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About the cover: Mitotic PtK2 cellsin late anaphasestainedblue for DNA and greenfor tubulin. Courtesyof Torsten\gittman. Library of CongressCataloging-in-PublicationData Molecular cell biology I Harvey Lodish . . . [et al.]. -6th ed. p. cm. Includesbibliographicalreferencesand index. 1. Cytology. 2. Molecular biology. I. Lodish, Harvey F. QH581.2.M6552007 57L5-dc22 2007006188 ISBN-13: 97 8-0-7167-7601-7 ISBN-10:0-7167-7 601-4 @ 1986,1.990,1995,2000,2004,2008 by tJf.H. Freemanand Company All rights reserved. Printed in the United Statesof America Secondprinting W. H. Freemanand Company 41 Madison Avenue,New York, NY 10010 Houndmills, Basingstoke RG21 6XS, England

www.whfreeman.com

To our studentsand to our teachers, from whom we continueto learn, and to our families,for their support, and love encouragement,

PREFACE

I n writing the sixth edition of Molecular Cell Biology I we have incorporated many of the spectacularadvances I made over the past four years in biomedical science,driven in part by new experimental technologiesthat have revolutionized many fields. High-velocity techniquesfor sequencing DNA, for example,have generatedthe completesequence of dozens of eukaryotic genomes;these in turn have led to important discoveriesabout the organization of the human genome and regulation of gene expression,as well as novel insights into the evolution of life-forms and the functions of individual members of multiprotein families. New imaging techniqueshave generated profound revelations about cell organization and movement, and new molecular structures have greatly increased our understanding of life processes such as cell-cell signaling, photosynthesis, gene transcription, and chromatin structure.

what we know. A number of experimental organisms,from yeaststo worms to mice, are used throughout so the student can seehow discoveriesmade with a "lower organism" can Iead directly to insights even about human biology and disease.This experimental approach, evident in the text itself, has also been thoroughly integrated into the pedagogical framework. For example:

New Author Team

r Updated Perspectives for the Future essays explore potential applications of future discoveriesand unanswered questions that lie ahead in research.

Two new authors have been instrumental in refocusing this book toward these exciting new developments. Anthony Bretscher of Cornell University is known for identifying and characterizing new components of the actin cytoskeleton and elucidating their biological functions in relation to cell polarity and membrane traffic. Hidde Ploegh, of the Massachusetts Institute of Technologg has made major contributions to our understanding of immune system behavior, particularly in regard to the various tactics that viruses employ to evade our immune responsesand the ways our immune systemresponds. Both authors are widely recognized for their researchas well as their classroomteachingabilities. 'We are grateful to Paul Matsudaira, Jim Darnell, Larry ZipurskS and David Baltimore for their exceptional contributions to the previous editions of Molecular Cell Biology. Much of their vision and insight is apparent at many places in this book.

Experimental Emphasis The hallmark of Molecular Cell Biology has always been the use of experiments to teach students how we have learned

r Experimental Figures lead students through important experimental results. r Classic Experiments essaysfocus on historically important and Nobel Prize-winningexperiments. r New and revised Analyze the Data problems at the end of each chapter require the student to synthesizereal experimental data to answer a seriesof questions.

showsthe locationof DNAand multiple microscopy Fluorescence et al, 2006,Science BNG Giepmans proteins withinthe samecell.lFrom 3'12:217 |

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New Discoveries, New Methodologies

Coiled-coilstalk

Motor head Microtubule

Necklinkers

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Methodological advances continue to expand and enrich our knowledge of molecular cell biology and lead to new understanding. Following are just a selection of the new experimental methodologiesand cutting-edgescienceintroduced in this edition: r Expanded coverage of proteomics, including organelle proteome profiling and advances in mass spectroscopy (Chapter 3)

I ForwardmotorbindsB-tubulin, ADP ^ releasing

p ForwardheadbindsATP

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r Expanded coverageof RNAi, including the use of shRNAs to inhibit any gene of interest in a cultured cell or organism (Chapters5, 8) r Updated discussionsof chromatin, including structure and condensation(Chapter 6), control of geneexpressionby chromatin remodeling (Chapter 71, and chromatin-remodeling proteins and tumor development(Chapter 25) r Evolution (Chapter 6)

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Conformationalchangein necklinkercausesrear head to swing forward

of chromosomes and the mitochondrion

r New molecular models, including pre-initiation complex and mediator complex (Chapter 7); annular phospholipids (Chapter 1,0);Caz* ATPase (Chapter 11); rhodopsin, transducin, and protein kinase A (Chapter 15); and myosin ATPase (Chapter 17) r Latest advancesin light and electron microscopy, including cryoelectron tomography (Chapter 9) r Reactive oxygen species(ROS) (Chapter 12)

ADB [ rue* forwardheadreleases ^ trailingheadhydrolyzesATP V and releases P.

r Role of supercomplexesin electron transport (Chapter 12) r Human epidermal growth factor receptors (HERs) and treatment of cancer (Chapter 16) r Myosin ATPasecycle (Chapter 17) r Kinesin-1MPase cycle (Chapter 18) r Use of retrovirus infection for tracing cell lineage (Chapter 21) r Axon guidance molecules (Chapter 23) r Somaticgenerearrangementin immune cells (Chapter 24) r Cancer stem cells (Chapter25) r Use of DNA (Chapter25)

Figure18-22 Kinesin-1 usesATPto "walk" downa microtubule

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PREFACE

microarray analysis in tumor typing

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For Students Companion Web Site www.whfreeman. com,/lodish5e NEW: Podcastsnarrated by the authors give students a deeperunderstanding of key figures in the text and a senseof the thrill of discovery. r NEW: Now available for your MP3 player or personal computer, more than 1,25animations and researchvideos show the dynamic nature of key cellular processesand important experimental techniques.The animations were storyboarded by the textbook authors in conjunction with BioStudio, Inc., and programmed by Sumanas,Inc. r Classic Experiment essaysfocus on classicgroundbreaking experiments and explore the investigativeprocess. r Online Quizzing is provided, including multiple-choice and short answer questions. -4), written Student SolutionsManual (ISBN:1-4292-01.27 'Wong, Richard Walker, Glenda by Brian Storrie, Eric A. Gillaspn and Jill Sible of Virginia Polytechnic Institute and State University and updated by Cindy Klevickis of James Madison University and Greg M. Kelly of the University of Western Ontario, contains complete workedout solutions to all the end-of-chapter problems in the textbook. NEW: eBook (ISBN: 1,-4292-0955-0)New to the sixth edition, this customizable eBook fully integrates the complete contents of the text and its interactive media in a format that features a variety of helpful study tools, including fulltext searching,note-taking, bookmarking, highlighting, and more. Easily accessibleon any Internet-connectedcomputer via a standard rWebbrowser, the eBook enablesstudents to take an active approach to their learning in an intuitive, easy-

to learn

For Instructors Companion Web Site www.whfreeman.com/lodish6e All the student resources,plus: r All figures and tables from the book in ipeg and layered PowerPoint formats, which instructors can edit or project section by section, allowing students to follow underlying concepts.Optimized for lecture-hall presentation' including enhancedcolors, enlarged labels, and boldface rype. r Test Bank in editable Microsoft'Word format now featuringnetu and reuisedquestionsfor every chapter.The test bank is written by Brian Storrie of the University of Arkansas for Medical Sciencesand Eric A. Wong, Richard Walker, Glenda GillaspS and Jill Sible of Virginia Polytechnic Institute and StateUniversity and revisedby Cindy Klevickis of JamesMadison University and Greg M. Kelly of the University of Ontario. r Additional Analyze the Data problems are available in PDF format. r NEW: Lecture-ready Personal ResponseSystem "clicker" questions are available as Microsoft'Word files and Microsoft PowerPoint slides. Instructor's Resource CD-ROM (ISBN: 1'-4292-0126-6) includesall the instructor's resourcesfrom the Web site' including all the illustrations from the text, animations, videos, test bank files, clicker questions, and the solutions manual files. 'l'-4292-0477-X) conOverhead tansparency Set (ISBN: for optimized text' the from key illustrations tains 250 presentation. lecture-hall

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PREFACE

'

