of the major tyrosine-phosphorylated proteins found at these sites. \A/hen integrins cluster at cell-matrix contacts, FAK is recruited by intracellular anchor proteins such as talin (binding to the integrin B subunit) or paxillin (which binds to one type of integrin a subunit). The clustered FAK molecules crossphosphorylate each other on a specific tyrosine, creating a phosphotyrosine docking site for members of the Src family of cytoplasmic tyrosine kinases. In addition to phosphorylating other proteins at the adhesion sites, these kinases then phosphorylate FAK on additional tyrosines, creating docking sites for a variety of additional intracellular signaling proteins. In this way, outside-in signaling from integrins, via FAK and Src-family kinases, is relayed into the cell (as discussedin Chapter l5). One way to analyze the function of FAK is to examine focal adhesions in cells from mutant mice that lack the protein. FAK-deficient fibroblasts still adhere to

Figure 19-52 Focaladhesionsand the role of focal adhesionkinase(FAK). (A)A fibroblastculturedon a fibronectincoatedsubstratum and stainedwith fluorescent antibodies: actinfilamentsare stainedgreenand activatedproteinsthat containphosphotyrosineare red,giving orangewherethe two components overlap.The actin filamentsterminateat focaladhesions,wherethe cell attaches to the substratumby meansof integrins. Proteinscontainingphosphotyrosine are alsoconcentratedat thesesites, reflectingthe localactivationof FAKand other proteinkinases. Signalsgenerated at suchadhesionsiteshelp regulatecell (8,C)The division,growth,and survival. influenceof FAKon formationof focal adhesions is shownby a comparison of normaland FAK-deficient fibroblasts, stainedwith an antibodyagainstvinculin to revealthe focaladhesions.(B)The normalfibroblastshavefewer focal adhesions and havespreadafter2 hours in culture.(C)At the sametime point,the FAK-deficient fibroblastshavemore focal adhesions and havenot spread. (A,courtesyof KeithBurridge;B,C,from D. llic et al.,Nature377:539-544,'1995. With permission from Macmillan Publishers Ltd.)

n o r m a lf i b r o b l a s t s

(B)

1 0p m

FAK-deficientfibroblasts

50 pm

1177

IN T E G R NISA N D C E L L _ M A T RAI X DHESION

fibronectin and form focal adhesions. In fact, they form too many focal adhesions; as a result, cell spreading and migration are slowed (Figure 19-528 and C). This unexpected finding suggeststhat FAK normally helps disassemble focal adhesions and that this Ioss of adhesions is required for normal cell migration. Many cancer cells have elevated levels of FAK,which may help explain why they are often more motile than their normal counterparts.

IntegrinsCanProduceLocalized Intracellular Effects Through FAK and other pathways, activated integrins, like other signaling receptors, can induce global cell responses,often including changes in gene expression. But the integrins are especially adept at stimulating localized changes in the cytoplasm close to the cell-matrix contact. We have already mentioned an important example in our discussion of epithelial cell polarity: it is through integrins that the basal lamina plays its part in directing the internal apico-basal organization of epithelial cells. Localized intracellular effects may be a common feature of signaling by transmembrane cell adhesion proteins in general. In the developing nervous system,for example, the growing tip of an axon is guided mainly by its responses to local adhesive (and repellent) cues in the environment that are recognized by transmembrane cell adhesion proteins, as discussedin Chapter 22.The primary effectsof the adhesion proteins are thought to result from the activation of intracellular signaling pathways that act locally in the axon tip, rather than through cell-cell adhesion itself or signals conveyed to the cell body. Through localized activation of the Rho family of small GTPases,for example (as discussed in Chapters l5 and 16),the transmembrane adhesion proteins can control motility and guide forward movement. In this and other ways, practically all the classes of cell-cell and cell-matrix adhesion molecules that we have mentioned, including integrins, are deployed to help guide axon outgrowth in the developing nervous system. Table l9-5 summarizes the categories of cell adhesion molecules that we have considered in this chapter. In the next section, we turn from the adhesion molecules in cell membranes to look in detail at the extracellularmatrix that surrounds cells in connective tissues. T a b l e1 9 - 5 C e l lA d h e s i o nM o l e c u l eF a m i l i e s OR HOMOPHILIC HETEROPHILIC

homophilic

actin filaments(via catenins) intermediatefilaments (viadesmoplakin, p l a k o g l o b i na, n d plakophilin)

junctions, adherens synapses oesmoS0mes

N-CAM,ICAM no

both

unKnown

yes L-,E-,and P-selectins

heterophilic

actin filaments

n e u r o n aal n d immunologicalsynapses (no prominentjunctional structure)

yes

heterophilic

actin filaments

manyrypes

yes

heterophilic

a6B4

yes

heterophilic

syndecans

no

heterophilic

focal adhesions actin filaments(via t a l i n ,p a x i l l i nf,i l a m i n , u - a c t i n i na, n d v i n c u l i n ) hemidesmosomes intermediatefilaments (via plectin and dystonin) (no prominentjunctional actin filaments structure)

yes

homophilic

desmoglein, yes desmocollin

lg family members Selectins(blood cells a n d e n d o t h e l i acl e l l s onty)

E,N,BVE

I n t e g r i n so n b l o o d c e l l s gLB2(LFA1)

immunologicalsynapses

Cell-Matrix Adhesion Integrins

Transmembrane proteoglycans

1'178 Chapter19:CellJunctions, CellAdhesion,and the Extracellular Matrix

Summ a r y Integrins are the principal receptorsused by animal cells to bind to the extracellular matrix: theyfunction as transmembranelinkers betweenthe extracellular matrix and the cytoskeletonconnecting usually to actin, but to intermediateftlaments for the specialized integrins at hemidesmosomes.Integrin moleculesare heterodimers,and the binding of ligands is associatedwith dramatic changesof conformation. This creates an allosteric coupling betweenbinding to matrix outside the cell and binding to the cytoskeletoninside it, allowing the integrin to conueysignals in both directions across the plasma membrane-from inside to out and from outside to in. Binding of the intracellular anchor protein talin to the tail of an integrin molecule tends to driue the integrin into an extendedconformation with increasedffinity for its extracellular ligand. Conuersely,binding to an extracellular ligand, by promoting the sameconformational change, leads to binding of talin and formation of a linkage to the actin cytoskeleton.Complex assembliesof proteins becomeorganized around the intracellular tails of integrins, producing intracellular signals that can influence almost any aspectof cell behauiorfrom proliferation and suruiual, as in the phenomenonof anchoragedependence,to cell polarity and guidance of migration.

THEEXTRACELLULAR MATRIX OFANIMAL CONNECTIVE TISSUES We have already discussed the basal lamina as an archetypal example of extracellular matrix, common to practically all multicellular animals and an essential feature of epithelial tissues.We now turn to the much more varied and bulky forms of extracellular matrix found in connective tissues (Figure 19-53). Here, the extracellular matrix is generally more plentiful than the cells it surrounds, and it determines the tissue'sphysical properties. The classesof macromolecules constituting the extracellular matrix in animal tissues are broadly similar, whether we consider the basal lamina or the other forms that matrix can take, but variations in the relative amounts of these different classesof molecules and in the ways in which they are organized give rise to an amazing diversity of materials. The matrix can become calcified to form the rock-hard structures of bone or teeth, or it can form the transparent substance ofthe cornea, or it can adopt the ropelike organization that gives tendons their enormous tensile strength. It forms the jelly in a jellyfish. covering the body of a beetle or a lobster, it forms a rigid carapace.Moreover, the extracellular matrix is more than a passive scaffold to provide physical support. It has an active and complex role in regulating the behavior of the cells that touch it, inhabit it, or crawl through its meshes, influencing their survival, development, migration, proliferation, shape, and function. In this section, we focus our discussion on the extracellular matrix of connective tissues in vertebrates, but bulky forms of extracellular matrix play an epithelium

f * basal amina

I I Co

r

* * c o l l a g e nf i b e r l

macrophage f

capillary U

e l a s t i cf i b e r

fibroblast

m a s tc e l l

F U

z z

h y al u r o n an , proteoglycans,and glycoproteins l

50pm

Figure 19-53 The connectivetissue underlyingan epithelium.Thistissue containsa varietyof cellsand extracellular matrixcomponents. The predominantcell type is the fibroblast,which secretes abundantextracellular matrix.

THEEXTRACELLULAR MATRIX OFANIMALCONNECTIVE TISSUES

1179

important part in virtually all multicellularorganisms;examplesinclude the cuticlesof worms and insects,the shellsof mollusks,the cellwallsof fungi,and, aswe discusslater,the cellwalls of plants.

TheExtracellular Matrixls Madeand Orientedby the Cells Withinlt The macromolecules that constitute the extracellular matrix are mainly produced locally by cells in the matrix. As we discuss later, these cells also help to organize the matrix the orientation of the cltoskeleton inside the cell can control the orientation of the matrix produced outside. In most connective tissues, the matrix macromolecules are secreted largely by cells called fibroblasts (Figure 19-54). In certain specialized tlpes of connective tissues, such as cartilage and bone, however, they are secreted by cells of the fibroblast family that have more specific names: chondroblasfs, for example, form cartilage, and,osteoblasts form bone. The matrix in connective tissue is constructed from the same two main classesof macromolecules as in basal laminae: (1) glycosaminoglycan polysaccharide chains, usually covalently linked to protein in the form of proteoglycans, and (2) fibrous proteins such as collagen.We shall see that the members of both classescome in a great variety of shapes and sizes. The proteoglycan molecules in connective tissue tlpically form a highly hydrated, gel-like "ground substance" in which the fibrous proteins are embedded. The polysaccharide gel resists compressive forces on the matrix while permitting the rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells.The collagen fibers strengthen and help organize the matrix, while other fibrous proteins, such as the rubberlike elastin, give it resilience. Finally, many matrix proteins help cells migrate, settle, and differentiate in the appropriate locations.

10lt. in connective Figure19-54 Fibroblasts tissue.Thisscanningelectron micrographshowstissuefrom the cornea of a rat.The extracellularmatrix is here the fibroblasts surrounding composedlargelyof collagenfibrils.The glycoproteins, and hyaluronan, proteoglycans, whichnormallyform a of the hydratedgel fillingthe interstices fibrousnetwork,havebeen removedby enzymeand acidtreatment.(From T. Nishidaet al.,lnvest.Ophtholmol.Vis. Sci.29:1887-'l 890,1988.With permission from Associationfor Researchin Vision and Opthalmology.)

(GAG)ChainsOccupyLargeAmountsof Glycosaminoglycan Spaceand FormHydratedGels Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units. They are called GAGs because one of the two sugars in the repeating disaccharide is always an amino sugar (-0y'-acetylglucosamine or l/-acetylgalactosamine), which in most casesis sulfated. The second sugar is usually a uronic acid (glucuronic or iduronic). Becausethere are sulfate or carboryl groups on most of their sugars, GAGs are highly negatively charged (Figure l9-55). Indeed, they are the most anionic molecules produced by animal cells. Four main groups of GAGsare distinguished by their sugars,the ti,pe of linkage between the sugars, and the number and location of sulfate groups: (l) hyaluronan, (2) chondroitin sulfate and dermatan sulfate, (3) heparan sulfate, and (4) keratan su$ote. Polysaccharide chains are too stiff to fold up into the compact globular structures that pollpeptide chains typically form. Moreover, they are strongly hydrophilic. Thus, GAGs tend to adopt highly extended conformations that occupy a huge volume relative to their mass (Figure f 9-56), and they form gels

cooc

cH2oso

cooc

N - a c e t y l g l u c o s a m i n eg l u c u r o n i ca c i d r e p e a t i n gd i s a c c h a r i d e

cH2oso

FigureI 9-55 The repeatingdisaccharide sequenceof a heparansulfate glycosaminoglycan(GAG)chain.These chainscan consistof as manyas200 units,but aretypicallyless disaccharide than halfthat size.Thereis a high density of negativechargesalongthe chaindue to the presenceof both carboxyland sulfategroups.The proteoglycansof the and basallamina-perlecan,dystroglycan, collagenXVlll-all carryheparansulfate The moleculeis shownherewith its GAGs. maximafnumber of sulfategroups.ln vivo, the proportionof sulfatedand groupsis variable.Heparin nonsulfated sulfation,while typicallyhas>70o/o heparansulfatehas a r repu l I ; u o urelord VSgl qr€g 'saua8 (VSAD eseqlu,{sasolnllac a}erpdeseerq} Jo stcnpord ale6uolaot lla) q)ea smolle6utuosoolllpM uralord aql surEluoJ pue .{r]etulu,,(spyog-xrs2 seq'ailasoJ ro 'xelduroJ aurtzua lla)'suorl)alrplle ut uloJtunsratnssard qJeE 'losotdc eqt ruo{ perrddns asornlS-dcfl aprtoalcnu r€3ns eqt a}erts to6tni q6noqt;V'slle/v\ raql ur s|rqUor)rur - q n s s l ! s Ps a s nq J r q M' ( a s E q l u ^ se s o l n l l a Jx) a l d u o J a u ^ z u a p u n o q - a u B J q t u a l u asolnlla)Josuolleluauolau tuarajJlpq]!M 'sletuluE -eusEld lnq (saqn)seoiaq uMoqs)sadeLls uI ueuornle.{q ,r(q le)tluapl aql e 1ac Jo aJEJJnsaql luoq lno unds sI qllMJJouers (8)pup (v) ur sllaraql ()'8) 'asolnllaf, 'pa}elf,as puB pue a{rl ale sn}EJpddp rBIoC runlnJr}eJ srruspld '(67-6; arn613 qllm areduo)) saln)alou 'selnJeloluoJJeru -opua qJrq xrrlEru Jaqlo eql ur apErx aJP A lsolu a)Iun xuleu Jo qannxalduot e'qltM uanomlalul '(09-6I arnSrc) IIe^\ eqt q trsodep pue Iq pa)url-ssollaresluqUor)tr.u faqr teqt slrrqrJoronu aso1nllar aq] Jo uor]etuarro aqt Sullorluoc ,(q dSoloqd aq1 uorlebuo;alla)Jo stxeaql o] -Jou eJntnJJraql alEdrJrluEaJoJalaqlsluEld uI slleS 'spuedxe pue laqtoueauo eq] qcrqM ur .relnllpuadrad o1 IIaJ ;a;1ered pau6rlpareslrrqUoJ)tut asolnlla)aqf uorlJeJrp eql suJaAoSIp^\ aq] Jo sJa.{ellsoruJeuul aqt uI slrJqrJoJf,rru eqt Jo uoll 'lleM -etuarJo aql'qloq ro 'paleJedasl.lapl^\ aJoru euroJaq Jo Jaqloue auo lsPd apqs llar paq)la-daoppue uazor;Ilprder e tuor; errldarpomopeqsp Jo qder6o.t)r.u Jaqlra lsnu slrJqrJoJJIueq] 'uJoJap ro qclaDs o] Ilel\i\IIac aql Joc 'ssaf,oJdeq] uorl)alaslql ul uMoqsst lla) lolJe) ur aloJ Ierf,nJsP ruaql sa^rSsrq] pue (qJlaJtso] alqPun eJeIIe,l aql uI slrJqrJoJJrru 6ultebuoleue ro llpM11a> fueu.rr.rd aql ur asolnllal lenpl^rput aql 'arnlJnJls aurllpls^rJ Jraq] Jo asnesag 'slerralBtu sluquor)rur asolnlla) ]o uotleluauoaqf (v) 'parrnbar ere sarlr^r]re Suqapourar-1eM 'uolle6uola IIP^\ ^\au Jo uoqrsodep eql se IIeM se lle) Jo uorl)ar!paql o)uanFu! sprq4or)nrtasolnlla)69-61 arnbr3 xaldluo] ']ualxa pue uortcerrp slr sura^o8 ]eql IIE^\ IIar aqt Jo ror^Eqaq aqi sl ll 'aloq,|/\e se 'uorsupdxa eql sa^rJp luu1d aqt;o ]nq IIaJ eql eprsur eJnssardro8rnl ruroJ IBUrJeql acuaq puP '.lla3qsPaJo adeqs pug aql saurrrrJelepuorsuPdxesrql Jo JauuPI'uaqJ'aunlo^ ur salul] puesnoq] e ueq] aJor.u^q ,{luoturuoc {1ecr1ur"u -eJp ^ oJB ueJ lr rpeleJeue8sr lI eJeqM uralsrJauraq] Uel seq IIaJ luBld p eJuO

tll/vlol9lla) lueld slolluo) uolllsodac lle^ -lla) palualro sua6oqtPdlsurebe pualap dleq 16ur1apou.rar pue la^oulnl lleMioj elqtsuodsa.l slleMllo) r!epuores satJolutet Jaul^lod jooldlelem 6uotls stutoJ leqt uorseqpella)-lla) 1s;;enn {reulud o} rlt6uol}sanrsse.lduol sanr61eq}l.rorvrlau)lllrldolplq ,paOrprl),(lanrle6ausu.roJ

asolnlla) lloMlau]snqololutsluq!J lutl-ssorl slle/v\ lleuo qlbuellsallsualJaJuo)sltJqu

suralordor{;6 suralold puesutalojd q)u-auIlord^xoJp^q'saLuIzua slor.lo)lP ;i(deurs pue';r{ra;ruor'g&eunor palurl-ssor) u;u611 sueuornpPle60uueLlJ puesueuoJnl)ele6ouor..l ulllad uPuueuipuP 'ue;{xourqereouoJn)nl6'ue)nl60l^x suer{16 6u1>1u;;-ssorl asolnlla) aso)nl6ro rau^lodleaurl

aql 8-6 [ alqel llen lla) lueld aql lo srar.u{;o6 66LL

IVM ttlf lNvtd lHi

olray ;o {sa1.rnot,g Cnserqs-olorur6n5 l6uruun9ueu8Jo {saunot'V) 'uolteouola loor Jo stxeaql ol sa;6uelq6;11eare s{ele esrensuel} leal) eqI'sllat 6ur1e6uo;a Ilprderasaqlur s{ele alnqnlol)tulleftllof arlua aql lea^alol'a)ualsalonuounulull ,{q ,paurelsuaaq seq tegt qsdoqquv 'llar aqt Jo dlt loot e ut sller;eutaprd3(B) paluauo lo srxe6uo;aq] ot IUeln)tpuad.rad arpsalnqnloJ)tt!asaql'auelqulaul euse;daql mo;aqrsnf6ur{;salnqnlol)tul ,sserb ;o Iele le)tlJo)e burmoqs Iqtourt uro4 llo) dll-toor e Jo uotl)as 6urzer6V (V)'llo) 1ue;de u; selnqnlor)lu 1o{erre le)!Uo) eq1 19-61arn6r1

u r d0 !

tull t 0

aqJ '(18-6I ernBIC) suralord pezrralrereqc .,(1rooddq araql pleq 'auerqueu errrseld eql aJPJ Jrloso]-{c aql 01 esoI} /talJa IaJPJqJ e uJoJ selnqnlor3lru Jo 'uo€ar ]eql ul ile^ ilec eqt q petlsodap Suraq r(lluaunc arE ]Eq] IBsrUocesaql slrJqrJoJsrru asolnlles eq] sEuollPlualro elues eql qll]\/\IusEldoilc IPJIUOJaql uI peBuErJE,(lluanbargaJe aseql 'sllaf, tueld ut sepqnloJclru eql Jo suollenJasqo uro4 arueJ uollBluelro srql selBlJlp tPqt luslueqJatu aql ol anlJ lueuodtul uv

uorllsodeclle A-lla)lueuo salnqnlol)lw '(O pue S08-6I eJnBICaes)lueraJJlpaq.,{.eruraq -Jea u1!\opppl are^\ leql aellaluel ralno aql uI slIJqIJoJcIuraql Jo uollelualJo aql 3ur1u8 q8noqlp 'uorle3uo1aIIaJ Jo srxp aq] ot JelnJlpuadrad aq dpouruoc s11ac -uola ur slrJqrJorJrupalrsodap rtlluaear lsou eqJ'aplslno aql uo ]saplo eql qlIM 'ae11aure1 pa8uerJedlleJrJluaJuoJJoslslsuoc llerlt eq] ]Eq] os 'euo snourardaql ol [1eura1ur palrsodap sr BIIeuel ,,t,rauqopg '(OZ-ef ernBICaas) luauru8lle arues aqt ssel Jo eJoru aleq slrJqUoJOIIueql IIB qJIqM uI 'EIIauBI Jo tefel e Suyuro; 'eueJq -ualu eluseld aqf SoaJeJJnsJBInlleceJXeeql uo lno unds eJeesaqJ'slIJqIJoJJIlu olur elquesse sureqs esolnllas lueJSEu eql 'pezlsaqluds Suraq are Laqt sy 'slseqlu1{s llPl\/\ IIac ^Jppuocas JoJaeJql luaJaJJIpe puP slseqluds [P^ IIeo Ileluud JoJpeJlnbeJ are saueSvsgl eeJql 'lrJquoJf,Iluasolnlles e Jo uollsnpord aql JoJlBlluessa sI

xulew lelnlla)erlxfaql pue 'uorsaqpvlla) 'suoll)unrlle) :6L laldeqf

002t

('urlaqaels MalpuVJo {salrno> ') pue 'arn)sqoureujaralqtsuodsaj B) saln)alouJ 6urlrauuo:oql lalnqnlor)tLr puPauelqurauruaaMlaqLuu0z lnoqp qlog ;o de6 luelsuo: aq1azrseqdr.ua 'uorl)as e smoqslaqlo aql ;eurpnlr6uo; alrqMuor])asssor)ut salnqnlol)tuiaql sMoqsauo'auerquror.ll euse;daqt qtlm salnqnloj)il..u le)tuo) aql Jo uotlet)osse uort)ala ]q6l] aqf nnoqssqder6or)rur oMl (l'B) uMopptplale s|rquor)tur Mauaqt qrrqm6uo;estxeaq] sautuualap -luasaro alPAoqiuaqM-salnqnlor)tu_t asaqlJo uotleluauoaql 'slauueq) auPrquraupouUapo1xa;duo: srqt sauUuo)]eql ^eM e ur auetqr,uaLu euseld oql o] paq)ellesrsalnqnlor)rur ]o Aeil? le)rljo) aq] asne)agauplqulauraql Jo aueldaql ur 6uo;exelduor aseqlu,{s aql saqsndpua ;eurxordaql lp uollebuola raqi pue 'lleMaql Jo atnlxa]aq] olul peterbeluraulo)aqsluqUol)tru #lts aqi Jo spuale]srpaql auprqutauleuseldaql JOO)eJralno aql uo sluquor)tulasolnlla) azrsaqlu^s {;snonurluotleqt sura}ord auerqLrLeur ;er6alurale saxe;duot eseqtuXs asolnlla)a6re1aq1 (y) 'salnqnlor)rur le)tl.tolJo uotlelualro aql ^q paurluralapaq lq6rul sluqqo,r)il,u asolnlls)pal!sodapI;anau1o uotleluatro aql /vloqro lapour auo z8-6 ! arn6rJ

*.

u^ oqs ueeq seq lI qSnoqllE 'palloJluo3 aJEsuorleluarro esaql ^ oq u^ oDI lou sl U 'tuaudola^ap luEld Surrnp suorleluarro alnqnloJJlru IBJIUOIJo luau^oldap paurallBd ,{.UBtq 'paleurprooc e uo spuadep ,{lqEunsard lueld rBlnllarulnlu B .{Soloqdroru errlua aql '(slle^\ Jraqt ,{q paureJ}suoJSuraq) a^otu louupc sllaJ Jo 'seFqnlorf,ru ]ueld asnpJag Jo .{.errepcrlroc Jraq}Jo uortBluelro eql uI a8ueqc uappns e ,(q uorsuedxeJo uorlcarrp rraq] a8ueqc upc sllac tueld ,{elrrsrqt uI 'a^oru UPJxalduoc aru,{zua aqt qJIqM urqlrlv\surelxopoJJrruauPJqruaruaurJepo1 lueseJd eJEsalnqnlorJrru IBJ -proc uaq.r,r.{lleupdspaureJ}suoJsr lr }nq lsalnqn}oJoru Jo .{FuepuadepurJnJro uBJ srsaqtu.{sasolnllal 'Mar^srql uI '(z8-6I aJnSIJ)}uaua^our auasoJureJlsuos o] Ieuec B Jo Dlueq aqt a{u tJP lBqt aueJqrueureurseld aq} ur salJPpunoqa}Ear:) uec.{aq} :a^our sauasor aq} q3rqM ur uor}Jalrp aq} ur aBuEqJe af,roJuEr ta^a -Moq 'lla3 eql aprsur selnqnlors[u paluarro 'llal aq] aplslno SIIJqIJOJSIIU pa]ua -rJo Surlsxaard aq1;o uorlf,alrp aq] SulmolloJsailasoJeql q]1lr,t,{lsnonard uarop prEI ra,/(elaq} se uor}e}uerJo etues aq} ur auBrquraru aq} tuo4 lno unds aq ol puat plnom slrJqrJoJJrruJo Ja^el qJEa .{Entsrql u1 '1em Surlsrxaaql ur paroqf,up sr aqt Jo puJ JPJJql qJIqM ur .4eMaql ^q paurJapuollJalp eql ur auerq IrJqrJoJJrr.u (lerJalptul au aq] slrsodap se a^o(u o] spq -Ixalu aq] lr Jo aupld aql ur Surla^eJ] pua Ieurxord '3ur.,r,tor3 slr le allasor aq] $lls sr IrJqrJoJc[uaq] acurs 'ilel aql Jo alnlxel eqt otur paleJSatursaruoJaq]r se seuo plo aqt qll^\ IellPJedur IrJqrJoJcru ^Aau aq] Surluarro '{lBrraleu Ip^ Jo ra,{EI snor^ard aqt ol s{url-ssor3 lcarrpur surro3,r(lqerunsardprqgoJJrur r{JPeJo pue P}sIp eq} 'spaacord slrrqrJoJcrruo}ur ,{lqruasse-yasJraqt pue salnJalotu esoFIIeJ;o srsaqtu,(seq} sV 'sJlncaloru asol -nllal Suol ]no urds auBJqruaureluseld aq] ur pappaqua sa]lasor Suzrsaqlufs 'lapotu -asoFIIaJ qll^\ aqt aJe suorlP^JasqoasaqJ aqJ 3ur-,rno11o; lualsrsuoc 'uoueluarJo ^\au aq] aurruJelepo] lueseJd aq saFqnloJJlu l3plur lEql saJrnbalslrJquororruasoFIIaJ Jo uorlrsodap aqt ur aBuEqJ^uE lnq 'salnqnloJJnu Jo acuasqE aql ur ua^e palPSBdordaq ueJ slrrqrJorJrruJo uorlBluarJo Surlsrxa -ard e leql suraaslI 'pa{oolq,{lqerrenur sr 're^e^ oq 'eplleluBle^rsseJrnsuaaMlaq rnJJo ^llBruJou plnoM ]pql uJaDEd IrJqrJoJJrruaqt Jo uorlEluarJo aql ur qJlrA\S ptuaudola^ap ^uV 'uorletuarJo SurlsxaaJd aql ur slrJqrJoJ3ruA\au ]rsodep ol anurluoc uEf,sllaf,saseJauros ur pue 'slrJqrJoJJlluesoFIIeJ ^\eu Jo uorlf,npoJd aq] ]dnJsrptou saop lualuleaJl SnJpaqJ'palJadxa aq ]q8rtu se pJe^\JoJlq8rerlssu lou ale la^a,l\,\oq'uorlrsodap asopilac luanbasqns roJ sacuanbasuoJeqJ'saFqn] -orJrur IeJruoJ Jo tuals,.{salrlua aq} alquassEsrp ol se os Snrp Surzrraur,{1odap -alnqnloJJrru qlr^ palsal E anssn e Surlear],{q aq uec drqsuo4elar srql ]ueld 'drqsuoqelar lesnEce SurlsaSSns'uorlrsodap IIeM-lleJ,{repuocas pue .,{reuud qtoq Suunp }uesard sr pue s11ac1ue1dJo sedeqs pue saddl dueru ur uaas sr (eprstno 1sn[ 3ur,r(1)slrJqrJoJJru asolnllac pue (aueJquraru eruseld aqt aprsu lsnf 3ur,r(psalnqnlorJtu;o derre IEf,ruoJeql Jo uoqeluarJo luanr8uo:) a u e r q u l a u er u l s e l d o1 paLllelle alnqnlor)rur

(v) LUri I 0

'losotA)

allasor ro 'xaldu.ror a s e q l u { sa s o l n l l a )

1

{l)

s a ln qn l o J ) ru l

{,ri

1

]fVdS

aueJquraur

uvt nttlfvulxl

6 u r l s r x a a rodl u r p a l e r 6 a l u r 11em 6 u t a qs ; r q r ; o : : r u ra s o l n l l a )] o s p u al e l s r p

LOZL

IV AIl)

INVtd lHl

'(9-6 uralqord)ulededqlrMuorlso6rp t Iq serpoqrtue961r,uor;sluer,ubellqeJJo uotllnpor6 1-61[ arnbrl slueu6erl qe3

i.T Ir ;l

,,,;\

A p o q r l u e9 6 1

T .l.

)

NIVdVd !'

b u r p u r qu a 6 r l u e JO]SalrS

'(gZ-Ot0 arnSrg)eceJrnspcrde aql ol pappe ueq^\ treJJaou seq lr :laaqs eqt Jo aprs lEreleloseqaql ol pappe sr]1uaqa,r.{pos}caJJe asaqtsaJnporduxol aql -{1sno -lrnl '(VZ-6I0 arn8ld) lno 1r 3urqse,u.,(qparolsar aq ueo aruetsrseraql lnq ruuol aqt Jo acuasardaqt ur.{lpcrleurerp sasearJapacuplsrsarpqaqlrdasuerl leql snoqs suorlcunf tq81] aqr ;o ,Qrr8alur aqt roJ ,{essepuorlcunJ V 'paqluprq ,{1q8q ssal are ,(aql pup 'iom} }noqe o} rnoJ }noqe uro4 srnoq tZ relo saseerraposle sllec eql {ull leql suortcunf tq8p aql ur spuerls Jo raqunu uuetu aqJ 'lcelur sreadde ]eaqs lp1aqllda eql pue,tqtpaq ureruarsllac eqt ,t-urpnelc 'srnoq 77 ulQllm alqetcalapun8uftuocaq Jo aJuasqeaql uI 'sreaddesrp 'tua{u8e{ urxot t-urpnelJ lpurrura}-l aq} qll.ry\peteqnrur sr leeqs prtaqtrda )lCW lJelur ue uaq \ ' s rq J r q ^ \ ' t - u r p n e l J pue 'urxol aqr dq punoq tou sr qJrqm rl-urpnelr :surpnelc orrnlssardxasflec):)cw 'eruplsrsar plaqlldasuerr qSrqqtyrt laaqs Iplleqllda lcelur ue uJoJ uec daql esneraq suorlcun[ tq8p Jo sarpnlsroJ ecroqf,uoruurot B ere sllecy3q4 'suoll -cunf lq8l;o sarlradord aql Surle8qsalurroJ lua8ear alqe -nle^ P aq ot ua^oJdsPqsurpnBlJaql ol spurq ]Eqt ur-xolaql 'auerqtuatu ;o uorlrod aqJ'ller eqt ruI leqt seloq Suturoy 11acluacelpe aqt o]ur uasur ot snurura]-N aql smolleurxol aql Jo snuluret-l aql '{ulpnPlce ol punoq uarliq'suotlrunf lqBll Jo stuenlltsuoJureu aql are qJrq ^ 'suralord;o .{pue; urpnelr eql Jo sraqruetu ot spurq leql urxol e saleur sua8 -ut{tad wnlprrlsolJ runrralceq SuruosrodpooJ aqJ L-61

iuone8erSSeIIaJ{rolq ot stueu8erJ qeC asn ol ^rpssacause^r 1r asoddns no,{ op ^qru'(I-6ID arn8ld) satrsSurpurqoml eql alpredasol'asealord P 'ulpdpd qlyv\ sapoqpue c3I aql SuFSaSrp[q palerauaSaraM sluaru -3ery qeg aqJ 'salrs Surpurq Iecrtuapr o^t1 qll A salnf,alou padeqs-I aJB qrrqm 'sarpoqrlup CBI ]rplut ueql reqlur '(sluaur8er;qpC pa[e]-os) alrs Surpurq a18utse r{}lm qcea 'sluatu8B4 punoJ sraqsrPaser dpoq4ue asn ol drBSSaJau lI aql 'sdesseSuqcoyq-uorsaqpeaql q 'uoltBBaJSSe 1ac >1co1q ol sluauoduoc aJeJJns-[ac]suIBBepesler salpoqpueSuISn perJrluapl,tleurSrro ereM salnsalou uolsaqpe IIeJ 9-6 t .,'sllal Jo tueells pexltu e ur punorS aql olul Jo /v\oupue alerSalutstpacuo lE plno^t sarpoq rno 'xlrletu relnllacerlxe Suproddns aql roJ pue raqloue auo roJ ssauDlf,rls 4aql asol o] sllacJo sadr$snotre,t aq] ere A,,'I8olorq1ac;o sraauoldaqlJo auo sB^\oq^\'sI^\e'I Suunoloyaql uo luauruoJ s*6 t uerregruorJ alonb 177611 'sua;qord 6urrvro;;o; aql ssn)srC 'sSurrds rsaf,Ileqn se relncaloru loe qJIqM Jo aq1 b-GL tuetuoc q81qstt ruo4 sa^rrapupseleJo r{r1cpsela 'sleuBrsreln -Jalou olur spu8rsIe3rupqJaruueluoc uec sutr8alul €-d t 'suralord Ierateloseqpue IPc -rde;o Suxtu lua,rard01surpuop aueJqueu JJoacuagrtaq] pue ,ltog relnllacuredtrlrlsar ol sllal ueemtaqacedsaq1leas ,laql :suorlcunJlrups1p o,r4 urrogradsuorlcunf 1q8r1 z-61 'uollerluacuo) +zeJuI sa8ueqcdq paleln8ar osle are suorsaqpeIlac-llac luapuadap-*zpl luql ,,(1e111 suraaslr 'uorlerluaJuoJ +zBJur sa8ueqc,(q paleln8ar aJBlpql sllal aprsursassacordsnoratunu aql ua^IC l-6t '1ou{qrvr.ro{qrvrure;dx3 eanr}ale sluaulo}e}sql!qM

SW]IBOUd

'apLlm a so ryo1d aqi 'lqaruMuln to tpua to uoqcatlp aLUaulwJapp IIar aqtto adaqs1au{ aql antataql puv uotsuodxa77ac urnl ut. t1ut1m'sptq1fotrru asoUIpJ pallsodap f1maulo uouatuauo aqt IoJjuoJ UDJ salnqruorJrrato[otto 1a4uoc y'tpoom puv 'praq tootdnrym waql Sul4oru patlsodap 'sapuoqJcastlod 's77am ulcad aq [ow u1u317 to xuruw pa4uq-ssoJ) 77ac,hnpuocasq [7q31qa u! pappaqua 'suac,{18 Sulqull-ssorr pua sytqtlotnut aso1n11ac lo 4tomlau n to pasodtuocst nnm aLlJ 'a1r\sat17s,rya1da to satnpat anbrun aqtlo [uaw rct a1qtsuodsat st l4rrl1m'lIDm lpi ro 'xuJow rvlnlpJouxa qBnol o [q papunotms aJo snac ruDId

{reurun5 'gt-gl arn8t4 uI paleJlsnfiI sE 'sadeqsJIISI -JelJEJeqJJraql asol sflac aq] 's]ue]nur €lzdtv uI se ')Jo/t\leu uI]Je IeJIuoc Jleql azrue?to]ouuec leql slleJ ul'LL-6I arn8tg uI rrmoqssllac leruJepldapue sauoqc -ID eleroqela paluquereJJlp ,i.ueur Surdeqs eqt sp qrns 'sllar 1ue1d 1eug ;o Jeal ^aeuJo uoU eql ol SuDnqrJluoJ (eJeJrnsIIaJ eq] uo salts ctycads lP IPIJeleIu IIU1V\ -rsodap eqt ]leJlp osp uuc sluetu€lu ul]f,E IeJIUOJJo IJOJIEJO'I'uoqlsodap lem aJuenlJulleql slueruala leralalsoutr r{1uoeq} talemoq'}ou ate seFqn}oJJIIN ' G M Z a r n 8 t ge a s )p I r B c r q a r a q q r 8 pue aual^,(qlese qJns srolep8ar qutor8 1ue1dlq8rarr,r-Jelnf,elou-^ o1 Surpnlcur 'rlnrurls JelnllaJeJlxe ol asuodsal ur ,{lptder lualJoeJ ueJ salnqn}oJf,Ilu aqt leql xfrlery relnlle)prlxf aql pue 'uolsaqpy lla) 'suolpunf ila) :61Jaldeq)

CAZL

END-OF-CHAPTER PROBLEMS

1203

19-10 The affinity of integrins for matrix components can be modulated by changesto their cytoplasmic domains: a 10,000 processknor,','n as inside-out signaling.You have identified a 3leldnoql sllalap leql ue)s ({qdetbotuol uotsslu.lo 1{qdet6ouor^et-y pelnduor) 136 e'a6erursrqluo preua^O'6utuuels g{poq aql lo e6eur punor6l)eqaqt'('lHN) 11 r(qpaurelqoseMsansstt eruoqdrur(; surl6pog-uou)rlelselaulqilM luolled e Jo ue)s Ipoq-aloqrt e sra6eur uorsnjsrqlut uMoqs'ele)lpelaol pJeqla)ue) aql 6ul)PLu 'saselselauol asua^!6111e>rdIr srounl 1ueu6t;eW'slselselaw 1-97 ern6;1 'Dutouolaut e 'upls uI aq] Jo slleJ el{l sEaJaqA\ III1\^ IIaJ lueuSld e uorJ pe lrep 'uDIs 'sluauelrJ ezlsaqlu^s ol enunuoc ,(lleJeue8 elerpeurrelul upeJe)ol-,(f, oqt ur IIaf, r.uels elboupeJa{ e Iuor3 pe^IJep 'Dutoulual naJ-Iosuq P Jo slleJ aql 'eydurexa 'snq;'ur8uo Jleql lJeger lpql scpslJalJ?JeqJ eABq sJeouec ]soIAI rog 'dlantlcadsar '"te DruoilDsoJp 'a8eppec ;o sJorunl lueu8qeur pue u8ruaq -uoqJ e pue araoJpuot4c e [peyu4s '(S-02 arn81g) otttourJruJouapo uE sI Jolun]

lueuSquru Jo adlr SutpuodsarroJ aql iuorlezruu8ro re1npue18 e qllm Jorunl pqaqlrda uSuaq e sI 'elduExe to! taulouapz ue :srolunl u8tuaq JoJ selueu Jo 'sJournJ JoJ selueu Jo les eql qrpralalpred u1 lueuBtpru les pelBIeJ E sr eJeql 'ri.poq 'Jorunt aql Jo eJuEJuadde ctdocsorsllx eq] pue aq] ur uoneJol 'add1 1ac rgrcads aqt ol SulpJotJP suoISIAIpqns,{ueur seq rtro8a 'selBls pallun aql 'alEr -]Ef, qll1v\ raqleSo] puP rleq] peorq qsEg qlPep ecueplcul 'uralsr{.ssnolJau eql ur uoturuoJ eJBleq] sJasuPJJo sad,fi aqt slv\oqs7-g7 arntlg '(slleJ cqetodoruaq) srosrncard Jleql pue Jo sllec ruo{ peAIJapsJaJuecse lle,vrse sllaf, poolq atrq^aruo{ peAIJap 'suuroqduufi pue sulrua{nal snoIJeAeql apnlJul 'sllac elJsnlu sarro8alec peoJq o^q eseq] Jo reqlla uI llJ lou op leq] sraJuEJ JOanssp e^IlseuuoJ [uo4 eslJesEIuoJJus 'suEIIInq uI sJacueJuoulluoJ lsoul aql 'eslJe re; dq are ,(aqt pue '.slleclelleqllda ruor; Sursrre sJecuef, eJPsuruouIJJBC ,(aql qcrqm uor; addl IIaf, pue enssu aql ol Sulprocce peUISSelJeJEsJeJusS 'alectpera ol 'luaqed JeJuBf, aql ilpl ]eql seselselaur rtlpraua8 sl ]l pue '(I-02 ,(poq aqr alntg) seluoJeq tI JepJeqeql 'speards reJuec e ,{1apu,rarour eqJ 's1assa,rcpeqd uI selrs reqlo lp 'sesulseleur palpc 'srorunl ,fuepuocas IuJoJ pue 'sllec JacuEJ cl}sualcemqc -urrtyro poolq Jelue 'asool {EeJq ol ueq} s1v\ollB lI Jo ppuasse uE sr ssauelrse^ul 'enssl] Surpunorrns epelul ol Ir111qeaql parpbce e^eq slle3 s1I1 ,(po (sI lPql 'lueu8guur sl lI JI dluo JecuEc e peJeplsuoc sI Jolunl 'aJnc alaldruoc E selalq3e dlensn d1eco1 sseru aql Surdorlsap ro Suyroruar V pue 'utpaq aq ol pIBS sI Jorunt aql JeAe^ oq 'eAISEAUIeruolaq lou op qlac rc tounl E o] asIJ cpseldoau aql se Suoy sy'quvror8 rlreu E '[11ercy1--npo.rd plno) slla)ds-uou lou dS aql'slla)ds-uoupue ds qloq patnpord slla)dSaqf sealoqMla^oalow 'sjot!nl uuoJpup (;enrsuelxa alela;r1ord plno) slla) uollelndodaprseq1I;uo lpql paMoqsslla) 6urureuelaqt qrlmslla)(dS)uollelndod aprsasaql6uueduor sluaulladx3 'uo11alut pale)tput ,pautets se ,{11q6;1 ,{1uooloJareqtare pue aIp aqr tno dund slla)aql,o o/ot'glnoqv .6ur1ros 1;ar{q pez{;eueuaql pue (Zt€€€lsq)eoH) auelqulauteurse;daql qbnolqrssedue> ]eql a^p e qllm pateoll aramloutnl euor;6 ler e ulolJpautplqoautllla) paqstlqelsa ue ur slla)oql ,alduexasrqlul .slla) 1o uorle;ndodapls llpuls e u! slla) uols ro)ue) sleanal6u1yos;;a1 g 1-gg a.rn6r1 +

uolsslrla par rq611 c o @ f

o

3 f

v

eql'[ec SuldJrldr.uelrsupr] E se qJns 'peleuuereJJrp ero{u sl }eq} IIaJ Surlera;11ord e ur a8ueqJ e q8norq] sr asrJe uef, IIoc tuels JeJueJ e leql ^e^a puoJes eql raJuBO e olur dolanap ol parmbeJ suorlelnru Jo Jeqrunu eq] elelnrunJou ol q8noua Suol JoJ elpJeJrloJdpue Lpoq eq] ut urcrueJ silec uals,{1uo 'sanssrleseq} q 'Ja^ourn} IIaJ ler}ue}sqnso8Jepun }eql puaq}rde ur dolanap,{.aq}asnecaq 'sllaf, ure}s ruoq esrJp z(lqeqord sJaJuEcueunq IBruJou fueru 'esrala{r'I 'JeJuec srq] JrlsrJa}JeJeqc uorlecolsuen eq} q}lm ,euros Jo -oruoJqc pqdlepplrqd aql upluo3 Lueru 'sluerled asaq] Jo ^ oJreru euoq aql ruorJ pegrrnd are (gglt 'd aasl sllac ruals Jperodouraq ppuelodpFru eqt uaqm ,snql 'asrre serua{nel snoueSola.{ruJruoJqc ,r(emaq}.,(pmgac }sorup sr s}uene 'aldr.uexaroC 'JaJuEJeqlJo Jor Er{eq Jo ecuanbes slqt Iprooseaq} JoJalqrsuodsar sa8ueqc ollaue8rde pue suopplntu aql selelnrunJJe dlenpe.r8 qcrq^\ ,llaJ uals anssr]IeuJou e ur saluurSuoJe3uE3eql leqt sJorunl etuos JoJJpelf,sl u 'saSueqccrlauaB tuoq upqt JeqlpJ cqaua8rda ruo4 llnseJ lsnru sacue 'saur1 -JeJJIp esaq] Jo ,{ueur ilec JacuEJpeJnllnc qllm paqrJcsep}sn[ slpsal eql ruoJC aq] ur silec dlrrofeur aql ruo4 sruJe] Jelnoeloru ur 1r saqsm8uq iJaf,uec Jo -slp lPqm puE 'llec ruels recupJ aql Jo eJnlBu aq] sr ler.I A iasrJEsllsc uals eseql op ^aoH 'suorlsenb Sum8ulur Jo Jaq{.unup sasreJlI pup ,}r Sunean JoJ pu€ JeJ -ueJ SulpuetsJepun JoJqloq 'suorlecrldrul lupuodurr flaruarlxe seq sllal ruels racuec 3o uo4epdod leurs e Lq paurelureru aJesrecupc dueru leql Surpug aql

slle) uals la)ue) oc MoH easrJv 'slInseJ Jelrurs uanrB aneq Jacuef, aql ur8uo anssr] eql ut sllec ruels pruJou ezru Jo Jo ,serpnlsreqlo uI -3ocar lpql sepoqpue puolcouour Sursnsuoqpuorloeq IIof, 's11ac SururcureJ eq] Jo seJnllnJ ur dolanap saop uouppdod eprs e sEaJer{M ltlalrugapur peure}umru lou sr pup p ,eJnllno sreaddeardlprder uoqelndod aprs peuJrual ol sr aurl raJuEJp ilptus IIao qf,ns ruo{ pa}Elosruorlepdod aprs aq} ueq^ teloeJol4tr'(gI-02 arntl$ tou saop 'aurl eq] ur slleo aql ,uoqepdod luelseJong Jo dluoferu ]sBAeqt sepnlJur qJIqM [UBlq Surureurareq] (uor]e]upldsuerl reUB sJounl srurogLppeal seurl eseq] Jo qJEaJouorlepdod aprsaql sBeJeqM:arnllncur srear(d1rrq]ueql eJorurog pale8e -dord uaaq a^eq leqt saurl ilec Jacuet eruos JoJasef, eql osle sr qql flqelJeurag 'eJrru luerorJapounru{ur o}ur uor}e}ueldsuerl regp sJeJuEJ ,tteu Sunuro; go alqedec s[a] eq] Jo 'lle ]ou Jr 'lsotu ureluoc ol punoJ sr rapodsuerf l11V uE q]pr uorlelndod eprs IIEtus e 'Ierr erues eql ur paz,{pue eJe sJeJuEf,eruos ueqlt 'lleJ eqt Jo 1no azi.paqt sdund z(1alr1celEql auEJqruaru eruseld rraqt ur (11 raldeq3 ur passnf,srp)ragodsueJl :)gV uB alpq rtaql asnecaq luaosaJong i(qeaa,r,,tpo ere sllar ruels aqJ 'slleJ tueosarong Lgeam; o uopn1ndod aprc lleluuLs B ur peqorrua ,{.1q8q eq o} punoJ uauo are enssr} aq} Jo slleJ ruets eql furvr slql ur pauos ueql pue a,{p aqi o1pasodxa sr enssq prurou B ruo4 peredard uorsuad -sns IIaJ p uar11\'(7-g arn8rg aas) Jeuos IIef, pele^r]f,e-eouecseJonge pue a.{p lua3seJonu alqearurad-1lece sasn sJecuEJeruos ur sllec Iuels JeJuEJa,rrlduns 'uorleBedord rournl loJ papaau -a.rdgo uoqepdod ileurs eqt qcuua o1dem aug firadord prcads aql e^eq uraql Jo lesqns IIErusE Lluo pue ,snoaueSoralaqaJe sllal Jacupc eql esneJeqsr srql 'sesec,{ueruur .lpql u aoqs e^Bq sllec Suqros ro; sa€o1ouqca1^aaN 'esnoru luerJrJepounruurruE olul ll Supueldrur dq ,aldurexa roJ-os op ot drgqe slr roJ petsel ueqm Jounl ^aeu E aleraua8 01 elqe eq IIIzr,r JeJUEJp ruo4 IIaJ IBnphrpur uesoqt ztluopuer p leq]-o6l u€ql ssel qcnur dle -nsn-acuuqf, llerus e Lpo sr eJaq] ]eql srea,{ 0t JeAoJoJrr,lrroDlueeq seq }I 'sJaJuEJ,{ueur ur sllal ar{l ,fi1roferu lse^ aq] dn a>1erupp Jo -uatod qpror8 palFull e qllm slleo aseql leql pazruSocar dlapur aurocaq fpuacar ,r(po seq 1I 'lemauer-JIasroJ dlrcedec petg4l e eABq]Bq] slleJ 8up,{SrrdureIISuEJ} 3upp1p fprder ol asrr an€ osp daql rnq ,IB^ auaJ-Jlasetugepur Jo alqedec are slleJ ruels eseqJ 'Jorun] e ur slleJ;o uorlupdod aqt Surumlureru JoJalqrsuodsar z{qcrerarq eql Jo dol eqt }e sIIeJ ruels JecrrBJ eJeJqllm dea,rpcrqcrerarq arues eql q pazrue8ro ele sJacuecdueur leql acueprna Sursearcursr eraqJ.uollep -ueJaJJrpo1 Surpeay,{ea,rqlede ol }rurruoc leql sraq}o Surcnpord el,[rllv',Qamaual -fias palet ssacorde) sllaf,ruels ureruer upJ leql slac ralq8nep aruos Burcnpord

ra)up):97 raldeq3

9LZL

'saltradord lla) uratsuraqla^r6leql sa6upq))tloua6tda ro suorlelnulauoolapuna^eqleql slla) palPlluaraJJ!p aroul ruo4 ro isnole)ue) ureqleleur ol suotlelnulpautelsns a^eLl ]Pq]slla)urelsleuilouLllorlasueue) slla) ulatsra)uel slla)6ut^,tldrue ltsueJlse q)ns slla))rua6llounl-uoualelaua6aultt auJesaql le pue'slla)uats ]ueu6tleul alnpolo ol Mauar-,las leuorlrppe ue) lPr]lslla)la)ue) asoqlse pauuap aresllal uals ja)ue)'slla) uals Ja)ue) urorJasue{eu slarue; 9 1-97 arn611

'luaq] ilpl o] puE sllal uals racuec aq] ]e8JElot s^emJaueq purJ ot I€rJnJJ,{lJealcsl U 'sreJuecoJnc oJ 'r(deraqto} asuodsal IeIlIuI JrlerueJpe Jaue JnJaJfluouuroJ sJeJuPf,.{qmuosear Jo[Elu P aq deur sIqI'JaJ 'palpJrpeJe -uEJ eJe slleo Iuals eql 'fuan Laqi eql eleJeua8al z(1a>11 lou JI 11t,u 'sllac reqto ilr{ 'slle3 uels JaJ ]Bq] slualulEarl .,{ueruol }uelsrsal dlunsnun ueq} e{elu plnoJ }I '@I-EZ -ueo arnBrg aes) ii.ern srql ul seuosoluoJqJ Jleql roJ esBJ eql osp sr srqlJI 'sllac ruels ,(po pue 'sllec urels lpql ;o uorle8ar8es aq] eteurpJooopeepur UEJ ]sa88ns sluarurradxa ]uaJag ;lleJ urels p urerueJIIr^ lerll 1ac ralq8nep auo aql olur pale8ar8asaq o] aruosoruoJqcqJEe ur pueJls aluldrua] VNCI plo aql smolle 'uorlucrldar leq] usrueqcau prcads e azrlln IIec tuals e Jo uorsrlrp qcea plnoo y1qq Surrnp esrre suorlelnu .{ueur arurs 'relnrrlred u1 'suorlelnu Suqelnur -ncoe ruo4 sllac ruals lcalord ]uql susrueqoeu JoJ pelJelas e^eq pFo^ uoqnl -ola leql pasodord sem lr 's0Z6I eql ul lenoerohl 'sluauleeJ] eseql ol elrlrsues ssayaq,r{eur,{aqt'{1.uoys aJolu qJnr.uaprnrpdlensn slleJ lue}s Jacuer eqt esneJeg 'rorun] E ur sllef, recueJ Superagrlord,{lprder }soru eq} 1q z(lppuarayard o1 z(1a4t are s8nrp cxololdc pue uorlerper se qJns sarderaqt Jaoupc luaJJnJ lsol I '(gI-02 arnS;g; qloq ro 'a8ueqc cpaua8rda 'uortetnur q8norqt p^ auer-Jlespaurelsns ro; dlrcedec aq] parmbce spq leql IIaJ p ruo4 esrresreqlo 'llal ruels e [uo4 eslJesJeJuEJuErunq ]solu alrqA\'teql ,{1a>91 srueas 'snqJ'llef, ruels papruruoJun ue ur ueql JeqleJ 'lleJ JosJnJerdal,boqdrudl g lI pallnutuoc e ur paleur8rJoJaJuEl aq] ]Eql SurlsaSSns'(gZ Jeldeq3 ur pessncsrp) saua8 .{poqr}ue raql ur sluaura8uerJeerf,r}aua8 arues aq} ur€luoc erua{nal e qons ur sllal aql Jo 11y,{erursrql ur asrJeol ruaas 'aldurexe JoJ 'serluelnal a$coqd -udl g auos 'llal Jacuer rrmolq-1pge eruoJeq 01pepeeu sa8ueqocqaua8rdapue suorlelnru reqto eql alplnrunJJe ol erup eq] e^eq IIIM IIaJ peJelle aqr 'uaql .{po pue 'uaqJ 'lemauar-Jlaspa8uoyord ro; dlrcedec eq] pue ,{poq aql ur paure}ar aq o1 ^,!rpqe aq] :seuradord 1ac-rua1s pnnJl o^ l aql JaJuoJ lsnur sa8ueqc

JOUrnl

l l a ) u r a l sJ a ) u e )

tI Ftte rF

i

I qyriror6penutluo:

I -\

llal uals ra)u e) tueuEtteu

lemauoJ '--.

i

I s a 6 u e q rr r l a u e 6 r d ap u e s u o r l e l n u r

lla) urals leuiJou

6tzL

1rsue.r1 11a:6urr{1r;du.re

sst)oud UVNoTIn-loA3ouftw v sv ul)NV)

poolq E ]JeJ]]e sJorunl 'senssp IBruJou a{q 'ezrs urEual E puo^eq qnoJS Jolun} JoJpeJrnbar sr 'slessel poolq Ai\auJo uorleruJoJ eql3lsauaSotBun 'snq1'sluelJlnu pue ua8z{xotuarJlgns sta8 }r }pql eJnsue o1 .{1ddns pooyq alenbepe uE }lnJf,eJ lsnu Jorunl e 'a8re1,uor8 ol 'paquJsep lsn[ sluaruarrnbar aq] IIB o] uoqlppe ul

e)npuls,rounl stseua60t6uv 'ap$1p pue anor8o1 slleoJeqlo uror; spu8rs uo luapuadep ssel oslp are daq;'auroq radord rraql ruor; Surdecsa rege aArAJns,tppuar eroru uec aJoJaJeqlpue sllal IBruJou qlyrr pared -uroc srsoldode o] ]uelsrsal uego eJEsrselselaur;o alqedec sllef,JaJueJ'(BI ral -deq3 ur passnJsrp)srsoldode o8rapun pue draurqceru qtpep iler Jleql alelllJe [aq] 'qeu8rs aseqt Jo paarrdap uaqrt lluaruuoJllue IeIuJou Jleql uI luepunqe ^,(1anr1u1ar eJE]pq] spu8rs IeArAJnsJplnlleJeJlxe uo puadap sllec pIxJoN ']uIEJlseJ leuorlrppe ue aq o1 l(1a>11sr saqilqedeJ IeAInJns ptcads JoJ paau eqJ 'srselseleruorJlru E Sururog Je{e uela 'uoupzluoloo eAIeqJEsllef, ana;os,(qm urc1dxappoan slleJ ruels JacuecJo dllrer aq1 ']ualuuoJllue uellp ue uI elt,uns pue aplas or irIIqE aql pue ']1u41]noqlul ap4lp ol uaql alqeue illv\ ]Pql Jel3eJeqf,IlaJ-ruals aq] qloq aner1JaJueJ lelldril p uI sflec aql Jo uopepdod leurs d-rane Lluo leql aq Leur ]l 'sI lEqI'papaau sauradord plcads aq] e^pq leqt slleo Jo ,!per aq1 slJager uollpzruoloJ Jo eleJ llrol aqt f1a41 aro61 ztla4lun sruaas slql 'ells uerp ue ur Suunor8 pue 'Surplas '3urnn:ns Jo ecueqo ,tup arues aql arreq lq8lu Iueerlspoolq aqt Surratua IIe3 raf,uer qrea lBql aldtcurrd ut alqtssod $ lI apq \ 'palorual sr Jorun] drerurrd eql paJnr aq uuc pue seluoloJ Jllplspleru punoJ JI o1 pa8eueru a,,reqdaql eroJaq peJe^oJSIp eJe sJacuBOdue141'saselspleruoJJlru ueq] erotu Surqltue uJoJ ot aAIAJns 'suotlltru uI euo sdeqrad 'spues -noql uI auo upql JaMaJ'eseql yo uotlrodord dun e dpo leql Moqs slueurradxa tanaanoll 'slassal poolq ro crluqdrur(1olur adecsaol a8eueur Jorunl B uI sllal eq] leql aJnlPInosPAeql ;o,,(uuur ler{l suees rr pue !(1ee1sr sJolun} olut srvr,or8 'luetuuoJllue irlreuaqt '(LI-OZ arn81g) uorlpzruoloo sB u^ ou{ ssecord l1nlUJIp e ur,{yanrsuaryaaleragqordpue eArAJns}eq} sllec ecnpord }snu seselseleluoJf,Itu asaql Jo aruos 'sseJo.rd crlelseleru eql alayduoc oJ 'slsulseleruoJJlru e sB rrr!\oDl slleJ Jo durnlo llElus e lsJrJ lE Sunuro; 'alIS ^\au eq] q ,rnor3pue fpoq aqt ur araq ^asla Iassel p uorJ ]rxe 'qdrudl Jo poolq eql Jelue ot sE os Ias -sal eql 3uru11prtaqlopua eql pue euruel Ieseq aql SurssorcLq lassarrcpeqd Jo 'l.pgssaccns ezlselselatu UPOlI -uL1 e ro Iassal poolq E alerleuad lsnu IIef, e aJoJag'll aAarqJEsllac .t,reJluorlerado ]ueIcIJJauIpue 'lr,rols'xalduroc e sr-sue8ro tuplsrp ur saruoloc Jo ]uauqslqelsa eqt-slselselaru ut dals ]xau eqJ '(Zi-Of arn8rg ees) tueur -dolanap prtaqttda atuos uI sJn3Jo qtlqm '(JWn uoqts senssll Surrnp IEruJou -unt11arufu4tuasara-ol-Ior1aquda aql salqueser a8ueqc slql 'seIuouIJJeJJoJtelel ssnosrp a,r,rsV'xrJleru Jelnllaf,EJxa eql ol pue sroqq8rau radord Jleql ol peJeqlal slac daal fletuJou leq] srusrueqJeru allseqpp aq];o uoqdnrsrp e sarrnbar L1urc1 -Jac]sorule sseuelrselur 'poolsrepun ilala lou are sa8ueqcrepcalou 8urflrapun aqr q8noqrly '16-67 arn8rg 'aldruexa JoJ 'aes) anssq SutpunoJJnseql olul suols -ualxe qlrm 'sJapJoq pa38er pue qutor8 go uralled paztue8rostp B ^ ot{s qJIWV\ 'senssp 'sJorun] lueu8rpur;o saqredord Sururgapeq] Jo auo snql sl ssaueAISuAuI Surroqq8rauSurpenurtuo4 luaql luanard pue saceldradord Jleqt uI sllal I€ruJou daa>1leqr slurprlsuoc Jo eaq )eaJq lsnu ]t 'azlsptselelu ol [ec t""y:;;i:i,"" aJepallolur srusrupqceruJplncalorueqlJo ]soru pue 'xaldruoc TIeslL,rlar rl.r",r" eseq] Jo qJEg 'salrs luelsrp ]e saluoloc Jelnllec r\/Ieuqsqqelsa ueq] puE 'slesse^ eql aleal 'uorlelnJJrc aq1 q8norql elotu 'slassal puu senssll Iesol epelul o] eluq 'uoueIpPJJIpezlleJol slleJ Jef,ueoaql :ssacord dalst]pru e JIeslI sI sISElseleIAI ro dra8rns raqlra ,tq elullppre o1 alqtssodrul lsourp seruoJeq raJuec u ,(poq aql lnoq8norq] Surpeards,rtg'sqleap paluloosse-JeJueoJo %06 JoJ alqtsuodsar Suraq tacuec 3o lcadse-poolsJapun tseal pue-dlpeap lsoru eql sl slselselel tr

luouruoilAul u6larol e u! alelar!lold pue a^r^rnslsnw slla) ra)ue) lueu6llew 'ezlselselewoI ro)ue3:97taldeql

OZZL

'JalEI ssnssrp e^asE 'ddeJeq] SnJp JoJ 1e3J4 snor^qo euo eJp sJorunl otut peloeJlle are ]Bql slessal leruJouqe eql pue ,{1ddnspoolq alenb -ape up uo serlal q$\oJB Jorunl telemoq ,{laruu4tln 'lueuBqeru aroru are daq} leq] sueaurLllensn qlrqm'suorlrpuoo lryssaJlspup qsreq aseql q eAIAJnsol elqe re1eq eft leql slleo JeJupJ luelnru JoJ slJeles 'uJnl ul 'exod[11 '(8I-02 arn8;g) erxoddq go suor8ar puoqrppe elearr o1Surdlaq 'rorunl eq] ul{tl,rlr ^aog poolq rEIn -3au ol peel 'sepcelou Surpu8rs Jo aJuelpq lerurouqp uB r.uo4 llnsar dlqeqord qrlr{ivr 'sarlqeurJouqe aseql 'spue peep qlrm seqcuerq ,(ueru aneq daqt pue '.{4ea1 pue 'Jaleruerp ur snoeueSoralaq 'snonpol eJe slassel peJnpu aql talel oH 'azrsplselaru ol slleo sll JoJelnoJ adecsa ue apnord oslp lnq 'ua8dxo pue sluerJtnu qlp\ rounr aql ^lddns dlaq dpo rou slessal eqJ ' (vE-tZ an -3rg aas; slasse^ poolq ,lreu qprorB aqt aleFrurls prre slleJ lelleq]opua ]Jer]]e Jo 'Utgn nlcnt qimoJ8 luryaqiopua JDInJSDI leql sulalord pelarcas ere eseqJ sB qons 'sJolceJcruaSor8ue-ord apocue leql saua8 go uorldrrcsupJl eql selelrloe 'uJnJur 'uralord srql ltz raldeq3 ur paqrJcsepuralord drolep8ar aua8 e'(nt-ilH) nt -toilol alqtcnpul atxodfg Jo la^al aqt ur esearrur uu Surxonord ,i.qd.yddnspooyq aql aseaJrur ol?4JflmsJrua&o1&unup selpllloe exodrtq eqJ 'JalarrrelpuI oml Jo Ja] -eurTlrur e puodaq sa8relua Jounl eql sE slleo eql peJJp o1 su€aq qJIqM'exoddq ol asuodsar ur pacnpo.rd are spu8rs asaql 'spu8rs eruaSor8ueSurlarcas,{.q,,{.1ddns

'alrsMau e rournl alqelralap e arnpo.rdol se os onssrlMou e azruolo) lp llrir,tueorlspoolq aLllralua ]eql slla) townl 1ueu6r;er.u puesnoql{ra^a ur auo ueql roma}reJIllerrdft leql Moqssleurueur sarpn}S'sase}se}au-r apou-qdu{1ol asu 6urnr6'IeMoql 6uo;esapouqdur(;u; padde.rl uauo lassan>rleqduI; er-uo>eq P paraluaa^eqteLltslla)rounf 'Luearlspoolq aql olu! (qdu^l) sluoluo)s1rsa6.reqrsrp,(lalPur]ln ]eql eql 6ursson{q 'sdeqrad,(;uor.uuorarou 10 'araqperuruer6erp se 'lassanpoolq e ;essenrtteqdu,(1e lo 1;ern oql raluo{eru s;;arrownl'ia^rl aql ol rappelqaql se qxs uebro ,o lleMaqr 6utssorlIq ^lparlp urearlspoolq ue ulolJlorun] e;o peardsaql salerlsnllr e;dwexasrLll'slselselaur 1ossaroldaql u1sdet541.-ggarn6r3 srselselauruMolq-lln] 6 u r u r . r o: 1 e n r 1a z t u o l o )

srselselauroJ)r [! r.r.rJo] ol lossa^poolq ulor] aoe)sa

r a ^ r lu r l l e M lasse^poolq ol ereqpe

a

/ -

(sasPlselaur urro] ol

e u r u P ll e s e q

.1.

a

a

a

a

a

I

a

u J n r l e q l r olee u r o u f u e l l r d e r r a l u a p u e a ^ t s p ^ u ta u i o l a q s l l a f

u n r ; e q l r d eu r J o t u n lu 6 r u a qs e n n o r 6s ; ; a r

LZZI

SS])OUd UVNOI.LNIOA]OU)IW V SVU]f NV)

'tuatudole^ap rounl ut tted lueuodull ue sleld sadll lla) leqto pue asaqtuoaMtaq )lPl-ssot)'slla)poolqaltq^^ItoleulurelJul p u e ' s l s e l q o l q u ' s l llae)t l a q l r dl a eln)se^,slla) ra)ue) 6urpnl)utsad^lllo) ^uerrlJo lstsuo) srounl'slsauebgrournl u1alor e s{e;d luauuorluooJ)!ut Jor!nl eql 6 1-97 arn613

sr"E)

-lv|/\louls

lla) poolqalrqM

l s e l q o r qr l

lla)relue)

sllallerlaqlopue

{re11tde>

'sa^lasluaq] sllac rounl eql 8upa8rEl o] uoulppP ur 'slleJ IBIuoJls palEAIlJB ]lqlqul o] peuSrsepaq plnos slueutear] reJueJ leq] ]se88ns sllnsar raqto pue aseqJ lv\0JBo] Jorunl uqs aq] JoJpeJrnbar srsauaSor8ueeql elElnlulls leq] xtJlelu JelnlleseJlxa eq] uorJ srolsEJ crueSorSuESursealar JoJ elqrsuodseJ sI esEeloJd aql jsfiec ]selu [EruoJls^q pe]arJas r(guurrou sl IEIII eseula]oJdolluleur KJleru cIJIs -ads e Suolcel aJrru ur papJelal ^lluerrJru8rs sr sJorunl ur)lsJo I{}lroJB aq} 'eldruexa puoces E sv 'op lou ill a slsElqoJqrJIeIuJou puP 'slsulqoJqlJpelElcosse-Jorunl eq] uo spuadep seuroulf,rEJ paluBldsuBrl auos Jo qlMoJS aql leql elef,Ipul aJrru ur sluerurJadxg 'slle3 leruoJls JEInsruBd sll uo ]uapuadep seuoceq Jorunl eql pue taqlaSo] aAIoAaeruoJls slr puE Jorunt eql 'sde^\ aseq] uI 'xlJteru JPInlleJ 'uoISIAIp puE qlMoJS -eJlxe eql Iapoual JaqunJ lPq] sasealoJd eleJJesdaql pue 's.[e^i\ snoue^ uI sllas IIaf, JesueJ aleFrurls leq] surelord PuSrs eleJsas ,(aqJ rournt aql uo {ceq }Je uJn} ur slleJ [EIuoJls eqJ'xIJlEIu rElnllaJeJue aql ltlpolu ]eq] saudzue Jlilloelord puE slle3 lBruoJls aql Jo rol eqaq aq] JellE tBql sulelord pu8rs qloq Surlarcasrtq euorls aql ut seBueI{JaJnpu slleo Jeoueoeql 'sassaJB -oJd purourJJeJe sV '(6I-02 arnS;g; sllao elosnru qloous pue saldJlJed luep -uaDe rraq] puE (slesse^crleqdurdl puE poolqJo slles lPlTaqtopuaaql 'slle3poolq alrqrr ,{roleruruplJur 'stselqoJquortur 'slselqorqg Sulul€luoc anssl} allltauuoJ leruJouJo pasodruoJ sr pue Jounl aq] JoJ{JoMeuB4 eql saplloJd eruoJ}seqJ 'Q7 pue pru,(qcueseu passncsrp; ur slac n1der43 sllar uo seller sue8ro plleqllde uaa,ulaq luaurdolanap ;o lelleqllda {let-ssorr 'PruoJ]sJoun] aql pue sllef, Jolunl eql ueeMleq IIE]-ssoJf, IEIuJou eql se lsnf uo sarlar Jounl p Jo lueudola^ep eql asnpoeq sl sHI 'sJapuels^q lueoouul ruo4 JEJaJe-BruoJls Jo 'enssrl a^rlceuuoJ Suproddns eql Jo esoql .,tletcadsa -Jorunl aql ut sllaf, Jaqlo eql 'stotunt asaq] uI suollelnru Jo sJeJBeqeql Sulaq 'leruJouqe ,{lssor8 ralueo eq} aII{ \'sllao JaJueJeq} o} uoll1ppu ur saddl ere sllaf, 'serlrlua xalduroc eJe seruoulcJeJ JaIIJEapauollualu sV 1ac -,tueruSurureluoc

lueuuol!Auool)!wtounl aql tuaudolo^acra)ue) sa)uanBul uu,rz

(^llouuol'f MalpuvJo^saunof) 'r(1ddns poolq rraql unirol6lnoa^eq slla) ra)ue) oql araqMsrsol)aujo sealeluto lerlua)seqsrselselausrql'6un;aql ul spue;6anrsaqo)6utulo; lltls/slla)le)ue) sMoqs lel)a.lolo)pale!]uaraJJ!p-llaM a)rlsanssrtsrql'6un; aql u! slselselau eurour)Je)ouope uolo) g 1-97 arn613

q.! .:tt

raloeq) zzzl rarue):02

EZZJ

'uo\saJ8oJd partnbal rowry rot aJ9 [IruarB I,)LUsa&uqlpJlpua&lda puo Jlpua? raqilnt aq|to uouqrulanna aLflsaruJalaJJa u asnu)aq'sJownl acnpord oJ alqo aJDlaup sIpJ Juorraqvlo sauo1caql ur tol panalas s! /ti!MD$u! cpaua?s1t11'adtaotta4 ut saBuaqcpua fuqtqolsut alaosoruoJqJoi poal 14)u4m's$oiltaSuunp uouaBatBasauosouoJllJ ut slcapp atD uowwoJ osyy'acuanbas '.spul4snornn sJoJrauoliac!1datpauoc o1rc aBawap VNe u! saBuaqcoi paal LlJrqm to y61q nadat oi [inlqo aLp u! sralap wott auta uac fu1yqa1su1 4tauaB aqJ'alqu$un [11ou1aua?ara sl1arraluoJ 77a[paau ]oqJ Sursrtdtns iou sI il 'srornvllaq Tonosolo uolpalloJ s1t1l ntuoc oi papaauata saBuaqccllauaBtdapuv suollDlnu fuaw acury 'uoauspoolq pua aLU Dm azlsolsopla aBnTmot8 oJrowru aqi monv pqj [Taalca1oJ'iuauuorrnuaorclru tourni. spssanpoolq mauSurpn1cut'slpclaruous pa11ac ar4tur ruasatd sllacraqrc uo spuadaposIDtI :sanpsuaL7snal JaJuor aqt ut saBuot1c uo [1at fidwts iou saop rolan] D to uoln1ona aqt tanamoH 'sluauuollnua uBptot ut uo4atatr1otdpua pnrntns sattnbat Lpnlm-azlsalsDplu oi uaLppuD anssrlToutBtto rlaqi u! aprntp puo 'motB'anmtns ot slpc ncunc to ,hruqo aI4JasuarcursaBuaqcasaql lo yy'aSowap VNe ro ssausIIaJ oi asuodsatut stsoidodannpu! ro uolszlrp 77acdols [puauoutnd ]nq7 slasruor4Jaua IoJiuoJ aLp ur puv uo4a4uatatllp u1 slcatapannbca sIIaJraJual 'uorssatSotdtoutnL lo ssacotd[touolm1ona aql lo ynd sy '1ot1uoc1tqBr1 rapun uouDJatl1otd71acdaatl[11nwtou LDLU ruauuoJlnua JnLp utott s1nuBtsaqt atouBr ot snal aqt Su4qoua's[amqlad Sutpu8rs lpc ut suoliuJaqoapryJw asaql'paatds puo '[1d4ptu 'anlonafaql sn sautadotd Tanadslo &auan a annbnt sryacracuaJ 'uouraps lDrnpu [q patuadwoccasaBuotqc cuauaBtdapuD uorruJruu oBnpun sllac JrJaruosqcrqm ut 'uolmpna to ssacotdayn -uDrulmroe pua D uollntado aql snaf [uaw sa4a1[11ansnuorssatSotdnwnl vcaTfat lo 'pmaua*t1asaltu{aput nt [ncadoc atp satrnbcnryr11 palarluatatllp arow a rc'[1a71u 77ac -{aput mauarfias o1[.11pqnatp LlJlmpamopuafpaat1as! ulJlqm'nn wa$ anssqDraI4]D aq faut JaJuD)u to urBuoto IIaJ aqJ 'snonlual aworaq o] 'siuanauiauaBrdapuv suorj -Dnu luuorirppo snotaunu Suumbat'saBuotlc nqlnt fuaut oBtapun $nM IIar snll to [uaSotd aqt inq'uouatnw Inl]tur uo paJuauadxasaqruqi paca18utsn wo{ a1au131to to [LaBtns[q alactpatao1qncSlp awocaq[aql o1ylSnoql aJasnJuoJ'uo!rulpailr 7oco7 'sasz$Dpu to 'storani [tapuocas o] asu SutntBfg 'Quou811nut ata [aqy'v lol4i) sua8rc rua$lp azruolor puq sanssuSurpunouns aponm o1 alqa aJa pua (ulsnldoau ata faql 'uotrruyfap[q tsnarraruoJ 's!ruqJ) sloouor puuou to acua{ap u1arytatqotd pua motB

{reururn5 'JaJuEc Jo luarudolanap aql alouord leql sJolceJIelueru -uorrlua aql se ilel se 'saluadoJd asaq] Jo eruos erlJapun leq] susrueqJau Jel -nJalou pup suortelnu aql euruexa aarrtaldeqc eql Jo suorlJasJelel aq] uI 'saJaluolel rlaql 3urzrnqels.1o.{e,rn raq}oup arrnbJe Jo 'aseJaruolalacnpord reqlre ^eqJ ' 6 alqetsun,{lpcqaue8 are .{aq1 ' 8 '(azrselselerudaql 's1 leql) salls u8raro; ut alera;t1ordpue e^rlrrnspue (e^rsp^urare ,iaql 'sr]Bql) sansslleuoq req] uror; adeesa.,{aq1 , L 'srsauaSorBue acnpur rtaql ' 9 '9 ']uaruuorrlue IeJol neql ul sfiec Ipruons IEruJouaq] uro4 dlaq acnpur [aq; 'a8eurep y1.1q uorsrnrp 1ac dols ro (exoddq sE qJns) sseJlsol asuodsar u1 ,fit1]uauerurad flerurou leql susrueqf,eu IoJluoJ JplnlleJprtur eq] q a^llJaJap are {aq1 ' h 'srsoldode 't o8rapun 01 sllar IpruJou ueql auord ssal are daql 'z 'spu8rs JelnllaJeJlxe flanrleyar are [aq; anrleragrlord-rlue o] alltlsuasur 'uorsuadsnsur ee4 Surleog pue runleJlsqns e o] ]uaJaqpe lou uaql uala alnqnc IIac ur aleraSqord pue e^r^Jnsuauo ueJ.{aqt 's11arleruJou lsoru a{llun 'aldruexaJoC 'uor}pra 'I -grtord pue qwor8 Jraql JoJ sllal upql aroru are ,{aq1 ]ualJIJJns-JIas IpruJou :praua8 ur sllec JaJuer sJorleqaq ,(a>1 aq1lo rs11 Jo 'sselequelaN 'sa4radord esaql uoqs e dn merp ueJ ellt Jo suo4purqruoo ]uareJ 'saAIoAa -grp arrnbar sJacupJ se-s[DIS ,nurau alrsJaAqnsJo uor]f,elloJ e ll ]uaJaJJrC ',{pea13 -satlradord lueJJaqeJo a8uer e arrnbcelsnu IIec e tecuec e ecnpord o1

qynorD snoro)ue) ol alnqtrluol [1Ier;df1 sa!]radord(u e61 v sv ulfNv) ssl)oudAUVNoil_nto^tou)rri{

('^lar)osla)ue) ue)tjeLuVurolJuotss!t!Jad LlllM'066t [1atro5]a)ue) ue)uor,uv 'sarn6r1 pue sl)el ia)ue) Luol;poldepe,g) 'ro1te1palltads q)ea ol alqelnqulle sase)ra)ue) letolJo aoelua)lodoql sMoqs alqe]aqf'satelspallun eql ut talue) uo al,{lse}llpue luotuuol^ue Jo sl)a]}e paleuillsaauos (B)')su le)ue) 6ulrel>lp ur '1arp6urpn;tut'srol)eJleluoruuoltAUa ;o aruellodturaqt lsa66nssuotlellasqo aseql rallloue ol uotle)ol auo uot, enou suorle;ndodtrlteds ua\M (sltrutD anlq)6ursearrep)o (stAoilD paJ)6ursear>ur re)ue)Jo satelaql sMotlspuoMaql Jo deLusrql (v) 'sa)uanlJu! leluauuoJl^uaol paleloJs! a)uap!)urraruel 97-97 arn6r1

LC !

nz 7-l

lPoJql'qtnouJ 'aletsord 'seer)ued'laMoq lseaJq snFeqdosa'q)euols r a p p e l qf a u p r 1 ' 6 u n 1 sadr{1snorien

(8) l o q o ) l ep u e o ) ) e q o l . s p o o +p e l r o r qp u p p a u l l a q r +M o l ' 1 e ;q b r q : 1 a r p. a rnM o l : ] a r po e 1 e - r 1q r u6 r q ' l l e s q 6 r q ' s a 1 q e 1 a 6 u peleler olleqol . atnsodxe;euolledntto . sjope,t a;r$salr;pue lelueuJuor^ue

sase)lelol %

(v)

s

s

v)tuilAtv HtuoN

,

1ir

'a8e uan6 'adorng pue salels pelpn aql q suolllpuoo lueJJnJ eq] tepun E Jo slBnpr rpur Suolue elEJ qlEep JeJuPs lelo] aq] saJnpeJ ,{lppuelsqns a;u 'lou sI sIqI Jo,{e.\,\esoqM sdnor8qns upunq aruos aJeeJeqJ'elqE}I aul ta^aMoq 'JaqlouE ol pesodxe ^,{1ensnsr Je8uep qJns euo sedecsa }Eqt uopepdod e pue 'sJolf,EJ {srJ le}ueuuoJr^ua }uaJeJJrpe^eq sJaJuPJ tueJa#Ip ,(1aleungo;un '@Z-OZ,arnBtC) alqEuodtsod tsEel 1Bro 'elqEpro^Paq ppoqs sracuPsJo %06-08 ]eql paletupsa (plueluuoJl^ua 01 ^Ilsolu anp eq sl ]r 's8urpurJqlns ruoJC 'sJolJBJ'crlaua8 lou o1 readde seJueJeJJrp eq] '[J]unoJ ]soq aq] 3o pcrd.,i1eJuepIJuI Jaf,ueJJo uJal -1ed aql ]dope o] pue] suoqplndod luerBru asneceg 'Jemolqf,ntu sI af,ueplcul aql ereqa.{JlunoJ JaqlouE sI aJeql ,{rlunoc euo uI uoruruoJ sl }eq} Jaf,u€cdJa^a lsolup JoJ :sarrlunoJ ]ueJeJJrpur aJueprsur JaJUEJJo uoslJudruoJ P ^q dlrealo ']Ea a^ApooJ 'sJa3uP3 lsolu pelBJlsuouap sl slqJ lsotu Jo ]asuo eql elBJelef,JE eql Suenlcur 'sJolJeJ.Iplueuuorr^ue leql e3uepl^e sI eJeql 'ssaleque^eN dolanap ol JecuEJJoJtuelcrJJnssuot]etnru Jo les e etu1ntu -nJ3Pflpnlue^e plno^i\sllal Jaq Jo srqJo auo ]seal ]P teql alqe]I^eul sI lI (qSnoue 3uo1 anl plnoc upunq E JI 'g raldeq3 uI pessncslp se tIBdeJ pue uoqeclldar VNCI Jo ,tcerncce aq] uo suollelltull lBluetuEpury Jo eouanbasuoc elqedPJsaul uP aJe daql asnecaq papro^e dlalnlosqe eq Je^au UPJ suoDulnlu :saJuPls -runJJrcJo sselpJeSeJ palcadxa eq ol sI JacuecJo ecueplsul punorS4ceq alqlJnp -eJJr ur€ue3 v 'aJII Jo de^\ pue ]ueuuoJl^ua Jaq Jo slq uo ]uepuedep sJeqlo 'lenpllrpur eqlJo uoqnlpsuoJ JIlaueB eqt uo luepuedap eruos-sJolJe; aldrlpul ,tq paurano8 qoee 'sdels ,tuuur sarmber .{llpJeue8 JaJueJ E Jo luaudola^ep aqJ

U])NV) IO S]SNV)]IBVIN]A]Ud]HI ra)ue):93raldeq3 vzzl

')rua60ur)le) osleaJesrqlsPq)ns slsalur )ruooptnur arPleqt spunodu,ro) Jo,(lrrofeuy 'yp6 a6euep teqt stua6eol alqllda)sns ,(;;er>adsa uraqtsa)eur1eq1(raurqrer.u reoar vNC raql ur l)alap e a^eq osleeualleq 3ql lsal aq1;o^lrnrlrsuas aql esear)ur01'aurprlsrq arnDar tou op leql erlolleqlueuanar6urlealr 'l)alap aql sasranar lPql auo6 s!ql ur e6ueqrraqunJp asnp)ue) suabe1n61 'srsaqtu{s eulpl}slqro; {ressarauaua6 P ur l)a]ap e Jo asne)aqunrpau aql ul oulpllslqo.lrnDar lPql eua])eqDilauowlos Jo urerlse sasnlsal aql'{1r>;ue6e1nu ro, lsal sauv aql 17-67arn611

vtul.l.)v8 .l.NrCNSdlCNl

-lNtcU_stH lo s ] | N O r Ol)N n O )

-':\

'?fiF-,-

[ . r ] ( a r . : 1 t r : : 1. j . . . i ]

\.-',1'ij,,d7 .-'-N]DVlNN

lNrctrstH 9Nt))V'r NntclN UVDVNO rno t-LV'ld CNVXIIA

Zffi

peJlxa Ja^rl pazrue6ou.roq

elleuoutles luepuaoep - a ur p r l s r q + oa J n l l n l

fl

: . : 1 : : i:: : : . j : - .: j r \ ' ::;':.;: .;:,:,:;5./

Fj:

ua6elnu Ierlualoo 'punooLUo) lsel

l{!-lr.jj-'

'QZ-OZ a.rntld) a{orus oJJEqo} pue JE} IPoJ uI }uesald IEJIIUaqJSutsnec-raluec e'auarltd[n]ozuaq pue tg utxotDl[D urxot IEBunJaql epnlf,ul{em slql uI pe]B^Ilce suaSourcJeJJo seldurexg 'clua8elnru {UBU aJE ]eql slsnpoJd saleJaua8 slPJ -rrueqJ urEual uo dlr^rtsu Jreqt '.{laleunuoJun 'spunoduoc palerJxe,,(psuapue ssalruJPqolur sulxo] pelsaSur ua^uos o] dlaq ^llPtuJou selu.{zua aselq.J-'sas?plxo Tgr-d aLuotqJoltJ aqt sE u^tou)I seudzua rPlnllesErlul Jo las e fq pazdptec tan11 aql ur sesseoordcrloqelaru ,{q urro; eAI]JpaJaJotu P ol pe8ueqc uaeq eneq.{aql rage i{po Sur8euep aruoJaq .{aqt l.{lecruraqJ UeuI z(1a,lpe1arare seuo }ualod erolu aq] ^,i.1praua8tnq 'VNCI uo ,tpcarrp lf,e suaSoulcrP3 eseql Jo ^ aJ V 'sua8elntu aJe suaSoulcJBf, Lueur teq] spurJ euo 'pazf1euu aJeseoJnossnoIJeAruo4 E]ep &lJlue8e]nru uaq \ 'sllec uerpluruEru uo pelsal ueqm suolleJJeqeeuosoluoJqJ Jo suolleln{u esnBc oslp prJelsBqur desse]uarua^uoJ pue prder sql .{q JrueSElnu se peJossspunod -tuof, aql Jo rsontr'(Iz-02 am8Ic) pernseeu uaql sl slrelJBq eql Jo aler uollPlnru Suppsar aq] ierJalceq1sa}pau8rsap,(lerceds;o eJnllnJ E ol peppe sI pue (^aolaq passnJsrp-lErurue ]3Plur ue ur sJnJJo lEql SulssaJord pcturaqf,olq eq] slrulru o]) sllec Jalrl leJ ruoq paledaJd lcpJxe Surle,ttlce uE qllllr pe4tu sI ueSouIJJp:) aq] ,ilrJrue8elnu roJ lsal uoruluoJ auo uI 'suollelnru esneo.{aq}-/ilJadoJd uour -ruo3 euo ]seal ]E aneq daq] 'seJnlf,nrlsasJaAIpeAPqsueSoulJrEl Ieclluaqc asaql q8noqllV 'se8 pJelsnu sE qJns slue8e 3u4e1,r{1p pue 'saultuesoJ}Iu 'saulrue srleurorB se qJns uraql Jo sa^rle^IJap puP suoqJeJorpdq clleluoJe Jo aSueJ e epnlcur saldurexg 'uDISJraql uo dlpaleadeJ paluled Jo slerulue ptuarulJedxa ol peJ are ,(aql uaq^ cruaSourJJEJ aJe slEtllueqc aleredstp almb due141 .{.ecape^l}JeoIpEJ rxoq selJlupd n pue s,(erl" se qcns 'uorlerper Surzruor sB IIal sE 'aulqsuns ruo4 rq8g AII-uoIleIpEJ Jo sruJoJsnorrel pue sprrueqo apnlJul sua8elnru Sursnec-racueJeseql'suoq -Btnur eleraua8 os pue ygq a8eruep]eql asoq] are puetsrapun ol lselsee eql lnq 'perJB^ pue,r(ueru are 'suaSoupJuJ se rr/v\oDl tarueJ esnPJ ueJ lBq] slueSe aql

'llv VNCa6euec sluoov6u!sne1-re)ue) loN lng'fue6; 'esuasp aqt Jo .{3o1orqJelnJelorueql pauruexa a^eq e^ JeUE'uollJes }sEIaq} q JecupcJo}ueu -lpeJl aql ssnosrp ilr\a aA 'JeJuEJueunq luanard ol sr(ert Surpur; uI 'saIllncIJJIp pue 'sqdurnrJ] aql Jo aruos JeprsuoJ ueql llrlr a A'JaJueJ esneo o] TI.'t,roDI are leq] stua8e leure]xe eqt ]noqe paurpal ueeq seq tEqM ]e Iool ]srIJ IIIM a i\'1ce daq] ,vroqqsnqelse o] Jo srolJeJ{srJ letuauuoJrlua crycads eq} dJlluapl ol-ocJeqo} 'paplole aq uago ;o uorldacxa aq] qlu^-sasec lsoru ur llnJrJJIp ueaq seq ]l uec Jacuer leql alef,rpursuoqepdod ueunq uo suollelJesqo qons q8noqrlV 'BJrrJVur suorlelndod ]uangJe fyalrqeyarureual ur ,rrrolosle sI eJueprJur JaJueJ 'prauaB ur suplrJaurv Suoruu eoueprJuraq] JIeq lnoqe fpo sr 'xas Iensec pue 'sBnrp 'sellareBrc'eeJJoJ'lor{oJle pro^E oqm'(A1ureJ erues eqlJo sreqtueru Surcrlce.rd-uou Suorue 1ou ,(puelrodun '1nq) qeln q suoruJolN ]crrls Suorue resueJ Jo acuaprJur aq] ]ng 'raf,ueJJo alp ily!\ aldoad aArJur auo flalerurxordde

ul)NV) lo srsnv)ilgvr.Nl^ludlHi

9ZZL

VNO

(s) eutofl esoalso : h l f l t G V U. eulotleqlosaur :SOI-S38SV . r a ) u e ) l a p p e l q ' s e u l o u t ) l e )u t l s :)tNlsuv . seru.ra)nal n)e :lNlZNlS . e u r o l r e s o r 6 utea n r ; :lctuo'lH)]ANtA .

se8upqr uepplq aq] 'a8pruep JrleuaB roJ toadxa tq8lu euo sV .pernpord seq Jolerlrur aql ]Bql IIat luelnu e ruo4 pepueJsep sllasJo euols elSursp Jo slsrsuos Burollrded qrea leqt ]q8noql sl tl :pe3npur sEruollrdedJo raqrunu aq] raBrEIeql tolerlrurgo esop JorJdeql JalBeJBeql'sDruonldud pellal sJorun]eIIIue a ,u8ruaq 'llplus duplu Jo ]uaudole^ap aql ur sllnsal uorleJaJlloJd srqt totenrur ue ol pasodxe uaaq ,{lsnor^ald sEq uDIs uor8er E uI '(pee}sulSuIpI^Ip enunuoJ o} }Eq} Jo Suppttp dols pue alerluaJaJJrp dleurrou ppo-rv\ leq] slleJ esneJ ol Jo) uorswp ilac eleFrurls 01sllas uo.,(pJalrpurpup.{.I}f,aJrp qtoq }Jp esaqJ'lueuuoJr^ua leJol eql ur sasEaloJdpue srotf,EJrl}lv\oJ8qloq Jo uorlaJJeseql Sursneo,asuodseJ^,fuo1eur -tuegut ue aJnpur ol ,{FueJeddesr Je}oruoJdJourn} eJo elerpeuur aql }JaJJe 'SZ-OZ aJnBIC)Jolerlrur trua8elntu qlua e lueu -]EeJtE JaUEparldde ale .{aq} ^luo dcuanbar; q8rq 1BsJatupJ esnel seouelsqns Jr eseqJ'(9I reldeql ur passncsrp;demqled Suqeu8rsrelnllef,ertur lolrsourldprleqd -soqd eqlJo Ued ale^rlJe ecuaq puB (JXA asourLulalotd J 1o srolB^rlJpIETJTJTUB sE a^Eqaq qJIqM '(VdJ) alplece yoqroqdldoupJepellal sp qJns ,aa1sapqtoqd .Jolerlrul are sJalouroJd Jorunl papnls dlaprllt aqJ Jorunl pasodxa lsou e o1 'Jrua8e}nru dlsnonard uDIs ur ,{1anr1ra1as Jef,ueo esneo ueJ sellasuaqr rou are slpJrruaqc'sraloruord Jorunl leql se u AouT seJuplsqns ol sqluoru porrad Jo B JeAoaJnsodxapaleadar flanrleurally.rarlree pauorlueu a.,r,r se ,1ca33a Suqoru -ord-racuer eql ur ued e ,(e1ds11ac droleuruegur leql tq8noql sr tr pue ,punom e ol pe]f,eJllB eJe slleJ poolq alrq ^ ,rtroleruruegul .punoM eql Jo aSpa aql ]e sllao eqtJo aruos tuo4 dolanap ol sraJupJ asnef,(uorJEJaJrIoJd 1ac Surlelnruqs dq 'uEJ Jolerlrur ue qJns ol eJuo pesodxa uaeq seq leq] uDISSurpuno,u.dldurrg 'Joluplul Jorunl .s}lnsur ,}ue e se slce ,{e,rn slqt ur Jacue3 spees aql s^ os uaSourJJe3 }eq} Jo V -JaJJIpalrnb taqlo urpual o] Jo uaSoursJeJ qll^\ eql slualuleaJl JaqunJ o1Jaqlre pasodxa aJE slleJ aql uaqm ref,uec pesealour ecuaprJur dpear8 e ro; a8els Jo aql les leql suot1elnru-a8eruep cpaue8 esneJ seop tualel 1l ,1a1rtlqerurouqe 3u4se1snorlqo raqlo .{ue Jo Jorun} p ol asrJalr8 y1as1r dq saop tanaa,roq,sua8 lou -etnru esaql uoqeclldde apurs y' (vsNC) aueserqlue Jo lelzuaq{qleurrp punod -ruoJ paleleJ aql ro auari{d[e]ozuaq se qcns uaSourcrecSurSeuep-y5c e qrl^1, UDISaqt 3u[urcd,{1pa1eadar,r(qaJrru ur palrolla aq ueJ sJacueJuDlS'uorssar8oJd rournlJo saSelseql a^rasqo o1^,(sea sr lr areq \ 'uDIsesnoru uo slEtrrueqcSursnec -JaJuel slcaJJeaq] uo o3e 3uo1euop serpnls uro4 saruoJ eJuapr^e Jo lsaJeelcaql 'sua8elnru are recueo Jo aruos Jo luarudola,rap aq] JoAEJleql saluelsqns [e ]oN

loN oclsJelouordtounl lylg6aoeureosroletllulJournl *rorj uoIss|,Jr radqr!M'z00z., o, I l1lfr:HJ l'rt"iil; ", ",,,, "

'jiJl

auag :luauuol^ul aql pue lafue) uolJ elep,g)]a)ue) luauruoJtAUf asnetsuabouole)luaraJJt6 tualaJJtp (B).aua6tossalddns tounl 1osed,(1 Egdaqt yo suottelnullsualfeleq) qllm pele!)osse srpue s>rdol1 aq1u1 ro)uel ra^rlJoasne:Itolnquluo) e aq ot lq6noqt st ll suotltpuo);etrdorl prunq rapun parolsalp r(eq}uaqmslnueadpue urer6uo snnolo}eq1 (aozltosnaollsnllt1tadsy) ploLup utolJutxo] e,lB ulxolDllDst ataq patellsnlll punodurotaLll'VNOqllrvr6urlrearIq suollelnu asnp)lltMIeql aloleq sua6ouole:lp)tu,laq) {ueu alenrltetsnul uor}eulo;suell)tloqp}aulV 'uotle^tl)pua6our:re1(V).suo6ou!)Je) u/vlouleuo5 77*97e.rn613

vNc NtlNtNVn901 cNnoS NlgONtfuv)

ICtXOdt-E',Z-NtXO-LV]lV

NIXO.LVIIV

(v)

I

N

ggp-6auorqroy{r

re)ue):g7ra1deq3

gZC.l

1227

CAUSES OFCANCER THEPREVENTABLE

CANCER

T

CANCER

NO CANCER

Figure20-23 Somepossibleschedules of exposureto a tumor initiator (mutagenic.l and a tumor Promoter (nonmutagr:nic) and their outcomes. Cancerensuesonly if the exposureto the promoterfollowsexposureto the initiatorand only if the intensityof exposureto the Promoterexceeosa Cancercan alsooccur certainthre:;hold. asa resultof repeatedexposureto the initiatoralone.

NO CANCER

+

TII

CANCER

caused by a tumor initiator are irreversible; for this reason, they can be uncovered by treatment with a tumor promoter even after a long delay. It is still not certain how tumor promoters work, and different promoters are likely to work in different ways. One possibility is that they simply provoke the expression of proliferation-inducing genes that had been mutated before the promoter was applied but were not expressed:a mutation that makes a gene product hyperactive will not show its effects until the gene is expressed.Alternatively, the tumor promoter may temporarily alter the way the cell reactsto the product of the mutated gene, either by releasing the cell from a counteracting inhibitory influence or by triggering production of a co-factor necessaryfor proliferation of the mutated gene product. lVhichever the mechanism, the result is that the mutant cell is enabled to grow and divide and produce a large cluster of cells (Figure 20-24). A tlpical papilloma might contain about 105cells. If exposure to the tumor promoter is stopped, almost all the papillomas regress,and the skin regains a largely normal appearance.In a few of the papillomas, however, further changes occur that enable cell growth and division to continue in an uncontrolled way, even after the promoter has been withdrawn. These changes seem to originate in an occasional single papilloma cell, at about the frequency expectedfor spontaneous mutations. In this way, a small proportion of the papillomas progressto become cancers.Thus, the tumor promoter apparently favors the development of cancer by expanding the population of cells that carry an initial mutation: the more such cells there are and the more times they divide, the greater is the chance that at least one of them will sustain another mutation, or an epigenetic change, that carries it one step further toward malignancy. Although naturally occurring cancers do not necessarily arise through the specific sequenceof distinct initiation and promotion stepsjust described, their evolution must be governed by similar principles. They too will evolve at a rate that depends not only on the frequency of genetic or epigenetic changes, but also on the local influences affecting the survival, growth, proliferation, and spread of these altered cells.

Contributeto a Significant Virusesand OtherInfections Proportionof HumanCancers A small but significant proportion of human cancers,perhaps 15% in the world as a whole, are thought to arise by mechanisms that involve viruses, bacteria, or parasites.The main culprits, as shor,m in Table 2O-1, ate the DNA viruses. Evidence for their involvement comes partly from the detection of viruses in cancer patients and partly from epidemiology. Liver cancer,for example, is common in parts of the world (Africa and SoutheastAsia) where hepatitis-B viral infections

n o r m a lc e l l s

|

'r'rrnro*

i s o l a t e dc e l l h a sm u t a t i o n b u t its growth is restrained

I eno'vort^RELEAsES REsrRArNrs I

mutantcellsgrowintoa largeclone in whicha f urthermutation of cells, mayoccur Figure20-24 The effect of a tumor promoter.Thetumor promotercreatesa localenvinrnmentthat expandsthe populatiorr of mutantcells,thereby the probabilityof tumor increasing progression by furthergeneticchange'

1228

Chapter20:Cancer

Table20-1 VirusesAssociatedwith HumanCancers

Papovavirus familv Papillomavirus (manydistinct strains) Hepadnavirus family Hepatitis-B virus Hepatitis-C virus Herpesvirus family Epstein-Barr virus

warts (benign) carcinomaof the uterine cervtx

worldwide worldwide

livercancer(hepatocellular carcinoma) livercancer(hepatocellular carcinoma)

Southeast Asia,tropical Africa worldwide

Burkitt'slymphoma (cancer of B lymphocytes) nasopharyngealcarcinoma

WestAfrica,PapuaNew Guinea Southern China,Greenland

Retrovirus family HumanT-cell adultT-cellleukemia/ leukemiavirus lymphoma type | (HTLV-I) HumanimmunoKaposi's sarcoma deficiencyvirus (Hlv the AIDSvirus)

Japan, Westlndies

CentralandSouthern Africa

Foralltheseviruses, th numberof peopleinfected ismuchlargerthanthe numbers who develop cancer:the virusesmu act in conjunctionwith otherfactors.Moreover, someof the viruses contribute to canceronlyindirectly; HlV,for example, destroys helperT lymphocytewhichallows a herpesvirusto transform endothelial cells.Similarly, hepatitis-c viruscauses chron hepatitis, whichpromotesthe development of livercancer.

are common, and in those regions the cancer occurs almost exclusively in people who show signs of chronic hepatitis-B infection. chronic infection with hepatitis-c virus, which has infected 170 million people worldwide, is also clearly associatedwith the development of liver cancer. The precise role of a cancer-associated virus is often hard to decipher because there is a delay of many years from the initial viral infection to the development of the cancer. Moreover, the virus is responsible for only one of a seriesof steps in the progression to cancer,and other environmental factors and

In some cancers,viruses seem to have additional, indirect tumor-promoting actions. The hepatitis-B and c viruses may, for example, favor the developmeni of liver cancer by causing chronic inflammation (hepatitis), which stimulates cell division in the liver, and enhances the viruses' more direct effects on cell

pylori, which causesulcers, appears to be a major cause of stomach cancer.And bladder cancer in some parts of the world is associated with chronic infection by the blood fluke, Schistosoma haematobium, a parasitic flatworm.

( ssard^]rsra^runproJXO[!oJJuorssil.urad qllM't/6 t 'zv-ta:09'lsultDuD) 'uoqewrew'B /lDN'r'uMor8r pue alol d rauv '816t 'ueuroarl'H M :o)sr)uerl u e S ' ^ l a r ) ops u ea ) u a r ) S: r a ) u e J ' s u l r p J ' f uorj) ]a)ue) lsearqJo luaurdola^ap atourordue)'ua60rlsaI1;eoadsa 'sauouiloqa^rlrnporoar suorleurqurol Jo urelra)ol arnsodxaleLllMatAaql uoddnsoslea)uoprAa le)r60lorL!aprda Jo saurlraqlo leranas'sauouroLl o] sosuodsal 'lseatq luanbasqnsrreqg6uue1;e aql Jo sllo)aql Jo uorlPrlualajJrp Jo atels aql ur abueq)luaueurad e ur 1;nsar {eu I>ueu6erdural-llnJlslUeql teq] rq6noqr s rl l ) s u a q t r a l e a r O o q l ' p l r q )] s r Ua q l ;o qlrlg ol dn sauouloq o^tl)npordalo] ernsodxa1opouadaqt loOuolaqf ueLuoM ssalplrq)p roJleql oi o^rtelaiIir;rqeqord aqt sMoqsqderbaql plrq)lsrUrall or qlrlq sanr6aqs q)rqM]e a6eaq1;ouorl)unJ e sPpailolo sreJrls,ueuroM e ur aurl auros ra)ue) lsearq1o,{lrlrqeqord 1e6urdo;anap a^rlPlaraql'la)ue) lseaJq]o Ist.taql uo 6ur.reaqp;rq) ro sl)arrf 97-97 arn6r3 plrql]sr] spqueuJoM qtrqnn 1ea6e :a

sz

sl o Lvd l

a

@ o

a

tt'

o

r ara a

90n

ll

f

o l a

oro t

ol

'

o o

0t5 .

a- _ -

UAtIlO/\^

.

ssalpllqr

a

f

a

aa

l v

ta

a

('I1ar>o5 ra)ue) ue)uaurvtrtolJuorsstu.uad qll471 '5002'sarn6Upup sl)pl la)ue) uolJ paldepv) aberaneuo'ra6uolanr;a;doad sesaleJra)ue) ur aseal)utalqe]t^aul aq] roj alesuaouro) o] papaauare asoqtelrl elep patsnfpe-a6y'lueulearl pue uorl)olap ut sluJr..uJ^oloult ol puodsoro){eu satelqleaplalue) raq}o ul suorl)npar]ua)au ratrDgo)tlaHqtllr'\ uorlfa1ur Jo suloDpdur lo letp ut sa6ueql o] palelarIlqrssod'latue)q)eurolsruotJ sLllpapur lleJaq] pue '6ur1ousol)pqol 'saxasqloq Jo uratled alll 6ulMolloJ roJra)ue) 6un; ur asrr)tleuretpaq] aloN 'saleLu (B) pue salpuraJ (v) rol palrold are ' 0 L 6 1u t u o l l e l n d o d a q r u o l l n q ! r ] s ! p sn Jo a6e aql o] palsnfpesa]prq]pappal)oles ' l00Z-0€Gt'satelspelrun'salerqleap re>ue>pelsnfpe-a6VSZ-OZain6rl

pue 'luaue euros ol pasodxe ilP erc a^\ qclq^t ol sluaSE ,{lqeqord aJE sJolJPJ aq] Jo^eJ]Eql sJolJeJlPluetuuor :sJaJuesuoruruocJo asaqlJo lueudolJ^ap lsoru (}sEJluoJuI 'auole ,{3o1o[uaprdar(q -r^ua ,{Bp,,fue^e 4puapl o] pJeq,{Je^sI }I 'uoqeFdod IeJaueBeql uI uolxluocun eJalv\Jeou€3Jo IUJOI sB^ uollJouuoJ eq] pue lecruaqJ aqt qloq esnecaq qsllqelse ol Lsea ,{.1a.tr1e1ar aql 1(8-02 amBIC aas) racueo rappelq padole^ep ,{1pn1uana(arnsodxa pe8uol -ord ot pelJafqns,{qaraqt ara^ pue) aunueldq]dpu-Z SuIIIusp uI pa,{.oldureueeq peq oq^\ uelu eql Jo IIe ^JolJEJqsrllJg euo ur 'aldurexa roJ '^rnlual qlallua^\l aql 'JeJuec puDI ureuac e Jo saseJaql Jo }solu roJ alqlsuodsar Jo Jo ued ,{Fea aqt uI sr uorlE8rlsa^urrapun lua8e aql uaqM puB 'lou atart oq \ esoq] puE lue8e eql ol pasodxa aJaMoq \ spnpwpul uaarUaq qsln8ul}slp ol Lsea sl 1l qclq ^ uI uol] -Elndod snoeuaSouoq,qJleJ e o] palldde uaq ^ lsaq s)Jo^\ qJEoJdde aq7'o7og7 sE qJnu sp ^q adoJna pue BJrJaruvquoN ur elBr qlEap JaJuPsIelot eql aJnpaJ ot ^E,ltE rrmoqsseq .{Soloruaprde 'aldtuexa JoJ'SuDlorusJo aloJ aq} Suqpa^al /tg 'atuoc o] aJoru sesrruoJd pue 'sassaJJnselqBlou aruos pa^ofue seq qJeordde ]r aqJ 'rasuEJ ueurnq Jo sasnec IEluaruuoJr^ue SupuIJ roJ Iool pdrcurrd aql aql-dSoloruapldg surEural-suollBlndod ur,{buanbe4 aseesrpJo srs,4'lPuP 'puru uado uE dea{ ol peeu e^\ 'ref,uec Jo sasneJ IpluauruoJr^ua aql dJpuapt 01 SuuduallE uaqM L1rea13 ']seaJq aql ur uorlBraJrlord IIaJ uo eJuenuul raql qSnoJq] esuaplJul Jef,uuJ '(gz-oz eJnSIc) Je3ue3 ]seaJq tJaJJp^lqeunserd sauouJoq e^rlJnpordar aq1 lsearq Jo acuaJJnJsoaql puB .{rolstq a^IlcnpoJdal uee^\}aq uoI}EIaJJoJ8uoF1s P sr erer{} pue '.aJIIJo saSe}slueJeJJIp}e pasnpoJd seuoruJot{ e^I}Jnpordar aq} ,4.qpacuangur .{1n;raltod sr Ja3ueJJo {srJ eq} 'uatuom uI 'snqJ 'salpoq Jno o} cIS -urrlur aJBracueJ aleJalasJBleq] srolceJaluos 'elsv puE EJI4V uI JaJuEcraAIIJo asner luPuodtur ue sr pue slnuPad IPJrdoDse qJns spooJalEulruPluoJ^lleJnlPu 'lseal s.{ESSe uIEl le tEql ISunJ,r{qpacnpord qryQZ-02 arnBICaas) Ig ulxo}eue sI -Jac/tq 'uaSoulcrecu^ ou)I luatod ]soru aql'plJo^ uJapou eql oi JIJIJedssueeru ou ,{q are }uE3rJru8rsaq o} U^\Ou)IaJeleq} sro}JEJlIuaSouIJJEJeq} Jo }soN 'GZ-OZ arn8gg)sreaL peJpunq lsed aq] ur qJnru ,{ra,LpaSueqJ lou seq a8e uantS E Jo slBnpl^Ipul JoJ sJaJuer uoruruoJ lsolu aq] Jo aJuaprsuI aLD'ttol[d Ja]JOqoJtlaHq]l^\ uolllaJul Jo saleJ SursearcaplJaual ,{eu 1uq} raJueJ qJPuols ur esearcap alqBlreuar E pue la^a,\^oq 'SuDlorus;(q pasnec sreJue3 ur aseaJJuraq] JoJldeoxg 'sotseqse puE aurruPl^r{tqdeu-7 se qf,ns frlsnput uI pasn slelJe]elu sIuaSouIcJeJ[UBlq aruos Jo uorleJrJrluepl eql uorJ ued ur aruos aABq'teu eapr aql ^\aIA slql uod -dns ol aJuaprla aln11sr eJeq] ]nq-uo os pue 'sall]Ippe pooJ Jo esn peJueque aq] 'uounflod ur asrr aql-aJII Jo ^B^\ pezqellsnpq ,(u8lq E Jo s]cnpord aq] aJp JaJuec Jo sasnpJ IElueuruorr^ua ureru aql leql rqSnoq] seurleuos sl 1I 'parJrluapraq ol uretual supurnq ur recueJ sasnec lEJruaqc luPuodrur flqeJed Jo -ruor Jaqlo 'sJaloruoJdJorun] pue sueSourcreJqloq suleluoc 11,{epo1plJoM aq} ur raJueJ Jo asneJ leluarxuoJr^ua luEuodr.ul ]soru eqt rEJ.,{qsr a4orus oc3eqoJ

re)ue) plo^v ol s^eMsleo^ausua6ou!)re)ro uorle)ltlluspl

(8)

r00z 066r 086r 016r096r0s5r 0r5r 0€6r 0

(v) t00z 065[ 086t01.61 096t0s6r 0t6t 0€6t 0 o

o

a

@ o f

J

o

0rl )^ alPlsoro . . qleuols snq)uorq pue 6un1 lPl)alolo) -

090

c

088

=

sltvn

lsealq . q)eurols snq)uorq pue 6unq lepalolo) -

09 E C

oR

-

sl'lvlArr

001

001

6ZZL

ul)NV) ro slsnv) StSvrNlAlud lHl

aseqlJo auo al8urs.,(ueJouorlsnpoJlur pu?-luerled rpur qsEeur las lueJeJ IEnpr -JIp ]eq \euos e ^,(llensn-sa8ueqoclleue8rde pue suortelnu Jo tas aloq ^ p uo spuadap reJuEJ pcrdfi y'xaldruoc,{yrulncrped sr eql reoueo roJ lnq 'ad.,{tou {sul -aqd luulnur uanr8 u Surrtl.rapunaua8 e rog qcreas rtue ur sesrJeuralqord 4ce1sl.eq -ul-alpeeu Jelrurs V islleJ snoJeJuef,aql u1 sauaBraqlo aql 1e Suorue sa8ueqc Suuourord-rarueJ eql qlurr saua8 Jo Jaqrunu fi€urs aq] ,fpuapl a^ op ,t,roq 'sJounl o] esrJSunr8 dq sanlasuaql ol uouueile ]ng IIEo pue uorlJeles IeJnleu dq paronegare daql ldseauago sr sllal ]uelalar aql Surpur4 'rorleqaq IIa3 snoJec -uuf, pue pallolur pue suop ol asrr daql moq sa8ueqc cpauaSlda a,r.r8 JeAoJSrp ol 'sllal orleruos -Blnru aq] IJlluepr ot paau a^a lelel JBInJelou B lB lr puelsJepun oJ ur sa8ueqc pelrraqurJo uor1plnrunJJeeql uo spuadap Jaouec'uees eneq ela sV

S]N]DIVf IIIU)-U]fNVf]HI.9NICNIJ 'acuapnut racuar pallo ruql stolcal Taluawuotmua Tadrcurtd aql lo ryatou81 [1a3ru7utvlaar am 'r{rtuapt ol qnc -t[rp ,{tanarD 4s1rncuac pallo lotll stoicat tuou asnulaq ranamoH'J Jo g slqrudaq io sasnunDruo1ydadsa qJns sasnJlnSulsnoc-tacuar Llnm uoqcatur ol [atd 3u177al puv oJJaqoi Sut4otus apnpm asaql 'tam qLIi ut pa{puap1 ato wtq stotcot 4stt 'Surnaq-p1ryc sutallad uloltac to laiuawuorrnua aql to fuaut pronn uuc slanpunpul pua a1&satysa qJns'sauuuaf)uuaLlJ lou arDioql uoltnl nnotun ual il pua 'qrom sLuaSolaluawuorrnua aqt moq Surmouqannbat rcu saopqcootddo 1nc18o1o1waprda aLIJ JaJuoJ uownq uo slcalla lDtuatuuoltllua asaLqSutttuuapl rcl TootTnttamod n aq uoc [Bo1otutaptdg'satnsodxa lotuauuorlnua raqio puo wa am pool aql Su8uaqc fq paptonn aq pruoc sfiJuoJ to fl1to[aut aLp ]DLU stsaSSns[73uot1s [trunoc $oq mau naqt to sutauad ncuac atlt JdopasuotlnyndodluntBnuun ruql Surpu{ aq1 'a1qaluanatd 'IorLuocrno a1dnuud u! ala sJaJuoJ to uotltodotd aBnTo npun ila asaql alaos puv JaJuoJuan8 n aql ol amq4luoc stolcot l.uautdo1anap lo lo 'uopalpatSutzruorpua 'uollalpat 1qBr7 1p 3u1pn7cu1 to lDiuawuoltltuafuntu asnnoag swtot snouan puo suaSoutctacpnutaqc Surpnpw'sua8ainu ata ruawdo1anapJaJUDJ asoarru!torli slua&alaluatuuorll1uaumouq atplo ,ttuo[aut aqJ rclvutu! aql fq palaatc q\acgalnu lo uouarudodaLflsasqaJcul saLqlnlarls'uorssatdxa pua 'uottvtatt\otd77ac auaB aatp iaql ruauuoJtnua pJol D ataan faql pua 'asuodsatfuolawwndut ua ainput sJajouroldJotunJ'slalowold Jownl Jo sJolqurulJouni sDblqla pD uoJ asaqJ 'uotssatSotdpua ruautdolanap Jouni to alot aLUaruJalano slua8n loluaruuolrllug

{reuun5 'elqeureilB 'raluec go sad-{l z{ueurJoJlnq deu fgpear aJoru aq 11 'Jacuec 'eJnJe ueql rapaq dluo rno ut aceld luanerd o1a133nr1s ]ou sr uonuelerd 'sseleqileleN Iertuao E aABq 1ps suaSourlrec Iprtuelod {puapl ol suoJJe 'plnolv\ JaAepooJ ur senprsalaprc€un; eq] ueql Jef,upoJo saseJeJorurEJasnpJplnom Ig urxol€Ue sp qons seqoqelau p8ury ltq pool Jo uoupurruptuoJ eql 'arnlynctr8e ur pasn lou eJa^ li'aqt JI leql palelnf,lpJ ueeq seq ll lng 'slsal Iptulup uI sasop qBH lu cruaSourcrecl.1plt- aq o1 readde 'aydurexaro; 'saptct8un; prnlpctrBe ureua3 'llus ]FlrJJrp eroru sr a3uelsqns aqt go fip1n aqt tsup8e ISIr slql Sutcue -luq E raJuec ueunq :]FcrJJrp e ulelre3 sr asneJ o1 ,{1a41 sr eJue}sqns }unorue Jo 'alqeldacce sr arnsodxa ueunq Jo la^el teqm Jo saseJ d.ueru ,r,toq Suqerupsg aprcep ot tpcrJJrp aq feru U 's]sel .{roleroqel ruo4 Jo ISoloruraprde ruo4 Jeqlle 'cruaSourcrecaq duur aJuelsqns B paulelqo sI aoueplle uaq,ry\uelg leql 'd1ts 'JeJueJ Jo ISrJ eql sespeJour,{1}uectyu8ts -aqo ol Surpeal 'pooJ qonu ool JarlJeapeuonueu se :lutod Lrelalp euo uo JEaIJ ,{prc; 'ranamoq'sr aluaprla pc€olonuaprda aql'serpoq Jno uo 1nd pue 'eqleeJq '1utrp 'lea arlt ]eq] saruelsqns raqlo ssapunoc aql JoJsao8 aruesaql'uollJeuuoc e lcadsns ol uoseeJrorrd aruos peq ellr ssalun ']no lI puIJ plnom alvrleq],(1a4lun sl U 'racuer lelJeJoloJ Jo {srJ aql pa^leq Jo pelqnop raqlta sa8uero Sutlua 'des 'acuapnur s,JaJuEJuarrrSe o1raqlaSo] elnqlrluoJ,(lqeqord ruaqtgo rtueru J1

re)ue):97raldeql

OEZt

FINDINGTHECANCER-CRITICAL GENES

into a normal cell is not enough to make the cell cancerous. In addition, the cooperation between different altered genes makes it harder to test the significance of an inherited change in any individual gene. To make matters worse, a given cancer cell will also contain a large number of somatic mutations that are accidental by-products of its genetic instability, and it can be difficult to distinguish these meaningless changes from those changes that have a causative role in the disease. Despite these difficulties, many genes that are repeatedly altered in human cancers have been identified-several hundred of them-although it is clear that many more remain to be discovered.We will call such genes,for want of a better term, cancer-critical genes, meaning all genes whose alteration frequently contributes to the causation of cancer. Our knowledge of these genes has accumulated piecemeal through many different and sometimes circuitous approaches,ranging from early studies of cancer-causinginfections in chickens to investigations of embryonic development. Analyses of rare but highly revealing inherited forms of cancer have also added to our understanding. More recently, sequencing of DNA from multiple casesof specific types of cancers has begun to give a more systematic picture of the genetic changes that are a regular feature ofthose diseases. In this section, we discuss both the methods used for identifying cancercritical genes and the varied kinds of inherited changes that occur in them during the development of cancer.

and Loss-of-Function of Gain-of-Function Theldentification MutationsRequiresDifferentMethods Cancer-critical genes are grouped into two broad classes, according to whether the cancer risk arises from too much activity of the gene product, or too little. Genes of the first class, in which a gain-of-function mutation can drive a cell toward cancer, are called proto-oncogenes; their mutant, overactive or overexpressedforms are called oncogenes. Genes of the second class, in which a loss-of-function mutation can contribute to cancer, are called tumor suppressor genes. A third class, whose effects are more indirect, are those genes whose mutation results in genomic instabiliry a class we describe as Dl/A maintenance genes. As we shall see, mutations in both oncogenes and tumor suppressor genes can have similar effects in enhancing cell proliferation and cell survival, as well as in promoting tumor development. Thus, from the point of view of a cancer cell, oncogenes and tumor suppressor genes-and the mutations that affect them-are flip sides of the same coin. The techniques needed to find these genes, however, differ-depending on whether the genes are overactive or underactive in cancer. Mutation of a single copy of a proto-oncogene that converts it to an oncogene has a dominant, growth-promoting effect on a cell (Figure 2O-271^)' Thus, we can identifu the oncogene by its effect when it is added-by DNA transfection, for example, or through infection with a viral vector-to the genome of a suitable type of tester cell. In the case of the tumor suppressor gene, on the other hand, the cancer-causing alleles produced by the change are generally recessive:often (but not always) both copies of the normal gene must be removed or inactivated in the diploid somatic cell before an effect is seen (Figure 20-278). This calls for a different approach, aimed at detecting what is missingin the cancer cell. In some cases,a specific gross chromosomal abnormality, visible under the microscope, is repeatedly associatedwith a particular tlpe of cancer. This can give a clue to the location of an oncogene that is activated as a result of the chromosomal rearrangement (as in the example of the chromosomal translocation responsible for chronic myelogenous leukemia, discussedearlier). Alternatively, a visible deletion of a chromosomal segment may reveal the site of a deleted tumor suppressor gene.

1231

1232

Chapter20:Cancer

(A) overactivity m u t a t i o n ( g a i no f f u n c t i o n )

s i n g l em u t a t i o ne v e n t createsoncogene

/t\

n o r m a lc e l l

activating mutation e n a b l e so n c o g e n et o promote cell transformation

c el l s e n r o u t et o cancer

(B) underactivitymutation (lossof function)

n o r m a lc e l l

second mutation mutation event event + + inactivates inactivates tumor secondgene suppressor copy gene no effect of m u t a t i o ni n o n e two inactivatingmutations g e n ec o p y f u n c t i o n a l l ye l i m i n a t et h e t u m o r S u p p r e s s ogre n e , p r o m o t i n gc e l lt r a n s f o r m a t i o n

t1\

Retroviruses can Act asvectorsfor oncogenesThatAlter cell Behavior Tumor viruses have played a remarkable part in the search for the genetic causesof human cancer.Although viruses have no role in the majority of common human cancers,they are more prominent as causesof cancer in some animal species.Analysis of these animal tumor viruses provided a critical key to understanding the mechanisms of cancer in general and to the discovery of oncogenes in particular. one of the first animal viruses to be implicated in cancer was discovered nearly 1.00years ago in chickens, which are subject to infections that cause connective-tissue tumors, or sarcomas.The infectious agent was characterized as a irus-the Rous sarcoma Ltirus,which we now know to be an RNA virus. Like all the other RNA tumor uiruses discovered since, it is a retrovirus. lVhen it infects a cell, its RNA is copied into DNA by reverse transcription, and the DNA is inserted into the host genome, where it can persist and be inherited by subsequent generations of cells. The Rous sarcoma virus carries an oncogene that causes cancer in chickens. This oncogene is not necessaryfor the virus's or,rm survival or reproduction, as demonstrated by the discovery of mutant forms of the virus that multiply normally but no longer make their host cells cancerousa process called cell transformation. Some of these mutants were found to have deleted all or part of a gene that codes for a protein called src (pronounced "Sarc"). other mutations in this gene made the transforming effect of the virus temperature-sensitive:infected cells show a transformed phenotype at 34.c, but return to the normal phenotype within a few hours when the temperature is raised to 39'C.

A large number of other oncogeneshave since been identified in other retroviruses and analyzed in similar ways. Each has led to the discovery of a corre_ sponding proto-oncogene that is present in normal animal cells.

Figure2O-27Cancer-criticalmutations fall into two readilydistinguishable categories,dominant and recessive.In this diagram,activatingmutationsare representedby solid red boxes, inactivatingmutationsby hollowred boxes.(A)Oncogenesact in a dominant manner:a gain-of-function mutationin a singlecopyof the cancer-critical gene can drive a celltoward cancer. (B)Mutationsin tumor suppressor genes, on the other hand,generallyact in a recessive manner:the function of both allelesof the cancer-critical gene mustbe lost to drive a celltoward cancer. Althoughin this diagramthe second alleleof the tumor suppressor geneis inactivatedby mutation,it is often inactivatedinsteadby lossof the second chromosome.Not shown is the fact that mutationof sometumor suppressor genescan havean effectevenwhen only one of the two genecopiesis damaged.

'raqloueeuo oo] ro uo dn alrd^aql leql os 'MoJbol anurluo) pue slurPrlsar Illensn asaqlpre6arsrp ' l s e r l u o )u r ' s l l a r) a ) u e ) ;uorlrqrqul l)eluo),,sPuMoul uouourouorlo e-slla) raqlo ql!M sl)eluo) ,(q palrqrqur aq ol pue'Llsrp aql qrlM l)eluo) uo puadapol suraosuor1erallo.rd :slla)Jo ralel ol6urse qllm qslp aql paladre>aneqIaql aruo 6ur1era1;o.rd ools slla)lPLrrrou lsow'arnlln) lla) u! uolllqrqurl)eluo) lo ssol 6J-97 arn6r1

'VNU slunourea6.re;saleu.r lo snr^ aqt asne)aqpa:npordreno I;1ear6 os;esruralordrr5-neql'(69-9 arn611 aes)aseurl uralo.rd>r;r:ads-aurso.rr(l e pue anrg>erad,(q se pale;n6arun rl 6ulleLl '(urvtoqs ulalordaqt e)uanbas lou) Jo pr)e ouil.!e aql Jalleleql suorlelnuJ sureluo)rJS-i'uorlrppeur .'rs-l Jo lno perr;dsuaaqa^eqrr5 rplnlla)ur luasard suo.ltutaql'pa^uap uaaqseql! q)!qM r-uol1 aue6o>uo-otord )/S.relnlla) aqt pue eua6oruo )JS-zt aql uaa/vrlaqdrqsuorle;ar aql (B)'suorl)un;6urssrr-u aqt sar;ddns t-:'ttgm'sru n nd 1aq6urturolsuerluou 'a^rl)aJapuoue qlr^^pal)a]u! {;snoauel;nwrs s! leqr lle) e ur {;uo se;rrlredsnor])a;Ur alereuabue) snrn 6urluio;suer1 oql aro;araqf'aua6oruo 6uru.r.ro;suer1 eq] setrnbtesnlrno.rlal aql uaqMtso;,(;lredro,{lloqMaresaua6 'sasnr^orlar leIA asaqlJo arourro auo rrua6oruoroqto ul'(uretordor,{;6 ado;anuaaql sarnpordqlrqM)^uf pue'(euoua6lsoq aq] olur au..rosourotrl) lerl aqt 6ulterbatulur pa^lo^uraur,(zua uP pue aselou)suellasle^alsa)npoJo r.l)!r.lm)/od'(suralold prsder aqt atetaua6 o] paleal) sr1eq1urelord,{1od e sarnpord qrtqm)6o9 :ap,(r a;r;;elrrrfueurp.ro aql ro4parrnbatseua6leltAaa.lqleql paurelarseql! leql ur seua6oruor{rer leql sasnrnorlaraql 6uoue lpnsnun srsnr^ euro)lessnou'(snltAetualnal auunu) snlrloJlar;errdIl arou e;o reqr qrlMpareduor seeuroua6ler^ aqt aqf (V)'snr^ puro)res 1ouorlezrue6ro snog aql ro arnl)nlls aq1 97-97 arn6r1

q s r pe J n l l n ) - a n s s)rrl l s e l o ur r.unrpauqyrrorb

'(62-02 arn -31g)aleraglord Laql sBre^{EIuodn Je,(eldn apd pue amqns aq] uI sllac peuroJ -sueJlun eql ^aoJSlnoduoloJ eql SuIruJoJslleJ paruJoJsueJ]aq] :uopBJeJIIoJdIIac aql pesEeleJleql eueSpeppe uE 8ul{.ue3 eqlJo aruos tuoq uo sloJluos IIaJ IPrsos 'lla3 elSursp ruo4 pelEulSlro ]eqt sllec auolJ B sI duolor qJeA 'uop Jo -BruJoJsnBJl auoSJapun e^eq o] rc pau)otsuaq eq ot prcs aJP^eql 'sarlradord snoJacueJ deldsrp saruoloc qJns SuIurJoJsllac eql asneJeq ireadde 01 ulSeq slleJ SurleJaJrloJd.|{.lleuuouqEJo saluolol IIEIus 'auaSoJuo uP sulBluoJ slualu -3er1aqt go due;1 's11ac peJnllnJ aq] olul peJnpoJlul pue 'sluaur8e4 olul ueloJq 'sllac Journl eq] tuo4 patJeJue sl VNCI ,{ea,rslql uI auaSoouo uE }Ja}ap oJ 'lJaJJa e aJnpoJd o1qSnoua eq ueJ eueSosuo el8 crlEruerp -urs p Jo uorlrppP aql 'uoseal slql Joc dcueuSqeur pJej\/\ol,{.u,,Vr aql;o ued luaql a{E} lPql suor}BJellE 3l}aue3 ulEluoc Lpearp o} }q8noql eJE 'eJn}lnl uI d.lellu -lJepul elEreJrTordo1 palJales dlsnornard 'sllef, aseqJ 'slsPlqorqg asnou luo4 pa^rJap sauq IIeJ pasn ^llPnsn r(aq] 'desseeql JoJ sllef, Jalsal sv 'seuq IIaJ sno -JecueJuou olq pecnpoJlur uaqM uortEJeJIIoJd pelloJluocun a{o^oJd plnoJ }eq} slleJ raruec uBrunq ur seruanbas vNC roJ paqcJBespue qf,EoJddElJeJIp aloru e {oo} sJaq}o'sasnJIAoJlaJur sauaSocuoJoJ paqf,rees sreqJJEaseJeruos eIqA\

seu-ouaD loJsaq)leesluelarJlo aues eql uo pabta^uo)aAeHsauabo>ug (s)

aua6oruo

tfilsl

-

: ,€

|

|

eua6o:uo-o1oldrr5-r ua))rtl)

uotlu I

u r e u J o po s e u r l6 u r p o r u eu o t b a . t

I

,s

I

uoxa

vNc llo) lsorl

(v)

6eg

^UJ Mffi

c

dE)

WV

snJ!Aeurolressnou ^u3

6eg

lod

,s OE)

WV

t

sleaoarreururra.'

s n r ^ e r [ ! a r n a ta u t J n t !

r €EZ

S]N]9 IV)IIIU)-U])NVf ]HI 9NIONIJ

Jo sPluoulJJec SurpnlJur TeJuBJJrpeJodsJosad,,{luoruruoJ lere^es ur Bursslu osp sI eue8 qU eqJ '(0t-02 aJnBIC)e8eaull ilec puper ay8ursu ur saruosouroJqJomt uo aua8 aruBs aq] alplllJeul leq] sluele luapuadapur orru arrnbar daql asnecaq arer ,,{rarrare spuolselqoupar ,tretperaquou eseqJ 'sardoc qtoq ut elrtceJep auroJeq eleq slleo snorecueJ aq] a[q t, 'qy;o.{doc Jaqlra ul }JaJepou ^\oqs sllac snoJeJuEJ

-uoueqr'1spr1uor dq 'aseasrp eqrJoruroJd-relrperaquou aqrqlt \dsr:"J:""11ir""

-nard aq] Jo uorlcunJ aql pelpurrulla seq leql ]uene Jrlpruos e Jo asneJeq qyr ;o sardoc qloq ur e^rlJeJapeJEsnoJecupJeJp leql sllaf, leurleJ aq1'aua8 aql;o ,r{docpooB euo urelal,{aqtJI os op lou op }nq 'snoJeJueJSururocaqo1pasodsrp -ard are slleJ asaqJ 'lleJ Jltetuos itrana ur aua8 qy aqt;o ddoc euo ur luasard uollelnru uorlJunJ-Jo-ssolJo uouelap e aAEqeseesrpaqlgo ruro;.,(JelrpaJaqeql Iuo4 JaJJnsoq \ asoql leql peJeloosrp ueql sea,r11'auaB qA aqt acuanbas pue euolJ ol alqrssod se,ti\]r 'uorlalap IeruosouoJqf, slq] Jo uoneool aq1 Sursn 'uorlelol leq] ul aueBpcqrrc e go ssoyLq pesnec se^aJaouuo eq] leql pa1sa38nsqclqm 'eseesrp d:elrparaquou eql qryv\ stuaued eruos tuor; slleJ Jorunl ur paJalunoJua osle are uor8ar etues slql Jo suoqeleq 'eseasrpeq] 01lenpr rput ue sasodsrpard'paluaqurJI '.leql tI euosouorqJ uo pueq cgrcads e Jo uor]alep e qllm 'adfiou{re>1Ierurouqe dlqlsyr e eAEqeruo}selqourler qtlm slen -pFlpul ^AaJv'ror.!n1 auo ,i1uo,{q pue 'petJaJJesr a.{aauo.,(po 'urroJ ,,{relrpa.raq -uou aq] ur lsada qtoq Supcage 'dpuapuadapur esrJe,r(lensn slorunt aldrlpru 'ruro; drelrpalaq aql q 'lou sr Jaqlo aq] pue '.fterparaq sr eseesrpeql Jo ruroJ auo 'pelJruJEsl 000'02 uI pllqJ auo lnoqv'eurler ernlBrurur aql uI sllaf, rosJnc -ard prnau uro4 dolaaap sJotunl pup 'pooqplrqJ ur sJnJco Eruolselqouueg 'srossarddnsJorun] sE alJas uago,,(aqt aJaqm'sreJuec crperods uoruruoJ eJotu ur luplalal eq ot punoJ {pouruor aJB salrroJpud.sracuec lprlrtupJereJ uI perJrluaprsaua8 'pe; uI 'acupleleJ peardsepmrgoaua8 e IEaAaJol lno peuJnl lr lnq 'asBJprcads e Jo uorleutuexa ruor; asore dlarrocsp eql 'd3o1orqur suaddeq uauo sV'suorlelntuJo Jequnu luurs dlensnun ue dq elels snoJacupcp ol paual 'EruolsslqoupeJ -uoJ aJE leql a'ta eql Jo purleJ aql ul sllec ruo4 sasrJBqclqM tacuec ueunq go adr! aJeJ B Jo serpnls ruoq arupJ aua8 rossarddns rounl aq] ot pal lpq] lq8rsur ,{a4 aq; 'araq} tou sr .{ldurrs leql ]srIJ aqt;o ,,fuazrocsrp Sutqlauros {Fuapr ol ,r(esseuorleuJoJsueJ] IIaJ e asn 'aldruexa JoJ 'louupc euo :a,u1cerad,r(qaruoJaq seq teqt aua8 B Surpug ruor; ,i3a1er1sluaJaJJrpe sa;rnbar IIac Jecuuc e Jo auoue8 aqr u1 pelelrlceur ueeq seq ]eq] aua8 u Surrlnuap1

sauagrosseJdd ns rournl pa!}lruapllsrrl sauorpu^sra)uP) {relrpare;1aJeuJo satpnls 'JaJueJesner o] dlaq leqr sauaSocuoolur suorlelnru Surlenrlce,{q paganuoc aq ueo lpq] saue8 'sr leql-sauaSocuo-olord Jo spalpunq Jo llrorDl,ltou elvr'salnoJ Jeqlo snorJe^pue 'paqucsap lsnf a^eq e^aesoql se qcns spoqlaru q8norq; 'saua8 'sracuec IecrlrJJ-Jaf,uec;o saldruuxa luelrodrul lsoru eqt Jo euo ureruer faql ueunq 3o a8uer ep4 B ul pelelnu are saue8 sz2yaql asneJeg 'lJeJJe uE aABq o1 a8ueqc ol speau sardoc aua8 oml s,lleoeql Jo euo [1uo 'sr leql-luuurruop sr lJaJJaslr 'anqcerad.,(quralord aq] sa{E{u srql asnef,eg aCC o} dIC punoq s1r8ur -z,rilorpr(q,{q JJoJlas}r}nqs louueJ teqt uretord seg arrrlceraddq e aleaJc leq} suop -elnur ureluoc srorunl ueunq ruoq palelosr saua8ocuo sz2yaqJ lurod 'JolJalur IIaJ aq] ol sroldacar aJeJJns-llac uro;; spu8rs lnusueJt dlaq reqr sasedlg JrJaruouoru are suralord sEU lpruJou '91 raldeq3 ur passnJsrpsV 'reoueJ dSolorqJBInJalou aqt;o SurpuelsJepunrno pa8ueqc saua8 Jo IesrlrJJ-JaJuEJ JOJaqunu pelruI e uI suorlElnu ,tq pasnec aJEsJaJuEJlEql uop -ecrTdurraql'3ur,r(;rrlJala sem snJrl Jorunl IEruruB uE ur pue slleJ Jorunl ueunq ur euaSocuo erues aqt Jo s086I ;t1reaaql ur draaocsrp srql 'sluepoJ ur suur -ocJESsesneJ]eq] snJrloJlal p ur auaSSursnec-Jorunleql aq ot punoJ ueeq peq aua8 vry palelnu E 'aro;aq d.p;oqs 'asnpoaq cpprueJp aJoru aql 1e se,u auaSocuo szry uerunq aq] go .{rarrorsp aqJ 'sJeJueJ uerunq eAUur euo lnoqe ur palelnru aq o] urlr.orDl,t,rousr aue8 szy aqJ 'srry auaSocuo-olord aql Jo uorsJel luelnru p 'auaSocuoueunq ]sJrJaq] Jo Surcuanbaspue uoBplosr eql ol pal .,(essesrql

re)ue) :02raloeLlJ vEzL

1235

GENES FINDINGTHECANCER-CRITICAL N O R M A LH, E A L T H IYN D I V I I

HE R E D I T A RRYE TNI O BL A S T O M A inh e r i t e d mutant Rbgene

,/*\

{J

,/

/

/l\\

N O NH E R E D I T A RRYE T I I ! O B L A S T O M A o c c a s i o n acle l l i n a c t i v a t e s one o{ its two good Rb genes

\\

,/

ffiG

o c c a s i o n acle l l inactivatesone of its two nnnrl

PA

nonoc

/

\\

I

o c c a s i o n acle l l I i n a c t i v a t eist s o n l y I g o o d R bg e n ec o p y I

L;

,4\.

,/ l\

the seconrl.oPY ot I Rb is very rarelY I inactivatedin theA same line ,/ i olcells /

cell excessive p r o l i f e r a t i o nl e a d i n g to retinoblastoma

\

'n*i'i':rfuih:'

RESULN T OT U M O R

E I T HI N H E R I T E D RESULM T O S TP E O P LW D E V E L OM P U L T I P LTEU M O R S MUTATION I N B O T HE Y E S

RESULTONLYABOLIT1 IN 3O,OOO PNE E E V E L OO N O R M A LP E O P LD T U M O RI N O N EE Y E

Iung, breast, and bladder. These more common cancers arise by a more complex seriesof genetic changesthan does retinoblastoma, and they make their appearance much later in life. But in all of them, it seems, Ioss of Rb function is frequently a major step in the progression toward malignancy. The Rb gene encodes the Rb protein, which is a universal regulator of the cell cycle present in almost all cells of the body (seeFigure 17-62).It acts as one of the main brakes on progress through the cell-division cycle, and its loss can allow cells to enter the cell cycle inappropriately, as we discuss later'

GenesCanAlsoBeldentifiedfrom Studiesof TumorSuppressor Tumors The Rb story illustrates how rare hereditary cancer syndromes can be used to uncover tumor Suppressorgenesthat are relevant to common sporadic cancers. However, only a few of the tumor Suppressorgenesnow known to be important have been discoveredin this way.A more direct approach to the identification of

tumor suppressor genes have already been well characterized and many more are known.

Tumor CanInactivate Mechanisms BothGeneticand Epigenetic Genes Suppressor It is the inactivation of tumor suppressor genesthat is dangerous.This inactivation can occur in many ways, with different combinations of mishaps converging to eliminate or cripple both gene copies.The first copy may, for example, be lost by a small chromosomal deletion or inactivated by a point mutation. The second copy is commonly eliminated by a less specific and more probable

Figure20-!!0The geneticmechanisms In the that causeretinoblastoma. lbrm,all cellsin the body lack hereditary one of the normaltwo functionalcopies gene,and of the Rbtumor suPpressor tumorsoccurwherethe remainingcopy by a somaticevent is lostor inactivated (eithermutationor epigeneticsilencing). form,all cells In the nonl'rereditary initiallycontaintwo functionalcopiesof the gene,and the tumor arisesbecause both copiesarelostor inactivated of two somatic throughthr:coincidence eventsin one lineof cells.

1236

Chapter20:Cancer

HEALTHY C E L LW I T HO N L YO N EN O R M A LR b G E N EC O P Y

P O S S I B LWEA Y SO F E L I M I N A T I NN GO R M A LR b G E N E

nondisjunction chromosome causes l o s s t, h e n chromosome chromosome loss duplication

mitotic recombination

gene converston

point mutation

mechanism: the chromosome carrying the remaining normal copy may be lost from- the cell through errors in chromosome segregition, or the normal gene may be replaced by a mutant version through either mitotic recombination or a gene conversion event. Figure 20-31 summarizes the range of possible ways to lose the remaining good copy of a tumor suppressor gene through DNA sequence changes, using the Rb gene as an example. It is important to note that, except forihe poinl mutation mechanism illustrated at the far right, these pathways all produce cells that carry only a single type of DNA sequence in the ihromoio-a region containing their /ib genes-a sequencethat is identical to the sequencein the original mutant chromosome. As discussed in chapter 4, normal human genetic variation makes each of our maternal and paternal chromosome sets distinguishably different. on average,.human DNA sequences differ-that is, we are heterozygous-at roughly one in every thousand nucleotides. \Mhere a large segment of one chromosomi has been either lost or converted to the DNA sequence in its homologous chromosome, as in Figure 20-31, there is a lossof heterozygosity (LoH): only one version of each variable DNA sequence in that neighborhood remains. Millions of common sites of heterozygosity in the human genome have been mapped as part of the Human Genome project: each of these sites is characterizedby a specific DNA sequence that is known to be polymorphic-that is, to occur commonly in two or more slightly different versions in the human population. Given a sample of tumor DNA, one can check which of the versio.rsbf ihese polymorphic sequencesare present. The same can be done with a sample of nruA from noncancerous tissue from the same patient, for comparison. A loss of heterozygosity throughout a region of the genome containing one or more polymorphic sites,or loss of a genetic marker sequencethat is seen in the non-cancerous control DNA, can point the way toward a chromosomal region that contains a relevant tumor suppressor gene. However, because of their genetic instability, cancer cells often exhibit a loss of heterogeneity for -any diff".ent chromosomal regions. Therefore the detection of tumor suppressor genes by this approach generally requires the subtraction of a large amount of rindom noise. Epigenetic changes provide another important way to permanently inactivate a tumor suppressorgene. Most commonly, the gene may become packaged into heterochromatin and the c nucleotides in cpG sequencesin its promoler may become methylated in a heritable manner (seeFigure 20-12).Theie mechanisms can irreversibly silence the gene in a cell and in all of its progeny. Given a catalog of possible tumor suppressorgenes,it is relatively easyto test their promoters for abnormal amounts of DNA methylation. Studies of this Rpe suggest that epigenetic gene silencing is a frequent event in tumor progression, and epigenetic mechanisms are now thought to help inactivate seneril different tumor suppressor genes in most human cancers (Figure ZO_JZ).

Figure20-31 Six ways of losing the remaininggood copy of a tumor suppressorgenethrough changesin DNA sequences.A cell that is defectivein only one of its two copiesof a tumor gene-for example,the suppressor Rbgene-usually behavesasa normal, healthycell;the diagramsbelowshow how this cell may losethe functionof the othergenecopyaswell and thereby progresstoward cancer.A seventh possibility, frequentlyencountered with sometumor suppressors, is that the gene may be silencedby an epigenetic change,withoutalterationof the DNA sequence, as illustrated in Figure20-32. (AfterWK. Caveneeet al.,Nature 305:779-784,1983.With permissionfrom MacmillanPublishers Ltd.)

GENES FINDINGTHECANCER-CRITICAL Figure20-32:The pathwaysleading to lossof tumon suppressorgene function in cancerinvolve both genetic and in epigeneticchanges.As discussed Chapter4, the packagingof a geneinto condensedchromatincan preventits in a way that is inherited expression when a celldivides(seeFigure4-52).As the changesthat silencetumor indicated, genescan occurin anyorder. suppressor

mutation

tumor suppressor

h e r i t a b l eg e n e s i l e n c i n gi n c o n d e n s e dc h r o m a t i n

qene

J!

-

+++

OR

I

= g e n e t i cc h a n g e

I

= e p i g e n e t i cc h a n g e

&

CANCER

r *i

m OR

p

a,

m

GenesMutatedin CancerCanBeMadeOveractivein ManyWays In the case of a proto-oncogenes it is gene activation that leads toward cancer. Figure 20-33 summarizes the types of accidents that can convert a proto-oncogene into an oncogene. (1) A small change in DNA sequence such as a point mutation or deletion may produce a hyperactive protein when it occurs within a protein-coding sequence, or lead to protein overproduction when it occurs wilfrin a regulatory region for that gene. (2) Gene amplification events, such as

produced. Specific rypes of abnormality are characteristic of particular genes and of the responses to particular carcinogens. For example, 90% of the skin tumors

proto-oncogene

DELTTION.]OR POINf IN MUTATION coDrNGstQurruce:

REGULATORY MUTATION

I

I

V

DNA

RNA E

hyperactive p r o t e i nm a d e i n n o r m a la m o u n t s

rl

I

I

V

I

fl

I-

: E normal protein greatly overproduced

n o r m a lp r o t e i ng r e a t l y overproduced

into an oncogene' Figure20-33 The types of accidentsthat can make a proto-oncogeneoveractiveand convert it

1238

Chapter20:Cancer

homogeneous s t a i n i n gr e g i o n

r + ,ri,;'l'J c h r o m o s o m eA

(c)

a b e r r a n tr e p l i c a t i o n

/

#*"#11':

chromosome B Figure20-34 Chromosomalchangesin cancercellsresultingfrom geneamplification.In theseexamples, the numbers of copiesof a Mycproto-oncogene havebeenamplified.Rmplification of oncogenesis commonin carcinomas and is often visibleasa curiouschangein the karyotype: the cellis seento containadditionalpairsof miniaturechromosomes-socalleddoubleminutechromosomes-orto havea homogeneously stainingregion interpolatedin the normal banding patternof one of its regularchromosomes. Boththeseaberrations consiitoi massively amplifiednumbersof copiesof a smallsegmentof the genome.Thechromosomes arestainedwith a redfluorescent dye,whilethe multiplecopiesof the Myc Aeneare detectedby ln sltuhybridizationwith a yellow fluorescentprobe.(A) Karyotype of a cell in which theMyc 9enecoplesarepresentasdoubleminutechromosomes (pairedyeltowspecks). (B)Karyotype of a cellin whichthe multiple Myc genecopiesappearas a homogeneouslystaining region(yettow)interpolatedin one of the regularchromosomes. (ordinarysingle-copyMyc genescan be detected as tinyyellowdofs elsewherein the genome.)(Cischematicof how gene amplification occurs'lt is thoughtthat a rare,abnormalDNAreplication eventproducesa chromosomewith extracopiesof one chromosomal region,as shown.The repairof this structurereleases DNAcirclesthat can replicateto form long t a n d e m l y r e p e a t e d s e q u e n c e s , p r o d u c i n g d o u b l e m i n u t e c h r o mA o ss tohme er ess. u l t o f a s e c o n d r a r e e v e n t , t h e D N A from one of thesechromosomes can becomeintegratedinto a new siteon a normalchromosometo producea homogeneously stainingregion.Otherpathwayscan alsoamplifygenes,suchasthat describedlaterin Figure20-41. (A and B,courtesyof DeniseSheer.)

evoked in mice by painting the skin with the tumor initiator dimethylbenz[a]anthracene (DMBA) have an A-to-T alteration at exactly the same site in a mutant Ras gene; presumably, of the many mutations caused by DMBA, only those at this site efficiently stimulate skin ceils to form a tumor.

1239

GAELN E S FINDING T H EC A N C E R . C R I T I C

overproduction appears to be due to a change in a regulatory element that acts on the gene. For example, a chromosomal translocation can inappropriately bring powerful gene regulatory sequences next to the Myc protein-coding sequence, so as to produce unusually large amounts of Myc mRNA. Thus, in Burkitt's lymphoma a translocation brings the Myc gene under the control of sequencesthat normally drive the expression of antibody genes in B lymphoc1tes.As a result, the mutant B cells tend to proliferate excessivelyand form a tumor. Similar specific chromosome translocations are common in other lymphomas and leukemias.

GenesContinues TheHuntfor Cancer-critical The sequencing of the human genome has opened up new avenues for the systematic discovery of cancer-critical genes. It is now possible, in principle, to examine every one of the approximately 25,000human genes in a given cancer cell line, or in samples of tissue from a set of casesof cancer of a given type, looking for potentially significant abnormalities, using automated analysis of either their genomic DNA or the mRNAs the cells produce. By analyzing substantial numbers of different cancers, it should be possible eventually to identify all the genesthat are commonly altered in human cancer.Although enormously costly, large-scaleDNA sequencing efforts have already begun to identify new human oncogenes.One example is the finding that a hyperactive form of the Raf protein kinase (discussedin Chapter 15) is present in a high percentage of melanomas and, at lower frequency, in other cancers. Straightforward DNA sequencing is not the only way to tackle the problem, however, and there are other powerful new methods for identifying new oncogenes and tumor suppressor genes that seem more efficient. Three different types of approaches appear particularly promising. 1. comparatiue genomic hybridization (cGH) uses fluorescent labeling of fragments of DNA extracted from normal cells and from cancer cells to identify regions of the genome that are amplified or deleted in a given tlpe of cancer (Figure 20-35). The labeled fragments are allowed to hybridize to DNA microarrays in which each spot corresponds to a known location in the normal genome. Different spots light up in different fluorescent colors,

Figure20-35 Comparativegenomic hybridizationfor detectionof the DNA changesin tumor cells.(A)DNA fragmentsfromtumor cellsand normal cellsarelabeledwith two different molecules(redfor the tumor, fluorescent greenforrhenormalcontrol)and in which hybridizedto a DNAmicroarray to a defined eachspotcorresPonds positionin the normalgenome'(B)The ratiosof red to greenfluorescencefor eachspot is plottedasshownbelowto definetho:;eregionsamplifiedor deleted in the tum,crcells.A redsignalindicates an amplification,while a greensignal a ,Celetion. siqnifies

rumorilssue

I

I g e n o m i cD N A i s o l a t e da n d fluorescentlylabeled

I

+

location of Myc ge^e

a

o2 a

I

t

.,t i

9ene loss

ti

Myc DNA

DNAmicroarray

(B)

amolification

I

on l

gene

100 80 60 40 d i s t a n c ei n m e g a b a s easl o n g c h r o m o s o m e

120

1240

Chapter20:Cancer

according to the ratio of normal to cancerous labeled fragments that bind to them. In this way, one can locate regions of the cancer cell genome that have been amplified or deleted. one then searches -or" ,ru.roirly for candidate geneswithin these regions that may contribute to cancer development. 2. DNA microarrays can also be used to reveal specific changes in gene expression associated with cancer. In this case, a cell's population of mRNAs is used to prepare the probe for hybridization, rather than its chromosomal DNA (seeFigure 8-76).

p. 571). This provides an efficient way, in principle, to identify any tumor suppressor gene whose loss promotes cancerous transformation in the particular animal or cell line being tested. \Arhateverthe approach used to identify a new candidate cancer-critical gene, it remains a challenge to determine whether the gene really contributes to cancer causation. Testing for the effects of an overexpression of candidate oncogenes or an inhibition of candidate tumor suppressor genes in cell culture assays can help. But more convincing tests use transgenic mice that overexpress candidate oncogenes and knockout mice that lack candidate tumor suppressor genes. In both types of mice, cancer development should be accelerated if the genes are real culprits.

Sum m a r y Cancer-criticalgenescan be classifiedinto two groups,according to whether their gain of function or their lossof function contributes to cancerdeuelopment.Gain-of-finction mutations that conuert proto-oncogenesto oncogenesstimulate cellsto increase their numberswhen theyshould not; loss-of-functionmutations of tumor suppressor genesabolish the inhibitory controlsthat normally hetp to hold cetlnumbersin check. oncogeneshaue a dominant genetic ffict, and many of them were first discouered becausethey causecancer in animals when introduced by a retrouirui that originally picked up a normal form of the gene (a proto-oncogene) from a preuious host cell. Oncogenescan also be identified by characteristicchromosomal aberrations that can actiuate a proto- oncogene.

suppressorgene.

wzl

uo puedep ]Inpe uB se esuBualuleru sll pue plulue JPlnlleJlllnlu E Jo ]ueuclo -le^ap aql qlog 'srsElsoauoq ureluleru puE JeAouJn]IIaJ IoJluoc o] dpoq llnpB IuruJou eq] ur alBJedo luaudola^ap 3luo,{Jqrua IoJluoJ }eq} susluPqJeu SuIlBu 'tZ -8rs a,,u sy'asr.rdrns ou sI qqt 'lq81spu1qqll6 Jaldeq3 ur ssnJSIp arues eq] 'sdem.qledasaql Jo lsEI aql Jo gud Suraq sEUqllm -saua8 pcrtrJJ-Jeouec,,(qpapocua sluauodruoo epnloul 1e sdeatqted 3u1pu -8rs aseuq aursor,,hrotdacar pue 'qcloN 'flgg;'Soqa8peH ']uM alfl i(ZZ pup gI sraldeq3 ur pessnf,srp) luarudolanap Surrnp eleJlunluruoc slleJ qtlqm q8norql s,,(ervrqtedSuqeu8rs rofeur aq] 1e ,,typcncerd;o sluauodruoJ apnlJu saldurexg 'luaurdolanap cruofrqure ur aloJ rlaq] JoJ rrlv\oDl dpearp sauaB ;o sSolouroq 'lxeluoo are 'aldruexa ro; 'saua8 rossarddns Jolunl pue sauaSoruo ,{ue4 aq o} lno suJn} uauo }l teJueo JoJIeOI}IJJse pau Jaqloue ur sarpnls ruo{ JEIITtUEJ 'sllal -puapr qJnru ,lrou>I arlr depol aJou \ .lrau E uaq os sr aua8 lnoqe leql 'sr(e,r,rqledSuleu8rs JelnllateJlul ;o luauodruoc e sE qg-sllal pruJou ut sutalord asaql lEJlueJ p sE seg pup Jolrqrqu a1c^,{c-gac Jo eloJ da>1aqr pelealal leql selpnls ro; ]utod SuDrets eql sem qJJEeseJJecuuJ 'seg pup qU qloq rog ',{-ralsdure spl\ sllec raoueJ Jo IPtuJou Jeq}la uI pauol}f,ury uralord seg aql rvroqlnq 'arn11ncut .{laleudorddeur pue ,tlazrtssacxaalera;qord 'seruolselqoulleJ ol slleJ llaJrp o] Ulr,rotnlsem aua8 luelnru eql 'szyJo asec aq] uI ur palelnu sem ]I ]eq] se^ rrrltoDl se^ leql 1e 'ayduexa ro; 'pauolc [1eu -r8rro sem aua8 qy aql ueq iysllar JaJupJ Jo JIlsIJelJEJEqcsJoIABqaqlueJJeqe aqt ol alnqlJluor aua8 aql uI suollelnlu ,lroq pue slleJ Ierurou ut suorlcury aua8 eq] ,lroq qloq puetsJapun o] paau allr SecueJ e uI palelntu sI leql aua8 e uanrS

seue9 le)!lu)-re)ue)Jo uoll)unl eql lo^o)un ol pedlaHe^eHe)!w qloSro serpnls pareeur6uS Ille)tlauagpupso^tqurf6urdo;ana6 'asJo^ sr lpql Jeqloue ol Jor eqaq peq ;o uraped euo ruoq eAIoAao] Jorunl B salqeue saua8 pctlrrc-JeJuec ur sa8ueqc 'aldruexa pepualxe uB sp JaJuBc uoloJ Surssnc Jo uorssaoJns p laoq Sulr,roqs -s1pdq uorlJes aql pue aM'paurruJalap eq ueJ JaJuEl Jo xaluoJ aql u1 saua8 asaql Jo suorlounJ er{l ,vrorl pue sauaB IeJI}rJJ-ref,ueJ uI suollelnru ruoq esIJB sJor eqeqsru f,llsFalf,EJeqJasaql l oq JepISuoJ a,lr 'uoIJJas slql q 'sdeldsrp IIeJ JeJueJE leqt sror^EqaqsrruJospuH eql palslTpuE snoJacuPJIIec B e{eu }uql sauradord praua8 aq] pazrJprurunse,vrtaldeqc slq] Jo uollJes ISJIJeql uI 'JarueJ Surpuelsrapun Jno poruJoJsueJ]seq d8o Jo -lorq IIaJ Jo slcadse crspq uo qf,Jeasar'Llasranuoc:qoJpeseJJeJuEf,Jo lcnporddq e sB peuJual uaeq seq ruaql lnoqe MouI e,vr leqm Jo qlnlN 's[ert xalduroc ur luapuadapJalur pup paluu are sLearqled snorJel aql'(6I raldeq3 uI pessnJ -srp) sJoqq8reu slr qlIM suollJeJalul IPJIuPqoeruJo 191raldeq3 uI passnJslp) sluarueloru s,lleJ eq] urano8 sraqlo illls 1(8I raldeq3 uI pessnf,slp)qreap ilac ro (11 raldeq3 ur passnJsrp)elJltJ IIer aq] Iortuor ]eqt esoql se qJns 'surerSord IeuJetur s,lleJeql ro; alqrsuodsareru sraqlo :191ratdeq3 uI passnJslp)tuauuoJ -rlua p ,{anuoc u1 srterr,rqled eql Jo eruos s,llaf, ruo{ spu8rs JeJueJ }uelrodrur 'sde,nnqtedrfuoleln8ar pue pcruaqcorq luaJaJJrpur lJe leql saua8 pcrlrrJ-JacupJ uaamleq 1nq 'sauaSocuo -olo.rd pue saua8 rossarddns-rournl uaamlaq lou sr uopcupslp luepodur aql 'uorlounJ sllac JaJuEr lrroq puets 'scrlaua8Jelnraloru Jo sruJet ur srql op ol pue 'sluauodruoc (rossarddns Journt) ,fuo4qlqq pue (aua8 -rapun ol sr urre eq] JI -ocuo-olord) [ro1e1nur1sq]oq eAEqsrusrueqJeu Jelnllaf,lsoru asnef,eq Io4uoc 'saua8 sselJ Jaqlra ur suorlplnru ruoq llnseJ ueJ Jorleqeq ilac uo slJeJJeJo Jo spur) alups aqJ 'uroJ atUESaqt Jo seprs ollu are ,taql '^\er^ Jo lurod s,lac JesuEJ e tuoq '1ng dlluarag;Ip-uraqt lJeJJe tBql sa8uuqc cpaua8rda pue suollelntu aq] pue-saua8 rossarddnsJoun] pu? sauaSocuosl arl Jelunq auaS-racuecaq;

UO I H ]B

ttl)-utf NV)ro srsv8uiln)llot^JrHr rl])-ur)NV)ro srsv8 uvtn)ltowlHl uorAVHrS

fallrue pessnJsrp'uralord sEUeql pue rotdeJeJCDg aql ale^rlJB ,{laleudorddEur }pql suorlE}nru uor}Jury-Jo-ure8 eq} epnlJur seldurExg .{yaleudorddeur uorsrlrp IIaJ pue 'uor1ec11darVNC 'q}nrtor8 1ac uo Suqclrrr,rs 'papeeu ]ou aJEslleJ aJou ueq ^ ue^a slBuSrseArteJeJIIoJdseteeJc]eq] ^B,\i\e ur sluauodruoc osaql JellBsuorlBlntu reJuEJ 'seprseqsuraloJdJosasselJraqlo LuEru puB 'sulaloJd d-rolep8ar aua8 'sasEuDIuraloJd 'suralord Surpurq-413 Jeln[acEJ]ur 'sroldacar eueJqrueusueJl 'sureloJd [Bu8rs pa]eJoas :Surleu8rs IIeJ ur pellolur ulaloJd Jo addt .{rana ,{lecqceJd Jo saldruexa sapnlcur saua8 Jossalddns rorunl pue seueSocuo-olord,{qpapocua suretoJdeqtJo }sI firy e pup 'saua8Sursnec-rec -uEJ roJ seqf,Jeasq8norql pauuuepr lsJg aJaMs,{.uarrqled Surpu8rs asaq} Jo s}uau -odluoJ aqtJo Iuuur 'tJpJ uI '(L9-OZ arnglg) serp ro 'selerluareJi;rp'sapplp 's^ or8 IIaJ e Jarpeq ^ IoJluoJ sllao SurJoqq8rauuror; saJuanlJurqrrqm qBnoJql etemqled Surpu8rs aqlJo sluauodruoc Tepcrued ur-purue uE ur slleJJo sJonpqaq lerJos aqt aleln8eJ lEql s.{E^qlBd aqlgo sluauodruoc apof,ua saua8 IeJr}rJc-JaJueJ^ueIAI

uorleraJtlojd lla) aleln6ausauegle)rlu)-re)ue)r(uew (.latnaslluloJ,uotssttliad

'sarderaql JecueJr!\eu dole^ep puP lsal qltM'186L 'sLv-99tt6Vila) 'P p ol qJrqM ur slapolu oslE ]nq 'uorleurJoJJorun] Jo srusrueqcau aql olur slqSrsur uurs'l rauv) ]e)ue) Jo]uaudola^apaql ro; parrnbar{;luereddeale'saua6o:uo apnoJd ,{1uo 1ou eJrru 'senbruqJe} eseq} Jo esn eqt qBnoJr{I 'aur} uesoq3 e oM] eql ol uo!t!ppeur 'sa6ueqt }e paJa^rlep(8nrp e) pu8rs IElueuuedxe ue 01 esuodseJur pelEaJJsr lceJep aq] 'passaldxa ale saue6 leluapu)eraqunJ peJaaurSua os aq upJ acru Jr}eue8aqt sltEldsrp-Ja^rl lEq] oMl aLllaraqMsonsst] aql ut slla)aqt Jo '(61-9 arn8rg aas)suorlElnu puoplp aql'^Bs-anssrl ro ue8Joeuo ^Iuo"{la^rlpuJallv'}caJap tEql qJns uorl.rodord ;;euse uor; I;uo pue {e1ape 'enssl} -uoJ,{JJeJ sno8dzoruoq o} a3rru Jaaur8ua uBf, auo uB ur suorlE}nru llnpE rageI;uo asueslounl aql'ssalaqua^aN 'lueudola^ep '{;aletedas pue aq] slqt aruoJJa^o oJ ualqoJd lJeJJe ]sal Jo IPruJou ur saua6otuo oMl aq} loJ 'e8pls saua8aseqtJo up qleap ol spEel saloJ eql SuIlJeUeJ Jruoftqua seteraqtlo urnsaql ueqtraleal6qtnu lE Ierluassa 'lllls 'eue8 eql aler reqbrq re; e uauo eue8 JossaJddns Jorunl e lp sloulnldo;anap Jo ,,(docpuo Jo serdoJ qloq Jo uoqele0 -Jes aql pale^rlJeur Jo lsol e^eq l(aq] 'sl leqt-^]lso3fzoralaq;o sprrq{qasaql'sutel}soml aql bulssolf ssol e Moqs ltilec ,{q paurelqoale .raq}a6olsaua6oruo 'auord -rdd1 pue dola,rap JaJuef, aJe aua8 JossaJddnsJorunl e sJorunl aq] lpq] qloq firel ]pqt a)rW'spue;6,{renr;es lo 'paledrcrlue sy '4y 3ur ;o ,r(doceuo Surssnu oJe leql suruJls luelnu eqt Jo ^ueru ,{reuureuaql ut uauolsot! 'lprulouueq} -pnlJur 'errru pe)ooDl uaaq a^eq saua8JossaJddnsrorunl snoJaunN ur ]no ,{lluanbarlaror-uq)nu, sJoulnldoqenep 'JerlJeapessnJsrppuE eruapr^a Jaqlo dq parroddns a)rruJo sure.rls qlog 'luaulalo{lo1e1n6ar se 'srseue8rrotun]JoJpaJrnbal aJe suonelnur eldqptu ]Bql 8uqsa38ns.,(y8uorls aurpsaql Jo lolluo) lapun aue6otuo 'snoJeJueJ auocaq ol se8ueqc paleJaue8 .{guopuer tequnJ o8rapun sDU aqt Idot palresut up satJle) lsnu Jo a u r lr a q t oe q l ' ( p u e ; 6{ r e u w e u a q t seueSo3uo o,ryUeseql Surssardxasilac ua^a 'snql 'slleJ snoJeJupJuou SuouE ur uorssa;dxarano:{;/y sanupuaqr q)tqlrr) sJorunl palplosr paJalleJs sp eleur8rJo sJacueJ eql 'urEBE']nq i(gt-02 eJnBIC) yp6 {roteln6alsnltAloun} {reuuueu ureJls IeluaJed raqlra uBq] eleJ raqSrq qcnu e lE puB JellJBasJeJuBJdolanap asnou aq] q\M )IW aua6otuo-olo.rd (auaSocuo szy e 3uilueJ auo qll,vr aueSof,uo JtW e 3uu{rrec asnoru crua8 aqt 6u;sn1,(q palPar)aua6otuoue1o pue JltW r+oq ssaldxa leql arrIAI fpoq {dor palrasurue saule)aug'pa1e.reua6 -sueJl e Surleru dq parq) sauaSoJuo sz2g, eq] ur aJaqMeurosesrJBIIrM raJueJ e ]eq] )srJ qStq e selBaJc1l esneJeq aJeueur lsrUalaMatru >rue6suell Jo sautl oml sluauuadxaasaq]ro1 'saua6otuo snorJesB sr euaSoJuo palrJaqur aql 'purrue aloqm aql Jo l arl Jo lurod eql ruo4 'ssalaqlranag 'sref,uec 'aua8suerl aql SurssaJdxa qloq 6ur{r.rer auo pup'auaboruo ol asrJ aArB 1ou op ta^emoq soy e 6ur,(.uerauo'eua6otuo r/yy 'sreJueJ sllaf, ol esrJ alt3 ol sa8ueqc Jequry o8Jepun IIrM sllac IBUoISBJco lso6 e 6urfuretauo'sutpllsesnourtrua6suell 'etull JaAo'pue 'uorleJaJqord 1ac pecueque rltoqs.,teurauaSoJuoarp ssardxaleql ;o sadr{taarqlur sJouln}Jo a)uapt)ul sanssrleql Jo atuos 'auaSocuosaY rc ttw B sserdxaol paraaulSuaaclru ut 'L1pct aql Moqssqder6aq1'a>1ur;ua6sue.r1 uq -d.{1 '1ac Jacuer e olur uorleioqello)eua6o>ug9€-OZarn6rJ IIar pruJou B uJnl o} }uaIJIJJns}ou dlleJaua8sI auaSocuo a13urse ruJrJuoJeJrru qJns Jo sarpnls 'eue8supJleql qlp paleloosseVNC leql s{epur a6e froleln8ar eq] uo Surpuadap ,vral ]f,alas e fpo ur Jo sanssrl dueru ur passardxa 00r 0 aq ^eru ,{um srq} ur peJnpoJtur sauaSoouo '8 Jetdeql ut pequcsap spoqtau ,tq paleraua8 aq ueJ sllal Jraql 1e ur auaSocuoue.{JJEo}eq} ecrru Jrue8sue{ 0 'ursrue8ro 'lryasn flrelncrged ue^ord a^eq aJlru ! o ]no)JoDI puB Jlua8suerl o aloqM E ur senss! IJaJJEsaueS[BJI]lJc-JacuPruI suouPlnru ^toq e]B8I]saAuIol ]ng 'stuarlEd reJu€c uErunq uo.g saldues Surururuxa.{qJo eJn}lnJ ur sllal Suldpnls,{q o @ o paurBlqo eq uec uoleurJoJur euros 'JaJuPcaql ot esrJarrrSleql senssll aql uI silac o ^ ou)I o] lrrEM e^a le^elv\oq dlaferupln o1 op saua8 asaql ur suorlEJel[B c teql\,\ f 'sauaB ^,tueur;osuorlJury IPruJoueqi ol deI o lEJrlrJc-Jef,uec + e sapnord snr4lsun&ap, pup afqdosoJ1 sB qJns sprulup Iapour Sutsn uago o o '.{3o1orqpluarudolanaq 'JacueJur pa8uerap arc leq} rolleqaq 1ac;o slcadse aq} 3 '!" )4iXr n uo 'spJol\AJaqlo ur-uorsaqpB IIaJ pue ']uarua^oru IIaJ 'qleep IIac 'uollellua :: 0O[ 6 IIE 'q;rvror8 -JaJJIpIIar 'uorsrlrp 1ac palelnBal uo pue uolleclunrutuof, llaJ-llac 11ac !,,

'-

l:!

f

-

::.:_

r:)

r,.ff:-t

ra)ue):67raldeql

znzl

1243

THEMOLECULAR BASISOFCANCER-CELL BEHAVIOR

Wnt +

-o

CYTOSOL

5

APC I

+ factors survival

NUCLEUS

*r,!,1*Akt +O-{OJ

c e l ld e a t h

lr

anti-growth factors

I

O

abnormality sensor cytokines i

The components in signaling pathways that normally function to inhibit cell proliferation often appear as tumor suppressors.A well-studied example is the TGFB signaling pathway (discussedin Chapter l5). Loss-of-function mutations in this pathway contribute to the development of several types of human cancers. The receptor TGFB-RII, for example, is mutated in some cancers of the colon, as is Smad4-a key intracellular signaling protein in the pathway-which is also often inactivated in cancersof the pancreasand some other organs.These findings reflect the normal function of the TGFBpathway in restricting cell proIiferation (seeFigure 23-26). Not surprisingly, mutations that directly affect the central cell-cycle control system feature prominently in many cancers.The tumor suppressorprotein Rb, discussedearlier, controls a key point at which cells decide to enter the cell cycle and replicate their DNA. Rb serves as a brake that restricts entry into S phase by binding to and inhibiting gene regulatory proteins of the E2F family, which are needed to transcribe genes that encode proteins required for entrance into S phase. Normall-v,this inhibition by Rb is relieved at the appropriate time by the phosphorylation of Rb by various cyclin-dependent kinases (Cdks),which cause Rb to release its inhibitory grip on the E2F proteins (disc u s s e di n C h a p t e rl 7 ) . Many cancer cells proliferate inappropriately by eliminating Rb entirely, as we have already seen. Others achieve the same effect by acquiring mutations that alter other components of the Rb regulatory pathway (Figure 20-38). In normal cells, a complex of cyclin D and the cyclin-dependent kinase Cdk4 (GrCdk) is responsible for phosphorylating Rb so as to allow progress through the cycle (seeFigure 17-62 andTable 17-1, p. 1063).The p16 (INK4) protein-which is produced when cells are stressed-inhibits cell-cycle progression by preventing the formation of an active cyclin D-Cdk4 complex; it is an important component of the normal cell-cycle-arrest response to stress.Some glioblastomas and breast cancers have amplified the genes encoding Cdka or cyclin D, thus favoring cell proliferation. And deletion or inactivation of the p16 gene is common in many forms of human cancer. In cancers in which mutations do not inactivate the pl6 gene, DNA methylation of its regulatory region often silences the gene; this is an example of an epigenetic change contributing to the development of cancer. Mutation in any one component of a given pathway is sufficient to inactivate the pathway and promote cancer. As expected, therefore, a

Figure20-37 Chartof the major signalingpathwaysrelevantto cancerin human cells,indicatingthe cellular locationsof someof the Proteins modifiedby mutationin cancers. Productsof both oncogenesand tumor genesoftenact withinthe suppressor Individualsignaling samepathways. proteinsare indicatedby redcircles,with componentsand the cancer-critical in this discussed controlmechanisms and chapterin green.StimulatorY betweenproteins inhibitoryinteractions asshownin the keY. aredesignated (Adaptedfrom D.Hanahanand With R.A.Weinberg,Cell100:57'70,2000. from Elsevier.) oermission

1244

Chapter20:Cancer

(A)

(B)

I I cyclinD-Cdk4 comprex

(a Cdk inhibitor)

(a G,-Cdk)

NON-PROLIFERATING C EL L

PROLIFERATING C EL L p 1 6i n a c t i v e or absent

a c t i v ep 15

I

J-

@ I

I dr*s*;

q,#F

I

t

transcriptionof target genesthat control entry into S phase

( a n i n h i b i t o ro f a g e n er e g u l a t o r y protein)

/z7y 11$

II

t

(a generegulatory protein)

P\ :

active Rb

a,. /P :

li

i n a c t i v eR b /

o*;*'i]',%*;*;n-

active E2F expressionof S-phase g e n e si s i n h i b i t e d

---jt L* expressionof S-phase genesis activated

Figure20-38 The pathway by which Rb controls cell cycleentry containsboth proto-oncogenesand tumor suppressor genes.All the componentsof this pathwayhavebeenfound to be alteredby mutationin humancancers(productsof proto-oncogenes,green; productsoftumor suppressor genes,red; E2Fis shownin b/uebecauseit hasboth inhibitoryand stimulatoryactions,dependingon the other proteinsthat areboundto it).In mostcases, only one of the componentsis alteredin any individualtumor.(A)A simplifiedview of the dependencyrelationships in this pathway;seeFigurej7-62 for furtherdetails.(B)The Rb proteininhibitsentry into the cell-division cyclewhen it is unphosphorylated. Theiomplex of Cdk4and cyclinD phosphorylates Rb,therebyencouraging p16 inhibitsthe cellproliferation. Whena cellis stressed, formationof an activeCdk4-cyclinD complex,preventingproliferation. Inactivation of Rbor p16 by mutationencourages celldivision(thuseachcan be regardedasa tumor suppressor), whileoveractivity of Cdk4or cyclinD encourages cell division(thuseachcan be regardedasa proto-oncogene).

cancer rarely inactivates more than one component in a pathway: this would bring no additional benefit for the cancer'sevolution.