ACKNOWLEDGMENTS In updating, revising and rewriting this book, we were given 'We invaluable help by many colleagues. thank the following people who generouslygave of their time and expertiseby making contributions to specific chapters in their areas of interest, providing us with detailed information about their courses, or by reading and commenting on one or more chapters: Steven Ackerman, (Jniuersity of Massacbusetts,Boston Richard AdIe4 IJniuersity of Michigan, Dearborn Karen Aguirre, Coastal Carolina lJniuersity Jeff Bachant, Uniuersity of California, Riuerside Kenneth Balazovich, Uniuersity of Michigan Ben A. Barres, Stanford Uniuersity Karen K. Bernd, Dauidson College Sanford Bernstein, San Diego State (Jniuersity Doug Black, Howard Hughes Medical Institute and (Jniuersity of California, Los Angeles Richard L. Blanton, North Carolina State (Jniuersny Justin Blau, New York [Jniuersity Steven Block, Stanford IJniuersity Jonathan E. Boyson, Uniuersity of Vermont Janet Braam. Rice Uniuersity Roger Bradleg Montana State Uniuersity IilTilliam S. Bradshaq Brigham Young (Jniuersity Gregory G. Brown, McGill (Jniuersity \Tilliam J. Brown, Cornell IJniuersity Max M. Burger, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland David Burgess,Boston College Robin K. Cameron, McMaster [Jniuersity \7. Zacheus Cande, (Jniuersity of California, Berkeley Steven A. Carr, Broad lnstitute of Haruard (Jniuersity and Massacbusetts Institute of Technology Alice Y. Cheung, IJniuersity of Massacbusetts, Amberst Dennis O. Clegg, Uniuersity of California, Santa Barbara Paul Clifton, Utah State IJniuersity Randy \7. Cohen, California State [Jniuersity, Northridge Richard Dickerson, (Jniuersity of California, Los Angeles Patrick J. DiMario, Louisiana State Uniuersity Santosh R. D'Mello, [Jniuersity of Texas, Dallas Chris Doe, HHMI and [Jniuersity of Oregon Robert S. Dotson, Tulane [Jniuersity 'Sfilliam Dowhan, IJniuersity of Texas-Houston Medical School Gerald B. Downes, (Jniuersity of Massachusetts,Amherst Erastus C. Dudley, Huntingdon College Susan Dutcher, V/ashington (Jniuersity School of Medicine Matt Elrod-Erickson, Middle TennesseeState Uniuersity Susan Ely, Cornell Uniuersity Charles P. Emerson Jr., Boston Biomedical ResearchInstitute Irene M. Evans, Rochester Institute of Technology James G. Evans,.Whitehead Institute Bio Imaging Center, Massachusetts Institute of Technology Marilyn Gist Farquhar, Uniuersity of California, San Diego

X

PREFACE

Xavier Fernandez-Busquets, Bioengineering Instituteof Catalonia, Uniuersitatde Barcelona,Spain TerrenceG. Frey,San Diego State Uniuersity Margaret T. Fuller,Stanford UniuersitySchoolof Medicine KendraJ. Golden, WbitmanCollege David S. Goldfarb,RochesterUniuersity Martha J. Grossel,ConnecticutCollege LawrenceI. Grossman,WayneStateUniuersitySchoolof Medicine Michael Grunstein, Uniuersityof California, Los Angeles, Schoolof Medicine Barry M. Gumbiner, Uniuersityof Virginia 'Wei Guo, Uniuersityof Pennsyluarua Leah Haimo, Uniuersityof California, Riuerside Heidi E. Hamm, Vanderbilt(JniuersityMedicalSchool Craig M. Hart, Louisiana State Uniuersity Merill B. Hille, Uniuersityof Washington Jerry E. Honts, Drake Uniuersity H. Robert Horvitz, Massachusetts Institute of Technology Richard Hynes, Massachusetts lnstitute of Technologyand Howard HughesMedical Inshtute Harry Itagaki,Kenyon College ElizabethR. Jamieson,Smitb College Marie A. Janicke,State Uniuersityof New York, Buffalo Bradley'W.Jones,Uniuersityof Mississippi Mark Kainz, ColgateUniuersity Naohiro Kato, Louisiana State Uniuersity Amy E. Keating, Massachusetts lnstitute of Technology CharlesH. Keith, Uniuersityof Georgia Thomas C. S. Keller lll, Florida State Uniuersity 'V/estern Greg M. Kelly, Uniuersityof Ontario StephenKendall, California State Uniuersity,Fullerton FelipeKierszenbaum, MichiganStateUniuersity Cindy Klevickis,JamesMadison lJniuersity Brian Kobilka, StanfordUniuersityMedicalSchool. Martina Koniger, WellesleyUniuersfiy CatherineKoo, Caldwell College Keith G. Kozminski, Uniuersityof Virginia StevenI(. IJHernault, Emory Uruuerstty Douglas Lauffenburger, Massacbusetts Institute of Tecbnology RobertJ. Lefkowitz, HHMI and Duke UniuersityMedical School R. L. Levine,McGill Uniuersity FangJu Ltn, CoastalCarolina [Jniuersity ElizabethLord, Uniuersityof California, Riuerside Liqun Luo, StanfordUniuersity Grant MacGregor, Uniuersityof California, Iruine Jennifer O. Manilay, IJniuersityof California, Merced Barry Margul ies,Towson Uniuer stty C. William McCurdy, Uniuersityof California, Dauis, and LawrenceBerkeleyNational Laboratory Dennis \il/. McGee, State Uniuersityof New York, Binghamton JamesMcGrath, RochesterSchoolof Medicine David D. McKemy, Uniuersityof SouthernCalifornia Roderick MacKinnon, Rockefeller(Jniuersity JamesA. McNew, Rice Uniuersity

X

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CONTENTS IN BRIEF

Part I 1. 2. 3. Part ll 4. 5. 6. 7. 8. Part lll 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Chemical and MolecularFoundations1 L i f e B e g i n sw i th C e l l s 1 C h e mi caFl o u n d a ti o n s3 1 ProteinStructureand Function 63

Genetics and MolecularBiology 111 B a s i cM o l e c u l aG r e n e t i cM e c h a n i s m1s 1 1 M o l e cu l a Ge r n e ti cT e ch n i ques1G5 G e n e s,Ge n o mi cs, a n d C h rom osomes 215 Transcriptional Controlof Gene Expression269 Post-transcriptional GeneControl 323

CeflStructureand Function 371 V i su a l i zi n gF, ra cti o n a ti n g, and Cultur ingCells 371 BiomembraneStructure 409 Transmembrane Transportof lonsand small Molecures437 C e ll u l a rE n e rg e ti cs4 7 9 M o vi n gP ro te i n si n to Me mbr anes and Or ganelles533 VesicularTraffic,Secretion,and Endocytosis579 C e llS i g n a l i n gl : S i g n a T l ra nsduction and Shor t- Ter m CellularResponses 62 3 c e l l si g n a l i n gl l : si g n a l i n gPathways That contr ol GeneActivity 665 C e l lOrg a n i za ti o n a n d Mo v ementl: M icr ofilaments713 C e l lOrg a n i za ti o n a n d Mo v ementll: Micr otubules and Inter m ediate Filaments157 I n t e g ra ti n gC e l l si n to T i ssues801

Part lV CellGrowth and Development847 20. Regulatingthe EukaryoticCellCycle 847 21. 22. 23. 24. 25.

C e l lB i rth ,L i n e a g ea, n d D e ath 905 T h e Mo l e cu l a C r e l lB i o l o g yof Developm entg4g N e rveC e l l s 1 0 0 1 l m m u n o l o g y1 0 5 5 Cancer 1107

CONTENTS

PartI Chemical and MolecularFoundations

E

The Diversityand Commonality of Cells

18

C e l l sG r o w a n d D i v i d e

WITHCELLS 1 L I F EB E G I N S

CellsDie from AggravatedAssaultor an Internal Program 19

1

!!|

InvestigatingCellsand Their Parts 20

All CellsAre Prokaryoticor Eukaryotic

1

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s

4

VirusesAre the Ultimate Parasites

6

Cell BiologyRevealsthe Size,Shape,Location, 20 and Movementsof Cell Components Biochemistryand BiophysicsRevealthe Molecular Structureand Chemistryof PurifiedCell Constituents 21

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

6

of DamagedGenes 22 GeneticsRevealsthe Consequences

EvenSingleCellsCan HaveSex

7

We Developfrom a SingleCell

8

GenomicsRevealsDifferencesin the Structure of EntireGenomes and Expression

23

DevelopmentalBiologyRevealsChangesin the Propertiesof Cellsas They Specialize

23

Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities

@The

Choosingthe Right ExperimentalOrganismfor the Job 25 BiologicalStudiesUse Multiple The Most Successful 27 Approaches

M o l e c u l eos f a c e l l

Small MoleculesCarryEnergy,TransmitSignals, a n d A r e L i n k e di n t o M a c r o m o l e c u l e s ProteinsGive CellsStructureand Perform Most CellularTasks

'

s|

on Evolution 28 A GenomePerspective

10

Metabolic Proteins,the GeneticCode,and Organelle StructuresAre NearlYUniversal

't1

The Genome ls Packagedinto Chromosomesand R e p l i c a t e dD u r i n gC e l lD i v i s i o n

Darwin'sldeasAbout the Evolutionof Whole Animals Are Relevantto Genes

12

Mutations May Be Good, Bad, or lndifferent

13

Many GenesControllingDevelopmentAre Remarkably 28 S i m i l a ri n H u m a n sa n d O t h e r A n i m a l s Human Medicinels Informed by Researchon Other 29 Organisms

NucleicAcidsCarryCoded Information for Making Proteinsat the Right Time and Place

E

Thework of cells

28

14

C e l l sB u i l da n d D e g r a d eN u m e r o u sM o l e c u l e s and Structures

15

Animal CellsProduceTheir Own External E n v i r o n m e nat n d G l u e s

16

CellsChangeShapeand Move

16

CellsSenseand Send Information

16

covalent Bondsand Noncovalent lnteractions

to CellsRegulateTheir Gene Expression M e e t C h a n g i n gN e e d s

17

the Structureof an Atom Determines TheElectronic of CovalentBondslt CanMake 33 Numberand Geometry

31

FOUNDATIONS 2 CHEMICAL ![

CONTENTS

.

32

E l e c t r o n sM a y B e S h a r e dE q u a l l yo r U n e q u a l l y i n C o v a l e n tB o n d s

34

L i f e D e p e n d so n t h e C o u p l i n go f U n f a v o r a b l eC h e m i c a l Reactionswith EnergeticallyFavorableReactions 57

CovalentBondsAre Much Strongerand More StableThan NoncovalentInteractions

Hydrolysisof ATPReleases SubstantialFreeEnergy and DrivesMany CellularProcesses

lonic InteractionsAre Attractionsbetween Oppositely C h a r g e dl o n s 36 HydrogenBondsDeterminethe Water Solubility o f U n c h a r g e dM o l e c u l e s 37

ATPls GeneratedDuring Photosynthesis and Respiration 59 N A D - a n d F A DC o u p l eM a n y B i o l o g i c aO l xidation and ReductionReactions

Van der Waals InteractionsAre Causedby T r a n s i e nD t iooles

37

The HydrophobicEffectCausesNonpolar Moleculesto Adhere to One Another

38

3 P R O T E ISNT R U C T U R ED AN FUNCTION

M o l e c u l a rC o m p l e m e n t a r i tM y e d i a t e dv i a NoncovalentInteractionspermitsTight, H i g h l yS p e c i f i cB i n d i n go f B i o m o l e c u l e s

3e

![

C h e m i c alB u i l d i n gB l o ckso f C e i l s

@

A m i n o A c i d sD i f f e r i n gO n l y i n T h e i rS i d eC h a i n s ComposeProteins

40 41

FiveDifferent NucleotidesAre Usedto Build N u c l e i cA c i d s Monosaccharides Joined by GlycosidicBonds Form Linearand Branchedpolysaccharides

44

PhospholipidsAssociateNoncovalentlyto Form the BasicBilayerStructureof Biomembranes

C h e m i c aEl q u i l i b ri u m

49

EquilibriumConstantsReflectthe Extentof a ChemicalReaction

50

![

Hierarchical Structureof proteins

SecondaryStructuresAre the Core Elements of ProteinArchitecture

66

Overall Foldingof a PolypeptideChainYields Its TertiaryStructure

67

Different Waysof Depictingthe Conformationof ProteinsConveyDifferent Typesof Information

68

StructuralMotifs Are RegularCombinationsof Secondaryand TertiaryStructures

68

S t r u c t u r aal n d F u n c t i o n aD l o m a i n sA r e M o d u l e s of TertiaryStructure

70

ProteinsAssociateinto Multimeric Structuresand M a c r o m o l e c u l aAr s s e m b l i e s

72

Membersof Protein FamiliesHavea Common EvolutionaryAncestor

72

![

BiologicalFluidsHaveCharacteristic pH Values

51

P l a n a rP e p t i d eB o n d sL i m i tt h e S h a p e si n t o Which ProteinsCan Fold

B i o c h e m i caEl n e rg e ti cs

@

SeveralFormsof EnergyAre lmportant in BiologicalSystems CellsCan TransformOne Typeof Energyinto Another The Changein Free EnergyDeterminesthe Direction of a ChemicalReaction The AG'' of a ReactionCan Be Calculated from lts K"o The Rateof a ReactionDependson the Activation EnergyNecessary to Energizethe Reactantsinto a TransitionState

xvi

.

coNTENTs

64 65

50

B u f f e r sM a i n t a i nt h e p H o f I n t r a c e l l u l aar n d E x t r a c e l l u l aFrl u i d s

63

The PrimaryStructureof a Protein ls lts Linear Arrangementof Amino Acids

ChemicalReactionsin CellsAre at SteadyState DissociationConstantsof Binding ReactionsReflect the Affinity of InteractingMolecules Hydrogenlons Are Releasedby Acidsand Taken Up by Bases

57

50

52 52

ProteinFolding

74 74

I n f o r m a t i o nD i r e c t i n ga P r o t e i n ' sF o l d i n gl s E n c o d e di n l t s A m i n o A c i d S e q u e n c e

74 Foldingof Proteinsin Vivo ls Promotedby Chaperones 7 5 AlternativelyFoldedProteinsAre lmplicatedin Diseases 77

54

ProteinFunction

78

S p e c i f i cB i n d i n go f L i g a n d sU n d e r l i e st h e Functionsof Most Proteins

7g

EnzymesAre Highly Efficientand SpecificCatalysts

79

An Enzyme'sActive Site BindsSubstratesand CarriesOut Catalysis

g0

SerineProteasesDemonstrateHow an Enzyme'sActive Site Works

g1

Enzymesin a Common PathwayAre Often physically Associatedwith One Another

g4

!f, 54 55 55 56

56

EnzymesCalledMolecularMotors ConvertEnergy into Motion

!E-

!l|

r.r RegulatingProteinFunctionl: ProteinDegradation

RegulatedSynthesisand Degradationof Proteins is a FundamentalPropertyof Cells

CentrifugationCan SeparateParticlesand MoleculesThat Differ in Massor Density

86 86

The Proteasomels a ComplexMolecularMachine Usedto DegradeProteins Ubiquitin Marks CytosolicProteinsfor Degradation in Proteasomes

RegulatingProteinFunctionll: Noncovalentand Covalent Modifications NoncovalentBinding PermitsAllosteric, or Cooperative,Regulationof Proteins

Purifying,Detecting,and Proteins Characterizing

85

SeparatesMoleculeson the Basis Electrophoresis Ratio of Their Charge-to-Mass Liquid ChromatographyResolvesProteinsby Mass,Charge,or BindingAffinitY

92 92 94 96

Highly SpecificEnzymeand Antibody AssaysCan Detect IndividualProteins Toolsfor Detecting Are Indispensable Radioisotopes M o l e c u l e s Biological MassSpectrometryCan Determinethe Mass and Sequenceof Proteins

101

88

Protein PrimaryStructureCan Be Determinedby ChemicalMethods and from Gene Sequences

103

89

Protein Conformationls Determinedby Sophisticated 103 PhysicalMethods

88

NoncovalentBindingof Calciumand GTPAre Widely UsedAs AllostericSwitchesto Control ProteinActivity 90 Phosphorylationand DephosphorylationCovalently RegulateProteinActivitY

91

ProteolyticCleavagelrreversiblyActivatesor lnactivatesSome Proteins

91

Higher-OrderRegulationIncludesControl of Protein Locationand Concentration

92

g

98 99

105

Proteomics

Proteomicsls the Studyof All or a LargeSubset of Proteinsin a BiologicalSYstem AdvancedTechniquesin MassSpectrometryAre Criticalto ProteomicAnalYsis

105 106

Partll Geneticsand MolecularBiologY 4 B A S I CM O L E C U L AGRE N E T I C MECHANISMS @

structureof NucleicAcids

111 113

Organizationof GenesDiffersin Prokaryoticand EukaryoticDNA to EukaryoticPrecursormRNAsAre Processed l RNAs F o r mF u n c t i o n am the Number of AlternativeRNA Splicinglncreases from a SingleEukaryoticGene ProteinsExpressed

A NucleicAcid Strand ls a LinearPolymerwith End-to-EndDirectionality

113

@

N a t i v eD N A l s a D o u b l eH e l i xo f C o m p l e m e n t a r y A n t i p a r a l l eS l trands

114

DNA Can Undergo ReversibleStrandSeparation

115

MessengerRNA CarriesInformation from DNA in a Three-LetterGeneticCode The FoldedStructureof IRNA Promoteslts Decoding

TorsionalStressin DNA ls Relievedby Enzymes

117

Different Typesof RNA ExhibitVarious ConformationsRelatedto Their Functions

118

@ T r a n s c r i p t i o n o f P ro te i n -C o d i ng Genesand Formationof Functional 120 MRNA A TemplateDNA Strand ls Transcribedinto a ComplementaryRNA Chain by RNA Polymerase

120

122 123 125

The Decodingof mRNAbY tRNAs 127

Functions NonstandardBasePairingOften OccursBetween C o d o n sa n d A n t i c o d o n s Amino Acids BecomeActivatedWhen Covalently L i n k e dt o t R N A s

@

StepwiseSynthesisof Proteins on Rabosomes

Machines RibosomesAre Protein-Synthesizing

CONTENTS

127 129 130 131

132 132

XVII

Methionyl-tRNA,tttRecognizes the AUG StartCodon T r a n s l a t i o nI n i t i a t i o nU s u a l l yO c c u r sa t t h e F i r s t A U G f r o m t h e 5 ' E n do f a n m R N A D u r i n gC h a i nE l o n g a t i o nE a c hl n c o m i n g Aminoacyl-tRNAMovesThrough Three RibosomalSites Translationls Terminatedby ReleaseFactors When a Stop Codon ls Reached Polysomesand RapidRibosomeRecyclingIncrease the Efficiencyof Translation

DNAReptication

!f|

133 133

E! 135

166

137

166

138

Segregationof Mutations in BreedingExperiments RevealsTheir Dominanceor Recessivity

167

ConditionalMutations Can Be Usedto Study EssentialGenesin Yeast

EO

139 140

Duplex DNA ls Unwound and Daughter StrandsAre Formedat the DNA ReplicationFork

't41 141 143

Recessive Lethal Mutations in DiploidsCan Be l d e n t i f i e db y I n b r e e d i n ga n d M a i n t a i n e di n Heterozygotes