DistinctPathways MayMediatethe Disregulation of cell-cycle Progression and the Disregulation of cell Growthin cancercells As described in chapter lz, a special cell-cycle control system ensures that

tumor-suppressor gene mutation, in many different cancers,is loss of the prEN phosphatase,whose normal function is to limit Akt activation by dephosphorylating the molecules that PI 3-kinase phosphorylates.

1245

THEMOLECULAR BASISOFCANCER_CELL BEHAVIOR

growth {actor

mrrogen

t

3 (

\

,,,.:.t1.,':rr''t lli:,lt:rt,l: iil.i:il:,:.il:.1':.',1'lr'rr.,,.

/tl

/

\

'n-1..

.i

PTEN

Pl(4'5)P'

:

i

I P

I PoP

P

CYTosoL

-*PoP.

/\

\

activation activation neededfor neededto d r i v et h e n u t r i e n tu p t a k e c e l lc y c l e a n d u t i l i z a t i o n

C?15

kinase \tyrosine ::dg..e Pl 3-kinase \

i

+

I

C E L LG R O W T HA N D P R O L I F E R A T I O N

I

increased pyruvare r

active ATP citrate lyase (AcL)

inffEased Protqifi sYflthesis

-A' acetylCoA

(A)

excesscitrate exported to cytosol

+ + +

fatty acids cholesterol isoprenoids

+

(B)

mitochondrion

The abnormal activation of the PI 3-kinase/Akt pathway, which normally occurs early in the process of tumor progression, explains the excessiverate of glycolysis that is observed in tumor cells, knor,r,nas the Warburg effect. Accompanied by excretion of excesspyruvate as lactate, the excessiveglucose uptake by cancer cells is used to locate tumors by modern whole-body imaging techniques (seeFigure 20-l).

ApoptosisAllowCancerCells Mutationsin GenesThatRegulate to SurviveWhenTheyShouldNot Control of cell numbers depends on maintaining a balance between cell proliferation and cell death. In the germinal centers of lymph nodes, for example, B cells proliferate rapidly, but most of their progeny are eliminated by apoptosis. Correct regulation of apoptosis is thus essentialin maintaining the normal balance of cell birth and death in tissues that undergo cell turnover. It also has a vital role in eliminating damaged or stressedcells. As described in Chapter lB, animal cells commit suicide by undergoing apoptosis when they sense that something has gone drastically wrong-when their DNA is severely damaged, for example, or when they are deprived of extracellular survival signals that tell them they are in their proper place. As previously discussed (seeFigure 20-14), cancer cells are relatively resistant to apoptosis, which allows them to increase in number and survive where they should not. Mutations in apoptosis-control genesusually account for this resistance.One protein that normally inhibits apoptosis,called Bcl2, was discoveredand named because its expression is activated by a chromosomal translocation in a B-cell lyrrphoma. The translocation places the Bcl2 gene under the control of a regulatory DNA sequence that drives Bcl2 overexpression. This allows the survival of B

.emtrana bi*sYntle$e, required'forcetfgrowth'

Figure20-39 CellsmaYrequiretwo types of signalsto proliferate. (A)ln orderto multiplysuccessfully, most to require normalcellsaresuspected signalsthat drivecell both extracellular cycleprogression(herered mitogen)and signalsthat drivecellgrowth extracellular (hereb/uegrowth factor).(B)Diagramof the signalingsystemcontainingAkt that drivescellgrowththroughgreatly stimulatingglucoseuPtakeand of the includinga conversion utilization, excesscitric acid producedfrom sugar in mitochondriainto the intermediates acetylCoAthat is neededin the cytosol and new membrane for lipidsynthesis production.As indicated,Protein Thissystem is alsoincreased. synthesis becomesabnormallyactivatedearlyin tumor progresslon.

'1246

Chapter20:Cancer

Iirmphocltes that would normally have died, greatly increasing the number of B cells and contributing to the development of the B-cell cancer. One of the genesinvolved in the control of apoptosis is mutated in an exceptionally wide range of cancers. This tumor suppressor gene encodes a protein that stands at a crucial intersection in the network of intracellular control pathways governing a cell'sresponsesto DNA damage and various other cell stresses, including low oxygen (hypoxia) and growth factor deprivation. The protein is tumor protein p53, and it is produced by the Tp53 gene,more commonly knora,rr as p53. As we now explain, when p53 is defective, genetically damaged dividing cells do not merely fail to die; they persist in proliferating, accumulating yet more genetic damage that can increase malignancy.

Mutationsin the p53GeneAllowManyCancerCellsto Survive and Proliferate DespiteDNADamage The p53 gene-named for the molecular mass of its protein product-may be the most important gene preventing human cancer. Either this tumor suppressor gene or other components in the p53 pathway are mutated in nearly all human cancers.\A/hyis p53 so critical? The answer lies in its multifaceted function in cell-cycle control, in apoptosis, and in maintenance of genetic stabilityall aspectsof the fundamental role of the p53 protein in protecting the organism against the consequencesof cell damage and the risk of cancer. In contrast to Rb, most body cells have very little p53 protein under normal conditions: although the protein is synthesized,it is rapidly degraded.Moreover, p53 is not essential for normal development. Mice in which both copies of the gene have been deleted or inactivated typically appear normal in all respects except one-they universally develop cancer before l0 months of age. These observations suggestthat p53 may have a function that is required only in special circumstances. Indeed, when normal cells are deprived of oxygen or exposed to treatments that damage DNA, such as ultraviolet light or gamma rays, they raise their concentration of p53 protein by blocking the degradation of the molecule. The accumulation of p53 protein is seen also in cells where oncogenes such as Ras and Myc are unusually active, generating an abnormal stimuIus for cell division. In all these cases,the high level of p53 protein acts to limit the harm done.

ther cell division, producing the phenomenon of replicative cell senescence(Figure 2o-40). The protection that p53 provides is part of the reason why mutations that activate oncogenes such as Ras and Myc are not sufficient to create a tumor. protein performs its job mainly by acting as a gene regulatory protein > Indeed, the most common mutations observed in p53 in human tumors are in its DNA-binding domain, where they cripple the ability of p53 to bind to its DNA target sequences.As discussed in chapter 17, the p53 piotein H Y P E R P R O L I F E R A T I VD EN A

srGNALs

Lltl

TELOMERE

DAMAGEss-onrirrrir,rtHYPoxlA

td:lllr s t a b l e ,a c t i v ep 5 3

CELLCYCLEARREST SENESCENCE APOPTOSIS

Figure20-40 Modes of action of the p53 tumor suppressor.The p53 protein is a cellularstresssensor. In resoonse to hyperproliferative signals, DNAdamage, hypoxia,and/ortelomereshortening, the p53 levelsin the cellriseand causecells to undergocellcyclearrest,apoptosis, or (Asdiscussed replicative cellsenescence. in Chapter17, a senescent cell progressively losesthe abilityto divide.) All of theseoutcomeskeeptumor growth in check.

'srossaJddns lounl lsol a^eqteql slla) Jo ro^ejur uorl)alasq]rM'saua6 Jo ssol P O] pPalosleue) raplosrpleurosoutolql eqf '(t€-02 aln6rl eas)seutosor.llorq) alnuru alqnopluapuedapul serPaddeos pue sn)ol leut6uo Iaql urorJpasr)xaeuo)aq-a6elParq puerlsvNc paredarunq6nolqllo lue^a uorleurquo)ale q6nolqtlaqlla-Ieu sardo)aldrllnLu aqf roqulnuIdot q6rqe ol poljlldLue sraua6aq] q)tqMut sluelnul ol pealsnql ;;rmuor6a.r leurosourorq) pal)aJJe aql ur aua6e;o sardo>;o sraqurnupeseal)utqllM slla)Jolo^ej ul uorl)olaSraqtJnJlltlsuor6a.r palel;dnp aql Jo saroo)jo raqurnuaql aspal)utue) (apl(rabpuq-uorsn;-abelearq palle)-os aql) abelearq pue'uorsnJ ;enbaun prleurorq)'uolte)!loar lo spunoJpaleadal 'pale.raua6 uaaqseqalaulolale 6ur1>e1 pue uorlerr;dnpe 6ur{let auosouolql e a)uo'uMoqssa)uanoesuo) eqr qllM'aseqd5 sralualla) anrl:a;ep-ggd aqt'abeutepaqt perredar qr;nt l! lllun asnedplnoMVNC pe6euuep aql ur lla) leruroue alaqM'e;:l{ruorsrnrp eql le 6ullleq lurodlreqt ]uapuadap-€sd 'uralo.rd ggd leuort:un;slte; Jo pealsul leql lla) e ur a6euep VNC leluapt))e qttrt su16aq ssarordaq1'sursrueq)oul alqrssod uel6erp leranas,oauo sMoL.ls aqf 'ssoleua6 pue uo;1e>r;r;dure eua6 se q)ns'satltleurJouqe auosotriorq) ol peal ue) y16 pebeuep ;o uoglerl;deraqt ^/roH1p-97 ern613

'saluelsruncJrc 01SurpJoJJeeJuelsxa saporu omt ueemlaq qJlllas upc sesnJIA Jo ^rpurpro Jo esrnoJ aq} ul vNC s,ilaclsoq eql q}I1!\Iel aseql 'selJ.{Juorsr^rp IIac (lalnb e sP aruoue8 'lleJ -leJedur 3ur1ec11dar ]soq aq] uI Ja8uassedpe^Eqeq-ila a Jraql alp8edoJd uEf, ,{aql :uorldo puoJas e aAEq uaql Jo eruos }ng 'rfu,r,rslql ur.,tpo acnpordar sasnrrl yl11qfueyq 'sseJord aq] ur IIar ]soq aqt SuIIpl ,!1ensn pue uorlecrldar yl11q uo slureJlsuoc pruJou eql q8norq] Suqearq 'preq 1t antrp pue ^Jaurq3eruslql Jaepueuuor ol seq snJr VNC eql 'lleJ lsoq elSulsB ulqllM salcrued snJr^ snorlcaJursnoJeurnu eJnpoJd oJ 'saluoue8 u1v\oJreql elPJrldeJ ot fuaurqcEu uorlecrldal VNC s,lle3 lsoq eq] esn sesnJr^VNC ,{upw 'sasnJr^ 'slseueSoutcrer IeJIAJo ed& sH] Jo itrolsrq eJIIeql puulsJepun ol ]ueuodrul sl lI puElsrapun oI'tgd uo puedep teq] asoql Surpnlcur'srsoldode puB elcltJ IIaJ eql uo slorluol s fiac aq] qU,,t,r SuueJJelur,(q.,(preru reJuef, esneJ sasnJIAJoruru VNCI

su!alord rossarddns rounl {ay ;o uoll)v eqrl)ol8 sasnr!nrorunl vNo 'llB tuaql ol alnqrJluoJ suorlElntu tgd lnq tor^eqeqsrur 1o sad,,! asaql 'sadr{l Jorunl eruos Jo raqlouE Jo ouo o] alnqrJluoJ uEf, suorleln(u Jar{lo,{ueIN ur uorlerpeJrr pue s8nJp JaJuBJrluE o] luelsrsal i{yalrlular slleJ sa{Eru lI 'r{UnoC 'ap4lp ,(aql se elElnrunJ3e ol suorlelnu Surlourord-raJuec JaqUry Suulolp 'slleJ JeJueJ JrlsrJatJeJBqJ,{1llrqelsulf,lleue8 aql ol speal ll 'saruosoruorqJ Jo pa8eurep qt!v\ sllar Jo uorsrlrp Surnlurad ,(q 'prlqJ 'slso]dode adersa o] ueql s^\olle ll 'puosas 'a1cdc 1ac aq] qSnorql enu4uoJ o] eSEtuep VNCI qlyv\ sllas s^\oll€ ll 'lsJrc 'suoseal ]uaJeJJrpJnoJ JoJ JaJuec eloruoJd UPJ lr esneceq snoJeS -uep -,tlensnun sr ^,$rnrlce$d p ssol aqJ 'snora8uep aq plnom ]l aJaq ^ seJuels -runJJrJ ur uorleJaJllord 1ac uo {ceqJ e sp 8urlJp 'sasseJlsIIaJ Jeqlo pup e8p -ruep ql$ dleJps adoc ursrue8Jo JelnllaJrllnu p sdleq ggd 'l.reruurns u1 VNCI 'uollEraJlloJdJreql 's8nrp llBq o] Jo arp ol luaql asneJ asr ^raqlo pFo^ qcrq,u JesuEJrluELueu ol puE uorlPIpPJJrol e^rlrsuas ssel qJnru sllef, JeJueJ eruos saleru osp ggd Jo ssol aqJ 'sauaSoouoJo uonelrlre o] pue saua8 JossaJddns rorunl Jo ssol 01 qloq peel ueJ uaqr(eru cpeueS qcns '(IH)z arntlgl euouaS paldnrroc e qlrM aleJaJgordpue aArAJns'asJoMrpJ to erp Jeqlle d.erufaql ']1nsare sy'a8eurep aqt Jredal o] Sursned tnoqtp\ uollportdar y111qolur Sur8unld 'Su1pyt1p uo IfJec deur ,(aqr 'pa8eurep sl VNC Jreql JI 'pue 'srsoldode adecsa ol puel ^eqJ 'sasuodser ssarts ]uapuadap-ggd eseqt r oqs 01 IIeJ tgd uI a^IloeJapslla3 '(gt raldeql ur passnrsrp) srsoldode SurloruorddqeJeq] 'pupuoqJolrru Jo aoeJJnsaql uo suralord 71cg crloldode-nue alelrlopur pue o] pulq osp .,teurll ]nq 'saua8 cpoldode-ord ^,(ueurJo uorssaJdxaaqt elelnrups uec 1r lxaldruoJ erour are srsoldode saJnpur ggd qcq,r,rdq susrueqJeu eql 'VNCIsll Su4ecrldar pue aseqd g Suualua ruo4 IIaJ eql stuanard uratord 17d aqr 'saxayduroc{pO esaq};o ,tlnqce asBuDIeql Suryrolq lg 'a1rdcIIeo aql qSnorql uorssarSordro; parmbar saxaydruoc{pl eq} stlqlqul pup ol spurq leql uralord € sapocua qrlqn'tzd lo uorldrJJSuEDaq] ecnpur 01 'tsual 'aycfc le ued ur VNCI ot Surpurq dq IIaJ eql uo stJeJJad]ollqlqq s]r suaxa

l'l)A) r9cru8-NotsnJ-l9v)vlu8 al!s a J a u o l e l anau1e a6e1ea.rq e 6 u r 1 r eu1l e 6 e1 n q 6urlear:'srsolru.r s a u a 6p a l e r r l d n pq ] y n r esn+saraurola] t e U P d ep e l l n d a u r o s o r x o r qe) s l r J a q u l aresprleuJoiq) ))el leql spua r a l s r sp a s n ] prleurorq) Jalsrs ; 1 a rr a l q 6 n e pa u o

sv

la

VNC s l rs a l e ) r l o a r a . / a u l o l ael 6 u r 1 r e ; p u e a s e q d etlosourorq) e sluaqur s s r o l u al l a l ; ; a : r a l q 6 n e pe u o

€zr

leorq p u e r l sp a r e o a r u n aldsap ygq slr sale)rloal pue a s e q d5 s t a l u a1 1 e :

luatu6e.r; aurosouroJql paqlPlap sJoqrj a l p ur o s )rlolrur

uorAVHtS llf-u:l)NV) lo stsv8ufln)lrow IHI

LVZL

'Qqbu)n>ue> e aleraua6o16urd;aqIqataql islla)lespq aq] ur uorsr^tp llo)Jo lortuot 3ql qllM sajaJlalur surolorolelrAro uotl)npold pele;n6arun eql'uorssardxa aue6 ler^Jo lorluo) lPrulouaql sldnlstp(asne) raqlo aurosI;qrssodlo) stql'slla)leseq eql ur sauablern aql ro luauuoltlua 'lsoqoqt aruosouJolq) aqt 6uua1;e Jo e olur pruse;de q)nsJo luau6er; e;o uorle.r6alur oql asnelue) sluapo)eajeU '(UaD aql qllM ]soqaqt,o sauosoLuorq) dalsdeal o] seos pate;n6arsruorletrldar asoqMsprLuselo se urnrlaq]roa aql Jo slla) leseqaql ur pJurelureu,(lqets,(1leu.rrou aresauosor.!orq)asaqf'slreoaptloallnu 0008lnoqPJo sauosor.rrorq) vNc leln)lt) papuerls-alqnop a^eqsasnl^euiollt0 ed 'xr^rs) oullaln aql ls)ue) ol asu Jo anr6ol lq6noql ele sasnrrneuo;;;ded uleua) MoH g9-97 arn611

3ur1e,lr1ceur;oacuelrodrul aql Surulpapun 'ggd pue qU llqqul ol susruuqceu relrurrs asn sasnrr^ rournl y5q rar{ro 'sarlllBruJouqEarolu 1al. alelmuncce pue 'aphrp 'aArAJnsol IIaJ leluJouqE eql Surrv\olle'(Ev-02 arn81g; uorlf,nJlsep 'tgd o1 Surpurq ,(q 1g9dsI raqlo aql 'aseqd ggd sra33rr1 uralord 9E IErl aql pallorluoJun smolle qclq ^ 'Jallrea pauoltuau suralo.rd CZA eq] S olq ^,fu1ua Surlrqrqur pue ot Surpurq ruoq qU stualard uralord Zg IEr.r eql 'qU ot Surpurq dq :qU sr suralord ]soq aseqt Jo auo [e,rnpa11o.r]uorunue ul ep4lp pue VNCI s]I alecrlda.rot aql Surllrurad os pue uoIlJE Jo ]no uaql Surltnd '[ac ]soq aqlJo IIaJ suralord rossarddns Journl .{a1 o.r,r,1 o} pqq.{aqt tepcrged ur '1nq 'suralord 1ac lsoq dueru qlp loeralut sauaSocuoIBJr aseq];o slcnpord uralord eql'lg pue gg palleJ are euelq o1 dpreur aJe tpql saua8 prn eql 'sasnJlleuopded u1 ' (Z7-OZarnElg; sauaSocuose Suuce saua8prr,r eq] qllm laJupJ o] peel UEJsrql'aprsal runllaqlrda eqlJo sllaJ ruels aql aJaq ^ te,{el IBSeqaql ul alrlJ€ aruoJeq pue eurosoruoJqc]soq aq] olul aler8alur o] ]saJJpa1c,ri.c-1ac tuanard ]eq] suralord aq] apoJue teql saua8IeJI^aq] 'ralamoq ,{lpuorseccg 'uem E uI se 'ssaluJeq d1a,tr1 sesnpJ rllaua8 B luaprJJe -EIar sr puP silac sra.r{e1 Jelno aql ol pa}srJlsal sl tJeJJeaql .{1ensn 'aurouaS ;o umo slr Jo uoqecqdar ^ olle ol sE os ]seJJEa1c[c-1ar sqt q]lm saJaJJa]uIsnJIAaq] 'aJpJJnseqt uorJ paq8nols Sutaq 1nq 'do1s.{lprurou ppoqs uorsrlrp IIac 'aJeH eJoJeqeterluaJaJJrp o1u6aq slleJ eselp Jo .{uaSordse 'sra,{e1pqaqlrda re}no eq} ur aseqd anlleclldar e o] qf,lr^ s e q8norql paleraua8 are salcqred snJIAsnollJeJul 'saruosouroJqJaql qrllt dals ut alecrldar qJIqM 'sptuseld leluosotuoJqoeJlxa se slleJ;o ra.{eypseq aql ur asuqd }ualel e uI sellesuaql um}uleru pue unII '(sracuec uerunq -aqtlda IIe Jo %g lnoqe) IEJr^JaJeql lraJul sasnJlleluolpdu4 {a1 e se lue};odrut,(llerradsa xrnreJeuuetn aqlJo seurourJJBJ ur JolJeJaArlesnBJ are pue aJp 'aldurexa JoJ 'sesnJllutuolgdud aql suem uerunq asnec eq] Jo 'sesnrll Jorunr VNo se paleu8rsapuaaq a^eq slueplcJe qJns o1 alqqdacsns sasnrrl VNCI eql pue taJuec o1 Surpeal 'ilac ]soq eql Jo uollere -gqordtuatsrsrad aqt uo qrtllrts uec ]uaplrrp yo adr! sql 'a1c.{cIIao aqt Jo .{puap -uadapur a1ec11dar o] VNC IeJrAaql ,ltolle o] aseqd anrleclldar s1rut asn r(geru -rou plnom snJrl aqt leqt suralord IeJIAaq] Jo auos elelllce dlarnleurard leqt JnJOouec sluaprJJe 'aseqd ]uelul E qll^ sasnJrl JoJlng 'ursrue8rotsoq eql 11t{o} sr snJrl VNC e a;t;o [ea,t qJI_qMra]lelu oN ]saralur slr ur ]ou s1]r '8urrr,roy1oJ 'aseqd tuelel eq] Suunp seruosoruoJqclsoq aql qllm dals ur salectldar teql pruseld leruosoluoJqJeJlxe ue uJoJ ol .&rlqe aql aneq rtaql Jo 'saurosoluoJqJlsoq aJolu Jo euo olut auroua8 IeJIA Jlaql aler8alur raqlla uolleclTdar prIA ;o asuqd ]ualel e ut sra8uassed tuacsarnb sp VNO rraql alecqdar ol ztnqe aqt a^Bq leql sasnJ-IA VNC esoqJ 'salcqred snorlJeJur Jo srequnu a8rel saleraua8 pue IIaJ lsoq eql s11tIleq] ssacord e uI sllar IEUoIS -pJJo ur Surlera;gord uaq] lnq 'aurt13uo1 e JoJ sseluJeq pue lualul Suturcurar l uonnl 1NVN9t'lvt

suraloJd l e r r n6 u r p o r u a aua6pele:6alur

IUVAAUO H]AAOU9N9IN]8

snr^eurollloeo lo auosouroJq)

u o r l e r a l r l o r dl l o ) s a ^ u ps u r a l o r d l e r ^ + o u o r p n p o r dp e l e l n b e l u n

rr^osoWouH)lsoH olNl .LN]IADVUI VNC]VUINIO I rVl-NlC l))V NO|-LVU 931_N

aurosorxoJq) lsoq

s n l r ^a q l , o u o r l e : r ; d a rp a l l o J l u o )l o J e l P r p a usr u r a l o r ol e i r A

Je)uP):02reloeq)

SvzL

'suraloldoMl atupsasaqltalsanbas o] 'ua6rluel abre;pe;;er,utalold asodtnd-;enp a;6urse sasn(slaluoru sl)ajur]eql snlrApalelale) snltA otAS aLlf (umoqslou) sls^loalordggd 6urrnpurleuupd Egds1r1ouolrel,(llnblqn ol speal6urpurqulatord93 ',{;anr1>adsat 'sureloldqU pue €Sds,lla)isorl aql tatsanbasq)lqM'll pue 93 'surago.rd ler^ oM] sa)nporosnJt^euolltded 'snr!^loutnl VNO e Iq uollelar!lord lla) Jo uolle^llry gy-97 arn611

zi.lprcadsasl 1l pue 'uorsaqpe Ilec-llec ur sr uralord urJaqppo-AeueJqruerusueJleql Jo uoucury rfueruudaql'aua8 uuaqpoJ-fl eqlJo uolssardxaur sa8ueqceJEuoIlIS '(6I raldeq3 uI pessnJslp) LroterBtu aroru puu ellsaqpe -u€rl slql Jo ilpaq aqt tV ssal Sururocaq tetf,eJeqf, ul U1qse o8repun qlac pqaqrlde qJII{ ^ ur'(IWg) uoll -lsun.ti Tnutqcuasaw-o11nqaqnda aql se rrmornl ssacord ptuaurdolanap prurou aq] salquasar dals srql Jarlrea pauonueu sy'raqlaSo] s11acpqaqrlda ploq dput 'sseualrselul pcol 'dals lsJU eqJ -Jou ssol e sarrnbar susrueqJau eql leq] Jo 'sasalsaput qsrlqelsa pue qdu,,t1Jo poolq aqt ErAsetrsluelsrp ol Ia^BJ]1smu.{aq} 'puocag 'q}eauaq .{1a1ep -arurur anssr]aql apelur o1ur8aq pue urnllaqlrda luared rraql;o saurJuooI€ruJou aql adecsa ]snu slleJ eql 'lsJIC 'ezlselselaru ol EruouIJJeJp JoJ partnbar uots -sarSordrorunt saseqd o,rnlqsm8u4slp or p;d1aq s1lg teqrea passncsrpe.tlrsV Jo 'runqaqlrda uE ruo{ adecsaor seq }pq} ileJ BruouloJeoB JoJuuql Jelseaeq ppoqs srselselaru'uorlelnJJrJaqr epl .,(poqaq] Sunueor Lpearp IIaJ poolq elrq ^ snoJa:) -uEJ B JoC'sllal Jo saddl lueragrp JoJsuelqord luara;;rp sluasard srselselahl 'ezrselsplaruol ^t4qe eq] ol selnqrJluoo loqg ,t,roq-{ltcexa talemoq tealc 1adtou sl }I &Uqe Jltetse}au rraq} {Jolq o} lualJ[Jns sr sllal asaq]ur,tFrlce foquJo uolllqrqur 'sesBJatuos u1 11ar paspq-ur]re alerpau o1 sdlaq qrlqa Jozly aspdlg,{.1urug-oqg eqlJo"{-lr1r1oru uorssardxa -JeAoue sr-Jenrl pue 'qcuruols 'lseerqJo seruounJeJ pue seruouelaruSurpnlcur -srotun] dlarren e uo{ slleo reJuBJ ol}Etseleruur a8ueqc relnJeloru euo Jo ^fienpr^rpul Je^ossrp 01 pJ?q eJPAeql ]Bql esuPlsuncJrc o1SurprocceparJpl os aq.{eru aseq} leq} }nq 'suoue}nru Ieuor}rppesarmbar 1eq1 'plaq rltepyrrtaJoru 'lvrernanrleu 1se1xaldruoJ pue UnJrJJrpe sr srsptsplaur lpql q -Jalle aql' \arl slql ]roddns rorunl drerurrd aql ur pue srselselarue ur passardxa sVNUru aq] aredruoc ol s-,terreoJJnu VNC Sursn sluaurrradxa etuos 'qlpep ruorswp 'qulor8 IIaJ puu 1ac ilac uo sloJluoJ lpruJou aqt ueTearlt ot papaau asoql puortaq sa8ueqc crlaua8 JaqUnJ ou sarrnbar ezrselselaru ol sllal JeJ 'Jrlelselaru aruoJeq ol arrnbce -ueJ A\erA eql sr erueJlxe euo &tUqe ]eql lsnur Jo 'paapul 'saselselaru ruJoJ IIao JaJueJ e sarlradord leqrr,rf.ypexa Jealf,uele lou sl tl pue ,{poq aql q8norql peards (senssl}Surpunorrns epelur o} sllef, lpurad ,t1ec -ryrads suoDelmu zlrluapr dpealc ol ]a.{ a,req eM'srselselatu pue ssaualrs ]eq] -enur suJaf,uoJJaJueJ Surpuetsrapun Jno ur du8 luecryu8rs ]soru aql sdeqre4 Jo

{lals{W e {;a6te1 lllrs arv srselselewol peol reql slle) rournl ur se6ueq) aql 'qJpa 's,{e,uqled ur q}oq pa}e^D luauoduoc auo r(po Supcage,{1pnsn -reur osp aleq sJeJuBJuerunq ge dlenlrpt leql 3ur4rr1ssl lI 'uoppJeJgord 1ac uo slureJlsuoJ leruJou aql Sursealarro; sf.emqled rossarddns Jorunl aseql Jo qloq snutAvNc A8 cl-Lvntl)v 'l'1t) NOIVU]Jt'lOUd

cl)fo'I8

Nolrvullt'loud'l't3)

N O t l d t u ) 5 t l v u tL Z d

+ I

u raloro leJl^ aArpeur

NO rdlU)SNVUi I Nlr)

VNC uorlera;r1old;1eruo a1e:q {1a1es seprnorduralo.rdggd anrpe

(uraloJdfuo1e;n6araua6) ./ol)e, u o ! l e r a J r l o Jlol a ) e ^ r p e u l

-)

t

T

roDe] uorleral;ord lla) a^11)e

l.l cNV 9r sr)ncoud NotDllNt snurn

6V7.1

JOpeluorlera]rloro l l a ) s p u r qu r a l o r oq u

]'E)]vnuoN

uorAVH3S Il)-UlfNVf

INl)stIno

rO SISVS Ufln)3tohtlHl

aql ^q sroun] ]ueu8rpu o] u8ruaq ruo4 SulsseJSord'1ac ]uPJJeqBelSuISE ruo4 ,r(lpnperBdolanap sJeJuEJ]sotu leql ,trESe^ 'raldeqc srq] Jo Suruur8aq aq] lV

s a6ueq) elq!s!n,ouolsse)lnse ern/(lMolse^lo^f sla)ue) lPllerolo) 'anssr]uSraJoJazruoloJ 'seSuEqcll]aueSlde Jo sllauaS ol slleJ ruels JeJupJ elqeua o] papaau aJe JI 'esB3,,(ueu1 'tz raldeqS uI "{uE lBuortlppe ]Eq^ lurodurd o1 pleq .{ran surerual tr 'leJaue8ur sllal ruals SulpuelsJapunJo urelqoJd JepIMaqlJo efual le pessnJsrp ued sr srqJ'saruadord IIac-uels lerJadsJraqt rueqt se^r3.{lle}uatuepunJteq} sl tl 'sllal luels JeJuesIJI} ]eqM puelsJapun 1a.{}ou op eN\'sJaJuEraruos JoJ}sEal}E -uapr dlaq ]eqt sre{rEtu ^\oDI art qSnoqlp lng 'paureldxe ,{pBeJIee^eq a^ se Jo 'sllaoIuals JacueJ JelJereql ',{rolsaqt;o ped 1sea1 1eaq }snru srql }eql sureeslI Jo prcads aq] aAEq]Eql uollelndod Journ] luaJed eql Jo sraquaru aJeJasoqt ]sn[ au6 aJEuorlezruoloJ Jo elqedBJ sllas eql ]eq] sI JaIIJEapassnJslp se 'dlITIqISSod 'lou op '3urql-{uE (lBq^\ ]eq] asoql uorJ azruolos leq] slle3 aql seqsmSuqsrp JI Ia^al Jelncaloru e lE JaAoJSrpol srql se qJns s.{essEasn ol sI eSuallEqsaqJ 'ftt-O1arn81g; Jrlelselau eruoJaq ot eJrnbJElsnu sllal reJuBc leqt &Jadord da>1e s (uoliDztuoloJ paIIeJ ssaJord eq]) anssll uBIa -roJ E ur alera;gord ot anurluoJ o1^]nqe eql ]Pql .{lrlqrssod aql SulsIBJ'sJourn} Suu,ror8 urro; r(aql dpo se 'saArlEIaJ Jllelselatuuou rlaql urro;radlno slac 'lurod slq] ]V'sllaJ puesnoql lualeduor-srselsElaur aqt IeJa^eso] uel SuIuIBluoJ e apIAIp sJeq]o m]s la]PJa saselsElaruoJc[u SurruJoJ'do1s uaqt puE serul] 1V\eJ -JnoJd o] IIeJ]nq enssrl uBIaJoJaql olul .[r]ua anr.trnssJaq]o ianssn uBIaJoJre]ue .{aql rage ,4.1a1eparurur arp sllaf, aruos '}ou seop }eql auo Jo sezlse}setau }Pq} rorxnl e luo4 auroc .{aq] Jaqleq AJo ssalpJBSer'enssll Sutpunorrns aq} o}ul lIXa puP 'slassa^Iprus ur a3po1'uorlelncJlJ aql uI aAIArnss1ac,{ueur }eq} sa^Jasqo auo sluarurredxe qJns q 'aleJ Jraq] Jolluolu ueq] pue 'asnoru e Jo lueaJls '(agg) ule]oJd -poolq ueerS Jo a^p luessaJo aql olur Iuaqt lualsaJonu tcafur (saIunJIJJIp -nu E qll^\ sllal eq] lselEeJSaql qll/!\ sllas JaJuBcluas IaqEIuer auo -ard srselseleu ur sdals Jelel aqlJo qJIq { Jalocslp o1 ,(pn1so} rapJeq qJntu are 'salrs alorual Jo uorlezruoloJ e^rllaJJaaql pue lassal cqeqdru.{1ro poolq e ruo4 'esEa qlII\ uopelnc aAIIPIaJ 1xa Sur,Llonur'srsplseletu ur sdals Sutureruar eqJ -rrc eqt q8norqr anoruuaql pue Jatua ol aAISEAuI aql dlaq aruoJeq aleq teqt s11ac .{eru slassan,vrauasaq}Jo ssauDlealpue .{lrt8er; IeruJouqe aql IJaIIJBapessnJslp se gCgA sE qJns srolJeJ cruaSor8ueSurlarcas^,i.qsanlasruaq]o] lJeJ]le sJorunl qJIqM 'slessal crleqdur'(1salultaluos pue slassa^ poolq Jo ,{1ddns asuap P Jo acuasardaqt .{q padlaq sr uor}elnJJlc aql o}q sllao Jorunl Jo ^,{J}ua IPUUI eqJ 'sseualrselur pcol .{.q Suqelqlceg 'aro;ararll'uuaqpoJ-,9 sso'I 's11ac prlaqlrda luaurdola,rap JaJueJ aloruord .,(eru Jo IeruJou a{rl aJoru aJaqoco1urSaqpue sJIlsIJalteJBqJeAIseAuIJIaq}Jo aluos asol daqt 's11ac JacueJ asaqt olul >1ceqtnd sr aua8 uuaqpac-fl IeuoDcunJe JI :arnllnc 'pencser -,(lerped eq ueJ utatord eql SuDIoPIslleJ rorunl JoI^Eqeq uI

]seel le Jo ('pfl letiual pawolgLuorJ uorssrLurad 'aua8 rossarddns Journ] e sp pelJlluapl uaeq ser4 auaB uuaq IpruJouqe aql qllM '0002'L0v-00v:z'saaD)uD) -paJ-fl eql'lseeJq aqlJo pue qf,EruotsaqlJo setuoulJJeJaluos uI'(9I-6I arn8tg ''p )sDilB ta sraqLueq)l'V u,ror,paldppv) ')rlelselau.r auro)aqo] alrnbrelsnurslla) ees) suortrunf suaraqpe q8norqt raqtaSot slleJ Ielleqllda Surpurq ul lueuodul leql saruadord{a1 are'anssrlu6rero; pue aArlrnsol Ilr;rqeue pue aql ur rvro.rb 'anssrl luaredaql uolj adetsao1{1r;rqe -L'tnfHltc aq] ]eq] suaasll slla))rlPtselauJuou uPqlssa)lnsralearbqrnu aneqol pa^rasqoaresllafftlelsplau {gq6rqteqt iiiip soals]ln)uJrpasaqlut s! ll 'lsolarepue t,-" (-l ur sdels lreJslla)jo sraqunua6re1q>rqan a.re,(aql lelll asuaseqt uriluor)UJaur, anssr] r o l l n ) U J rdpe ' ^ - O Z e t n 6 r 1u r lassa^ olur {:1ue u6tero; paurllno'ssa)ord )rlplsplauraql ul sdals sasne) qvrtorb 1o u rs l l a ) q)r[1MMoqssase]se]aur 6urqsr;qetsa I ssouaAlSe^u arualsrslad +olPArArns pue'uorlelnlr) aq] 6urralua'allsloLun] e 6urnea;slla)rounl palaqpljo sarpnls 'srselselaurol srarJreqeq177-97 arnbrl

l,u

ra)us) :02reloeqf

o9zl

LgZL

JdV puu ggd pue 1,(puel aua8 sry aq] Jo raqrueu saua8 rossarddns rorunt aql e) saA-X auaSocuo-olord aql-palelnru ,tpuanbar; ]soru se lno puels eerq] le3uec IelcaJoloJ ur pe^lonur eq ol paralocsrp JeJ os saua8 esoql JO aslue^a Jo ureqo slql acnpord ol erup qlyr elppruncce leql suorlelnru eql eJE ler{l\\

sre)ue) lel)alolo) Jo uoll)erl a6rel e ol uouruto)arv suolso'l)lleueg {ay me1y 'slassal poolq Er^ sue8ro Jeqlo pue '3un1 tanrl pue 01 slasse^oneqdurtl ern sapou qdurrtl ol Surzrselselaru.{1pug pue '(ggV-02 arn8rg) 1nBaqt spunorrns teq] elJsnru ;o raf.eyaql q8norqt Surpeards ueql 'purruel leseq prlaqlrda aql q8norql Suoparq lsJl; 'alrsplur aruoJeq sllec Jolunl aql 'aseesrpaql ur sa8ulsralel tV 'sllao snoleuouape Jo auolc pufuo aq] ulqllv\ auopqns luplntu p sE uesrJeaneq.{aqt q8noqr se 'snoJecupcz(gea1c3ur -readde Jeqlo eq] ut asoql pue lpruJou dlanrlelarSurreaddeeeJeauo ur slleo eql qtyvr 'dr{1oda13urse ulq}Im peqsrn8uqsrp aq ueJ seeJelourlsrp aJoru Jo o^ l ,seuru -auos 'seJnlrnJls pazrue8ro ,r(lpurrouqe ruJoJ pue palequeJaJJrpun,{.lprurouqe erou eqt 'd,,(1odaq} ra8rel eqJ 'lpruJou }sorup {ool lpq} slleJ uretuof, o1s111.{1a111 readde -,(1ensnurnllaqlrde eql ul luauaSuerre Jraql pue sllaf, er{l teleu€rp ur ruJ I ueq] Jellerussddlod yo suor]tas crdocsorclu u1 dlsnot.a;d passncsrpuorssar8 -ord rournl uoueuouaqd aql;o aldruexa Jpall e sapnord JeJuec uolo3 Jo 'asrMJaqloeq plno.{ ll ]eqmJo ragenb B upql ssal 'sarpnls auos o1Surprocce luo1t(rel sr JaJueJ IElJeJo -loo Jo arueprJur luanbasqns aql-eJnpaoord pc€rns ,tsea pue lrmb e-adocs -ouoloJ aqt qBnorql paloruar are sdrlod eq] pue ser4u rraql ur ddocsouoloc 'tq pauaarcs are eldoad uaqr 'snqJ lueuBrpur peuJnl lari.lou seq lnq alqelJalap sr rorun] 3umor8 ,r(1m.o1s eql qorq \ ur sread 0I tnoqp 3o porrad e dllBclddr sl aJaq] l^ols ftan,fi1ensn sr aseesrpar{} Jo uolsserSord aql asnuJeg 'sJecuet Ie}JeJo -loJ Jo uoqrodord a8rel e Jo sJosJnJeJdaql aq o] pa erleq are sd,{.lodsno}Btuou ' -epe p (g7-gT eseqJ arn8rg lygT-g7 osp ees pellpo arn81fl dQod enssrt Jo ssuurBur -pnJloJd e urroJ eql uI runqaqlrda 1nBaq1;o 'EurouapEJo .Jotunl u8uaq Jo IIEtusp slea^eJuago adocsouoloc B q]!v\ sl1npe IeruJou Jo uolleurrupxe euqnoJ tanairnoll '(gg Jo a8e aqt ralle rnrf,o 0606)eJII ul alEI lpun pasou8erp ,r(1pnsn1ou ere Laqt 'sJacuec 'JaJuEJruo4 sqleap ,sa]Els pe]rufl aq] uI lsour eT'I [B]ot Jo 060I lnoqe Jo rea,{ e sqleap ggg'99 fyreau SursnecdpuarJnJ 'uoufiuoo aJpsJecrreJ[B]ceJoloJ 'sJacupJ lepeJoloJ ]soru JoJuorssa.rSord JounlJo ssacordaql ur8aq spu8rs Surzrue8roprurou aql ldnrsrp leql suopelnlN 'qcJeesarJaJueoruorJ euroc e^eq leq] slq8rsur ot $lueq] ped a8rel ur sr srql'gZ reldeq3 ur paurcldxa se 'poolsJepun llalvralrnb aq o1 Suruu€aq are urnqaqlrda aql Jo IEMaueJ pue uorlezrue8ro prurou eql IoJluoc pue sllal tuels aql urel -urpu teqt spuBrs aql 'sldrirc puusatul pallec 'runrlaqtrda aql;o slalcod daap ur aI leql silac ruals uo spuadap le^aeual aq] suo€ar qtoq uI '1aaqsprlaqlrda eql Jo lsoru aceldar r(yalaldruoc 01{aam p lnoqe 3uopl 'a1erprder l.lrJpurproeJlxe uB ]e paMeuer arp runrleqrlda rn3;o suo€ar qlofJ'(z?vr-gstl 'dd) 97 raldeq3 ut lplap ur passnJsrp 'eurlsoJurlletus eql Jo teqt ol Jelrurs ,{lpeorq sr enssl} slqlJo uorlezruu8ro ael'(ln8 aql;o luau8as Ipurure] aql) unlceJ pue (euusalul a8rel aqr) uoloJ eqt Suruq runqaqryda aqr tuo4 asrJe sJaJuec IBtJeJoloJ 'saua8 repc4rud;o uor]f,urg aq] ur sa8ueqopelrJequr qluvrsa8elspcr -Soloqdrou rqrcads alerJosseo] sn smolle 1l pup ,lueudolanap Jorunl eJnleu Jo asuldals aql Suuerlsnp .rogru8rpered e se selJes ,espesrpupunq eql Jo surJoJ palrraqu pue crperodsqloqJo serpnls uo pesuq ,leporu srr{J'le^el JelnJelotu aql le parpnls f1ryarec pue (runlcar pue uoloc erpJo JorJa]u1eq] SurmarnJoJ eJrlap cqdo-raqg e) adocsouoloc e qlrmo/?rnw pawollo! ueaq aleq uorssarSordJorun] 'aldurexa Jno sE JaJrruJ .sruJal esaqt ul ;o sdals eql qcrqm ur IBpeJoIoJ alel el asEeslpaql;o ,trolsrq IeJnlBu aql Jo asuas eTpru ot Surldruaue ,paJnpoJlur e^eq e^ srusrueqf,arurplnceloru pue saldrcurrd praua8 eqtJo aruos uodn a8relua pue elpJlsnl[ ol lr Sursn ,{1eso1c aJoru sJaouef,ueunq uoturuoJ eql Jo auo eurruexa ,,t,rou el1'Jor^pqaq snoraf,uec ot alnqrrluoc faql l'vroquaas pue sruJal JEInoaIou ur are sa8ueqJ puB sluaproJe asaql Jo eruos ]eqm pessncsrpa^Eq aM'sa8ueqc cpaua8rda pue sluaprcce crlaue8 luepuadapur Jo raqunu e Jo uorlpFrunJoe

uotAVHlS ttl)-u3)NVf lo stsv8uiln)ttow tHl

r a r u e ) l e n p r ^ r puup u rp o t e t n u t ss t u a u o d u l o l J q t l o a u o l ; u o a r o l e r a q l p u e ' , ( e r , . r q l e d a u . r e s a q t u r t l e l d t l l s l a d n s o u J e s a q t q l r , r , t s e u a

(uor1e;{qlau {q paruelrsuago) 5t 0€ 0t 09 08< 0[-9 0b

rredarqrleuslu VNO 6u1geu61s $391 6u11eu61s {191 abeuepVNCpue ssarlso] asuodsar 6utleu6ts 1un4 6ur;eu6;s lu1111 6u;1eu6;s aseurl-aursor{l ro}de)ar

(l(rrilqe1s )l]aue6) rounl rossarddns rounl lossarddns roun] .rossarddns rounl rossarddns rounl iossarddns aua6oluo aua6oruo

seua6rredarqlletrJsrtu vNc raqlopuelHlw z'PDwS 7tltoldatatfl191 a)v

t )dv ,u1ua1o>f,

s0a-x

slla) Ja)ue) lepalolo) ul paDelac sa!l!leulouqv )lleuaD aruos z-02 alqel 'raJueJ lulJeJoloc ol uoqrsodslpard r{relrpalel{e ^\oqs }eq} serutueJaJeJesoql uI o1 sI qceoJddBJaq}ouP 'reIIrEapessnf,slpsv slsaJep3r1eua3eq] u^ op {JBJ} 'gga suouelep Jo suol] Io -elnu Surlenrlceut a^eq %0g lnoqe pu€ 'eueSocuo ue aruoJeq o1 1r Supenpce SaA-N ur uorlelnru turod JUIJadse aleq sJacueJlelsaJoloc p %0V lnoqe ]eq] uI paAIoAuIeq ol pelpaleJ spq Suruaens clteue8 Jo ed.rilsIqJ 'ereqmaslesJef,uEO palcadsns Jo u^ ou{.{pearp saua8uI saplpluJouqE JoJslleJ aq} ueeJJsol sI JeO -ueo qJeoJdde euo ol .{ra,tocsrp eq] suollelnru roJ alqlsuodsal Jo IelJeJoloc '(z-02 elqB.L) 'pegpuapr eq ol urEruersraqlo flps '(,&\olaqpessn3slp) sJacupf,uolos Jo sraqrunu ra[elus uI paAIoAuI aJe sJeqlo

u l nr l e q l r o a leulou

t l

('spreMpl;ne6;o,{sa1rno3)'ra,{e; apsnur 6ur(;repunoql epe^urol 6uluu!6eq sl lpql eurour)rp)V (g)'leulrouqe,{1p|rru s.readde dllod aql uo r-unr;aqlrda aql iurnrlaqtroa )ruolo) leuilouq]!Mpara^o) sruolol aqlJo lleMaqlJo lsaraql 'uolo) aqt aprsura>edsaql-uaunl eLll dI;od eq1'uo1o)aql uror1 olur sapnrlo.rd d,{;odsnoleuouapp uV (V)'Ja)ue) uolo) ;errd{1e;o luatudo;anepur sa6e1saql 6ulmoqssuoll)osssorl gy-97 arnbrl

raf,ue):gTraideq]r zgzL

ESZI

'Je3uB3 IelCaroloJJo uoqesnBceql ur ^Ilueu[uoJd os eJnleaJ suorlElnrucdy ftqu,uruldxapFoc paJJaSuporuord-raJuB3luepuadepur'lEuoll -1ppeqql 'epplp slleJ uaqm seplleruJouqperuosoruoJqJot 3upea1 'slJaJep alpurdsrpolgr 3o ,tcuanbar;eql esualJuro] sreaddecdygo sso.J'seFqnloJcrur Surpnlcur 'sluauodruoc IIaJ Jaqlo snorJel qll \ oqe ]nq uruetec-d eql qlllvr dpo 1ou stf,eJetut11pue a8rel sr uralord cdy aql iref,ueJ [BpeJolocug llrdpc uoruruoc lsoru eql qonru os repcqred ugaua8cdv er4lsr dqrrn'os Suraqaq; 'os op ol ll JoJa8elue pe an4qaduroJou sr eJeqlsp 'srs -aue3lrounl ol sr e ur lueuoduroJ euo rrEr.D eJoruur suoll leq],furr,rqled [BclllJc -elnru sarnbcu dlarpJ JeJueJ lenprlrpur up luql eldlJuud 1ereua8aqt qlpvrpJoJOp ur sr srr{J'suleluocde,rnqled eql l€qt aua8rossarddnsrournl ro auaSocuoa18urs due ueql reqtuJJecueosrqtJouortelllq eql roJIucrlrJf,srleql ,(emqledSurpu8rs tu1 eql q dll ltcu elrssef,xesr]l 'snql 'pea$uIuluajac-j ul suo4elnu 3u11e^pce peq uoqrodord q8rq B 'suopelnurcdy ener4roup1pluql srounl ,lral eql Suorue 'srorrrnl lelceJoloJJouopoelloJu ur pacuanbasseirnaua8errya1nc-f,ar4uer{ ,\ '(EZ-EZern8tgaas)qddrc Ipunselu eqtJo ezrspue Jeqrunu eql q eseeJJurelrsseruE pue uopplndod 1ac-uels eql 3o uorsuedxepelloJl -uoJun up ol snq] pue urualpo-flaarlJo sseoxeue ur sllnsalcdygo sso.I'tZ Jel 'elels -dnqC pessncsrp ur se llec-uels eql uptu1eru o1roleln8ar puoqducsuerl 'snalJnu E sB slJp 1l aJar{,r,r eq} ot 3ur1ur31ru ruoq uruelec-d aqt sluerrard 34y 'd.earr 'uoqepurBap srqt ur urualuc-$ Suqrqrqur ,,{.g s1r eonpur pue ,,{e,rn o1 sdlaq 'uratordurualoc-j aq] ol spurq '(9I Jel -qted aql raqloue ru11l1 lueuodruoc lI Jo -deql tn pessnrsrp)[nmqlnd 3u17nu31s tuAy eqtJoluauodruocfrolrqrqur ue aq o] rl,l,rornllvrousr uralord ody aq;'uoqplnru ro; sla8rel aq r{eur,oo1,saua8asaql luql uorlelcadxe aqt ur 'aua8 Ipcrlrro-ref,ueJrr Aou{ e ,,(qpapocue uralord aq1 qtlm lJuralul teqt suralord apocua1eq1saua8]E {ool ol uaeq seq qceordde 1ry

-esn I eqroue' papue q suoll3uryrlaur dxasE aua' .Pue-s fl -"::it#Tl Jliriy *,

-pug JoJ sta8rel FUrlnrJ aq ol rtla>11eJe pue sJecuef,letoeroloc ur sure8 Jo sessol luanba4 ^\oqs oqp saruosoruoJqo Jaqlo ;o sged cgrcadg ',furrqted dACf aql Jo slJaJJe,{-rolrqrqur-q1r,ror8aql qtolq leloruer s1rlsracuec uoloo Jo %0t lnoqe ur pelelnu sl r{JIq1!\'aua3 vpnug eql sepnlJur reqlouv edy sapnlcul luql uofar eql sr ueqlJo aug 'saua8rossarddns Jor.unlroqreq deru suolSerasoql teql slsa8 -3ns ,,{1rso3.{zoretaq ssol srqJ 'seuosoruorqc pelrJeqq ,(1pura1ed pue dleu Jo -Jelpu aqr qloq uo sacuenbas erues eql dlasrcard aleq Lpuanbe4 seruos VNC -ouoJqf, urelJeJ go suo€ar peloeles ]eql slueleJ sreJueJ leloeJoloo Jo Jeqrunu e8rel e Jo ueos cpuruals.{s V 'slleo Jorunl aql Jo uonnlole eql Surrnp parmbce sa8ueqc cqauaB ro; Surqrreas r(q urrop peIJeD eq ueo saua8 rossarddns rournl 'aruorpuds ,fuelrparaq rrivrornl ou sr alaql eJeq A uale tarlJee peureldxa sy 'JecuBc lelcaJoloJJo sluerp -ar8ur prlual perJ[uepr er{l euo sB uaeq suq euaB cdy eql Jo uoqe]nur ,Er.uo] Jo .snqJ'arulleJq rraql Surrnp -selqourtal JoJpessnf,srp a,vr ot JEIIrms alnor dq e lpqt parmbce suortetnru qBnorql auaBcdy aqt go qloq pele^lpuur anpq (slleJ sardoc .uorlrpuoJ .,fue1rperaq Ierurou Jreq] ]ou lnq) sJeJuec Jraql Jo o699ue{l eJor.ulng eql a^Eq lou op JeJuEcIplceJoloJ qlun sluaqud lso141's&(1od u8ruaq eqt ul uele 'sJournJu1 &1so3rfuorelaqJo ssol Moqs pue silec Jleql IIB ut aua? cdy aql ;o ,tdoc euo Jo suonelep Jo suonplnru Supenpceul e Bq dVC rlllm slenpprpul .eruoJp -uds aqt JaUEpaueu 'cdy aueBrossetddns Jounl eqlJo uorlplrpeul Jo uouelep p 01 peJeJt eq ueJ espesrpaql 'sread. sr JacuEJ;o srsou8ep aql or sd,tyod ZI 1o uollJelep lsJU eql ruoq arup a8erane aql lluuu8rpru eruocaq ot ssarSord s^,terup lsoru1e [u eJoru Jo euo 'pelorueJ lou eJu daql Jl pue ,aJIIlppe dyrua ur readde o] uels sddlod asaql '(9trr{7 arnt;g) uoloc aql go qr8uel aqt Suop dolanap sdri.yod spuesnoql ro speJpunq qrrqm uI ,@Vt) t1oc stsodrt1od snolaraou Jo -apa gnlpual searrpazdpue eq ot aruorpuds Jacupc plcaroloc drelrpeJeq lsJrJaqJ .eIa matpuy;o{saUno3) fspualvllew pueal1;{11 paleuaql q]!MpaMe!^uaLlM JaMoutlnet r(urte 6ul;qtuasal q)eo,(gt-02 arnbrluruotl)asut unnoqs) sd{god6ulrtalordJospalpunq,(q paranot ,{lalalduorstuolo)srsod{;od aql (g).a)e;lnsq}oouls1nq6ur1e;npun ,(;tuabe srllemuolo)leulou aql (V).uolo)leurrouqlyvrpereduo) snoleurouepe luolledl;or srsod{;od lplllue}}o uolo19g-97arn611

uolAVHtB ttl)-ulfNvf Jo stsvSuilnfttow tHI

fspreMp]lnedpuPueuqeu -lapqv lae^ ^selrno))'sa^ptua)saronu e qllM Jo uorleurquro)luoraJJrp paurels6uraqaurosorlojq)ueunq q)ea l o v N c a q l ' € [ - 0 2 e l n 6 u u t s ep a u t e l s aroMarn6r,s!ql ur sauroso[r,rorqr aql vNc ur sl)aJapIq ,o llv lredarq)leursrur paleal)uaaq6urneq'alqrsrAUr ,(;lsourare sl)ajap sll 'sarleuouPleuJosor.lorLp /naJ qllM tuaualouro) aurosouJojr.p alqets e seqleql rounl e Jo ed^lotuplaqf (B) '(uMoqslou) llar ot llal uor, lsrxaosle pue uef uorlPuen alqeraprsuoJ'ernlrnrls requnu auJosoruolq) u! sarlrleuilouqe ssor6r{ueurs,vroqs ratuer lerrdrt e;o ad,{lofte1aql (V)'&lllqelsu! )!roua6 ,o spurl luarar,lp 6ulmoqssra)ue) uolo) Jo (sad{torfue>l)slueue;dutor sruosouorq) 19-97 arnbrl

'uoPeJeJIIoJd SuUEInBeJ eJnlPaJpJaue8 P 'passnJslpLIsnoIAaJdsE 'JaAoaJoW IIeo aue8 B ur eq uBJ ll 'sJaqlo uI laua8 JIBdarq3telxslur VNC E uI eq ueJ uolleln(u 'sasBJatuos uI lBql l ou{ el 'snql :a}noJ auo ueq} arou fq asIJBUEJ lsJU aql JesupclulseJoloc esn€Jeq te,\/\sueelSuISou sI eJeqJ illeJ JaJuBf,aqlJo rol^Eqoq 'elelnur seuaSpJI IPrsoseeql ol e{Eur ueq}Jo qJBe seop uopnqlJtuof, }eq a pue -lrJJ-Jef,uEJlelJeJoloc parJlluapl Jeqlo aql pue'Jdv'ggdsoa-N op Jepro lPq ^ ul

suorlelnw)!il)ads qllM palelalto) ag ueuo ue) uolssal6or6rorunlro sda15aq1 'srte,vraldqpur ut zllqtqelsut slq] eJrnbf,eueJ sllal JaJueJ leql pue-asneJ dJolnqlJluoc P lnq rol^eqeq luEu -Srleru;o pnpordrtq pluaplJ3e uE lou sI .{l$qe}sul clleue8 teql aleJlsuouep 'qloq r(larer qcleursnu e^pceJapro dlpqelsur dlrealc s8ulpug asaql lnq-rledar 'JIeclaJqslBIUSIlu pruosoruoJqc Jeqlla Moqs seluoulsJBc Auetu 'peapul VNC ur lJaJepe s]JeUaJpue aluJs railetus qonru e uo sJnJJo.{tlTrqetsureql sreq}o aqt ur sealaq ^ 'saruosoruorqopeJalle z(lqrsrnol speal leql &lllqelsul puosoluolqJ 'sr(em oan] ]sPal lE ur parrnbce Jo ruJoJ e ,r(eldsrpsJacu€J aql Jo .,(}lJo[Bu aqJ 'snqJ aq ueJ sJacuec lelseJolos ,tueur ut punoJ dllllqelsut JIlaueS eq] 'uorlelnur SutsodsrpardpellJequl ou qll^ aldoad uI 8uu -JnJJo sJacup3Ipl3eroloc aqlJo %gI lnoqp 1rrpunoJ eJellrsauefiJIedaJqcle111sllu ur suorlelnru 'pazruSocarsem sluarlpd lldNH uI loeJapaq] acuo "''VCVf,Vl ro ' ''WW sEqcns sleedeJaplloelJnulp pue -ouolu suollceJluoo pue suolsuedxa Jo uoqs pup sepuoalJnu IEnpIAIpuI ut sa8ueqc .,(1qe1oulsolu-seurosoluoJqo aqt ut saBueqr elqlsl^ul sacnpord ,!ryqe1sur cpaua8 Jo ad,{f repcrged slq; dcueu8qeur o1 ssarSord o] sllef, Jo sauolJ nlrollpleql uollceles Iurnleu pue uot1elnru ;o sassarord pJepuels eql q8norqt paads,{.lqeurnsarduec ^ ou sllac elqe}sun r(lecqaua8 asaql'(9 raldeq3 ur pessnosrp)eJour Jo ploJpaJpunq e dq alEJuollelnru snoaueluods aql elelela itlalepaulur pa,r ,(doc aua8 pooB Sututeurar aqt Jo uone^llJeul ro ssol pluep -rJJpaq] asnPJaq'ISglp ate Spnpt^lput aseq] ,tlsnonard passnJslpSe'Ja^al oH 'sllel s,lueued aqt ul JnJJo lurll sJorra uollpJudar ygq elqetl^eul eq] alorueJ ol alqu 1ps sr rua1s.{sJlpder eq} os 'eAI}JeJapsl eue8 peAIoAuI eq} Jo sardoc oml aq] Jo euo f,luO '(OZ-E arn8tg aas) tseal. pue PIratJBq uI seua8 SnW pue 1mW 'utalsts trudat qcryw aql ol uollJunJ pue aJnlJnrls ut snoSolouoq aJe qc1q \ -sllu VNO eql Jo sluauodruoc prlual JoJapoJ leql saua8IeJaAasJo auo uI Jncoo asodstpard ]Eql suopelnru eqJ rerueo IetJaroloc ol spnprlrpq lldNH 'lL7-Ozarn8gg) puJou ueql seuosoruoJqJ aroru fueru Sur,req se 'suorteJJeqeraqlo pue 'suoqolep 'suollef,olsue4 aldrlpu qllrlt'sapleru IlalrlrsE -Jouqe leruosotuoJqc ssor8 eleq 'lserluoJ uI 'slualled CIaXH-uou uI sJolunl ]sorulB Jo) IBLuJou e anuq faql IEloaJoloc go z$rro[eru aq] ladd]odrDl (letuJou '(seurouepe) 'lensnun aJEslleJ JaJuer aql 'raAoeJoIN s&t1od lelseJolo3Jo leql q Jaqurnu aql ul esearrut -,(uelnoqlll peseeJJulsI JeJuEcuoloJ Jo &Iilqeqord aqt 'OJ1NH) )aJual lalraro1ot srsod[1oduouftnltpataq pelql'uoprpuoc sH] uI aVC roJ paqlJcsap aABqa^\ auo aql luorJ sJaJJIpslua^a Jo esJnoc eql qJIqM uI eluou -rJJeJ uolo3 o] uorlrsodsrpard .{relrpaJaqJo puDI 'uourluoJ aJolu 'puooes e SI eJeq] 'suorlelntlltdy qtp peleloosse (dVC)esuaslpfrelparaq aql ol uopppe uI

iledeuq)leus!w vNc ul st)aleca^eHsla)ue) lPpeJolo)euos

re)ue):0zraloeqf bsc.l

'sJa)ue) luararJrploj leltuilsuauo ale asue sa6upLl) asoqlq)tqMut repto lelaua6aql pue paratleareleqt sleMqted6ur;eu6rs aql qloq leln)rtredu; 'sula11ed uoulruo)auos erearaLll'a1r;e,{1}rexa aq o1pa1>adxe lou ele sJoulnlleLltos ,stselselau o1do;anap,(aqlsestor.unluolo) lenpt^tpul ur palatleaq ;;ran saua61uata11p,{ueu allq/!^'leqr areslurodrofer"u eq1'{;;etal; ua)el eq lou plnoqs]! lnq la)ue) uolo) ;o luaudo;anapaql ul slue^oumoul aqt lo auos raue palapoulsr uet6erpsrql.la)ue) premolsessar6oldlr se selelnuln))p sllaftoulnt Jo auol) 6utnlonaauo leql suorlelnuraql ale)tputol uMelpuaoq a^eq (sauup)tua^ palop)) sqted oalLll aql '])aJJa aql ure6ol qonoua uauo st aua6 lln, s1r6uua1;eAq(xoqtolnbuol:a.1.) uralordeuo Iue 6ur6ueq> 1eq1os ,(ennqled ;e>rtuaq>orq e sluasaldaruer6erp strll ut n,rol q)el 'la^al leln)elout aql le uolssel6ord rounl Jo arngeulereuebeq1 69-97 aln611

E lla) r.!oJ+ Jotlnl

z llo) ruor+ Jo[!n]

I lle) uroJl r ournl

q,

1 feanqled ut suotle]nul

y r{emqledur suo!}e}nur

^ e M q l e d€ s d , o s s o l

^emqled fl391 ur suorlelnu I e n n q l e d6 u r 1 e u 6 l s e s e u r ) - El d u r s u o t l e l n u /6 u r l e n r p e ^ e m q l e d6 u r ; e u b r s s e U - )u r s u o r l e l n u 6 u r l e n r p e [1r1rqelsur :rlauab o1 6urpealsuorlelnu.r

llli:lall|l:l &id

uor1e1{qlaulad{V q N O^ q p e M o l l o l ' u ! t e u J o J qpl e s u a p u o ]) o s u r J o J a l q e l u a qu r e s e a r ) u or s n e )l e q l s u o t l e l n ( x

) d v u r s u o r l e l n u ro u r l e ^ r l ) e u l

fdv

a

'suorlelnLu,o uanbas saf lualaJJtp q6norqr ssa.rbotd ue) sJafue)uolo) luara#rppue'alemele{ lou areaM q)tqm 1o saua6luelnul .raq1oI;urella>a.re areql lng 'raqta6o11r;uet luatudolanap louJnl pue uotletnul,{ervr aq1;o eapr parl;durs ;ereua6e saplnoldr,uel6erp -la^o strll .euou!).te) lel)atolo) e;o luaudo;anep aq16uu(;.rapun sa6ueqrr11eua6 1oetuanbes lerrdll pelsa66n5gtr-o7 arn6r3

I tlJ

z't'll)

[

'l-r]l

'(6ru2 aJntIJ ruopueJ lou sr JnJJo suorlpJalle asaql qsrqM ur JepJoeq] puB 'sursrupqJaru lplnJrued elqEsrpteql suorlE IoJluoJ ,ueu -Jalle srlaueS urclrac seJrnbeJ srsauaSoursJBc eql ol JatuEs [Bl lBparolo] ,rfueurrunsu1 -caJolos auo tuoq LJeA IIr ^ suorlBlnrx Jo 1eslsuxe eql qSnoqlp ']saJre el3.{c-llal pue srsoldodE pro^B ol pue suorlelnu puorlrppe alElnr.unJcp ol sllac JecuEc s,ry\olle uoncury $d p ssol tarlJea pessnosrp sV '(g7_1)ZaJntIC) seruourJJeo ur uotu (ilr]s -r.uoJ sdf1od ur aJErare ,taql se Ja]el atuoJ tgd ur suollu]nlu SurlE^rlJeur lnq pue aua8 JosseJddnsJorunl eql sso.I 'unlpJlsqns e ot aBeJoqJuElno tpeurs Jo -qtlm etereJrlord o1 drruqe aqt sE qJns 'slleo peurJoJsuen Jo sernteeJ 1ectdir aql 1v\oqsa8Els slql le sllaf, eruourJtpo IppaJoloJ peJnllnC 'uJe]]ed pcrSoyolsrq pue uorlBrluaJeJJrpIIaJ ur secueqJnlsrp ^ oqs lEqt seuo JaSJEIur uoruruoc ]nq sd,{lod IIErus ur aJ€JeJe,,taql se talel rnrco euaS sr2u-) eq] uI suorlelnu Surle^rlJv 'sdals relel eql e^rJp ot pepeeu eJe ^aql aJUIS 'uorssarSord Jorunl ur dlJee esrJu o] osp dlaTII aJe dlqrqelsur cqauaSrda pue rrleua8 01 p€al teq] se8ueqc palrJequl 'srorunt lueuS{el.u aBJBI ur se sd,{lod u8ruaq q8rq uI ,,tcuanbar; elu€s aq} te pelJelap aJe ,ri.aq1se llE(xs (lsJg ar{} aq o} Jeedde aue? 'da1s[1rea .,fuane JdV erll Surlenrlceur suorl 1sea1]p Jo -elnu 'sasEJ ur (snqJ 'JapJoJrlsrJelJeJpqJp ur JnJJo daql pue teouPJ IEI lsour ,ssslequa^aN -caJolor ur uourruoJ .{lrepcqred aJEsuorlelntu Jo sles ureuac 'saua8 ]ueJegrp ur suorlelnru q8norqt 'sder,rgortapen e ur esrJeupJ dllBtxJouqP elBJaJrloJdo1,tcuapual e Jo dlrlrqElsur srleuaS sp qlns PaseeJ)ur +o uorlrsrnbre

srselseleu.l p u e u o r s P l u r-

-l

e u l o u l ) l e )-

tl

"1o;0" "nf,::'" T"'ffi:l,1H, T sroSsaJoons rounl rsqlo puP tpeus +o ssol

unrlaqlloe )rNeldrodl{q

[rJnrlaq]rda leurJou

-t

I

s P U) + o u o r l P ^l l e

sszt

)dv + os s o l

uolleralle

uotAVHlS ttt)-ul)Nv) lo stsvSuiln)ltow tHI

'papaau aq ol enulluos sluatulPen JasuPcpue uoluluoJ eq ol anul] III1V\ 'etuoJ ol sJeel{l{ueu JoJ '.snql 'eAIpeJJe -uoc IIro\ asPesrpluBuSlplu uMolq-ilru dpca;rad aq Ja^eu uec .{aq} lnq 'a1EJqleep recuec aqt Sulcnpar ro3 slcadsord aterpeuur ]soru aq] rago ,{lqeqord aldrsag ut sa8ueqc Q}lm SuoP spoqlau Suruaarcs ur sacuelpv 'stsou8etp asnard eJolu pue JellJEe q8norqt luaruleall relnJalolu pup uouualard ra11aqroJ sallrunuoddo,ueu elEeJJol astruord sr{.esse e^Illsues ,tUBtH 'sJeJuec IeJIAJaJJoJl Ps el su 'pazlselsEleru aleq daql eroJaq peloruer eq ueJ qJIq {'srorunl ztrerulrd lerus roy Sutuaarcs r(q pnq aql uI peddp 'Suolorus Sutptone dq .{lprcadsa 'palualard aq eq uego uEOsreouec 'Janoatotr41 uer sJaJueJdueru taqrua passnJslp se 'pue 'eJnJ ueql ratlaq srterurpsl uopual -eJd 'luerulpeJl pue 'stsouBetp'uotluanald :slalel eeJql 1e eseeslpaq] uo {celle rno uadreqs o1raouec;o r{Sololqaq}Jo Surpuelsrapun Suntor8 rno rtldde UBJa A

]UNTNICNV1N]S]Ud:INIWIV]UIU]f NV) 'palpaalq aq ual JaouDJlswa&o sasualap 'tlpalnadat Jno rpnlm u1 sfom to nqtunu parywll o [1uo s! alaqt wtll &u!tsa88ns panlunocua ata saBuaqclo sad& atuzs aIU to fuoru 'ssaplqJanaNLncco saBunqc pailraLlul asaL7Lpnlm ut [om uopuur aqt Suucadat lacuur lo ad& aruDSaqi LUlm vualwd ruatatllp m uanapuo ncuoc to sadrQryatatlry u1punot ato saBuaqc4rcua81da puo suotpntu lo suotloutqutoc ruatatllp mg 'a1duuxapoo$Japun-lsaqaLllSutptrrotd raiuox uoloc r4i.tm'sauaB rcssatddnsnunl 4ficads alollryrau! pua sauaSocuotyfnads aruapo ruqt suolivJnu ql1muotssatSotdtounlto sdalsaql aruIarJoJualto uat a14 Lacuoc1suru&osn &ulpatotd ut s1stamr,padasatqlto qcL)apiualavpunt moq 'an)ra Sutptlsnly 'sraruar uaunr4 tsour ur uncco os1o[omryad qg atll to uolwnllJaul -lt1otd o1anultuor puo stsoldodoadacsaoi sIIaJpaSouap ,[11o4lauaB smollv 11sa'sno 'tnmqlad -nBuop r(Tlarcadsa s1'uacual uounq snoauntuods77afytaau u! stnlJo t6uqm ggd aql lo uortonuluu! to ssol'sassartsIIaJ Jaqlo pua a&nutopVNQ oi asuodsatu1 sts -oidodarc $atn a1ct(c-11ac sarnpur[lpnatou 14xn4m'unlordggd ary puD'uo$mrp IIaJ uo a4oJqo sDspa [11autou LlJulmlularcJdqg aLUryqltlu! pua purq sulaptd luJrnaqJ 'sauaB nuny autos rcssatddns lo vcnpotd arp llqrqul ruql suraptd Sulpocuafq ncuoc VNo to gatudo1anap aqt apwoJd uac sasnnnawo1pdndsa qrns sasnnnrowru 'umouTun 'awouaB [1aBto1IIUSarn azszisz]aru oi sJaruzc moIID lDlll saBuoqcJDInraIou aq1 aql to fryBalw aL!] utztulatu d1aq,,'sauaBaJuoualulow VNo,, so pazuoBalac[1asooy 'sauaBnt4tg 'arp Jo 'alalluatafirp 'ap!(t!p 'motB s1.lac uaqm aw1n&atwql s,tamrgad IoJiuoJ Tnnosaql to vuauodutoc nt apoc munc u parulnu sauaBnssatddns nunj 'sauaB pua sauaSocuoaql to [uo1,41 laluuc-JaJuar to nqwnu aBta1o lo suorlcunl aq1 pua sofuqua Sutdo1anap to salpng IDanarol pad1atla(toll arnu pataautBuart11oulauaB

(reurun5 'saseeslpsnollJaJul qlul s1uarledro; sursrue8roorllru Jo uolleJguuepl aq] sel\ se JaJrrEJJo luaruleer1 puu stsoufietp aql JoJ]Irepodurr se aq o1arrorduoos 'luaqed ualr€ Lue;o slles JoIuru eql uI deru uorleruroJur qJns ']xeu ssncslp aA sV 'pele]nru '.pe]alep'pagldrue are saua8 suslueqJau crlauaSlda l(q paleln8arstu Jo 'aldrcutrd u1 qcrqa,r,{lastcardlno pug o} sloot aql sapnord ,i8o1orqJelncaloru 'luaruleorl ol sasuodseJ]ueJeJJIpo1 puods -aJJoJuago sa3ueraJJlpcpaua8 esaql pue 'suollelntu Jo suoqeulquoc luaJeJJIp e6eq sluerled luara;;rq 'sauafi rossarddns Jolunl pue sauaSocuo-olord IIa1SDI 'aldruexa rcJ'olutou Jaqlo alg lseel le pue 'cfyg 'qV 'ggd uI suouelnru spug euo -rual llac-na&rs se umou{ raJueJ 3un1 go urJoJ eqt uI 'suolsal cpaua8 3o -'(erre 'saJn]eal uotuluo3 auros altdsap rrmo Slr [q pazualceJeqc eq ilI A JapJoslp qcea 'ct1q1d :sJepJoslpJo uoucelloJ snoauaSoralaqE sI Jaluec 1o adrir a13utse uarrg -rurs sr sJeJueoJoUOIIPOIJISSEIS leuoplpeJl eql TecuBcIElJaJoloJAq pelEJlsnlll sV

suo!sa'l )llousg ro ^erlv uMo sll {q pezlral)ereq) sl ra)ue) to ase) q)ef re)ue):93 taideq3

gSZl

CANCER TREATMENT PRESENT ANDFUTURE

TheSearchfor CancerCuresls Difficultbut Not Hopeless The difficulty of curing a cancer is similar to the difficulty of getting rid of weeds. Cancer cells can be removed surgically or destroyedwith toxic chemicals or radiation, but it is hard to eradicate every single one of them. Surgery can rarely ferret out every metastasis, and treatments that kill cancer cells are generally toxic to normal cells as well. In addition, as described earlier,whereas the great majority of the cancer cells can often be killed by irradiation or chemotherapy, the small population of slowly dividing cancer stem cells may be harder to eliminate in these ways; if even a few cancer stem cells remain, they can regenerate the tumor. Moreover, unlike normal cells, cancer cells mutate rapidly and will often evolve resistanceto the poisons and irradiation used against them. In spite of these difficulties, effective cures using anticancer drugs (alone or in combination with other treatments) have already been found for some formerly highly lethal cancers, including Hodgkin's lymphoma, testicular cancer, choriocarcinoma, and some leukemias and other cancers of childhood. Even for types of cancer where a cure at present seemsbeyond our reach, there are treatments that will prolong life or at least relieve distress.But what prospect is there of doing better and finding cures for the most common forms of cancer, which still cause great suffering and so many tragic deaths?

TraditionalTherapies Exploitthe GeneticInstabilityand Lossof Cell-Cycle Checkpoint Responses in CancerCells Anticancer therapies need to take advantage of some molecular abnormality of cancer cells that distinguishes them from normal cells. One such property is genetic instabiliry caused by an abnormality in chromosome maintenance, cell cycle checkpoints, or DNA repair. Remarkably, most current cancer therapies work by exploiting these abnormalities, although this was not knor,r,'nby the scientists who developed the treatments. Most anticancer drugs and ionizing radiation damage DNA. They preferentially kill certain kinds of cancer cells because these mutant cells have a diminished abilityto survive the damage.Normal cells treated with radiation, for example, arrest their cell cycle until they have repaired the damage to their DNA. This cell-cycle arrest is an example of a cellcycle checkpoint responsediscussed in Chapter 17. Cancer cells generally have defectsin many of these checkpoint responsesand often continue to divide after irradiation; this causesmany to die after a few days becauseof the severegenetic damage they experience. Unfortunately, while some of the molecular defects present in cancer cells enhance their sensitivity to cytotoxic agents, others increase their resistance. Some of the cell death that DNA damage induces, for example, occurs by apoptosis, and cancer cells often acquire defects in the control systems that activate apoptosis in responseto such damage.For example,as we discussedearlier,DNA damage induced by anticancer drugs or irradiation normally activates p53, which can then trigger apoptosis. Thus the inactivation of the p53 pathway that occurs in most cancers makes certain types of cancer cells less sensitiveto these agents. Cancer cells vary widely in their response to various treatments, probably reflecting the particular kinds of defects they have in DNA repair, cell-cycle checkpoints, and the control of apoptosis.

NewDrugsCanExploitthe Specific Causeof a Tumor'sGenetic Instability As we become increasingly able to pinpoint the specific alterations in cancer cells that make them different from their normal neighbors, we can use this knowledge to design therapies that kill the cells in the cancer without killing normal cells. One of the characteristics of cancer cells is their genetic instability; as we have explained, this is one of the features that helps them to evolve

1257

1258

Chaoter20: Cancer

and proliferate dangerously.But it is at the same time a defect-a vulnerability that we can exploit to kill them. We have seen that cancer cells are forced to walk a difficult line as they evolve toward metastasis:they need to have a defect in their DNA maintenance processesthat is severeenough to allow them to accumulate new mutations at a significantly increased rate, but not so severethat they kill themselves by frequent loss or genes necessaryfor cell survival. Since there are hundreds of different genes required to maintain DNA sequencesand chromosome structures with high fidelity (discussedin Chapters 4 and 5), we would expect there to be at least dozens of different ways for a particular tumor cell to acquire its genetic instability. Moreover, these ways should be mutually exclusive: once a cell has become genetically unstable to a modest extent, it is likely to increase its risk of death if it inactivates additional DNA maintenance genes.Those cells that do so will die and be lost from the tumor population. Detailed studies of the mechanisms for DNA maintenance discussed in Chapter 5 reveal a surprising amount of apparent redundancy, with multiple pathways for repairing each type of DNA damage.Thus, knocking out a particular pathway for DNA repair is generally less disastrous than one might expect, because alternate repair pathways exist.We have seen, for example, how stalled DNA replication forks can arise when the fork encounters a single-strand break in a template strand, and how cells avoid the disaster that would otherwise result. First, they have machinery to escape the problem by directly repairing single-strand breaks; and then, if that fails, they can repair stalled forks by homologous recombination (seeFigure 5-53). Supposethat the cells in a particular cancer have become genetically unstable by acquiring a mutation that reduces their ability to repair stalled forks by homologous recombination. Might it be possible to eradicate that cancer by treating it with a drug that inhibits the repair of single-strand breaks, thereby greatly increasing the number of forks that stall?The consequenceswould be expected to be harmless for normal cells, which can repair stalled forks, but lethal for the cancer cells, which cannot. lVhile such a possibility might seem too good to be true, precisely this strategy appears to work to kill the cells in cancers that have inactivated both of their Brcal or Brca2 Iumor suppressor genes.As described in Chapter 5, Brca2 is an accessory protein that interacts with the Rad5l protein (the RecA analog in humans) in the initiation of general recombination events.Brca2 is another protein that is also required for this repair process. Like Rb, Brcal and Brca2 were discovered as mutations that predispose humans to cancer-in this case, cancers of the breast and ovaries (though unlike Rb, they seem to be involved in only a small proportion of such cancers).Individuals who inherit one mutant copy of Brcal or Brca2 develop tumors that have inactivated the second copy of the same gene, presumably because this change makes the cells genetically unstable and speedstumor progression. Drugs that inhibit an enz)rynecalled PARP (poly ADP-ribose poll'rnerase) have a dramatic effect on cells of such tumors, killing them with enormous selectivity.This is attributed to the fact that PARPis required for the repair of single-strand breaks in DNA. Perhaps surprisingly, PARPinhibition has very little effect on normal cells; in fact, mice that have been engineered to lack PARPlthe major PARPfamily member involved in DNA repair-remain healthy under laboratory conditions. This result suggeststhat, while the repair pathway requiring PARPprovides a first line of defense against breaks in a DNA strand, these breaks can easily be repaired by a genetic recombination pathway in normal cells (Figure 20-50). In contrast, tumor cells that have acquired their genetic instability by the loss of Brcal or Brca2 have lost this second line of defense,and they are therefore uniquely sensitive to PARPinhibitors (that is, they are missing repair pathway 2 in Figure 20-50). PARPinhibition is still only under trial as a cancer treatment in humans, and is likely to be applicable to only a small proportion of cancer cases.But it exemplifies the type of rational, highly selective approach to cancer therapy that is beginning to be possible, and thus it offers hope for many other cancers. To extend this approach widely, we will need new tools for determining the precise

12s9

CANCER TREATMENT: PRESENT AND FUTURE

.

N O R M A LC E L L

T U M O RC E L L

DNAreplication

DNAreplication

al

I occasronal accident I

a

a I occasional t"'^'"""' V

a reparr pathway 1

')

(,:

reparr pathway 1

reparr pathway 2

,.^ continues

.-^ continues

NORMALCELLTREATED WITH INHIBITOR OF PATHWAYI

continues WITH TUMORCELLTREATED INHIBITOR OF PATHWAY1

occasional l/y'- accident

a

a reparr pathway 2

occasional qr: accident

a

a

(,'' ?

ttoro

replication

n o D N A r e p a i r ,c e l ld i e s with fragmented chromosomes

cause of the genetic instability in individual tumors, as well as the development of many more drugs that target alternate DNA repair pathways.

More GeneticInstabilityHelpsCancers BecomeProgressively Resistant to Therapies Genetic instability itself can be both good and bad for anticancer therapy. Although it seems to provide an Achilles' heel that therapies can exploit, it can also make eradicating cancer more difficult. An abnormally high mutation rate tends to make cancer cell populations heterogeneous,which may make it difficult to kill off the entire population with a single type of treatment. Moreover, it allows many cancers to evolve resistance to therapeutic drugs at an alarming rate. To make matters worse, cells that are exposed to one anticancer drug often develop a resistancenot only to that drug but also to other drugs to which they have never been exposed.This phenomenon of multidrug resistance frequently correlateswith amplification of a part of the genome that contains a gene called Mdrl.This gene encodes a plasma-membrane-bound transport ATPaseof the ABC transporter superfamily (discussedin Chapter ll), which pumps lipophilic drugs out of the cell. The overproduction of this protein (or some of its other family members) by a cancer cell can prevent the intracellular accumulation of

Figure20-50 How a tumor'sgenetic instability can be exploited for cancer therapy.As explainedin Chapter5, the is so maintenance of DNAsequences criticalfor life that cellshaveevolved multiplepathwaysfor repairingDNA damageand avoidingDNAreplication forkwill a replication errors.As illustrated, a breakin a stallwheneverit encounters DNAtemplatestrand.In this example, normalcellshavetwo differentrepair pathwaysthat canfix the problemand therebypreventa mutationfrom arising DNAsequences. in the newlysynthesized Theyare thereforenot harmedby treatmentwith a drug that blocksrepair pathway1. ln contrast, of the inactivation repairpathway2 was selectedfor in the it evolutionof the tumor cells(because unstable). madethem genetically onlythe tumor cellsare Consequently, killedby a drug treatmentthat blocks repairpathway1. lf a treatedcelldoes its daughterswill not die by apoptosis, die becausethey inheritfragmented, incompletechromosomesets. ln the actualcasethat underliesthis example,the moregeneralschematic functionof repairpathway1 (requiring proteindiscussed in the text)is the PARP breaksthat occurin to removeaccidental a DNAsinglestrandbeforethey are by a movingreplication encountered fork,and pathway2 is the recombinationfor repairingstalled dependentprocess in Figure5-53 forksillustrated replication (requiring the Brca2and Brcal proteins; for details,seeH.E.Bryantet al.,Nature and H. Farmeret al., 434:913-916,2005 Nature 434:917-921, 2005.\

1260

Chapter 20: Cancer

many cytotoxic drugs, making the cell insensitive to them. The amplification of other types of genes can likewise give the cancer cell a selective advantage.The gene that encodes the enzyme dihydrofolate reductase (DHFR), for example, can become amplified in cancer cells treated with the anticancer drug methotrexate.Methotrexate binds to and inhibits the ability of DHFR to bind folic acid, and the amplification greatly increasesthe amount of enzyme, reducing the cells' sensitivity to the drug.

NewTherapies AreEmergingfrom Our Knowledge of Cancer Bi o l o g y Our growing understanding of cancer cell biology and tumor progression is gradually leading to better methods for treating the disease,and not only by targeting defects in cell-cycle checkpoints and DNA repair processes.As an example, estrogen receptor antagonists (such as tamoxifen) and drugs that block estrogen synthesis are widely used to prevent or delay recurrence of breast cancers that have been screened and found to express estrogen receptors. These antiestrogen treatments are also being tested for their ability to prevent the development of new breast cancers.These drugs do not directly kill the tumor cells, but instead prevent estrogen from promoting their proliferation. The greatesthopes lie, however,in finding more powerful and selectiveways to exterminate cancer cells directly. A variety of adventurous new ways to attack tumor cells have been shown to work in model systems-t)?ically reducing or preventing the growth of human tumors transplanted into immunodeficient mice. Many of these strategieswill turn out to be of no medical use,becausethey do not work in humans, have bad side effects, or are simply too difficult to implement. But some have turned out to be highly successfulin the clinic. One strategy depends on the reliance of some cancer cells on a particular hlperactive protein they produce, a phenomenon known as oncogeneaddiction. Blocking the activity of the protein may be an effective means of treating the cancer if it does not unduly damage normal tissues. About 25% of breast cancers, for example, expressunusually high levels of the Her2 protein, a receptor tyrosine kinase related to the EGF receptor that plays a part in the normal development of mammary epithelium. Monoclonal antibodies that inhibit Her2 function slow the growth of breast tumors in humans that overexpressHer2, and they are now an approved therapy for these cancers. A similar approach uses antibodies to deliver toxic molecules directly to the cancer cells.Antibodies against proteins like Her2 that are abundant on the cancer cell surface can be armed with a toxin or made to carry an enzyme that cleavesa harmless'prodrug' and converts this into a toxic molecule. In the latter case, one molecule of enzyme can then generate a large number of toxic molecules on the surface of a tumor cell; these molecules can also diffuse to neighboring tumor cells, increasing the odds that they too will be killed, even if the antibody did not bind to them directly. Antibodies are hard to make in large amounts, are very expensiveto produce and buy, and must be given by injection. The ultimate goal in cancer therapy is to develop small molecules that kill cancer cells specifically.The PARPinhibitors discussedabove are one example; but to tackle the majority of cancerswith simple drug treatments, we shall need a large collection of different small molecules, tailored to the many different types of cancer.

SmallMolecules CanBeDesigned to InhibitSpecific Oncogenic Proteins As our knowledge of specific molecules involved in the genesis of particular cancers builds, there is an increasing effort to devise targeted therapies directed against the oncogenic proteins that are essential for a cancer cell to survive and proliferate-thereby exploiting the phenomenon of oncogene

CANCERTREATMENT: PRESENTAND FUTURE

1261

addiction mentioned previously. A particular dramatic successof this type has raised high hopes for the general utility of such targeted therapies in the future. As we saw earlier, chronic myelogenous leukemia (CML) is usually associated with a particular chromosomal translocation, visible as the Philadelphia chromosome (seeFigure 20-5). This is the consequence of chromosome breakage and rejoining at the sites of two specific genes, calledAbl and Bcr The fusion of these genescreatesa hybrid gene that codes for a chimeric protein called BcrAbl, consisting of the N-terminal fragment of Bcr fused to the C-terminal portion of Abl (Figure 20-51). Abl is a tyrosine kinase involved in cell signaling. The substitution of the Bcr fragment for the normal N-terminus of Abl makes it hyperactive, so that it stimulates inappropriate proliferation of the hemopoietic precursor cells that contain it and prevents these cells from dying by apoptosiswhich many of them would normally do. As a result, excessivenumbers of white blood cells accumulate in the bloodstream, producing CML. The chimeric Bcr-Abl protein is an obvious target for therapeutic attack. Searchesfor synthetic drug molecules that can inhibit the activity of tyrosine kinases discovered one, called Gleevec, that blocks Bcr-Abl (Figure 20-52). VVhenthe drug was first given to patients with CML, nearly all of them showed a dramatic response, with an apparent disappearance of the cells carrying the Philadelphia chromosome in over B0% of patients. The response appears relatively durable: after years of continuous treatment, many patients have not progressed to later stages of the disease-although Gleevec-resistant cancers emerge with a probability of at least 5% per year. Results are not so good for those patients who have already progressed to the acute phase of myeloid leukemia, known as blast crisis, where genetic instability has set in and the march of the diseaseis far more rapid. These patients show a response at first and then relapse because the cancer cells develop a resistanceto Gleevec.This resistanceis usually associatedwith secondary mutations in the part of the Bcr-Abl gene that encodes the kinase domain, disrupting the ability of Gleevec to bind to Bcr-Abl kinase. Second-generation inhibitors that function effectively against these Gleevec-resistantmutants have now been developed. Ultimately a cocktail of multiple agentsthat cooperatively block BcrAbl action may provide the key to successfultreatment, by preventing the selection for drug-resistant cancer cells at all stagesofthe disease. Despite the complications with resistance, the extraordinary success of Gleevec for the patients in the chronic (early) stage of the diseaseis enough to prove the principle: once we understand precisely what genetic lesions have occurred in a cancer,we can begin to design effective rational methods to treat it. This successstory has fueled efforts to identify small-molecule inhibitors for other oncogenic protein kinases that could be effective targets for new anticancer drugs. A second example of such a therapy is provided by a smallmolecule inhibitor of the EGF receptor, currently approved for the treatment of some lung cancers. 8 c r g e n eo n c h r o m o s o m e

A b l g e n e o n c h r o m o s o m e9 q

J

A

breakpoint

b r e ak p o in t

T R A N Soc L ATION

5'

I

3

fused BcrlAblgene

fused BcrlAblmRNA

I TRANSLATION

I

V B c r - A bfl u s i o nP r o t e i n

Figure20-51 The conversionoftheAb/ proto-oncogeneinto an oncogenein patientswith chronicmyelogenous leukemia.Thechromosometranslocation joinsthe Bcrgeneon responsible chromosome22tothe Abl genefrom chromosome9, therebygeneratinga chromosome(seeFigure Philadelphia 20-5).The resultingfusionproteinhasthe N-terminus of the Bcrproteinjoinedto the of the Abl tyrosineprotein C-terminus the Abl kinase kinase;in consequence, active, domainbecomesinappropriately driving excessiveproliferationof a clone of hemoooieticcellsin the bone marrow.

1262

Chapter20:Cancer

(A)

(c) BCR-ABL ACTIVE

Bcr-Abl

ADP

B C R - A BB LL O C K EW D I T HG L E E V E C substrateprotein +

no signal

+

NO LEUKEMIA

Figure20-52 How Gleevecblocksthe activity of Bcr-Ablprotein and halts chronic myeloid leukemia, (A)Thechemicalstructureof Gleevec(imatinib). Thedrug can be givenby mouth;it hassideeffectsbut they are usuallyquite tolerable.(B)The structureof the complexof Gleevec(solidgreenobject)with the tyrosinekinasedomainof the Abl protein(ribbondiagram), asdeterminedby X-raycrystallography, (C)Gleevecsitsin the ATP-binding pocketof the tyrosinekinasedomainof Bcr-Abland therebyprevents Bcr-Ablfrom transferring a phosphategroupfrom ATPonto a tyrosineresiduein a substrateprotein.This (8,fromT.Schindler blocksonwardtransmission of a signalfor cellproliferation and survival. et al.,Science 289:1938-1942, 2000.With permissionfrom AAAS.)

Tumor BloodVesselsAre LogicalTargetsfor CancerTherapy Another approach to destroying tumors does not directly target cancer cells at all but instead targets the blood vesselson which large tumors depend. As discussed earlier, the growth of these vesselsrequires angiogenic signals such as VEGE which, in animal models at least, can be blocked to prevent further tumor growth. Moreover, endothelial cells in the process of forming new vessels expressdistinctive cell-surface markers, which might provide an opportunity to attack developing tumor vessels specifically, without harming existing blood vesselsin normal tissues.Clinical trials with various inhibitors of angiogenic signals are now taking place, and severaldrugs that inhibit thevEGF receptor have recently been approved for treating kidney cancer. similarly, a monoclonal antibody againstVEGF has been approved for treatment of colon cancer in combination with chemotherapy, even though the added benefit is only modest.

Many CancersMay BeTreatableby Enhancingthe lmmune ResponseAgainsta SpecificTumor In recent years,we have realized that the body's normal immune responseshelp to protect us against cancer. Mice lacking important parts of their immune system have elevated levels of several types of cancer. Similarly, humans who are

's6nlpqroq ol luelsrsar areleql sauol)llo) tuelnur;o uort)alesaqt iol Molleuauo ^lletluanbas ua^t6ale ]eql sluautleaJt6nlp al6urs/alqelnulladlLl ale slle)lounl asne)aB'Ideraqgraruet toJ sluau,tleatl ;egluanbasueql a^ll)a1Joaror,uaq {eu sluaulearl 6nrp11;nuIq14 gg-gg arn6r3

(8) I puP v ol luelslsal lla) = v o l l u P l s r s alrl a ) = I p u e V o l e ^ l l l s u a sl l a ) =

s w

c3un) ur)NV) r,Jl,:10,,u;:l;J

tt li

:^l)

I puev s9nuc Hl_o8 HTIMIN]IA.LV]UI snolNV-L']nNts

(v) JOUrnlsuJJot 'g 6n.rpo1 luelsrsat 'lla) luelnuraleJ

s9nuc HIoS 01 INVI-SIS]U U])NV) t'l8v'l'loulNo)Nn

loulnl sutJoJ lue$lsaJ

a&l-

lnurerer

tr$$h

ffi w

@

rffi

t

i

I V 9NUC Hl_tA -LVIUt

8 DNUO Hl_rAA IVSUi

'sJaJuec ^,tueu rog pau8rsap aq ueJ s8nrp aldrlpur qlp{ lueruleall snoauellnurs Suhlolur sluauleaJl eArtJaJJeleql fla)H dlSursearcur seuo3aq Jr 'sesearf,ur sllec JaJUEJ lsurc8e s8nrp alqrssod Jo tueureruJe Jno sV '(tg-02 aJntIC) asrJe IIu eruelsrsal alqnop qrlm sller teq] l|le{ll tl SuDIeurfqaraql 'sraqrunu aBJEIol d1dr11nuro] Snrp lsrg eql ol tuel -srseJsllac ivral eq] .,t,rolleUEJ sluauleeJl Snrp pquanbas ,1ser1uocuI .]ualaJJrp aJp leql s8nrp o.r4 ol luelsrsal IIaJ ou eq pFor{s ereql .}uau}eaJ} SnJp auo dup o] luelsrsal suoqepdod ler]rur eql ur slleo eruos aq sdertp ,{eur araqt speJaq ^ :srolrqrqu asealord lueraJJrp aaJql Jo IrpDIf,of, e qllv\ SOIV-AIH Jo tuaruleer] luaJJnJ eql purqeq leql se erues eq] sr cr3o1aql i{.ysnoaueqnrurss8nrp arour ro oml ql!v\ stuarled Surlearl fq paluazrard eq uec uralqord slql ,saseceruos uI 'a1era;r1ord ol Surnurluoc [q rounl eq] aleerJal l.lunluana sllal asaq] pup '(srotunl 's11acurals JaJuEJaq] Jo ^\erl teq] ol Surprocre Jo etuos) uorlepdod IIeJ rorunt pu€rro aql ur sllef, tuelnru Jo uouceq duq e page ol palluJ dlqeruns -ard seq lueuleeJl Snrp pqlur aq; 'SursudJnslou sl slql 'sllao Jorunt Jo eJnluu alqelnurraddq aql ualrg 'lnJssacJns os lsJg le sem leql SnJp aql ol ]uelslseJ sr Jaouec aql JaIBI srea.,tro sqluou 1ng .read ]Bql uJoJ peJelle ue ur readduar III,vr ,]uarulea4 Snrp pqrur -desrp o1 Surtuaas slleJ Jorun] alqe]f,elep aql Jo IIp qll,r,r ue.,(q azrs ur pacnpar ^,(lecrleuerp eq upc sJaJueJauros ,paqucsap dpearp sy

{delaql ta)ue) roJsa6e}ue^pvlettuolod seHr(lsnoauellnutss6n.r6lela^osr{l!Ms}uelledOut}eall 'anssrlu8rarog araru.daqt sE peleururle eq 01 sllaf, JeJueJ asaq] ^ ollp ruv\ sHl lEqt sr adoq aql I 'sJotunl crycads uo {f,enp aq}.{Jrsualur ol asuodsar eunturur aql e}epdrueru ol rroq Surureal are qs€oloJuo '(gZ pue VZsraldeq3 aas).,!rumuur eleuur pup a^p -depe qloqSo srusrueqJeurxalduoc aq];o SurpuelsJapunpesee.rcurJno q8norql :ala^ oH 'Journl aq] yo ,$rcrua8rlue eqt af,npal leqt sa8ueql elqetrJaq apnlJur o1lqSnoql sr uorssarSordrounl;o ssacord aql pue ,acuelrarrrnsounrururJorunl leaJap ueJ {cE}}B eunrurur o1 arnsodxa peJnpal e qlp sllal Journ} luerJp^ 'uaql spJeMol eJuPra -1o1dolanap ol dlrunlroddo aql peq Jeleu suq rualsr{.saunrurur eqj leql os ,ureJq eql sE qJns eaJe pa8apnrrd l.lpcrSolounruur Jeqloue ur Jo sofrqua ur fpo passardxa.,tlpurou are daqt asnpJeq asuodsar eunrururolnE uE aIoAoJd sural -ord asaql leql alqrssodsr 1r'r(lanrleuJatlv'sllal Jorunl repc4rud aql ut slunourp a8rel .{lpnsnun ur passardxa are daq} esnecaq uals^s aunurrur aq} Jo uor1ua}le aql 1JeJ4E,{eru sasec asaql ur pazruSocar ale leql sua8rlue-;yasaqJ .sJorun} pllos ureueJ Jo saler raq8rq ploJ-aalql o] -oml a^eq pasruordruocounrurul

EgZL

]UN]NJONVIN:IS]Ud:IN]WIV]UIU])NV)

eJoJeqeq tl ilF 3uo1rnrog 'srsoldode q8no;qr epIJIns ]FuruoJ ol e{BIu sllal leql uorsroap auou-ro-ile eq] allJapun ]Eql sassaJordrepcalour pellelep aqt;o 3ur -pu€lsJepun radaap qonlu e paulelqo aAEqe^Asrear(luacar ur 'aldruexaeuo elel oJ 'lueuruur sr saseasrp;o dnor8 slql Jo lueuleeJl aqt u1 ssarSordro[eur 1eq1 'Sutpuulsrapun Lupol rtlrumuruoo qoJeesalJaJuecaql q asuesIBerE sI aJeql pue 'seld ur luerualoJdur SuqeralerJe ue s8utrq a8paprnoulyouollelnunf,Je aql lng -pJeql Jeoueoelr]oeJJaolut palPISuBJluaaq seq JecuPJ;o e8papr,rou)peseerJul ,(pear8 Jno qclq a le aced ^ ols eql qllm uolleJlsnrJ elqepuelsJepun ssardxa ,{lpuorsecco sdnor8 asaqJ 'qf,JeeseJIeJlpauolq sISEq uI lue(u}selur cqqnd JeJueJ roJ selef,olpe luelrodut uaaq 3uo1eAPqselru€J Jleqt pue sJoAIAJns 'esl^ Jeqlo-uaq] 'ge; z(1aleruqF IIIrv\sarderaq] Jno JIaq}Jo slseq J€lnoeloru pulJ ol paeu al ztletcnrc lsotu ,,:ssautue}s,, 11qo1 s.{err,r 'uleql elelosl eq] pup scllslJalJeJeqcptcads Jleql auluJelap uec alt,rleql os 'asea Jallaq peau arr 'sSurql raqlo Suoruy pue slleJ ruels JaJuPseulJap ol sr(e,r,r -srp pcrSololq-iler pqlal slqt ranbuoc ueJ e ^ aroJaq raldeqo slqt q peqlrrsap sassacord aql Jo 1e ,r(1reau;o SurpuelsJepun J3]1eq qJnu u paau ilIls e^ pue 'sJacu€J ueunq uorutuoJ ]soru aql Sutpuelsrapun d1n; ruo4 JeJIIIIs aJEa^ 'sluarulperl 'pa1a8re1Llastlard esllep o1 alqtssod aq o1Sutuu€aq st ]ng IBuoIIEJ 'suoucunJ ll IpruJou JIaq] pue sauaBpcrltrf,-racuec dueru;o seppuapl aql lrlrou>I a,vrasnBlag 'sellrleurouqe ctlaua8 ,(a1 auros aleqs eseeslp eqr Jo suro; ,tueu 'lle teue 'eJu eraq] leql pulJ ol Sur8ernocua ]pq] pue saldrcuud praua8 auros uaaq seq tI 'JaJuBJJo srseqJelnJelotu aql o] sanls JoJ{Jep aqt uI SuldorS;o era ue ol pua ue lq8norq saua8 peplrJ-racueJ Jo Jeqrunu a8rel e;o zfua,tocstpaql 'luaued IEnpIAIpuI aql ol ,(lalerncce eJolu qcnru ,(deraql Jollel o1 sn Suqqeua .{q }uaru}earl JeJuEJ tuJoJSuPJlo} sasttuord lef,uec ;o sts^,(puuJEInJelo{u aqJ

oc ol atow Ll)nwlllts sl eraql 'sluaruleaJ] Surpm8 JoJ eJnlnJ sr }l }nq 'sJaJuBJ}sour ro; padola,rapSuraq eql uI esn peardsapurrpug ol .{1a>1U 'alrlrJns pue ^,(deraql sr Suqr;ord uotssardxa aueg 'lou op sJeqlo sealaqm 11t1s o1dlarrrlca;;a puodsar aseeslp slql qll7v1sluaqed a1uos]eq] uollp^Jasqo snor,rard aq] Jo sseulnJesnaqr Suqsll aqt roJ uoqeueldxa ue Sutpnord pue uoIlEJUISSels -qelsa 'srsouSordrallaq e qllm raqlo eql pue stsouSord rood e qlyr palelf,osse sem ssBIOaug ,{.1ecr3olo}sqpaqsln8uqslp aq lou ppoc sesselcaq} q8noql uana 'salgord uotssardxa aua8 uo paseq sasselcaleredas o^u ere aJeq] ]eql peleJls -uouep 'aldruexa ro; 'seuoqdu,{1 1ac-g a8rel asry;rp Jo uol}JalloJ e;o srsz(puy 'luaped aql JoJsacuanbasuor luelrodrut qllm ^lsllaJd eJotu sJolunl I;1sse1co1 dlaq osp ]nq 'saseouleuatun uI Jo{un} e ,{;puapr or dlaq -'t1uo1ou uec Suqgord uorssardxa aua8 ,snq; ,(puaragrp a^Pqeq ]Pql sJalueJ raqlaSo] drunl uec daql ]eqt lv\ODI^\ou aA\ pue sts'(pue pcrSololsrq uo ,(1arL1urcur seluaqls uolleslJls -sel3pJppusls .uollpsrJrsself, Joun] ra11aqqSnorql aslcard arour srsouSErpelelu 'saseesrp uorlJelloc snoauaS o] Iool e sluasardar Suqgord uorssardxa euac Jo -oJelaq dpreurproerlxe up ale 'uees a1eq ala sP 'SJaluES'aAIpeJJasI luelulpaJl e 'luaued Jeqlaqm a8pnt ot sluaqed;o uopepdod e uo slelrl e^llceJJa1onpuof,ro 'etuoolno sl1telpard 'sasneJsll JeAoJ uenr8e JoJluaruleeJl alerrdordde eq] loales -slp louueJ aarr'.{1}carrocaseaslpu d;rluapr louueJ arug 'stsou8ulp a}eJnJceuo spuadap ssarSordIpclperu 11VilnJasn aq sap;ord relnJaloru qcns lq8lu fqq 'sernleeJap;ord uoru -ruoc ereqs sJaqrueu asoqm 'sesselc]Jullslp Jo Jaqlunu ilerus e olut padnor8 aq 'lenaa,to11'algord uec .{aq} lpq} smoqs sluaped ,{ueru;o say;ord aq} SulredruoJ uolssardxa aua$ grvrosll 3AEI{IIuA 'JelueJ }suerq sB qJns lef,uec;o ad,ft uanrfi E JO eSeJqf,eg .anssp loJtuot pturou ur sauaS e(uEs eql 3o uorssardxaJo uJel -1edaql qll,lr lI aredruoc pup relupo e 3o aldues a13utse ut flsnoauellntuts saua8 aq UEJ (8 reldeq:) Jo spuesnoql;o uorssardxa5o uraged aql euluJelep ol pasn 'II.Prappaluapacardun ,(erruorclu yNIq 'aldruexa JoC ur passnJslp) ,ri8o1ouqce1 ur lelel JBInJalou u lB JaouEJIenpIAIpuI qoea ezlJalJeJeqcol alqrssod^aou sl ]I

sdno.r6qn5 In,6utueaw{1;e>;u;13 uolssaldxfauag olur sra)ue)^J!ssel)d;ag ue1 6u;1;1o16 la)ue):97 taldeq3

VqZL

1265

END-OF-CHAPTER PROBLEMS

we can analyze the abnormal signaling network in an individual tumor well enough to allow us to select a tailor-made cocktail of drugs and growth factors that specifically causesall cells in that cancer to die? Looking back on the history of cell biology and contemplating the speed of recent progress,we can be hopeful. The desire to understand, which drives basic research,will surely reveal newways to use our knowledge of the cell for humanitarian goals,not only in relation to cancer,but also with regard to infectious disease,mental illness, agriculture, and other areasthat we can scarcelyforesee.

Su m m a r y Our growing understanding of the cell biology of cancershas already begun to lead to better waysof preuenting,diagnosing and treating thesediseases. Anticancer therapies can be designedto destroycancer cellspreferentially by exploiting the properties that distinguishthemfrom normal cells,includingtheir dependence on oncogenicproteins and the defectsthey harbor in their DNA repair mechanisms,cell-cyclecheckpoint mechanisms,and apoptosiscontrol pathways.It may also be possibleto control the growth of tumors by attacking their blood supply and depriuing them of the help they requirefrom stromal cells.Wenow haueproof that, lry understandingnormal cellcontrol mechanismsand exactly how they are subuertedin specificcancers,we can deuise drugs to kill cancerspreciselyby attacking speciftc moleculescritical for the growth and suruiual of the cancercells.And, as we becomebetter able to determine which genesare altered in the cellsof any giuen tumo\ we can begin to tailor treatmentsmore accuratelyto eachindiuidual patient.

sarcomasare relativelyrare in young children (up to age 9) and in adults (over 20).\tVtrydo you supposethat the incidence of osteosarcomadoesnot show the same sort of agedependenceas colon cancer?

PRCBLEMS Whichstatementsare true?Explainwhy or why not. 20-1 Genetic instability in the form of point mutations, chromosome rearrangements,and epigenetic changes needsto be maximal to allow the developmentof cancer. 20-2 Cancertherapiesdirectedsolelyat killing the rapidly dividing cells that make up the bulk of a tumor are unlikely to eliminate the cancerfrom many patients. 20-3 The main environmental causesof cancer are the products of our highly industrializedway of life such as pollution and food additives. 2A*4 Dimethylbenz[a]anthracene(DMBA) must be an extraordinarily specific mutagen since 90% of the skin tumors it causeshave an A-to-T alteration at exactlv the same site in the mutant Rdsgene. 20-5 In the cellular regulatory pathways that control cell growth and proliferation, the products of oncogenesare stimulatorycomponents and the products of tumor suppressorgenes are inhibitory components.

20-6

20-8 By analogywith automobiles,defectsin cancer-critical genes have been likened to broken brakes and stuck accelerators,which are causedin some casesthrough faulty serviceby bad mechanics.Using this analogy,decide how oncogenes,tumor suppressorgenes,and DNA maintenance genesrelate to broken brakes,stuck accelerators,and bad mechanics.Explainthe basisfor each of your choices. breast

,I E; F: { E g-

1

or,

Discussthe following problems.

2o--7 As shor,r,nin Figure Q20-f, plots of deaths due to breastcancerand cervicalcancerin women differ dramatically from the same plot for colon cancer.At around age 50 the age-dependentincreasein death ratesfor breastand cervical cancer slows markedly, whereas death rates due to colon cancer(and most other cancers)continue to increase. Why do you suppose that the age-dependentincreasein death ratesfor breastand cervicalcancerslowsafter age50?

(A)

i..'

1

32

40 50 63 age(years)

79

Ozs (B)

32

40 50 63 age(years)

79

9s (C)

32

40 50 G3 79 age(years)

In contrast to col

whose incidence '".,""":"'."x'.""1 ;:[:tr,'J:::[J"::l',;,5;:?":i.1';J];lll,,?."$:,':f ::?5ffiH,f"1:ffii3::ll matically with age, osteosarcoma-a tumOr that occurs most commonly in the long bonespeaks during adolescence.Osteo-

Thedata in all casesare plottedas log of the deathrateversusthe patientage (on a log scale)at death.Thestraightlinesin B and C arefittedto the datafor the earlieragegroups,whereasthe line in A is fitted to all the data points.(Datafrom P.Armitageand R.Doll,Br.J. Cancer Ltd.) from MacmillanPublishers 91:1983-1989, 2004.With permission

1266

Chapter20:Cancer

FigureQ20-2 Cumulative riskof lung cancermortalit; for nonsmokers, smokers, L @' and formersmokers (Problem20 9).Cumulative 6 f riskis the runningtotalof E deaths,asa percentage, for S eachgroup.Thus,for o continuingsmokers,1olo died of lung cancerbetweenages F . 45 and 55;an additional4olo :o ) c died between55 and 65 6 (givinga cumulativeriskof o 5olo); and 11olomore died between65 and 75 (fora cumulativeriskof 160lo).

never stopped

1q

stopped age 50 stopped age30 never smoked 45

55

65

75

85

age (years)

20*9 Mortality due to lung cancerwas followed in groups of males in the United Kingdom for 50 years.Figure QzO-z shows the cumulative risk of dying from lung cancer as a function of ageand smokinghabits for four groupsof males: those who never smoked, those who stopped at age 30, those who stopped at age 50, and those who continued to smoke.Thesedata show clearly that an individual can substantiallyreducehis cumulativerisk of dying from lung cancer by stopping smoking.\A/hatdo you supposeis the biologicalbasisfor this observation? 20-10 The Tasmaniandevil,a carnivorousAustralianmarsupial, is threatenedwith extinctionby the spreadof a fatal diseasein which a malignant oral-facial tumor interfereswith the animal'sability to feed.Youhavebeen calledin to analyze the sourceof this unusual cancer.It seemsclear to you that the canceris somehowspreadfrom devil to devil,very likely by their frequent fighting, which is accompaniedby biting around the face and mouth. To uncover the source of the cancer,you isolatetumors from I I devils capturedin widely separatedregionsand examinethem. As might be expected, the karyotypesof the tumor cellsare highly rearrangedrelative to that of the wild-type devil (Figure Q20-3). Surprisingly,you find that the karyotypesfrom all 11 tumor samples are very similar. Moreover,one of the Tasmaniandevils has an inversionon chromosome5 that is not presentin its facial tumor. How do you supposethis canceris transmittedfrom devil to devil?Is it likely to ariseas a consequenceof an infection by a virus or microorganism?Explainyour reasoning. 20-11 Now that DNA sequencingis so inexpensive,reliable, and fast,your mentor has set up a consortium of investigators to pursue the ambitious goal of tracking down a/l the mutations in a set of human tumors. He has decided to focus on breast cancer and colorectalcancer becausethey causel4% of all cancerdeaths.For eachof I I breastcancers and l l colorectal cancers,you design primers to amplify 120,839exonsin 14,661transcriptsfrom 13,023genes.As controls,you amplify the same regionsfrom DNA samples taken from two normal individuals.You sequencethe pCR products and use analyticalsoftwareto comparethe 456Mb of tumor sequence with the published human genome sequence.You are astoundedto find Bl6,986putative mutations. This represents more than 37,000 mutations per tumor! Surelythat can'tbe right. Onceyou think about it for a while, you realizethe computer sometimes makes mistakes in calling bases.To test for that sourceof error,you visuallyinspect everysequencingread and

find that you can exclude 353,738changes,leaving you with 463,248,or about 21,000mutations per tumor. Still a lotl A. Can you suggestat leastthree other sourcesof apparent mutations that do not actuallycontribute to the tumor? B. After applying a number of criteria to filter out irrelevant sequencechanges,you find a total of 1307mutations in the 22 breastand colorectalcancers,or about 59 mutations per tumor. How might you go about decidingwhich of these sequencechangesare likely to be cancer-relatedmutations and which are probably "passenger" mutations that occurredin geneswith nothing to do with cancer (but were found in the tumors becausethey happenedto occur in the same cellswith true cancermutations)? C. Will your comprehensivesequencingstrategydetect all possible genetic changesthat affect the targeted genes in the cancercells? 20-12 Virtually all cancer treatments are designed to kill cancer cells, usually by inducing apoptosis.However,one particular cancer-acute promyelocltic leukemia (APL)has been successfully treated with aLI-trans-retinoicacid, which causesthe promyelocytesto differentiateinto neutrophils. How might a changein the state of differentiation of APL cancercellshelp the patient? 20-13 One major goal of modern cancertherapy is to identify small molecules-anticancer drugs-that can be usedto inhibit the products of specificcancer-criticalgenes.Ifyou were searching for such molecules, would you design inhibitors for the products of oncogenes,tumor suppressor genes,or DNA maintenancegenes?Explainwhy you would (or would not) selecteachtype of gene.

(A)

Tasmaniandevil (Sarcophilusharrisii)

a (c)1

3

4

s

6

M3 M 1 M 2 M4

FigureQ20-3 Karyotypes of cellsfrom Tasmanian devils(Problem 20 10).(A)A Tasmanian devil.(B)Normalkaryotypefor a male Tasmanian devil.The karyotypehas 14chromosomes, includingXY (C)Karyotypeof cancercellsfound in eachofthe 11 facialtumors studied.The karyotypehas13chromosomes, no sexchromosomes, pai, one chromosome6, two chromosomes no chromosome-2 1 with deletedlong arms,and four highlyrearranged marker (M1-M4).(FromA.M.Pearse chromosomes and K.Swift,Nature 439:549,2006. With permission from MacmillanPublishers Ltd.)

REFERENCES

REFERENCES General (2004)Howto Winthe NobelPrize: Bishop.lM An Unexpected Lifein ScienceHarvard University Press: Cambridge MA StillmanB & StewartD (eds)(2005)Molecuiar Approaches to Controlling CancerColdSpringHarborLaboratory Press: Cold S p r i n gH a r b o r WeinbergRA(2007) TheBologyof CancerGarland NewYork Science: Cancer as a Microevolutionary Process MW,WichaM et al (2004) Al HajjM, Becker Therapeutic implications of .:n.cr (rpm ccll1p rlsEqaas---€ulPIIP 'snrnl pue txotlloA'DllatlN apnlcul saldruexg 'stust -ue8ro cqeqlu,,tsoloqd cttofrucna relnflacqlntu pue relnllar

-runaldrurs;o ol pasn aauerapu e aqtlcsep H"?rfilj"i.yi** (L11'd'l-Z laued) 'loqorle ue 01pacnpal lo plrE ue gJ pezlplxo aq uec 'apdqapler_acd13:e1d^tuu -xg 'dnor8 t oqr supluoc leql punodtuoc ctue8r6 l),C-l oap(qapp ('9-9I arnBIC)'(S)d) g aseul) ulal -ord palec oslv'aAIArns pue,rnor8 ot sllar Sugpu8rsur dlerc -adse pa,rlo.rur dervrqted Surteu8rs JelnllaJerlul UV/aseuD{ -t Id aql ul slrP leql aseull utalord auluoarql/eulras (S)d'g aseupl ulalord; p1y 'uorlezlu -nrurur JaUe arull go a8essed aql qlyv\ ua8rlue Sutztununut aql roJ salpoqllue ;o .ftrurge eq] uI aseeJcur a-ttssarSor6 uopernluur z$1u1gu (gt-t arn8rC)'g pue '[g][V]/[gV] V uaamleq Surpurq aqr rarq8p aql ra8rel sI pue .,{q uanr8 sl }uplsuoc uorterrosse aqt 'gV = g + V unlr -qrlnba Surpurq e pue g puu V sluauodtuoc rog 'xalduoc e ur stuauodtuoJ oq] Jo Surpurq ;o qlSuarls otlt Jo arnsealN (9I) (ruulsuor uollulrossu) luelsuor zfulugge (€I-B alnBIC)'xIlJPIuaql uo paurclar sr uralord aql leqt os 'paqrette are ulalord pelnbar aql ro; spue311cr;tcads qJIq^ ol xlrtetu e talo passed st paglrnd aq ol arnlnru utalord eqr qtlqm ur ,{qde-rSoleruorqc;o adl.1 fqdu.rtoleuorqc ft ;uggu 'alrsSurpurq a18urse 1epue8q slr ot alnraloru e 3o Sulpurq 3o qt8uarls aqJ ri1;uggu

'(zo) ua3[xo JEInJaloruJo acuasardeq]

'Eurrrnberro 'u1Sutunccg JIqoJau

'rallltusuEJlornau aullueloqsalPc e osle sl U 'slalal re8ns poolq pup ater ueoq uI aseorrul uP sapnlrul qcrq,,r,r'asuodsar ,,1q3rgro rq8g,, e aleulpJooc pue alsluul ue3 'sUCdC cr;rceds ot spulq lpqt 'ssarls ot asuodser ut dletc tI -adse 's11acurJJeluorqc pue13 puarpe ltq paseela: auoruroH (au1.rqdau1da)eugeuarPe (Zg-Z arnBIC)'uoll -e1,,{roqdsoqda ItPpFo se qcns ssacord SuperaueS-fSraua uol{nr dIV seletauaBag 61y;o aleqd ud .{q pa1e1,,{-roqdsoqd -soqd punural aql Jo srsdlorpdq ,{q pacnpold aplloalcnN (apqdsoqdp-,9 eulsouape) dOV 'stsPlqorqrJpaJnllnc Jo oseJrnsra1!{olaql uo slJel -uor IeroJ pue s11ec pllaqlrda luacufpe Suqull stleq uolsaqpe apnlour selduuxg 'sluaruellJ UIIJP ol paqlelle sI auelqruelu

UVSSO-ID Zi9

G:3

GLOSSARY

anchorage dependence Dependence of cell growth, proliferation, and survival on attachment to a substratum.

surface of an epithelial cell is the exposed free surface,opposite to the basal surface. The basal surface rests on the basal lamina that separates the epithelium from other tissue.

anchoring junction Cell junction that attaches cells to neighboring cells or to the extracellular matrix. (Figure 19-2, and Table l9-1, p. I133)

apical meristem The growing tip of a plant shoot or root, composed of dividing undifferentiated cells. (PaneI22-I, p. l40l)

angiogenesis GroMh of new blood vesselsby sprouting from existing ones.

apoptosis Form of programmed cell death, in which a "suicide" program is activated within an animal cell, leading to rapid,cell death mediated by intracellular proteolytic enzymes called caspases.

Angstrom 1A; Unit of length used to measure atoms and molecules. Equal t o l 0 - ' u m e t e r o r 0 . I n a n o m e t e r( n m ) . animal pole In yolky eggs,the end opposite the yolk. Cells derived from the animal region will envelop those derived from the yolky (vegetal)region. (Figure 22-68) anion Negatively charged ion.

aqueous Pertaining to water, as in an aqueous solution. Arabid.opsis thaliana (common Thale cress) Small flowering weed related to mustard. Model organism for flowering plants and the primary model for studies of plant molecular genetics.

antenna complex Part of a photosystem that captures light energy and channels it into the photochemical reaction center. It consists of protein complexes that bind large numbers of chlorophyll molecules and other pigments.

archaeon (plural arch[a]ea) (archaebacterium). Single-celledorganism without a nucleus, superficially similar to bacteria. At a molecular level, more closely related to bacteria in metabolic machinery but more similar to eucaryotes in genetic machinery. Archaea and Bacteria together make up the Procaryotes.(Figure l-21)

antibiotic Substancesuch as penicillin or streptomycin that is toxic to microorganisms. Often a product of a particular microorganism or plant.

ARF (ADP-ribosylation factor, ARF protein) Monomeric GTPasein the Ras superfamily responsible for regulating both COPI coat assembly and clathrin coat assembly at Golgi membranes. (Table f 5-5, p. 926)

antibody (immunoglobulin, Ig) Protein produced by B cells in responseto a foreign molecule or invading microorganism. Binds tightly to the foreign molecule or cell, inactivating it or marking it for destruction by phagocltosis or complement-induced lysis.

aromatic Molecule that contains carbon atoms in a ring drawn as having alternating single and double bonds. Often a molecule related to benzene.

anticodon Sequence of three nucleotides in a transfer RNA (IRNA) molecule that is complementary to a three-nucleotide codon in a messengerRNA (mRNA) molecule. antigen A molecule that can induce an adaptive immune responseor that can bind to an antibody orT cell receptor. antigenic determinant (epitope) Specific region of an antigen that binds to an antibody or a T cell receptor. antigenic variation Ability to change the antigens displayed on the cell surface; a property of some pathogenic microorganisms that enables them to evade attack by the adaptive immune system. antigen-presenting cell Cell that displays foreign antigen complexed with an MHC protein on the cell surface for presentation to T lymphocytes. antiparallel Describes the relative orientation of the two strands in a DNA double helix or two paired regions of a polypeptide chain; the polarity of one strand is oriented in the opposite direction to that of the other. antiporter Carrier protein that transports two different ions or small molecules across a membrane in opposite directions, either simultaneously or in sequence.(Figure l1-B) antisense RNA RNA complementary to an RNA transcript of a gene. Can hybridize to the specific RNA and block its function.

ARP (actin-related protein) complex (ARP2/3 complex) Complex of proteins that nucleates actin filament growth from the minus end. ARS-see autonomously replicating sequence asexual reproduction Atty type of reproduction (such as budding in Hydra, binary fission in bacteria, or mitotic division in eucaryotic microorganisms) that does not involve the mixing of tvvo different fenomes. Produces individuals that are genetically identical to the parent. association constant-see affinity constant aster Star-shapedsystem of microtubules emanating from a centrosome or from a pole of a mitotic spindle. astral microtubule In the mitotic spindle, any of the microtubules radiating from the aster which are not attached to a kinetochore of a chromosome. ATM (ataxia telangiectasia mutated protein) Protein kinase activated by double-strand DNA breaks. If breaks are not repaired, AIM initiates a signal cascadethat culminates in cell cycle arrest. Related to ATR. ATP (adenosine 5'-triphosphate) Nucleoside triphosphate composed of adenine, ribose, and three phosphate groups. The principal carrier of chemical energy in Cells. The terminal phosphate groups are highly reactl;e in the sense that their hydrolysis, or transfer to another molecule, takes place with the release of a large amount of free energy. (Figure 2-26)

APC/C-see anaphase-promoting complex

ATPase Enzyme that catalyzes the hydrolysis of ATP Many proteins have ATPaseactivirY.

apical Referring to the tip of a cell, a structure, or an organ. The apical

ATP synthase (FoFr ATPase) Tiansmembrane enzvme complex in the inner membrane of

APC-see adenomatous pollposis coli

G:4

GLOSSARY

mitochondria and the thylakoid membrane of chloroplasts. Catalyzes the formation of ATP from ADP and inoiganic phosphate during oxidative phosphorylation and photosynthesis,.respectively. AIso present in the plasma membrane of Dactena. ATR (ataxia telangiectasia and Rad3 related protein) Protein kinase activated by DNA damage. If damage remains unrepaired, ATR helps initiate a signal cascade that culminates in cell cycle arrest. Related to ATM. atypical protein kinase (aPKC) An atypical form of protein kinase C (PKC) that does not require both Ca2aand phosphatidylserine for activation. One such aPKCis involved in the specification of polarity in some individual animal cells. auditory hair cell (sensory hair cell) Sensory cells in the inner ear, responsible for detecting sound by converting a mechanical stimulus (the vibrations caused by sound waves) into a releaseof neurotransmitter. (Figures23-13 to 23-15) autocrine signaling Where a cell secretessignal molecules that act back on itself. autoimmune disease, autoimmune response Pathological state in which the body mounts a disabling adaptive immune response against one or more of its own molecules. autonomously replicating sequence (ARS) Origin of replication in yeast DNA. autophagy Digestion of worn-out organellesby the cell'sown lysosomes.

isms used to analyse the molecular basis of genetics, and are now widely used as cloning vectors. Seealso bacteriophage lambda. bacteriophage lambda (bacteriophage t lambda) Virus that infects E coll. Widely used as a DNA cloning vector. bacteriorhodopsin Pigmented protein found in the plasma membrane of a saltloving archae an, Halobacterium salinarium (Halobacterium halobium). Pumps protons out of the cell in response to light. (Figure 10-33) basal Situated near the base. Opposite the apical surface. basal body Short cylindrical array of microtubules and their associated proteins found at the base of a eucaryotic cell cilium or flagellum. Serves as a nucleation site for growth of the axoneme. Closely similar in structure to a centriole. basal lamina (plural basal larninae) Thin mat of extracellular matrix that seoarates eoithelial sheets,and many other types of cells such as muscle or fat cells, from connective tissue. Sometimes called basement membrane. (Figure 19-40) base A substance that can reduce the number of protons in solution, either by accepting H+ ions directly, or by releasingOH ions, which then combine with H* to form H2O. The purines and pyrimidines in DNA and RNA are organic nitrogenous bases and are often referred to simplv as bases. (Panel 2-2. pp. l0B-109)

autoradiography Technique in which a radioactive object produces an image of itself on a photographic film or emulsion.

base excision repair DNA repair pathway in which single faulty bases are removed from the DNA helix and replaced. Compare nucleotide excision repair. (Figure 5-48)

autosome Any chromosome other than a sex chromosome.

basement membrane-see

auxin Plant hormone, commonly indole-3-acetic acid, with numerous roles in plant growth and development.

base pair TWo nucleotides in an RNA or DNA molecule that are held together by hydrogen bonds-for example, G paired with C, and A paired with T or U.

avidity Total binding strength of a polyvalent antibody with a polyvalent antigen.

basic (alkaline) Having the properties of a base.

ttxon Long nerve cell projection that can rapidly conduct nerve impulses over long distances so as to deliver signals to other cells. axonal transport Directed intracellular transport of organelles and molecules along a lerve cell axon. Can be anteiograde (outward from the cell body) or retrograde (back toward the cell body). zlxoneme Bundle of microtubules and associatedproteins that forms the core of a cilium or a flagellum in euiaryotic cells and is responsible for their movements. BAC-see bacterial artificial chromosome bacterium (plural bacteria) (eubacterium) Member of the domain Bacteria, one of the three main branches of the tree of life (Archaea, Bacteria, and Eucaryotes). Bacteria and Archaea both lack a distinct nuclear compartment, and together comprise the Procaryotes.(Figure l-2I) bacterial artificial chromosome (BAC) Cloning vector that can accommodate large pieces of DNA up to I million base pairs. bacteriophage (phage) Any virus that infects bacteria. Phages were the first organ-

basal lamina

B cell (B lymphocyte) Tlpe of lymphoclte that makes antibodies. Bcl2 family Family of intracellular proteins that either promote or inhibit apoptosis by regulating the release of cltochrome c and other mitochondrial proteins from the intermembrane space into the cytosol. benign Of tumors: self-limiting in growth, and noninvasive. beta-catenin (F-catenin) Multifunctional cyoplasmic protein involved in cadherinmediated cell-cell adhesion, linking cadherins to the actin cltoskeleton. Can also act independently as a gene regulatory protein. Has an important role in animal development as part of a Wnt signaling pathway. beta sheet (p sheet) Common structural motif in proteins in which different sections of the polypeptide chain run alongside each other, joined together by hydrogen bonding between atoms of the polypeptide backbone. Also known as a p-pleated sheet. (Figure 3-7) binding site Region on the surface of one molecule (usually a protein or nucleic acid) that can interact with another molecule through noncovalent bonding.

GLOSSARY

G:5

bi-orientation The attachment of sister chromatids to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when they separatein anaphase. biosphere All of the living organisms on Earth. biosynthesis-see

anabolism

biotin Low-molecular-weight compound used as a coenzyme.Also useful technically as a covalent label for proteins, allowing them to be detected by the egg protein avidin, which binds extremely tightly to biotin. (Figure 2-63) bivalent A four-chromatid structure formed during meiosis, consisting of a duplicated chromosome tightly paired with its homologous duplicated chromosome. blastomere One of the many cells formed by the cleavageof a fertilized egg. (Figure 22-69) blastula Early stage of an animal embryo, usually consisting of a hollow ball of epithelial cells surrounding a fluid-filled caviry before gastrulation begins.

calmodulin Ubiquitous intracellular Ca2*-binding protein that undergoeja large conformation change when it binds Ca2*,allowing it to regulate the activity of many target proteins. In its aciivated (Ca2t-bound) form, it is called CaZ*/calmodulin. (Figure l5-43) calorie unit of heat energy, equal to 4.2 joules. one calorie (small "c") is the amount of heat needed to raise the temperature of I gram of water by l"C. A kilocalorie (1000 calories) is the unit used to describe the energy content of foods. Calvin cycle -see carbon-fixation

cycle

CAM (cell adhesion molecule) Protein on the surface of an animal cell that mediates cell-cell binding or cell-matrix binding. CaM-kinase Serine/threonine protein kinase that is activated by Ca2*/calmodulin. Indirectly mediates the effects of an increase in cytosolic Ca2'by phosphorylating specific target proteins. (FiSure l5-43) CaM-kinase II Multifunctional Caz*/calmodulin-dependent protein kinase that phosphorylates itself and various target proteins when activated. Found in most animal cells but is especiallyabundant at synapsesin the brain, and is involved in some forms of synaptic plasticity in vertebrates. (Figure l5-44)

blotting Biochemical technique in which macromolecules separated on a gel are transferred to a nylon membrane or sheet of cAMP-see cyclic AMP paper, thereby immobilizing them for further analysis. [See Northern, Southern, and Western (immuno-) blotting.l cAMP-dependent protein kinase-see protein kinase A (Figure B-38) cancer Disease featuring abnormal and improperly controlled cell B lymphocyte-seeB cell division resulting in invasive growths, or tumors, that may bond energy spread throughout the body. (Figure 20-37) Strength of the chemical linkage between two atoms, measured by the energy in kilocalories or kilojoules needed to capsid Protein coat of a virus, formed by the self-assemblyof one or break it. more tlpes of protein subunit into a geometrically regular bright- field microscope structure. (Figure 3-30) Normal light microscope in which the image is obtained by Ca2* pump (calcium pump, Ca2* AIPase) simple transmission of light through the object being viewed. Transport protein in the membrane of sarcoplasmic reticubrush border lum of muicle cells (and elsewhere).Pumps Ca2* out of the Dense covering of microvilli on the apical surface of epithecl.toplasm into the sarcoplasmic reticulum using the energy lial cells in the intestine and kidnev. ofATP hydrolysis. buddingyeast Common name given to the baker's yeast Saccharomyces cereuisiae,a model experimental organism, which divides by budding off a smaller cell.

carbohydrate General term for sugars and related compounds containing carbon, hydrogen, and oxygen, usually with the empirical formula (CH2O)n.

buffer Solution of weak acid or weak base that resists pH change when small quantities of acid or base are added, or when solution is diluted.

carbon fixation reaction Processby which inorganic carbon (as atmospheric COz) is^ incorporaied into organic molecules. The second stage of photosynthesis. (Figure 14-39)

Ca2*/calmodulin-dependent

protein kinase-see (CaM-kinase)

cadherin Member of the large cadherin superfamily of transmembrane adhesion proteins. Mediates homophilic Caztdependent cell-cell adhesion in animal tissues.(Figure l9-4, andTable 19-2, p. ll35) Cae no rh ab diti s eIcgans A small (-lmm) nematode worm used extensivelyin molecular and developmental biology as a model organism. caged molecule Organic molecule designed to change into an active form when irradiated with light of a specific wavelength. Example: cagedATP CAK-see Cdk- activating kinase calcium pump-see

Caz* pump

carbon-fixation cycle (Calvin cycle) Major metabolic pathway in photosynthetic organisms by which CO2 and H2O are converted into carbohydrates. Requires both AIP and NADPH. (Figure 14-40) carbonyl group (C=O) Caibon atbm linked to an oxygen atom by a double bond. (Panel2-1, p. 107) carboryl group (-COOH) Caibon atom linked both to an oxygen atom by a double bond and to a hydroxyl group. Molecules containing a carboryl group are weak acids-carboxylic acids. (Panel 2-1, p. 107) carboxyl terminus-see

C terminus

carcinogen Any-agent, such as a chemical or a form of radiation, that causescancer.

G:6

GLOSSARY

carcinoma Cancer of epithelial cells.The most common form of human cancer.

cdk-activating kinase (cAK) Protein kinase that phosphorylates Cdks in cyclin-Cdk complexes,activating the Cdk.

cardiac muscle Soecializedform of striated muscle found in the heart, consisting of individual heart muscle cells linked together by cell junctions'

Cdk inhibitor protein (CKI) Protein thit binds to and inhibits cyclin-Cdk complexes,primarily involved in the control of Gi and S phases.

carrier protein-see

cDNA DNA molecule made as a copy of mRNA and therefore lacking the introns that are present in genomic DNA.

transporter

cartilage Form of connective tissue composed of cells (chondrocl'tes) embedded in a matrix rich in type II collagen and cirondroitin sulfate proteoglycan.