171 ComplementationTestsDetermineWhether Different 't7 Recessive Mutations Are in the SameGene 1 Double Mutants Are Usefulin Assessing the Order in Which ProteinsFunction 171 GeneticSuppression and SyntheticLethalityCan RevealInteractingor Redundantproteins 173 GenesCan Be ldentified by Their Map position on the Chromosome 174

D N A R e p a i ra n d R e co mb i n a ti o n 145

!!|

p|

DNA Polymerases IntroduceCopyingErrors and Also CorrectThem

145

C h e m i c aal n d R a d i a t i o nD a m a g et o D N A C a n Leadto Mutations

145

H i g h - F i d e l i tD y N A E x c i s i o n - R e p aSiyr s t e m s Recognize a n d R e p a i rD a m a g e B a s eE x c i s i o n R e p a i r sT . G M i s m a t c h eas n d D a m a g e dB a s e s

147

MismatchExcisionRepairsOther Mismatchesand S m a l lI n s e r t i o n a s nd Deletions

147

NucleotideExcisionRepairsChemicalAdducts That Distort Normal DNA Shape Two SystemsUtilize Recombinationto Repair D o u b l e - S t r a nB d r e a k si n D N A H o m o l o g o u sR e c o m b i n a t i oC n a n R e o a i rD N A D a m a g ea n d G e n e r a t eG e n e t i cD i v e r s i t y

s f th e C e l l u l a r @ V i r u s e s : pa ra si te o GeneticSystem Most Viral Host RangesAre Narrow Viral CapsidsAre RegularArraysof One or a Few Typesof protein

147

148 149 150

154

'

CONTENTS

DNACloningand Char acter i z ati on176

RestrictionEnzymesand DNA LigasesAllow Insertionof DNA Fragmentsinto CloningVectors

176

E. coli PlasmidVectorsAre Suitablefor Cloning lsolatedDNA Fragments

178

cDNA LibrariesRepresentthe Sequences of Protein-CodingGenes

179 cDNAsPreparedby ReverseTranscriptionof Cellular mRNAsCan Be Clonedto GeneratecDNA Libraries 1 8 1 DNA LibrariesCan Be Screenedby Hybridization to an Oligonucleotidp erobe 181 YeastGenomicLibrariesCan Be Constructedwith ShuttleVectorsand Screenedby Functionar Complementation 182 Gel Electrophoresis Allows Separationof Vector DNA from Cloned Fragments 184 Cloned DNA MoleculesAre SequencedRapidly by the DideoxyChain-Termination Methoc 187 The PolymeraseChain ReactionAmplifiesa Specific DNA Sequencefrom a ComplexMixture 188

154 154

VirusesCan Be Clonedand Counted in plaqueAssays 1 5 5 LyticViral Growth CyclesLeadto the Death of Host Cells 1 5 5 Viral DNA ls Integrated into the Host-CellGenome in SomeNonlyticViral Growth Cycles 158

XVIII

GeneticAnalysisof Mutationsto ldentifyand StudyGenes

R e c e s s i vaen d D o m i n a n tM u t a n t A l l e l e sG e n e r a l l y HaveOppositeEffectson Gene Function

D N A P o l y m e r a s eR s e q u i r ea p r i m e r t o I n i t i a t e Replication

SeveralProteinsParticipatein DNA Replication D N A R e p l i c a t i o nU s u a l l yO c c u r sB i d i r e c t i o n a l lfyr o m E a c hO r i g i n

5 M O L E C U L AGRE N E T T C E C H N T Q U1E6Ss

ff|

UsingClonedDNA Fragments to Study GeneExpression

191

HybridizationTechniquespermit Detection of SpecificDNA Fragmentsand mRNAs

't91

DNA MicroarraysCan Be Usedto Evaluatethe Expression of Many Genesat One Time

192

ClusterAnalysisof Multiple ExpressionExperiments ldentifies Co-regulatedGenes

193

N o n p r o t e i n - C o d i nG g e n e sE n c o d eF u n c t i o n a l RNAs

SystemsCan ProduceLarge E. coli Expression Quantitiesof Proteinsfrom ClonedGenes VectorsCan Be Designedfor PlasmidExpression U s ei n A n i m a l C e l l s

Or ganization Chr om osomal of Genesand NoncodingDNA

196

222

223

s o n t a i nM u c h G e n o m e so f M a n y O r g a n i s m C N o n f u n c t i o n aD l NA

l d e n t i f y in ga n d L o ca ti n gH u ma n Disease Ge n e s

DNAsAre Concentrated Most Simple-Sequence in SpecificChromosomalLocations

224

199

DNA FingerprintingDependson Differences DNAs in Length of SimPle-Sequence

225

D N A P o l y m o r p h i s mAsr e U s e di n L i n k a g e - M a p p i n g H u m a nM u t a t i o n s

200

SpacerDNA Occupiesa Significant Unclassified Portion of the Genome

225

LinkageStudiesCan Map DiseaseGeneswith a Resolutionof About 1 Centimorgan

201

FurtherAnalysisls Neededto Locatea DiseaseGene i n C l o n e dD N A

202

f[

Show One of Three Major Many InheritedDiseases Patternsof Inheritance

198

]f|

M a n y I n h e r i t e dD i s e a s eRs e s u l ft r o m M u l t i p l eG e n e t i c 203 Defects

ffl

Inactivatingthe Functionof S p e c i f i cGe n e si n E u ka ryo te s

Normal YeastGenesCan Be Replacedwith Mutant A l l e l e sb y H o m o l o g o u sR e c o m b i n a t i o n

204 205

Transcriptionof GenesLigatedto a Regulated PromoterCan Be ControlledExperimentally SpecificGenesCan Be PermanentlyInactivatedin t h e G e r m L i n eo f M i c e

207

SomaticCell RecombinationCan InactivateGenes in SpecificTissues

208

D o m i n a n t - N e g a t i vAel l e l e sC a n F u n c t i o n a l l y l n h i b i t S o m eG e n e s RNA InterferenceCausesGene Inactivationby g RNA D e s t r o y i n gt h e C o r r e s p o n d i nm

6 G E N E SG , E N O M I C SA, N D CHROMOSOMES

][

EukaryoticGeneStructure

(Mobile)DNA Transposable Elem ents

Movement of Mobile ElementsInvolvesa D N A o r a n R N AI n t e r m e d i a t e

226

Are Presentin Prokaryotes DNA Transposons and Eukaryotes

227

BehaveLike Intracellular LTRRetrotransposons Retroviruses Transposeby a Distinct Non-LTRRetrotransposons Mechanism RNAsAre Found in Genomic Other Retrotransposed DNA M o b i l e D N A E l e m e n t sH a v eS i g n i f i c a n t l Iyn f l u e n c e d Evolution

@

215 217

Most EukaryoticGenesContain Intronsand Produce m R N A sE n c o d i n gS i n g l eP r o t e i n s

217

n nits S i m p l ea n d C o m p l e xT r a n s c r i p t i oU A r e F o u n di n E u k a r y o t i cG e n o m e s

217

P r o t e i n - C o d i nG g e n e sM a y B e S o l i t a r yo r B e l o n g t o a G e n eF a m i l y

219

HeavilyUsedGene ProductsAre Encodedby Multiple 221 Copiesof Genes

229 230 234 234

236

or ganelleDNAs

M i t o c h o n d r i aC o n t a i nM u l t i p l e m t D N A M o l e c u l e s

210

226

mtDNA ls InheritedCytoplasmically The Size,Structure,and Coding Capacityof mtDNA Vary ConsiderablyBetweenOrganisms Productsof MitochondrialGenesAre Not Exported M i t o c h o n d r i aE v o l v e df r o m a S i n g l eE n d o s y m b i o t i c Bacterium EventInvolvinga Rickettsia-like

237 237 238 240

240

MitochondrialGeneticCodesDiffer from the StandardNuclearCode

240

Mutations in MitochondrialDNA CauseSeveral G e n e t i cD i s e a s eisn H u m a n s ChloroplastsContain LargeDNAsOften Encoding M o r e T h a n a H u n d r e dP r o t e i n s

242

Anal Ys i s Genome- wide Genom ics: of GeneStructureand Expression 243 SuggestFunctionsof Newly StoredSequences ldentified Genesand Proteins

CONTENTS

243

xtx

Comparisonof RelatedSequences from Different SpeciesCan Give Cluesto Evolutionary Relationships Among proteins G e n e sC a n B e l d e n t i f i e dW i t h i n G e n o m i cD N A Sequences

244

S m a l lM o l e c u l e sR e g u l a t eE x p r e s s i oonf M a n y BacterialGenesvia DNA-BindingRepressors and Activators

273

244

TranscriptionInitiation from Some Promoters RequiresAlternativeSigmaFactors

273

Transcriptionby osa-RNAPolymerasels Controlled by ActivatorsThat Bind Farfrom the Promoter

274

Many BacterialResponses Are Controlledby Two-ComponentRegulatorySystems

275

T h e N u m b e ro f P r o t e i n - C o d i nG g e n e si n a n Organism'G s e n o m el s N o t D i r e c t l yR e l a t e d to lts BiologicalComplexity S i n g l eN u c l e o t i d eP o l y m o r p h i s masn d G e n eC o p y Number VariationAre lmportant Determinants of DifferencesBetween Individualsof a Species