Cdtl Protein essential in the preparation of DNA for replication. With Cdc6 it binds to origirrrecognition complexes on chromosomes and helps load the Mcm proteins on to the complex, forming the prereplicative complex.

cascade-seesignaling cascade caspase Intracelrular protease that is involved in mediating the intracellular events ofapoptosis. catabolism General term for the enzyme-catalyzedreactions in a cell by which complex morecuresare degraded r" ri-;;;;;; ;i;t release ofenergy. (Figure 2-36) catalyst

cell adhesion molecule-seecAM cell cortex Specialized layer of_ cytoplasm on the inner face of the yl3,t-" membrane' In animal cells it is an actin-rich layer responsible for movements of the cell surface' cell cycle (cell-division cycle) Reproductive-cycle-of a cell: the orderly sequence of events

substance rhatcanrower the,acrivation energy:li.:T:jr?:

(thus increasing its rate), without itself being consumed by the reaction.

catastrophe factor Prot-ein that destabilizes microtubule arrays by increasing the frequency of rapid disassemblyof tubulin subunits from oneend (catastrophe).(Figure 16-16) p-catenin-seebetacatenin cation Positively-chargedion. caveola (plural caveolae) Invaginations at the cell surface that bud off internally to form pinocytic vesicles. Thought to form from lipid rifts, regions of membrane rich in certain lipids. cD4 Co-.receptorproteinfound on helperT cells,regulatoryT cells, and macrophages.It binds to classII MHC proteins (on antigen presenting cells) outside the peptide-binding groove. CD8 Co-receptor protein found on cltotoxic T cells. It binds to classI,MHC proteins (on antigen-presenting cell) outside the peptide-binding groove. cD28 Co-receptor protein on T cells that binds a co-stimulatorv Bz protein orr dendritic cells, providing an additional signal required for the activation of a naive T cell by antigen. Cdc6 Protein essential in the preparation of DNA for replication. With Cdtl it binds to an origin recognition complex on chromosomal DNA and helps load the Mcm proteins onto the complex to form the prereplicative complex. Cdc20 Alltyaling IAPC/O.

subunit of the anaphase-promoting complex

Cdc25 Protein phosphatase that dephosphorylates Cdks and increasestheir activity. Cdc gene (cell-division-cycle gene) Gene whose product (a Cdc protein) controls a specific step or set of steps in the eucaryotic cell cycle. Originally identi^fied in yeasts. Cdk-see cyclin-dependent kinase

:t[[:T,ffi1'.Xfft:'#"i,li::lllil",#fff,[l*

usuailv' the

cell-cycle control system Network of re-gulatory-protein.sthat governs progression of a eucaryotic cell through the cell cycle' cell division Separation of a cell into two daughter cells. In eucaryotic cells it entails division of the nucleus (mitosis) closely followed by division of the cytoplasm (c1'tokinesis). cell-division-cycle gene-s ee Cdc gene cell fate In developmental biology, describeswhat a particular cell at a given stage of development will normally give rise to. cell-free system Fractionated cell homogenate that retains a particular biological function of the intact cell, and in which biochemical reactions and cell processescan be more easily studied. cell line Population of cells of plant or animal origin capable of dividing indefinitely in culture. cellmemory Retention by cells and their descendants of persistently altered patterns of gene expression, without any change in DNA sequence. Seealso epigenetic inheritance. cell plate Flattened membrane-bounded structure that forms by fusing vesicles in the c)'toplasm of a dividing plant cell and is the precursor of the new cell wall. cell senescence-see replicative cell senescence cell signaling The processes in which cells are stimulated or inhibited by extracellular signals, usually chemical signals produced by other cells. cell transformation-see

transformation

cellularization The formation of cells around each nucleus in a multinucleate cytoplasm, transforming it into a multicellular structure. cellulose Structural polysaccharide consisting of long chains of covaIently linked glucose units. Provides tensile strength in plant cell walls. (Figures 19-78 and 19-79)

'aoEJJnsslr uo PIIrJsnorerunu dq pazrJelJeJeqr(uuozolord) rusrue8rocrlodrecna pe11ac-a13urg aleIIIr 'adocsorclur tq311eqt ur alqrsrl arnlrnns a)ll -po: lceduoc p olur pasuapuoc saruocaqauosoruoJrp qJpa qcrq.r,rSurrnp 'srsoreru Jo srsollru SuloBrapun sllal Iprurue pue 1ue1dur luaprla ,{lprcadsg 'rusrue8ro up Jo uolleuroJur dretrpereq aqt Jo (lle ro) lred sarrrec teqt suralord palerc -osse pue elncaloru yg6 3uo1,4rane;o pesodruoc arntrnrts aruosoruoJrlc (tI-B pue g1-g sarn8rg) fqderSoleuorqc prnbl arueruroJ -rad-q8rq pue '-rQrurgeVNC '-,ir1urgeosla aas'splf,u rlalf,nu ro suralord Jo sarnlKru aluredas ol pasn 'aseqd f,reuopuls e pue asuqd Sunour e uaemleq uorlrlred ot alnuJru eql 3uI -,r,rolp,(q,&radord raqlo aruos ro rsarlruuJeSurpurq tuale^oJ -uou ',!rcrqoqdorpfq 'az1s'a8ruqcLq paleredassr sa]uetsqns Jo arnulru e qcrq^ ur sanbluqrat lpJrrrraqJorqJo sselJpeorg t(qdurSoluruorqc 'uolldllrsuerl uI pa^lo^ul 'suralord raqlo ot -,(1qe1ou VNC _aso_ql aqr 1o ftpqtssaJce eql SurBueqc 'saruosotuoltlr rttofuBcna ur suopern8guoc VN6l-auolsrq sratp teqt xalduor auu&uE xalduroc Sullaporuar ullpruorqr (Z€-Zarn8rC)_'ura1_ord punoq aqt tsure8edpoqlluE ueJo suealu .{q t1 3upe11d1card.,(qpegpuapl pue patelosl aq, uuc_uralord relncrtred e dq punoq VNC pruosoruorqc qc1q,r,r dq anbluqcel (dIrIC) uopurldgce.rdounurul ulleruorqr 'apeul AJeSauIOSOtuOlqC qslqM Jo lelralPlu aql 'llar c1lo.{recnaP Jo snalrnu aql ul punoJ sulatord auotslquou pup 'sauotsrq 'VNCIJo xalduo3 ulluruoJrlJ

'e11a8ug IErrelJeqJo uonelor aql Jo uollsnpord 41y se r{3ns 'ssacord Surrrnber-.(3raua ue aAIJpol pesn sI auelqluaru e ssoJce(luolper8 uolo:d ro '1ua1p -er8 gd e) suor ueSorp,(q;o luarper8 e qcll{,rr uI ruslueqralN (slsorusolueqc) 8ulldnor rllousoluaqr ('puoq ueSo:pdq 'puoq rruor osl, aas) 'spuoq ]ualeloruou pup spuoq luop oc apnlsur sllec 3ur^rl uI punoJ saddl'raqlaSo] uaql sploq teql sluole o^\l uaaMtaq ,{ltutge leJlruaqc puoq lBrluraqr 'tr dpnls ot pasn aq uaql upc leqt pue ssacord pcrSolorq repcrued e ]cage leql slerlruaqc I;rluapr ol s,tesse pcrSolorq ur selnralotu ilprus Jo spuesnoql Jo sperpunq ;o Suruaercs alecs-a8re1sasn lpqt ,48elerls e o1 ue,rt8 eueg ,ito1o1q pcptaqc 'urnlsauSeu Jo 'runrcleo 'uorl se I{Jns uol Ielalx e qtl^\ lt]ruuJP q31q qtprtdpnsn'dlqrsraira-r aulquoc oJ olBlorlJ 'peecord ol IIec eql roJ alqetrns are suortrpuor lltun patluq eq uec elcdc eiil q8norql sijl3brd araqryr'ajcfc.ibrrrnip-nei crlo,fuecna luloa "qi,r! lqodlieqc .(dsHJ sura}ord lcoqs lpaq sapnlcul .Burplo;s1u pro^e uaqt sdlbq ro ,suralord inqio go Blrpiog'radord bqi abFC rat"q rnqr uiar6ra lauoiaaifuc rulncjlodr; iuoradeqc .ralrOdsuerlalnduoS .ssBduec ,uor ue .,(1pnsn,e]n1oscgrcads e qsrqM qSnorqt auerquau aql .r, arod snoanbb ,rn i-rog reqi ut'alord 1rods.r"rt ".rnrq-"61 ulalord lauueqc

(t-II pue g-11 sern8rg)'rafelrq prdl aqr ssorcu,tlalrssed esrygrpol sepceloru Iprus reqlo ro suor crue8rour smolle leql xalduoc uralord auerqruatusue{ (IouuBqJ auBJqruau) IauuBqc

(7-91 ern8rg) 'sllac Ierulup Jo sauprqruaru etuseld eqt;o luauodtuoc 1ue1:odruruV 'aJnlcnJls proJals Surr-rno; crtslralrepqc E qtlm alnoalour pldl luepunqe uV Ioralsaloqr

(StI I'd' I-6I alqel pue'Z-6I arn8rC)'eletusapotuseld dq paruro;rad sr uorlJury Jelrrursp 'slueld u1 'sutalord urxauur Jo ugeuuocJo pasoduroJ'sanssuprurue uI'ilac ol IIaJ uor; ssed ol suor pup sapralotu ilprus JoJ .{enra8essede sap4ord pue s11acluacefpe;o use1do1,{caql qull leql uoncunf [ec-[a:) uollcun[ SupuoJ-leuueqr

'araluorlual aql le pauro[ are 'sprleruoJqcJalsrspelleJ 'spltptuorqc omt aql 'eseqd g Surrnp uorleclldar ygg dq parxJoJaruosoruorqc paleclldnp e ;o sardoc omt aql Jo auo pperuoJrlJ 'aSe1uec;o xlJtetu aq] selarcas leqt IIaJ anssll-alrlJauuoJ (11acateglrec) efcorpuoqc

'pr1se1dloruJoJpazrleroadsy'srsaqludsotoqd lno sarrrecpuE ldqdorolqc suleluor leqt sluu1dpue ae31euaar8 ur alaue8rg lseldorolqc 'ae31epue 's1ue1d'erralceq ur srsaqlu.{soloqd ul lrpd Iertuer u sdeld teql tuaru8rd uaerB Sulqrosqe-rq8lT p(qdorolqc 'r8un;;o sllem llac aql pue suolala)soxa lcasur ;o lueuodruoc rofeur V 'aulruesoJ -n131,4racu Lr go aprreqcces,{1odcrreu.(1od crue8r o luepunqV ulrlrlr uogtut;d;cardounrurul upeuoJqJ aas-dlrtC ' lres out ato dut o3'saloB fz tua]oJJrp u or; Surleur8rro' (sad,{l -oua8 a:oru ro oml) sllal Jo suorlulndod luaJaJJrp,(lecrtaua8 aroru ro oa,q;o a1e8er88eue tuo{ pauuoJ rusrue8ro elor{r\\ uJerulrlJ 'uollpulqruocar cqaueS;o tuJoJe 'Jalo-SulssoJc Iptuos -oruoJqJ alrs e sluasardag 'srsoreu Supnp sauosotuoJr{J Jo snoSolouroq perred uaa.r,qaqolqrsh uollceuuoc paduqs-y (ulurusulqr p;n1d; eursu;qc 'Pcruraqr alqrsnJJrpB ru04 .4.e,nero spJemol rusrue8ro Jo IIat p Jo luarxalotu palcaJrq slxPlouaqJ 'rualsdsaunrurur aql Jo SuruorlounJaql ur luelrodtul 'aJrnos slr spre^ ol e^oru o]'sllac poolq allqM se qcns 'sllaJ stceJ]]e 1eq1 uralord palarces leug 'aur1oilc JllJelouraq3 ouDloruaqJ

l.roirqeu 1t1og slc aas-NCC 'pe1e1.,{qleuunureuror r{leraue8 suorSor esaql lsacuanbas 93 go ,firsuep a8erarreueqt rateer8 e qrrM VNC;o uor8ag

puBIsIcc

'salorrtuac;o rrcd GZ-LI pue 69-91 sern8rg) P sumluoc 1r slles lerurue lsolll ul 'srsolltu Surrnp alod a1p -urds aqt se stoepuu (l)OIhl) raluac Surzlue8ro-alnqnlorcru prulue Jo a1aue3;o paleco1d1er1ue3 .,i:eurrrdeqt sr leqt s11ac aruosoJluac (09*t arnBIC)'alpurds crlotrru eq] ruo{ salnqnlorc[u sarn]qec leql suroJ aroqco]auDl aql araqm VNCI aqt uo atls aqt oslv 'raqlaSol splleuorqc ratsrs sploq tEr{l euosoruorr{c cr}otrtu e ro OrtrjltjJrl?r.r"" "ottil (I€-9I arnSrC)'sllaf,lprulue ul auosorluac e Jo ratuar aqt le puno;,(1unsn sr salolrluarJo rrcd y ,(poq psPq P ol arntJnrls ur rcpurs dlasolc 'salnqntorrrru ;o ,{erre pcrrpu11,'tctroqg aIolrfuec 'AOrteuI auoq pue snul{qt aqt are asaqt 's1eturupru tlnpp uI 'sllac rosrnca;d uorJ paJnpord are saldcoqdurdl qcrq.tt ur ue8r6 (uutro ploqdudl fruu4rd) uutro ploqdudl lprluac 'sllac IeIu -rup lsoru u1 luasard tog 'r8un; pue 'ae31e'eapqJJu'erralceq 'slueld lsoru ur luaulruoJd 'eueJqrueru eruseld sU aprslno 11ace dq palrsodap xrJleru Jplnllacurlxa Suorls ,tlecrueqcary IIErtl IIar

AUVSSOT9

L:'

tuaruuorr^ua-aua8 ro/puE saua8 eldlllnul Jo uoilJelatui aql ol anp aIP sUerl qJnS 's^\el uellapuahl eldtuls daqo lou saop IuaSoJd ol uorssItusueJ]asoq^\ JrlsrJa]JpreqcalqelrJaH l[Brl xaldruoJ (g9g-t99 'dd 't-g 1aue4)'saua8tuareJJrp ro arues eql ur arp suollplnur o^\l roqlaqM Jo lsal e Jo srseg 'parotsar uaaq spq ad,ftouaqd (ad,rit-pp^) Iprurou oqt qrlqm ur Suudsgo ur stlnser 'ad,{louaqd prxrouqp ue Suunoqs qJea 'spnpt,ttpur o^ l;o 8ur1eu aq] qJrq \ ur uouoruouaqd (uopplueualduroc clleua8) uopuluarualduroc VN(r aas-VNq

freluarualduroc

(91-7 arnByg)'patptlllceJsr Surpuoq lualuloruou ]eqt os 'dn ,{rreu }eq} sarn}pal leJnlonr}s Jo lecruaqJorq Supteq :alerlsqns sU pue aurdzua ue se qJns 'solnJoloru 3urlce:alur raqlo JO e) ft-n a:n8yg) 'raqlo qrua qtg,nxeldnp parred-aseq 1ce;rade Sururrog;o alqedec :sacuanbasprlu rralcnu JO (I) lrelueualduoc 'srsoltco8eqd traqt Surtourord ,(q ro srsdl rlaqt Sursnec dltcarrp ,{q srusrue8roorcnu crua8 -oqled eleurula sdleg 'sursrue8roorcrurdq ro saxaldtuoc ua8rlue-,(poqpue ,(q palplrlce suralord unJes Jo rua1s.{g ualsds luaurayduoc 'sllar poolq Jo uorlPrluaraJJrp IoJluoc leql salnJalou leu8rs snorerunu JoJ arueu IpJauaD (gg3) .ro1cu;Supulnups-,{uo1oc (99-6I pue g7-g sarnSrg)'apurtuel Ipspq ur ]uesard sr n1 ad.,i1le8e11rec ur punoJ q 11ad! :auoq pup 'uopual ,uDIsur punoJ sr ,uoru -ruoJ lsoru aql 'I ad,{l :surro;,r{ueurur s}sxg 'qlSuarls alsual Surrra;uoc 'spurrue ur xrrletu Jplnllacellxa aq11oluauoduoc Jofetu e sl ]eq] aulloJd pue aurc,rtpur qcrr uralo.rd snoJqtC uatulloc 'srautred ar{l auo ro; cgrcads,{poqrtue ue Sursndq raqlo Jo qrpa qtlm xalduoc p ruroJ teql surelord Surtelosr;o poqlahl (dI-oJ) uo11u1;dlcardoununul-or (6-9 ern8rg) 'raqlo qJpa punore palroc seJllaqeqdp arour ro o.tg.,{qpaurroJ aJnl3nrls uralord a)llpor a1qels,,{lercadsg IIor-paIIor (77-21 arn8rg) 'uolturedes rraqt aroJaqqt8uelrraql Suop raqta8ot sprleuoqc ralsrs sploq 1eq1surelord ;o xaldruo3 xaldruoc ulsaqoJ'ulsaqoc &1lrllce s,aru.,(zueue ro; parrnbar arudzuaoc.ro uor crue8rouy rolceJoJ '79-TanBtg pue 'VoC 1[1aceoqn aag'sdnor8 aq] ul pasn alncelou IIeuS (yo3) y auriizuaoc

1,tce ;o raJsuerl Jltpu^zua

'y au.{zuaor pue '-CVN 'utlorq apnlJur saldruexg 'alprlsqns eqt ol puoq luale^oc e SunuJoJ dq uego 'saz.{p1ec aur.{zua aq} teql uoltcear aqt ur sa}edrcrl -red req] audzua ue qll A palerrosse [1]q3u a1nca10jlirHr"o" 'uruqc aprldad,{1od Sur,tror8E o}ur prrp ourrue cr;1cadse;o uorlerodrocur JoJuorlJnrlsur eql sluasardarleql elncaloru VNUrU ro VNC e q saprloelcnu aarqt Jo acuanbag uopoJ 'suralord raqlo uorJ aperu aJesJaqlo 'uuqlelc Jo aperu aJEslpoJ aurog '(1rdpalpoJ) oueJquau Jo uot8 er paleoc e ;o 'areJlns crlosot-{c slr uo (teoc aq}) JJoSurqrurd aq] dq paurroC suralord;o a8ec e qlun elaue8ro papunoq-eupJquau [erus elJIsaA paluoJ (19-2 arn8rg) 'aue8 eJo uorl -dpcsuerl alelrlce ol suratord ,troleln8er eua8 punoq-ygq raqlo uo salquasse tnq vNC purqJleslr tou saop leql utatord JOIeAIIJUOJ

(69-9 ern8rg) 'pa1ec11dar aq ol luau8u4 'lla3 tuerolcor aq} olul pauolc vNo eqr selqeua qcrr{^\ pup aq ot vNC Jo luetuSP{ ar{} ftrer o1pasn sl qclqM 'pruseld ro a8eqdorralceq B ruo{ pe^rrep,(1ensn 'alnoalotu VNC IIutuS .rolceir tuluolc 'sanbruqcal ygq 'pararrocer eq uec qcrr{,ll urorJ pue 'paJnp lr ]ueurquocar Lq -onur uaoq suq aua8 aql qrrqm ollur Apc'g s€ qcns) slac rarrrec Jo auolr e Sur,r,ror8[q eua8 e yo seldoc e1dr11nrue1e -arJ ol suparu ,,aue8e auolc ol,, :qJoAe se pasn oslv'roJsacue uoruurof, e tuor; uorsrlrp (lenxase) peleedar dq pauro; (susrue8ro ro sllac) slenprlrpur Iurrluapr Jo uorlelndod euoIJ (B_92 arn8rg) 'ua8rtue aqr sUJ lpqt roldarar e ^(eldsrp leql sauolc ailcoqdtu.(1 asoql .{1uo (stcales) se}elrlcp ue8uue uBIaroJ ua.tr8 e 'srotdacar cgrcads-ua8rlup paleJeue8 fluopuer ;o arrolradar lse,t e qlrm sal-{rorldudl;o uorlelndod u uorg dervr cgrcads .,{1q8rqe ur sua8rtue tuaraJJrpJo suolllrur ot puodsar uec ruals.{s eunurrur arr.qdepeaqt .ttor{ sureldxa reqr -{roeq; ^roeql uopcalas leuolc 'qt1!\or8 lnoqtl-{\ sllao rallElus ^ueu olul papnlpqns saruoraq ut uaas uols1,tlp[ac 1ac a8rel e,{qaraqm sodrquo '{1.ree,{ueur pcrs.rtq4(1) go ad.Qpazlprcadg (Z) 'o^\t olur IIar e;o 3ur1r11ds etBABaIJ i9-91 ern81g)'alcrsaApaleoc-ulrqlelc rplnllarprlur ue uroJ ot srsol^copua Aq JJo spnq qcll{^t 'lro paleoc-urJqlel3 e rrrroJol sP os auPJqrueu e Jo epls cll -osoilc aql uo a8ec prpaq{od p otul salquesse }eqt ulalord ulrqlelr (17-97 arn8rg)'uolleurqtuorar qollll,ls-ssEI:) pallec luaua8uerrear VNC se^lo^u1 'asuodsar aunrulul anrldepe ueJo esrnoc aql 3u1rnp o8rapun slac g dueur leqt (931 'alduexe roJ) sselo roqtoup Suqeru ot (ru8l 'aldruexa ro;) ulpqopoununur Jo sself, auo Suqeru uorJ a8ueqC qclyr{s ssBlc (09-92 arn8rg 'sllac Iredlaq o1 saptldad u8raro; slueserd U araqm's11acSurluaserd-ue8que snolrel 'uralord lHI trJo sesselcomt Jo auo Jo arpJrns eql uo punoC ugalord CHtr\III ssulr (99-97 arn8rg) 's11acI crxoto1.{cot (smrn e se qcns) aqoJJrru JelnlleceJlur 3urlca;u1 ue tuor; peAIJep sapqdad luesard uec tt ereqm 'sod,(lIIar aterqaual IIe lsorup 'utalord lHI trJo sesselco^\t Jo auo Jo acPJrnsaql uo punoc ulalord f,Htrr1lI ssulr u1elo.rd rollqFlul {pf, aas-Dlf, GZI-ZT' dd' e-Z loued)'PIrp -uoqJolnu aql ul sJncco11slac crlodreJna uI'zo] elsell,\pue 'ZHCVC pue HCVN srarrrer pate^pce eql SuI}e AJD auros -reua8 'salnralotu pooJ lrorJ pallrap sdnor8 ldlece seztpxg 'srusrue8ro Jrqorap ur punoJ fu,'.qled clloqe]au prlual (alctlc sqary'a1c[c (y31) plcu cq,(xoqrucpl) alcfc plcu c;.4;c (92-tI pue 'snteredde t-€I sarn8rd) f1o9 ro unlnrllar crurseldopueaqt ur punoJ sP 'tuaruuedruoc papunoq-auPJqtuelu pauollelc (auurelslt prnld) uuralslc 'aprsreau ro arues aut ro "r" 'suErunq ur alcdc ssauln;elem-doalsaql aldruexe roJ 'srnoq tZ punorp;o porrad e qtl,r,tursluu8ro ro IIec e u1a8ueqc relncluud e secnpord leqt ssacordpc11cr(cpulalul (urqilqr rrelpecrlc) {tolr uulpurrlr 'tunllaBeg atodutoS 'sluauta,Tou 3u11eeqpeleadar uro; -rad leqt sraqunu e8rel uJeluoc sJaqlo e1q.tt 'runIIIc aluotu -uou a18urse uretuoJ slac [ue6 'salnqnloJcltu Jo alpunq a:oc e Sururetuoc IIac crlo.{recna p Jo uolsualxa a{llrlpH (e1gcprnld) unIIIr

UVSSOID

8:9

'sPuSls relnllaJprlxa snolJe^ o] asuodsar Ilerus se slce lI r{}ll\^ sa}eroqulloc lPqr ro}dacar e :d1ereua8 arolN (19-92 ur aselJ^J l,{l^uape .(q 41y ruor; paleraua8 sr leql aprtoalf,nN ain8rgl'a1,icoqdurd1aq] atu^lce sroldacar Surpurq-ue8tlue (dWVr) 4141yc11cttc aqt sdlaq pue salnreloru leqlo ot spulq lnq_uaSltue pulq lou saop luql sllar J ro sllao g uo roldecar :.43o1ounuru1u1 roldecar-oc (69-9 ern8rC)'aufzua asusodsuer]IerJedsB .{q e11s,r,reu e o}ur peuesur pup VNC aqtJo lno tnJ sr tuaruala aqt qclq \ ul tuaruala alqesodsuerl e Jo luoua^our;o ad,,[ uo111sodsue.4alsud-pue-1nc

(I-t arn8rd) 'dnor8 (ggg3-) ldxoqrec aa4 e salrrer ler1t urcqc appdad.fiod e Jo pua aql (snuFural l{xoqmr) snulrural C 'suqp{qr uerpecrrcul pe^lo^ul sr tr'sprurue uI'sprulue pue s1ueld qtoq ur punoJ 'rq8q anlq ol anrsuodsaruralordo,,relg auorqcoldr{.rc uopcun[ fepg11o11 aas-atueqrxa

prrurls-ssorJ

(0I-IZ pue 9-17 sarnBrg)prleur -oJrlJ IeuJaled snoSoloruoq e Jo luau8as Surpuodsarroc e ro; pa8uuqcxa sr prleuorqJ [euro]eu e;o luatu8as e alar{,vr seruosoruonlJparrcd eql uo elrs e 'uorleulquooar crlorau uI (Er.usE!qJ)Ja^ossoJc (626-826'dd'Z-gt 1aue4)'(dlartlcadsar 'sapqnlorclu Jo slueruelrJ urlce olul palquasse "a'D urat -ord aqlSo uroJ palquesse eqt qllm rungqllnba ur sl tpqt 'ull -nqnt qcns ro urlce se tatuouoru urelord e Jo uorlertuaJuo) uollDrluaruoJ IBJIIIJJ 'auprqruau prrpuoqcoltu rauur aql Jo uo1]uu13enu1 (oelslrr prnld; ulqrc (62-9 arn8ril 'aua8 aqt oslrxa ot salerado ar3 pale^ltrp aql qrrqm lu 'saruanbas xol parnportur Lq palueg sr paldnrsrp eq o1 aua8 aq1'parrnbar se lr ele^rlce IIIM 1uq1ralouord E Jo Iorluoo aqt rapun pecnporlul sr (arC)aseu -lquocer cqrceds-a1rsV'arull cqrcads e le ro anssrt cgrcads e ul peslcxa aq uec eue8 1e3re1eqt qcrq^ ur sluelnru puo1} -lpuoc acnpord o1 pasn ualsr{s uoleulquocar cgtcads-altg xoI/arc

(z0r-90r'dd

'suorlcolo srrud arotu ro auo 3u1 I I-Z Iaued pue g-Z arn8rg) Jo -reqs Lq pacnpord sruole omt uaamtaq {ull Iurruaqc elqe}S puoqluaP ot (I9-Z arn8rd) 'raqto aqt alrrp ot salras auo l{q pesealar

.{3raua oa4aqtr{rrr{'*ur suorlrear Ierrruaqr r""ffil"B:Hfr"." (g-1 1 arn8rg) 'alncaloru puocas P Jo raJ -sueJl lerluanbas ro snoeuellnurs aql uo spuadap alnJelou auo Jo raJsuerl aql qorr{ \ ur ssacord uodsuerl auerquolN (godsuu.4 peldnoc) l.rodsuerl -oc (tg-g arnSrC)'aurosoqrr e ruor; se8raure lr se arnlJnJls drurlral pue ,{.repuocas slr olur urelord }uac -seu u Jo 8up1og aqt pup '(;E-ZI arn31g'uorlecolsuerl leuoll -elsuerl-or) pazrsaqtu,(sdlalalduoc sr uIEr{cepqdad.!od aqt aroJaq runlnJuar cluseldopua aql olul utelord e;o lrodrut aql apnlrur saldruexg 'spaecord uollelsuerl se Suprnccg lPuoIlBIstruJl-oJ 'urrads rar{unJ Jo dnue aq1lua,lard ol se os tuoc 33a aql ralp 'uollpzllqral rage srsol-{coxafq pesealar 'salnuer8 Iecruoc aql Jo s}ualuoc eql 'spruueu Jo asoqt Surpnlcur's88apazllqraJunJo euerquatu etuseld aq] Japun luasard alolsel drola;cas pezlprcadg elnuert lerluor (19-2 arnSrg)'aue8 e;o uols -serdxa aqr rlqqq o1 sulalord.{.ro1u1n8ereueB punoq-ygq raqlo uo satquasse tnq VNCI purq{eslt lou soop leqfj!l:l*r_r" (21-91 arn8rg) 'suecdp -oalord aoeJrns-llal pue (-{e-trqredSulpu8rs urualec-fl 71u1,1 aq] ul) dUT are selduexg 'sula1ordpu8ls pelorcos o1puodsar 'rotdacar acpJrnsllec puouualuor raqloue IIaJ aql Surdlaq

Gz-gr

arn8rg) 'pue8q e o1 asuodseraql suedreqs 1l eral{lw'stpnqns Jrralsolp eldpptu;o pasoduoc sroldecar pue saur,(zuau1se 'saxeldtuoc e8rey;o dlqruasse eql uI uees 'selnr_alour IIa-r\ sp pue311anrssaocns;oSurpurq aqt satoruord alncelou la8rel e ot alnoelotu pue8rt auo;o Sulpurq eq] qcll{.trul uouauouaqd zbl,rlleradooc @2-77anhg,)'raqloue uI IuIrLIs pue uolsuarulp euo uI puotxa o11l sasnecleql enssp e ulqlp sllar Jo lueuaSuurreeg uolsuolxe luatrarruoc '1rede slec ralq8nep ozr'$aq1 qcurd i6g-11 arn8rg; ol stJertuoJ 'uoIsIAIp 1ac Suro8rapun slloo IPIuluP Jo areJ

-rnsaql rapunsuroJ1eqlurso.{ur pueuttret",l?;""tilJgr"r"

(g9-g1 ern8lg) 'luasard are spu81s Sutlros raqlo ou JI auerquau etuseld aqt ol olnor llnPJap eql 'auerqluau Pluselo aqr qlll\^ asru tpql selcrsa^ ur snleredde r31og eql luoJJ euerquaru erusuld aqi ol pararrllap,{lpnurluoc aru sutetord auerqruaur_eruseld se qJns salnceloru qcq,,r,r ^,{qsflac IP u1 luasard ,{e1v\q]ud (durrrqred rpugep) femqrud,irolarJes a pnlllsuoc 'paluln8er SuprnccA ;o alrsoddo :sacuelsruncrlc;o ssaypre8ar'.{1pee}s e^pnl.rlsuoJ 'luegodurr.{.1puollrury sl rl reqr seldrur 171-9arn8gg) 'uorltsod qJee ]P punoJ uago acuanbes e Jo uope tesard tsoru plce ouJtuero appoalrnu eql saleJlpul'sacuenbas u1a1 -ord ro 'ygg rVNC pelelarJo dnor8 e ul suollelrel roulur qlp,t pacnpordar s1leql acuenbas E Jo uroJ lecldr! lsoru ro a8urarry ecuenbas snsuesuoJ (79-61 arn8rg) 'ssed uuc selnoaloru llerus pue suol qclqm q8norql Iauueqc snonulluoc u uroJ ol utof s1lacSururofpe oml tuo4 suoxau -uor :uorJJuntde8 u go1eg 'sllunqns urelord ulxauuoJ xls Jo 3ur-re,,tq paruroJ ouerqruaru eruseld aql u1 arod pelU-r4PM uoc (79-61 ern8lg) ,'lauuEqcluaq, ro 'uoxauuoc B ruroJ ol auerq -rueur uruseld aqt q alqruosse surxauuoc xIS 'ulalord aueJq -rxorusuen ssed-rno; u 'suotlcunf de3;o luauodtuoc ulaloJd ulxauuoJ 'enssll a^llcau -uoc asool pue 'a8eltlrec 'euoq sapnlcul 'xlnelu rulnllecerua e uI pappaque s_llarJo slslsuor Jo tunorue e8rul d1azr.tlu1ar pue sanssrl raqlo uaaMleq sall leql anss11Suproddns duy

t",rorr.r"-r'-o r"Jl:ili::l"IEJI'do,, ,,'uoIlJas

aueld aqt ssorce suecs pue uopeulrunlll Jo alJnos lu.rodurd 'tcafqo pllos e uIqlIM aueld uenr8 e se ueeq rasel p sasn ll e 3o a8etu'r reelr e secnpord 1eq1 adocsorenu 133rt"rr.lriff*r*" (999-r99 'arnleradurat,rnol.,i.llensnun ro q31q dlpnsnun 'dd 't-g laued) ue le se qtns 'suolllpuoc otuos Jepun.{1uoparalp sI uoncunJ slr leql os alnsololu vNg ro uralord e saSueqc leql uollulnlN uopBlnlrl Puolllpuor 'slso}ilu ol rotrd 127-21 atnfu1)'{pC-I I ro; te8rul uorlusuepuoJ etuosorxorqc uI paAIoAuI sulalord ;o xeldruo3 (xaldruoc ulsuepuoJ) ulsuaPuoc 'JalE1v\

pa>1u11 Jo alnrolou e Jo lelotuor aqt qtyvr sdnor8 116- q8norql ,{puapzroc arc solnoalotu oml qJIq \ uI uoI}JEor IBOIIrraqC (uollruar uollDrpfqap) uollcsor uollesuepuoc

UVSSOTD

6:9

plJe .,{npJo^\t Jo pasoduoC 'spu8rs IelnllecErtxa ol asuodsar uI sprdlloqdsoqd Iotrsour Jo a8e^Balr aql ^q pacnpord prdrl (CVO lorar^ISdcep 'uorleltua_raJJIp 1ac;o ssacord palJelap .{lpear arour qJnru eql sapacard1rpu€'llal eqlJo JalceJeqclEuJaluraqt ur a8ueqc e slcagal uorleunuJalaq 'luarudola,rap;o qled pazrprc -ads relncrlred e ol panruuoJ etuoJaq spq ll JI peulurolap aq ot plEs st 11accruo,{rqua ue '.43o1o1q ptuaurdola^ap uI

'sluauelrJ alelparuJelul pup'salnqruotcrru'sluatu -elu urlcE aJe sluauoouroc luepunqE ]soru sll 'luarue^oru patrorrp ro; dlrcedec aql pue adeqs IIac aqt salrB leql IIac cqodrecna e;o useldolr aql ul stuouelrJ uralord;o tuals^S uo1e1a4so1tc 'sureqc apls aurso:d1uo suJat -ord crurseldolltc la8ret Surteldroqdsoqd ri.q spre.r,tuopu8rs roldacar aql trusueJl 1eq1(sroldacar pelerJossp-aspuDI-aurs -orr!; sroldacar aceJrns-ilac ulpuac dq pa1e,r.r1cesatudzug asuurl aursolg cpusuldopb

,r-u,",";,'o1t:t:iil"t"o

slec Sururofpe oM] eqt ur s]ueruelu atelpauratul qf,rr{^ao}ur urelord;o senbeld esuap dq pazlralrereq3 .sllac pllaqllda omt uaamlaq pauroJ dlpnsn 'uortcunf lloc-llac Surroqcuy aruosorusep uolluldepe aas-uolluzlllsuesap

'ue8e11oc ul qolg 'snureprda aql qteauaq 'uDIs aql ur anssrl a^r]tauuoc ;o rar{e1 3ur,{Fapun >1crq1 srurJep 'aprsur anlen a.nrlu8eussal e ol prluelod auerqruaru s,llac p ur ]JnlS uoltu4.relodap 'asoqu atndtuoJ .'O0IHEJ .HO upq] raqlEr uorlrsod uoqrel-Z aqt lp H Surneq ur esoqlr ruo{ sreJJrC 'VNC Jo ]ueuoduroc aprJer{JJesouotuuoqlpc-al1J aql asoqlilxoap

'snalonu aql oprslno 's1ec cqod-recna asec aqt ul 'lnq auerq Jo -urau erusuld slr ulqlp\ peurcluoc are lpql 1ac e Jo slualuoC rusuldoldc 't-Zt 'aseqd '(srsolrrust GLOI-ZLU'dd tt{ Jo Ued 1eue4) qcrqM) snalcnu sll Jo uorsr^rppelElcosspaql tuo4 lcullsrp se 'oMl olul flec IPIIIlue ro 1ue1de;o useldofr aql Jo uorsr^rc slseuplolric

(gg-91 arn8rg) '.{e.r,rqtedSulpu8rs JVJS-IeI eql q8norql strp pup auoru -roq ro auqoldc cgrceds E spurq lpr{t rotderar areJrns-ila:) roldacar euqolb 'uorJptrunururoc ilec-llac ur lolerpalu pcol e sp strp leql appded ro uralord pu8rs relnllaceryg euplolac (92-tI am8rC)'roldacce uoJlcala ue se ua3,,{xoJ€lncalotu 3ulsn raleru sateraua8 pue r aruorqJoldc uor; suoJltala stdecap lI 'ureqc ,ftolerrd -sar or{t ur sdund uolord ualrrp-uor}cala eerqt aql Jo prrqJ xaldruoc eseplxo auro.rqcolib

arn8rg) 'auournbrqn ruo4 suotlcele sldaecy'ureqc197_71 drolerrdsar aqt ur sdtund uolord ualrrp-uortcala aaJqt oql Jo puoces xalduroc lc-q auo.rqco1lb

'uortcunJ pcr3010rq;o ssol eql ur sllnsar .,{1pnsn'slpcrruaqcot arnsodxa.,(qro 8ur1eaq,{qpasnec prce cralcnu .ro uralo:d e Jo uorleruJoJuoc ur a8ueqc crlpruercl uolleJnluuep

(97-71 arn8rg) 'srsoldodesatertrurosp ecedsauerqtuoruratur prrpuoqJotnu aqt ruo{ 1oso1.{crr{} olul esealal stl .upqc uoosueJJ-uoJlcalapupuoqcolru aql Jo lueuodruoJ alqnlos c aruorqcofc

VNO aas-pJre era;onuoqrilxoap 'sllal I 01 uorleluasald ro; 11sassaco.rdpue ua8rlue dn se{p} qrrqm '1ac Sultuaserd-ua8rlue;o adft aql lualod lsotu IIar rlllrpuap 'sllac o^]au Jaqlo ruo{ Ilnurls sa^recaJ . leqt 'paqcuBrq Alalproqela uauo ,llar o^rau e Jo uorsuaug alrrpuap

etueqc,{t.raua-aa{

pJpprruls aas-d

pllop

etueqc,{8.reua-aar; aas-{ Bllap 'aFcalour aleJlsqnsp urorJ suoJlcala o^A]snld uot -ord e o1luale,trnba '(_11)uor eprrp,{qu salotuar tpqt eudzug asuuoto.rpfqap uollJuar uoppsuopuoJ aas-uorlJpal

uollurpfqap

'alduexa toJ 'prJe ourrue atues aql roJ apoJ lEr{l (suopoc) saspq apr}oalJnuJo suorleu -rq_tuoclaldrrl luaJaJJrp:8urql eruesaql ot lunotue lEql salpls aldrtlmu Surqrrcsapa.rrlcefpBue ]nq luaur8pnl leroru e tog alu.reuatap fumqlud rkolaroes alrlnlrlsuoc aas-,{emqlud froloJJes llneJap (9-91 arn8rg) 'pue31 relnllarerxa str spurq U ueq \ srsoldode o8:apun o] IIaJ arll pu8rs uec leql ulalord roldecar aueJqruausue{ roldacar qluap

aqrorr'nba ^","-f"i';8ii3:J'#il1:,:?i::tfl; rossp."

uolPp

lorac{tybupaas-CVO 'ua8oqled relnllaoerlur Jo ad& .raqlouero snJrl P r{]I^{ pa}laJur sllac SullF-{ roJ alqrsuodsar tsot{ 1ec 1;o addl (llar I rallpl) 1ec I c1xo1o1b

'srsaq1u.{soloqdpue uorlerrdsarSurrnp suortJala sJaJsuerlleql ulaloJd Surureluoc-autaq palolol) aurorqcolr(c xalduroc turlouro.rd-esuqduue aas--aurosoyc.rb (9I-ZI arn8rC)'sula1ordla8rul cgrc -ads Surleldroqdsoqd [q alcfc uorsrlrp-llar ar{] uI sdats tuo -rag;rpre33rr1sexalduroculcdc-1p3 luaraJJrq'lJe ol rapro ur uraloro urlc^c e qlr.^M paxalouos aq ol seq lEql aseuDluraloJd ({pf, ) asuuq ruapuedep-u11cdc (€90I 'd rI-ZI alqe1 pue'1I-LI pup 9I-ZI sarn8rg) 'pale^llrE Lluped seuoraq uaqt ({p:)) aseuq tuapuadap -ur1cr{c y'sasearcur urlc.rtcrelncrlred e;o Ia^al aql se alcfc 1ac ctlodrecna aqt Suunp dlecrporred pauroJ xaldruoc uralord xeldruoc 1p3-u11cfc 'lxau aqt o1 alcdc flec aql Jo e8ets auo tuory uorssarSordlorluoc dlaq,,tqaraql pue (sIpJ ro 'saseuDIurato-rd]uapuadap-urlcdc palpc) saseuDlulatoJd Iercnrs ale^rlf,e su11c,,t3elJfc IIar Jrlo^rerna aq] qtllv\ dals ur uopertuocuoJ ur slleJ pue sasrrdlpcrporrad ]eql ulalord ullr c 'spu8rs lplnlleJu.rlxasnorJel o1 asuodsar ur esu1c.{c1d1.,{uen3.,{q dIC uro4 patereua8 sr }eq} apuoalcnN (dIAICc)4y419c11cfc (g€-9I arn8rg AhlVcrlc^r rplnlacertur ur esrr e o1asuodsarur suralord 1e8re1saleldroqdsoqd ]Bqt atu^zug (V;1d 'V aseur)I ulalord; oseupl u1elo.rd luepuadap-4141y cglcfc

-su1dopua.seqr,.,rr"11".rn'.,:'';o$3H"tt-tH'3r1-#llt'dl.lii',t"# 'ruse1do1.,{caql Jo luaut:eduroc

ureru aql Jo stualuo3 IosolAJ

edq4ytryolpazd1orp.,{q.,'?l;3r:;'f;tl;il:i31Tiffi f :::$ -4yqyc 3ur1e,r.r1ce dq.{lureru'iolnoalotu Sulpu8rs rc1n11acErtur

AUVSSO]9 01:9

'alr^r IIac al{t Jo aseqd-S aq} o}ur .,fu1uarog parlnbar sugalord apocua 1eq1saua8 dueur uo saqr4ms 1eq1uralord fuole1n8ar auag ulalord g7g 'suaSoqled aleurrulla dlaq reqr sailco8eqd pue sa{coqdur,{1 palenrlce are'alduexa roJ'tuel 'ssecord relncrl -sds aunruur aql Jo sllal rolraJJa ureu aqJ -red e ur uorlJunJ purJ ro asuodsar aq] sarrreo ]no leql ilal IIar rolraJJa 'ualsr(s sno^rau pue sluJap -rde aqt;o ros:ncard aqt sr teqt anssrl Ielloqllda cruo.{-rqug ruJapolJe lloc qqclraqcsg aas-!|oc' g 'salnqnlonru Suop luaru -aloru ]uepuadap-dlv sao8rapun teql uralord roloru a8rel ulauap 'uorlpuroJ alcrsanSurlaldtuoc ul paAIolur sr qJrq/r puu 'auerquau aqt tuo4 Suppnq;o ssecord eql ul alJlsa^ paleoc-urrqtPlc e Jo {rau aql ot spurq tpqt asedJc f,rlosol^c ulrrrBu p G L6-BL6'dd'7-91 1a-ue4) 'luauPIIJ urlJe ro elnqnlorc[u e se qcns ]ueuelrJ ulalold e ur 'esJeAaclApue 'a8elurrqs ot ql,r,ror8tuorJ uorsraluoc uappns ,ir1lqursul cprew(p

Iapou V

'dg

'vNCI papuPrls-alqnoc ygqxeldnp 'sJrleue8JPInoaloluur rusrue8ro tpr; p palpc dluoruuoc 'dg lerus ;o sarcadg nlsaSouo1au qgqdosotq

(9-7 arn8rg) 'xllaq e olur punoM arp 'saspqaqt uaa^{laq Surpuoq uaSorp[q ,,{q raqtaSot pleq 'sureqc ygq 1a11erud -llue o^\t qrrqM ur 'VNC Jo atntrnrts puorsuaulp-aerqt eql 4laq alqnop (99S-r99 'dd 't-g 'aua8 eqr;o ddoc 1aue6) Ipulou e;o ecuesard eql ur uala adlloueqd uorlcun;-;o-ssol e Sulsnec pue.{1r,rr]ceaua8 3ur1co1q 'adftouaqd aql slraJJp.{ltueuruop leql uor}etnhl uollelnru anpetau luuulruop (S99-t99'dd'1-g 'alrssarar;o alrsodd6 ']uasatd aru salaile qtoq q8noqt1aue4) ua^e {lou sI alalle raqto aqt allqrvruslueBro ue;o ad,ftouaqd aqt ur passerdxasr tuqt salallpJo rled e;o raqrueru aq] 'scrlauaSuy lrrEulruop 'surelord lueregrp .,tueu ut pezruSocararp sureruop sno3o1oruo11'ureqc eprldad.,(1od ;o suor8ar alqxag poqs .,{q txau oql o} pa}rauuoJ qcea 'sureruop pasodtuoc ,(leraua8 are suralord ra8rel 'ur o slr lprales;o Jo arnlcnJls drerlral e seq lpql uralord e Jo uortrod (ur-Pruopu1a1ordl ur-Btuop (gt-02 am8rfl 'srorunt asnec uel ]eqt sasnrr^vNC luoraJJrp;o ,fiaFen e JoJ rural plauac

'ape.., srarnraro..,vNC eJo,iao,q'qtl;ijtt#. " (11-9 arn8rg) 'srsaqlu,{sVNC roJ raruud e Surcnpord ,e1e1d -urol VNq p uo VNHJo puprts troqs e sazsaqlu,{s 1eq1eulzug esuurgldypq 'aprn8 e su ale1drue1ygq e Sursn reqtaSol saprloalrnu Sururof dq VXC sazrsaq]ufs 1eq1 arufzug asereur^Iod VN(

osID aas'uollrsodsuerl alsed-pue-1nc dq anoru^#ilq"ir?l a;l s1rlnoq8norqt VNCI sp stsrxo teql luauala alqesodsuer; uosodsuurl fluo-VNO

(tZ-B orn8lc) der -rporrlru aql ol pazrprrq,{qsr urnl ur qcrqnr'y56c o1 palrair -uor sr sllal lsel uorJ palelosl y1quru :dlsnoeuellnuls saueS 'uoddns alqe Jo spuBsnoql;o uolsserdxa rolruoru ol pasn -lrns Jaqlo Jo aprls adoJsorsftu sse13e o1 punoq (acuenbes umou{ Jo qcea) salnraloru VNCI lroqs Jo derre a8rel y ferruorcgu ypq 'etels a^rloeul ue ur saua8 daal ol salErqaua^ ur pasn sI secuenbascc ur aseq aursol,{c aq} 'VNC ol sdnorB lfwatu Jo uol}ppv ;o uorle/.qlau e^rsuelxa uope{qreuVNO 'pueJls VNCI snonurluoJ P a{eru 01 puoq }uoP^oc e qlr^{ raqle8ol vNC Jo spuels o1vrtJo spua eql sulof ]eq] elufzug asetgytrlq '(drerq11y111qc) 1ac e .{q pacnpord y1qgtu aql Jo saldor ygq .,{reluaueldruoc Jo (-fuerqrl cruouaS; aruoua8 arllua up raqtra Supuasardar 'salnJaloruVNC pauolJ Jo uoltrallo) ^TBTqIIVNO 'uoqecqdar y111qro; spuerls a13urs slr otur xlaq VNC aqt Suruado ur pello^ur sI leql atudzug asBrlleqvNo (62-Z am8ril 'spurq uralord Surpurq-ygq e qcrq^ ol ecuenbas VNC aqt 3u1u1ura1aprog anbruqcal tupul.rdlooyypq (97-2 ern8rg) 'paqcelle ere sluatu8er; VNC ete -rrdo.rdde aql qcrq ^ ol xlrletu e o1 Surpurq rraqt ,{q suralord Surpurq-ygq cr;rcads-ecuanbas 8ur.{;rrnd ro; anbruqce; ygq fqdurtoluurorqc,$p1ge (ZII-9II 'dd'g-ZIauBd pue g-7 ern8r4) 'uoneraua8 ol uorle;auaB ruo4 uollpruJoJur slql Jo JerJJeceql pue IIaO p urqtr^\-uorteurogur-{relrpaleq Jo ero}s aql 'slrun aprloelc -nuoqrr'{xoap pe{u11dpuap^oJ ruo4 paruroJ apqoalcnudlo4 (plre rlolrnuoqFiixoap; yNq (97-g arn8rg pue '/0I 'd'1-7 1aue4)'s11ac crlo.fuecna 'uralord atues Jo unlncller cruseldopua aq] ur paurog eql 1o slred ]uaraJJrp Suqurt ro raqlaSot sulalord orru 8ul -urof ;o.{e.n uoruuroc e 'surelord relnllacertxa rog 'seurelsdc uo sdnor8 1.{rpdtglns omt uaam}aq paruroJ a8e1u11luap^o3

(-s-s-) puoqapulnstp

(tt-€ em8rC) 'tuelsuoJ runuqnnba 'luels -uoc r{lrur;;e osp aas 'fy ;o pcordrcar aq} sr (qy) }ue}suoc uonercosslp oqJ'g pue V uaamlaq Surpurq aqt ralq8p eql rallerus sr tr pue 'tSV1/tSllvl 4q ue,u8 sr luetsuoc uorlprcossrp aqt 'gV + g + V runrrqllrnbe Surputq aqt pup g pue V slueuod -ruoc roC 'alercossrpo1xalduoc e;o ,{cuapual aql Jo arnspalN (DI) luElsuoc uopulrosslp ' proldeq atn du.to,' (sncol crlaua8 ro aua8 qJea Jo sardoc o,r,r]acuaq pue saruosoru -orqc snoSolouoq Jo slos omt) atuoua8 alqnop e Surureluo3 pIoId!p 'luarua^oru lutuJaq] uopuEJ ol anp uorluJlueruoc Jo^ ol Jo uollcalrp aql ur selncelour Jo lJrJp laN uo!snJJIp 'ad,{1 1ac pezlprc -ads dprano ue o1a8ueqc e seoBrepun1ac e qcrq,r,r.dq ssacor6

's11ac peurelsun ^\ap01pasn'saxapur Bur,rll

"^';r"Jl|itll::** -raJJIpJo IIar p Jo slred q8norqt sassed tq311uar{,lr rncco tpr{l slJaJJaocuereJralur eqt slloldxe 1eq1adocsorcnu 1q311;oadd; adocsorcrru lserluoJ-eJueJeJJelu!-[BllualaJJlp (0s-B

arn8rC) 'Surcuanbas y11CJo poqlaur crleurikua prepuets ar{J PoqleurrkoaP;P (Bg-gI arn8rC)'(D)d) C eseurl uralord alenrlcp dlaq o1 apc -a1ou Sulpu8rs fierus p sp salJas 1r '1orac.4.po] pa{u[ surcr{c

[ [:9

AUVSSOI9

'luarudolerep Ierulue 8ur;np suoxe flac alrau pue sllar Jo uoper8rur eql aprn8 1eq1 sasuodseruol}cerllp ro uorslndar elelnluDs ,suollJunJ raq]o ,,tueu Suoue 'leql (s)IU) saseupl aursordl roldacar qdg eql ro; spue8rl uralo.rdpunoq-euerqruaur;o dpue; p Jo euo ulrqdg (29-9I arn8rC)'surrqdg sazruSocarleql ()IU) aseuH aursordl rotdacer go ad.ft snorarunu tsou aql .roldacar qdg (9I-gI arn8rp'roldaca: aqt ot Bupurq pue8rt rulnlacerlxa ue r(q petepru-rls sr dlnrlce cueurrkua aql 'asec Jaqlra ul 'atu,{zua relnlleJprlur ue qll^\ palercossp sr ro ,{1vrr1cec1}eurtzua seq teqlla leq} urcruop crurseldolb p spq teql rotdecar arpJrns-llal go ad,! rofetu y roldaco.r peldnoJ-ouilzua 'uollJear lecruaqJ crylcadsu sez,{plec }Brll ulalotd e 'llec aq] tuo4 spnq snrr eql ueqm auerqueu 191-y7_arn8rg) etuspld IIaJ tsoq aqt ruo4 pa^uap sl r{olq1!\ ,(adola^ue eqt) auerqruau ra.{epq prdn p ^q papunorrns plsdec e qtu\^snrr^ sn.rprpadolalrua (6II-BII 'dd'Z-ZIeupd) 'raproslp eqr raruar8 aqt .{dorlua aqr raqBrq aqt luralsds e ur ssauruopuer ro rap -rosrpJo aar8ap aqt sernsearuleql dlpuunb crureudpouraq; g) fdorlua ,{e,r,resrred aseq Jo spuesnoql .{ueu aq uec leq} auaBprnlcnrls e ;o uoudrrcsueJl Jo aler eqt Sursearcur,purq sutalo:d,{rolep8ar aue8 qcrqm ol acuanbesylrlq.,{rolelnBag JaJIIUqUa

slassel clreqdru,{1pue poolq 1e 8ur -url (urnllaqlopua e{l) taaqs e sruroJ }Bq} ed.{r flac pauallplC IIec IBIIaqropua 'uorleper8ap ro; saruosos.,{1 o} uo uoql ;o rtueu sassed pue slsolrtcopua rtq palsa8ur dl,rnauslelJaletu sarJJBcleql sllac 1erurueur ellaue8ro papunoq-auuJqrualN aruosopua (99-71ern8rg)'apuu ere sulalord fuotarces pue suralord punoq-auprqrueu pup pezrsaqlu.,{sare sprdq aJaqM 'slleJ cportrecna ;o urseldolrtc aql ul tuaulreduroc papunoq-euerqruau au1qlu1r,{qe-I (U[) tunpcpa.r cpusuldopue 'oseelonuoxaalnduo3'urcqt aptloalcnudyodarputqum spnp JralJnu selealc 1eq1atufzug asualrnuopua 'sue8ro pale -rrosse pue ln8 eqt rosrncerd aql sr teqt anssrl cruodrqrug Jo ruJapopua 'srsoldco8eqdpuu srso{courd oqo aag'alrlsal popunoq -auerqureru E ur uorlezrpuJaluJ sU pue eueJqruau euseld aql Jo uo11eurSeaurue dq 1ac e otur pualeu ;o a4u1dn slsolrbopua (9-9I pup 3-gI sern8rg) 'auoruroq cgrcads aq] purq leq] sroldacar a^pq lpql slec 1e8rul tuelsrp ot palrrer pue ureerlspoolq aqr olur spue13auJJJopuodq paseelar sauoruroq er,r Sulpu8rg tugeuSls oulrropua 'pue13 drelrnlrd ro pro-rdqtaqt se qcns 'pue13e 1o lred dlpnsn 'poo1q aqt olur euouroq p seJeJcaslpql IIaJ lerurue pazlprcadg fiac aulJJopua 'uroql etarcas teql spue13aqt ro souoruroq o1 8urle1ag aulrcopua 'lurulue cruaBsuert e dolarrap o1 1si(colse1q e otur pauasur pue 'parJrporudlpcrlaua8 'aJnllnc u1umo.rBaq ue3 ,{poq aqf ur sllal aq11e o1asr:8uvr.r3;oalqede3'odrqrua

uulprulllelu dlrea ar{tJo ssEruIIes reuul oql uorJ pa^Irap IIol) (IIer Sfl) IIar uals cluofrque fqdurtouol adocsorcpu-uor1ca1a aas-fqdertoruol 1419 'ureqc apqdadfyod Sur,r,ror8aqt ot splce oultue 'pue eurosoqlr aql Jo uorlrppe eqt a up i119 3u1z1ppqdq,Q ol pulq srolJeJ uorle8uola 'uorlelsuprl uy 'Surlercossrplno -q4,rt VNC Jo saqclarls 3uo1 eqlrJsuert ol tl lvrollppue aspralu -d1odyNg qllm atelrosse srolceJ uorleSuola 'uotldtrcsuerl uI 'uoJlelsuPJl puu uorlducsueJl I{}oq uI pasn arnlelcualuoN GA) rolcuJ uopetuole 'ygq u8taro; oq] ot alqeaurad Lypurodrual auerqruaru IIaJ eql seTelu lcoqs clrlrala Jalrq e qclqM ul 'eualrpq flercadse 'silac olul y111qSurcnportul roJ poqlantr uopu.rodo.rlcala 'plau rlrl -ca1a8uorls e ol petcafqns uaqm tunlperu snorod e q8norq] uoller8nu ;o paads rraql Jo srseq eql uo (splJe clalcnu ro sulalord ^r{pcrd.rtl) salnraloru Sulleredes ro; enbruqce; sgsa.roqdo.rlcele 'srsaqluLs 'gI-tI pue dJV a^Up €-tI sa.rn8rJ,{/lnnadsaaag ot pasn sl ]eql ouprquau aqt ssorre luelper8 uolord e ale -raua8 slseldorolqc Jo ouerquratu pro>1e1i(ql aql q pue (upqc .,{rolerldsar aql pallec) auerqrrraur Ielrpuoqcotltu tauul aql u1 luaserd suteqc lrodsueJl-uoJlcalg 'sessacordsnolrel ra,r,rodol pesn oq ueJ luarualoru uoJlcela Supnp peszalar ,{3raua rarwol ,{8:aua eqJ alncalotu roldacce purJ e o} 's1a,ta1 .,tla,ussaccnso1 raq8rq e uorJ {Ieqc aq} ur!\op, suoJlcele ssed salncalolu JerJJeJuollcala qclq^\ uI suolceeJ Jo solros uJeqc godsrrun -uoJlJala 'a8eur puorsuaulp-aarql e an13o1 ,tleuotletnduroo paulquoc ere smar aql'raploq uarurcedsaqt 3up1p,{q suol}Jarlp luereJJlp ruo4 ualel arp smarl eldrtlnur qclq \ ul adocsorcnu uorlcela aql uI suorurJads puolsuaurlp-aalql Suunarn ro; anbtuqcel (fqdurtouol 1419),{qdurtourol adocso.rcpu-uorlcala 'a8eut aql alearJ ol suoJlcela Jo lrreaq B sasn 1eq1 edocsorcl6l edocsorcJru uoJlJala 'ssacordaql u1 pazrpuo Surtuoceq'uortrala ue dn senr3.{1rsea }pq} alnralol Jouop uorlJala '99-7 arn8tg pue 'upqc lrodsuer uoJlcela osf) aas'salnf,aloru roldacce ol salnJe -lorrr Jouop ruo{ ruaql sJeJsuerlpue suorlJala seIJJecqcll{rvr ZHCVCpuE'HdCVN'HCVN'c etuorqcolf,c sEqcns elncalol Ja!JJ?J

UOJIJaIA

'peJnper Sururocaqpue uoJlcala uu Suturu8 rtqaraq] '.{lrpear suorloela dn salu1 lpql elncalotu lo tuotv .roldaccu uorlcale (1-7 arn8rg) 'ruole ue uI snalrnu aqt strqro tuql alcrlred rlluoluqns pa8reqc d1a,'rrle8a111 uoJlrele 'luerperB IPf,Iruaqrorlcele rlaql umop ,,{1uodla.trssed a^olu uec selncolotu pa8reqc ro suol '(lpuuatod auerqurerul auBJqruetu aq] ssoJceecueraJJlp a8reqc pcrrlcala aq] pue ouprqtuatu e Jo sapls o1\Aluo uol ue JO uorlerluacuoc aql ul af,ualeJJlp e Jo aluanuul paulquJoc 1ue1pu.rtpcpuaqrorlcala 'sanssu a^llcauuos ur (sJequ crlsela) sraqrJ alqlsuaua suroJ leql ulaloro relnllacellxg ulls?Ia 'acualllsar pue &gquqclarts rleql uraql san13qctqivr's8un1 pup 'slasse^poolq 'upls ur se qcns 'senssF o^IlJauuoJ [eru -pz .(ueur ur ups€Ia utalord aql ^q pouuoJ raqg elqlsuax[ raqu rIlsBIa ro csJ uollBll1u1 cl1oi{recna aas-dle

AUVSSOI9 Zri'

'alnralotu puooas e ol pa)ull uoqm sralsa 'dd't-Z laued) uro; dlensn sdnorB aluqdsoq4 'dnor8 ctprce ue qtvvr dnor8 Ioqocp ue Jo uollsEar uollesuapuot eql fq paruroJ "t.t"to1"r."

'g31ro '1€1 '931 se qcns salpoqllue Jo sassulr luaraJJrp -ro; cr;rceds ere sroldacar r{ luaraJJrq 'e1nc -alou (ullnqolSountuurr) dpoqpue ue;o (uo18arcg) uor8er p Jo auo lel aql purq teql sroldacar arpJrns-llrr ;o ,,(11ueg roldacar eg

(IZ-I arn8rC)'(eu -alJeqapqJJe)eoel{Jru ol uollcuUslpPrluoc uI 'euo}ceq an{ EIJaIJBqna

u.9-€ puP 99-t sarn8rg) 'r{enqted erues aqt ur uorlceer snor,rard e rlglqul ol IJeq spaal uorlJeal e Jo lcnpoJd e qcrqM ur ssaJoJoeql uolrlqHu! {ruqpeaJ

(17-1 arn8rg) 'eapqJrv pue elratceg Suraq o,r'q raqto aq] 'pporvr 3un11 'snelcnu aql Jo suorsr^rpureu aorql aql Jo euo Jo JaqruaIAI ]Jurlsrp aleq teql sllal aJoruJo euo;o pasodtuoc urstue8rg " (a1o[.ru1ne) elor{rucna

(IZ-Z arn8rC) '+CIVNot HCIVNJo uolsra^uor aqr qlp\'loueqta ro elelJel olur paua^uos sl alp^nild 'asuelsul roJ 'srs^loc^I8 crqoreeue uJ .{Emrilud Jrloqplaru 3urp1ar,,t-,,i3reue crqorapuv uollEluaruJaJ

(9I I-tII'dd'E-Z1eue4)'sprdlloqdsoqd;o stsaqt -uds aql ro; lurod 3ur1re1se pue ruslloqelerx 3u1rnp,{3raua;o acrnos ro[eru y'llet uoqrprorpLq 3uo1e qllm prce ctfxoqre3. plre ^llBJ '1orecd13qtrm paglratsa sprce ft1u; 'sllac ul pldl a8urols-.{3reug ;o pasodtuo:) IPJ

-saprrac,tlSrrt

(9-91 arn8rg) '(pue311sug) pue3l relnllacertxe str spulq tl uaq,vrsrsoldode salElllur leql roldacar qleap auEJqueusuerl (roldacar qleap seC 'u1e1ord sug) sug assupl uolsaqpB IBJoJaas-)vd (gg-7 arn8rg) 'zHCVd rarrrpr patelltrp aql Suruoceq ut suolord orru snld suoJlcala o.tq sule8 CVCJo elncaloru auo 'a1cdcprce JlJllc aql ur suorlJunJ teqt ruals.{sJaIJJEcuorlJalg (appoelrnulp au.IuapP auluapB uI uU) ZHOV{/(JVJ ul^uu pernpar/oplloalrnulp

lrodsuu.rl anlssud aas-uoFrqJlp palerllruJ 'roldacar aql SurssardxaIIar aql Jo ,irFtlrce aq] saleln8ar pue sroldacar ol spurq leql pu8rs Iprltuaqo areJrns-llar ro palarcas ,{uy elncalou putls rBInIIeJBrtxe 'pappaqura are sllac aqt qcrq^a ur pue 's11ac.{q petarJes (ua8e11oc se qcns) surelord pue (osolnllacro suecdlS -ouruesocdlS se qcns) seprreqccesdlod ;o ryo,r,r1euxalduo3 xlJluru JuInIIaJBJlxa (97-g arnBrg)'acuanbasaqt r(q pepocue uratord aqt Jo srsaqlu.{s eql stcerrp ereql pue IIac }soq alqe -llns e olur acuanbas VNC e sarrJec lur{l prurseld ro snrrl V rolcarr uolssardxa 'eseelrnuopua ataduoS'saprloalcnudlod ;o spue aql ruoJJ aull p }e auo seprloelcnu se^eelc leql otu^zug asualJnuoxa 'uorlul uu pallEc lueu 191-7arn81g) -8as ygg Sutpocuou e o1 luace[pe ,(1ensn sl uoxa uV 'ulel -ord aql ul splce ouFuz aql apocua suoxa 'saua8Surpoc-ura1 -ord u1 'alncaloru VNU arnleu ra_qloro 'pruosoqlr laJsuurl IPUrJaqt ul ro vNuru ur petuasaroer aq IIV!\ teql seplloelcnu ;o acuanbas p Jo slslsuoc teqt aua8 cqor{recnae;o luau-ttag uoxa 'aplslno aql ot sluoluoc raql asealorpue auerq -uaru etuseld eql qrlm asnJ leq] salJrsal papunoq-auprq -ruaru olul pe8elcud are salnJalotu eseqJ 'llac crlodrecna e IIIoIJ polarces aJP salnJaloru lsour qJIqM ,tq ssacor4 slsolaJoxe rpudar uolsgcxe eseq aas-rpdal uolslJxa 'ulleuroJqcoJa -leq pasuapuoJ arou eql o1pasoddo se 'urleruoJql ,,pruJou,, :.r{lesqgp surets }eq} eruosoruollr aseqdralur ue ;o uor8ag ulluruoJrIJna 'uoJlelsuer1 Surlurlrur snql 'atuosoqlr aql ol uo VNUI roterlrul peol sdleq leql ulalord (dJa) rolcuJ uolleFlu.r crlor(recno

(z0r-90r

'qf,reaser pclpatuolq ul pesn dlapp,r pue sletutuptu raqlo puu supunq Jo uoloc aql ul punoJ Allerurou runlralrsq a{llpou ftloi'iil lloc DlqclraqcsT IIar rrrals cluofrqrue aas-llar

Sg

'^\orJeruauoq ul sllec poolq parJo uollsnp -ord aqt salulntups teqt faupDl aql fq pacnpord auouroq y upagodorqfra (6y-11 arn8rg) 'sanssll 'ruor; epxorp uoqJpJ pue 'ol ua8dxo suodsuerl leql solprqaua^ Jo IIec poolq Sururuluoc-u1qol8otuaqfiurus (ilac poolq par) aybo.rqtl{.re .{r1ua ragu asuprlded pu8rs dqgo pa^Eel3 '(U[) ulnp]Itar cluseldopue aql Jelue ol sutalord slJaJIpteql ecuenbaspu8rs pururel-5 acuenbes put;s gg 'araqt uorlcunJ pup Ug aqt ul luaplsat aru leqt sulalord uo punoC '(Ug) unlnrpar crurseldopua aql Jo lno Surrour ruorJ lr slualard 1uql u1elord e uo acuanbas plce oulrue lroqs putgs uopualar Uf, 'Usuerl ur dluo UA aql ul lueserd aru teql su1a1o.rd o1 pasoddo sp 'eraql uollJunJ sll lno salrJet puu sauuJqruatu str ro (Ug) unlncFer cluseldopua eql ul suletuar teqt ulalord ulelord luaplsar Ug '(ug) tunlnrrtar cruseldopua aqtJo euerquau aql ,tq pasolcue acedg uolunlut run1ncllar clursuldopua aas-gg (€t-€ ern8r{) 'lu"lsuor uollElcos -srp 'luelsuoc rirlur;;u osp aas '(gv = g + y) uopceer Supulq eldurrs e roJ gt) luelsuoc rllurge ro uorlelrosse eq1 o1 pnbg 'uollJeal puP preMJoJ Jo ollPu aJPJ asJa^ar e roJ sluelsuoJ 0,) luulsuor rungqlgnbe lueulruralap cluetgtue aas-adotgde ,finec e 3ur -url Jo eJnlcnrls e Jo aJeJJns:alno aq1 Sut.taloo sllac Jo laaqs (eqeq11daprnld) runllaqrgda '11ac-toruslue8Joarues aql ur luasard are suoltelntu qloq uaqm aue8 raqloue uI uopelnru e Jo lcaJJaaql s{seu leql eua8 auo uI uollplnur B seqlJcsaq cltutslde aulleuarpe aas--eulrqdeuJda (98-Z pup gg-7 sarn8rg) 'uolteruroJ ullpruorl{ror -alaq Sursnuc suorlucglpou auotslq ro uo11u1,{q1auVNCI se qcns urleuorqJ ur suorlergrpou alqelrJeq01ro sutelord.{ro1 -eln8ar auaS;o sdool lceqpee; e,rrttsodol anp aq uel 'VNC ;o acuanbes eprloelcnu aql ur sa8uuqJ ruoq llnseJ lou op tuql uslue8ro ro IIaJ e ut sa8ueqcctd.fioueqdgo eJuellrequl arrrullraqu! cpauatgde 'snureprda aql pallec os1esI anss! luu1d;o ra,{e1 ratno aql 'sdnor8 prulue luaraJJlp uI sarntrnrts luareJJlp sug .4poq aqt Jo areJrns ralno oql Supa,roc ra,,(e1prtaqtrdg sgturapgda

AUVSSOT9

€[:9

'tuals.^.s sno^reu Ielluec aql aprslno palpcol .sllacprF patp -rcossppue sarpoq IIoJ IeuoJnauJo dpreur SurlsrsuoJralsnl3 (e11tuet prnld; uo113uet

rraqlsdecpuesalnqnloJcrrx JoJolpalc.,,,.rnrrrrrT$::l$il suralordraqlo pue urnqn]-1,Surureluoc xelduocuraloJd (lgry/t) xaldruoctug

'urseldolb aql ul stuauelrJ urtrp ol'suleloJd raqto q8norql rpe{ull eru qcrqm'surr8elur se qcns sugalord aueJqruerusuprl dq paleparu sr lueuqcellv 'xrJleru Jplnllac -Prlxa aql ol paror{cuEsl ruq} IIac raqlo ro lselqorqu e Jo aJeJ -rns aql uo uorSor lplus e Sururo; 'uorlJunf IIoJ SurJoqcuv (enbuld uolseqpu 'lJDluoJ Ieco;) 'uo.rsaqpu pco;

(97-g arn8rg) u11nqn1-ururuu8 'sllar ur (uorlreralur rraqt snql puP) salnsalotu pelaqel flluacsarong oml Jo ssauosolcaql Surrotguou ro; anbruqcal (If,UC) ra;suer1 i{traua oJrreuosal acuacseJong

'11acutn8 os1oaag 'uollcnporder Ienxes JoJ pazrprcads pue srsoratu dq s11acrura8 prprou -rrd uorg pauroJ @3a ro tureds e) 11acproldeq pazrpicadg alaurut

(9q9-t99'dd'1-g 1aue4) 'lueurruop dpnsn'saJuelstunJJrc alerrdorddBur ur eAIlJe lr saletu ro 'aua8 e ;o .,ftrnrlce eql saseaJcul leq] uollulnlN uopulntu uo;1cury-;o-uJe3 uuc,fitouluresoefp ulalord g frolelnrups

aas-gyg aas-sg

.{emqted 3u11eu8rsprdrtoqdsoqd Iotrsour aqt ote^rtce ol $-3 asedrloqdsoqd o1 sgld5 saldnoc 1eql ulelord C Jo sspll

bC

ulalord I {ro1Jq1qq aa5-t9 atuuqc ftraua oa{ pJppuuls aas-orv atuuqc dtrau a oat! aas-+)V d8raue ea{laas-{ 'auaB lueurquorar e ruorJ pecnpord .sepqdad,,tlodeteredas .{1eu.rou arorrr Jo oml saurqruoc leql uralord pereaurBug ulalo.rd uolsrg '€ung dq pasnec aJe saseaslpprurue Jo Jaqurnu IIErxs d1anr1u1e: e pue saseasrptue1d,{ue6 'sruoorqsnu pue ,splou 'slseed aqt sapnlrur ]eql susrue8ro cqo.{recna ;o urop8ur; (1turg prnld) sntunJ re;suer1 dtraua acuuuosal acueJsalong aas-Jilgd 'pasodxe salpJ roualur aqt qtyv\ sra,{elouotu o.Mt aqt olur 1r Surluredas tefelrq aql Jo Jorralur aql Suop paJnlce4 sr IIac uezorJ e Jo aueJqruau aql qJrq,l ur 'arnlJnJls auerqruau 3ur.,(pnls ro; anbruqca; ,{.docsorcpu uoJlJala eJnlJu{-azaa{ -tueru.,tue o] paqreneun losolr{c aql ur aa{ q ,nql

"-"::;iuq aa{ euosoqlr

'a8euup VNCI relnllarprlur qrnu roJ elqrsuodseg 'uortcala parrzdun euo lspal le Sunpq stl Jo anul^ ^q azrrlceerdlauarlxa sr qJrqM alnsalou ro tuolv lBJIpEr aa{ (6I I-BI I 'dd' L-Z laupd) 'rncco o1.,(cuapuelSuortse seq uorl -ceer aqt teql setuorpurgVJo anp,r anrle8au a8rel y 'salncalou Surtruts aql Jo .43rauaae{ aqt snuru salnralour lcnpord aql Jo,{3leua aarJaq} :uopcpar e Surrnp,iSreua aa4 aq} ur e8uuq:) 19y) atueqc dtreua-aa{ (6II-BI I'dd' Z-Z1aue4)ddorlua pue d8raua qloq ur sa8ueqc lunocle olur sa{eJ .suorl -ceer alrJp ol ruets.{se uorJ palJerxe eq uec 1eqt.,{8reuaaql @treua aa{ sqqlC) (9) ftraua aar; tulqcealqoloqd

ra1;e i{ranorar eJuarsarong aas-dVUC

tupugdloo; ygq aas-Supupdloo; (77-17 arn8rg) '33e ro aldcoo Surdolarrap e punorrns 1eq1sad,,{1IIaJ aql Jo auo IIar alcllloJ 'surr8etur Jo sltet crurseldoldc aql ql!v\ uorlploosseur (suorsaqpplpcoJ) suorlcunI xrrleru-llac le ]uasard aseupl aursor.,t crusuldo1,{3 (XVC) asuupl uolsaqpu luroJ

'tqBII rosel tueaq e .(q paqcealq Jo IIac aq] Jo eere ue olur alorrr salnooloru uralord luacsaronlJ ,uoq Surzdpue .i{q urelord pJo sJalaluercdcllauq eql SurJolruoruroJ enbpr{ceJ (dygJ) Sulqcealqoloqd regrp ^ra^orer eruersarong 'sarsaJonu a.,tpaqt uaqM pall[ua sq]8ua1aaelr.asoql dpo 1ca1asot 'a^a eql seqcearll aroJaqsratlrJJo tas raqloue q8norqt pue 'adp aql atlJxa tpql sqlSualo^em asoqt tJalas o] 'uaurrcadsaqt aroJaqsrallrJ Jo tas auo q8norqt passedsr 1q311 Surleururnlr aql lnq adocsorcru IqBII p ol Jplrrurs 'sar{pluac -saronu qlF peululs Ielreletu ^ er^ 01 peu8rsep edocsorJrl edoJsorclur eJuecsaJong 'nlrs ur soJuanbas yI\Ig ro VNCI crJrcadsol azrprrqdq saqord prsp clelcnu palaqel dltuecsarong qcrqM ur anbruqceJ (HSIf, ) uopu4pytqfrlwgs ut acuacseronu (7-g arn8tgl 'aJuacsaronlJrraql ol Surprocce sllar suos leql aurr{JelN (Sf,Vfl rafros IIaJ palE llrp-aruarsaronu 'uorlprper paqrosqp aqt ueql (qtSualanelr Je8uol) ,{3raua rorl ol p 1e sfernp sr uouerper pallftua aqJ 'alels.(8rauara.&\ol F(uJou Jlaql ol uJnlaJ'tq311;ouorldrosqe r{q palrcxa uaeq Suneq 'suorlcele rleqt se (sauorqcor -onlJ) sacuplsqns eruos dq palrqrqxa uorssrure 1q8r1 eJueJseJonu zgqygTgyg aas-eppoelcnulp

euluepu u! EU

'utntyc andwoS'uoqce Jo rusrueqceru pue uopcnJlsuoc uI '?rlrc luaraJJrp,tleteldruoc pue ralptus are e11a3egPrratceg Jo suorsral ra8uol are elle8eg crloI:ecng 'tunrparu pmg e qBnorqt '3uo1 Iac e a^lrp suo[eppun asoqm uolsnrlord a>111drqrvr (eletug prn1d1 tunlleteg 'uonexrJ palleo sr ssecord eql pue ,'paxg,aq ol pres aru stua8uarasaql qlrm patearl saldueg 'r(dorcso-rcrturoJ sllao azrrasard ol pasn aplxoJlal runluso ro 'ap,,tqappre1n13'ap,{qeppruroJ se qcns }ua8ear lecruaq3 a puxrJ 'sllar pezs-pnba o,r,uarrr8ol sopNp 11'aqutodsactut -otqrlnasozlqcS ruslueSro lapour 1sea.{eql roJ arueu uoruruo] lsBea uolssu uo;ru4plrqfq

r?r!sil.r aruarsaronu aas-Hsld

ILV1I ern8rC)'llar Ietulue Sulmerc e;o a8pa Surpeel eql uo palere -ua8 'aroc tuauel.rJullce ue qllll uolsnrlord a41-a4ds 'uq1 (a{.rdsorclur) (uJpodolg prnld; urnlpodolg

roJ srordorarare areJrnsIIar eqr .ro ,,rr#'r111i"Y;:3ll -a3o.{rqrua Surrnp sllac Surter8ru;o acuepln8 pue xlrtetu aql ol sllal Jo uorsaqpB ul pa^Io^ur ulaloJo xrJletu relnllacel}xg uprauorqu

'alnunJ anssll ul puP anssrl papunoM ur .,(Ipear salera;11ordpue saler8ry4l'selnceloIuoroetu xlrletu relnllacenxa raqlo pue ueSelloc ul q3lr xlrlelu l"lnllarerua uE salanas 'anssrl e^rlceuuoc ur punoJ adft 1ac uoruruo] lselqorqu asuqlu(s dlv aas----asBdlv I coc 'lenpr^rpur ^\au e olur sdola^ap qcrq,u.'e1o8r{zproldrp e ruroJ ol lproldeq qfoq) atatue8 aprual p puu aluru eJo uolsnC uoIlBzIIIUeJ

AUVSSOI9 VL:9

G:15

GLOSSARY ganglioside Any glycolipid having one or more sialic acid residues in its structure. Found in the plasma membrane of eucaryotic cells and especially abundant in nerve cells. (Figure 10-lB) GAP-see GTPase-activating protein gap Sene ln Drosophila development, a gene that is expressed in specific broad regions along the anteroposterior axis ofthe early embryo, and which helps designate the main divisions of the insect b ody. (Figure 22-37 ) gap iunction Communicating channel-forming cell-cell junction present in most animal tissues that allows ions and small molecules to passfrom the cytoplasm ofone cell to the c1'toplasmofthe next. gastrulation Stage in animal embryogenesis during which the embryo is transformed from a ball of cells to a structure with a gut (a gastrula). (Figure 22-3) Gr-Cdk Cyclin-Cdk complex formed in vertebrate cells by a G1-cyclin and the corresponding cyclin-dependent kinase (Cdk). (Table l7-1, p. 1063) G1-cyclin Cyclin present in the G1 phase of the eucaryotic cell cycle. Forms complexes with Cdks that help govern the activity of the G1/S-cyclins,which control progression to S-phase. GEF-see guanine nucleotide exchange factor gel-mobility shift assay Technique for detecting proteins bound to a specific DNA sequence by the fact that the bound protein slows dornrnthe migration of the DNA fragment through a gel during gel electrophoresis. (Fi1.rlre7-27) gel-transfer hybridization-see

blotting

geminin Protein that prevents the formation of new prereplicative complexes during S phase and mitosis, thus ensuring that the chromosomes are replicated only once in each cell cycle. gene Region of DNA that is transcribed as a single unit and carries information for a discrete hereditary characteristic, usually corresponding to (l) a single protein (or set of related proteins generated by variant post-transcriptional processing), or (2) a single RNA (or set of closely related RNAs). gene activator protein-see

activator

gene control region The set of linked DNA sequencesregulating expression of a particular gene. Includes promoter and regulatory iequences required to initiate transcription of the gene and control the rafe of initiation. [Figures 7-37 (procaryotes) and 7-44 (eucaryotes)l gene conversion Process by which DNA sequence information can be transferred from one DNA helix (which remains unchanged) to another DNA helix whose sequence is altered. It often accompanies general recombination events. (Figure 5-66) gene expression Production of an observablemolecular product (RNA or protein) by a gene. general recombination, general genetic recombination-see homologous recombination general transcription factor Any of the proteins whose assembly at a promoter is required for the binding and activation of RNA polymerase and the initiation of transcription. (Table 6-3, p. 34I)

gene regulatory protein " Geieral name for any protein that binds to a specific DNA sequenceto influence the transcription of a gene. gene repressor protein-see

repressor

genetic code " Set of rules specifying the correspondence between nucleotide tripleis (codons) in DNA or RNA and amino acids in proteins. (Figure 6-50) senetic engineering (recombinant DNA technology) " Colleciion of techniques by which DNA segments from different sources are cbmbined to make a new DNA, often called a recombinant DNA. Recombinant DNAs are widely used in the cloning of genes, in the genetic modification of organisms, and in molecular biology generally. genetic instabilitY

Abnormally increasedspontaneous mutation rate' such as occursin cancercells. genetic mosaic-see mosaic genetic recombination-see

recombination

redundancY senetic The presence oi two or more similar genes with overlapping functions. genetics " The study of the genes of an organism on the basis of heredity and variation. genetic screen " Procedure for discovery of genes affecting specific aspectsof the phenotype by surveying large numbers of mutagenized individuals. genome " The totality of genetic information belonging to a cell or an organism; in paiticular, the DNA that carries this information. eenomic DNA " DNA constitutingthe genomeof a cell or an organism.Often used in contrast to cDNA (DNA prepared by reverse transcription from mRNA). Genomic DNA clones-representDNA^ cloned directly from chromosomal DNA, and a collection of such clones from a given Senome is a genomic DNA library' genomic imprinting " Phenomenon in which a gene is either expressed or not expressed in the offspring depending on which parent it is inherited from. (Figure 7-82) genomics " Study of the DNA sequences and properties of entire genomes. genotype " cliretic constitution of an individual cell or organism' The particular combination of allelesfound in a specific individual. (PanelB-r, PP. 554-555) germ cell " A cell in the germ line of an organism, which includes the haploid gameles and their specified diploid precursor cells' Germ cells contribute to the formation of a new generation of organisms and are distinct from somatic cells, which form the body and leave no descendants. serm laver " One of the three primary tissue layers (endoderm, mesoderm, and ectoderm) of an animal embryo. (Figwe22-70) germ line " Th" cell lineage that consists of the haploid gametes and their specified diploid precursor cells. GFP-see green fluorescent Protein Gibbs free energY-seefree energy

'auerquau eluseld aql uI slauueqc uor ro seu^zua Ilelus ol sUCd_Daldnoc dlureru pue strunqns luaraJJrp aatql .sa11tue; a8rel ;o pasoduoc are suralo.rd C Jlrarrlrl e8rq omt olut IIeJ sasedJg aCC ol dlg sue^uor teqt atudzue uV asudlc (SB-7arn8rg) '8ulpu8rs IIac pup ,srseqlu.r(s uralord Alqu_osse elnqnlorcnu ul elol IerJads e seg .dnor8 eleqd -!oqd pulurat stl Jo srsdlorp,,(quo ,,{3raue aa{ Jo lunorup '(areqdsoqdlp aursouenS; a8rel e_sesualar { AIV a{IT dCICJo uortel^{ror{dsoqdaqr fq pacnpord atEqdsoqdut oprsoalf,nN (aleqdsoqdpl-,9 aulsouent; 6;9

(gZ-tI am8rd) 'sllal lprurup ur suucdlSounuesoc -.,t13xrrleru relnllecerlxa pue stueld ur saprreqcces.(1od 'peuos pue perJr IIe^ IIac [ueru yo srseqlu.{s Jo atrs aqt sr ]I -poru eru runlncrler crruseldopua aql luo4 parreSsue4 sprd11 pue suralord qcrq Aur 'secedspepunoq-eueJqrue{upauellpg 's11ac cqodrecna ur alaue8ro xalduo3 Jo Ioets e uo paralual (xaldruoc .rt1og) snlu.redde Jt1o9 (tI-ZI am8rC)'alpurds aql uo luauuSlp auosoruoJqf, ol peal leql sluola crlolnu ,{1rea aqt 8ur-ra38r.rlaroJaq uo4ecrldar yl{6 yo uorlelduroc roJ s{ceqc IIec eq} qcrqm 1e alcdc IIer cllo,4rerne eq} ur lurod lulod{raqr tr\l/zC rorllru IdC aas-ror.lrue 1ol.rsoup(ppuqdsoqdfsoc{t 'elncaloru prdr1ro uralord e ol sJp8nsaroru ro auoJo uoruppv uopuldsoczfiS

rpsrreJra^arsrr.rrrua",rnr(-:,'";'Jr"rll"lt];*Xi;*i;fi it'"::l

'gelg q8norql uorsserSordra33rr1 sdlaq dqaraqt pue a1c.{c 11acc4odrecne ar11Jo IC atel q qpl selplrtce teqt ur1cdl) ullJ1b-S/IC (EgOt'd 'I-ZI alqul pue 1I-LI arn8rg) '(4p3) aseul{ luapuadap-urlcdc Surpuodsarroc aqt pue urtc.{c -s/rD E ,||q sflec atetqeua^ ur pauroJ xelduoc 1p3-u11c.r{3

rpc-s/Ic 'Gccd) -tolce; qynor8 pe^lrap-]elalpld pue (CCg) rotce; qyrror8 pu -raprda apnlour seldruexg 'a1era;r1ordJo alrlrns o1 s11ac3ur -lpln_rullsSurpnlcur 'lla.ir\se suop]unJ reqlo a^pr{ uego,,{aq1 'ir.or8 ol IIoJ e alplnturls uec lpql uralo:d leu8rs relnlacergg rolcu; qyvrort (90I-9I arn8rgl 'atrrpuap Jo uoxp IIaJ arrreu 8ur,r,ror3e;o dq alloru 3ur1er3r6 auoc qynort ',43o1orq ur lalreu IIao e se pasn ,{1app1'qsg:(11at e ruor; patelosruralord luacseronlg (4gg) u1o1o.rdluacsarong uaart (Z€-€Z am8rC) '1ac rolruaSord (1419)a8eqdorceu/ailcolnuer8 e urory sasrry .sflqdoursoa pue'slqdoseq'slqdo-r1neu sapnlcul'salnue:3 crurseldo{c snoncrdsuoc ,r(qpeqsrn8urlsrp IIar poolq alrqM;o dro8ale3 epbolnuer8 (gt-tI pue gg-71 sarn8rg; 'srsaq1u.{sotoqdgo suorlcear Surdde4-1q8rl aql Jo atrs eql alp pue l.rtqdo-rogc urpluoo ]eq1 slseldorolqs ur (sproledql) srslp auerq{uau poTcBls (unuert reln8urs) uuert (0t-9I pue g1-91 sarn8rl) 'euerq -uaur eruseld aql ur lauuegJ uor ro aur.{zuaue laqlre salpl -nce 'urnt uI qctqrrt'uratord p satplrlce 'pue311;e1n11acer1xa C slr dq palelrlce ueq,&\'luql roldacar aJeJrns-llaJssed-ua,rasy (UldC) roldecar paldnoc-u1a1ord-g (616 'd 'S-gI alqel) 'auerqrueureruseld Jql ul slauusrlJ uot Jo sau-,{zuaol suf,dD saldnor lpql ^rl -^Itce asedl5 crsurrlur qtp,l ureto.rdSurpurq-419 clraurrt V (ugalo.rdtupulq-dlC clrarulrl) u1a1o.rdg ((9)6I-0I ernSrg; 'tunlnrllar crurseldopuaaq] qSnorql ..ra)urI apu Ialerl str Surrnp roqcue lolrsoulldplteqdsoqd e ot -eqccuso8goue en'paurof sr urelord aql'auerquau aql o] punoq are suretord auerqruatu aruos qcrqn dq a8e1ul prdrl (roqrlru lorlsoullfppuqdsoqd(soc{3) roqcuu 149 i7-21arn8r1 'slsotrru yo Suruur8aq aql pue s1saq1u,{s VNC Jo pua aql uaa.&qaq 'e1c,r(cuorsr.Lrp-1accrlo,{recna aq} Jo aseqd 7 deg esuqd zC (7-21 arn31g)'srsaqlu-{sVNCIJo ue}s aq} puE slsouru Jo puo aqt uoamleq'a1c.{cuorsrnrp-1acc4o.{recnaaqtJo eseqd 1 deg esuqd tg 'slleopalerlueJeJJlproJ elels 'luJueru;ad saturlauros,uoru -.ruoJy'aseqd tg aql uorJ uorssar8rp]uacsernb e olur drlua ^q alr^c uorsr^rp-llac crl^orprno eql ruo4 IpMelpqlr^ Jo o" ffiu

asu/socfp

y11q aas---ose/soc[1t

(TII-ZII'dd 'saprruqcreso811o pa1ur1-1-rpue palull-O aos 'n-Z 1pued) 'suralordocdlSpue'sprce cralcnu'seprJer{ccesdlodSurpnlcur sarnlrnls snoJerunu ur punoJ eJe spuoq Jrprsocdlc 'Je3ns Jaqloup uauo 'alncaloru Jaqloue puB auola{ aqt ro apr{qapp aql sarrrpf, lpqt IuolE uoqrEJ reSns aql uo dnorS l,,ixorpdq e uaa^\laq uorlcear uorlesuapuoJ e dq pauroJ puoq pcnuaqc puoq clpgsocdlS (q9-6I arn8rC)'urredaq pue 'upuornledq'a1u;ps uuorp -uoqr apnlour saldruuxg'suec^Foetord xrrleru relnllecerua ur aror ulalord E ot pe{ull [puep,roc puno; .,(1uretr41 :e8ns oulue up sLe,rnp sr qcll{,tt Jo ouo 'src3ns Jo rred Supeada-r pa8reqc dlq8q 'reaul '3uo1 e;o pasodruoc aprreqcces.,{1od (OVC) uer{touguesocf18 'surelordocdlSare euerqruau euseld aql Jo acpJJnsJatno eql uo pesodxa suralord lsolu pue surol -ord pelerces lsoIAI'surpr{oaprs prcp-ouruu o1 pa4url ,r{1lua1 -eAoJ surpqJ appeqccesoSllo aroru Jo auo qlp{ uralord ztuy qelordocdlt ' g-Z 1ZI-OA'dd Ieued puu g1-7 ern8rg),,'3u1t]1dsre8ns,, ,t1pra1r1aJV Jo uorlcnpord qlr,r,tpeper8ep flaleldurocur a;e sre8ns qcrq,rnur losofc eql u1 dervrqledclloqetatu snolmbrqn s1sfioil13 (gII-tII 'dd'S-Z lauud) 'paqrelle aprruqcceso8rlo ro anprsar ;eBns e qlpn alncalou prdrl pldqoczip (tII-ZII 'dd'V-Z Iaued pue g2-7 anfug) 's[ac elcsntu pue ro ll uI luppunqp dlurcadsa are ua3oc.{13yosalnuer8 a8rel 's1acptulue u1.,{8rauaarols o1 pasn'slrun asocnlS;o r{larnsnlcxapasodruoc aplreqccesrtlo4 uetoilp (Srt-trydd'9-7 1eue4) 'sprdlloqdsoqd Surpnlcur 'llao eql uI selncalour leus ,{uetu ;o punoduroc luared eql sl ]eql elncelour ctue8ro luurg 1oracf18 (tI I-ZI I 'dd'V-Z laued) 's[ec 1ue1dur qrre]s se pue sllaf, prurue ur ua3oc.{13se uroJ crraru.{1odur parots 's1ac 3ur,,rq Jo rusr1oqelarueql ur alor rofeur e sdeld leql ru8ns uoqrec-xtg asocnlt 's1ana1 'ullnsur reSnspoolq ur Jo teql ot etrsoddo sl ]caJJg asrr e ol Surpeel 'esocn18o1 ua3ocd13parols ualuoJ ol sllec raAII salplnrurls leql seercued aqt ur paJnpord euoruroll uotucnlt 'urals-{s snonJau preqdrrad aql ur sllal uueA\qJS pue ruelsds snolrou IBrtuaJ elerqaual aql u1 sal{corlse pue saltcorpuapoSllo sepnlcul 'rualsds snolreu oql Jo IIaJ lernauuou Sutgoddng 1ac pqt 'GOI) uoluq auo Sultouap xrg"rd_rtrt

AUVSSOID 9I:9

G:"17

GLOSSARY monomeric GTP-binding proteins (also called monomeric GTPases)consist of a single subunit and help relay signals from many types of cell-surface receptors and have roles in intracellular signaling pathways, regulating intracellular vesicle trafficking, and signaling to the cytoskeleton. Both trimeric G proteins and monomeric GTPasescycle between an active GTP-bound form and an inactive GDP-bound form and frequently act as molecular switches in intracellular signaling pathways. (Figure 15-19) GTPase-activating protein (GAP) Protein that binds to a GTPaseand inhibits it by stimulating its GTPase activity, causing the enzyme to hydrolyzes its bound GTP to GDP (Figure 3-71) GTP-binding protein-see

GTPase

guanine nucleotide exchange factor (GEF) Protein that binds to a GTPaseand activatesit by stimulating it to release its tightly bound GDB thereby allowing it to bind GTP in its place. (Figure 3-73) gnanosine triphosphate-s

ee GTP

H+-seeproton H--seehydride

helix-turn-helix

from the helix-loop-helix motif. (Figures7 -10' 7-l l, 7 -I2) helperT cell \pe of T cell that helps stimulate B cells to make antibodies and macrophages to kill ingested microorganisms. Also helps activate dendritic cells and cytotoxic T cells. heme Cyclic organic molecule containing an iron atom that carries oiygen in hemoglobin and carries an electron in cytochromes. (Fiq.rlre14-22) hemidesmosome Specialized anchoring cell junction between an epithelial cell and the underlying basal lamina.

ion

hair cell-see auditory hair cell haploid Having only a single copy of the genome (one set of chromosomes), as in a sperm cell, unfertilized egg, or bacterium. Compare diploid. haplotype Haploid genotlpe. haplotype block Combination of alleles and DNA markers that has been inherited in a large, linked block on one chromosome of a homologous pair-undisturbed by genetic recombinationacrossmany Senerations. haplotype map (hapmap) Human genome map based on haplotype blocks. Intended to help identify and catalog human genetic variation. H chain-see heavy chain heart muscle cell-see muscle cell heat shock protein (Hsp, stress-response protein) One of a large family of highly conserved molecular chaperone proteins, so named because they are synthesized in increased amounts in response to an elevated temperature or other stressful treatment. Hsps have important roles in aiding correct protein folding or refolding. Prominent examples are Hsp60 and Hsp70. heavychain (H chain) The larger of the two types of pollpeptide immunoglobulin molecule.

a flexible loop to a second, Ionger alpha helix. Its structure enables two HlH-containing proteins to dimerize, forming a complex that binds to DNA. Distinct from the helix-turnhelix motif. (Figure 7-23)

chain in an

Hedgehog protein Secreted extracellular signal molecule that has many different roles controlling cell differentiation and gene expression in animal embryos and adult tissues. ExcessiveHedgehog signaling can lead to cancer. HeLa cells 'Immortal' line of human epithelial cells that grows vigorously in culture. Derived in l95l from a human cervical carclnoma. helicase-see DNA helicase q, helix-see alpha helix helix-loop-helix (HLH) DNA-binding structural motif present in many gene regulatory proteins, consisting of a short alpha helix connected by

hemopoiesis (hematoPoiesis) Generation of blood cells, mainlY in the bone marrow. (Figure 23-42) hemopoietic stem cell Self-renewingbone marrow cell that gives rise to all the various types ofblood cells, as well as some other cell types' hepatocyte The major cell type in the liver. heterocaryon Cetl wittr two or more genetically different nuclei; produced by the fusion of two or more different cells. heterochromatin Region of a chromosome that remains in the form of unusual$ condensed chromatin; generally transcriptionally inactive. Compareeuchromatln. heterodimer Protein complex composed of two different polypeptide chains. heterophilic binding nindlng between molecules of different kinds, especially those involved in cell-cell adhesion. (Figure l9-B) heterozygote Oipioia cel or individual having two different alleles of one or more specified genes. hieh-enersvbond " Covale"ntbond whose hydrolysis releases an unusually larg-e amount of free energy under the conditions existing in a cell' A sroup linked to a ilolecule by such a bond is readily transfeired irom one molecule to another. Examples include the phosphodiester bonds in ATP and the thioester linkage in acetyl CoA. liquid chromatography (HPLC) high-performance q,pe of chromatography that uses columns packed with tiny U'ehds of matrix; the solution to be separated is pushed through under high Pressure. histidine-kinase- associated receptor Transmembrane receptor found in the plasma membrane of bacteria, yeast, and plant cells, and involved, for example, in sensing stimuli thaicause bacterial chemotaxis. Associated with a"histidine protein kinase on its cytoplasmic side' histone One of a group of small abundant proteins, rich in arginine

G:18

GLOSSARY

and lysine. Histones form the nucleosome cores around wlich DNA is wrapped in eucaryotic chromosomes. (Figure 4-25)

homozygote Diploid cell or organism having two identical alleles of a specified gene or set of genes.

histone chaperone (chromatin assembly factor) Protein that binds free histones, releasing them once they have been incorporated into newly replicated chromatin. (Figure 4-30)

hormone Signalmolecule secretedby an endocrine cell into the bloodstream, which can then carry it to distant target cells.

histone code Combinations of chemical modifications to histones (e.g., actylation, methylation) that are thought to determine how and when the DNA packaged in nucleosomes can be accessed(e.g.,for replication or transcription). (Figure 4-44) histone Hl 'Linker' (as opposed ro 'core') histone protein that binds to DNA where it exits from a nucleosom-eand helps package nucleosomes into the 30 nm chromatin fiber. (Figure +-S+I HIV Human immunodeficiency virus, the retrovirus that is the cause of AIDS (acquired-immune deficiency syndrome). (Figures t3-19 and 24-16) HLH-see helix-loop-helix hnRNP protein (heterogeneous nuclear ribonuclear protein) Any of a group of proteins that assemble on newly synthesizf.^dRNA, organizing it into a more compact form. lFigure 6_33) Holliday junction (cross-strand exchange) X-shaped structure observed in DNA undergoing recombination, in which the two DNA molecules are held iogether at the site ofcrossing-over, also called a cross-strandeichange. (Figure 5-61)

housekeeping gene Gene serving a function required in all the cell types of an organism, regardlessof their specializedrole. Hoxgene complex Cluster of genes coding for gene regulatory factors, each gene containing a homeodomain, and specifying bodyregion differences. Hox mutations typically cause homeotic transformations. HPLC-see high-performance

liquid chromatography

hyaluronan (hyaluronic acid) Type of nonsulfated glycosoaminoglycan with a regular repeating sequence of up to 25,000 identical disaccharide units, not linked to a core protein. Found in the fluid lubricating joints and in many other tissues. (Figures 19-56 and 19-57) hybridization In molecular biology, the process whereby two complementary nucleic acid strands form a base-paired duplex DNADNA, DNA-RNA, or RNA-RNA molecule. Forms the basis of a powerful technique for detecting specific nucleotide sequences.(Figures5-54 and 8-36) hybridoma Cell line used in the production of monoclonal antibodies. Obtained by fusing antibody-secreting B cells with cells of a B lymphocyte tumor. (Figure B-B)

homeobox hydride ion (H-) Short (lB0 base pairs long) conserved DNA sequence that A proton (H+) plus two electrons (2e). Equivalent to a hvdroencodes a DNA-binding protein motif (hom-eodomain) gen atom with one extra electron. famous for its presence in genes that are involved in orchesl hydrocarbon trating development in a wide range of organisms. Compound that has only carbon and hydrogen atoms. (panel homeodomain 2-r, pp. 106-107) DNA-binding domain that defines a class of gene regulatory projgins important in animal development. (Figures 3_13 hydrogen bond Noncovalent bond in which an electropositive hydrogen and 7-13) atom is partially shared by two electronegative atoms. (panel homeotic mutation 2-3,pp.110-rll) Mutation that causes cells in one region of the body to behave as though they were located ii another, causing a hydrogen ion A proton (H+) in an aqueous solution. The basis of acidiry. bizarre disturbance of the body plan. (Figures 22-42 {nd Since the proton readily combines with a water molecule to 22-127) form H3O+,it is more accurate to call it a hvdronium ion. homeotic selector gene ln Drosophila development, a gene that defines and pre_ hydrolase General term for enzyme that catalyses a hydrolytic cleavage servesthe differencesbetween body segments. reaction. Includes nucleasesand proteases. homolog One of two or more genes that are similar in sequence as a hydrolysis (adjective hydrolytic) Cleavageof a covalent bond with accompanying addition of result of derivation from the same ancestral eene. The term water. General formula: AB + HrO -->AOH + BH. coversboth orthologsand paralogs.(Figurel-25) Seehomologous cnromosomes. hydronium ion (HeO+) Water molecule associated with an additional proton. The homologous form generally taken by protons in aqueous solulion. Genes,proteins, or body structures that are similar as a result of a sharedevolutionaryorigin. hydrophilic homologous chromosomes (homologs) The maternal and paternal copies of a particular chromosome in a diploid cell. homologous recombination (general recombination) 9enetic exchangebetween a pair of identical or very similar DNA sequences,typically those located on two copi'esof the same chromosome. (Figures5-51, b-53, S-59, and 5-64) homophilic binding Binding between molecules of the same kind, especially those involved in cell-cell adhesion. (Figure l9-B)

Dissolving readily in water. Literally, "water loving.', hydrophobic (lipophilic) Not dissolving readily in water. Literally, "water hating.,' hydrophobic force Force exerted by the hydrogen-bonded network of water molec.ules that brings two nonpolar surfaces together by excluding water berween them. (panel 2-3 , pp. I l0--1l l) hydroryl (-OH) Chemical group consisting of a hydrogen atom linked to an oxygen, as in an alcohol. (Panel2-1, pp. 106-107)

'uorlEzlpuqAq tu1ru1.rdru1 clurouet aas-tupu1.rdru1 ' uollelgdtcardounturul .(q anssq ro ilac E ur alnJalou VNUru ro aua8 p alecol ol pasn s1aqord VNCI ro VNU papuerls-a13urse qolqm uI enbruqcal -oc pue uorleltdtcardountutul ulluluoJqc osla aag 'xe1d uopuzlplrq^q t4!s il! -uroJ eql atetrdrcerdo1 sulalord aql Jo auo rog cgtcads dpoq -rluu uu Sursn ,{q slcerye 11ecu1 sugelord SultJere}ul Jo saxeld (6€-SI pue -uoc ,{;rtuapl uec anbluqcal eI{J 'uonnlos Jo }no uaSllue gg-91 sa.rn8rg)'runlncrlaJ cruseydopua aql ruo{ +zeJ asPalar urelord Surpuodsarrocaq] Merp ol dpoqrlue cgtcads e Jo osn o1stcy .{e.uqtedSurreuBrsprdqoqdsoqd Iotrsour aqlJo uollel (dI) uoperldlra.rdounrurul -r1ceSurrnp pacnpord alnralotu Sulpu8rs relnllarerlul ilerus (tdl) arBqdsoqdslrl-9'y' 1 lollsoul (91-97 arn8rg) 'esuodsar aunrurul .{repuoces ra8uorls pue plder eroru (6t-9I pue B€-9I e aloirord IILtr ua8tlue elues aql qll.&\ Jalunocua luenbasqns se:n8rg) 'J aseuDluratord alplrtlp ot sdlaq DVC aqJ'euerq qJrq Aur 'sue8tluu Lueru o] asuodser aumuul &eutrd e smol -uau euseld aql q spldloqdsoqd ruo4 (DVC) Iora lolrsour -loJ leql urats,ts aunrurul a,rrldepe eqt 3o ,,firadord palq-3uo1 -c,,{131dcerp pue €41;o uorterauaBaqt pue 3 asedlloqdsoqd;o ,iroruaru pcltolounurtul uorlelrlcp aql qlrm slrcls leql.{e.r,rqted Surleu8rs relnllacpJlul ,{emqred tulputls pydqoqdsoqd 1o1;sou1 (79-97 arn8rg) .{1 -rue;radns 3y aq1 eur;ap pue uotlluSocar ue8rlue puu suoltoe -ralul IIaJ-llac ur paAIoAursurelord raqlo dueu u1luaserd are 'sulpluop (aIII-8I) e41-uJpqolSounuul se Ul\\otDI'sulPtuop relrurs 'surcqr.,i,reeq pue tq31 ulpqolSounrulul uI punoJ sI lpql splre ourue 00I tnoqp Jo uleuop ulalord cDslralluruq3 (ur.europ 31) ur.Buop ulpqol8ounruul (Zg-gI orn8rfl 's11ac crlor{recnaur uollcnpsupJl Ieu -Srs.relnlacerlul JoJpue saupJqrueu luaraJJIpSuqreruap ut 'eueJqruau eurseld aql;o luauodtuoo roulli\ luelrodurr lnq 'alrtelrrop lotrsour paleldroqdsoqd e Surureluoc prdrt y (applsougoqdsoqd) pldqoqdsoqd 1o11soug 'sprdloqd -soqd lolrsour;o lred Sunuro; elnJaloru re8ns padeqs-3utg Iol!soul (BB-ZZam8rC) 'pa^uap sr ^,{poq }lnpe oqt Jo aloqM aql qf,rqM uo-r; o,{rqua

'sasuodset ounlurul antldepe uI aloJ ]uereJJlp e qlrm qree-1q31 pue 'CBt 'ABf 'CBI 'Wl-u1nqolSoumuul 'e1nca1oufpoqllue uV Jo sassEIJa^rJaleq salerqapa,Traq341 (t1) ulpqopounurul

uerTPrurueru,{.1rua aq1ur sllar palur}uareJJrp"".d?J"jrt;t%"",

tuplolq

aas-tuplolqounruu!

'arrrldupe pue oleuul-saleJqeua^ ul sluels^s ounl'ulul 'uollceJug tsule8u esueJap sapn ;o sad-,i1o1!u ere eraqJ -ord teqt Lpoq aqt uI sllal reqlo pue sa{coqdru-!;o urals,tg

'asuodsar aunrurur anrldepe up ol 'ueSoqled lserluoo ur eql roJ cgrceds tou sr asuodser e qJnS 'sy1accrlrtco8eqd pue salncalou Terqorcrru[ue apnlcur qcrqlt'(rualsrts eunrutul alpuur aql) .{poq aqr;o sasuaJapSurlsxa-ard aql sa^lo^ul leql pue sprulue qloqJo) asuodsaraunrutul uaSoqled e o1 (s1ue1d esuodser aunruru! efuuul

*t

'sasuodser aunuruy,{rep"""";ti:Tf

-rrd 'arrqdepe 'aleuul oslo aa5 'asuodsar euntuutl arrpdupe ue ol sJaJeJ.{1ensn:dpoq aq} sralua urslue8looJJltu Jo ecuels -qns u8rero; p uaqm ruelsds aunrutul eql ,{q apuru asuodsag

(71-9 arn8rg) 'IVNU]-lal xeldruoc aql Sururo; 'euluolqlatu plsB oullue { eql serrrec s.4.emply 'uoFplsuurt satullul leq] VNur lercadg vNulroluplul

'."r,r"";tlt;#:*t qtlm sllao put8tro aql Jo uolsnJ Jo ro uolteturoJsuerl IPIIA ro suollelnru Jo llnsoJ aql aq ueJ 'suoJsl^IpIIaJ JO laqrunu patrullun ,{puaredde ue;o alqudec auII IIor e Jo uollJnpord

(72-9 arn8tg)'uouelsuerl Surlertrur snqt 'auosoqrr eqt ol uo rvNgt-tahl peol dleq sgla 'salodrecord ur 91 'salodrerna ur dIJ palpnaJqqv'srsaql -u.{s uralord;o uortertrur eql ro; parrnbar sI pup VNUtu qtlm seruosoqrrJo uorlulcosse radord aql salouord leql ulaloJd GIa puE CD rolceJ uollElllul 'prlualod

arn'rg

oqdroruelall"rr":::**t

i 1e-77 )'srs 'arnlcnrls llnpe uortertuaraJJrp lrarrg '3ur.r,r'3e1 'eda ''3'a ue olul dola,repIIIM qrll{lr pue o.,{rqruanpqdosotq etqtuy 'palunuaraJJrpun dlluarudde'eplselas areleql s11ec 3o dnorg

cqppqtuurl alc uortru up uorleraua8 aqt ol spual snql pue tlqlqul IIac Jo -sntu ro alrau e Jo eueJqruau cr1deu.,{s-1sod aql ul slauueqc @L-92 enfu il'uolluSocer ua8 -lluB Jo suorlceJalul llaJ-llat uI poAIoAuI aJe lsoIAI 'sulPluop +) ro -lJ pareS-raultusuerl suado ]eql JallltusueJloJnaN ralllrusrrurlorneu fu or1q1qu.t a4r1-urlnqol8ounrurul ro suleruop ullnqolSounurtur ulpluoc leql sulalord go dlure; asre^lp pue a8rul ,figru;radns 31

(616 'd 't-SI alqel) 'uralord gosltt aas 'auerqruau etuseld eql ur aselcdc 1.,{[uepe au.{zua aq} lrqrqur pue slauueqJ uo1eleln8ar ueJ leql ulalord 9 crrarur{ 0C) ulalord g iiro11q1qul

fpoq.rluuaas-t1 rolrDJ uoIlEIlIq aas-dl

fgurul 4y1 aas-,i.gurug sgsoldodu 3o rorIqFIuI 'auruPlsrq sB qJns sJolerpeIu 'sllal poolq alrqM Iesol snorru^ asEalal qJrqM Jo uorse^ur 'ssaupal anssrt dq pazr .{q pasne3 'ured puu '1eaq 'Surqarr,ts -JalcpJpqr-uorlJaJur ro .,{rn[urol anssrl e;o esuodsar pcol asuodse.rr{.rolutuurugul

'slsoldodu ;o srollqlqul ulalord relnllecenul ,fi1uru; (slsoldode ;o .rolgqlqul) dVI 'slsoruso dq 1ec e olul aloru ol Jalel\ asnec ol salnlos Jo uolleJluacuoc ,uo1 ,tpuarcg;ns e Suureg cluoloddq

tgr-zzpuelvzz

fiE-92 arn8rg) 'a1rsSurpurq-ua8rlueeql yo &1cg1cadsaql aullurelep suor8ar asaql 'a1ncaloru 01 alncelotu uror; ritlpqerre,r 1seq31q eql sMoqs lPqt ureqc roldecar IIar I ro ulpqolSounuur ue ;o uor8ar alqelrp^ aql ulqlpr suor8ar flptus aarq] Jo fuv uolte.r elqe;.re,r.re&(q

sarn8rg)'uorlJpJaluralrlJnpur ue pallpJ sr ]JaJJaup qJnS'ans -sll Jaqtoue qlrl\ uollo?ratur ue ,{q pJsnpJ anssrl auo Jo aleJ ptueudole,tap aql ur a8ueqc ,{8o1orq pluarudola,rap u1 _e (uollrurolu! a llrnpul) uollrnpu! ''3'a) snlnurlls pcrsdqd ro Gv-zz a:r.fuil'({f,oqs leaq relnJalou relnclued e dq uo paqctrms aq o1 aua8 polelcosse ue Jo uolssardxa s,r,ro1pluql acuanbas VNC .{roleln8er y ralourord alqlrnpul

'slsoluso ,(q 1ac e;o lno oloru ol Ja]e,rlr asneJ ol salnlos Jo uouelluacuoc q8rq ,(1lue1cg;nsu 8urrre11 cluolrad-r(q

AUVSSOT9

6[:9

'salJolec 7Z'0 ot pnba dlaleurlxorddv'ectnos ramod ge.u -1e,{q puoces euo ur pala^rlap ,{3rauaeql s1alnof auo .tua] -s,{spuocas-urer8o1q-ralaruaql ur .{Braua Jo ilun prepuels alnoI ' (SJVJS)uoqdrrcsuerl ;o srol -e^It-repup srernpsuert pu8rs puu ,(s)VI) saseuDlsnuel cltu -seldolr{cseAIoAuI-'uor}drrcsuerlaua8 relp ol snalrnu aql ol auerqrxau etuseld eql ruo4 alnor prder e Surpnord ,sauoru -ror{ auos puu seuolo{c fq palerrrlce ^,{e,,rnqled Bulpu8rg femqlud Sugeutls 1y15-yy1

ro arqersraqrraaq.{etr1'.{r's*uaqce(ups

'suorlcela pue suolo.rd roqunu "u, "r"i:1t';itf# etuus aq] e^eq tnq lq8ram Jo rrlxole ul raJJIp leql tuolp ue Jo sruroJ Jo roqunu e Jo auo edolosl (9II- tII 'dd, S-Z Iaupd) .loqrllop pue plcp Jrourlal apnlJur seldruexg 's1run euardosr uoqrcc-elrJ alorllntu uo paseq uotela{s uoqrpf, e r{llm alncalou prdrl (p1oualdos.ufiod) plouerdosl 'ase;aurosrodolosp aas'apcaloru alBurs p urqll^{ spuoq 1eql eru.{zug Jo luaua8uerrear aql saz.,(1e]ec asBraluosl (SII-ZII 'dd'V-Z leupd) 'uor] -eruroJuocpuorsueurp-aerql tueraJJrpp 8ur^Eq tnq reqtoup se sa8eluq pue sruole atues aqt ruo{ peruroJ rt.r"tlT*o., 'seua8 -snoSolouoq dlasolc;o las e Jo sJaqtuou tuaraJJrp ;o slcnpord se ro ]drrcsueJl uoruruoc e;o SurcqdsalrleuJallp ,{q raqlra pa^rrap 'urelord e Jo sruroJluerrel Jo tas e Jo euo ruJoJosl 177_garn8rg) 'plarJ JrJlJala ue ur oloru ]ou saop aroJaJaql puu a8reqc culcola tau ou seq uorlnlos ur alnJaloru E qJrq^ le gd aql (1d) 1u1odcl.aralaos! (97-71 arn8rg) 'suralord uodsuerl-uorlcelaJo sselcp ur punoJ'sruolp JnJInsJoJaqunu pnba uE ot punoq stuole uoJr rnoJ Jo oml JeqlraJo Surlsrsuocdnor8 SurlrodsupJl-uoJlcalg Jalual rrulns-uoJr a1.rs,{.r1uaauosoqlr [EuJelq aas-sguJ (69-91 arn8rg) '1osoilr ,€4y orlosof.c 8ur eq] otul +zeJ parots Sursealar -purq uo suaoo auplquau ug eqt ur Iauupqs +7ecpalec teqt (leuuuqc esualar-+zuC pefut-e4fl roldeJar edl eluqdsoqdspl-9't' I lollsou! aas-e41 'suor crue8rour cgrcads ol rblllqearurad rreql sasearcurpue sra^Elrq pldll q sa^lossrp leqt alncelolu crqoqdorp.r{q 1eur5 aroqdouol

(rrr-orrdd

't-Z '9-Z arn8rg; 'puoq tua1e^ocuouJo edz! aug Iaupd pue 'e8reqc aarle8aup qlr^\ Jar{lo aql ,a8reqcearlrsode q}lm auo 'sruolP o^{l uae^\laq uoqcprlle sllElsorlcela ol anp uorsaqoJ (uollcuralul cluoD puoq cluol uor palut-ra11lursuu4 aas-roldacar

Iauuur{t paldnoc-lauuuqc-uol

(IZ-t I arn8rJ) 'sluerper8 pcrruaqtorlcala rraql umop osnJJrpuec suor crue8 -rour cgrcads qcrqa,rq8norql raz{e11q prdq aqt ssorre Iauueqr pallrJ-ralem p sturoJ teql xaldruoc ulalord auerqruausue{ IauueqJ uo! '_D pue alduexa ro; la8reqc *e111 e arrnbce ot suoJlJalolsol ro paure8 taqlra seq leql tuole rO ,ro,

q,, roJurluT'urslue8roro IIecttptur ue uI ,,'aJII ontnuL

'snJaln s,Jaqlou aql olur peJJeJsup4 uar{] aJe so^Jqrua Alrea aql pue 's^ep ^ral e JoJ paJnllnJ are s33a pazrpra;,{lnyssaccns :,{poq s,Jeqlotu eql aprslno ureds rfJ,r,r pazr1luoJ are s33a qclqm ul lueuleart ,{r111ua;uruy Gr\D uolr?zllluol orlln u!

(,,'ssep

ur,, roJ urlp'I) 'sursrue8ro ]celu1 ul salpnts tuo{ satn}lnc ila:) ur sarpnls qsrn8urlslp ol pasn sarullauos oslp tlac 3urrr11 e ur o1pasoddo se 'lJerlxe ae4-llar palplosr ue ur eceld Suqeg o4!n u! (999-tgg 'dd't-e Iaued) 'paua ur sr aruosoluorqc ;o luau8as e qorqm ur uorle1mu go add; uorsJaau! (91-y arn8rg) 'vNu IeuorlcunJ raqlo ro vNuru aq] Jo uollsnpord 8ur;np 3urc11dsVNU,iq pasnxa uaqt sI lnq elnJalou VNU ue olur paqlrcsuprl s1 teqt aua8 cr1o,{-recna e yo uo18ar Surpocuog uoJluI (I-9I arn8rC) roterpau relnlleJprtur flelus p sa]e -reue8 ro ,{e,r,rq1edeql ul ulalord uau aq} sole^rtce .{1ensn '[ac aq] aprsut .,terrrqled8u11euBrse ur pallolur urelord ulalord tugeu8ls rplnllarurlul

lI

(98-9I arn8rC)'e1odraqlo aql uror; Surleueua salnqnlorrru aql qll-A rolenba oql le 8ur -]ellSrpralur alnqnloJcnu e 'alpurds Jrloraru Jo Jllolrru aql uI alnqnlorrrru rulodrelul (7-21 ern8rg) 'eseqd zg pue 'aspqd g 'aseqd Ig sapnlcul'lxeu aq] pue srsolftu auo uaam]eq elJ.{c flac aq] Jo porrad 8uo'I asuqd.relul (B0I-Z arn8rC)'patprtruraq uec uorlElsuert qrlq^ le 'pua 'vNuru cllodrecne e ur alls cgrcadS ,g aql ]e uEql raqlo (ggg1) e11siirlua auosoqlr leuralul (696-896'dd't-gt Iaupd)'sluauep prelalsoilc go sed.41 'sllec pturup ur $lromlau a{ll luaurruold tsotu aarq] ar{lJo ouo -ado: sturo; leql (ralauprp uru 0I-) luoruelg urelord snorqrC luatuulg elulpeurralu! (gOSt'd 'n-gz alqeJ-)'sasuodseraunlulul puu uorteruruplJur Surrnp (sa{cornal) sllac poolq alrqru ueemleq suorlceralul lerol salerpau dpreur leql eup{o!{c petarcas (.ID uplnalralul (gg-97 ern8rg) 's1ac patcaJul-snrl IIDI ot sllao ralp-)IIernlpu puu sa8eqdorceu 3ul -lelnrults pue uorlucqdar pru, 3u1llq1qur'sasuodsarprrlrlue acnpu suoJeJreluJ'sllecJ pelP^I]JsJo sao l ulPuas pue sflac pelceJur-snrrl dq patarcas saur{oilJ Jo sselJ e Jo raqrualN (NCI) uoraJralu! (99-77 arn8rg)'raqtaSor page;8 aJe aJnlcnJls e ;o slred paqcleusnu oMl ur slleJ aqt uaqm sanssrl Surssruraqt ur sllrJleql uoperaua8ar;o addl uolluJauata'r r{rupc.ralu1 (Setf 'd 'Z-6I alqelpup't-6I arn8rg)'raqlo qcPa ol puP xlrleru relnllecerlxa eql 01 slles Jo tuaruqrPlle aql ur pa^Io^ur sr leql ureloJo uorsaqpe eueJqruarusue{ ugrtalul (6I-0I arn8rd) re.{egq prdrt aql ur pappoquro are ro ueds leql suleuop aroru ro ouo Jo enlrpr dq auerquaru p ul paulelal sr lpql uraloJd ulalord arrurqrueur pr8elug (gg-€ arn8rd) 'slprurue ul tuslloqplau asocnlS aleln8ar dlaq o1 searc -ued aqt w slloc d dq p"tercas sl lpqt ouoluroq apudeddlo4 ullnsul (Zg-Z am8rC) 'saua8luacefpe;o uoqdrrcsuerl aq1Surcua -ngur ruo4 aua8 auo;o uor8ar lorluoc aql ul VNC ot punoq ulalord droleln8ar aua8 e slua'rard ]eqt aJuanbas VNC luaruala JolBInsuI

AUVSSO]D 0Z:9

(?I-0I arn8rC)'lorolsoloqc pup spldlo8 -urqds ur paqJrrua auerqueu euseld eql ;o uor8ar leug rPrPldq 'ra1e,r'r (gII-'II'dd'9-Z laued pue ZZ-Z arn8rg) o1 pasodxe 'premlno speaq cllqdorpdq rreql pue premur Surturod spel crqoqdorpfq rraqr q]l,tr pa{rud are selncelotu pldll Jo sra.,fu1o,r4 aqJ 'sauerqruatu IIar IIp Jo arntcnrls eroc aql sruroJteqt selnrelou prdloqdsoqd Jo laaqs alqnop urqJ (rafu11q pgdgoqdsoqd) ra,{u11qpldq ( g II - t I I ' d d ' S - Z I a u u d ) ' s a u e r q -ruaru pc13o1orqJo srseq prnlcnrls aqt suroJ 'sprdqoqd -soqd aqt 'sse1cprcads V'stua^los crue8ro ruyoduou ul a^los -srp ol spuat lnq ralem ur alqnlosur sr leqt alncalotu crue8rg pldII 'eprrecd13rr1 e ;o,{latour loracd13 aql ruo{ sprcu d11e;;o a8e.tealc aql sez.{p1ec 1eq1aur,{zug asudl1 'lq311o qlSuale^em eql dq paururalap trrull e 'ddocsorcnu tqBII Ieuorlueluoc roJ ruur Z'0 rapun lsnl'aleredas se pallosal aq uec slcefqo lurod oml rlJrr{,rt le lrede aJuelsrp lsalprus aq1 ',{docsorctu u1 uopnlosarJo IFUII 's1ue1dur anssp z{poo.u pue ura1.{xJo sllem IIac aql lnoq8norq} {romlau 3ur}roddns e surroJ teqt spunodruoc cgouaqd pa{ull-ssorJ Jo {rom}aN uJutg fiz-92 arn8rC) 'ulpqolSounuur ro (ZZ-9I arn8rg) urso,{ur se l{ons uralord trunqnsrtFru e ;o saprldaddlod relerus otll Jo auo (u1eqr 1) upqc 1qt;1 'spuoq lalsalp -oqdsoqd q8norql pua-ot-pua raqlaSol solncolotu VNCI oml ygq 'ssacord luapuadap-d8raua surof 'aldruexe ro; 'ase311 up ur sa1ncalorx o,tr] (sale8g) raqleSo] surof 1eq1 aurfzug asut11 'pulq or 'ato317ultel rxorC 'apcalotu raqto ro urato-rdp uo alrs cgrcads e ot spurq ]eql elncaloru .(uy puBtII 'sllac poolq elrqMJo rarue] sFua{naI

ur-eqc lqtq aas-ur-Bqt T (79-7 arn8r4) 'aruosoruorqc aql Jo srxe reaull eql lxo4 lno Surpuelxa 's33e uet sdool grls a8rel sruro; ulluuorl{r aq} qllqm ut

-qqcirularnruurrut uI stsoIaluuI aurosoruor3illo:ii: i."fj[1.,*, (tt-6l pue 77-61 sarn8rg) 'Iro1v\teuaTllleeqs e sluroJ u aral{^\ 'eeuftuel pseq uI punoJ ule]ord snorqu xlrletu ruInllecel}xg ululurBI uFrrel JBaIJnU aas-uFrrel

(ZB-9I pue gg-91 sern8rg) '11acPrulue 3u11.uurc 'sluauellJ ullce e ;o a8pe Sutpeel aqt te papualxa sI qrlqm

ro{ro^rqsaue'tqpalroddnf i.iil#,?lJH,%l'"ffi"Jfi [r"_,, upqurel a8uqdol.relcuq aas-ePqrrrel 11-9 arn8rg) ,{puap,toc paurol:ale1 are leql sqfual snonurluooslp uI apetu sr puerls 3ur33el-aqJ'{roJ uopucrtde.l e tp punoJ vNC Jo spuels pezlsaqlu^s .{l^{au o^\] aql Jo auo __ puu.4s tupllul eycdcplcu cpllcaas-e1cfc

sqe.ry

'aua8 e;o uolleln{u 3urlen1lcuu1Jo uollelap paraeut8uauy lno{JoIDI (629'd'€-II 'llal ,,8utlsar,,e ul ua^e uado suteuta: leq] sllac ptu IauBd) -lueJo aueJqruaureurseld aql ul IauueqJ uol Sutlrodsuerl-*y IouuDqc {EaI +) 'aruosoruorqce Jo oroqloleuDl aq] ol alod elputds aql slcau -uoJ teql alnqnloJJlur e 'alpurds sllolatu Jo cllollru eql uI aFqnlorrlur aror.lrolaupl (gg-21 ern8tg)'eraluoJluec atuosoluorl{3 eql uo slurod'sa1odaql ol saluosourorqJJoluaua^oru aql uI 'I{celle seFqnloJJlru qJIq ^ ol aruosoru tred anrlce ue sr{e14 -oJqc cllollru e uo suralord ruo4 paruroJ arnlcnns xalduro3 eJoqrolaupl (89-gI ern8l$ 'selnqnl -orc1u Suop alotu o] srsdlorpdq 41y;o ,,{8reuaaql esn leql sulalord Jololu Jo sasselJ uteul oml aI{] Jo auo Jo JeqIuaIN ulsaupl

'esEuDIurelotdos1a aas 'sepcaloru IIar poolq el1qm aas-efrornal o] sdnor8 ateqdsoqd Jo uopppe aqt sazdplec 1eq1aurf.zug (61-2 ern8rg) 'ratulp ulal ASEUDT -ord e Sururro; 'Qaddve aIrI raqter) Iror-palroc e ur raq1a3o1 '1ac cqo,{recna paurof ere suralord alerudas urorJ sacrlaq eqdp o,,vqqctq.r,t ur suralord Surpurq-ygq ,{ueu u1 uees Jllotu prntJnJls raqloue 3u1p1 -{lrcarrp ;o elqedec 11eccllo-{recna duy raddlz aulcnal IIar reIIpI 'uoruod Jplnllacenxa s1ru1 sacuenbes 'sdnor8 />1P UOI d'fZ 1eue6) leadar qcg-eurcnel Jo ,{erre urapuel e suleluoc }eql omt ot pa{ull dnor8 lduoqrec e Suturcluoc alnlaloru cruu8rg s1ue1dur aseupl euruoeJql/eurJasroldacar;o addl uourtuo3 euolal (u1a1o.rdgg1) ugalord luader r{rlr-aulcnal 'sraqleaJ pue (999-tS9 'dd't-e Ieued) 'lr surcluoo lpr{l 'slreu lreq uI punoJ ere sulleral paztrctcadS 's1ac pleqrlda usrue8ro aql ro IIar aqt Jo qleap aq] sesneJ teql uoltelnl ur rtpreu 'slualuellJ alelPetulatul ullera{ surloJ lPql ulaloJd ulluro{ 'elrtadde Butssarddns ^, ;:'';;:Jtt"t 'Jaqrunu pue 'adeqs 'ozls ol lcadsar qlpt aql aleln8ar sdlag 's11ec1e; ,{q petarcas auorxroq eprlda6 pa8uurre 'llac e Jo sauosoluorqc Jo las IInJ aql Jo APIoSICu1rde1 ad,$od.re1 'sllar Jo aJEJrnseql uo rueql lcalap ot ro surelordoc,tlS .tr-rnd ol stua8eer,trurge se pasn aru spaas lue1d uorJ sultral 're8ns cr;1cads e o1 .,tpq81l spulq leqt ulalord tuepunqv ullral aulloqc$ppuqdsoqd

(€-t laued) ,41pg1ea,ro1dlanpelar qll^{ alerlsqns sl] ol spulq 'aler erudzua aql ]eql atuclpul .{1ensn {y ;o sanp,t a8rel rumurxpru sU JIpq le $lJo-ll atu.,{zueue qclqm le e}eltsqns Jo 'luelsuor uotuaru-sllaEqJlhl aqJ, uollerluaJuor aq] ot pnbg

aas-uFlrlrel

'uorlceJrp [-g arn8rg) ,g-ot-,9 aql ur stseqlu.{s snonurtuor ,{q aperu sr puerls SulpEal )roJ uorleclldar 9qI e lP punoJ vNo Jo spuerls pezlsaqlu^s AI^\aU o^rl eql Jo auo puerls tulpual

n)

luelsuoc uollulcosslpaas-D1 lnelsuoc l11ugp aas-Ex aas-Y lrrulsu oJ runI.rq.rl.rnba

UVSSOID

LZIU

(1-91 arn8rg) 'llam sp sallauuBro.{ueu ,sllac crlof-recna ur ,pue sllaf, IIe sasolcua]eqt suralord pelercossusnld re,telq pldl eql arluJqrueur 'rreq pup uDIs uorlptuatu8rd eql ro; 11-97arn8rg) Jo ayqrsuodsag'uruuloru luaruSrd {rpp oq} secnpord teq} [e:) alAJolruleIu (9-IZ am8rC) '1ac proldrp p urorJ sllac ploldeq Surcnpord dqaraqt 'uorlecqdar VNC Jo punor auo AIuo qlrl\ suorsr^rp Jealcnu a^rssaccns o^^l saAIoAuIlI .uoll -cnpordar pnxas ur sJnJJo uorsrlrp ler{l 11ac;oad,ft prcadg slsoJaru 'sessacordcrurseldo{c eV{Z pue gt-€Z sarn8rJ) 3uo1 uor; slalereld JJo spng 'erntpur uoq ^ rurorreruauoq aql

p qrr^rraco,"Jit$i3jrlr"* ursu*uerreq]snarrnu paqorrrrnu

ulalord acrrelsrsar tn.rplllnru aas-u1e1ord gq141 aql salouord

(91-11 arnSrg)'slsolrruJo stuala ]pql sllar cr1o.,{recnaIIe ur punoJ uglc.tc y ullc c-w

'sIroJ uorterlldar y1qq Sur,rour le luesard aserllaq aqt uroJ ol tq8noqt juortecrtdar VNC Jo uolleqlul aqt IoJ paprau 11ar:r1o-{rern" "Ut "ljrli"j""jd_"^ (€90I 'd 'I-ZI alqel pue 1I-LI arn8r{) .(Ip:)) aseuDl tuapuadep-urlcdc Surpuodsa;roc eql pue urlcfc -4 ue ,{q sllar atErqaua^ ur pauroJ xalduroc {pC-ulcd3} (>1p3eseqd-4) >tpC-ltt

sepnrru.sural ord x rleu saperg ap leq]*;:il"r1iffiHr"rll

aql u1 tuasard aurfzua cr{loalord luapuedap-*ruz ro -+zEJ esualor-Io1pleurxlrluru 129-71arn8rg)'pappaqua aJp sllec qJrqn ut saprreqccesdlod pue suralord palarcas .xrJleru relnllasprtxg Jo alrsoo(uoc Jplnllasellxa aqJ ,]se1d (S) 'etuoJls oql se u^\ou{ ,{luoruuoc arour -orolqc p ur Surpuodsarroc aql (Z) .uorJp luarulJpdtuoc -uoqcollu aql .paruroJ Jo ]uauUeduroc Ipuralur aBrpT (I) s1 Surqlauos r{rnlm urr{trm runrpeu Surlroddns ro aced! xIJlBur (99-2 arn8rg) 'llar tspedploldeq eqr 3o (e ro D) adzbSurteu aql saururratapleqt snrol aqi ,tsea,{-Surppnqu1 (snrol lBI tr)sncol ed,$-tupuur 'aua8 luelntu eql palrraqur lou sEqJlastr o.r{rqruaaql Jr ua^a ofuqure aql ;o luaurdolanap .33a aql ur suralord aql ]ca$e suortelnu lceJJa-leuraleyrtr pup SVNUtuIeuraluu.lrcads ol raqlorx oql ul slrp leql auag auat lcaga-puJoluru (IZ-B ern8rfl 'sapqdad.fiodSurcuanbaspuu surelord bullJrluapr JoJ Ioot InJJaMod.oqer e8reqc-ol-sseru aslcerd rraqt Jo srseq aqt uo spunoduroc Surf;rluepr ro; anbruqca; (94) d.rleruorlceds ssuur 'spu8rs ru1n1 192-91arn8rg) -laceJlxa o1 asuodsar ur uralord seg e ,{.qpa}elrlJp d1ecrd.4.1 'prql aqt sE aseupl ,acuanbas ur 3urlce ,saseup{ dVIAI qtln ulalord aalqlJo pasoduroc alnpour SurrcuBrsrelnlacerlur uy (alnpottl asEupl ulalord palu ltcu-uato1;ru) aFporu asBupl-dvtr{l

-ruaueurseld aqr..,oqspugrs s"r^"rr;tliFJ^1#11,?#:i;

Eurlpubrslueuoduro3-eeJql e Jo pua eql lp asBuDIuraloJd (asuupl ulalord pa1ul11ce-uatolpu) asuupl dvtr\l ulalord palulJossp-elnqnlorrlru

aas-dvl4l

(€t-€I aln8rC) 'seuososdl roJ pauqsap suralordoc.,(18 aruos uo saprreqcceso8r1oartr1ot par{Jellp Ja{reu enbrun (49141)aluqdsoqd-g asouueru

(6-97ern8rg)'JaJuecp srJorxn]lueu8rpruy'slsulsptoru o8rapunol olqeJolpup alrselur:sllal Jorunlpue sJo{unlJO lueut.rpur xaldruoc aas-xalduoc zgqlqpudruocolsgqro[uu CHW (gg-77arn81g)'sasuodsar aumururur suorJourg Surluasard-uaB -uue pue ra8uarrecs qloq seqlI 'rueoJol alqplnq sanssulsoru ur luaplser 'salitcouourpoolq uorJ pa uap 1ec cr1,{co8eq4 atuqdo.rcuur esuqdtr41aas-tr41

surpqraprpqccesdrodloe,ror.r'51ir"trt#i"TltfrTXt#;J awizost(1

(zt-tr

pue Z-ZI se-rn8rg) 'saruosos.{1Jo uerunl aqt ur puno; gd plce aql lp a,tllce tsour l.11ecrd.&are qcrqlt 'sarur{zuaa,rrlsaBrp Suluteluoc slac c1]odrecneur allaue8ro popunoq-auerqtuel4l etuosov(1 'llar aql Jo rltpap aqt pue urseldoldc;o aspalar aqt ol Surpeal 'auerqrueu euseld s,llec B Jo arnldng sls l

ur se'poorqeql ur ueqrreqrpr)supgro

or"uoJi",llo?l3j f1uruu are slleJraruer ar{}qcrqmur 'sap{coqdru.(1;o rarueJ uruoqdur^{1 (g-97ern8rg)'sue8ro proqdu.(1leraqdrradur ue8rlue o1 puodsar pue suu8ro ptoqdudl ,{reurrrdur pacnpord aru saltcoqdrud.I'sal,tc -oqdur.,{1 Jo uollJunJ ro uopcnpord aqt ur pa lo^u1 ue8rg

-,#,.1, .:;l;*t o-n,

-r1ue SurlelncrrJ Jo uoqcnpord aqt -ro; alqrsuodsar are pup spurtuetu ur 1r^orrerueuoq aql ur dolenap s11acg '.{lrunuur palelpeu-ller ro; alqrsuodsar are pue snurdqt aqt u1 dola,rap sllac J'sllec palcaJulqlIM pue tuals.{seunlutul ar{tJo sllaf,ro} -raJJaraqlo qlurr..{ltcarrp tceratur qcrqm 'sllac pue dpoqpue I acnpord qcrqm 'sllal g :sad,,i1 upru ontJ'sasuodsar aunrurul alrldepe;o Ilrcr;rceds aqt roJ elqrsuodsar IIor poolq alrqlv\ alboqdudl

(g-97 ern8rg) 'slassaA cpeqdrudl ur punoJ sa1,{coqdur{ Sururcluoc pmg ssolJolo) qdurq uo;1epua1od urral-tuol aas-dJT 'sllac olur dn ua1el pue poolq _ (09-tI arn8rg) aqt uI pagodsuerl sr Ioralseloqc qcrq ^ ur urro; aql'sprdrl raqto qt.r1v\raqlaSol 'selnralour Iorelsaloqc paqrralsa fueru pue alnJalou uralord a18urse 1o pasoduroc xaldtuoc a8rul (1q1) ulalo.rdodq fi lsuap-mo1 .o^rssaoardlensn (999-t9S'dd,t-g 'aua8 u;o dlrnrlce aql seqslloqp ro laued) qcrqm seJnpar uoqetnntr uoFulnu uollruru-Jo-ssol (77-1 1 ern8rg) 'suornau rlldeudsard aql ur Surrg a,rrlrledar 'urerq aqt ur sesdeu.{suteuao Jo lsrnq uoqs e , q pecnpur Jo ,fintlrsuas aqt ur (s)aa ^ o1 s.{ep; aseercul Surlsel-3uo.I uo;lupua1od u.ra1-tuo1

aqt ddlcco aue; eues aqr Jo saralerue'aJJrp ti}t?,XHt"J

e ur'alduexe roC'aruosouroJqJe uo uoursod eq1,scrlauaBu1 snJoI .s1ac dqreau uo d1eco1 srce leql alnretou

(7-91 ern8rg) ,""rr:.r$l;:r;r*".,

(6-91 arn8rg) 'salncalourprdloqdsoqd;o uorsuadsnssno -enbe ue ruo4 paurroJ alcrsan raf.elq prdlloqdsoqd IerJrJruV aurosodrl clqoqdo.rpdq aas-cqlqdod;1

AUVSSO]9

ZZi,

(gg-1 arn8rg) 's11ac crlodrecna ur dIVaqtJo tsoru sarnp -ord pue uorlel.4.roqdsoqda.Lrleprxolno sarrreJ ler{l 'unrral -Jeq e Jo ezrs aqt tnoqe 'allaue8ro papunoq-auerqualN (Elrpuorlroqu p:n1d; uoppuoqco4u 'puerls a1e1dualaqt ot arua 167-9arn8rg) -JaJarqllm acuanbes lcaJJoJaql qlll paceldar pue pe^oruar sl apltoalrnu paqrleusru aql Surpnlcur ylrJq pazrsaqtu,(s dpreu ;o r{ctarls uoqs V 'uorlecrldar VNC Suunp _pauasul saprloalcnu paqf,lPrus[u slJa]ror tPql ssaroro rrPoar vNC rr-edar qcluurspu vNu orclruaas-vNulru GL6_BL6 'dd'Z-gt 'pua palurod aql se u^ ou) osp sr tuaruelg loued) ullJe ue Jo pua snu[u aql ']ualuplrJullrE Jo alnqnloJcrru aql aql :^lrpeaJlseal sJncro sJeruouoruJo ;o pua ,,8upror3-,u,o1s,, uorlrppe eql qrlqM ]P tuauelrJ urtJe ro alnqnlorsftu P Jo pug pua snullu (Ig-/I arn8rd) 'slPru -rue ur sllal ralq8nep oA! aql uaamlaq raqla] p se arull aruos .ro; lslsrad upc leql a8elealf, Jo pua aql te paruJoJ aJnlJ.nrls ^poqpFu (99-91 ern8rg) 's11ec pllaqlrda Ipupsalul Jo eceJrns a^lldJosqp aql uo sraqunu a8rel dlercadsa u1luasar4 's1uau -elIJullJe Jo alpunq aroc e SurureluoJ llal leturue ue Jo aceJ -Jns aqt uo uorlcaford paJaloJ-auprquau leolrpull.b urql (1111 orrpu 1e;n1d)snllyrorcpu ,rnor8salnqnloJcru qcrq ^ tuo-g ,{poq pspq e Jo oruosor}uac e se qJns 'llec p ur uor8ag (IOIW) raluac tul4ue8ro-elnqnlorc.rtu 'aseqdeue ur ateredas.{aql reue spueruorqc re} 117-21arn81g) -sls luaua^our premalod arp aleraua8 ot sdlaH 'spua snulu Jo rIaID lp ullnqnt Jo ssol .{q salod aql spremo} alpurds aqt ;o

sapqnlorcru aqrutsaFcororu urnqnrTEnpr to"t*jl:jfiei"#"",_

l,'aseuDldVI tr,Jo (aseuplurelord palen -rlce-uaSolnu) ,dvhl, aqt qlIM pesnJuocaq ol toNl 'ulaudp su qcns 'suralord Joloru pue 'ZdVIAIsp qcns 'suralord leJnlcnJls Surpnlcur 'punoJ uaaq a^pq spuq tuaraJJrpduel4 'saruadord rreqt sarJrpou pue sepqnloJorru ol spurq ]eql uralo-rd .,{uy (dVI I) ulerord palulrossu-alnqnlorrlru (896'd'I-9I 1aue4)'uo1a1e1so1tc aql Jo sluatuelrJJo sasselcJoleur aarq] aql;o auo sr lI 'urlnqnl uralord aqt ;o pesodtuoJ arnlcnlls pcrrpu11,{c ,l,ro11oq 3uo1 aFqnlorJlru (19-71 arn8rg) 'pazruaSouoq are sllal uoqm uorlelueur8er; ,{q pecnpord

sr ]pql unpcrtar rrrxseloopuauorJ pa^rrap "rrtr"xJJnoHrrr* 'uortplsuett str ur 1711-2sern8rg) )Jolq p o:

aqrJouorcnrrsap o] Iaqrrapparuer svNurrr,rli,?NT,"Jil

'yggur qtmr Surrred-eseqdlel Jo ]ualxa aqt uo Surpuadaq -ueruelduroc q8norqt uorssardxaaua8 aleln8ar leqt 'atuoua8 aql uI papoJ stdrrcsue;l VNU pazrlerJads ;o Surssacord aqr dq pacnpord 'sygg crto,{recna (oprtoalcnu 9Z-IZ) uoqs (VNUIru) VNU orclru Jalatullllru g-0I Jo Jalau g_0I ol pnba luaruaJnsearu Jo llun (JelatuorJlru ro rurl; uoJJIru luarrrBlu ullJe aas-luauuluoJJlur q aas-r(e.r.reo.rc gru f urru orcgu y111 ly aas-luplsuor

ualual^tr-sllaBqcllN

(09-92 pue 'ulalord 'uralord 6p-97 sarn8rg) lHhl II sselc CHI l ssplc osID aas'asuodsar aunluurr enrldepe ue aJnpur ol sllac J ot uaq] ]ueserd pue sua8rlue uBraro;;o sluaur8er; aprtded purq suralord 3g6 .{puuyradns 31 ar{l Jo sJaqruau aJp qcrqM

'surelordocdlS arpJrns-ilec aterqetran;o fllrueJ a8rel e auO Jo ulalord 3116 (Ig-gz am8r{) '(suralord JHI ) sulatordocdlSece;rns-1ec ;o ,r{lueg e8rel e ro; Surpoc se}erqaual uI saue8 Jo xeldtuo3 (xelduroc zilllgqpedtuocolsq roferu) xelduoc a9tr41 '(tHl) aupqlau uorJ pa^rrap dnor8 pcrueqc ctqoqdorp,tg (cHf,-) dnor8 piqraur (ZI-02 pue 1-97 sarn8rg) dpoq aqr ur salrs raqlo ol ur8rro Jo alIS rlaqt tuo4 sllec reJuec Jo peardg slselsBloru 't-Zt '71-11 ern8rg) G2OI-ZLOI'dd Iaupd pue 'lurod srqt 'eseqdeue o] paacord te patpq sr a1c,{cflac aql o1 trpear lou sr IIar aqt;y 'aseqdeue le uotleredas p1letuorl{f, -Jelsrs SurpaJard alcr{c 1ec cr1o,{-recnaaq} uJ lutodlcaq3 uoplsuuJl asuqduue-o1-asuqdulau GLOI_ZLOI 'aseqdeleu 'dd 't-Zt le pauoqtsod aru saluosotu 1aue4) -orqr qcrqm ur aueld oql :salod alpurds aqt uaamleq dervrpnu pue alpurds crtolrur aqt ot sa13uutq8lr 1e eueld fueur8erul alBld esBqdulour 't-Zt yaue4)'sa1odatrsoddo pre,r,ro1 pele8 GLS1-ZL11'dd -ar8astad lou a^eq lnq rolenba s1r1e alpurds crlolru aql ol paqJpllp ,t1urr; ere saruosoruoJr{Jqsrq \ lP srso}rru Jo e8E}s aseqdelaur (gt-Z arn8rC) 'uslloqeue snld ruslloqeteo Jo IIV 's1ac 3ur.t11 u1 aculd a1el ]eqt sassacordIerluaqc aqt Jo lelol {uns aqJ IuslloqBlaur (ZZ-gpue 17-9 sarn8rg)'sauosoqlr dq pez(p -lec ssacord e ur uratord olur palplsuerl sl lI 'VNC ;o .ri.doc drelueuraldruor p se aseraru,tlod VNU ,{q apeu alnceloru 'uralord VNU ue;o Surssacorddq selodrecne ur paJnpord u ;o acuanbas prJe ourue aql sagrcadslpql apcaloru VNU (VNUur) ygg ratuassau

(oL-zzpueE-zz

sarnBrg) 'sue8ro Ieuratul aqt Jo duetu pue 'uotalals 'anssll anrlJauuoJ 'alJsntrtot .ros.rnca.rd aql sr lpql anssrl luofuqug ruJaPosaru 'xlJle(u rplnllarerlxa urr{] B ul pappequa sllar Jo 3ullslsuoc 'spru -Iue ur onssrl allloauuor Jo ruroJ pazlprcadsun 'aJnle(urul auraqcueselu '90?I-t0tI 'dd'z-zz @u-zz arn8rCpue 'sloor pue stooqs;o sdrl aql le sruolsrrau pcrde eqt Iaued) are saldruexg '1ue1dSupemog e Jo sue8ro pue sanssll aql ol asr: ang8salrlenrJap asoqm sllac Surpr,trp;o dnor8 pazrue8r6 rualslJaru (t-II pue g-11 sern8rg) 'slauupqo puE (sesEauJad ro srerrrec palpc osp) sralrod -sueJJaJp sasselculelu o^\l eql 'euerquaru e ssorce solnca -loru ro suor Jo eSBssedeql seterpeu leq] ulelord auerqtuel^l ugelo.rd godsrruJl arreJqrueru (t-II pue t-II sarn8rg)'uralord trodsuert auerqruaru e Aq palerpau 'auerquau P ssoJcesalncaloruJo luarua^ol^l godsrmrl erruJqruaru (ZZ-II e.rn8rC)'(prng Surpunor -Jns aql ol e^qeleJ enrleSau eprsuo Aur 09- sr eueJqrxau euseld ilec Ieurue uB roJ Ieltuelod euerqueu pcrddl y 'raqto aql uo suor anrleSau pue aprs ouo uo suor elqrsod ;o 1o ssacxa 1q311s e ol anp auuJqruaru p ssoJopaJuaJaJJrpa8ellon lBpuelod ouuJqruaru Iauuur.IJ aas-IaurrBrlJ

errEJqruoru

(17-71 arn8rg)'unlncrlor cgurseldopua eqt ratua teqt surelord ;o srseqtudsJo etrs aql 'runlnf,ltar cruseldopua aqt Jo aceJc11oso{caqt ot paqcelte auosoqrg auosoqlJ punoq-auBrqruaru

€z:9

AUVSSOt9

0g-9I ern8rJ)'stuatuellJ upce Suop eloru teql sulatord roloru Jo sselc e8rel e go .{riy ulsofru 'tuslueqcatu tuaruelg 3urp11s e dq slcurluoc leql sllac elrsnru Jo urseldol,tc aql ur surelord reqlo pue 'ursodru 'urlcp Jo elpunq pezrue8ro dlq8rq ,3uo1 urquo ru 'enssrt telnpuep ur pue a,ta aql go srrr [7-g7 ernBrg) eql ut '3'a 'ellaqtrda ur punoJ IIec altsnu palBrrtsun Jo ad.r{I 1ac pqaqrldaoftu (Bt-€Z am8rC)'s1selqo,,ttu eldrlynu Jo uorsnJ ar{} dq paruroJ sr elcsnu p}ale{s IIac 'llar ,pelpalcnuouol4l roslnJerd alcsntu palellueraJJrpun V lsBlqoatu 'a{coqdtu,{1 e ueqt roqlo 177-97arn8rg) 1ac poolq alrqrr,r.{uy " IIar plola^ur 'tua1s.{ssnonrau praqdrrad 179-11arn8rg) aql ur sllar uuemqcs ,tq pue tualsds sno^Jau lerlueJ aql ur sel{Jorpuapo311o ,{q peJnpord 'suoxe atprqaual punorp padderm auprqrxa(u IIec pazrl€rcods ;o ra.{e1 3ur1u1nsu1 qleaqs ullaaur (79-21 arn8rg) 'ql^{or8 IIar alel -nrulls lpqt asoql Surpnlcur 'saueBdueu;o uorldrrcsuerl aq1 selE^Ilse11'spuSrs re1n11ecerga.{q ap4lp pue,r,rorS01paluln -Iurls sr IIar u uaqm palelrlJe sr 1uq1urelord r{roleln8ar eue5 " J^IAtr (ggg-tgg 'dd'f-e laued) 'atuos -oruoJqc e;o acuenbas oprloalJnu aql ur e8uuqJ alqelrJeH uopBlnru (1p-97 arn8rg) 'sllac alrsnru qloous pue ,uEaq ,plalaTs erE sasselcureru aarql aqJ'uorlceJtuot roJ pazr1erJadsad,,irIIaO IIaJ alJsnru (6I-0I ern8rg 'acuo ueql arou radelq prdq aqr sasson urcqc aprldadllod aqr qJIqM ul ulalord auerqrrral4l u.raloJd aueJqrrrarusrruJl ssudpptu 'llunqns euo uuql arou 3u1upluoc :suralord;g Jlreruplnru

'etautlrlt atnduoO 'srsltpue cllaua8 pp or rtlateraqqap epeu eq uec ro 'sanssll mau o1asrr a,rr81eq] silac ur uonelntu p Jo llnseJ e se 'dlernleu esrrc ueJ ssresol I 'aloBfrz a18ursu uror; pedole,rap tnq'sadftoua8 lueragp qum sllecJo erntxnu e Jo apetu rusrue8ro pnpr,rrpur ue ',i3o1orqpluarudole,tap u1 (Jr.BsourJpeuet) Jr.Bsoru (91-77 ern8rg) 'satpJ luaraJJrp ldope ol saculd lueragrp uI silac Sursnec dq sllac Jo plag p uo urelled e esodul uer leqt elncalou pu3r5 uaSoqdroru (SII-ZII 'dd'V-ZIoued) 'B ot 'u(OzHJ) elnuroJ praua8 aqt qtr.u re8ns aldturg € = u ereqM oplJur{crusouoru asudlc aas-asBdlc

clJeurouour

'(raurdlod u) alnf,a -lou ra8rel u uroJ ol adr! arues eql sreqlo o1 pe1u11Sureq Jo 'rlunqns e se anras upr teql 4co1q 8urplnq rnt.."tojln#otro_ (Zt-tZ arnBrC) 'sanssll u1 a8uqdorceur e olul sernletu puE luearlspoolq aqr sa Bal lPql ilac poolq elrqr,r ;o addl elaJouoru (B-B arnBrJ) 'lecrluapr [e are 'llal rournl e13u1s teql selpoqque sacnpord europrrq,,{q qrBe e qll1\AIIac g a18urse;o uorsry aqt dq palereueS st etuoptrq.(q eql asnecog 'aul ilac euoprrq^q e dq pelerces dpoqpuy ApoqllrrB lBuolJouour '(uolpp auo ''a'l'Jzi Jo rxole ueJo sseruaqrJo ZIi I ot alnelar'L11crrts; tuole ua3orp.,{r{p Jo sseu aq} ol a^Felar alncalotu p Jo ssEI I (sseru rulnrolou anpular) lqtgan.repcelour auo n dlvqt a as-auoJedur{J Julnceloru 'ecuelsqns aqlJo salncelotu g70I x Z0'9 Jo slslsuoc alolu v '(tq8larvrrelncalourl sseru Jelncaloru aApEIaJsll sr X aleqm 'acuelsqns u Jo suer8 y alolu 'uopnlos ratll Jo I ur pa^lossrpalnlosJo ololu I suPluor uollnlos (nI) ,relotu auo, v 'uopnlos Jo ralll rad aln1os E Jo salolu Jo reqlunN (W) Irlruloru

'seldlcuud pcISol rusurdoilc aqlJorno(s;nrp racuec-o* ,JX3'J',il*jXEil; crqoqdorp.{q drund uec }pr{l ulatord rouodsuerl 3gy3o addl -orq plueuepunJ Sur lrap roJ ,,lapolx,, e se sa^ras snql puP (u;elord gq6) ulelord acuelslse.r tn.rpglpru parpnls porrad ^lalrsualur ueeq seq salcodsV u ralo 3uo1 leql tusguutro lapotu (99-9I pup 79-91 sarn8rg)'UV aseu11ulatord aql alp^llre 'auroua8 aql ur uonrsod JaIDoue ol sdleq raqlo aql alrqm'qlmor8 IIar setulntuns pue urcLruedur Snrp aqt ol elrllsuas sr qcrqmJo auo 'sexelduoc ulelord lcur1 ;1as1r.{docro aloru upc leqt (luau8as y1116)tuaruala rltauaC -slp omt aql) ued sl (UOJ pellec sr Solotuoq tsead Jo UOIur luauala rpauet euqou Le.trqludSurpu8rs l4y7aseup{-t Id aqlJo tuauodruoc ruparts 'f -uaop e sl l€ql aseupl uralord euruoJaql/auues uellururupl{ QLOI-ZLOI'dd -Zf Iaued pue 97-21 arn8rg) 'lrede seruosoruoJqc peleclldnp eql olo(u ol salJes puu sls (ulcduredur ;o 1a8ru1uelpurureru) UOJur -olru Suunp flac rr]odrecne e Jo salod a4soddo eql ueemleq raluac tulznre8ro-elnqnlo.rrlur aas-COItrAI sruJoJler{l salnsalou pe}elcosse puu salnqnloJcllu Jo AeJrv alpulds rllolgu ypg ratuesseru aas-VNgrrr 'splleruoJrIJ aseqd-6 {p3-tr{aas-{p3 ralsrs sp eJeruo[ual ar{] lE JeqleSol plaq IIIls saluosoruoJqJ 't-Zt '7-11 arn8rg) ,trau oml aql qlIM otuosoluorqc pelecrtdnp pasuapuoc,(1q8rg GLO1-ZLOI'dd laued pup 'apmp useldoltc pue snelrnu aql pue pasuepuor are satuos oruosouroJqJ Jlloulu -oruonlc qcrq,r,rSurrnp alcdc rllo^recna aql porrad 1ac Jo Gtot-ztot'dd eseqd 141 'I-ZI 'saruosouorqc pasuapuoJ eq1 ;o ecuereadde Ioued) aluqdsoqd-g asouuutu aas-dglAl e{rpeaJql aql ot Surrra;al 'pealql e'so1lut laarD uoJc 'slas IeJrluepr orvrl ruJoJ ol sotuosoruorqc patecrtdnp aID Jo uou -eredaspue 'saurosoruoJrloalqrsn olul VNC aqlJo uonesuep -uoc Surnlorrur '11accrloftecna e Jo snapnu aql Jo uolst IC slsollru '(alnJolour crraru.{1odraq}o ro luaruBIIJ utalord) {rprl rpaurl e Suole JIastrledord or srsdlorpdqateqd -soqdr4 eprsoalcnu ruo{ pe^rrap ^48rauasesn tuqt ulalord ulalord roloru

'su1a1o-rd;or{lelrerr u uI pazru -3ocar aq uec teqt u1eruop lerntcnrls IIEus e dlecr;rcadg 's1xaluoc dueru uI sJnJar leqt uraupd Jo arntJnrls Jo luaualg JIlolu

aseupl dvtrAIaas---€suuq u1a1o.rdpelunpce-uetolpu 'oleJo -;rtord o1 sllar salelnulls lpql alnralou pu8rs relnllacerlxg uatolpu

AUVSSOID VZ:9

arn'1g;.slJac a rauurpunoJlua.'plg arn,o"_ili3J.lrn$rrrr"., 177_91

(67-p1 arn8rg) 'er:alceqouedcpue s1ue1du1 HdCVN pup dIV qtoq sacnpord teqt ssacord crlaqludsoloq4 uolluf-roqdsoqdoloqd cycf cuou

e Jo leurruJal uoxe ue ueaalaq asdeuds lesrruaqJ pezlercad5 uollcun I rulnJsnruoJnau

JrEde qJIqM ur srsoraruSurrnp.{lleuorseccoSurrrncJo tualg uopcun[s.rpuou

(I I I- 0I I 'dd ' t-Z Ieued) 'salnf,aloruuaamtaq suor1]eratul cgrcads,{1q8rq'8uo.rlsacnpord ot raqlaSot urns upr,,{aqr rnq 'Iua^tAIaAIlelaraJuspuoq 'pareqs arP suorlJala lualP^oJuoN ou 'puoq lualP^oc e ol lsPrluoJ ur 'qcrqM ur puoq IeJIuaq:) (uollruralul luale^oJuou) puoq lualp^oJuou luele^oJuou'uollJuJlle

aplxo rlruuaas-oN '(1gyq)SurBeur atupuosar tltau 167-9arn8rg) -8etu ur sasodrnd crlsou8erp 1ecrparuJoJpasn oslp aJpUINN ;o saldrcurrd eqJ'salnraloru llpurs raqto pue surelord lpus Jo aJnlcnrls leuorsuaurp-aarql aql aururalap or IIaprM pasn sr UIAIN'relf,nu aql Jo ]uauuorl ue IEJrrxeqcaql ]noqu uollpuroJul sapnord unrlcads HhiN eql'slueuou elodrp crlau8eu rrar{t Jo uor}etuarro aq} Jo Surddrg ol enp /pleg crlau8eu e ur ralJnu cnuolp dq ,{cuanbar; cgrJads e Je uorl -erper crlau8euorllala ;o uorldrosqu lueuosot aql sr UhlN (,{docsorlcads 91,1lq'acueuosar cllauteru JualJnu) UNN JalaruorrEu aas-tuu (Ig-ZI pup 69-71 sa:n8rg; aprreqccesoSllo pa4ur1-g atad?roJ 'anprsal aur8eredse ue Jo ureqJ aprs ar{t Jo dnor8 zHN aqr q8norqt uralord e ot paqrelle sre8nsgo u1eq3 aplruqrrusot[o pe{ull-N IIar rallpl lernluuaas-ilec

xN

'sallloqplaru Surureluoc-ua3orlru snorrerro1.{1un1ue.re Surpeal 'eruoruure ol (zN) uaSorlru cpaqdsoule saonpar leql plJalJuq ureuac ,{q rno perJJBJssatord lpJruaqcorg uolluxrg uatorlp 'stueld pue sleurup qtoq ur uorlprrunru 171-91a.rn8rg) -IuoJ IIaJ-llal ur pasn.{laprrnsr lpql alnJalour pu8rs snoeseg (ON) apr-xo]IDIU aas-eluqdsoqd

Hd(rVN/+dOVN apltoelcnulp auluepu aplruuullorp

HOVN/+OVN aas-aplloalrnulp

euluepu epllrrEullorlu

(62-91 arn8rg) 'lueurdolanap 1erurueur salol tuetrodur seq oslv'sasuodsar ssaJlslo ',{roleruuregur'aunrurur Surrnp palelnurls eJp sllal uaq.tr sr{.erulqledSurleuSrs JulnllaceJlul snouel dq palearlce sr 1eq1uralord ,fuoteln8er aue8 luele.I ule1o.rd glg11 'pauegur ro palcaJur ftZ-gzpue gg-77 sarn8rg) aruoJaq leql sanssrl sralug 'srsolLco8eqd dq lerreteu atel -nrlupd Jo a1eldn aqt roJ pazlprcads sr 1eq1flac poolq a]rqr\\ gqdo.rlnau (1-7 arn8rg) 'snapnu Jrurole ue ;o lred suroJ teqt alcrlred Jrruotpqns ,{,teaq pa8reqcun uoJlneu 'saprldado:nau dueur pue'aurcrt13 'vgvg 'ateuelnlS 'aulloqcldlace '1ac apnlrur saldurexg crldeu,{s1sodaql ol leu8rs aq} ^elal o1 asdeu,r{s IelrruaqJ e lp IIac alrau crldeudsard aq] ^q patatras alnJalou pu8rs lptug JelllusrrBJloJnau 'eJaqMaslaro sasdeuds le raqlla alnJalotu leu8rs e se suolnau ,{q pala.rcasaprlda6 apFdedornau 'sleu8rs ft6-ZZ pue 96-77 sern8rg) llrusuerl pue rlJnpuoJ 'aAlaJoJor paz;prcads sassacorda.l,rs -ualxa qlrM 'ualsrts sno^Jau aq] Jo IIec Surlcnpuoc-as1ndur1 (IIaJ a Jau) uorneu

(6€-rr

pue g€-II sarn8rg)'ilac alJsnu plole{s p pup uoJnau roloru

(99-77 arn8rg) 'rosrncerd IIac alrau eruodrqtug lsulqoJnau (90I-9I arn8rgl 'atlrpuap u Jo uoxe ue sr ssacord aqt raqtaq,l,r.{;ycads1ou saop }eql ruJal rrraue8 V'arnllnJ ul IIar alJau u ruor; Suwror8 ssecord 3uo1 ellJneu (g2-77 anh g)'o.{rqua eterqatral e ur pror purds puu urerq aqt uroJ llrrvrteql rurepolce Jo aqnJ aqnl lerneu U6-ZZ pue' VB-ZZ'I t-6I sern8rC)'smeI pue areJ aqt Jo sauoq aql pue ruDlseq] Jo slloc luoruSrd 'uralsrts sno^rau pra -qdrred aqr;o e13 pue suorneu Surpnlcur 'sanss1}Jo /torft^ € ol aslr an13 o1 a1er31u sfiec lsarc lernaN 'ortrqua alerqet -ral aql ur srturaprda Sulpunorrns eql tuo4 JJo seqcurd eqnl IPrnau aql araqa eull eql SuoP polecol sllac Jo uollJolloc lsoJJ luJnau _ (02-6I arn8rg)'salerq -eue^ ur ]uetuqcetle IIaJ-llaJ ]uapuaoapur-+7e) lo rolPrpau 'sllal a^rau tsoru Surpnlcur sad.& 11ar,{ueur [q passardxa v '.{pue;redns urlnqolSountuur aqt Jo alncalotu uorsaqpe IIoC 0lMN) alnralour uolsaqpe iloc prnau pltuatod uo11cuaas-aslndrul uornau aas-llal

a^Jau a^Jau

(oLq'd'Z-lr 'auerqtuatu aqt ssorre aruaraJJrp a8ellon aql ol auerq Iaued) -urotu alqeau-rade 1o aprsraqlra uo uor ue Jo suorlerluacuoc Jo orler runrrqllrnba eql satelar leql uorssardxa alrtetrluen} uollunba lsu.reg 'de,uqled arups aql u1 dals rarlrea ue slrqrqur ferr,r.qled Jo uolcpal e ;o lndlno aqr .{qalaqm uslupqJaru IoJluoC {JBqpaaJ a,rllu8au 's11ac ]acueJ aruos puE slloo palJaJur-snJrl 129-77arn8rg) IIrT ueJ 1eq1 tue1s.{s oumurur a}puur aqt ;o 11acrrxolopt3 (llar )N IIar rallpl prnluu 'retoru 'salleueSro IIas 6-0I = (ull'i) releuorcru €-0I = Iuu I pue salnJalou aJnsearuol pasn .{luouruoc qfual Jo lrun (tuu) ralotuoueu (tI-II am8rC) 'srs,tlorpdq '11acoql olur dJV ruorJ po^ltap ,{3reua Sursn +) Pue Jo lno +eN sorxno lEql sllec Ieurue lsoru Jo auPJq -uau eruseld aqt uI punoJ ulalord ralrJeJ aueJqruausue{ (asedlv +)-+ptrf dund +)-+sN (99-7 arn8rg)'sde,uqted'rrloqplec ueql reqler 'cltaqtufsorq anllcnpar ur ,{laalsnlcxa lsoup pasn 'HCVN/+CVN palelrr ot ,{1aso1c uatsfs rar]rpr uoJlcelg ]nq (aleqdsoqd appoalrnulp auluapu apruruullorp pacnparTaleqdsoqd oppoelcnulp euluapB apFrruultorlu) HdOVN/*d(VN (97-71 arn8rg) 'HCVN 'ureqc prrpuoqJ tuo4 suoJlJala sldaccu rtrolurrdsor 11 -olru eql ur sdund uolord ua^rJp-uorJsaloaarql aql Jo lsJrc xaldtuoc asuuatorp^qap HOVN (99-7 arn8rg) 'uorteldroqdsoqd azrrleprxo ;o ssacord SurlereuaS-41y aqt ol suorlsala ,iSraua-q3rq sll saleuop paruroJ HCIVN aql 'HCVN JarJJBopalenllf,p aql aruocaq ol (suoJlcola o.tq snld uolord e '_H) uor apgpdq e yo luap.runba aqt sldaf,re +CVN 'salnJalou pooJ 'suollsear uorl Jo uorlepuo eql sE qtns -cnpaJ-uoltepr-xour saledrcrlredleql uralsdsrarJJecuoJlJalg (appoalcnugp auluepu aprrrrBurlorlu euluapu apFrrBullorlu) HOVN/*OVN parnpor/apFoalrnulp

sz:,

AUVSSOI9

(ggg-tgg 'dd 't-g 1aue4)'aua8 e 3o I1r.trlce aq] saqsrloqe dletaldruoc leql uorlelnu uortrunJ-Jo-sso.I uopslnu IInu 'sarxosoruoJqcolur pazrue8Jo ,s11ac VNCI 8ururc1uoc crlo,{recna ur allaue8ro papunoq-auetqrueru luaunuoJd snelJnu (97-g ern8rg) 'rredar uorsrcxaaseq atndwo3 'e1e1drua1se puerls pe8eruepun aql Sursn U Suras -eqludsar pue puerls auo uo uor8ar pa8eruep aqt lno Sunlnc .,tq 'a8eurep rul ro pcrueqc ,{q pesnec asoqt se qcns ,xllaq alqnop VNC aql Jo a8euup stcarroc teql rpdar ygq go ad,,{; rr.edar uolslcxa aplloolJnu

IeuueqJ snoanbp ue 8u1uro; arnlcnrls uralordDlnu a8rcT (34trf xalduroc arod ruelcnu uIAIN aas-aJrrBuosa.r cllauturu JBaIcnu (9I-ZI pup II-ZI sarnbrC)'saxalcl -uoc arod rpalrnu aql q8norql Iosollc aqt ruor; snalcnu aqt olul uoclsuert a^rlcalas rlaql salqeue leql snelcnu oql roJ paurlsap suletord ur punoJ qcled puSrs ro acuenbas puSls (9111)pu8ls uollezlletol ruelrnu 'suruel rPallnu rxoJJ paruJoJ slueluelIJ elelpeIu -Jatul Jo {Jomlou p Jo dn operu sl }I 'aueJqruaru Jeelcnu Jeuur aql Jo aceJJnsrouul aql uo sulaloJo Jo {Jo^ qseu snoJqlc Bulurul JEeIcnu

(91-71 arn8rg) 'sexeldruocarod realcnu q8norqt Iosol{c aql ol snalrnu aql tuo4 pauodsuer} are 'stlunqns puosoqlr.&\au pue sVNU se qcns 'saxeldruoc teqt pue salnsalou Jo ernlcnrls aql ur peurPluoc puSls Suqros putls godxa Jualcnu

'urleu OZ-, pue g7-p sern8rg) 'suralord auolsrq -oJqc Jo lrun prnlonrls plueuepunJ aqg Jo eror lrraruetJo up punore padderrr VNC Jo qfual uoqs e ;o pesodtuoJ 'ull€urol{c JI}o^r€Jna ur eJnlonns alnpeog oruosoalJnu

'EUrruelIeelcnu aql ruroJ ler{l s}uauellJ alelparuralul eq} Jo llunqns ule}ord ururel JEaIcnu

OI I-9II'dd'g-Zlauud)'saprloelcnu Jo sraru -d1od ere VNU pup VNC ,{ralour ru8ns aqt o1 sa8eluq ralsa ut peuro[ sdnor8 eleqdsoqd ororu ro auo qtr^\ eplsoalrnN ePpoapnu

(ZI I-9I I'dd' g-Z1eue4)'re8nsasoqrr.,ixoap ro asoqrr e ol paIurl ,{puap,roc aseq eurpr{urrdd ro aurrn4 aplsoelJnu 'saxaldurocerod rualcnu dn a>1eru}eq1 suralord luereJJrpJo Jaqrunu e yo ,tuy urrodoalcnu (6-t am8rC) 'palquasse ere slrunqns Ipuos -oqrr puP paqrrcsuprt sl vNur araq ^ snalcnu aql ul arntf,nrts snIoaIJnu 'snloalrnu z [7-9 arn8rg) 3o ped o1 asg sa,tr81eq1 saua8yggr;o Jalsnp e Surureluoc atuosoruorqc e go uorBag ra4uut.ro reloalJnu

(B-ZI pue 6-7 sarn8rC) 'tunlnrllar clus-eldopue eq] qlF snon_ulluocsr auPrqualu Jalno aql'saJod JealJnu Aq peleroJrao sl pue auerquaru Jauul pue Jelno ue Jo slslsuo] 'snalcnu aqt Sulpunorrns (sra[u1q oml) auerquau alqnoq (auerqueur Jealcnu) adola ua rBaIJnu (1-g arn8rg) 'dnor8 ourue-n aorJ e salrrpc lErD uleqt apqdad.,{1odp Jo pua eqJ (snulural ouFrre) snuFural N '[UVNS-1 e pue SUVNS-^e kz-il pue BI-€I sern8rC) go xeldruoc e salquessuslp reqt Il1nIlrB asEdIV r{}Iir''ulalord (rolruJ a lllsuas aplruleptudqra-N JSN xaldruoc eJod JDoIrnu aas-CdN

'alPrras pue Pllac sP r{rns sulal 192-91arnSrg) -ord ace;rns-11ec are spuu8rl sll 'urnllaqllda plurapotce IuoU sllar a^rau Jo uorlecglcads eql uI alduexa roJ 'lueudola^ap IeruluB ur sacrorlr aleJ-llac dueur u1 paAIoAuI (utalo.td [ro] -eln8ar aua8 lualel pue) ulalord roldacar auetqtuorusue{ qJION

uolrezlplrqziq aas-uolle4p1.rq(q pleu rlalrnu 'spuoq ratsalpoqdsoqd pauroI dq raq1a3o1 seprtoalcnu 'VNC ro Jo ureqJ e ;o SurlsrsuoJ alncalotuoroeru e VNU pIJu JIeIJnu 'ssacorddlqruasspuE ur dels 3ur1nu11-aler aqt flleraua8 erow (626-BZe 'dd 'Z-gt laued) 'uortezuaru -dlod ptder alelllul o1 luaura8uerre llarror aql ur sote8erSSe sJaruouoru Jo ralsnlc lletus e qcrq A ur 'alnqnlolJ[u e se qJns taJnlonrls crraudlod e;o dlqruasseaq] ul a8pls leorluJ uollualcnu 'ase -apnuoxa pue aseelJnuopua os122 aas 'sapl]oalJnu uaaMlaq spuoq Surzdlorp,rtqdq sprJe cralonu s1r1dsleqt atu.,{zug asualJnu

(26-77 enfu g,)'uunlor Iurqal -JOAaql o1u1palerodroJul sa{uoceqse}elqaua^ uI 'so.4Jqlue eleproqr IIE Jo srxe pl]uar aqr Sulugep sllor Jo por JJITS pJorlJolou

(61-91a;n8rg)'prsduco -alonu aql spunoJJnsadolanue eql 'luasatd sr edolenue pr4 e ;1 'prsdec uralord Surpunorrns slr snld prJe crelcnu Iprr1 plsdEroalrnu

(9I-ZI arnBrC) 'roldacar lrodxa.tealcnu ro roldacar podrur JEalcnu:snalJnu aql Jo tno Jo olul raqlra selncaloruoJJerusuocsa leql ureloJd (u1raqdof.ro1) roldacar lrodsuerl reelcnu 'uorr 19-gatn8rg) -cafurorcru trq raqloue o] flac auo uorJ snolcnu e Jo JaJsue{ uollulueldsuuJl JBalJnu (71-91 arn8rg) 'snapnu aql ul Jolf,pJuorldrrcsuerl p su slrp xalduoc pueSll-JoldaJaJ aql'prJe crourlar pue sauorulor{ pror^r{} pue proJalssp qcns salnoaloru pu8rs crqoqdorp.,tq ro; sroldace.r relnllacertul flpre;radns .roldaJer reelcnu (6-ZI arn8rC) 'useldoilc pue snalcnu uoomtaq aloru ol sapcalou palJolos smolle tpql adolanua realrnu aqt q8norql (arod reelcnu aql)

(gZI-gI arn8rg) 'aurut8re;o uoll -purueap aql .{q (ON) opuo rlrtlu sazlsoq}u.{s teql au^zug (soN) eseqrurts oN 'eqord prce cralrnu paleqel e qtrM uorlEzrpuq.{q.,tq parce}ep ueq} sl vNu crJrcadse pue 'laaqs raded e uo pezlllqotutul are slseroqdol -rale ^q paleredas stuatu8erJ VNU qclll\,\ uI enbluqcel tulrrolq ureqlroN (gg-9 arn8rg) 'urelord olur petelsuPrl eq uEc faqt aro;eq suopor dols purelur eurer; -ur Sururcluor sVNUru luprlaqe Surper8ep roJ tus1uugral4 fucep ytr1gru polulpau-esuesuou uosodsuerlo.rlal aas-uosodsuu.rlo.rlar [eJlaoJlaJuou (III- B0I 'dd'e-Zpue Z-Z slaued) 'ratem 'e8reqc a u ur alqnlosur dlereua8 aft salncalour teloduoN -e8eu pue a,trlrsod;o uollulnrunoce cplaunu.{se ,{ue 3uo1ce1 (rulodu) ruloduou (epue JVZ-V7 arn8rg) dluo prsdec urelord p pue eroc plcp rlalcnu e Jo Sultslsuoc snJIA snJl pedola uauou 'seruos -ouorqJ AraJool ro .{ueru oot roqlla seq IIec u:a8 Suqlnsar aql luql os aleredes ol sllPJ saluosotilorqJ snoSoloruol{ Jo

AUVSSOID 9Z:'

G:27

GLOSSARY

nurse cell Cell in the invertebrate ovary that is connected by cytoplasmic bridges to a developing ooclte and thereby supplies the ooclte with ribosomes, mRNAs, and proteins needed for the development of the early embryo. (Figxe 2l-24) occluding junction Tlpe of cell junction that seals cells together in an epithelium, forming a barrier through which even small molecules cannot pass-making the cell sheet an impermeable (or selectivelypermeable) barrier. (Figure l9-2, and Table l9-1, p. ll33) Okazaki fragments Short lengths of DNA produced on the lagging strand during DNA replication. Rapidly joined byDNA ligaseto form a continuous DNA strand. (Figure 5-7) olfactory sensory neuron The sensory cell in the nasal olfactory epithelium responsible for detecting odors. oligodendrocyte Glial cell in the vertebrate central nervous system that forms a myelin sheath around axons. Compare Schwann cell. oligomer Short polymer. oligosaccharide Short linear or branched chain of covalently linked sugars. (Panel2-4, pp. I l2-l 13) O-linked oligosaccharide Chain of sugarsattached to a protein through the OH group of serine or threonine residues. Compare l/-linked oligosaccharide. (Figure l3-32) oncogene An altered gene whose product can act in a dominant fashion to help make a cell cancerous.Typically,an oncogene is a mutant form of a normal gene (proto-oncogene) involved in the control ofcell growth or division. (Figure20-27) oocyte Developing egg, before it has completed meiosis. (Figures 2l-25 and2l-26) oogenesis Formation and maturation of oocytes in the ovary. figure

2r-23)

open reading frame (ORF) A continuous nucleotide sequence free from stop codons in at least one of the three reading frames (and thus with the potential to code for protein). operator Short region of DNA in a bacterial chromosome that controls the transcription ofan adjacent gene. (Figure 7-34) operon In a bacterial chromosome, a group of contiguous genesthat are transcribed into a single mRNA molecule. (Figure 7-34) ORC-see origin recognition complex ORF-see open reading frame organelle Subcellular compartment or large macromolecular complex, often membrane-enclosed, that has a distinct structure, composition, and function. Examples are nucleus, nucleoIus, mitochondrion, Golgi apparatus, and centrosomes. (Figure l-30) Organizer (Spemann's Organizer) Specialized tissue at the dorsal lip of the blastopore in an amphibian embryo; a source of signals that help to orchestrate formation of the embryonic body axis. (Named after H. Spemann and H. Mangold, co-discoverers)(Figure 22-74)

origin ofreplication Site in a chromosome where DNA replication starts. origin recognition complex (ORC) Large piotein complex that is bound to the DNA at origins of repllcation in eucaryotic chromosomes throughout the cell cycle. (Figure 5-36) orthologs Genes or proteins from different species that are similar in sequencebecause they are descendantsof the same gene in the last common ancestor of those species. compare par' alogs. (Figure I-25) osmosis Net movement of water molecules across a semipermeable membrane driven by a difference in concentration of solute on either side. The membrane must be permeable to water but not to the solute molecules. (Panel I l-1, p. 664) osteoblast Cell that secretesmatrix of bone. (Figure 23-55) osteoclast Macrophage-like cell that erodes bone, enabling it to be remodeled during growth and in response to stresses throughout life. (Figure 23-59) osteocyte Ndndividing cell in bone that develops from an osteoblast and is embedded in bone matrix. (Figure 23-55) ovulation Releaseof an egg from the ovary. $igure 2l-26) ovum Mature egg. oxidase Enz).rynethat catalyzes an oxidation reaction, especially one in which molecular oxygen is the electron acceptor. oxidation (verb oxidize) Loss of electrons from an atom, as occurs during the addition of oxygen to a molecule or when a hydrogen is removed. Opposite of reduction. (FiSure2-43) oxidative phosphorylation Proceis inbacteria and mitochondria in which ATP formation is driven by the transfer of electrons through the electron transport chain to molecular oxygen. Involves the intermediate generation ofa proton gradient (pH gradient) across a membrane and a chemiosmotic coupling of that gradient to the ATP slrlthase. (Figures 14-10 and 14-14)

p5s

Tumor suppressor gene found mutated in about half of human cancers. EnCodesa gene regulatory protein that is activated by damage to DNA and is involved in blocking further progression through the cell cycle. (FiSures 20-37 and 20-40)

^pairing (homolog pairing) Inlmeiosis, the lining up of the nvo homologous chromosomes along their length. (Figure 2l-6) gene pair-rule ' ln Drosop,hila development, a gene expressed in a series of regular tiansverse stripes along the body of the embryo and wfiich helps to determine its different segments. (Figure 22-37) sequence palindromic Nucleotide sequence that is identical to its complementary strand when each is read in the same chemical directione.g.,GATC. (Figure B-31) Par3, Par6 Scaffold proteins involved in the specification of polarity in individuil animal cells; Par3 and Par6 form a complex with aty?ical protein kinase C (aPKC).(FiSure19-31)

G:28

GLOSSARY

paracrine signaling Short-range cell-cell communication via secreted signal molecules that act on neighboring cells. (Figure l5-4) paralogs Genes or proteins that are similar in sequence because they are the result of a gene duplication event occurring in an ancestral organism. Compare orthologs. (Figure l-25) parthenogenesis Production of a new individual from an egg cell in the absenceof fertilization by a sperm. passlve transport (facilitated diftrsion) Transport of a solute across a membrane dovrmits concentration gradient or its electrochemical gradient, using only the energy stored in the gradient. (Figure ll-4) patch-clamp recording Electrophysiological technique in which a tiny electrode tip is sealed onto a patch of cell membrane, thereby making it possible to record the flow of current through individual ion channels in the patch. (Figure ll-33) pathogen (adj ective pathogenic) An organism, cell, virus, or prion that causesdisease. pattern recognition receptor Receptor present on or in cells of the innate immune system that recognizesand responds to pathogen-associatedmolecular patterns (PAMPs)-such as surface carbohydrates on bacteria and viruses and unmethvlated GC seouences in bacterialDNA. PCR (polyrnerase chain reactlon) Technique for amplifying specific regions of DNA by the use of sequence-specific primers and multiple cycles-of DNA synthesis,each cycle being followed by a brief heat treatment to separatecomplementary strands. (Figure B-45) PDZ domain Protei!-binding domain present in many scaffold proteins, and often used as a docking site for intracellulai tails of transmembrane proteins. (Figure l9-21) pectin Mixture of polysaccharides rich in galacturonic acid which forms a highly hydrated matrix in which cellulose is embedded in plant cell walls. (Figure 19-79) pentose Five-carbon sugar. peptide Short polymer of amino acids. peptide bond Chemical bond between the carbonyl group of one amino acid and the amino group of a second amino acid-a special form of amide linliage. Peptide bonds link amino acids together in proteins. (Panel3-1, pp. l2B-129) peripheral lymphoid organ (secondary lymphoid organ) Lymphoid organ in whichT cells and B cells interaitwith foreign antigens. Examples are spleen, lymph nodes, and mucosal-associatedlyrnphoid tissue. (Figure25-3) peripheral membrane protein Protein that is attached to one face of a membrane onlv bv noncovalent interactions with other membrane proteins, and which can be removed by relatively gentle treatments that leave the lipid bilayer intact. (Figure l0-19) permease-see transporter permissive (nonrestrictive) conditions Circumstances (such as temperature or nutrient availabiliW) in which rhe phenotypic effecrof a conditional mutarion will be absent:that is, the phenotype will be normal. (FigureB-55 and Panel B-1, pp. 554-555)

peroxisome Small membrane-bounded organelle that uses molecular oxygen to oxidize organic molecules. Contains some enzymes that produce and others that degrade hydrogen peroxide (HzOD. (Figure 12-30) pH Common measure of the acidity of a solution: "p" refers to power of 10, "H" to hydrogen. Defined as the negative logarithm of the hydrogen ion concentration in moles per liter (M).pH = -log [H*]. Thus a solution of pH 3 will contain l0-3 M hydrogen ions. pH less than 7 is acidic and pH greater than 7 is alkaline. phage-see bacteriophage phagocyte General term for a professional phagocltic cell-that is, a cell such as a macrophage or neutrophil that is specializedto take up particles and microorganisms by phagocytosis. (Figures 13-46 and 13-47) phagocytosis Process by which unwanted cells, debris, and other bulky particulate material is endocytosed ("eaten") by a cell. Prominent in carnivorous cells, such as Amoebaproteus, and, in vertebrate macrophages and neutrophils. From Greek phagein, to eat. (Figure 24-53) phagosome Large intracellular membrane-bounded vesicle that is formed as a result of phagocytosis. Contains ingested extracellular material. (Figure 24-30) phase-contrast microscope Type of light microscope that exploits the interference effects that occur when light passes through material of different refractive indexes. Used to view living cells. (Figures g-7 and

22-r0t)

PH domain-see

pleckstrin homology domain

phenotype The observable character (including both physical appearance and behavior) of a cell or organism. (Panel B-1, pp. 554-555) phosphatase Enzyme that catalyzes the hydrolltic removal of phosphate groups from a molecule. phosphatidylcholine (lecithin) Common phospholipid present in abundance in most bioIogical mebranes. (Figure l0-3) phosphatidylinositol An inositol phospholipid. (Figure 15-37) phosphatidylinositol 4,5-bisphosphate (pI(4,5)pz, plpz) Membrane inositol phospholipid (a phosphoinositide) that is cleaved by phospholipase C into IP3 and diacylglycerol at the beginning of the inositol phospholipid signaling pathway. It can also be phosphorylated by PI 3-kinase to produce PIP3docking sitesfor signaling poteins in the pI 3-kinase/Akt signalingpathway. (Figures15-38 and 15-64) phosphoanhydride bond High-energy bond linking phosphate groups in, for instance, ATP and GTP (Panel2-6,pp.116-117) phosphodiester bond A covalent chemical bond formed when two hydroxyl groups form ester linkages to the same phosphate group, such hs between adjacent nucleotides in RNA or DNA. (Figure 2-28) phosphoglyceride Phospholipid derived from glycerol, abundant in biomembranes. (Figures 10-2 and 10-3) phosphoinositide-see

inositol phospholipid

(g7-17 a.rn8rg)'aleraueBap.{1un1ua.La sarpoq relod eql 'Jlastrrunlo ro a{coo aql Sureq ralq8nep (a8reDraqto aq] 'ailf,oo fuepuocas ;o dreurrrd B Jo uorsrlrp cp1auru.,{se ,{q srsoraurSurrnp pacnpord slac ra}q8nep o-t,r}Jo rallprus dpoq rulod (60I-80I 'dd'Z-ZIeued pup g1-7 arn8rg) 'IalP-/rl uI alqnlos aq ot .,(1a1r1 Jre salnJaloru Jplod 'suoJl -cala uorlnqr[srp ua^eun up Jo Jo unsar p se rarl]o aq] ple^rol a8reqc anrle8eu pup pua euo pJp.&\olpalerluacuoc a8reqc enrlrsod qlrm (elnoaloru ro 'dnor8 leJrruaqc 'puoq I€Jrruaqc e'eldruexa roJ) alnlcnJls e saqrrcsep'asuasIpJullala aql uI rulod (999-t9g 'dd't-e laued) 'VNC ur 'aua8e13urs e;o lred 11eurs .,{rerre ro lred aprloalcnu a18urse ;o a8ueq3 ropulnu lulod (626-8Z6 'dd 'Z-gt 'pua paqreq ar{t sp ulrrou>Iosp sr lueu laued) 'tuaruplg urlJe to alnqnlorcrru -BIUurlrp up Jo pua snld aq1 e Jo pua ,,3ur,r,ror3-1se;,, aq] :^lrpeal lsotu slnJoo sraruouoru Jo uorlrppP r{Jlrl^\ lp tualuplrJ ur}Je ro apqnlorsrru e Jo pua pua snld 'oml ueql eroru aAEqsruslue8ro pro1d,{1od11asa13ursp aleq sursrue8roproldeq :sllar rrtpruos Jleql uI slas ol\l aleq srusrue8ro proldrq 'auoua8 e ur saruosouorql;o slas snoSolouoq elaldruoc Jo Jaqrxnu aql fplo1d 'pale^rlcE sr aspuDl-t uaq^ eupJqtualu Id etuseld aqt ol uralord 3ur1eu3rsaql Sur8uuq 'aseuol-g 14 dq pacnpord aleqdsoqdsrrl-9't't Iotrsourl.,{prleqdsoqdo} purq sulalord Sulpu8rs relnllaopJtur ur sureruop Hd aruos 'sural -ord Suqeu8rs JplnllaceJlur aruos ur punoJ urpruop uralold (u1europ 114) upruop,{Solouoq up1s1ce1d 3 asudlloqdsoqd aas-314 'parn[ur aJe slassal poolq uaq^ 3uq1o1cpoolq atertrur sdlag 'uea.rlspoolq aql ur slaqrunu e8Jel ur punoJ sr pue ,troJretu auoq eql ur aldcor{rele8au e ruo{ JJo slearq leqt 'snalrnu e 3uqce1 'luaru8e.r; 11a3 1a1aluyd 'sprlse1dere slse1doro1q3 'petuauBrd uauo sr pue ,auetq VNCI umo slr sauJpt leqt -uaur alqnop u,{q papunoq 's1ue1dur alleue8ro crurseldo/.3 ppseld 'sllem IIer rraql ur arod 11eurs e q8norqt sl1acluacefpe olr1 slJauuoc eueJqruau euseld .r{qpauq use1do1,{cJo Ieuupqr e qJrr{ ^ ur slue1d ur uor}cunf 'uorlcunf de8 e;o Ilac-llal Surlecrunruuro3 lualu,rrnba1ue14 (Blausapouseld prnld) urusapoursuld '8uruo1c VNCIroJ slotJal se Iyanrsualxapasn a:e spruseld palJlpohtr'auoua8 aqt Jo ^lluapuadapur selec -r1dar1eq1alnJalour VNC leurosoruoJqJurlxaJelnJJrcIIeuS pnusuld

aql qll^\ Suop sauosopu-a olul pa olu pue salclse^ [PIxs ur lueruuorr^uo aql tuorJ on ua)el Allenul]uor arP slelJalelu alqnlos qsrq^t ur srsol,(copua;oadd1,,'8ur4urrpIIa3,,'d1pra1r1 slsopbould asuull-€ ap.rllsouloqdsoqd aas---esuuq-t Id 1u1odcplcalaosgaas-1d QL-Vrpue gL-v sarn8rg)'aar1c11aua3o1.,{qd e se ruro; ueqr uI paluasa.rduago 'stuslue8ro;o dnor8 ro rusruBS.roue;o frolsrq freuoqnlolg fuato1l(qd (Z9-ZI ern8rC)'a1e1d11aceqtJo uoneuro; saprnB pue IIaJ 1ue1de;o uorswp Jo eueld enrtcedsordaqt ur sruroJ leql sluauelrJ urlcP pue salnqnlorcru Jo apeu oJnlcnJls lsuldouturqd (g7-y1 ern8rg) 'ISraua Jo sruroJ InJesn ol lr sue^uoc pue lq8rtuns Jo dSraua aqt sarnl -dec teql srsaqtudsoloqd ur perrlo,rur xeldruoc ure1ordrl1nytr urelsfsoloqd (B€-tI arnBrg)'HdCIVN pue dJV Surteraue8 'aueJqruau e ur ureqc lrodsuerl-uorlJala ue Suop e,Lotu suorlsala qJrqM ur srsaqlur{soloqdur suorlcear uarrup-1q311 raJsrrErl uorlrala cpaqlu^,tso1oqd (B€-tI pue g7-7 sarn8rg)'tate^ pue opFolp uoqrBc ruo4 salnraloru rrue8-ro;o srsaqludsaqt a^rrp ol tq8Iluns Jo d8raua aql asn prratreq aruos pue aeBp 's1ue1dqcqm .,{qssecord slsaqlu^soloqd 'aBetots -ro; a1erp,,{qoqrer uortrnpord aqt }noqll^ Jo pelereqrl zOD pue dn pasn sr zO rlrrqm ur zOJ l,rol Jo suoq -rpuoJ ur s1ue1du1 sJnJro l€ql ssacord cr1oqelau InJelsEM uopu.4dseroloqd 'tq8n ol alrllsuas sr leql aFcoloru ro Iipr"UU (97-71 arn8rg)'s1saqlu.{so}oqdur,{3;aua pcluaqr otur d8raua tqSll sua^uor 1uq1ua1s,{soloqd e ;o tred aql raluar uollruar pcpueqcoloqd (0t-6 arn8rC)'llar aql ur lods asrc -ard e te tqBIIJo rueaq pasncoJe,{q palenrlce uaql sI pup 'llar aql olur poJnpoJlur sr lsaJalul Jo alnJalou e Jo ruroJ e^rlceul ue qJrqm ur sassacord relnlloJeJlur 8ur.ri.pn1sro; anbruqcal uo11e,r11ceo1oqd (09-9I arn8rfl 's,{e,r,rqledSurpu8rs repllacertur ur uotuuroc are sappJsec qJns 'ureqJ aql ur eseuDl lxau aql sale^rlce pue sa1el,{roqdsoqdqcrqm go qcEa 'saspur4uralord;o sarres u [q palerpau suorleldroqdsoqd uralord prluanbas;o serrag epucser uo.rlu/roqdsoqd 'olnsaloIu Jaqloue o1 paldnoc rtltuaploc s1dnor8 aleqdsoqd E qcrqm ur uorlcpag uo11u$roqdsoqd

(9II-tII'dd'E-7 1aue4pue'€-0I arnBygl'sdnor8relod;o fta .1acBu^llp spunorrns (1_slarn8rs) rnur q8norq} ""T#r!Tr"#r"Jl*r",u-rrel E Jo auo o1aleqdsoqd iaurso8urqdsro) 1orac,{13 paIuII sprrp d11e; o.r,r1;o pasoduror .4.1prauag 'sauerq -uaruorq lJnJlsuoJ ol pasn sprdq go fro8alec ureru aqJ pldgoqdsoqd

'sproralsourssprqaqt pup 'prJp crsrJsqe,suru11o1dc 'surllaraqqr8 'sulxne 'aua1.,{qta aJe saldurexg .tuaru -do1e,reppue qu,ro.r8aleurprooc sdlaq leqt alncalour pu8rg (ouorurorl lueld) roluln8ar qfmort luu1d 179-61a:n8rg)'unllaqtrde aqt ;o aueld aqt ur palualro rotJel .,firrelode seq IIac qo€a leql qJns 'ertaqtrdaaruos ur uaas .{rlaurur.,tse relnlac;o ad,{1 ,g1ru1od lac reueld C osuupl ulalord aas-C)4 uvaas-g)d asuupl ulalord luapuadap-4tr41y-c;1cdcaas-y;4 'srso1,{coBeqdatoduto2'salncalou

(97-91 arn8rg) punoq-auuJqtuaru

6Z:D

(99-9I pue 6t-92 sarn8rC) 's)JU dq patplrtoe sl,tl'Id allq.r\ 'sulalord g cr;rceds et,t sgldC,{q palerrlce sl dC'Id '.{e,r,rqredSulpu8rs prdloqd -soqd lolrsour aqt ur pue €41acnpord o1 sprdl 1oracd131.,{cerp -oqdsoqd lotrsour saleelc 1eq1 au.{zua punoq-auerqruahl (C.Id) C osud;loqdsoqd (tg-gl a"rnBrl) 'su1a1ord SurleuBrs Jelnllaf,eJlur raqlo JoJ auerqruaru aqt uI satls Suqcop tdld ecnpord ot Sulr Iollsoul aql uo uorl -rsod g aql tp ateqdsoqdsrq-9'7 sa1e1 1o1rsour1.,{prleqdsoqd -froqdsoqd 11 r{e.uq1ed8u11eu8rsrulnllarer}ur t{V/aseuDl -t Id aql Jo luauoduroc p sr leql atur{zuepunoq-aupJqurolN (asuupl-E Id) osuupl-t apltgsoqoqdsoqd

AUVSSOt9

(91-y7 arn8rg) '(uoud) uralord paplo; fpeurouqe ,snortraJur uu ,{q pallrusueJl pup pasneo sl leq}-smoJ ur (,aseosrp^ oc peru, ro 'ggg) dqtedopqdacua u:o;r8uods aurnoq pue ,daeqs u1 aldurcs 'sueunq ul (CIl) aspasrp qo{el-plaJztnarl pue run) se qcns-.r{qledopqdacua ruro;r8uods alqlss[usue{ esuesgpuogd (96-9 pup 96-9 sa;n8rg) 'arnlrnrts luerraqe aql tdope ol edlr aures eqlJo suralord purou Surcnpur .{q tsoq aqt ur peleclldar sl tpqt ulatord e ;o urro; peplog .,tlpurouqe 'snorlreJrl Toga (0€-IZ pue 'EZ-IZ'Ll-rc sern8rC)'salaruu8 o1 astr err13lPq] sllal ol rosrncard E sr leql o-fuqua ue ur IIaJ lec urrat pgprorugd 'ase.rar-uA1od e,4q puerts dreluauelduoc ,lrau e;o srsaqlur(saql salouord pue pueJts VNU ro yI\Iq aluldutal e qlIM srred leql appoelcnuoSlj)rurrd

esuupdypq rr"----".n*ird 'srselseleru,{qaraqmesladolanap srounl z{repuoceg

'asorelsru roJuer p qrrq^ lu alrs put8rro

"Urrlhilga_r'

(17-g arn8rg) 'suollecgrpotr leqto ro 8ur -cqds auo8repun seq lr aro;aq 'tdrrcsue4 pazrsaqlur{srtlqsarg (ldpcsuurl ypg fruugd) ld.rrcsuurl iireug.rd 'uralord e;o acuanbasplce ounue aqt se qcns larudlod e ul sUun JetuouoruJo acuanbasreaurl arnlcnrls Ireulrd (91-97 ern8rg) 'ua8rlue reqt qlIM relunocua lsr.rJ uo appru sl leql ue8rlue up o] esuodsar aunrurur a,trldepy asuodsar eunururl d.rurulrd 'tunr1rcdruurrd aql 197-91 arn8rg) ul paluJtuaJuoJ arp sureloJd Surpu8rs auos 'saddl ilec IEtu -pu ,tueu;o aJeJJnseq] uorJ slcaford pue elorJluac B tuo4 saslrp teqt ureudp 3uqcu1 unrlrc 'alrlouuou 'elSurs 'uoqs unlgc i{.ruu4.rd (62-6r arn8rg) 'qlmor8 IIaJ roJ ruoor 8ulr,ro11e'alqualJ pue ulql sl tI :llac 1ue1dSurdolarrap u .r{qpacnpo.rd II"^\ IIaf, }sru aqJ 1e.r* lac frurupd (€Z-ZI pup 77-11 sern8rg) 'uor1ecl1der 'xe1d VNC Jo uorteurur luanbasqns aql pue -nIoJ uollelllutard e;o,{lquassp aql asuaJIIo1alrsrnbararde :elc^c flec aq];o saseqd IC flrea pup srso]lu a1e1Surrnp uorl -ecrtder;o surSlrot€ polqurasses1teql xelduoc ura1o.rd1t1n1atr (Cg-erd) xayduroc enpuclldarard (29-ZI arn8rC)'uorsrlrp IIec pue slsolru o1:opd auuJqruaur eruseyd aql rapun 1ec lue1d E punoJp sruroJ ]eql sluauPIIJ ullre puP salnqntoDrru Jo puEq IplluaraJunf,rlS pueq asuqdordard (67-61 arn8rg) 'ural -ord e ot dnor8 prdrl prouardosr ue Jo lueuqcelp tualelo3 uollul^uard 'Sulssecord

sa8ulsale1parura1u1

VNU Jo uI .vNHra8uesseuo1Joril?ii$_"r. IIE sapnlcur,saloi{recna 'spuerls 197-21arnSrg) VNC eqt otuo seru,tzue uoltecrlder reqlo pue seseraur,{1od y111q Surpeol pue xllaq VNC aqt Surpurmun ,,{qsrsaqluds VNCI sateqrul 'a1cdc 11acc1lo.{recnaaqt Jo eseqd g aql Jo lasuo aq} lu uollucltdar ;o urBrro aqt uo palquasse sI teql xayduoc uralordqlnu y xelduroc uopullplord (77-97 anfu1)'llac g u Jo rosrncard alerpatutul lac g-ard 'uor1e1r{uard puu'uogtelfroqdsoqd'uor1e1.,{qlau'uor} -u1dsoc,{y8'a8enealc'uol}Bl^tarpare saldruexg'pazlsaq]u.{s

sr lr raue aperu uralord p ot aSuer{3 pazdplec-audzua uy uollurglporu lBuollBlsrrurl-lsod (76-2 arn8rg) 'un8aq suq uopdlrcsuer] JaUea8els e ]e pauaxa sI lpq] uolssaJdxaauaBuo IoJluoc ^uV IoJluor puopdFcsuerl-lsod 'uorlelllce ro uotlcnpord u.lro sll setulmurls dumqled Jo uorlcear e Jo lsnporo pua aql AqaJeq \ uslueqcau lorluo] {cuqpaaJa plsod 'anle^ puousod stl pa[Pc sl uol]euroJul lPuolllsod su Jo proJor puralul s,ilec v'urslueSro relnlloJrllnu u ur uorlrsod rteql o1 Sulproccu slac dq pessessodro ol pallddns uolleuroJul uollBruroJul Isuolllsod (gt-t am8rC) 'uorw&auon patla uoutsod ot asrr Sunr8 'oar8ap alqulrE^ e ol eua8 aql llaJJP o1 peerds u€r ulteruorqcoralaq aqlJo acuongur Supenltcuul aql 'ulleurorqcoJaleq ot txau paculd sr eua8 alllce uu uallttv\ 'auosoruorqJ aql Suop uIleuoJI{J eql Jo a}e]s aql q sacua -raJJrpslcolJar .,{lqeqord pue aruosourorqc eql uo auaS aql;o uortrsod eql uo spuedep leql uolssardxe eue8 ul acuaraJJlq paJJa uoltlsod 'slseldorolrlr pue'elrpuoqf, ot!tu'EI.l -alreq Jo sauerqruaruralno aql Jo sulalord Suru,uoJ-lauueqJrrod (6g-t pue gg-7 sarn8rg)'reqteSol padels aneq serdoc ,tueru aqt pue uotluclldar peleadar auoSrapun seq VNC aql qJIqM uI aruosoluorqc luelg eruosotrroJqt auau(1od (911'd'7-7 laue4) 'asolnllac pue 'prrP cruornp,tq 'qrrels 'uoSocdp apnlcur salduexg 'saplrpqccesouoruJo raru.{1odpaqouprq ro reoun apFeqccust\od (g2-g arn8rg) 'slsaqlu^s ulelord ur pa8e8ua seruosoqlr Jo raqunu e paqcPlle are qrlqM ol elncalour vNu reSuassalN (auosfi od) eruosoq.rrr(1od 'sauosoruorqc snoSo -louoq Jo slas ox\l uEql aJorusuleluol leq] uslu€8Jo ro IIaJ pgold{od

Gzr-Bz|dd'.r-s

'seplt 1aue4) -{lqea8ueqcralul pesn aq uer sruret olvr}oqt pue -dadrtlod a8rel are sulalord 'splce oulrue Jo ratudlod reaulT appded{od

'uorlulndod e u1 ,buenberl q8rq le lslxaoc luql selaile arotu Jo or.q qlrru aua8 e saqlrcsaq c1qtuouri1od ucd aas-uolpEer

uleqr asBreu{od 'eseretuflod

y111g'aseraur.{lod VNC osp aas 'VNU pue VNC Jo slseqtufs aql se qcns suollreer uollezpau.{1od sazdplec leq} ouilzug assreur^Iod 'raqla3ol (sraurouotu) sllun rulluls ro prrtuapr a1dr11nuSuUull ^pueP^oc,{q apeu elncalou a8rel .reur,fiod 'ad,,touar{d aq} ol uoltnqlrtuoc lletus e sa{eu qJIqMJo qcee 'saueBa1drl1nu.{q pacuengut sI }uql cpslratcereqJ olqellraH llBrl cluat lod

(€z-9

ern8rg) 'selorfueJnauI lou lnq PlJelceq uI punoJ .{luoruruoc -suralord tuaraJrp lpralas sapocua rpqt VNUUr IBnpI 1pul VNUIU c1uorls1cd1od uopu$uepe{od

aas-1;er y-,{1od

(Bg-9 pue IZ-9 sarn8rC)'alnoaloru VNUIU ]uacseu e Jo pua ,€ oql ol (ye1y-z(1odoql) sapnoalrnu y;o ecuanbasSuol u Jo uoll1ppv, uopulduapuflod

AUVSSOI9 0€:9

G:31

GLOSSARY

probe Defined fragment of RNA or DNA, radioactively or chemically labeled, used to locate specific nucleic acid sequences by hybridization. procaryote (prokaryote) Single-celledmicroorganism whose cells lack a well-defined, membrane-enclosed nucleus. Either a bacterium or an archaeon. (Figure l-21) procaspase Inactive precursor of a caspase,a proteolytic enzyme usually involved in apoptosis. (Figure lB-5)

Ca2*,phosphorylates target proteins on specific serine and threonine residues.(Figure l5-39) protein subunit An individual protein chain in a protein composed of more than one chain. protein translocator Membrane-bound protein that mediates the transport of another protein acrossa membrane. (Figure 12-23) protein tyrosine phosphatase Enzyme that removes phosphate groups from phosphorylated ty'rosineresidues on proteins. (Figure 25-71)

processive Of an enzyme: able to proceed along a polymer chain catalyzing the same reaction repeatedlywithout detaching from the chain.

proteoglycan Molecule consisting of one or more glycosaminoglycan chains attached to a core protein. (Figure 19-58)

programmed cell death A form of cell death in which a cell kills itself by activating an intracellulardeath program

proteolysis Degradation of a protein by hydrolysis at one or more of its peptide bonds.

prometaphase Phase of mitosis preceding metaphase in which the nuclear envelope breaks down and chromosomes first attach to the spindle. (Panel l7-1, pp. 1072-1073)

proteolytic

promoter Nucleotide sequence in DNA to which RNA polymerase binds to begin transcription. See a/so inducible promoter. (Figure 7-44) proneural gene Gene whose expression defines cells with the potential to develop as neural tissue. proofreading Processby which potential errors in DNA replication, transcription, and translation are detected and corrected. prophase First stage of mitosis, during which the chromosomes are condensed but not yet attached to a mitotic spindle. (Panel l7-r, pp. 1072-1073)

erulalrrre- see protease

proteomics Study of all the proteins, including all the covalently modified forms of each, produced by a cell, tissue, or organism. Proteomics often investigates changes in this larger set of proteins in'the proteome'-caused by changes in the environment or by extracellular signals. protist Single-celled eucaryote. Includes protozoa, algae, yeasts. (Figure l-41) proton Positively charged subatomic particle that forms part of an atomic nucleus. Hydrogen has a nucleus composed of a single proton (H+). (FiSure2-l) force proton-motive The force exerted by the electrochemical proton gradient that moves protons acrossa membrane. (Figure 14-13)

protease (proteinase, proteolytic enzyme) Enzyme that degrades proteins by hydrolyzing some of the peptide bonds between amino acids.

proto-oncogene Normal gene, usually concerned with the regulation of cell proliferation, that can be converted into a cancer-promoting oncogene by mutation. (Figure 20-34)

proteasome Large protein complex in the cy.tosolwith proteol)'tic activity that is responsible for degrading proteins that have been marked for destruction by ubiquitylation or by some other means. (Figures 6-89 and 6-90)

protozoa Free-living or parasitic, nonphotosynthetic, single-celled, motile eucaryotic organisms, such as Paramecium and Amoeba. Free-living protozoa feed on bacteria or other microorganisms. (Figure l-41)

protein The major macromolecular constituent of cells. A linear polymer of amino acids linked together by peptide bonds in a specific sequence.(Figure 3-l)

pseudogene Nucleotide sequence of DNA that has accumulated multiple mutations that have rendered an ancestral gene inactive and nonfunctional.

protein activity control The selective activation, inactivation, degradation, or compartmentalization of specific proteins after they have been made. One of the means by which a cell controls which proteins are active at a given time or location in the cell.

(plural pseudopodia) pseudopodium Large, thick cell-surface protrusion formed by amoeboid cells as they crawl. More generally, any similarly shaped dynamic actin-rich extension of the surface of an animal cell. Compare filopodium, lamellipodium. (Figure 16-94)

protein domain-see

pump Transmembrane protein that drives the active transport of ions or small molecules across the lipid bilayer.

domain

protein kinase Enzyrne that transfers the terminal phosphate group of ATP to one or more specific amino acids (serine, threonine, or tyrosine) ofa target protein. protein kinase A (PKA)-see cyclic-AMP-dependent protein kinase protein kinase B-see Akt protein kinase C (PKC) Caz*-dependent protein kinase that, when activated by diacylglycerol and an increase in the concentration of cytosolic

-purifying selection Natural selection operating to retard divergence in gene sequences within a population in the course of evolution by eliminating individuals carrying deleterious mutations. purine Nitrogen-containing ring compound found in DNA and RNA: adenine or guanine. (Panel2-6, pp. 116-117) pyrimidine Nitrogen-containing ring compound found in DNA and

G:32

GLOSSARY

RNA: cytosine, thymine, or uracil. (Panel2-6, pp. l16-117) pyruvate (CH3COCOO-) End-product of the glycotytic pathway. Enters mitochondria and feeds into the citric acid cycle and other biosynthetic pathways. quaternary structure Three-dimensional relationship of the different polypeptide chains in a multisubunit protein or protein complex. quinone (Q) Small, lipid-soluble mobile electron carrier molecule found in the respiratory and photosynthetic electron-transport chains. (Figure l4-24)

recombinant DNA Any DNA molecule formed by joining DNA segments from different sources. recombinant DNA technology-see

genetic engineering

recombination (genetic recombination) Process in which DNA molecules are broken and the fragments are rejoined in new combinations. Can occur naturally in the living cell-for example, through crossing-over during meiosis-or in uitrousing purified DNA and enzymes that break and ligate DNA strands. Three broad classesare homologous (general),conservative site-specific,and transpositional recombination.

Rab (Rab protein) Monomeric GTPase in the Ras superfamily present in the plasma membrane and organelle membranes. Involved in conferring specificity on vesicle docking. (Table l5-S, p. 926)

recombination complex In meiosis, a protein complex that assemblesat a DNA double-strand break and helps mediate homologous recombination.

Ran (Ran protein) Monomeric GTPasein the Ras superfamily present in both cFosol and nucleus. Required for the active transport of macromolecules into and out of the nucleus through nuclear pore complexes. (Table l5-5, p. 926)

recycling endosome Large intracellular membrane-bounded vesicle formed from a fragment of an endosome; an intermediate stage on the passage of recycled receptors back to the cell membrane. (Figure I3-60)

Ras (Ras protein) Monomeric GTPaseof the Rassuperfamily that helps to relay signals from cell-surface RTK receptors to the nucleus, frequently in response to signals that stimulate cell division. Named for the ras gene, first identified in viruses that cause rat sarcomas.(Figure 3-72)

red blood cell-see erythrocyte

Ras superfamily Large superfamily of monomeric GTPases(also called small GTP-binding proteins) of which Rasis the prototypical member. (Table l5-5, p. 926) Rb-see retinoblastoma protein reading frame Phase in which nucleotides are read in sets of three to encode a protein. A mRNA molecule can be read in any one of three reading frames, only one of which will givb the required protein. (Figure 6-51) RecA (RecA protein) Prototype for a class of DNA-binding proteins that catalyze synapsis of DNA strands during genetic recombination. (Figure 5-56) receptor Any protein that binds a specific signal molecule (ligand) and initiates a responsein the cell. Some are on the cell surface, while others are inside the cell. (Figure l5-3) receptor-mediated endocytosis Internalization of receptor-ligand complexes from the plasma membrane by endocyosis. (Figure l3-53) receptor serine/threonine kinase Cell-surface receptor with an extracellular ligand-binding domain and an intracellular kinase domain thal phosphoryIates signaling proteins on serine or threonine residues in response to ligand binding. The TGFB receptor is an example. (Figure I5-69) receptor tyrosine kinase (RTK) Cell-surface receptor with an extracellular ligand-binding domain and an intracellular kinase domain that phosphorylates signaling proteins on tyrosine residues in iesponse io ligand binding. (Figure l5-52 and Table t5-4, p. 9231 recessive In genetics, the member of a pair of alleles that fails to be expressedin the phenotype of ihe organism when the dominant allele is present.Also refers to the phenotype of an individual that has only the recessive allele. (panel B-1, pp. 554-55s)

redox pair Pair of molecules in which one acts as an electron donor and one as an electron acceptor in an oxidation-reduction reaction: for example, NADH lelectron donor) and NAD+ (electron acceptor).(Panelf4-1, p.830) redox potential The affinity of a redox pair for electrons, generally measured as the voltage difference between an equimolar mixture of the pair and a standard reference. NADH/NAD+ has a low redox potential and O2lH2 has a high redox potential (high affinity for electrons). (Panel 14-I, p. 830) redox reaction Reaction in which one component becomes oxidized and the other reduced; an oxidation-reduction reaction. (Panel la-I, p. 830) reduction (verb reduce) Addition of electrons to an atom, as occurs during the addition of hydrogen to a biological molecule or the removal of oxygen from it. Opposite of oxidation. (Figure 2-43) regulative Of embryos or parts of embryos: self-adjusting,so that a normal structure emerges even if the starting conditions are perturbed. regulator of G protein signaling (RGS) A GAP protein that binds to a trimeric G protein and enhancesits GTPaseactivity, thus helping to limit G-proteinmediated signaling. (Figure l5-19) regulatory sequence DNA sequence to which a gene regulatory protein binds to control the rate of assemblyof the transcriptional complex at the promoter. (Figure 7-44) release factor Protein that enables release of a newlv svnthesized protein from the ribosomeby binding to the ribosbme in the place of tRNA (whose structure it mimics). (Figure 16-74) replication-see

DNA replication

replication fork Y-shaped region of a replicating DNA molecule at which the two strands of the DNA are being separated and the daughter strands are being formed. (Figures5-7 and b-19) replication origin Location on a DNA molecule at which duplication of the DNA begins. (Figures 4-2I and 5-25)

sr lI 'aleldua] vNC u uo pazrseqlu.{svNu Jo qc}ar}s uor{s rerulrdvNu (g-9 arn8rg) 'sros -rncard aleqdsoqdrrl aprsoelcnuoqrr uorJ aleldrual VNC e uo alnralotu VNH uE Jo srsaqluds eqt sazdpler 1eq1erur{zug

r""J;;rH::,t* 1-2arn8rg, 1e1

-lelrlceur pasn Llapr,vr Jo sttaJJaaql fpnls o1.4.1e1uarurredxe sr acuaJaJralur VNU 'aruoua8 urwo sllac eql ur papocua are qf,rq^\ '(sVNUIru) syllgorcru .{q uorssardxaeue8 ;o uo1} -lqlqul aq] apnlrur osp ot dlpeorq pasn uruo sr rVNU rxrel aq;'a8enealc crfloalcnuopua .,(qpacnpord lsyggrs) sygg Sutra;ralut Ilerus pepuprls-alqnop lroqs qSnorqt spaecord 'sa1o,{reona pallesuot ur .{1q8rqsr qcrr{llr 'rusrueqcaur aql 'sVNUru dreluaualdurof, Jo uorlcnrlsap clgrcads-acuanbes saJnpur VNU papuerts-alqnop pacnporlur dlletuau .paqrrrsap .{lpur8rro sy -rradxa qrrq^a uB ,{q usrueqceru ([VNU) acuaraJretu! VNU 'sepltoalrnu '3ur1a1ap Ienpnrpur Surralp ro '3ur1rasurdq pazrsaqlu.{ssr ralJu tr ldlrcsue;1 ytqgtu-erd e;o acuanbes aprtoalrnu aqt sratlp leqt Surssarord y11ggo adLl tup;peygg ( Z I I - 9 I I ' d d ' g - Z I a u p dp u p ' 9 € S ' d ' I - 9 a l q e l ) V N U r a J 'y111g -suerl 'VNU ra8uassaarosIDaas 'sJaurouoru Ipruosoqu aprloalJnuoqrJ pa{url r{1}ua1e,tocruorJ paruJoJ rarur(1o4 (plcu clelrnuoqlr) VNU ,(1!rlpe cltr{l€lpt rltlm VNU oumzoqlJ (91-1 arn8rg) 'VNUru ,(q paplrord uorleurro;ur Sursnuralord;o srseqludseqt sazdIB -ter leqt suralord pruosoqu pue sVNUrJo pasodruoc alclupd aIIIosoqlJ '(VNUr ,VNUr ,'3.a) luarcrg;aocuolt SS SBZ -eluarurpas rraql .{q paqsrn8urlsrp uaUO 'sura}ord go srsaql -u.,(saq1u1aledrcrlred pue aruosoqrJeJo aJnl3nJlsar{tJolred IuroJ ler{} salnralou ygg cr;rcadsJo requnu e;o auo .{uy (VNUr) VNU lpulosoqlJ 'VNU

'asoqtr,{xoaparoduto2'sOoIHs:) Jo tueuodruoc aprJeqccpsouou uoqJpl-aAIJ aql esoqlr

vNu aas-plru rrelcnuoqlr 'spuoq ralsalpoqdsoqd stl arotu Jo ro auo 3urz,r{1orpdq.,{qalnralotu VNU up slnf, leq} eul{zug asealcnuoqlJ (6t-9I arnBrC) 'lourtar dnor8 cqaqlsord a,trlrsuas-1q8rleql sureluoJ 'purler olerqaual aqt ur sllac roldacaroloqd por ur rosues IqBII p se g34g aq};o uralo-rdauerqruau ueds-uenag stoe teql ,{11ureg uJsdopoq.r (926 'd '9-9I alqeJ) 'Ztrpl pue ,reg ,oqg sapnlcul 'uo1a1a1sofcaq1;o luaure8uerrpar ar{l SuIpuBIs ur pa^lolur LlrrupJradnsspg aql urqlt,r\ saspdJC crJaruouou;o dpureg (rtgrel u1a1o.rdoqg) oqg 3u1put1s uralord g go rolepter aas-SCU (JZL-6I arn8rgl 'surelord relnllacerlxe raqlo aruos pue urlrauorqlJ ur luasaJd lsurr8alur loJ alrs Surpurq e surro; lpql prce cqredse-aurcd18-aurur8re ;o acuanbes aplldadul

'uralord olurperersrrer srvNupuevN"

o";::il:I:""

'eu'9op p4uac dq paqrrcsard "rq 1eq1o1uorl VNC letll sploq qctqm -carrp a rsoddo arp ur sr sHI'VNC ot VNU uro4 uo4ducsuerl uolldlJJsrruJl asJeaaJ

'uorlrunJ s,aua8eql azf1eue ol sluelnru salearr ueql pue lrnpord urelord str pue (eue3) yNq aql ruo4 strPls leql uotcury eueS Sulra^ocsrp o1 qceorddy scllaua8 asJaAaJ ( 12-g arnBrg; 'VNC s,llac aql otur peler8elur seuocaq luq] alnrolotu vNC papuPrls-alqnop P uaql puP alerpauratur vNC-vNu up Suqeru 1srq,4.qflec E ur salecldar ]pqt snrrl Sururetuoc-VNg snJI oJleJ

(Brs'd's-s a1qel)'suosodsueJloJleJleJrlorlaJuou pue suosodsuerloJleJ a)ll-prr^orlar :sedr! om1 ere arer.{J 'etuoue8 aqt ur alaqm -asla (,pasodsuer1-or1er,) pepasur pue aseldrrcsuer1osreler ,{q VruC o} peuo^uocer ueqt sl leqr.,{dor y51g up o}ut peqrrcs -uerl lsrrJ Suraq ,{q salotu }eql tueruele elqesodsuerl;o ad-{; uosodsrrerlorlar (29-ZI arnBIC)'uorsrlrp IIac puu uorleclldar VNCI o1 uorssar8o-rdSuqcolq snql 'sulatord CZg eqt Surlrqrqur pup ol Surpurq ,{q a1c,{cilac crtofrulna oql eleln8eJ o1 sr /tr,\Ilce pruJou sll 'sJorunl reqlo duelu uI se ila^ se 'euolselqoullal racuec aqt ur palelnhl 'uorsr^rp IIac Jo uolleln8er eql ur pellolur ulalord rosserddns rorunJ (qg) ugalord euolsulqoullor (S99-tg9 'dd 't-e Ieued pue '99-g arn8rg) 'luaprire eq IIIM uoltptnru Iuuortrpuoc e Jo lcaJJecrdrlouaqd aql qcrqm ur (dfryqele,re lualJtnu ro a;nleradurel se qcns) secuulsruncrrC suoplpuoJ (a,rlsspureduou) a llJlrlser uuls aas-lulod

uopcrrlsar

(Z€-Bpup 1g-g sarn8rg)'-{3o1ouqca1 'srnoJo saprloelcnu VNC lueulquroJar ur pesn .{lallsualxg Jo asuanDas uoqs crJrcaos e eJer{^\ a}ls Aue le alncalou VNC p alealo ueJ leql sasealcnuJo Jeqrxnu a8rel e;o eu6 (audzue uollJlrlser) osEalcnu uollJlrlsar 'seru.(zua uopcrrlseJ snorrezrdq a8ulealc;o selrs eql Surlecrpur alnJalou VNC e Jo uopeluasaldar crleuurer8erq duru uorlcr.r1se.r '(s)aur.,{zua uoucutsor Jo uollce eqr ,tq pale.rauaS VNCI Jo luauSerg luaurter; uollJlrlsar 'prlualod auerqueu osl?2 aag 'aueJqtuau euseld oql ssoJcesuor Jo llrog ]au ou sr eJaql rlrll^a ul suorllpuor unrrqrlrnba ur prluelod auprquolN pllualod arrurqtueru Supsar (92-tI pue tI-tI sarn8rC) 'auprqruour rauq eql ssoJop ]uarper8 uolord aql aleraue8 o1 sdrund uolo-rd uelrJp-uoJlcala se lJe leql uleqc ,4.ro1errdser Iprrpuoqcolrur aql;o sexaldruoc ureloJd rofeu aqt Jo ^uV xaldruoc aurr{zue r{-ro1ur1dsa.r upqc lrodsuuJl uoJlcele aas-uJeqc r{.ro1u4dsar (17-7 arn8tg) 'slcnpord olse^\ sp OzH pue zOC Surcnpord aFI/\,\ zO yo eqeldn aql sallnbor pue salnralou crue8Jo Jaqlo ro sre8ns Jo rrn op)earq alrleprxo aql sallolur lur{t sllar ul ssacord SurlereuaS-d8rouo ue roJ rurat plaueg uopu.rldsar 'euaB luace[pe uz 3o uoqdrrcsuerl tuaaard ol VNCI;o uor8ar cglcads e ol spurq teqt urolord (.rosse.rda.r [BuolldlJJsrruJl'u!aloJd Jossardar euet; rossarde.r (02-B ern8rC) 'alltleur to alrtce sr lsaralur Jo eue8 eql Jar{laq A selecrpurlcn[suoJ aql Sururel -uor IIac e ur (,urelord ralroder, eql) lonpord srqlJo ecuasqe ro acuasard aql 'lcnpord alqetcelep-^Isea ue ro; Surpoc acuanbas B ol palull sr lseretul Jo aua8 e;o VNC droleln8er

'1cnrrr"o3.rT}"1"1?od", aqr3o.{docE qcrq^ ur 'lerrrJnru .41ensn

'st eq d.lqrsralarrr,{11e a1e1dtua1 e ruo4 {d"r o*a'T}i3L"rT vNu papuprrs-al;urs u g pup lrmop smoysu orle:a;rlord ; -alqnop E saIeru sasntl orlal poraloJsrp ur audzug lpql lsrg IIac qJrq Aur sornllnc IIas AJptulro uI po^Jasqo uouauouaqd eseldpcsuu.4 esJaAaJ eJuerseuas oslz aas---€JuecseuasIIaJ alpeoJlde.r

Et:9

AUVSSOID

fiV-ZZ pue ZS-ZZsarn8rC) 'lueru8as .{poq qcee ;o uorlezrue8ro ropalsodoralup aql 3ur.4;rcedsur pa^lolur aua8 e 'luarudola,tap n1rqdosotg u1 euat,$1.ru1od-luarutes (rOrt 'd 'fzzpued) 'teoc alrlcalord preq p ur pasolcua 'arols pooJ qlp\ e Suop 'ol{rqua lupu]op aq] SururpluoJ arn]cnJ]s eql 'slueld uy Paas (77-21 arn8rg)'uolllsuert aseqduue-o1-aseqdetatuaql 'srsoJrrudpea ur raqlaSot sprlpruorqc 1upedorlsap sr urJn3as retsrs plor{ teqt sa8e1u11uratord aql ;o a8e,realcs}r s}uo^ -ard,,{qaraqlpue eseredasesealord aqt ol spurq }eq} ulalotd uIJnJes (99-€r pue tg-€I sarn8rg) '1ca[qopqos Ipus e se elqrsrl ellaue8ro oqt alptu slualuoc Sururels.{qrep asneJaqa1nuer3rfuolarcas pallec saturlatuos 'asealal ol rorrd parols aJe uolleJJes JoJ paurtsop sa1ncaloruqcrq ^ ur allaue8ro papunoq-euerqrual a1c1sa,r d.rolarces

(l.r-9r

pue '+zpJ 'sdl AWD crtc^,bIjINV crtc.{c arn8rC) '1orac,(131,(cerp apnlcu saldruexg 'llac oqt qqlyw pu8ls aqt .{u1a: o1 sdlaq pue pu8rs relnllaJpJxa ue o1 asuodsal ur uortcp JoJpaspalal ro paurroJ sr leql elnralo{u Surleu8rs relnllecerlur 11erus (rolulperu rBlnllacunul lpurs) rotuessatu puocas ' s t a a q s - $p u Es a J l l a q - n' s u r e l -ord ur lalncalotu crreur.r{1od e;o uralled 3urp1o;pcol reln8ag arnlcnrls frepuoJas (0I-92 arn8r{)'asuodser ounrurur dreurrrd aql ueql re8uorls pue tasuo ur prder eroyq'ua8rlue uanr8u qlIA JalunoJua luanbasqns ro puocas € uo appu sr ler{l ua8rlue uE o} asuodsar aunturul anrldepy asuodsar aunrurul drupuocas (JLL-6I arn8rC)'qtmor8 rraqt palald -ruof, aAEq leqt sllac 1ue1durelrac ur Ilellr 1ac dreurrd urq] aqt qtpaurapun u.&\opprcl sr leq] IIB.&\ilac prSp lueueura4 luin 1ac ifuepuoces 'srsaroqdorl -aala osf) aas 'slrunqns aprlded.{1od eqt eleredes pue 'selnJelolu Jer{lo r{ll^\ uouerJosse luo{ tuaql aorJ 'sura}ord aql ploJun lua8e Surcnparpue lua8ralap aql'1aB ap[ue1.{-rce -.{1ode q8norql unr Suraq aroJaq'(loueqlaotdecraru lua8e f,1 Sutcnper p qllm pue (gqg) lua8ralap pa8reqc dla.Lr1e3au1n; -re,r,rode qll1\^palEaJ] lsrr; sr paluredos aq ol arnJxrru uralord eql 'azrs^q surelord aleredaso1pesn srsa-roqdo.r1ce1a;o ad.,i.; (slseroqdorlcala yat appreli.rcu{1od-a1u;1ns $capop unlpos) 1ICVd-SCS (91-21 arn8rg) 'pa.(orlsapare sur1c.{casaql qclq^ raue 'srsolrru alEI Irlun q8rq uretuar slala'I 'uorlpJ -lldnp auosourorqc pue uorleclldar y11CIat€prurls dlaq deql l1re1gq8norql uorsserSordraue uoos s{p:) pulq puu aseqd 15 a1e13ur-rnpoleFruncce teq] surTcfcJo ssplc p Jo raqruel qp1(c-5 (Zt-II orn8r{) 'aldcorpuapoBqo atodtuoS'tuals-,(s snolrou leraqdrred oql ur sqleaqs urladur Sunuro; ro; alqrsuodsal IIaJ IeIIC IIaJ urrP.0rqcs 'uorlecrlda; @Z-LI pue 6Z{ sarn8rC) VNC pue s{pO-S Jo uorlelrtrp aq} Su1toruord snql pup ID atel ur s{p:) -S Jo srolrqrqul Jo uortrnrtsap aql Burlcarrp 'a1cLc1ac crlo -drecna aqt SuuelnSor ur pa^lolur sr euo 'suretord tuarogrp IeJaAas;o xaldtuoc e se pauroJ sase8rl urlrnbrqn ;o ^{1ureg (ugalord CtS) Cf,S 'serpnls lueJeJJrp,{uuru ur pasn usrueSto Iapou e sr'a41Au1 -atac sactruototlccag tseaf.Bl-rrppnq aq] q]IM Suop 'aqtuod'g 'uorssg .,{reurqfq ecnpordar leq} s}sua,{padeqs--porJo snueg saJLutoJnqccasozlqcs

sac^uroJoqcws aas-ao!s!na$c's (t90I 'd 'iI-ZI alqpJpue 9I-ZI arn8l{) '(Ipl) eseuDl luapuedap-ullrdr Sulpuodsarroc aqt pue urTcf3-Sup dq sllac alprqeuo^ uI pauroJ xolduroc 1p3-u11cd3

{pc-s

'lcafqo uP ecBJrns Jo eq] Jo a8erul ue sacnpord leql adoJsorcttu uollrala;o addl adocso.rclur uorlJale tulurruJs (21-91 arn8rg) 'llor aqt ul uonesol cgtcads e lp xaldluoo eql SuuoqcuE uego 'xalduoc Sulpu8ts e olur surelord Sulpu8rs renllaJprlur Jo sdno$ spulq ]eqt ulalord ulalord p1o;gecs (67-7 arn8rg) 'uoncunJ aroruorluor ur lred e.,fu1do1 rq8noql pue 'sa1o.{-recnaraq8rq ur (selrs raqlo se lla-tt se) saJeluorluac lE lueserd ,,{pcrdLl raruos 'uortrsodruoc aplloelsnu I€nsnun sll,(q alqelJt]uepl -oruorqc crlo.{recnee tuo4 ygq e.tqrladar,ri.p8q;o uor8ag vNo ollllelus 'uolletlrxa alcsntu [2-91 arn8rfl Surrnp losot-{c aq} olul paspalar sI }Ptl} +ze3 perelsanbes Jo suorlerluacuoc q8rq suleluoc leql sllec alssnlu Jo useldoilc aqt uI runlnollar crurseldopua yo adr! pazrtercadg runlnJ.rlal cyusuldocres (72-gI arnSrc) 'scsrpz luacefpe o^\] uaaMlaq sluatu -elrJ (urtre) urql pup (ursodur; lcrqr Sulddelralo Jo ,{erre ue go pasodtuoc 'llal alrsnu e ur lrqr;o.{tu e Jo Uun Surleadag AIAIIIO'JBS

'anssrl alllrautot

ro r"t#lr?"*,

'sacttuotnqccasonqcs osIDaas 'r{3o1orq 1ac cllorfuecne ;o dpnls eq} ur usrue8ro laporu e1d -rurs e se pasn dlapun srao1slnancsac[utorDqcJDs'8uqeq pue 'uotle8nfuoc dq dlen Suwrerq ug luelrodurr dlectuouocg -xas ro Sulppnq .,(qdlenxese acnporder leqt slsead;o snuag sactutotrtlctog 'reBng aplrEqJJBs aseqd gaas-g 'Uld dq pagrrdue ueq] aJE svNcc aql pue 'uolldrrcsuerl asJeAaJUIA svNcc otur paualuoc sI sVNUruJo uorlepdod e qctq,r,rut anbruqcal (uollreer ulerlc asu.reruflod-uolld.rrcsue.rl asralar) UDd-IU asuupl au1so.rd1.roldorar aas-XJU '(VNUJ) VNU I€ruosoqrre sagrcedsleqt eueD eua8 yggr vNu luuosoqlraas-vNur 'sulalord punoq -auerqruaru puu palercas;o srseqlu.{saq} ul paAIoAuI 'aJeJ -rns crloso$c sll uo satuosoqlr qtlm IunFcIlaJ cnuseldopug (gg qtnor) runFcllar clursuldopua qtno.r (21-97 arn8rg) 'rqBI rutp uI uoIsIArolocuou roJ

aqlHj:ij:l*:3311l3l$ al'Iqaur^ srreqreunal alqrsuodsar r., 'svNu raqlo pue ra8uessaruJo uopeluJoJ Supnp snalcnu aql uI sldtrcsuerl VNU uro4 pospxa ere sacuanbes uoJlul qJIqM uI ssasoJd tu1c11dsytr1g '8utd 'uope1.{uepedlod '3u1r1papue '3urcg1ds'e8elealc ,g ,g 'urro; arnJetu sll seqcear 11se sao8rapun -dec .9 apnlcur r{u141

snolreltut totHi:lJ"".1lno*" ldrrcsue4VNUuEsuollprlJlpotu 's1saqlu.{s VNC rlaql lrc}s ol sase:aur.(1odygq dq pertnbar

AUVSSOI9 V€i'

G:35

GLOSSARY selectable marker gene Gene included in a DNA construct to signal presence of that construct in a cell, and making it possible to select cells according to whether they contain the construct. selectin Member of a family of cell-surface-carbohydrate-binding proteins that mediate transient, Caz+-dependent cell-cell adhesion in the bloodstream-for examole between white blood cells and the endothelium of the-blood vessel wall. (Figure 19-19) selectivity filter The part of an ion channel structure that determines which ions it can transport. (Figures 17-23 andll-24) senescence (l) aging of an organism. (2) replicative cell senescence:phenomenon observed in primary cell cultures in which cell proliferation slows dornmand finally halts irreversibly. sensory hair cell-see auditory hair cell separase Proteasethat cleavesthe cohesin protein linkages that hold sister chromatids together. Acts at anaphase,enabling chromatid separation and segregation.(Figure l7-44) septate iunction Main type of occluding cell junction in invertebrates; its structure is distinct from that of vertebrate tight junctions. (Figure l9-28) sequencing Determination of the order of nucleotides or amino acids in a nucleic acid or protein molecule. (Figure B-50)

signal-recognition particle (SRP) Ribonucleoprotein particle that binds an ER signal sequence on a partially slmthesized polypeptide chain and directs the polypeptide and its attached ribosome to the endoplasmic reticulum. (Figure 12-39) signal- relaying junction Complex type of cell-cell junction that alows signals to be relayed from one cell to another across their plasma membranes at sites of cell-to-cell contact. Typically includes anchorage proteins as well as proteins mediating signal transduction. (Figure 19-2, andTable l9-1, p. 1133) signal sequence Short continuous sequence of amino acids that determines the eventual location of a protein in the cell. An example is the N-terminal sequenceof 20 or so amino acids that directs nascent secretory and transmembrane proteins to the endoplasmic reticulum. (TabIe l2-3, p. 7 02) signal transduction Conversion ofa signal from one physical or chemical form to another (e.g.,conversion of light to a chemical signal or of extracellular signals to intracellular ones). single-nucleotide polymorphism (SNP) Variation between individuals in a population at a specific nucleotide in their DNA sequence. single-pass transmembrane protein Membrane protein in which the pollpeptide chain crosses the lipid bilayer only once. (Figure 10-19) single-strand DNA-binding protein Protein that binds to the single strands of the opened-up DNA double helix, preventing helical structures from reforming while the DNA is being replicated. (Figure 5-16)

serine protease Type of protease that has a reactive serine in the active site. (Figures3-12 and 3-38)

siRNA-see small interfering RNA

serine/threonine kinase Enzyme that phosphorylates specific proteins on serine or threonines. (Figure 15-70)

sister chromatids Tightly linked pair of chromosomes that arise from chromosome duplication during S phase. They separate during M phase and segregate into different daughter cells. (Figure 17-26)

sex chromosome Chromosome that may be present or absent, or present in a variable number of copies, determining the sex of the individual: in mammals. the X and Y chromosomes. SH2 domain Src homology region 2, a protein domain present in many signaling proteins. Binds a short amino acid sequence containing a phosphoryrosine. (Panel3-2, pp. 132-133) p-sheet-see beta sheet side chain The oart of an amino acid that differs between amino acid types. fne side chains give each type of amino acid its unique physical and chemical properties. (Panel 3-1, pp.

r28-r29) signaling cascade Sequenceof linked intracellular reactions,typically involving multiple amplification steps in a relay chain, triggered by an activated cell-surfacereceptor. signal molecule Extracellular chemical produced by a cell that signals to other cells in the organism to alter the cells' behavior. (Figure l5-l) signal patch Protein-sorting signal that consists of a specific threedimensional arrangement of atoms on the folded proteins surface. (Figure 13-45) signal peptidase Enzyme that removes a terminal signal sequencefrom a protein once the sorting process is complete. (Figure l2-25)

site-directed mutagenesis Technique by which a mutation can be made at a particular site in DNA. (Figure 8-63) site- specific recombination Type of recombination that occurs at specific DNA sequencesand is carried out by specific proteins that recognize these sequences.Can occur between two different DNA molecules or within a single DNA molecule. skeletal muscle cell-see muscle cell sliding clamp Protein complex that holds the DNA polgnerase on DNA during DNA replication. (Figure 5-lB) Smad protein Lalent gene regulatory protein that is phosphorylated and activated by receptor serine/threonine kinases and carries the signal from the cell surface to the nucleus. (Figure 15-69) small interfering RNA (siRNA) Short (21-26nucleotides) double-stranded RNAs that inhibit gene expressionby directing destruction of complementary mnNas. Production of siRNAs is triggered by exogenously introduced double-stranded RNA. (FiSure7-1 15) small intracellular mediator-see

second messenger

small nuclear ribonucleoprotein (snRNP) Complex of an snRNA with proteins that forms part of a spliceosome. (Figure 6-29) small nuclear RNA (snRNA) Small RNA molecules that are complexed with proteins to

G:36

GLOSSARY

form the ribonucleoprotein particles (snRNPs) involved in RNA splicing. (Figures6-29 and 6-30) small nucleolar RNA (snoRNA) Small RNAs found in the nucleolus, with various functions, including guiding the modifications of precursor rRNA. (Table 6-1, p. 336, and Figure 6-43)

spindle assembly checkpoint (metaphase-to-anaphase transition checkpoint) Checkpoint that operates during mitosis to ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation starts. (Figure l7-14, and Panel l7-1, pp. 1072-1073)

smooth endoplasmic reticulum (smooth ER) Region of the endoplasmic reticulum not associated with ribosomes. Involved in lipid synthesis.(Figure l2-36)

spliceosome Large assembly of RNA and protein molecules that performs pre-mRNA splicing in eucaryotic cells. (Figures6-29 and 6-30)

smooth muscle cell-seemuscle

splicing Removal of introns from a pre-mRNA transcript by splicing together the exons that lie on either side of each intron. See a/so alternative RNA splicing, and trans-splicing.

cell

SNARE Member of a large family of transmembrane proteins present in organelle membranes and the vesicles derived from them. SNAREscatalyze the many membrane fusion events in cells. They efst in pairs-a v-SNARE in the vesicle membrane that binds specifically to a complementary t-SNARE in the target membrane. SNP-see single-nucleotide polymorphism snRNA-see small nuclear RNA solute Any_moleculethat is dissolved in a liquid. The liquid is called a solvent. somatic cell Any cell of a plant or animal other than cells of the germ line. From Greek soma,body. somatic hlpermutation Accumulation of point mutations in the assembledvariableregion-coding sequences of immunoglobulin genes that occurs when B cells are activated to form memory cells. Resultsin the production of antibodies with altered antigenbinding sites. somite One of a series of paired blocks of mesoderm that form during early development and lie on either side of the notochord in a vertebrate embryo. They give rise to the segments of the body axis, including the vertebrae, muscles, and associated connective tissue. (Figure 22-Bl) sorting signal Amino acid sequencethat directs the delivery of a protein to a specific location, such as a particular intracellular compartment. Southern blotting Technique in which DNA fragments separated by electrophoresis are immobilized on a paper sheet. Specific fragments are then detected with a labeled nucleic acid probe. (Named after E.M. Southern, inventor of the technique.) spectrin Abundant protein associated with the cytosolic side of the plasma membrane in red blood cells, forming a network that supports the membrane. Also present in other cells. (Figure r0-41) Spemann's Organizer-s

ee Organizer

sperm (spermatozoon, plural spermatozoa) Mature male gamete in animals. Motile and usually small compared with the egg. (Figure 2l-27) spermatogenesis Development of sperm in the testes.(Figure 2l-30) S phase Period of a eucaryotic cell cycle in which DNA is synthesized. (Figure l7-4) sphingolipid Phospholipid derived from sphingosine. (Figure l0-3)

S. pombe-see

Schizosaccharornyces

Src (Src protein family) Family of cytoplasmic t),.rosinekinases (pronounced "sark") that associate with the cytoplasmic domains of some enz)'rne-linked cell-surface receptors (for example, the T cell antigen receptor) that lack intrinsic tyrosine kinase activity. They transmit a signal onwards by phosphorylating the receptor itself and specific intracellular signaling proteins on tyrosines. (Figures3-10 and 15-70) SRP-see signal-recognition particle standard free-energy change (AG) Free-energy change of two reacting molecules at standard temperature and pressurewhen all components are present at a concentration of 1 mole per liter. (Table 24, p. 77, and Figure l4-18) starch Polysaccharidecomposed exclusively of glucose units, used as an energy storage material in plant cells. (Figure 2-75) Start (Start checkpoint, restriction point) Important checkpoint at the end of Gr in the eucaryotic cell cycle. Passage through Start commits the cell to enter S phase. The term was originally used for this checkpoint in the yeast cell cycle only; the equivalent point in the mammalian cell cycle was called the restriction point. In this book we use Startfor both. (Figurel7-14) start-transfer signal Short amino acid sequencethat enables a polypeptide chain to start being translocated acrossthe endoplasmic reticulum membrane through a protein translocator. Multipass membrane proteins have both N-terminal (signal sequence) and internal start-transfer signals. (Figures 12-45-12-48) STAT (signal transducer and activator oftranscription) Latent gene regulatory protein that is activated by phosphorylation by IAK kinases and enters the nucleus in responseto signaling from receptors of the cltokine receptor family. (Figure 15-68) stem cell Undifferentiated cell that can continue dividing indefinitely, throwing off daughter cells that can either commit to differentiation or remain a stem cell (in the Drocess of selfrenewal). (Figure 23-5) stem-cell niche The specialized microenvironment in a tissue in which selfrenewing stem cells can be maintained. (Figure 23-27) stereocilium A large, rigid microvillus found in "organ pipe" arrays on the apical surface of hair cells in the ear. A stereocilium contains a bundle of actin filaments, rather than microtubules, and is thus not a true cilium. (Figures23-13 and 23-15) steroid Hydrophobic lipid molecule with a characteristic fourringed structure; derived from cholesterol. Many important

'pue'sopl 'uorleclldal )aarD ruorc Jo punor I{3eaqll^\ uauoqs ol asrMtaqlo auosoruojqc eqlJo ^Juapual aqt stceratuno3 ,{e.r,r prceds e ur paleclldar sl tpqt acuanbes 'aruosouroJqce VNCI Crlsrralsereqcp qlJM pelErcosse Jo pug alaurolal 'saruosoruorqccrloz{recna spua Jr{l le JnJco Jo I{JIqnr 'VNC ur sacuanbes araruolal sale8uola tpqt aru.{zug asBJaurolal tn'Jr? -ads-ua8r1ue .{q palerpau esuodsar aunrurxr asuodsar aunruurl""rttt}A* polulpeu-llar-I 's11ac droluln8er pue 's11ac radleq l 1 'slla3 I Jrxolol^t sapnlJur sselc aql :sasuodseJ aunrulul anrldepe palerpatu-llaJ-1 ro; alqrsuodsar a1i{coqdtudlyo edz{1 (afcoqdru{ a) IIar I eycfc plce c1.r11c aas-e1cdc (p1cucpfxoqrectrl)

VCI

(zI-9 pue 'pelprlrur sr uorldrrcsuert qJrqm uorlrsod eql 91-g sarn8rg) lu sagrcedsocuaq pue rolce; uorldrrcsuerl praua8 p spurq leql saua8 crlo,{recnadueu;o uor8ar ra}ourord eqt ur acuanbag

xoqvJvr 'uorlerederd arnd r{rarre ul }lnseJ laqto aq] uaqt pue 8et auo 3u1snlsrg 'uorlecgrrnd IlruUJe Jo spunor o.trJ'uorlergrrnd dt{derSoteurorqc,!rur;;e ro; s8el se lre leqt uapue] ur sureuop ulalord o.{{t ot palurl sr lserelul ;o utalord aql qJrq-l ur uralord uorsnJ e Jo uorlJntlsuoc uo paseq 'uollecr;rrnd urelord loJ poqlau luarogJa dp8tff (tuFflul uoglucgFnd flIuUJD rrrapuel) 8uptu1-du1 'lou saop auole uo11elmua13urs Jaqlra sparaql 'qleap IIaJ ur llnsar taqlaSol saua8 luelnur oml aql qJrq^ ur saue8 luelnur oA l uaaMlaq uorlceJalur uV &;1eqral rllaqlu^s 'tapJo atues aql ur sauaB arues aql qll.tt saruos 'sarcads -oruoJrlJ luaraJJrpur ,acuasard aql ;o suor8er;o fuetw(s 'tou seop IIar aql lnq ap4lp ralcnu eq] qcrr{,rrur salc{c uorsr,rrpalaldruocur Jo sJues u Jo ro uorsnJ 'auerqrxaru eurseld e13urs IIec Jo raqlle tlnsar aqt .{pcrddg e dq pasolcua relcnu .{ueru Surureluoc useldo{c Jo ssel runllbw(s (6_IZ pue B-IZ sarn8rg) :alo Surssorc;o sdels 1eurJaqt salourord pue srsorau ur I aseqdord;o aua1,{qcedur raq1a3ol ,t11q8r1 sauosoruorqc snoSoloruoq parred sploq leqt arnttnrls xalduroc pruauoldeufs 'lPurruJaluoxP aql soqseaJ prlualod uorJt€ uB uaqlr. srsofcoxa .{q galc cr1deu,,(saq1 olul pasPalar oJp slualuos sll 'sllaf, a^Jau Jo slBururlal uoxe aql ]p punoJ aprsaA drolarcas pallrJ-Jalrrusuprlornau IIBTuS alclsair cllduuds (6-17 arn8rg)'saruosoruorqcsnoSo -lotuoq pau31p dpq8p o-ru uaamtaq xalduroc IpuauotdpuLs e Jo uorlpurroJ aql 'srsoraruq (z) (gg-g arnSr{) .saruosoru -oJqJ uaamlaq rano-SurssorcJosalrsle slnclo ler{l salnJaloru vNC luaJaJJrpur spuells vNq r{reluaualdruoc uaaivrlaqsrred asBq Jo uoqeruroJ Ierlrur aql ,uol]purqtuocal cr]aua8 uI (I) slsduu(s (79-61 ern8rg) 'suorlcunf de8 ern sllar oM] aql;o surseldo{c aq} ueemlaq apeu sr uollrauuor lcarlp p ,asdeui(slecrrtoalaue q (ZZ-6I aJnBrC) 'ralnusuertornau alqrsnJJrp e .{q parrrec sr pu8rs aql 'esdeudslecrrlaqJ e uI 'llal raqloue ol IIaJ olrau E r.uo4 ssed ol spu8ts smolleleql uollcunf 1ac-11acSuqecrun*Tlpr*. (g-11 arn8rg) 'uorlcaJrparues aql ur auprqrxau eql ssorceelnlos Jo sadlt oml spodsuerl ]eql uratord Jerrre] repoduils

/E:9

(91-1 arn8rg) 'e8u1 -uelpe al-rlJalas urral-8uol e a^rJap qloq qcrqm tuo4 salcads tuoraJJrp Jo sr.uslue8Jo oml ueemlaq uollercosse alerunul slsolquns (71-91arn8rg)'srsoldode 3up1qqul ,r{qp,rwns 1ec selouord leql IEuBrsrplnflacer1xll

",",",il:;'J;

-Iqourul aql Suldrrec aoeJJnsJosuasolq e pue salncaloru 3ur -purq prtuatod;o uortnlos snoenbe ue ueeMlaq af,EJraluraql go 1q311yo ueaq e Jo uortJol;ar aql Sulrolruoru dq palcalap arp suorlrpralur Surpurg 'VNC ol sulelord ;o Surpurq eql pue'8ur1dnoc rotdacaJ-pue811'Surpurqua8Fue-^poqrlue se qcns 'suorlJeralur reln3oloru ezrJelJEJpr{J ol pasn anbruqcel (UdS) aJueuose.r uoruseld acu;.rns (999-tg9 'dd 'r-e laued) 'letu -rou stuees IEnpr^rpurluElnru alqnop aql leql os 'uopelnur raqloue Jo loeJJacrddlouaqd aqt sossorddnsleqt uollelnlN uopulnru Jossarddns (97-g arn8rg pue '291-991 'dd 'I-Z 'puoq epgpsrp e acnpord o1urof uec sfrp.,i.tgps laued) o^\I 'salnsalolu raqlo pue aulalsrtc prte ourue aql ur punoJ lua3orp.,{q pue rnJlns Surureluoc dnor8 pcrtuaq3 (HS-'loFD) /rp,{rgps (tI I-ZI I'dd 'V-Z1pved) '(pa)u1 asolcnrJpue asocnlS; osorJns aprJeqJcesrpaql pup 'asouueru pue asol -cnr; 'asocn18saplreqccesououraql are saldtuexg 'u(OzHJ) elnruJoJ lerauaS;o trun Jaruouoru E qllm alprp,(qoqrec flerus tns (tI I-ZI I'dd' V-Z 1pued)'s1aclue1d uaamlaq pauodsuert sr asocnlS qJIqM ul ruroJ rofEur aqJ ']lun esol -rnrJ auo pue tlun asocnlS auo;o pesodruor aprreqccnllt""r* 'saraqpp IIas e r.{crq^ ol arpJrns pllos runlDJlsqns

'slcu aurdzua ue qcrqm uo alnJalo4 aluIlsqns 'uorssardxa aua8 aleln8ar rBqr VNC;o suolSer ruor; paqsrn8urlsl(l 'uou -cun; crlerudzua ue seq ro arnlonrls e;o ged sruJoJleql alnJ -alou VNU ue roJ ro ulalord p roJ sopoc reqr VNC Jo uor8eg auat 1ern1cn.r1s (17-71 arn8rg) 'sre8nsolur 263 alerodrocul leql satu.4.zuaSurureluoc '1seldoro1qc e ;o aceds roualur a8rel aq1 (d Gl-02 arn8rg) 'anssrtaql ulqtl^r sllal Jaqlo Jo luaudolalap aql JoJ dlesserau luaruuoJrlua aqt apvr.ord sllao Ipruorts 'poppaqua sr unllaqlrda .raqto ;o re1npuu13e qJrqm ur anssu alllJauuoJ aqr :,3urppag, (1) BruoJls (72-91 arn8rg) 'saldurexe are salerqaual 'slu Jo elJsnur upaq pue plele{S -qgodur (peleuls) padrrls dlasrensuerl pesoduoc alcsnl^ ;o alJsnru polurJls GV-ZI pue 97-71 arn8rg)'auerq -ruatu aql ur ulErIJ uralord oql SulJorlJue snql (aueJqruau runlnorlar cruseldopua aqt q8norqt ureqc aprldad,{1od e go uollerolsuerl stlpq ]eq] acuenbas prre ourrue crqoqdorpdg pu31s .ra;suur1-dols 'salqprrel '.4111qeqord'ecueqc 'ruopueg ruopuer ro SuFIo uI r!lsurlrols ('616 'd rg-9l alqel pue ,uralord !osID aas) Atr{V 3IIJ^J Jo uopcnpord oql satBlnurts snq} puE aselcdc 1,,i1 rpo}B^rtJe uaqm'lpql uralord g -,{uepe arur{zue oql salplrlce (sg) qelord 9,{.ro1e1nups

(sri-vrrdd's-z

'sroldaoer rualcnu rc1nfle]Ertur otelrlce leql sprore]s 1eue4) aJe'auoJalsolsalpue 'ueSorlsa'1osr1roc Surpnlcur 'sauoruJoq

AUVSSOID

G:38

GLOSSARY

telophase Final stage of mitosis in which the two sets of separated chromosomes decondense and become enclosed bv nuclear envelopes (Panel 17-1, pp. 1072-1073) temperature-sensitive (ts) mutant Organism or cell carrying a mutation that shows its phenotypic effect in one temperature range (usually high temperature) but not at other (usually low) temperatures. (PanelB-1, pp. 554-555, and Figure B-55) template Single strand of DNA or RNA whose nucleotide sequence acts as a guide for the synthesis of a complementary strand. (Figure 1-3) terminator Signal in bacterial DNA that halts transcription. tertiary structure Complex three-dimensional form of a folded polymer chain, especiallya protein or RNA molecule. TGFp superfamily (transforming growth factor-p superfamily) Large family of structurally related secretedproteins that act as hormones and local mediators to control a wide range of functions in animals, including during development. It includes the TGFp/activin and bone morphogenetic protein (BMP) subfamilies. (Figure l5-69) TGN-see trans Golgi network thioester bond High-energy bond formed by a condensation reaction between an acid (acyl) group and a thiol group (-SH). Seen, for example, in acetyl CoA and in many enzyme-substrate complexes. (Figure 2-62) thiol-see

sulfhydryl

thylakoid Flattened sac of membrane in a chloroplast that contains chlorophyll and other pigments and carries out the lighttrapping reactions of photosynthesis. Stacks of thylakoids form the grana ofchloroplasts. (Figures 14-35 and 14-36) tight iunction Cell-cell junction that sealsadjacent epithelial cells together, preventing the passage of most dissolved molecules from one side ofthe epithelial sheet to the other. (Figuresl9-3 and 19-26) TIM complexes Protein translocators in the mitochondrial inner membrane. The TIM23 complex mediates the transport of proteins into the matrix and the insertion of some Droteins into the inner membrane; the TIM22 complex mediates the insertion of a subgroup of proteins into the inner membrane. (Figure t2-23) T lymphocyte-seeT

cell

Toll-like receptor family (TLR) Important family of mammalian pattern recognition receptors abundant on or in cells of the innate immune system. They recognize pathogen-associated immunostimulants such as lipopolysacharide and peptidoglycan. (Figure24-51) TOM complex Multisubunit protein complex that transports proteins acrossthe mitochondrial outer membrane. (Figure 12-23) topoisomerase (DNA topoisomerase) Enzyme that binds to DNA and reversibly breaks a phosphodiester bond in one or both strands.TopoisomeraseI creates transient single-strand breaks, allowing the double helix to swivel and relieving superhelical tension. Topoisomerase II createstransient double-strand breaks, allowing one double helix to pass through another and thus resolving tangles. (Figures 5-22 and5-23)

totipotent Describes a cell that is able to give rise to all the different cell types in an organism. trans On the other (far) side. transcellular transport Transport of solutes,such as nutrients, acrossan epithelium, by means of membrane transport proteins in the apical and basal faces ofthe epithelial cells. (Figure 11-ll) transcript RNA product of DNA transcription. (Figure 6-21) transcription (DNA transcription) Copying of one strand of DNA into a complementary RNA sequenceby the enzyme RNA polymerase. (Figure 6-21) transcriptional

activator-see

transcriptional

repressor-see repressor

activator

transcription attenuation Inhibition of gene expressionby the premature termination of transcription. transcription factor Term loosely applied to any protein required to initiate or regulate transcription in eucaryotes. Includes gene regulatory proteins, the general transcription factors, coactivators, co-repressors,histone-modifying enzymes, and chromatin remodeling complexes. (Figures 6-19 andT-44) transcytosis Uptake of material at one face of a cell by endocltosis, its transfer acrossa cell in vesicles,and discharge from another face by exocltosis. (Figure 13-60) transfection Introduction of a foreign DNA molecule into a cell. Usually followed by expression of one or more genes in the newly introduced DNA. transfer RNA (tRNA) Set of small RNA molecules used in protein svnthesis as an interface (adaptor) between mRNA ind amino acids. Each tlpe of IRNA molecule is covalently linked to a particular amino acid. (Figures l-9 and 6-52) transformation (I) Insertion of new DNA (e.g.,a plasmid) into a cell or organism, such as into competent E.coli. (2) Conversion of a normal cell into one that behavesin manvwavs like a cancer cell (i.e., unregulated proliferation, anchoiage-independent growth in culture). transforming growth factor-B superfamily-see superfamily

TGFp

transgenic organism Plant or animal that has stably incorporated one or more genes from another cell or organism (through insertion, deletion, and/or replacement) and can pass them on to successivegenerations.The gene that has been added is called a transgene. (Figures8-64 and 8-65) transit ampliffing cell Cell derived from a stem cell that divides a limited number of cycles before terminally differentiating. (Figure 23-7) transition state Structure that forms transiently in the course of a chemical reaction and has the highest free energy of any reaction intermediate. Its formation is a rate-limiting step in the reaction. (Figure 3-46) translation (RNA translation) Process by which the sequence of nucleotides in a mRNA molecule directs the incorporation of amino acids into protein. Occurs on a ribosome. (Figures6-66 and 6-67)

pauroJ sr leqt puoq tualeloouou (1eamu{lpnpwpur) Jo addl uollJErllu slee arap ue^ 'ruroJ uec Iuole aq1lPql spuoq al3uts;o raqunu aql ol IEnba sr Iuole ue Jo acuelel aql'llaqs ralno slr IIU ol (raJsuerldq ro Suueqs dq Jaqlra) asol ro ure8 lsnu ruole uB lpql suollcale Jo JaqrunN eJuaIPA

-;11ord-11ac ur traJape ruor; Surlpser sllat Jo ssetupturouqV JOIIINI

tu1.r u1pqnl uururetaas-(3gq/) pup '896'd't-gt

(69-91arn8rg)'aun 's1ec -lo^ IIar aql Jo prlqr p ueqt aroru 8ur.,{dncco ,,t1pcrd,Q IeBunJpup tueld lsour ur punoJ alcrse^pallg-prng a8rel d;an aIonJEA

laued)

'sapqntorf,lruJo

xalduroc xelduoc tugr qpqnl-/, (11-91arn8rg lrunqns urelord aq1 ullnqnl

f,uvNsaas-gwNs-r luulnru allllsuas-a.rnluradruel

(69I-Z9I 'dd'g-g 1aue4 pue gt-€ arn8rg) 'uorltear crleru.,{zueue Jo aler runtur.xpl4l *z1 'srsaqluds uralord selprlrur leql uopof, uels aql ol duc ,g ar{l ruo{ spuaxa UIn,g eqJ 1991-2ernSlgy'pely-d1od aqJo uets aql ot srsaqlu,{suralord saleunuJal }pql uopoc dols aql uor; spuatxa UIn ,t aqJ 'alncelotu VNUUr ue Jo uor8ar Surpocuog (uoltar palulsuertun) UIfl 'taqlo aql ot auerqtuatu aql 1g-11 arn8rg) Jo aprs auo ruo4 alnlos a18urse sgodsuerl leqt ulalord JarrJuC ralrodpn (99-ZI ern8rC) 'satudzua a,rrleper8appue sauo;adeqo UA Jo uorldrrcsuerl pasearJur saAIo^uI 'urnlncrlal cruseldopua aql ur sulelord paploJsrruJo uorlelnrxnotE uu .,(qpareSSrrtasuodsa.rrulnlleC asuodse.r ulalord paplo;un (62-g arn8rg)'uorlel,&rnbrqn pallec sr ase311urlrnbrqn e ,(q paz\e1ec ssacord eqJ 'aruos -ealord B ur uorlcnJlsap JoJ ll Suureu uago ,uralord u o1 ulllnbrqn qoelle ]Bql satu,4zua;oJaqrunu afirel e;o euo.{uV

aas-luulnu

sl

vNu JeJsrrDrlaas-vNul loradpltbelq aas-(ulelord

tupulq-;;g

rlraulrl)

aas-apgarf1tJ.rl ulalord g ulelord g clrarulrl

eIJ^r plru culrcaas-a1cr(c (VCI) plcu cqikoq.recgl (9I I-tI I 'dd'g-Z lauud) '(palernlesun dlureru are sprce'{1e; eqt araqm) s1o alqele8enJo pup (patprntes are sprcu,file; eq1 eraqm) sanssrlprurue ur slaldorp tpJJoluanlrlsuoJ ulpru aql '1orec.r{p paglratsa o} sprce,,{11e; pasoduroc alnralolN aarqtJo (ap;railp;.r1) 1o.recd181l(cu;.rr 'raqlo aql te slrunqns Jo ssol pue GL6-BL6'dd'Z-St Ieupd) pua auo te slrunqns urel0rd;o uouppe dq ql8ua1luelsuoc le peumlulptu sr tuauelrJ urelord cgau.,{1od e qcrq,u,.,{qssacor4 tu1lgupee.r1 (tt-g arn8r$ 'VNUru ue uroJ o1 raq1a8ol pautol are sldrrcsuerl ylqg a]eJedaso,,r,r] ruo{ suoxe qJrr{ \ ur srusrue8ro crlo^reJna ^\aJ E ur luesard 3urc11dsygg go addl tulcqds-suu.r1 luaruale elqusodsrruJl aas-uosodsuurl

(76-9ern'rg)'"*"r"rr;:":l;::::-"

-rnrlsap rlilloelord rppllarprlur ro; uralord e 3e1uec aursdl e qJns ol surlrnbrqn Jo urer{ctJor{se Jo luauqcelly .suralo.rd Iaqlo Jo saurs,,{1 o} paqcpDp ,{lluapnoc seuoJaq tpq} sflec cttodrecne 1p ur luasard urelord palresuoc ,(1q8rq ,prug ulrpb;qn 'aseuDl eursor.{t crurseldofc oslz aag 192-91 arn8rg) 'saulsordl uo suratord cgrcads saleldroqdsoqd 1eq1aur.{zug aseuu au;soil1 (g-77 arn8rg) .tsoq traql Jo sllal aql olul suralo-rd crxol Surraarlap -ro;ruels,,{slprra}ceg rualsds uoparras nI edib uralsr(s pgq(q-orrq lsuar( aas-rualsds pgqiq-orrq (tgg 'd 't-t t Iaued) 'sa^Ealpup sruats1ue1d;o ,ir1pprr aql sulplureru 11pue qu,lor8 1ue1dur uorsuedxa 1ac Surrrrrp af,IoJ aql sr lr :srsourso ,tq rale.tr Jo a{elur aq} Jo llnsar aql se 11aclueld E aprsur padolerrap arnsserd crlelsorpdq a8rel e.rnssard rotrnl 'snoJoJupc slJaJur 1rIIaf, aql a{eru uec lpql snJIA snJr^ Jolunl 'rarupr 127-97anfug) Jo tuaru -dolarrapaql Jo^eJsaua8qcns ur suollelnu uoqcunJ-Jo-ssoT 'racueo e Jo uorleruJoJ lua,rard dleq o1 sreeddp lpql aueg auat .rossarddns Jorunl

(rr-02

pup 6-02sarn8rg)tacuecumolq-llnJe otur saAIoAa,{lenper8 ror^EqaqIIec poraprosrp^1pllruprlrur ue qcq.n.{q ssecord uorssarto.rdrotunl (62-91arn8rg)'sasuodsar fuo1 -Brurupgur Surcnpurur luelrodur.{lprcadsasl reqt euplor,(C (DCNI) rolruJ slsorrau rorunl (t-02 arnSrc)'(1ueu31pur,snororupc) a^rse^ur ro (uBruaq)alrseluruou aq uEJ rorunl V .loJluocuorleJa

(BIt'd'€-9 alqpl)'reqtoue 01 alls auoua8 auo ruor; acuanbas VNCI e Jo luaruenol (uolluulqurorar puolllsodsuurl) uorlrsodsuerl (69-9 arn81g)'uodn slce tl tpqt uos -odsuerl eq] ^q papocua dlensn sr asesodsuerleql 'a1rsla8 -JEl^\au P olur pauesur eq ol atuanDas VNCI aql sasnpcpuu aruosoluoJqoe ur acuanbes uosodsupJl u slnc leql arur{zug asusodsrrerl (BIS 'd 't-9 elqel) 'uoqrsodsuerl ,iq raqloue o1uollrsod atuoue8 auo uorJ aloru uec lpLOy111q;oluau8ag (uosodsuurl) luauole elqesodsue.rl (t-I I arn8rC)'ural 'uralord -ord godsuerl auerquau os/z aag lauuerTt atodutoS 'trodsuen alrlcp olrrp o1 srsdlorp.{q dJV sp qcns .{8raua Jlloqelau Jo ecJnosp o1sa8ueqcpuorlpruroJuoc aql {url upc ro luerpur8 pcrueqcoJlcala ue [r aop ,tlanrssed selncaloru ro suor uodsuurl uec sregodsuur;'se8ueqc IpuorlerxJoJuoJ Jo sarrese Suro8rapun,{q euurqruatu aql ssorcp}l suodsuprt pue etnlos p ol spurq 1eq1 urelord trodsuerl euprqual4J (esearurad,u1e1o.rdrapruc) .ragodsue.rl (9S-II pue 91-91 sarn8rgl '114ccqdeu.4.s1sod eq1 ur pu8rs pcrrlJala Ierol e Jo uorl -eraua8 oql ol speal suor Jo anogurSuqpsar eql'tatlrursupr1 -ornau ruln_llecerlxa cgrcads e ;o Surpurq aql ol asuodsar ul AIuo suado'sllac alJsnru pup a^rauJo sauerquau eurseld cqdeudslsod eql ur sasdeuds lpJrruaqc le punoJ Iauueqc uol (roldacar cldo.rlouor lauueqJ uo1 pa ut-.ra11ilrsrrerl toldacar peldnor-IauuuqJ-uoD

(61-91 arn3lJ) 'auerqrualu arlJo eprs rarfra uo sseu s1r;o ged rpl-^ ledplq prdq eql q8nortp spueua teql uralord auerqruel4l urelo.rd euerqruerusrrerl 'euprqruaur e ssorce uralord e Surrra;sue4;o ssacord eq1 (7) (9-97 ern8rg pue I-B leued) 'rarpoue ot paqcepe pueJJo uoTorq sr aruosoruorqc auo Jo uolgod e qJrqm ur uorlplmu Jo odrtl (I) uollecolsuerl

6€:D

AUVSSOID

'33a pazppa; y'alarue8 alerual pue aletu e Jo uorsnJ .,{q pacnpord 1ac proldrq atotAz rleq uolseqpu aas-sueraqpu Elnuoz

'salr{couotupuu'se$copuer8'sa1,{coqdrudl sapnlcul'urqo13 -oruaq Suolcel silac poolq palealcnu aqt IIe JoJaruuu preuaD (allbornel) ilat poolq euq 4.

'a1cdc 1ac aqt;o aseqd y41olur drlue 'alls eAIJce Surleln8ar ur tueilodtul )pC eql uI splJu outtue 3ur1e1.{roqdsoqd[q &4pce )p3t s]lqqul reql eseupl ulelord IEAAA

:aqlaSo] uoyleturoJuocurato-rdeql ploq dleq leql surole curz aJoru Jo euo alerod.rocursJrlorure8ug curz 11y'suralorddrol -eln8ar auaB .{uetu ur luasard;qoru Iprntcnrts Surpurq-y111q .ratug culz

,{poqpue palaqel e Jo suearu ,,i.q,(1pnsn 'paz1.l1eue uaql pue ]eaqs reded e uo pezlllqourt4 pue sls -aroqdortcela .(q paleredas are sulelord qc1q,udq anbruqcel (Euprolqounuul) tq11o1q urelsalA

(ZZ-IZ am8rC) 'sercadsssorrp uorlezrllgal ol ralrreq e ualJo sl U '33a pazllruaJun aqt Jo aceJrns aqt uo radel uralordocdlg eplcn11adeuoz

'sqderSorcgu ur aull asJeA f,uvNsaas-[wNs-^ -suP4 'anlE^ plor{srrqr P lsed P se uaas 'par{Jelle aJBsluauPlrJ urlcE spue )Jep Jo '(s1ec snld aqt I{JItl.tt ot arauroJJesalcsnru e;o uor8ar a{Ilaleld auurqtuaru ul ulqs e oJ esuodsar ut suadg (auII Z) rslp Z Ipltualod apsnu pue '33a 'auucopua 'alJeu se I{Jns) sllac alqullcxe 'sprurup truuru;o s33aaql ur 'sapp .,{lucrrlcayaJo sauerqrueu aqt ul punoJ Iauueqc uor;o eddl luasard -eqccesdlod pue sutelord'sprd11ur qrlr salrasar IauuPqJ uo11ucpalut-ate11o,r IpuortrrlnN {IOA (91-77 arn8rg) 'ilac ol IIoc uorS Sutpeardspue (77-g arn8rg) 'suorlJeralur y1qq-uratord pue IIar lsoq e uHllm 3u1lecl1dar;o elqedec pue leoc ulalord e ur pesopua (VNC ro VNU) plce rlelcnu Jo Surlstsuocalclued ulalord-u1a1ord3uf;rluapr ro; anbruqcal crlaua8 rclnralol snJlA uralsrh ppq(q-olr1 1sue,{ 'oseo -srp asnec ol dlr1lqe s,urslue8ro ue ol selnqllluoc leql auag auat acue1n.r1,r 'se1o.{recna ;o lseldrurs aq} Suotuy'sarcads cruaSoqlud se JoJpasn sarcadssepnlJul 1e,r,rse'Suqeru-pearq pue SuLe\aJq 'r8un; relnlacrun Jo sarTrrueJIEJaAesroJ erueu uorutuoC lsea;( 'aluosoruoJr{J auo puP auo uleluoc uaru x I 'spruulelu Jo saruosouoJqJ xas o^u aql Jo euo euosoruoJrlJ I

Jo sllac aql

(BZ_B arn8rg; 'alnraloru aqt;o pts.{:c e q8no.rq}Sursseds.{er-y;o urapBd uortcerJJlp aql uo paseq alnJalotu e ur sruole Jo tuaru -a8uerre puorsuaurp-aarql aq] Surururalap ro; anbruqca; (uopcerglp dur-y) fqdurSolprMrc fur-X 'spJemlnospeardspue paleuul sr uollE^Ilc€ul qclqM lP oruosoruorqJ x uP ur alrs (CIX) reluer uollB^llruu!-X 'sl€ruluelu alErual sllaJ Jo Jrleruosaql ul auosoruoJr{3x aqlJo Aool auoJo uoFe^Ilceul uopu IlJBul-x raluac uolle^llruq-x

aas-Jx

'luarudola,rap alPJqaueA dpea;o sarpnts q pasn {puanba:; (peot tou) Sorg;o sarcedg (puol pa rulr uucl{V qlnos) qlraol ntdouay .auo,,i.1uo uPluoc ua(u Jo asoql alrqM 'saruosoruoJqr X o^\l ureluoJ uouro^ 'Sletuluelu JO SIIoCeqJ Jo saruosorxoJqc xes o^\l aql Jo ouo aruosoruoJqJ x (ZZ-9I arn8lfl 'raruec ol peal uer .riemqteduruatec-fl71u11ar{} Jo uoue^r}ceralo'uoqera;1ord pue uollelluaraJJrpIIec 3u11orluoc seueS;o uorldtrcsuerl aql 'snelcnu aql ralua o1uruelec-$;o slunotue sa1e1n3ar 1reJeqm poseorcul sasner uorlp^rlce'qcuerq (lecruouer) rofeu aq] u1 'saqcuErq aq1'sroldacar areJrns-ilocslr IPJa^asseq ztervrqted Surrcu8rg ot urelo-rd lul e Jo Surpurq .{q pele.rrlce ,,(e,,r,rqted du,rnqted tuleu81s 1u7y1 'sanssrl

(gl-tz arnBIC) 'alctlred snrr,t a18ursy UoIJI (91-77 ern8tg) 'pts -decoalcnu aql sasolJug 'snl,t padola,Tuaue Sutra,toc'aueJq -ruau euseld flac-tsoq tuo4 pe^Irop 'ra,{e1q prdqoqdsoq6 adole,rua pryr 'ureqc epr1dad.,i.1od roldacer IIoc J ro utlnqolSounruurt ue ;o uor8ar elqelrel eql Jo tsoru Sulpocue luau8as aua9 _ luetu8es auet n 'sluaur8er; elaueBro ro salclsaA se r{Jns sollelpaturalul papunoq-auerquatu ;o sueaur ,4q Jaqloue ol luaulreduoc ilac auo ruor; sulalord;o lrodsuerl podsue.4 rulnclserr 'pcrraqds uago tlec ctlo.4.recna 199-11arn8rg) 'papunoq-auerqruaru '.iletus e;o tuseldoilc aqt ul a1laue3.ro aIr!sa rolcu; qyrmr8 lBllaqloPue rBlnrsu aas-Jc&\ 'a1odprurue aql eqsoddo pua aqJ 199-77arn8rg) '33aprurue ue uI patecol sI I1o,{eq1;o lsoru qrlqm le pua aql _ alod plete,r (6g-g arn8rg) 'tolceA uolssardxa 'ro1ce,r3u1uo1c oslD aas'ursrue8ro Jo IIac p ol Ielraleru cpaua8 llusueJl ol 'ISoloIq pesn (pnuseld ro snrll) lue8e ue Jo VNC aql ,t"" ;t;r""r, (Bt-92 pue 'n-92'9E-92 sa.rn8rg) 'roldacar IIar J ro uqnqolSoumuul ue Jo UPI{J apqded-{1ode roy aue8 puollcunJ e urro; o1raq1a8ol lq8norq a;e slueu8as aua8 qcrq-'lL.{qssacorduoleuqluoJal cIlPuoS uollDulquotar [(o)A (99-97 ern8rg pue'EZ6 'd '7-91 a1qel; 'slassa^poolq;o qvrtorS aq] solelntulls leql ulalord pelalras (CCfln) rolcu; qlno.rt Pqaqlopua rulnrsu

pue gg 'd'I-z elqel) 'sruole reloduou uea.trlaqeSuer asolc le

'(plu aqluil arnleu ur punoJ uroJ aql :tusrue8ro ue Jo rxroJ luplnruuou 'leruroN ad&-pg1vr

(IS-92 pue gg-97 sarn8rg)'a1rs3ur -purq-uatrlue oql sruroJ pue elncaloru ol alncalotu tuo4 sroJ jp lirqr ureqc ,4,teeqro lq8q uqnqolSounurut ue;o^uot8ag

'uollera tlnpe pup sor{rqtua Ipurue ur uorssardxaauaS puu -Sr1ord'uorlepuaraJJrp 1ac 3u111or1uorur salor tuaraglp dueur a^pq ]Eqt suratord pu31s palarcas go .{pue; E Jo raqual (ugalord ru A) ru \

(irr_orr ..";1;;""t:.'*" UVSSOID O?:9

(s)uuaqpe)aassuuaqpPl uoxe t€L6'tL6'erp-) 8 ' 0 [ ' a i n ] ) n r l s 'a>ro1 Itbtt'znLL'(suaraqpe euoz)llaq uorseqpe 6urqsnd uorlezuau{;odutpv aas^lqulasse 6961 's^Pile t S € ! t , ( s ) u o t l l u n sf u a r a q p V l6E0 r 9OO[ 'g€0t-l€01'uorsnrlord auerquJau puJseld / [ 9 [ ' S u o x p u t l u a u i a ^ o u rp e s e q - e l n q n ] o l ) t u l J Z nLL ' Z t ) J ' s u r u o l e le ! ^ '6ulleo)un 109t !90s t PueItlua 9 76'y 16'sts(lotplqaprloaPnu J 0 . LLL' 1 L l 1 . ' l u a u q r e p eu u 6 a l u r ,(sa)snlt^ouapv ,uor1eln6ar Jg6t [ 955,-955 Ztl I itrplour lla) pue suotl)unfsuateqpe dlv aasaleqdsoqdulaursouapV l6t0r'8€01-r€or IS€ il 'luaualour 't€lt dCV aasaleqdsoqdrpaursouapy lla) Jo lapoul uorsnrlord l _ t € L[ ' t u a u q ] e l l e u o r g : u n6f u u o q t u e 'salei uo!])eel 'euetqurau euseld ge I [9] (s)uralord6urpurq-ur1tyaassurelotdr{rossa>te 966-966 aursouepv ltSt't8t'(suvcv) (s)asputureep l g t S t ' 9 [ S [ ' t u s r u e q ] e u rp e r n p u r - u a 6 o q l e d LL9'slueuelu utltP/utl)v 7S96'l 'elpodrllaurel uralotdTaua6 srsodllod 1r1or 966 Il06 tE08'(auouroq )rqdorlo)rl.ro)ouelpp) Hf )v 'g6ZL 'L6ZL'uoq)ed autosor)v snolPuouape) aas r;or srsodr(1od snoleuouepy xa;duo:6gy 16y JL66'elc,: l66ZL ',uorteallnu g0z [ /uot]ruuap,euouapv lE16 €62[,al]tsa^ uosol)v l e 'uot]tuuep'eulouDJelouapv 902[ 1 6 0 9t ' 8 0 St ' s s a ) o r du o l s e ^ ulte u e l t e q eruera;ol1e:r6o;ounuullos/Daas ,alnllntls /aruelelol z€€ parrnbry lr s r s r Utg t ;err6o;ounur,ur , u o t l e u tvNU ueap ' 9 [ 9 t - S I S [ ' ] u a u e ^ olu9l lre9 l n l l a l e t l u lts u a l ) p q I [0€ n lH oslDaas '(sclv) euro.rpu{sItuar:uepounutul uorlezuar,ul;od urlry parrnb:y t89 t 'uDlurourt)v 'aselruo)v f98€ lzz [ (suv0v) (s)aseutulpop autsouapvaassuvcv r9€tt poy OgL-6LL'seruososi(;'saselo:plq 't€t toldepv 776' 669's(enqted 6ur;eu6rs,surelotd l e l u t l u t t ) v t I t € t t ' u o t s e q p ex r . r l e u - l l a p SSI'sreuo.rlal'sJoldalelasploJp^qpt)V '1.00 '1766'6ur>1red aAtldepP,uals^s/asuodsaJ 'sapup^que ]uauelu u tl)e 'uturDv-n ll.OOt t pDV 110t aunulull aasasuodsa:aunululte^tldepv ',Jale/v\ (ro1:e;6ulzrreul;odap ul l€9 url)e) urlUoJaasro])e, uotlpzrJau^lodaputDV l 9 S l ' s a l ) t s a np a l e o ) - u U q l e l t , u r g d e p y 9€'['q)euols'uorlei)as 6 [ 6 ' u O t ] l n p s u P l le n s t ^ ) 0 L 6 ' 0L 6 - 6 9 6 't s e a ^u r ' s a l q e )u t l l v aat luauleJnseau, HO gggl 'suralord6uuanas loldalal 0e6' J€.06'uo!lerlsanbes 160r '€9-zs'(s)p!)v '000r-566 'pue uorle6uo;a 026 !t06'uorre^!l)Pu! rotdarar lueue;r; 1000t 1666 99EL'aua6 apDQV totdara.r l€06't06'uor1e;nbet-unnop l Z 0 0 t ' € 0 O t - 2 0 0 ! ' p u e s ) t L u e u ^tpu a u e l U # LL' ln LI !gZ9'pt)e )tuturernau;{1e:y-11 '600 ,9gg ,6uru.rrog-pa67qar,a I 16-L L6'sloldaleloloqd 1600t t-gOO 1 1 l€8'ernllnrls ,1.00 ,6ur1punq 069 llOO r t_9OO 1 api(t pr:e ru11 osyaaas /leuueq)uor unrsselodpalp^tl)e ,{OO ,6urlurl-ssor) uot unDle) t-9OOI ll.09 I LLg'fL6'96'uorlxpord leupuoqloltur 'uollePlxo ??6'istxelouaq) 6urpurq-urlry 800 l-900 I' leual)eq Jn66' J€86'(s)urelord ll6 Plle ^DP, 'e6ero1s uorleldepV urso^Wos/Daas i(6reua 96 'sureloJdJolou.rpaseq-urlrv '(V t6€l'uorleututle esdeulsluapuedap-rgrnrl:y t lO t- I lO I L6-96'Lne eu^zuao) l^la)V) Vo) llla)V 'saq)ledurl)e (s)uraloro)au)e) oslDaas lsee^ J0L6'696 989-tg9'slauusrlr pa1e6-laprusuerlse 'srouodtun Jgsg'999 ) 6 L 6 ' 6L 6 'J J L L 6J' L L 6 '0 g 6 - 9 L 6 , 6 u r 1 ; r u p e a r g 989',ernDn.rls 659\959'999'sralioduils 086'(punoq-dlv) urroj-f lggg lapoul leJn})nlls 9 S 9 ' ^ J e p u o ) a s ' s{ r, e l uud [/6',s]runqns Alt^tt)eles s89 LS9-9S9,uanuptue;pei6 uo1 sL6' 1896'896'Jin L'arnlrnrls Sg9'letluelod6uttser 'lrurlolru slauodrluVaassJeuodtlue OL6- 696' aorsutatat sa>Iwon q>to5 9 t6 ' Z e g L' p u e a ) u e l s l s e i l l l o l q l l u e J E Z SL gg9-Zg9'uotte^ll)p letluanbes 996'saruadord 'ZZg'Jn99' 'pue euturel uorlezuaui(lodutl)V aasuorlezuaui(;od tEZg L99-€59' uodsuerl anrl:V 89[ t les?q ,sauosoerrlds g 161'luaulanoururso(r.u 7gg J Lg9'999-ng9' uorpunf re;n:sntuornau ,uralotd6uldder J99L'nSL'sosealordau!ras 9661 9[6')ruue)snu srs{;e1eteu{zu3 osloaas ,uorDe apout puasnlo sg9 Jo 'srs^lelP)ur aloJ 'uotlrsodap 99[ Z0Zt 11em ;;a: lueld tg9,/tuolstq 9 9L ! 9 9 L 'J b 9I ' a w A z o s ( l $ . n o L 'J e b j L ' € n oL- a n o I 1gg9'sa6ueq:leuotlpulloJuo) 'alrs 'sDa#a ')PU 'uotleztue6to '(suqfv) a^rl)v rvL'EL-ZL z')p) pue oqu srolda:araul1oq:;rta:y t6€ t '(clv) aseutLupap pa)npur-uotle^!l)v 89St 1L99[ I t L 6 ' bL 6 ' a \ s 6 urp urq - e p r l o a ; :un ls88'a.rnl)nrls ,au.r(zos(; uollezuatui(1od utllv aasuotleol)nu 991_S9L (sUq)V)sroldaretaur;oq:|,Qa:yaass.rotda)al '09t 'lEl ,saul^zua 'sauo] ^q 6urrar*o; quro.r6 109[ g0g 118€[ ta$tLusuerlorneu ;euotnau tgg'uorl)unJ '988',sllo) lE L' ZL' A6tauauotlentl)V 8ZO1-920 t'uorllertuo) al)snut uo sl)aJJeluaraJJ!p ruataJJtp ls88 'urlnpouodol] 'pue snutul uautol >gt>adsoslo aas €00[ tlldeu^s u! uo!lerlue)uo) 288'rjall ',suiloJosl SZB-tZ8'€g-Z809-67'6ur;dnoruorlrear ggg ,aptxo)tJltuslessa^poolq 886-286 srsl;o>{19osyoaas uotlezuaur{;odut}lv oslDaas (q)V) aurloqrl^la)V 'srsi(1orI16 176 l 9 [ s t ' u s t u e q ] o u lu o t ] e e l ) n u 06Z'IEZZ'sauolsrq'uotleliqalv 'uorlPuroJ JSISt'919r-9t9t l a l as c - l v 'ustloqplaut61-8l 'pelnpur-ua60qled',(lqr"uasseTuorltnpur 'slle]f g [ € , s ] u e u{lossarry ut slaure) pele^tDv !n8'98-6L 099[ I,iolda>al 16 79'6 79'6utdwel: e6ellon g 191'ursor{uu'ttuoila$D)oqaowotlluDv J6L9' 6L9'8L9'sluoL!uadxauoxe1uer6prnbs enrldrosqy lgEn L'lLEb L' LEt L'sl?o (reproq-qsnrq) ')rleureq)s 1919 llEE'uorlpllu! uorldu)suprla^tuoqV 1619'6urpro:ar JL9ZL 'L9Z 'eluialnal snoua6o;a(tu)tuoJrll 699'uorl>npuorfto1e11es ut ,aua6/gy I JLLq'LLg'uote5eddd 999-S99'uralorde)uelsrsel6n.rpr1;nr,u l6g9 lerrualod rrldeu{s}sod l 0 9 9 , u 6 t s a pl e l n ) a l o u r /uOrlerlrul 689 lg99')tlo^lelno.s lpual)eq 919',uotltuuap 1999'lrodsuerg{rer;rxne ll619'(s)lerluelod uorr>V S 9 9 ' 6 u r p u rdqt v l . € 0 1l 9 t 0 t ' x o u o ) q ) u - u r p v ua6t]ue Z g 9[ ' u o t ] e l u e s a r d ,099 ,(s)raUodsuerl L99_899 t0Z )8v

,,,iqi:;iljtis;ifi:,,

zz0 L,1661166,(duv) :!;)?ijiB"jil|T* [16'sltunqns

/spuasnutul/sn;d agetqynotb 916 1o E96-286'uotlenlesuo)ft euotlnlone 'uorlelnbaruralord 6661 6193 'sutelo.rd lq uotle)!Jrpoul'uol1e6uo;a t-666 OOO Lt} L' 996lot^eqoq truteui(p Igg6' Lg6'6urpages6nrp 086'(punoq-dclv)r.uroj-c (ro1:e;6urzuar.ur(;odap urpe) utlUoJaasutluo) 1 9 61 ' u o r l e z u e u i ( l o d a p ,960 /pue 1960r [ voqu t60t-€501'996'(stseurlo}/b) 6uu eltl)prtuo) ,xalror l.€0t l9E0t lla) l€00t'€O0t'6urdder l 0 6 6 ' 0 6 6 ' 6 0 l o u o ql e u e l ) p q

v

'uosuedu_ro:TareduJo) sueau s ialqel e ol leJajf e q1M staqunu a6ed :Ila^lln)asuo) Mol1o;leql saln6rlol ll uP qlriv\'arn6r1e o1te;e.rI up qlrm sJaqurnue6ed tifuluaaql uorssn)srp Joleure ol rararererploq lxal ur sraqurnue6e6 ro

xepul

'suteloro pto;i{u-ty )SLL 06s | L6t-96E'uorle6el66e'utalold l€ 'suotltsueJl 'J€Ll 'nLL-tLl e^lleraoool lnLL 1551-06St'uolleluasa.ldua6lluV l8 t€ 'slueurala:r1aua6a;rqou.r'a)uelsrsar ulllllldulV tOt'saulAzoqu 1 6 t s t ' 0 2 9 t - 6 t s [ ' u o r ] e u el ^r u a b r t u v ' s a l n ) a l o u:,t l t q d t q d u Y 'gts '(sadolrda)slueutuilaleplruaotluv Jl.€8'9€8-988'sdurnduoto.rd lggg t I 8 t9 '!Lst'9sL'arnlrnrls s1 ['paletparu-uolrelfuoqdsoqd t9s t-09s I 6 S - 8 9s p r d r l o q d s o q d go?-tgt'elue)Ulu6ts,fueuotln;ona J ZL L ' LL L ' s a 6 u e q l:e u o t l e u t l o ; u o ) It95L gLJ)ualsollV ILL'uotgeln6er selnleloul)tqledlqduv 1 6 9 9 t J t S SL ' I Z S S L ' e i s t ' a t r s6 u r p u r q - u a b r l u v ' s u l a t o r d] u a l s o l l V 'Jaqunu saDadspnpt\tput lsndouayosloaas E L L - Z L Ll'l [ lueututalop l€ss L uotlelnoel qsnlqouel S S St ' f ) ) l u P r s u o )I t r u r r e JSt(.' Jb€Z'saulosoutoJq) )ualsollv aassuolllsuPllaAllelaooo)lllelsollv [ € - 0 t ' a z l sa u l o u a 6 I S S S t! € S SL ' L S L - g S t ' 6 u r p u r(qp o q r l u e - u a b r l u v auou-ro-llV t r, 696'asuodsar'(s)Uer6ollV lueurdofa^ap s/ 60S-g0S'(1or;r:ads , 6 t S L ,leuol)ouor..u 9S ,(s)ua6rluv ";:( ;:!i"j:; " ", S1S t LSSL ! !6relle/I6rellV t L g gL 'L S St ' 9 S S[ ' s u o r p e a' ur] o 6gt L-vgt l'uorlelnllse6 1 6 9 €6 9 € ' , e l q q o M r s n l l x al l l e l l v sofuqua 9 9 SI L L E ' t 6 9 tl 8 9 t ' 6 9 € - 8 9 € t 1 ' 9 ' ( s ) u o p o r r l u V uorpolaslelnleu suerqlqduy ( s ) u r l n q o l 6 0 u n u Laual ss a r p o q r l u V lz99' z9s- lgs'sl)are 'suralord ''g€ 'sJoltqtqutsrsaqlu,(s fHW Jo 919I uralord V d W V t 6 9 ' s n d u e ) o d d r q ' ( s ) l o l d a ) a r lggt ltg€ '98 'fn6n uotllnpord JZ99'$l)olq ad^roldeq'i(>uanba.4 ljlv u.ro.r1 L'uorl)eJo sursrueqrar,u/sla6.rel 198 )aag | 'reJsuPrl 8 S St t s S ' ? t € ' ( s ) a l a l l V 6Yr;Y:r1l[1aasrtPI: €zs t-zzs L 'ainssarda^rl)alas aulsouoPe) )sL8'nLS',uollnlo^e dY!V €zs | laleqdsoqdouotu 'aseostp peards 7 g'ae61y s,alteuuor6al;o 80S[ f t Z S t ' Z Z St ' l r n p o r d a u a 6 - g 'se{p exaly 's]uaurale JLgg'LBS lllaua6 alrqou € 9 9 ' u o t l e l n 6 asrt s o t u s o l8 [ € 'sesoplv ' 6 u t l m e l r 'ra;suer1 lz I t'oseloplv aua6 9gg; ;14> 77 leluozuoq l0z [ l€E'aeqaoL!v f E Z gL ' e a gt ' u o r l e r a l l ea r ! ^ z u a ut uot}eptxo'sapi(qaply 176'srsllo:,{16 6 L E L ' ) e sl r l o r u u l v ,zs l- EZsL'sJro^iasor leluauruorl^ua '06 'uotleluauraJ ^q uolDnpold 'loqollV J06 vNU iaJsuerlos/Daas J € Z SI ' Z Z SL ' 6 u r d u r n da n r l r e 'ernl)nrls 'aseua6otp(qap ;oqorly ltt I t97'seLu{zoqrtllleqlu^s nzsl-lzsl'o)uelsrsar 'asnsrur 0 ' s 6 € S ' u o r l e z u a l ) e r e qalu a 6 ' u r u n q l y ) EL E 'aL t - LL t ' a r n l l n r l s tzs [ 'slrlorqrluv u.tstutqly 981'(uotleluau6tdodi(q) f ZL t ' Z L E ' 9 1 6 ' 6 u t p e a t ; o o r d zzs l- Lzsl 'arnllnlls 'au!uPlv .E6n ,xPrqluv J6Zt L 1 | LE' I LE'uorl)eal)!illelPl )E6bt llzL')e)uP) ul BL E -LL E 'ZL t - LL t ' ^ ) e r n ) r P JLntL'JgV€l '&€1-nnga'saua6 rol)alas )rloatr.roq alo.r l r a l o l d )l l v t€6'(g asPuru a LE-OLE'OLt'saselaqlu,{s vNUl-l^lPourr.rv ,o ggt |'-nen I',{6o;olstq's^er*ttY J 9L E 'I L t ' 6 u t p u t qa r u o s o q p - y 1 6 t - l I l e o u t l u y 9 9 91 ' o f t q u a u e r q r q d u . r e nlH aassnrl^sclv spDDouruo pnptAtput!(s\utaldd osloaas salerqal.ra^ 'sureqa ) pls n lH oslDaas L Z t - S Z Lt 0 9 JENEI'JZVEL ,l6s '09-6s 'arnlrnrls ' t 8 s t l8z t LnEt-fiEL'saua6 .rollalas)!loauoq Jo alor ' '6s 'srlunqns utalo.td SClV 71y 1' @wotpuAs{ruar:yapounutu I parlnb)e) l9z t lt€€t tZ€€t t tttt't€€t-t€91'olqdosotg '1-y111 'uu6y 'alol ulalold 991I arnllnJls srxerorralsodoraluy luau-rdo;anap LEL-SZL ltSt t !z8t t'z8t t't8L t'uera'r66y L ' s l ) a J Juar P q ) - a p r s J b sL ' s s | _ n s'elol l 8 t 8 ' 8 ? 8 ' x e l d u r o )e u u e l u v ,a)ueJ os/Daas utalotd z5 [ lggg'ur>(uosruy '6uruauoqsaraurolalol uo!lelaJ ' 0 8 [ ' a1euot1:un; lor 'uoluv u l a ] o i d 6 u r P l o J n6z-€.62 lott 1?'uo!l!uljap 'atnleureld 'sllajap rledaJ 'srauosl gbnL'aZZa-OZZ1'pue VNC qwot6 tounl fS6Z L l8z le)lloo 6ur6e7e6y 1 0 1 - O O t ' a 1 : r u{ ra 6 o t l t u J g b vL 'g n nL ' a 6 e u r e pp u e 6 u r p u n o r o' nt a s u o d s a r 'stsa.roqdorl:a;a asote6Y t€s ;e6 J 6 aL ' l L e l ' 1 p a 6 i e q > u nt e1l o d J 6 V nL ' L n VL - 9 W [ ' s s a ) o r d 'srolrqrqul 1pp;'aua6 snowobY f6e L!LZl'reloduou 3971 Xderaqtla>ue) ur ssdoptqoJvsnowDOV )t LnL'tL, t'ruelnw l 8 z L ' , 8 z' t)'r,p) sr )eeq t L r v t ' 9 v v L a l o rs l l a )l e ! l a q l o p u a 'ssaullts 6utdaalsue:trly tSt J 6 Z L ' r g Z L ' 8L2 'sarlrLUeJ J L W I ' L n i l ' 6 u r l n o r d s( r e l p d e r . gz t 6 Z Zl ' ! 9 Z ZL ' S e aL t 8 u r x o l e u v 6 V V| - g v n L ' 9 t t L ' s r s a u a 6 0 1 6 u V 't0 'lPrluassa 99SJ'9991 t I t0 t 00€t ^ureds^lod 'Z9g 'uo!l)npotd r(poqrlue'uotlernlPurAllultv el)^) pt)e )ulll oslDaat l. 6Le | -g LZ L'uorDunfsIpuou llorau ( p o q tlue-ua6t1ue '66 'sa>rnos {6-raua 8 S S[ ' 6 u l p u r q'(e)) {proldnauy t0 t luelsuo) IlluuJV l8s t apo) )!lauaD oslDaas 9V9'ZSl'etwauv 3 91 5 ' 6 u r 6 6 eur r a r o r d JOOZ' 00Z-66| 10, 6urPo) Z t [ [ ' l e ] a l a l s o ] ^ > ' s u r a l or o dq : u y sbel uralordos/paas :(s)uuaqpe)osp aas sadlt)tlDadsa(s\uubalul J O 9 ' ( s a n l e)^d )' sal s6^r el equ) e lsts'tls-€1s vgz'ePPaleluollelnu s € t t1 € € t t ' ' O 9tl- € € t ' J Z E \ tr' Z ulalold )6ZV'6Zn-BZi'uorlPlUundluo!leredas vNU rarsuellos/Daas E t [ ' ( s ) u o6r ]ur u on qf ) u v €zs'suollleJalululaloJd-ulelold ) LL E 'OL t ' u o t 1 e ; { l e o u t u e ) 6 L SL '6 L S| - 8 L St ' u r a l o r d 'saldrluud 6Z?-8Zt 'sa)ulPur s z L 'o 9 - 6 s ' s'sdno.rb ' ( s ) p r roeu r u r v 3 1 1 yoy16 u r p u r qa p r l d a d ' p r r eo u r u e J o q l u v autuY l?€s 3tg 1 ! 9 L t L ' 9L l a - Sf L l ' a l u a p u a d e p a 6 e r o q : u y ' s t s { 1 o r P ,' P { qu o qa p l u v l 6 Z V '6 Z b - g Z V ' s u t e l o l6du t p u t q - y 1 6 3191 J g'uo!l)nrlsap V ( 'L n Z ' 6 u r ) e Jrlo l s a ) u V !, L9'z ts Xqdet6oleuotql,{1tutgY ulJn)as $ e z l ' s z a t'urlrueurv-n 'lsal saurv '90 [ 'lueururalap )tue6tlueloJ Ipoqllue '^lluUJV 'surl)^)-S BS9L fs8€ ?90[ 'azrs 'surll^)-w auoua6 'nutad uruIdotaY f8 [ rz0t t9o I ' '26€'sale6a.r66e orlnlo^e v L g - t L 8 ' l Z ' Z | ' u',lerral)eq utalord'aseastps,taulaqzlv t 00 t 6 9 0t - 8 9 0t ' u o l D n D s a pu t u t t u a 6 ' p u eu o r l e t l r uur o r l e l r l d a r (.reuuretu l6€8',688 J L Z 7L ' g z nL -L z n; ' P u e 1 6 VNC 8901 't101 / u o r ] ) p r or u s r u e q r a u r T s l a 6 r e l 86-16'uJslloqelaul)tqolev 39gpy'y2p1'6un1 00t t !S90['180t ' ? 9 0 1' u o r l e l n b a r rloa^lv JL6S t0t t-00t t l_9901 ' L6S-965'uo!lPllua)uo)uol lelnlla)ellul'uuonoav n € nL ' l / l s a d l r '' ss l1u1ealueral ol aa n ; Y 1 9 6 € ' S 6 € ' s r s ( ; o a 1 op.ar lde ; n 6 a r nlv z6s'0uoptADuonDav )€zE'zzE l 0 0 t t ' t 0 t L - 0 0 [[ ' p u e a s e q dt D 'aua6 lrqdollolluo)oualpv utso{t'uodor}-n l-106!€08'(HI)V) auoulloq 08n' 16fi ' 1880rt/80t'Laot'9901 'loliuol al)snu qlooLrs 'srsolrru 6ur)llds 0€0t ' s l e u 6 l s f 6 f f i 6 n E'997 Jo uorlaldu.ro:pue uorleredasplleuorql 'uot1eln6al t06'sloldalai pa1ut1-utalo.rd-9 36gY LggO l' r9O t'()/)dV) xalduor 6urlotuordaseqdeuy 006'uMoPlPerqua6ol,(16 ggg1 'pue lurodpeq: r(1qu;asse a;purds t6LV ' I / 0 6 ' ( a u u q d e u l d aa) u r l P u e l P V aua6 Ogn-6tn'uot}e1n6a.r ;euorld;t:suerl-1sod 0 6 0 1 - 6 8 0 1 ' 8e s e q o e u e '08 alv urorJuorl)npolo 108 J6LV'OgV-6Lb'sunned 0601-680t'v aseqoeue 'uc :]ut 086'url)v ruroj-c !6Lv leuotldo 1 6 8 0t 3 € 1 . 0t''1?1s 0 [ ' s r s o t r u r 'olrer dcv:dIV vzg-Ezs !6Ll'uoxa leuolloo 3 1 6 61 a s e q d e u e o u a P ed)C V l a t e q d s o q d ;aPu r s,(s)rue^n[pV Lntr9nll It60t'[60['l aseqdeue ' ( W V ) N ) 6€S[ a l n r a l o u u o r s a q p lel a ) l e r n a u stsotaul sllal IPJaassail)odlpv aseqoeuv J6l?'suoxa a^lsnl:xar(11en1nu.l ':rua6oqled rsst-099t aruanbasuo.t1u1 6gy'{1rn6rqr"ue 169I 'uorlpln)Jt)et 'atls eil:oqdu-tr{1'sloldaloluolsaqpV elrlds osloaas stsr{;o:r(;9 J6Lt ;eulalut uotltut;apaue6 ) w L L ' a V l l ' ( s u a r a q p ee u o z t) l a qu o r s a q p v L8?-O8t'uolle)utpor.u 659'sts,(1o:(16 '80s 'eua1:eq,(q uoll]npul stso/:o6eqd 'll8 'uolleplxo woosg t [ p l ) e ) l u l r o J l60s 161tr'aua5 I u8 'ulnllaqlldeo] uolsaqpe 'sa6elue^PP lPllal)eq zOs| 6Lv'8n€ ZLa-OIA' Z L'a:ue:r;ru6rs(reuorln;ona sursaqpv g 7 g ' 6 u r r ; ; das n l l e u l a l l Y l 6 € 8 ' 6 € 8 ' s t u s t u e b llot q o i a e u V ' a u o t P u { sY o d ; Y uolsaqpella) oslDaas stseqlu^so!g aasustloqpuV 1 9 1 1 'a)uaJn])o ztI L uorle;n6atl)eqPaal osloaas JSL'DuaDqouv ,711-y7y'a6e4wr X 1 t l t l o t u 7 9 1 1 l l e ) J ou o l l e u l p J o o ) 771y 196',(lquasse ' s u t t 6 a l u t l a q l l d al e u u i n l o ) J n EI L 'V F . lt' u'rsnu! o srsorel)s;eragel:rqdor1o,{tuy u It luaurelUornau'(SlV) l D e r a ] uul l u a l e f 'tt8 'slseldo1{utY os1 o aas Z?[[ (suorl)elalu!ura1ord1,!tntletadool I t?8

X]CNI

z:l

'uosueoulo)/aiedtuo)sueeu s^ jalqpl e o] lajal t e qIM staqulnua6ed l^la^llnlasuo)MolloJleql sain6Uot ll ue qlrm 'arn6r;e o1ra;arI ue qlrM srequrnua6ed 1^lluaoqlro uotssn)srp lxel lofeu e o1ra;ararerploq ul slaqunu a6e6 'J9t6'S 'uorlel^roqdsoqdolny Z(.6 t6 1uogy S t ' u o r l r u U a p , t q 6 ; a:m €,9€L- LiE J'lueLudola^ap,sa6epuaddy 'srsoldode 9Z t t :rtoldode-ord [ 9 ' , S n r p e) rru l o ] v E ZLt - Z Z Lg ' s u r a l o r d .sutelord J n gL ' J t BL ' E AL - Z gt , ^ 6 e qd o l n V 6 Z S ' , U l ^ l N ' , t a ll)tnuul o l v sz L L-vz J l, dvl J V gL ' E gL - a BL , ( s ) a Lous o 6 e q d o l n V I t S - 1 Z S ' s u l a i o J d ' 6 u r l a p o: ruur: o 1 y n1lJ-lZlJ,suralotd 7;:g '009 '(1413y) (do:sor:rlu a)ro, lrurolv rLSZ I t09 tZ LL-ZZL L'sutalord:rtotdode-rlue'982'(SUV)a:uanbas6ut1e:r1da.r I;snoujouolnV uotlelnoaJ etrt'aue6 Iqolv 'uorlelnur l 0 S S ' 0 S S ' 6 u r : u a n b aVsN C p a r p L ! o l n v (s)asedseJ aassaspdse)'epe)se) 90 t t )tilloelold '6€5 'aseastpaunululolnv , p u ea 6 e u e p y 1 6 6ts L t 9 9 1 1 l Z 9 ' a u u a s l l p t leqdsoqd 'sl)ejap 1g g ' 6 u r 1 e u 6ar su t . t ) o l n V t0€ f96z S LLL ' l g L , s r s o i l ) o b e q d 'JL'alll ,srs(;e1e:ogny ,suolneu agt aseurl ureloJdwlv lo ur6r.ro 06€[_68t t /a)uplalol ' ,sanbeld )tloral)soraqlv 8 8 St 00S L t61 9 9 g , s u t a l o l dl p u p u o t l ) o l t r u 'utafold; e > r 6 o ; o u n u u r 't61 ,stso.lallsojaqlv (lUlV) Joleln6aj aunurultlnV g L[ [ , l e t ] u a l o da u e l q u a u r 00s L '?0€ ? l 0 t , s a s P u t le . l o l n v g T y g, p u e 1 {6t e u u e r . u u r 90t t 196z',uorlelnur ,aseutl g e r o r n v ?g0t n $ ' L s 6 z ' ( l v ) e r s e ] ) a t 6 u e leallx e l v 1 9 1 ' 6 u r 6 u a n e ras6 e q d o r r e u lJr€tL JVSE'bst - tSE'aurosoelrlds)v-fv L6V 'I ' L z v L, s l l a ) 'suralord l t , L ' Z E vt - O E nt ' 6 Z nl ' s l l a ) I p q , { r o g r p n y u j a l s qleep-lla) uorltnpotd palelpaul-SlUl Jtart Jo ,(1u e ; o d 9911 leseq J 6 g ZI ' 0 6 Zt _ 6 g Zt , s a l I ) o o ) 9 2 LI ' J n ZLL ' 1 Zt l ' ^ e M q l e d) t s u u l u l - o ) r d el e r l a q l l d a ' ( f ) d e ) a s e u r lu r a l o t d ,srsarodoulaq f ;e:rdrey 1 6 6 0t ' G 6 O l ' u o t s t ^ t pl l a ) ) l l l e u j u ^ s v J9; g gll- LLV'uooe16et auab leua])eq,uotlenuallv ! 6 g Zl ' 6 g Zt ' s a t ^ ) o o ' u o t s t A tl pe t u l a u u { s V lZ I l-OZ I t',(eirtqled)tsuulxa 'pue ,aseutl '9601 'lapoul a6euep ' a 6 eurep uralotdg1y 961.L VNC uorlelnultls 1960t lglLL'LLt t vNc ',lepouruotlexplerleJlsv srs{1o:(;9:aseqluAs 41yosloaas !9601'1601-960t elodpel lerlsv JLl t t' 9 11.1'srsoqd.ror.ueleLu ,9[ ,sMeda_snour llz58 ! 9 L OL ' 9L O t ! S € 0 t ' s a l n q n l o r l r u lre r l s v 19t t L tt '€S8-OSg'uotlelfi .009 .eurq]sv oqdsoqdoloqd:r;:{ruou Jr€s [ t LZEL'suDbala.) g g g ' u o r 1 e ; ( r o q d s o q d o l ot qr ld: l > 'luaLudola^ap l 6 E L- 9 6 € t ' 6 u r u re a 1a ^ r l e r ) o s s v 9Z [ [ 'auetquau eulseld gzs lggt,ater uotleDossv _ €S8-0S8 l56'56-76,>rgaqlui(soloqd 1 1 11 '(ry) uorlel{loqdsoqdaAtteptxO aasleupuoqlotLu tuetsuo) uorler)ossv l89t SItt,sa6ueq)relnlla) 'ainl]nrts ,pr:e:rlledsy 't88 '6ur;eu6rs srs(;o>(19 osloaas l6z [ ls88 ;1a: 'ornl)nrts ) L Zt ' f t 6 3 2 6 ' L 6 X e M q r p :d1 1 1 o r ^ 1 6 g t lvLL'tLt ltt L l l - l t f I ' l e ) t u a q ) o t q , u o r 1 l u 6 o : a; 1r a : 'v 'ulstueq)au.l L1g-0lg,uotleluau.|aJ )tl,(lele) ls Ll LL-ELL Ll, aa-gJ ll,uorlPuil..ullella) S IA-ZLA'eual)PqouPI) g n z L - s n z t ' l g L z L' j L Z L - SL ZL 'L a t L l p u e) ; D u e ) f i L L ' t L L - t L l ' s u o t l t s u e lal A t l e t a d o ottu a l s o l ; e /saqolaeue aselloueqte>sueltalelledsV zlg-1zg l z I [ , s a s e a s tp a ] e t ) o s s e /uollnlo^a /ainl)njls ',(pt)pltuedse)eleuedsv 9t8-Ol8 l6z t I Z L L ' f j e L Lt 6 t Lt , 8 t [ [ , s e s e d s e : o r d 'slrlabiaua 'atnt)nrls 'aut6eredsV 08 l6Z t ( s ) u t e qu) o d s u e Jut o l ] ) a l l ' t E L S l ' L L 9 L ' 0 2 ' r o ] d a ) a rb 6 9 l u e 6 1 ; L ' r r c J L n l ' 1 5 1 ' u o r 1 e l r ( s o r { 1 6 s e 1 7 p se3 t [ jslualppr6uolold ,aDPruep urelord'(s)apueqr)eso6tlo pa1ur1-aur6e.redsy leltueq)o D)ell osp aas 9 0 1L , l j veNd cv 9gg,6ur1dno>un 9 l t ' J 9 L t ' ( 6 u t p u r qa u l o s o q ua) l r s - V r ( . z t L ' L a tL J LZg'EZB-lZ8'slua!Per6uolord t a 6 1q s y l t Z O L ' € 2 0t ' u r a 1 o . rrd{ r o 1 e ; n b a u J 8 0 tt 1 6 t 8 ' 6 1 8 - Z f g ! 9 9 1 . ' u a n r - r p - y o d s uueol lr l ) e l a t 6 9 ZL '6 g el ' , u o t ] r n p o l d alle n x a s v ' 8 0 I l - U 0 1 t ' u o t l e l n u t l sl t u e 6 o l t u Jl e u i l o u q e 0 8 ' s r s e q l u Adsf v uot])nput/uotleAtDe srsaqlu,(s 61y lstsIlorp(q dIV osp aes l9rtt 'aint)nrls ' -9 lzzS pauuer6otd) srsoldody tzns 6Zl t L L l'(qleep lla) 'fl8odv 0'g_6€g,ua^t.jp_untpos st9 r 'lueura^ou 'ots(tdv LZB-gZg'srrla6laua 0t0 L /eual)eq ' € 9lD t 'looqs 6€8 JllbL'jlnF60b t !J80't !tort r J9a8' tz8-9z-8'A! |!qlsrenaj !LjvIE9Lt'roo) J lzg'ZZA- IZA'uorlel^roqdsoqda^tlpprxo ' 6 u r ; e u 6 rest e n e l JpJuj tev L ' o L n L - 6 0 b 1 l s s S ' Lz g ' u o t l e z t l e ) ol el u p u o q ) o l t u l l a q t s n q{ q a ) u e u a t u t e u EZg-(Zg,uot])PJo r!stuPq)aul $ L n L '0 L n; 6 O V t 3 8 0 t L ' € O tt ' u r a l s u a u l e ) r d v , a u a 6p p p d v J S S g ' u o t l e z t l e )los le l d o r o l q ) 5rtr[ ',leual)Pq 6€8 l€.lb L'tlV t'ruelnu.jssdop4oty gololady ,E ,999 ,aseqlu,(s It t8 tg 31y l?8'uotlel^roqdsoqdpalerpaur-61y .saulosoalt ZS€_t 9t , { 1 r 1 r 1 ouur a t lds ds 9671 9 t Z - Sl Z , 6 u l p t l sa L U o s o a l ) n u L I L - 9 t L ' t i o d u t u t a l o l dl e u p u o q ) o l t r x ' o s e q l u ^a surueln;6 J tg JgZg'S(B-VZq 1 9,89,99 8 , t 9 , s ) r l a 6 r a u o , s p t ) e) r a l ) n u J18

u"'"'o#eiilvil;i ", l,'],, o

szg-nas '!8 'suort)ear ltlaqlui(sotq ot 6u!ldno) t 1.8 'uorle)rldar JtLZ'tL( V N Co r p a l d n o ) € ? Z ' u o t l e s u a p u oafu t o s o n l o r q l J S V q ' S b g , u o r i e xuuo q r e l J g Z g ' 9 a g , a s e q l u ^dsl v l l t ' s a s p l a q l u ^ sV N U I - l A l e o u t u e 'alnot a^tleulallP 198

/ s l u a u369,srs(;orp,{q l p l tu,r l l e , s d p l61y t 6 L 6 ' 61 6 dlv su1a1oft7sdund t4oads oslo aas uor)sasedlv s99',099-659',(Uodsuerl veg-tzS,orled r cv:dfv 8 9[ ' ] u a u r a i n s e a uuro r l e z r l r ] n 'ttodsuerl zz8 srsaqlu,(s 31y aassrseqluxs ItOg,6utlaqelotper srsi{;orp{qdIV aar sts{lorplq ,sl)adse 06 lp)tJotstq ,81 'JZg't9')a'JJD A6)eua tB-08 ')lg ,alnl)nlls JLI L , t g , a l o l) t l alqel)ut u^ s. roatqq) I tg , ( a r p q d s o q ld8u' la r) suoeupaupneq)e a u 6-g dfV lt'sa)ualeA / s l a p o u6 ur1;r;-a:eds II (s)uorlrell aassuol])ela 7gv'gv_sv'aJnDnlls)tuole ,snlpel)tulole Is gt-gt,suotl)Plalut )il.llole !8n'! h' 9v'8t-gt',sLuorv

' t t S ' s r o l l a ^6 u t u o l l ,sauosotuolq) VNC lpoUtUV sat)adslDnpt^tpurosp aas pa11ru.rsue.r1 suotl)aJul z0g t- L0gL "{qL 'sapueq))es^lodWfl 081 spooolqtJV 'suolllaJulltuolq) 'stluquv 00s L 15trtr9Xrauy !LgZ '982'(aruanbas6ul1e:r;darIlsnoLuouotne) , , u r l sSUV ailv t o z 6 t z 6 _ 0 2 6 , 6t 6 9 L S! ' ] u a u l e ^ o u lu a 6 o q l e d l B t 0 L ' S t O t , e r p o d r l l e uurre ; JL66'166'(xeqwo) €/Z dUV)xalduo) dUV Z Z 0 L 1' 6 6t t 6 6 ' ( u r a l o l d p a l e l a r - u t l t ed)U V 9 6 n 'f 7 6 n ' V 6 V , u r a l o tadl n e u o 6 r V 'arnl)nils 'aururbtV J8Z! L9LL'63L'(s)uralordIUV outpueqvNc os/Daas tLW'grV-SnV' (s)utalordlprn]ratrq)rV 'ezlsauoua6 'snp16p1snqol6oaoqty lg l tzg'uotusoo|.l1o)auelqu.leu Zt9-0t9,utsdopoqroual)pq L g L L' l ' J 9 t ' 9 t ' ( e u a D e q a P q l r e a ) eqlrv ',)ruabsuell 69S 's6urlpeasguelnur 379y1 56€ t-86€ l'69S-89S' 6€.-L€.'9e',1ue;d;apou.r fiLbL lamolj ur uotssetdxaaua6 lo1:a1es)tloauoq 'azrs f8 t 0 0 t l - 6 6 € ! ' 6 9 5 'L VL ' 7 t r1 ' a w o u a 5 c 1 vL ' J 6 6 E L , u a a r )l st l a u a 6 'u! suralotd i(roge;n6at aua6 100tt 'suotlelnu )tloauJoq l€ tt t lzltt'sJaMou J Z O 7I ' Z O n1 , 9 6 9l , s r s a u a 6 o fqi u a 'J9E'(ssarr s$doplqotv 1186€L !6€ le,/'\ DuDuDqt 'ajll 19 L ,o aerl ul ,azts aulouab lg | xaJ!nDv ttL9'tL9'lEEg'?JnDil$ ,tlg /uot])e apouJ 9lg Jo J t L 9 ' S L g - Et 9 , ( s ) l a u u e q u r uodenby 3 6 6 7 , s a gtrrsu r p l r u u { d y ',salts )tuundv l66Z ,29€ 'aua6snotaldy €St t t saiopuaddottloads osp aas 'salerqolra^ 95€L-9S€t (.981-99tt'SS€L-ltt t ',strasul pe ad urer6-aur; Z9€L'8SE[-9S€t'6ururag

),uralotd vtrl'eua6 zDpladv 16gy1 6urpurqluauala-anrsuodsar-aua;(qra77ele1edy fEW I' tLVL'tuetnu"t sBdoptqorvZopladv '86Z'(Ulg)ltedar uotsDXeaseqt66Z /aseellnuopua dV J B V Zf 'L V Z ' l t Z ' s d r q s u o r l e l (at-eru o r l n 1 o n e , 1 s 1 a d y E S ZL 'I Z S ZL , r a ) u e )l P l l a r o l o ) aqzL'os6'6v6 'uralojd/auaO(tlot stsod/{lod snoleu.louape) JdV (f/)dV) xelduo) 6urlotuo.rdaspqdeuVaasf/fdV J e aI L ' L ZLt , s r s o l d o d eg,l e d y 1 6 6 7 1 ' s u r a i o t^dr o l e l n 6 ael u a bd g l u l / Z d V 'sal)rlloJ z6z L lerluv 'ulnlastluv 6gs +95 ' t 9 s ' s u o r l e t n ua A t l e 6 a u l u e u t u o p , v N Ua s u a s t l u v 'tooqs^Jeelol reMou Z€ 6uru.uo;suel1 uotlelnLu'(uo6etpdeus) wnuLpt4uv sad,fitqoads oslo aas lS9'la6ueq)xe uotoid-ulnrpos 0?8-6€8 'E99199'(esedtv)dund utntssprod-urnrpos gsg-lsg la6ueqtxa ateuoqle) ua6orpr(q-apuolq)luepuadapuruntpos 999-LSg,ra6ueqrxe aleuoqre)ua6orp{q--apuo;q)ua^tJp-urntpos 0gg loDueq)xaulnt)le)_ulntpos 'ursrueqlau ,9S9 ',sralrodtluv 999 J 9 8 Z t g g et ' a u o ( x j o q u e u a l l n w - r l u v aluetstSal 's)tlorqrluVaasa)uelstsel's6ntp lerqortrr.urguy (s)abeqdor)eW os/Daas 'salnralou rtole;nurlso: JZIS L L ' Z' sLlS J ' J Z V S 'IZ V Sl t'L09t S l aa)6- tuLrS luasatd-ue6r1uy | t 's1;ar-1tad;aq .sr >rxoloilr S8St -t89 t ,uotleluasaJd ssol) tgS !

€:l

06sr '8zc)

X]CNI

$ L n L '6 9 | | - n g J L \ Z t t t ' L t LL ' e u r u r el el s e S S f Z t ' r a ) u e ru ra l o l ' a u a i ^ d o z u a g 'J6Zv (tol:e;1o t'er;aqtrda OEnL ) L S S '9 S S ' s l u e l n ul re r o r ^ e q a S '6lb 'srullaproa | J6LnL lgl'oolot66ag s l l a ll e s e S l € s ' ' u r a i o r d 6 u r p u r q - r o t e l n s1uyr3 g 'Puloul]re) 't e 1u so s p e a g , , Is6 lla)lPseS JZ1Z'ILLZ tZ'urteurorql,,6uu 'uolle^ll)eu!-X'^PoqJreg €It J L g Z L 'aue6 'L9Z 'erurelnal snoua6o;a,(uL :ruorq: ;:g 1 8 1 9 ' u o r l e A ! ] ) esulla u u e q l [ 'pue ra^aal9 u o ru r n r s s e l opda l e 6 - a 6 p l l o A ' l a p ouLrue q r - p u e - l l e g lz9z L ' s 6 u ur u e r q l e g '69t 'yodxa Y1gtr J6S€ 1 9 7 1 ' u r a l o r ldq y - r : g ZZLL'srsoldode'le8 E ZLl - Z Z Lt ' ( l r . r 6 a t uar u e r q u r a u r ' u r a l orryd- ; r g ' 6 u r l e u 6 rass e u r l - € l d ' u t a t o t dC V g fi86'rE6 s v z l L z l L ' r a ) u e u) l L0sL'saptotapDS l t z LL ' n z l J - J Z I [ ' s u r a l o r dz l ] 8 ' s l u a ^ o6 u r l e u 6 r s rsdopoqrorra})eS Lng r i0v9'zt9-0t9'u 1 9 6 St t 9 L t t 'Y1a6eqdottal:eg '6urlsar 177 ' 6 u r 1 e u 6rrosl d a > a3:rly y' u9o; r 1 e ; n 6 a . r 6St'sllnlll) uolldulsuPll 969; l g g p ' '8S''alels 19y'a6eqdotd 0 9 sL 'sa1>r11o; )lill proqdu{1 qdu.r{1'uorteln)rl)ar apou l85t 'uotlet6a1ut 'Atr)UDedsouoLu ISZE'r(t 999L-S9SL '^rouau ) g S V ' 8 9 b ' a l e lo s' ul qoel sl rl lrxaaq 9tS t tz€ 1 1 6 91 ' a s d e u i (1se : r 6 o 1 o u n u u . r t 'a1>(:a;t1 opuq(q 1979 l 6 0 S ' G O 9 - 8 O S ' u o r 1 : n p oe.ur d rossardarepquPl llossalda)d) osloaas ( s ) u r l n q o l 6 o u nuLl l or s p a a s g lt lossddal epqLuel r89v' Lsv' I8 Lr' JBgsI' Egn-ZBv'6urq:lmssse;: ' u o r 1 r u 6 o : aalr u a n b a s 1177 l99S t 'znL 'uollezuaurlp '99S 'uorl)alas ib l l-S9S f ;ood-aue6{poqrtue t .t] ) 8 9 n ' \ S V - L S n3 1 7 7l o s s a r d a o I '?sst-€sg t Itss 'vnsJ-Ensl ']uaurdola^ap 'zssL'J€nsL 1gg7'157'uotssa.rdalo> aua6 ;igsn'ASn-LSn'uot1e;n6ar 1965L' 569L-b6S;'s.ro1da>ar-or .r6a1ut l s z E ' v z E ' 7 6' a3S' aesUeO 9 6 9 L - 5 6 5 L" s r o l d e ) e rl l a ) - l lSllxa '965 'rotderer 9Z€ 1-565 1 !9 t? t 1965t lla)-8 ' s l l a r JSZE 6 6 9t ' s l l a )z 8 'tZ€ 'uolleutquo)al f Ul)ads-allsenlle^lasuo) t8 6 6 9t 5 0 9 t ' u o l s a q p el e l r a l ) e q €8t-28?'lorluor ;euorldu>suer1-1sod 'srsaq1u,{s ePquiele6eqdolrelleg {poqrlue 3551" sasnlt^lonpt^tputoslDaas JL6ST 'g6S e 6 b l L 6 i L ! a ; s u e l 1a u a 6a ) u a l n r l n L SL , ( H )f s l l a ) _ rf a d l a q t _ L 6 9I ' SL 9L _ V uolllsodsuellos/Daas g 9 7 - 7 9 7 ' 6 u r q r l u vsts e l : J s e t ' n z E ' u o t l e l 6 a l uat u l o u a b J96SI'565 l',roloa)orIlal-8 s o s[ ' u o l s'(s)a6eqdouaireS a q p el e l l a l ) e q l 9 9 St ' 'Z6n 'uortlalas '99S {poqrlue | !ZZ t Z JZ6bL l-S9S ! lood-aua6 ',189s ',899 v6nL-t6bl loJ ulals^suotla.rlasnl ad(l t t 1969t 'Jrss n O S l ' ! v 6 v I ' E'ot 6 t L ' ) o !u a l s ^ s u o l l a l a s I l l a d ^ t I ' c s s I ' J E v sI ' n n s J - E n s l ' u o r l e ^ r l ) e '(s)lla) anp uoll:nPordlano3Y1Y: €OSt Ots t I v6v L-E6VL'uollre jo ulslueq)aur ZZLI'stsoldodP'xeg ' ( x e r q l u e u) l x o l l P q l a l €6tt l l s s t ' 9 s sr t z s ? L ' s l r q d o s e S '(xerqlue) u l x o le u a P a €6?L 'o] ' anp eaqllelp )rseg [6t L '26? zgt r' gst L'suralordtr'1rol *,t,"tu%lor-xrraq 'eraloq) [ sseS 909t-tos L 9 t z ' ( s ) u o r i e l n uur o r l n l l l s q n a snilt)o8 t6b I'sD2lLlluD 3 6 9 9 ' 6 u r r r e d - a saelqq q o ^ 'surxol z6t L lellal)e8 3 g E g ' 6 u 1 p ; yo5; g '692'lensnun uotldtllsue.tloslaaas z€.t arnl)nrls vNU jvNU leuosoqruoslDaas 8Lv- LLI'uotlenualleuotldtlrsuell (uo otwuaq)sl) uo,edo )D1 osloaas 19 g ' u - r s r u e q : a6uu r : r ; d s 'ztt 'pue E 9 t Z 9t !€€?'suorado vNU lr€€ l€E€ lsaq)trMs)tlauaD oslDaas (s)uraloidrossardeU 1 , 9 7 ' a ;1r o 1u r 6 u o 'SrOssalool iuor}e)rldal oslDaas 9 Lt slsaqlu{s VNC VNC 'uo!llu6olaa r ) u a n b a sv N C f8l.t 6 g z _ g g e , l g g zg,g z , p u es r s a q l u ^Vs N C 'suralold aua6 ,{-ro1e1n6al alnl)n.llsvNC os/Daa' 9 [t ' 6 6 1 * L 6 t ' p u ea . r n l ) n r l s JSVE'J6EE VNC 1 1 8 6t 1 8 6t ' g € € 9 € € ' u o t l e l n b a uJo t s s a l d xaau a 6l e t t a t l e g ( s a p r l o a l ) n u6)u t t t e d - a s e g eururellese8aasaueJqtllaurluaujaseS l e)l l u l u el e l r a l r e S a s s ' L v g ' ( s l v 8 )s e u r o s o L u o i q swstuobto>tloadsosP aas l 9 0 € ' 9 0 €l O 0 € ' 8 6 2 ' 6 u t d d t ; ; - a s e g aasjo sasnJ!A (s)a6Pqdoualreg J 6 6 Z ' g 6 Z -L 6 Z '( g 3 g )r r e d a ru o r s l l x aa s e € 'sJol)e; t6t[ P u es a u a 6a l u a ; n t t n OOE,VNU ' L g t ' l o l l u o )^ l r l e n bu o r l e l s u P l l 't9'autpttuur(d )Lgt l9L | uolldu)suerlJapunaasuollolt)suPll ( s ) a s e aq u u n da a sa u l i n o aassulxol sulxol 6 u u r e d - a s ea6a s6 u u t e d lPlla]le8 ' t 9 ' a r n t l n r l se p r l o a l ) n u Ll'wl'sazts l g LL 'lPrnleuun t'sadPqs 1 0 6 ? L! ? I t0€ 'sauosoqrJ lz [9 JOLZ' 692-89e'sreuolnel 'lernieu l 0 6 t t ' 0 ? 8 - 6 € 8 ' a u e r q u a ue urrus)p l o I t0€ ' ( a e u q u rlrd 106 '[ 1 9 6 ( ' 9'66 6 ( ' e 6 e w'VNC e? 'euerquaui lelno l06t t L-16t l186 r !86 t e 1 ; a 6 ea 1a 3 se 1 ; a 6 e g ( p D e) r a l l n u )( s ) a s e 8 'lle!\ lla) L'St !O6Vl'?[ Hd aasluauralnsPaul '€s '(le)lLuaql) ;rns-;;al (s)ase8 [ ' s u o l t ) a l o r da : e l 0 6 t 160L 'salnsde] 'srsaqru(s Z € 9[ '(s)ernDnrrs 5 9LL 'S9 'ua6ellor ad,{r 106?|'saq)llMsoqrr LL At LgJL-ggat 'su ruluJel 61t-8l? 99lL-SglJ uorlelsuerllslsaqlu^sulalold os/Daas )L9tl 'sauosoqu ' lz [9 sodtuo)/arnpnlls r g g | | l s g Lt ' , 5 91l,' , u o r l r'pue16 (reurLueu s)llolqlluv oslDaas g7y 1 ''8€'srollqrqul l-s8€ l 8 9 t L ' 6 9 t t - 8 9 1[ ' p u e u o r l P r e u e b a r 0 8 €! s ? € ' s l s a q l u ^ su r a l o r d 59 LL lerluPqleul ' J r g L L' S g t l - v g L L ' s n l n r a r , u o, ;{ 6e u p t 1 ) L l L ' L l L ' u o l u e s uul l r o o Lgl | eual)eq 69LJ-rgtL'IlrstantP letral)Pqoue^)osloaas a;drn6:srsaqtu(soloqd 8 9 [ [ ' S U O I ] ) U nr aJ l l l e q 'salol s La-z La' t 98'o?8')!laqlu^soloqd tgt L leuolDun} ggy-797'luar,ua6uerle VaNJC ' u o l l P u ee^ s e q d 9 Z n l ' l z v l ' e l u e u e l u ! e u l l a ) - u a l sl e u r . l a p l o a 6 6 7 1 ' s u a 6 o q l e)dl l s l u n u o d d o ) b 9 | I ' 5 9 L L - n gaL ' , a ) u a r n ) ) o / u o ! l n q u l s l p

a1e6t;qo t6v L-o6nL'sua60qred ^s^suo6oqleduou )(.6Vt 'uolse^ul I ts r-los I JZ6VL' uo|}e1ue6lo autoua6 ^l]e]ln)P] o 6 t [ ' s u a 6 0 q ] e da'rrue6oqled n6nL-6gnL L 0 s [ ' 9 8 t t ' P r o ul'VNUtu eurou '08€ r [8€ t9t€ ':tqdo:loql!l 's]uaA lp z[ 'slla)lsoq uolse^ul '80s leultaqlolpi(q 1-z0s [ Jo 1609t LLSJ-n LSl'luaula^oLulelnlle)erlul LZSL 106t L'.a^rlrsod-uer9 Les I' Jo6nl'e^tle6au-urer9 88'[ 18['Zl'azrs 'pe]uano€sa6 fg [ uolleln6ar aua6 lPtlal)Pgaasuotle;nbalaua6 uorssardxe uollolllsuell os/Daas ' u o r s s a t d xaau a 6 35yg Z1t's)!laua6!da 809 t-los I 'srsolr(:o6eqd Iq s1;arrsoq olut (rlua 'sa>.rnos,(6laua 6Eg (s)ureqluodsuerl uorl)ell os/Daas /uollnlo^a z/8-lz8 l€18'llodsuell uoll)ala 'lapour sello) 3 )sz'sz-vz ' s l u e l n u t l s o u n u u ls es a ) u a n o e V s NC 1 Z St uolle)lldel vNC os/Daas vN r C J q S ZZ ' 8 Z ' 0 8 2 ' u o l l e )' r l o e s [ lrsra^lp 'uor1e:tldat ut6uo ;o JtgZ'eBZ ql ! L6n L' a1z'saLuosotuoi l 6 € g ' 0 t 8 - 6 € 8 ' 6 u t l d n o l) l l o u r s o l u l a q ) le)luleq) l€9'f9s'uolilsodl.!lol 'uorseqpea6eqdorralreq s0s t ' g L ' s As a l o ( l e : n ap u e e a e q l r e lLl ,sla6relpue uot])e )ltolq!]ue l?6'[ ,88V ,J9l ,9 ,PuaDeS t t 06vt_68nL Z S s 'f t s ' s r o l l a n 6 u ! u o l ) vNc'(saurosoulolq) lel)ulue lPuel)eq)sfvS JOa'I8 L'sIlqqnssnilt)D8 €6VI'st)Dttlluosnilt)og I8L'J9t'snilt)08 '!ll!)e8 l06t |' 'uolle^ll)e llal-I I t65 t e 6 s l L 6 9 L ' 6 u t p u t7qy 1 1 3 r]ue 0 6 s[ ' u o t l e ] u a s a'lsduuar 6 elord 18 J g 6 9 t ' n 6 9 1 ' f z 6 st l€zt'sluauala l8

s y g g ' 6 u r p u r qa s e p r x o euolqlol:'aptzy 6 8 9 ' s l a u u e qu) o l ' ( s ) l ) o l l l qu o x v r E 6 ZL ' E 6 a'ls' |u. ItlaJua(dPs ' a u J a u o x v 'E€O 'g t 1 61 leuauoxv Jt€gt ',sasnrl^ saoraq t9LSL'LtSl-919 [ 8?0!'aPel6orlar s,raPelboralue lrodsuPrllPuoxv (s)alllpuacos/Daas '6urleu6tsolneu 979 1 0 8 9 ' 8 1 9 ' q l e a qusr l a ^ u r 'uol]e]uelloalnqnloJlllIl 1'0 [ 'uorl:a(o.rd lel)alou!lar €6€t- t6€ t ' 6 8 t t - 1 8 €t l 8 8 Et ' a:ueptn6 L ' 0 t lt ' s u r s l u e q l a u L L l l ' L n L'salupuap uloJJuollellueleJJlp 98€I 'luaudola^ap ' rLgt L L8EL-98€[ uodsuerl Pvoxv oslDaas 'uolalalsol^) 090 1-lt0 I (s)leltueloduolllv os/Daas

rzle,uorre6edo_r.fi 1",j!gJ,?lili_"

6t6'ulxv I I t v I ' O L vL ' P n q( r e 1 1 1 x Y 0 tt t_'ltxv 9t-9''raqulnu tolpeoo^v 1 9 5 - 6 5 6 ' s l u e l du l u o d s u e r l l90t t l6s6'p!le )rlale-€-alopul 60t 1-80tt' njv L-tlv t'6s6'urxnv LL Zl ' I V S S ' € l t '' (asu)rapuurno-sHo€l n v €09 figz, E8z_zsz,aurptrll^ql_H€ JZZi'lDVd-OZ lngz' t8z-z\Z'eUoJ uo!le)lldarrtlofue:na 1969'i(dorsotlttuuoll:a1a lE09'€09-ZO9' 199 "(qdet6otpetolny

XSCNI

vil

'uosueduo)/aJpdulo) supaul s jalqel e ol la]al I e qllM sJaqulnu abed i^la^lln)asuo) !\ollo] leql salnbu ol ll ue qlrM /aJn6U e ol laJar I ue qllM sraqunu abed :ftlua aql Jo uolssn)stp uat rofput p ol ra]ar a)erploq ut sraqulnu a6ed

'tz€ 'ila) ' l a p o ua 6 e l r U e ) llrz€ t t l t L V t ' L L n L - O Lty l y7g1 '6uruta11ed audola^ap z L bL- o L v t ' l u ' f a z E L' z z E J ' ' a 6 e a u r;;; a r ,uotltsooulo) vzEl lZtL 0Lv L_6gnL 'sa1ol.re:na 'arej 1 ; a : (s)lsepoalsO i(s)lselqoetso ]uaraJJtp os/Daas lggg I zzEt ,qreoplla) u r s r a q u a u { ; r u e ; ' s u l a l o l d, { l o l e l n b aal u a 6d l z g lLZt l'gZeJ-tZE L lla) )jLrt'LgtL's|a) '^q IEZEL''ZEJ-€,ZE!',uotst^tp e 6 e ||1te> l u a u a l e l d a t tos ;1a:rrrlar.ur"ur(se JLLbt'tLnL-OLV L 'qder60llr.u '0Lb 'rlnlrleue) arnllnl llsl /seutllla) Ieltourul ZAg t * z E L ' l n z € L ' r r € L 1 l a )d 8 v L JOLV | 'ller '(s)auo8 J J V Z E L ' J V Z E l , t[z t e 8 v 6 6 VL 'I S Z Zt ' u o r l e s n e )l e iI n ?{t t-6,9t r BZEL- LZ€l'luauldole^ep suoba1as4tpqoqtouao3 6 t Z t ' a u a 6 r { W 6 u r 1 e n r l :ueo t } e l o l s u p r } LbNL '9gg 'I11ue;(utaloldlrlauaboqdrou tazts euoqdu{; s,11r1tng auoq)dWg 1 ' u o t t l n p o r6gbl der (s)lla)g aassal^loqdu^l g ! Z €! s89 lenxas 'srolda>ataurloq:lrta:e uo ' u r 6 u o' e r u a 1 _ naay; { r o q d u l l g g g zl ' r z g z L ' u o t l e ur u l l a t a px a s llaJJa,utxoloJeOung-o 6131 uotleztpuq^qvNc osl, aas ! LL S ' t L S ' ( t V N Ua)) u a r a J r a l uVtN U 9t8'sllal qleeqs-alpung gg 1 'sdeu uotllejalut ulalold aotsAatatsail,wotDqDoSosp aas 3 6 1 9 ' 6 u r p o l qu r a l s a r r 'JLSS'sluetnur 'sal)^l aA!l)npoJdal ]t99 J€ tt l 6 € S , 0 t S _ 6 € S , 6 u r 1 1 uo r1aqq l n o s 'al)^l uraql.roN llorll ZZ€L 11 9 7 '9 9 7 ' s u t 6 u ou o t l e l t l d a J l 6 € S , 6 t 9 g E S , 6 u r 1 1 o 1 q 'alo^te)na 'SeuerquJaul se 6 € 'L t - 9 t , u . r s l u e 6 l o t€-E€ l a p o u ls e 8€S 'salnqnlor)tul / a t P l sp r o l d r p ' lapoLu sptoldeq 6urttolg lz86 't ,saltpolqdeujeq 'f8 'auoue6 'sJaplosrp ledel VNC 'aujojpui(su-roo1g tz€L t€ [ f96Z sadft tloads oqo aas 295102 18t'auroua6 iE's^ llo'1 6urqtlrnns ad(1-6ur1ey1 osloaas luauloole^ap s r s a u a 6 o r 6 uaya s6 u r l n o t d sp u e u o t l e u l o l suobalaswpqoqtouaD))apun osp aas ad^i llel s l l a )l e r l e q l o p ual a ss l l e l l e r l a q l o p u a JLSy'JqSV'fgn-gg9'uor1e:r,u:ads ' l u a u l d o l a ^ a rpe l n l l a ) t l l n u 'luaurdola^aP roJ tZ€t 0 g t - 6 1 ? ' 6 u l l r l d se A t l e u r a U e 9tt I t g t t L ' u s t u e q r a u6 u r q : u e r q )9 LV SLb' lot1r-suaduo>a6esop l l E ' ( s ) i s e a ^6 u r p p n g ',uosodsueJl 'JSnn 'OSna-SnnJ,slassa^ sauab poolS 1 8t € ts8 JgrV | L /sesnuts eua6 pool€ lggy 1'sutaloldi{rogeln6a.r auoq ut t E E b L ' L E n l ' 9 0 8 ' r a p r o q q s n r 8 I bsbl'Md)ew 'slapiostpIedat ,luatlpd y61 1.,uolleperbapy113 luauoola^ap 156Z vNc uggt 'asrnol'uMorg ' s u u 6 e t u t s t' p s 1 u a 66 eu r 1 1 o ; : - r 1 u e suoba1as4tpqoqtouaoSaasiuatudolanap L0€ L € 6 L L 'srequnu 'lej uMorg uorlPlnoPo)poolg L r €L 888 l l o ) ,])l ,srso]d o d e /elorq)uorg pnpulpu sadfi 8t t [ oslo aas lsEt t ,(uLoleue 'suorDunJ ( a u r p u n ^ x o a p o u o l qn)p l g a a sa u t p u n ^ x o a p o u o r g 791 1;npe l€st L 'gZEa-IZEL 'f6t 'tL€1'suobala srlrpqoqtouaol plaglq6ug lZZt.l stsatooouaHaasuotleullol Egg Ido:so.r>rr.u sadn )tJDadsosp aas 776' olqdoso.tg'luelnurssa/uaaslo-apUg L 'eseurl.roldaterglgg 'JZSVt'tSnL-ZSV ' € Sl€s? t t - r 9 r r ' , s l l epr o o t S g l t t ' p u eu o r l e u t o ;a s d e u { s 196 Lt L 9 r t rPlnlla)erlul z t LL ' s u t e u l o p 7 g g 1 ' a u a 6s s a l q l o a t g €ttt'r!18 'qtMol6 g E11 ' s a 6 u e q rl e u o t t p u r t o ] u o l J80ZL le)ld^l s 9 € L! € 9 € [ ' , e l n r s e l 8 'Jllouluretuop uuaqpPl l€ L! 'i(rolsrqanrl:npotdaruo) 6 Z a t ' 6 Z Z t aluepoul ' 1 g g' s r s X l e uuer a l o r d' l u a u u b rllrea€:5u a n b a s J 6 t I I ' l L E I L 'L t I t - g l t L ' , a r n l ) n l l s luapuadap 15y1g ' 'sl)ajap redar 9 VLL L € , 1 " J - g E l,Ls u o r ] l u n 6j u r ; e u 6 r s ts6z vNC JLgtt'A9ELL9€,1'99€ t'arodolselS 1 8 € LL , l € t t - 9 E t t ' u u a q p e ) _ I l s Lz L ' s a t l t l e u t o u qaeu t o s o t l t o r q l 1 6L EL ' J 9 9 EL , S 9 €[ , a r a u r o ] s e 1 8 '6urleu6rs ,t ,urrapolselg 9711 0 l € , s u t a l o r d z ) e J g / P \ g l 0 € € t € € t _ 0 € t | '1Bt ra)uPl lsealg - 1 8 €t t ' g t L t , s u u a q p e ) o l o j d 6 1 €t t-6LtL,ls/{)olsptg 'zosI t 8 € t ! 1 ltuaooqledoln ,suoll)alul gztL 'uotst^tp al)snut S t t t I g t L t , 1 9 11 , u r a 1 o .or6du r u e 1 3 l€Os t t 3 tuotst^tp l l a ) "/oJ 6 Z ( . 1 ' q Z ZtL0 [ Z t , r a ) u e ] J _ g €[t' 1 € t L , s u t a t o r]de l ZZVI '650 'JSSZ ' S g a ' s r slle) { 1 eleullaptda au ;e> { : ; ; a r 1 8 €t t ' 9 € t t , s u r l a 6 o u r s a p 1 6 5 0t t .rappelS (aurpun^xoepourorq) 1',Sleadal g €LL ' g €t L , s u t l l o l o u l s a p nprg lzz9' az9'sauuqurau llelg ,tlZ ,uotleu]lo, uteLuopuuaqpe) 1ELL 0 I € ' s u t e l o r dz ) q g / L P ] ) g ! 9 L Z L ' J V L |e ]uelp^rg [ ',uraloro l-8€LL,€Zuuaqpel terS L9€L J|VEL,ZnEl,xeldu_rolxproql!g ' u L l I ' I g Et I ' L t LL - 9 t I l ' s u u a q p e l)e ) r s s e l ) u o u EOV t L96'sprotalsoutsselg 3 13 1 ' a l e r a t i ( l 6 o q d s o q d s r g1- g , ,98€ ,leuornau,s^epqll|g 8 8 t t ! 9 € LL ' 9 t ! t ' a n s s rsl n o ^ r a u l o s € ' ( d 8 8 )u r e t o r d6 u r p u r q - l u r oqdr u e . r g l9g€ t t_98€L l 6 € t t ' 6 t t t - 8 € Lt , i a l d r ) u u do j ) l e n , , lzt€ !80€'80€ lloE,uorler6ruq r ruerg L9L'gEL'9EL'd!8 , p u eu o t l e 6 a r 6 aasn s s t t 'urlorS lVtt-1Vl ssalqtuotg L I L 9 L ' L 9t tt8 'Z8t ' s r s aTggy'aua6 u a 6 o q d t o6uu. rr q : u e r g g g 1 1' f i r u r g e6 u r p u r q lZgt L t - L 8 €1 . UodsuerluraloJd:srsot^)ox1osp aas 'saq)le ' ; o r 1 u op: d l S t t t ' 6 € LL - 1 € Lt , u o r s a q pter ; r q d o u o q s?€ L letq)uPtg ) o LL'OLL g g g1 ' a r u e p r n 6l p u o l n e uu t , s a u a 6 t t z 6 t L ' 5 6 €t - L 6 t [ , s d e u r^ r o s u o s , u r e r g 008 ,slaqLueul 'ularord(,,llel ' ^ltue, lggt t uoqs,,)^rn^qlerg i(rolartas-)|1aq1u{sorg 0l€ L L' LgL-6n L'(s)^e/\^qled JOS 'tELL_9€.JL ( 1 r s . r a n 1 p (drd) sutelold uoud osp aas _ t 8 € tL t l E t t strouqlodtgr>adsosloaas /9€t l 9 € t 1 , 9 € t| t , u o r l n q r . j l s r p '868-L6t'(lSg) 86rl lg-tg'sluaulaJtnbat r{6_raua ,saln)alouolleu ^ q l e d o l e q d a r u uar o ; r 6 u o d sa u r n o g €g 'rlb ' , t u a u j )uZo V s sLaeL^ l ] l a l a s sluauralalolelnsu;aasslueula;a{Jepunog Ll t n l|-on l [ ) 0 1 l ' j L l . u o t t r q r q ul )t e q p a a J 'sseullt)tuclt4)'uaJtopfunq lott t t'otll-6€tt'uotsaqpe a^tllalas alang 00St l€8'lolelsaloq) 'l ! 6 € t 'JOVL 't0g 'stssrytadollapptog n5 ustloqelP) 8 8 €t t t t | L ' O b L t ' t u a u r d o l a ^ al e pr n a u t0S L t rlg su : r l a u a 6 o q d r o ua u o g 1 9 €t 1 , 9 € [t , u o r l r e d u o : o ( r q u a lWt'gEEL'1s6y19 1rarord l B - S 9 , r a p r o; e : r 6 o ; o r q 'uo uotssaloxa stauD) )Ut)adsoslDaas J g s V l ' 9 S v L - ; s V1 . 1 : a g aX e t - y , l - 8 € t leluaudole^ap '99V 'uotletueldsuer] ' 9 ? LL t'uuaqpe)_3n $SnL | JLg Lg'16L'staure)pale^tl)e 'arn])nrts 'gsn ,uorsnjsuell l6€ [ [ J99Vt L 99',srsaqtu^sotg g y 1 1' 6 u r 1 e u 6 r s 6SVl-99' L'alo|slla) leuorls L6S-q6S,alue)sauIr! n lotg l - 8 t t t ' 9 € Lt ' u u a q p e r - d sltuJeu^pouJtaql os/Daas J 9 9 nL , Z 9 nL _ $ , t , ^ 6 o l o r s r q ' s a s n u lps o o l q 8 8 € t - 1 8 €Lt ' 9 € tL / u u e q p P ) _ N tgtt _79'79'Adogua stsstooouaHoslDaas o s z L - 6 v z l ' t t t t l _ 8 t It / 9 €t L , u u a q p e ) _ ] g9-99,sltueu^poLulaql 6StL'lorluol ls9 !t9'lernl)nrls 'Ztl /uolalalsol^) utl)e J Z V T L ol lu!l ulualel I J l VSI ' J E S V t ' g g nJ - e S nL ' u o t l ) np o r d Il a ) p o o l q t 8 - S 9 , r a p r o; e r r 6 o ; o l g ,sleadaluteulop 'srsoldode 'xalro) 's;;a:uanup{;tpln)outg uueqpe) z€ Lt 1 1 1I 96€l-s6€ L lensr^ur MOIJe|'ll aUOg u L t L ' I g ELL ' , 9 tt L ' , s u u e q p el el ) r s s e l ) 27.6 ' s u r r q d ap u e s r o l d a r a rq d 3 ' 6 u r ; e u6rs 8€L[-1€rr leuottlalptg '9€ I 1 - S €I t ' u o t s a q p el l a l - l l e l p a l e t p e u j - + z e ] ' z t t ' s t s o r d o d ep l g ' ruoqealprol z n L L ' 6 n 6 , p u eu r u a l e : _ f i EZOL-ZZjI L8V-98t vNUur 8 0 9 t ' p u Pu o l s e ^ ulle l l a l l P q r6vn'6bb-gw lotentl)e aue6 a a 0Stt-€gtt,(s)uuaqpe) t(.0 1-ZZOL,luaudola^ap o1tqdosotg , € € E , u r a l o r dp r o ) r g (aspdlv-+ze))dund Lunt)le)aasdurnd *.e3 ) n t E L ' V | E L! € € € t I , s r s o l d o d,es u r a l o t d g g g utlnpoule) )apunaasutlnpoulet/+ze) J t a t t ' Z Z t 1 . ET (asedfV-+zef) d(xnd unllle) aasase31y*)e] n Z l L - € ( | L ' s t s o l d o d e , s u l a l o(j1du o - g g g 'suralordXrole;n6er aua6 lra6uryu7i91 lggyg alnl)njls utelold oslDaas J LVq' LV8-9n8'slueldt) JteV'ZZn,6urpurq_ypq ',S€ 'JVEL ' JlvS' Lb8-9V8'slueld€J J9€[ I leeqs-e]a8 agl Jr]otr.l 'sutaloJdi{to1e;n6at aua6 pa6url uz) aHZ) f00t I I n g LL ,V 8| L , u e ) ^ l 6 e l e 8 'WV '(6utlal)as-utlnsut) l_00t r sllal-etag JWV L | 'sulalord{ro1e;n6a.r aue6 y1y97pa6uU uZ) Z)Z) sula|old)uDads osl2aas St9'ZEg 'surelordauelquraulsuellJse,sutatold lolleq-elag

)

'aJnl)nrls lolt t vLlL'nede) 'asuodsejssal]s t1?[ € 6I 't ' u o l l e p e r 6 a py \ l l l l E L VL ' EL bL- Z t V 1 ' 6 u r; a p o u r a r t O L bt ' 0L Vt - 6 9 Vt ' ( p r o a l s o p) a U r ) l e r u n 'suratord L 1 ?L l e u 6 r s; o a : r n o ss e 0LVl-69V t'slselqoatso^q uotlalf as 'slsel)oalso^q uotsoJa L/t[ xuleul ' 'luatldo;enap,6uo; Jl LVL LLb I-OLV1 'aeun]Pl olt [ 77y1-y7yy'slutol 0 1 ?t l e r p u o q ) o p u a ' u o t l e u l r o J I L Vl - 6 9 b l ' 8 9 t L ' o l u o t l p r u l o l s u esr l s e l q o JtqJ 6lLt'uor])npordwfl