246

@ St r u c t u r alOrg a n i za ti o n of EukaryoticChromosomes

][

ChromatinExistsin Extendedand CondensedForms Modificationsof HistoneTailsControl Chromatin Condensationand Function NonhistoneProteinsProvidea StructuralScaffold for Long ChromatinLoops A d d i t i o n a lN o n h i s t o n ep r o t e i n sR e g u l a t e Transcriptionand Replication

247 248

276

250

Three EukaryoticPolymerases CatalyzeFormation of Different RNAs

278

254

The LargestSubunit in RNA Polymerasell Hasan EssentialCarboxyl-Terminal Repeat

2s6

ChromosomeNumber;Size,and Shapeat Metaphase Are Species-Specific 257 During Metaphase,ChromosomesCan Be D i s t i n g u i s h ebdy B a n d i n gp a t t e r n sa n d C h r o m o s o m eP a i n t i n g 25g C h r o m o s o m eP a i n t i n ga n d D N A S e q u e n c i n g Reveal the Evolutionof Chromosomes 259 InterphasePolyteneChromosomesArise by DNA A m p l i fi c a t i o n 260 Three FunctionalElementsAre Requiredfor Replication a n d S t a b l eI n h e r i t a n c eo f C h r o m o s o m e s 261 CentromereSequences Vary Greatlyin Length 263 Addition of TelomericSequencesby Telomerase PreventsShorteningof Chromosomes

263

7 TRANSCRIPTIONALCONTROL O F G E N EE X P R E S S I O N 269 Controlof GeneExpression in Bacteria

W

271

TranscriptionInitiation by BacterialRNA polymerase RequiresAssociationwith a SigmaFactor 271 Initiation of /ac Operon TranscriptionCan Be Repressed and Activated 271

XX

O

CONTENTS

276

RegulatoryElementsin EukaryoticDNA Are Found Both Closeto and Many KilobasesAway from TranscriptionStart Sites

M o r p h o l og ya n d F u n cti o n aEl l e m ents o f E u k a r y o ti C c h ro mo so me s 257

@

overview of EukaryoticGene Controland RNAPolymerases

279 RNA Polymerasell InitiatesTranscriptionat DNA SequencesCorrespondingto the 5' Cap of mRNAs 280

E

Regulator ysequences in pr ote i nCodingGenes 282

The TATABox, Initiators,and CpG lslandsFunction as Promotersin EukaryoticDNA

282

Promoter-Proximal ElementsHelp Regulate EukaryoticGenes

282

Distant EnhancersOften Stimu late Transcription by RNA Polymerasell

284

Most EukaryoticGenesAre Regulatedby Multiple Transcription-Control Elements

s @Activator s and Repr essorof Transcription

286

Footprintingand Gel-ShiftAssaysDetect protein-DNA Interactions 286 ActivatorsAre Modular ProteinsComposed of DistinctFunctionalDomainsand promote Transcription 288 Repressors Inhibit Transcriptionand Are the FunctionalConverseof Activators

290

D N A - B i n d i n gD o m a i n sC a n B e C l a s s i f i eidn t o NumerousStructuralTypes

290

StructurallyDiverseActivation and Repression DomainsRegulateTranscription

293

TranscriptionFactorInteractionsIncrease Gene-ControlOptions

294

M u l t i p r o t e i nC o m p l e x e F s o r mo n E n h a n c e r s

295

TranscriptionInitiation by RNA Polymerasell GeneralTranscriptionFactorsPositionRNA Polymerasell at Start Sitesand Assistin Initiation

296

@

GENE 8 POST- TRANSCRIPTIONAL CONTROL

Formationof HeterochromatinSilencesGene at Telomeres,Near Centromeres, Expression and in Other Regions

299

Can Direct HistoneDeacetylationand Repressors Methylation at SpecificGenes

303

ActivatorsCan Direct HistoneAcetylationand Methylation at SpecificGenes

305

FactorsHelp Activate or Chromatin-Remodeling RepressTranscription

306

HistoneModificationsVary Greatlyin Their Stabilities 307 307

Transcriptionof Many GenesRequiresOrdered Binding 308 and Functionof Activatorsand Co-activators The YeastTwo-HybridSystemExploitsActivator Flexibility to DetectcDNAsThat EncodeInteractingProteins 310

E

312

ResponseElementsContain Inverted Nuclear-Receptor 313 or Direct Repeats Hormone Bindingto a NuclearReceptorRegulateslts 313 Activity as a TranscriptionFactor

![

RegulatedElongationand Terminationof Transcription

314

Transcriptionof the HIV Genome ls Regulatedby an Antitermination Mechanism

315

Pausingof RNA Polymerasell Promoter-Proximal Occursin Some RapidlyInducedGenes

316

@

f[

other EukaryoticTranscription Systems

TranscriptionInitiation by Pol I and Pol lll ls Analogousto That by Pol ll

315 316

of EukaryoticPre-mRNA325 Processing

The 5' Cap ls Added to NascentRNAsShortlyAfter TranscriptionInitiation

325

A DiverseSet of Proteinswith ConservedRNA-B|nding 326 DomainsAssociatewith Pre-mRNAs SplicingOccursat Short,ConservedSequencesin Pre-mRNAsvia Two TransesterificationReactions

329 330

with Pre-mRNA During Splicing,snRNAsBase-Pair Assembledfrom snRNPsand a Spliceosomes, Pre-mRNA,CarryOut SPlicing

330

C h a i nE l o n g a t i o nb y R N AP o l y m e r a slel l s C o u p l e d Factors to the Presenceof RNA-Processing

333

SRProteinsContributeto Exon Definition in Long Pre-mRNAs Group ll Introns ProvideCluesto the Self-Splicing Evolutionof snRNAs

334

3' Cleavageand Polyadenylationof Pre-mRNAs 335 Are Tightly CouPled DegradeRNAThat ls Processed NuclearExonucleases 336 Out of Pre-mRNAs

Regulationof Transcription-Factor 311 Activity

All NuclearReceptorsSharea Common Domain Structure

323

298

o f T ra n sc r iption M o l e c u l a Me r ch a n i sms 299 and Activation Repression

The Mediator ComplexFormsa MolecularBridge BetweenActivation Domainsand Pol ll

317

296

SequentialAssemblyof ProteinsFormsthe Pol ll TranscriptionPreinitiationComplexin Vitro In Vivo TranscriptionInitiation by Pol ll Requires Additional Proteins

Mitochondrialand ChloroplastDNAsAre RNA Transcribedby Organelle-Specific Polymerases

Regulation of Pre-mRNA Processing

337

AlternativeSplicingls the PrimaryMechanismfor RegulatingmRNA Processing

337

A Cascadeof RegulatedRNA SplicingControls DrosophilaSexualDifferentiation

338

and ActivatorsControl Splicing SplicingRepressors at AlternativeSites

339

of RNA Editing Alters the Sequences SomePre-mRNAs

340

]f|

Transportof mRNAAcrossthe NuclearEnveloPe

341

NuclearPore ComplexesControl lmport and Export 342 from the Nucleus Are Not Exportedfrom in Spliceosomes Pre-mRNAs 345 the Nucleus HIV Rev Protein Regulatesthe Transportof Unspliced 346 Viral mRNAs

CONTENTS

'

xxi

CytoplasmicMechanisms of posttranscriptionalControl 347

f!|

Micro RNAsRepressTranslationof SpecificmRNAs RNA InterferenceInducesDegradationof precisely ComplementarymRNAs CytoplasmicPolyadenylationpromotesTranslation of SomemRNAs

Localizationof mRNAsPermitsProductionof Proteinsat SpecificRegionsWithin the Cytoplasm

357

347

Processing of rRNAand IRNA

358

349

f[

351

Pre-rRNAGenesFunctionas NucleolarOrganizers a n d A r e S i m i l a ri n A l l E u k a r y o t e s

359

Small NucleolarRNAsAssistin Processing Pre-rRNAs

360 363

Degradationof mRNAsin the CytoplasmOccurs by SeveralMechanisms

352

ProteinSynthesisCan Be GloballyRegulated

353

Self-Splicing Group I IntronsWere the First Examplesof CatalyticRNA

356

Pre-tRNAsUndergo ExtensiveModification in the Nucleus

363

357

N u c l e a rB o d i e sA r e F u n c t i o n a l lS y p e c i a l i z eN d uclear Domains

3G4

Sequence-Specific RNA-BindingproteinsControl SpecificmRNATranslation SurveillanceMechanismspreventTranslationof lmproperlyProcessed mRNAs

Partlll CellStructureand Function 9 V I S U A L I Z IN G, F R A C T ION A T IN G, A N D C U L T U R I NC GE L L S 371 Organellesof the EukaryoticCell

lll

372

Phase-Contrast and DifferentiallnterferenceContrast MicroscopyVisualizeUnstainedLivingCells 381 Fluorescence MicroscopyCan Localizeand euantify S p e c i f i cM o l e c u l e si n L i v eC e l l s 382 l m a g i n gS u b c e l l u l aDr e t a i l sO f t e n R e q u i r e tsh a t t h e SamplesBe Fixed,Sectioned,and Stained

T h e P l a s m aM e m b r a n eH a sM a n y C o m m o nF u n c t i o n s in All Cells 372 EndosomesTake Up 5oluble Macromolecules from the Cell Exterior 372

lmmunofluorescence MicroscopyCan Detect Specific Proteinsin FixedCells

38s

Confocaland DeconvolutionMicroscopyEnable Visualizationof Three-Dimensional Objects