9:l

X]CNI

'sasnllAollar t€z [ t099' 099-659' 1999'sdrundua^up-dfv 6 S 9 - 8 S 9 ' u O r l n q u l srtepl n l l e l u t a L u u t l s e J g t EL ' S E ZL - V E Z L ' a u ag6u 'auaE sog lgg_?sg,l.rodsuetle^!l)e 7SZJ-EEZL '(ltsobIzotelaq ssol 299-299'(s)utalotdroureJ 9€ZL Jo sezJ-tgzl'sotuolpuls Ja)uP)^lellparaq tsl'(s)lolda)ar o6Ie) '(aua6losselddns lzEz [ JLgL'JOEL'6VL'o6re) 'aseasrp rounl sn aua6o:uo)aAlssa)aisAIueutulop Jeln)se^orpre) 00s [ z E zL - L E ZL ' s a l l l l e L u l o u qa eu o s o u o r q l J898'L98- L98'urdrlorpre] 'saiauo)Jes E € ZL - Z E Z L ' I e s suPo r l e u l o J s u e lrlle ) 920t 'sl)e#e Jnz voEz l'uorlel!Iluop! I [€0 | ' e u p uleuor]elnur oqtoltru nnzL-znza'uolleln 6ar uolleiaJllordlla) l9 tg ' a t t u -ttt u a 6 s u e rul l s l s ^ l e u e Znal-WZL lEgtL'VgVt-EgtL's|p) 'sofuqu.ra 6urdolaneput slsl;eue ztL L'suorlrunrsueraqpe ZyZFWZI 'saua6 ' a l ) s n u) P l p r e ) Itz l-o€z t ;et;l1tr-talue;t l . t 0[ 'Arnt]reasetauola] '9oz 'euouDreJ 1[Z t ntzL t 6lzL-Llzl'sllar uats 6ZZ\-SZZL'sad(t u e 6 e l nu lnoql!M uolpnporoaj LOZL-9OZa'lulellsaJ J S Z ZL ' S Z Zl l r ): r l o q e l a u JNLb'ZLV ) s z z t ' s z z t ' u o r l e nr"l(fe 's(erreor:tu.t ' s l s r .6r u r q 6 r a n n VNC'uo!]ezuetleleqllPln)aloul O € ZL leulsnpur ' u o l l l q l q ull) e l u o l s s o l lEtz t Jo ogzl-6zza'ef ueproAere)ue) roJuorlelrrrluapr 'setuadordalqetueq 'IOLZL'O 'arnsodxa ralJe ra)ue) 90Zt t ZL 1ZZL 'suaDour)ie) 802[ JSLZL'8tZt'ralroslle) pale^lllP-a)ualsalonu y116'sa6ueqrrtlauaEtda g 171 'uor1e;(qraul g 9 71 ' s r s a u a 6 o u r : r e 1 'a:uapuadapuor :urz 'aseprldad,(xoqreS lozJ-gozl'lurerlser lnoq]!M uollezluolol 19 1 'spl)eiqlej'sdnotb f90s'saurllel It t t ;{xoqleS 'aleJ uolsl^lplla) pete;,{xoqrel 602L lt8'u!lolq '1eu6rs ggg1 '6ul1metr11a> re1nl;a)relur sp 'eptxououruoqre) 699 'sa]eruorpear'aserp^que)ruoqre) d n sP o o l q J 9 3 1 ' a : u a P u a d aX p l d'sllal I [9 [ ra)ueJ sszvLnzL JSng'InA-tnA'JL L' ap/.ouorlexU-uoqreJ stDuD )tlDads:(s)uollelnWoslDaas eleqdsoqdsrqasolnqu JnVg'nng'esel^xoqre) l9z l'saloPlaql Puoll!pell $b8' &a-nng'![1'suoll]ear 886 loxel l9r8'sr8'HdcvN '^l!^!lrsuas 'uorlnlo^a 6 !Z L sL8-ZL8 o9EL- LgZl'sardPlaqlMau s{8-€{,8'0t8'eua})eqoue^) '^deraqlor a)uelslsa.l uollnlo^a 'dund zOf 65ZL Jo Ln8-9n8 'surelord:rua6oruo 6urla6relse!poqllue '€1 '(aplxolpuoqrel) zof O9ZL sng-bn? sgzL-9SZa'luaurtearl 8t8-€t8'69'slsPldorolq) 'satuad-oJd 6 tz L J S ' 8 ' S t 8 ' s r s ^ l o r p ^dql v '€[ 'uotlexttuoqle] 6 , l z l 8 1 2 1'ulDlro 69 alzl-IJZJ'sllal ulals Ln8-9V8 'a)uarln)er 'uorlexUuoqre)'dunO aprxorpuoqre) 6 [Z t 1zg3; 'uoltP6edord 'elor ap^) pr)P)ulr] os/Daas 8 tz t lla) urals €,lzl-zJz L'uorl)alaslelnleu 66-86'uortlnpord :r ;oqe1eu.t uorlexuuoqreJaasuorlexu J Z Z ZI ' Z Z Z I , ' l u a u l u o l l A u a o J ) l t l l (zo)) aprxorpuoqtPl s l z L - v L z a ' l : a g a , { } r l r q e } slur tra u a 6 'spuoqelqnop uoqrP)-uoqre) l s s z t ' s s z 1 - o s z[ ' l a ) u e ] l e D e l o l o l l90L 'suolalels g 171 'uorsset6o.rd 190l. 'uaai\^laq spuoq alqnop 7971'Atewud 190[ oEzFnzzL'uollueAaJo ss-ts'sluauoduro) lla) 'alnlel)uauou 902[ l 9 ? ' a r n l ) n r l s) r u o l e '{}tleuou-t uoqrel f6ZZL'VSn e se sre6n5lsapueq::es{1o6 osloaas nzzl-soza'ssalord ^Jeuollnlo^aor)!u '9ZZ 'uorlelllu! uorlexuuoqJel os/Daas | LZZ\ 'salltuno) ueaMlaquollPllPA 'srsaqlu^sorq lSzz I tt8 'seterp^qoqre) t 6 z z l ' 'suouuow tLozL'vsn ss 'g9L'6ur;auun1 gzz[ L9L !99 L eteqdsoqd;i(oueqre3 reln)alou.r'aseqtu(s J 6 O ZL '6 0 Zl ' a 6 e r o u o r l l u n ]s e 'uralord ) n z a L 'v | z L ' s l o p e j l e l u a u l u o r j ^ u a ) u l 969I 7de1 ZESI 'sL!srupqreur eruelsrsalsrsol{:o6eqd'salnsde; ' 6 u r q : 1 e udse l 1 1 91

€r9r

' L 6 nL 'J 6 8 nt ' l 0 S [ ! 6 t t ' r g 7t ' 8 ' L ' ( t e l | ^ () s ) p r s d e f 'tuaulelUutlle 'utalold 6ulddel €00L iln€'gvE'uorDear 7 g g ' 6 u r r a p rV o NUtu-ard 9 t € ' u o ! l r e a re s e l e q d s o q d 'uolr)earaseraJsuerl 9?€ l^qlau 'uorl)earasera;suer1 9t€ ;(uen6 'suoll)u nJ Lnt-gbE 76y'6urdderap uro) 6urpurq-de: Lv€-gv E'xald )9nE 'ItE-gtE 'tt€ 'vNur.llrrlo,te:na ,toEurdde3,9 sluauelu urllp/urllv aassluaurelUurlre ro '6urdde1 srseua6or6uy osloaas JgW L' gbb l'6ulpuno/v\ol esuodsar ! 9 n v L 'L n nL - q V V[ ' s a u e l l r d e J L V | - 7 V E ' ( ) 8 ) )x a l d | . U o6)u r p u r q - d e ] L6ZL'16ZL'uorlelDedeJ l L 9 l ' s u o x eu l aurueJ ]uaura^ourpasPq-elnqnlor)rrJ'snrl^o^rPd ra)ueJ S€ZL-v€Za'fuetrparaq'seLu-orpu^s sauaD pnpwpu !(srsl\ seue6rossarddns ioulnl 1(s)aua60luoi(s)uorlelnwoslDaas gtzL-vtZL 'uo!]elnul sad(1par.ren 'SOt ' uJo r a 1 o r d 7 e uqay6 t-tOt t JnntL'SEZL-rtZt 'puPuralo.rd/aua6 €Sd 90 t t

7 1 7 1 ' a 1 eqr y n o r 6 O Z z l ' ) o p e !6 u ! t ! t u !sl e u a 6 , ( x o € az L-z Lzl'uol])alas lernleu saselselawaasslselselaul saselselawos/Daas 'ssaua^lse^ul 0zz L 'uottePet6aP 6113 5611, I JZJ-SJZa'uoUe!lueraJJrp Ila) a^ll)erap 9 JZI-g LZr'qleap Ila) a^rl)alap ' s a t u a d o r d6 u t l n q t l l u o : € Z ZL 8 0 r 1 - l o I 1 ' u o l l e l a J r l o rl lda l o Iz !'uorssa.r6ordTqrmo:6 t t LL ' s u o t } | s u ellel L U A q ) u' as s, ta u - l e l l a q l l d e ' e 6 u e q :r t l a u a 6 ) ! l a u a 6 ! d a 19171 o E Cl - 6 z z l ' 9 9 7 ' { 6 o l o r u a P t d a J V 6 l l ' V 6 l 1 . ' u o t l e p e r 6 a6p1 1 3 slla) Ja)ue) oslDaat J60el 'gOZa-LOzl'lla) t'uot;ut6tro a;6uts tuelnu ' J Z L Z '\E I Z L - Z l Z l' u o l l n l o ^ el e u o l ) LZZL AlrlPuol) slla) la)ue) aasslla) 1s951)saua6 rosserddnsrounf j(s)aua60)uOos/Daas 60ZI'AOZL'S9Z'suo!lelnur)!lPuros ogz l-nzz L'a lqelua^aro saua6;e:t1tr:-lalueJaassauo6 €azt'80(. t'a6ueq: ltlaua6lda r r a z L ' b z z L ' s l o l l e ,l e l u a u l u o r l ^ u a ltedal VNC oslDaas LLZa-9LZr

' 1 9 6 Z ' 9 6 a - 9 6 2 'L8Z -L L a ' s l r a r a pr r e d e vr N c sua60ullle) aassua6outllel g g 71 ' s t s a u a 6 o q l e d / s a s n e ) ' 1 u e u 6 t 1 e sur tu 6 t u a q tLoz\'gozt 'srsoldode t71 1

uI s! 8ffir'eauro) :"rfff lt"fi

'rln)rleue) ", zth [ 6yr1yrt1:,(1aas3yt1y3 ' ( o s e rul ) 9 1 6 ' l S t 6 ' 9 L 6 - 91 6 uralordluapuadep-urlnpoulel/+ze))esPull-y\Jef a1:i(:uotlex;;-uoqle)aasall^l utAleJ 6EL-g€.L'uorllunJauoredeq)ulolotd'ulln)llaJle) s) e ' u l x a u l e J L gL ' 6 E L - g FL.' u r a l o i de u o r a d e q'ernl)nrts t ' u lltt lnpoulel/+.e) t t6 'bulpulq 0 8 0[ +7eJ ggg '6urpurq ollatapiog asep(> 1 ' s ' l l l(l(uape l s t 6 1 6 a s e u r l - y l e Ju o u o l ] ) e gL6-V16'urlnpoLulef '909-t0S 'sllal ]ueld 'uo!lPurjoJsnllel 899 ' Z ' s l l a )6 6 e ' e n e r vt ur n t t ; e 3 66ZL'JzL6 l6 7 1 5 ' . r a b u a s s ar ue l n l l a l e J l ustn o l ! n b ! q n Z L 6 ' s a s d e u llse I L 6 ' p u ef ) d uollelll)so 915-3 15 "(ruanbel, JZt6'ZL6 Mol slanel+ze) ltlosol^l 6urdaal stustueq:au-t is)prdrloqdsoqd lolrsoulos/paas lo |6' o L6'pelelpau-€dl (s)lauueq)ulnl)lel os/Daas Zt6-01,5 losor^) olut ,(rlua o € 0[ ! 9 t 6 ' J n L 6 ' 9J 6 - n$ ' p u e u l l n p o u r l e ) ' a s e a l a ' 0 1 6 ur n l ) l e ) €[6 z ! l l u'saltds a J ' a ^ e+M zeJ 6 6 Z J ' J ZL 6 ' ZL 6 ' u o ! l e'€ lE t6 t6 +zeJ n J6-Z 16'suol]el[)so+zeJ #. L6''esealai ,zel- pa)npul-+zPl) t l6 t!nllleJ 9 L6-Z J6',bulleuDls lualsalonu a^lllsuas-unl)leJ lz 16'z L6'sJO]€)lpul I 199't99-099'elnllnrls allsnul 099'Uolllelluo) 'utslueq]aur I [99 999'uotlelrtoqdsotldolne '(asedlv-, 199-099 ze)) dund tllnllle] Z 16'slolclalelaulpo-ue^l p la ] e o - t d l [ [ 6 ' 0 t 6 ' s l a u u e q ]e s e a l a r - + z e E L 6 ' Zt 6 ' ( s ) l a u u e q|). U n ! ) l e l EL6_ZL6 '969'769'sesdeur(s 1easea;aliallltrJsuerl gZ )lulseldo)lPs'a6elols L-92 L'LUnln)rlal 6 u r ; e u 6 tusn r ) l P Ja a s6 u ; 1 e u 6 t s 'uottellua:uot losoy(l;o uolleln6ar € L6-zl6 ut uorlellue)uo)Jo uolteln6ar t L6-ZL6';oso1i(: la6uessaullelnlla)erlulse 9 t6-Z 15'luaulalnsPaul ' lelnlla)eJlul JL6S L69-965 ud o t l : u n Id e 6 rla Z gLL - L gLL ' { 1 r ; r q e a u'uorlenttre 66a 6621, 099'suotlPllualuo)l!losot^) 9€t1-S€1t 'uolsaqpe lla)-lla) palelpau-ulloqpel (+ze))uol ulnllle)/Lunl)leJ 069'(s)lauueq)uln!sselodpelPAll)e-uln!)leJ 1 Lne' LVZ'satpoq1efel lb6s' tgg1'ng€-Egt '969-n6S'se1nra1ou.r pa6e3 1969lS6S geil'JnZtt '17g1 Iennqred6ut;eu6rslutr11 'ulalolo esen 'zE[ g g zL ' J z q zL ' , u o ! l e!uu l a l e p x a s 'uorsr]aro zzE[ szEt't-dod 'lPu6r6 s utztre;od € Z €L 'salnuPrD d J t z E t ' n z E L - E ( .t

tt rt,'rrr,'u"",

J*ZE.'ne€t'slla) sw rfizE l'nztL'suolDerelul lla)-llal a^ll)npu! lzz€ t lnp €.zEl' tzE L-Zeil's11artura6 sauabpnPntPut osloaas '\utuDLld (Natd) dod SZEL lo g|tL'(wlaposaw ilow) wow nzE J-€z€ L'])aJJeleulaleur tgz€I ' L z t L - g z t L ' 6 u 1 t u tp1u es a u a 6r r u o t q r o t a l a q _ 'seueD €z€ t 'suralordr0o1e;n6alaua6 f66t t 'sllarrepunoj zz€ [ 95lt-s9tl 'sL!srueq)aLu ue;od ft lelloqllda leseq-orlde 'lle) swl )lnzEl'lvzEl'tz€t g7g1 11ar66a

X]CNI

9:l

INDEX

conformationa l a n g e s6, 5 3 F6, 5 5 ,6 5 5 F ch coupledcarriers,656F couplingto proton gradients,822-823,823F evolution,655 k i n e t i c s6, 5 5 , 6 5 5 F p u m p s ,6 5 6 F light-driven l o c a l i z a t i o n11 , 51 mechanism,655F membranetransport,652-643 reversibi lity, 826-827, 826F specificity,652 seed/soABCtransporter(s); Activetransport; specificproteins Cartilage,1468-1469 b o n e r e p a i r1, 4 7 1 cells,1467, 1468-1469,1469F seeo/soChondrocytes erosionby osteoclasts, 1472-1473 growth, 1468-l 469, 1469F d e f e c t i v ei n a c h o n d r o p l aas,i14 71- 1 4 7 2 , 14 7 2 F m i n e r a l i z a t i o 1n 4, 7 ' l 'models'in b o n e d e v e l o p m e n t1, 4 7 0 ,1 4 7 1 F r e p l a c e m e nbty b o n e ,14 7 0 - 14 71 , 14 7l F C a s e i nk i n a s e1 , 9 4 9 Caspase(s), 1118-1 I 20 a c t i v a t i o n1,I 1 8 ,111 9 F 1 , 120F seeo/soProcaspase(s) caspase-3,1119 caspase-8, 1573F c a s p a s ree c r u i t m e ndt o m a i n ,111 9 h u m a n ,I I l 9 T i n h i b i t i o n1, 1 2 4 - 11 2 5 ,11 2 7 i n t e r l e u k i n - 1 - c o n v e r tei nngz y m e 1 , 11 8 s i g n a l i n gp a t h w a y sI,I l 9 Caspaserecruitmentdomain (CARD), caspases, 1119 C a t a b o l i s m8,8 - 1 0 3 activatedcarriers,79F a n a b o l i s mv s . , 6 7 F definition,66, 88 o x i d a t i o no f o r g a n r cm o l e c u l e s7,0 ,1 0 0 F citricacid cycleseeCitricacid cycle g l y c o l y s isse eG l y c o l y s i s oxidativephosphorylationseeOxidative phosphorylation s u g a r s5, 5 - 5 8 , 8 8 cataboliteactivatorprotein(cAP),418r,420F,436 Catalase,144F,721 Catalysis autocatalysis and origin of life,7F,4O1 catalysts,73, 158-159 seeolso Enzyme(s);Ribozymes i n c o n t r o l l e de n e r g yu s eb y c e l l s , 6 5 - 8 7 energybaftierc,72-73 by RNAseeCatalyticRNA;Ribozymes C a t a l y t iac n t i b o d i e s1,6 0 , I 6 l F CatalyticRNA origin of life,401, 402,4O2F self-replication, 404,407F ribosomes,378-379 spliceosomeactivesite,352 seea/soRibozymes; Self-splicing RNA Catastrophe factors,1080 C a t a s t r o p h ien, d y n a m i ci n s t a b i l i t y1,0 0 3 ,1 0 8 0 Catenin(s) p-Catenin cadherin binding, 949, 1142 c o l o r e c t acla n c e t1 2 5 3 p 1 2 0 - C a t e n i ln1, 4 2 y C a t e n i n( p l a k o g l o b i nc)a, d h e r i nb i n d i n g ,| 1 4 2 c e l l - c e lal d h e s i o na n d i n t r a c e l l u l asri g n a l i n g , 949, 1142,1145 c l a s s i c ac la d h e r i nl i n kt o a c t i nf i l a m e n t s1, 1 4 2 , 1142F C a t i o nd, e f i n i t i o n , 4 T Cation-transportFgATPases seeCalciumpump (Ca2+-ATPase) CouIoboctercresentuS cytoskeletonshape,991 Caveolae,790,790F Caveolin,790 C B C( c a p - b i n d i ncgo m p l e x )3, 4 7 C - C bp l rotein,926 CCR5,HIVreceptor,1505-1506,1505F CD3complex,T cells,1590F,1592f,1599F cD4 protein,1580-1581,1580T,1581F,15921 1599F antigenpresentation,1590 HIVreceptor,1505, 1505F r o l ei n v i r a le n t r yi n t o c e l l s , 7 6 5 F

l:7

CD4T-cell(s), 1580,1592T,1599F negativeselection,1587,1587F positiveselection,I 586, 1587F seeo/soHelperT-cell(s)(TH) cD8 protein,1580-1581,1580T,1581F,r 592T, 15 9 9 F antigenpresentation,1590 CDST-cell(s), 1580,1592T,I 599F negativeselection,1587,1587F p o s i t i v es e l e c t i o,n1 5 8 6 ,1 5 8 7 F seed/soCytotoxicT-cells(Tc) CD9,sperm-egg binding, 1298-1299 cD28, 1590, 1591F,15927 1594,1595F,1597 CD40ligand,1590,1592T, CD40receptorprotein,1590,15927 on B cell,1597,1598f on macrophage,1593F,1594 cD80, 15927 cD86, 15927 Cdc6,288,289F, 1068 Cdc2O, APC/ C regulation, 1064,1066,1066f, 1087, 11 0 0 C d c 2 5p h o s p h a t a s e 10, 6 3 ,1 0 7 1 , 1 0 7 l F Cdc42,1042F,1043,1156,1516 Cdcgenes,1056-l 057 temperature-sensitive mutants,1057,1057F C d h 1 ,A P C / Cr e g u l a t i o n , 1 0 6 4 1 ,0 6 6 T1, 1 0 1 Cdk (cyclin-dependent kinase)seeCyclind e p e n d e n kt i n a s e (sC d k s ) Cdk4,in cancer,1243,1244F cDNA,542 clones,542,543,543F,544 P C Rc l o n i n g 5 , 46F libraries,542,543F,544 synthesis,543F,574 C d t l , O R Cb i n d i n g ,10 6 8 Ced3gene, 1327,1327F Ced4gene, 1327,1327F Ced9gene, 1327,1327F C.elegansseeCoenorhobditiselegons Cell(s) behavior cytoskeletoninvolvement,1025-r050 seea/soCellmotility/movement chemicalcomponents,45-65 c a r b o nc o m p o u n d s5, 4 - 5 5 s m a lm l olecules,55 freeenergy i n f o r m a t i o tnr a n s m i s s i o 8 n, i n o r g a n i c h e m i c asl o u r c e s( l i t h o t r o p h icce l l s ) , 12,13FF l i g h ts o u r c e s( p h o t o t r o p h icce l l s )1,2 f r o m l i v i n go r g a n i s m (so r g a n o t r o p h ci ce l l s ) , 12 genomeseeGenome(s) isolation,50l-517 m i x e ds u s p e n s i o n5s0, 2 s e p a r a t i otne c h n i q u e s5,0 2 ,5 0 3 F5, 0 3 F F originsof life,400-408 polarizationseePolarity/polarization procaryotes, diversity,14-15, 14F,15F 7F self-reproduction by autocatalysis, 11, 0 3 t e r m i n adl i f f e r e n t i a t i o n tree of life a r c h a e a cn e l l s1, 5 bacterialcells,14F,15-16, 15F,25,25F eucaryotic cells,14, 26-32 unit of living matter,1 universalproperties ATPas energycurrency,8-9 store,2,3F D N Aa s h e r e d i t a r iyn f o r m a t i o n g e n ef a m i l i e si n c o m m o n ,2 3 ,2 4 7 p l a s m am e m b r a n e9, - 10 , l 0 F proteinsas catalystsand executivemolecules, 5-6,6F ribosomalmachineryfor proteinsynthesis, 6 - 7, 8 F RNAas intermediaryin informationtransfer,4, 5F s m a l lm o l e c u l e as n d f u n d a m e n t acl h e m i s t r y , 8-9,4s-123 vehiclefor hereditaryinformation,2 seeolso entriesbeginningcell/cellulor;specific components;specifictyPes Celladhesion,1111-1204, 1177f , 508 b a c t e r i ai ln v a s i o no f h o s tc e l l s 1 c e l l - c e lsl e eC e l l - c e lal d h e s i o n c e l l - m a t r i xs e eC e l l - m a t r i ax d h e s i o n s tractionfor cell movement,1040-1041 seealso Celladhesionmolecules(CAMs);Cell

j u n c t i o n ( s )E; x t r a c e l l u l m a ra t r i x( E C M ) C e l la d h e s i o nm o l e c u l e (sC A M i , 1 1 7 7 7 c a d h e r i nsse eC a d h e r i n ( s ) c e l l - c e lal d h e s i o n1,1 7 7 f Cell-celladhesion seea/soCadherin(s); cell-matrixadhesion,1177f seeo/soCell-matrixadhesions;Integrin(s) l C A M s4, 11 , 1 1 4 6 1 , 592r immunoglobulin superfamily,1145,11 46-1 147 i n t e g r i ns u p e r f a m i lsye eI n t e g r i n ( s ) selectins,1145-1146 synapseformation and,1147-1 148 T - c e lflu n c t i o n ,15 7 1 seeolso specifictypes C e l l - c e lal d h e s i o n a s ? y s ,11 3 5 Car+ro|e,1135-1136 c e l la d h e s i o nm o l e c u l e sI ,l 7 7 T c a d h e r i n lsl ,3 3 - 1 1 5 0 , 571 d e n d r i t i cc e l l s 1 i m m u n o g l o b u l i snu p e r f a m i l y1,1 4 5 , 1146-1147 integrins,1134, 1145, 1146 selectins,1145-1146 c e l lo r o l i f e r a t i oann d ,115 3 - 115 5 h e t e r o p h i l ivcs .h o m o p h i l i cb i n d i n g ,11 3 8 F scaffoldproteins,1145,1'l 48-1 149 s e l e c t i va e d h e s i o n1, 1 3 9 - f 1 4 0 ,11 3 9 E11 4 0 F s e l e c t i va e s s o r t m e n It ,1 4 0 - f 1 4 1 , 1 1 4 0 F1, 1 4 1 F , 1142F s i g n a l i n g1,1 4 5 f o r m a t i o na n d ,1 1 4 7 - 11 4 9 , 1 1 4 9 F synapse seeo/soCelljunction(s);Cell-matrixadhesions; matrix(ECM) Extracellular Cell-cellcontact actin polymerizationvia Rac,1047 bone marrow 1458 c a p i l l a r sy p r o u t i n g1,4 4 8 s e eo / s oC e l l - c e lal d h e s i o nC; e l lj u n c t i o n ( s ) Cell coat (glycocalyx\,636,637| Cellcommunication,879-903 adaptation,902,920,920F autocrine,881 budding yeast,mating,880,880F c a r b o nm o n o x i d e , 8 8 9 receptor(s) receptorsseeCell-surface cell-surface c o n t a c td e p e n d e n t8, 8 1 ,8 8 1F Notch receptorprotein seeo/soEphrin(s); differentresponsesin target cell types,885, 885F endocrine,882-883, 882F,883F seeo/soHormones evolution,955 signals,combinatorialactions,884, extracellular 885F gap junctions,884, 884F nitricoxide,887-889, 888F paracrine,881, 882F,883 plants,955-962 speedof response,886-887, 887F seeo/soCelljunction(s);Neurotransmitter(s); Recepto(s);Signaling Signal molecule(s)/pathway(s); transduction;individualsignoling moleculesond pothwoys C e l lc r a w l i n g1, 0 0 6 ,1 0 0 8 , 1 0 3 61, 0 3 6 F activitiesinvolved,1036 l e a d i n ge d g e ,1 0 4 0 F seea/soCellmotility/movement C e l lc u l t u r e , 5 0 f - 5 1 7 anchorage d e p e n d e n c e1,1 75 - 11 76 , 1175 F bacterial,cloningvector production,540,541F c e l l si n c u l t u r e5, 0 4 F definitions,502-504 historicalaspects,504 mammalian 1 ,0 5 9 F c e l lc y c l ea n a l y s i s , 1 0 5 9 ' i m m o r t a l i z e d1j0 5 9 1059,1107 replicativesenescence, plant,504-505,568 primarycultures,504 secondarycultures,504 tissuesegregationin, 1140-1 141,1142F seeolsoCelllines;Tissueculture C e l lc y c l e5, 5 4 F1, 0 5 3 - 1r 1 3 , 1 0 5 4 F an al v si s a n i m a le m b r y o s1, 0 5 7 - 1 0 5 8 ,1 0 5 7 E1 0 5 8 F BrdUlabeling,285,285F,I 059, 1059F 1065 DNA microarrays, flow cytometry,I 059-1060, 1060F

l:8

INDEX

m a m m a l i a nc e l lc u l t u r e1, 0 5 9 ,1 0 5 9 F p r o g r e s s i oann a l y s i s| ,0 5 9 y e a s tm u t a n t s1, 0 5 6 - 1 0 5 7 ,1 0 5 6 F1, 0 5 7 F arrest,1061 a b n o r m a pl r o l i f e r a t i osni g n a l sa n d , 1 1 0 7 - 1 1 0 8 .t 1 08F D N Ad a m a g ea n d ,3 0 3 - 3 0 4 1 , 10 5 - l 10 6 , 1106F G op h a s e ,| 1 0 3 m a l eg a m e t e s1, 2 8 1 c a n c e rc e l l s1, 1 0 7 - l 1 0 8 checkpointsseeCellcyclecontrol chromosomechanges,208-209,208F,209-210, 209F controlsystemsseeCellcyclecontrol n u c l e o l acr h a n g e s3, 6 3 ,3 6 3 F overview,1054-1 060 phases,208F,285F,1054-1055, 1054F,1055F Ge(Gzero),488-489,489, 1103 elF-2regulation,488-489 G 1p h a s e2, 8 5 F1, 0 5 5 ,l 1 0 O - 1 r 0 1 , 11 0 0 F m i t o g e na c t i o n s1, 1 0 3 G 2p h a s e2, 8 5 E1 0 5 5 interphase,208F,209,1055 M phase,208F,285F, 1054,1071 cytokinesisseeCytokinesis mitosisseeMitosis seed/5oMeiosis S phase(DNAsynthesis), 285F,1067-1O7'l c h r o m a t i np r o t e i np r o d u c t i o n1,0 7 4 chromosomd e u p l i c a t i o n1,0 5 4 ,10 6 7 , 1068F,1059-1070 DNA replication,284,285,1067-1 069 histonesynthesis,289-290 l a b e l l e dc e l l s 1 , 059F m e i o t i c1 . 0 9 0 .1 2 7 2 s i s t e cr h r o m a t i dc o h e s i o n1,0 7 0 - l 0 7 1 t i m i n g ,2 8 4 , 2 8 5 seeo/soDNA replication universalcharacteristics, 1053 s t a r t( r e s t r i c t i o np)o i n t ,1 0 5 5 ,1 0 6 1 ,1 0 6 6 ,11 0 5 '1056 timing, seea/soCelldivision;Cellgrowth;Cell proliferation Cellcycle conrrol,l77-178, 1050-1067, 1066F a n al y s i s a n i m a le m b r y o s1, 0 5 7 - f 0 5 8 , 1 0 5 7 F1, 0 5 8 F i m p o r t a n c e1, 0 5 3 m a m m a l i a nc e l lc u l t u r e1, 0 5 9 ,1 0 5 9 F y e a s tm u t a n t s1, 0 5 6 - 1 0 5 7 ,1 0 5 6 F1, 0 5 7 F cancerand, 1216-1217,1243,1244F c h e c k p o i n t s5,0 5 ,10 6 1 d e f e c t sm a k i n gc a n c e rc e l l sv u l n e r a b l e , 1216-1217 DNA damage,303,I 1O5-1107 G2lMcheckpoint, 1061, 1062,1066, 1105 m e i o t i c1 , 281 m e t a p h a s e - t o - a n a p h tarsaen s i t i o n1,0 6 1 , 1066,1071 s p i n d l ea s s e m b l cy h e c k p o i n t1, 0 8 8 ,1 0 8 8 F start (restrictionpoint), 1055, 1O6t, j066, 11 0 5 cyclicalproteolysis,1064, I 065F,10667 APC/CseeAnaphasepromoting complex (APC/c) SCFenzymecomplex,1064, 1065F e u c a r y o t isci m i l a r i t i e 1 s0, 5 6 f u n c t i o n sI,0 6 0 - 10 6 1 i n t r a c e l l u l at rri g g e r i n go f c e l l - c y c leev e n t sD , NA replication,1057-1 059, 1068F m o l e c u l a r / b i o c h e m i cs a wli t c h e s1,0 6 1 , 1065-1066, 1075 regulatoryproteins,10667 CdksseeCyclin-dependent kinases(CDKs) c y c l i n ss e eC y c l i n ( s ) E 2 Fp r o t e i n s1, 1 0 3 - 11 0 5 inhibitoryphosphorylation,1063-1064 p53and.1105 R b p r o t e i n sI, 1 0 4 - 11 0 5 ,I l 0 4 F u b i q u i t i nl i g a s e s e eU b i q u i t i nl i g a s e ( s ) seeolso specificproteins resetting,I 069 a st i m e r / c l o c k1, 0 6 0 ,1 0 6 1F t r a n s c r i p t i o n rael g u l a t i o nl ,0 6 5 , I 1 0 4 ,11 0 4 F C e l ld e a t h apoptotic5eeApoptosis(programmedcell death) c e l ln u m b e ra n d ,I 10 2 defectivein cancer,1215-1216 n e u r o n s1,3 8 9 - 13 9 0

seealso specifictypes Celldetermination,1311-1312,1312F combinatorialcontrol,465-466 Celldifferentiation. 411. 454-477 cancerand, 1215-1216 p r o c e s s e4s1, 2 common differingresponseto extracellularsignals,415, 464 DNA rearrangement, bacterialphasevariation, 454-455,455f genomeconstancy,411-412, 413F patternsof gene expression, 412, 464-465,464F, 465F seeo/soCombinatorialcontrol proteindifferences, 412 s p e c i a l i z a t i o4n1,2 t e r m i n a l1, 1 0 3 seeo/soDevelopmentalgenetics;Gene expressionregulation Celldiversification, role of Notch,1362 C e l ld i v i s i o n1. 0 5 3 .1 0 5 5 F asymmetric,1099, 1099F C.elegansembryo,1323-l 324, 1323F oocytes,I 289-1290, 1289F plant development,1400 cell death balance,1102 c e l ln u m b e ra n d ,110 2 control,11Ol -1 112 d e n s i t y - d e p e n d e(ncto n t a citn h i b i t i o n )1, 11 0 , 1110F DNA damageresponse,1 105-1 107 mitogensseeMitogen(s) seeolso soecificfactors coordinatedgrowth and division,1 t O8-1110, 1109F cytoskeletalrole,966-967,967F delay,I 103 density-dependent inhibition(contact i n h i b i t i o n )1,11 0 ,1 2 3 3 - 1 2 3 4 l i m i t s .1 0 5 9 .l l O T density-dependent, I 110, 1233-1234 seeo/soReplicative cell senescence plane of, 1095-1097 plantcell(s),1195 s t e mc e l l s1, 4 2 5 t o t a lc e l lm a s sc o n t r o l 1 , 1I 1 - 1 11 2 seeolsoCellcycle;Cellgrowth;Cellproliferation; Cytokinesis; Meiosis;Mitosis Leil oocrflne.5/9

Cellextractsihomogenates), 510, 5 12 Cellfate determinants,asymmetriccell division, 1099 Cellfractionation,510-51 2 cell-freesystemsseeCell-freesystems c e i lr v s t s . 5 tu chromatography, 5 12-5 I 4 electrophoresis, 5 I 7, 518E 521-522 m a c r o m o l e c u l e / o r g a n eslel ep a r a t i o n5,10 - 5 1 1 , 5 11 F m i t o c h o n d r i a8,l 7 F u l t r a c e n t r i f u g a t i o5nl ,0 - 5 1 1 , 5 10 F ,5 I I F ,5 12 F seed/io Proteinanalysis Cell-freesystems b i o l o g i c apl r o c e s sr e c o n s t r u c t i o5n1, 1 - 5 1 2 ,5 1 6 c e l lc y c l ea n a l y s i s1,0 5 8 ,10 5 8 F cellfractionation,5 16 vesiculartransport study,752,7 52F Cellgrowth, I 053 anchoragedependence,1175-1176,1175F c o n t r o ll ,l O l - 1 1 1 2 c o o r d i n a t eg d r o w t ha n d d i v i s i o n1, 1 0 8 - l t 1 0 , 1109F organ growth, 1108 o r g a n i s mg r o w t h ,1 1 0 8 seealsoCellcycle;Celldivision;Cellproliferation C e l lh o m o g e n a t e ( s5)1, 0 ,5 1 2 C e l lj u n c t i o n ( s )1,13 1 - 1 2 0 4 a n c h o r i n gj u n c t i o n s1, 1 3 2 ,11 3 2 F1, 1 3 3 - 1 1 5 0 , 1133T,11357 c e l l - c e l l1, 1 3 2 ,1 1 3 3 - 11 5 0 ,11 3 3 T1, 1 3 5 7 adherensjunction seeAdherensjunction(s) desmosomeseeDesmosome(s) i m m u n o g l o b u l i snu p e r f a m i l y1,14 5 , 1146-1147 selectins,1145-1f46 s e l e c t i va e d h e s i o n1, | 3 9 - 1 I 4 0 , 11 3 9 F , 11 4 0 F s e l e c t i va es s o r t m e n1 t ,1 4 0 - 11 4 1 ,I 1 4 0 F , 1141F,1142F s i g n a l i n g1,1 4 5

seed/soCadherin(s) c e l l - m a t r i x ,113 3 1 11 3 5 1 1 1 6 9 - 1 1 7 8 a c t i n - l i n k e d1,1 3 3 T1, 1 3 4 ,1I 3 5 7 focaladhesions,llT0 hemidesmosomes seeHemidesmosome(s) seed/solntegrin(s) i n t e r m e d i a tfei l a m e n ta t t a c h m e n t1,1 3 3 7 seed/soCell-celladhesion;Cell-matrix adhesions junctions,1132, 1132F,1133T, channel-torming 1158-1154 g a pj u n c t i o nr e eG a pj u n c t i o n ( s ) p l a s m o d e s m a t1a1, 5 8 ,I r 6 2 - 1 1 5 3 ,11 6 3 F e p i t h e l i a l1,13 3 - 113 5 seed/soEDithelia t u n c t i o n acl l a s s i t i c a t i o1nI ,3 2 ,11 3 3 7 h o m o p h i l i cy s .h e t e r o p h i l i cI ,1 3 7 ,11 3 8 F o c c l u d i n gj u n c t i o n s1, 1 3 2 ,11 3 2 F1, 1 3 3 T ,

rr50-rr58

s e p t a t e1, 1 5 4 - 1I 5 5 ,11 5 4 F t i g h tj u n c t i o ns e eT i g h tj u n c t i o n ( s ) j ugn c t i o n s1, 1 3 2 ,11 3 2 FI, l 3 3 T signal-relayin chemicalsynapseseeChemicalsynapse(s) i m m u n o l o g i c asly n a p s eI,1 3 2 t r a n s m e m b r a nsei g n a l i n g1,1 3 2 ,I I 3 3 7 transmembraneadhesionproteins,1134-1135, 1134F,11357 cadherinsuperfamily,f 133-1 I 50 i m m u n o g l o b u l i snu p e r f a m i l y1,14 5 , 1146-1147 integrinsuperfamily,1134, 1145, 1146, 1169-1178 selectins,1145-1146 seea/soCelladhesionmolecules(CAMs); specific types seeo/soCelladhesion;Extracellular matrix(ECM); specific types C e l ll i n e s eucaryotic,505,5067 hybridcells,509F seeo/soHybridomas immortal,504F,505,5061 1059 primaryys.secondary, 504 transformed.505.5067 seeo/soCellculture Cell-matrixadhesions,11331 1134,1134F,1135f, 1169-1178 a c t i n - l i n k e dI ,l 3 3 l 11 3 4 ,11 3 5 7 C A M S1, 1 7 7 7 f i b r o n e c t i na n d ,11 9 1 hemidesmosomes seeHemidesmosome(s) seea/soCell-celladhesion;Celljunction(s); Extracellular matrix (ECM);Integrin(s) Cell-mediatedimmune responses, 1540, 1540-1 55r, 1540F,1559-1 589 i n t r a c e l l u l apra t h o g e n s1,5 7 2 transplantationreactions,1575 seealsoMHC(majorhistocompatibility complex);T cell(s); T cell recepto(s) Cellmemory,454,458,458F,466 seeo/soCelldifferentiation C e l lm i g r a t i o n developmental,1140, 1140F,1373-1375 ECMdegradation , 1193,1194,1194F externalsignals/guidance molecules,1045, 1140 gut epithelialcells,from cryptsto villi, I 440F I n l e o n n s a n oI .t / u - t t / l n e u i o n a l1, 3 8 5 F seeo/soCellmotility/movement Cellmotility/movement i n a n i m a ld e v e l o p m e n t1.3 6 3 - 1 3 7 8 seeolso Cellmigration;Development c o n t r i b u t i o no f m y o s i nl l , 1 0 3 9 F c r a w l i n gs e eC e l lc r a w l i n g microscopy,533 protrusion,1037-1038, 1039F traction,1040-1041. 1041F via actin polymerization,1037-1039 C e l ln u m b e r ,1 1 0 2 C e l lp l a t e ,1 0 9 7 Cellproliferation,1053 abnormalsignals,cell cyclearrest,I 107-1 I 08 anchoragedependence,1'l7 5-1176, 1175F cancercells,11O7-1 1Oa,12'17 DNAtumor virus proteins,1248,1249F seea/soCancer coordinatedgrowth and division,1I 08-1 1I 0 d e n s i t y - d e p e n d einnth i b i t i o n1, 11 0 ,111 0 F integrinsand, 1175-1176 l i m i t a t i o nt,e l o m e r el e n g t h ,2 9 3

INDEX

l:9

requirements,1244,1245F scaffoldproteinsand junctionalcomplexes, 1153-1155 t o t a l c e l lm a s sc o n t r o l 1 , 1 11 - 1 11 2 seeolsoCellcycle;Celldivision;Cellgrowth; Replicative cell senescence Cellrenewaland turnover epidermis,1417-1428 in liver,1443 mammarygland,1426-1428 smallintestine,1436-1438,1436F,1439 seed/soRegeneration; Stemcell(s) C e l ls e n e s c e n c e m a c r o p h a g sec a v e n g i n g7,8 7 replicativeseeReplicative cell senescence telomereshortening,293 Cellsignaling,879-974 all-or-noneresponse,901 generalprinciples,879-903 seeolsoCellcommunication;Signaling molecule(s)/pathway(s); Signal transduction C e l ls i z ec. o n t r o l .11 0 9 - 111 0 Cell-surfacerecepto(s),891-895, 893E 894F,936F enzyme-linked, 956,958F G-proteinlinkedseeG protein-coupledreceptors (GPCRS) intracellularreceptorsys.,881,881F i o n - c h a n n e l - l i n k seedel o n c h a n n e l ( s ) pathway(Tollfamily),1530 in NFrt2l

molecularweight determination,522-523 sedimentationcoefficient,511, 522 centriole(s),993,993F,1076, 1076F replication,1078, 1078F zygote,130l, 1301F Centromere(s),22A-23O chromatinstructure,231-233, 231F heterochromatin, 228-229,232F histones,H3 CENP-A variant,230-231,232F m e m o r yc i r c u i t s2, 3 1 ,2 3 3 F seea/soChromatin chromosomereplication,209-210,210,210F, 1076,1076F DNA sequence,210, 229-230 plasticity,229-230, 230F structure,229-230, 229F Centrosome.1076, 1076F center-seeking behavior,996F composition,992 d u p l i c a t i o na n d s p i n d l ea s s e m b l y1,0 7 8 ,1 0 7 8 F ,

1079 m a t u r a t i o n1, 0 7 9 microtubulesemanatingfrom, 992-996, 993F reorientation,in cell locomotion,1046 'search and capture'ofchromosomes,1082, 1084F Ceramide,biosynthesis, 744-745 Cerebralcortex,1386F h o m u n c u l u s1,3 9 1 n e u r o n am l i g r a t i o nI, 3 8 5 F somatosensory region,1392F Cervicalcancer.1211 -1212. 1211F Cesa(cellulosesynthase)genes,1199-1200 C e s i u mc h l o r i d eg r a d i e n t s5,11 CG (CpG)islands,434, 47O-471, 1527 D N Ad a m a g e3, 0 0 - 3 0 14, 7 0 evolution.434.470 r o l ei n i n n a t ei m m u n i t , 1 5 3 0 CGNseeCisGolgi network (CGN) C h 4e l e m e n t s3, l 8 T C h a g a s ' d i s e a s1e 5 ,0 9 C h a i n - t e r m i n a t i nngu c l e o t i d e sD,N As e q u e n c i n g , 550 ju nctions,1132, 1132F,11331, Channel-forming 1158-1154 seeolso specifictypes Channelprotein(s),652-653 conformationa ch l a n g e s6, 5 3 F passivetransport,653,654F seealso lon channel(s);specific types Chaoerones b a c t e r i aG l, roEL,390F 1 5 ,71 6 F , 71 7 e u c a r y o t i c3,8 8 - 3 9 0 3, 9 0 F , 7 m i t o c h o n d r i aplr o t e i ni m p o r t ,7 15 , 716 - 7 17 , 716F proteinfolding role,130-131,388-390 seeolso soecificmolecules Charcot-Marie-Tooth disease,1048 CheA,943,944,944F Checkpointsin cell-cycleseeunderCellcycle control Chemicalbiology,527 Chemicalbonds,46-50, 48E 106F 46-47 electroninteractions, e n e r g yc a r r i e r s6,1 ,6 9 F (adenosine triphosphate) seeo/soATP , 07F C h e m i c agl r o u p s 1 Chemicalreactions,free energy,75F Chemicalsynapse(s), 682,684 acetylcholinereceptors,684 c e l ls i g n a l i n g8, 8 2 excitatory,6S4 inhibitory,5S4 mechanism o f a c t i o n6 , 83F s e ea / s oN e u r o m u s c u ljaurn c t i o n( N M J ) ; Neurotransmitter(s) C h e m i o s m o t icco u p l i n g8, 13 - 8 1 4 ,8 1 4 F ATPproduction,817-819, 819F seealso AfP synthesis bacterial.839-840. 839F seed/soElectrontransportchain(s) C h e m o k i n e ( s1)5, 3 3 - 1 5 3 41, 5 5 0 - 1 5 5 1 ,1 5 5 0 F in inflammatoryresponse,1453-1454 p r o t e o g l y c a nasn d ,11 8 3 receptor,HIVbinding,765F C h e m o r e o u l s r o1n1, 4 0 C h e m o t a x i s . 1 0 4 I51, 4 0 bacterial,941-945, 943F,945F growth cone guidance,1388, I 388F neutrophils,1045,1045F Chiasmaformation, 1274, 1274F,1276,1276F C h i c ke m b r y o l i m bd e v e l o p m e n1t ,3 1 2 - 1 3 1 31 ,3 1 3 F1,3 5 5 seea/soLimb buds (vertebrate) neuraldevelooment,1384F s o m i t e s1, 3 7 2 F Chickenpoxvirus,1516-1517 Chimeras,mouse,1380, I 380F,1381F Chimericproteins,transcriptionactivatorproteins, M2F 247, Chimpanzee(s), evolutionaryrelationships, 247F,248F C h k l p r o t e i nk i n a s el, 1 0 5 ,11 0 6 F C h k 2p r o t e i nk i n a s e1, 1 0 5 ,11 0 6 F Chlamydiapneumonlde,I 500, 1500F Chlamydiotrochomatis,1511F,1512F,1513 Chlamydomonos, flagella,1032, 1033 Chloramphenicol, 384,3857 C h l o r i d ec h a n n e l s6,6 6 ,6 73 , 6 74 F Chlorophyll(s),848F

photochemistry,847-848, 848F,849-850, 849F, 850F Photosystem(s) seeo/soPhotosynthesis; seea/soChloroplast(s) Chloroplast(s), 30F,840-855 bacterialresemblance, 857,863 l roteins,635 I barrep biogenesis,856,856F,867 biosvntheticreactions,855 cell-freesystems,511 development,698,699F d u r i n gc y t o k i n e s i s1,0 9 8 distribution electrontransportseePhotosyntheticelectron transportchain(s) 842 energyinterconversions, evolutionaryorigin,29, 31F,840-841,859-860, 874,875F endosymbionthypothesis,859-860,863-864 maintenance, 868-870 organelle-nucleargene transfer,859-860 function,696 geneticsystem,868-870, 869F genome,855-870 c o p yn u m b e r , 8 5 8 diversity,8571 859 evolution.859-860, 868-870 genes,863,864F gene transfer,864 higher plants, 863-a64, 864F introns,863 liverwort,864F maternalinheritance,866, 866F mutants,867 replication,35S variegationand,866,866F seeo/soNon-Mendelianinheritance glycolysis, 854-855 growth and division,857-858 l i p i ds y n t h e s i s , 8 6 7 mitochondria vs.,842-843, 843F proteins,867 tissue-specific nuclear-encoded photosynthesis seePhotosynthesis proteinimport, 7 19-7 20, 720F proteinsynthesis , 856-857,856F,869F starchgranules,94F,95,841,842F structure,713F,842-843, 842F,843F transport,854-855 seeo/soChlorophyll(s) Chloroquine,P/osmodiumfalciparum resistance, 666 in enteroendocrinecells,1437 Cholecystokinin, Choleratoxin, 629,906,1492,1493,1504 Cholera,transmission,1491 Cholesterol 83F,743,744-7 45,791 biosynthesis, membranes,626 02 ,3 structure,620,620F,62OFF transportseeLow-densitylipoproteins(LDLs) C h o l i n e1, 1 4 F ECMproduction,1179 Chondroblasts, Chondrocytes,1468-1469, 1469F 1187 Chondrodysplasias, Chondroitinsulfate.1179, 1388 Chondroma,definition,1206 definition,I 206 Chondrosarcoma, Chordates,1370 Chordin.940.1336 ChromatidsseeSisterchromatid(s) Chromatin,202,365F 290 chromatinassemblyfactors(CAFs), 243,286,288,1070 condensation, seeo/soChromosomecondensation; Heterochromatin euchromatin,220 heterochromatinseeHeterochromatin 431-432, 432F immunoprecipitation, nuclear sites,239-240,239F,240F,241F packing,243,244F remodelingseeChromatinremodeling structureseeChromatinstructure seeolsoChromosomestructure;Genome(s); Nucleosome(s) 290 Chromatinassemblyfactors(CAFs), Chromatinremodefing, 215-216,M2-443,443F -228 "barriersequencesi227 'tode-writer enzymei 226-227, 227F,228F compfexes,215, 215F,343,344 histones,216,216F,432,433F 216, 432,433F nucleosomes, positioneffectvariegation,226-227

l : 10

INDEX

RNAinterference(RNAi),443 R N Ap o l y m e r a s e ( s ) , 4 3 3 t r a n s c r i p t i o n ar e l ,p r e s s oprr o t e i n - m e d i a t e4d4, 5 , 446F Chromatinstructure,211 -21A,21 1 -2'19 3 0 nm f i b e r ,2 11, 2 1l F ,2 1 2 F , 2 1 6 - 2 1 82,1 7 F2, 1 8 F , 222 seed/soHistone(s); Nucleosome(s) " b e a d so n a s t r i n g , " 211, 2 1 1 F , 2 1 2 F centromericseeCentromere(s) direct inheritance, 230-234,232F , 069-1070 d u p l i c a t i o nd u r i n gS p h a s e 1 effecton replicationtiming, 285-286 epigenetics,4T2 h i s t o r i c aal s D e c t s2.2 0 inheritance,473-476 seed/soHeterochromatin i nterphase,loops, 234-236, 235F mitotic chromosomes, 243,243F,245 regulation,2l9-233 s e ed / s oC h r o m a t i nr e m o d e l i n gC; h r o m o s o m e structure Chromatography, 512-51 4, 5 13F,534F seed/soProteinpurification;specifictypes Chromocenter, Drosophilopolytenechromosomes, 237F L n r o m o K t n e S t nt u S,,/ / Chromomeres,234,235F Chromoplast(s), development,699F C h r o m o s o m aclr o s s i n g - o v se er eH o m o l o g o u s r e c o m b i n a t i o(nc r o s s i n q - o v e r ) C h r o m o s o m ai nl s t a b i l i t y1,2 17 seed/io Geneticinstabilitv Chromosome(s), 195-l 96, 2O2-2O5,554F a b e r r a nst e eC h r o m o s o m ae b n o r m a l i t i e s analysis, 195F, 196, 196F,202-203, 203F,203FF, 237FF,285F,534,535E590F seeolso specifictechniques a u t o s o m e s1,2 7 1 . 1 2 8 4 F bacterial,202,282,283F,1491F b i o l o g i c af lu n c t i o n s2, 0 4 evolution.207,208F gene content,204-205,204F seea/soGenome(s) historicalresearch,195-196, 196 h o m o l o g o u s e eH o m o l o g o u cs h r o m o s o m e s (homologs) human,202,203F,203FF chromosom3 e e v o l u t i o n2, 0 8 F c h r o m o s o m e12 t r a n s l o c a t i o 2 n0 , 4F chromosome22, 205F.206f evolution,207,208F g e n eo r g a n i z a t i o n2,0 5 F mousevs, 249-250,249F r e p l i c a t i o on r i g i n s2, 8 7 - 2 8 8 replicationrate,283 s e ed / s oH u m a no e n o m e lampbrushchromolomes,234,234F,1288 mitotic seeMitotic chromosome(s) p a c k a g i n g1,0 6 9 DNA,202-21 8. 202-219 post-mitoticchromosomes,1090 seed/soChromatin;Chromosome condensation;Chromosomestructure polytenechromosomesseePolytene chromosome(s) pufts, 220-222, 239, 239F seec/soPolytenechromosome(s) r e a r r a n g e m e n tIs2,3 1 seed/soChromosomeabnormalities; specific reorrongements r e p l i c a t i o sne eC h r o m o s o m ree p l i c a t i o n sexchromosomesseeSexchromosome(s) speciesdifferences, 204 -205,205F structureseeChromosomestructure seed/soCytogenetics; Karyotype;individuol cnromosomes C h r o m o s o maeb n o r m a l i t r e s analysisseeCytogenetics; Karyotype cancer,12 l 5F,1231,1254F meioticerrors,127A-1279 seealso specifictypes C h r o m o s o mb e a n d s2, 0 2 2 O 32, O 3 F F2,3 7 F F Chromosomecondensation,243,244F,107SF A T Ph y d r o l y s i s , 2 4 3 cell cyclevariation, 208-209,208F,209 chromatincondensation,1070 chromatinpacking,243,244F c o n d e n s i nsse eC o n d e n s i n ( s ) M - C d kr o l e ,1 0 7 1

X-chromosomeinactivationseeX-inactivation seed/soMitotic chromosome(s) C h r o m o s o m de e l e t i o n cancer role, 235F,1234-1236, 1236F genomeevolution,246-247 C h r o m o s o m de u p l i c a t i o n c e n t r o s o md e u p l i c a t i o nv s , 1 0 7 8 S phaseof cell cycle,1054 c h r o m a t i nd u p l i c a t i o n1,0 6 9 - 10 7 0 regulation o f 10 6 7 ,10 6 8 F Chromosome puffs, 220-222, 239, 239F seeo/soPolytenechromosome(s) Chromosomereplication,208-209,209-210,209F, 210F centromere,209-210, 210, 21OF chromatincondensationand timing, 285-286 seeo/soChromosomecondensation controlseeCellcyclecontrol d u p l i c a t i o nd u r i n gS p h a s eo f c e l lc y c l e ,I 0 5 4 r e p l i c a t i o on r i g i n ,2 0 9 ,2 1 0 F segregationduring seechromosome segregation sisterchromatidsseeSisterchromatid(s) s t r u c t u r acl h a n g e sn e e d e d 1 , 067 t e l o m e r e2, 1 0 , 2 1 0 F seea/soTelomere(s) yeast,2 I 0 seea/soCellcycle;DNA replication;DNA s y n t h e s i sM; i t o s i s Chromosomesegregation ( h o m o l o g o u cs h r o m o s o m e s ) , meiotic 1276-1278 failure(nondisjunction), 1236F,1278-1279 mitotic,865,865F,1089-1 090, 1089F seeo/soMeiosis;Mitosis;Sisterchromatid(s) Chromosomestructure,554F celI cycle changes, 208-209, 208F,209, 243F seeolsoCellcycle;lnterphasechromosome(s); Mitotic chromosome(s) centromereseeCentromere(s) c h r o m a t i ns e eC h r o m a t i n DNA packaging, 202-21 8, 202-219 g l o b a l( h i g h e ro r d e r )2, 3 3 - 2 4 5 chromatin5eeChromatin condensationseeChromosomecondensation loops, 234-236, 234F,235F polytenyseePolytenechromosome(s) l i n e a r2, 0 9 - 210 , 2 0 9 F , 2 1 0 F replicationand,209,210F,1067 seeo/soChromosomereplication;Replication origin(s) telomereseeTelomere(s) X-inactivationseeX-inactivation C h r o m o s o mter a n s l o c a t i o 5 n2 , 8F c a n c e r o l e ,12 6 1 P h i l a d e l p h icah r o m o s o m e i n C M L ,1 2 0 8 , 1208F,1261, 1261F,1262F translocationactivatingMyc gene,1239 c h r o m o s o m e1 2 a n d , 2 O 4 F D N Ar e p a i rh, o m o l o g o u sr e c o m b i n a t i o n3 ,0 9 genomeevolution,246-247 C h r o n i cm y e l o g e n o ulse u k e m i a( C M L )1, 2 0 8 ,1 2 0 8 F , 1210,1218,1261,1261F,1262F Chymotrypsin,138F,144F C i l i a 1, 0 3 1 - 1 0 3 4 basab l o d i e s 1. 0 3 3 ,1 0 3 3 F o f e p i t h e l i acl e l la p i c a dl o m a i n ,8 0 6 f l a g e l l ac o m p a r i s o n1,0 3 1 in left-rightasymmet(y,1376F,1377 microtubule a r r a n g e m e n1 t ,0 3 2 F m o t i l i t y1, 0 3 1 , 1 0 3 1 F polarity,of beatingin respiratorytract, 1435F p r i m a r y1, 0 3 4 C i l i a r y( a x o n e m adl )y n e i n ,1 0 3 1 - 1 0 3 21, 0 3 2 F1, 0 3 3 F hereditarydefects,1033 Ciliatedcells,respiratorytract, 1434-1436 C i l i a t e s2,8 F Circadianclocks,460-452, 461F,462F CisGolgi network (CGN)(intermediate compartment),771F,772, 778F C i s t e r n aml a t u r a t i o nm o d e l ,o f G o l g i t r a n s p o r7t ,7 8 Citratesynthase,122F Citricacid,98, 98F,122F Citricacid cycle,97-99, 1O2F,817 e l e c t r o ng e n e r a t i o n8,l 7 pathway,98F,122-123FF,122F,123F Clamploader,274, 275F,276F C l a s s i c ag le n e t i c s e eG e n e t i c s , c l a s s i c a l C l a s s w i t c h i n g1, 5 6 7 - 15 6 8 B cell activation,482-483

V(D)Jrecombinationvs.,1568 Clathrin coat(s),751,754-755,755F,756F pit(s);Clathrin-coated seed/soClathrin-coated vesicle(s) pit(s), 743F,790F Clathrin-coated LDLendocytosis,791-792, 791F,793F pi nocytosis,789-7 90, 789F C l a t h rni - c o a t e dv e s i c l e ( s7)5, 1, 7 5 4 , 7 5 4 F , 7 5 5 F , 758F a d a p t i n7, 5 6 F cargo receptors,7 54-755 formation,7 55-7 57, 756F pi nching-off, 7 54-7 55, 7 56F,758F regulation,T95 structure,T55F vesiculartraffic,754F Clathrin,structure,7 54, 7 55F C l a u d i n s1 ,1 5 3 CLAVATA 1,957F Clavatalprotein,1410,141oF CLAVATA 3,956,957F C l a v a t ap 3 r o t e i n ,1 4 1 0 ,1 4 1 0 F Cleavageand polyadenylationspecificityfactor (cPSF3 ) ,s 7 - 3 s 8 ,3 s 7 F Cleavagefurrow, 1093, 1093F Cleavagestimulationfactor F (CstF), polyadenylation, 357-358, 357F, 482-483 Clonalanergy,1548,1548F seeo/solmmunologicaltolerance C l o n adl e l e t i o n1, 5 4 8 ,15 4 8 F s e ed / s ol m m u n o l o g i c at ol l e r a n c e C l o n ael x p a n s i o n1,5 4 6 C l o n a il n a c t i v a t i o n1,5 4 8 ,1 5 4 8 F Clonalselectiontheory adaptiveimmunity,1544, 1545F C l o n i n g5, 0 7 - 5 0 8 D N Ac l o n i n gs e eD N Ac l o n i n g reproductiveseeReproductivecloning therapeutic,507F,508 vectors(DNA)seeunderDNA cloning Cloverleafstructure,tRNAs,368,368F C l u s t ear n a l y s iosf g e n ee x p r e s s i o, 5n 7 5 , 5 7 5 F CLV1(CLAVATA 1),shoot meristem,957F cLV3 (CLAVATA 3), 9s6, 9s7F Coactivatot(s\, 445,447F Coatcoloration,maternaleftects,474, 474F seed/soX-inactivation Coatedvesicle(s), 7 51, 7 54 formation ARF-proteins, 759 coat-recruitment GTPases, 7 59-760 Sarlprotein,759 vesicu l a rt r a n s p o r t , 7 5,l7 5 4 , 7 5 4 F seealso specifictypes Coat-recruitment GTPase(s), 7 5a-760 seed/soGTP-bindingproteins(GTPases) Coaxialstack,RNAstructure,403F Cocci,1490F Cockayne's syndrome,300 Code readercom plex, 225-226, 225F " C o d i n gp r o b l e m i ' 3 6 7 Codons,6, 367,367F,368F CondidaCUGcodon, 383 initiationcodon,367F,380,489-491 mitochondrialgenome,861-862 redundancy,246-247 s r o pc o o o n s3, o / t , 3 6 1 synonymous,247 wobble,369,369F seea/soAnticodon(s) CoenzymeA (CoA),83-84, 83F,84f,117F seed/soAcetyl CoA Coenzyme Q ,8 3 1 ,8 3 2 F8, 3 5 Coenzymes,167,167f seeolso specifictypes Cofilin(actindepolymerizingfactor),994F,1001, 10 0 2 F l a m e l l i p o d i a1,0 3 8 F nucleotidehydrolysis,1002 Cohesin(s), 1O7O-1O71, 1087, 1272 N - t e r m i n adl e g r a d a t i o n3,9 5 structure,I 070F Coiled-coilmotit 135, 135F,145,349F C o i l i nn, u c l e a rl o c a l i z a t i o 3 n6 , 5F Co-immunoprecipitati4 o5 n8 , ,5 2 3 Colchicine(colcemid),987,988f, 1021 C o l l a g e n ( s1)1,3 1 ,118 4 - 1 1 8 6 ,11 8 6 T1, 4 6 7 i n b o n e ,1 4 6 9 degradation,1194

uosupourof/aleouro) supau 54 :alqel e ol JaJalf e q]!M slaqutnu eDeo 1(;ant1n:asuot molloj leq] saJn6!j ot ll ue qltM /arn6r; e ol la1a.rI ue qllM sJaqurnu ebed :filua aql ]o uotssn)stp ya1 tofeu e o] laral a)erploq ur sraqulnu a6e6

g L6/aselalsatpoqdsoqd slPU6rs fuolelnurrlsof 6yr19-:r1:i(1 lduor l)elle auerquaur J 6 Z SL '6 Z 5l ' x a'sllal ,ONol asuodser poolq par ur suorsal 1888'1BB L 9 6 ' , u r a l o lzol P l s o ] l6zs L 'stuauodrJo)atel JL t 6' gl6- LL6'srolda)arotoqd I l6g'.roloa)aJ l6zs t /lauueq) ' u o l l e ^ r l ) eD l 1[6 € 9 ? ' u o l l e l n b aul o l s s a l d xaau a 6 *ep pale6-6y19->r1:(> rlsS [ '688 '€9t 'los!uo) ( d W 9 ) )d W 9 ) t ) ^ f gZSt 'slueuodulol ^Uea 1688 8 0 6 ' ( l U f ) l u e u a l a a s u o d s adr W V - ) r l ) ^ f L,ape)se) lE08'(HlfV) urdorlolruo) J S Z St ' 6 Z SL _ q Z S 'oo€ '662 'uorl)ear p>{1 JLO6'906'saseralsarpoqdsoqd 6yr1y->r 100€L I ! 6 a s L - 8 Z S L ' e tpf u eq € ) le)ruo) /uorle^rDe aueJqueur'(s)lerol J L O 69' 0 6 - s O 6 ' u o r 1 e p e . rp6uaeps r s a q l u [ s L V 9 ' 6 u r u o r l r l . r e d J g n g ' 8ZS L €) '806'V)d uorle;n6al 'SaSnr^PuoroJ r lrd e p e 1806 ro 1 9 6 5 t ' 9 6 5 1 ' Z S S t - L S S t ' A l r u naunr u l96t L '5zs 'uorle^rl)e 906-S06'utaloid D'uor1e;n6a.r r q w L 'g r n l ' s r s e u a 6 0 r 6 u e ' i o r r n t t-8zs I l et a u r o ) lSzs 'i{q uorlenrl>e 'ulatsls auaO azrel/\laasuro) luar,ua;duol ZIS t 'slauueq)1696 'xa1du.ro: uorle: pa1e6-6y1y-tr1ti(: (suort)pralururatord)^lr^rleradoo)oslDaas .roldalar-ol6urpurq-luar,ua;d r-uo1 / [6 9691. ,uotl)e utxol '6SV-8gn'asn-LSn 'JLVb'S??'(s)iossardarof gss 'lgzs 'slseluotleluaualouroJ €0st JSSV lPual)eq 'rolda)ar-o] I t S S t l - 0 8 9 t ' t 8 S t - 0 8 9 t ' l l af ) lSzs'uorleluauralduro) )||rx, iLo6'rso6' 6o6-so6'rLt t'iltigrl 1 6 9 Z0 ' 9 Z - 6 5 Z ' u o r l n aa u o u e 6 VNCr aasVNC {te1uaualduo3 'ajrl ayuv ' u o r l e r lJoer^A a q u r n u^ d o ) u o t l ) n p o l d al re n x a s J9 L Jo aarl ul 8SZ !LLZL'ZLZI-JLZI'pue 0'g tlZS,srsaqlulsoroqd JN6EI 'n6t S LA-ELa'a:u etrlu6rs{teuorlnlona rLsL'tsL')6sL'rbsL'rrr'(')t"toi:"i1""T."i:r":33 L-€68L'r(roluralrrldeu,(sroJsuoxeuaaMleq eual)eqoupAJ uorlrlaouo) ! h s L ' n 9 L ' ! s 1 , ( s ) e l ) r s a ^p a l e o ) - l d o f 'urqol6ouaq y g g ' 6 u r p u r qa s e p r x o auolqloy{>,aplue,{3 s l u a u r u e o u r ol )e l n l l a ) p l l ual a 5l e l n l l e l e i l u l J99Z'9SZ 'zs€ 's)srp r6eur ^J uorlelrldar f L g S 'L g S ' a l u a ) s a . l o ngU 9 l z ' j v L a ' L Z ' \ t o ! E t s € ] € s € t I v N c ;eu ;o g(; uorle;nbatlrJolsollVoslDaas JL9S'LSS'a:ua>saton;; toz-s69'llal lo uo!lPzrletueurueduo) '90s ',918 ',ru)xf '99€ '(s)luaurlreduol l90s t r-90s L r J SL t 1 nL l ' , t E L L ' LVt - E L L ' , s u o r l r s ul uear tl s o l l e t-z9t l. lzs€ L suosodsuetl(;uo-VNCos/paas (suorllerelururelord)iQrrrletadool L V ( ' u o r l e a i fa a J l) t l e u a 6 0 l l q d ) L V Z ' ' u o r l r s o d s u eal ll s e d - p u e - i n ] 't '6urluudloo;:r1aua6o;,{qd l 0 Z € ! 6 L€ l S t S ' l . L S ' 6 u r u r eulrsa l o r d ' a n laqr s s e u o o ) I I €t €t '89€ 'uotsualxa /asnou s^ ueurnq arntln) a a ss l l a ), a J n l l n J luaOla^uo) I 169€t l6t. ' u r o t o r d( t f )l lsonl l d n l t a l u sr n l t q n ) srsaurloy{3aas6ur-rap1:e.r1uo1 JZS6'196 t g r ( ' L n z ' a a z u e o u l qs) / u e u r n q sluaureluurl)e/ur])v os/Daa5 a^rleteduol l Z 6 t t ' a 6 e q d o u a l r e qO X f f ogz-gvz' Loz'zz's)truoua6 '100 'salpunqalrl)erluoJ -y111 1 7 6 9; ' u o r 1 e l n 6 a1t ; a r - 1 ' u r a r oyr d ll00 t L-900L t 6 t z L '6 t z l ' u o r l e ) r r r l u e pl lra ) r n o u r n l ' ( H g f )u o t l e z t p u q ^lqt u r o u a 6a ^ t l p J e d u r o ) z 6 sl - l 6 s L ' 6 u r p u r q1 9 ' y y 1 1 1 J b € z l ' E E z !l 0 t t t ' 0 t . t L ' u o r r r q r ql u) er l u o ) '0t 'a)ueprn6 zbt-wt lletuo) 88€t-1.8€t IL t lZ-0[Z'6ur6e)ireV d N C ' o r r eu r ot]leduo) '11 a s e r a u A ; oV d N U ' ( C D )u r e u t o pl e u t u l l a l - J uralordrolda)al q)loN 1(s)uuqdloslpaas ) 6 L E t '6L EL , u o r l ) e d u r o J ',auoq lOlt'ularord lflf l)eouo] I L 8 g 't 8 9 ' 6 u r ; e u b rrsu a p u a d a p - 1 r e 1 u o 3 J01tL srsol^)oxloslDaas lolsalue ^leuotlnlo^euoululoJ €8V-ZgV 298 ' '009 ,{emqled,{ro}el:asa^unlt}suoJ ' S € .L-S € . ' ( il o l l e J u o r l e l n u l l sa 6 e n e a p3l t s 3 ! L S Eg I t0g 8[SI plo] uor.ur.uo) ' u r e q r( n e a q6 1' u r e u l o p 'uralotdssalalnssil.uruo] ^qdel60llels^il ^et-y aas(er-y luplsuoJ t t 1 0 9 9 ! 6 S 9 1 69g 'uorllala 'gg€ '(s)uoxe p a ^ r a s u o J z 1 9 -t t 9 L O Z ' ^ u a l u ^ s t J S O Z ' lg8€ t lelnsstu.lruo] 'uorlsnqiuol (qder6o11e}stu; f g e E 'g Z E - g Z t ' s u o t l e l t l d d>pr u a 6 s u e t l l0z8 9 7 9 ' s u l a l o t d ' u o r l e z r ; ; e 1 s { . r ; J S Z 1j'7 a t ' t Z € ' l u a u l a 6 ue i r e a rV N Ca l q r s r a ^ a l s6n-b6v /9€tt'aurlsalur 'ursrueq)au '(VNUlur) JtWl't9t7l 9ZE-SZE V N U o r ) r u r ' u o r 1 e ; n 6 a ro l e u t q u r o ) loJluo)aua6 l e u ' s u e q rl lae6t u u sei ol , ss11dd{{tr31 '9 :r;r:ads-alrsaArlen resuo) l g z St nZE LE'uo!leurquro)ai ;o St ' s u r e q ir( p o q r l u e ' u o t l e ) t r t€s9t a uotreAtasuo) po l e u t q u l o ) t96'aLuorqlold^lf l^et u 'SS€ ' 7 g g' s l e u 6 r6s u r > r 1 dt rsl d ( r 3 ifueuorlnlon3aasft euollnlone'uotle^iasuo) ISS€ !79g 1 O S V ' O S V - 6 nu bo 't l e r 6 a l urta l o u o J d 'surprd,(r3 ' 7E71 $ f f 6 V € ' s l e u 6 r6su r ) r l d s ttszv'#zn'szn-tzt'uotlezuar!tpoJaleq '^dolsol)tLri 'salts uorltalao,(l1 0 L9 1le1sasetau{1odylg lZt€ J6rb' OSV-gbb'eua5ary o1tqdosotg tLilL'gstL l g € € ' 6 € E 9 5 5 ' s l u a u r a ;taa l o u r o r d l f L n n ' L b b ' u o r l e u r ox; a ; d u o t '^]uelod 'SgV-VgV',uorlerluaraJJr squ.rn.rl p Ila) lpseq-o)rde;ellaqtlda,xaldu-rot J L S t 'L S E ' s ; e u b u r so r l e ; , ( u a p e , { 1 o d JSgn s a ) u o n o aS s nsuasuo) O € G ' s e s e udr lV h lu a a m l a qu o r l u a n a r d , ) l e ] - s s o l ] 9 9 ? - S g t ' u o r l P u r u t a l alpl a ) g a sa 6 u e q : x ap u p l l s - s s o l ) s u o r l t u n (f e p r 1 ; o a l 0 9 l l ' 1 9 1 1 - 6 9 1l ' s u o x e u u o ) 9 Z t ' l o r l u o )l P u o l e u t q u l o J 'uorleluasatd ,uorleluasatd ua6rlue ssotl ?8SI 0 6 e t! 0 9 t 1 ' t 9 l 1 - 6 9 t1 ' s u r x r u u o ) L 9LL - O st t ' t g . t ' e ! l a q t r drap u u n l o 1 '92-urxeuuo) (rano-6utssoll) t 9 t t I Z S €L ' uorleutqurolal sno6o;or.uo1-i aastano-6ursso.r; erlaqlrolos/Daas 9 S ZL - V S Z l ' a S ZL - O S L Z ' u o r s s e r 6 o rudr s d e l s (wJl) xr.ileultelnlleleilxl os/, aas J L 6 t . 'l 6 t ' s l u a u l e l ue l a q - s s o r f I ' s s z L ' s u o l l e l n ujlo a ) u e n b a s ) s s a '156Z 'LLZ'sDe]€p)tedarq)]eurstu tossatda:epqup'l osl, aas 0 L v l - 8 9 v t ' a ) u a n u u ru o r l e r l u a r a J Jl lrap) 'SZL ' l l a n ' J U L ' Z t ' r o s s a r d aorr ] 'xutPLurelnlla)ellxa tBSV'ggv-LSb L s6 L t-gl I I 992[ ^rlauaooraleq 'pouad g t b I ' L S e l ' € s z L - L s z l ' s a l l t l e u r o u q e) r l a u e 6 ( s ) u a 6 e l l oa) a su e b e ; ; o r 9 6 9I 1e:r1rr1 , l ) i l ) ' 6 u r u a e r l sp u e u o t D a l e p sadft ilD pnpt^tpu! osp aas 9 6 L" ) . H s D U P l l Z9Zl-lsaL ' 'sed(t 86F.-L6t'(C f)) aspasrpqo)er-lplajzinarf llgtL LgV| lzgE I'SSZI-O9Z ['la)ue] le]leloloJ , g 9 S - L 9 S , u a 1 sx{ so 1 7 a r 1 'uotletlualaJJtp WnL'gbt t ra)ue) aas ra)ue) uoloJ 0Lv l-89b | le])arolo) 'n 'slla) ^poqtlue',suoxauorbarI JJ€.951'299t'saua6 JLgbt LVL-Lgt L 909'^dolsotltuu l orl:a;a'p1o6 leptollof ',saseuaoellof e u r u r elle s e ga a sp u e e u t u e l l e s e q 1 8 0 6 ' 8 0 6 ' ( d 8 l )u r a r o r d6 u r p u r q - 9 3 9 3 t6 L| 1 8 0 6 ' 8 0 6 ' ( u ! a l o r6du r p u r q - 3 g 1g13 g 3 t g L LL ' J Z tl | , L €I [ , a n s s t ta ^ t ] ) a u u o ) t 9 8 r L ' S 8 rr ' S 9 rr 1 1 A Xa d ^ l t 5 6 Z ' s l ) a r a pI e d a i v N C ' f t r l e u r o u q el e l t u a 6 u o ] 1 9 8 tt ' S 8 r r ' i l A Xa d ^ l 'uorlpzrrerads lselqo,rr,"r.r'1J.f&oqd aurlearl ?€?-€€t'rossardaiupqdoldfit 68tt's8t r'ilx ad^i 'ly )ll)^f) lU) 806'ouauralaasuodsardU\JV lgob'lol'sewtzoqt) 1 9 9 11 a d ( l ' e r 1 6 u ei 6 g 6 t ' J L 6 E' ( d i d )u r a l o l du o u d ( t o s u e sl e t u p l ) 1 1 6 8 r[ ' 6 8 1 L ' S 8Lt ' X ta d ^ l '3875g€E- l 9 t ' ( r o t r e J lls€ JZLI f98tt'981t'ilAad^l 'tI llDrI:ads uolie;i(uape,{1od pue a6enealt)1563 t ' ( u o r l r q r q ul )r p q p a a l )P p q p a e l a n r l e 6 a u 198tt'Aad^t 1 1 9 y ' u o r s s a r daxuaa 6u t q o l g - d , l d f 6 L 0t - 9 t 0 L ' ( s ) u r a l o rrdo l o r . u t-98[ L ' ( s ) j a u u e qu)o l 's8L spuelsr(Ddf) 93 aass;rlou 9d3 1899 t ' 8 9 1L ' J L g L LL' g t t - 9 9 1 t ' S 9 rr h t a d ^ i sad/fi L l I ' Z L l J Z J O L I J 'suu6elul l . 8 LL ' t 9 8 tt , i l t e d ^ l ' r o l ) e 1u o r l p 6 u o l en f - l l ,r/rads :uotle)utpoulleuotlelsuet]lsod osp aas L L E ' l t g t ' t 8 L - O gt 6 9 V L ' L gLt ' f 9 8 rr ' i l a d ^ r 'Zgl-9gl 'sutalold ',suuaqpe) l_9gt Jo uorlelutpoulluale^ol 8 ELI l 8 L I l _ 9 8 L1 ' 9 8 rr ' t a d l r sadll pnp^!put osp aas uorleln6ar)ualsollVaasuorle;n6at)ualsolle 6 8LL ' 8 8 tt - 1 8 [ t ' q ] 6 u e r l sa l r s u a ] '29- 'ralsar ,saprldadord poqdsoqd sa6ueq)leuotleuloJuo) 19 9311. ' 1 9Jt6 ' s p u o qa p r r p , i q u e o q d s o q d l€6 )269'f L65'Z6S-06S'{dorsot:ru.r L Al l ' 9 81L , u a 6 e 1 1 o > o . r d le)oJuo) ,sar6laua 'ado)soJ)rrlr p u o q gg[[,suteq)?o_ord 196 I t65 leloJuo) ,18 ,sluqr;ua6e11o: aleqdsoqd J Z tV I ' Z t b L ' ( s r o l d a ) a . l o l o qedu o ) )s a u o J t 1. 188t t _ ' 'JLSS' 'aAtltsues-atnleradu.ta] gg I L 'stsaqlu^s r g t " s s p u o ql t u o l tLS}l LSOI LSS l g z s ' u o t l e l n u ll e u o t l t p u o ) J l . gLL ' g g l l ' u o t t e l u t p o u l; e u o r l e ; s u e t 1 - 1 s o d g'[ 'utalordsno.lqu St O t ' J S V Z ' J n n €e n' Z , ( s ) u r s u a p u o J i € 5 ' 6 ? - 8 1 ' q r 6 u a r l sP u o q 179'99'ste6ns J 6 8 tt ' 6 8 1t ' 1 t 8 L L ' L g t l .t8t ! 5 8 t t ' S 8 t t ' s l ! . i q ! J lts'8t'rll6ual puoq 158'18-t8,(uorlPzuaLU^lod t't9v L'JnnL,arnl)nlls l s 8 Lt 'suorletntU alnlalouor)eu) suotlfeai uo!lesuapuoJ 1 9 0t I t s ' J ) s ' 0 9 - 8 V ' L V ' s p u o ql u a l e ^ o f 18 t t 6 u r l d n o tu o t ] l e a ua a ss u o t l l p a jp a l d n o 3 uotlesuapuof 98tt-s8l |'uortnto^a gtg L 'uotleltl)e arl)oqdul^l aurosourolqJaassaulosouloiq)'uotlesuapuo) 98[t-t8il'saua6 i s l l e l) t l u p u a p u o r t e l l u auf o ) L 1 SI 559'luarper6 6 8 1L - 8 8 tt ' s p a l e t f o s s e - l D q t , ',Slrerl pue6rlgy61 :suralotd7g oslo aas xalouroJ E9S 198tt'68t 1-t81 I lellilqr] aldLuof J VLL ' SL L - €L, L ' ( s ) a p u e q ) l e s o 6 rxl o 6 8 L L _ 8 8 t "[ s r e l l u q U 881'alor'srsolIlo6Pqd 1 6 8 tt ' 6 8 t 1 - 2 8 1t ' S 8 t t ' p a l e D o s s e - l u q U

"

LLSl'tLSt !ZtS L

''*';fY"f1""ffi13

' s l l a r6 ul r€l u6asrs a r d - u a 6 r 1 u e

I t:l

X]CNI

/uralold6oqa6papyasa6 nu{qt ut uot})alas l l S s L ' 9 8 9 l - s 8 s 1 ' s'sl)aJJa 056 /t-uoraJralul uotleloepvaasuotlezt]tsuasac z6s L ) t L s L 'EL s a - Z L g [ ' u t s r u e q ) a6uu t 1 1;t11a r L L bt ' l g l v L ' 8 [ t [ ' s r u r e c 9 8 9 t ! t 8 5 t ' t 8 5 t - O g S t ' a l o rr o l d a ) a r - o8l C J 6l L[,aie]lns uPleuJlac ^s^s1lar-1 'aellrded tadlaq vSsl lZZtt leurac ua6!lue l€gst'zg9l-lgsl'uotteluasard J 6 6 Z! 8 6 2 ! L 6 Z ' 8 6 ? L 6 7 . ' 9 6 7 ' u o r l e u u n d a 6 '999'{dotsorlrr! uot}lala ,p;ar; qrdaq S L S l - VL Sl ' ( H f ) s l l a ) - Ir a d l a q ' u o r l e ^ r l ) e 969 1o (s)aseleqdsoqd utalold oslDaas zSsL-t8st'ELgL-ZISJ , t 9 S t _ O 9 S,LS € S t, ( r l ) s l l a r - fl x o r o i ^ f J 9L l ' 9 L L ' u o r 1 e ; ( r o q d s o q d a 6 'J9 'asoqu,{xoaq Il69',aurnlo^ || )ZEt ' 3 5 9 - 4 9 9 ' u o t l e 1 n 6g ad t zgg-Lgg _ ' 9 9 2 ' ( s d l N p s) a l e q d s o q d ual p J89Z tsoallnuoqu^xoac 969'losol^) nloJ)tW sluauodwe it|Dadsj(s)alnq vNC aa5pr)P)ralrnuoqu^Xoac 'I 'uorte6nJutua: 1(s)luaurelu alerpa[uJaluI islueurelu l u a r p e t 6{ 1 r s u a 6 Z Z S1 Z L 9 t S (s)ellrsa^fuolar)aSaassal)tsaAeJo)-esueC lelala)tsol^JistuaurelUurlle/urtrv.os/Daas 596',^lrlrqPls Lgn 'uorlel^qleLu /saseraJsueru^qlaur o ouaO JL b g '9 V 9 ' ( u o l e l a l s o l ^a) u e i q u e u r )u u l ) a d s VNC ' t € S ' ( U l f ) s r o l d eVNC ] a ra l r l - l l o f EZOI-ZZOL'uorlezrlelolvNU 9 € S[ t € t 0 1 - I t o l ' s u r a l o l oo q u l 6 s [ ' u o r t e u r u r l al l a ) - fa ^ r D e a r - J l a s s96'seluadord Z ; v L 'L B! ' s r s o y { : o 6 e q d LlsJ-t ls L'uorlez!l!]nua6oqled I LSL'9F.5l's.rolda>atuorgru6o:aruralled (s)uralordiolow aassutaloldlolou-t 'suraloldloqlue lelnlla)-Pllul Zt LL t 0 L l L ' o L LL ' l u a u l q l e l l eu u D a l u l o 6 EL ' LL 9L ' u o r s s o l d x a 'ior^eqaq ' u o r l e )r H n tW eu 6urruengut oso l-szo f 11a: us [ 'tt /slueLuelu 'rt6st 'Jan9 'zvs 'suorl)unJ t It L lel!leq v6st L | ',uoll)unj 'uorlnqulsrp 996 Lls | glg 1 'suralordfuo1e1nurls-o> V n O t ' 6 9 6 ' ^ r u e l orde l n l l a ) ' t € t ' l u a u r q ) e l lueo r l t u n [ 1 ; a : / s a l n ) a l o Luuo r s a q p e I Z V L 1 ' Z V L t t'€so l1s t '92 'uoiala>lsolll ' , u 9 I ' , ulol ar )l e- l^l ar l)) e l-s95 ls96 L 6 5[ - 0 6 5 L 'ZLSI-aLSL 'ZV9L'EESL'slla) )rtupuac j(s)]uauielr, (s)elnqnlorlrw rLL9t '6urleu6rsorneu lsluauelU url)P/url)V os/Daas alerpeu.ualul 919 'salrlatxorq)lolsllunqns €00t-200 t 1 8 8 9 ' u o r l e l n d u r ol ef u o l n a u 'uorlezrlq-els 'uorlelueuoalnqnlor)rur 200t- t00 t 6t0 I 'uo!lezlueDlo '1.8€ 800L-s00t tl8€ L L - 9 8 t| 1 0 9 0 t ' L r j L ' , ( s ) a r u p u a o 'ulelold 'uollernleuaC u ro l l e e l l n u e;n6a Or Ol - Z 5 G ' u o r 1'raL6'palebuola 0t t ^e/.^qted 916 'ror^eqeq 't rruieu^p 6urleu6rsq)loN :urelordrolda)al q)loN os/Daas t -200 t €00 66-996 1'6ur>1ut1-ssotr 8 0 0t - 9 0 0t ' 1 9 6 '€00 llSsE t llt6' Lv6-9b6'uo't lqur leralel '6utdde: 'luaudola^ap allsUqAtosuasoltqdosot6 t l€00 L GS€I y7Ey'o(qwa Apeasuobala'1 696-99G1or^eqaq ut]:v osp aas uorlezttaur(1od uraloroellac ^ e M q l e d6 u r 1 e u 6 qr s) t o N a a s6 u r ; e u 6 rqs) l o N - e l l a 6 JL6-OL6'sllunqns (9V) 9 elle6 a6ueq:,(6rauaaarl aas(9V) eUaC 166-S95'Alqulasse-Jlas ' u o r l r p p e '9u r e q lg l l u n q n sl o a l e r /16 uSl'iolda)er lla)-f uotlalapaulosoLUolqJ aasleurosourorq)'suorlalac r l e )anl u 2 6 6 r' 8 L 6 ' E l 6 ' u o's)llaull €/6 s e a I ' s a p a s s eu>o r l a l a C 0 l S ' s r s a u a 6 e l n tl u 1886'686-t86'sa6ueqP > axPur-6nrP 6 8 9 ' ( s ) l a u u e q+)) p a ^ e l a c t!l tsuotpee'tuorleua6orplqa6 uolle|.UroJ/^lqulasse eL L ' '!!Vg LVg'sluauPlUleialalsol^) ESOl-596 1 9 6 € ' ( s ) u r a l o J d ' s 1 e u 6or sl e p e r 6 a 6 z € € ' a j n l ) n J l sv N U E E SL '9 Z SI - S Z SL ' s u r s u e ] a C srsol^loxl os/Daas J L O E ' r 8 6 aJ'L 6 Z ' 9 6 2 ' u o l l e u r u r e e p 'OOgXPMqrPd y g y ' 6 u r 1 p a1 - o r 3 tlnejec I t0g 3 9 6 g ' 6 u u t e d - a s_e q l _ t 8 LL 3 Z 8 tt ' , Z 8 t - L 8 t L ' , u u o ) e c f L 1 € 'L 6 L! 9 [ t ' a u r s o r ^ ) 367'6urdde:a6 'saseurlaursot(]:ruseldol() 9f6 l € s €L '€S€ 'St€ '(ddq) a ) u elllaqul u r e l o r d r r 6 a l d e l u a d e : a 6 I ! S € €t L aasa)ueltraquttttuseldol,tl uerlapuay!-uoN ggg g 'aua6 (dd1) )o4dDtuadDa] 'sutaulp :rulseldol^) ; g91" J j e t I ' L z a L - o z a t ' s r s o l d o d e ' ( s ) l o l d a )qalre a c l g 6 Z L ' v 6 at ' t O 6 ZL '0 6 Zt ' s a 6 p t t q> r u r s e l d o v ( ; t l0ztt'0zLt'srsoloooe '(fSlC) osP aas sluauodwo )!]Dads:losol() x a l d L u o ) q l e a 6 6 u r 1 e u 6 6 r s u o n p u r ' s l l a rr u e l du l s p u e l l s qleap lla) aar (sllelJo)qleac lz81 '(LUn^o) 664 l8Zt t 0 € - 0 0 €! 8 6 2 ' L u s r u e q l a usrl s a u a 6 e l n u 'aursoiltl{qtau-9 srseutloy{3aas;o uotstnlP 91; 9 6 9 'f SL ' SL - n L ' u r s e l d o Y { ] ) n g n 'n S nj 6 9 E ' 6 9 € ' u o r l ) n p o i da u r s o u r '00€'uotlrnpo'tdau!qluexod,iq g E S ' u o r l r q r q uuro l l e ) o l s u e J l t I t0€ ' 1 0 € - 0 0 €' a l o ru o t t e l ^ q l e u r ulalo.rdI ssplr)HW'(AW)) snrtnole6atlov(3 I t0€ VNC 'sasnrtl 'sl:ara :tillol{f ',96Z',uorleurureac 96t L Jo JLOE1L6Z 'suor]elnuJ 'ssaujeec srsolrnios/Daas [9 I I 9Z-urxauuo) '6uttltl s 6 0[ 176tr'76y'Ae>ap VNUr.!pelerpau-uor1e;(uapea6 auelquleu eurseld S08'tuaua6.relua lz6v '76p' Ae>ap J 8 6 0L t l 6 0 t VNUr! tuapuadapur-uorte1,(uapea6 'stue;d 'g61a-t61l 'Bg€ 'uralordiolda)ar raq6rqur alor 1se;dou6e.rqd t )JC 188€t 8 6 0t ' u o r t n q l r l s l pa l l a u e b r o 96Zl-S6aL 'uoltelnbarsrsaua6oleurads' ualotd./aua6zoO )160t'J9601 '960 'lapouruorlelnulllsalpulos lellua) t [€s-0€s'saseqe]ec p o u ru o r l e l n u r l sl P r l s e 1 9 6 0t ' 9 6 0 t ' l e'lapour Egg Ido:so.ortu plarrye6 uorlexelailPllse )9601'1601-960| l 1 8 S ' e ) u a r s a r o n lud v c 'slla) 2601-s60t lZ6?'eu^zu eN V C ' , u o r l r u u a' upo l l e c u r u o r s r ^ r p a u e l dp u Pe l p u l o s) l l o l l u l l e u r u e J o 9' 't8 'saue6a41-{11e67i(11eq !v601'n6ol ApoqprLu tt8 t t L1. '{lquasse alts 'riiuelod 'snosq)Do 1691 ;o 85 LL 1|a:reue;d l s 6 o L ' 9 6 0 1 - ? 6 0 [ ' a l o rv o q u 199y'eua6(punseDp))DO )qt u e u ^ P 9 9 6 ' r o t ^ e q a'Alqujasse 't60 t-€60 [ It60 t urso^lAlsluaLueluu!])e/ulllv os/Daas t60 l-€60 I 'sroleJauaba)Jojsp utso^u pue ull)e ll '6uueltpetluol 39661 '€60 'morn; a6enea;: L

d,fl,ffl:llfzffi

o

(s)lla)-ISclr os/Daas 'sllar palralur-snlr^ I t89 t 'sluauuadxaralsuerl J L8St

l€60 t

'660I 'r1l1auu,(se 1660t J E 6 0t 'IO!t-Z6Ot ! E l . O Lt S S O t ' 9 S 0 t ' ' 9 0 t ' s t s a u t l o t ^ 1 j(s)urlnalJalul otD aas sa\)alow 2tJDeds ',slle) 289[ I 'asuodsarftoleuruPlJul t€S t-€€9 t 'slla))rlupuap €65 t-Z6g t !t t' t 'papo)ua-leit^'slstuo6elue t€9 t (s)au!)ol^) lnOZ' ilEoz' #Oe' Eoz-zoz's:rlaua6ol,{) 9ZZL 'uolle^rl)eua6out:ter'aseplxo 0St-d aulolqlol^) w€8'Vt8'l€€8'arnDnrls ygg'6uruosrod l € € 8 ' t € 8 - Z € 8 ' u o r p n'UerleurLueur p a ru a 6 , { x o tE8 t€8'ralua) Jeddor-uorl 'arnleu '€8 le!luasse aLuorqloll1 3ggg'7gg'xaldtrol eseptxo'arnl)nrls ltt | 'aleJuollelnul s9z J Z Z L ITL Z L L , t Zt t ' g t I t ' s r s o ] d o d e l€€B , aLuorq)ol^) l9t8'ernllnrls 'alrr(r-6 9Eg-9gg g E g ' 6 u r d u n du o g o r d L r - qa t u o r q : o l { l } l € € g ' Z € g ' x a l d u o l 'zeeg 'arnl)nr]s euorqlo]^J ll€ [ JZ98 'I58 'll uals{sotoqd 'xalduro) }-eqaurorq)oilJ sawoJq)olb pnpt\tput osp aas 'tt8-z€8 '!€8 '628 '(s)aLuorrl)ol^) lzsS t EE8 'urseqeq>orll 1996 zs9'eunurls^J 999'(UIJ)) utalotdroleln6a.l elupl)npuol aueiquautsuell slsoiqU)lls^) )rls^) 89L' 999' O9S'slsoiqU ' 16Zl L0L'aurals^) lZZt'suralotd la6ut; >utzstg-s; g-s{1-si(1 (l/ldv) xalduo: 6utlouro.rdaseqdeuyaasauolsolll) 198t't8€'aprurxaqoll^) )tlt)adslsaseurlautuoelqVeullaSos/, aas sasDuDl '290 ']sea^ [ Itgo l. '290 'saleiqaua^ t lE90 t ' u or1e;nba.r jLLL 'BLtL L L qdsoqd L g g ot ' J v g O L ' t 9 Ot - € 9 0 I ' u o t l e l l t' o a s P q d1 9 10I l-o0t t uolllqlqul/uolle^ll)eul 29 7 9 9 1 ' l u r o d l' ruaoqr :]yn1l 7 o^a 111[ saxalduo: 1p;r-utp[1 aassaxelduo] utl)^l Z 9 0 t ' I t r'1990 n r l : eu r s a 6 u e q >1 e : l ; > { : t !€90 L rvLoL'sLoL-nLOl ' u o r 1 e l i ( r o q d s o q d^aqpu o l l e ^ l ] ) e 'tgol ' LLL'391 € 9 O t - Z 9 O t ' ( s l p ) )s a s e u rl)u a p u a d a p - u t p ( 1 t0t t 1990t lt90t't90t-€90t '(sl)l) sureloJdIrolrqrqutaseull luapuadap-ull)^J 1 9 9 0 tt € 9 0 t ' € 9 0 t '()v)) asPurl6urle^rlle-aseulliuapuadap-ulpIl (slp)) saseurlluapuadap-utll^l i(s)utl)^)osp aas '5901-490I '990 '290 ',lpf-s L t 890r 'uoll)e jo usrueqlau.t '€90 t-290 t lz90 t 080t 'r6LOL'6LOl-8Lg 'a;ot,(lquassea;putds 1 'pue s]tueuIP alnqn]ol)lLu 090[ 'slso]ll.l.l olul Nlua nLoL'Jlo] *L0l ' {q uorlentl:e 9LOJ-nLOL'uor1e1{roqdsoqdep 990r'290t'lpf-w '990t '(.901'lp)-slt9 90I t-€o r I s o t 1 - € 0 11 ' 9 9 0 1 ' 2 9 0 t ' l p l - r D t90t-t90t'ourpurq l)l J € 9 0I 'lZgOL'ZgO 1-ur;:i(1 t j L L L ' g L L - L L I ' s e x a ; d u o :1 p'lsPa^ t l€90 'sa]erqaua^ lE90 [ ) L L L ' g L L -L L I ' a ) t ^ a p6 u t l e i 6 a l u;te u 6 r s t g O L ' V g O t ' 7 919' s u r p { : - 5 ' Z q O L5' 6 € ' s u r l ) I ) - W t Ot t ' 1 8 0t ' n L O L ' V gtO 1 9 11 ' u o r s s a l d xaau a b 'su!l]^)-S/tD 290[ Z9O['sull]^r-t9 l e:t;:{: t g 1 L ' a g oL ' s a 6 u e q 1 aassaxa;duor 1p1 saxaldurot>1p1-ut;>[1 /80['pue )/)dv 290['(s)url]^) € 9 8 ' u o ! l P l ^ l o q d s o q d o i o lqr dl l ^ f L t 6 ' g L 6 ' s l a u u e qul o l p a l e 6 - a p l l o a l ) n u - ) l l l I J

X]CNI

ztil

l : l3

INDEX

Desmin,9851 987 D e s m o c o l l i nIs1. 3 6 .11 3 8 7 D e s m o g e l i n1 s1, 3 6 , I 13 8 7 D e s m o s o m e ( s1)1, 3 4 , 1 1 3 4 F1, 1 3 5 1 11 4 3 - l 1 4 4 , 1144F,11777 keratinfilaments,986 D e s m o t u b u l e1,1 6 2 Detergent(s), 517, 517F,638F,639F i o n i cv s .n o n i o n i c , 6 3 6 membraneproteinsolubility,636-640 seealso specificdetergents DeterminationseeCelldetermination Detoxificationreaction(s), smooth endoplasmic r e t i c u l u m7, 2 5 Development,1305-1 320 asymmetriccell division,1099,1289-1290, 1 2 8 9 E1 3 1 3 - 1 3 1 5 , 1 3 r 4 F C.elegonsseeCoenorhobditiselegons develooment c e l ld e t e r m i n a t i o1n3, 1 1 - 1 3 1 21, 3 1 2 F c e l lf a t e , 1 3 1 1 - 1 3 1 2 t r a c i n g1, 3 1 0 - l 3 l1 , 1 3 11F cell ineage c e l lm e m o r y1, 3 0 51, 3 1 2 1, 3 1 5 cell migration,1140, 114OF cleavageof egg, I 307 descriptiveembryology,13 10 DrosophilomelonogasterseeDrosophilo develooment epithelialfolding,1142, 1143F epithelial-mesenchym t raaln s i t i o n s1,14 1 e v o l u t i o n a rcyo n s e r v a t i oanm o n ga n i m a l s , I 306F,I 307 e x p e r i m e n t aelm b r y o l o g yI,3 1 0 ,1 3 1 0 F 1305,1306F four essentialprocesses, g a p j u n c t i o n sl l,6 l geneticsseeDevelopmentalgeneticsand gene regulation germ layersand gastrulation,I 307, I 307F i n d u c t i v ei n t e r a c t i o nasn d s i g n a l s1, 3 1 0 ,I 3 13 , 13r3F,13167 l a t e r ailn h i b i t i o n1. 3 14 . 13 15 F m i t o s i si n a b s e n c eo f c y t o k i n e s i 1 s ,0 9 9 - 11 0 0 m o d e lo r g a n i s m sl 3 ,l I m o r p h o g e nasn dg r a d i e n t s1 ,3 1 6 - 1 3 1 71 ,3 1 8 F m o u s es e eM o u s ed e v e l o p m e n t nervoussystem,1383-1 397 seeo/soNeuron(s),development plantsseePIantdevelopmentand growth positionalcontrols,I I 11 positiona l l u e sl ,3 l 2 - 1 3 1 3 , 1 3 1 2 F1,3 1 3 F va r e g u l a t o r yD N Ad e f i n e sp r o g r a m1, 3 0 9 , 13 0 9 - 13 10 , 13 0 9 F s e q u e n t i ai n l d u c t i o n1, 3 1 9 ,I 3 2 0 F t i m e k e e p i n g1,3 19 - 13 2 0 ,13 2 0 F t o t a lc e l lm a s s1, 1 1 1 vertebratesseeVertebratedevelopment XenopusloevisseeXenopusloevisdevelopment seed/soEmbryo(s)/embryogenesis; Signaling m o l e c u l e ( s ) / p a t h w a yi (nsd)i;v i d u a l organismsand processes D e v e l o p m e n t agle n e t i c sa n d g e n er e g u l a t i o n , 450-451,451F,1308, 1309 cell differentiation,464-465,465F combinatorialgene control,464-4695,465F Drosophiloseeunder Drosophiladevelopment g e n e ss p e c i a l lrye q u i r e d1,3 0 8 oeneticscreens.1308 orqanformation,465-466,466F regulatoryDNA definesprogram,1309, 1309-1310,1309F transcriptionalsynergy,464 seeolso Celldifferentiation;specificAenes Diabetem s e l l i t u st y p e l , 15 4 9 D i a c y l g l y c e r o9 l1, 0 Diakinesi1 s .2 7 5 . 1 2 7 6 Diapedesis seeLymphocyte(s),recirculation Diarrhea in bloody, dysentery,1491 enteropathogeni c E.coli,1504 Solmonella entericdsDread.1518 soreadof infection.1487-1488 , N Ai n t e r f e r e n c(eR N A i ) , 4 9 6 D i c e rp r o t e i n R Dickkopfprotein,1316T Dictyostelium,16F chemotaxis,1045 m y o s i nI a n d l l l o c a l i z a t i oinn c r a w l i n ga m o e b a , 1041,1041F DideoxyDNA sequencing,549-550 Didinium,28F s ,i t r o g e n1, 0 1 D i e t a r yr e q u i r e m e n t n

Differential-interference m icroscopy,583 microscopy seeo/soPhase-contrast Differentiationof cellsseeCelldifferentiation patterns, Diffraction 528, 528F 163f, 169 Diffusion-limited enzymecatalysis,163, D i f f u s i o nr,a n d o mn a t u r e7, 4 , 7 5 F D i g e s t i o n8, 8 , 1 4 3 6 seeo/soLysosome(s) Dihydrofolatereductase,cancertreatment,1260 D i h y d r o u r i d i nteR, N Am o d i f i c a t i o n3,6 8 F3, 6 9 F Dihydroxyacetone, 112F phosphate,120F Dihydroxyacetone D i m e rf o r m a t i o n DNAdamage,296,298F proteins,142F seedho DNA-bindingproteins Dimethylbenz[a]a nthracene(DMBA),1226,1238 2 , 4 - D i n i t r o p h e n o8l3, 6 Dinitrophenyl,1545,1545F D i p l o i dc e l l s genetics,554F classical sexualreproduction,1269,1270F seed/soMeiosis yeast Iife cycle,34, 34F Diplotene,1275-1276,1280,1288 D i p o l e sc, o v a l e nbt o n d s 5 ,0 D i s a c c h a r i d e5s6,,5 7F ,113 F Discslarge(Dlg)protein,1148-1149,1148F,1154 genelprotein,planarcell polarity,1158, Dishevelled r 359 D i s i n t e g r i n Is1, 9 3 Dislocation,proteinsseeRetrotranslocation, misfoldedproteins Dissociation constant(Ka),158F Dissociation rate,158F,526 D i s t u H e sgse n e ,1 3 5 1 ,13 5 1E I 3 5 5 Disulfide bonds a m i n oa c i d s 1 , 2 9 F , 1 4 71 4 8 electrophoresis, 518F proteinstability,147-1 48,147F Diurnalrhythms,460-462, 461F seea/soCircadianclocks Divergence(evolutionary) mutation rateanalysis,264 phylogenetics, 247, 247F,248F s e ec / s oG e n ed u p l i c a t i o n ( s ) Anton van, "Diverseanimalcules," Leeuwenhoek, 5 0 1F p r o duction, s t e m c e l l D i v i s i o n aals y m m e t r yi n, 1421,1421F ) ,u t a g e n i c D M B A( d i m e t h y l b e n z l a l a n t h r a c eansem c h e m i c acl a r c i n o g e n1,2 2 6 ,12 3 8 , ,3F D N A( d e o x y r i b o n u c l eai c i d ) 3 a m p l i f i c a t i ovni a P C Rs e eP o l y m e r a sceh a i n r e a c t i o n( P C R ) a n a l y s i s5,3 2 - 5 5 3 b e n d i n gs e eD N Ab e n d i n g c a t e n a t i o n1,0 7 1 c e l lm a c r o m o l e c u l6e2, F c h e m i c asl y n t h e s i s5,4 8 chromatography, DNA-bindingproteins, 428-429,429F c l o n i n gs e eD N Ac l o n i n g c o m p a c t i o n , 2 l 0 - 2I 1 complementaryseecDNA d a m a g es e eD N Ad a m a g e fingerprinting,546,547F h e l i x - p a s s i nr e g a c t i o nD, N At o p o i s o m e r a sl le, 281F historicalresearch,195-196,195-197,329 identificationas geneticmaterial,195-196, 196F,197,198F pneumonioeexperiments,196F, Streptococcus 19 8 F s t r u c t u r e l u c i d a t i o n1,9 5 - 19 6 , 1 9 6 ,1 9 7, 3 2 9 hybridizationseeDNA hybridization informationcoding,199-200, 329,330F,331 DNA makesRNAmakesprotein,33I , 331F relationto proteins,199-200,199F as universalinformationstorc,2, 297, 4OB seeo/soDNA sequence;Geneticcode; Genome(s) labeling,534 l i n k e r2, 11 location, 200-201, 2O1F seea/soNucleus maintenance,263-265 failure,263,265,1212 seeo/soCancer;DNAdamage;Mutation(s) genomeevolution,246-247

seeo/soDNA repair m a n i p u l a t i osne eR e c o m b i n a nDt N At e c h n o l o g y n o n c o d r n gs e eN o n c o d i n gD N A packaging,202-218, 202-21 9 chromatinpacking,243,244F seeo/soChromatin;Nucleosome(s) c o m o a c t i o n2, 10 - 2 11 Chromosome seeo/soChromosome(s); structure polarity,3F proteininteractionsJeeProtein-DNA i nteractions r e c o m b i n a t i osne eR e c o m b i n a t i o n regulatoryseeRegulatoryDNA r e p a i rs e eD N Ar e p a i r repeatsseeRepetitiveDNA replicationseeDNA replication structureseeDNA structure synthesisseeDNAsynthesis t e l o m e r i c2. 1 0 seeo/soTelomere(s) 3-4, 3F templatedpolymerization, t h e r m a ls t a b i l i t y , 2 9 6 unwinding r e p l i c a t i o n2,7 3 , 2 7 3 F t r a n s c r i p t i o n3,3 3 seealso entriesbeginning DNA DNAarray(s)seeDNA microarray(s) D N Ab e n d i n g nteraction2 s ,14 F n u c l e o s o m e - D NiA proteins,enhancesomefo(matio, 446,447F proteins DNA-binding seeo/so D N A - b i n d i nd g y e s c, e l lc y c l ea n a l y s i s1,0 5 9 DNA-bindingmotifs (proteins),416-454 p sheetmotit 422,423F D N A - b i n d i nh g o m e o d o m a i n sp,a i r e dd o m a i n s , 14'l gene regulatoryproteins,418-419, 418T helix-loop-helix morif, 425-426, 426F motif,419-420 helix-turn-helix homeodomain,420-421, 421F seeo/soHomeodomainproteins recognitionhelix,419, 42OF structure,4l9,420F leucinezipper motif,423,424F protein-DNAinteraction,417, 418-419 b a s e - p a irre c o g n i t i o n , 471, 4 17 F seed/soDNA structure zinc fin ger motifs, 421-422, 422F,423F seed/soProtein-DNAinteractions DNA-binding pr oteins,427F b i n d i n gs i t ep r e d i c t i o n4, 2 6 dimerization,420,420F,422 142,142F Cro repressor, f u n c t i o n arl o l e , 4 2 3 424-425, 425FF heterodimerization, 424, 425F homodimerization, DNA-bindingmotifsseeDNA-bindingmotifs , 19 4 ,16 - 4 1 7 ,4 1 7 F 4 D N As t r u c t u r er e l a t i o n s h i p seea/soDNA structure helix-turn-helix protei^s, 420-421, 420F h o m e o d o m a i np r o t e i n s e eH o m e o d o m a i n protelnS histonesseeHistone(s) leucinezipper proteins,423,424F,425F major groove-binding, 423, 424F mi nor groove-binding, 423, 424F ic,430F sequence-specif affinity chromato g:raphy,428-429, 429F c h r o m a t i ni m m u n o p r e c i p i t a t i o4, 3 2 ,4 3 2 F DNA sequencedetermination,429-431,430F gel-mobilityshift assay, 427-428, 428F s ,2 7 F z i n cf i n q e ri n t e r a c t i o n 4 DNA-bindingproteins SSBsseeSi-ngle-strand (SSBs) zincfinger proteins,42l -422, 422F,423F seea/soDNA replication;Generegulatory protein(s);Protein-DNAinteractions D n a Bo r o t e i n , 2 8 3 F DNA catenation,1071 DNA cloning,532,540-541 cloningvectors,540-541, 541FF,542F genomiclibraryproduction,540-541,542F liqationreaction,540,541F rdstrictionfragments,540 reversegenetics,575 seea/socDNA;DNA libraries D n a Cp r o t e i n , 2 8 3 F 295f,296,297F DNAcrosslinks, DNAdamage,296,296F,297F

l:14

INDEX

a g e n t sc a u s i n gs e eD N A - d a m a g i nagg e n t s a p o p t o s i s1, 1 0 6 , 1 1 1 7 ATM/ATR s i g n a l i n gp a t h w a , 1 1 0 5 b u l k yl e s i o n s , 2 9 8 cancerand, 1216-1217,1225, 1246 seed/soCarcinogens; Mutation(s) c e l lc y c l ea n d ,3 0 3 - 3 0 4 c h e c k p o i n t s3,0 3 ,I 1 0 5 - l 1 0 7 ,11 0 6 F cross-linking, 296,297F failureto repair,1106 seeoisoCancer;Mutation(s) r e p a i rs e eD N Ar e p a i r replication,296,298F seea/soMutation(s) spontaneousalterations,296,296F seeolsospecifictypes seed/soDNA repair D N A - d a m a g i nagg e n t s c a r c i n o g e nsse eC a r c i n o g e n s i o n i z i n gr a d i a t i o n3, 0 0 - 3 0 1 seed/soUltraviolet(UV)radiation sensitivity,DNA repairdefects,2957 seed/soMutagenesis D N A - D N Ai n t e r a c t i o n 3 s ,0 6 F h y b r i d i z a t i osne eD N Ah y b r i d i z a t i o n D N Ad u p l i c a t i o n ( s ) c h r o m o s o m esse eC h r o m o s o m de u p l i c a t i o n e x o nr e c o m b i n a t i o n2 ,5 7 g e n e ss e eG e n ed u p l i c a t i o n ( s ) g e n o m ee v o l u t i o n2, 4 6 - 2 4 7 w h o l eg e n o m e , 3 8 - 3 93, 9 F2, 5 5 DNAexcision,site-specific recombination,323,324, 324F D N Af i n g e r p r i n t i9n, 5 4 6 ,5 4 7 F DNAfootprinting,429-431,43OF DNA glycosylases, 297-298 mechanism,299F recognition o f D N Ad a m a g e3, 0 0 - 3 0 1 , 3 0 0 F Dnacprotein,283F D N Ah e l i c a s e ( s2)7, 3 ,2 74 F assay,273F defects,2957 D n a Bp r o t e i n , 2 8 3 F i n h i b i t i o nD , n a Cp r o t e i n2, 8 3 F mechanismof action,273,282,283F TFIIHtranscriptionfactors,340-341 D N A - h e l i x - p a s s irnega c t i o nD, N At o p o i s o m e r a sl e l, 281F D N Ah y b r i d i z a t i o n , 3 0 6 chromosomepaints,202-203 D N A - R N Ah y b r i d i z a t i o n 5 ,3 5 - 5 3 75, 3 8 F N o r t h e r nb l o t t i n g 5 , 3 8 - 5 3 95, 3 9 F h e l i xn u c l e a t i o n3,0 6 F h y b r i d i z a t i ocno n d i t i o n s5,3 5 - 5 3 65, 3 7 F m o d e lo f r e c o m b i n a t i o n b aa l s e - p a i r i n3 g0 , 6F n o n p a i r i n gi n t e r a c t i o n 3 s ,0 6 F recombinantDNAtechnology,532 S o u t h e r nb l o t t i n g 5 , 39-540;539F seeo/soDNA probe(s) D N Al a b e l s , 5 3 4 DNA libraries,540-542, 54't -542 cDNA,542,543F,544 g e n o m i c5 , 42,542F cDNAvs, 543F,544 in yeasttwo-hybridsystem,524 seeo/soDNAcloning D N Al i g a s e ( s ) DNAcloning,540-541,54tF DNA repair 2951 302-303 L a g g i n gs t r a n dD N As y n t h e s i s , 2 7 2 reactionmechanism,273F D N Al o o p i n g , 4 3 7 F 4 ,3 8 F transcriptionregulation,438,438F,480-481 D N Am e l t i n g 5 , 37F DNA methylase(s) seeDNA methyltransferase(s) D N Am e t h y l a t i o n , 4 T 2 F c a n c e rc e l l s 1 , 213 CG(CpG)islands,434,470-471 damagerecognition,300-301 enzymes,284F,467 eucaryotes, 278 gene expressionand,467-468, 468F genomicimprinting,46A-47O inheritance,467-468, 467F methylatedDNA binding proteins,468 5-methylcytosine, 467,467F procaryotes,277-27 8, 282,284F restriction-modification systems,532 recognitionfunctions,277-278, 532 r o l ei n m u t a t i o n3, 0 0 - 3 0 13, 0 1F

strand-directedmismatchrcpafi, 277-278, 277F, 284F uncontrolled,296F DNA methyltransferase(s), 284F,467 D N Am i c r o a r r a y ( s ) , 5 7 4 c e l lc y c l ea n a l y s i s1,0 6 5 gene expressionanalysis, 537, 574-575 cancercell typinq,414F,1240,1249,1264 clusteranalysis,575, 575t 1 h u m a ng e n e s , 4 2 metastases, 1249 m e t h o d o l o g ,5 7 4 , 5 7 4 F replicationfork analysis,285,286F sizes,574 DNA mismatchrepairseeMismatchrepair D N A - o n l yt r a n s p o s o n 3 s ,1 8 F3, 1 8 T3, 2 3 cut-and-paste ransposition 3 ,1 8 1 3 19 F ,3 2 0 F DNA polymerase(s), 266 5 f 3 ' c h a i ne l o n g a t i o n2, 6 7, 2 6 8 F catalyticmechanism,267F,268F cooperativity,275-276 D N Ar e p a i r , 3 0 2 dNTPsubstrates, 266,267F,268F eucaryotic,280,281F,293F,294F,2957 fidelity,269 movementalong DNA,273-274 proofreading, 269-27O,27OF seed/soMismatchreoair RNApolymerases vs, 334,335 slidrngclamp,274-275, 275-276, 275F,276F structure,26SF T7 polymerase, 269 thermophilic,544 seeo/soPolymerase chain reaction(PCR) viral,1517 seeolso specificenzymes D N Ap r i m a s e s2,72 , 2 72 F ,2 73 , 2 76 F b a c t e r i adl n a G ,2 8 3 F eucaryotic,280 mechanismot action,282,283F DNA probe(s),534,536F nucleicacid detection,539-540,539F seeo/soDNA hybridization DNA-proteininteractionsseeProtein-DNA interactions D N Ar e a r r a n g e m e n t ( s ) phase bacterial variation,454-455,455F conservativesite-soecific recombination heritable,325-326 reversible.324.324F seea/soRecombination; specificreorrongements DNA recombinationseeRecombination DNA renaturationseeDNA hybridization DNA repair,263, 295-304 baseexcisionrepair(BER), 297-298,299F cell-cycledelay,303-304 c r o s s - l i nrke p a i t2 9 5 7 defectsin seeDNA reoairdisoroers directchemicalreversal, 298 DNA polymerases, 302 D N As t r u c t u r ei m o o r t a n c e2.9 7 double-strandbreaks,2951 302-303, 303F enzymes,296,299F e r r o rp r o n e , 3 l 0 - 311 f a i l u r es e eM u t a o e n e s i s homologousrecbmbination,295T,305 identification,295 imoortance.295 mismatchrepairseeMismatchrepair multiple pathways,295,297-298 nucleotideexcisionrepair(NER), 295T,29A,299F RNApolymerasecoupling,299-300 transcriptioncoupling,299-300 translesionsynthesis, 2957 seeo/so DNA dam age;individuolrepoirpothways DNA repair d isorders,277, 295-296,295I, 304,1106 seealso Cancer:specificdisorders DNA repeatsseeRepetitiveDNA DNA replication,3, 200, 201F,263, 266-295 analysistechniques,282-283,284F,285,285F bacterial,280, 282, 283F end-replication,292 D N Ac a t e n a t i o n1,0 7 1 DNA synthesisseeDNA synthesis DNAtemplating,200,201F,266 end-replicationWoblem,292 eftots,246-247, 269,271-272, 276-277 seea/soDNA damage;Mutation(s) eucaryotic,280 chromatineffects,285-286

chromosomeendsseeTelomere(s) i n i t i a t i o ns e eD N Ar e p l i c a t i o inn i t i a t i o n multiple replicationforks,283,284F replicationforksseeunderReplicationfork replicationotiginsseeundetReplication origin(s) replicationrate,283 phase 5 of cell cycle,284, 285, 285F, 1067-1069,1068F timin9,285 seeo/soCellcycle f idelity, 269,27O,271Fr seed/soDNA reoair historicalrcsearch, 266-267 incorrectmodel of DNA replication,269F i n i t i a t i o ns e eD N Ar e o l i c a t i o inn i t i a t i o n machinery,266-281, 276F cooperativity,275-276 DNA helicases, 273,273F,274F D N Al i g a s e2, 7 2 , 2 7 3 F DNA polymeraseseeDNA polymerase(s) D N Ap r i m a s e2, 72 , 2 72 F ,2 7 3 ,2 76 F DNAtopoisomerases, 278-280, 279F,28OF primosome,276 s i n g l e - s t r a nDdN A - b i n d i n p g r o t e i n s2, 7 3 , 274F,275F mitochondrial.856-857.857F nucleosomeassembly, 289-290 originsseeReplicationorigin(s) phylogeneticconservation, 280 proofreading,269-270, 27OFF replicationfork seeReplicationfork replicationoriginsseeReplicationorigin(s) semiconservative nature,266,266F,268F strandrecognition,277 procaryotes methylationin seeDNA methvlation nicksin eucaryotes,278,278F seeo/soSingle-strand DNA breaks seed/soMismatchreoair transcriptionys.,333-334 "winding problemi'278F seeo/soChromosomereplication;lndividuol comDonents DNA replicationinitiation,2a1-282 bacterialchromosomes, 282,283FF,284F eucaryotic,289F,1067,1068F preinitiationcomplex,1067 p r e r e p l i c a t i ocno m p l e x1, 0 6 7 - 1 0 6 81, 0 6 8 F ORC(originrecognitioncomplex),287 originsseeReplicationorigin(s) originsof replication5eeReplicationorigin(s) proteins,281,282,283F,1067 regulation,2S2 DNA-RNAhybridization,536-537,538F seeo/soRNA-DNAhybrids DNA segmentshuffling,19, 19F DNA sequence,199-200 alterationsin genomeevolution,246-247 analysistechniquesseeDNA sequencing bacterial,as immunostimulant,1527 evolutionaryconservation,207,208F,250 h u m a np - g l o b i ng e n e ,1 9 9 F , 2 0 0 F recognition,418-419, 418f seea/soDNA-bindinomotifs structural effects,41 6-417 DNA sequencing,548-550 alignment,530-531 automation,550,550F c h a i n - t e r m i n a t i nngu c l e o t i d e s5,5 0 gel electrophoresis, 534,535F g e n o m i cs e eG e n o m es e q u e n c i n g historicalaspects,549-550 human genome,142,205-206 comparative,207,247, 247F,248F intron-exonrecognition,551 open readingframe prediction,551 proteinsequenceprediction,I 39,550-55I , 550F recombinantDNAtechnoloqy,532,534, 535F DNA structure,62F,195-201, Iill-ZOl backbone(sugar-phosphate), i97, 19g-i 99, 19 8 F base-pairing, 197-199, 198F,417F complementarychains(strands),197-199, 266F antiparallelnatwe, 198F,199,267 roteIn reparr,29l r o l ei n r e p l i c a t i o, n 2 ,6 6 2 O O2, 0 0 F , 2 0 1 F deformation,coupledto packaging,213,214F double helix,197-199, 198F,199E338F complementarychains(strands)seeDNA

l:15

INDEX

replication hydrogenbonds,197-198, 198F,199F,417 major groove,199F,416F , 9 9 F4, 16 F m i n o rg r o o v e 1 repair,296-297 seea/soDNA repair rotation,278,278F s t r a n ds e p a r a t i o n2,7 3 turns,198-199, 199 elucidation,195-196,196,197 h a i r p i nh e l i c e s2, 7 3 mechanismfor heredity,199-200 seed/soGene(s) nucleotides, 62, 197-199, 198F seea/soNucleotide(s) phosphodiesterbonds,199F p o l a r i t y1, 9 8 F 3'to 5' pola(ity,197 p r o t e i n - D N Ai n t e r a c t i o n 4 s ,16 - 4 1 7 ,4 1 7 F seeolsoDNA-bindingmotifs (proteins) RNAvs.,408 sequenceeffects,416-417 seea/soDNA bending X-raydiffractionanalysis,195-196, 196 seeo/soDNAtopology D N As u p e r c o i l i nsge eS u p e r c o i l i n g DNA synthesis,266-268 analysis autoradiography, 282-283,284F,602-603 Brdu staining,285,285F,1059,1059F,1422, 1425 ATPrequirement,86, 87F chemistry,268F 5 ' - 3 'c h a i ne l o n g a t i o n2, 6 6 - 2 6 7, 2 6 7 F , 2 6 8 F , 2 71 - 2 7 2 , 2 7 1 F triphosphates, 266, deoxyribonucleoside 267F,268F DNA replication,266-268 5 L 3 ' c h a i ne l o n g a t i o n( l e a d i n gs t r a n d )2, 6 7 , 267F,268,268F,27 1-27 3, 271F,276F l a g g i n gs t r a n ds e eL a g g i n gs t r a n ds y n t h e s i s ( D N Ar e p l i c a t i o n ) primer strand,267F,268F pyrophosphaterelease,267F,268F template stand, 267F,268F seea/soDNA polymerase;DNAsynthesis initiation,28l mechanism,26SF seeo/soDNA polymerase(s) relationto histonesynthesis, 289-290 seed/soDNA replication 278 DNAtopoisomerases, catalyticreaction,279F DNA replication rcle, 27 8-280 seeo/soDNA replication lambdaintegrasevs.,324 mechanismsof action,278-279,279F,28OF topoisomerasel, 278, 279F topoisomerasell, 278-279,280F, 281F DNAtopology s u p e r c o i l i nsge eS u p e r c o i l i n g transcriptionelongation,343-345, 344F seeo/soNucleosome(s) DNAtransferseeHorizontalgene transfer DNA tumor virus(es),1247 -1249, 1248F DNAvirus(es),1496F,1497F in cancer,1227-1 228,1228I seea/soDNAtumor virus(es) triphosphates dNTPsseeDeoxyribonucleoside (dNTPs) (lRS-1), D o c k i n gp r o t e i n si,n s u l i nr e c e p t o sr u b s t r a t e 924 D o l i c h opl h o s p h a t e1,1 5 F Dolichol,proteinglycoslation,737,738F,747F D o l l yt h e s h e e p 1 , 287 Domainfusion,activatorproteins,442F D o m a i n sl,i v i n gw o r l d ,1 6 ,1 6 F Dominantnegativemutation(s),528F,564F antisenseRNA,564F geneticengineering,564 R N Ai n t e r f e r e n c e , 5 6 4 F D o p a m i n eg, a pj u n c t i o np e r m e a b i l i trye g u l a t i o n , 1162,1162F Dormancy,Epstein-Borrvirus(EBV),1499 Dorsallip, of blastopore,1367-1368,1367F Dorsalprotein,of Drosophila,1334-1335, 1335F Dosagecompensation,473, 475-476, 476F D o u b l eb o n d s ,c a r b o n - c a r b o n1,0 6 F DNA polymeraseproofreading, Double-checking, 269

Doublesex(Dsx)gene,Drosophilosex determination,481F,482F Double-strandbreaks(DSBs) h o m o l o g o u sp a i r i n g / m e i o t irce c o m b i n a t i o n , 3 0 5 F3, 12 , 1 2 7 5 , 1 2 8 0 orooucflon.JUU-JUI repair,302-303, 303E304-305,308-309 defects,295T seeo/soHomologousrecombination (crossing-over) topoisomerasell production,280F viruses,1534 Double-stranded RNA(dsRNA), Down syndrome,meioticnondisjunction, 1278-1279 gene/protein,I 335,1353, Dpp (decapentaplegic) 13 5 3 F1, 3 5 5 D r kg e n e ,9 2 8 F Drosophilodevelopment,1328-1 341 anteroposteriorpatterning, 1341-1 347 seeolso Drosophilamelonogosterhomeotic genes c e l lc y c l ea n a l y s i s1,0 5 8 c e l l u l a r i z a t i o n . ' 1 019190,0 F e a r l ye m b r y oa n d g e n e s i so f b o d y p l a n , 1328-1341 b l a s t o d e r m1.3 3 0 - 13 31. 13 3 0 F F 1332F,1333-1334, body axisspecification, 13 3 4 F dorsoventralpatterning,1334-1337,1335F, 13 3 5 F F e99,1330,1331F egg polaritygenesseeEgg polaritygenes (Drosophilo) fate map, 1331F,1332F p f o l l i c l e r o v i d i n ge g g - p o l a r i z i nsgi g n a l s , 13 3 3 F gastrulationand mesodermformation,1335, 1336F,I 340 m o r p h o g e ng r a d i e n t s1, 3 3 3 - 1 3 3 4l ,3 3 4 F F , 13 3 5 F1. 3 3 5 F F1,3 3 6 mRNAlocalization,487,488F,1333-1334, r 334F oocyte,1333F p l a n a rc e l lp o l a r i t y1, 1 5 7 ,1 1 5 7 F regulatoryhierarchy,genes,1338F segmentationgenesseeDrosophilo melonogostersegmentationgenes sexdetermination,1286 s y n c y t i at lo c e l l u l atrr a n s i t i o n1,3 3 0 - 13 3 1 syncytiumformation,1099,1100F s y n o p s i s1,3 2 9 F vertebratebody plan vs, 1336F geneticcontrol/generegulation,4OA-4O9, 447- 450, 448F,449F,449t F gene Even-skipped seeEve(Even-skipped) Ey gene,466,466F homeoticgenesseeDrosophilomelonogastel homeoticAenes Rasrole in eye development,927 ,927F segmentation genesseeDrosophilo melanogastersegmentationgenes seeolso specificaenes o e n e t i cm o d e l .1 3 2 9 !enetic screeningfor earlypatterning,1332 g e n e t i ct e c h n i q u e s1,3 2 9 i m a g i n adl i s c s1, 3 3 1 seeolsounderlmaginaldiscs key to developmentalmechanismsin other animals,33 oogoniaformation,1290,1290F o r g a n o g e n e sai sn d p a t t e r n i n go f a p p e n d a g e s , 1347-1362, 1347-1363 c o m p a r t m e nbt o u n d a r i eas s s i g n a l i n g c e n t e r sI,3 5 3 -13 5 5 ,1 3 5 3 F c o m p a r t m e n t s1,3 5 2 - 13 5 5 , 13 5 2 F F1,3 5 3 F eye developmenr,466,466F i m a g i n adl i s c s1, 3 4 9 - 1 3 5 1 ,1 3 5 0 F1, 3 5 6 - 1 3 5 7 individualityspecifiedby homeoticselector g e n e s ,13 5 1 intercalaryregeneration,1354, 1354F s i z er e g u l a t i o n1,3 5 3 - 13 5 4 parasegments v5.segments,1330,1330F sexdetermination,481-482, 481F,482F Drosophilamelanogoster adult anatomy,1328-1330, 1328F a p o p t o s i s1,1 2 5 , 1 1 2 6 chromosome2 s ,3 7 F 2,330F chromosome gene-chromosome relationship(proof), 3 7 - 3 8 .3 8 F

polyteneseePolytenechromosome(s) s e xc h r o m o s o m e s , 4 3 l circadian clock, 461 -462, 462F cytoskeleton,967, 967F,983 developmentseeDrosophlladevelopment gene expressron alternative splicing,479 chromosome oulls, 220-222 dosagecompensation,475 gene regulatory protei^s, 418T,447,448F, 14007 position effects,221-222, 221F gene regulatoryproteins,I 4007 genome informationcoding,329,330F mobile geneticelements,318T,480,556 s e q u e n c i n g5,5 1, 5 5 2 size,18T seeolso chromosomes(above) homeodomainproteins,429 seeo/soHomeodomainproteins a s m o d e lo r g a n i s m3, 7 - 3 8 mRNAlocalization,1022-1023 multicelluf ar developmentand,447 -45O,MBF, 449F, 449FF, 465-466, 466F oocyte, 1022-1023 photoreceptorassembly,927F proteininteractionmaps,188 RNAinterference(RNAi),571 vertebratehomologies,1306,1306F,1308 w i n g h a i rm u t a n t s 1, 1 5 7- 1 15 8 ,1 1 5 7 F Drosophila melonogdsterhomeotic genes, 1341-1347 bithoraxand Antennapediacomplexes,1342, 1342F Hox complex, 1342-1347 seeo/soHox complex;Hox genes m e m o r ym e c h a n i s m1,3 4 4 modulatedrepetitionstrategy,1341-1 342 mutations,1342 Polycomband Trithoraxgroup genes,1344, 13 4 5 F Dositionalvalues,1344 sequentialHox gene expression,1343-1344, 13 4 3 F vertebratehomologies,1344-1347, 1346F, 1346FF seealsospecificgenes Drosophi la melanogdstersegmentationgenes, 13 3 6 - 13 3 8 , 13 3 7 E13 3 8 F g a p g e n e s1 , 3 3 3 ,13 3 7 - 13 3 8 ,13 3 7 E13 3 8 F pair-rule genes1 , 3 3 3 ,13 3 7 - 13 3 8 ,13 3 7 F regulatoryDNAcontrollingpattern (evegene), 447- 450, 448F,449F,4 49FF, 1339-1340, 1339F,I 340F segmentpolaritygenes,1333, 1338F,1339 seealso individualgenes Drug-inducedchanges,cytoskeletalfilament polymerization, 987-989, 9887 1521-1524 Druo resistance, Dscdm gene,alternative splicing,479F Dynamicinstability,cytoskeletalfilaments,980, 981F,982F c a t a s t r o p h e , 9 8100, 0 3 catastroohevs.rescue,1080 of microtubulesseeMicrotubule(s) 979F nucleotidehydrolysis, r e s c u e9, 8 0 , 9 8 1F Dynaminprotein,756F,757 -75a, 7 58F D y n e i n ( s 1) ,0 1 4 - 1 0 15 , 10 15 F ,10 18 , 10 19 F A T Ph y d r o l y s i sl 0, l 9 organelles, attachmentto membrane-enclosed 1022,1022F 1 0 1 5 axonemal, ciliarybeatingand left-rightasymmetty,1377 c y t o p l a s m i c1,0 1 4 forcegeneration,1018 l i n k e rr e g i o n 1 , 019 cycle,1019 mechanochemical mitotic spindles,1077,1077F,1079 p o w e rs t r o k e 1 , 0 1 9 ,1 0 1 9 F Dysentery,epidemic,1491 congenita,294 Dyskeratosis Dystrophin,1466

E E2Foroteins,1 103-1 1O5,12MF E6viraloncogene/protein,1248,1249F

l:16

INDEX

EZviraloncogene/protein,1248,1249F E a re, m b r y o n i co r i g i n ,1 3 8 4 F1, 4 2 9 Earlycell plate,1098 Earlygenes,chromatincondensationeffect, 285-286 Eastere n q u i n ee n c e p h a l i t ivsi r u s ,14 9 6 F Ebolavirus,cell entry,multivesicularbodies,797 E - c a d h e r i n11 , 3 6 ,I I 3 8 7 in cancerinvasiveness, 1249-1250 epithelial-mesenchym t raaln s i t i o n s1,14 l E.coli seeEscherichia coli Ectoderm,1307,1365 Edema,1493 E d e m at o x i n ,1 4 9 3 rooe enects.5a2F E D T Ac,e l ls e p a r a t i otne c h n i q u e s5,0 2 EF-1elongationfaclot,377F EF-2elongationfa ctot,377F EffectorB cells,1543, 1546,1546F,1552F EffectorT cells,1543,1544F,1 546, 1546F,1552F Effluxtransporterproteins,auxintransport,959 E F - Ge l o n g a t i o nf a c t o t , 3 7,73 7 7 F EF-Tuelongationfactor,I 81, 181F conformationa l ange1 ch , 8 0 1 8 1 ,1 8 1 F3, 7 7 , 8 0 - 18 1, 1 8 1 F3, 7 7 G T P - b i n d i npgr o t e i n 1 proteinsynthesis , 180,377-378,377F EGFseeEpidermalgrowth factor (EGF) Egg (ovum),12A7-1292 a c t i v a t i o n1, 2 8 7 , 1 2 9 A Ca2+increases, 1299 c o r t i c agl r a n u l e s1,2 8 8 corticalreaction,1299,1300, 1300F spermbinding to, 1270F,1298-1299, 1298F, 13 0 0 F seed/soFertilization C elegans,1323-1324 developmentstages,1288-1 290, 1289F,1291F oocytesseeOocytes PGCsseePrimordialgerm cells(PGCs) timing1 , 288 e n u c l e a t i o n4,l 1 ,4 ' l 3 F parthenogenes1 i s2,8 7 size,1287, 1287F,1290-1291, 1290F s p e c i a l i z ecde l ls t r u c t u r e2, 8 8 E1 2 8 7 - 1 2 8 8 , 1291F,1298F totipotency,I 282 Egg polaritygenes(Drosophilo),1 332-l 335, I 332F, 1334F E h l e r s - D a n l os sy n d r o m e1, 1 8 7 elFs(eucaryoticinitiationfactors),380F elF-2, 488-489,490F,1535 e l F 4 E3. 8 0 . 4 9 1 e l F 4 G3, 8 0 regulationby phosphorylation, 488-489, 490_491,490F s e ea / s oC a p - b i n d i n cgo m p l e x( C B C ) Elastase 13, 8 F E l a s t ifci b e r s 1 , 4 6 F1, 1 8 9 - 11 9 1 ,I 19 0 F E l a s t i n1,1 7 9 ,1 1 a 9 - 11 9 1, 11 9 1 F structure,146-147, 146F,1190-1 191, 1191F synthesis,1190 E l e c t r i c aalc t i v i t yi,n s y n a p s em a i n t e n a n caen d e l i m i n a t i o n1,3 9 3 - 13 9 7 Electricalpotential,neuron(s),675 E l e c t r i c as ly n a p s e sg ,a pj u n c t i o n s1, 16 1 E l e c t r o c h e m i cgarla d i e n t s6,5 4 F E l e c t r o c h e m i cparlo t o ng r a d i e n t s6, 5 3 ,8 13 , 9 2 1 F , 827-A40,853-8s4 a c r o s sb a c t e r i am l e m b r a n e s8,3 9 .8 3 9 F production, ATP 662, 8l7-A19,8i9t, a22-824 seeolso AfP synthase bacterialflagellum,821-822,823F c h e m i o s m o s i8s1, 3 - 8 1 4 ,8 l 4 F couplingto activetransport,822,823F generation,100, 100F,820-821,821F seec/soProtonpumps membranesand,640,655,820-821, 82I F m i t o c h o n d r r aplr o t e i ni m p o r t ,7 1 6 7 1 7 , 7 1 6 F n o n c y c i l cp h o t o p h o s p h o r y l a t i o8n5,0 - 8 5t , 8 5 2 F p H g r a d i e n t8, 2 0 - 8 2 18, 2 1F proton-motiveforce,821, 839F Electrogenicpumps,662-663,67l Electron(s) atomic interactions,46-48 seea/soChemicalbonds electronshell,46,48F,49 orbitals,46 Electroncrystallography,61I -612 Electrondensitymaps,X-raydiffractionanalysis, 528

Electronmicroscop, 604-61 3 3-D reconstruction, 606,607F,611-612 EM-tomography, 611F,612,612F autoradiography,603F p u f f s , 239 chromosome contrastgeneration,606,610 colloidalgold, 606,607F heavymetal stains,606 metal shadowing,608-609, 610F n e g a t i v es t a i n i n g6, 10 , 6 11F cryoelectronmicroscopy, 6l 0 depth of field,606,608 electronlens,604 electronscattering,605 freeze-etch, 6'l0F grids,605,605F history,604T imaging,6l0-612 immunogold,506-607, 607F microscooe,604 molecularlocalization,606-607 resolution,604-605, 604F.608 samplepreparation,605-505, 605F scanningelectronmicroscopy(SEM), 607-608, 608F,609F SEMseeScanningelectronmicroscopy(SEM) s i n g l e - p a r t i crl e c o n s t r u c t i o6n1, 1, 6 1 2 F6, 1 3 F surfaceanalysis,607-609, 608F,609F tomography,611F,612,612F,613F t r a n s m i s s i osne eT r a n s m i s s i oenl e c t r o n microscopy(TEM) Electron-motive force,814, 8l 5F Electronscattering,electronmicroscopy,605 Electronstains,606, 607F E l e c t r o nt r a n s f e r7,1 - 7 2 ,1 0 0 F , 831, 8 l 4 F energetics,820-821, 829,830F seeolso ATP(adenosinetriphosphate) m i t o c h o n d r i vas .c h l o r o p l a s t8s 1 , 4 ,8 l 5 F NADH/NADPH carriers,82-83,82F,818-819, 8 18 F seeo/soActrvatedcarriersin metabolism o x i d a t i v ep h o s p h o r y l a t i o n I 0, 0 ,8 19 , 8 19 F photochemicalreactioncenters,848,850F,852F protons,827-828,828F redoxpotentials,829,831,835,835F,852F,853 seea/soElectrontransportchain(s);Oxidative phosphorylation Electrontransportchain(s),a27-a4o A T Ps y n t h e s i s1,0 0 ,1 0 0 F8, 1 7 - 8 1 9 ,8 l 9 F proton gradients,821-823, 821F proton movement, 828, 828F,829F uncoupling,836 seeolso ATPsynthase;Electrochemical proton g r a d i e n t sP; r o t o np u m p s bacterial,839-840,839F,8734F e v o l u t i o n a riym p l i c a t i o n s8 ,7 1- a 7 2 chloroplastsseePhotosyntheticelectron t r a n s p o r ct h a i n ( s ) cyanobacteria, 872, 873-875 e n e r g e t i c sS,2 0 - 8 2 1 evolution,87O-876 anaerobicbacteria,871-872 cyanobacteria, 872,873-875 f e r m e n t a t i o nS, T 0 8 7 1 photosyntheticbacteria,872 i r o n - s u l f ucr e n t e r s ,0' l1 mitochondriaseeMitochondrialelectron t r a n s p o r ct h a i n ( s ) ( s ) photosynthesis seePhotosyntheticelectron t r a n s p o r ct h a i n ( s ) seeolsoNP (adenosinetriphosphate);Electron tranSler E l e c t r o p h o r e s5i s17 , a g a r o s eg e l ,5 3 4 cell fractionation , 517,518F,521-522 g e l s t a i n i n g5, 1 7 ,5 1 8 F5, 3 4 ,5 3 5 F p o l y a c r y l a m i dgee l s e eP o l y a c r y l a m i dge l (PAGE) electrophoresis proteinanalysis,518, 521-522, 521F,522FF proteindenaturation,5 17 p r o t e i np u r i f i c a t i o n5,17 , 518 F E5 2 1- 5 2 2 ,5 2 2 F F pulsed-field,534 r e c o m b i n a nDt N At e c h n o l o q y5, 3 4 b l o t t i n gs e eB l o t t i n g D N As e q u e n c i n g5,3 2 ,5 3 4 ,5 3 5 F5, 5 1 p u l s e d - f i e lgde l s ,5 3 4 ,5 3 5 F Electroporation, 565-566,598,598F Electrostatic interactions,54 Elements(chemical), 45, 46,47F,48F E l o n g a t i o fna c t o ( s ) ,1 79 - 18 0

EF-GseeEF-Gelongationfactor EF-TuseeEF-Tuelongationfactor transcriptional, 343-345 translational, 376F,377-37A, 377F Embryo(s)/embryogenesis anteroposterioraxisseeAnteroposterioraxis develooment cadherins.1136.ll36F gene function analysis, cancer-critical 1241-1242 c e l lc r a w l i n g1, 0 3 6 c e l lc y c l ea n a l y s i s1,0 5 7 - 1 0 5 8 ,1 0 5 7 F1, 0 5 8 F descriptiveembryology,1310 dissociationexperiments,1139-1140,1139F, 1l 4 0 F e x p e r i m e n t aelm b r y o l o g y1, 3 1 0 ,13 1 0 F G1phaseof cell cycle,1100 gastrulation,1364-1369 plant,1400, 1401F,1 4O2,14O2F polarity,1364 stem cellsseeEmbryonicstem (ES)cells seeo/so Development;individuolspecies;specific stoges E m b r y o n ig c e r mc e l l s 1 , 283 E m b r y o n i sc t e m( E Sc) e l l s , 5 0 5 - 5 0 71,2 8 3 controlof differentiationby differentfactors, 1481F OerlVaIlOn. 5U5-5U/. 5UtrF

differentiatedcell productionin vitro,1481 drug discovery,1482 immune rejectionproblem,1481-1 482 mouse.567F.1480 "personalizedi' 1303,1303F,1481-1482 " t h e r a p e u t iccl o n i n g ,5' 0 7 in tissuerepair,1481-1482 E n c e p h a l i t ivsi ,r a l ,I 5 0 2 E n d o c h o n d r ab lo n ef o r m a t i o n1, 4 7 0 E n d o c r i n cee l l s8, 8 2 gut (enteroendocrine cells),1437,1437F i n r e s p i r a t o reyp i t h e l i u m1, 4 3 5 E n d o c r i n sei g n a l i n g8, 8 2 - 8 8 38, 8 2 F8, 8 3 F Endocytic-exocytic cycle,789 Endocyticpathway,749,7 sOF,751F,795F Endocytosis, 749,787 -799 earlyendosomes,782,792 fluid-phase,789 l a t ee n d o s o m e s , 7 8 2 LDLs,791-7 92, 791F,793F receptor-mediated seeReceptor-mediated endocytosis s r o n a l/.9 J se?a/io Endocyticpathway Endoderm,1307.1365 Endoglycosidase H (EndoH),oligosaccharide p r o c e s s i n gT,T 5 F E n d oH - r e s i s t a notl i g o s a c c h a r i d e7s7,5 F Endoplasmicreticulum(ER),723-7 45 anlroooysyntnesrs, /ouF a n t i g e np r o c e s s i n g / p r e s e n t a t i1o5n8, 3 F c a l c r u ms t o r e ./ 2 5 - / 2 6 ceramidesynthesis,7 44-745 cholesterolsynthesis, 7 43,744-745 COPII-coated vesicleformation.757. 767F development,702,704 distribution d u r i n gc y t o k i n e s i s1,0 9 8 evolution,T00F gfycolipidsynthesis, 7 44-745 glycoproteinsynthesis, 7 36-7 38, 737F,747F glycosphingolipidsynthesis,744-745 lipid bilayerassembly,7 43-7 45, 7 44F l i p i dm e t a b o l i s m , 6 2 6 m e m b r a n ea, m o u n t s6, 9 7 7 membraneproteins,486,630,635 microsomes,511, 726 microtubulemotors,membraneorganization, 1021 processing, oligosaccharide 775F phospholipie d x c h a n g ep r o t e i n s7, 4 5 phospholipidsynthesis, 7 43,743F p h o s p h o l i p i tdr a n s p o r t7, 4 5 p o s i t i o ni n c e l l , 6 9 7 p r o t e i na s s e m b l y , 7 3 5 GPIanchor attachment, 7 42-7 43, 742F v i r u sa s s e m b l a y n d ,15 13 , 1 5 1 4 F proteindegradationseeProteasome(s) p r o t e i nf o l d i n g , 7 3 6 misfoldedproteinfate,739-7 42, 741F unfoldedproteinresponse,740-742 seed/soProteinfoldino proteinglycosylation, 73:6-73a,737F,747F

l:17

INDEX qualitycontrol,767-768 r e s i d e npt r o t e i n,s7 3 2 F , 7 3 6 , 7 3 7 F4, 7 F , 7 6,7 770-771,770F selectiveretention,771 retentionsignal,736 retrievalpathway,769-77O,770-771,770F rough ER,725F,726,726F smooth ER,724-7 25, 725F,726F seed/soSarcoplasmic reticulum 7 44-745 sphingomyelinsynthesis, structure,696, 723, 724F T-cellreceptorassembly,768 unfoldedproteinresponse,7 4O-742 v i r u sa s s e m b l a y n d ,15 13 , 1 5 1 4 F volume,697T Endoplasmicreticulum(ER),proteintransport, 723-745 c o - t r a n s l a t i o nt ar al n s l o c a t i o n m.e c h a n i s m7.3 2 F to Golgiapparatus,7 66-7 87, 7 68-769 M H Cc l a s sI p r o t e i n si,n h i b i t i o nb y v i r u s e s1, 5 3 6 misfoldedproteinfate,739-7 41, 741F m u l t i p a stsr a n s m e m b r a nper o t e i ni n t e g r a t i o n , 734-736,735F post-translational translocation,732F,736 qualitycontrol,767-768 retrotranslocation, 391,739-7 40, 740F Sec6l proteintranslocatorcomplex,730-731, 730F,731F 727 -73O,729F signalrecognitionparticle(SRP\, mechanismof action,728,730 s i g n a sl e q u e n c e 7 s2 , 6 - 7 2 7 ,73 3 - 7 3 4 s i n g l ep a s st r a n s m e m b r a nper o t e i ni n t e g r a t i o n , 724-725,733F SRPreceptor,728 sional,732 start-transfer stop-transfersignal,733, 733F p - E n d o r p h i np,r o d u c t i o n8, 0 3 F Endosome(s) I 583F antigenprocessing/presentation, early,782,784F,791 , 794F a o i c a al n d b a s o l a t e r a7l 9 , 9F epithelialcells,798,7 99F materialretrieval,7 92-7 94 function,696 late,7 82, 784F,794F,798, 799F m e m b r a n ea m o u n t s , 6 9 7 T p r o t e i ns o r t i n g , 8 0 7 F recycling,793F,794,794F,797 -79a, 797F,798F transportto lysosomes, 791-792 volume,697T seeolsoEndocytosis Endosymbionthypothesis,organelleevolutionary origin,859-860 Endothelialcells.1 445-1450, 1447F e m b r y o n i co r i g i n ,1 4 4 6 inflammatoryresponse,1534 structure,1445 t n o o t n e i l n - J I. J / 5 E n d - r e p l i c a t i opnr o b l e m2, 9 2 Energy ATPas carrier.61. 62F seedlsoActivatedcarriersin metabolism;ATP ( a d e n o s i nter i p h o s p h a t e ) c e l lu s e catabolism.70.88-1 03 catalysis,65-87 energyconversion,813-81 5 oxidationreactions,70 seeo/soChloroplast(s); Mitochondria;specific uses chemicalbonds,48-49,49F,69F electricalenergy,69F forms,69F free energyseeFreeenergy heat energy,67-68,69F seeo/soThermodynamics interconversions. 69F.813 kineticenergy,69F l i g h te n e r g y , 6 9 F seeo/soPhotosynthesis p o t e n t i ael n e r g y , 6 9 F Engrailedgene/protein, 1306F,1340-1341,1340F, 1352,1352F proteinstructurevs yeastalpha2protein,138F 438F,1309 Enhancers. Enhancesome,447, 447F proteolyticprocessing, 803 Enkephalins, E n o t a s et z, t F Enteroendocrine cells.1437. 1437F Entropy(5) 66-67,67F,119F

Envelopedviruses,I 497, 1505 seealso specificviruses E n vg e n e ,H I V( h u m a ni m m u n o d e f i c i e n cvyi r u s ) , 1521 Environmentalreservoirs, antibioticresistance, 1523-1524 Enzyme(s), 6, 62-63,72-7 5, 158-159 antibioticresistanceand,1522,1523F catalysisseeEnzymecatalysis classification, | 59, 1597 167 coenzymes/cofactors, energetics,72-73 mechanism o f a c t i o n7 , 4F multienzymecomplexes,168-169, 169F receptorcouplingseeEnzyme-coupled receptor(s) r e g u l a t i o n1, 6 9 - 1 71 , 1 7 O F cooperativity,172-173, 173F phosphorylatio n 75 - 1 7 6 ,1 seea/soAllostericregulation;Feedback regulation structure,72 site seeActivesite active regulatorysites,I 70 tetrahedralintermediate,160 t r a n s i t i o ns t a t e ,1 6 0 seeo/soMetabolic pathway(s);individuol enzymes 159-1 69 Enzymecatalysis, catalyticantibodies,160, 161F d i f f u s i o n - l i m i t e1d6, 3 ,1 6 3 f, 16 9 energetics,72-73 activatione^e(gy,72, 73F,160, 16OF free energy,72 heat energr T4 enzyme-productcomplex,164F,166,166F interactionsee enzyme-substrate Enzyme-substrateinteractions , 6 ,7 8 F e q u i l i b r i u mp o i n t s 7 f l o a t i n gb a l la n a l o g i e s7,4 F kineticsseeEnzymekinetics lysozymecatalysis, 163-165 74F,159-160,160,164F,166F mechanisms, moleculartun neling, 167-168, 168F receptor(s), 892,A92,893F, Enzyme-coupled 921-945 classesol 921 seeolso individuolreceptors Enzymekinetics,I 59-160, 162-l 63FF double reciprocalplot, 163 Kcar,162F,163 K m , 1 6 0l 6 , 01 162F,163 M i c h a e l i s - M e n t ekni n e t i c s1, 6 2 F1, 6 3 reactionrates,159-160,160F,161F i n h i b i t o r yl i g a n d s1, 7 3 ,1 7 3 F multienzymecomplexes,169 steady-state,162F-1 63F t u r n o v e rn u m b e t 1 5 9 - 1 6 0l,6 2 F ymax,159,160F E n z y m e - l i n k ei m d m u n o s o r b e nats s a y( E L I S A ) , 588-589 Enzyme-substrate interactions,74, 164F diffusion,74,75F,163 fit,160 kineticseffects,159-1 60, 162F seeo/soEnzymekinetics lysozyme,164-1 65, 164F substratebinding, 159-160, 164-165,164F Eosinophils 1532,1532F attackon schistosomes, 1556 cytokinesis, l i n e a g ea n d f o r m a t i o n 1 , 452F structure,1452F u p r e g u l a t i o inn p a r a s i t i icn f e c t i o n1, 4 5 1 E o s i ns, t a i n i n g5, 8 5 F E p hB ,g u t e p i t h e l i acl e l lm i g r a t i o n , 1 4 4 0 - 1 4 4 1 , 1441F Ephexin,931 Eph receptors,922,923T,1392-1393 Ephrin(s), 922,923r, 931 receptorsseeEph receptors s i g n a l i n gg,u t e p i t h e l i acl e l lm i g r a t i o n , 1440-1441,1441F seealso specifictypes EphrinA2, retinotectalaxonguidance,1393 EphrinA5, retinotectalaxonguidance,1392 EphrinA proteins,1392-1393 Ephrin82,capillarysprouting,1447-1448 EphrinB family,gut epithelium,1440-1441 E p i d e m i c s1,4 8 7

Epidermalcells,1419-1 420 Epidermalgrowth factor (EGF),1103,1238 uptakeby Epidermalgrowth factor receptor(EGFR), endocytosis,T94 Epidermis,1 417-1 428, 1418F 1418 associatedstructures/appendages, cells,1419-1420 granularlaye\ 1419,1419F 1418-1419 interfollicular, p l a n t s 1, 4 0 2 ,1 4 0 4 F renewalby stem cells,1417-1 428 associatedsignalingpathways,1426 rcte,1420 s i g n a l i n gl ,4 2 6 s t e mc e l ld i s t r i b u t i o n1, 4 2 2 F waterproofbarrier,1418,1420-1421 E p i d e r m o l y sbi su l l o s as i m p l e x9, 8 6 ,1 0 0 5 E p i d i d y m i s1,2 9 4 Epigeneticphenomena,219,471-47 3, 472F bacte(ia,472 in cancer,1208 chromatinstructure,472 DNA methylationseeDNA methylation eucaryores,4T2 oeneticinheritance vs.,219F histonemodificationseeHistonemodification i m p r i n t i n gs e eG e n o m i ci m p r i n t i n g inheritanceseeInheritance 472-473, 472F mechanisms, 473 mono-allelicexpression, positivefeedbackloop, 471,472F proteinaggregationstate,472F silencingof tumor suppressorgenes,1236, 1236F twin studies,473.473F seealsoGenomicimprinting Epigenome,473 voltage-gatedcationchannels,682 Epilepsy, E p i n e p h r i nsee eA d r e n a l i n (ee p i n e p h r i n e ) Epistasis analysis,558-559 Epithelia,1131,1l32F bacterialadhesion,1503 c e l l - c e lal d h e s i o na n d c e l lj u n c t i o n s1, 13 3 - 113 5 , 1134F a n c h o r i n gj u n c t i o n sI, 1 3 3 - 11 5 0 a p i c o - b a s aplo l a r i t ym e c h a n i s m sI ,1 5 5 - 11 5 7 , 1155F,1156F o c c l u d i n gj u n c t i o n s8, 0 6 ,115 0 - 1 15 8 p l a n a rc e l lp o l a r i t ym e c h a n i s m sI ,1 5 7 - 11 5 8 , 1157F Cell seea/soAdherensjunction(s);Cadherin(s); a d h e s i o nC; e l lj u n c t i o n ( s ) ; Desmosome(s) c o l u m n a r1, 1 3 3 t raaln s i t i o n s1,1 4 1 epithelial-mesenchym folding,1142,1143F host defenses,1525-1526, 1526F m i c r o b i ael v a s i o no l 1 5 0 2 - 1 5 0 4 m u c u sa n d .1 5 0 2 , 1 5 2 5 as permeabilitybarriers,1150 sensory,1429-1433 structure,1134F,1150 seedlsoConnectivetissue E p i t h e l i acle l l ( s ) apicafdomain,798, 799F,806,8O6F endocytosis,798,799F glycolipids,62S lgA transport,I 556F membraneproteindistribution,645 microvilliformation,1007 polarity, 798, 799F,8O6F surfacearea,1007 t i g h tj u n c t i o n s8, 0 6 t r a ln s i t i o n s1,1 4 1 Epithelial-mesenchym Epitopes(antigenicdeterminants),1545, 1558F E p i t o p et a g g i n g 5 , 1 4 ,5 15 F ,5 1 6 F seed/soProteintags virus (EBV),1228T,1499 Eostein-Barr Epulopisciumfishelsoni, 14F Eouilibrium c h e m i c a l7, 6 - 7 7, 7 7 F free energychanges,76, 77F Equilibriumconstant(K),157-1 59 r e f a t i o nt o f r e ee n e r g yc h a n g e s , 7 6 , 7 7 f , 1 5 7 , 15 8 E I 5 9 F E o ui l i b r i u ms e d i m e n t a t i o n5,1 1, 5 1 2 F protein, transport,767 ERto Golgiapparatus ERGlC53 929F Erk(MAP-kinase), ERM(ezrin,radixin,mesin)familyof proteins,1009, 10 1 0 F

l:18

INDEX

ERp57,protein folding,737F,747F E r r o rc o r r e c t i o nD, N Ap o l y m e r a s e2,6 9 - 2 7 0 2, 7} F F Erythroblasts, 1459,1459F Erythrocyte(s), 1453r,1459 c o m p l e m e nlte s i o n1, 5 2 9 F cytoskeleton,646, 1008-l 009 development,1454,1457F,1459,1459F seeo/soErythropoiesis lifespanand turnover,1459 membrane,618F,646 phospholipids,626 osmolarityregulation,663,663F r r y l n r o m y c t nJ,6 5| Erythropoiesis, 1292-1293 colony-stimu lating factorsrole,1459- 1460,1462 erythropoietinrole,1103,1459-1460 E r y t h r o p o i e t i nI 1, 0 3 ,1 4 5 9 - 1 4 6 0 c e l l sr e s p o n s i vteo , 14 5 9 synthesis,1459,1460f target cellsand receptors,14607 E Sc e l l ss e eE m b r y o n i sc t e m( E 5 )c e l l s Escherichio coli,14F d o u b l em e m b r a n e , 6 6 5 F enteropathogenic, 1504,1504F f l a g e l l at,u m b l i n g ,9 4 2 ,9 4 3 F g e n ee x p r e s s i orne g u l a t i o n4,16 , 4 3 3 F , 4 3 94, 3 9 7 lactose(ldc) operonseelac operon (Escherichia coli) genome,25F,282 c h r o m o s o m e1,4 9 1F replication,282,283F,284F s e q u e n c i n g5,5 2 size,18T as model organism,24-25,25F mutations,264,276-277 phylogenetics, tree of life,16F P p i l i ,1 5 0 2 p r o m o t e rs e q u e n c e s3,3 8 F RecAproteinseeRecAprotein replication,282,283F clamp loaderstructure,275F D N Ap o l y m e r a sset r u c t u r e2,6 8 F refractory period, 282, 284F strand-directedmismatchrepai, 277 t r a n s p o s o n3 s ,18 T u r o p a t h o g e n i c15, 0 2 ,15 0 3 F E-selectin, 1146 Estradiol,SS9F Estrogen,receptor,891F E t h i c ails s u e sr ,e p r o d u c t i vcel o n i n g I, 3 0 2 - l 3 0 3 Ethylene,957 -959, 958F Ethylenediaminetetraacetic acid (EDTA), 502 Ethylenereceptor,958 Etioplasts,84l EubacteriaseeBacteria Eucaryote(s), 14, 16F cellsseeEucaryoticcell(s) epigenetics,4T2 evolutionary origin, 26-27 generalfeatures,26-32, 27F genomes D N Am e t h y l a t i o n , 2 Z S hybrid3 ,O R e g u l a t o rDyN A , 3 t - 3 2 s i z e1 , 8 1 3 0 - 3 1 ,3 l F n o n - c o d i n gD N A ,3 1 , 2 0 4 seeo/soNoncodingDNA predators,26-27 t r a n s m e m b r a nper o t e i n s6, 3 5 E u c a r y o t icce l l ( s ) cell cycleseeCellcycle c e l ll i n e s5, 0 5 ,5 0 6 7 c e l lt y p e s , 4 '1l seeo/soCelldifferentiation c h e m i c acl o m p o s i t i o nm, a m m a l i a n6, 3 7 c o m p a r t m e n t a l i z a t i o1n 6 ,9 DNA localization,200-201 seeo/soNucleus i n t r a c e l l u l amr e m b r a n e s1,6 9 seed/soOrganelle(s); specificcomportments d i v i s i o ns e eC e l ld i v i s i o n DNA packaging,202-203 seed/JoChromatin;Chromosome(s) D N Ar e p l i c a t i osne eD N Ar e p l i c a t i o n gene expression, 345F,412 coordination,462,463F regulatoryproteinsseeunderGeneregulatory protein(s) transcriptionalregulationseeTranscriptional controlof gene expression

seed/soGeneexpression; Transcription gene structureseeGenestructure,eucaryotic motAh^lir

rria

1AO

mRNAJeeMessengerRNA,eucaryotic protein phospho(ylation,176-177 proteinsynthesisseeTranslation RNApolymerase(s) seeRNApolymerase(s) r R N A s e eR i b o s o m aRl N A , e u c a r y o t i c transcriDtion seeTranscriotion E u c h r o m a t i n2.2 0 . 1 0 7 0 5eed/soHeterochromatin Euglena,tree of life, 16F Eve(Even-skipped)gene combi natorial conlJol,448-450, 449F modularcontrolof expressionpattern,448-449, 4 4 8 F , 4 4 9 F3, 13 7 ,13 3 9 ,I 3 3 9 F Evolution Algae,874,875F a n i m a l - p l a ndt i v e r g e n c e9,5 5 ATPsynthesis, a7O-876,87 1-A72, a72-A75 process, canceras a microevolutionary 1205-1224 carrierprotein(s),655 conservationin seeEvolutionarvconservation electrontransportchain(s),876-876 gene(s),16-17 , 1 8 5 - 11 8 6 c o l l a g e ng e n e s 1 duplication/divergence seeGene duplication(s) seeo/soGenomeevolution g e n o m e ss e eG e n o m ee v o l u t i o n Hemoglobin (Hb), 256-257, 256F h o m o l o g o u sr e c o m b i n a t i oann d ,3 0 5 i n n a t ei m m u n es y s t e m1, 5 2 4 major events,874F metabolicpathway(s), 870-872 aerobicmetabolism,12,27, 873-874 carbonfixation,872-875 molecularseeMolecularevolution m u l t i c e l l u l a r iat yn d c e l lc o m m u n i c a t i o n9 ,5 5 mutation rateanalysis,264 seed/soMutation rates m y o s i na n d k i n e s i n s1,0 15 - 1 0 1 6 organelle(s),697 -699, 7}OF seeolso specificorgonelles p a t h o g e n sI ,5 2 0 - 15 2 1 plant(s),36, 840-841 proteins5eeProteinevolution s e x u arl e p r o d u c t i o an n d ,1 2 7 1 - 1 2 7 2 ,1 2 7 1 F tree of life,15-17, 16F,23 seeo/soOriginof life Evolutionaryconservation,I 7, 17F cytoskeletalelements,982-983 genomesequence,39-40,207,208F,246,250, 292 h i s t o n e s2,13 homeodomainproteins,138F,420-421 m e i o s i s1. 2 8 0 ,1 2 8 6 m u l t i c e l l u l adre v e l o p m e n t1,3 0 6 F1, 3 0 7 Evolutionarytime, units,265 Evolutionarytracing,proteinfamily binding-sites, 15 5 - 15 6 , 15 5 F E x c i s i o n a speh, a g el a m b d a 3, 2 6 Excitatorypostsynapticpotential(EPSP), 688 Exocytosis,750F,799-809 constitutive,800, 801F defaultpathway,800, 801F e x t r a c e l l u l amr a t r i x 8 , 00 neurotransmitters seeSynapticvesicle(s) proteolyticprocessingof cargo,803 regulatedseeRegulatedsecretorypathway secretoryproteins,800 secretoryvesiclesseeSecretoryvesicle(s) Exon(s),346,347 gene structure(eucaryotic), 206 l e n g t hv a r i a t i o n3, 5 2 ,3 5 3 F r e c o m b i n a t i oinn e v o l u t i o n2, 5 5 s k i p p i n g3, 5 2 ,3 5 5 ,3 5 5 F " E x o nd e f i n i t i o nh y p o t h e s i s3i 5 ' 2 ,3 5 3 F Exon-junctioncomplexes(EJC), nonsensemediatedmRNAdecay,386 proofreading, 269-27O,27OF Exonucleolytic Exosome,358.485 Experimenta e lm b r y o l o g y1,3 10 - 1 3 11 , 13 10 E 1311F Exportin(s), HIVmRNAtransport,485,486F Export-readymRNA,327,357,358-359, 359F (DNA)sequencesseeExon(s) Expressed Extracellular matrix(ECM),117a-1195 b a s a l a m i n as e eB a s alla m i n a

cell adhesion/interaction seeCell-matrix adhesions cell secreting/synthesis, I 179, 1179F components,1165 fibrousproteins,145-146,147F,1179 seeolso specificproteins shapes/sizes, 1166F seeolso Glycoprotein(s);speciflc mocromolecules compressive forcesand, 1180-1 181 d e g r a d a t i o n1,1 9 3 localisatio1 n1, 9 4 matrixmetalloproternases, 1194 serineproteinases, I 194 d i v e r s i t ,11 7 8 exocytosis, 800 matrixreceptors,1169 seeo/soIntegrin(s) m e c h a n i c ai nl t e r a c t i o n s1 ,1 8 9 ,11 8 9 F1, 1 9 0 F f i b r o n e c t i nf i b r i la s s e m b l a y n d ,I 1 9 1 - 11 9 3 , 1192F p l a n t ( s )I,1 8 0 ,11 9 5 seea/soPlantcell wall tensileforcesand, I 042F,1187-1189 tissuemorphogenesisand repair,I 180-1 18t seea/soBone;Celladhesion;Celljunction(s) E x t r a c e l l u l sairg n a m l olecule(s)/pathway(s), 880-881,880-886 c e l lr e s p o n s e , 8 8 5 c e l ls i z e l n u m b erre g u l a t i o n1,1 0 2 classifiedby rangeof action,881-883 co,889 c o m b i n a t o r i aalc t i o n 8 , 84 c o m p e t i t i o nf o r ,1 l t 0 development,gonad specification, 1283 growth factorsseeGrowthfactors hydrophobic,S89 i n h i b i t o r y1, 1 0 2 ,11 0 3 ,1I 1 0 ,1111F mitogensseeMitogen(s) Nq 887-889,888F survivalfactorsseeSurvivalfactor(s) seealsospecifictypes/pathways E x t r a c e l l u lsapr a c eI, 1 6 4 Eyo(Eyesobsent)gene, 466F Eyecolor,as polygenictrait,563 Eyedevelopment, genetic control 466, 466F seealsospecificAenes gene/protein, 466,466FF,1306F,i352 Ey (Eyeless) Ezrin,1009

F FqFlATPase seeATPsynthase Facilitateddiffusion,653 FACSseeFluorescence-activated cell sorter FactorV,secretionfrom ER,767 FactorVlll,348F,767 Facultativepathogens,1490 FAD/FADH2 citricacid cycle,98, 817 electroncarrier,818-819, 819F fatty acid oxidation,97F structure,99F F A K( f o c aal d h e s i o nk i n a s e ) , 9 3 71,1 7 6 - 1 1 7 7 , 1176F F a m i l i aal d e n o m a t o upso l y p o s i cs o l i ( F A P )1, 2 5 3 , 12 5 3 F Familialhypertrophiccardiomyopathy, 1031 FanconianaemiagroupsA-G,DNA repairdisorders,

29sr

F A P( f a m i l i aal d e n o m a t o upso l y p o s i cs o l i ) ,1 2 5 3 , 1253F (fibertracts),neuronal,1387 Fascicles Fasciclin3, synapseformation,I I 47 F a sl i g a n d ,1 1 2 0 ,1 5 7 1 ,1 5 7 3 F1, 5 9 4 Fasreceptorprotein,1571,1594 FASTA sequencealignment,proteinanalysis, 53 1 Fat(t composition,58-59 seeo/soFattyacids digestion,96 energysource,91, 94,96,824f storage,8F,91, 94 structure,97F seed/soLipid(s);specifictypes Fatcells,625, 1474-1476, 1475F,147 5FF g e n ee x p r e s s i orne g u l a t i o ng,l u c o c o r t i c o i d4s1,5 Fatdroplets,1474 Fatproteins(cadherinsuperfamily),I I 37, 11387

uulricuuuJ/d/YuuuJ

5ucdu

54 .dlqcl

c ul rdrdJ f c q+!m srdLluilu

ducu

:^la^rln)asuo)MolloJleql sarn6uol ll ue qllm 'arn6!Je ol raler I ue qlrM sraqurnua6ed i&lua aql jo uorssn)srp lxa] rofeure o] raral a)erploq ur sJequrnua6pd '186b 'aueD L ' J S ] Vl t S € ' € 8 € ' 6 u r g r q s a u r e r ; L 6t t J 9L LL 'L L Lt - 9 L t I ' OL LL ' s u o r s a q pl Pe) o l 'lz6L '€5 'sl)alje uorsual L I 1- 16I t t9Ltl Jt 6t t l e u o r t e l s u e r l ' s u t e l o( p r dt s d e )l e J t A6)e g ' L t t t g L L J ' 1 € 6 ' ( ) V l ) a s e u r )u o r s e q p e 989'sl)arJa6nrp anrl:eoqr(sd's.rotda:at Z6tt'puesuu6alur . 0 0 9 t , s l l a rul er er ooll ',tgg'alo.lrelltursuPlloinau ^lquresseluqu J 0 0 9t 808 ggl ,ansstl tllauuo) a ^ (pDe)u^tnqourLue-,t) | r L|S' L}S-q8S'sa^plua)saronll VgVg ,^rlsruaq:o1i(> g g t t ' P u t u e ll e s e q zg 6o;eue aP(r aas aP,{r aseqd 1uarsa.ron13 t69 'r65-t6S'J96'9ZS ' 9 9 0 '1|a] 'ur])auorqrl Z g O t' L 9 O L , lla) ] u r oJo dlraq) l5 [ [ l96S S 0LL t '(IlUl) ' 6 u r p u r qa l e ; l n su e r e d e q ' s u l l l AW l - S/ 2/ L9D rajsuerl^6rauaa:ueuosara)uelseronll € 8LL 2 9 0L 'Eng'J965',569 '28€ ' r u a u d o l a ^ a p6 u n 1u r ' 9 1 3 9 3 )ESL1bV9 lz8€ t t - t 8 €t s 0 I r - € 0 I 1 ' 9 9 0t ' 2 9 0L ' l p f - s l 1 9 '(dVUl) 6urq)ealqoloqdraUeIra^o)ar a)ua)salonll loJluol a1:(r 11a3 la;:{: aasal)^) aseqd lD 1 0 0 9 ' 6 6 9 ' , l e u r a lluerl o l 1;a; lla) }o (l9l) rorleJ qlmor6lselqolqrl 9 l g ' s r s ^ l e uupo r s s a r d xl a a)-al6urs 290!'sull)^)-LD '18S'slop lrunluenb Lgbl itedej punoM ul 1885 s0t r-€ot 1'9901'290t'lpt-rD ,slla)iej ol a;>It 11a3 aasa;ti() lla4o oseqdoD l88S'185'sal)ruedoueu lslt t ' 9 8 S' s a d o ) s o r ) r r u (9V) 9 e]lac a6ueq) i(6.lauaearj aas(9V) 9 eilac og tLVt-g97L'auoq pue a6e;rg.rer I t65 !98S uorleu.uojsueJl l 6 8 S 3 8 8 S ' , 6 8 9 - 8 8tSl 8 9 ' a ) u a l s a r o n U o u n u r u r l '18s-98S'sa^p /slpadar)uor.uolol'a)ua)sauas aArle)rldar 9 lua)saronu €62 llSs /solnqn]oJ:rur uorlezrue6to /le)oluo) l8t0 [ )z6s' J L6S'Z65-06t ;o 'arnleurur 'suorle:rldde pue eJnlPtU tgg 89t L suotsnJ lsOe]utalotdoslDaas lonptNput 69S-9gS'(dorsoltru.ra)ua)salonll 89bt- LgnL'suorDun, 'uorsnj 'snltA auelq|.Uaul 59L-VgL s13 'fin9 J 6 L Lt ' 6 L LL , s r s a q r uy( 1 ' t t 9 ' ( d l l l ) 6 u r q : e a l q ol gEoSqL s u r a l o r du o r s n 3 udr s s o la ) u a ) s a i o n l l 6 911 , u o r 1 e z r u e 6y.1r o3 3 g 9 nf L 9 nL ' V g V ' s a 6 u e qp)u e u o t l e t l u a r a # t p au.losoruolq) L€tL'DltrldosoJo'luelnunzuDt tqsnl 6EZ',sJJnd 'suratord '(HSll) uoud uorlezrpuq/.J4 nl$ ut a)ua)satonll a]^)ojpuoql 86t 69VL-ggVt'otu I uorlpurjoJsuerl '>rua6oqted 95tr'1 ItOS'^do)soDil.u]se.rluo)-aseqd'ernlln) LZL'8rZr llel 'Z0S'(S)Vl) rarros I Spale^rt)e-arua)saronll ,6ur;nner: g E - 6 7 ' u r 6 r . (r toe u o t l n l o ^ a Jt0S 9961 llar ;1at y 6 9 1' u r s r q d . r o u r p (s)iselqorqll 98S a)ualsaronll 'uorl)npord ) r t o r q r l u e t8€ EB€ ,92'ap) uorlelnu'sap!ldadouuqtl y97'saprldadouriqU'uorleArlre'ua6ouuqrl r6ung '(]Ul) .uorlPrxloJ,uuqtJ LLS-695'slnoI)ou)i pa66e1's:ltuoua6;euor1>un1 laorPleseurquro)ardll 8t€ t 'gz ' r z z L' a l e re u n l lEal t6tt-06tt'u!lluq!l J 6 V tL ' g F t l ' e s e u r q u r o ) adrl l 'asereurnl (s)tueldaasslueld 6ur.ramo;3 l€z I l s 9 t ' u o ! l e z r l e r orl e a p n u ' u u e l l u q u 'sauabio]lalas )r]oauoq {q uorle>r;oads (saduqil n6nl) qsUraJJndaassaduqil n6n! (s)ue6ellofoslDaat It t' [ sdurndad^i-l 1099'099-6S9'(sasedlv) n at L-Ew a'€ Lt L'luaurdole^ap 6 8 t l - 8 8 [ t " s ^p a l P r ) o s s P - l u q u 't66-6g6,ura1ord '(s)raMoll l1686 1686 7s13 I t0t t 1 9 8LL ' G g lt - t g 1 1 ' 1 s 1 u a 6 e 1r1eol 1: u q r l '090 '919'^rrauorl) Mog 9L E ' ! 9L t ' ( 6 u t p u r qa u i o s o q ue) l r s - l L-6SOt 1090t 1 6 8 tt '98tt ^poq '68lt-1,81t'S8t Jatso60uDlau Dlqdosotgaas$1ynt1 ueunq Jo 'lprqoj)il.uleuuou 'e.rolj t0SL t ' ( s ) u a 6 p l l op) a l e D o s s e - l u q U 'lpuornau'(sal)!)seJ) stlert reqU t t'aln])nlls'asol)njl l Z'areqdsoqd-g lgt t J88€L'88€t'a)ueprn6 'pror 'aleld 'ureloro qlaror6 asol)nil auo: ur lool3 l0z t u€ I leurdsJo 8l9l ' a l e q d s o q d s r q'-L9a s o l ) n i l (l9l) ror)ej qlmorolselqorqu aasl9l I O ZL 9 9 n ' f g g v ' l S t ' q ) r r M sd o l r - d r l l 'suor])asuazorl 'ra^al ( d l l l ) 6 u r q r e a l q o r o qudr s s o la ) u a ) s a l o n U ' d l ' l l s89 '€st SgtL sAeDlsndouaXoslDaas a1oBA7oslo aas ) 2 0 9L ' Z O Sl ' u o r s s r u J s u ea.nr l6 e l d ' s p a l l 'uorleutol alo6Iz j € t t , LL r , ( s ) 6 0 r l 8 9 t t ' s t t t l 8 t t t ' l € L t ' u r a l o r do 6 u r u r e l l 6971 'SSb-nSn's 'ofuqwa sndouay 6 g g1 ' , { e m q r e6du r l e u 6 r s - l t r . r e ; o d luauta6uelear aua6 JSgn 1991 'urlla6ell g g 1 ; ' { 1 u e | o dl l a ) l e u e l d j z 0 € [ ' u o t s n ]t e l l n u o r d € € 0| 'VZEI'luaurdo;anap 'u)eds ',uo!le]!)ede) suo6ala SZtt 7 162[ E 6 Z L ' E 6 Z L 'uralold/aua6 palzzul 'sernpelordpalelarpue f9 t € L €0€ l- to€ I I tt0 t Alrlrlor.u llAD otltAut 'saspla;suertl(so:,{16 '10€ 'saloulua)paleuop-ulads 1t6 L E 0 [ ' , U O S u e d ueor)l r ) 1o (;ruel a6uul I I t6t t U-lul) l6€8 l z 0 € L t L 0 € t ' 1 0 €L ' u o r s nr,e l ) n u o r d 'OtB-688' 'salerqa]ra^ur la;suer1'(6rauaa)ueuosara)ua)seJonllaarllUl auueur f t Z 8 '( Z g - L Z S ' s l u a r p e ur 6o l o . r d l6Z [ uoJtlalaqlla-azaall uorsnl66a-ullads 1919'(do>so.r:rr"u t'!En6'k6'Jb 1 0 6 ? t ' € € 0 t ' l e u a l r e q 66Zl-867t'ursrueqrour ' t € 8 - z € 8 ' s l e l r p ear a r l '00€ '662I 'uotl)ear ztst'vL8 t€01-1€01'ellaDelJ 100€I I le)ruol gL- LL'suotDe4 ;erluanbas 00t!st'€1'€L'ueborlru 66ZL 'S09 '99g 'uorleredarda;duLes ' l ( 1 6 ' Z L 6 ' u o r l e ^ r l l e6 6 a p u e s a s e a t : u r {dorsoLlru.r lszS'sz8 t81 !sI'6urldnof uorrfear lS09 ' 8 6 2 t ' 1 6 Z ' u o r l ) e a ra u o s*or le: )e u o r l e x uu o q l e ) a a sa p t x o t pu o q l e ) 9l'sl)aJJa uotleJluafuol uorlleal t )6621 uollexu fll'91'sluPlsuo: utnuqr;rnba )8621'a6ZJ 'raJsueJl 'LgZL uorl)ala 6Zg I O L Z l ' 6 u r p u r qt r r a d sp u p u o r l e n r l : e6 6 a ' p u eu o r l ) n p o r d a r J l L a l ' t L e l sa 0 € 8 ' s l e l t u a t oxdo p a l ' u o r l e l n l l e ) 1 6 ZL ' v 6 Zl ' p u e u o r l e t n p d e ) l p n x a s ' ( , { l e u o t l n l o s^ a ) ulU '(DV) ellaCa6ueqr i(6tauaaar3 aquod sa>lwotoqDDsozlps osp aas J6lJ'aL-SL D €o€.1-L6zl'uortezrlruaJ 'pa)npal '^ttltual eoeqs Sgt ^)etn))puotlelsuell L0EL Itz sluelnur ,sqdorioqltl '190 'el)I) '7q,A 'U 17q,Q lXOOUral Z! 1950I t-9s0 L lla) sn sqdotloioqd sn sqdotloue6to.roJ,sallnos uorssrl lsee^ I Zs8'ese]rnpr-rlj jCVrrrlu,iofji,, tl 'uolle)Urpourasdeu(sto; 'a;nr6urr13 srs{;o>{19 osyoaas I L aJ!lro]aruPuoon_rl 56€[ '6u11:ed url)e JSZS'SZS-Vaq luaLuelU 1 7 6 6 196'sriemqred alqero^ejunsnalqelo^eJ urpe 391',SuOtlleal rLo} L' Loot'6ur>1ur;-sso-D 0 6 ' u o r l ) n p o J dp r ) el r l ) e l 'slla) ( 9 V )9 e ] l e 6a 6 u e q :{ 6 r a u aa a r l a a su t s a 6 u e q : uuquru tsarFPa t18-018 srs,{1e1e: au{zu3 osloaas 106?t'aPuquu 6€8'06'(s)uotlplueural 'elnl)nlls 'ulelord lgl'ZL'sts(lel€) 68€ r-28€ I 9t9 Vdal , 9 4 - 9 4 I 6 r a u ae a r 3 'Lgt lJGl l-gt f L'6n1l'euo: qMnor6;euornau6uuaals 1 8 [ € s ] u e u r a l Ia 'srsaue60r6ue ( d V U l )6 u r q ) e a l q o l o q d 6tt t !6Sr' 65l'sttrD)u uorldu)suell'sdoolpJeMroJ-paal 'uorleal)nu tarye{tanotat a)ualsatonll aasdVUl saoP)sef l u e u P l r Ju r l ) e 966 ,900 ,erpodolu (ro1eln6ag1(uorlrqrqut 6urgrqsaLlet1 ItO t [ l)pqpeaJ) '800t ^ltltlor! l e u o r t e l s u eat fa sl e u o t l e l s u p l l , 6 u t U t q s a t u e j l 1600f llel leurouqe'ssol 1>eqpae;anrle6aNiuotlpln6etlualsollV os/Daas 'uorleuro] qaM uolleuorl)PrJlla) aasuorleuotltpll luaureluurlle 600t-800 t l)eqpaaJe^lttsodaasa^tltsod '(s)llo) 'tdxot '6utlled t19 L tuauJelUutl)e t ^rol€!6e.l lt66 8 € 8 - l t 8 ' l L t - O L t ' s ( e r n q l e dl r l o q e l e u r E e t l Z Z E l ' s u o 5 a l a) u t , l l a )l o p u n o l ururPlu L 11 - 9 11 . ' u o r 1 e 1 n I6)ae.qr p a e l _ 'suosuedulo: ,plo)aj aarl 11aue6o1r{qd stueuelt, 8?Z ltssol letalalsol,^) 6 5 5L 1 9 9 9r '9ZS '9gg'auruorqlau;,{u.rrog 'ssst'!n99 lsluaruelUurI)e/urllv aaslelalalsoll)'sluaL!eltl ItS t | L l z s g t ' a l n r a l o L u{ p o q r r u e ' u o t 6 a)rl ,J666'866 ' u ! a l o r du u 6 6 e l l l ',SrSOlA)SUerl t60t !000t 900t J16l ' , u o u e p !t66,urrillol t xpoD e ) r u l l o l ' s u n 6u o t s s t u ap l a t l ggl'alol srso/>o6eqd ILLg tLg 609'VO9 '965 '9SS '(s)ro]da)errl anssrt'ap^qapleLulol sutalotd)UDadsosp aas 58S'uotlexu r tSSSL 1665L !96S L [ 'uot]eln6at 'suteloldxoq-l (y\l)l) xuleui lelnlle)ertxl aasw)l llts,s)tlaua6 )tsualol t90 [ IJS ,16 ,a6ero1s 'yo1 ( s ) u a 6 p l l oaf a su a 6 e 1 1 o t 95,_95, 96 1i(:ei{ge3 l ulstloqele)os/Daas g g I t / P u t u l el e sadll )!]pad.sosp aas seq '€O '^6raua6ururelqo surelorosnorqrl 100t t-gg SS8',srsaqtuAs 'asno1i(11;rqe 'lpadar uotlnlo^a,alqeluaula]uou 719 u r l r a u o r q l l a d , { 1 ro t 6 L L ' 1 6 t L J L 6 ' L 6 9 6 'uotPptxo ' a r1n1l l1n r l s seln)alourpool )z6ll'l6L L J96'uorlezrltqou (s)prdrloqdsoqd 1(s)auerquaW oslDaas 0'6'u!rersrllol L 6 L t , L r , | o r a l q n l o s ' s a l n p o uul t a ] o l o z 6 zt ' ( H s l ) a u o u J l o q6 u r 1 e ; n r , uar1l s> r 1 ; o 1 [tt l 6 S ' 6 S - 8 S ' s l u e u o d u l oa)u e l q u r a ulll a ) , s l l a a) l ) r l l o l ' J l r o u I e r n6 u r p u r qu u 6 a t u r 's6 '5s-89 'spr)e^llel J O 6 Z \ ' t 6 Z l - O 6t Z €6l f 699 lls I t-t t I !8s

#feJ;J,11'3:%i'."