386

Lysosomes Are Acidic OrganellesThat Contain a Batteryof DegradativeEnzymes

Graphicsand lnformaticsHaveTransformed Modern Microscopy

373

Peroxisomes DegradeFattyAcidsand ToxicCompounds 374 The EndoplasmicReticulumls a Network of InterconnectedInternal Membranes 375 The Golgi ComplexProcesses and SortsSecreted a n d M e m b r a n ep r o t e i n s

376

P l a n tV a c u o l e sS t o r eS m a l lM o l e c u l e sa n d E n a b l ea C e l lt o E l o n g a t eR a p i d l y

Eil

ElectronMicroscopy: Methods and Applications

388

Resolutionof Transmission ElectronMicroscopyis VastlyGreaterThan That of Light Microscopy

3gg

377

T h e N u c l e u sC o n t a i n st h e D N A G e n o m e ,R N A SyntheticApparatus,and a FibrousMatrix

CryoelectronMicroscopyAllows Visualizationof ParticlesWithout Fixationor Staining

399

378

MitochondriaAre the PrincipalSitesof ATp Productionin Aerobic NonphotosyntheticCells

378

ElectronMicroscopyof Metal-CoatedSpecimens Can RevealSurfaceFeaturesof Cellsand Their Components

390

ChloroplastsContain Internal Compartmentsin Which Photosynthesis Takesplace

379

!!| g

L i g h t M i cro sco p y: vi su a l i zi n gC ell Structureand Localizingproteins W i t h i n C el l s 380

The Resolutionof the Light Microscopels About 0.2 pm

xxii

.

coNTENrs

381

Purificationof Cell Organelles

391

Disruptionof CellsReleases Their Organelles and Other Contents

391

CentrifugationCan SeparateMany Typesof Organelles

392

Organelle-Specific AntibodiesAre Usefulin P r e p a r i n gH i g h l yP u r i f i e dO r g a n e l l e s

393

l s o l a t i o nC , u l tu re a , n d Differentiation 394 of MetazoanCells Flow CytometrySeparatesDifferent CellTypes

394

C u l t u r eo f A n i m a l C e l l sR e q u i r e sN u t r i e n t - R i c h M e d i a a n d S p e c i aSl o l i dS u r f a c e s

395

P r i m a r yC e l lC u l t u r e sC a n B e U s e dt o S t u d yC e l l Differentiation

396

P r i m a r yC e l lC u l t u r e sa n d C e l lS t r a i n sH a v ea F i n i t e L i f eS p a n

396

TransformedCellsCan Grow Indefinitelyin Culture

397

SomeCell LinesUndergo Differentiationin Culture

398

H y b r i dC e l l sC a l l e dH y b r i d o m a sP r o d u c eA b u n d a n t M o n o c l o n aA l ntibodies

400

HATMedium ls CommonlvUsedto lsolate HybridCells

402

organelles 407 Cnsstc ExprRturrur9.1 separating

ET R U C T U R E 1 O B I O M E M B R A NS s[

409

B i o m e mb ra n e L s:i p i dC o mp o sition 411 a n i za ti o n a n d S t r u ctu raOrg l

P h o s p h o l i p i dSsp o n t a n e o u s lFyo r mB i l a y e r s

411

B i l a y e r sF o r ma S e a l e dC o m p a r t m e n t Phospholipid S u r r o u n d i n ga n I n t e r n a lA q u e o u sS p a c e

411

M o t i f s H e l pT a r g e tP e r i p h e r a l Lipid-Binding Proteinsto the Membrane

427

ProteinsCan Be Removedfrom Membranes by Detergentsor High-SaltSolutions

427

$f,

sphingolipids, Phospholipids, SYnthesis andCholesterol: Movement and Intracellular

429

Fatty AcidsSynthesisls Mediated by Several lmportant Enzymes

430

SmallCytosolicProteinsFacilitateMovement of FattyAcids Incorporationof Fatty Acids into Membrane Lipids TakesPlaceon OrganelleMembranes

430 431

F l i p p a s eM s o v e P h o s p h o l i p i df sr o m O n e M e m b r a n e Leafletto the OppositeLeaflet

431

Cholesterolls Synthesizedby Enzymesin the Cytosol and ERMembrane

432

Cholesterola nd Phospholi pids Are Transported BetweenOrganellesby SeveralMechanisms

433

1 1 T R A N S M E M B R A NTER A N S P O R T OF IONSAND SMALLM OLECU LES437

E

overview of Membrane Transport

l lasses B i o m e m b r a n eC s o n t a i nT h r e eP r i n c i p aC of Lipids

415

M o s t L i p i d sa n d M a n y P r o t e i n sA r e L a t e r a l l yM o b i l e in Biomembranes

416

O n l y S m a l lH y d r o p h o b i cM o l e c u l e sC r o s sM e m b r a n e s 438 b y S i m p l eD i f f u s i o n

L i p i dC o m p o s i t i o nI n f l u e n c etsh e P h y s i c a l Propertiesof Membranes

418

Membrane ProteinsMediate Transportof Most Moleculesand All lons Across Biomembranes

Lipid Compositionls Different in the Exoplasmic and CytosolicLeaflets

419

s l u s t e rw i t h S p e c i f i c C h o l e s t e r oal n d S p h i n g o l i p i dC P r o t e i n si n M e m b r a n eM i c r o d o m a i n s

420

ProteinComponents Biomembranes: 421 and BasicFunctions

@

438

439

uniport Transportof Glucose and Water

441

Most TransmembraneProteinsHave M e m b r a n e - S p a n n i nagH e l i c e s

SeveralFeaturesDistinguishUniport Transportfrom S i m p l eD i f f u s i o n GLUT1UniporterTransportsGlucoseinto Most M a m m a l i a nC e l l s s F a m i l yo f S u g a r T h e H u m a nG e n o m eE n c o d e a TransportingGLUTProtetns

M u l t i p l e B S t r a n d si n P o r i n sF o r m M e m b r a n e - S p a n n i n"gB a r r e l s "

424

TransportProteinsCan Be EnrichedWithin Artificial M e m b r a n e sa n d C e l l s

CovalentlyAttached HydrocarbonChainsAnchor Some Proteinsto Membranes

424

$[|

ProteinsInteractwith Membranesin Three Different Ways

s re A l l T r a n s m e m b r a nPer o t e i n sa n d G l y c o l i p i dA A s y m m e t r i c a l lO y r i e n t e di n t h e B i l a y e r

421

OsmoticPressureCausesWater to Move Across Membranes AquaporinsIncreasethe Water Permeabilityof Cell Membranes

CONTENTS

O

44'l 442 443 443 4M 4M

xxiii

pumpsand the ATP-powered I n t r a c el l u l al ro n i cE n vi ro n me nt 447

@

Different Classes of PumpsExhibitCharacteristic Structuraland Functionalproperties ATP-Poweredlon PumpsGenerateand M a i n t a i nl o n i cG r a d i e n t sA c r o s sC e l l u l a r Membranes MuscleRelaxationDependson Ca2*ATpases That Pump Ca" from the Cytosolinto the S a r c o p l a s mR i ce t i c u i u m CalmodulinRegulates the plasmaMembrane Ca'* PumpsThat Control CytosolicCa2+ Concentrations N a - / K - A T P a s eM a i n t a i n st h e I n t r a c e l l u l aN r a* a n d K * C o n c e n t r a t i o nisn A n i m a l C e l l s V-ClassH* ATPases Maintain the Acidity of Lysosomes and Vacuoles BacterialPermeases Are ABC proteinsThat lmport a Variety of Nutrientsfrom the Environment

N o n g a te dto n C h a n n e l a s n d th e potential RestingMembrane

SelectiveMovement of lons Createsa TransmembraneElectricpotential Difference T h e M e m b r a n eP o t e n t i a li n A n i m a l C e l l sD e p e n d s Largelyon Potassiumlon MovementsThrough O p e n R e s t i n gK + C h a n n e l s

467

Na*-LinkedCa2*Antiporter ExportsCa2* from C a r d i a cM u s c l eC e l l s 447

448

449

451 452 453

454

The Approximately50 MammalianABCTransporters PlayDiverseand lmportant Rolesin Cell and O r g a nP h y s i o l o g y 455 C e r t a i nA B CP r o t e i n s" F l i p ' ,p h o s p h o l i p i d s and Other Lipid-SolubleSubstratesfrom One Membrane Leafletto the Opposite Leaflet 456

El

BacterialSymporterStructureRevealsthe Mechanismof SubstrateBinding

SeveralCotransportersRegulate CytosolicpH

468

A PutativeCation ExchangeProtein Playsa K e y R o l ei n E v o l u t i o no f H u m a nS k i n Pigmentation

469

NumerousTransportProteinsEnablePlant Vacuolesto AccumulateMetabolitesand lons

469

ft|

Transeprrnerar Transporr

Multiple TransportProteinsAre Neededto Move Glucoseand Amino AcidsAcross Epithelia

471

SimpleRehydrationTherapyDependson the OsmoticGradientCreatedby Absorption o f G l u c o s ea n d N a +

411

ParietalCellsAcidify the StomachContentsWhile M a i n t a i n i n ga N e u t r a lC y t o s o l i cp H

472

Cusslc ExprntueruT 11.1 stumbting Upon ActiveTransport

477

12 CELLULAR ENERGETICS 458

@ 458

470

FirstStepsof Glucoseand Fatty Acid CatabolismGlycolysis : and the CitricAcid €ycle

479

480

(Stagel), CytosolicEnzymes During Glycolysis ConvertGlucoseto Pyruvate

481

460

lon ChannelsContain a SelectivityFilter Formed from ConservedTransmembraneSegments

The Rate of Glycolysis ls Adjustedto Meet the Cell'sNeed for ATP

483

461

Glucosels FermentedUnder AnaerobicConditions

PatchClampsPermit Measurementof lon M o v e m e n t sT h r o u g hS i n g l eC h a n n e l s

485

463

U n d e r A e r o b i cC o n d i t i o n s ,M i t o c h o n d r i a E f fi c i e n t l yO x i d i z eP y r u v a t ea n d G e n e r a t e ATP (Stagesll-lV)

485

464

MitochondriaAre DynamicOrganelleswith Two Structurallyand FunctionallyDistinctMembranes

485

464

In Stagell, Pyruvatels Oxidizedto CO2and HighEnergyElectronsStored in ReducedCoenzymes

487

Novel lon ChannelsCan Be Characterizedby a Combinationof Oocyte Expression and P a t c hC l a m p i n g N a - E n t r yi n t o M a m m a l i a nC e l l sH a sa N e g a t i v e Changein FreeEnergy(AG)

Cotransportby symportersand Antiporters

@|

N a * - L i n k e dS y m p o r t e r lsm p o r t A m i n o A c i d sa n d G l u c o s ei n t o A n i m a l C e l l sA g a i n s tH i g h ConcentrationGradients

xxiv

.

coNTENTs

465

466

Transportersin the Inner MitochondrialMembrane H e l p M a i n t a i nA p p r o p r i a t eC y t o s o l i a c nd Matrix Concentrationsof NAD* and NADH MitochondrialOxidation of Fatty AcidsGenerates ATP PeroxisomalOxidation of Fatty AcidsGenerates No ATP

491

@ Th e

El e ctro nT ra n sp o rtch a i n a n d Generationof the Proton-Motive 493 Force

the StepwiseElectronTransportEfficientlyReleases EnergyStored in NADH and FADH2

493

ElectronTransportin Mitochondria ls Coupledto P r o t o nP u m p i n g

493

ElectronsFlow from FADH2and NADHto 02 Through Four Multiprotein Complexes

PhotoelectronTransportfrom EnergizedReactionCenterChlorophylla Producesa Charge Separation

514

I n t e r n a lA n t e n n a a n d L i g h t - H a r v e s t i n g C o m p l e x e sI n c r e a s et h e E f f i c i e n c yo f Photosynthesis

515

494

MolecularAnalysisof Photosystems

517

ReductionPotentialsof ElectronCarriersFavor ElectronFlow from NADH to 02

499

The SinglePhotosystemof PurpleBacteria Generatesa Proton-MotiveForcebut No 02

517

ExperimentsUsing PurifiedComplexesEstablished the Stoichiometryof Proton Pumping

499

The Q CycleIncreases the Numberof Protons Translocated as ElectronsFlow Through C o m p l e xl l l

LinearElectronFlow Through Both Plant Photosystems, PSlland PSl,Generatesa Proton-MotiveForce, 519 02, and NADPH

500

A n O x y g e n - E v o l v i nC g o m p l e xl s L o c a t e do n t h e L u m i n a lS u r f a c eo f t h e P S l lR e a c t i o n Center

520

CellsUse Multiple Mechanismsto ProtectAgainst Damagefrom ReactiveOxygenSpeciesDuring PhotoelectronTransPort

521

CyclicElectronFlow Through PSIGeneratesa Proton-MotiveForcebut No NADPHor 02

522

I and ll Are RelativeActivitiesof Photosystems Regulated

523

The Proton-MotiveForcein Mitochondria ls Due Largelyto a Voltage GradientAcrossthe Inner Membrane ToxicBy-productsof ElectronTransportCan D a m a g eC e l l s

[[

Harnessingthe Proton-Motive F o r c ef o r E n e rg y-R e q u i ri n g Processes

502 502

503

[[l

The Mechanismof ATPSynthesisls Shared Among Bacteria,Mitochondria,and Chloroplasts

505

ATPSynthaseComprisesTwo Multiprotein ComplexesTermedFeand F1

505

During co2 Metabolism Photosynthesis

524

RubiscoFixesCO2in the ChloroplastStroma

525

Synthesisof SucroseUsing FixedCO2ls Completed in the Cytosol

525

506

Light and RubiscoActivaseStimulateCO2 Fixation

525

ATP-ADPExchangeAcrossthe Inner Mitochondrial Membrane ls Poweredby the Proton-Motive Force

s09

Which Competeswith Photorespiration, ls Reducedin PlantsThat Fix Photosynthesis, CO2by the C4PathwaY

527

R a t eo f M i t o c h o n d r i aO l x i d a t i o nN o r m a l l yD e p e n d s on ADP Levels

510

Brown-FatMitochondria Usethe Proton-Motive Forceto GenerateHeat

510

Rotation of the Ft 1 Subunit,Driven by Proton Movement Through F6,Powers ATPSynthesis

@

1 3 M O V I N GP R O T E I NISN T O A N D O R G A N E L L E Ss33 MEMBRANES

Ph o t o s yn th e siasn d L i g h t-A b sor bing Pigments

ThylakoidMembranesin ChloroplastsAre the Sites in Plants of Photosynthesis

511 511

Occur Three of the Four Stagesin Photosynthesis O n l y D u r i n gl l l u m i n a t i o n

511

EachPhoton of Light Hasa DefinedAmount of Energy

513

Comprisea ReactionCenterand Photosystems Complexes AssociatedLight-Harvesting

514

$[

of secretoryProteins Translocation 535 Acr ossthe ERMembr ane

A HydrophobicN-TerminalSignalSequenceTargets NascentSecretoryProteinsto the ER

536

CotranslationalTranslocationls lnitiated by Two Proteins GTP-Hydrolyzing

537

Passageof Growing PolypeptidesThrough the Transloconls Driven by EnergyReleasedDuring Translation

539

CONTENTS

.

XXV

ATPHydrolysisPowersPost-translational Translocationof SomeSecretoryProteinsin yeast

Insertionof proteinsinto the E RM e mb ra n e

$[

540

542

543 InternalStop-Transfer and Signal-AnchorSequences proteins DetermineTopologyof Single-Pass 544 M u l t i p a s sP r o t e i n sH a v eM u l t i p l e I n t e r n a l TopogenicSequences 546 A P h o s p h o l i p iA d n c h o rT e t h e r sS o m eC e l l - S u r f a c e Proteinsto the Membrane 547 The Topologyof a Membrane ProteinOften Can Be Deducedfrom lts Sequence 547

A Preformed/V-LinkedOligosaccharide ls Added to M a n y P r o t e i n si n t h e R o u g hE R O l i g o s a c c h a r i dSei d eC h a i n sM a y p r o m o t eF o l d i n g and Stabilityof Glycoproteins D i s u l f i d eB o n d sA r e F o r m e da n d R e a r r a n g e db y P r o t e i n si n t h e E RL u m e n C h a p e r o n eas n d O t h e r E Rp r o t e i n sF a c i l i t a t eF o l d i n g and Assemblyof Proteins lmproperlyFoldedProteinsin the ERlnduce Expression of Protein-FoldingCatalysts U n a s s e m b l eodr M i s f o l d e dP r o t e i n si n t h e E RA r e Often Transportedto the Cytosolfor Degradation

550

ss2

coNTENTs

569

Largeand Small MoleculesEnter and Leavethe Nucleusvia NuclearPoreComplexes

570

lmportinsTransportProteinsContainingNuclearL o c a l i z a t i oS n i g n a l si n t o t h e N u c l e u s

571

ExportinsTransportProteinsContainingNuclear-Export S i g n a l so u t o f t h e N u c l e u s 573 573

1 4 V E S I C U L ATRR A F F I CS, E C R E T I O N , AND ENDOCYTOSIS 579 Techniques for Studyingthe SecretoryPathway

s80

Transportof a protein Through the Secretory pathway Can Be Assayedin tiving Cells

5g2

YeastMutants Define Major Stagesand Many Componentsin VesicularTransport

584

Cell-FreeTransportAssaysAllow Dissectionof I n d i v i d u aS l t e p si n V e s i c u l aTr r a n s p o r t

585

![ ss2 555

556

and Chloroplasts

.