Jl:i:HI i] 68 1t !,"rr?3;38,s"

6[:l

X]CNI

l g g s ' 6 u u a e u t 6 u a) t l a u a 6 ' l u a t x a l e l d aaru a g suDtotd)lJDadsoslDaas uorssardxa eua6Jo lolluo) aasuot}eln6aluot}du)suell leuortdr.r)suP{ (lllf ) lereua6,srol)ej uolloulsuerf aasslolle,|uotlou)sueJl saq)lrMs)rlaueDaassa]rsuot]tu60lal lolluo) ler/oleurqurolaar suorl)eralururalold-utaloid suorlreralurvNc-utalold :(suralord)sJ!rou6urputq-VNCoslpaas t6Lb ' 6 L n - gb ' J L L n '9 [ ? ' s u o r ] ) e r a ] uvtN c - u l a l o r d (s)rolp^rl)eleuotldu)suplfos/paas JqEV'V€r' lo Jluo)e^rlrsod (s)utaloidrosseJdau os/paas ) 9 E VV ' w' ldruo) a^rle6au suraloJd^roleln6alaua6 lualel aasluelpl lorluo) leuoleutqulo)os/Daas tszv'szn-nzn' uollpztJautpoJalaq (lllI) leJoua6's.rol)eJ uorldu)sueJfoslDaas

s9v-n9n

'JWV' EVb-nnV'A6rau,(s leuolldrrtsuell (s)utalold.rossardag aass.rossarda.r ' 'uor1eln6ar Jlsb L;V-OSV luaLudolanapoyqdosotgosloaas J99V9 ' 9 n ' p u Pl u a u d o l a ^ e pr e l n l l e ) r l l n u r J$nn'gnr- Lnn'Jztt'zti'dnpu relnpour 'J;W 'Snn '18€' 'sr!srueq)eu )gnn lZtt rurel.rofer,u 1gg71.'sar1 lEgn' E9r'uraloid roldelar pro>r1.ro:o:n;6 ' 'auosaluequa )LVb LVn-gnV t9t-€9t'seuapeq aueb ueatgsumop l€'Alrsra^rp )finn 18W'8tt'uorssardxa leluauldola^ap JV9V 'v9v'Jtgv' t g v - z g t ' l o r l u o r e u a 6p a l e u r p r o o ) lorluo) leuoleurqwo) osloaas 99r-n9n 'JJ xa;dtuo: Lbb'LW' LVn-g7y(;qu-rasse (s)JoleArlle leuorldursuerlaassJolpAtl)e wn-jnn' vEv' 6ot-65€')rlo^rPrna s11or.u 6urpurq-yNCoslDaas r0*' oE -6zn'uorleuruilalepa)uanbas gz?,uotl)tpalo ' a)uanbasVNC Ig ft 6Ln-g h' uor11u6o>ar 9 [t'Ara^o]srp J g Z n ' B Z n - L Z (Ve s s e1 ; r q s{ g r p q o u; a 6 J0*'09b-6zn'uorlPurujralap a)uenbasvNc JZw /Z€t'uorle]rdDerdounulu.rr u rleuoJq) t €t $Zn' 6ZV-gaV(qder6ogeuo.rqr(1rur;,re s a n b r u q ) asl r s ^ l e u e ISLN'VN-6LE 'ltrE' E v F . ' z z z ' ( s ) u r e l of tr do l p l n 6 a ra u a g ls€'9€ '6ur1apou l e ) ] p t ! a q l e u ' s l r n ) r ) ^ r o l e l n 6 aar u a g uotleutquo)aJ sno6o;or,uop aasuorleurquoler lereueD '^60lorq 'uorlellr)soauaD ]r]eqluls I [9t ll9s'uor])nporo /89S-99S/asnou )899'JLgS J LS-69S's)r.uoua6leuor])unJ 019'sallasse)uollalap slnolloul aua9 sa!llure,u!alojd os/Daas sauaourqolDos/Daas )9SZ' LSZ-?SZ'seur6urqol6 S 9 Z ' a ) u a 6 r e ^ rppu e u o r l e ) r l d n p lz-51'uorlnlo^e

Lnz'€z

'salo^re)napue 'eualleq /eaeqlleol uoururo) s a r l r r ! Pa,u a D luetudo;anap oltLldosd1 :sgnd aurosor.uorq)os/Daas uorssardxa aua6 aasleuo!ldUlsuprl Jo lorluo) ;euorgdr.trsuerl (s)urelord {ro1e;n6arauag aassurelordLrole;n6a.r Slb'J6 Lt'lorluo) ^lr^rl)p uralord uorleln6ar aasleuorldu)suerl-lsod leuorldr.r)sueJl-lsod '19? 'se)r^aptuo]Pllr)so I [9] LLV-9Lt ,:astou,, E6n-Z6n'SLn'J6l€'lorluo) uorleper6apyggtu fiSn'gEb'sa)t^op^loulaut gZE-SZ€, 'uorleurquo)ar r U r ) a d s e l r u s o r lelnd!ueur rEgn'tgv 'uorleln6arpro>rl.ro>o>nl6 'leuoutoq L? '9

tt

'Z

6urddeu lrlaueg aasuotleztle)ol saqllrMs)llauag aat saq)ltms:l}aua6 aaspatuudtul 6urlurrdu-r)ru.rouaD (s)uor6aJ aua6 auag aas suor6er 1ot1uo: lorluol 'uortnlo^a spoLlpw )ut)aos oslDaas ott-6€t /l0Z'uorlelUrluepl ' a l o ra r n l ) n r l s ' Z€ J O 9 96' Z V 6 vNc ) L L b 'L f t - g f t 'n9n 'Jzgb 'Egl-zgl 'lo,tuo) peleurproo) g n z 'i l Z ' J O Z 'L Z - O Z ' s 6 o l o u o q Jbgn 61,-gl'saua6 6urlslxa-ardu.rorjuotlelauao s w't6LE',soalsalqellolluol '(6o1ouroqa)uanbestuor1uorlrpald 71 isgn '?€t '6urdaolasnoq 'sgn 'jEzb 'gav 'qzl-tzl 'lorluo) 0l' leuolPulquio) g l S ' s u o r t P l n up e l a 6 l e l JLgn'66n-LW'uOlleln6eJuotssaldxaauag uorlelsuerfluorld|Jsu".l]-osp aas 01s'slnol)oul pa66et ESS ztt'sad^l lla) uaaMlequollel.lPn '71-97 'ad(louaqd luelnu uo.l, uolDnpap g$'fvtt'sl!un uotldtt)sueJl 'pessaJdxa auoue6 9 7 9 7 ' s r s ( l e u e lelruraq)orq z It Jo uoluodotd 'a)uelsrsar )rlorqllue l8 t€ l5t€'lrlo^re)ord ' ( A l d )u o r l e 6 a u el^l a # a u o r l l s o d (s)uorl)unJ Jlzz'LZa uotsSaJoxe aue9 aasuolssaloxa J L ( Z 'j z a ' s l ) a J J au' uoer lur jsnoqd 6 l ' u o r l P ^ o u uftue u o t l n l o ^ e z It epo) )rlaua9:elnlrnrls vNc os/Daas 6 u r : u a 1 rasu a Dl e u a u i o u a q d 'pue ernllnrls rlaua6rdl luorle;IqlatuVNC oslpaas vNC zoz'00z-66l [8t-08t'uorle]rJrpoLu 9 9 V 'l g s n ' g s n 'n g ? ' ^ l rl r q e l u a q I g;-6gy'1da:uo: jo ^lolslq )rlo^rerna ! € 9 VZ ' g VZ' t b 'J S ' t € ' '^)ueDUJo r n s g '| n n ' b o z ' s 61 , 8 _ l , u o l t l u u a p lZtE t €€ 6utddeu )tlauag oslDaas (s)uot6ailorluo) euag oslDaas 'uorldu)suprtrol s;eu6tsy116 J V g St'b ) Z ' S 0 Z - t 0 Z ' u o r l e ) olle u r o s o r ! o r q ) ot€-9€€ saua6;e:r1u:-ra:ue)aaslelrur)-lalue) J g g VB ' gVL _ g n ,p u e u o r t e l ^ q l a uV N C aasu]d (ufd) uorl)eerureq) asPraur^lod uorlelnoar 'seua60)uolaluPl u! uorssardxa auag aasuot1e1n6ar7;otluo: 6EZl-L€ZL uorler!]rloure urleurorq)oralaH (s)auaD 1 6 u D u a ;ar su a g l u o r t e s u a p u o ) s e r p o qp a | o ) J o r u r u r a 6S) W 3 9 auosot!oJqJ iutlPulolqJoslDaas J g g € v' g t ' V g E- t g t ' ('fl)dv ^q uor])nilsap'uru!rua9 690t-890 t 0zz' alc, ullPtxolq)olalaq 6ulllo;g aasra;suel11a9 982-982'uollesuapuol u lleuiojq) 'urloslaD 000t 3t66 6EZJ-LEZa'puera)uP) gtqs rQtltqou-;a9 a u a 6r a l l o d a l lBZv'gzn'szn- L7y' Aesse J Z L g ' ' L S - Z L SX e s s e l s € s ' u o ! l e ) u l l n du l a l o J d J s L g ' S L 9'SLS-V ' s t s ^ l e ruaPl s n l l 'IVLS Itt9 sa)uleui L9 JSLS ' uotlelll!r-laD Jb LS'a L9'Iqdel6ogeuorq: L t s ' l v l v ' a l b ' s € - ? € ' s i ( e i l e o r : r r uv N c '900 'suralold6ultu.to;-;a6 s)llaua9 os/Daas t 1600t 08tt-6Ltt'pues9v9 t99'saq)eordde (s)le9 f i L V ' e L V l 9 V'srs^lPue d-CZ l 0 g ' r a l u n o lr a 6 r a g 9LS-899 !l' tt ro])eJa6ueq)xaaplloallnu auluengaasll9 r00z'J66L'9'uolssardxaaua9 'utalo.td saua6opnas6 tu:D t6b-O6V'JVaV'Igti ( s ) l e u u e qu) o l a a ss l a u u e q lu o t ' 6 u l l e D i^luepunpal )rlauag lsarlrure, auaDos/, aas gg7 'sautoua6qsUelqaz tLoL'001 'tuseldol{r pue snal)nuuaom}aq'uodsuerlpa}eD l6€'6€-8€'ar"uoua6sndouaY gsrL !r9? r-8Li't-V]VD auaA 6 € - 8 8 ' u o l l n l o ^ aa l e i q'seter luaudo;anap srrraDlsndouaXaassndouaX ssz 'urqlrn ees t l l v L ' L vL - O Vt ' 6 u t g ; n q sa l n p o L u r a l o r du l ' ll0€ 'uor]nlo^aaseull ulalold ur6uo 1n6 JLOETLOtL'(q 'uol]elnllse9 9l [ l0E [ v s z - t s z ' u o t 1 n ; o neau r o u a 6 'elnrlsPg sallluej aua9 os/Daas l€9€ L 'uo!]e!)osseuo[d ]aDoqollaH 'la)ue) )u]se9 €os L SSTvSe'gvz'JOl' ;D''latua^!pleuorlrunj 'Uol]erlaspl)e lll]seD 'srle)!lonpjo orej 9€'[ 9SZ foot L'salo(.re:naluaJaJJtp 9 8 1L - S 8 tL ' s a u a 6u a 6 e ; ; o r ,tole1n6eraue6 6gy9 g V a ' ! 0 V ' J 6L ' 8 l . ' ( s ) u o r l e r ! l d napu a 9 ur sraqueur r(;ttue;'suralold / s s o l a 6t o s s a l d d n rso u n ] u l a u 0 9 t L - 6 9 tt ' d ! q s u o l l e l eur o l l l u n J - a r n l ) n r l s l9€z [ 'uotslaAuo)q]]eulsll.rj surxeuuo)oslDaas lS [€ 'g 'suotltun[ , { e P 1 ; 1 o ; l09t t !65t t't9tt-691t'6SI L'ern]rni]s St € - ' t € 1 g ' u o l l e u l L u l e l aapz l s 1 6 9 t t ' 6 s LL^sA I t t - t L€ ' u o l s l a ^ u o e ) ueD elPulsapoulseld (s)utatold^loleln6al auag oslaaas t9 LL ' s l e u u e u ru l e 1 n 6ra{ 1t; tt q e a u r a d l z g LI ' Z g t J - l g L 1 ' u o ; 1'lla) J Z S ,\ l S V ' ! j s n ' € S ? - O S ' al)rlloJ-eM)oo 06ZL J6tt'q7r'@ua6 a^a)orydosot1 ul salnpotu(.ro1e;n6al G S It - 8 S t 1 ' 6 u t ; d n o :)' sr luooqrel ]luenuJ tgl [ sluaulalaialouloid aasslaloulojo 'zst-09? 3u)l 6s t l-8S I r'6utldno)le)rrllele I t s? t're^ouJnl/s)lLueu^p logt t'L9t t-o9t u €9t lzst'srolelnsul ' n r l a q l t d ar e u t u n ; o : I n E L L ' n E t1 lSEt',sraruequa '(s)uor6ar / 9 9 ' s u r a t o r dl a u u e q ) !Lnn'ovv!8€t l o l l u o )e u e g 6uruo;:y1r16osloaas 1t88',t88'uollP)lunuulolle) t t ] u n [d e D t9 t f-8St t'(s)uo 775'qreotdde,,un61oqs,, '8€€ ' E E E ' s a u e 6d e 9 [ l-It€ t l8€€t !1€€ t ) g V S 'g n 9 - r n 9 ' 6 u r u o ; :9 1 6 (sdV9)sulalord6urlenrt:e-asedlgaasdV9 3 y 9 ' 6 u r u o 1ar u a g 829'seueiquau I L9t'))oP auaD 629'!8a9'Lw\ t9t-€9t'uralord i { r o 1 e 1 n 61aer : r l r r : , { p lslaprsor;6ue9 q a } ? u r p r o o l ' s a u a u Paqu a g (pr:e :ur{1nqoutue-1,1 (s)ioleA|l]e aassuralordJole^llle auag vSvD aas(v8vD) pDe ru^lnqouluie euluieg ;euorldrr>suerg eozoleuiad5 :1u.tnno\661 osloaas t1aua61aua66utuwbaqsauluaosloaas aasuorsrntp1;a:i(q uoll)npold srsora1A1 9 E € ' s l t u nu o l l d ! l ) s u e l l ' , a ] e l ps l o l o e q ernl)nJlsaua9 aasalnl)nlls L S Z | ' Z L Z I ' 6 9 2[ '(s)elaue9 692L 6ut:uanbesyp6 aas6ut)uanbas aassaua6oPnasd seue6opnas6 l€11'sapueq))eso6tp ; oa 1 u t l - 1u1t ' a s o l l e l e g l8z9'aprsolqererol)elP9 117'197'y7-97's5oprcd 'anbruq]el Svn/t]V9 109€[ J L Z f' o e ' 1 Z _ O Z , s 6 o 1 o q u o 'uo;l;u6o:al a)uanbasvNC 1g 17 fi02'noa'sraquinu 'urelord/aua6 ( s ) u s r u e 6 i :ot u a 6 s u e r l bpD los€ t i^6olouq)el VNC lueurquro)auaasuotlelndtueul rngs'gss- Lss !8zs'(s)uorlernuruorl)unJ-Jo-ureD aassgyg (sgV9)suer^l60uruesor,{19 saua61e:t1tlt-la:ue)osq aas ' s l l e )i a l u e ) ' s s o l ILZSL z € zt

X]CNI

0z:l

'uosrJeduo)/aJeduo)su?au s jalqel P ol rajer e qllM sJaqunu abPd f 1,(lantlnrasuo: trto1lo1 leql sern6rjoi ll ue qllM 'arn6!,e ol Je;arI ue qtrMsrequnu a6ed l,(rluaaql ]o uorssnlsrp]xal rofetup o] iaJaraterploq ut sraqunu a6e6 'JL;V ' 'sralsnl:aua6 I St-ost JZiV Iorluol snlol os/Daas '!$, 1s1uor6ar 'ZSn-69y 'uor1eln6at uolssatdxa fagn s a u a 6u r q o 1 9 /etnl)nlls lgtt sarlrleuJouqeurqol6-fl'eruasseleqloslDaas zsv'JssE'1882,8gz'suorlelnuj I t z € t o s z ' e s n o u ls ^ u e u r n q 'a)uenbas J00z 366 t vNC ueunq aua6 f L9n' Zg;-OSn'uo!leln6aruorssardxa ggZ'alnl)nlls utleuloJql l S S t ' 6 u r s s a ) o r yd l g u - a r d l p t l J o u q e 9 9 7 ' a u a 6u t q o l g - d sadfi 4oads osloaas ' a a r 1( r e u o r l n ; o n p a u es a r 6 o ; o u l o q fgSZ\tZ saua6urqo19aasseua6 ) 9 9 Z ' , g S ZP 1 3 l 9SZ'(s)urqolD ' u r a l o J dr l 9 ts6 '(lDl) .ro1:e;q1r'ao.r6 'euolselqotlg 8tZ t ;er.u.raprda J S 8 €L l e r p e r 8 1 9 ' u o r l ) n p o r du r l a ^ | . U t 8 € 1 - € 8 €t ' , l 0 8 9 ' , s l l al re r l 9 1 9 7 1i { d e r a q t e r u a ) t n esl n o u a 6 o ; a , {)uu o t q l ' ( l S l - l t s ) ) a ^ a a l g IVN)l g J98L'SBL'aseraJsuerloqdsoqd t l [ [ ' a s e a s t ps , u u e u i z u e l g '(Agl srunuog-ua1sd11a^al relnpuelg 66Vt (6raua aa.r1aas,(6taueaat; sqqrg €0'I ls6'surllaraqqr9 '(€VDp ) r ) e) r l l a r e q q r 9 J90tL 'aJ!l edlUt'otptotg l9 [ ]o (s)auJosouorq)auarllod aassauosoujolr-l)tuptg (6191urarordtualsaronUuaerg aasdlg ur uralold esel tZEt 10 uorleurul.lalap v z t l - E z € . , ' ' a 1 os ra ; n u e t 66 g1S's)tlouabasJa^al 'S9Z'bgz'suorlelnur I L9 (s)ursrueOJo :rua6suerlaasluaula>e;dataua6 'suorlrun, ' s a u a l6Isp9ozq l l u p , V N C 19991 aurl-r.!reD '99t 'sja^elLurag lS9€ L t-99€ L S 6 ' s a n t a s a( 6l t e u a ' u o r l e u r u l t a g 995t 'uorlernleL!^ltuUJe^poqtlue'slelual leutullaD i(s)alaLueg eozolpLuiadS 1(ulnAo) 66f osp aas 9 9 5 - S 9 S ' 6 u u a a u r 6turel a u a 6 ' u o t u a s uat u a b J Z S ZL ' ( 8 2 L ' s l u e u r t l J e l a p EeEFZZE I's uo6a1asty pqoqtouaol ' ( s ) l l eu ) raD [/z L 'ay11 6u;;an;Xbrauale)tureq)oa9 71 'tSS 'ad^louag ltSS I L9-695's1no1:ou1pa66e1 ' s r s { l e uae: u a n b a sa u o u a 6 759 slrtuoua6anrleleduoSaasanrleledr"uot slrr.rJouaD L Z SL - O Z S t ' s a s n J r A o i l a r ' i ( l r t r t s)et ul do u a D uotle^tltput-X:6ut)ualtseuog os/Daas OLI-69t'a-)ope] qlrvror6a>1r;-urlnsur teuawouaqd>rlaua6rdase tLb-eLV L 8 ZL ' J 6 9 v O ' L V - g g y ' 6 u r l u u d t)ul .l . u o u a D vns'Jwg',vNc) s lzts ueLunq z t s ' s e u e r q t vl N c ) t u o u a g '6uruop )gni libi'gng-ntg 936 zts'ouol) vNc )rurouag uotlnlo^Jau.iouagoslDaas l 0 9 S ' s u o r s nuJr e u r o p ' s e q ) l e a1s6 o l o r , u o q 102'suor6arpallasuo)uou s^ paniasuo) s:rtuoue6enrleteduol aasanrleleduo: ' Z V t ' I B t ' 6 u t ) u a n b a sa u l o u a D Z S S -t 9 9 'uorlua^aio ls t€ fS6Z'st)oJaplredarVNC ^ l r l r q e l s uaru r o u a g 'qsuPrqaz 992 , E a E' I O S Z ' l 6 b z, s u o s o d s u e r l J'ZE 's>l:olq,{uaruXs 0SZ ' S a b u e qa] z r s [92 s>ruroua6anrlereduol osloaas ' J L V ZL n Z ' s a e r:lr r a u a 6 o l ( q d ieln)aloul 6VZ-gbZ'srsaqtod^q l)oll 'asnouJ-ueunLl l6tz l g b z ' L V Z ' 6 ur ) e r lr o t s a l u e 4 p 7 ' a > u a 6 l a n rap: u a n b a s 6u1:uanbaseulouag oslDaas 6 S Z ' ( s d N Ss )t u s t q d r o u t ^ l oadp r l o a l : n ua ; 6 u r s 0?-6€'u! uo!lenrasuol'sleuruleul 'LOZ'r{ua1u{s paruasuo: OSZ'JSO. JZSZ

' a:uanbaspanrasuol ESZ-7;Z'uorle)Urluapr gnz' o r 6 t ' u ollP^lesuo) g n z 'L O Z ' s r s ^ l e u ae )uanbas 'uor]e.r6alur alnpou uralord 0'[ '098-5Sg 'rel1aue6ro J 198!098 8 9 e -L S Z ' s u o l l e l n ul e l l n e u salPruorlelnw oslDaas (s)uorlelnwaasuo!lelnu S S Z t' V r _ } n t " ' 6 € - 8 8 ' u o r l e ) r l d napu o u a 6 ( s ) u o r l e ) r l d napu a Da a su o r l e : r ; d n pa u e 6 '6urg;nt1s uoxa 197 'su-rstueq)aur 9tz 8SZ',o9Z',sassol 8t€'erntrnJls aua6 uoxa-uo.rlur 'qsr;ra;;nd s ueunq 3 1'53 ',asnou5 ueunq $na'09e-6ve 'ltz'aazuedurql s ueunq $vz'rLva u ^e u r n q J 6 S Z ' 0 9 e - 6 5 (0. '9 2 - 8 S Z ' u o r l e u P 'r(1rxa;du.ror srrraqunu aua6 9y7 b E Z -E g Z ' su o r l e :1r dnp a ua 6 l 8 0 z ' L j z ' ( s ) a u o s o u r oqrl 'spue|g(9d)) oLv'lt Dl ',0SZ',suorlrppe 892 )rsz' t sz'a6ueqr palerala)re ogz-snz'uorln lonaauoua9 pnpntpur osloaas sar>ads €z€'slueura^ouruosodsuej] uorlnlo^aeuoue, osloaas LL9-019'0t '(DLusoldDIW) luauroldruo)auaOunururur ' 0 1 ' a r ! lj o J I t[ r^u 't€-0€ ' s u ol er u l errul rea zrs LSZ'9VZ'OO(. I8t 6ur>uenbas auiouag aas6ur>uanbas uorle)rloaJ VNC luotle)tldaiauiosoulolqJos/paas 'uorlelrloaJ 002 eurouableupuoq)olrWaar leupuoq)olrlrt auroua6'(s)lseldorolq) aaslseldorolq:^ sallaueDJo J Z t ' Z E - l E ' 1 o r 1 u ol:u a u r d o ; a ^ arpe l n l l a ) r t l n u - r asnowaasesnou auroua6leupuoqrolrWaaslpupuoq)otrur apol lrteueg :uorssardxe aueg j(poe :ra1:nuoqulxoap)VNC oslDaas uorlnlo^aauroua9os/, aas 8t€'lerlualoo uorlelsuPrf:uorlou)suPrf :y119ra6uassa6laaseur6op;erluar I t € € ' t € € ' J O t € '6 Z E ' 6 u r p ou) o r l e u r o j u l a u o u a o u e u n H : a u r o u a 6u e u r n Ha a su p u n q lsSt ntH uorlnlo^aauoua9 aasuollnlo^a 'sndouay' p (s)uorle)ldn 6E-g2 9Z-l [',A]rsra^rp panrasuo> OSa'JgO('LOZ',(ua1u,{s ',uo!l!sooulo) 6Z€ ' Z S €' q l 6 u a 1u o x a T u o r l uurr u o r l ? u e ^ J€S€ y p 6 6 u r p o > u o pa a ss u o r 6 e6r u r p o : u o u 'su.rsrue6ro lt98'?98-€98 ur sraqunu aua6 luaraJJrp 18t t L v - O L t ' s p u e F( 9 d l ) 9 ) 1 Xlrxalduo> SOZ-1OZ,77 ^lalalduo) 1g 1'su-rsrue6.ro-pa)uanbas s a u e r q rvl N C : o u r u o l )v N c o s / pa a s '6uruo1> 7 y g -1 7 9 (s)aurosouorqlaassauosouor ql Zbl'ouo!lDtllstsdoprqoty 002'8'(s)auoueg g g 7 1' a 6 p u1 e 1 r u a 9 (s).role^r])e aassrolenrl)e;euorgdu>suelg ;euotldu:sue.r1 :ossardaUaassrossardar lslurelojd 'rtE 'vEb-tEt 'srolerado rn€n J9En'nSn_ZEn,surstueqrau 'uorlnlo^a 0tt-6tt s;;roeu6urpurq-yNCorlDaas I L t ' , s e l u a n o evsN c aua6 (paddt\s-uata)atj osp aas JgW'8W- Lrn''eluaudola^ep xeldu.ro) saq)lrMslrlaua9 tLss'9s9 leior^eqaq tuelnur LSS-gSS'uOrtPrUrluapr s s s ' € s 9 ' € s s ' s u e a r l s) l a u a 9 6uuaaut6ua rllauag:6uruolV l N Co s l Da a s glg 'stsaue6elnur o.rlrrr ur .sanotuq)a] ngs_tgg paDarp-al!s 399g'srseuabelnur z l s ' ( ! v N U )a r u a r a J r e t uv rN U 'uotleJalle atuanbasulalotd 9 1 9 ' S U O r l e l nat U u rl-Urra6 lggg

LZ:l

(s)ursruP6ro os/Daas :rua6sue4luorleurquro)aU aua6 3199'luar.uere;da: luelnur uorlrun;-;o-ure6 1799'uorle.raua6 lltgS'uorleraua6 luelnu.ranrle6aulueururop sL9' I9S-EgS'€99'€SS'asra^ar slsal uolleluaulalOtUOlt OslDaas 'Zauo saua6onnl ro lgzg uorleu!quiolauaasuo!leu!qu.roler 'ad,(1ouaqd llES'€SS (s)uotlelnwaasuo!lelnul 1 9 9 9 ' s r s i ( ; e au 6e e 1 u r 1 1799'alo6Izouoq L6 L-96L' 96 L-56L',sl)aosele)r.lolsrq ,a1o6Izora1aq 1799 stsotawoslDaas 3 7 9 9 ' a 1 : i (p: r o l d r p - p r o l d e q ESS'ad{loua6 (s)auegaasseua6 ',slsaluOlleluaualoulo) 8SSl8Z9 (s)euJosouoJqJ aassau..rosor.uorql LZS-'SS'€S9'lelrssell s)!laua9 5 € ^ ) u e p u n p a r) r l a u a D uo!trPu!quo)au aar uorleurquro)ar)rlaua9 )!leua9 8t€ ['luaLudola^epDlqdosoto')resoLu 6ur:uanbasy1r16osloaas 'O9S-69S'srs,{;eue e6e1ur; l099 I t s s ' u o r l e u r q u o ) a r) r l o ! a u u o p e s e q 6urddeur:r1aua9 'uorlua^ard l9 [€ '6urueyoqsareurolatieue ssolg'gdurorl I 131 's1re;aprredery16 1963 JVSZ L ' n q e t ' S! Z t - t t Z [ l a ) u e ) ( 1 ; r q e g s ur rrl a u a g 86rL '(suoud)pDe lral:nu ur paporuar(llresselau lou /e1rl elnleaj 6ulur;ap Z Jo uorletr.lJolur )rlaua9 908 'uo!leulquro)arsno6olouoq'e6ueqlxa )rlauaD sanbruqtalty>ads :^60lour.l)el 1s)rleuegoslDaas tueurquJoleu VNC rrua6suejlaassurslue6ro:rue6suerl 1s1u.rsrue6ro 'sbe1aruanbas 919 lSgS'Sgg-tgg'uorleJallea>uanbasuralord t9S'sralou.rordelqDnpul aua6 ;pa>r,u.ra6 999-S9S'uoluesur 3 9 9 9 ' 1 u a u a > e l da u t a6 JJNgS 'g9g 'uorlerauab luegnu anrle6aulueururop '7gg'6uueaur6ua:rlauag 899-t9S T6LZL ' rrlauag O9ZL-6IZL' L LZL'ap) srsorau'(lrsrenrp 80s'uorleluelosuerlreallnu l!lPr.1ros n i l F Z L L J ' s u o t l e l n uu u 6 a l u l aseasrp)rleua9 uorlelsuerfoslDaas l t 8 € t € 8 € ' E g g ' 6 u r p o r alrp u o r t e l s u e r l I Z 9 8 ' 2 9 8 'E 8 t ' L g E ' s u o r l e u el e^u p u o q l o l r u r 'uopo) €8€ tn) oplpuo) 'suollelre^ €8€-28€ l9t lesra^run 'VS€. ' B9E'LgE LnZ'Iruepunpar l B g g ' 9 9 € , s e u r p6r ;u r p e a r 80n- L}v'00€'uortnlo^a 'IrlParaq 66 t Pue arn])nlls VNC suopol aassuopol o0e-661'apo) tlauag E9r_29, !€€?,suorado

tt6n| 'z0z'seurosourorq) f[?,rr.rxro

6ur:r;d5osloaas pgu-ard 1 y g ' 6 u r s s a : o .yr d (s)uo.tlul aassuollul gyg'uorlezrue6rouoxa-uoJlul '1902'9oz' 'ueunq rgnt l0z-902 (s)uor6ar lorluo) auaDaasuor6at;otluor aua6 (s)uoxlaassuoxa ')rlo^telna JgVt'LVE arn])nrls eua9 jVNCa^rUladaU uorle^rDeur-X i6urluudur )ruouag:uorlel^qlaurVNC os/Daas losselddnslournl Jg€eL'9EZL'seuab JLSt' 99V'see( ur 6urq>ymsad,{l-6ur}eu . z ( . a l o ru t l e t u o l L l ) o l a l o q J Leeo 'a6euep 'spuelst (9d)) D) 1gg-99E VNC D U r ) U A lar U Sa9 (s)ura1old rossardagaassuralordrosseJdar auag

X]CNI

'ltt '(slla)auulopuao.latua)sllo) eut.Dopua L ,JLjtL'l0€ ']uaudola^ap IZZIL [ L E VL - g t b t ' r a ^ o u l n l Il a ) finn L' ftn lowt,uotlelbru lla) lng l ? 8 t ' € 8 t ' 6 u r r r p av N U t z g E . t 9 E , s v N Ua p r n D sasel)Il l^li(uenbtolda)au os/Daas 683 1888'uorleln6ar asel)I) l^l^ueng '6urdde:ypgtu ,aseta;suel1 ilrE'gnE ,g 1luen9 laleqdsoqdul a u r s o u e n b6)1 9 a a sa l e q d s o q d ual u l s o u e n g 'uotle^ll)e s60 L voqu 6 1r ! 1 9 s e u 'uolleulrojelputds )ttouru 180| bvoFEnj l'uorlet)ossa elnqnlor)tu l08t'6lt'alor lPuorllunJ )O6V'6gn-g9r' 6urp{rar 7-11a 1968 '969'(ll9) tol:e; a6ueq:xaaprloal)nu autueng tE6 ' ( s l c a )s r o l r q r q uuro t t e D t o s t a p p t l o e l l n ua u t u e n D 'arnl)ni]s I t0€ 19 t [ z E t , a t n l ) n j l sv N U 'uorleurueep I t0€ '6uured-aseq 1 9 6I 'aurueng 1 6L 'sro1:e;a6ueq)xa / 13719 1 6 ' s a l n q n l o n t u l ' s d edfD > J 6 L 6 '6 1 6 619 ragotd6urpurq-619 1sase61y7su )uaulul) (s)uraloidD l(s)utaloldg aar)lauul Hl9) r o 1 : e ;a 6 u e q > x a p ! l o a l ) n ua u t u e n D :(sdVg)surelord6urlenr]:e-esedl.osp aas ' 6 L L' u o ! l e l n 6 a r )o8l 'LB 'slolelauaO t8 t t-61 t luatlanou sasDdlg)tJDadsosp aas 96g "s (sutaloJdD) )uauul '(^lquesse l e o ) r e l n ) t s e ^t)i e S f69L'65L (s)espdfgoqu aasAlrLueJ oqu (s)asedlgseuaas^ltrueJseu J 0 L1 ' , J 6L0' 6 0 L - B OL ' a s P dD f ueu '(6url)op aptsa^){1lure]qe5 191-Ogl '16Ll '08l-6Lt 'f ueLuouor.u 091-8SL'lO8t l0g I'sn uotlel^roqdsoqd ' 6 L l - 8 L l ' s l o 1 e ; n 6 at e r ln;1ar 1 0 8t ! 6 1 t 1 8 1 - 8 1 , 1 ' ( s a s e d fsDu)r a l o r d6 u r p u r q - 6 1 9 '969 '61 '(sdv9)surelord1968 6urtenr])e-asedfg t tO8 t (sasedfD)suralord6urpurq-dl9aasasedfg J66',arnl)nrls u ralo.rd 6L t -g LL'uor1e;(.roqdsoqd '(alPqdsoqduraursouen6) 86 619 '0€)sD

rz96'0s6

'uotlep.lela.l 'qlMotg l_s6z sr)ajaplledar vNc a96-ts6 /(sauouuoq qlrvrotg lueld) slupldur sroge;n6at stoDo1)l1Dads!(s)ua60ltWos/Daas 1 6 0 tt 8 0 t | ' u o r ] ) e s u . l s t u e q l e u l ' rJooj u o r l r l a o L u o ) 0111 'sJol)PJq]/v\orD z0t t 6 9 €l - Z g € l ' a l u n a u 6 u r d o ; a n a p . 6 u u a a 1 s esoello) ' s a r ] r l r ql zr s€u6e sa 6 u e q ) 6 9 €I Jo JLNNL198T1 '68€l-98€1 t6t0t'8t0t'leuolnau,sauo) ql^ org '9S€'usrueqlau.r 'suorlul dno.r9 6ur>r;ds-;;as l9S€ 11 l g S E ' 9 S € ' t € € ' s u o r l uI rd n o r 9 I V O VL ' Z O bL ' s l u e l dJ o ' a n s s tpl u n o r g 'auoradeq) 106€ llorD l S O g ' S 0 9 ' ( d o : s o t l t Luuo J l ) a l a , s p u g 'uodsuer] uorllele'eual)pq JnIns uaarg lElS 'sarpnls u o o s u e l ll e l n l l e ) p l l upl u e l e l n ) t s a ^ l€sl l€65 arnl)nrls (tlUl) ralsuerr^6rauaa)ueuosar a ) u a ) s a r o n lal a su o r l e z r l e n sl teAl n l l a l e l l u l aua6 LLl-9 LV' uol;.eq6eruorssatdxa '(dl9) urarord luarselonUuearg fr6s'|6S-Z65 3 Z 6 ' , u r e l o l zo - q r D J t L S L ' I L S L ' a s e e l o rIde r u ^ z u e r D ' s l l a )e s o l n u e r g 8 6 Z t ' J L 6 Z L ' L 6t Z 09nl 's1;a> rolrua6ord(Wg)a6eqdoDeruTafi:olnuet9 t L g VI 'GS)-WD) I09t t 353 a6eqdor:eu7al>o;nuer9 '(lS)-9) t09t L lS) el^)otnuerg (s)lrqdoJlnaN :slqdoursol lslrqdospgos/Daas I09t t'09t t'(s)al^rolnuerg Z € S [ ' s l l a )> r / : o 6 e q d ' s a l n u e r D

'leur]sa]u! l9z9 [ J 6 L nL '6 l b l ' s r r - u r a p r d ; oal a l e ; . r e ; n u e r 9 ',J06V',euetleq e^llrsoo-uei9 zll'snleiedde r6lo9 LZSt L 9En'LntnL'slla) ralqog LZSI'JO6nl'eual)eq entle6a_u-uel9 'ue:rdi(;9 'uor])ala, ;g 11 s1s [ uer9 '(s)UD)saseurl.roldararpalur;-uraloid f Z ZL ' Z ZL ' ( s ) e u r o s ^ x o ^ l 9 JOZ6'0Z6 9 sJoldDal)UDadsosP aas 771'apb a1e1AxoA19 gg I 1'suer,(16oa1ord a:e;rns-;1e: l 8 1 6 ' , 8l 6 - L l 6 ' u o r ] ) n p s u e rl ]P n s r ^ LOg'TNL'ETL-'NL f O Z 60 ' ( 6 - 6 t 6 ' u o r l e r t r r l d t ulP eu6ts ' gtg' Otg loq)ue (ld9) 1o1rsour1,(prgeqdsoqdl^sor^lg 916'slauuequ ) o r6 u r 1 e 1 n 6 a r 'sro]da)aio]oqd uor1el(so:i(;6 uralor6aassuragord I [6-Z !6 'sroldaler Irol)P,lo LLL-g IL'uor1e;{so:I;9 I l6 SnL-nnL'(Ul) Lunln)rler)rurseldopua'srseqlu^s JZ6-6J6'JJLL6 ' p u es p r d r ; o q d s o q d uralord 4gg-999'6uryos I Lt 6 l _ Ot 6 ! O t 6 ; o r r s o u r gy o6uruuelg 1 0 8 ' s u e rp r d r l 1. 1.'suralo.rd (s)prdrlo6urqdsorllD J O Z 60' Z 6 ' u O l l e z l lul sa s ep sadlQ)tlDadsosp aas I L O 6 ' J L O 61 9 0 6 ' 8 0 6 - 9 0 6 ' p u ed W V) t r ^ l l 0 8 t t ! 6 1 . tt ' 6 1 [ [ ' a r n D n r ] s rso6' Jn06' Lz6-no6' v06 ! €06 lE68 '268' 912'uor1:npordue:Il6oa1ord L V g ' ( s U ) d gs)r o l d a ) e rp a l d n o > - u t a l o r9d aastuaurq>elleuralo.rd (suldD) siolderar 1s)uer,(16oalor6 pa;dno:-uralord9 aas6urldnor.toldarar t8 t t-08 t t'sa)roJ a^rssaJdtuo) ' e u r u r e l ',SUrelOrO )ueU,rOUOLu s9t t leseq 968 I SASesedf9 '(s9Vg)suerll60u!uJ 'uorlPAll)eu! eso)i(|9 08 t t-5t I t r906 i l n L \ L E1 , 8 € t _ 9 € Z , s t s a q l u ^ s ) 1 6 1 6 ' 81 6 ' ( u r l n P s u e rrlD 9 8 9 ' u o r s a q plel a ) - l l a ) 16 L6' LO6-9O6'(uraloid D fi olelnurls),sD 9€9-S€9'(s)uralordo)^lD 16t6'"D )JtZL_OZL,JE6,JZL 66 , t68,^e/,^qled 1 6 1 6L' L 6 ' t t o t ' o 9 6 8 ' a l o rH c v N / + c v N t6l6 l or q e l a u l z 0 [ ' s o r q s u o r ] 9 l a i . r al r] u I6t6' 106-906' (uralordD ^iotrqrqur)19 SS',uOrlnlo^a 906'ztt sawlzua tloads oqo aas z 0 6 - 9 0 5 ' s l a u u e q u) o l u o '6u J J t Z t - O Zt ' t Z 6 ' L 6 '6 9 - g g ' 6 9 - 9 9 ' s a u i ( z u a 11 5 - 5 9 6 r l e u 6 rps r dr l o q d s o q d - l o l r s o u l 3 7 6 ' 1 5 ' 6 u r ; d n our o r l > e a{.qr a 6 e r o } s{ 6 r a u a 906-S06'dWV) sl)aJJeureaJlsuMop L6',S)rlaojeue s98-r98 1906'906-906'uollenrpP 'aleqdsoqd-E 'S06'xelduo: Lfl ap(qaplera:,(16's1se;doro;q> J906 ',906',lrunqns '69 '(ute6 n lau) uorl)npord dfv 3 171 J906 (s)uorleluauJalos/Daas 906' z968'61L'(sesedfv/suralord ,88 ')rqoraeue 6urputq-619rttauul) (s)uraloldD 6€8 ;srs{1o:{19 roqlue 15,-gg S V L - VLV, U l u t s t s a q l u ^ s 1 1 6 911o 1 r s o u r l ( p r l e q d s o q d l ( s o r I 1 9 'ainl)nils aassutalordparoq)ue-ldD ls L| 'ulalord 9 € 9 ' ^ l r s r e ^ rupr e q )a p r s OZI dD r99l r8a9' 629-829'souerquaur LtE t' DtILldoso.t O'rue$w,funqasoog 'sa1e6ar66e 1911 )982t 'S8Z 'luaudolanep salselpue aua6/tS (s)prdrlor^19 l-€BZ t '(dilSD) J6r6'6n6 s g u ' f E g ( .L ' g g 71 ' u o t g e t 6 t t3t t9 6 d€ aseull aseqtu{sua6o:(19 '(s)peuoD lueudo;anap 1y6'p6's6'ua6oil19 1799'9gg'x\1e>o>A19 1169'aLunlo^ 808',t89',alorraDrusuerlornau 8IL-LLL 'aurr{19 'q6no.rq1 1671 lrodsue.rlJo'lapoLugrodsuerlrelnrtsan ',rotoa)ar JIZL n8L-tSL d9w '16'eseua6orp,(qap 'uorlrppe ateqdsoqd-geplqaplera:r(19 s8l d9w 'srs{1o:{16 NllD S98-t98 1 9 8L ' 9 8L ' a s e r e ] s u e r l o q o s o)qv o ol tlodsuPll $bg'Sng'al)^) uorlexU-uoqrP) lgg L' LgL-6 LL'saurosos^l srsol^)ox3oslpaas ?98'a)Jnosalerp{qoqre: 'u.rorJ ol a)ejrns 9b8 uodsuPrl 6O8-66L lla) 'JLZt 'JOZI 't6 'arpqdsoqd-gap,{qaplera:,(19 !26 aLL- ff4'q6notql uodsuerl 'ap,{qaplera:(1g (ul) unln)llar )lt!seloopul oslDaas 11 ' s17 Ls-vLs 'Ul urorj llodsuell l9t9 ts Ls LAL-ggI 's6el uralord'(lSg) eseralsuerl-S auolqlelnlg (N9f) )ro/v\rau g g g , e s e l n u l s tapu o t q l e l n l D t61ogsuotl aar (NDl) IroMlau '6pg suotl '(eleJ llxa) a)elsuul rSLL'ZLL L 't L L - aL l ' 9 6 9 ' a r n l l n r l s J S L L ' J L/uot]ua]ar utalold entl>a1as IEl g LL-SLt'(;qu.rasseue:(16oa1o.td ',llalur uorlrsoo 169 L L Ll S L L 'S L L - € . L L ' 6 u r s s a l o rudt e q :a p r . r e q : r e s o 6 t 1 o e :; n r a l o u € 1 1 ' u o r l p z r l p l u e u r l r e d ul o

taot

'uorgezrue6ro aue.rquaur'srotourelnqnlor)tLu g g 9 ' u o r 1 e 1 i ( s o : ( 1 6 ' s u r aatuoer d Jqt!au 1169'SlUnOUieauelquleul l S l l ' u o r l e z r l e l u a u r u e d' LZU oe,)uao) e rD unJ LlL lJr x a JSLL ;g7'a>uelrodtul'saLu^zue 3 6 9 1 . ' s r s a u r l o i6l lu u n p u o t l n q t t l s t p J9SL ' as L-ng l'.uorlPrlJojallrsa^palPor-uuqlel) 944'q6norqr uoosuerlJo 'lapoul uotlelnleuJleulalsll l 8 I L ' I I LL ' ZLL - | L L ' e e u r a l s r ) (ND)) lro/vuaur6;o9 sr3aas(NDf) lro^ reu t6;o9 st: zll'uorlnqulslprelnlla) 6llue l € 8 9 [ ' u o l l P ] u a s a r d / 6 u t s s a : ou,ar d 9 9 2 ' ( x a l d L u orr6 1 o 9s1n l e t e d d et 6 1 o 9 Itll arnl)n[s '9* 'u nrlaq]1da ,(rolelldsa.t t lsEv L ' Z l l ' u o ! ] a i ) a ss n ) n u LEnL'JVLL

lS09'909'S8S'ap^qeplerelnlD 'srsi(lorp(q 'aseqlu,(s autueln19 119 61y 'alnl)nJls reuluretnlg 16z | 'arn])nJ]s l6z [ 't69 't89 'alorreluusuerlornau 808 (pr)e)rurelnl6)alPurelnlg 'uorl)unJ6u;p1o1 uretotd'eserelsueil;,(sorn19 6€1 aso>nlD 961'srsoilrsue.rl'rauodsuerl 'aleqdsoqd-9esolnlg 1631 8 6 1! l s 9 ' u o d s u e r t ',ornllnrls )zLL'J9S srsl;o:{19osyaaas 'plarI MB'€Zg '100I6raua 1e1o1 '19 'uorleprxo t l0z [ 3 1 5 ' s r s a q t u , { s oetpqr J e q ) r e s r p asolnl9 'srsaua6oauo:n;9 76 1

J € g r ' E g b ' 'spro)ruo)olnlg SLn 'Z 'uolteln6aruorsserdxa aua6 tt 'g97'uotle1n6et 1E97 aua6'utalo.tdioldo)at pto)tlJo)o)nlg uorssardxa fl06'uooelnl9 'qlnq {lor:e;|o 'rlnrauJolg o€' t ;o 157'saua6opnasd 379y'197'urqo;6-1" t a s v ' L S vL' S Z - g g 7 'ruq o l 6 - g aua6 utqol9-$aasu;qo16-$ ',urqolD-D L9Z-99( )gSZ' 49Z-9SZ'uo;te:t;dnpi(q uorlnlo^e

X]CNI

zzil

'uosueduJo)/areouro) suPaurs^ :elqele ol rejerf e qllM srequnu abeo l^la^rln)esuol^ olloJ]eqt sern6uol ll ue qllM ,arn6r1e ogralar I ue qlu!^sJaqunua6ed i^llua aqlJo uorssn)srp txegrofeure o1raar arerploq ur sraqunu a6e6 0 z z ' ( ) u l u e r )) u e u o r l u a ) lztz E69J-Z6S I'S LS L'J' LS t'17H11 7 adlr ' ' zEz- lEz'v-dNl) iueueAlr;r:ads-arauolluar I Z Z - 0 Z Z ' s u o r ] l u n1;e : r 6 o 1 o r q #65L'h69L-Z6SL'JtLSt. sa 7u1 ' € Ha u o ] s r H 'uOrle^rl)e llupuap oL0 L' 6€Z-8€Z'ZZZ-OZZ'ut]€worqroralaH [ [z J € 6 5L lla) 'suotle)utpoul lg Lz ) € v 9 ' t v 9 t 6 0 g ' ( s ) u o f te r o r e r a H ls6st 'aueb ' a L e' s u o t t ) e l o l u l 's5s 'sa6eqdorleurjo uorle^rDP , saH I l€ L )b Lz 1-t6s I '1 l9tst'lt5t-9LsI 062 la|.UrpvzH z6sa'sL9L jorg 1 1r111 ad,(l 'suoxeur luaura^ouJpaseq-alnqnloj)rr! I L Z ' 8 Z Ha u o ] s l H ) E 6 SI ' n 6 S L - Z 6 S 1 " ' JL,SL ' $ t o q : Z H I ' s L H t ,9gS ,snu,{qtut uor})alas sLv-v Lv' lvzz'sluerJeA ed o l o ^ u a J t I S[ ' u o r ] r s r n b ) a t-SgS 1 llgg t 'suolle)ulpoul l?[s[ ^lquasse J9 LZ 1165['esdeuls ;err6o;ounuru-rr '(sa)snr^sadraH J Vl ( ' Z L a s u o t ] ) e j a t u l l96t I -L86StIq uorllnpord eurlol^l 'sJo]da)ar ' 6 u r q > 1 r rsusse l : s0s [ 062'rarlrp 9991 ' v z H a8uzoHl s r H ',[85 ',alorro]oa)ar-o) 9 E St - S € SL l .t z [-08S [ tc] I t85 t ' u o r l r q r q uuro l l e l o l s u P rul r e t o r d s s e l l : 8 l Z ' , 8 t Z ' , I Ha u o t s r H I IHW a 6 s F L 6 SL ' SL St - ' L St ' u o r l e ^ t t ) el l a ) I sadreH r ! 9 n n ' Z Z Z ' ( s ) V C Hs)a s e l ^ l a ) e eapu o l sH 96nL' ggbl- LgnL'(nSH)snr^ xeldLurs s 8 9 t € 8 S l ' u o r l r u 6 o l a ru e 6 r l u e 'suD6ala 'alrporqdeurap , s L s | - n L g J , o 1 9 , ( H t )( s ) l l e r _rlo d l a H JqZZ'9ZZ-'ZZ'srseqlod^qapor auolsrH t(€l 1 r s 8 s t _ € 8 st (slV)) srot)eJ a)uelrJaquloslpaas surerord 'a]rl ain]eal 6uluuap ^lqLuasse urleuiolq) aassauoladeq:auogsr;1 ureuopoauroHaassuratoldureuJopoauroq [ ]o 'A]rparaH olsrH J € n t ' E V E ' e a z$' I V i l s e s e r a l s u e r l ; i ( 1aau: e s6 [ Jrlourxrlaq-uinl-xrlaHaasJrlour6urpurq-yp6 ' sauolstLl b s z L L L z ' ( l l d NH ) lonpt^tputoslDaas l 0 z t ' O z t ' s u r a l o j ox r l a q - u i n l - x r l e H reluel lelraJolol srsod^loduour{relrpara;1 l0z?'arnl)nrls ',(deraql ' 'uor1ru6o:ar re:uel ur 'uralordZraH e)uanbas 6971 JLZn IZV-OZ7 ' J | L Z ' E l Z' ( L ( . ' a r n l ) n r l s tttt'(JDH) rol)p; qlmor6 al^loledeH J nL Z J O Z VO' ,Z n - 6 W ' | l o u r x r l a q - u r n l - x r l e H : rlu s e l d o p u aq l o o u r s Etz-z lz'suorl)etaluI y56-uralotd 921'LUnln)tla lgzn' grn-San'surelordxrleq-dool-xrlaH 'suorletnur € lz I/69'slunoue aue.rquau'sallaup6ro $zn'gzn-san'llour xrlaq-oool-xrloH uoilP)urpourauolslHaasuotlp)utpout ( s g S Ss)u r a i o r d6 u r p u r q t9n'stv 'srseua6elnur 'ZIt'uorssardxaaua6 pa]erpaLU-pro)r].ro)o)nl6 -VNCpueJls-al6urS o-r.1r,l ur aassuraloid6urzrlrqelsap xr;a;1 917 a ;: u e l t l a q u t uoJ]lalo f L 6 Z 'L 6 Z - 0 6 Z ' u o l l e ) l l d 6 au i tmo1;o Jg6g'qdei60r)rur l9'['sarlradord 'aseqo 'sseupepuPq n v nL - E b bL ' z t t t ' ( s ) a l ^ r o l e d a H 0101 €t [ 'srotoe]e.r ' u O t l e ) u t l u sa p l arnl)nrls a a s( v N c ) a l q n o p s0s [ J Zt Z v N c 'arntrnrlsrelnlalour uou.ruro) J V t Z ' et ( , ^ i q L u a s s e 8 Z ZL -L Z ZL ' p u er e ) u P l r a ^ ! l stl €'t XIIAH z l(' t t('iauel)o auolsrq 0 0 s t ' s s e u l l)rr u o r q ) (sa)snrAsrlrledaH 6g3,saua6 6 Z Zl ' 8 Z Z L u o l l e s n e r) a ) u e ) q l e u o l s ',azrs auJouao Jttz'ztz'ptol ILZZL'8ZZL-LEZL f8 [ 'uorlpsne)ra)ue) 'qleurotsjo uorlezruolol ra^rl'uorllolur g sllrledeH € l Z ' u o r l e ^ r a s u o{tr e u o r l n l o n a €09t ' 7 1 L Z' s t s \ e u e 'sr}leooH / s s a u l)l rr u o r q l tZ LZ s05 t 0 0 st ' a l e j l n su e r e d o H 0 6 Z ' ( u o r l e ) t l d a r l s oVdN ) Co t u o r l t p p e uolld raD)qDUaH € 8 1I ' t 8 8 'leJr^la^al ]r6eq.uou.reg 9 l z - l l z ' ( s ) a u o t sH r Z0S[ 9 Z t [ ' u r l su l 'sa/:oqdu{1 'sanssrl)r}arodoL!aH 'ln6 ul EEnL-6ZVl'e!laq]!da ^rosuas 5y51 ;o ur6uo Ltt L ,€SE 'gyE1'olrqdosotg srsarodourag osloaas ,llJ-zrtL')a^tl L l€S€ L 'leMauer-,las tttL-g€na'ln6 89t t l u a u d o l e ^ a pl e u J t u e l g L€ t ''stsdoptqDtV 'slenpr^rpur gztJ-LanL ,sil.ulaptda palerpeilrJo an)ser ut luasqe 9StL OOVI /slla) 'u!6rroa]Itoqdu{1 'ruarodpnld Iennqred6ur;eu6rs6oqa6peg osn l-snn a E?S[ letlaqlopue 996 'lerlualod{deraqt aua6 0 s t l - s t t 1 ' s l a s s ap^ o o l q 9St L 0 S 6 ' u t a t o t d6 o q a 6 p a g 's'(erute ' e l n rq q a H 9gg 1-9gg 1 S S t L ' A I PU J OlSl^lp 96tt 'uor]e]ueurpesunrJqrlrnba'sedolosr^^eaH (6o1o1sr1-1 l8s' t [ [S '69V1-89V1'l)eluo) '5ss 'surPurop (xaldLrJor ^lrlrqrledu.ro:o1srq tofeu) )Hli! oslDaas alqeue^ l l a ) l e u o J ] su o a ) u a p u a d a p 1-8ss t l09s L j6ss t 'lueurlruru-rol S l S t ' a l n l a l o r u^ l r l t q t t e d L u o ) o l s t H 8S' L-gSb| 6gs [,sutPtllop6u;leada.r ',Wbl 1 S S S6' 1 5 ' S S S - t S S ' 6 u r u t el el sl r t u a q r o l s r H 99t t'sJaileu ele]rns-llal l 9 9 St z g b L - v q v l M o r e u r a u o qu ! l € 9 9 Ll o o d l u a u 6 a sa u a 6 ' Z Z 6 ' s r o l d a l apt a l ezi6-w6 '5ss 'sureurop t)osse-aseutl-autptlstH €9tt-os?1'slle) urals)rla!odouraH ]ue]suol l.-8ss [ l09s t 1699t ',eurprlsrH 8'S l8Z t z9b | - LgVL'sllarlrlarodor..ueH Jtss I /t08 /uorlar)as 'lSS ' ( s e r p o q r l uu e r) p q r( H )^ ^ e a H slle) uratslrlerodouaH :srsarodoJql^llos/Daas ll.ss L lt08 t -€99 I !1S L 't08 'aseelar 'slla) uals u.ror; Ieoel vNc os/Daas 1ualodr11nu lt08 95?L 'sllar ' ( a u a q ) s )u e r 6 e r pa 6 e a u r l ]seu swapld ) tlDads:sauoredeqlosloaas ll55 t J l S t t ',t08',auruielsrH 'slurod 999t I l9t L lorluo) aldrllnur 8 8 t ' ( s d s H )s u r e l o r d ^q !S8Z'aae^aD) sD^wuDtp)DS'auabgp )rleurospe)npul LgnL-6SnJ's.ro1:e;6ur1e;ntrrls-Iuolo) 8tfl.'uo!]eurquro)ar 'sroldera:a1eu.reln;6 'sndu.retoddr 'srsotdodP p ^q 269- 169 zgf[ lloqs-lPaH 'lorluo] y1 'srs(1e1e: l € S S t ' Z S S t ' a l n l a l o{upro q r } r r e ' u o r 6aa6ru r p atri(zua tst [ s11e: 6urilrldue irsuerlos/Daas gEg'anssrlesodrpeuanotq T L\ '()dH) ^qder6oleuorq: prnbrlatuer.u.rol.rad-q6lg ,99-t9 ,(6raua g9nL-99nL',ul',]uauj]rurrllo) seag l8 t L !69 * L L ' n4 ' ( s ) a p u e q l l e s o 6 t ; oa s o u u e u t - q 6 r g al)snu )erpreJaasallsnur lleaH z 9 ' I u ! q ] e e pl l e ) 'suorl)eralul €zs 9Sn|jESVL'Molleuauoq El E ' J L g '9 8 ' u o r l e z u a u r { ; opde a ; i u r a l o r d - u r a l o J d ' s l e lul et a l o J d( l r s u a p - q 6 r g Egna-ogn a',srsarodouraH l90t ptoJACH 'sasoxaH (s)VCH)sasel^la)eapauolsrHaasSIVCH lz [ ! l8t€'suorlelnu'erlrqdouaH (sfvH) saseralsue.rl;r$a:e auolsrgaassIVH z t 9 t - t t 9 t'saua6ol()ouow or;a.;sr7'urs(;ourag 69t S0Zt',aseur)ioxaH ',tV ',suor])eJalur lrunqns l t S s ' u o r l ru u e p ' a 1 o 6 { z o t a l a g s d e u a d ^loldeH L l-ZV | J€V Z 9 S ' 1 9 9 ' ( s d e u r d p q ) /uorsnJ auerquaur :rd{Iota}aH (s)urqol9 oslD aas 891 L g S ' s l ) o l qa d ^ l o l d e H ! 2 9 9 ' 2 9 9 -s^ 'lsPe^ ) r l r q o o u o q sdno-r6 ar.uag aassdnoJbauaq l S E Lt t€ 9 y 11 ' 6 u r p u r q: r ; r q d o . r a l a g srsorawos/Daas J 7 S ZL' S Z - 9 9 2 ' s u r e qulr q o l 6 '!99L 'JV' 'arnllnrls 8S€ LgZ-gSZ t t 8 zL 'zLzL'10 ! € S € ' s u r a l o r d o a l l n u o ql e u a l ) n us n o a u a 6 o l a l a g J 9 9t ' 9 9 L ' u o r l e u r p r o ou)o J l L z L ' I L Z l , - 6 g Z L ' u o r l l n p o r d al e r n x a su l saua6urqo19osloaas J n l t ' v l t ' u o r l e u t q u . t o l a)lt l o t a u l ' x a l d n p o l a l a H ?€ "s alels ptoldtp '90t 'slurofxaldnporalag JSd.'VZt 3SOE alels proloPH 9 7 ' s e r 6 o ; o u r oaqu a 6 J g a EL 'L Z t | - g Z E t ' s a u a 6) r u o r q ) o r a l a H J9SZ' 4SZ-9SZ'uorlnlo^a 19t'aj!l Jo ae,r ut'xualopH '( 8tZ LdH)1 uralotdurleuJorqloielaH J g S a9 ' 9Z,Ilr^rleradoo) J0b9'zng-ong' w ntDurtosw nuaDDqolDH ,(qH)urqo;6or.uag ', ',saooroslolpeJ 'aJll-rleH 9lt-tlt'uorle^tl)eut-x , tZ I I 09 [09 ' s l P a d aul a p u e t 'sluauleluulleral ' 'aue6tuDH uoqs llls 986 I LEt L€€.1 ' V N U' s a r n l r n r l su r d r e H s8z'uolle)rlda.r ) | L I l'fiv I l'lueulq)Plle luauJelualelpaul.lalul lEot'8€t ZZZ-OZZ,uorlezrue6ro IV.t L 9 Z n L1 8 L V t ' , a p r l l oI ej H '01 J g t Z ' 6 E Z - 8 tZ ' s u l o l a ; d r 1n1u t L t 9 € LI ' r 7 t I L ' r t | l ' ( s ) a L u o s o r . u s a p r u r e H I ' Z E n L - O E FJ ' 6 Z nL ! 6 0 9 ' s l l a rr e H JJTEV uulqdrod 6tz-8t z'uotle)tJtpoulauolstq t€8 3 0 € 8 ' , s o u u z9s-r99 'r(1;rqeluaq 'a)uanbasauroua6'aozuan gu sryrqdowao;1 Jtzz'lzz t 9 9 L ' 9 9L ' u o l l e u l p r o ou) o r l sluauJalalolelnsul16ur)ualls auag orlDaas (uorord'+H)uor ua6oJp^Haas(uolord) 1 g g3 9 g g ' s d n o r 6a u a g +H 'uorle^rl)euFX s8z J9gS,9g9,url^xoleruaH ' !lzz Laz'uorle6eue^ 6t? ['eulotselqor6ueura;1 H g>agauorlrsodpue slta;;a uorgrsod (s)llarf 1(s)lla)-t tO) osp aas ! l z e ' O Z t , 6 u r : u e pasu a 6 1E6SL'V6St-Z6S L'!t L9 t' sn yu1 'uot]P^t])e )t]!ipua_p t S V - ( S 7 ' S W ' 9 9 g ' u o r g e l n 6uaor r s s a r d xaau a 6 l€6s [ €0s L Jo uorlezruo'o)aotapq) ouqtA lla) 677'S)u.reu(p r 6 5 L ' , s a u r l o)l rgEnL' nnn a-ggt ['9tt I X6otorsrq 'slle) (s)arauorlua)os/Daat 869[ I JOuor]e^r])e JLtVt

o6z-6sz "#tt5tl5't';":1r"ilii

€z:l

X]CNI

'stsdoplquv ' s a l r s P r er d elnlla)erlul zl ! u'suot]leal uotleua6olp^H JELTL I t s t - 8 0 5t 'EZ9'1901'(s)uoqre)orp^H 'sasualap uorse^a uorlelntrJlrloauroH JEZq t09 !-zos I Jo 'slaule) uolllala ,suotapup^H os/Daas saua6xog lsuralordurpr.lropoauoH zg l 6 0 st ' l 8 0 s L ' t t s L - 1 0 9 t ' e u a l ) e q '169 'suaboqted xalouro) xoH aas xalouro) xoH i(q uotsenut J6gZl' 692L'uorl)npordelpnxase'DtpIH S0St-tos L 1. s l l a )l s o H l60s'609-805'seuropuq^H * W I ' n J n ; - € L n t ' l u e u r d o ; a n a pr e i v r o p 'se1oi(re:na uotlpztpr.iq^q Ilrurel luaJa;Jlpur s.raqr.uau vNC aasvNc./uolrezlpuqlH 169y1 8t5!',pearos sauaDlrloauoq ua6oqledalelrlr)eJol uorle]|Jrpour lor^eqaq lsoH l60s'uorl)npoto seulopuq^Haasseuopuq^q nts o6ouolaw olqdosotg aasol qdosot1 sauoLuoqI0nptArpu!osloaas _ s l l a lp u q ^ H u o r s s a r d xaau a 6 nnEL-€v€t 'saxalduo) xoH ur 6uuapro 'asepruornle^H Itgv' tgr' I Ln'z[t'uor]eln6ar L 8[ [ leuJosoruolq) z88',SauouiloH ' U O r l e ^ o u ^urre u o r ] n l o ^ a l08tL'1811-08tt Lnel-an€,J 6[ '61 '(a]euolnle^q '0Zt'saua6 ' u o l l e l J e ^ i p D e (xoqoaruoq) )tloaruoH l t u o l n l e ^ q ) ) l u a 6 l l u e u e u o r n l e l H [[ !B€€ t 0 z s L ' a s P a s rsp, r e F n H aua6 l0ti['laq)snM s81 zzaz' ril'raJsuerl leluozlloH 's1ue;d ',sn ln)unuJoH o g s ' L 6 E ' a s e e sst p, u 0 ] 6 u t l u n H 6691. L 6 Et 6Z?'uOrlPlosl I L g Z ' S U O S u e d (ar l)oula n b a s ' u t l 6 u t l u n H Itss'uolil uljap'alo6^20tlloH /aseestp :rd{lou.rog L Z r ' l g E t ' g g1 ' s a r 6 o ; o u o q sgl trolunH t6g L'89 L'uorsnJauerqLuaLu (suatdDs owoH) sueuinHaasJualdosowoH lSZt'uotlezuaultpotelaLl J6VV '05t-8'''uorlP^rl)P aua6 a a 'utatold Vv-Ozn' Jgt l'uorlenrasuo:ft euo!lnlo^e l8€LL's rr;rqdorelaq l)eqqlunH ( s ) u r l n q o l 6 0 u n L uj (usl ) l l ag) o s p a a s rllqdouloH ro*'a€v-gzb'J Lzt' Lzf'-ozv'6u!putq-vNc 6 E t l - L t l L ' u o r s a q p) e 'ASUOdSar gp7'197'A6o1owog aUntrjt.ut 89S1-Z9St Izt-ozt'sula]old ureuopoa[uoH ',arrl leloulnH jsrslleue suraloro ureuopoauroHoslDaas osp aas srsoraw a6e1u11 J9 L ,o aarl ul ' u o l s e ^ u pl u P l l s 0 ? ' u r s r u e 6 l ol a p o u lS e Jlu s0€ ' 6urpurq-yN6ureuiopoauoH auloua6 ueunL.taasauJouao s u o r l ) u n^r P p r l l o H a a se 6 u e q l x ap u P r l s aZV-OZn's;r1ou-r '80€-20€ '10€-90€ 'v)au (suatd0sowoH) suewnH sauab(xoqoauoq))rtoauoH aassaua6xoqoauog ll0€ ',Uorlezuauos! suraloJq uDwnH 66't'la)uel le)tNa)'snJt^DwoltrdDd I I [€ l z I t 't s t € - t t € ' [ [ € ' u o l s l a n u oa ] ua6 n l H a a ss n l t A^ l u a t ) u a p o u n [ ! [ ! tu p t ! n H 0 8 2L t o o s ' u o ! l e r 6 ! uqr ) u e r qa l q n o p 'tL€'uot1e1n6al auouab uelunHos/Daas fZrc'2rc O g Z t '902'902-soz'llalord arloueg ueurnH ' s u o r l : u n{[ e p r ; 1 o ; . 1 ' s L z l ' x a l d u r o )u o l l e ul q u o ) a r zts Jat€'L LE o S z L 'azls 8 5 €l € S € ' s d N u u q ?tt-€ t€'sre^ossoJ)-uou f8 L ' s l o d sp l o r p u e s l o d sl o q ( ) ) d N H )r a l u e ) Z t S ' 9 0 ( ' 9 O Z - 9 O Z ' 1 r a f oaru6o u a g u e u : ng o8zt ' ' )gVZ !LV( Lr( LOZ,a^rlerpdLuo) le])arolo) srsod{loduou(reltpatag aas))dNH ln Lt'n lE'xaldnporalaq 9 0 t [ ' u r a ] o r dw c - v l H 7 O C S O Z , 71y, 6 u r : u a n b a s 16LZl 'OSZJ-6IZJ' ' p u e^ l r s r a ^ r p (xelduol )rlaua6 J 6 0 Z ' 1 9 0 2t 9 0 Z ' a l e ) s tlZ ! ioteLu))HW ,{}tltqtledeuoro}stq ) l s a 'J g V ( ' u o r l ) n l l s u o t a l , { u a 6 o 1 ( q d t L t ' u o r s i a ^ u o )a u a 6 ' s l e a i qp u e r l s - e l q n o p aasuabplue(paler)osse-eillolnal-ueLunq) a) tl-lel|Aorler uou zzt- LzE'suosoosuerlorlar VIH z I E!508 ' u r 6 u o j t 9 8 ' 2 9 8 -t 9 8 l e D p u o q l o t r u r pupuorlelursran!p'z-AlH I 1 3 91 o t l r u ni if e p t ; ; oagl q n o p l € t € ' Z L € ' u 'a)ualaJlatul 'tzs 'L-AtH la^ossol) r z s L t I L?'uorlet.re A 082[ ' ( t e t q r ly 1 6 l er rnuprto^ut pau6l sadtl )rJDadsosp aas JZng'LVS tL€'lolluo) la^ossorl ']uauldola^ap ' 9 L Z L' l V 'n ' u o r l e L U oej u s e r q l aullleA arntfnrls auag oslDaas LZS[ Lat LaL J 9L a t '90('1502'102-902'arnt)nrls 'rv 's 'L 'rz60L l t S E ' € 8 € ' 6 u r g r q s a u r el p r ;u o r l e l s u e r t I90a J 0 8 Z t L Z t L Z t , L ( t L ( t 'z60l'vlE-ztt )llolaur 8 l t ' u o r t e n u a l l eu o l l d u ) s u e J l 902'aas !s0€'uollP ' uuol rqsuel o ^ ufparul e J l s lsSt'81t'uralord lel 902'^roleln6er 90€ l 9 8 V j S S n ' S g n ' ( l U Ur)u a u a l aa ^ l s u o d s a^la u 1902'Ztt'seue6 1 9 0 € ' s l a p o u . r ' ( s r s d e uu o^ sr l)e z r p u q ^ q yp6 6urpo:uop osloaas s u o r l ) u n^[ e p ! l l o Ha a ss u o r l ) u n^[ e p r l l o H 98n-S8t l€tt'uralord ^aU J98V'JSSV' 'aJnl)nrlsase1dl.t:suerl '90t 'sluroIxaldnpola]aq asrana.t sluauale )rlaua6 altqow os/Daas I LZ€ 80€ 'slueutala 'uotle.r6ru..r '80€ qluerq a l r q o u !98V lsqb € z t l _ 8L €t l 0 z 180€ ltgg 'uodsuerlreal:nu pale;n6a.r 'ggn-Sgn ' s l u a u l a l an l v Slttt 90€-90€'6ul.rred-aseq J€ZE '90s 'sro]da)er t-909 [ lsOs [ rl0z'uorlrsoot!o) 3tgg'6utddt11-aseq uslueq)atU l 5 8 t ' u r e l o r dJ a N 9 O Z - 9 0 2 ' ZLt ' ^ l r x e l u dr o ) (Mopq) uoDoutqwo)at)Dotau oslo aas L..SI-OZSl'alel uorlelnu €6Z',oool-l 'SUOXe 'alerJ uolle)rldar 's0€ 'lueul.tosseTa6ueqtxa ur ]uaula^Ourpaseq-alnqnlorlil-u :t1aua6 I LSL €82 8t€ t ) g g'nJ 'gg6gnnL'J L a t ''aapuIo: uaa; 6r; SgZ-LBZ'sur6uo uoile)rldar 9 0 t ' l ! r a u a q^ r e u o l l n l o ^ a '0 lleo-ar,, ua6 t z g L ! g g y 3 5 9 7 ' u o r l e z r u e 6ar o [ E L € l0 ,,'sallo])e] 'aptldad uotsnJ IOSL Igo(' Jsjz'zz aulosouloiq) LL € - 0 [ € ' u o r l e l n b a r (ltso6i(zo-ralaq 1 3 9 1 ' a : u e l s r s6 an r rp lt0z'uolle)olsuerl t aurosouroiq) LE'ssol z O 'sroldalar-o) '909 ',srlaJap orql t-sos L lsos t J#02' r1z'saLuosour l96Z '6utputq rolda:ar autlotuaqt ueurnH 3991 I7OZ' 9OZ-SOZ'aurouob l60t'60€-80€ ts0€'s0€-r0€ '€09-20€ lredar )jearqpuerls-alqnop / 6 / ' s a r p o qr e l n ) r s a ^ l l l n u . l 9 9 9 ' s > r g a u au6e u n p t€0€ '190s '90s '(sUVH)suor6arpalera;a:)eueurnH js t€ 'uoll)alro) q)leusll.! el^ lorluo) r L-90st 10s L €SZ ' 6 u r l e o r u np u e ( r l u a ' l g g L ' S g L n g L 'salnlPajuoLuLuol Ha r J t l o uH r fH 11a: S0€ , r l o u x t l a q - u r n l - x t l aa lSzz I pue ra)ue) LLL'TgLL'SIL l s 0 € ' s 0 € - t O € ' s u o r l ) u n J1 e r r 6 o ; o t q ' u o d s u e r lu r a l o r d L g sL 'r 9 6 VL ' 1 s n r n , { : u a r : r ; a p o u n u uu rP! u J n qA) I H 9r€-t0€ l e u p u o q ) o 1 r u ' u r a 1 o6. lrdds g '(ra^o-6urssoJ)) uorleurquro)arsno6o;otuog lv6b L' v6v L'wnqnsdD 0wsoldolslH 1 0 6 € ' 6 8 € ' g g g ' u r a l o rgdl d s g r dg d s H l8€z'sauosot!ol q: auay{;od l l l l r o d u r u r a l o r dg e u p u o q r o l t r u ' u l a l oO ) t z ' J o z ' l Z - o Z ' s a u as6n o 6 o ; o t u o g J t z z ' z z z ' u o \ e ) l J r p o u lrr P le u o l s l q 6 8 € ' g 8 t , u r a 1 o rgdg d s g E 0 € ' l l e o alrP e r q 'suraloid p u e r l s - e l q n oVpN C ' 6 u r u r o L p usan o 6 o l o u . t o 1 1 J9nn 106€ OtdsLl 'zzz' ',ureloro 'uollrun!stpuou $)voH]| saxa;duo: aselrQleapauolsrq 8ZZ-LZZ 'sH 6 L ( \ - q L Z L3 9 € ZL J w t ' t n E ' z z e ' ( s l v H ls a s e r a J' lseuuP lo^rrllal o) u ea) suuoel sr ltq f L f t L ' a I Z a - gL Z I ' u o t l e 6 a t 6 a s: t ] o t a u tt9 '(Iqder6oteurorq)prnbrla)ueLuro,lad-q6rq) (ia^o-6utssoJl) Et€ )ldH 'S?€ 'saua6 osloaas xog uorleurquio)aisno6o;otro;-1 !9ea' gzz-izz'srsaqlod^q epol auolslq 99€t t 171tr'77y'>4aua6tda n n E l - t V €t ' u o r s s a J d xaau a 6l e r l u a n b a s J L L ZL ' 9L Zl ' s l u a u l P l Ua s J a ^ s u e l t 'paiedu.lol selorleuorl)un, l9t€ t f i L Z I ' nL Z I ' p u e s e r a L u o l a t leuiulPl.llpue ])asul 3 g E y ' 7 E 7 ' 6 u r l a p o u rual tr e u o r q ) 1gtrg1' oI r.ldosotgur uralled uorssardxa T9LZL 'g /asnou u t u o l s s a r d x a T EN LZL-SLZa'|p 77 y'xaldwot leu.rauoldeu{s lgt€l '7gtr'stsApue ' 9 L Z L' s r s d e u ^ s urleuolql gng a-ZVEL'uorleurulalepsrxetoualsodotalue uoqleltdt:ardounurur! JgLZI 'zzz'sureqa ) p l sp l ) e o u l u e 'sauosouloJq) xas xalouro)xoH Jeee s1z I 'salts6u!tted gnea-zngJ O6Z'uoltellla)eaP7uot1elr(1e:e tlZ t 'uorleururralep 'uorl)unfslPuou srxe roualsodoJalue'epor )Eee' nzz-zzz'uorlelurpour auolslH 6 L Z L - B L Z!I9 t Z L XOH 'uorle^rDeur-X'slueUeA 'asnourur uorssaldxa'xaldulolgxoH 'sa6ueqr 9If l9'€t 9131 1e:r6o;oqdtoeu ' s a u a b6 u t d e a l e s n o H u o ! l e ) p ot u L 6 Z ' | 6 Z O 6 e ' s ) U r O L I ' V w ' s p u e F t( 9 d l ) 9 ) J 6 L Z I 'OSZJ-6LZL'yZ 'pue (]rs.ran!p )llauao rua6l1ue ',suorl)eraluI ISLZ',817.-LLZL ',a t ' , Z l Z,rauerlar 0ZgL-6tSt'uorleu> en I 'sua6oqledIq uorsp^a t6z_062 tH tOS l-ZOSI 0 8 z L 'I L Z L ' q e a r qp u e r l se l q n o p ' ' l Z ' u o r l e u r o Jl u a l e ^ r q 9 2 9t - 9 2 9L , s a r e J i nlse r ; a q l l d a I 1 9 L Z lj b L Z L ,sasualap 'JV 'SLZI-VLZJ lsoH t- t0s L 19LaL LZI 'sa6ueqr 209 ' 1 o 1 a u rr 8 t St-1.t SL v L Z I - t L e L 't L aI ' Z 6 0 t - L 6 O L ' 6 u t t t :et d loqelaulpa)nput-snln 'sarol ' U M O p - l n quso r l d u l s u P r ] tL6Z I L S| 9/Z I lerxe 'suorlPlurpoLu ' ' J g z z ' 9 z z ' l L g t' gg71 'sua6oqtedol esuodsar LgL 16z-06z LL Z L ' f i L Z ' , el z ' s u o r l ) e l a l u l 060l'(oz'(s60louroq) sauosor.rorqfsno6o;ot-uog ItL9L'ttSt-11S1 'stsaqlod,(q 'stolda:aj 'sarbalprlsua60qled 'sar,(>oqdr-uI| oPo) auolstq 6utu.tog pup )U1ellauelquaul fSeZ' gZ1. 0SSL 'reLuerlal 's09 'sasnrr^ tH l6z-062 [ lzvE L'zvEL'oltqdosoro 1909l.

sez' €Lz'* *' " :':'',i::iilii:l'

X]CNI

tT:l

'uosueduro)/aieduo)sueau 5 jalqel e ol ralar e qlli\^sroqu.ln u a6ed I :^lantln)esuo)rurolloJ a6ed :fitua aql Jo uorssnrsrp e)erploq ur sraqulnua6ed ieqt sarn64o] lJ ue qllM 'ern6ue ol re;erI ue qtrMsJaqLunu lxal rofpure ol JaJe.r ',Surxauul 6 5[ [ L nLL - 9 V| ; ' 6 u r > r l d a s ^rleuralle ageuur'ualslsTasuodsa.r J66SL 'oo9 '09s '^|rueJradns aunururlaasasuodsataunuluJtaleuul urlnqol6ounuurl t-65s I L ' v N U ]r o l e ! l ! u l 'auPrquaursuerl 08€ 1669I 'uorlP^rlleluaualduro) (stol)eJuotlPtltul ?ss t lrlo^re)na) slla aas()tlo^.lelna)slolleJ uotletlrul ! g 9 SI ' L 9 Sl ' 6 u r q ) l r M ss s e l ) 1 y g ' l e u o r l d u t s u e 4 ' x a ; d u .ur o rt l e t ] l u l usst l 6 t - 6 8 t ' 0 8 8 ' JL 9 t ' u o p o ) u o r l e r r r u I ! S S S [ ' t S S t - € S S l ' ( W 6 t )h Ju r l n q o l 6 0 u n u r u r l 8 8 9 ' ( d S d ll)e t l u a t o d> r l d e u l s l s o d , t o l r q r q u l 6 6 S t l 0 9 S t ' u r e u r o pa l r l - u r l n q o l 6 0 u n u r u r l 'ernt)nrls uretordg fuo1rqrqu1 106_906,1rg1 I L95L '6urq:1rnns sse;r S ZLt - V Z I L ' ( s d V ls) r s o l d o dJeo s l o t t q t q u l 1991 sg€-ngt,uotlPlnuJ 9 9r ! 6 s s r 'IL'St '9SSL-tSS '€9S1 0'(961) uralo.rdp615'i(qdotlele;n>snu paluaqul urlnqol6ounururl I t 9 leurds ' p l o ju r l n q o ; 6 o u n u u . r l J g 6 E ' 8 6 € ' l s e a A , ' a l u p l u a{q; u lo - u r a 1 o r 6 , l0t f 'uorla.r)as aururelsrq 998',leureleu llss L ' u r e q l^ ^ e a q 1998'998'(s)tse;dotolq: € s sL ' 6 u r q ) l r M ss s e l ) 1998'598-?98'uerlapuew-uou 1 9 SL ' u o r s s r L u s uueor ]r l e u J o ] u l s- [ 1 1 5 91 ' s u o r 1 > erar.6r- r a 1 1 e J l 6 e ' l 6 t - o 6 z ' s u o l l e l u l p o ual u o l s l q t 1 9 g t ' 9 S g t ' ( 3 6 13) u r l n q o l b o u n u u l ' u r e q l A^eaq ! L 9 b 'g g V -L 9 r ' u o r l e l ^ q l a uVr N C fss L '6urqr1rrurs sse;: 9L V - EL n ' J Z E ( ' V € Z - O E Z ' , a l n l ) nurtllse u l o l q ) 199I v o L ' z o L 'l 6 z ' ) r l a u a 6 r d a € s 9 [ ' ] u a u r d o l a ^ a pl l a ) - 8 I l s s t ' ( c 6 t )6 u r' ul nr eqqo); 6 o u n u u l A^eaq € s sL ' 6 u r q ) l t M ss s e l l L UOrlelr]rluenD 1 9 SL '9ss1'(v6t) urlnqol6ounu]uJl ( p D e) r a P n u o q u ) aas L l . s s t V l l9s9t v ' aN1U1 1 V N U (s)lla)g os/oaas ) Z O b ' t O V - Z O V ' 1 6 7 1 ou r 6 u o '60s-8os 'seuoprrq^qet/\utr\ ut ( p D el r a l ) n u o q u ^ x o a p ) aas V N C l60s V N C seln)-eloLu l 8 9 l ' u i n l n ) r l e r ) r u s e l d o p u eu l ',Sfrlaoraua srsaqlu^s 8 'aJ!l ^lradord 6urur;ap t 6 9 qr ro 'uol6al uotssIul suetl uotleut.t (lJ) oJU L ):) r lz:: r lcjj L l::: ' l r o d s u e r ul t x n e' s u t a l o r dJ a l j o d s u e lxl n U uI l ' s u r e u o pp a rl lreel d 696 171 ,s)ruapued ( s a r p o q r l u eu )r p q r( l ) l q 6 ! l a a ss u r e q )( l ) l q 6 r l )zz9L'tasl 'SUOXe ur lUAUraAOtx paseq-alnqnlolltul ol6ounuiurl / LSL l o t t ' p l o l u r l n q'surPr.xop 'burleorunp_ue '6ss ^rlua 6l 10SL tgOSL L l09s t 1699t 'Allua sgL lla) r9sr-09sr '65S 'uor6aa i lqeuenrad(q L a sL ' J 9 6 bl ' ( s a ) s n I ^P z u a n u u l L!1S L t 6 S St 'srolerpeur(ro1eu.ru.re;;u1 'ZSS 'uor6aa i 6urq t€S t-€€S L t l ? 9 9 t ! € S 9t (satpoqtlue) € 8 [ L ' p u es u e r ( ; 6 o a 1 o r d ureq) (H),{neagaasurPqr(H)X^paq lr€sr '?€s]-€€sr ' 'lood L Z St ' 9 Z St ' a s u o d s a{rr o g e u u e l ; u r T u o l r p u l u eIB u ]uaur6as-aua6 l€9S t a l r s6 u r p u r q - u a 6 r l uava ss a l r s6 u r p u r q t0€ t Alr|l.raJul 6 6 t [ ' S r S O A l ) n u O uS Ont uO t l ] a J u l l09s t ',l8ss ',69s 'JLSL',gSL',arnl)njls swstuD6Jo/suo1)a1u t >ty>ads os1o aas 36S5t t t-8SS t gg7l- LgV| 'suroldu{s7su6rs sadft tloads oslo aas 'alnor 'g8S'fuepuo:aspue ^reuud z o st - 1 0 5L J9?St 'Ots 'sasuodsar urals,(sTasuodsal t J0'9 t aunuurl :sesuaJap s a r p o q r l uleP u o l ) o u o wa a sl e u o l ) o u o L u lsoH aasot asuodsal sua6ouled L S SL ' w ) o l p u n o q - a u e r q u a u r '(d6t) ur;nqo;6ounu.rur r p l n l l a l e r l ual a ss u a 6 o q t e dr e l n l l e l e t l u l 166Sr f, ' s a s e a s r) pr u o r q l 0 0 st - 6 6 t t ' ( D 6 l )n u r ; n q o ; 6 o u n r . u u . r r 1 6 6 5t'€9S 'z9s 'sluau6as t Z S t - t O S 1 , , { 6 o 1 o r1q; a r s9s t-t9s L !l€9s L I I ' S U a 6 O u D rsee> 4371 0 1 8 ' u o l l e l n 6 a;le u o t l d t . t : s u e l l - l s o d l99S L _ . (s)uorDalul 'luaurdola^ap slouDts)ur)ads osl0aas lla) I'uorl)eles lood-eua6 s a u aD 0z€1-61€t'lelluanoes ' a l o ru o r t e l n r ] s e 6 1 8 9 €[ l / s z ' u o r l e u r q u r o ) aur o x e ' u o r l n l o ^ e '69S 'uo!1e)Ursra^rp g 1 g1 ' l u e u d o ; a n a pu r ' s ; e u 6 ras^ t ] l n p u l l89S L l-Z9S I y 9 g ' s r a l o u o t da l q n n p u l 6 u r q : l r m s s e l )a a s6 u r q r l m ss s e ; : sadh )Upadsosp aas J 6 V t l ' g n t l ' u o r l p u t q u i o f a )r t l e u o s p a l n p u l 9 8 t - S gt , { I l r w r x o t d p e t n p u l I L 9 9 I ' L S S t - € SLS' s e s s e l l 1 8 8 9 ' , 8 8!S1 8 S ' , a l u a ) s e . l o n u o u n ulu) ta l | p u l 8SSt nltpt^e /satpoqtlue 9 9 6 ' u r a l o r d6 o q a 6 p a pu e t p u l 609 leuol)ouot! 9 8 L - 9 8 L ' , ( a s e o sl lrap) - l )a s e a s t p !865' L6S'uorl)e[uror)rur llel-uotsnl)ul 6 u r l u u d u l) t r r t o u e D a a s6 u r t u r . r d u l l e E v ' z t t - L E t 'u o ! l e l ! d ! l e r d o u n u | . U l 88t t'srsoln)laqnl'uotssalddnsounulLul 58S-889 g'^dolsor)il.ualualsalonUounulul! ' p a l e o o s sLeZ- uSat 6' Lo(qSl pt d- 9' s2l u9e1l n u l t l s o u n u l u l 'tlB 'Ado)sol)rruuorlralaounulul! lLOg tO9-9O9 gSV'fztV 'ZEn'uorlelrdr)aldounulul lua)saronU I 8 8 S ' Z 0 S ' 6 u r 1 a q e 1 lSrs t Jtas 'gts '6urlrpe ' s a n b r u q : at € 1 0 s ' z 0ass' s f v t roldalar u o ! l e r P d JS1St t 799 llal 'leraqduad g8S suorle:r;dde 8 St ' s l l a l) t l u pLu- 1 ,^]tut#e a pp a l P ^ t l l e u o u us t 8SS[ '8ts 'uorlalap 'tVSSt'IZSS lSts L t It99 L L leuoll '89S g g g l ' u r a l o r d( I U I V )J o t e l n 6 aat u n u i u t l n e t - Z S 9t ' t 9 S ] - Z S St ' O t S t ' ( t u r l n q o l b o u n u r u r l '(d6t) JL b SI ' L V SI ' L V SI ' J q V Sl , p a l n b ) e l 6 6 S t' ( n 6 l ) f , u r l n q o 1 6 o u n u u . r l n ur;nqo;6ounr.uu1 6 V SL -L t S L ' a l u e r a l o ;i e : r 6 o ; o u n u u ; l66St Z €t t ' s u o t t r u n f6 u t I e ; a l - 1 e u 6 t s l 6 t S , gt S , 6 u r g o ; q o u n u u . r l 'slla) srso/to6eq6 osloaas 1 1 6 SL I t a d l e q / s l l agt 'tIS 'asdeu(s 'elol srolda)ara)tl-llof f 6St t 0€s L ;err6o;ounu.ru; 'sasnr^ot asuodsar JgrS l'gng L'^rouleul ;etr6oJounu-rr"u 9t9 t-tEs t J llL09' Iog-gog'{dorsoj:rLu uotlta;a p;o6ounuruI 8ZSJ-9ZSJ'sua6oqled1o uollru60lar 'uorllunj naquaw tloads 1(s)utlnqol6ounLuwl osp aas tz9 t 'sarnl)n.rls 'vzgL 'uoilnlo^o ll'[ [ Its | t 0 t t ' p l o Ju r l n q o ; 6 o u n u l u . r r Las L' LZS| 9eSI sJolt)tta l/sz'uorlnlo^a l l 0 't 9 t ' 6 t 9 t ' s a n r l d P p e '6€s 'elPuul g n | | L t I l ' p u eu o t t e L l l oaJ s d e u ^ s llo?s t t LEsL-izgL'98t [ ( s ) l l a1) : ( s ) u r l n q o l 6 0 u n u i ju( ls ) l l aI) o s l pa a s u l L L ' L i l a - g n I I ' S t [ [ / u o r s a q pl lpa ] '^Jepuo)essrrfieur.rd y 1 y y ' s p u e 6u4u 6 a l u Z rd 9t9 [

",,:f,t";,i;i';t5#''

(xalduor Itrlrqrledulo)olsrq ro(eLu)fHtA aassuralordJHV\I '6€s t "s eleuul ll0's t t.!ats^sluauralduro)aastuals^sluaua;du.ro: au-lla) l g t s t ' 6 t S L ' {'JZvS p o q r g'Znglu es p a } e r p',a^rldPpe sngL L LnSa ura]s^slasuodsaJ eunururl 'saxalouo) aunuru.rl l€ss t ^ qu P r ] sl e u o u r u r l J ; Z Vl ' 9 Z nL - V Z V I ' s r s a q ] o d p lortuol ell^l lla) os/Daas 6 5 0[ r/saurl 190S'lt0s'uorlezrleuourur '(s)aua6 llar lelioururI ^liea alerpauurl 626 € s s [ 1 l a )I a ^ r e ua r n ] e u u r l ',oDeurl 6ZEI ')s!p l g s Et l e u r 6 e L !6ru r ^ ,lorluo) qlMoj6 ts€ L-ts€ L 1 S €t - 6 t € 1 ' s ) s r pl e u r 6 e u r l 1q6r1 l'8s't8s-€8s "{do:sor:ru-r uorl>a;a z l9-o J9 {do:so.r:rr"u 6 u r s s a : o rad6 e u ; ( s ) u r l n a l r a l uala ss l l €96 uralordg)rl l yl t 9 6 ' ( ) ) t ) a s P u r8 6 9 6 ' u r e l o t d6 o 9 1 (W6l)W urlnqol6ounr.ur,u; aas616; ( 9 6 19 ) u r l n q o l 6 0 u n u u l a a s9 6 1 ( l 6 l ) I u r l n q o l 6 o u n u u la a s3 6 ; (C6l) u r l n qol6ounuu;aa6 s 6; C ( V 6 l )V u r l n q o l 6 o u n u u r ; a ays6 1 (s)uorejralulaar sNll (sv!vf t) s a l n ) e l o uur o r s a q p e l l a ) r e l n l l a l r a t ual a ss w v ) l I 'tZZt '(LItH) 6n L-BnnL t rol)e,alqr)npur-erxodl;1 ' u o r ] e A r ] )ser s a u a 6 0 r 6 u' eer x o d ^ H L Z Z[ '00€ 'aulqluexod^H J t0€ Igt'l)ol) uerpe>rlr'snute;eqlod(g 9 8 1 ' ( r u s r u r q lueo) l l e l u a u r 6 r d o d ^ H J g L bl ' g L nL ' ( r e ^ esl n o e u e l n l q n ssl r u r a p o d { g '(poqrlue'uor6a.ralqpue^rad^H 199l-099 t (IH-S'aurueld,(.ry(xorpXq-g) uruolorasaas(fH-S)aurueldfu/xorplg-5 0 6 t | ' J L 9 LL ' 9 911 ' s a u r l o r d l x o t p ( g J L BLL ' 9 g LL ' s u a 6 e 1 ; o : ' s a u t s ( l I x o r p I H 'uo! J60l l^xorpIH /z /stua^ Jl€ [ [ leultaq]orp^H releM osl, aas 169_66g,suralotd ' Z S' s e l n ) e l o u)rr q o q d o r p l H J L Z q ' O Z 9 ' g L1 9 8 0t t 8 t t t t t ' s u o r t r e r a l ulrt q o q d o r p ^ H Z t9 XqdProoleLlorq))rqoqdorplH relPM os/Daas ' Z S' s a l n ) e l o u:rr l r q d o r p , { g f L Z g ' O Z 9 ' g L1g8 0t 9 ' Z € 9 ' ( s ) u r a l o iadu e i q u a u s u e rIlt' E s l o l d^ q l e d o r p ^ H l 8 Z St 6 0 t ' € 9 ' Z S ' u o ! u r n r u o r p i i l l JZL€'ZLt 'saselaqlu{s :r1(;orp,{g VNUI-lllPourure'6urlrpa '9s'sre6ns s9L-bgtlls srs^lorp^q'(s)aprloal)nN aasaprloapnu 1g-yg's:r1a6raua'selnrelouor)eu sts^lolp^qdlv aasdfv 399'srs{1otp(g 'sauJosrxoJad 'eprxorad ua6otp(g I Zl sdurnduo1or6aas(dund +H)dLunduor ua6o-rpXg '8Z8',ralei!^ l8z8 96g'luaularnseau Hd sdLunduolord lsluarper6uolord lerruraq:orltall os/Daas ,sE8'8(8ls€g LZS,luaura^our 9 6 9 ' u O r l p J l u a ) u ou lo rr e l n l l a l e t l u l 'srsaqlu^s anup o1sluarper6 100t 61y 'a6ueq:xa 369;

zs,spr)e

(uoto.rd'+H)uor ua6orpXg l 0'tat lln8t0: nt l1l sa l e M u l uralotd !1tt'LZl'92 t ' n S L ' t S ' 6 u t p u r qp u e 6 r ; - u r a l o r d fVSl t '6urp1o;uralord 1691 ' ',suotperalut JLtt LLl ' J 6 6 1' t 8 6 1' 8 6 1 ' avNc-utalord rnl)nrls L6L VNC 1€S'ql6ua.rls/qr6urlpuoq

LW

l:1":". 11 B: "''' "o#rl;i

'tS ,spuoqua6orp{g l0 [ I !80 [ 'alnl)nJls)rulole 'ua6orpi(g l9t

9Z:l

X]CNI

;:1', t:i '", ostl!"ii,"i:fr;

' 'ialual rn,lns-uotl 298 t €8 t0t8 t6l'alerdn :r1(:opua,uor; vE8-a€8 'xa;duo>eseprxoaulotq)oy(: lalua: laddot-uorl (sslul) sarrs^llue oluosoqulpulolul aassslul (€dl)aretldsoqdltl-g't'Ilo]rsoulaassdl aAtl!suas-uol JL6S',96S',srOlp)rpur (s)lauueq)uol aassroldelat ldollouol uotlptpel(nn) os/paas lalot^ellln [ 0 € - 0 0 € ' s l u a 6 e6 u r 6 e u e p - y 1 r 1 6 ' u o t ] p t6puetlz t u o l , 6 u r p 1 ou1r a r o t d 3691 ^s spuoq luale^ol Jgt puoq lgg'q16uar1s7q16uel I t [ [ ' s u o t ] n l o ss n o e n b e ll[l'lt'spuoq)ruol dralotd l gEg'uorletgunu lt€s saluleuJ ' l S ' { q d e r 6 o l e u o t q >a b u e q : x a - u o ; J VL S a 'pate6-eptloal:nu-rl;:i(3'slauueq)uol I t6 sadh pnpt^tput osp aas 8/9-9t9'pale6-a6elton 199,,{:ual]Ja trodsuel} '^lr^rllalas 699-L99 'ZB9-Og9 Jl 189 ! lgg,s6urprorarduel>-q>red ',suorlelnur 289 (s)lauueq)uor pale6-pue611 aaspa1e6-pue6r; ' 8 9 9' 6 u ! ] e 6 ItEnt'tEvt 1899 ',/99',uorl)unJ 899 fz69'sarIurP,t 9 t6'pare6-aplloa;:nu-ltll{> l899',saDueqlleuo|leul.lo]uo) 899',^)uepunqP 269- L99'(s)louueq)uol suot)lJr)adsosloaas (s)lauueq)uol aasslauueq)aueJqulat! sluarper6uolo.rd;e:lu-raq:olt)elloslDaas I Z S 9 'J 1 6 9 ' 9 6 9 ' u o r l e l l u e ) u o l 1t'(s)uol 6[]['urj]nlo^ul 'uorlruuap'o^,^u/ t0s-€0s gjb'!r}V'sluauluadxa uotl)alasVNUoJJ,iu/ 'g 'srsauabetnuu dtll ul 919 13 t ' € . OL JZOE e - a O EI ' L 6 ZL 'saJnpa)oJd palelar pue (ln l) uotleztltlre]o.rll^u/ t0s-€Os'uortrulJap'o.rllu/ 'lS 'suosodsuerl 'sleeder peua^ul ,{;uo-y16 L€ 16t € Lnz-gne'uorlnlo^aau-loua6'(s)uotsla^ul 'urse^ul 8 0 5t 'ura]ord f 6 L 9 L ' g L S l - L L S[ lgyrf ia1:odlueue^ul 't89 'ureqr luPue^ul J 9 6 St t t S S ' u o r l r u 6 o t au.or x a - u o l l u l 1 g g 9 ' 6 u r d d e tuuo x a - u o J l u l gg7'llnbrque a>uanbas 6ur:r;d5oslaaas s ; e u 6 r 6s u r : r ; d 5a a ss ; e u 6 r 6s u r r r l d s ' € s E - z s €' r a p r o lEs€ gng-LnE'gn1-SVt '6ur:r1dsy119,(q ;e,rotua.r LSZ'eloJuotlnlo^autaloJd t98 lellauebro 898'leuorldo I t 9 € , Z 9 t , 6 y g , u o r 1 e u eqn1 6 u e 1 ' s a u a 6u e u n q I g O a ' 9 O Z! 9 0 2 l 9 9 E ' 9 S € 'd 1n 1o r 6 j g s t ' 9 s € ' y g g ' 1d n o r 6 'ernt:n.rlsaue6 902 ggg 'sleu6rsage; 8t€'uorlnlo^a (s)uorlul 1 6t ' B t ' ( s ) u o l r P l n r:.ruu a 6 e r 1 u ; '20€ '(ls)l) uoll)elul ureds rruseldoy(ret1u1 lz0€ L I (s)uralordrotoW juolala)so1A1 osloaas ZZOl- IZOl'sa1;aue6topasol)ue-auelquaut 'sarpnlsuralorduotsnJ-dlg l€51 u o o s u P l lt P l n l l a ) P l l u l slonqlodysalntalow ,lllrads luotllnpsuell 1eu6r5 osloaas (s)aseu rl aursor{1aasuor1e1,{roqdsoqd aurso.r(1 't06-006 'asuoosaialtl-ploqsarql I t06 l l 6 8 ' u o r l e r 6 a l u rl e u 6 r s salDapw )UDads l(s)^eMqred/(s)aln>a;or.u 6ur;eu6l5osloaas 6urleu6rs 669- L6g'fn6g'€68'suralo.rd fg6g' L6B'saxalduor6ur;eu6rs 459'luaura:uequa ;eu6rs € 6 9 ' s r e 6 u a s s apuu o ) a s ',l]eqpaaJa^rtrsoo I [06 (s)aseutlutalold oslDaas (s)aseurlaursoji(]aassanprsaraurso.rll

,L09n1,09, ,(E]) 'Durpurq-d]D t €_urlnatratul )LgnL 108[ J) uor1e1{roqdsoqd I t6S[ lo]da)ar Z-urInaua]ul '169 '(Zll) 'sua6olrtu t I t 6 9t Z-urlnaualul €0 LL '(lll) aurr(zue 6urttanuorl-urlnapalul 8t t t l g L t I ' U t t - g L L 1 ' 3 111 ' s u t t 6 a l u r sut\nauatu!)a!)ads oslo aas (s)aseurlu!aloJdoslDaas ',sJo]oeraJ I [65 [ I L t - L L t ' s a l r ^ e pl n d l n o - l n d u l 1 8 6 9t t t 6 9 t ' ( s ) u r l n a F e t u l Jr68'668-E68 reln)rlloJralul 6 L nL - 8 L nL ' s r u r a p r d a t 0 B g ' ( s ) ^ e M q l e d / ( s ) a l n l a l;oeLuU6 r s. r e ; n ; 1 a : e r l u ; 'uo sDeJJa'suorl)elur j(s)rolde)aJ relnllelertur'(s)roldareu 190?t ' Z 6 5 l . ' u o l r a r ) a s ler^ auouroq reallnN aassroloelar relnllo)?rlul 1869t1969t lla) [HI ']uauldola^ap sa^Plua)salonll os/Daas 289[ lle)-l 'uo slrejJa 'slla)-I )lxoloil) seoolosrorpPU oslDaas z6s t (,tNlt)l,-uorarralul 969-799'6ur:er1:rdolosrorpet ,S€S ,(dNJt) 9t9 t t_r€9 r d_uoraJrelul )865' 865- L69'uOrllafuror)rtu gESt-t€S t '(nNlt) D-uoralatul tL6S' 16S-965'uo!lellua)uo) uo! (s)uoralialul (dl9) uralordluarsaronuuaeJ9os/DaaJ 't8s 'sllaJJaa)uaraJralul lzSs €65-Z6S'dlD JLLZ'LL(, l86S'869'uorlerodoJl)ala 'ernl)nJts u!leuroJqr'lepou prouelospagelr6lpragul uotlezrlensrn 'vgt-EgE'JLVZ lsgt 665-969'uorlelndruPur ' sassa)olo JelnllelPllul Itz'(selllads) sralsnl:a;nuer6urleurorqlralul E t S t t t t S [ ' a u o s o s ( ; o 6 e q du r l e ^ r ^ r n s aua6reruozr.roH arrr"u,jiiili'iu rernta)rarul I t t s t ' t L st - E t s 1 ' s a s n r n( q u o t l e l u t p o u t IZ65t'9nLL J ZL St ' Z L 9L ' e u a l ) e q^ q u o r t e ) U t p o L u ' t ' ( s w v l l ) s e l n ) a l o uur o r s a q p lel e ) r e l n l l e l r e l u l t? Z L S L ' J L L 9 L ' u o r t u a n au rodr s n Jl e u o s o s ^ l ) b s t L 'n S EL ' u o r l P J a u a O{ laer; e t t a 1 u 1 lLtst 'zLs tuol; ade:sa 1 6 8 ' 6 u r l e u 6 rl sl e ) ' ( s ) u r e u o pu o r l ) p r e l u l L- LLSL'auosos,{1o6eqd ' 6 9[ [ ' s ] ) a # au o r s u a ] J L L 'J0LL LL ' I L LL'ZtJL-OLtt ILLSL'ttSl-llSl 'ajnrrn.rls ')UJpileuerqurau L L lsoq lsule6esa!6elerls ILL 'uotle^tl)e ul-epls]no 'sallseled 'slla) zl[ [ lsoq Jo uolse^ul I 1Sr-80S I 'aspasrp)rlaua6 pue uorlplnur nilL-ZLJL 9[91-SL9L 'uorlezuaur{;odurl>e6ursnluaua^ou g J y l ' p u e ; 6f t e u t u e u u t ' L L L I - q I L ' Z l ' 6 u r l e u b rrse l n l l a ) e l l u l 'S0S rslla]rsoq uolj ]lxa/^i]uo I [[ JgLLL t-?0s I JZt9 t 'sua6oqledrPlnlla)erlul z l [ [ ' u o l ] e ^ l ] ) e] n o - e p l s u l 90s t-tOs t 9 711 ' 6 u r p u r q: r ; r r q d o r a l a q LJS|-NLS' '(lquasse 'sasnrApue euaDPq'luauia^ou,r ur]lauorqU relnllaleilul J 6 11 ' 6 u rl u pq uU r qu r l ) a u o J q U 96t 1 I Z 9 9 ' J L 6 9 ' 9 6 9 ' ( s ) u o r t e r l u a f uuoofl l e l n l l a l e r l u l 'uortlunJ ^lrsra^rp 'IELL 'VLL osloaas L L - Z L LL f n L LL Jo 1s1a11eue616 's.rolda)ellaluno] 'a)ueualuleul tl [ [ 99L-OSL 'suorsaqpe '(ervrqred 6uor1spue 6uttalsn;: :r/ropua Sil,L-nLJJ 1 ;.gt J 9L L L ' g t J t - 9 L l ! ' a r u a p u a d a p Jg6g'an L-s6g'sluauuedu.to: lelnlla)Prlul pue uorte.raJrlo.td a6eJoqlue lenrnrns 1 9 7 5 1 ' s u a 6 o q t el sd u r e 6 e ' s a s u e Jlaepu l l s a l u l ' p u eu o r l e . r 6 r lla> (s)uortulaassa)uenbas(VNC)6utuanlalul ILLL-OLI1 ;u; e : 6 9 11 ' 6 u r 1 e u 6ltesu o r D a J r p - r q 6 L O L ' J 9 L O L ' S l 0 [ ' s a l n q n ] o r )l e ! ul o d l a ] u l ql aua/1o6 (s)auosotuor iarnl)nlls auosouorqJ 1al)Il llaJ os/paas y111'surr6e1ur7fl 99z-96z IuaY(|od ' g 7 I 1 ' s u r r b a l ugr f , JZZVL'Z(.nL JSEa'gEZ-ttZ'6utdool qsnlqduel l Z L LL ' Z L LL - | L L [ ' u o r l e l n 6 e] ur e l s o l l e 882L lStZ lbEZ'V€T.',sauosourorq) 9 nLL ' n € . 1 l ' s u o r l ) u n Iu o l s a q p e uu r olslq ) g E Z ' S tZ ' , s u o l l e ) u l p o a urleurorq)oialaHotlDaas ) o L l I ' o L L l ' a 6 P l u r lu r l r e ue6lo utleuotq: LLLt t,E Lt t,gLaL_6gtL,(s)uu6alul EIZ' J7EZ'uorlezr 6e1ut epqu-t-e; JSZq'VZE'eser 6 0 Z J' 8 0 Z ' ( s ) a u o s o t u o l qa)s e q d r e lIu 'aserDalul 'sleqlJ ssalls 18 L€ €60t '(alnlrrsqnsutls1et6a1u1 's]r.!eu^p alnqnloJlll.u l/tl. 080[ (s)auosouroiqlaseqd.ralul aassaulosoulolq) )0Ln'oLn-69n '6urluudtu!'(Z-lDl) 'aseqoJalul Z-rolreJqvuo.r6a1t1-ur;nsu1 ss0 t apoujalul bZ6'lEZ6')oldDa) LOnL j86E l'(urels lueld) 'suOinauJalul n E 6 'V Z 6 'I t Z 6 ' ( t - l D t ) t - J o D e Jq l M o r 6a l rl - u r l n s u l J98€| JnnnL'vfr L'(sllarg) uorlai)as LL9L 'l6t'(sS3Ul) s e l r s ^ r l u a a u r o s o q t nz6' Itz6 1tz6'!86 L',861',.roldarar ! t6f lre u r a l u l otd J Z sL ' L sL ' a 6 e n e a ;t rt / ; o a l' u 9 Z ' S a U e l q u l a luel u l a l u l rlnsul ltt I L/6'slrunqns Jt si' Esr'lzsh'Est-zst'sluauiele lolelnsul l€86 !896'896'ernlrnrls 'uolsl^lp 6u1pu;q-tole;nsul JESV'ESn-ZSb'sue1old L96-996 llel ul eloj ',srelsul 6Z€[ S96'sarlredoJd 'sJJnd aurosourorql'(HSll)a)ue)satonU eplrded(1od 6€Z t86'arnl)nr]s'uorlez!uPDro I EL S '€ L ] - Z L S ' ( H S lu)o l l e z t p l rAq. l n t / su l 900 t-900 I (s)uosoosueJf os/, aas L86-SA6'^]rlrqels le)rueq)aur g S S J' } Z S ' s r s a u a 6 e l nl e uuotlrasul rJn86'586-E86 ' s | o l p a l r o ) , o6 u r l s r r vprul e 6 u t l p u n q @so^ouolaw olqdosotg oslo aas l e t a l e ; 'sua6oqled (s)ullela)aassulleJal letqol)rurloj stolten se ZOst- tog t (s)l)asul s86'suiloJofl J 6 L L ' J L L L ' 8t -11 1 L ' s e s P u r l € 8 6 ' u o l l n q u l s l pl e q o l o sa)t^aplndlno-lndul uraloJd'(,,sdrqloJlrt!,,) lzl6'paleouola sadtr1t4oads osloaas 9 9 6 ' J o r ^ e q atqt u e u i ( P 'uo!le)ol osloaas r e l n l l a ) I esedr;oqdsoq6 996 '1 '{emqred 6ut;eu6rs ll t t6 !o t6 16-565 J V g 6n' B 6 ' S tt ' ^ l q u r a s s e 'sraLuorlaJ (s)auosoulsacoslDaas 9S1 - €a p r l t s o u t o q d s o q d I V Z L ') $ u e ) u r ' e s e u r6l 0 )lLtl'finlL'wlt'tnLL ' 6' [ z d ( s 't€ I r r6 !0 r6 t L ' l E E tL ' l u e u r q r e l l eu o r l r u n f6 u u o q > u e 't)ldl areqdsoqdsrq-9'; (s)]uaulel!J elPlpeulalul ;olrsourl,(plreqdsoqd (N9)) f i r c ' E nL ' 1 1 6l o1r l s o u r l l p ; r e q d s o q d l8s/'uorle)ollelnlla) lroMlau r6;o9sp aasluaugreduroragetpaurelu; €€6 n 6 sL ' u t l ) I [ - u r l n a F e l u l ' '(€ dsoqd rsoul r o L 6 ! L SL ' L SL ' l L Z g ' n Z 9 '( s ) p r d r l'os q tll) t [-urlneFelul € 6 S[ a u e J lqout a ur 016 t 6 S l ' ( Z [ ' l t )Z l - u r l n a l i a ] u l 1 8 6 5t ! 5 6 5 L ' n 6 SL ' !' S 1S '(0 '€69 6 6 Z l ' e p ) u o t l e n t l : e6 6 a t t [ll) 0 l-urlneFalul 1 8 6 5t l l t t 6 ! 0 t 6 ' 0 t 6 ' ( € d Da l e q d s o q d r r l - g ' t l' oI l r s o u l (9]) g_urlnaualul , € 6 5t ,'(ill) '865 1 8 6 5 r '€65 J V S VV' g V!'6 9 8 ' 6 9 € ' a u r s oIu r ?-ur{naua}ul t l€65 t

80s'eu, ;:[elxi];5i,]i:;

1865t

X]CNI

9Z:l

'uosueduior/ereduo)sueauJs^ ialqel e ol ralal e qllM slequlnue6ed I i^la^tlnlesuol Mollojleql satn6!]or ll ue qlt/\^'aln6ue ol ralal I ue qllM sraqunu a6ed:i(rluaaqlJo uorssn)srp ]xollofPul e ol ralal arPrploq ur sraqulnue6ed j 0 9 s L 6 9 5 t - 8 S 9 [ s u r e u ] o p] u e ] s u o ) 8t5-9t6',q)loN ',9s9 't95-zs5 '8)rlN L l8s9 r 11ss r ogs t t€s6 ' J V S S I ' 8 9 9 \ - Z S S | ' ( s a r p o qur rl ue pq ))( t ) r q 6 r t $n6' os6-8n6'6ut;eu6rslurvr'utualel-$ (s)ase6rl sutalord(toleln6ar aue6 luale'l VNC os/Daas Z I Z ' ( u o r l e ) r l d aVr N C )s r s a q l u ^ps u e r l s6 u r 6 6 e l 984-S8Z 'OVS'6uruopy516 'l)aJJauorlesuapuolulleuolq) 'saua6alel JLng uorle6rl lasel J66S'665',sJazoaMl '209 'uorl)assrpo.l)ll.! alnloPl lasPl 9 9 6 ; ' p 1 oa 1s e 6 r 1 l€0s '6t€ 's)srp ( s ) l e u u e qul o l IOS€L'pueqt leur6eull pa1e6-rallrrusuer1 aaspale6-.rallru.rsuerlornau ut:a6 papualxa lO€€t I l€t t't€tt'pale6-(llerrueqrau,r t t v EL '6 z t L ' @ | q d o s u o )e l J e l '1yg '6ur:r;dsySgu-ard 'ernl)nls leuel l € 6 8 ' 2 6 8 ' s r O t d e l apr a l u r l - l a u u P q ) - u o l JOS€tBlS (s)lauueqruor pa1e6-pue6r1 slla) )rtupua6 aas;1ersueq.ra6uel 6urpurqau(zua'(s)alellsqnSoslpaaJ qsnrqdurel 882t tS€Z' JbEZ'nEZ'saurosourorql 'sa]rs6urpurquragord 'arnl)nJ]s €S[ J99! [ ( s ) l a u u e qul o 1a a s6 u r u a d ol e u u e q lu o l 89I I TWN ' a l o re : u e p r n 6 (s)pue6rl 8 9 €t leuolnau / L/ s a l n l e a j 'suolle]nu ll-l 19[ l, lesra^run '(ururuel 17'ssa:o.tdrt}^;e1ero1ne 9 9t t - s 9 l | l e l r s s e l )l',su-DoJOsl ), - u r u l u l P l ,alt t 99 [ I IZ65I 3551I lesPq-o)rde lellaqlrda '( uralordpalpoosse-uor])un;-ay(:oqdLui(1) L ;y11 L gLL - g gI t ' 6 u r p u r qu e 6 e l ; o r ) L g I I '"{1ue1od 'luaudolanap 's11a: 6rp,{a1 9971. 9 9 1 l s 9 t t ' e u r u r P l l P seq ' u r a l o r dy x a 1 (s)urururel lZ'''uorsnJ ureurop /sr)alap.rreder '8€0 'xe;duro> yp6 qaM 196Z t dUV^q uorleLuroJ l8€0 L 'a6pa 6urpealle 6utp;nj (lylf) er|.Ualnal tto t 30t0 t 'a;purds snoua6o;a(tu)ruorqJ aassnoua6o;ar{ur rruorqr lllolr.u ]l uollezlue6Jo 6gg1 ' u t 6 t o ' ? r t u a l n aal r b o q d u L l lg 'Lusrueq)aur lua[ra^ou.l 6;75 8€0t-l€0 t eruralnal 6 t 0 t ! o ? o t ' s a u o' ruql l M o . r 6 Lt8'slsPldo)nal l8€01 urlrro) g1 uorsaqpe a1(rolnal 1 1'fuuarrr;ap ' 6 u t ; r v r e r; ;: a r l 9 € 0 [ ' 8 O Ot 'uorseqpe 'sad^l €9t t- t.St I 0t0[ lle) 'arnl)nrls lz9t L 8 0 0 [ ' s l a ou r ] ) e J b S bL 'n s nL- $ V 1 ' s a u e ; 1 r dLer>o . ru1o r l e r b r r u 966'uOrleal)nuluerrteluul])e 'suu6a1ur 'erpoorllaue'l 71 11 l€0 [ l n s v L 'v s v t - t s n t ' a s u o d s a r , { r o g e u u e ; ; u r 1 19y 16olorq)rteqluls ol uorsaqpe xrlaq-urnl-xrleq , g Vl ' , s l l a ) f i g v L 'E l e r l a q l o p u a l 0 z t ' J l r o u 'utalo.td,(.roge1n6ar aua6 - L € SLt ' t s t t ' ( s ) a ] ^ ) o r n a ' l 9 [t 'uor1ru6o:ar aruanbasyp6 J ; Z V ' f n z n 'E Z n ' s u r a l o rrda d d r za u r r n a l 1g 17 'eJn]lnris'aur)nal [ l g S V '8 9 V -L g V)' o s s e r d aer p q u P l ' 9 5 5 ' s u r a l o r d( u16z u l )l e a d a rq l u - a u D n a l epqurele6eqdouag:egaase6eqdo!ratleqepquel 19s6 /utxol ',IuelnurzllJlel €6?L leqlel 999 'stauud l S Z S ' ( s ) u o r l e l n lLeuq l e l YPg JZLZ'ZLZ LZtl'aua6 lla]. - g z ' s 1 u a u r 6 er l.er ;z e y 6 t L B a ' ZL Z ' J 6 g Z ' g g z L 'aua6oldal SIZ t z/z'uorleD!l ')r]ofte)ne 9 L n ; S L V L ' s l l a )] e j u o l , u o l l a l f a s lLgZ'OBZ aseJauolalos/Daas J 6 n Z ' f i n Z ' s u o s r . r p d r uao: u > a n b a sa u a 6 ! 9 L n l ' 9 L Vt - SL n[ ' s ] ) a J J a / u o r ] ) u n J 7 6 7 ' u a l q o t du o ! ] P ) r l d a r - p u a lgltt'^)uar)Uap )9LZ'IZLZ'J69a 'urtdal '892'(uo!le)rlder S1t | VNC)s!saqru^spuerts6ur66e1 y6g 1-9651 (sordal 1 8 9 9 ' 1 5 9 ' a s e a u J aads o l ) P l t6S t-€6S t'(so.rda1snogeuo.rdal J L a ' t ' g z nL -L Z Vt ' u o r l e l ) P t sene^a srs^lol^lgoslDaas 1q6r10s1aaas l Z S S ' Z g 9 -t 8 5 ' e d o r s o n r u1 q 6 1 1 06'uolDnpolo elellel (s)ureloJd oslDaas JossaldaU '09'ado)sor)rrx uoJl)ela (sa)sue'l 3 197'(6o1orq)rlaqlu^s 'srserueurqsre'l a:uanbasyp6 88t L 19 1.7'uorgru6o>a-r 'aseasrps,areuuor6el 80S[ 9 tt Ara^orsrp 'uorssruisuell ',.rossardar re1 8 0 sL ISL7'9LV36 (s)uralordrossardeUos/Daas lzrst'€[st-zLsL ')r;Jei]euerquau (6a]et]s lsoq lsure6e ! [ [S [ l 9 € t ' 6 u r q ) ] r m sl e u 6 t s - o r u 1 -{ROq | ',ROCI Sltaf ]SOU lO UOTSeAUI t9z'suorlelnur rossaroer)el aaslossaloal)el 9 E n I' O Z VI'8 L t ' u o r l e ^ r l ) ed v f 'uolreln6araua6-ro1 l9€t t6 g E 7 ' 6 u r d o oyl 1 1 6 uoradotDJ ISLV'9Lnj6'uQ DtLpuaq)sf) ' , x a p uDr u r l a q e l 650[ stuaurala( n) lNll aassluauala [l 'l oty qdow naud o11a uot6a1 7791'aua6$4a1 tLE L-9te l'luaudolanap {r1auu,{se 1q6u-rya1 9 5 6 ' s u r a l o r (dl o l e l n 6 a re u a 6 ' [ 0 s ' u e ^ u o l u v ' l a o q1u3eyM1n3e3a' 1l I l0s '(entqged uorlenlt)e ur1ra1 tuaure;dr"uo> 6791 '4oMlau '6pg-suot1 B08 '6utputq-ueuueut 6 Z St 9€9'uor])erelurx(;ero:,{;6 /uorleArl)P luaureldurol 6ZSL '(s)url)e'l It LL 9 6 7 ' 6 u r u u e >, s( 1 e e 1 J9LZ 1 t L z ' E r z - L r z 1 6 9 2i g g z ' B 9 a ' ! L 9 z '197 '1uor1e:r;dar y16) srsaqlu,{s puerls6urpeal (slcl) suralordodrl^lrsuap-Molaass'lc'l )6b6' 6V6'(dU-l)uralordpalelar-roldarar-161 (s)uor6arlorluol sn)ol aassu)l saseurlaursol{1uragotd;o(lrue; t.r5osloaas 1069r'r8sL'l)t -L886,l86,urln)untlel J 6 t V I ' O n nt 6 E , 1 ' 6 u r 1 e u 6qr s) ] o N - ] u M 8 5 €L - l S €t 'luaudo;anapalls!rq fuosuasoltqdosotg qrloN-prlaC JL r 6 ' L V 6 - 9 V 6 ' 6 u r l p u 6 r s u o r l r q r q ul e r relel auerqueur)uorsnJJrp J9V9'EV9'(suralord lerolp'l ,56_9n6,srs{;oarold

166'L'nrn) 'slla] raJJdn) l€??[ 'utalord J6w'Osn-gw'I8 ti laddnr) LEt l' Dt rLldosor?'tuetnuJpddnJN L Vt ' J O vt ' u r e u r o pa ; 6 u r . r y e;:i(>pr>e:rr1r;aasa1:{: sqery

sru-) sszI' IzszI'zszL-tsat'auo6

slnolloul aua9 aassleuruPlnol)ou) yXyy 611 Xennqted lenarJler '89tUl'aruanbas /91€ 'uralord]r) t [ l8s' t I t 0 ? ' a i n l ) n r ] s y 1 1 g ' s u r d . r r6euqr s s r y 'uotluelaruletold anrl>a;as 'uotl!u6o)al ut) t€l 'JaqueJoqro]aul) t80 [ 'arnl)nrls l€80 I 9 9 6y 1 u r o d 1 : a q r( ; q u a s s ee l p u r d s 't80 'uorsuat t 980| '9LOL ')!roltru !S€0 t !9101

,uo?19f. r"r,o, !e8or!sgor,r8or-ssor l t 8 0 [ t E 8 01 , 2 8 0 [ , ] u a L u q ) e u e solnqnloJ)rur 'l S L Z L - L L Z Ls r s o r a u rq L' 9LOt-9 LOf i(1ue;od JgLO'!9101 '0LZ'(s)aroqrolaur) €80 1-Z8OI IZOl-OZOt'sulalord rolour sr;laut1au{zu3 aassts^le}elaul^zue s)rleur) (s)uralordroloW 1(sdU)) suralord j(s)urseur) palelar-ursaur) aasx;rr.ueJladns urseury l ? [ 0 [ ' t t 0 t ' ( s d u ) ) s u r a ] o r dp a ] e l a r - u r s o u r ) sutsau!\l0nptAtputoslDaas 'arn])nr]s l? [0 [ '&rArsse)ord OZO[ 'paadslueulenou 020[ 'vtot'salpu rds:rlolru.r '6LOL' LLoL lzgol alnqnlorlil.u lS66'^lqLuassestp 'alts 'v 6utputq-alnqntor:ttu LOt 'JnLOL l 'sa1:,{: l8 t0 t t0 t-9 t01 ; e : r u'auqo:to6uaer q ) a u r ra1ut1 9 y 91 6 t 0 t - 9 t OL ' u o l l e l a u e 6a l t o , 'ur6uo^rPuorlnlo^a 9 L0 t-s t0 I p u ea i o ) 9 1 9 1 ' u o r l e r a u aa6) l o ;'lJodsueJ] l e r luel lz0 [ lPuoxP 391613ts,{1orpr(q 61y Itt0t'tt0t'1ZS'(s)ursaur) (s)aseurlurelordaassasPur) L9LL'JNgII 'SgLL-bgl 'puruel [ l e s e q ' s n ; n t a u o 1( a6 u p t y lz I ['sapueq)]esouol.lJ'seso]e) lEz ['xalduo: aseue6otp(qapale.re]nl6o]a)-n Jt e | \ze L'66'elerelnl6ole)-n f LEOL'L€OL'ftoler6ru g z nt ' z z nL ' a i n l l n )u r u o r t e r l u a r o J JJ o r pl o r l u o ) 6 t v t ' f i L nL t 8 € o t ' s a l I : o u r l e r a ) $tft / 6 L t [ ' s l l a )a l l ) u d t]eJa) l e u i J e p r d a ' s ] u a u eul U s'['ullela)i-D 1 9 8 6r 9 8 6 ' 9 8 6 - S 8 6 ' ( s ) u ! l e r a ) 6 / t t ' a l e J l n su e l P r e ) 'aluanbas llO) 971'Aenqledle^arJlarUl '0ll 'urue]e) '000 [ ts66 I t00 L lrlolrl l os/paas (s)e|.Uosouorql J s o z 'J n o z ' . r a arp'eufeguuJnnuq l € 0 2' s r s ^ l e u e ll€02 €02'ad(ror(re) gg61'auorpu,(ss,raua6elrey I S Z Zt ' 8 Z Zl ' e u r o l r e ss , ! s o d e )

v yyq burporuol osloaas 'vNo 098'ija lunr 'esollnqsrsi(;ouraprde 59[ [ ' 6 u r u r o f leuorl)unf 9991.-p991 luatu6esaua6 Ipoqrlue'uorle)Ursra^rp lpuorl)unf (s)uralordplo#e)Sos/, aas 'Jgb '6tJL-gnta'uorlezrue6;o || !6vl | /pue uorleraJrlord 95 I 1-€5 t t lla) I t S t t ' € 9 t t ' s e x a l d u r o'srsdeu^s lrP u o r l l u n f lS0€

lsz€

/'Z€'uorleurquolar )UDads-alrs eAlle^resuo) (uotleutquo:at)saln)aloullutof

l s s s t ' t s s'v6l ['w6l lgss|

u r e q l ( 6 u r u r o ff) '(ler) eseurlsnuPr 686-L€6 '595-496 Xerurqred 6ur1eu6rs 1y15-1e1 l_6€6l8€6 '(aseurlsnuer) 686-LE6 lPr I ',uralordournzl 862[ saoolosrorpeu aase^rl)eorpej t [ 0 9 ' 1 0 9'sre6ns 'sado]osl I1 '37 s p l ) eo u l u l e l8z t 9S'siaurosl 'elnt)nl]s 'aut)nalosl 16Z[ d - C Z ' 6 u r s n r rorJ. r l : a l e o s l J Z Z S ' !L Z g 't Z S ' l D V 'aseua6o.rpr(qap alerlr:os; 3971 '1ZZ ',alerlDosl | JEZI Jtnn L' VnnL'sueqra6uel;o sle;s; 'aua61a1s1 9961

X]CNI

LZ:l

'uorlellqlaui 'uorlernleur vNo'aseleJsuetlllqlaulatueualutpw J 181 /srezaaM] )tlau6elA 1ZS l l b S L ' ! t 8 L ' E 8 L ' 2 8L ' l r o d t u r; e r r a l e u L 6 9 ' u o t l e t l u a l oudr a t - 6 u o l , u n t s a u 6 e w lbqL' o8 L-6tL'uorlsa6!p relnlla)erlu! 'sauroso6eqd qllM uolsnj aua6 xoq 56y1ir1 n LbL' IOO,L'sutelordi(ro1e;n6ar z€s l- ttg t ',969',uorl)un, ( l S g )^ q r e d o l e q d a ) u a 081-611 ',srsol^)oxa ru.ro;r6uods aut^ogaasaseastpMo) pew 98/ 'uorlnqulsrp L8l-081 'ggg '1urod1:aq:i(;quasse uortepe-r6ep 1 3yg1'si(emqred 7p.61 ,saulosoutdoJlew "1ffilr,"rora g0g pr)e t 081-6LL',saselorp^q lg7t-09n1'alor uotltnpord a6eqdotreu l 1 gt ' L gL - 6L L ' ( s ) e u o s o s ^ l 9AL-SgI'saseasrpo6eJols;etuososr(1 109? t '(lSf -W)rolleJ 6ur1e1 nu r1s-,{uo;o:-a6eqdor>e61 981'UOrlaJ)as leurosos^l sJ;at6urluesard-ua6r1uy osyoaas J98L lSS L',aselorp,{qleurososll n 6 9L - t 6 St ' 8 0 9 t - l 0 s t ' s r s o t n l a q n l JSSt'99''epquel a6eqd Xue6os^l/srs{'l ,(ull) srotda)eralrl-llol tEst l l S l t ' 9 8 1t ! 8 Z t ' o u r s ^ l 'srsPlselau'sur16pog-uou 9* fnEn L'l)etl tuoleldsal l90Z L 'st)ajepredar 'sroldara.r Z€sL- ttS t 156Z vNc euroqdul^ls,l]t)iJng aasqllllrng EES|']Duatuaqlauou4Ds, g0St-l0S t 'stsoln)raqil wnuaDoqoKW (s)euoqduir{'i 'SSS 'pale^rlrp-Ipoqrlue snuJ^qlj(s)apouqdu{1 :nnolreur euog oslDaas l_199t !999 t L 'J6vSt '6nS '€v9 'tpreqdued Z € 9L - t € St ' J B B I 'L g L ' s r s o t r ( : o 6 e q d JtSS| | | 'lereqduad's z€s|xaloL!or€sep!;:JiqSYlj €tS L lerlua) t € v ; L ' E V gL ! L ? 9[ ' t P r ] u a ) 'OtS '(s)uebroploqduIl I ttS L I '0SS 5apt11o1 ptoqdtu,{1 I tSSt t Isilt^ (W)l) s!l!6urueurorroq: :r/:oqdu(1 Jg6t L IZ69I '([Vll) ura]ordpa]er)osse-uor])un;-ay{:oqdu(1 L (s)ller1 1(s)llar I oslDaas l'uorlPln)rr)aj J6bSl' lsg J-6nS'JLnS.L '(s)urbuo EnSL ',slla)e^reu 9?SL 'sllal ^JoureLu 9t9 t 6991's.roldaral6uruloq LSnL-7SVl'(eir,rqiedproqdui(1 ol lue|.UlrLuLuo)'sllal ura]s)rlarodouaq 'a)ueialo] 8ts [ VrSt-€VSL'luaurdo;anap;;a: 1 '{rouratr 9yg g ;e:r6o;ounuru.rr l l s t [ ' u o t ] e u l r o Jp u e a b e a u r l 09'['ra^outnl pueuedsartl ,{sordal n691_9691 . 1 8 6 5 t A l t ^ t l t s u apsu e u o t l a r ) a su t l n a l j a l u l 'uorl)aJur l8s I AIH zgt['uor])unJ 'sllar ueoJ 00s I 888 oN,o l)eJja '6urlarrer> 9gg1 ' s r s o l 1;a: dode 'slle) 1 1 1 1 ?€s [ Jo ]ueuj]tnl)aj J 0 € St ' a p u e q l ) e s i { l o d o d r 1 ,lE6S , S l S , ( H 1s) 1 ; a r _ 1 r a d l a q l 5 6 SL t L 'uorle^ttfe s6s 1-t6s I (s)aOeqdoDeW sadfi tloads oqo aas 'slrunqns zs t-8t I l€9 !29 ll9' t9-89' uole:t;l:ads adeqs L g - V g '9 5 ' u o t l e s u a u ^ l o d suorl)pralutlpln)aloulot)ew aassuotlteJalul 'uotleuorl)erj I ts-0 ls llal g ? L , s l t u n q nus t a l o l d /slla) f€9 l e u a l ) e qs u p t l e u u e u r g€€-s€€,uotltsoduro) 'aluepunqe lz9 E 9 - 7 9 , { r l s r u a q :; ; a t ,98 ,uorleluauo l/g /sluaualtnbat r(6taua /g-ig '€g,srsaqlu^sotq ls8 z9t-gt1 Alqulasse (s)aln)alourorlew spoqlau )rJDadsoslDaes suotl)elalul pue6r1-ura1ol6 aassuotl)elalutpue6r;-uralotd suotl)elalul vNc-ulelold aassuotl)erelurvNC-uralord 'gs /suot]letalut 189t t tgg luale^o)uou /suot]lela]ur:tqoqdotp,iq I Itt suorl)eJaluraletlsqns-au,{zul aassuotl)elelutaleJlsqns-auj^zuE uotlpztpuq^qVNC os/Daas l 9 0 t ' s u o t l ) e r e l u !v N c - v N c SUOrl)etalut teln)eloulollew (d9W)aleqdsoqd-g-asouupW aasd9W

tll (s)aulosos^los/Daas 'Jnb 'ernl)nils JVgt | ,suotl)unJ ngL-Egl 8tt'spuoq-epulnsrp l S 9I ' J V gL '9 9 a _ V g J1, 9t , s r s l ; e l e > aulzosll aw(zos(l oqo aas za L- Ja t'salon)eAlsea^ 'uolsp^ut,znD1169'eurnlo^ owosouDdful [ [9 t JSSL ' VgL-€gL' LaL-6LL'sngeledder61o9Luor; u.rorJ J96L'tS6L'Z6L-L6L',seurosopua uoosuerl l08l'arnl)nrls !o8L'OgL'dund uolord zgL-13L,sa;on>en1ueld /sauerquaut ll69 'sa/:oue;ar.u 991

znsL-Lnsa

'uals^s eunLuuraleuuruo a)uapuadap 'uoll)alas tts t leuol) nns L-tvs L'luaurdola^apllal I ' , { g r u n u uar n r l d e p e t t S t - O t St I S Sl - O t S t ' s a 1 d r : u u d; e r a u a 6 'slla)rol)aJJa 9'9 t 'srsoldode':rxolo]{: 0Z L| ( r o 1 e ; n u r l s o'ru o r l e A r l l p gyg I 1 e u 6 r s I E i VL ' t 9 VI - ( S Vt ' ( s ) a l ^ ] o q d u r ^ l t ' ( s ) a p o uq d u ^ l J tSSt'0SS /uor])un,/lassan>rteqdur(l 9??[ 'aseasrpaur^l 00s [ 6urzrurelnl a 6 Z L ' L L O 6 ' ( Hal )u o L u r o q g € nL - n E nt ' s € t L - t € t t ' ( s ) 6 u n t lua)saulunl L6S-965'salnraloul l o s s L , 0 s s t , s t It , u r ] r a l a s - t ) UI l 9 S 6 ' 9 5 5 ' ( s u r a l o . lreda d a rq r u - u D n a lU (sdt) apueq))es^lododtlaas561 uels{s xo17e.r3 xol aas}uauralauor}eurquJo)ar It6L'Z6L ' J t 6L ' Z 6L - L6l ' ( s l C ' l ) s u r a t o r d o d I (rl r s u a p - r u r o 1 JL6L 'n6L-l6L'srolda)ar (lCl) uralordodrl Iysuap-ruro1 ',aseasrp s,Duqe9nol /86 g E a L ' O t €' ( H O l )I l r s o 6 ^ 2 0 r a l aJqo s s o l 899-1SS!8ZS'(s)uorlelnLuuolt)unj-jo-ssol JZ69' t691)a!!a'(dfl) uo!le!iualoduia]-6uot '(Cl'l) uorssardap ura]-6uol 169lorluol )ldeu,(s sluauela ( t l ) l N l l a a sl u e u a l a r e a l ) n up a s r a d s r a l u6ru o 1 fZSn'ZSn'ZSn-Ogy'1s1uor6ar lorluo) sn)ol l9sLr'9sLL-99tt ftue1od;eseq-o:rde;erlaqlrda'uraiordTaua6 1qry7 'arnlrnrls l€?? L (s)ailroledaHaasslla) 6 Z Z L ' 8 Z Z l - I Z Z L1 a ) u e ) 1a^t1 tttt-zttL '9t 'Zl ,(s)qdorloqrll 'srolda)el 80s [ lS L

lztst '€ 'uorDnrlsap auerquau t9 t-Z t9 t so6eqd '80s 'slla)lsoq; e u ouorse^ul lz t s t t Jo 'uorl)p pue ursi(;oulaq uorlar)as ZLgL-llgL lll9[ 'ZLSI-LL9L'aurosos{;o6eqd u-ro.r; ade:sa }so11 l9tst '9tst ! 9 t s t ' t L s L ' l u a u a ^ o u rp a s e q - u r l l e sauadojLtouowoualsrl 3 3 9 I q d e r 6 o l e u o r q >p t n b t l l€08'urdorlodrl-1.

3ggg'urdotlodrl-fl gg9 uralordauetqurau firlrqn1os J S ( 9 'J Z Z q ' Z Z 9t-Z 9 ' ( s ) a u o s o d r t 'arn])nrls llzs [ '^q uolle^lpe a6eqdor:etu JO€St /29 ['uor]elnuJ]sounurul l06tt'(Sdl) apueq:resi(;ododrl /uotleutoJeeloaAP) 061 u s se l'z b L ' w L _ z bL , r ( 1 q a '929'(s)Uerptdn JLOS 108t9Z9 sasDut\)ut)aos oslDaas /(s)aseurlprdrl lz9 'J029'ainDnrts zz9- Lzg 129-029'uolleuioJ snoaueluods 979'6urleu6rs lEsg'Jasg'rfirIqeeured enrle1a.r , ,firpqeauiad ZS9 L€9,y79 17gp'9gy-9gy'ut6uo JtZg'ZZ9'Artltqow 1s;ure1o.rd auerqueWaasuo!le!losseuralordauerqurau.r n 9L ' , J t gL ' , s u r e l o rl o uvNs'uorsnj rleg'nag-azg'^\p'r'l! sz9-?29'uolleLuroJulPulop zs9'zz9'uotsn]'ltp *29' nz9-729' uortrsodrro) /seuerquau zzg ))elq ' u o r t ) u n ,J a ureq ts9 )929' LZ9-929'h lauru-rIse (s)prdrloqdsorld os/Daas *b L'St L-Ei L'ZZ9' LZg-OZg'Alq|.l.lesse '629-l '6 '(s)re^ptrq prdrt t9 )SZ9 jglg 0 € 9 ' s u r e l o re d u e r q u e u ' ( s ) r o q l u ep r d r l sadh )tJDads:(s)tel osp aas 199'srsaqlu{s t 9 Z 9 ' 9 2 9 - S Z 9 ' t S| | ' r L 6 ' l n 6 ' s l a l d o t p (s)auerqLUaW lalelrq prdrl osloaas n z g ' 6 2 9 -L t g ' ( s ) a u P r q u a ul l a r (s)ralelrqprdrl aasuorleur.ro; rar(e;rq 'sa]e6ar66P .S [ [ lsrr'89'(s)ptd!l r lu11 9 t o t ' ( u r s e u r l ) ' u o r 6 a. r. a 'vNc ialurl [ [z JZLL 'z L l - LL L ' S u O l D e i a lpuul e 6 ! l - u l a l o i d ' a 6 e > | u t 1 (ra^o -6ulsso.r:) uorleurquro)alsno6oloruo;1 osloaas l 0 9 S ' 0 9 9 - 6 S S ' 6 u r d d e ueru a b l 0 9 S ' O 9 S - 6 5 S ' u o r l e z r ; ea: ou1a 6 0 9 S - 6 S 9 , s r s I ; e au 6e e 1 u r 1 vNc e^rlrlaoauoslpaas 3 g 3 g ' a u o u a 6a s n o u r ' s u e u r n q 3 1 6 7 ' a u o u a 6u e r . u n q ' 1 8 l . €' s l u o u a l a( t l ) laz€'zzE-Lt lNll TZZEL ' ZZ€L'suobatastrpqDqlouaD)'6uoer1a6eaurl 'aua6 p pq 79791 97gS'aua6yutl 'euo6 u],1 s8t [ 'Ogt 'lMau 'uor]elaua6a,qu tl t JOg?[ J n r c L ' r g s t t ' ( a l e i q a u a ^ )s p n qq u r l 1 6 6 l L ' 9 6 1 1 ' 5 6tt' l l e M1't!nluautoul ; a l:u e ; d ' u r u 6 t 1 66S-g6S 'z8s'sl)oJJaeruereJralul t8s lzSs '695'aseqd t8s lzSs ',l t85 3285 t85 8t5 st)eJJauorllerrjrp 'uorlelaua6 lsprluo)l?gS !€99 ',sa^eM lqDrl lJZ89 I S 8 S ' S g S ' 6 1 9 ' S S S - y S S ' u o r l e r e daal rddu e s uoJt)ala )609 "s^ ^do)sot)il.u ,€gS-0gS,uorlnlosar 06S!289 t tBg {do>sor>rtu}sej}uo)-aspqdaastsej}uo)-aseqd ',l8s ',aoo)sor)rur lt09 I l8s 'tgS-€gS '6urssa:ord ltSS _a6eurr 619 Arolsrq Xdo>so.r:rtu a)ua)saronlJaasa)ua)saronU ggg'{do>sor:rrua)uara}Ja}ur-ler}uaJaJJrp €SS',plauUep 't8S 'uorlereua6lserNo) lngi'ng9 lt89 €S9 pletjrqDuq 1 0 6 9 ' 0 6 9 ' u o l l n ; o n u o : eap6 e u t JZ6S'L65'Idorsor:rrule)o;uo) 0 6 9 - 6 8 S ' 6 u t 6 e u6t - g €8S- 1Bg'Idorsoortu gq6r1 1g7g'gyg'saxaldtuo:6urlsanreq-1q6r1 slsaqlu^soloqdos/Daas 66S-g6S169'^6raualq6r'l '6ss 'surPuropalqeue^ 1-gsg L l09g t l O 9 9t ' 6 S 5 l ' s u r e u r o p6 u r l e a d a i 995L-Z9SL'r{lrsranrp ;o uorlelaua6

X]CNI

8Z:l

' u o s u e d u r o l / a r e d u r ouf e a u s i e l q e l eo l l a l a l I e q l l M s l a q u l n ua b e d s l'{la^r}n)asuo)Mollo}leqr sarn6Uo} ll ue q}tM'arn6r;e ol ra;arI up qlrM sJaquJnu a6ed i,t}ua aq} }o uorssn)srp ua} rofeu e o] rarala)erploq ut srequtnua6e6 6 u r : r ; d 5a a s6 u r : r ; d s 9St-SSt'(lsea^)ad& 6urteW 865- L69'seln)alourlueaL!raduI s.rol:eJ6urleW (llto^re)na)6urssaroldylg aas6urssa>ord uoosuPll 99'-SS''0seaI) LSg'ssor)e 269prdtl osloaas (s)ra^elrq uoosuerl l S € ' 9 € - 9 'auoua6 € ' 6 u r 1 l a p o lue l l e u a q l e 1 i \ '6n9'einlrnrls :rurseldol(>-real)nNJapunaaruodxa realrnu 998 tS98 lellPuoqlotil.u Ltg'Ang- rlg J8L9 'ql6uarrs 'L8r-98t 'uorlezr l99g'998'auoua6lse;doro;q: 8t9-9t9 )Lgv le)ol a ) u e l r J a q ul er u J a l e w L IL69' P) aull)oxa )lleal)ued ' t 6 t ' ( S l U le ) l r si ( r l u aa u r oLsLo S q u1 e u l a l u l 99V'JSZV I 8' l b ' . u r a l o r dz D l e w ! L6? 1 1 6 9 ' ( a l ^ l o l e d a ql)l a r r a ^ r l 'sad^i 'sla^aluotssaJdxa 99t',ureloro [D]ew Z t' ITLSL llal ueurnq 't (.role1n ggy-ggy'6ur1rpa 6a.rralseyrl 799 1,'surago.rd t S l - f l 9 t ' s a r 6 a l e r gusa 6 o q l e dr e l n l l a l e r l u l 'sauol) o r l o r ) a su r l n a u a l u l 1 8 't08 6 9[ ' u'uorlar)as nns'JEvs vNCl IL69',869L - 6 9 ' , 6L9' , g zl e l n l l a ) e r l u r y5gr.u:r1o{rerna;o 6urdde3,Eaassde>-,9 aururelsrq l/s9 t tt08 J69L' 89L' t9 L' tEgL.'uotsnl (pua ,€ VNUrU)uor1e1{uapei(;o6 osyoaas 9 S S I ' L S n Ls l l a ) ] s e w zz9-129'^ltptnu ,Lr.iapue] 'uorrprauabpua I zs_6t s Ean-ZAn'fLS€.'g;t lS€ lerlualodeuP.rquaWoslDaas ,t eue uralord fOaS' JZS-619'srs^l 88V-Lgn'l9V€')rlo^rerna 269- I99'sa tuado.tdlP)ut)ale /a)ua.raJralur l0Z9'saldr)uud S6t 0[s'uorlonrsrp VNU auerqueu (s)uralordeuprquraWaassuralojd t Z 9 ' u o r l s o d L u o l 176p'76y'sAenqted (erap ypglu palerpaur 0a9-6t9 tor-tctvw l 9 l ' s a p r l r s o u r o q d s o q d r q rp r n b r ; -asuasuoNaas^e)ap palelpeul-esuasuou u l a l o r do l p l d l l Z Z g ' u o r l e u r q u o(:q d e r 6 o l e u o'^)ern))e 6 2 9 ' o l 4 sprdtllonpt^tput 6 LS E6n-Z6n 'sw'!6Lt'uotle16$31 (rlauorl:ads sseW prdrl osloaas l(s)prdrloqdsoqd :(s)ra^elrq u o t s s a l d xaau a 6 (s)aue6reUodaUaassaua6raueyl sSt uorlepPrDap ) s v 9 ' 9 n 9 ' 6 z ' 9 -t r 9 ' ( s ) p ! d ! l 'salerqeua^ur 'ur 'spl)e uor]ezrlrual euuew AUeJ l6z [ 6S-8S ' e u r o r p u ^ssu e j r e w uorlrsoouro) t6t t-06t t (s)aueJquraw n f f i L - | 6 E L! t 6 € t ' l e l r a l o u r l a r 9 ' 8 ' s l l a l ' ( e u r ^ q ) u a r e dl l)A q d o s a y l '(urerq) sdeW 6aulr l l a W JZ6Et'E6el-16€,! 9t8'sllarllAqdosaW 3 1 g g ' a . r n l e r a 'duut ru olelal 'l)ol) uerpe)r) (sdVW)suralordpelpr)osse-alnqnlor)rW aassdVW s n n L ' s g t t ' s € €Lt l O € t ' 1 0 € L ' u r a p o s a w t9t ',aur^qluasaw guauodwot t4oads oslo aas L'saurosouelew L?LL f i 2 0 L ' J z z o l ' z z'0uLo'9! l8! u l J a'pe u o u e l a w g9t[ 'slla) uals € 0 1[ t 9 0 2 leu{q:uasayrl '|6e6' slla) leu^q)uasalA L€6-AZ6 XeMqled6ur;eu6rsaseurl-dvl^ 89t t'(slsplqolqtJ,,atnleu|.Ut,,) 3ggg'uorlrnpord '(9561)auouLroq6ur1e;nur1s aseurl-aseurl-aseu!l-dvy\l ay{:oue1ay1 16z6' 6e6'()))dvw) 9 9 8t U U I W aassaurosoueleul SauioSouelaw r!626' 626'0)dVtll) eseurl-eseurl-dvlA 0ttL-60t['azrs g g 9g ' l u a u d o ; a r a p (s)ased19 seuos/Daas 981'stsov(:oxa leurosos^l b a s ' L A ' t f^ q ;ueorri luea; n n6ar 8S6'stuetd ,a0t-€zo,t'Llsu JLS6'956 'sl)eJJa 'sal^)ouPlew 0€6 IDN l8 l.t [ E911'eleu.rsapou.rse;d 'ottl-60?L 'a)ueualureu 0€6'leuoJnau l0 tlt 1 6 z 6 ' a s e u r))a w 'uollnlo^e 'iaMol, (ra^o-6urssor)) uorleurqurolar Jll L t It[ uralsuau le)tdVaasle)rde snooolouroH.rapunaa5uorleurquro)erlrlorew 1626',626'>t3 srsolrlAoJ/Daas 16z6'spaJral9l tott's6t L'996'(s)Lualsuew '082 'sa:uaragtp6utu-ttl )9€.6'1626' lE6-aZ6'826'(s)aseurl-dVW L / LS'loueqlaolde)raw-d I t8Z t ,100[ 'sa)ualaJJrp '(.touaY1 xas l[00t ! 9 6 6 , u r a ] o r dz d v w 96€ I ^r!)!]seld:tldeu^s L8ZL-082[ ',uorlelnDar s ; 1 a(rl o L u a y l j g V S l ' g V S | X t r u n u r war r u 1 d e p' e ) leqdsoqd-9-asouueW bgL'SS L ' ( s ) r o l d a ) a(i d 9 W a 1821-O8Zt 'a.lnl)nrls ltSl z 6 z t ' 0 6 z l ' 8 8 2t ' 0 8 2t ' s a l ^ ) o o 9tS['slle]g(.rouLay1 's.roldalar (s)uralordlauupqf lUodsuerleAtl)Vos/Daas nBL'SSL J L L Z L^ s ' L L ( l - g L a L ' J t L a t ' a L Zj L slsollur lrodsuetgreln:rsa1aaslUJelllplnltsa^ L'SgL'6ur1.ros fgg [ 6 0 [ leurosos(; ( s ) l a u u e qul o l : ( s ) u t a l o l lda u u e q ) ,8 L-tq L'vodtut ;euosos{; 8 l Z [ ' s l u a u u a d x au o r l e l n d r u e u r o r ] r u (d9W)aleqdsoqd-9-asouueW i(s)urafoJd .laure)luodsuet]aArl)VoslDaat )rlorau.r 0 6 0 1 ' e s e q d 's, l l q L eL ' V L Z I ' r t L Z L ' a 6 0 tI t 6 0 t ' , 0 6 0 6 2 9t ' s u r t ) e l6 u r p u r q - u e u u e y r l L srsoraur lOL'owtluou o6otoutaql 0 t - 6 ' t l o d s u e t l a ^ l l ) e p u e s u r a l o ; dl a u t e t JLeVI'sler lPrlaqlrdaolur O g Zl ' g L e L ' s a x a l d u o )u r a l o r dl U o e d s )LEVL 269- lsg'saln)alou IlPr.us ) 9 L Zl ' 9L Z L - ; L Z L ' s a 6 e lIsa s e q d o . r d Jo 'gzvl-gavl'sap(: gL al - Ll z l ' s a i o q ) o l a u r l p u e u o r s s a r 6 a r l u e u r d o;anap uoosuell uralordluorle)olsuerluralotdi6utuos 8 Z ' [ ' e u r L u elle s e q ZL 1 6 0 t ' , 2 6 0 t - 0 6 0 [s' r, ts o r a u r J TL Z L ' n L Z L - E L 3 'gzbL'.loa^le urarord1(Ul)unlnltlat ltutseldopul os/Daas LZt L )LZtL ItEZ'sau.rosor!orq)qsnrqdurel puelo Aleururew 6LZt-8LZL 9 € Z - S 6 9 ' s u r a l o rJoo 'seldr)uud jLLZ 6 o l o e u o q 9 1 9 9 9 9 , 6 u 1 1 urrdtur L ' A L Z a g L Z l ' u o r l e 6 a r b a s tS9- LS9 ruorle^rasuo> :rlaua6 (ra^o-6urssorl) 0t-68 99L- OSL'r PlnIle)PrluI gy'surago,rd ,{.rote1n6ar aua6 uorleurqulo)ar snooolouroH LS9',a)ueuodurl lg gg9-gg9'suorber;orguo:aua6 6olouoq l.roosuerlauerquraw np un aasnno-6ussorl/uortpurquro>ar s>1taua6 uPrlPu.ruJeW (s6o saurosor.u orqf I Lt g t , t t S t - f t S 1 , s a r 6 a l e t 1 s louroq) ua6oqledrelnlle)erlur'6ur1:rge,r1 saoadspnpurrpurltuaudole^ep asnoWos/Daas sno6o;ou-rog .rapunaas6uted 6olouoq auetqu-rayrl 'oftqua Ipea saryedordanrle;n6ar ?81'(s)rolda)ar 996 t ;o J 6 L Z I ' JL Z l , ' p u e , ( ] t s : a n :t tpl a u a 6 ggz L '08(.1'uorteAresuo) ( d 9 w )a l e q d s o q d - 9 - a s o u u e u ' a l i l naquse r q u r a w fueuorlnlo^a 1 9L EL ' L L t t - g L t 1 f t l e u u r ( s e1 q 6 r r - g a ; suolotdysadfitgoads oslo aas l[8€[ 6tzL-8LZa'eol)e 't8€ ' ( s l l a ) s l l a )u a l s ) r u o ^ r q L u a (s)uralordauerquraursue4 aasauelquleusuell L - 0 8 €t l8z L sturool)aql Sl) 'arnl)nJls 9t t t6Ltt'08t1-6LEL tazPzLzl 'saueiquaru>ruollquaerua pue sa6e1s 'n i(1.rea I z J - z L z L ' 6 9 Z t ' ? : 6 0 1 - 0 6 0 I1t S g ' , t € ' , s r s o r a w 979-9g9,(1rlrqnlos y 7 9 ' 6 u r 1 r e .ar 1; > r g . r e d - a 1 6 u r s (s)Ielareld :slla)pool8 os/Daas lz8€ t 'SSv 'vsnL 'IES7L'666 'sa1(:o,(re1e6ayr1 '28€ '(6un 6urq:uerq l 9 t t8€ t 0t9'leiaqduad l Jo)srsaua6oqdrotu JLSnI 'luaudo;anap uerleuue6 'suralordTsaue5 €.n9-zn9'saxeldurolluauoduro)runu 71a141 €g€ l-gZ€ t 3y9y ' a ) u o ] s a u a as ^ t t p l t l d a J $ v 9 ' 9 n 9 'L S V - g g n ' u o r l e z r l e ) o l 6S0t l9S€[ 'ourlrpa 'sallsuq^JosuasouPqlau.i'(s)rolda)a.loueq)aw t8t 0€9'palurl-prorl vNULrl ' s p u o qa p u l n s t pu t e q l l a l u l 9€g 6 S 8 ' V N Cl e u p u o q ) o l ! u r l . 0 t L ' 1 9 0 t ' s 0 L L ' u r a l o r zo L u p w v8z- E8(.'J lSZ',uorleltloal vNc L 0 tt ' 1 8 0 t ' n L O L ' V 9 O t ' 7 9 6 1 ' s u t 1 : , { r - 6 1 0t9'ler6alur TZNL lEg'uotllsodulo) leltulaq) B9Ot'fgsn' f69a'suralordurl4 ' /arnlln) '6LoL-8IoJ 'a1o.r {lqu.rasse a;purds €nL-Zn L'.roq)uelolrsourl^p!leqdsoqd 1 6 9 0 t ' 6 5 0 t ' s r s ^ l e uael l ^ ) l l a l 080t !610t llsol^16 lla) 'pue ( s ) l l a )u e r l e u i u r e w s)rruPu^palnqnloDrur 999-g99'uorlelIso:(16 080L g9t'ulnlnlllal )truseldopua ra:ue3 aasr(>ueu6r;e61 n L OL ' L L O a ' s r s o t roulJu r^ r l u a an9-9v9' Jt v9'st9-zt9'uorsnJJrp 0ZS-619 Xrlaurorl:adssseullOt-lC-lVW )VLOL 'arnsodxaelPJrns 'aseua6orplqapareleW 's 0€9 Io J-v Lo l'uortel,toqdsoqdep,{quor1en11:e 1g7I lla) 'ra^elrq prdrl qtlM uolter)osse Jl9g'OE9-6a9 990r'2901'Ip)-w l€zt'lzzt'aletew ',slUnOUre utntodo 1o1utn powsold osp aas 629 l066,6g6,ura1o.rd ;qyr1 '6n9-629 '(s)uralord ' p e a r ds ' l l a ) a ^ l e ua l n l e w auerqulaw !0€9 7 g g1 - 1 9 9I € s st I ( s ) u u 6 a l ual a ss u u 6 a l u r I L 9 ' aL 9 - 6 9 9 ' 6 9 9 ' 6 u r 1 s a . r L ' S 6 n I ' S 6 V L b 6 b t ' e u e'ew J S 6 n (xalduro)Ilrlrqrieduo>olsrq ltag' lz8-028'auPrquJaureuut leupuoq)oitr.Lt 6 9 1| ' , S J O l O a )XaUr I e W '(sdww) rofeur))HW aasxa;duror{1r;rqrleduo>olsrq saseurelordollelaur xulew rofe61 t l z g ' J LL 9 ' 6 9 9 'E S 9 ' l e r l u a l oadu e i q r l a w t6 Lt auplquiaw arn])nlts vNC os/Daas J 6 Z Sl ' 6 Z 5l ' x a l d u - r o1: : e 1 1 e 0 2 9 - 6t 9 '{rsauorl>ads(lOt-lClVW) suotlDol/sad /{l ) t}r)ads:auerqulau }q6tU l j z n ' 6 L v 'l l l p w x r l a q - u i n l - lxol q puseld j(s)uer prdrl :(s)re^elrq ptdt psls!sse-xulew uorldrosap rasPl aas ) n e V 't Z V j 6 l V ' s u r a l o r d6 u r p u r q - y y q JO-aurl-uo!tez!uor .1.osp '6 6ur:ua;rsauag luorsJa^uo) auag oslDaas t?9'AISO)sr^ 16 Lt tt'uorlru6o:ar anoo.rbrofeyl 'f8 ',azrew uoosuPll t L L n L ' t f t L - O t b[ [ E 3 9L JBS7!L1n'J99v'g;v-;sr'6urqrlrms ad,{l-6urleW reln)tsan:uoosuell auplqulaw os/paas L9V lLSv' g;n-Ssr' allasse>ad,{l-6ur}eW

bEt, tE€.,r,(r d,i,i;rT]#',3:,'"^

6Z:l

X]CNI

.s ,uotlPzrlelol lg t8 tg t g L SL ' S r 9 t ' S L 9L ' LL SI ' 6 8 9 t - 5 9 S I 9L OL '9L 0t - SL O[ ' s r s o ] r u l '(xelduro) 's J'uoltPr)osse ptdtl rofeul 1gy1 eupuoq)ouur 898-1.99'ljodLLI ftr;rqrleduro:o1srq l9 t8 t8 !l6E t 'rossoider triodut utalold leupuoq)olty!os/Daas ursautl ley! l€Zt ,lloru laaqsd l?tOt'als 6urpurq gtl'olur lrodr,ul ) L V E ' g n t ' 6 u r d d ey: p g u r , g lS€0['5€0t'relodra]ul ,sl)adse g (s)aserarsuerlllqlaur qynor6 aasVNC snurtuTsn;d,to aler tg_s tg L6' 9 L6-S lP)uolstq VNC J9 L6'spue 'uorlezrue6losnle.redde (s)aseraJsuerll^qlaW t6109 tzg-gzg'alueualuteu,lotlel dcv:d-LVq6!q I zo L 'aln]lnl]s 'stselqorqu g€g uotl)npolo leaq J [0€ l8t0 t 'uorlezrue6ro uinln)rlar:u.rse;dopue lg9g,gs8-lsg,uotst^tp ueqlMor6 1361 JLgt' L9t' uotl€l(qlaurVNC 'elorauosoJluaf g g g ' a s e p t x o l aadu o t q l e l n l 6 0Lb ll}E,uotlPUtureap 9t0 L ,aursoybl{qlaW_S auoua6 a a sa u t o u a b l e u p u o q ) o t t w 1g7y l L 8 0 t '080[ '086'696'(lrlrqe1sur uotleuotllelj lla) os/Daas (s)aseraJsuerlllqlal/! os/Daas :rueu,{p tz86 I t86 , u o t l e u o t l ) e J j ' [ 9€ 'vNUr l l t g LI s t0zt'680t !980t'990t'uorrpzuaurllodep lz9€ 'a)uelqu.laser 6Sg Lgg Z Z Z ' s l r eel u o l s r q leua])eq J € Z Z ' € 6 6 ' l P J l s e ' u o r t e J n 6 U u o l uorlPl^qleurvNc aasvNc relnlla) $18'nL8 6 9 6 ' u o r l P ) o l 'uolalalsol^r uotlelAqlaw 1 0 9 8 ' 0 9 8 - 6 5 8 ' J O O6L6' 9 ' t 6 Z ' A Z -t Z ' u o r l n l o ^ a 9tO I g9y'uor1ellq1au stsaqtu^sdIv osloaas u r l ) eq l r Mu o r l e ) r u n L ! L l r o ) ' u o r l e z u el lleold y116'suralord6urpurqy56 paiel{qray1 (Molaq stsoltwosloaas OZg'xalduo) eui(zua,tolerrdsa.r uorlelr(toqdsoqd 9 6 g ' s a s e p r l d a d o u r ua eu r u o r q l a W 6 9 6 ' p u eu o r s r ^ rlpl a ) ,auruotqlan '6urdder aArleprxO aasuot1e1{roqdsoqd eAtleptxo t6zL'LOt 9661 't00 'sdVW 'salpunq ^q uo;lezruebro )618'tL6',saln)elou pooJJo uorteptxo IgL'JgL'sD)oouDt4taw JZ00t L ' u o r l e r a u e '6s u o t 1 : a ;(e6 : a u a - q 6 r q 'LunuaDDqouDt4law urlnqnl juorleel)nu'(s)elnqnlor)rw os/Daas 41g lgL ,6 ,s)tla6roua '816 ^ l q L u a s s e 16tg t8 I l z ( .L 'o z z t , ( s d a l s s) s a ) o r d SL6'vL6 'ot 6utPea;suotlelnu (s)(s)uteq u )o d s u e r l oszl-6tzl fS66' a66'suratord^rossaf)P 'ouuolruor.! '(s)alnqnlorllli! uorllele aas uojl)ala uodsuell leupuoqlo]tw 0sz I 1596 's89 'aruolor)ll/! e;:(r pr:e lult) aasa1r{t pr:e :u1o 6 n zL ' o z zt ' s s a u a A r s e ^ u r / u o r s lee^)uorl ls85 ' u o r ] e l n ) r ) g7g1'suobala2 ,X,ra6.rnsor:r61 rlua LZ9-S13'uo tslo^uo)^6teua O S Z I ' J I Z Z L ' 0 Z9[Z6 ' u o r l e poe]ru6ra^ p ' ' 'elpuodsoj)rt^ gI atloua6 letJpuoq)oltWaasVNC 61;3 h6tt'n6LL I t t I t Lt s t-0 t s L ' s r s a u r l o y {6t u u n p u o l l n q u t s r p 'sda1sllnrlJJlppue Isea 't 9 6 91 39971 r g eL ' 9 a L t 9 ' ( s ) a u o s o r ) r l / ! 'S8S'uorlezrpuq{qnlrsut l u e l s r pJ o u o r l e z r u o l o l l6€8'6€8-8€8'lursrloqPraur o s ( . 1 ' o z z L ' s a n s s r l 1989 l l a ) '0zz 'siaureq '08s 'ate)s t t S ' s u a l s ^ sa a l l l l a ) I l0sz [ l08s g9g'6ur6e ozzL J : d : : b : o z : : : > i 9 9 ' 6 u t u t e t s 1 1 a > ' l g 9 B ' 8 9 8 - 1 9 8 ' s r s a q l uu^ st d r ; o r p r e : O e eI ' J q O ZL ' 9 0 2t ' ( s r o u r n ^l i e p u o ) a s s) a s e l s e l a W S 0 9! S 8 S ' 9 g g ' 6 u r u o t P a s ' ,9Sg,srsaua6orq '909 '989'uorlexu JLgg I9A-999 !9S8 LSOT'LLOL 1909 ' 9 9 0 t ' l 9 0 t ' u o r t r s u e j ggg,suralord;arreqfl el s p q d p u e - o l - a s e q d e l a W ues S 8 S ' S S S - t S S ' u o r l e . r e daal dr d 'areldaseqdela6l t z Lt ' l z L L ' a s e a l aul t a l o t d 980t I L85laMod butnlosar (s)aurosoLuorqr )lotrl/\ os/Daas ,(do:sorrru.r 8 t I t ' l e l l u a r o da u e r q u l a u l 1q6r; ]q6!l aas,{do:sor:ruL f S ( t t ' * Z LL 'J Z aLL 'J t Z I L ' ! Z t [ ' a l o r s r s o l d o d e 6991'srrueu,{palnqnlor)rur l t S g ' t 8 9 - € 8 9 ' 6 u r s s a : o r da b e u r /err LZS-SJA' 969'162 puoqrolrw 'arnl)nrls auosourorq) J E V Z*' ( .']Uatrla^OUr I 1 0 9 t 0 s ' s t ) a o s el e ) r J o l s r q slaptosrprredatVNC os/Daas Or,uOsOurorq) {do>sorrrruuorl:a13aas{do:soJ)ruruor})ala 980 [ 'ngjL ,aseqdelaW 'srs^leue aJnl)nrls I96Z JaL0l J6S-6LS '962-56Z'g ' s r s ^ l e uael l A ) Ilal L Z -LL Z ' l e l u e ) p u e u o l l ) u n j s i ( p q'stsor.ldrouelaW Z g t L ' 6 Z t l ' o l q d o s o t'6urwroppqs-le]a!\J 6 S Ot llal '609 'Ido:sor:rur uorl:a;a JLL' lO [9 619 Xdorsonr6l 'gLZ-gIZ'7 77' rcdat q)leustul paDaJtp-puprts 7 19 Xqderboleuorq:{1rur;;e;e1ey1 llts'(sUI-NA) e;qeuenrad(q'VNCaillleleso.DrW (s)^emqled)rloqelaWos/Daas )OLZ' OLZ'6urpeal;oord:rg(;oe;rnuoxa r n 6 v ' s 6 n E 6 n ' 9 € € ' ( v N U ! r u v ) N u o r ) r w 'sa)uara#rpanssrl pa1eli{q1aul pa}eutuleap E0€-20€'saursoL{: Z0t l86S 'uo1le1n6el '86S]ueauradur'uorDafuro.r:ry1 7 7 7 ' t e d a tq ) l P u r s r W zo 1- 1ot L6g'saln)aloLU 'suorl)eratul 691-891 ISL€ l08s L ' 'saxa;duo: aur{zua11;ntu q ) l e u s tW Iq sasearluraleJ u e 6 t l u e / u t a t o .ItHdW ' u ! l n q o l 6 o J ) r W - z d 9 L t- 9 L E ' ! LL ( . '6L Z -LL ( ' 6 u r p e a t ; o o r d 'uorleJaooo) a^u or rr u,;?' rrt..lr: slueureluurlre/ull)v aassluaueluor)rw rsrr,uo,sr z0 t " r!srloqele] aasursrloqele) ;l",iJ;]J 1fr l L 6 t l ' 8 6 a L - L 6 L L 'L 6 LL - 0 6 1L ' s l u q U o r ) r y \ J 'a)ueleq z0 [ vb! 'EZt'suretor6 d u r p u l q - y g 6 ' 6 u lvab ' l u a u a i n s e a uur o rr e l n l l a ) e r l u r ' ( s ) a p o J l ) a l a o D r W r p u r q - a n o ot o, ru6r y l srsaqlu(so!8aas:r1aq1u{sorqTusrloqeue raDie) pJle^rll? t L S - O L SO' L ' l u a u l l d u l o )a u a 6u l n r . u t u t y r l J6L',9A-6 I',saln)alOur l E 0 S ' Z O S ' s a n b r u q )uaol r l ) a s s r p o r l r W ' u o r l a la f s p u e u o t l ) n p o r d/ l l t w eualleS oslDaas ilznL'Lzbt 99',LUsrlOqelaw uorlel6rru alej )rloqPlaw aasuol1el6161 (s)ua60qred aas:rua6oqled L' l-59 L',luaua)uPqua 69 69 ;;a1 slouulod r v 6 o L ' n 6 ot , ( s r s a u r l o i l ) {) p o q p l w L0st'98tt'leurou / u o r t r s u eer ll n l s e l q - p r w :srsaqlur(sorg osloaas :goads :u.rsr;oqelel erou lerqorlrli\J s 9 €[ ' ' J 9 9' 9 9 ' u o r l e z r u e 6 r o 'JZOL 'ernl]nr]s lOl ffZOL 1800[ 3 7 91 ' u o r l e n b au a l u a W - s r l a e q ) r W 'lLZg 'OZ9'!S '(s)alle)rl/\l 6 9 9 ' U o d s u e tal u e l q u a u l 0 0t t 1 9 ' d r q s u o r l P l a r a l u l Ltg LL 66a zL8-0L8'uo!lnlo^a 9 LSa-SLSJ'suo!l)eeruo!lPlueldsuell ' ,l sgt6uZaLu'lge 6 l uzr lt)'e( u nenl po u) n q u l0L6'696 J O L LL ' L L - 6 9l ' u o r 1 e ; n 6 a r 9 8 S1 - S 8 g t ' u o r l ) e l a s l Jo 'suoll)elalull l a ) au.r(zua 9 6 6 ' u o t l e a l ) n ul u e u l e l uu t D e 199's!sr{le}er 685 1-595 I lla) f (s)^eMqledlrloqelaW J 6 L Sl ' 8 L SL -L l S t ' ] a l r o d & r ) U r ) a d s ,t89 ,90, ,uorl)ulser auoso.,ruar J Z OL 'JL 9' a' (]su)eclleeqL) rul o q P l a w L s8s t_r8s L t '9lS 'tustqdrouj{;od )rloqP]aw z0[ 88St I uorlelsuerll(vNUl)vNU rarsuejloslDaas osloaas suorl)eraluru ralord-ulago.r6 'suorDerelur Lg[-09 [ VNUre]suer]-VNUra6uassa6l t 'J6LS '615t-LLSt 'fi19t l08s '6urpurq-aprldad (s)auosoqrUlsrseqlu^suralordoslDaas L 'sad{rqns LLSL JgzSt 'l6t '(SlUl) ' J 9 L S L' a r n l l n r l s a1;s,{rluaaurosoqulpura}ul ! t6t L L S| 'srolda:aro:i(q uorlru6o>a.r 88t 3 t8€ J LgS[ ' g g g ' a r u o n b a ' 6 u r p e oa; p r l d a d os u r p 6 l p C - a u r ql S S g Sr - € g SI 'VNULUe u a l ) e q ' t B s ' u o r l r u 6 o r arro l d e r a r o : I t8€ leual)eq t I t 8 9t '€89 ',l gL E 'f S L t ' a 1 s - 6 upr u r qa u r o s o q u r r89 t l665 r ' 1 9 t g , q 6 n o r q lq l e d r 0 8 sI ' a ( s F L L g a ' L L S a ' S l SL ' i l s s e t )f H W 'sad'{rqns suortlerelu!auosoqlJ-VNUra6uassa6l Jgls [ uorlplsuerf:uorldu)suerlos/Daas J 6 L SI ' J SL SL ' g L Sl - L L SI t g l S [ ' a r n l ) n r ] s ' s l l a ) u rosr l e l s u P r l r e l l r l €B€'OB €-GZ€'sleu6 9 € St - S € SI ; e r n l e uX q u o r l r u 6 o r a r 'suorl)eralur 'stolda:aro: i(q uotltu6otat t8 [-08 I vNU rajsuer] I LgSt ,Z6V,^rtltqe$ aptldad E6t_Z6t l E S g L ' Z B 9 t - 1 8 9 1 ' 6'ul et p ) les so el l ) u o u 6 u r : r ; d 5a a s6 u r r r ; d s 1ls [ 'slla) pat]eJur-lerlA suoD)eJelur aurosoqrJ-vNU uo uorssaidxa 9€9[-S€St 'I 'uoluu6o]arrotdararo: rebuessawaassuorl)eraluraurosoqu I t 85 t gS[-O8SL 'OBE'!gVE ')rtofiP)ord 'Z8S 88i t tg€ t - t 8 5[ 1 6 6 5t ' (pue I ' A L SL -L r S J ' SL SL ' t s s e l )) H W IOSS aasuorlel(uape(;od ,€ vNUru)uorle;lueper(;o6 J 6L 9l ' 8L 9t ' l e l r o d l u e r J P ^ u l at1l,{quor}ez!le)ol €ZOL-ZZOL'uo1e;a1so/: B B SL ' J L L St ' s a u a 6u e u n q ',uOrl)unj 19€€ 9 1 91 ' u a 6 r 1 u(eZ - I l r l r q r t e d u o ) o ] s lZq-)H ' s l l a ]) l t u p u a p 8gt'lgt'suor6arpelPlsuerlun Lls[ '08€-61.8'salls lrPls uollelsuell 08t 0 8 9 1 - 6 1 , 9t ' u o l l ) l l l s a l s s e l l

"flf:i;'^ 266'()Oly\J)reluar 6urslue6ro-alnqnlor)tW 'S80 ,xnualnqnlor)rw 680L 1980t [ (s)ursaur)os/, aas 9t01-?t0t '(s)uratoJd Jolou tuapuadap-a1 nqnloj:ryr1 '(sdVW)sulaloid palet)osse-alnqnlor)tW 080L !s66 lt LL6' Zg6-9f6' 6ur;1rurpea.rl /strunqns u6 urlnqnl os/Daas nrls * L6'SL6-nL6 3896',896',arn}f s96'saruedord J g L 6 ' 9 1 6 - n L 6 ' s p usen u r u lp u es n l d It0zt ',z0z '8601-L6ot ',s1;a: r-oozt lue;d 3002r LLSI-9lS l'uo paseqluaulalour ua6oqled nt o I' Jz66'266'uorleat)nu l8t0t'8'0t-1?0['[20['suornau (s)utsaut)osl, aas tolou.r S lO t-t lO l'sadll urago.rd alpurdslrlolrw :stso]twoslDaas 6LOt 'l/Ol-SLOt 'S€0 'uorleulloj alpurds L lS€0 t '280 ']uauqletle aroqlolaur) l?80 t !€80 L t I t g 0t , 0 9 0 l , s l r L u e u l p / I l r l r q e t s u l 't9601'160 'pue srsaur4o/: )1601 t-560 t 't80 'uo sl)aJJaauosouloiql t lz80 t

X]CNI

0E:l

'uosueduro)/eleduro)sueauls jalqPle ol lajal e qluvlsloqulnuabPd I 1^le^ttn)asuol^ ollo] ieql saJn6t,ot ll ue qltM',alnbrle o1ta;ar I ue qlu!^srequrnua6pd j^JluaaqlJo uorssnlslplxa] rofeu e ot reJale)erPloq ut staqulnua6e6 atuoua6leupuoqlolrWosloaas (s)ulelordroioW 1(s)alnqnlo DtWosp aas 0 € 0 r - 9 2 0 1 ' u o r l ) e i l u o la l ) s n L u '080 'StO '^lr||qelsul uorlelftoqdsoqd S € O[ - t € O l ' s l u a u a n o u l a l p u r d sr r l o l r t u t t I t80 t 't€0 'sassel] ' 6 L O I' J L L O L a^rleprxOlureq)Uodsuerluorl)ell os/Daas [ )6LOL ls€O L ' A;qwasseelpurdstrlolrtu ; 7 9 ' u o r l e r r d s aurr a ; o r 1 8 0t 12gy'uot1>ury1 'pue s)rueu^p elnqn]or)rur ' XO e rt t er e ; o d t q ) L LL ' L LL ' u o r u a s uurl j o d 080[ LLOI'gLOl-SLOL'S€Ot-t€ 'tZ0 '1.rodsuer1 l g t 8 ! l t 8 ' 9 L g ' € . a t ' €L 1 ' a u e r q u r a ur a l n o l8to t t ls€0 t'5€0 t-?€o 1 ' 1 8 ' s u o r l ) u n ;; e u o x e l q u r a s s e 1 / 6 9 ' u o ! l r s o d u r oel u e r q u a u r a Z O t - L Z O L 't00 t L 6 L O L ' J 9 "L(O L ;err6o;orq 'a)eosxuleur ' srs^lorp^qdIV osp aas € ll LLOJ-SLOI'aurq)eu paseq-alnqnloJ)ulse '9 'xrrleur ',qloual t8 L8 €98dl8 6/0 L l l L 0 t ' 6 t O t - 9 t 0 1 ' s r s { l o r p I6q1 y ' a : e sa u e r q u r a u r a l u r ' 9 ' € (s)urso^W a a sp a s e q - u r l r e l8 t8 tB tZ 6€0[ "s elpoolllaulpl 'ssor)ellodsuerl '28 '(s)uretordrolow elPrpaulralul ZZ8 5ZO1-OtO l t- 18r L96-996',alol',sluaurelrJ '028'saxalduo: au(zua r(rolertdsat 's8€ '889 '(s)uoinaurolow 'sso Ztg- t €8 198€t t 1889 060[ L-tso t ,{lquassesrp 'uorsr^rp g g g - y g g ' 6 u r l a u u nulo r l ) a l a zosL-rosr l5O l-560 t Jo aueld srseur)iol^) 't6t aloqlolaul) oslDaas J rcg' tZg-OZg'1uarper6 lP)ruraqlorllela [ ' u o r s s r u ] s uDew n r y o w s o l d ' s a o ] r n b s o W 'pue l 6 L E L' e ' n . ) o w paq)eDeun seu.rosourorq) J L Z St 8 t 8 ' ! L L g 'L L g ' € [ l ' a u e r q u a Lrua u u l 880t 76tEL sua6oqdtow>rloadsoslo aas l1t8'1[8 ae]sul 6 8 0 t l S 8 0 l ' l g g f - g g 6 t ' u o 6 u r l l es a ) r o , '980 'ursrue!l)auruorleluauo-rq srrslse;doro;ql Ln|' Zr8J€n8' ZS€L')s!p leur6eurr6uu* oltqdosotg l-€80 I lt80 t 'S€€ '€80 ']uaurq)e]teaurosouroJql ' 6 6 ao l r y d o s o t g r 8 l 8 l9€€t 3t€€ t t - € € €t t t-280 [ lt80 /uorlrurrap puoq)olrw I L' ELL'dnDills \ L L8' tag-s L8'1E 8t€! 1 6 6 0t ' 6 6 0 t ' u o r s r ^ r pl l a ) ) u l a | . U u l s e ' l s p a ^u r ' u n l n ) r l a , lelr '988 'uolltsoduo ]uapuadapalej l8S8'198 leupuoqlotrW t 1988 880 t tz80 lla) xaldtuo: ylgg aasxa;duo: y1g1 ( s ) r u a r p e r>6r u a 6 o q d r o y l 'f6LOl '!SLOt'JLLOt'Z9OL-LLOI !Z I I '^lqurasse ' a l p u r d s) r l o l r w xa;dulo>6111aas xa;duo: y111 t s o L! 8 0 2 9 7 6 ' u o r 1 e 1 , { u r 1 r n b r q nJo u o t 'a)uanbasrajsuPrl-dols 'xapur)rlolrw I L1 650I tt I I' J L9'SS'saprreqr)esouow (sasedfD)suralord aroq)oleu!) os/Daas n b alse u 6 r s J , LL ' n I L ' s e'rxuaal o uo) 'pue p a q ) e D e u n 6urpurq-619lapun aassasedlg )uauouoW s a u o s o u l o r q l s [l wvs 8801 s I L-n J L'sJole)olsueilu!alord J L S Vt ' I t S vt ' J a S nl ' a ; v L ' s a / > o u o y 1 6 8 0t '€ 'suratord rosinfard SL1 u 9 7 51 ' 1 a / : o q d u ^ l ) a s u o d s elre u o l ) o u o W 1 9 8'980 0 t! s 8 0 1 ' 1 8',uisrueq)aur 0 1 - 9 8 0 1 ' u o6 u t t > es a : r o ; uorleluauo-rq r XO J g LL ' 8 t L ' S I L ' J Vt L ' x' saul dr surruoeV l-€80 J lt80 t 68S q)au efua)saronlJounurul llg L/ l88S ll8S'^dorsor)rLu lt80t'€80t-2801'luauq)elle alpulos ' A d e r a qrla r u e : u r JSIL'6'L-LLL € 8 0l - z 8 o 1 ' t u a u t q > e u a elPurds 697; 'a)eds '880 '180 'uorleiedas rauurolul aueiqtuarurelur/euprqureur t !180 L t J880L 1 6 0 9 ' 6 0 5 - S 0 S ' , s a r p o q rlleuueo l ) o u o w g 7 y ' s r r l a u a 6 r d a ' u o r s s a l d) rxl a l l e - o u o W Itg0 [ '590 ]-€801 'u.rsrueq)euuorle]uauo-!q J 9 LL ' L I L - 9 1L ' a s n1 6 r a u e 'sluelnu (u.uaposaui ralsrs elow) wow LL ' J SLL ' 9 LL - St L ' s a l r sl ) e l u o ) ! 8 SZ€L VZ' Eia'sprleLuorq) JJT '88€-l8t ,a1nqo16 ua11oyr1 L l L ' L t L ' ! 9 LL ' 9 LL ' s u r e l o redu o r a d p q ) 0 6 0 1 6 8 0 1 lSgt 'g-xed 'luaurdola^apaXa'1sn;1o141 ' f L B O l ' i l g LL ' 6 J L - E JL ' u o d t u ru r e l o r dl e' suppruooaql )l n ou lrW LgOL ! S 9 8 ' S 9 8 ' u o r l e 6 a r 6 a s T u o r g e r e d a s J90€L (lelnllar'llp!us) 698 JE7Z'ftz'aseqdela|.U 'pue s a l n ) a l o u1l 1 u e 6 r 6 a a sr r u e 6 l or p l n l l a )l l p u r s 9 9 8 ! 9 9 8 ' S 9 8 - t 9 8 ' a l u P l u a q u ru e r l a p u a w - u o u 1 1 0L lpf-w 'alor 'uOrleluasaJda.r u r s u a p u o ) 998 Saseas!plPlrpuoqlollw s u0 1 LS 'uorlesuapuorulleuorq) (s)alnlaloLuoi)eW aasa6re; 010L '298'ueunq [98 I [98 J SL Ol ' l V V a ' € t Z ' u o r l e s u a p u o ) ZS')rqoqdorp^q '19€'uorleue^epo: :rlaua6 I(98'298 8 1 0 1 ' u o r l e ) l d n pa u r o s o j l u a ) Zg'lrlrqdorp^q ',uOrlOu)suerl €98 Z g Ol - t B O t ' ( l q t u a s s ea ; p u r d sr e l o d r q 8?'(s)aln)alow 'sanotuq)al e g g -L g S! t 8 t 'aler ' € B t ' u o r s s a r d x aa u a 6 snz'rvv7.'JEnz tzg-zzg 'EnZ' relnralour u o r l e 6 n p l u a r ' u o r l p u r u J a l al qp6 r a l vt re l n : a ; o y 1 298 l)ol) 6 0 Z ' J 8 0 26' 0 Z - 9 0 2 , ( s ) a u r o s o u o r)q! l)o l ! W 'alueualuleur (s)alnqnlol)lw eln)eloW 0/8-898 S t ' u o r l r u r r a p ' t q 6 r arM '098-658 'uorlnlo^a :srsoraW lsrsaurlol/iflallI) llaJ os/Daas swalotd pnputput osp aas J 198 'LSS-998'uorlerldar ,(lquasse sasDqd)Ur)adsoslDaas 1eo:relnlrsanul 898 tl98 VNC 09L-6SL l6s8'sazrs E v j l - Z v j t ' s a s e d f g^ l r u e , o q U O G O t I € l o L ' 9 9 6 1 ' a s e q d o l a l 'raqurnu 'vSOl'esPqdord 'ased19ueu Ido: BSBtlSB lZLOt 60t-801 'aseqdelau.rord .6Ll ,sasedlg 3g99'599'(lrsranrp 33191 t6Lt '9901-S9Ol'190 t 'n901'r*e 'Enz'aseqdelaLU ' l o r l u o ra l ) ^ ) 8 S 8l a q u n u ^ d o ) S10t JCL0t lla) r esl n ) a l o w 1698 ls68'1,68-S68'968'saq)trM 16801'0501-680t'JtLol'ni}t'aseqdeue 'OZg-B9s (s)uralordioloW aassrolorurelnlaloW t t98'lsg-9s8'aLUoua6 ;errpuoq:o1r61 llzl0L'110['saseqd uollelXroqdsoqd a^rleprxOos/Daas J SL ' SL - b L ' s u o t t o urle l n ) a l o w l e I L ' a l L - 0 1L ' u o r l e r ) o s s r p ' e u r uJreeal l ) n u 'L8E a l p u r d s) l l o l ! Wa a sa ; p u r d s: r l o l r r u 9gg'6urldno:un lz8€ '(leuorlelsuert) g€g ^ olj uorl)ala asla^at s r o l ) e J r l n q n l o r ) r u r a s e a l a r ' ^ J l t [ ! t u i e l l n ] a l o W Itg0t'O8Ol'pue ^lr|qelsua 'f '1y7 's:riaua6o;,{qd g€g-1,€glolluot IiolPltdsal JLL(T fiVe LVZ '9€B'slerlualodxopar 'LLZI-qLZ\'ltLZL'ZLZI 'eupuoq)oltuJ lS€8 ! 1 . 6 t0 " s s r s o r e | . U zgg ' p u ea r o q ) o l a u r l 8 € 8 ' ( l e ju M o r q )l P r n l e u € 8 0t - 2 8 0 L 6vZ-8vZ's!saqiod^q)j)o p ' s a s e u ral l r l - o l o d saxalouro) ? 1 0[ ogz-gnz'uorln lo^a i elnlalolA '080 's)ruJeu^p aLu(zue(.rotp.rrdsau aassaxaldurorau^zua alnqnlol)tul t 298'6nZ-8nZ'(s)lrol) r eln)alow I LB0t 'a6erols,{6raua -W sauoradeqlaassauotadeq>.re;n>a;oy1 t Z8 6LOJ-8LO L ' I t O J - nL O L 't Z 0 [ ' p u e l p f '6 's)rlaoraua /uor]ruUap'AllJeloW l0Z8 t8 n q oL 'bL 0l ' s a s e u l le i o r n v 9t ,utsaow olu! Ailua v Z g - t z g ' 6 ur ; e u un 1u o r l : a ; a 600t ' p u eu l s u a p u o ) saxa;duroraui(zua ^rolerdsau os/Daas re;npoy11 6 € €t ' a u a 6 a l ' u o r l e z r u e 6 r o szo I slapowlDnptAtputoslDaas (s)auosouiorq) lZ€g' tt8'{do:sorl:ads )llolrw aasarnl)nrls euosouorq) t € 8 ' s u o r s r l l or). u o p u P r It€'t€-€€'rsea( t t 8 ! 0 € 8 ' s u r e l o j dr n ; ; n s - u o . r r 060 l-6g0 r'uorle6ar6eseurosourorq) r€?'0t-6€ ' sasnour ueunq (s)auorq)oM) os/paas 0 6 0 t ' 1 1 0 1 - O l O t ' 6 u r 6 e 1 raeudo s o u r o r q ) lEt'0? L € 8t 0 t 8 ' 6 2 8 ' s a u o . r q ) o l ^ ) l9z'sz-nz'!lD'l lorluo) al)^l la) osp aas l Z € 8 ' t € 8 ' ( a u o u r n b r q nO) e L u ^ z u a o ) B g O t ' t 1 0 1 ' 9 9 0 tL' 9 0 t ' s 1 u r o d 1 : a q r 6Z€1-82€ t /6zg 'sraule)uorl)ala aurosoJlua) l€g ! rc' AE- L€.7atso6ouolaw zryldosu1 9 4 61 ' u o r l e r r l d n p ' u o r l ) u n Je l o u l u a ) f Lgt' LE-98'suo6ap sutpqoqtouaD) l S S S ' t S 8 - € 5 8! S t 8 ' t t B " s s l s e l d o r o l q ) 9 1 0t '199'99 'stsdoptqoty 66€ r-96€ [ 6EA-teg lLZg',oz8-6 r8 1 0 1 1 0 0 1 I ' u o l l e ^ ! l ) e u ! l p l 't /uorsr^rp )uleuu^se S Z ' ( s ) u r s r u e 6l a r op o w l 8 ' ( s X s ) u r e ql i)o d s u e r lu o r D a l al e i l p u o q ) o l r W 650l llar 'uorlalouro) pue l/fdv sluawala)Ur)ads auroua6leupuoqlolrWaasVNC leupuoq)olrW 1880t l,8o I 'srsaurlol^r 1(sa)snrn 1(s)uosodsuerj :vNC a^tlrladauos/paas ul 9 9 8 ' s a s e a s rl epu p u o q ) o l r w OOI t-6GOI Jo a)uasqe ',t90 ',srsolrw uotleutquJo)al I/69'aunlo^ z60J-aloa 3s90t t 3802 ,adiq -,..--l^..^ ---...( e m q l e d6 u r ; e u 6 rass e u r l - d V W )!Jr)oos-alrs aas susrueq)aui ^l!l!qeue^ uo!uasul ls8 lla) qllM',al)Al earn aas6ur;eu6rsaseurluralordpale^rlle-ua60lrW 198 l10z'slNts g 9 g ' a s e l n u l s tapp t x o l a d n s (s)asPur) ll0z'sluau.ralaalrl-ler^o[ar -dVWaas(s)aseurluralordpalenrl:e-ua6olrW arnlfnrls leupuoqlo}|lAaasarnlfnlls ll0z'stN|l sua6oltwtry>adslsrotleJ qlmorD oslDaas l 1 0 Z ' s u o s o d s u eV r lN C 1 7 g 7 , g g y , 6 u 1 I pVaN U auoua6 l J € Z t ' J O S Z , ^ l r L Une €0t t-z0t t'sallr)urlads l e r J p u o q ) o l ro Ws / Da a s l VJ urerord LglE'JLOa'auouabueurnq )695' Lgg t9S8'1.S8-9S8'slsaqlu^s 0rr-zolr ' l 0 ' € 0 1 ' s il(8e0mr qr 't 8 e6d o6ouo1a w o1tqdosotg aasuodLururalold u r 1 e u 6rresl n l l a r e r l u l uodLrlruralord 19 yg't a1s L t I lpupuoq)olrlA 'leualreq /uolelnurls 9€8'?t8'suosrod s 0 r 1 - € 0 1! l8 tE lp)-slr9 a a su o d u l u o d u r u r a l o r dl e u p u o q ) o l r W 9 t t ' s l u a u , r a l :ar 1 a u e 6a ; r q o 6 1 90r l-€o t t'uorlelnu'rr s LD ' l o j u o l t !l]pa)d- u r o ) papo)uareal)nu (s)aurosouJolq) )tloltw oslDaas rr;rrads-anssrl 199'suralo.rd 011t ',S ',uorler)osse '(s)ua6olrw alnqnlor)!ur ) 5 6 5 ' 9 6 9 - b6 5 ' u o t l e z t l e n sut ^o r l e r l l r e o l o q d € O tt - Z O r I l9 [8 !8

[€:l

X]CNI

'6ur1enr1:e gztL'uorle!lueralJrplla) Luals uo!lelolsueJletuosoruoJq) 96'ale^nt^d ul04 uotl)npotd 697I al)snur aar lelalals ) E 6 'r Z 6 ' 6 8 ' s r s ^ l o r ^ 1 6 lelolols 6931'euoqdu.r{1s,11r)lJng 'uor]e^lauur 'eurout)let ur patltldule 'uollPraua6 uornaurolor.lr s8€[ 028-6 l8€Z t Lg 'apsnur 'salor gg6 'ura1ord7aua6 :/yy lq6rg llasul llZO t €g leuotl)unJ 'suralordLroleln6araue6 q^W el)snur)erpJPJaasuPaq 19gy1 +cvN/HcvN oslDaas '6 ',€8-28 'srner6 (s)raquellsnw aassraqu eruaqlser(61 laure) uoj]rala 028-6 [8 !8 t8 t8-8 L8 6y9 1 '86 'al)^) pr)e )r.rlr) t9?['ro]elrp I t8 3 1 1 7 ' u t a l o t d5 1 n 6 1 j(s)lselqo^woslDaas (s)urelord:rua6o,{yr1 lllz'ularoio llnw L,+CVN/HCVN ' Z € 8 ' x e Jl dZu6o'E)a8s_e2u8a8' 6 'Jrgn 'ngn 'luaurdole^ep gln61 0 r p ^ q aHpc v N 9 € 8! € € 8 n9n L 1117'uralord ' l a p o u ' 8 2 0 olPlnW lueuelU-6urprls t - 9 2 0t f 0 0 t [ ' s u t e l o l d , { t o 1 e 1 n 6aau.ar 61 y p l 8 Z 0t LL Z - 9L ( . ' ( s ) a u alO '+ze) (VNVN)pr)e )tletSaaspt)e )tutulerneul(}a:e-ry alor 0€0t-820t vgz- €92'uo!leu,rlsarapun '620 'uruodo.rtr{q Jo 'v9z's])4!a uorlralas t l0t0 t lo.rluo: sgz leJnleu 'uo!l)elluo) N 0€0 t-gz0 I 2 9 8 ' V N Cl e l l p u o q ) o l r r . I l '€9? 'sasset) 'lueuernseaur l€9t I [ t9z (s)lla)al)snw aasslla) '9Z sleuuiPur 'slauueql 'etuoto^W 'SaUa6 uorle> pale6-a6elloA al)snui )erprPJaasle!prel 289 997 lertuossa Jo raqLUnuuo uorlelrurl Puil.uPl ,{r!l!qersul )r}auaDiJa)ue) os/, aas 01 t L'suotl)unIsnourpualo,{61 [ lt9[ lPseq 'a)tul ut uot]elnul lggt[ l9t1-€9tt'alrsnw s9z'saseaJ)ur '(sUqfVLu)s.rolda>ar y97'aurl-ura6 aur;oq>;(1e:e :ruuersnyl J99nl' 99nL'lorluor qlMol6 allsnL! 9 t6 'saprldedouuqU ggnL- ggvL'sl)eJJa,uotlelnLu aua6 ls0z t9Z 'SOZ-hOZ'seruaJaJJrp aurosoruoiq)laap refiunyl Itttt'otil'ullelso^w V 9 e '6 v Z - B r ( ' a ) u e ) U t u 6 tf suP u o t l n l o ^ a 'snr^ sournw 'ID DttPuaq)s3 0 € 0r l96t[ ngz-Egz ' 'si:a;ap rredaryg6 J S Z 1L 'n e 1t ' ( ) l l W ) a s e u r lu r e q : - 1 t 116u1r s o I 6 l r L 6L ' 1 9 6L ' t S 6L ' t 6 t - 5 6 L ' s e r p o qr e l n ) r s e ^ r l l n w 967 '(s)ua6rrue y97's uo6a1asry pqor.UouaD) ruale^rllnW z z o L ' o z o[l 0 ' €t u r s o ^ t ^ ISS t , l u e u e l1u lz t0 L 961'uorle;I1rnbrqnrg1n61 ngz-Egz' gVe sale)uorlelnw l)!ql sadfi >qr>adsosloaas It[0['arnl]nr]s ! z 9 z ' E s z - z g z ' s e ) u a n b apsa A J a s u osle D e d s r l l n w (s)llaluals aasslla) uats lualodrllnW L 9 (I ' r 8 Z 9 ' s u o r t P l o l s u e J l ' S Z O L - V a O t ' u o r 1 e 1 ( r oJqSdlZsqO oL q d 'snour{uour{s uorgeln6ar 9 2 9 ' u o r l e u r ; a l u o p ' s r s o J aal 1 ) sd r 1 1 n 6 1 1y7 g79g '{lrnrssa:o.rd (s)uroloJd auerquausue{ aassu;a1ordssedrl;n61 lSZSlossarddns 8 Z 0 t - g z o l ' u o t l ) P . t t u o la l ) s n u l 660t 6 0 2t ' 8 o z L ' s 9 z ' ) r l e u r o s 'sall^) 'srsaurloy(> Jl l0 L lnoqlr^ srsolrru'slle)pateel)nurllnW le)tulaqtoupq)aui '9Z',lualrs '020 'ure ra^al 'puP '6uruearrs (sallqurasse) saxa;duo: urolordos/oaas l0z0 t t t Z O € 1 4 ; '691'691-g9L'saxe;duro> 'aArSSa)aJ eurlzuarl;ny1 9 7 61 ' l u a u a 6 u e l e l u a u e l U 8S9 !69 L 6nrpr1lny1 salPruollelnw aaseleJ 999-599'a)uelsrsar ' s 6 0 t ' t 6 o t - € 6 0 [ ' ( s t s e u r l o ] ^ )t 9 )66u0ur a ; r 1 > e t 1 u o : (s)ansslljluouldola^eoos/Paas lg9s'uo lle#a'ureloJd ruorlnlrlsqnsaseq'uorlelnuLlu!od tt0t !6Eot fi;1;1ou ss6'uorlnlo^a 9?Z ,ll 1 1 a : , uolte!luaraJJlp t t0 [ utso^l^l lSzs'ilnu llaJ aasuollezllellaoslla) 'suollelnu (wf3) xuleurrelnlla)erlxf l E t O t ' € L 0 t ' 1u t s o r { y 1 I I lellneu sadfi pnp!^tput osp aas l ( s ) u o r l l u nl fl a l : u o r s e q p el l a l o s l Da a s srsauabelnylosloaas ' s u o r l l e ratur ueqlau.r Itt0t'€t0t'sed^lqns z € LL - t t LL lla)-llel Jg6Z'96Z',sr.Ilst '899-lss 'uorlrunj-Jo-ssol ^luelnlla)llnw 9 [ 0 t t S t 0 t ' t t O L ' ZL O L ' a i n ] ) n r ] s t Ezt !8zs 'a]e]s.ro6rr 6 [ Ot L E VI ' S t n L ' J VL L ' Z L L ' u o r l a r ) a s lSz9,leqrel 'aloJls raMod 'z0s r.Ilstupq)auJ 'uorsraAul asuaJaplsoq 9 L0L szs t t lgZ9 '8 '>ruabe-rlur snlnw € L 0 t - zt 0 t ' a l ) s n u ] - u o u 16t L suaar)s)rlaua9 os/oaas 820 t-gzo l'uotl)eJluo) allsnu.l € 0 St ' S E t I l o g e ; e > s a , { . r e r l r o r n y l 'paads ' W 6 l' u r e q ]n u lueulanoul 020[ €SS[ 098-6SS' LSS-gSS'uorlPlUrluepl 'ppaq ^l!^!l)e ro]oul ',sauotsrq lZ [0 t )b$'n$'aseurl ureloro'uolw € LZ Jo ,uotleraua6 'uolllnpord 'Hsw 'bgz 'aul1-ur.ra6 a)Jo, 9 t0 t lE08 ]ge ypg ra6uasse6laasVNUW 9 L 0 t - gt 0 t ' u t b u o ^ l e u o t l n l o ^ a L E U 1 n 9 9 '8 S SL- S S! 8 Z S ' u o r l ) u n J - J o - u r e F 'ngz 'uorl)alas y9y'uralord fto1e;n6e.r aua6 ypyrl 7 1g 1'uorlsa6rp:rteu-ti(zua Sge leinleu ^q uorleururle 'sa6ueq: (s)uorlelnu 9 lO| 686'uraloid SaJy\l ;ernlrnrls;o altr(> ap/o 1a1npun aasa;:l: 11e> anrle6aulueuruoC aasenrge6eu f L L OL ' a l 0 t ' s r s i { ; o r p r6{ q1 y lueuruop 1o aseqd 61 ,(s)urso^W /lueuruop t t0 f - t tO t It8€t 8SS ' t 8 € l - O g € l ' ( s l l a ) s l l a )u a l s : r u o i ( r q u a )tu I'I LZ'67,urqo;6o,(y1 SJ) n LZL'S9Z'snoualalap/6urztlrgelsep 'urelotdr(tole;n6at aua6 urua6or{y1 t9t O A i l ' J 6L Et ' 6L €L ' o ( t q w a lSzs uorlolop surelord(to1e1n6a.r aua6 oslo aas uorlnlo^e eurouat oslo aas 6ttl'66e 6o,{yr; JVgt' t9t' (s)uatotd trua6o(y1 6Ltl 9 b Z ' 1 6l ' B L ' u o r l e n o u u( tre u o t l n ; o n a 's1se;qorqrlo(y1 'sauerqLuauJ rruortquaerua pup so6elsIFea saue6;e>rgrrr-ra:ue) lialuel oslDaas 9971 '9201'luqUo^y! '991. 'iuaudole^ap ra)ue) JLZOT lz8€ t LLaL'OIZL-6O21 1. 'zg€ '(6unl 'vgz'se)uanbasuof 6urq:uerq )E9nL' r9n L-Egnl'!LZV L's1prlerlaqlrdaor{61 L- Lg€L 0Ls ,o) srsaua6oqdroru ' 'JL;S 'L99'anrlsuas-ernleradual asnol v9vt € 8 €l - 8 2 € 1 ' t u a u r d o l a ^ a p JLSOL ISOL 'JVqV'ngr' 'leuor]rpuol aua6 6o{61 !8 lb'suralotd,(roteln6e.r 3 7 9 9 ' u o 1 1 : n p oa r ds n o u > r u a 6 s u e l lSzs y9p'aua6 1otryy 8lEL'sueunq sa's:r1aue6 Lg'I'LLZL '08€ 'seraurrq) '012t-602I'602 998'(lUU] ) L I t8€ t t08E t t ' 8 0 2 I ' 9 0 t 1 ' 9 9 2 ' r e ) u eu) l gL€L'Jw'On-68'r.l.rsluebro aseasrpraqUpar pa66etpue (sda;rda:ruo;rol61 lapou se J 8 ( . 5t' g z ' l t - 9 L ' ( s ) u o r l e l n w slnolloul eue9 oslDaas (s)uollelnwoslDaas bL E | - E L t L ' u t 6 u oa l r u r o s 'lsea^ 99Vt-Sgn L'uotleuioJ raqu eltsnur lelalels l 8 9 S ' 8 9 S - 9 9 S ' ( s ) r u s r u e 6) rruoo O s u e J l ols 'uoll)npojd 9 9 t [ ' q u ' r o t 6 t a q Ua l ] s n u ' ] u o u r l t n J f e J ll9s Slg'srsauabPlnuo.rlr u, 'Slla) (uels) alrllales slnolloul sp efualstslad 99tL 1 S 9 S5' 9 9 ' p a l ) a r p - e l r s '€zt'suosodsuerl 9 9 y1 ' s l e u 6 r s ' a ) u e u a l u t e u . i tEzE 959',€99',uropuer 'lgv 'uotsnl )!urouag os/Daat 6urtuudrur sluaulala oslo aas >rgaua6 aIqoy1 ltgl L L 'uorlertuereJJrp t9t t 191y,j69y'6ur1uudu-rr 9 5 5 l S Z St E 0 € ? 8 6 2 ' 9 6 2 ' s u r s r u e q ) e u r ' s r s { 1 e uueo r s s a r d xaau a 6 pe^resuol 1y69'qder6otlu-relnlln) lla) OSZ'tgOZ'797Xua1u,(s 9gg gOZ 'srseua60ur)e) s'slluroue6 anrlereduo: t9t'(s)rsPlqoXW l 6 t z ' u e u n q ! 'arnl)nrls stsauaoelnw b6z-t6z'sauosouoJq) 1089 (s)lerlueloduott)V orlDaas auJouao 9 9 bI ' L'69 t ( q d o r l s r ( pr e ; n > s n y 1 t L L [ t ' 9 0 0 'uorlelnur uorlre. luauroola^epesnow aasluauroola^ap J6L9' Og9-gL9'uorlp6edotd;er1ua1od sg€-tg€ '(leurdspatuaqur)Iqdorlp reln)sny! qleaqsurla^w esnow ureloid ;r1y1g sadfi >qoadsoqo aas t L S - OL S 'I g L ' JLL ' O J 'w nl p t t u a bow s o 1 d o i l 6 1 l s g n t ' s g n L - n g nL ' ' 9 0 t ! 9 2 0 t ' ( + a q u a p s n w ' b u r z r l P n s r A SZOI-€ZOt'uorteln6ar 00SLttt'owsoldo>Iyy s z ?[ 'uorssil.usuet] 'uorsr^rp (s)urseur)l(s)ureu^oos/Daas palpulproo)un pue q]Moi6 10s [ 60[ I Z I S [ ' u o t t u e ^ a l du o t ] p J n l e ua u o s o b e q d s l0 t-tt0 1'paseq-alnqnlor)rur ?9t['un!]bu^s 'slle) 'ost '888'q]oours oz01-9ro 1'srusrueq)aur 80s t-los L t rEgbt'n9nL-€9n[ >rybo6eqdeprsurle^rAJns pue stsoy{to6eqd aPsnLrilelalals aaslelelels 1201-0201/s)rlautl 'sur6r.ro 'a)uals!sred g9t['suilojosl ulalolo r(reuorln;ona 80s t-10s L 9 tO l-S lO t 'se6ueqr (s)uratord:rua6or(61 1(s)lselqo^W os/paas J Z I S t ' Z L S L ' p u el u J e r le u e i q u a u l l e l l s o q 6 t0 t-g t0 t leuorleuroJuol elpurdslllouhl :srsolrwoslpaas ogv I'n9n L 1b9l' l9l'uot]€tluaraj]rp 'uotle:edasalod alputds 680| nvtsoopr;Zln;:";,i#;2"":n* , 9 V t E g V L ' ) e t q e ) ( s ) l l aa l l)snw EZOI-ZZOJ318?',uorlezrle)ol vNU '6201 'ainl)nils ZZOa- aZOL'l.rodsuerl allaue6ro J6ZOI \gajl'|LZ\L'920 | ' a u a 6 o > usoe J e V a L ' Z y Z L ' 6 t ge E e L ' 1 99; 'pup uottentllpua6olru_t €0 t L

XlCNI

zE:l

'uoslreouo)/areourorsueau s :alqel e ol rajar e qllM sJaqunuaoeo f :^la^rln)asuolMollo,ieql sarn6rJot ll ue qlrM 'arn6r;e o1ra;arI ue ql!/\^sraqurnue6ed i^rlua aql,o uorssnlsrplxal rofeu e ol ralar etetploq ur sraqurnua6ed '09€-85€ 'lrodxa €8t !6S€ t6Z€ ' u o r l e lVNULU noar r [ [l ) o I L ' J i OL ' a OL - t O L ' s r o r d a ) a r J OI L ' J 6 OL ! 8 0 L ' 6 0 L - g O L ' s u r s r u e q l a u r ',t.looull o 14-S0Z J60L' 60 f-AOL'slrsrueqraurl.rodxa 1 , 6L0' 6 0 L ' ( 1 r ; e u o r l r a t r p l i o d s u e r l) r u s e l d o y { r - r e a ; r n p g g g y ' 6 u r d o oV1N C ' u t a l o i d^ l o l p l n 6 a J) 4 N 9 6 € - 9 6 € ' u o r 1 e p e . r 6l paupt u i i a l - N 't9l'lSN t69L!V9L ( s a ) x a l d u oa) r o d. r e a l ) n N a a ss ) d N €z[['srsoldodP'DXoN 'o^Jqu.ra^ll ea suobala'1'u q:go1r1 ralord tZ€ L , q u r l l ;eu6rs u r sst L lerqaua^ 9 Z t l . u r l su l L L s€ '8S€ ' ] u a u dl loSl a ^ a p!a9;sl st t l q( l o s u a su r t - 9 S €t tttI sear)ueo 'ansstl Z 9 €t l e l n a u - u o uu l '19€ ' s r s a u a 6 o r n auul 1 0 9 €L L - 0 9 €L 'uorlerluareJJrp ) 6 € . V l ' O n V L - 6 tLV l l a )t n 6 Stlt'slla) lerlaqtopua te )ZSt L'ZgtL'ur6reur6urMD/,qdosoto 00t L'a)uasqe'sBdoptqoJv lgl€ Lluaudola^ap ' s r s a ulaP6uol u r 6eu e 6 y y1 6ur;eu6rsq:1o11 BV6-9r6'^eMLllPd 8 S €r - 9 S €t 'J8V6' L n 6 ' 9 V 6 'L L L ' u r a l o r rdo l d e ) a rq l l o N 6 Z Vl ' ! V g El ' u t 6 u o l r u o l i q u l a ' a s o N S'6€9-8€E ' u o r l e z r p r . r qp( o qle6 )Et a l ) n u ' 6 u ! u o l qu J a t l l J o N '98?t ',elolj L0sL letqol)tuileulJoN 198€'l,g€-Sg€ {erap ypgur patelpaul-asuasuoN 'suosodsuel] 19 [€ lelt^ot]eluoN 1998 '998 uerlapuey\l-uoN 3S98',S98-t98',a)upruaqui '€0€-20€'ledai It0€ learq p u e r ] s - a l q n ovpN c ' 6 u t u t o f - p u as n o 6 0 l o u o q u o N 'srsplselau'euoqdu'(1 surl6pog-uo11 l90Z t 6L ZL - 8L Z| ! 9 t Z t ' u o t t : u n f s r P u o N ,t6 ,srsaqlu^s LSg HdCVN L S BJ' S 6 '5 6 - n 6 , s r s a q l u ^dsl v t lq) Idl u o N J I Z S S ' E S 8 - O S B ' u o r l e l ^ r o q d s o q d o )l o 'suottlelalut l0t ! )eaM sa)roJ sleeMrap uen aassuotl)el]le sleeMlap ue^ l8s L '95 ' 'rV9 'V9 'r99 'selnralouror)eur L LZI-92I s p u o qJ t u o la a ss p u o q) t u o l I LLL ' s e l r o J) r q o L l d o r p ^ q spuoq ua6o.rp,{;aasspuoq ua6otpi(q l l l I t - 0 1 1 ' ? s - E s ' s u o r ] ) e t e l ultu a l p ^ o ) u o N sadhvNa llPads osP aas ,ypg 6urporuop t9€_09€ anrlrtadat VNC a^rltladaUaassluaLUala 0gg'eupuoq)olil-urltoJJ (s)uollulaassuollul /ut t€€ q)u aulouab ' 0 2 ' L € ' V N C6 u r p o : u o p '^do)sollrLrl €8S tlsupuloN lserluo) a)uaJa/atut-letluare#tp '(dotsot)tru uolllala 'astoN 119-g L9 t'0t6'uraloid ur66oN - L' Z9€t € S 'suralord 9 € SL t - L € SL coN ,lat^ueu sapoN l0gg,0gg ' s l u e l ro du r ' e p o N LOnL'J86€l L L t t _ g L El , u t a t o r ol e p o N Psp o ) o N ' 0 € S' J 6 Z S ' O E S - 6 ZLSZ' 60'€t^1d8o8)6s 'oarltol az d 10tS t UWN 269- 169'srordara.ry66111 llll'alnl)nlls leuotsuautp-aatql J 9L L ' g EL ' u o r 1 e 1 { s o r {u1r6a l o r d ' u o t l l u n J6 u r p ; o u 1ralord 16€l w LL ' SLL - VL L ' u o r l e u r l o J l s l a p u e q : : e s o 6 rp l oe 1 u t l - N ( s ) l l a () ) N ) r a l l r l a a ss l l a )) N l e r n l e N ggg 'auuarXl6oty11 '€ 't 'uortexrJ ua6or1r5 00 t !s L t ! 'ap^l ua6olltN r01-001 J 6 € 5 ' 6 € 5 - 8 € 9l 6 l 5 ' s a u e l q u l a u a s o l n l l a ) o j l t N 1 8 8 8 ' B B 8 ' a s e q l u ^asp r x o) r l t N '888 '588-1,88'(ON)aprxo)ulrN Ost L /S9 'eururel ',ue6oprN J L g L L ' 9 9 1L [t leseq '(sUq)Vu)srogde:al aurloq>1,{1a:e trurlo>rp 9 t6 +dOVN/HdCVN a a sa l e q o s o q oa p r l o a l l n u t pa u t u a p ea p t u l e u t l o l t N +CVN/HCVN a a sa p r l o a l l n u t pa u t u a p ea p t u l e u t l o ) N

JOL€L' LLE L-OL€,]!/9€ ['ateld lerneu lOtl'uorieper6ep urelordpaplojslur'aseue)^16-N aas uorleJ6ruilla) lsar) lernaN-os/D (lgN) roltel qlMorb a^raNaaslgN 'uorleiorur 'surelord ls8€ ! leinau o€9t !t96'ns6-zs6 SXJN '6ur;lapourar 'O8tt 'uorlelaua6arqurl 'lMeN luapuadap-^lr^rl)e 96€L-€6€L JOg?t )E6EL'JZ6q.L tSSt'(Ult) s.roldarara1r;11o1 '26€ 'uollpulrojder! 'arnl)nrts t- !6€ t !l t6€ t lerneu lzgt L 'sroloefar uorler6rlulla) lsar) lerneNaarslle) lserr lerneu z€9 t- t €9 L 'srsoldode{q re^ourn}prder l 9 8 tt 79y;. '988 'uo!le)utsre^rp puP s^epqurqlernau t-S8t t l9t0 t tStgt'uorlezuelod , p u es u u 6 a l u r 'ZES ' J 8 8 1' 1 1 11. I L S S| L-tESt L8L'srsol/()o6eqd 'xalouo)aseprxo l € 6 €L ! 8 8 € L ' 8 8 €t - 1 8 € t ' O t t [ ' e ) u e p r n 6 z € sL HdovN ' u o r l e u r o Jp u e e 6eaurl ilnnL L g V l - O g n tLl 9 t t ! 9 8 € r , 8 8 € 1 _ 9 8 €I ! 6 1 0 r , 6 7 91 . , s e u oq>v n o r 6 t696' 696'luaura6uelleeruolalalsol^) '6urUv\er) [ g t t , s u o r p u nd f e6 9€0t lla) 'srsord o d e L6EL_9681 1 111 'JS6t 'a)uauadxa '(s)lrqdorlnaN l'968l-S6E l ro sl)aJJa tSt t uorler6rurlla) )saJ)le.rnaN oslDaas '(SNd)urals^ssno^rau ?8Et staItMsuDI)o)nai#fi l;',]i#,|'#" lereqdued 'lsar] tl€ [ (s)leuueq) lejnau 'o^rqura u o rp a l P 6 - r a u r u s u ear fa s6 u r l e 6l e u u e q )u o l Jt8€ L l)rql ' ( s N ) )p r o ] ll.9€[ l e u r d sp u Pu r P r q J888'288'n89'E89'289'(s)raUrursuerlornaN /uor]ruUap gggl. 'sur6r.ro )ruoX/qua ^Uea aua6'DtodsotnaN [8? 'saJaqosoinaN LL ' J O L VL ' f t LL - O 't t ' p u e s u u a q p e ) J L n 3lt L 'aurjl)op ll8€ t tos lPuornaN '18€ -98t '€8€ 'l 'uorl)urlsrp alrrpuap-uoxP t t L saoAl :(s)rallrriJsu 1 , 6 €t - € 8 € 1 ' l u e u d o l e ^ a pl P r n e N )ll,.)ads1(s)asdeu^S eJlornaNos/Daas JbLET 1908 908',slle)pazueloo 'nLt 'JOV '0? '9€O 'uor1er6ru.r lsat) lernaN t L It tt l 6 t 0 t 1 8 8 9 ' 9 L g - S L 9 ' d r q s u o r l e luaor r t l u n l ' ( t A V f N )a l n ) a l o u ru o r slla) aqpe LnLl9nLL (s)elupuacaasselupuep llal lernaN (a^oqD)uolala\soli(: aasuola;a1soy(r 1 8 8 € t ' 8 8 €t ' 6 ? 0 t ' u r e l o r du u l e N sadh ilD/suotba) ,r,uo*orrr"r.,xlli.,r, )rJDads:pJnau/-otnau6utuwbaqsauluaosp aas (s)uornaNaassuornau l 0 t t L ' 6 0 tt ' l o r l u o )a z r s luaudola^ap ;ernepaasluaurdo;anap lsl9'l!]eulaq)s J g t LL ' 9 8| L ' u o r s s a r d xueu a q p P l st9'auerquraureurseld 'suorl)auuo)ralul gtLL-Sttt'srsotdodP 989 t-s8€ ! ' s u o l D u ndf e 6 urelsls snontel l ' 9 1l '. (s)lPriualoduorl)V oslpaas 60tL ' 0 t 6 ' 9 a 6 'L E Z 6 ' J t e 6 ' ( l D Nr)o r ) e Jq l M o r 6a ^ r a N 059-889'sa!lradoJole)l.lDala (s)uoJnaN aassllal a^ieN lueuroola^aplelnaNoslDaas 0 1 9 ' 6 9 9 ' u o ! l e n b las u r e N s 8 € 1 - € 8 €r '09€ '(srsaua6ornau) Jalue) aas(Journl)urseldoaN uorle:r;r>ads t !09€ t 'uollel6lu uorl-eper6ap leurural-Naasalnr pua-N lSSt l' 'luaurdola^ap suDDala stltpqDqtouaDJaassepoleuraN z5€ l-€8€ I 9gl,ZZ'aDaoq)ouo6 oltass 1a11 0so1-2t01 'uo1a;a1so1(r uo uorlezr;er>ads o e s L 'DuassraN luapuadap 'ggt 'lolluo: /salnqnlo.lrru uorlezrue6ro arr1e6el lo6t ;euorle;suetl JBtOt ;o altle6eN (s)luauJelu alPrpeuralulos/Daas SVt'!Vn€'6ur;ro:.radns '6ururels J t l9'O19'^do)sor)r.! uorl)ala'uorl)alasenrle6eN 8 t 0 t ' s 0 0 t ' 1 L 8 6 ' 1 8 6f 9 8 6 ' s l u e u r e l u o r n a u 'LASJ-ggS 'snur^qt '^trlrqetsur a,rle6ay lil-uPu^p ! JLSSI 6t0t , '6st 'slrnrD uorldu:suert uolalalsolA) l 6 s t ' J O L L, u o r l e l n 6 a r 'luauru6lssP lal)eieqr LL L 989 l-98€ I 'ZLt'uolllqrqur elPr 'srsotdode JELL Jo f 9 e L L ' 9 Z lL (s)uornaN ;lr%l"il*r',13 JzLt' LLL'6,!F"t"rrtr ' 6 9 1 1 - g 9 l ' p u pe u r t u e l l 8 9 t t uorlelnDerlrJelsollvos/Daas L leseq'uollereue6ar raLL'LLt,fta1so1;e 989-t89'sleuueq) uol 'pue 'srolda)or a ^tle6eN euruel 2 0 6 ' ( u o r l r q r q u r aurloq)l^la)e J Z 0 6 ' l ) e q p e a r ) ) r e q p a a J 89 t t lPseq 'srsor)eN ' ( r w N )u o l D u n IJ P l n ) s n u o i n a N s l il lt89 'urlnqaN l8z0t 820L 8 n l L - L V t t ' u o r l e u l o ,a s d e u { s ' u r 6 r ; o r n a l ' ( a l n ) a l o u ur o r s a q p e (s)luaurelualerpauraluloslDaas 88t[ n a u )w v ) N ' 9 E l ' ( u u a q p e ) l l a ] l e lu ggg 'uorlezrue6ro 8 8 t t 1 8 € [ t I lernau) ueqpe]-N 1. uortnlo^l os/Daas 8V0| 1 186' 186'ISS6',sluaurPlUornaN 'uorssat6oldJolunl u! /srsolPurojqlJornrN €LZa-ZLZL 0 t0 t sasDastp )Ur)adsoslDaas LL Z L ' p u eu o t l ) n p o J d al Je n x a s gOV-rOr'saln)alou6urte:rldat-jlas saseastp uoud oslpaas 1 ' Z ' u o r l ) a l e s6 u r { u n d L6t-g6E'sale6ar66euralord lE l'uotleulloJuol utalold 8to l'uorlelaua6aporneN 'slselqornaN (s)uorlelnw orlD aat L9€t-09€ t 'ngz 'suotlelnul 'uor]ruuap'salunaN sgz /t0 L 'z9s'salalle lz9s t9s + n I ' E bl ' u o r l e ! l o s s et r u n q n s ' a s e p t u r u r e l n o N z l , - 9l ' u o r l ) a l e sl e r n l e N 1 9 8 € lt L l . € t ' o L t L ' J t v L [ ' 0 ttt' a q n ]l e r n a N 9 9 g 1 ' u o ; 1 r u 6 o : lal a r ) pal)a;ur-snltA o q bL '6 h L - 8L VL ' s l l a lu r a l sl e r n e N 'uorl)nrtsap pal)aJut-snIA eurlauaaseullar lernaN 9€St-9€S t ' u o r 1 rlla) u 6 o : a sl s e l ) ' ' 'a1e;d 9 € St - S t S t I fHW J t L t - t LL E 1 , - O LI E1 911 lernap '(dNlt) 'uorle^rl)e '€6€ 'sdeLu 9€St t - t 6 €t I t6t t lernaN ' I E S V L ' E Sf,-uo.ra;.ra1ur n L ' ( s ) l(l e r )r e l l r l '6tl yN 9 € St - S € St lernteN lSit t 1 - 8 t t I ' s u r a l o r op l o J J e ) s 'swv) a)uerelollelrDolounuul oslDaat 8tl]-ltl t 'alueralol '16] 'uorteuroj asdeu^s ItS | ;err6o;ounululr ;ern1e1 t6€ t -€6€ [ tt ',aporelo ' ! V 6 E I' S 6 t JtSt L J S 6 Et \-E6EL ' b t t ' € g Z ' u r a l o r dsleseN '6ur;apotua.r oueN pue uorleururlaasdpu^s t l € € €t t J V E E L 'esuodsel '€8€ 'saseqd eunulLut'slla)enleN JgnSL'9bSl t t 'xelduro) ' J O L E'L l t 8 € 'uollelninau asPprxoHdcvN z€s [ IL€l lLt l-0LEL '06t 'sro])ej)rqdor]oinau rr;:i(>uou €Sg-0Sg'uorlel^roqdsoqdoloqd 106€[ t-68€ [ 'rl]Pap uornau Z 1 g ' a l u e ) U t u 6 ts,(leuorln;ona 06€[-68€ [ lstS'st8'uorlexrj-uoqre) sSgt-€8€1 109€['09€t'srsauaEorneu ,srsaqlulsoloqd 'slselqornau tr6 09€I '(s)aluneu t8'olor leuotl)unj t ' ? 8 € ' J O L E | 'l06E }LEL'aqnl l z 8 ' 8 8 - 2 8 ' s J a r x eu r orl)ala t lerneu lz8'€8-28'81'+dOVN/HdCVN ? 6 € 1 - r 5 € t ' , { t ! : ! J p a d sl e r n a u J58tt

€€:l

X]CNI

[ 0 t ' l u a u r a l t n b a st t s ^ l e ] e ) o l n e saln)alou 6urletrldal-;1as srsoqlod^qppoM VNUaasppom VNU uratoJd B}n- LOV'stsaqlu^s J Z O yt' 0 V - Z 0 np' p o my p g - a r d 9 0 t - 9 0 ? ' s i a ^ e l rp qr d r l ' Z L g - L L B' u o t l n l o ^ eu r e qsla-zlg rl t o d s u e l lu o r l : a 1 a g6t 'leualeul(.retrparaqse VNC ggt-oot 'a;r;;o ur6ug 'sqdorloue6ro z[ suDbto)tJDads:tueudo;ana6 osyoaas e616 €.98L- LVEJ'srsaua6ou 'o€? 'r]roJ ue6ro I t €t t [ ]o '99€ 'ltezrue6tg s,uueuad5)razrue6rg 0l€ L t ,{1rre1 n;1a>rg;n61 iluaurdolaAeC oslDaas ztlt-tttl'z0t t ' l o r l u o ls s e ulrl a )l e l o l '0t Jt t t f t t ' s r o l l e J^ i o l r q r q u i ' 2 0 ' p u eq l M o r 6 8 0 1I Lt lle) qlMor6 usrue6iO s l e 6 n 5a a ss t e 6 n s g1-99'srsaqlu{s:r1aq1u,{so1or;d (s)aptloel)nNaassaptloal)nu 99',r!srloqeleur spt)e^llel aassprre{11e; sL-bL'la) ut aler uotsnJJtp 'sl)olq6urp;rnq se l9s (s)pt)eoutLUVaar sprfeouil.!e ' ( i e l n l l a 'l ; 1 e t u ss 1 a ; n : a ; o u:jr u e 6 j 6 ss gy'r{:gsruaq:true6.rg sallauo6totrloads osp aas I/69 969 arunto^ 3 9 6 1 ' s d r t l s u o r l e 1l aer: r 6 o ; o d o g ',uOrlrsoo 169 /Sl'septltsoutoqdsoqd eupuoq)oltw aaseupuoqloltuJ

' u o r 1 e 1 { s o : (u; 6r a } o r d ' ( s ) a p u e q r ) e s o 6prel ol u ! l - O t tz'vNc ralurl 'slladse 0zz JL nL ' J L €L.' l € / ' a s e r o l s u P l l ^ r P q r ) e s o 6 l l o ' 6 u r p u r q le)uotsrq 1gauolsrq JgL(.'qLZ 9€9'^]lsia^ru p l P q la p l s tuoilppe pr)e lrlels ls La'9 LZ-SlZ'alnl)nlts lluleulp JVLL 06z- 682'082'uorle)rldai vNc TSLL '6urpuaq 'suorl)eralur 'rt It tZ VNC VNC LL's LL-ELL'6ursse:ordsnleredder6lo9 '6urssalold (s)auolsrH os/Daas ul lsll ' t L z - z J z ' a P r u e da r o l 'xalouro) Kle l€ [ [ J € t b ' Z E ' g L Z - 9 1 7 ' 6 u r l a p o uuarrt e u J o r q ) l€ t t'9s-99'(s)aprreql)eso6rlo u!leuolqJ os/Daas J6Zb 'g7p',(qder6oleurolqr (lruUJe'sap!loal)nuo6rlO l e q UL u u - 0 8 lZLZ'JLLZ I LZ- atZ' €.LZ-t aZ'(s)aurosoal)nN I L9 \VOS'(s)al^ro.rpuapo6rlo antldepe'ua1s{s/asuodsar aunurur oslDaas t € E ' u o t t e z u e u r l l ovdN U ' s a l e q d s o q d ualp r s o a P n l I 'asuodsal 901'(s)uuodoal)nN sls [ lPuol]o6llo (s)aurosoqrU aassrseqlu^sauosoqu J6ZnL'OEnJ-6ZVL 'l 'suornau (rolda)ar)xrosuasitolrel;6 wgt'ttgE' {98' tgt-ZgE',snloel)nN [6 698'sproelrnN ogt t la^ournl 'qlnq ^ro])eJlo ILOE leuornaupue sllal uralslelnau 'suorl)eialur vNc-uretord vrau'sluaueluoal)nN l t I I ' p r r e] r e l o l16t t'lPrA'prsde)oellnN J L S Z ' Z L Zt 6' 9 Z ' 8 9 2 -L 9 Z ' s l u e u r 6 errl te z e l o 'a.lnDnJls ll [ [ O t t l ' e r u e p r n 6 u o x eu r ' s u r a l o r rdo l d a l e rl u e r o p o 'saua6rolda)ar ( p r ) P] | a l l n u o q u )v N Ua a sv N U ]ueropo 0E' [ 'suurnlo) 6uured-aseg aas6uured-eseq a l u e u r u J o i p e l n ) O 1 9 6 €t 16y'saryadord I t 9 ' ^ l r l r q n l o su r a l o r da u e r q u a L U ' a p r s o ) n l 6 l ^ l ) O 'uralord lall;o ur6u6 osloaas srsaqlodlqppo/vl nod ureLuop vNU tlro lszt t8 tt 'arrl ur6uo sad/fi)UDadsoslo aes zo?- tot 'slrunqna s pJo !]oalrnu z9- t9 l _ € Ett l z € t L ' z € tt ' ( s ) u o r p u n6'fuu rrppnnll))l )oo uorlezrprrqlqvNc os/Daas € s LL l 6 € 9 ' O t 9 - 6 t 9 ' 6 u l r l o l qu r a q l n o s L6bt-o6v t'sua60qled ete6rlqo yJ16lueulquroler JLt S'75S'16o1ouqral S L tI ' S L ( L ' ^ t t s a q ] l 6 t s ' 6 € 9 - 8 € 9 ' 6 u r p o ; qu i e q u o N ' 9 € s - s € s' s u o r t r p u ou) o r l P z r p u q ^ q ll€s . uorlezrpuqAq y116_ (pDe)ral)nuoqu,{xoap1 aasy16 J L r l _ 9 L Ln r l s r u a q ) l6€s '6€S-8tS'6urgolq jsrsaqlu^sVNC os/paas srsaqlu^s'VNU u i a q u o N ' s e u e r q u a uur o l / { N f L g '9 9 ' J S g ' 5 8 ' s r s a q l u ^ s o r q l9t9t'9LSl.'luaua^ou ua6oqled'dsvm-n ( b l l ' ) a ^ l ' u o r l d r o s q el u a l J l n N l l l . I ' ( s ) p r r e) r a l l n N (s)alnqnlor)rW luorlpzilaur^lodurllv os/Daas srnN J O 6'arnuade Z I ' 0 6 Z L ' , s lal e l 't8s 'sualedo)sor)rur 2 6 6 ' l gL 6 ' EL 6 l a L u ^ l o dl e l a l a l s o i l )J o ' u o r l e a l ) n N lzSs le)uaurnN '(s)JoldalaJ '8s€ 'ulaloid qurnN Uodsue[ real)nN 80/ L l69t t '[ l € L t [ t ' u o r ] P ] u e l o s u eiJel e l ) n N l 8 z 9 ' ( s ) u o r l 'snepng e l n Ll lun N '(|.Un^o) 6 u 1 ; u n qrse a P n p 66a O Ll - 6 0 L ' ( s ) u r e l o r d l8Z L xulPr.xreellnN aasplo#els real)nN napnuT-oapnu 6uruwbaqsauluaoslo aas fa6g' f L6g'069-69g'^llLue/ednsrolda)ar real)nN 1169'aunlo^ (sa)xaldLr,rol erod JPal)nNoslpaas l.lodsuPll :ruseldo/r-reapnN aasJo lno/olur lrodsueJ] J 6 O 9J' L } Z ' 0 0 2 ' s a r o rde a l ) nN 'uollezllenslA I L OL ' L 0L - 9 0L ' u or l e zlrP n s r ^ i59€ t 9 0I ' s 0 L ' 1 6 s € ' 8 s €a r n l ) n r t s J S g €n' g E - t9 t ' s a l ) p a d s s n l o a l ) n Na a ss n l o a l l n u ) 9 0L ' S OL ' u o l s n l l x ee z r s 'asedIDueu 601-80L r99E'VgE't9t-€9€'SWl9 'J6S€'8S€ 'liodsuPrl n, LgV VNUUr !62€ 99E-V9E'suorllu L 0 L - g O L ' u o r l )jeo u r s l u e q ) a u J S q En' g E 'V 9 € . - t 9 E ' s a r p ol eqt P ) ' ( s e ) x a l d u oa) i o d r e a l l n N S0Z'8St l 9 9 t ' 9 9 € - € 9 € ' , s e r p o rqe a l ) n u q n s r90L \s0 L't01'euerquiaurreellnN 'arnl)nrls ) l o z ' L o z - o o z 'sulalord ava' (ploJlDslxuleurieellnN 698-8S€ ,i^^-^..--f... ^oorsoJl)aos uwN aas fl69'slunouP auerquau i(dorsoll>eds(UWN)alupuosarlrlau6eurree;rnJ1 ( p r ) e) r a l ) n u o q u ^ x o e p ) s o r l e z r l e l or el e l ) n N oslDaas ) 9 O L 'L O L - g O I ' ( s S l Ns)l e u 6 r u vNc :(s)aLUosoLuoltl) l _ s 8 6 ' t 8 6 ' , s u r u rl eela l ) n N [ 0 2 - 0 0 2 ' u o r ] P z r l e )voNl c 0u aJnl)nrls J Z v Z ' t L V aE' n Z -W Z ' , s l u a u i u e o u . r o ) snal)nN tzlL'zLL-LlL'stsoltw 'qder6o.r:rur ' ( U l N )r p d a r u o r s l l x aa p l l o a l ) n N uo.r1:a;a 3; ;1 J 6 6 ( ' g 6 Z1 9 6 Z saplloal)nulDnptArputosloaas J 9 OL ' J S O L ' VL0' ! L 0 ( . ' 0 0 2 ' e u r u rreele P n N , V N UU T! JgOL'aOL-LOt'(s)rolda)arlrodr.urrea;:np IOIT ftv'suralord ra6ur,trurz Jt(.V'ZZnL 6 l ' t 9 L L ' ,tl 9 l 9 ' , a r n l ) n r l s 'suralo.rd fuo1e;n6ar eue6 ( p l r P) a l ) n u o q u ) 9 ' a199y r u a 6 r a n rppu e u o r l e r r ; d n a pu e 6 JssZ'vg7 vNU i(pr)e)rapnuoqrr^xoap)vNc oslDaas (s)rolderaJauourroqreal)nN z t t ' L 6 L ' 2 9 '1 9 ' s t r u n q npsD e) r a l l n u '(s)leu6rs 'arnlelluaulou yodxa rea;:n5 BOI J/ LL '(s)Jordalel leellnN uodxa srs^lorp^qdIV osP aas 801 eururPlreal)nNos/Daas LL 6 ' gL 6 ' 6 ur ru .pr e a r l ',arnDnrls ' u o r l e r aIu aab: r o ; u r a l o;1r dr o l o u r I t0z l9ot'JsoL 6L0t-9t0t 'JZt '080 '0801-61,0 ' t t tLot L'sller]|lolrur 060t 16L6'6L6'?16 xlquressealnqnlorlil.u n 0L ' l l o z ' L o z - o o z ' 9 2 ' a d o l e ^ ure e l r n N ZsU 6 8 6 ' u r a t o' ur d rluo] Ot L-6OL'sutelotd6u1;unqs 2 0 0[ (.OO url)e J | | L ' OLL - 6 0 I ' u o 1 t e 16na . t t ' V L 6 ' A l q u a s steu e u r e l U s!sllorp^q 801'srolda)ar (aleqdsoqdr.ll aursouape)dIV osloaas JOt L' 160L' 60 L-gOI'esedl9 ueU 901'SUUOdOelrnU 1 9 ' s r a t t l e1: 6 l a u a sarooreal)nNaassarooreelfnu J L 6 Z ' J g 6 Z'stsaqtu^so!q '962'a6euep (sa)xaloulol L0[ 'E 'E '(s)ep!loal)nN arod reel)nNaassaxalduro)arod reellnu JJLJL-9JL'Z9-L9 it JgOL J SL Z ' 9L Z - ; L Z ' 6 u t p t ;ast u o s o a ; > n 1 ' sleu6rsuo!lez!lerolre^allnu urleuJojq)os/Daa5 LOL-SOL'(sS'lN) 0[/'uor]elnoar tt€'s])aJJa leuolldl.l)suell 'uorl)prJJrp ^pJ-X'uorteururelapainl)nrls tLSV'88n-98n'sr.!srueq)euuorlezrle)ol lZ tZ 198?',98?-S8'^lH l Z L e ' f LL C 'LL Z ' u o r t e z ! u e 6lJeol n l ) n r l s ' 6 u t p u t qu t a l o r d aue6 98t-g8t'uorleln6ar uorssa.rdxa 5;7 '6S€-8S€'LgE'$et 'y11gr.u ' s u ! a r o l da u o ] s l q u o u Ipear-rrodxa l6St

o

"'-iBTi,'Ji,:iH

/uorlnlo^a J001'6,6,9-169 eupuoq)oltU\:(s)rseldorolq) os/, aas g ; g ' 6 u r y a n u o tr ( 6 t a u a 'luaudole^ep voL'z0L ' p u es r s e u r l o l ^ l 860l (s)lseldorolq)aasslseldoiolq) z t s - 6 o s ' s p o q l a u .sr r s ^ l e u e 169-569'(s)elleup6rO ()UO)xalduot uorllu6o:atut6uOaasfUO 'suorlut €gg lPUotldo '16€ 'urnD a l) ! l d o t 6 €t L '6ot; ur I'uorleraua6a; ,antau:r1d6 [6t t lZ9',SrazaaMl lPlrldo f Z 6 S ' ! 0 6 5 ' Z 6 5 - O O S ' 6 u r u o r l r; e a> s rgdg L S S ' , u o r l l e r JlJer)pt l d o llss I 9st'sss t'uorlezruosdo ! s €€tL ursoo 'sua6oqled)tlstuntroddO 96gI (tle otLl,uaesl) uoJado)Dl osp aas €9t-z9t tt€?,suo.rado 8St-lst'epqLUPl 'nEn 'rEEv'(s)rolerado ' 0 6 ' ' (Jb€b s l u o ) s a u r e r6; u r p e a ur a d 6 tss rDuassaN [1rue; uralordeuerquaut ed6 0ZSl , O 6 Z t , g g 7, e r u o 6 o 9 1O6ZL 1 Jt6Z I j6g( L'06ZJ-9AZt",srsaua600 'L6ZL-O6Z 'srxstueq)auuorlrsrnbteazrs J06Cl J Z 6 Z l ' f 6 g ( . 1 , 6 9 I7 L r e p u o : a s Z 6 ZL ' JL 6 Zt ' 1 6 9 7y 'g g 71 ' A t e w u d z 6 z l ' 0 6 2 t ' 8 8 2t ' L 8 z t ' 0 8 2 [ ' s r s o r a u ] l L 6 C t ' Z 6 Z l - J 6 Z J '6 8 aL - 8 8 2L ' u o r t P r n l e u r 8 8 2t ' s a u o s o u r o r q rq s n J q d L U e l 'uorssardxa aue6 6631 IL6ZL 'JO6Z L ' Z 6 Zt - O 6 Z g ' p u es ; ; a :i ( . r o s s e l ) e / a l ) r l l o J f 0 6 ( t ' 0 6 Zl ' g g 7 1 ' u r n r u o E o o ' D l t r . l d o s o r 1 IL6ZL '06Z '(srsaua6oo) ruaurdola^ap l68Z L f-ggZ [ ) 6 g aL '6 g at ' u o r s r n r ;pe : u t e u u { s e '8821'sailloo )L6at 'sesnl^oi]al 'stsaue60luO Z€Z[ swalotdysauab pnptNpu :seua6leltul-Ia)ueJ oslDaas zEZl'P)t^ 6te I LEZL'uorsseroxara^o 'uotle)tjtluaptJo spoqlau /\(eu 6tz L 'uorle)rJrluapl z€z I ZVEL_LTZL ' 6te L-SEZ I ' n E ZL - L E Zl ' , l o [ [ ' , u o r ] e ^ r ] ) e LtZLL ' Ot t ' ( s ) a u a 6 0 ) u O 'saleruot])eal 'asellxoqJelap dWO J9LL'9LL'SEL I [9[

LI Z

X]CNI

t€rl

uu)!rcuwuJ/drEu

)/r .olvE+

E v+ )otd

rc

Ylrrvr )ioYar

ru uuEu

l^la^rln)asuo)Mollo,reql sarn6!Jol ll ue qlrM /arn614 e o1ra;a.rI ue ql!/\^sraqunu a6pd j^Jluaaql Jo uotssn)slptxel rofeul e o1ta;al atetploq ut slaqunu a6e6 ipozotoldlsalrsered :r6unl leual)egoslDaas 1 | LL ' s l s o t d o d e ' s a 6 e q d o t > e u LZtt'EZlL'srsoldodP 'a6euueP 'g0S 'eualrpq ^q uorl)npul ggd ansstl uralordTaue6 88t | J60SL t 'peajds 'uorle)r]rpour ror^eqaq uotl:a1a lsoq I ts t 3gg1'sqdet6ottru.r 7 V Z L J' 7 O L t ' 9 9 ; .1 . ' u r a l o r d 7 a u1a76' d 'ggl ,alor 'lsoq ur {;dr1;nu o} s}uaua.rnbar 'uralojd lueualdLuo) lStt 6Z5J-AZSa l)f (t)Nl) 9,d t t z l ' ) e D u c . ]u r 'sller 'sllar 'zd 'ofiqtua i(q uorlru6o>a.r s u o q a l a ' 1 u s ! l l e r lsoq lsoq ogur(r1ua leual)eq 8OSl-109 1 azsl-gzsl 1 t ' S Z E L t e g J f t Z t 1971-ggy1fteurrd I S S St ' S S St ' p a l e ^ r l ) e - ^ p o q r l u e '(grsra,rrp :r1aua6o;,{qd d 68' t-88t L It€9t 'S '6gfL| | fgl'JrgL'JgZ'117'97'stso1Ato6eq4 z€9t- t€s 1'srso/>o6eqd l(s)a6pqdoDplA os/Daas 0 6 t [ ' > r ] s r u n t . r o d d o 1s1lrqdor1na5 'ur>o/x6 9371 l 6 v L - O 6 vl ' l e r r a ] ) e q ' a l e 6 r ; q o t € 9 t - t € S t ' s ; ; a >> r i l : o 6 e q 6 6urft.rer-ua6(xg (le)rLuaq)) (s)ase€i(s)pr)Vos/Daas LSZ-qSZ'saln)alou.r )Z6bl "s^ sua6oqleduou s u a 6 o q t e dr e l n l l a l e r l ual a rr e l n l l a l e i l u l L 9 Z - 9 5 ( . ' s a l n ) a l olui o d s u e r l 969'rPlnlla)erlul 'spunoduro::rxol 'ssalordsnorllelul 7gg1 Z0Sl- tOSt r L(8' LZ?-Oa9'luarper6 'ZS'Hd usrloqelaurlrqorev os/Daas (s)uotl)alulaassuotllerul J60 L '16-96 'ursrloqeler ro,rluatrarrnbar ua1s,{sTasuodsar 8 L8-l L8 f E Z FL. ' t Z F .l ' s a 1nu e . r 66 aunuul aas0l asuoosaraunu.rull IttS J t Z Z ' L Z ( . ' ( u o r r p b a ule) a^ l J au o r U s o dA) l d 'rEg-Ztg 'aseprxo 'uollnlo^e auoiq)ol^) ^q uorlrnpar ueulnq 99 11'atuorpuXsstaq6al-z1na3 LSlL'uo sl)aJJa 'uorleraua6 ggb tsoq V L B ' E t g 298-998'sluelnu alllad l e l r p e ra e r J lLBn l'su.rotdru,(s/su6rs'asuodser 'SLA-€Lg'aruerrlu6ts(reuorln;ona 'sa6ueq:)r;Jertauerqureurlsoq '106-906'urxolsrssnuad )VLB t tS f- t 15I €0s L 'srssnlJed r;,reuorlrala €€8-Z€8'iQru LqnL-98nL'suoll)eraluI lsoq €0s L 'pue uola;a1soy(:rsoq .ra6 )b L8'' Lg-EL8')uaqdsor.!]e LJgL-nLil 379y'ura1o.rd ',t ',puPseuerqurau] lsoq ua6{x6 (s)aulos^xo^lg oslpaa5 I LLSL lS 1- I 1SI llel I LL8' Z r8- O18'uortnlo^a 8 [ S [ ' u o r ] e ) r ] r p o uJro r ^ e q a lqs o q fl69'aLUnlo^ u r e q )u o o s u e J ] 06tI a^r]e]ln)P] LZ1'arnl)nrls uort)ala leupuoq)olly!aasl.roosuPJl uoll)ala 1 2 91 - O Z S1 ' ( p l d e r u ) orlnlo^a EZI- lZt'uodulr uralord 'sasueJap 'uorlrnpo.rd tsoqJo uotse^a L ' 8 6 ' s r s a q l u ^dsf v t z g , J Z Z qZ , Z g -L Z g J' O O tos 1-z0s I 3572 '6 'uollel^roqdsoqd anrleprxg lozS t6 t8 L8 v z s l - l z s t ' l u e l s r s ao l nip L71'sua6o;eurse;d 'tsoqjo uortezruolo) sts,{1o:I;9 osp aas 994'93 uror;lrodLurprdrloqdsoqd t zos L- tos,LESL_SA9J ' 9 6' s r s I l o r r ( | 6 ,(s)ua6oqled ,689 176 r 6€Sr 67's:r1a6.raua s179'6u11se1,{1rura1e6 'saporl)alaqlled dslloqelaul a^lleplxo auelquaut lz9 f169'stuno|.UP a^rleprxO ; 7 7 ' a p r x o t a du a 6 o r p { q ) 9 6 2 '9 6 2 ' y 1 1 6 ' a 6 e u e p l z 9 6 ' 0 9 5 , ' u r a l o l dp a q r l P d '289-089 (s)lerlualooxopeuos/Daas z(L-L(L 969 suorl)unj I 189 ' s e r p n l ss l a u u e q )p e l e 6 ' ( s ) 6 u r p r o l a y71'sawAzua d ru e ; : - q : g e 6 L€8 ' . rr l r a l a 6zg-gz8'suorllear(xoper)uortlnpar-uorleprxo 9 ' l r o d s u eat ln t s s e 6 J t e L 'J l Z L ' q d e r 6 o . r : r u o f i 5 9 ' E S'96n 'SatrOSrxOrad ',sasnrAon Jed (s)eurosrxorad 774 | L )96n 'Zl 'staJsuPtl ,srsaua6ouaqlte6 (Z I'ltodu uralordauosrxo.rad'(s)utxolad uolDala 828 1971 '066'urelord uorlelrdsagosloaas (s)uralordreure) aassaseauJad 1066 wred ' t 1 9 l t ' 9 9 1 |' 9 9 1 1, u e r a F a d swalotdysauab >rlDadsosp aas € Ol - 8 8 ' 0 1 ' u o r t ) n p o J di ( 6 r a u ar e ; n ; ; a r Mt | /srlruoluad 7 7 1 ' s e u o s r x o r a d ' u o r l e p rf lx o 98t t ) L S t t ' J g S t L ' l SLL- S Stt 'uorlsnquro: '(tue;od szr;e:r6o1orq l0Z8 l06tt'a)eds )il.usPldued ;eseq-orrde;er;eqlrda'sura1o.rd7saua6;o3 'u 'suorl)eal ',a)ue]alot uorleprxo 88S[-l8S L I 11 l/06'auouroq pro-r(qtere6 ' ( s ) u e 6 rp o r o q d u , ( leiaqouad { n S l ' € b S l ! L ? St l t z L l z z t ' 6 6 'J 8 6 '8 6 ' , a l e l a ) e o l e x o s6n|.n6nL'ueozolord ;1e r a q d r . r a 6 l 8 LL ' 8 | L ' Su ' x a l d L u o rv x o 6 8 t I ' , s U l J O lrl l l s p r e o J 6 ? ' a l q ellr p o u a d 'uorsen (un^o) 661 aasuln^o u I lla) lsoq 1elnllalerlul I I St-8OSI l s o l a ) e o sl P a l t n u u a d salrsered z6zt osv l'lgwL'saM)!rad 'xuleLuleloulua)uad 'O6ZL'Oge 'uorlelnAO g g 2y ' o 1q d o s o 4 ' s l u a u l 6 a s e l e d l'reue uorlelduro)srsoraur 910t 'a,!l adt ut'wnt)awuqd 'ELSJ',uuoJrad 1 9 1 1 , s u o r 1tunfde6 t IELSL l9 ,o 'S8Z 'luaudola^ap ' , s a u as6n o 6 o ; e r e 6 882t t 1 7 9 7 ' a u a 61 > o 1r>a g )LZ lOZ' IZ-OZ 'sl)aJaprPdar laluel auurered quosoqlt-y11gl-1(pr1de6 ! 9 L E ' sL € ' 6 u r p u r a € 8 9 ! Z B B ' 1 g g ' 6 u r 1 e u 6 r s 'J6Lt '6Lt '91€'aseiaJsuerl ' Jfs6z 0 6 Z L' Z 6 Z L - O 6 Z' s l lvNc a )A r o s s e ) ) e 'OSS 'apou qdur^l'xauorered Ljn | t SSt t l L 6 ZL l^prlded /llpM ' ( sI ) u o r t ) u n r saueAo ] r l 6 r l' l r o d s u e rrl e l n l l e l e r e d L Z ; L ' J O 6 |V zsI t lla) leilal)pq'(s)uel^looprlded )(?St'Z8SL € 9 9 ' s l ) a 1 Jdau n d u r n r s s e l o d - u n r p o s ' u r e q e n O iLsll'Lstt 9sLl ' 6 u r p e os; u r a l o r d ' s r a y o d s u e tal p r l d a 3 'r(1ue;od y19 1 'srsorodoalsg )HW I ssell ;eseq-o:rde;erlaqirda'u;atotd /aua6 gtod yly 1 'srsorladoalsg l Z o t ' Z o t ' ( V N d ) p D e ) i a P n ua p r l d a d 95LL-SS[[ 'r(gue;od (xalduro)^lrlrqrledtuotolsrq rofeut)111y1 osloaas t 0 L v L '0L nL _ 6 9 nL , p r o a l s o /aua6 ilod leseq-o:rde;erlaqlrda'u;alotd 'slla) :rua6oalsg L 9 €t 1 0 8 9r lglt [ ' 'uolslAlp J 6L SL 1 8L S| ' 6L SL -LL SL ' , x a l d u r o) )H W - a p r l d a d 7 g11 ' e 1 : a ; r a d usr r s a u a 6 o a l s g llar lselqornau'xeldLuol)\de /qed/ iled aprlde6 98Sl- l gS t'asuodsat aunuJu.tt's1uar-u6e.r; Lvt'gt7 1'say(:oassg f ZL v L ' J O lLSLt'LStt-9SLL 'srs^lorp^q 'auoq 6urlapoua.r 'r{gue;od L9L l€lt L E.ro4 Jo ;eseq-o>rde ;erlaqlrda'u1alord7aua6 '6urlMet) 'reaurs oed L j n 1 g el , u o t l P u j l o j 9 € 0L z Iz [ lla) 'uorsora a6elrye> (sdvd) l l z t ' s u o t l e l t u l t l ) u a l s ' s a l 6 u ep u o q RLnt'ilbt-ZLv I are;lnsoqdsoqd-,E-aursouapeoqdsoqd-,€ aar SdVd 1 0 9 ' 6 S ' s p u o qa p r l d a d al)^)-lla) jo uorsranqns t6LSL 6nZL-8n(.1 lorluo) '!8lSl' 'auoq 6urlapor.uat '(/3 pue gl) saua6o>uo L L S t ' u t e l o r d) H W ' a n o o . r 6 u r p u r q - a p r l d a 6 l€lt L J6V(L'\VaL 'sasolued ' ]o s l l a )r o s J n ) a r d lz L[ loltt 8 V (t ' I S Z ZL ' a l o ir a ) u e l 'urllDrued ' u o r ] e u J o p, u e a b e e u r ; ' g Z Z L' s a s nri^ e u o l l r d e d z z s L -t z s | JgZal llgt[ 'sn6rqdua6 ; y 11 LZZL-gZZL',seurol 6711,uor1tnpord6133 lroed /sluauiale 99S'08t IELt Z I Z L ') a ) u e ) l e l r ^ r a ) ' a n b r u q ) ant o e l o ) r u p d e d $ L nL 'O L Vt - 6 9 ' r . ' 8 9 ?t . ' ( s ) l s e l q o a l s o d ' s l t a ) q]aued l - 6 6 t t l g 6 t t ' 8 6 1L , l l e i !; ^; a :1 u e 1 d , s u r 1 t a 6 flLnl'(.LVt-0LV['ssa]oid'uorle)rrrssO J q Z S L ' j r r t I L E V \ ' 8 E t - L E n[ ',suraloJd 'Jn99'V99'999'uo11e;n plorets 'suteuiopzcd 6ar ,{lueloursg 8?t t !Jr99 L Z S L ' L a St ' s n r ^ p z u a n U u r ' s ) r u r a p u p d 'stunourP flcd) aseraulostapulnslputaloldaaslcd 1909 fl69 ' S 0 9 ' u o r t e r e d aar dl d u e s (lDCd) rol)eJ qlMor6 parr.rap-1a1aleld aaslDCd a u e . r q u . r a u r ' s a l l a u e 6 r o ' (as u) lul a) o f x a) r l e a r ) u e d W l ' a p r x o r l a lL u n t L u s O (ufd) uorl)earureq: asetau(;o6aas936 9 0 9 ' s u r e lis( d o : s o r ) r uur o r l ) a l a ' u n r L u s O vtlL'seeDued 'g€€ ' g g gg ' s a u a 6a l n r -r e 6 lslz'uretord vNfd ll€t t t-l€€ 1 #LTL'JCLT,L 'tLtL-l aas u r u o r s s a r d xaau a 6; o 1 8 € t t 9 € L L , u u a q p e ) _ d (lgvd) srsaroqdorl>a;a apruLe;i(De,(;o6 L E L ' s r s a u a 6 o l r t u o s lgvd ;e6 ',aua]Aq)ed s l ) o l ) u e r p e ) r Ja a su e r p e l r l vgvt'Lxed 9/Z [ gts l- LtsI uorlellr)so l 9 9 t ' 9 9 t ' l u a u d o ; a n a p a { a , ( s 6 o l o u o qp u e )g x e d 'suralordsnJrAJounl bgvL'Exed ! L Z ' f O ZL' Z - O Z , s a u as6n o 6 o ; o q l r 6 VNC (q uorlenrlreur t € S t - O € Sl ' s u r a l o r da 1 r 1 - 1 ; o 1 1531 ,ideraqlra:ue: ur palaorel(luar)llap 688 'sa1{:obeqd ',re)uPl '(;rtreyadnsloldetar leal)nu'sJoldaletueqdl6 (q uorlru6o>aruaOoqled 1g51 RS(L ZSZt-LSZ I lellerolol / s l l a )) r l u p u a p Ils l'gts | L Z L L ' 9 0 lL l a ) u e l u r p a l e l n u rl o s s a r d d n si o [ u n ] 890r !682 ' ',passardxara^o l lalle6 su.uo,luelnlrj Z n SL - L V S l ' 1 Z S [ ' s l o l d a : a ru o t l t u 6 o : a u 9tz t J g g Zg' g z ' 1 g Z ' ( l U O x) a ; d u o t u o r l r u 6 o : aut r 6 1 1 6 'suor]erurpor! (s)ur6uouorle)rldagaasuorle>r1da.r 5 8 €1 - S 8 €I ' J 8 8 1L 'L L I I ' l V l l ' O V I l ' , l e u o r n a u rl8 [ ;o ur6ug 'S?Ol 'lla: 6urte.r6rr.u 'arueprn6ler*qle6 uorlnlo^l orlDaas O?LL $ n 7 . L ' 9 b ZL ' u o r l l el o s a p o L u Jo 'suor]reJalur 'sluanarofeur'aurleurl uraloJd l l 6 VL ' L 6 V| , s p u e ; sI lrr l u a 6 o q l e 3 s0 | L zurpw lt18 ,uortruUap ,srsaua6oqle4 'S0L 06vl-6gV L l90t f L ' t u r o d l l a q l a 6 e u e py p 6 VNU)rl^lete) os/Daas 'saprlded,(1od e v ' I g L v ' 6 u r p u ryqp 6 16y l v e v ' E' 6 uruapoqs 1171 Z 0 ? -t 0 t ' s a P r l o a l ) n u ^ l o d got-tot /uott)alasleinleu araurolelol asuodsarur lsalle a1:{:-1;a:

zzt-tzt"?I,ff#i:9I;

' I d J r to e hl-il []i3'"p:T' "

6ESa'L(.SL '9ZS /sluelnultlsounuJLut paler:osse-ua6oq1e3 L (sa)snrrn

s€:l

X]CNI

J L 6 LL stz'(ul) unlnlllar l 8 t 8 ' 8 t 8 ' x a l d u'(s)uarslsoloqd ro) euualue ' pue prdrloqdsoq6 saloJ : r u r s e l d o p u a ' s u r a l oaJ6du e q > x a L6LL-961L'uotleztlpt)ods 8t8 / s l l e M I r eleuotltunJ 's1er1ue1od puo)as salD4ow )ry)ads oslDaas xopar 96ll'96LL llal 1799 '96 'slleM ^reuud J86LL'86aL-L6t l tt 9VL_wL,53,srsaq1u(s 7y9'xaldu-ro:ll uals^soloqd 'uor]rsodapjo lla) '0S8-6t8 !aJsuPrluorl)ala uottpluauo 002 1-66 t I 8 1 9 ' J bLL t 6 S ' 6 S 8 S'ornlrnrls J098 'z0z 'pue salnqnlor)lul 'aVg'leualleq l00z t 1-002 I 029'sartradord JZng ruor]rsodLuo) euprqureLu z0z L'pu? sluaujeluutlle 029-g [9 lvsg' $ng'8tg'srelue) uorl:ea.rr;1aq1u{so1oq6 'qlMor6 pup (s)tuPldaasslueld J96Ll'L6Jl-96t I luaudola^ep 9 2 9 - 9 2 9 ' s l a l d o l PP r d r l ,861 ,sura]ojd l_66t[ I l z 9 ' s a l n ) e l o u o r ) e u lJl e l s L g - CL 8 ' t t 8 - 0 t 8 ' u o r l n l o ^ a jeual)eqoupl) otlDaas g9S-L9g'8S'srsaqlu^sorq euapeq e1d.rn6 1 6 6 rr 3 8 6 1| ' , 8 6 t[ ' , s u l l ) a d prdrl osloaas (s)rar(elrq ! 6 6 11 , 9 6 t1 , 9 6[ [ , u r u 6 r l sLB-ZLA'uorlnlo^a t-66r I J L O ng' O n - S O !v6' 9 ' s r a ( e ; r q €98',0t8',eualleq 'lg6t '96[ gyg'stusrue6.ro>!laqlu^soloqd qledlqdue t'86t L [ ' s u p ] ^ 1 66 u r 1 u r ; - s s o . r > 6 S - 8 S , s a l n ) a l o lul'sale6ar66e (s)lseldorolqf:uorlexUuoqie) os/paas lS I t 186tt 'lL6tt'a6la-t6tr'96t t'08t t',asolnllal 8 l9'6'(s)Prdrloqdsoqd 298 auiaq)sz 'pue a d r Q 16t t-S6! I 6 6 Z L ' J 0 1 6 ' 6 0 5 ' ( l l d )I a s e d r l o q d s o q d 1|ar €18 !ZS8' tS8'arui(zua6urDrlds-lalervt /aln])nl]s/uot]tsodu.jo) trJoadsoqo aas sasoduorldsorld J66[ [ € s 8 l z 9 8 ' s l e l l u a l o dx o p a r '(s)esedrloqdsoqd 'lleM '098-6?8'8?8'sralual uorl)ear zz9 zozL-s6aa J0s8 lla) iueld '?98-€S8 salon)eAluPldaassalonlPA J L SL ' L SL ' , s a s e ulrol l r s o u r o q o s o q d 1998 'JSlg "s^ Uodsuertuorlrala 1 6 11 ' a t n s s a t rdo 6 r n 1 l L s L ' L SL ' ( t d )l o l r s o u r o q d s o q d leupuoqlolrur 'uorleuilojsueJl 'uollnlo^a l01S 9LB-ZIA l s l p r d r l o q d s o ql odu s o ual a ss e p r l r s o u r o q d s o q 6 '^lualodrlot '€s8-058'sJauleluoJllela 89S J L SL ' L SL ' , s a s e uar lp r l r s o u r o q o s o q d lzsS ,799_ ,spr1seld (s)prolel^qf aas oslD 1 173 179 t € 6 ' l a s e u r lu r a l o . rldu e p u a d a p - a p r l r s o u r o q d s o q 6 '^lr)rlseld 'auerquraurprolellq] ssor)e saln)alou lDnptNputoslDaas 89SS lZgS 'g '(s)apua)Il60qdsoqd 'OZ9-8 (s)iseldoiolqfaassa;;aue6to>r1aq1u(sotoqd t9 [9 l6 t9 JI,L8 'eselnuragera:l;6oqdsoq6 '098-6?8 'JVng'nnB-Eta I IZ [ ILOZL tzs8 l0s8 'JOOZL'Zj|L-1OZ'860 '[ 'eseurlalera:I;boqdsoq6 'salnqnlor)il.u L L-160t I LZ [ 6 ! 1 2 8 ' ( s ) u r e qllr o d s u e r lu o r l r a l a: r 1 a q 1 u ( s o g o q 6 sluauodwo) )!]pads osloaas 9 ? 8 ' s l l a rl l ^ q d o s e u I L ZL ' f Z 6 ' 1 6 ' a 1 e r e : ( 1 6 o q d s o q 6 - g 'aiere:(16oqdsoq6-7 '69'sa6e1s 3 131 6 7 ' u r 6 r . rIot e u o r l n l o r a 191 'uorsr^rp ',01'uollelosal ol uolielal 9 6t [ l 0 Z l ' a s e J a u l o sats o t n ; 6 o q d s o q 6 J01 gue;6aas ql/\^oi6pue (s)uals^sotoqd aas sulals^soloqd luaurdo;anap lueudo;anap [ ' a s e u l lo])nl]oqdsoqd l 0 z 'alennri(dlouaoqdsoqS uorlelfioqdsoqdoloqd 1 8 6 0 t! 1 6 0 t ' 8 5 0 t - 2 6 0 1 ' u r s r s a u r ) o l ^ l 3 171'spuoq '29'S0S-t0S'arniln) ragsarpoqdsoq3 >rp(:uo51aasuor1e1fi oqdsoqdoloqd)rl)^)uou 19 166L'!t6 899 'l)Pl aurosor]ua) r r e d a ru o r s D x a s e q ' a s e r a l s a t p o q d s o q S t80 [ l 6 6 Z ' ( U l g ) 9L8-ZL8'leual)eq / [ 9 ' s p u o qe p u p ^ q u e o q d s o q d lE6 l l P Ml l a ) l u e l da a ss l l e Ml a ) €58'Lt8-0?8'uorlnlo^a 'uorlenba ]9LL'9LL 01 J€L' 'I 'uor]enreserd '(SdVd)erellnsoqdsoqd-, g-aursouapeoqdsoq6-,9 g4-99 'srrla6raua auoua6 'uot]etluaJaJJtp lt lle) ' g g L ' 9 2 9' J 6 t 9 ' 8 1 9' a u u a s l ^ p r l e q d s o q d srsaqlu(sotoq6 (s)ureq) L LLt iuorlexUuoqle) aasuollexlj uoqlp) rt;oeds-;oltsoutl,{ptleqdsoq6 uodsuprl uorl)ala rrlaqlu{so}oq6 0€9'l asedrlorldsoqd aas(suorl)eartq6rl)relsuerl-uoJl)ala 9t8'slla)qleeqs-alpunq J 0 t 6 ' t d ( t ) t d la l e q d s o q d - ;yo l r s o u r 1 { p r l e q d s o q 3 '606'606 /(s)llarrueld r 1q>dI r ll r r6 t0 L6 9'8 5 5 9 ' u o r 1 e 1 , { r o q d s o q d o :l o ' t z d ( S ' t ) l dal s e q d s o q d s r q - 9 ' ; J OL S 'J 6 9 9 ' G 9 S - B 9 S ' : r u e 6 s u e r l ;o1rsour1,{prleqdsoq6 t5g-€ gg'a)roJa^rlour-uoloJd J F . tgL ' E g JL - Z g a[ ' 8 S L[ ' e ] e u r s a p o u r s p t d 1 6 0t t ' g Ot L ' 6 u r 1 e u 6 r s . r o lqr ey ;v r o i 6 Ita98 '€gg-098'uorlel^roqdsoqdoloqd (sser)lle^ ) rt ;:,{ruou 1779'779' 6uxeu6rsre;n1 ;arerlxa g n j L ' J € n j L ' u o r l e z l u e o ruor l r P DUDlpqtslsdoplqo.ryaassursrue6tolapou se ( s ) u r P qu)o d s u e r lu o i l r a l a 'ur6uoIteuorlnlo^a '6uuaa,ro;; :r1aq1u(so1oq6 aaslrodsuerl uoll:a;a 9g lua)al sE6-Z€6 '(espurl-€ eseurl-,€ )rl)^r s 6 LL ' , 0 8 [t ' , x u ] e ul e l n l l a l e r l x o ld) ;olrsourl{prteqdsoq6 Egg'uor1eli(roqdsoqdoloqd 0t0t tt8-0'8'9€'uorlnlo^a JnrS' 8t8-€n8'(s)lseldoiolq) ' [ . d ( t ' E ) l d ]a l e q d s o q d s r q - y ' g qvno.r6pue luaurdo;a,rap q) os/Daas (s)urals^soloqd :(s) ;o1rsour1,(prseqdsoq6 ll^qdorol tueld itua|.Udola^ap lue;6aastuauldo;anap EE6 J 8 r 8 ' 7 1 n 8 ' 8 V 8L-n q ' ( r l s r u r a q r o l o qldl ^ q d o i o l q ) 't606' uollPXu L 6 L - 9 6 1E ' nL ' L a g 'Q d l o l r s o u r l ^ p r l p q d s o q d JLNS ' '^uoleup enrleleduto) uoqreJ aas(suorl)ear)llep) uorlexuuoqre) Lng-grg'slueldt) s^ €l E VL 1 6 L g '8 L g 'a u u e l o u e q l a l { p ; l e q d s o q 6 (s)]ueld eual>eqa;drn6ieual)pqoup^)oslDaas r E nL ' w L ! 6 L 9 '8 L 9 ' a ur l o q : 1 , { p t t e q d s o q 6 (s)asereqdsoqd urelord:(s)aseurl 1 0 9 €r sL a - zl a ' t 9 8 ' 0 ? 8 ' l e u a l ) e q '8S€ 'f '(iue;od autsouape)dIV osp aas ulelord1(aleqdsoqdul t LSLL'ast J-tS1 1 !6S€ t 1098 llar reueld aasuor}e;{roqdsoqd uor1el{roqdsoq6 ) o 1 6 o 1 e 1 6 '0s8-6'8 lttS's98-ot8'01-69'srsaqtu^soloqd 7 7 11 ' 6 u r p u r qu u e q p e r ' ( u r u a i e ) -u1r. q '16 'stsr(|or{1 6 uorlexrJuoqreJ os/Daas 376 JaOS L'ZOS L'>ruoqnq'an6e16 'sapo)eld ollelIosaroloqd J gL l ' g L L ' u o r t e ; { r o q d s o q d a p LV8-9V8',u l?g€ t /Plualeld (spor)srolda)aroroqdpou jsauo) oslDaas l?g'09'suorl)ear ralsuerlateqosor.ld 61€t (f)d) ) aseurluralordaas))d 9L L j L O l ' s d n o r 6a r e q d s o t l 6 )TENT'EEN|-ZENL '6Zt '(slla) .splau rotda)eroloqd)s.roldareroloqd L 9l a l s a r p o q d s o q d r t d f E 6 ' 2 9 t t8 t6 z9t t ' t 9 ' s p u o qa p u p ( q u e o q d s o q d It6 f 6 8L ' 0 6 L - 6 AL ' 1 8 1 ' s r s o ] { > o u r d [96-096',u1a]ojooloqd (s)uJals^soloqd os/Daas l € 6 ' , s e r b r e upau o q t 9 t ' l ) o l l u e r p e > . r r > ' p u ;ee1a6u 1 6 'DuassDN turalordullld l/0['spuoq ateqosoqd 0Z9l l l Z S S ' € 9 8 O S 8 ' u o r 1 1 ( r o q d s o q d o l o q 6 'l e[ualslso]oqd '!ltd (s)aseleqdsoqd uralordaas(s)aseleqdsoqd 1 0 6 r[ lySS 'sralouroidrournl se 'sra]sa 'lersuerluorl)ela 9ZZt u :a u 6 1 6 loqJoqd r v e o L ' v z o L - t z o l ' q s r ; ' s 11; a 6t8 (aseurl-€ J L 9 Z LL' g e Lt 8 0 Zt ' 8 0 2t ' e r u e l n a l td) ts8-€s8 !098'098'lPual)eq etqdlapeltq6 aseurl-,€lolrsour;i(prleqdsoqd aasaseutl-€ld l n s q ' ! 6 1 8 ' 8 n 8 ' s J a l u au) o r D e e rl e ) r u a q ) o l o q d snoua6o;a(u )ruoJLlfut'aLUosoLUortlf 'sauouoreqd ( l d )l o } | s o u r l ^ p r l e q d s oaqadsl d I t6 l96S tS6S'S6S-V6S'uorle^ll)eoloqd 'arn])nr]s'euruelel^ueqd '(al!s l6Z [ f 196' J96-O96'euro.rq:oy(q6 8rd 1668'868 'ureuop rf t;tqeltiaq :ttuouag aas 6urlurrdtut l 0 9 S ' 0 9 S ' 6 u r d d e ue u a 6 ; e r r s , ( q 6 8 f d ) u r e t r o p6 u r p u r q - a u r s o r ^ l o q d s o q d 'lLve ' '(s)aart suasr)sfrlauaD oslDaas utelold rrraua6oli(qd uo;le1(roqdsoqd uralold aas SvZ LVZ'J9L 'srs^leue ' '6urlurrdioo;:ryaua6o;(q3 uortelfuoqdsoqd e^!lpprxOaasa^rteprxo ls5-9ss I t €t | €t '€SS'adXlouaq6 uotlel^loqdsoqdotoqd ltSS 860r-2601 ' s p r ) e 's1ue;draq6rqu! srsaurlolX>'lseldou-r6erq6 l l a l ) n u ' s u o r l ) e i l x al o u a q d rr1:(ruop aasuorle;,toqdsoqdoloqd:1p(ruou 8ES ttaNluo ])aJJa ' z z z ' s l r eel u o l s ! q ',r!srdorloloqd lllL lelrqreqouaqd t96 # e e '5^ uorleurquro)arluDaos-allsos/Daas 6urpurq-dl9 196'urdoJloloqd 108[ 'sapelse) 'sqoorloloqd tssv s6g zt 'uorletleA 'SSFtgt lpai:ra6uerrear 'palerperu-d]v aseqd (s)auorq)ol^l j(s)ll^qdorolrlf oslpaas VNC lt8 uo!lel^roqdsoqd E Z 9 ' S a U e l q u r e u r ' u o r l r sauseer q ]d €s8'sdrqsuorlPler nl)nrls 'slettuelod lseJluo)-aseqd xopor Egs!t0s €s8 tzsS t € s ' l o q e lv N c ' z € - s n l o q d s o q d "(do:sor:rurt 8 s ' L 8 5' a s e q d slalua) * n L ' 9 nL - n nL ' l 2 9 ' s e s e l q u r e r ) s ',lS6',urprolleqd uorl>ear:r1aq1u{soloqd aassra}ua)uor}leaJ 1886 9 VL - E nL ' 7 7 9 ' ( l q u a s s e: e ^ P l l qp r d r l '€98 'saseddrU 'il Z E St - t € St ' Z S Vt ' g gL - L gL ' E gL - a BL ' ( s ) a u o s o 6 e q d ItsS !ZS8 I t58't58 uals^soloqd fitL'VtL '€s8 'Zsg't zgst-[€st Its8 t58 uals{soloqd 1J9'srsoldode lzsS '€ (s)role)olsuerlplolloqosoqd t SI ! t t 9 t ' Z t S L - L L S L ' a u . r o s o s ( ; o 6 e q 6 l gtg'gtg'saxe;du-ro>6urlsanteq-lq6r; /]srnq ^ro]eJrdsar Z€g[ S ? l ' s u t a l o t dr a ; s u e rp1t d t ; o q d s o q 6 €s8'uorlnlo^a 'suJsrueqfaLx a)uelslsar prdrloqdsoqd (s)uteq) LttS t Z€S u o o s u e t l J n n L ' s v L b b L ' l 2 9 ' a s e l q u e r ) s 'ssa]oro uorl:a;a :rlaqlulsoloqd aasJaJsuerluoJ])ala zEs[- t €s t rnnL'vvL '898 ' ( ; q u a s s er e , { e ; rpqr d r l ' ( s ) a s e d dprtUd t l o q d s o q 6 Z€Sl_l€St,sua6oqled ItsS 30S8leuar)eq

X]CNI

9€:l

uu5llcuuuJ/drsuuuJ

sucdu

)4 .dlqcl c u+ JdJdr 1c

qllm 5Jdllu'ru

duEU

1^le^rln)asuo) MolloJtpqt sarn6!,ol ll ue qlrM'eJn6r;e o1ra;arI ue qllM sraqunu a6ed j^rlua aqr Jo uorssnlsrptxel rofeul e o1ra;arerelploq ur srequnu a6e6 , 6 g e t , ( p o qr e l o 6 ' , 6 L V' , s l l aal l ) i ) u d or/Daas (s)aurosoqru | r6BeL J 6 L VL 'uorl)unJ /rJlaposau )tlrr!osald peards]urod L1EL 065 oEL'8aL!28€'28€-18€'(s)aurosoqu^lod /suorlPlnuJ lurod 9tz lt6'urIuasard €08'(s)uraiord^lod 'p;romypg-ar6 'uollpue^ lrue5}ue 'iluuD stlsi)ownaud (s)uratord aassaprldad^lod tO1-ZOV'allllo ur6uo 6 Lg ! /slapour gg7'xe;dLuot ) r e P n u a p r l d a d v) N d anrle>rldar-a.r6 uralord auoqpeq eprldadllod t 396 lZ€ J Z O V ' ( 0 V ' ( p D e '990 '()U-ard) 't00 ',uorleuroJ xaldLUo) (s)pr)e lrallnN os/D aas uorle)rlda.rard t-190 L t 3s66 1890L lt00 L 1u as n ; 6 (pr)P aas a l n q n l o l l r . !' ( s d l l + )s u r a l o r d6 u r 1 r e r p ) r a l ) n u o q r . r ) tog'(s)uralord-ord-ard V N U V N U 'ur6rro 6 u l t r ; d 5a a s6 u l : r 1 d s tot'saruadord € ? st (pua,€ a l ^ ) o q d u ^ l ' s l l a )u i a l s: r l a r o d o u a ql u a l o d u n l d slsaqlod^qploM vNU oslDaaJ aasuorle;Iuape{pod urDuo VNUrU)uorlel^uape^lod [ 0 ? ' a J l l J Z L L L J o ( p D el r e l l n u o q u ^ x o a pv)N c a a sv N c -8t'uorleue^^prold 8 5 € ! € S € ' 6 u r b e 1 > e6dp g u q t L t LL ' t t t t ' 9 S Z ' 1 6 € ' 6 €'900 ',urrrald saprloal)nuAlod t 3s66 V N U U)rr l o f i e ) n a l o 6 u r d d e 3 , 9a a s6 u t d d e l 1900t 'ureuop (96) {Eolotuoq uuls)i)ald (s)lrqdorlnaN suolle)ulpotuvNUur-ard aassal^)o)nel real)nuoqdior!^lod €€6 's1ralaprredoJ '6uta6ealnleulard '91S 'suratord 'tusrqdtou-t(;o3 09vl'Lvvt'(.011 1967 VNC t 88SL 1961 ' oslDaas (s)alnlaloLuor)PW 1 9 61 ' x a l d u r o ru o r l e r ] r u r a i d JSZ6'IEZ6'(l9Cd) rot)pJqlmor6 panr-rap-}a1are16 'arrolradat(poqrlue aunuutrat6 699g 1 1 8 ' 9 8 ' u o r t e z u e u l l o d1 r e 1 'l ' ( s ) a p eu q r r e s o 6 r ;,or o s r n r a r 6 JLbL'lLtL tl EL E 'JL 8 ' 9 8 ' u o r l e z u a u . r , ( ; o d p e a q 'lla) lzsS'urue^lolseld €ss t. 8-ard ',stsPloolnal '96n1'sasnrAXod Lt8 r96V| 'urso^L! 'alorls raMod g s g - 1 9 g , u o r s r npr p ueqyno.r6 uorlezuatU^lod ur]lV aasur]le 9 L0L Jo gsg-s99,sauoua6 suorl)eeluorlPzlraur^lod ' ( s l a u u e q ) l s z t 'sallanul luneJql s) ' u t euLrunot sps-entoodd sautLzua tryoadsluorlelrldarVNC.osloaas 599 669'uollnlo^a )eal+)) leal (s)lauueq) tt8'slsPloorla t t s ' l r l r q o o u . u e q l '^lrsJa^rp pale6-a6ellonaaspale6-a6ello^ u.tnrsselod (s)aspril.!^lodVNUaasVNU I Lt8 I lr 1n r 1 > a 1 a s u o r l e r r ; d aVr N Ci ( s ) a s p r a u ^ l oVdN Ca a sV N C ft8' 1669'869'luauldola^ap 1E79'ra11 'slauueq) (s)tseldorolqfaasslseldorolq) sasPraurAlod 699 )Pal ZLg'6uw6 lLnS' Lb?'slseldol^urP JLrS'r9VS /aluel)npuo) ' tl9 zng- ang'969'(s)pllseld J9n9'gng-Vng'(Ufd) uoll)ear ureq) aseraL!^lod '(s)prouardosr^lod euelewos/Daas I t l g " s l a u u e q )a p u o l q l l 9 LL '€.19',aJnl)nrls ',leualleq 'slrPrl )rua6^lod L19 z o s t - 1 0 9 t ' s 6 v l n 6 v L ' r o l ) a ^p u e u o r s s r L u s u e J l JZL9 €99 l 9 6 t l ' u o r t d u ) s u e rl u e p u a d a p - a u r l 0 a u u P q )+ ) ) ( s ) l a u u e q|l' U n t s s p l o d ) s n E L ' v r El ' d n o r 6 q u o l ( 1 o 6 suotlDtltpow )ur)adsoslDaas ( s ) ur l n q o l 6 o u n L uI u 6 9 8 ' V N Cl P u p u o q l o l i l . u luJals^s/osuodsaJ aunururlos/paas e o g l ' J S 6 V t ' a l ) ^ ra j r l l88E'IUale^oruou '18 'uorleln6al uorl)un, l_98t t-981 6 L g t ' u o r l e u e)^r u a o r l u e s ' 9 t ' a s u o d s a jl e u o l l ^ l o d /urisela 56, ln6n I' w ruodo1o1w ntpowso14 0 6 LI 0 8 € ' v N U u lrr u o r l s r ) ^ l o d uorlelrtoqdsoqd (pua,t VNUUr)uorte;(uape,{1o6 aasslrelV-^lod J t g LI ' E 9L J - 2 9 1 1 ' 8 St ! ' e l e L u s a p o u r s e l d uralordiuotlel^sor,{;6uralot6osloaas y-{1o6 ua60utLUseld t6 L t'srole^ll)e 8 S € ! 1 5 € ' a s e r a u { ; o d ',uaooururseld t6 L| 188€'luale^o) JZLT. 'ururseld ' t6 [ t l18tt'9811'sua6ello) I L F . - O L F . ' u o r g e l na bu a r6 l e u o r l d u l s u e r l l s o d l 8 8 t ' u o r l e ) r l r p o ulrP u o r l P l s u e l l - l s o d tz6bI 8 S E! l S € ' a s e r e u ] ^ l o dv - ^ l o d 'Jt6n 'z6bt' J J L 7 S' ' '(s)prurseld uorssaioxa | L6nL LVS-OVS IZ lls€'uraiord 6urpurq-y-(1od a u a bJ o l o r l u o ) l p u o r l e l s u eaJal sl e u o t t e l s u e l ] (s)aueJquawos/Daas 93E vNUurpaierpau-asuasuou 'S '16lt 'lorluor "ierap uodsuerl VNU 98t-S8t tt Ol-6,^UlpstaAtun o s o d s u e lj e l JrAoJlaiuou f 8 t € ' s u 'pua,€ '?SE-LiE 6ur>r1d5 osloaas 6€0 1-Z€O1 JLSq VNUre6uassar! 'uorlezuau^lod palerpaur-uor1e;{uapeap ull)e ^q ue^up uorsnrtojd S g t - L L n ' Sl V ' J 6 L t ' ; o r l u o r6 u r s s a : o . yr dp g )Z6V'Z6V'Ae:epVNUr.u ,sue:,{16oa1ord ':rr.use;doy{: s l , 1 6L E ' , l o . l t u ou)o t l e z t l e ) ovlN U tg t t-€g L1. 959 'gSE, ,utalord J ZL E ' LL : - 1 L E ' u o r l e 1 { u a p e { |yopdg L u Lgt lSoblLov' gon-sot',urbuo ILqE lls) '8S€-19€'ura1o.rd ( s ) r e ^ P l rpqr d r la a ss p r d r ; tgV-ZBr'01€'sauau 6r;nqo;6ounr.uur ll9€ 9563 's08 '(srsoMroxa) O A n6- L n , 6 u o r; d sa , r r l e u t a l ; e l9o8 JLS|'qst-LSt'9t€'(pua,t vNUu) uorlelluape^lod '6ututels {emqled rtotar>aspelpln6ar'luatua6Jelua !lLn' 66n-tLV' uotteln6allpuorldr.r>suel-lsod JBts'Lls 'suorllunIlq6p]'suretuop 'JgtS 'l 't9Vd-SCS :r1deu,{sgso3 1689'(dSd)1er1ua1od t S LL Lt9 rI '675 'a;nuan,(re;1rder-1so6 ' ( e s s eg r q s( 1 l r q o t r - ; a 6 jgzv'LZb tuetUPluurl)e I t ss L !6t5 t I 866-966'uorteal)nu 9 7 y ' s l a 66 u r ) u e n b a s S n t 'l V b F . ' 6 urro1: r a d n sa n r l r s o 6 lsts'vEs J 0 L 0 L ' , O 1 0 t - 6 O O'6 l . ' '(s)auelqulaul , l l e l ul uoe) t u P l luJ r l ) e vNc euseld 1[g l 1 8 Sr zzs-Lzs w'twd-Qz '9BS 'snu^ql ur 'uorlralas '(l9Vd) srsaroqdorlralai naprurel^r)e^lod anrlisod 3989L t-98S I L LS ) Z eL , t aL , ( s )u a 6 0|e r u s edl la6 ' a p r L u P l ^ r ) e ^ l o d (s)lla)I os/Daas l6st'6st'sltn)I) uotlotJ)sueil I L9 Z S SL 1 1 5 9 ' u r a l o r d6 u r p u r q - y - , { 1 o 6 ' ' 6 ufli1LdVn' loL>V ' s l l l a u a 6 l d a ' L V S L ' J b b S l ' € t S' ( s l l a )g r o l ) a J J as)l l a l e u r s P l d ) Z L t Ll l L lEuotleuJoJuo) l ? B E ' t B € ' 6 u r 4 r q s a r . ulee.ur o ; r l e l s u e J l ' s u r a t looJdd 'pue eleursapourseld 'sasnr^ ' s a s e u lal l l l - o l o d 99VlgSV'g9n'tuouiau lla) lueld 7 1 0L €9 t t ',uailod St t€ t'r(rlatuut{se;;a: ) 2 8L ' r L 8L ' Z 8 L - L 8L ' s a l o n r e ^l u e l d 692t 3269 't06€ t-t 'lo.rluo) /a6e^eal) 106 z L 9L 8 0 t t - 1 , 0 t 1 ' s a l n p o ul u e l d l)eqpaaj a^tltsod cllll ' J 'arn])nl]s J L Z Z t z e ' ( A l d ) u o r t e 6 a u el^) a ] J au o l l ! s o d l68t [ ' € O t l ' l S G ' ( s J o l e l n 6)90n1'90nL aqry n o r 6 )s a u o u r o q uotlrsod lupld Jlee' lzz'st)aJJa I [9 t JJo-]nqsuol]du)suel] lla) ]soq ' 6 u r ] e o ) u np u e ^ r ] u a a r n s s a r ldo 6 r n l F.61 € -. e 6 E t ' E1 €l - Z r g I ' a n l e ^ 6 6 1 L ' L 6 L L ' p u e l 0 S [ ! 9 0 S t e 's)slp , u o t ] e L u J o ]l u lu o l l r s o d 'prsoe) 'tot ' , s 6 u r l p a raosJs l e u o r s I S €t Lur €ot t I 3 6 6 91 leuorltsod l6' [ 'ssaullr l e u l 6 e'sllottbut6 /uorsrnrp ( q g g t l )tucaq) sououLotAtldto4 p a l u a u o p u e b u rdeqs 00St t9t'tL5 t-t LSt J S ! 9 6 ? [ ' s n r r ^ o r l o d ' s d n o t ba u r a q' s 6 u r .urr . r ( q d t o 6 ' s l l a )pl lrao)l d e q' u o r l r n p o r d e r 'uorleuDle^^q uorle)rpeta'srltla^uorlod tt8 l0€8 0 1 2L l86t L ;enxas . s € 0 . x n l jp r e M a l o d J L LL ' L t L ' l L 0L ' L 0L ' 1 9 9 ' S Elgt t t ' ( s ) u u o d L € O t l ' s u r a l s r . r a ^uq ' l e r l u e n b e s l5t0 t 'sluetnu (xu^leqd j g O t L ' 6 u r u i a l l e da A r l r l a d a r SZ€L Jo /{luald)dod € 9 - Z E ' s a l n ) a l o ur e l o d ' u t a l o i d{ l o 1 e 1 n 6 earu a 6 L d o d S Z €L I ' w O l ' g g g , 6 u r p p n qr s e a l :l[[?[ JS7O '(s)ua6rlue 'ZWl-OJnl 'azreu ur suorlelnu {ro1e1n6al 'sanssrl 1ue;en(;o6 lss L s 9t t ,lr0 ,(s)ila) 96l'uorlel^irnbrqn^lod r Son1-LOnt €1Sr I 'erplouruduor; qlMor6 alnpour sJJnoauiosoulotq) oslDaas tueld VIOl'aas!^ilD sa26wuoqDDS 'UOrlezrlensrA 'z0z 'pue salnqnlot)tLu 9€Z l-002 I l9t0 t 3sto L',(s)lrqdorlneu I toz t ,srsaqlu^s (s)uralsuawaassuels!laul l6EZ VNU Z g Or - t B OI ' a I O J - L L O L ' a ; p u r d: rs1 o 1 r u - r ernlfnrls aurosor!orq) otlDaas rl t 9 L 0 l ' 9 L O t S L O L ' a r o q ) o r a u J90Vl 'gDn I ' E O il ' 2 5 5 ' o 6 u e r - 6 u o l ' s l e u 6 lresu o u r o q 908-SO8'lojluo) stsol,(loxe 't '1969'(do:so.r>rr.u 6urpurq-rolPlnsul 'J€W 'nltL-ELna 'luaudola^ap raMoU l€St tt LVL | L Jn ) L S I 'AS l g € z ' s u o t l e ) u t p o ra. uu o l s t q LL -L Sa a ' s u r s r u e q ) a A u luelod reueld € 0 t L ' s a ) u a n B ul er l u a u l u o l t ^ u e . s l l al l )a l uab Z Z a - \ Z Z ' u o r s s a r da xa J908'J661,86/ f Z O nL ' ! t O Vt ' g 6 y 1 ' s r s a u a 6 o { l q t u e letqla!oa l 0 6 S ' ^ d o l s o l ) t uu t ollnlo^uo)ap I 9 S L L3 S S L L 0 0 ? t ' u o r s r ^ r)pu l a u r u ^ s e ' ( a 1 o 6 I z616 a 'l,S 'sursrueq)aLu ^tuelod leseq-olrde IIPM JtLtz'8€.2 gtz I l-SS t t ' /uoilPr.uroj o6ouo1auLo1tqdosot6 erlaqlroa LEZ-qE('196'gg t a1s lla) lueld Japunaas196t t llpMllal (aZ- 0ZZ'sJJnd aurosor.!orql o 1 t 1 - 5 0 t 1 ' 6 u r 1 e u 61r1s a : l € ' s p u e r l sv N c aual^lod (ssarr llpM)ouolpqt slsdoplqorvaasssdoprqorv 8EZ-9€Z'(s)auJosoutorq) luawoola^apoltqaosuo )apun oslDaas 'leluaLudola^ep g6E1 Xturads{1o6 '9t t tott ,uorlsaOrp '9LnL-a6EL'qlmor6 pueJyuaudo;anap 9 9t _ ? 9L 3 8 8 9 n oL ' v r o L ' sL 6 - vt 6 ' 6 9 6 ' p u P u o l a ; a 1 s o y { > lue;6 '€91L-Z9t lzg'saln)alouJot)Ptu Zt0L'uorlezuelod/^luelod l€9t t , 9 8, s r s a q l u l^l sa o) ! q ' 9 6 [ I ' e l e L u saal rpsouuas]e l d 'S80 'alroj lSg 66Lt'L6Ja L'ql6uar]s 1980t ' 0 s 'Ls p u o q uottlafa relod 'a)ue]srsaluorsse.rdLuo) It t t'9S'sapueqrres^lod l u a l e ^ o rr e l o d I tg 961[

, LIL'r.oLL'.0L i Jilt"ji$i :

,"'t','fi5oot;; 5'";"nl;

LEII

X]CNI

z g s - 6 1 9 ' u o r l P ) u l l u a pull a l o r d (Ul) unln)ttat )tutspldopul oslDaas ; 6r a l o r 6 f L nL ' ! L t L ' g e L - g eI 3 g gg ' u o 1 1 e ; { s o r I u ',Otqsuotlplal uotl)unj-alnl)nrls 9St-?S I suotlfelalul p u e 6 r l - u r a l o Jada s6 u r p u r qp u e 6 t l - u t a l o l d uor1e1,(toqdsoqd uraloidaasstlaJJauotlel^Joqdsoqd (s)aLUAzul aassaulzua 96L-ZSl'uoll)unj utatord 'srsaqlu{sol uottelar lgg seuoladeqJos/Daas I L 6 € ' 1 6 €I t € L ' t € r ' 6 u r p 1 0 ; a r '16€-05€ 8 9 L - 1 9 L! t 6 E lorruo)^]llenb (drd)sutelold uoud osp aas 6 6 Vl - g 6 n l ' s u t e l o l du o u d 3ggg'slemqred l o l L ' L ZL - 9 2L t t 9 ' t 9 ' s u o t l l e r a l u I l u a l e ^ o t u o u J L nL ' ! L t L ' a 1 our o r 1 e 1 { s o > (p1a61 u r l - 1 r '6uuolruor"u 6t L-|tL 1 8 8 € ' 8 8 € - 1 8 € ' e ; n q o u1 a6 l l o u r lOVL' Ln L-6€ L'suratordpaplo1srru J6EL' 6EL'a1olasela;sue.r} ;,(so:n;6 1 6 € l ' , a l o lrg d u l 9€Z,U] 1€t-o€l's:r1a6raua l 8 8 E ' g g € - 2 8 €l e u o r l e l s u e r ] - o ) 'slulPJlsuo) 9Z l. sauotaoeql os/Daas 'J6€L'6€.t-8EL 'O5g-88€',seuoradpq) L9L J06€ 6 El - g t L ' u t x a u l e )p u e u t l n l t l a t l e ) 'uotleat) alrs 6urpurq lts | '0€ 'aloi pt)e ouil.le ureql-apts t lOt t sts^loeloldi(drd)suralojduoud osp aas 't6E'leLurouqP 86t-L6t I t6€ 'O6E-L8€. '92 '6urplojura]ord 66nL-86Vt [ t^^,,,, -^A/, -,,,-^)saQN :satlrueJ )uDaos aueg os/Daas '6€ 'aluelquasel lg€ t L-8tt a)uanoassAaluelqulasal nllS '6€ ' 8 € [ 6 o l o u o q alelnlf )uanbas 1 6 €t t !g€ L t '9S ' 6 u r ) e r ^l l e u o t l n l o ^ a t - S St J S St 6 E L -L t t ' s a r l r u e Ju r a l o r d 'gtS '9tS 'sJol)a^ uotssaidxau!elold llB?S 9 9 7 ' a u r 1i ( . r e u o r l n l o l^tau n 9 5 [ - S S t ' 6 € L] g € L ' g g 1[ 6 o l o u o q a ) u o n b a s satlil.!e,euaDos/paas 9 5 t - S 9t ' 6 El - L t t ' s a r l r u r eu;r a l o r d bgz'sa|u uotlelnt! ^ p u r e fa d n su r ; n q o ; 6 o u n u u o q s l oa a s 'satnpour L q Z 'f t l - O r t gnt'LSe '6urrr;dspue suo.rlur,(q palelrlt)e; ' '!Ob ' '6urg;nqs ureLrLop fLSe LS( L LvL-On| l 0 g s , u o t s n Ju t e u o p S 9 Z ' n 9 Z ' l €L ' s e 6 u e qp) r ) eo u r u e zn v9€.1'uorlnlo^a uralord surato.rd 6urpurq-yy6 :s;r1ou.r 6urpurq-yNCoslDaas 8€t'l€t-9E€ 'pue sa6upqr 'saselau.ri(1od ylg lernl)n.rls z g z ' u o t l e t l t uut o l l e ) ! l d a j 'uraloro l/0€ v)eu ll0€'uralord tspeu Etz-zLe,sauotstq J6Ln'6W-8tV jL lV' L ft-g Lr'suralo.rdrfuole;n6alaua6 'awosa)uerqa tLbb'Lbv alnl)nlls vNc oslpaas 's]lajJearnl)nJls 1 [t-9 lt vNC Lar' 9Zl'suo1'l)elelur uoui r.lrof 9 Z ' ' u o t l r t p a l da l r s6 u l p u l q l t t t ' s u o r l l e r a l u v! N c - u l a l o l d 9 € l ' ( l c d ) a s e l e u l o sarp u l n s r pu r a l o r d '6€ 'saseqelepuretord l6t t I suorllerelu! uralord-urelord:saurq)pL!uraloldos/Daas 'EVL-ZVl ' 6 u r p u r qu l a t o l d - o l - u l a l o r d )wl ! tt L )JLNN'1" 'saxa;dLuor suralord,{to1eln6al-aue6 :r1ofte:ne uorleln6ar)rJatsollvos/Daas ) € L I ' t L L - ZL L ' ( l r n r l e r e d o o : 'sasnr^ 6VL'rgnl'8t L Lgt-6tl ^lquasse-llas 6t['saurosoqu /sl)aj]auorle;ritoqdsoqd S1t tztL'zvl eewtp IsL'sro])eJ^lquessP ,(1qr,uasse ZSa-gnl $ b l ' € nl a v L ' ( s e r l q u a s s se a) x e l d u r oul r a l o l d 'll 'uorldrrrsuerl'vNU6ulpo)-ulalord l-ott lod ylg uotleluuno utalold

luollplrlluapr uralordl^do)sor)lWosp ees J S Z S6' Z S -I Z S ' 6 € [ ' u o r ] ) e r # r pA e r - X

t't"i;;:"orot$fl: 6Er 'o)ueuoser '6€L'serpnts 0€9-629 uwN

z E g - L J S ' , 6 t [ ' u o r ] e u r u r a ] aapr n l ) n i l s 6ur:uanbasy56 osyoaas ' ' ] u a L u u 6 l e l u a nb a s I t € s L€ s lP tzs-6ls ^ilaurorllads sseur / u o r l e l u r l u a part r s6 u r p u r q - p u e b r l S SL '6€ 'saq)reas(6o1otuoq l6€ t t 6 ur : u a n b a s 6ls's)ruroalord ISZE'929-SZS 'JVZE' I urelord-utalold 7Zi-ECS'suOrDeialu fo6' lzs-6ls'i(rlaurorlrads sseu-t )rueu^porp^q EZS-ZZS'slueureJnseew 1 6t 9 ' 8 t 9 ' 6 u ; l 1 o 1uql a 1 s a t r i 1 l z z s ' 6 u t s n l o Jl l l t l e l a o s l J J Z Z S ' J L Z S ' Z Z LSZ- S ' Z Z Sl-Z 9 ' t 9 V d - C Z 'srsaroqdoll)ale ll8 ts +6E ' 9 6 5' 6 u r 6 6 e } 339 ^q uollezrlensr^'s>tLueu{p I S t 5 t t LS 'J EL S ' nL g - Z L S ' { q d e r 6 o l e u o r q : urelord srsAleue saseas!p uoud os/Daas 371y'srrlaua6tda'ate1s 168'a)uelstsar)tl^loelold - 6 t ' e s e o s rupe t x n q r L 6 E ' 8 6 EL 1 6 9 f t t 1 ; q e t a u ; n5n1 3 uorle6ar66eulalold lrodsuer]utalordaasuotle)olsueil srsaqlu(suralordaassrseqlu^s suorlfelaluI isexalduo) uralordotlDaat uralord-ura1o.r6 ,l s nL 1 * I ' I n l ' * r z n L I t [ ' s r r u n q n s aJnlfnrlsuralordaasarnllnrls ,Lt t,^lrlrqeis llnL 6uruosuralo.tdaas6ut].tos ainl)nrls uralordosl, aas ',ezrslaoeqs L€ L srs{;eueuralor6.rapunaas6ut:uanbas ' t 6 € - 0 6 € ' l o l r u o ]^ ] ! l e n b I L6€ suollfelaluI uralord-uralor6aasuott:elalut utalold uorle;{roqdsoqdulalordaasuo!}el^roqdsoqd L9 | -99 t'slUauoduro)uralojouou ' r 8 1 - 5 1 , t ' ( s a 6 u e qa) d e q s s) l u a u a ^ o u l I t8 L (s)utalo.ld rolow aaslolot! sureuop'arni)ni]suralordos/, aas L g L ' SL L ' u o t e l A t o q d s o q d ) o b I ' L nr o n L ' J 9 Et ' 9 t l ' s e l n p o u r 'slrunqnsJeln)alouror)eul It t saurq)eu uralordaassaurq)eulse uorle)urluaprulalordaasuorlelr,rluapl uorge;(so:{16 ulalo.rdaasuotlel,iso:,i16 '€t 'relnqolb t st t 6S'€65-Z65 ' s 1 ; a6r u r n ru; r u o rJl V e z r l e n sr!oAJ6 u r 6 6 e 6 11 9 (s)ureloro (roge;n6ar Iq uolleln6aJ aua6 auag aas uoll)unJ ulalo.ldaassuoll)un] aas6utp;o; 6urp;o;uta1o.r6 sad,Qtqttads osloaas 9tt-st['snorqu sarlrtueJ uralordaassalllueJ uorlnlo^aulaloid aasuorlnlo^a suo!l)erelu!vNc-uralord aasuorl)EralulvNc 1 L S ' 0 €L ' u o l l e l n l e u a p aasuorgepe.r6aP srs(;oa1or6 '979's1e1sft: lgJg saEueu: aassa6ueqrleubrleu.ro;uor leuorlpr!.roluoJ 'uolle)ulssPl) 6 Et - 8 € L 9 7 5 ' s r s a q l u rl{Ps) l u l a q ) (s)euj^zuloslDaat '9-S 'slsi(lelerse l9 (sarlqurasse) saxaldLUo) uralord:alrsa^rDVoslDaas 'ztl '(s)a}|s6urpurq t9l"-€.9|,, g ' s a ; d r > u urdt s e q (sarlquasse) saxaldLuo)u!alordaassatlqulasse srs(;eueuralor6aasspoqlaurstsr{1eue uorteln6ar)ualsollVos/Daas ' ')uelsol tLl-zLl ILL lP uorte6ar66euraloldaasuotleOal66P ' s l l a )u ! e ) u e p u n q e lz9 n6J-gZL'(s)uralord ^eMqiedurlrnbtql osloaas t z 6 t ' E 6 t - z 6 E ' enr l ) n r l s

LgL' E6e- J6€'lo.rluorllrlenb uralo.rd )Z6E'(6E'srsIloalordyo ifrnrssaro.rd 36y2'uorleper6apuralo.rdpaplo1srur l € 8 9 t ' z B s [ ' u o r ] e ] u a s a . r d l 6 u t s s a ruoar 6 dtlue L6€,'(s)auosealord JZ6E'r6e- 'srolrqrqur gg 11 'uor1e1n6ar asealord (s)aurosealord o5/paaJ J V 6 L L ' n 6!l' € 8 [ [ ' ( s ) a s e a ] o r d t9t['uratordoradsol6

'i:;'t" j,!3,,',nt?rif "rp",d,

'610 'tso 'srso]rtu L-810t tzlo [ t 080t ! 9 L ZL '9L a L - SL Z t ' VL Z L ' 2 6 0t - 1 6 0[ ' s r s o r a u - r eseqoord 'lslaprrdad-or6 969 )!llloatoJd'uruo)ouelaLUordoord 3ggg'6ursse:o.rd redar qrleursrlAl osyaaas y16 r(q 6urpear;oor6 IJOLZ'OLZ-6qZ'ase.raru,(;od l Z O EL t t 0 €t ' [ 0 8 t ' u o r s n r] a l ) n u o j d '9S'alqDnpur'srolou.roJd JOSV'Osn-6rn' uorter6alu! leuotldr.t:suerl 6 t € ' s a ) u a n b a ss n s u a s u o f LLV-OLt' rEV'spuelst1qd11_91 ott')rlo^re)na ,6€€_8€E'LEE-9Et,llt .leual)eq l8€€ 'ftlaururlse t6EE'6tt ',9€€',slua|.Uala retourord 6EE-g€€ 'uorleurquJoler snonlsru.rord 9 t t-s L€ 9 8 0t ' 0 8 0 t j z 1 0 [ ' a s e q d e ] a u r o r d l L g L L ' L g LL t 6 Z [ ' a u r l o . r d 'le])alourlar'uorl)aford [6€ t iZ9L',t9t'urlnsurord (qleep lla) paLULUer60rd) stsoldodvaasqleop lla) pauruler6o16 (s)lla)urols os/Daas g;n L g s n L ' 1 9 9 91 ' s 1 | ar:o r r u a 6 o 1 6 1 0 0 0t 1 6 6 6 ' 0 0 0t - 6 6 6 ' u r l u o r d L a i l ' g gt 1 ' u a 6 e ; ; o > o . r 6 JS6r'V6V'$atpoq-d)sarpoq6urssaror6 bLsl ')tLSL' L Z n ! 0 Z LL! 6 t t t ' g g I g ' l s l a s e d s e : o r 6 eaeqrV oslDaas euelleg :(eual)eqaeq)Je) 'uorletlruruollelsue{ lz5 ! LL( lteoaJ qrleuisrurpalrarp-puPrls 08€ !St€'srsaqlui(suralord

9€n-v*

"snsatoi(re:na'lolluol leuorldu)sueJl uolloulsuerl-rapunaasuorlorJ)sueJ] (s)uralordrossardeU aassrossardar '08€ 88r t [8t S9rE',VNUru 'uorssardxe aua6 397g uorle;(qlau VNCaasuorlel^ql9u VttC 'Alrsra^rp s! ls[ 3t[',st.-?t',slla) 'salo^Je)ord t I arnl)nils 66nL-B6Vl'uo!Ie6ar66epup 'uor]e)rldar 86t [ 'salorrelnlla)aAr]rsod 86€ 86t-L6t illd lelnlla) leurou ! 6 6 V 1 ' g 6 V l ' o la n p u o r l e r a u a 6 a;pe r n e u 86€.-L6€'uo!le6ar66e/6u!ploJs!ur g6t_L6E,Alr^rDalul 86€',rOunJ B6t' J L6t'e6ueq: ;euotleuro;uo: 66nFa6tJ'68t t'(drd) suralorduoud r L6t' 868- l6t'saseaslpuor.rd I E S Z 'Z 8 Z ' 9 l Z ' a u o s o t ! r l d €8ZL-ZaZL 'sleurueurur s;1ar6uuoqq6rau,(q uotle>t;trads JZSZL 'z8z 'slu?ur.uretep tura6 (q uotle:gt:ads l. 1;ar gg71 '(:ualodun;d 88eL'SqZL ' g(. olut uotlet6tur Jt t' €g1a'peuo6 6urdo;anap ggzl-Zgzi '(s)9d) slle) uta6 letprotuu6 l l o Jl e r p r o u i l r d ) L 6 eL ' L 6 ZL ' , s l l aa) l l r'puens 'srsaqlu{syp6 raLu!.rd lggz'lLgZ L g vt - 9 8 n L ' s u a 6 0 q l P^dr e u u d '882 'sa]^:oo,{teur1.t6 [ 168Zl. ueoJo proqdu,{1leJ}ua)aasueOroproqdu^l Ireuud eunuuJl os/, aas anrldepe'ualsi(s/asuodser gns l'gns l'asuodsaraunurut &eut-t6 'sainlln) ^lPulud t0g ',Urnrlr) A.rPurrd 668 1 8 6 tt ' 8 5 1 f - l 6 l l ' 5 6 t t ' s l l e M; ; a r( r e u r t r 6 eunuu.rI os/Daas anrtdepe'uralsi(s/asuodser 'esuodsat{poqtlue ,fueutt6 19791 ggS'^do)sor)rru I'selpoqllueAleulud a)uolselonuounu.Iul

X:lCNl

8€:l

1(;anr1n>asuor airoloj ol ll upqlrM'arn6r; eo1.ra;er reqlsarn6u I ueqrr,*srequrn, ,;;H;;ffi;;;;ilil; "6rd 0gg'alol'uotlnlo^a lpupuoq)oltur Itl-969'seuerqureur ssor)e e u a l > e qa ; d l n 6 uoosuPrluralojd 'tB€ 'ur)^urornd lSSE s LL-n LL'srole)olsuerluralord 'urnr)le)lelnlla)eilut ';;a: afur1.rn6 (ul) urnln)rlar 1165 eutuPngjautuepvosloaas :turse;dopu3aas[rJnln]r]ar)ruispldopuaul '962'uottpuundap yodu.rruragord oslDaas )L6Z leupuoq)olrW ' 1 9 ' ( s ) a s eaqu u n d l9 [ t 8LL-t a I'saue.lqueurleupuoq)olrurssor)P oslDaas 4 t Z ' u o l l l e l a s6 u r , i ; r r n 6 Uodur uraloJd'(s)lseldorolqf 6ZEI'Dllrldosor]'ednd ozl-6L L'sauuquleul lseldotolq)ssoJ)e (s)utaloldlaure; aassdun6 l€01'uorlerolsuerlurato.rd 6ur66et adolrd3:,(qdel6oleuorq)^ltuUJVoslDaas ',bPlaurprlsrq J a 0 9 '2 0 9 ' s l u a u u a d x e s e q l - a s l n d 8'S ) tS('! t€.'OE'(saduqiln6ry) qsrr.raJJnd l9r9'9r9-rrs'1s9 '26(.t'682 ',^l.raqnd 'alqe^eal) E6Zl t - 8 8 2t s t9 (asedlv)dLUnd lS I S'^qder60letuorq)-IlruUJe unrsselod-untpos ld|.Undunt)leJ oslpaat sDeluralojd '6S9'(sasedlv) sdLUnd ad^l-d uorlelsuerl:6urplojurelordos/Daas 1099 '(rossa.lddns ' , 1 8 €' u O r l e u r u J r a l lounl; aseleqdsoqdNlld €€6 J l8€ vNU la6uassawjapo) ' ( u r e L u o6pu r p u r q - a926'J668'868 ursot(loqdsoq ud t e) u r o pg t d )rlaua9 :uorsserdxa auag aasvNc ol uotlelal 0 6 9 ' 9 8 9 ' s b n r pa , r r t : e o q r ( s d 1 8 9 € ' g 9 t ' s a u e . r6; u r p e a . r 9 L t ' J 9L t ' ( 6 u r p u r qa u o s o q u )a l t s - d 0 8 €! 9 t € ' : r l o ( r e r o r d uorle)urpourleuorlelsueJl 1 8 9 € ' u o t l e ) t r t p o vuN l Ui a j s u e l l/ a u t p u n o p n a s d ',erpooopnasd -lsodaassuorlejatleleuotlplsuptl-lsod 1€0L ' 6 9 ' u o r l e L u j oplu o q e p t t d e d L6Vl'Dsow6nDDsDuouopnasd J€lE'SZtt09 (s)auosoqrUaasuorte)ol l€0t'arnllnlls vNU,loulopnasd LSZ'SSZ'977'saua6opnas6 6 6 9 ' u o r 1 e ; n 6;aor s ; e n a 1 'url)alas-d 9VI t-SVIL 108€'08€-61€'uorler]rul sltlotqtluv oslDaas 'a6pa 8t'['ura]oJd Ixotd ' ' 8 8'srolrqrqul 6 u r p e a l 3 6 9 61 fs8€ 'suratslsaar;-;1a:'srs{1eue le lJoMqsaururlle,|o 'luauJa^oullla) ut ,uotsnllold Zt S lelueuluadxa gon- Lob'uottnlo^a t t s t - 8 O SI ' S 6 n l - n 6 i L ' s a u s e r esde €€-zt 382'eozolord l 6 6 t ! 0 8 €'08€ts'€')rlo^rerna ( u l ) u n l n r t r a r) r u s e l d o p u lJ a p u na a sp u e u l f L E ZL 'L E Zl ' 19 71 , s a u a 6 o : u o - o 1 o . r 6 saxaldLuo)au{zua fuo}etrdsaE osp aas J9Lt,9Lt_SLE,sda$ 'S€8'sletlualodxopaj l5E8 f 6 L E ' 6 L t ' 9 l t ' e s p r e j s u p rl l p r l d a d 't ,srsaqlu{sotoqd tsg Sg ) L L E '8 L € , -t L E ' 0 8 l - 6L l ' s r o l ) e Ju o r l e 6 u o l a JjgL'0gL,sauososr{; J L L q ' l 9L E ' J E L 1€' L t ' e ; > X :u o r l e 6 u o ; a 'S8'srseqlu^suralold 3 t E g , u . r s t u e q t al ue r a u a 6 00t-99€ !98 'orqsuorlelar uorl)unJ-aJnl)nllsulaloJd 118'uortnlo^a ss t-ts t 6urp;o;uralor6lspuoqeprlda6oslaaas Zng-Ong'utsdopoqloual)eq 'lrelep)il.uolP s€8 LVL-9Vl'sureql aprldadl(;odpa.rnpn.rgsun 'arnDnJls^rellrel 9€8 ftersolle 9€ | 'Ev 'rtv 'J\n 'strunqns J 6 7 . 9g' Z g -L a S , s d t nr d u o r o . r 6 )tbl l-zv t | L '.Orqsuorlelar ts8-t98'J Lag' lzg'a)Jo, a^ttoul-uolotd uorllunJ-eJnt)nrls 5St-'S t sguarpel6 rLv l'8v rLn L'lt r ^rltlqels uolold lelruaqloll)all aassluatpet6uolo.rd ) € . z n ' z z n ' J s E t ' s t € I t t t ' 1 € t ' s l a a q sd (uolold'+H) uor ua6otp(;1aar uolold l s E t ' s € t ! t € L ' ! € 1 ' x r l a qn 'aJnllnJls^lePuo)as t z L 6 ' zL 6 _t , l u e u r P l u o l o l d 9€ |, .9€t L 6.suUaqpe)olojd 'alnl)nils ,{teutalenb l_8€t t t 9€ L 't€-z€ 'stsrlord 'uort:rpatd l€€ t sg-ogs gg ! 'slr!uoalotd apol )rlaua9 oslDaas (eaaqledullrnbrql :(s)aulosealord os/paas J O O ( ' ! 6 6 1 ' 6 6 7 - 616' 6 u r p o > 'arn1:nr1s 9 6 € - S 6 E , u o r l ) u n^,r o l e l n O a r fteuud 99 1 pp ale;n6at 196€'95€-56€',uorl)nrlsa S Zt ' a u o q l ) e q a p r l d e d { | o d 'aluelstsatale6at66euralotd ,6s ,snutuljat_N l6E t lgz lzs[.tsL,urlnsul LZL-gZL'setroJluale^oluou g gg ' s u o r 6 a6. ru r u u e d a s uelqueu 1 9 6 € ' s l e u 6 tuso r g e p e l 6 a p tgol'lorluo) allr(l 1|a: LEl-g€t'sraqunu ploj pell|.U!l 'sseupapuPq (s)esedsef €r t aassasedser'(qlpap lla) pauLuer6ord)stsoldode 16 19'{dotsotrru.uoil)ala ',AlqurassP !6€'srs^loalold JSt [ sadfl t\oads oslo aas slueuelu url)P/url)v os/Daas 'urt]e I t S LL ' Z 8 t - t 8 t l9t [ ' ^ q u o t l p l n 6 aulLt 'a,lsoal dz r s €gt t-Zgl t ) s n t ' s n L ' f i n L ' E nl ' s l u a u e l u l e ) r l a q 'sa^arsreln)aloulse ,uteurop €g Lt-zg t L €HS l9€ t 9€9,sutalojdeuplqulau.r 9 5 [ , S 9 t l 9 t t ' J r t t t _ z 9 t ' t € t ' u r e u o pz H S ',61 ',sansslt a^tl)auuo] z8 I 1- l8 I l, I[ ttt'paJled 'sJolda)at-o)a)e;rns tgl t-€gl I lla) t o b I ' L v L o v t , s a tnpoul rureuiopaseurla6le; s 9 LL ' P u r u i e l l9t t 'lB ' s t s a q l u ^ s / ^ llqeusaPsqs e 'uorsn, I t8 t t t l'9LL-SLL ,sase6ar66e ' 6 u r ) e l ]^1099 l € 8 t t ! Z 8 t 1 ' 7 9 11 teuortnlo^a J L S | ' f S S l ' 9 S t 9 S t '[€ 'slepou l l € t j l t t r - z Et [ ' 9 E L ' L t L ' s ucr-e€u r o p 8rt l g Z S '6 Z 9 -L Z S ' 6 t t ' u o r l ) e r J J r^pe r - X

Ji:"dii"T;?ll: tEs'rr,",oqao!,liJ

j',1i,:]fff3]:j: 6€6-8€G'(sd-Ld),",.,.ud33,11

(s)aseutlaursorll aassaseurlaursoti(1 uraiot6 uodsuell Jeln)tsan:1s1ura1otd j6uruosutatotdos/oaas auerqLUaulsuetf q6no.rqt LAL-6nL'(ennq1ed,{lo1at:as l€01'sarpnls J B Z L ' J L e LI Z ' L - 9 ( L ' s r s e q l o d ^lqe u 6 r s Uodur uralord'(s)aulostxolad aasseuJostxolad l0Z-6,6,9'sal laue6rouaamlaq tlodsuPrtrtLuspldoil)-Jeal)nNaassnaltnu uooul| utelold letjpuoq)oltwaaseupuoq)olil.ll 'mo;; 31.91 reua6 '991l e'pa1e6 3 162 Uodsue.llutalold ,(Ul) unln)ltar )ursploopul aas[rJnln)t]al)tLuseldopua UodLururalold'1s1gseldoro;q1 aasslse;dolo;qt

I rs 'l9vd-sos

z g s _L l s , 6 € [ , u o t l P ut u l l a t a p f n n L ' f J E t l - Z € L ' 6 ur t : t d a P E A n - Z B nJ'9 Zl , 6 5 , s n ur u J a l - J l9€t'5tt'Ig['ern]lnrls aro) sa6ueu) leuorleujroluoJluotleln6erlualsollV orlDaas J S S tl t g t ' S S L - n g L ' t € r - 0 € I ' u o r l e u r o j u o l '6€ 'srs^leue 16€t t leuotlelndLuo) 'sluauod-uo) l9Z L 'srs^leue 6 €I (s)pDeoutulv os/Daas LE l-gza'aruanbes pt)e oul.up zs L-szJ'09-6g,alnl)nlls utelord

"1;;"i;il;#iil;;;

#;

' 'sutelsutaloJd lgLs LLs'anlqetsseuooJ /IroMlau r61o9suo.r; llOg L ' Z OL - a OL ' 6 6 9 ' s l e u 6 r s J t oL ' I Z Ot ' J L O 1 0 8 ' s u P rp r o r l 4gg'sprdrlo6urqdso:,(16 'saLuosopua 1108 l0g,utPtuop l e i a l e loseq 208-908'ureL!op le)de DUTUOS U!alord sureloldlupurqurolauisrs^leue utalotdos/Daas 1 6 t S , 6 u r l l o l qu r a r s a M 'tzs'Jl8L9 ' 'srsoroqdorl)ele Jtzzs tzs Lt9 I S € S! S t 9 ' n $ - Z L S ' { q d e r b o l e u r o r q : uorge:rpndure1o.r3 'EVL-ZnL'tlnL '7ObL',slrunqns !8n | L a s - g z s ' s a l n ) a l o ue l 6 u r s (ureuop )rS) ureuropZHSaassuteuropZHS Z ^6oloLuor.{ 'arn])nr]s^reura]pnb 9€ [ 9ZS-'Z9',spoqlau le)tldo '06 'uorlrnrlsuo) deu 'ZS t-88 t f g i L ' 9 5L , s a d ( 1a : e p a l u r 'uorlezuaultp z' I sls(;eue LZS-EZS'spoqler.u 'suottlprelu!utelotd-utalord OGl-l,g t 'uor1e;n6at utolordqU 79 11 9L L - SL t ' 6 u r p u r qu r a l o t d Jo, leu6rsuorlu6o:ar se alts uotlellloqdsoqd LL L'sapb uorlelr(roqdsoqd (sasPdl9) suralord6urpurq-dlgaaspaletpeul-dlg 6L L-gL t'uor1e1fioqdsoqd ol a^rteurallese aEueq:xadCg-dlg /OSt'suralord{to1e;n6a.r aua6 I t9t ) L 8 t ' L 8L - 9 81 '9L L - SL l ' s l ) e j j a l p u o r l ) u n , (s)aselPqdsoqd ureloJd:(s)aseutlutalordos/Daas 1 9L l , 9L L , p a ^ l o ^ ust a L u ^ z u a 's)tlablaua // [ ltg louop aleqdsoqdse 31y oqdsoqd urelot6 a LL-g Ll'uor1e1rfu aursor{1uralo.rd 6€6-B€G'(sdld)saseleqdsoqd gt/nt'JLVE'6urdde> VNUur,g ',€90 ',uorle^rlre sLoL-nLoJ 1990 t !€90 t t lpf ' J 9 L t' '(s)aspleqdsoq ud ralord 56A LLt-9Ll lg6€'86€'1sea(iarueluaquri(;uo-ura1ot6, (suralord)sjrlour6urpurq -VNCaassjtlour6urpulq-ypq'sJltou uralord 9 L L - SL l ' L ' l j n L , s e l n p o Luut a l o r d gg utalold l's^elJpol)tru (sarlquasse) sexelduo) utalordoslpaas ',arnl)nr]s ?8 [ 9 8 t - S 8 l ' S B t - t 8 t , s u r a l o t dp ; o g e : s l 9 8 t ' 9 B l - S g t l l a ) u ru o t l t s o d 5 8[ - t 8 L ' s ] r e da ; q e a 6 u e q l e l u r ,uotleztutululaua6 Sg[-]g t gg l-s8I ,uotle^rpe gg t t g l , s a u t q l e u lu t a l o r d suorl)eralururalold-utelold lsuor])eralur os/Daas VNC-uralo.rd 'uotsnllxeJaIPM tg L '^r!ru!)ads €s | uorgeln6ar :ua1so;1y aasr{ta1so;;e ' u o r l enl 6 a r oLL-691

I

' E s ' s u o r t ) e ilss atu! luate^oluou lvst'vsL lEs L t 'zLL'a6e\ul lrzLL |Lt suo!lleralul alerlsqns-aulIzu3aasalellsqns-au{zue uorle;n6atluelsollV os/Daas J €L L ' €L. L - ZL | [ l r n r l e l a d o o r (, luPlsuo) u,rnuqrltnDl os/paas 8 St - l S t ' q ] 6 u e l l s 6 u r p u r q l s s t t ' 9 t ! € s [ ' € s t ' s a t t s6 u l p u l q 6 u r p u r q , { p o q r l u e - u a b r lauaysi ( p o q r l u e - u a 6 r 1 u e 'ss ' s u r p q l - o p rpst ) eo u r r l e lss t t?9 t t-?9t /suotl)etalutpue6tl-utalold €gt ' S 8 L' ( f ) d ) a s e u r lu r e l o r d LL 6 ' 9 e 9 ) '(8)d l ralord $lV) I asPuru WeL'VE6 sawlzua pnpwpu osp aas (s)eseutleursor{1aassaseurlaurso.rIl ' L b L, a r n t ) n r t s tgLt a>uanbas J L L t '9L L ' { 6 o 1 o u o q J 6 L L ' !L l L ' B Lt - L L I , i s d ' L . p o l ] | L u , , , u o t l p l n 6 e r ' 9 L L' , u o l l n t o ^ a JLLL '?90 ,uorle^tllpur 1990t ltgo L t-€90 t lpJ 5 6 A ' aL L - gL t , ( s ) a s e u ru) r a l o l d 106t t 6 g t ' 0 6 1 - g g t ' s d e u u o t l ) e l a t u ut t a l o r d uollelUundutalold lsts^leueu|atc,dosp aas tZS-6 LS,fulauotl)ads sseLrj

6€:l

X]CNI

/6u!sol s^e/n aasrUrlads-alts uorleurquorai :r;r>ads-alt5 882'uP|'Unq J9€Z[ ]o ')rlo^re)na ',snon)sll.Ilo.lo yg 11 'uor1e;{toqdsoqd r(q uotleln6ar JSS.'t8Z 9 t t-s LE l(la^o-6urssoJ)) srsoralA z S T l S Z , s t s a q t u ^vsN c zlzL'$tw ul lno-l)oul 'lnoL 's0 -€0 'lorluo) ap^r aas)llotau uorleurquro)ersno6o;ou.tog 8 8 al L S e ' 9 8 2 ' L q Z ' 0LZ ' s e l u a n b a s v N c t L t t t llel )rvu ,{6o;ouq:a1 J 0 L Z '6 0 2 ' l e u r o s o u i o r q ) S E a t ' r € . 2L ' 9 0| L - t 0 [ [ l a ) u P ) gU uralord/aua6(eurolsplqou!lar) ) v g a ' z 8 ( ' u o r l el ^ q l a ur u ra q VNC tueurquro)agaas6uuaaut6uattlaua6 ,Z8Z ,lerrarrPq gLLL't0tt (ra^o-6urssor)) uorleurquola.t JJtSZ '1626' sno6o;ouro11 aas(sno60louroq);etaua6 L g OL 'r Z S e ' ( s ) u r 6 uuoo r l e l r l d e u oE6-826' AeMqled6urleu6lsaseull-dvw-seu uotle)tloelvNc os/, aas €o€'(Iedar 18(6'LZ6 '002 'pueils '(llg spU)rol:e; a6ueq:xaaprloallnu autuen6seu g S C )6 u r u r o l p u as n o 6 o l o u o q ' a l oIre d a l V N C aleldura] J99Z'992 I tOZl00Z '892 ',arnllnrts 'ern]lnrts sgsl-v9s l'sureql Apoqrlue t69Z 108[ sasPraurosrodot VNC 1(s)ase)tlaq lt99 !s0t'€92'uo!leulquo)au VNC os/Daas J 8 Z 6 'L l t 6 ' s 1 3 9 ' u o ; r e ; n 6 a j 'auaoo)uo glz 'uotleredaspueJls uolle)ulJnoulalold os/Daas se 101,[ 'suretordseu gv1''gLS-blS's6eluralord r L q ZJ' 9L Z 't 6 9 Z ' 8 9 ( ' s r s a q l u ^psu e i l s6 u r p e a l t€z t letlJou '!(La 'FEZL-E€ZL'10 'sia)ue) ul pa]e]nur uolssaroxa )l8z'J9Le JSiS' 9n9',srolfaA [[ ftzL ' q n s ' 9 V 9' s u o l l P l r l d d e - 9 a ' s r s a q l u ^psu e r l s6 u r 6 6 e l aL z ' 1 6 9 2 ' 8 9 2 L t ' u o r l e ^ r l ) eu 3 6 o ] t t u € 0 [ 'suuoJa^lllPul pue a^lllP (s)ut6uouotleltldaUos/Daas sulalolo lueulquJolau I L8L 'uorlerlrul sanbruqtaltloads lsureloid uolle^llre rzSz'z8z t € 6 ' e s e u r l € l d ] o aseurl-dVW uolle^!tle lueulquro)eUi6uuaaurbua)rlaua, osp aas 0 8 2 ' u o r D e l a l ual t r i o s o a l l n u 3676'{ennqred }o jOSt '6LL'(s)esPd19 (s)ursrue6ro :rue6suerlaassusrue6ro:tua6suetl seU 06z-68a'i(;quasse aulosoalI nu LZ6-926 J9€6' 'aua6sog 'yEZL-EEZ1' 'ZVZL' 9Zt-57.E t8z'aler luaura^ot! 76yy L L7ZL IZVZ 'l 'uerleu|.Ueur 'suorte)rlddeuolleu!qLuolarrt;t:ads-alts '(llg-ueU) .ro1re1 a6ueq:xaauruen6-ueg v8z-t8z L8z 801 utalotd 6utlentlte-asedlg-ueU 0 6 2 ' u o t l P t ) o s saeu o l s t q 9 ? 9 ' u o l l r n p o l dv N U 801'(dV9-ueU) g y g T ' s r s { l e ul e uorplllser e}uauuedxa JVtS' EES-ZES'Z€9'saur^zua 1 , - 3 0 Z ' , l r o o s u el er le P n u J OLL ' t 6 0L ' 6 0'uolleLuJoJ 'asuodsalabeLuep (s)aua6rel.rodaU aassaua6reltode.t a;putds:t1o1;tu SOI t VNC t80 t suralo.rd urleuorq) lueurquo)aUaasuorlrnpotd utelo.td 062'(slvl) s.iolreJ^lqLUasse 1601'uollezlleluauluedurol 'OSZ')rto^relna (s)asedlDueu LgOL JgbS'jsns'gng-nng'Ald 'uollezlPuq^q nl,su/ ^erpor)ru.rVNC Z€9 JggZ'SBZ'srs^leue I u iil_9-ueu ' u o r l e z ! p u q ^pqr l e r r a p n u 9L Z - gL ( , { l r n r l e t a d o o r 801 !lD-ueu 1 6 € st 8 € s ' J L E S ' Z E i auouab ueunH oslpaas z 8 z ' LL Z - gL Z ' 9 9 2 ' l e u e l r P q 80/ iiv9-ueu '€SS g 8 Z ' u o r l e ) r j r l u au p t 6 u ou o t l e : 1 1 d u aP t ulnq 892'992 Xrlatutulse 9 9 S ' z € s' s l > a d s e 'luauaouPlle ' s r s ^ l e uueo r s s a l d xaau e 6 ' s t s a u a 6 e l nuulJo p u e U 1€€s ;e:tlolstq r9lz ' 1 o ; du e r p u e q : e u l e g srsaroqdorlral3 aasstsaloqdotg>a;a J 6 9 Z '8 9 2 -L g Z '9 9 ( ' \ o ! u o t l e ) ; d a 5 1 7 7 1 ' s a 1 6 upeu o q a p r l d a d s NC lteoal q)lPtustwos/paas 6 u r : u a n b aV s N Ca a s6 u t ) u e n b a V o o 9t ' J v 9 9L 'V 9 SL ' E g Sa ' u r a l o r dD V U 'ZLZ'sJu)a uorlelrldaU 1 E S ' s ^ e l r e o r l l tVuN C LLZ-qLZ | LZ'692 6 E a l 'J626' sadtrt)lJDads!(s)uosodsuerfoslDaas eseulljeu (eseuDl-aseurl-eseull-dvw) vNC Lvs'zns-ons'sauPrqrl J€.ns'Jzbs'zns626' /urxrpeu (s)aqordVNC oslDaas ( 6 2 ' Ol Z ' s e l u a nb e sl u a u r o l a l 600L ' 6 u r ; a q ey;5 6 )gES'VE; lltg'saltllelesor)tur l E O g 'E O q - Z On7g' g ' 6 ut ; a q e l o t P e g 'luauialnseaul 6 u r u o l :y 1 1 6a a s6 u t u o ; :y 1 1 6 alqeue^red^q'slpadai[uapue]Lloqs [09 'saldt:uud (qder6otperolny : t s e q osloaas 7gg 9 V € . ' O ni l l o o v N U € S S - Z € SX 6 o l o u q : ay1p 6 l u e u r q L u o l e u l S l E ' u o r l e u r q u o l a rp a ) n p u r - l e a d a l ft1g lZOg' EOg-ZOg'ZO9'V€9'suorle)rldde '[ 'sadolosrorpPU (98'sDuowoutPaa 3g 7 g ' a u o u a 6u r u o r l e l r l d r l l n u l I t 09 09-009 8 S S ' ( s ) u o l l e l n au ^r l s s a ) e u r t z t ' r L o z ' a u o u a 6u e u r n q 6ZS'UWN'^)uenbar]orpeU / O € G - 9 2 6 ' u o r l e ^ r ]^)eem q l e ds e U uorlerper(nn) lalot^ellln oslDaas llts'suINA'sJa)|leur )rlaua6 il626' Jg(6 'se]uanbes)uauror]uaf 'uo!le!peu 'sl)ejla rsc6'*26 [ [z-0 Lz vNc L0€-00€ '9z6-?26'suratordureulop-7115 q1M 6ulllop vNc a^rlrlaoau 1 8 0 t , 8 0 € , u r a l o r zd9 p P U 'luau.rdola^ap pe)nput-lpadaU ulaloro v)eu os/Daas t€ | l l9 leulue lS t€'uorlua^ard'uorteurquola JSZ6'5Z6'ilV26'IEZ6 l0 r€ ll0€'uraloro !speu '0 /uorleurquiolaisno6olouroq '6uuaalsauo: qv,rrol6 saseut)joursor^llolda)au redeU,, L€ 1 \EZ6''Z6-ZZ6'UlU) ,'sauol)eJ 1gE 'uotlnlona'{lrule}aseutl autlasloldarag 'seruaJeprsoq 'sasnr^oeu s€s L 3t1 1 l ' A u e l o d lee l l a q l l d a 9 5 t t ! 9 S L l e s e q o r t d 'suort)auuorrrldeu{s '6ur;apoLuag (s)rolda)auaassulaloldlolda)au uo l)aJJa €6€| L V O I - I V O !LZ t O l ' u o t l e z t u e 6 l ou t l : e 'uollenllle ', / ( l P u o r l e l s u e;oi ] ) 'alor s r o l ) e ] a s e a l a u salnqnlorllul ! L 6L ' 8 6L - 1 6L ! € 6 1 ' s l s o l ^ ) s u e l ] lz8€ I t8€ L8€ €?0t Z 6 l ' s e p t l d a dl e u 6 t s L € 6 ' ( s ) a s e d l)9P U s/sa-tL9l 'ro]oa)or 'oLs 'vvs '1s111e: 90s [ J t 6L ' 1 6L - 2 6L ' 6 u t p , { l a tl o l d a ) a r t-69st t-wst 1,tore1n6ag g l 9 L ' L L S | l 9 6 t L' s n r ^s a r q e u paterpar.u s 6 0t ' u o l l e l ^ r o q d s o q d Jb6L' 5utuos uteloJdauelquiaul -oqU'(flWU) ureq) lq6!l urso,{r,u qeu i(.ro1e1 n6ay 1 g 6 1 ' u o t l e P e',alelon l 6 al P l l e l e u . l I t g L ' L 9L ' t 9L - 0 9L ' ( s ) e s e d f D 'salo^re)na ^lrler)adsse uorl qeu t61 Z€- t€ I L9L' 19L'(s)ror)ajJa Jo '60€ 'pue 6 u r u r u r p J b o i d t u a u d o l a ^ a p , o t€.61 t JZgL'Z9L'SqeA 1 6 0 €t 'Z6L'ofuqua oyqdosotgSo6urura]led L6I'uodut eln)aloLuol)eurlelnlla)Prlxa 7gg1 'uorlezrue6j0relnpour auab a/a U t6l'eleldn l9l 3gyE1 66L- 16L'yg1'srsoy{ropuapatetpauJ-lolda)aU 9 O Z ' 8 -L ' V N C^ r o l e l n 6 a U (sdId)saseleqdsoqd aursoJ^lutalold os/, aas I 8 n ' E q b - Z q n ' Ul ' s a p P ) s P^) r o l e l n b a u J Z S S ' t 5 8'arn])nl]s ' 9 €t8Z € 8 ' ( s ) a u o u r n O 7 7 6 ' a s e l e q d s o qedu t s o r l la l l l - r o l d a ) a U '/s r-9sr'suonerUrpoLxJ L S T ,(teu.ra]enO 9€ [ 776'sasepAt1(;{uen6 rolda:ag saoAl ;rsol^roxl oslDaa5 "i:i{HL1r:tt:!:9tu l 8 8 S ' 1 8 9 ' s l o pu n l u P n o )uDads lstsor()opua palerpau-,roldaleu os/, aas JELS 1 9 0 8 ' s 0 8 ' l u a u a 6 J e l uaau e i q u r a uer u s e l d 'vL;-€LS '(Uld-IU)uorl)earuleq) ( s )l a uu e q ) l?08'?08'uorlezrlP)ol eslanale^tlet!luenO aseraw{1odase}dt.t>suell u o rp a 1 e 6 - p u e 6ar 1 a ss l a u u e q u l o rp a l e 6 - p u e 6 t ; r08 ! 108 'OOg'(srsoi^roxa) (emqled paleln6ag auoulroqreal)nNos/Daas ^rolal)as 9€8-S€8'al)^)-O 1s1;o1da:at I Lg g , L S S , r P l n l l a ) e i t u l Igt L-Egnl'al)snu lelala)s 'anrau a ) u e l a l o l : r l d o 1 e r t 6 o ; o u n u l uo;s l oa a s L6tL'6q! ur S9St,99S t ! 8 r S r , 8 r Sr , 6 u r l r p a fisEL'VSEt 'uorleln6al-umoP S61 l 8 9 LL ' 6 9 tt - 8 9 t t ' p u P e "u0r eu1r el el: rsae1qu r 'aseurlelennt{d aasat911lj:111r--,, - Lz[ (s)rolda)aro)eJrns-llaJ uorlerauaoau '96 'xa;dr,uo> 'uorlp)rldar40) .f 'pouad {rolrer;ag alennt{6 aseua6otpi(qap 396 t)rrv+uoJod fngZ'ZBZ 196'esel^xoqre)epalennr^d 6g'>t}aue6'i{:uepunpag 'asnutlotq 'asellxoqle: alerrnrr{6 g(.9'JL ltB L ' e L - | L ' s u o r ] ) P auro r l ) n p a u 'trodsuerl zz8 L€8 'Vo) ' Jg6'96 ;I1a:eot uorleplxo 628-828'suorlfear(xopar)uorleprxo-uorlrnpau f LZL'J68'gg'srs{1o:(16 f0€8'prepuels lzzl'apA> ptle ltJltl 8 ' 08€' r8e' Jl useuue,rrla r n s e a u . r ' S € 8' t €l 08€' 6 2 1 0 6 ' 0 6 - 6 8 ' u M o p l e a r lqr q o l e e u e uorl)ala E S 8I Z S S! S € 8 alennr{d 0 € 8 ' u o r l e l n l l e9r v J 8 9 Z ' s ! s a q t u ^vsN C ' e s e e l aar l e q d s o q d o r { 6 6 g g ' s a 6 u e q u: o r t e r l u a ) u o ) jaurul^ql jautso}^1osloaas lDern l o € 8 ' 6 2 8 - 8 2 8 ' 1 s 1 1 e r 1 u xao1poadg 't9 '(s)aseq e u r p r L uu^d l9 [ [ 8 Z 8 ' , S J r xeodp a u lzot'(vNU-d) ylg-;,{soueri(6 (s)ar^rorql^rlaas(s)lla)poolq paU 'snd tts t lr98 !0gg'0S8 !128'sralua) uorl>eat>t1aq1u,{soloqd Jn9Sl '6ururoI f ( C ) n ' s a ) u a n b a; se u b r su o r ] e u r q u o ) a U g g 71 ' x a l d L u ou: o r l e u r q w o ) a u

o

ll0t'suoll)eralu!vNo-uraloro OL€'sutalolde)uq/ LcJ)a 1 8 0 €l l . 0 t ' 8 0 € - 1 . 0 € ' s o o l o u r o q 110€'80€-10€'srsdeu^s tLot' LlE-got'uolleulquro)al snoDolouoq uralorovlau '89€'srsaqlu{sulelord lg9€ (sluo) saLuerl6ulpeal uado aasuado sauJerjDulpeau ,(6lauaaar3osloaas 1 0 8 ' l a p o ul e ) l u e q l a u l 15'srs,{1o>(16 t 8 ' a l o rd f v lallle) pale^llle 08-61'uolleLUroJ S Z q - n Z gt 8 l ! 5 1 ' 6 u r l d n o )u o r l ) P a u

X]CNI

0t:l

uu\rJcuuuJ/drcuuuJ

)uEdu

)/l .dlLls+

c u+ /dloJ

a c q+!/v\ >rd9ur

ru duEU

:^lenrtn)asuo)Mollol lpql sarn6!Jol ll up qlrM 'arn6r;e o1ra;arI ue qllM sraqurnua6ed li(rluaaqt Jo uorssn)srp lxat rofeu e ol rarare)erploq ur sraqunu a6e6 'atrslrillelel 6LE-gLE 3 1 g g ' a q : ( :u o r l d u r s u e r l ll€€'aJnl)nrls ) SL E '9L € - SL E ' s a 1 r s - 6 u r p u r q 'ase]du)suerlesraAeJ I6EV'6€.V'9EE'srolle,eur6ts Ig It J6L€, 'sult '6Lt-8LE 'rn ',€l€',arnlrnrts l 0 ? € ' 0 t E - 6 € €" s l l l o d V N U l_8tt 18LE ft'SLE-VLt 'rLE€.' 'Ul q6not rStn LEE-gtt'leueDPq 3 1 6 7 ' e u r o u au 6e u . r n q s'L-nZL J S € €! ? S t ' 7 E g ' l s l a s e r a u l l o dy 1 5 lzzE 6 L€;g f€'(1nr1re aul,(zoqu 'sraur,{;od 'OZ€ '1 'suosodsuellor]er alrl-ler^orlau uortelsuerlisrsaqlu(suragol6osloaas al!I-VNU )ZOv'ZOv'ay; ur6rro l-81€ [€ / u o r l ) n r l s a p ,ro p a l l a l p - v N U s p suosoosuerlorlar elrl-lPrAorleuos/Daas utalold s € 5L vNUss BBv'g LE-ELE'stsaqtu(s lelt^ 'uorle)urluapr (s)awosoqu^lod aas(saLUos^lod) saurosoqr.r,(1od otzt €Z€ l-g t €,suosodsuerlorlau llal rounl /p 96t'uorllnpal]uauJalalqpsodsuell rjvL'onL-6€.L J6ZL' 8ZL unoq-aueiquiaur ' ',anoruqta] g 6 y ' 6 u r u u e >Isl e a ; paplo;srru'uor]e)olsuer]orlau [6€'suralo.rd zL s - LL S 's)rlaua6asra^ar '(S3Ul)alrs,(rluaaurosoqu 16t ZIS J g SL ' 9 5L ' 9 5L - S SL ' s r a L U o r l a U leuralul 'uorl)e ujsrueqlaur ' 'uoll)eford tl9 t 8 € ' s u o t l ) e r e l u)ll q o q d o i p ^ q It6tL L6EL lpllalourtaU ro l 6 t ' a s e a s r pu e u n q u l J V 6 tL ' 9 6 t t - E 6 E[ ' l u a u r a u u e r ' d e uI P I ] a t o u r l a u 16ZL'8ZL'AdI tsuo\e)rldde 'roldatar pt)p )toutlau lbgs' L6t 3 169 f VL E 't g E _ Z g Et,9 € _ 0 9 t , > r s o ( r e : n a lelueuluadxa '068 'pr)e )rourlau 'uor])nr]suo)ar y19 'oltqdosotg 982t !688 lz [9 (e!!olselqourlaJ) uralo.rdlaua6 a l ) t u e d - a l 6 u t^sd o ) s o l ] | u tu o r ] l a l a 9 6 y ' u r a l o r dr a > r 6 '^)uar)UJe g y y ' 6 u r l a p o u a -urt l e u o l q ) gg aasuralordTaua6 eurolselqourlag S1€ s4rpqoqtouaoS f I LS' LL9'suo6a1a ['t0 [ l'eurolselqourlau ) q E ZL ' S t ZL - V E Z VNU :suralordleurosoqrU aasuorl_rsodurot z L s - a L s ' t 6 t - s 6 t ' ( ! v N U ) a ) u a r a l r a l uvrN U Z E nL ' r c EL ' s ; ; a :u o r ; 6 u e 6; e u r l a y lpu,rosoqrU 96r,(SJU) 1 , 0 n 9t 9 9 r , 9 9 [ , t e u r ] a u a.€b J V L E ' s ^l t t o ^ l e ) n a x a l d u o >6 u r : u a ; t sl e u o r l d r . r : s u ep ta1) n p u t - V N U rz* I' Ew L-e* L'arnt)nils IsB€ !?g€'t8t'6urpurq )rlorqrlue '€6€ J V 6 V ' V 6 V ' ( ) S l xUe) l d u r o r6 u r : u a 1 rpsa l n p u t - V N U r - 2 6 €r l€6€r J 6 L € ' J 8L t ' L St - 6 V I l e t r a l r e q 'deur snloal)nNos/Daas JLbE lpl)alourlarpue ^IDUr)adsleuornau '9yg '11aserau{;odypg '1da:uo>,,(ro1te; 'fVg€.'t9t-Zgt')lo^re)na ypg,, pue i(6o1o1srq 9Lt-V LE E€n|-ZEtL leMauar 1|a: ' s u o r g r udnef 6 ,leua]teq l z 6 v ' z 6 v ' a s e a P n u o p uvaN U ! ( 9 L L ' Z 9 t1 ls l_6t[ svNUeprn9 os/Daas PUrlau 88' Alquasse '(s)aurosoqru l t S t ' € g t ' V N U t u l e u p u o q ) o l t ual L ! o s o u e d ^ l l s0il'990r lv LE'J8L ' -9 '[90 'SS0 'aP{: )698' 69t 189€'VNUralsuerr l-0?€'9€€-s€€'lt€€'uorldrr)suerl t [ ;1a> ;o lurod (ue}s)uor}]u}seu ' 1 9 E' 6 u r s s a r o .yr dl g . r aserau{1odVNU:l asereu^lodypg oslaaas )Zgt'Jl9E Z€S'sualsr{suor}elUrpour-uorl)ulsaU 111 'srsaqlu^s tgt'vNUr.u uPtleululeut €tg ^lDtJDadsa)uanbas l9€-09€ svNuousos/Daas S 8 t - € 8 ? ' u o r l e l n b aul o r s s a r d xaau a 6 LVS-OVS'JVtg '€€S-Z€9 '7gg',(6o1ouq:a1 lueurquro)ar t 8 t - € 8 t ' a ) u e ) r r r u 6 r s( r e u o r l n ; o n a VNC J Z g t ' Jl g t ' t 9 t - 0 9 € ' 6 u r s s a r o r d g g 7 ' 6 u r 1 r pyap g L g t ' t 9 € ' s u o r t e l r J r p o u €t9-ZE9'uorl)u_n1 J Z 9 E ' l 1e:r6o;orq seu^zua uorl)ulsau l 8 € S ' 1 € g - 9 € S ' 8 g g ' s p u q iV ( qN C - V N U 0 6 z l ' 8 9 8 ' € 9 8! 1 9 € '1 . 9 E - g 9 g ' s a u a 6 'snfnur [??-6€t'uorleln6at a6eneapy11g 9 Z 9 l ' Z 0 S t ' r ! s r u e q ) a ua s u a r a pl s o q s e 8l€ 19€€',suorl)unJ 'uorllunJ , t 9 € - 0 9 8, > r 1 o , ( t e : n a uotldulsuell os/Daas zos I I t9€ 'J8LE',leilal)eq ,zos ,uotlezruolo) l6€Z'(s)aulosouloiq)aual^lod t €09t ' 1 - 9 ')6LE '9w 'urnr;aqlrdar(rogeldsag (VNUr) 0lZ ^luenber, roila | l t L E ' I 9 J9EV V N U l e u o s o q r u '{qder6orperorne '3gg 'xalduor aseua6orp(qep 769 9€8 !€€S 88t'lorluo) leuorlelsuerl 96y1r1 arnl)nJls os/Daas 80r,9tt_s€€,srsaqlu^s 16LE'6 Lt-q Lt',uorle)ol leupuoq)olrw '1818leurosoqu ',leua]req arn])nJlsvNU aasatnllnils l€t8'z€8- [ E8'uorlez!le)ol )6LE 6 u t r r 1 da5a s6 u r > r ; d s z € 8 _l € 8 , u o ! l e r ! r ! l u e p ! € l € ' s u r a l o r dl e u r o s o q r u 'S8S'uorlezrpuqi(q 'asoqru n;rsur sdurnduolord os/Daas l98S J Z E | ' a t t ! 9 I L t Z l .[ g r s ' 9 V S ' { 6 o l o u q r ayi p 6 t u p u r q u o l a t ( p D el r a l ) n u o q r /v) N Ua a sp r ) e) r a l ) n u o q t u l 9 € 8 ' S € 81 6 t 8 ' 6 t 8 ' s ) ! l a 6 r a u a (lrlo^te)na)6urssa:ordy5g aas6urssa:ord lr€8'r€8 lzSt t'azrs'aseapnuoqru ll l5's-t'uotleulloluo).lpln)aloul !tt8'ttg-Z€B'xalduro: eseprxoauorqroil: ILE L'JZtt's;apou uralo.rduoqqrg 'sarauJoquroqu 88n-98V'StV 9t€ t l9€8 '9€8-S€8 '7gg'xa;duo: L)-g euroiq)olI) (s)asedl9voilu aasvoqu t 6 l t ' u o r l e l n b a l u o l s s a l d xa u a 6 ' u o r l e z r ; e : o ; 3€€g uotgdt.rtsuerl oslaaas aur(zua(roleirdsag t v o t z r o t ' L € 6 ' ) u a u o u o u r 6€g-l€B'OZ8-GlB'saxelduJo) '6uuaalsauo>qyr,ror6 I t € € ' t € € ' r a j s u e ru] o r l e u r o J u l 18€[ B€g-l€8 lorluor ^lolerdsau g g g ' ; e u a l e w: r 1 a u a 6 (s)ureq)uodsueil uoJpall aasureq: (rolerrdsag s?0 t-€?0 I 'IZVO 'srsoil:o6eqd'rsJnq(rolerrdsau srsaqlod,{q plloM VNUos/Daas urllp I' EFOL-Lf.OJ'uorlezrue610 7gg 1 'g0t '00€ 'a:ue>rllu6rs '91 'srsaqtu,tsoloqd ,{reuorln;ona (s)asedf9oqu oi uorlele.r lggt 191' s a l n ) a l o u r 6 u r l r p ay 5 g a a s6 u r l r p a r l u e b r o ; ou o r l e p r x o 0l s60 r !t90qu s t s t ' ( v N U s p )p a p u e r l s - a l q n o p Slg-b Lg's1:adseft euorlnlona J 9 9 L ' 9 9 1 ' 6 u r p u r;qe u r g a r '€6n-Z6n 'SSn'S 'J6LE'uorleper6ap uorleJroseu S6n lV 3 9 E t ' u o r 1 e . r 6 aat uure J q u J a u l ' u r a u { po 16 u r pU '996'(1rlrqe1sur g€€_gtt .uotltsodL!of urq :rueui(p u! 'an)sau 7 7 91 080t ! t86 ' f g r c ' g g 6' u r s d o p o q g sad{t )Upadsosp aas sauowtoLl2llpaososloaas a*t 'auruiepoqu 'aruapDul lZ9€' r9€-09€'6urporuou 6 Z ZI /8S-98S ypg la6uassayrl aar VNUu.l ralue) uo l)e#e 'seuourJoq anrpnpotdag 6 t Z t ' s r s p l s p l a uu r ' a u a 6; o g g 'sad{r 'daaqseqr 111o6 relnlla: L L t | ' ! 9 6 0 1 ' 9 0 0 t - t 6 O t ' ( s ) e s e d 1 gV o r l U tg7 1 ' 9 8 f9€E ' S 8' s r s a q l u ^ s o r q ll8 t58 L ' ( G l ' 1 : n p o t d a u a 6 - g J € 0 E t ' € 0 €t - Z g EL ' J L O SL' O g ' 6 u r u o a ; :n r l r n p o r d a g ZS ' 6 u r p u r qu r l1r E auorqr;-uu6alur J Z g ' 8 - n ' ( p D el r e l ) n u o q u )v N U 6511 l 0 6 t ' 8 8 t ' s r o s s a r o alre u o r l e l s u e r l C D U t r l o r u ' s s a u p u t liqa ^ t u iossaloal e ^ ! s u o d s a^ra u 3[g t 1 9 8 ?! 9 8 t ' S 8 t ' ( l U U ) i u a L u a l a uetldold{r1aasrossardarueqdold(t1 96t'(xaldu.rol $8n 'JS9n'98V-S8V'U 6 u r r u a l r ;se u o r l d u t s u e p odsuPrlVNUrUnlH 'uralord^au r la ) n p u t - V N US)I I U JVEV'nw-ntn'6urpurq roletado 1 p 6 y ' y 6 y ' l x a p w o : 6 u r : u a I sp a ) n p u t - V N Uf )S I U tZS'uals^s puq^q-oMlasreAeU ItZ?'rossardarlaW 'urso{ru 'a1e1s lo6rg rossejoerPpqurelaasrossaroalppquel asPlaurolalos/Daas 6 161 ;o .alnl)nJls rossaroarlel aasJossaJoel lel 199g'ur:(ueyg I tz€ nlH ,suosodsuellollaj rossardarorl aasrossatdatotl 9tst-9tst fg [ € 'luaua^otU paseq-utlle' otslla\)uo tslla>pr )tor{le:old J E 7 S ' Z V S ' 6 u r u oy;pr6 r ' s u o r l e : r 1 d d e A 'azlsaulouab 'il\azDMud olsltaoply 'sbn 'rgEn'rttt 'vEt-€€., 'swsrueqraur f8l z 6 e '* 9 2 ' s a s e l o u ) s u e r a l sJa^au $nn esla^al'sftlouag aass:r}aua6aslanag 9 b g ' f n n 8 ' t t 8 ' e s p l ^ x o q r ee) l e q d s o q d s raqs o l n q r U J J L W '! L n n 'L r r - S w ' S t t ' S t b 5 l l o f t e ) n a 'asolnqru J ZLL t E L S ' ( 1 8 ' 8 8 8 ' M o luJ o r l l e l ea s l a ^ a u JtZb'JtZn'JOZV I g' 1 y ' 6 u r p u r q - y p 6 suosodsuergollar alrl-lpr^orlauos/Daas VNUlillele) oslpaas 9 lt Are^olsrp (s)uralordrossardaU 80, 3r0?,r!laqlu^s J t 8 € ' E 8 t ' 6 u t g r q s a t u elre; u o r l e l s u e r l '797'6u;p;o1y11g saseldr.r:suer1 asranagaasaseldr.r)suell aslanal 1y99 ) z b b ' ?o1 (dlD) uralordlualsaronUuaaJDaardlD IS0?'suot]leal pas{;e1e:-au{zoqrr lt8€ 'sauosoqu 'ggg'6urllqsatuerJ uorssatdxa aua6 6LE-8IE lZ LS' EIS-Z LS'srsi{;eue lpuorlplsueJl'suralo.rd 1o6 'sl sls^lPleruraloJd ' s r s a u a 6 o : u o (s)aua6raUodaU tot 7 9 71 'aleJuollelnul lgon' VOV'sa6ueq> leuotlpul.loJuol 0s?t L L ZL ' J90''^ratsolle L OLL '6 S 0L ' 5 0 9 't 6 Z ' a ) u a ) s a u a ;s; a >a n r l e : r ; d a g f 9 O , \ J V O v '6t E - g L t ' s a u ( z o q rg ,8tEgZ /seq)lrMsoqtu 'sur6uo u o r l e : r ; d e r r r l o { r e : n a ' s 1 r u ) 6 8 V ' l gL V '6L V - 8 1 ' uno r l e : r 1 d a g lsntrni(>uar:r;apounututueunq) AIHaasAIH 'lol)eJ 6ur;t,{>a.r auosoqtU uorlrsoosuetf os/Daas I tg€ 1 9 6 1 ' 6 u r p u r (q) U - a r d x) a l d u r o ru o r g e : r ; d a t a r d 'l!unqns ',Oursue)rl 51€ 3 1 . 3 E ' u o r 1 e r 6 ea1uuoru a b 1 9 0L ' u o r t P ) ol l le u s 'auoue6 '982 'aD$t^atD'S J8Lt'8LE vNU 1179 l88Z'tL8Z J 6L t ' 6l S - 8 L E ' u o r l e ) oul l a l o r d ng(.-€gz'stlunuotle:r;de.r l186 ' l l u n q n sa 6 j e l 'ggg'6urryrqseure.U 'LgZ'(lUO) xalduor uorlru6o>arur6uo SIE Jgge l e u o r l e l s u e l l ' s u l a l o6l de g

,",n,norr"!"11fi1?i"o

"'.,u"tr1!::""'';f#l

LV:l

X]CNI

'00€'srsaqlod^q '(s)uornau^rosuas 'JtZ'aqwod sD/ftudotp)DsoztLps ,/90 ppoMvNU JSOF'lz}n'nOn-OOt 06€t t98€ L t-9S0 L 'sarnl)nrls 'l 'lla) leqlou ^losuas 'sauosotstqls 'a€S J96t[ llgst L SEt-998 l. t')ilelle |qdoutsoa )ZESL '62't'prlaqltda^rosuas LZSL-OZSI'alPl.uollelnul gzzL,uorlesnel E€VL-6Z'J ja)ue) rappelq 'wnrqolDwaoLlowosolsil.ps sadll )t|Dadsosp aas ll6tt'sauouaD 't€9 '(VNUsp) papuPr]s-alqnop 'sla)npsuellse 'slla)ftosues 'gg 'sdeu uorlreralururalo.rd t VNU 9 € 9[ - S € SL 6Zt L t 106L '9S€ 'saltsuqfuosuas l9S€ t t t 1990 '$g1t 'ngOL ,Sg -tg ,xalduo: a)ua)sauas aasa)uatsauas aui9ua lla1 135 t L L !99 'snl^ ,(tollej (sa)snI^vNu lsaioj rlrluras l0S t Slt t llo)-ulels)lfs 'uor]eln6ar 'selnqn]snoJa;rutuas 0 [l aasaosttat>'5, aotstrata> sattrwotoLp)os €62t 'uolle)lldar 'lp)-S aasteal)nu lrodsuerl:rurse;doy{:-rea1:np )992'992 V N Ca ^ r l e ^ . r a s u o ) r u r a s B90t'690t-t9gl'9901 ',ura]orouuoqoeuras l88t'88t-18?'Uln ,E 68€t !88€ [ !909 1609 ' 'sursrueqlau ,gO9-1,09,(l ,{dotsortruruorl:a1a6uruue:5 ,{dorso.Drr.u uotl:a1a6uruue:SaasWIS lLgn LSn-ggn lS) 88t-98t v N U) l ^ l e l e ) o s / D a a s 1 0 8 5 ' 0 g s ' a l p r s 'uo;le1n6al 'g8t-98n's'' aua6 uorssardxa !619 JgSt'LS€-qSE'y5g6uorlds-11a5 l ? [ , s u r e q ] a p r l d a d {, l(osd) upoarrln) u } )nn[t } s u n 'saprldadr(|od vNU 88t-98t'lroosuerl ] q 6 t r 1gy S SI t - € S r I ypg ra6uassa6laassldl.t:suetlVNU L0?'soprloal)nu^lod fivLL'6n[-Ana1'pueuorleuto;asdeu(s ,VOV_|OV,fievar g t l ! , s u t e u J ozpc d JEOa? 9 0 t - t 0 t ' u o t ] ) a l a sl e r n l e u ''0t'VNU rrl{leter ,9tt , L 6 A ' V 6 9 , ( s ) u r e t oprldo J ] p r s tldlllsuel]'Jef I L V ' a l d u o r l e n u a l l eu' 8oE ll.Ot t !L€6,086 l8tL L E' s u r d r e q , p t s d e ]s n l ^ s t s o l f a uo ) l e q o l a ] t l l a ] e s l E O t 'tot '^repuo)es L 0 ' ' ] u a u i a r n D esr r s ^ l e l e ) o t n e l6t[ ,slla)altllales ,al)snu uortnlo^a/saln)aloulbutle)tldal-,las I €0' 99t t lplalals 'uorlruuap ' g z L - s . 1 , 0 9 9, u l n l n ) r l a r t r u s e ; d o r r e 5 J Z € € ' e u o q l ) P qr a t s e t p o q d s o q d I |€ ,'vNc qsl,las,, 6(0l ' 6 ' s r a ( e l r qp r d r lX , g z g t , 9 Z O t' e r a u o ) r e s (s)aplloal)nNotl, aa5 ;quasse-1;e5 39; lSZOt '29 'saprloellnu ' € 8 €' 6 ur p 'gOZ o r a rl e u o u e l s u p'lal u r a 1 s , ( r o u a 1 a 5 z€t lESt l,(s)euortes etzl ' s l a u u e q ul o r' ( s ) . l a ] l U ^ l r ^ r p a l a S 189€',89€',sVNUl ZL9'999 I t e s J 6 S L ' 6 5 L , u o t l e u j o l a l ) t s a ^ p a l e o ) , u r a l o r d 'a)uelsrsarlrlorqrluv 'arnssatdanr1ra1a5 'xaldr"uor y1y5 Ejv' Jv9' v9'suotllelelul luale^oluou tZS L 914 '3gg '6u;p101 'ernllnrls vov itov'vov-861 jgEE JgVll'grL L-Stl I 089 /url)alas-l ,uorle6edord r NU'pepue.rls-alqnop 9 6 ' ' ( ! V N Ua ) ) u a r a p a l uV uoll)e'uotl)npuo:,totelles l0SSt'0SSL'Sttt l e l t u a l o d 'Sn 'suorpunj 'SSV-nSn 'uorleuenaseqd'oilauowlDs 8 0 t ' u o s u e o u o )v N c lgh | | LL JSS7 ' Z € €' 6 u r r r e d - a s e q € O ?! E € € ILLLT'9naL-9nl['(s)uri)alas J Z | S t ' Z L S L '6 'VNC'6u1pnqs ,)UJe]lauerqLuaul]soq lsure6e(6a]er]s l € E t t z € € ' € t E - z € € ' z 1 z ' a i n l ) n r i sv N U 1uaur6a5 L L t ! [ [S '18€€16'ggg 'saua6i(1rre;od-1uau6a5 6 u r : r 1 da5a s6 u t : t 1 dysl g g [9 L ,peards 6€€L t 1 'olqdosote'uorleluaubas g g 9 1' s r s o / t o 6 e q d Z S t - [ S t ' s r s { 1 o r p r6{ q1 y 6ZEl ,spuelsr ts€-t9€'euosoe)rlds JV-fv €L€1-ZLSJ'1:o;: uor1eluaur6a5 fir>ruaboq]ed I [6t | , 8 0 5 , s l l a )l s o qj o u o r s e ^ u l I t s t l u 5 t ! l t - u s t "w" s t u e q ) a u6l u l l t l d s taLt t'zl€.a-Lt€.L'tgv9l'sn€L'salerqeua^ t l60s t tgE,sdNUus luaudo;anap olqdosotg osloaas t6Vt'Dualua Dilauowlos ,62€ ,^poq slueula6uerreet VNU-VNU lrasu! l0€€ L t tlL€Z 'fLEa'gEZ-gtZ'(ua/1od oltqdosotg'pue16(tenrle5 6 u r : r 1 da5a s6 u t : t 1 d s sauabtloads osloaas 'uot1e1,(uapeI;od ,6ur:uanbas,autoua6'aqwod sauabuorgeluau6as 97E sattrwotoq>>o5 7gg 'ttoL,iooLuqs,, nlsodouolawolrqdosotgaasoltqdosotg 9 9 € - ? 9 € ' s u l e u o p q n sl e a l ) n u l?tot 'B€€ '6ur;;o.r1uor ,uola;a1soy(r a;o.r 9 7 g ' 6 u t d d e :Y P g u . t saua6 ]-9E€ I J8€€[ lltE t OL6-6g6XruelodI e1n11a: 1o 6uot;dsantleutallVoslDaas uotleluaru6a5 u o r l > e r a l uurr a l o r d 1 0 6 t ' g g L ' s d e u 'sluelnL!stsdoplqDJv 6ul>ttds tgt-Llt l€ott lgz'sluelnur 'sabueq) ' u r s r u e 6 rlo1 a u a 6 g6y1's6ut1paa5 €8t-z8t /€ ngg'LgZ-gB( lapou se s a n -L L n ' EL b 'J 6 l € ' u o t l e l n 6 aur o t s s e t d xaau a 6 o?t'(s)paas 1 9 9 7e u a b s r g ' I u a Dlrt#0et to' Z ' a u o u a 6 s8?-c8t'Dul]rpa u) o r l p l u a u r | p a s ZZS'|lS ZSSlLgZ'LgZ-ggZ gnE-Snq''()rlo^re)na) 6urssaroldypg 180t'9901'VaOLlDdVAquorl)nrlsap'uun)as JLSZ'Lgz-g}Z'uor1e:r;dary56 ' a r n l ) n r l s l € 9 ' ( s ) e q o r dv N U aiarlolluer S 0 9I S S S !61Z'6ZZ 'SBS'uorlprpdarda;dues {do:sot:rru '6uruorl>a5 'uotlezrre;od l z L Z ' ZL Z ' u o l l e ) l l d evJN c ' s l a L U l JvoN U 7y61 ;;a: 'a1:i(r '1S0 (s)al)rsa^ L 9 €l - O t E ' 6 € t ' l l la s e r a u r ^ l oVdN U )rldeu^Saassasdeu,{s t-9SOI l9S0 t ;1a> 'r.!reds '€96'sutalordlaqlo ql!/v1 8 S E ' U O r l e U r u rueol l l d l l ) s u e r l suoll)eralu!'url)e €62L l€96 '€Og'o6rer;o Eurssa:old €t€ lolPrparu;euotldu:suer1 >ry(1oato.rd aotsnatatsat'Quotoqtto5 l€08 Onnlt Vt' *tZnE'siole^ll)e leuolldlllsuer] 5 6 9 ' 6 u r 1 : ( r aar u e i q u a u i 8 0 t t ' , ( ) g s )a s e u r l9 s 'uorsn,auerqureur 'urso(tu'luau6e.rj g7g'sutalold 6ur(Ipou.t-utleuot q: €OB [S 6161, 'ZVE'208'uorleLuroJ f t E ' u o r l e ! l l uu! o l t d t t t s u e t t J E n €1 ' . f i 108 I t08 '(Cff) 9 V € ' I V E 's^ u r e u l o pl e u r u r a l - ) S rz68'srsolboxa '0t€-6€€ aserauJ^lod gBZt '(tun^o)66a leualleq J0tE 'arnllnJls l0tE 0 0 8 ' ( s ) a l l r s e^^r o l a r ) e s 'suor]>unf 'stsol,{:oxa'(s)utato.rd , & o t : ey;p g , , (epllloH '8AnU i(ro}er:as I L b E 'g V E , l d e : u o r ,'^lrArssa)ord 008 lZ [€ 'suolrru nf {PpllloH'VAnU (s)al)rse^ 8S€-1S€ fiolarlas aas(s)alnuer6i(ro1alra5 lZ t € 'sllat a6pa 6urpealle '6utg;ng 'leuruual-) ^roiarlas (llll) leJaua6'srol:e1 uotldt.ttsuerlosloaas €8t-Z8t l0t0 | Jo 'luauoduro:,(.ro]a.r>as lelaua6 asel,{xoqje:aleqdsoqdstqasolnqlUaaso)slqnU ZnE-On€'srolre;uotld' t.t:suerl J9SS[ 11 d55 ()1u) saseull I O 7 E ' 6 E E a s e r a t u ( ; oY lt6'aselarlas-/" (dWV))dtVtt1>A1os1oaas autsot,(1loldalagaas(aseullautso.r(1lolda:al1y15 t 9 E ' O 9 EI' O r € '6 E E ' 1a s e r a u { | o dY 5 g sawAzualDnptLtpu!oslDaas aasVNUI puo)as VNU €GB'(s).la6uasseui 'zEaLleuosoqlu 'snJt^euro)lessnou '6821'se}/(loo - n h t ' u o t l P r e u a 6u o t s u a l l e l l l aqladns ,{.repuola5 J S V E ' S v t 66n| 1$zl 16821 'aJnllnlls 'uta}ordro}delal l'€€ ue6to proqdu,{1 }noqepunou t 68€ t !88€ q 6 n o r ' 1 9 3u1l n l n ) t } a l t € € ' t u s l u e q l a Lu ! oltleel ; e r a q d r r aa6a su e 6 r op r o q d u L ,{{r1e p u o : a 5 q6nog 8tt anrldepe'Lralsr{s/asuodsar auntuurloslDaas :ruse;dopu3aasurnln)r}ar:rr-useldopua ' L E E - 7 E 'E6 u r p u r q - y 1 6o l p a l d n o l l96tl'sasnllnelou 9 9 9 1 ' a s u o d s aa. u r n u r u r( l e p u o > a 5 'sutelotdaue.lqulaur'uolsn;Jlp sa6ueq> ygg 'sa.rngln: l e r n l l n r l s ' u o t l l e l a l uVt N C - u l a l o j d fuepuo>a5 leuollelou Zt9 g6LL'56LL's11err11ar,fuepuo:a5 S€€-t€€'6urPear;oo.td nzl'6611'aseqlui{sasolnlle)'sa}}asou 'asuodsar(poqrlue ,tepuora5 raloulold 1 6 9 ' 6 u t P u',uollelualro tq loou JL17L'JSOTL'slleq 19791 '6€€ ' , d e ) ' p u e 1 6 l6€€ s n o e l e q a 5 l o o u llott 1 ' u9o1r7l e1l n r l s e'bu r q r r ne a 5 t tal p t s o a ; r n u ? € € ' s a l e r l s q nasl e q d s o q d 'OLZ'Att)ept! lLjnL'dlt llot | S€t-t€€ lsovl-b)tl'arnlrnrls r € s LL '00t 'tuaudola^ap 'JZSL '6 '(s)uorlrunf 'spuerls u! l s € € Jo uorlnlo^a l u e l d 6 u r 1 e a 5 L lq611 100' L L |t uolldli)suP{ os/Daas (lllf) (l9vd) leraua6'srol)e, ltOtt'€9tt'ualsrreu le)roe uotldtl:suerl (s)roou 677'uor1e;nba.r srsaroqdorl>a1a d osp aas 1a6aprue;(r:e,{1o l - 0 t g ' 6 € € ' 1 1a1s e r a u { | o dY 1 5 l8t6'lq6rl olasuodsat LEg'JgLg'Its'l9vd-scs 1 0 ' € ' 6 € € ' 1 1a s e l a u { | o dY 1 5 u t a l o r da u e r q u L a u r l € € t l l e M a u a l 159 'luaul6as 'EEn ' "$r1tqn1os e t a t u { ; oY d 5g lalno l g t € ' 6 € € ' 1 a s'6€€ unrpos) scs L',leMauar r*v t JL:S LLi'1a1e11ns |r(rapop 'suoll)unJ 'apP)sel 'Z9Ot'surl)^r-S l-ot€ |SOL'vgOL J0e6'026-616 ',E?€',slleJJaurlPr'uorql ' a u a 6a ; n : 5 nnE 1*t ut uorDnpsuerl 999; 1 ou o t l e ) l ; l l d u e l e n s t n 1 e u 6 r s l 0 ? € ' O t € - 6 8 €'s)!laDraua ')rlo^re)na 9g11xrvn:5 # t L ' z t b L ' ( s p o r s) r o l d a ) a r o l o q d p o g l€€ s60l'aseulll)ou !Li\L'qSLL ',{trrelod 'xa1dr"uo> rol)e, uorle6uo;a vvE-EnE'uollel)ossP satulzoqrgiVNU)ri^le}e) oslDaas a;qqu:5 leseq-o:rdelerlaqtrda 0 O € - 6 6 2 ' 6 u r l d n o. r)r e d aVr N C 86tt'86€-16€'alde.l)S € 0 t - 2 0 ' ' p p o'197 i \ ^ V'ua1s(s NU-aJdurorluol]lsuer] 'leluno) d NC s ^s e s e r a u ^ l o V 6utle:1;dal-11as 9 € E ' b t € ' uollelltlut)S 109 37gy gg9'6utlaPoura ul l e u l o J q ) 9 0 t - ? 0 t ' u o l l ) e l a sl e r n l e u 8 1 9 ' u o r l ) n p o r du r l a ^ u r ' ( s ) l l au)u e / v \ q ) S uorldu:suetl l l a u e 6P a l t u l ; OVb'I6En' 6€n-ggy'uor1e1n6a.r L O I ' L a P CrD :9501

i8f-iYT"'"'J:{1:

X]CNI

ztTl

'uoslteoulof/atpoulot sueau s :alqe] e ol lalel f p qltM slaqunu abeo 1^laArln)asuo) Mollo, leqi sarn6U ol ll ue qlrM 'arnb4 e ol ra;ar I ue qlrM sraqurnu a6ed :^Jlua eqlJo uorssn)stp lxal rofeul e ot ralal arerploq ut siaqulnu a6pd

y9g'6ur;:{rer uotleutquo)aJ)uDaos-elts tSzL I t S € ' S S € - t 9 E ' l e u o s o a r r ; d3sy - 1 y l S g S ' S 9 S ' s r s a u a b e l npue p a r p - e l r S \ L ZL ' L ZL - 9 2 L ' U o d s u e rul r a l o l d ' s r s a q r o d ( q 1 5 's801-€80 '1uauq:e11e ' r L t € ' 9 E E' s r o l : e ;el euur b a;purds 615 399g X;qurasse Jt80 t 1 I6EV'6E 'uorleuuoJasdeu(s'suralo.rd qllapts 098-6t€'sdNUus 1? Lt 1680r'060r-6801 aulosoa)tlosos/Daas ! 8 8 0 t ' 8 8 0 L 1 1 8 0 1 'L B O J ' , 5 1 0 L ' , u o r l e r e d a s 9 6 ' t ' P t u l a u el l a ) - a l l l t s ' 6 V t' V N U u s 'uor]nloseJ 'ure]oro EgE'ZSE'tEt 9n sl0 [ l0[ | l)rs 'VNUus 'LtoL-oLo 'tso 'uorseqo] a9€'6VE 9n l0L0l t. [ 'VNUus 6?€ tn ll€tz €tz')rlol!r.u 'vNUus ']rloreur esE'6nt zn €lz [ 199,7yg,6uruo;5-aiu'ao,,irholoqil z9€'r9€'6',8'VN r nU u s v s oL ' n z ' ( s ) p l l e u r o r qr )a l s r s l-6011 /sgSS J J t ' J J g j b L ' J L }JnLLL' €V9T ''0u lr V alsuaul IOVE'9€€'uo !ld!t:suerl JbLZ' t La | 'L Jo l)a;Ja'VN6 puerls-a16u15 l e ) r d el o o q s pallaJrp-pueJls ireoarq)lPr.usrru o5lDaas l t s € ' v N U U st s I' w0 L']sPa^Jo/oourqS,, lvvj sdNUuS aas(sdNUus) saxa;duo: uralotd J6LZ J 6 B ? ' g g ? ' O g € ' a : u a n boaus t e 6 ; e 6 - a u r q 5 'g L Z ' I L L Z ' J S g t ' n 9 t ' u o l l e z l l e ) ol el a l l n u L L a '( s ) j . t us\ l e e r qV N Cp u e . r l s - a ; 6 u r 5 1 1 6 y1 ' p r r . u s eal dl u a l n J t ^ /arnllnJls 6 u r : r ; d 5o s l oa a s l9lz ? z s[ ' l u e l s t s a r - ] t l o t q t ] u e /uorr)unj ')rlo^re)na 'g 'luauran paseq-utllp oul 19€€ 0gz lg t s t t s L 'uauxago11abtq5 l NC 15. g ' s u o r 1 : u n>;r / 1 e 1 e : J 9L Z ' J VL Z ' g t 7 ' f ua u r q : e u u o t l e l t l d a V 16tr1 'J9tL 'arnl)nlls 05€-6t€'svNUUs 9 0 € ' a l o ru o l l e z r p u q ^vqN c LnL J VL Z ' EL Z ' r ( t r n r l e r e d o o : J gI L 'gL t ' a 1 o r , { - r o } e61anr (sgss)suralo.ld6urpurq-y16pue:}s-a;6u15 986'926 'uou)unJ '368 '(ureu.rop {6o;ouoq )rS) ureuop (s)uralordauprquausuerf aassutalordssed-a;6u15 JZqE'I9tt !668 E EHS t9t'svNUous l 0 9 S ' 0 9 9 ' 6 u r d d e ua. ur a 6 1 e : r s I q d J 9 Sl ' 9 5 l ' s u o t l ) e l a l u t6 u r r 1 s - a : e ; t n s 'a>ueprn6 sllolq adltoldeH os/Daas l r o d s u e t lr e l n t r s a r 994 J V n LL ' n Ll g t t l l E € L - Z € t ' t € L ' e r n D n r l s J t 9L ' E 9L - 2 9L ' s l U V N Sa p r s a ^ l Z g S ' 6 u r l ) e r le ) u e l u e q u l 1 8^ ' I L L ' a l o r , { . r o l e l n 6 a r 'la6tel utxol snuelal 'uorlnlona auoua6 09/ 6 9 7 9 1 1 ' u o r 1 e ;of tq d s o q du r a l o r d 'Sg '6uoet1i(reuorlnlone a u e r q u r a ul a 6 r e l t J t g L ' E 9L - 2 9 L ' s l U V N S 0 9 9 ' ( s d N Ss)u s t q d r o L u ^ l oadp r l o a ; > n u - a 1 6 u r 5 t L ISS 'uotl)eralutrol)ejja qeu '8€6-t€6 'slvts lgl 1 9L S '9 L S - Sl S ' s r s i i ; e uueo r s s a t d x;a1 a : - a61u r 5 l8E6 J 6 9L ' g gL ' ! E 9L ' , V g L - 2 9L ' u o r s naJ u e r q u a l r j J B L L ' g L L ' A l r L ur rPsJ ' ( s l u a u a l al e a l ) n up a s r a dZ(,8 s i a l ul ilo q s )s l N l S )i9L'n9L ^lquassesrp ll0Z 986 'prsdP)'(0? 'fSZ6'926-nZ6 'g68 'suralord6ur;eu6rs sureloro l6?t )0 t s n l t Au e t u r s 1668 ;;a> 'SUOrltsUel] luvNs ' u r 6 u oS pue L t LL ( u t e u i o p3 , { 6 o l o u o ql r s ) u t e u o p z H S 3 1 3 91 'sa:uare;rpi(6a1ergs l e t r ( q : u a s a L l - 1 e r ; a q l r d a , u r a l o t d Tyaouuagl u o r l p ) U r s r a A' (rnpl S )s n r ^ I ) u a r ) U a p o u n u u lut e t u J t s 79J 1 'uott)e ggzl-saz J'saluarajrrpsaDads 0€0[ l ur a l r s Jo lustueq)aul ?92'(s)uorlelnu '988/slassa^ poolq Ost t uorlPuruualapxes aasuorleutultalapxos JL96 '[96-096 /sura]ordoloqd ,Mor^ra^o (.LZL_6921 ttgtL'v9rt-E9tL'sF) 1ueld's.ro]da>alotoqd / x n l ] uu t rnDle) 0€0L aua6 I 168 77 1a;suer1 ' s l l a ]p r o l d r pletuoztJor.l allsnur qloours r o l d a : a rr e a ; : n u p t oldeq s 1 6 8 8 ' 1 5 8 6 g 9 ' I ; r u e ; r a d n s L ' J L Z I 6 9 2 ) j L e [ 1 9 5 , u r a 1 o rpda u a q l o o u t s (s)Dd) 0 € sl ' s r o l d a ) a ra l l l - l l o l ' 8 ) { J N qloorus'(Ul)r!nln)rlar ralord s1lartua6 aas Ll.ra6 luatrdoganap 1sase61y7su lprpJoruud ;;a: )ruseloopul aasunln)tlal ltujseldopuaqloou_lS 6urpurq-619>r.raurur) (s)uieloJd9 aassuralotd9 eozoleurad5lsrsoraW l(urn^o)661 osloaas s9t-b9E'uraiord (suolnauloloul Jo le^t^rns)NWs 606-S05 ijWV )rl)Al n L Z L - ZI Z J ' I' L ' u o r 1 : n p o ta d1 a u e 6 6 9 2 0 6 0 '97 7 ' s u r a l o r d orcL'fbbz uotleztlllJalaasuotleztltueJ 15 J 0 Z 60 ' ( 6 ' Z 0 6 ' ( u o r l e z r l r s u a s aupo)l l e l d e p P ' s n r r nx o d ;1; ey u 5 j 6 L Z I ' O S Z L - 6 I 2 1 '1 4 71 ' , { l r s r e n r) p uorllnpsuell 9671 1aua6 ' / s r a ) n p s u e lrel u o r s s v N U o u sa a ss v N Ur e l o a l ) n ul l e u l s t68 L?9 ! l L ZI ' Z L Z L - L L Z 1 ' a 6 e g u e n paen r l r l a d u t o > ;eu615 S v N U UaSa ss v N Ul P a l ) n ul l e u l s anrlradstedr{reuorgn lZ01'sad^r lona sdNUua Sa ss u t a l o i d o l ) n u o qiup a l l n u l l e u l s L'L-gZL VOEt-692 L'uotl)npoldaJIenxes 'gtodurr 'e)tu pasla^aJ-xas uralo.rduJnln)llaltrulse;dopuaqbnor 6-9'rfir;esranrun ?gz L auelqulaulsuell 1g9'ltodsue-r1 ZZL'ttodu uralold sauosrxotad t(qb'tLsv r so r l e z r l e r or el a l ) n u 'uolleu rLurelapxas o1tqdosot ( i e l n l l e )' l l e u r ss) a l n ) a l o uttr u e 6 t 6a a st r u e 6 r o g g 1' s 1 e u 6 u g' aue6 (FS)IDLllafxas saln)alour xas L L ' t l l e u s l n t L ' s u t e l o l d l p u p u o q ) o l t u r 068',auourioq au l nI ) e l o u l - l l e u s t LZS LZg',sJolrqtq lspldorolq) OZL-6 LL'surelo.rd l g Z t - O 8 Z l ' u o l l p l n 6 a ls t s o t a L r . i ' s a ) u a t ax#atsp autlselur ) g t n l ' Z n n F g E n 1 X 6 o l o 1 s r/ q (s)9d) sller u.ua6lerptotur.16 osloaas # 0 L ' f L 0L ' Z O t - L O L ' 1 s 1 a : u a n b1aesu 6 r 5 l l e u l s ggz L 'sat)adsuaaMlaquolleuP^ sadll ttJDadsosp aas 9 r€ € ' ( V N U l sV)N U6 u r . r a l a l ulrl e u _ r s 'suortelnu p o l e t ) o s slea ) u e t 6 u n ; 1 ; a r - 1 1 e u 5 9 S ZL - L E €tt t Z € t t ' Z € t [ ' ( s ) u o r i ] u n 6f u r l e l a r - ; e u 6 1 5 J 9 8 a t ,arn]lnrls '582 ppr.us W6' On6-6€.6,^ltLueJ lSzL.gzL !€82 t l - € 8 Z L ' E g Z a ' t u a u r d o ; a n appe u o 6 6 u r l e u 6 rlsu a p u a d e p - p p r u s 1976'(entqied oltrqdosot1 O tL ' B e L 'O E f -L Z L ' U o d s u p rul r a l o l d JZBV'fLgn'Zgn-Sgtr7a1so6ouo1aw ( d U S()s ) a l ) t u e du o r l r u 6 o : a t - 1 e u E r 5 ggz l-zgzl,'uotteutulralap xas S S Z tl a l u e ) l o ) ' a u a 5y p o w g , sl e u ]o)rel tl o su e r l Lt I L auosoL!oJq)-AlaurosouloJql-X os/Daas L Ol - S OL ' s l e u 6 ruso r l e z t l e ) ot pl a l t n u u lo r l t p p ed g w ; e u r I q : u a s a u - ; e r l a q l r d a , u r a l o t d T a6una165 xas 9SZ',uotltuoo)a f i g z l ' LL ZI ' , p u eu o r l e u t u r e t a p '?s€-€5€ ' 6 u u r e d: r g o l a l u rrsE iiNU J L OL ' ( O L - L O L ' ( s e ) q r l el e du 6 r S 9131 ' u r a l o r dt S 1r;g 6 8 SL 3 8 9 €t ILLL l b s g ' EL v ' ( 0 ( ' $ ) a u l o s o r u o r qx)e s j L g Z ' l SL ( , y 1 7 d L r e p r6 u r p r 1 5 'I L L -L L L ' I 6 o ; e u e , , d l q > o r r t u t , , ' s l r o M 6u l aru; e u b t 5 La6'0ltLldosuO'u taloJd^aS I W L -L L bl ' s l u a u a r r n b a slonqtodTsadtrl l e u o t l l u n Ja n l y o d d n s lZ6' DI rLldosoi3'luelnul ssa/UaaS /slla)rloltos luau.tolat'a)ua)sauas enlle>l;dat €62'sleada.r )UDads!siuauodwotyslonqwd >ry>adsoqo aas !99(.L'SgZl-VgZ1'lueuldo;anep ',uetleurulew l8 Lt L laDlulsuerlolnau l l 8 8 tg9'alol 'uorl)unJ 'Z8B-989'saln)alour 'unr;aqtrda ggtr 6ul;eu6rs I tt t ,(lolelrdsar;o s;;a)auu)opua I ;o alel ranournt gzal IS6a la)ue) u o r l ) n p s u e r;le u 6 1a5a su o r l : n p s u e t;ge u 6 r s ueld,(.rMxo-rp^q-S) uruoloras ?06'(-LH-S'aur 'gLV ,alnl)alrq)te g g 6 ' s a s e l e q d s o qadu t u o a l q V a u u a S JgLVL L l € 9 - 0 8 9 3 1 7 9 ' s u r a l o l d6 u r ; e u 6 r s ,sue:{l6oalord sasout\ Egt t lDnpt^tpurosloaas 'sa)r^ap 'uorlelnbel lndlno-lndul se sesput)i JII L ll I 'palurl-ro]daler (s)i(_eM qied/ (s)aIn) aIo u 111[ l e u o t sl e l n l l a ) e r l ual a sl e l n l l a l e r l u l 6S6_9S5.slueld (s){eMq}ed/(s)aln)aloLU J O V 66' 8 6 ' 6 u r ; e u 6 ;r;sa : l e u D rise l n l l a l e j u l a a si p l n l l e ) e t l x a 1 t 6 ' s a s e u t la u t u o a J q l / o u u e s ,aln])nl]s ,autJes 0zI t ')stc 16z [ 'g€ 'sat6oloLuoq (s)luarPer6 lg€ t t-1t t lprnl)nrls >rua6oqdroyliluaLudola^ecoslDaas JOt t 'h '6€ 'uolrnlora{11Lre; J8t€t !l tt t'Zt€ t-9t€ t,sua6oqd.rou.r ulalotd t1n L t-B€ L t 6 [ [ u o t l e p e r o oxpU ] e L tue l n l l a l e J u a 'alls a^rDe ls9 L ts t (s)aseatord auuas luaudole^ep oltqdosdo osl?aas 0 Z €t - 6 t € l ' u o t l ) n p u t ' 6 u u a a u r 6 u>ar l a u a 6' s 6 e;lae trul uaannbbaaS5 619 'surldas 1 6 0L ' 1 : o q s: r 1 d a 5 7 g gI J t S LL , 9 St l - ? S [ 1 , s u o r 1 : u naf1 e 1 d a 5 'srsdes ? € s[ ' 'aseiedes S L Z LL 8 0 L ?[t[']ue]nur srsdoprqoty'o1o11oda5

)sLL'rvLLrrr'!V)YuJ, ffiaffi :

1'.il;il ,rrr,,ilii.r; 1,",:i

'lLalt',gzol ',",nrrn.,ill5 J6ZO:'6201 !z LoL Lgn J-EgnL,uorleraua6al lgn J-egn l, uotleInpoul ,slsaue6 4gg 1-ggj 1 (s)reqtJ al)snw os/Daas ,slla)arrllales 99t t /uorlettuaraJJtp lt9t'tgt ',azrs E 9 n L ' $ Z j L ' 9 2 0t l l'slla) al €9?| (s)urso^tAJ lsluaupl!, utllp/ut])V os/Daas '€00 ,stuaLuelt, uil)P z L0L t I g n L - € 9 nJ , a P s n u l e t a l a ) s asesodsuelfiaset6a1u1 oqo aas uotltsodsuellaas;euorlrsodsuetl gZE-SZE', w Z t ' n Z €.'e^rlP^lasuo) sursru eq)atx 'SgS'suotlelnul l99S I S S V ' S 9 V ' l r 1 t ' 9 7 6 ' l u a w a 6 u e , e aar u o u a 6 'lolluo) aua6 9Zt-SZE sa)uanoasuo) gZE-SZE,suorletrldde

l8r€t '6191-gl€ 'peards f ; e u 6 r1 s os r u s r u e q t e u g1€t-lt€l,slolrqrqul .7 gZE1-SZEL'suo6a1a l _ 9 t €t , 9 t € l , l e l u e u d o l a ^ a p z L l L ' s n aL ' I E J L - g E t t ' p u e u o r s e q p e . l l a ) 6[[[',seseose) 'alor arue) r ItvzL'gvetzvz L t Z [ [ ' s u r a l o r d^ l u o - € H g (s)IpMqred/(s)elnra;ou 6ur1eu6r5 0 8 8 , ( s ) l l a6) u r 1 e u 6 r 5 S 5 g ' s a p e l s p 6) u l ; e u 6 1 5

€t:l

X]CNI

'Pupuoq)o]luI 'aj!l ee,t,snqophns, spuoq apulnsrcaasspuoq s-s l9 [8 l9 t Jo 'S8z 'sgz 'ura1ord7aua6 eD6nspn pt^tputlsapDeqllps^lod /.r5 L 6 ZL ' V 6 eL ' u o ! l P l ! ) P d P l t t-€gz f JggZt 'zs€ 'sulalord uo!lPz!l!lraloslDaas :sapueq))esouowisaleJp^qoqipJoslpaas €s€ us '662 'pu? uollleej (dUS)(s)al)rueduortru6o:er-leu6rS aasdUS L le)luo) 100€t l€ t t lz t L'lL9'gs-ss'ernl)nrls aurosor)e q)lets oslr"as L6al'uopear L'862L' 166Z [ ' a r n D n r ] s J S L \ ' 1 9 € L ' 9 € '66a or 6urpurq 'aDerols '8Ll'uo!leln6aj tOlZ I J86ZL'662L-8621 l?6 )8Lt 'fuanrlap y16 gs 'uotlPzuau^lod ro; uorleldepe E6ZJ-z6zl L L ' a l o i 1 6 L l ' 8 l e u o l l l u n J '89-S5 'a)rnos^6raue uralorolls 962 l-Z6Z J'eozoleurads IbzS'L6-96 188 'eruo6oleurad5 ' a s e u re l lll-lrs ',Sa^rle^uap '6ulleu6ls 9671 It I I 1 9 6 5 | l l a ) 8 u r e u o p € H Sa a s( € H Su) r e u o p g I 6 o ; o t u o qr t 5 J g 6 aI ' g G Z| - V6 Z ! ' e r i l : u r ( s 9 5 ' s l ) o l q 6 u r p p n qs e srseqlu^solorld os/Daas u r e u o p Z H Sa a s( Z H Su) r e u o p 7 i ( 6 o 1 o u o q: r 5 t-€62 I'so6els ! 5 6 Zt h 6 Z L ' t G Z'eruo6oleurads 'arnl]nJ]s g671 lsvg'9n8-Eb8 l8l [ '69 'srsaqlulsoloqdernsrsaqlu{sorq sali(:oleu-r.radS aassaM)oleurads Jraloro)rs aaslrs !01 'E6Zt'sprleurads 'gLL'uorle1n6ar b6zL ll€r t_Zl t,gg_99,re6n5 JsLL g6al- 962L'6ulle;n6a.rsaua6 9t8',1ls'aso.Dns [€9'sauerquieul 'uorlnlo^a 'ese1aq1u(s srsaua6oleulrad5 yo1-;i(ur::n5 1971 111[ 'F.6Zl'fuPPuo)es 'ra)ue) s u o t l e l a p o 1 6 u t p e a l b6ZL z € z| J E ZL ' J Z ZL ' Y o ) 1 , { u t : : n 5 ' J z z L' a t e u r ) l n s 'saseurl ^lluleJ )ls uralord;o aursoj,Q 9 671'Atewud tEzt 986 ' u rs r s o r a u r ' ] u r n b s '(saln)alourorlPur) slrunqns t8z[ zs t-8t 1 9 6 €[ 'sa1{roleurad5 (s)pue6rlos/Daas 9671 t6L9 '6Lg'ILg'stuawuadxa 'uoxe1uet6prnb5 n 6 zr ' t 6 z L ' s p ! l P u l l a d s 09 t-6S t' g7-7 7'6utpwq eurlzua'(s)ateitsqns lp)rssel) ' s e L U e n b S ' 6 L V l eozoleutadsaasullads lSttt J6LnL ' 'sra!rPl uorl)alo '8 'urls (srLulapod^q) la^el snoaue]nlqnS (uds)alueuosaruorlrseloa)P,lnsaasuds [? t lz€8 I €8 ]o 'arnl]nrls '(s)alods 'uorl)p apou 'autuql^rls [ 800"{dolsorlleds t89 692 t Jo 'uorssar6ord rournl ur alor JLtg' gtg' uopplso1l(r auerquau ZSS'aqwod'S 3771 'uorleLUJoJ 'utatord]Ut)ads-stsotaLu qaM lueurellJullle 800t I tods J 1 ZL ' O ZL - 6 Ll , u o d s u e r l u r a l o i d 082[ ' 6 u r 1 : e d ' g77g'pue;6(retutueu t u a u e l Uu t D e 1y66 l 9 9 E ' 9 9 € l' z s t ' z s E ' J 6 f i 6 n E ' s l e u 6 t6su t r r ; d 5 ' b u r l u r l s s o ju)r l l e 'JEbg'Zbg'OZL-6t 'slseldorolql Jl00 | ESg L t1 l69E'69€',VNUl ' , l o r l u otslEl s e r o d o u a 'qs l l a ) uul)aos ) 8 9 b 1 ' 6 S n L - S St V J y S E ' S S E '-379S5€5'' 6 u1 r >t sr ; d s - s u o . r t 'ral)od ,(1t:t;t:ed5 'utelord sa 'eLUoJ]s 'ES€-ZS€ art;ds [ ]HW J6LSI'\LSL-LLS s9 [ t J€S€ t '679 'sleubrs 'sraqussarls ,gZ sa]€) uorlelnur'a)ua6te^tpsat)edS €60[ 3679 '997 'uot1eln6al /asealar 'sserls 6t-8€'st^aDl snoouax €9? 369tr 'SSE'Ilr)rlseld losrl.rol 'uDIuroldertS 9 8 Z L - 9 8 2 J ' Z 8 ZL ' , u o r l ) n p o r o el Pr n x a s Ogt fSg€ 'uollPulLuralap 'snDooldals 'surorosr xas L 982 t05t ItL s9tt lla) al)snul q) s aUtlelts 902-bOZ'saurosouror JSOZ' tttt'ulntlaqllda snoulpnbp LVt',uorle)uualsasuell JSrt' . sa)uaraJJrp saDoos yyg 6ur>r1ds-11e9 aas6utrtlds-1;as 'urPr.uop ' g 0 t ' u o r l e u r q | . U o :san. o r 6 o ; o u o90€ uralordzHs p tu e J l S q'uotse^u I l€€ I r 9 E 'ZS€' t 9 E - 0 S €' s l u a u a 6 u e r e a r ltt t'salnralour uralord fS€ VNU-VNU JLLZ 'gLZ-g 'asPalar 1tr91'awAzosAl tZ'Z LZ'.redalqlleulstut pal)al|p-puetls z9€ (s)e|.UosoalrldS aas{rautqleu.t 0 s ' s l a P o u6r u t 1 1 t ; - a : e d 5 Lqt \L9t'suopo) dols 'utalotd '1t€ 'arnl)nr]slerrel 1d5 19 1y'uotlru6otalaruanbasy116 9?8'eleurols J0s€ !8t€ '6urrred-aseq .8zz ,sra)tn gBZt 'lueudola^ap selsel'uraloJdpua6 6xo5 1g g €0st t g€?L ,uJntlaqttoa l 6 € S , 0 t 9 - 6 € S , 6 u r s o ;u qr a q l n o s ltgE l09E'8t€-lt€'sursrueq)au .rredaryy6 osloaas t09r'6zzL atg- Ln€,1e^oLuaruoJtul 'asuodsar '8ZZ 'uollel)osse uo1,(d npoqotlaH laluel I [€-0 Lt sos t ilgn 'uotleztuolo) ' ssa/uaas-Jo-uos 826'olu.ldosoJo'luelnul €0s[ J LgV'Zgb- Lgn'uollPu!ulralapxasDl tldosuo lPua])eq a g: t u o 5 qleuols uotssaldxeaua6 0 s 6 ' u l a r o r d6 o q a 6' P Lgt- LLI' uorlerl.Ear 'LLEI (s)alruos gyg'a6eluenpe,iJeuottnlona urelotdg {to}elnurls IzLEL 1616'LO6-906,1s91 ',u!lelsoleulos 'uorlnlo^a ,Ideraqr 806 1931 19€-SSE qlterure)nelsnoua6oleIu.r )ruorql'(le^aalg) L'xalJor lZ6€ ;e.rqala:'uot6at,{losuaso}Pu.loS L l € ' i l o , ) e z s t ' [Sl-lIS l s s € ' s s E ' J Z s € ' 'uotlP)ollelnlla) l l 0 S ' 8 O g - 1 0 s ' u o t l e l u e l d s u et el la l ) n u) r l P u r o S 0 2 9 - 8 1 9 ' ( s ) p r der lu e r q u a u ' ( s ) l o i a l s 99€ (s)uotlelnWia)ue) oslDaas l9 t ['arn]lnrls'sprorels I t s € ' t 9 € - € 5 8 ' 6 u t : t l d s1 Y - 1 Y 6ur:r;dsanrleutallVaasa^tleulalle 6 0 z L ' g o z l ' s 9 z ' ( s ) u o r l e l n ul r l e L u o s 1609'PrlI)orJets 'spuoq aplldad 'suoltellull '9t€'6ur)rldS )lrels I L g SL AnE-tn€ 11z L ' ',satls ',UOrSr^ >t}euos e)rlos Oarels tgs J-ggg a'sarpoqrluP'uottelnullad{q 109€ 96€L 6 0 ZL ' S O Z a ' 9 9 2 ' s u o r l e l n u r ) t 9 nL ' r o t b l ' 0 € ' | ' e r l r ) o a r e t s lt5€'99€-t9€',euJosoa)llos-suDJJ 'zs€ 'sulalold raJnl)nrlsy5g 'sdool-ua15 J6Vt l'gni L'uolleulqLuolelpa)npuI €s€ us l€0? 'ZSt'sluaue6uerlear p e ) n p u ll l o q s - l e a q 6ur1eu6rs'eq)tu ula}S 8 t t [ ' u o r ] e u r q u r o ) a i VNU-VNU JZVVI' Lnnl'sr{eir,rq}ed €S€3 t S€ t S€ ' 'uollre apour '(l)s) rope, lla) urals ls9z'suorl)unJ sIE t lOs€ Ls€-Os€ Jo lla) '(s)lla))rleulos t1z [ 6 1 7 . 1 ' g l Z J - L J Z1 l e ) u e l u r a l o r Z S € ' s t u s t u e q ) e^ul ll l a P U 'releM'(s)tua^loS '!lzn 'zzb 'uorl)npord t 160 vzv| LSE-SSE'uorlnlo^a L l- LznL 'aua66o5 g9tl-ogtl')!latodoureq'lualod!llnuJ aat svNUus os/D 9ggI ',sluauootllol (s!F)oaws)os J99t'aua6 lSZn L' gZVlnzn 1'srsaqlod{qpuei}slp}.roulurl zs€ 309t SVNU:ry{;eler s;;aruuals:rlarodouragaasrrgarodoulaq Ll9',AlrArl)elas Z S € lzgg'ap(t 6utdund dlv L E nL _ q E lV, u n l ; a q l r d al n 6 zs€-ts€ srs^lorp^q 'uottelndod 699'uot1e;n6at:t1ou-tso l?z?L ltg€',?s€-€s€'aulosoe)llos)v-fv ; ; a :l a p u n o ; Jagg' €gg- Jgg' (asedlv)dund untsselod-ulntpos OS€'^lquasse EZn|-llnl leuilaprde 'alrsaArl)e 6 S 9 ' r a u o d u l l sp a l u t l - u n r P o S z5€ EgnJ-9 Ln a'6 urraaur6ua '(s)auosoerrlds ' gs9're6ueq)xaleuoqlel slla) (Sl) Lr.rals )ruo^rqLulaas(Sl) )tuo^iqua Jnst J9E-6FE'L9t-6Vt 'srsI;euea;:{r u a 6 o r p , { q - a p t t o ; }qur a p u e d a p uut n t p o 5 Dpstds s z v L ' 0 z r L ' a l e ru o r s r ^ r p 9961 11ar'}uaudo;a^ap L€9'J8tS llt'euseldoltds J L Z Vt ' L Z nL ' s a 1 e11; a : - l a 1 q 6 n ;eop, { t 1 e u t u , { s e '1 '(l9vd-scs) srsaroqdolllele 'aJnlrnlls'salaeq)orlos ts v a n a -L a n J ' ( s ) l l a )u a l s l06t I '(rolreJ 1a6aprueli(r:e{;odare;1nsl^lapop runtpos S1t t €66 (poq a;od alPutd5 llo)-uals lllS) torle, lears ' p r ) e) u e a l s (alPJlns g g g 1 ' l u t o d p a q : ^ l q u a s s ea l P u r d S l t [ [ .uruqrels untpo5 aasa}ejlns SCS ;,{:epop 1,{:apopr,unrposl sgE-bgq'uorlelnu] 000t 3s66 (s)lauuPq)urnrpospaleo uralord11y15'paluaqur'^qd:]lt,1Tl::iL.-Y]ds E 9 ' ' a s e a l al o s t U o ) ' u o t l e ^ i e l s 'aJnlln)anssrl 'ursrueq)aul -a6e1;otr unlpos aaspale6-46^ello^'(s)lauuPq) t09 J€€/ 'uodsuerl uralordg3 '(s)leu6ts '98€ 'luauidola^ap tejsuell-uelS 099'labueq)xaulnl)le)-ulnlpos L Zt1 t [-S8€ l98E 6ur:r1dg:sypgu5osloaas pro) l e u l o s s0tt'990r ' t 9 0 t ' S S 0 l' a 1 > ( : €98 Alquesse S nL - W L ' 8 Z g! 6 1 9 'I L g 'a u t s o 6 u r q d 5 ; ; e r ; o t u r o d( u o r t ) r r l s aur )e l s g6's;saqlu(s tq d 5 I t s € 3 0 9 €a N u u s9 n s vL - n vL ' L g 'g L 9 ' ut f 6 ; a l u o 6 u 'alnl)nlls sadfi >tltads osloaas I t s € 3 0 9 8 d N u u s9 n lt6 g a g 'O Z g - 8 L g(' s ) p r d a r lu e r q u i a u ' ( s ) p r d r l o 6 u r q d S l 0 9 E d N U u st n t Z n S ' t V g '5 6 t ' 6 ' s l s e l d o r o l q ) apA> yal npun aasaP^l lla) Jo oseqd S l0S€ ijNUusaldr'rl9/9/tn )bg' LnB'slseldol^urP ^ 'dNuus zn aasuoozolPulladS t s€tost 'dNUus PozoleurJadS sernuelD I 'Is€ ,srsoqtu^solq tn ts€ tOs€ I 9tg ! E 6 ZL ' t 6 Z L - Z 6 Z[ ' a r n ] ) n r ] s 'rustueq>au.r 6ut:t1ds qlrels aat uotl)nPotd srsaua6oleurtad5 zs€109€ 'alnor 'sD)ool{rldDis urs n)ald aas s.losl sailroleuladS uorllerur ls9€'suraloro f 0SL 'saleruorl)eeJ'aseal)nu aUprquleuleulselo lr9€'tsg-€g€aNU'lS I l9 | )9V9'SV9'SuteuJop lerrorol{qdel5 '€62 '^lrlrlotu VNU-VNU ZS€-t S€']uauaOuelleal [ l€62 t lsSs'6lS'SSS-'SS'lerureq)olsrq'6urure15

X]CNI

tvil

uosueuuoJ/dJeuuoJ suedu sA :elqel e ol laJeJI9 qllM sJaqunu ooeo l^le^lln)asuol ^ olloj leql saJnbu ol JJ ue qllM /arn6u e ol raJar I ue qlr/n sreqLunu a6ed i^Jlue eqlJo uotssn)stp lxal roleu e ot lejal ererploq ut sreqLunu a6ed

'JLnL '€€€ L ' l V l t - 0 ' [ [ ' u o t l p 6 e r 6 a sl e l u a u l d o l a n a p l?€€ l Z V t ' J J L b t ' O V t ' ( d E l )u r a l o r d6 u r p u r q - y 1 y 1 't9 'w 'JE'S-E 'V-t.'uorssruJsuei] ensStlantl)auuol aasa^tl)auuo) uotletrjlorul 8LV ' 9 9 2' r t o ( ' { O O Z ' 0 0'2V N C 'alor 8 0 I I ' Z O LL ' p u eq l m o r 6; 1 e : uorlenualle uorldu)suell'aJnl)nJls 1892'JLge'J992 VNUleI (hlll) xuleLurelnllalerlxl l € ' t - E ' u o r l e z r r a u { 1 o dp a 1 e l d u a 1 6 t t t - g t t t ' s r s o l d o d e ' s u l e l o t1da 6 t e 1 '6ur66er-del 1(s)uotDunf lle) luorseqpela) osp aas l1s0 r 9 LS-9tS ' ' I(,NLL L S O1 I L S g ' l S S ' ( s ) u o t l e l nauA t l t s u a s - a J n l e J a d L u a f Z Z S t ( . 5 - 6 L S ' ^ l l a u l o t l l a dsss e u u e p u p l Ido>sor:rruuorl)ele uorssrulsuell aasWlf 3 t t t t ' , l t l t - 0 ? l l ' , l u e | . U u o s sep^ t l ) e t e s 9r s _ tss '0t 'uorsaqpe ' 9 9 6 1 ' a s e q d o l a l a^rlreles Jot t t l6t t t I 1-6€ t t uapuel 0601lElo t {6ur66eydet)6ur66eluorle:rJund(1ru;;4e ,uosoclsuel] a ) u a ) s a u a lsl a l a ^ r l e ] | l d a r t z t I L ' z E LL - L ELL ' s u o r D e r a l ul lra ) - l l a ) L 0 LL ' € 6 2 ' , p u e Ig [€ €uJPf PUtU.lel aseraurolefos/Daas lesegaaseututelleseq 101[t'0ltt'u!tel'e6Z'sdoofl (s)anssrl aln)alour{poqrlue'uor6at:g aasuor6ar1re1 JE6e '6ur1no.rds ^lelltdel 1la) drf 1y7;. ' L Z ' u o r l eItrd e r J n6 Z 'Z 6 Zl'O L Z O l l g ' 9 9 ' u o t l e z u e L u ^ l o d l t ef lzgt,utalold ssalauJtf pa66e1 € 6 2 ' s s oal n r l e r r ; d e . r I LS-699'slrruoua6 leuotllunr'slno)i:ou1 6 qL)Le'uJ'8UTo1Ld9uLt I ' L T L _ 9 L L Z6Z'uotle^lesuo:{teuolln;ona 693'6urpear;oord'aseraurl;od 11 'ursrue u t a l o l dl e u p u o q ) o l t u l 0 t Z , s a l u a n b aV s N Ce A l l ! l a d e t 1 J 7 ' a 6 e q d o u a l > e4q l t u ' u o t t e t ) o s s ea u e l q u l a u l f V L Z I ' n R L ' p u e 6 u u r e d6 o ; o u t o qr r l o ; a u l sL/-nlL.xeld|.Uol wlI )V6Z'E6Z'$ee( I 3 7 9 y ' a u a 61 : o 1 ti u 4 L 0 LI ' t 6 ( ' a l c ' la f u a fs a u e s 8u'€zwu_ 7 6 Z - E 6 Z , u o r 1 e ; n 6qa1r6 u a 1 gg11 'suralolduotsaqpeauplqulausuell urleLuorqlolalaHaaselo.tutleuoJqlojalaq trlaqgu,(5 699,ad^louaqd ',ra)ue) I t 5 t t t s t L ' l r o o s u e Jl le l n l l a ) s u e l l I IZ | 69S'^l!leqlel11aq1ur(5 '(s)araurolal g9p' A5olotq:rgaqlur(5 l E s Lt l z s L L ' t 9 t t - z s t t ' a r n l ) n r l s t6Z-Z5Z 1 19tr' 'asnou-ueunq saseldr.r>suetl aslaAeUoslDaas S S t t - € S t t ' s u r a l o r dp ; o g e : s Jg0z ' s t a u l e qr ( 1 r ; r q e e u l .sr e 'eJnllnrls ' 'po^rasuo) ad lzsll'zsl !- tg t t lz6z osz l0z 'uotlnloneeuoua6 'sl)olq eloallnu z s I [ , u o d s u e . lr] e l n l l a ) e l e d € 9 € ' ^ l q u r e s sr P OSZ 'pue a]uetseuas I t S t t ' S S t t - € S l l ' s a x e l d L l o l; e u o r l : u n f ^ualu^S 1.0[ t lla) €s11-09t1,suorl)unj sos' n6z-z6z'aseraurolaf Itgl'tuvNs-l'urxeluls '908'sllar;erlaquda ',spr)e)roqlref /uotlelnu snou{uou{5 L S LL - 0 S L I'r7tll'Ett t 129! ItZ /azts 'stlsltoqtaulg 8 9 1! - O 9 t t ' ( s ) u o r l ) u n [ ] q 6 1 1 auoua6 t6tt-z6EL fgl 'dpu lellalourlar pue r(1rrr;Deds ?8tt-€8tt'suelapu^s ' 1 6 € ' : r leuoJnau 1 d o' u n 1 r a 1 '[-uP)apu^S ru+weJol f tod t I t 6 Et tt8 [ t 'slla] ptoJ^qf 1106'069'auoulloq el)snru L € ? L I ! 0 8 ' , t ' 0 € t L ' a u p r q u J a u r t9t[ l e u o ] l a l 'o^rqure 98St-ggs l , u o r t ) a l a s Dlqdosu] l l a ) I 106€'auoradeq) O€€ t l-d)f t8tt 'ery{ruI5 8 8 S l - Z g S l ' u o r s s a r d xuar a l o r dt r ; r > a d s - u e 6 r o 6€Vt 6661 ,uorlezrue6to 'sa)tnos^pt-y la)uel lpllaJolo) 'utalold ^Joleln6araue6 y331 3ggg1. uotlotq:u{5 6Zg 'uottelutpour 9 8 5t - S 8 S t I [ ? S t ' , s n u r ^ q I t L sI ' l o L s L ' t L s t - o t s L ' a r n l r n r l s 96€t-€6€ t 'uolle)Utpout 'uotlrunJ 666 luaulelUutlle ,utsoullq-L 6 u r ; e u 6 r s asdeu(s ul {1rnr1te 1 0 6 5L ;e>rr1:a;a;oIuotq:u,(5 lZ€€lt0€ t9L l'ernl)nlls 1 0 8 9 rt 6 l s r ' 6 1 9 1 - L L i l 9 8 Z ' s l l a lp a z t u o r q l u ^ S '9LZa-SLZL'!l 'xaldwo: t 16z' 96z'uotlerr,oj lau.ltp tflS['suorl)erelurua6rlueTulagord fS LZI LZL ;euauoldeu{5 '6uured-aseq ' u rlegqy)y^ 196; lls L It9L'IUVNS-^'ur^arqoldpu^S 'aurLu^ql /uot]eDosse xalduJor€C) (s)JallrursuerlornaN 16 t os/Daas l06S [ 809-ao9'Jngz'tgz-zsz 965t-S65 t "s roldelar llal-g 1608'aJnl)nrls '(qdel6orpetolneaurprr.u,(ql-gr,aur 'Ul u! ^lqurassP prtu{q1 891 tog'uotsnJaueJqulau.l 'ol uolleluasaldua6tlue l€t8'€'8,a)pds pro)el^qf trldeu{5 069t B0g-209,(s)al)tsan 'LIga-OtSt'(s)rolda)ar 969'uotielaua6,salltse^ J669t !0BS t lla) I l Z g g ' Z g 8 ' 6 u r ; e u 6 r: rsl d e u i ( 5 'esuodsaiaunurultpaletpaul-lla) utelord J j Z L ' O ZL - 6 | L ' u o d s u e r l )or l d e u I S otg I L 6 E L 9 6 t 1 ' 9 6 9 1 ' 6 u r l a p o u a i ' s u o t l ) e u u f 'ua6r1ue lzsS l€t8'tvg'JE IL,auerqrrraur l u a p u a d a p u t - l l al ) 6 6 SL - 8 6 St 2 8 9 ' u o ! l ! u u e p ' ( s ) u arl|)l d e u ^ s ',]uaudole^ap ,srsdeu{5 t V g ,€ v g - Z n g0' Zt _ 6 t L , ( s ) p r o l e l l q l tL89L'L8St',98s[ llo) I J g L Z L ' S L Z1t 9 0 €! S 0 S ',eln])nJls f 6 6 SL ' 6 6 5l ' u r a l o j d 1 { q 1 (s)el)tsa^)tldeu^Saassal)tse^ l66S t ',urquJorql (rf) sllal-I )txoloIA) osp aas 65 L Z89,Uel))rldeuls 'ainDnlls 'auluoalqf 16Z|' I [8St t-089r'r8S1-089]'8C) JZBB,Zgg,1gg,6urleu6rs (Hl) (s)lle)-1radlaHosp aas 6 €t ' s r s ^ l e u pu r a l o l dl p u o t l e l n d r , u o : , 6 u r p e a t q l L 6 EL - 9 6 81 , 5 6 91 , 6 u r l a p o u a r '691g-9911 ,uorletaua6a.r t6r9t'6vs t']rnp )r)Proql I r89t J_089r'185t-089r'tcf 1 8 9 [[ urso^y!os/Daas srolda:ar-o: 1;ar1 l6tI L'eJnDnlrs/uorlezrue6to (s)loloa)aJ lzL0['s]uauelu)iltql ls6€ L I8 t' !0 L' oLulluoLuo6010wtaq1 ftrArlre le)ul)ale uo atuapuadap'uotle)trtpoul lla) 1:(s)lla)t ^Joleln6au1(rt)slla)-l 'aj!l ae,l,wnqoDrwowtaql 'suort>unf6uri(e1ar-1eu6rs rxoror^) i(s)llar-fgC) l(s)lla)-ftC) os/paas lg t lo Z€[ [ 1e)t60lounuult '6tt l9t,ajll Jo adl,wry!]owraql L l S t ' s l l a )) ! l u p u a pp a l e A l l r e u o u ' , a ) u e i a l o t lStt t 1 - 8 t 1 [ ' p u e s u r a ] o r dp l o j J p ) s (6taua aall oslaaas 'pue saln)elou uorsaqpe JL89L anlJ-lvaL lla) 'Melpuolas '98S -Sgs '98S 'snur^ql u! uorDalas JStt'L9_99 f f t-98s t v 6 E l t 6 t | t t ' , uorleurroj .99_19'mel 'ourlser 's6€ 3 6 t 'uo!]eulurla rsr!l ,,, , l}'s | l?6€ t [-86€ [ 'tss 'uollelnrr)aj 'leluDala Osst t-6i,s t t9 t t ' 1 9,19'79_99'Adogua -99,s)lueuApo(Ulaqf ll6l l-gl l (s)asdeu^s aasleltueq) s l , s l ' 6 8 s 1 - 6 g s J ' u o t t f e l a l u tu t a l o r d le)tueqJ .6t,suotloul 'uotl)trlsajssel)) H W l6t le|.Ulaql 08gt-51s1 )HW 289'(s)esdeui(S 'sessel)ureul g 0 S! 1 0 9 , 1 9 9, 6 u r u o ; t: r l n a d e l a q t (s)raUodLu^S €?sI 659'J999, 959' 'a:uapuadap 'suornau:riaqledr,ul5 zsr'lssE Itsgt'apou rldur^l 96g1 lgN ,uor16ue6 ! 8 8 2 ' 8 8 2 ' s a t t r l e u l l o u quer q o 1 6 -,fe r u a s s p l p q l :liaqledu,{5 fiLl L'VLLL'suu6alul 396g1, (Hf) (s)lla)-tiadleH aasredlaq g 7 - 4 7 ' s u r 6 r r ol e u p u o q ) o l t u r t6s['slla)ltHl ,sur6r.ro (HI) (s)lla)-fradlaHaasH1 gse;dorolqr I S Sl - O t g t ' s a r y a d o r d; e t a u a 6 57 . 'sla6Jelol uorl)arp (NDr) )iloMraut6;o9 suo.{ aas191 stsotqulAs Oggt-Gls I 'ruauldole^eP '6ur;eu6rs 'aseurl \ turPJ(dl9t) . It?S t 19691 1|a:-g 1{5 aasi(llurepadnsflg91 fl-ro1>e;q1mor66uru.uo;suer1 s sr se p , s a : u a n b aqs: g l n n 5 t L S L 'l V 1 L ' u t u o t l e z u e l o lde l a l a l s o i l ) 9991'6urq>1rw 'suralold6urpurq-619/xtleqq)ltMs (Hf) (s)llar-tiadlaH aastC) ;eraue6'slolre; 191. uotldu)suell aass.lolle,uotldu)sueJlllll 9 4 91 ' u o r g l u 6 o : eul a b r l u e 1886'aprloqutMs [?t-ot€,slol)eJ uotldu]suelt Hlllf l L 6 9 [ ' 2 5 9 r - 1 6 5 31 0 6 5 1 I t0tt !00't,00?[,rosuadsns ' ' Z 0 t '(s)rolreJ 3 19y X6olorq:t1aq1uIs,Josseldel ]ef I Z L i L ' Z L g J - J L SI ! € t S t t [ [ Z ' , u o r l e ^ r l ) e JLZLL'9att t le^r^rns 'srolda)alIlossa)le 662l_g6z L'utuedse.ltef LgSt-099 t J99SI 'O'SL'60tL-2681 ',(s)iler '99S '€SS 'sureq: 9ZZL g-ard 68S1-695 I l-999 t f L lqbr; a1e6or_rn5 1la) 'nEv 'luelrP/ns 1 8 € tt , l € t t _ 9 E tt , u u a q p e ) _ l tsEn L L all^t pt)e )tllt1 aasa;:I> y1 a)ueuosaruouseld alp/nS +SZS'9ZS-SZ9'(UdS) 'euoralsolsel (lf) slla)_lltxotoll) aas)1 1688 t 9 ? ' , l ) o l ) u e r p e ) r ) ' , ( N f ss) n a l : n u: r l e u s e r q : e l d n 5 ' E 6 Z L, a r n l ) n r l s (d6t) ureloid 6urpurq-ylylaas691 )v6(L l g f g ' ( s ) u o t l e l n ur o s s a t d d n 5 J 9 6 ( I ' l b 6 z I ' n 6 ZL- t 6 ZL , s t s a u a 6 o l e u J t a d s a)uetalol ;e:r6o;ounuu; osloaas t9gz I'sa? |-€,921'luaudolenep gyg1'sa{:oqduIl anrltear-;las,uorssalddn5 salsal ! 6 2 , L '6 Z nL ' u n r ; a q l r d a{ r o g r e ; l o , s l l a6l u r l r o d d n 5 ' 8 € €' l € € ' u o r l d u ) s u e J' sl l u e u o l a roleururaf 6€€ €€g 1p)tpetaptxojadnS O z t L ' t 0 t t , u o t l e t l u a l a J Jlrepu t u j l a l 898'098'(OOS)aselnursrpaplxoradnS 'aua6 ( LLt L tq! fpaq>uotqatwsoat g9g ,6ur6e,eptxotedn5 J n n E ' s v E - w t , u o r s u a;le : r ; a q t a d n 5 lnn€'svt-bnE'6ur[o:radn5

,19te^Yr:{ql

,ralourord.rournl ser""*

l.r1l"lg,,f,X., ir?!:liii

tot,;lut'u''?3f., :?i:i oLz'6sz*"0otf,

uorld,rs uerr:uo,t'l'ij3l :o";:::"t otrtJ

, t 0 0 t , J S 6 6 , u r e l o rnde f JZ00t 8l''uotlenualle uorldu)suerl'uralordlef eo Jrldrr:suell J Z n t ' l L r € . 'L V t- O v € ' 6 u t p u t q - r o ] ) u 0'€'uorle)ol lztt'a)uanbas snsuesuo) xoq vfvl_

st:l

X]CNI

uoqssardxa aua6;o ;orguo: Lr9['de]vNuur ! 8 6L ' 8 6 L ' r o l d a r a lu l l'nVsDull '6uruuelsI)jeal leuotldu)sue4aasuollPln6aluotssa:dxaaua6 1 9 9 9r 06t '(slul) alrs^Jluaaulosoqu aso)nl6 bEE'OLZ'h|pptt l5t 8 6 1 ' s l a l r o d s u e l l , v N U ]rlPuralul uolldlDsvetLosl?aas (llll) lereua6's.ro1:e; ole!ltu! ! L 6L ' a 6 L -I 6 L ' ! 8 6L ' t 6 L ' s r s o ] ^ ) s u P r f 08€ yp5 gt€,-sng'6u tssa>o.rd (s)aua9os/Daas 1 0 6 ? ' 6 8 ? - 8 8 ? ' 0 8 € ' ( s j t sar)o ] ) e Ju o r l e r l r u l (s)aseJau^lod VNUoslDaas L L n 'g t E ' ( s ) l l u nu o l l d l l l s u e r f 06t-68''08€'uopo) uorlerlrul '0t€-6€€ '6€€'sasereurllod vNU l-ot€ ZVE-LbE'xa;duro:uorletltutuotldt.t:suet1 og€'autuotqlaul llullo, '08t 'satotue)na € 9 8 ' V N Cl e l . l p u o q l o l l u l 88t-l8t !08€ M € ' U O l t P l l l u lu o l l d l l ) s u e l l ',Pual)eq # n € , J L n t , u olletltu! l8€? 88t',08€ 'uo!le6uola ' J Z n t' J l b € ' z n E - w t ' L n t o v E ' 6 u t p u t q - x oyq] y 1 snE-EvE J08t,o8€_52€,uollell!ul 'r!lo^rPrna 'JEnt 'JL7E'9nE-6E€.' lSbb'uor})etalututalotd rossardar 6€€ f66E ls8€'t8€ srolrqrqul )gll 'alnlel)uaulou lwE' gnE-En€'uorle6uole L9t--99F.'slladse otg lP)uolsrq uorssardxe eua6Jo lolluo) 1E€'sialoulolo 0'E'uorl)un, uorleln6a.r uolssardxaaua6 6€t-8tt'i(lraua6oralaq leuorlelsuerlaas l8€t'uotllelelut utalojd lolentl:e l 8 € € ' 6 € € - 8 t t ' s a ) u a n b a ss n s u a s u o ) ZnE-OnE'(1 J nL E 'E 9 E - 2 9 €t.9' € - 0 9 8 ' s a u r o s o q u 911)leraua6's.rollejuorldu)suerf O t € - 9 € € ' s l e u 6 rys1 1 6 OOE662'.ledarpa;dno:-uotldtrlsuell 1 6 6 €l 0 8 t ' 0 8 € ! 9 t € ' s a t o ^ r e ) n a 'J6SV'O9b-6St'sllnlr ll uolldlrlsuerf J6€€'68€'r(lrleuorlrarrp vNU rarsuertiapol )tlauag os/, aas J09V y15 re6uassayrl osloaas UOltol.llsuelf J 8 9 € ' 8 9 € ' s a u e j6 j urpear 8Ll- L LV'UODenUaUe 9 € € - 9 € €l t € t J ZL E ' LL t ' s a l n r a l o ur o l d e p e Sgn-bgb'Jnnn'Srv-vw' AEtauAs ;euotldt.t:suer1 'ntf.'EEE'€€€-Z€€'1gg '6urporap 6 u o u a l t sa u a g a a s6 u t t u a l t s; e u o l l d t t r s u e t l 3 9 ' s a l d t r u t t dr t s e q )Lgt'Lgt (s)uralordrosseldagaasstossatdat lSt€ !t€€'€t€-t€€ !t'S-t'uorldu)sue{ lt'8-t'saldr)uud )rseq ;euotldt.tlsuetl (6raua aar; ggg 'arngrpuadxa lolelpau I t St t ' t S t t ' ( s ) u o r l ) u nl qf 6 r l € ? € l e u o l l O l l ) s u e { '6S9-859'vodsuetl re;n1 qLt-LLt 'elor nl-ll (s)aseiaul^lod 1a>suell 1659 VNUos/Daas 'uorl)erf 'luaua^oul 'tt0 'OLa'^lern))e aasslossaldal (s)urelord.tossardag [-0]0 [ lla) I tto L agt 'EgE-998' '8-t 'uorlelsuerf (s)ulaloro r 0 L nL 'oL nL ' a u o ql e l n ) a q e j f JqLE | €€ !' gzz t 'ralourord (ro1e1n6a.r auag aassutalold(ro1e1n6al n9L '09 '09 'srs{1e1e: rourn] se'(alele)eloqroqdlAoue)apeller) vdf (tto aeuaeq) uorado)Dl aasuotado)Dl eur^zua'uortezrlrqe}s t L-65t '09 's6o1eue uralo.ldleuaoEgdaasgdt saloi(re:na t 9t V-VEV"s,l ' t9 t 'aua6 (ssaP(a uUAl)Iot 'r6tb '09 '{6raua uorlenrl:e Jo 1997 t 8Lb-LLV lO9 L 'uorlPllldal '605 ' pue uolleurJojalon)e^ t J0 LSt 0 9 L ' , a l e l us o r l r s u e J f J 6 E V6' E V - 8 € v! 1 9 € ? ' ( l e r . r a i ) Psqa) l o ^ r e l o r d q r o o u s ' ( U l )u n l n ) r l a r r r u s e ; d o p u 3 l68tI arnllnJls l85t'89?'^]rlrqelrreq '609 'sllel 'uorlnlo^a ]soq Jo uolse^ur t aasurnln)rler)rr].lseloopua l0 Lst ')!JJell 0tt-6Et , u n r l aleuorllsuerf qlrda euerqLuaullle) ]soq (lllf z L9[ 1n6 1 g y1 ) leraua6'srolreJ 'prouo) g7y1 X6a1erlslorluo>qyvrorb 60s L uorldu)suprlls,rlou.r 6urpurq-yy6 osloaas 'slUauaq tupuobowsoldoxol sgv-vgn 9 Z Vl - S Z bL ' s 1 1 a6>u r [ r l d u e ' n6v l€6b l'uoll)P Jo L!slueq)atx lrsuerl J Z Z y L ' n a n L - Z Z11 Jnnb'SrV-nw' A6rau,{s ;euotldt.t:suer1 z6?t'eraloq) srsolfloxl os/Daas B l t ' u o t t e n u a l l el e u o r l d l l l s u e r l sulxol le!lal)e8aasleua]leq xoq vlvf aasxoq vfvf fggL' Lg t-6LL'Uodsuerl 'arnDnlls €6't'(s)urxol flsn I t// '^lualodllof 's11a: 's,(entqled 'ZS?-OS''(sUf'l)suotbat lueld sn:o1 999 6url.rosuratotd ll0g ! l0g ;o.t1uo> 1009'669'(ltur) tg1'sutalord lolda)ar d9tA €st'sJotPlnsul 'sruaLuala '108'^lqruassP prdrl erua)saJonUuotl)aUalleulalul lelof ^dolso.r)il,u ,tole1n6a't Ott !8€' 1108 Uer /uorlpzrleluarul.lpdLuo) aas oslo t ' l o r l u o ) ' s s e u l l l a l l elof slueulala ialoulo)d z l t r r l t t ' z 0 t lSll lpuorl)unJ 'urelord 't€€ ',6tt',sralouold oslof t !€€€ I J?€€t 0w 188v ^t 3 1 1 1 ' q d e r 6 o . r : ruuo. r l : a 1 a 't€6 'aseurl g€v-vtb u01 salo^re)old 80 t t zll'(N9I) ilorralau161o9suo.l_1 sasereurostodol aasseselaLUoslodol (s)uralord(role;n6a.r slnolloul auaDoslDaas vNC g9Sxo17ar3 aua, )apun aassuralotd(toleln6a.reua6 J Z 8 l ' s a l o n l e ^l u P l d ' ( s ) l s e l d o u o l LgS'ualsi(s JZL9 3 g 9 7 , 6'{itxalduo: u r d o oyg1 r 1 6 9Z€-SZ€'uolleulqulo)a.l)Ul)ads-a]ls 'aLg'JL '(W1)adorso;ttu..t uoiue;a '(qdet6otuol lg 9gy-757 l0ls'89s-99s'uorl)npord '?Ot | L ' f i LL ' S LL _ nI L , x a l d u r o )y l o l f g LL j 9 | L , L Ll _ 9 'ptsde: t ' s 9 o l ' l o r l u o ) a 1 : , ( r1 ; a > t0 Ls'J695'695-899'slueld ItoL L 'snltAlunls ^qsnq oleuof r t.rJr?'i];;"" 1671 l8€t,,'a)uelsrp 8 9 S - 9 9 9 ' ( s ) L u s r u e 6: rruoa 6 s u e r l 'asuodsat 'ulls ftoler.utuegut t€S t-E€s L 9Z' L 9 € St ' t € 9 t ' s l l a'uorle^rlfe )) r l u p u e p (s)roleArl)e ;euotldtt:suet1 aas slole^llf e S l 9 t ' ( s ) l l a )t & o l e l n b a r t€s t-tgg t LLn-ZEn'SW €8[ ['pue suer{;6oa1ord '1s511) ',t€s ',urlelsoAr! stolda:ar a1t1-11o1 t-g€s t 99t I ! 6 1 g ' u o r s s a . r d xa au a 61 o; o t l u o r 1 e u o l 1 d 1 l : s u e t 1 ztg t 30€s L 'suorlelnur 1 ' s u r e l o l da l l l - l l o l 5llo,{.re:o.td t € s t o € s uorl)unj €tz L JgEb'gln'gEV-S€nlOZn' I8 lb'sursrueqraut Jo ssol '(nwi) snrlA)lesoulo)leqof '^lquasse 6t | ,las lg€? I t t t t ' O t t t ' E g 11 ' s 1 e u 6rrosl r q r q u t 'qder6or:tur 'luaurdola^ap o)leqof arnllnl Dltqdosuo lla)'sllel 3tr69 VNUos/, aas 9€€L 11asetau.-t(;od 'jo (nlNl) D-tol)eJslsor)eulot!nfaas nlNf OOtL a)uasqestsdoprqoty Jnnn'Sbb-nnn'A6tauIs;euotldtl:suet1 'suosodsuell tul f8 l€ s;tlotu6urpurq-yNCoslDaas 1 9 1 . g fi u a u d o l a ^ a pl e u i t u e ' (s)llart j(s)al^)oqdurll aas(sllerf) sall)oqdu^l t JZW'ZVV'lll'arnpills JOn6 Ln6-6E6,^eMqled I lossaloal JE6Z'Z6Z'sdool-I 6urleu6rs uortenrl)e l u a p u a d e p p e t u 5 tgw'sbl'suotDeralu ; o yg 11 i(1rr"ue; utalotd d[ i(1ru e; (f,191) f,-rol: e1qvrrol6 6u ru.ro;suer1 l 8 € ? ' u o l l ) e i a l u la u ^ z u a o l o q '820t ull!1 l8z0r I JL V ' ' L w ' u o q e u r r oxJa l o u o l lZSf ! [8t'uor]eururJa]ap '(Vdr)role^t1:eua6ou;use;dadll-ansstf xas o rqdosotg' aue6 (ul) )auJoLsuut t6 t t lLVl';Vb'eoleNpeo) 1 ansstl fSgS'6 LS'SSS-7SS'6utute1s l9€?'l!lo^lP)na ttzL 'Z0S'sanbtuq:a1uotl)asstpor)ll.!'salllsanssll 'atz l e u o r ] d r r l s u e r - [ l€0S I ' I q O S ' s l l ar)a ) u e l ' a d ( l o u a q d , p a u r o J s u e, r f 5 € ? t € ? € ' w E - Z V E ' I t € ' ( s ) r o l e ^ r ] ) e N6LL apol lrlaua9 os/Daas s-t'saldl)uud lesra^!un '(sdlAll) saseutalotdo;1e}aut 1osrollqlqul-ansslf 6 € € ' L E E' s' uaoulal eDu l u r r a l t98'9€€-9€€'uo!ldlr)suer] 'slueldxeenssll 't0Lt '89€ 'arnlrnrls tOs vNUr I L9€ t€Lt 189€ 'uo!leluallo '6€€ ainlln) llal os/, aas y15 aseraul,{1od J6€€ )EL€.'gLE-s Lt'6urpurqar,uosoqrr-y1191-1,(prldad t09'pro) leulos t€€'r uorlelrloar l 0 L E 'l 6 9 E ' 6 9 t ' 1 8 9 8 ' u o r l e ) r r r p o u r ' t € € ' s e l e r 'svNUl t09-€0s'slueulallnbaJ rolerlrul o L € ' L E t 08€ 'sl)aose t09 le)uolslq I Lv- LLI' uonenuaileuotldt.t:suetl 19€€'uorllunJ 'slueloxa 'sJoleulullal t09 6EE'L€E 8 O t - 1 0 ? ' a l u e l r r r u 6rrtse u o r l n l o n a gt eu61s tos-€09'arnlln) ansslf (s)uopo)rluvaassuopo)rlue E ' s . t o 1 l e ; r L $ ' sanssn)ut)aos oslDaas Y55 JLEE' Ltt-gEE'aseratl{1od ) sL t '/€€ 'srelou'lord ' [€L[',^laue^ l8€€ tl€€ 9Lt-S LE'6urpurqaurosoq;r-y5gg-1{reourure n B nL -L J n J ' p a z l l e l l a o s saselaqlu(sVNUI-l^)eouluVoslDaas l6€€'6€t s 0 9! s 8 s l 9 t € ! 6 € € "t (S1trE1 e u o t 1 r a l ; p f Z L t ' t L t - C L t ' u o t l t u 6 oef r a s e l a q l u ( s '5gg'uorlerederd '68€-8EE r{do:so.rrtu'6utuotl:as a;dtues 10?'urDuo l l E E ' 8 € € - 9 € € ' ( l e r r a } ) P:qt l)o , t u e : o r d IE6LL'rPda) 'I>ernrre LLVt oslD aas reloulo)d sluaulala ELE-LLt 'leMauol ngnL-LfiL '(luelod (s)loleAll)e l ) e aul O r 1 e 1 , { : e o u r u l e ) z L E ' J LL E ' L L E - OL E ' u o t '9'(VNUI) sg [ [ 69€-89t ll VNUrelsuerf leuorldrr)suerfaass.lole^tl)eleuollo!.llsuell 's.lalou.lold 0 t LL ' a l u e u a t u t P uplu e u o l l e z t u e 6 l o 6€E-8€€ v6LlOIOa)d uUralsueJl /srol)a^6uruop yp6'uorlla;suerl lla)erlxa 0 t t [ ' z 0 t [ ' s r o ] ) e ]r e l n'slueloxe lypp'iuPeat aPol auolslq ttS-gtS )ne '0s ' V t- O V E ' 6 € € ''lxl oa ^l dr e J t W ' J L V €Z Iq'E'8'E_LVE uo) '6ur>r;ds etleqrtd3aaslellaqllda ypgur-ard'suorllearuo!le)!Jlrelsasuerf z n E L v E '6urraaut6ua ',sra)npsuerf 6 1 y1 ll€€'a^ruoqe 6Zt t JZNLL L€t-gtt,uorlell!u! 861'saulosopua6urlr{:a.t

X]CNI

gttl

'uosueduro)/aJpduro) sueau s lalq?l e ol JaJarf e qttM slaqunu a6pd l^la^rln)asuo)Mollojleql sarnbuol ll up qlrm 'aln6!Je ol ralar I ue qllM sraqurnua6ed :Illua aql uorssn)stplxal rofeul e ol taJalatefploq ur slaqunu a6ed Jo pnprNpur osp aas swalotdTsaua6 'Dursol sulsrueq)eul uotlerppr(1;1)ra1ornel111-1 aastq6rl41-1 l9€z | Jo g t a t - s E ZL ' u o l l P l n u^r q s s o l suor6atpale;sue.llun aas '(Vdn)lole^tlle ua6ourr-use1d Uln aseutlorn ?6 t t sEzl-tEzl'uorle)urluapl gEZJ-n€.7L'saulorpu^sta)u e) ^reltpelaq ' ' a 6 u e q l: r l e u a 6 r d as ) r t a u a 6 aurpr | 9€'suorlerurpo, l 1 € Zt o*rr,uo,i.?rt"urosr n g g 9 X q d e t 6 o r p e t o l napu t p u n - H € J Z € Z 1 L' E ( . 1 ' ( s g s fs)a u a 6r o s s a r d d nrso u n l 'asearn L t Z L '9 Z Zl ' ( s ) r a l o u o r dr o u n l €0s[ 'eupuoqlottu.t ,al)^) eeln l99 ' 'earn (o NDo, oll3frsrl soneu rouJnr z s t' 6 JE s 6' E s 6' r L 0L 999'surelsIdorsol]|ul uolllala,ulntup.ln L Z ( l ' g z zL , s J o l e t l It luo L U n f suotloto 11sadtr1 ttlDads ie)ue) aas(s)JoriJnf It€€ lzE€'ztt'J9 | I'|Dc.Jn 6LOt'LL1t 06t'slUOueatlsdn ' g g 7 ' u o r 6 a pt e t p l s u e l l u n , S xa;duo: 6uu ur;nqn1-/" lv 66'JZ66'266'()Un1-,1) 'ggt 'ygtr'uot6atpalplsue.llun (s)alnqnlo/)twos/Daas lSgt ,€ suor6atpale;suet1ul'1 8 6 6 r' Z 6 6 '2 6 6 ' ur l n q n r - 1 . a ; r ; ; ou l 6 u g a a sa u t la u l l , a s l a n t u n 086'uIoJ-O pue ultoj-f 'uotlnloneulaloJd,aultlXleuotlnlo^a 'alnilnlts s9Z ltun El6 '959'(s)reUod run l t ! 6 ' a s r n o la u J t l ' u o t l e z u e u ^ l o d 1999 l 9 6 S ' S 6 S - t 6 S ' u o l l e z l l e n suloAl l e n r l l e o l 0 q d 8 € 8 ' 9 € 8 ' s 1 u a 66eu r ; d n o : u 1 ' 1 ggg 'saua6 :up 1 zS6',SurJOJosl ggg1 'aua69:u4 S Z G - € Z G ' ^ l q u l a sl suea u e l U ,s)un Ileuotlnlona €86-286'uotlenJasuo) 6g€L lolda)er uulau ,HSlun 6 9 €L l o l d a ) a t u u l a u 6 9 6 ' 6 o 1 o u o ql e u a ] l e q 'sl)a;apledat y16 ,Ilnlllsues (s)urlnqnl 156Z 'surroMeqnl ltedal VNC iaGpuepVNC osloaas l€ [ s$olnraqn wnuaDDqD/{Wosp aas 8 6 2 '! L 6 Z ' 9 6 2 ' a 6 e L u evpN C '80s '88t 'srsoln]raqnl u o t l P t p e(ln n ) r a l o t ^ e r l l n 809t L-10st [ l 0 L S 't t S - Ot S ' a 6 n J u l u a ) e l l lenn r l e t e d a t d n6SL-E6SL',(sotdalpro;n:laqn1 'ellraqnf nd auloso.l)tu.l 971'uor1e:r;u los | S]UVNS J t t 5 ' t 1 s - 0 t s ' u o r l e u o r l ) e rl,l e l auerquetu la6iel'suralordIUVNSaassIUVNS-l uorle6nlr.rgua:et11 1 l € Z Z ' s l ! ea l uols!q'uollel^lrnblqn 209[ Al] asrasl (s)auloseatold os/Daas 676I' o1tqdosotg,sluau.r6as 1 'urlrnbrqn l0zt J!]oruxrlaq-uinl-xrlaq )0bL't6E losserdarueqdoldlrl fntn'JttV'n*-€€t J S 6 E , 6 u r l a querl a l o j d v6t-E6t'6urta6,reiaulosealold 3g6y'uoradoueqdoldfil 9 6 € - 5 6 € ' u o t l e P e r 6 al epu l u l l a l - N #*'16zt'uPqdold^r1 'sanbruqral 't uorlelos!llel 'utsd^ll 7gg Jn6E ,sau^zua 'v6t-t6t6 €/uorlP^rlf e h6t z O sI saruaslasr 'uotletieA ltua6tlue $ 6 8 ' f 7 6 t ' t G E - € 6 € ' ^ e M q t p du r l r n b r q 1 0 Z SI 3 6 t St sawtzua )UDadsosp aas (s)eurosouedl{ 88r'ls8L'S8r-t8L I t [ 9 t ' t L St ' 6 9 5 51 z r u ot w o s o u o d l t l ,t90 ,lo.rluo)al)^] l f s 1(OS 1Dilq DLuosouDd(4 _1990 t !S90 t 0ZSt !6 tS t | L lla) ! 9 6 t ' J V 6 E ' E 6 E ' 1 s ; a s eu6r r11r n b r q 6 1 ) n q b ' E g Vr' n s t ' b s t ' ! 9 L ' D w o s oD ud^4 f i 6 E ' t 6 t ' a L u ^ z u a6 u r l e n r l t e - u r l r n b r q l L00L'Jb66'lgn'l6VE'utso^urodorf .utlnpouooo.tf rjn L'$t-z6E'u rlrnbrqn t00 [ 'ullselaoooJf O aul^zueo) aasauoutnbtqfl 06 [ t l OL E '6 9 t ' a s e a l ) n u o p u6eu o rl d s - y 1g1l 'luauata (aruanbas orrrnilit.l""lrrantsvn vNU rarsuerfaasvNUt ' u l s r u e q ) a u6l u r : l 1 d s '(rolda)ar l0S€ 06EL 3911 uralordroldalar VilI 1-1 ' u i s l u e q ) a u6l u r r r l d s2 p g u s9 y1-1 l0St 1EZ6',Vryl_ 'ulstupq)aul6ul:rlds2 p g u s l0S€ 1€9 l{rrlrqnlosutalordauelquteLu,uoluf Spgus 71-1 ,utolord dnor6 xeroqiuf l0S€ 1 l e u o s o a ) t l d s l V Z n ' u s t u e q ) a u779 6 u r : r ; d sg p g u s a 1 d r r 1 l 0 S € ' u l s t u e q ) a u6 u D r l d st y g u s 1 1 - 1 l0S€ ad : r.r1 l g 9 6 ' l 5 6 ' 6 u t lp a a s6 u r a r r o l 6 , a s u o das 1 JZLI sasoul I L 9I ' l j a L ' a s e r a u o sa! l p q d s o q d a s o u l (sasedlv/su talord 6urpurq-619 /a.lnllnlls,aursot(1 )uaurut) (s)uralordg :(s)uteloldg 16Zt paleDosse-aseutl-aursot(1 g) sutaloid 6urpurq-619ruauul 9C6' aas (suralord ZZ6'roldalal saseul)jautsol^l ulalotd ;o ,{1rr,ue; ,S osp aas s;ola:X;61I:eu1 aas(s)apua:I16rrt ' 6 u t l e u 6 tjso t d e ) a l /slnoltoul asnou'(qdotlsXpo!tlloqlt{ l 0 6 St l l a ) f l89S 'eJ!ljo adt'sDuowoq)ul ()ru) l9l saseurlaulso.r{1 toldatag aaspa1ul1-toldatat tqt |'uilnila)tJl ,6utleu6ts l o l d a r a r g al)^) poe l!rl!) aasapll pr>eiilrxoq'je:ir] 1 0 6 5L , ( s ) a s e uat;l1u ar s: o t ( 1 'tL6'arnl)nlls lll [ )b | | g1,y'uol}e1n6a.r gS 'a6erolspr>e(11e; uorssaldxaaua6'asetalsuellourLue aursol,{1 1 9 6 , g 6 , a 6 e r o 1r {s6 t a u a 'slorar{;61,{reul vjst'Jb6vl 1g11,azts '€6tt 'sutxol ,u.rals,{s uorlattaslll ad(f letJa])eq aulouab 1g t 1g tE,uosodsuettollall^l p r i l o dD w a u o d a r l ,€J b l ' w n,€ ,a,rl aarj tz6s 9 t_s I t_z I t_z t Jo '269'^dolsol)ttri leloluo),1ta;rra,,uo1oqd-orvr1,, il LL6' 0g6-9 L6'6ur;;rurpea4 * Z S ' n Z S - tZ 9 ' u a l s ^ sp u q ^ q - o M f JLLZL'9171 '6uured6o;ou.roq)tloteuJ'sluauelU suotllejalut utalold_utaloldoslpaas asla^suel1 ' s p o q r a up u q l q - o m 1 wZS'VZSEZ9 ) V S € . ' V S t ' s o t u o s o u e d^rt 'tS '85€ 1779'kldeftotpelolne,uottezrlenstA Irs€ iiNU 'ders tzs-lzs uot1 39y6-565 ltgt'99t-?9€'6vt>11ds-s ' d a t sb u r s n t o > l u1:alaosr lzzs'lL6'l zE sluauap lonptAtpu, iasesodsuejloslDaas ' Z t V , s l s { ; e uueo r s s a t d x a eua6 uorlrsodsuelfaasu.ls!ueq)eul fi tV uotltsodsuell z e s - L Z s ' ( l 9 v d - c z )s r s a r o q d o r l t a ; a J € Z € ' a s n o u ' s Iu e u l n q ' u o r l e l l e:nr 1 a u a 6 o ; I q d 1 a 6a p r u e l A t t e { 1 o;de u o r s u a u l p - o r n 1 9 9 S ' J g Z S ' s r s a u a 6 e llneuu o t u a s u l JW6 1 1 9 7 ' a u o u a 6u e u t n q ' 6ur;eu6rs 7p6' walsAs 1Aeiu.qted luauodruo>-orvr1 3 9 9 7 ' s e u a 6u l q o l 6 - f , S t t t ' t ? t I ' u l a 1 o l d 7 a u a; 6 sr,n_1 €Zt'sluaua^our r(reuorln;ona I t L n ' t L r ' s : t l a u a O t d a ' s a r p nul tsM l suosoosuellollal 'srs,{1e1e> eu(zua ,loqLunuta^ournl 09 t-6S L alll-lplr^ollaujsuosodsuptlollaE osp aas 'l 'suosodsueJloltar 6 6 t L 'L 6 LL , l g l , a l n s s a t dr o 6 r n 1 19 t € t € l 8 t t t ' 1 t t t ' s ; ; a >: r l o l d o d e , a n b r u q ) a l - l l N n 1 t s r € ! 8 t € ' r 1 g[ 1 u o - y 1 1 6

n

Lt:l

,r,rr8,j,r",Hffil?i":?';

6 6 5L 'l L t ' ( s ) u o s o d s u P l ] uotltsoosuell aasuorleurquo)el lur)aos-e]tslpuotltsodsuejf 'uor1er6e1ur 3 179 letrn 'suosodsuerlorlaj 18 t € uorleurquorerlur)eos-alrsaasustueqlau.t sawtrzuatloads osloaas 'sau^zua 1 8[ € J j a t ' J 6 l E ' r ! s l u e q l a u ra l s p d - p u p - t n l 'suosodsuell^luo-VNC fg [€ g 'uotltsodsueJf It 'saua6 l8 t€ 'l 'suosodsuerl Iluo-VNC l_8t € t € 6 g y ' 6 u r > r ; das^ t l e u t e l l e lStt'lLE'asesodsuerl (s)uosodsuerf aassluauralaa;qesodsue.rl 0 9 L ' 6 VL ' ( s ) a l ) t s au^o d s u e j t (s)utalord iarJJPJ aassulalorouoosuelvsreuoosuetf /uOlDeaJuollPlueldsuP.tf 9 IS l-S LSL stolda>attloads osloaas Lg9-999'st)aJJa6ntp anrl>eoq:(sd € 9 9 ' s e 6 u e qItl t l t q e a u l e d '989-tgg 'suotllun[ ]eln)snurorneu 889-1,89 t39',uotteztlP)ol 999-tg9'se srolde:al aur;oqt1{1a:e uol pele6-rall!uisuerf 268'n89-289' G);auueqr l 0 [ 9 ' 6 0 9 - 8 0 9 ' 6 u r m o p e q;se 1 a t u ateyns GO9-809'srs(1eue l S 0 9 ' 9 0 9 - 9 0 9 ' u o r l e t e d a tadl d t u e s 1t09,909-t09,edotsol:ru.i ' '^dolsol)tul uoJlleleounuJt!l lL09 IO9-9O9 tL09' 909'uo!Dnllsuo)erc-t fi09 '109-t09'(Wll_) {do>so.Dru-r uolDala uotsstu.Jsuerf sadlt/swatdd )t|Dads:(s)uuodosp aas /saln)alout L5 9 lleus,Uodsuerl f E EL ' n E L - E e L! 9 € 9 , 2 € 9 , s s e d _ a t O u r s JSEL'98.L-NEL 'S€, L-nEL' tsg'ilvEg'tnt9'799'ssedrllnu Z€9,slold,(qredorplq 7 9 9 ' 6 u r p u o qu a 6 o r p l q t€g-z€g,suotl)PJalutxtlaq-D lzEg' zgg- )€,9'uotleuloluo) xtleLl-D z€9'sureurop (s)uu6otulos/Daas ',69 ',uotseqpe xtrleLu-lla) ILLL L LL (s)uueqpPfoslpaas €9tt l - s € tt t t E t L , s € Lr - t € [ t , u o r s e q plel a ) - l l a ] JStg'SE9-n€.9'Zgg'uorleulto;uor lalleq f, t€9'AlquassP l0€9'0€9-629'(s)uteloa r du e t q u a L u s u p t f '(s)uorl)unr E SI t t q 6 ! l ' s u l e l o r du o r s a q p e u p l q u i e u l s u e { uolle)olsuell euosouroJqJaasleulosoruo.lqt,suotle)olsuerf xaloulol lAof aas auerquiauJleupuoqlottulrelno aql jo ase)olsupll xJloujol wlf aas a u e j q t u a ul e t J p u o q l o l t Li auu u te q l j o a s e l o l s u e { )z8E ',(r>rruru-r reln)alour'(llUa)tol)p, aseelaluotlelsuerf 1 0 6 ' ' g g t ' s r o s s a J o el el u o t t e l s u e r l J t 8 € t E 8 € ' E g E ' 6 u r p o : al re u o r l e l s u p r f l t 8 € ' t 8 t , 6 u r r y r q s a r . u el er ;u o t l e l s u p 4 (VNUul)VNUra6uasse6losloaas '88V'a^rte6eu !06n JZ6n'E6n-Z6t' uorleper6apyJrlgm lG?'(SlUla ) l r s^ l l u a a u o s o q r rl e u r a t u l )O6b' J6n-6An'uollelnberuorlelllul g59'uol1e1(uapei(;od :rurse;doy(: 88t '9Ln'16LE'uorssardxa aua6,o lolluo) leuotlelsuerl (s)eulosoqtu :stsaqlu^s uralotdj(vNUul)ylg ra6uassa6losloaas S t s t - 1 t s l ' v N Ul e i r ^ ',19€',uolleululal I t8E

^e)ep w0",.'0",,-"rr"133K vNU H'J:ii'

'l8E-S8€ '€8E-Zgg ';orluor Jl.8€ ftrlenb stsaqlu^sutalotdoslpaas !6 Lt' 6Lt'9 Lt-S LE'aseralsuell ;{prlded g LE-L L€.,srot)eJ uorlP6uola J 9L E ' J SL Et t l t ' u o r l e b u o l a u r e q )a p r l d a d 1lst'sasnr^ 775y'>r1o(n>otd 6 1 g ' s l e u 6 rysp g r u

X]CNI

'^ruat)Uap') ultrjPlln 'suralord{.ro1e1n6a.r ,(1rue1 aua6 (re6uquZ) l)UM 98 LL 'alnor uor]larul 069'0t9'v uruelrn 209L- tos L 'glD_Llo oslo aas surwDltA elor 9Ztt ' Llonpatput g L ' s a u ^ z u a os) e ILgl 9av l-szn L'ra^ournlller jo lo]luol (s)uruelln g y y1 ' s r s a u a 6 o 1 6 u e (spor) 6urleaq iullsaJlvoslDaas ) g r V t ' g n nl ' s r s a u a 6 o 1 6 u e srolda:aroloqdpou lursdopoqu 'slla)loldalaroloqd ( s ) p u n oA 8 L6 suobalasrypqoqtouao) aasu)oM JOZ6'026-616 's.rolda:a.roloqd po.tut ;eu6rs1o uorgettlt;due 8 L SL ' l t St ' p e a r d sl e ) r u a ^ '026'uorleldepe l0z6 lL LEL',L lS t',luaule^ourpaseq-alnqnlor)ru.r 8 1 , 5 - Z[ 6 ' , u o l l ] n p s u e ll]e n s l n I t s L ' l u a u l l e a Ju] o l ] ) a J u l 'suulnlol 'uor]e)urpoullot^eqaq g 196€I ]soq [5 [ a ) u e u r u j o pr e l n l o u o e l u a n l r u l' a l u a l l a o x el e n s l n Drrl)DqlolA ' s u u n l o ) a ) u e u l u r o pr e l n ) o ' x a ] i o ) qqoM lensln ls6€ [ 1 6 9 9 ' 6 9 g ' 6 u r r r e d - a sael q ' u r ) su l 1 9 6 €t 9Zt L '969 'slndur pue -1q6trJo uotle6ai6es a^a14a1 g g 11 ' ( 1 u e ; o d; ; a : . r e u e ; d 1 'a)uatladxa^q peploLtr '96€ tr96t [ ls6€ t t r b t - g E nL 'e)ueuelu!eur L uorlelndod 9 6 € t 5 6 € 1nb 1;ar-u.ra1s ' x a u o f u e t l p t u u e uul t ' 5 u o l l ) a u u o l 'raluel lensln €sz | lel)arolo) 'pue s osp aas tllaJu ) s ws t u odtolsu o u o r s e q p e UDad s t LL llel-lla) 'slol:an r{q uolsslLllsuel} ros L 8 5 ? L M o r e u A u o qu l 'leruaqds los [ 3 6 7 '6l u' 9e5L5u-d9o9l5e'^uar p uale:-$ 6tt lilqurosse,las 1 9t € L leurue 'Ptsde>oa1:nu ur tuesqe 31671. 'lerpaqesoll oolL'stsooprqDtv Iennqred6ur;eu6rs z 1 9! LL 9 1ug €LSt l € E [ ' s l a p o u lu l a l o l da l l l ' L 6 nL ' # 8 b t ' l o s I ' J 6 ' | 3 8 ?t ' , 8 t t ' , s p r s d e ) Z S € . tL' O Z L ' u r 6 l e 6 uurnn '6V 'L9nL'alnllnlls 6urleubrslug os/Daas 96nL g9g1 '(enaqled6ur;eu6rsssal6urntr 96Vl'azts (se)snll^vNU aassasnrlAvNU {ennqted6ur;eu6rstu6osp aas 'qu!l aa5 sasnl sesnJlAollau lAoJlal a t e r q e u a ^ u ! sstt 'uorleluau6as 16t['uo!]e)!loar oltqdosot1 ut 0t€ ! ',sroloafar 'ESt 'u ralo-rd7aua6 (61y,,) ssa16ut1111 909L-S0St t I €S€L '1 ' s ! s a q ] u ^usl a l o r d ')s!p L t S | 9 € S [ S € 9 JgsE[ l e u r'pnq 6 e u r6 u r 1 n 'sula]oro l6t [ 6urtr1 3g9E1, '1ue;d 'puPeleutsaPoruseld 'uorle:rlderyy6 6urpurni1,, €9 | L 1977 'podola^uauou ' urxol"'ualqord srssnuados/Daas LOSI L6Vl '(srssnlrad g6v q )6 n o : l ' { 6 o l o q dtotu L ' 6 u r d o o q M ! g 6 n € O SL ' u o t l t q t q uut o t s s a t d x as s e l ) )Hyrl I sadll 9 € S L S t S t ' s a P r (a: ; t ; aasslla) poolq auqM ! 9 6 n t ' L 6 n L - g 6 vI ilD lonptAtpur:(s\al^)o)nal 'luaua^oul reln '8 '6urllolq uralsag LLSJ-n19l lla)erlul 16tS tS : l s l l o q e l a ul sl o q 8 L St - l t S L ' s a 6 u e q u f 9 6 Z ' s r a p r o s rJpr e d ayr p 6 ' a u o r p u ^ s r a u r a 6 'aseur)i )n19l laeM ) v L o L ' n L j L ' € 9 0t 't 'sa6ueq: ^C )UJel]auerquaur llal ]soq LS[-t t g L 96 L saue|uoslPM lsluaruala u 8 ' r a 9 8 ' 19 g ' a u . r , { z u6au r } l r l d s - ] a t e M uorleurquio)arlr;Dads-a1t5 'lzs 'aln])nlls :r1aua6a;rqoy1aasuotler6alutauloua6 180[ 'lua^los J L 6 nL 'L 6 VL ' 9 6 71 ' e u r o u a 6 160L lzs l-ozst'uollnlo^o l8zg'828'ror^eqau qolord 'suralord'salor I L gL -L t S l ' p a P o l u a s a L u ^ z u a trgI leuor])unJ ' 'a)rnos uorl)ala l ? r s L ' Et s t ' s o i I ' J L 6 bt ' l 6 t t ' p a d o l a ^ u a tl8 Ls8 ' S 0 S ' 6 u t l e o : u n 'saluadold t t P u ei ( t 1 u a 1 9 0 9 Z S -L g lP)ruaq) ' ( s a ) s n r rl ^o r u n l vNc ( s ) l a u u e qul r . l o d e n baVa s( s ) l a u u e q ) )gnzl'6nzL-Lnz [ BB'I Xttstantp zs-Ls'luaiuol lla) 'sl:arJarrilloil> ,raleM 96tt ll6,0 t-80I (sa)snrl^loulnl vNo os/Daas d5y11 lgts t'9t9t'E761 se " i | r u6tnqlelq e1 SZZa-LZZ L'sua6oulfief ;VZL!J4)e 'euroqpoolq 209L Duo\Dqts!sdoplqDlyaasssal) lleM aaslellallPq (s)aOeqdoual)e8 ' s l l a )] s o qo l u o l s a q p e sL 9 0 9 t s o'A6ta-96n1'88? tvl '(sa)snrrA t 9€5 1-t€S I ',spruselo ) z 6 v l 2 l 6 n l '/,lle6ttr aL1 : P' sqa u a 6a l u a l n r r n a:ua;nrt1 L6tt 099'(sesedfV)sdund ad/ii-n sulxo]lPlrel)egoslDaas L' t 9L-29 L',SI S-A ' a u o r p u ( s JEg n e p u r l - l a dUVN sl lI a d , { } d r ;u1o 1 v o gl ' l v 6 b l ' € . 6 tL ') o ! L u a l s , (uso r l a r : a/xerqiue 6771 119n1 E6tl JLOS'xo^loA 'srol)eJa)ualnlln l6t L LL9-919'sel€$ ' u o ! l l u u a p' u o u l n 96t [ l 8 9 ' s u o r l l u n fr P l n ) s n u o r n a u sw stuobtoysuotlta|uI ilJDads:(sa)siltA osloaas 9 1 9 ' u i s r u e q l a u6 u r l e n r l r e u r '86V 'uorleul))e^ (s)lauueq)r.unrpos pa1e6-a6e1lon | L )86n ')o lg1v L s1:agat-uora;ta1ut 819'sluelnur (s)uoll)aJullelln lgl9'lapour ureql-pue-lleq'uorle^rlleu! llselquln f 8 8 6 ' ( a u l l s l l l u l Aa) u Z89'drqsuorlPlar,teuorln;ona 'uriuaur!n /986 IS86 399 Xsdalrda /9€t 'gL9'pa(elap [ ' a u l l s a l u ! l l e u r sr o ' l l l ! n lw I'J9t, L 689 1 6 6 1 ' 6 u t 1' suat 1u-as6suolrlll e ' u l l l l n l l g ' ( s ) l a u u e q l L r n r s s e l opda l e 6 - a 6 e 1 1 o 1 alualnr!A sadfi tloads oslo aas 1 6 91 ulxol elaloq] aasulxol I L9-9 L9'(s)leuueq)uor pa1e6-a6e1;o1 'arnllnlls l68t L 8 8 9 - 2 8 9 ' 2 8 9 ' ( s ) l a u u e qul . r n D ; epra 1 e 6 - a 6 e 1 ; o n L 6 ? [ ''sd)eruu e p u e o t 9 s 1 ' a s e u r q u l o l eri( c ) A 'uorlrnpord ureqr aua6 >r'ssa>o.rd 6ururof 1 3 7 6 y lq6r; ;-1 l€9s t 'seruanbes uorleurquoral q XI3 1 7 6 ; 1 ' a 6 e q d o u a i r PQ 1y9g1. 1eu6rs ,ln6 'lojluo) uo!lezluolol €0St ro'aurosoulorq) 9991-S9St. L lz6t 'a6eqdouelleq 89S[ "s^ 6utq:]tanssse;r gg9 l-;,g9J'6ututof ;1614 r6t L 's09 'a]! snoar]rn )vL'aualotl) ouqtA 0 [9 ' r a ^ eal u r l l a l r n J 6 9L ' 8 9L ' u o l s n Ja u e r q u l a u r 182[ 'snleiedde !610gol Ul ulol, uodsueil 1 6 8 8 ' € Cu r u e l r n rrd(louroq '691 i(emqledlenaulet JoLL'!69L 11,69'roldarar 169L'691'uolleLUloJ 0 6 8 ' Cu r L u e l r ^

JZLL' J69L'qdelbor)rtuuorl)ala 69 l-89 L'sialsnl)Jelnqnl lPln)lsan suraloJdluvNs aaspue sulaloroluvNs (s)al)rsaA fuolaJlesaassal)tsaA^lolal)as d r q s u o t t e ;aa;r; a u e 6 l o 3 1 'pJ1Z' S L'ZgL'suotlP6rtsa^ul J t SL ' € gL gLL_LLLIPPOUJ lgl-6zZ'sauososll ol stsol^)oxf os/, aas 'a)e/ns 608-66I llar ol lrodsueJlsnlPreddet61o9 stsol^)oxl os/Daas 's!sot^)oxe 608-66t 'snlerPddet61o9o1g3 Igl-ggL z gL - L gL ' L gL - 0 9L ' f t r r g t : a d s6 u t 1 : o p paleol aassal)lse^paleo) (s)al)rsa^ L ' g g L - O gI ' r L o L ' L OL ' U o d s u e lrl e ; n > t s a n JOS saPadspuo sassDotdlonptNputoslDaas luaudolanap slrtaqsndouaxaasstAaqsndouax , qnl lerneu J L nLL '0 b I L ' u o l l e u l r o' a ]sarl lott t'0?t [ leinau luaurdolanapesnoWaasasnouJ luaudola\ap st^aq sndouaxlapun oslDaas l z L E L ' ZI t L - L L E' SI 'Luto r l e l u l oaJl ! u l o s l ' s ; ; a :l u a t u 6 t d )gLEL 'proqrolou LL€a-OLEI ) z L t L ' r oL t L ' LL EL - 0L E L ' u o r l e l n r n a u L't I€J'lsarl lerneu f i L E'uorter6tur 'n tse;qoItu lVLEL Lt L-ELt [ sL €L - EL t L ' s ; a > , t o 1 e l 6 t t u 'uoltlsuellelnlselq-plLu s9€t Xtlauu(se 1q6u-r;a1 JgL€ t' tLEt-gtE1 'uolle;nrlse6 'JggE l'69€ l-s9€ I JLgtl ' e ; q r q d r uuet s a t l l a u r u r ^ s6e6 a )VgF-l'ngtL 'uorsuelxa '69t luo6ie^uo) t-89€ I 169€L 's9€ 'a6enea;: t 199€t )OLEL ' O L E L - 6 g E' lln o - 6 u r u o sp u Pu o t s e q p e lla) 1 9 9 €t ' s 9 € t ! € 9 € L ' e l n l s e l q 8l€ 1-€9€ I 'ue1d,{poq 6utdeqspue s}uaualou lla) 1o (s)ulleqpel)os/Daas JLNLL \ o n I L ' L nLL - 1 vl L ' 0 t t t - 6 € Lt ' p u e s u r r a q p e r 'luaurdola^ap alerqolran 8Z€ t-€9€ t 'a>ua6rertp ,fuPuo!lnlo^a Jo seull 1gy alerqalienaasluauldole^ap luaurdo;arrap (s)aterqauan l Z I S ' t t 9 - 0 t S ' u o r l e l u a u r l p a^sl ! ] o l a n y 1 11 ' u o r s a q Puer r 6 a l u t 1 6 € tt ' 6 € t t - 8 E L[ ' u o r s a q p ue r r a q p e ] ,,a;douudor:;a1,, 7ggf i(q uolssttusuellsnllA l l 8 t s ! 8 t 9 ' } V S ' 9 V 9 ' u o l s s a l d xuel e l o l d Z 0 S[ - t 0 S [ ' s u a 6 o q l e d; o 6utuoll Y516osloaas I r L n s ' w 9 - o v 9 ' 6 u t u o 1y: 1 1 6 srolran 9 ' t t l - 8 € t[ ' u'Uu a q p e ] - l n 1106 rssaioosen lb}bt'z}nL'slueld Jo poolSaasslassa^poolq slessaA a n s s l rl e l n l s e n o u J n lu l e l o l 1 7 7 1 ' u o t s s a i 6 ol.nr d 6 V bL ' 8 W t ' s l l a ) l e ! l a q l o p u a € 8 [ [ ' b u r p u r qy \ l f ] ' p u es u o l ] ) u n f llel-lla) S t L[ (lDlA) rol)eJ qlMorb lelleqlopualeln)sPn l o r s a q p lel a l r P l n l s P n 9 i L t ' 6 W V f A ) s a l n l a l o uu 'sutalord{1r'ue;esel Z8zL )998'998'uollebauen 'luauranou L19 l-9LSl lalsoz elleluen paspq-alnqnlol)lLu'snl!A '(sUlNA)sleedeJuapuel Joqulnualqel.lPn llts r 1l q b t ; l09St!699t'sureq6 'sureqr 61,fieaq l09g t !655 t ',saueD €9SI u r e u o pa l q e u e n ' [ 9 ' s n l p e ls l e e M J a pu e n l0t t '6urp;o;urarord 1991 puoq 1E5'qr6uatrsTq16ual uen leeMraP S ' s a l l o ,s 'ull^Luo)uen l 0 t t ' t S ! l g ' l'atuelslsa, EZSI 'unllen 'uoll)e Luslueq)auJ 989 Jo 'aullen 'ajnl)nils 16z | ' s n r r ^e l u ! ] ) P n [ L L g L ' j 9 L S tLt [ 9 'lueudola^aP'uol]ua^alon lH IZg l !86V l' 86nL'uolleul))e^/saul))en

n X]CNI

8t:l

l:49

INDEX

i

membersin differenteucaryotes, 14007 W u s c h epl r o t e i n ,1 4 1 0 ,1 4 1 0 F

x I

I

X-chromosome(s\, 202,473, 1271, 1284 inactivationseeX-inactivation , 275 m e i o t i cp a i r i n g 1 Xenografts,transplantationreactions,1575 Xenopuslaevis,39,39F developmentseeXenopusloevisdevelopment g e n o m ed u p l i c a t i o n3, 8 - 3 9 speciesdifferences, 38-39 Xenopusloevisdevelopment,I 363F c e l lm o v e m e n d t u r i n gd e v e l o p m e n t1, 3 6 3 effectof blockingNotchsignalingin, 1360F egg,1364F cell-freesystems,1058, 1058F fertilization,1057 growth, 1058F microtubule d y n a m i c sd u r i n gm i t o s i s1, 0 8 0 , 1081F embryo c e l lc y c l ea n a l y s i s1,0 5 7 - 10 5 8 ,10 5 7 F cleavage,1058F gastrulation,1365-1 369, 1366F,1367F,1368F, 1369F cell movements,1365-1367, 1365F,1366F, 1367F cell packingchanges(convergentextension), r368-1369,1369F s i g n a l sc o n t r o l l i n g1, 3 6 7 Xenopusruwenzoriensis, 39 Xenopustropicolis,39, 39F Xerodermapigmentosum(XP),2951 1208 center),474 XIC(X-inactivation X-inactivation, 473-475, 473F,474F Barr body,473 d o s a g ec o m p e n s a t i o ,4 7 3 ,4 7 5 - 4 7 6 histoneH2Avariants,474-475 histoneH3 variants,475 histoneH4 variants,475 mechanism,285,473-475, 473F m o s a i c s1, 2 0 9 F randomchromosomechoice,473-474 s e eo / s oG e n es i l e n c i n gG; e n o m i ci m p r i n t i n g X-inactivationcenter(XlC),474 X I S TR N A4, 7 3 F , 4 7 4 XMAP215, microtubuleformation,995F,1004, 1004F Xpd gene/protein,mouseknockouts,568F

-528 x-tay(s),527 sensitivity,DNA repairdefects,2957 synchrotronsources,529 X-raycrystallography DNA,I 95-196, 197 electrondensitymaps,528 historicalaspects,5307 l g Gs t r u c t u r e1,5 6 1F proteinstructure determination,139, 527-529, 528F

m i t o c h o n d r i aDl N A , 8 6 3 inheritance,864-865,865F petite mutants,866-867 deletioncassettes, 570 mutagenesis, Yersinio pestis, 1502, 1502F,1520 pseudotuberculosis, I 1 508, 520 Yersinio Yolk,1287, 1290 Yolkgranules,1287

Y

Zap70tyrosinekinase,1590F Z disc,1026,1027F Zebrafish,255,556,592F,1391F Zellwegersyndrome,723 Zigzagmodel,30-nmfiber formation,217,217F Zinc finger motifs, 421, 421-422, 422F Zinc finger proteins,421-422, 422F,423F D N A - b i n d i n g4,2 2 F sequence specilicity,427F structure,42l, 422F ty pes,421|-422, 422F,423F zinc finger repeats,42I Zinc protease,lethaltoxin, 1493 Zona adherens(adhesionbelt\, 1142, 1143F Zona occludensseeTightjunction(s) Zona pelfucida,1287,1288F,1291F,1298,1298F, 1300 ZOproteins,1154 Z P 1g l y c o p r o t e i nl 2, 9 S ZP2glycoprotein,1298,1300 ZP3glycoprotein,1298, I 300 Z - r i n gf,o r m a t i o nd u r i n gc e l ld i v i s i o n9, 8 9 , 9 8 9 F 852 Z scheme(photosynthesis), zygote,1269 c e n t r i o l e s1,3 O l , 13 0 1F reconstruction(cloning),genome preservation, 4 1 1 ,4 1 3 F zygotic-effectgenes,I 337 seea/soFertilization Zygotene,1275 Zygotic-effectgenes,1337 Zymogens,793

YACsseeYeastartificialchromosomes(YACs) Y-chromosome, 202,473, 1271, 1284 , 275 m e i o t i cp a i r i n g 1 Srygene,1284 Yeast(s), 33,606F,1494 budding seeBuddingyeast(s) cell cyclecontrol,1056-1057, 1056F,1057F, 1062, 1063F r e p l i c a t i o n2,10 chromosome E.coli vs.,34 fermentations,90 seea/soGlycolysis pombe fission yeast seeSchizosocchoromyces g e n ee x p r e s s i oann a l y s i s3,4 - 3 5 genetics5eeYeastgenetics genome3 , 4 ,3 18 T m e t a b o l i cm a p ,1 0 2 F mitochondria,857,858F,867F DNA,863, 864-865, 865F,466-867 as model eucaryote,33-34 'protein-onlyinheritancei398,398F proteintransportstudies,703F reproductivecycles,34F s e x u arl e p r o d u c t i o nh,a p l o i dv s .d i p l o i dc e l l s , 1269-1270,1270F "shmoo:'1044,1044F in tree of life,I 6F two-hybridmethods,protein-protein i nteractions,523-524, 524F u t i l i t ya s m o d e lo r g a n i s m s1,0 5 6 vacuoles,lysosomalproperties,7A1-782 541,542F Yeastartificialchromosomes(YACs), Yeastbudding cellularpolarity,880, 1044,1045F mating types,1044 signalingpathway,880,880E 1045F Yeastgenetics gene regulatoryproteins,DNA sequence recognition,418T

z

T:1

t

U C A G

n

r

Ser 5er 5er Ser

Tyr Tyr STOP STOP

cys cys STOP Trp

Leu Leu Leu Leu

Pro Pro Pro Pro

His His Gln Gln

Arg Arg Arg Arg

lle lle lle Met

Thr Thr Thr Thr

Asn Asn Lys Lys

5er Ser Arg Arg

Val Val Val Val

Ala Ala Ala Ala

Asp Asp Glu Glu

Glv Glv Glv Glv

v

L

Phe Phe Leu Leu

n

I

\l

I U

c

A G U

c

A G U

c

A G

A

Ala

Alanine

c

cys

D

Asp

Cysteine Asparticacid

GACGAU

E F

Glu Phe

Glutamicacid Phenylalanine

GAA GAG U U CU U U

G

Gly His

Glycine Histidine

G G AG G CG G GG G U

lle

lsoleucine

AUA AUCAUU

K L

Lys Leu

Lysine Leucine

AAA AAG

M

Met

Methionine

U U A U U GC U AC U CC U GC U U AUG

N

Asn

Asparagine

AAC AAU

P

Pro

Proline

ccAccccccccu

a R

Gln Arg

Glutamine Arginine

CAA CAG AGA AGG CGACGCCGGCGU

S T

5er Thr

Serine Threonine

AGCAGU UCAUCCUCGUCU ACA ACCACG ACU

V

Val

W Y

Trp Tyr

Valine Tryptophan

G U AG U CG U GG U U UGG UAC UAU

H I

Tyrosine

GCAGCCGCGGCU U G CU G U

CACCAU