Transportinto and out of the Nucleus

s[

552

A m p h i p a t h i cN - T e r m i n aSl i g n a lS e q u e n c eD sirect Proteinsto the MitochondrialMatrix 558 M i t o c h o n d r i aP l r o t e i nl m p o r t R e q u i r e sO u t e r - M e m b r a n e R e c e p t o ras n d T r a n s l o c o ni sn B o t h M e m b r a n e s 559 Studieswith ChimericProteinsDemonstratelmportant Featuresof Mitochondriallmport 550 T h r e eE n e r g yI n p u t sA r e N e e d e dt o l m p o r t P r o t e i n s into Mitochondria 501 Multiple Signalsand PathwaysTarget Proteinsto SubmitochondrialCompartments 561 T a r g e t i n go f C h l o r o p l a sSt t r o m a lP r o t e i n sl s S i m i l a rt o lmport of MitochondrialMatrix Proteins 565 ProteinsAre Targetedto Thylakoidsby Mechanisms Relatedto TranslocationAcrossthe Bacterial I n n e rM e m b r a n e 565 xxvi

s68

549

Sortingof Proteinsto Mitochondria 557

E!|

P e r o x i s o m aM l e m b r a n ea n d M a t r i x P r o t e i n sA r e Incorporated by Different Pathways

Most mRNAsAre Exportedfrom the Nucleusby a R a n - l n d e p e n d e nMt e c h a n i s m

Pr o t e i nMo d i fi ca ti o n s, F o l d i n g, and Quality Control in the ER

Sortingof Peroxisomal Proteins sG7

CytosolicReceptorTargetsProteinswith an SKL Sequenceat the C-Terminusinto the Peroxisomal Matrix

SeveralTopologicalClasses of Integral Membrane ProteinsAre Synthesizedon the ER

s[

s[

@

M olecularMechanisms of Vesicular Traffic

Assembryof a protein coat DrivesVesicle F o r m a t i o na n d S e l e c t i o no f C a r g o Molecules A ConservedSet of GTPaseSwitch proteinsControls Assemblyof Different VesicleCoats T a r g e t i n gS e q u e n c e os n C a r g o p r o t e i n sM a k e S p e c i f i cM o l e c u l a rC o n t a c t sw i t h C o a t Proteins

585

5g6 5g7

588

Rab GTPases Control Dockingof Vesicleson Target Membranes PairedSetsof SNAREproteinsMediate Fusionof Vesicleswith Target Membranes

591

Dissociationof SNAREComplexesAfter Membrane F u s i o nl s D r i v e nb y A T PH y d r o l y s i s

5g1

589

@

EarlyStagesof the Secretory Pathway

592

COPIIVesiclesMediate Transportfrom the ER to the Golgi COPIVesiclesMediate RetrogradeTransportwithin t h e G o l g ia n d f r o m t h e G o l g i t o t h e E R

ss4

AnterogradeTransportThrough the Golgi Occurs by CisternalMaturation

595

@

Later stages of the secretory Pathway

597

VesiclesCoatedwith Clathrinand/or Adapter Proteins 598 Mediate SeveralTransportSteps

l: SIGNAL 1 5 C E L LS I G N A L I N G AND SHORT- T ER M TRANSDUCTION 623 RESPONSES CELLULAR [[

Signalto cellular FromExtracellular 625 Response

Signaling S i g n a l i n gC e l l sP r o d u c ea n d R e l e a s e Molecules

625

SignalingMoleculesCan Act Locallyor at a Distance

625

B i n d i n go f S i g n a l i n gM o l e c u l e sA c t i v a t e s Receptorson Target Cells

626

D y n a m i nl s R e q u i r e df o r P i n c h i n gO f f o f C l a t h r i n Vesicles

599

$[

Mannose6-PhosphateResiduesTargetSoluble Proteinsto Lysosomes

600

ReceptorProteinsBind LigandsSpecifically

627

RevealedKey Study of LysosomalStorageDiseases Componentsof the LysosomalSorting Pathway

602

The DissociationConstantls a Measureof the Affinity of a Receptorfor lts Ligand

628

ProteinAggregation in the trans-GolgiMay Function in Sorting Proteinsto RegulatedSecretoryVesicles 602

BindingAssaysAre Usedto Detect Receptorsand DetermineTheir Affinities for Ligands

628

Some ProteinsUndergo ProteolyticProcessing After Leavingthe trans-Golgi

603

SeveralPathwaysSort Membrane Proteinsto the Apical or BasolateralRegionof PolarizedCells

to a SignalingMolecule MaximalCellularResponse UsuallyDoesNot RequireActivationof All Receptors

629

604

Sensitivityof a Cellto ExternalSignalsls Determined by the Number of SurfaceReceptorsand Their 631 Affinity for Ligand

Receptor-Mediated Endocytosis 606

631 ReceptorsCan Be Purifiedby Affinity Techniques ReceptorsAre FrequentlyExpressedfrom Cloned Genes 6 3 1

!f|

CellsTake Up Lipidsfrom the Blood in the Form of Large,Well-Defined LipoproteinComplexes

608

The Acidic pH of Late EndosomesCausesMost Receptor-Ligand Complexesto Dissociate

610

[!|

DirectingMembraneProteins and CytosolicMaterialsto the Lysosome

MultivesicularEndosomesSegregateMembrane ProteinsDestinedfor the Lysosomal Membranefrom ProteinsDestinedfor LysosomalDegradation

627

606

Receptorsfor Low-DensityLipoproteinand Other LigandsContain Sorting SignalsThat Target Them for Endocytosis

The EndocyticPathwayDeliverslron to Cellswithout Complex Dissociationof the Receptor-Transferrin in Endosomes

studying cell-Surface Receptors

611

Highly ConservedComPonents Signal-Transduction of Intracellular 632 Pathways GTP-BindingProteinsAre FrequentlyUsedAs On/Off Switches are Employedin Protein Kinasesand Phosphatases Virtually All SignalingPathways Carryand Amplify Signalsfrom SecondMessengers Many Receptors

612

612

RetrovirusesBud from the PlasmaMembrane by a Process Endosomes 614 Similarto Formationof Multivesicular

14.1 Following a Protein Cnsstc ExprRturruT 621 out of thecell

s!|

633 634 634

of G ProteinGeneralElements 535 SYstems RecePtor Coupled

G Protein-CoupledReceptorsAre a Largeand Diverse 635 Familywith a Common Structureand Function G Protein-CoupledReceptorsActivate Exchangeof G T Pf o r G D Po n t h e a S u b u n i to f a T r i m e r i cG 637 Protein Different G ProteinsAre Activated by Different GPCRs 539 and ln Turn RegulateDifferent EffectorProteins

CONTENTS

.

xxvii

G Protein-Coupled Receptors T h a t R eg u l a tel o n C h a n n e l s

640

AcetylcholineReceptorsin the Heart Muscle Activate a G ProteinThat OpensK+ Channels

641

Light ActivatesG..-CoupledRhodopsins

641

Activationof RhodopsinInducesClosingof cGMP-GatedCation Channels

642

[f|

Rod CellsAdapt to Varying Levelsof Ambient Light Becauseof Opsin Phosphorylationand Binding of Arrestin

644

G Protein-GoupledReceptorsThat Activateor Inhibit Adenylyl Cyclase 646

$!|

Adenylyl Cyclasels Stimulatedand Inhibited by Different Receptor-Ligand Complexes

646

StructuralStudiesEstablishedHow G,,.GTpBinds to and ActivatesAdenylyl Cyclase

646

cAMP ActivatesProtein KinaseA by Releasing CatalyticSubunits

547

GlycogenMetabolismls Regulatedby Hormone-lnducedActivation of Protein KinaseA

648

cAMP-MediatedActivation of Protein KinaseA ProducesDiverseResponses in Different Cell Types

649

S i g n a lA m p l i f i c a t i o nC o m m o n l yO c c u r si n M a n y SignalingPathways

550

S e v e r aM l e c h a n i s mD s o w n - R e g u l a tS e ignaling from G Protein-CoupledReceptors

651

Anchoring ProteinsLocalizeEffectsof cAMp to SpecificRegionsof the Cell

ActivatePhospholipase C

PhosphorylatedDerivativesof InositolAre lmportant SecondMessengers C a l c i u ml o n R e l e a s e from the Endoplasmic Reticulumis Triggeredby lp3 The Ca2*/Calmodulin ComplexMediatesMany CellularResponses to ExternalSignals Diacylglycerol(DAG)Activatesprotein KinaseC, Which RegulatesMany Other proteins Signal-lnducedRelaxationof VascularSmooth Musclels Mediated by cGMp-Activated Protein KinaseG

Clnsstc ExprnlveruT 15.1 TheInfancy of signal Transduction-GTPStimulationof cAMP Synthesis 663

15 CELL-SIGNALIN l lG : SIGNALING PATHWAYSTHAT C O N T R O L GENE ACTIVITY 66s

sl|

IntegratingResponses of cells t o En v i ro n me n taInl fl u e n ce s

'

CONTENTS

668 668

RadioactiveTaggingWas Usedto ldentify TGFp Receptors

669

ActivatedTGFBReceptorsPhosphorylateSmad TranscriptionFactors

670

NegativeFeedbackLoopsRegulateTGFB/Smad Signaling

671

Lossof TGFBSignalingPlaysa Key Role in Cancer

671

sf|

cytokineReceptors andthe JAK/STAT Pathway

672 672

CytokineReceptorsHaveSimilarStructuresand Activate SimilarSignalingPathways

673

JAK KinasesActivate STATTranscriptionFactors

674

654

ComplementationGeneticsRevealedThat JAK and STATProteinsTransduceCytokine Signals

677

654

Signalingfrom CytokineReceptorsls Regulated by NegativeSignals

678

655

Mutant ErythropoietinReceptorThat Cannot Be TurnedOff Leadsto IncreasedNumbersof Erythrocytes

679

553

6s6

656

657

Integrationof Multiple SecondMessengersRegulates Glycogenolysis 557

XXVIII

Activation of Smads

A TGFBSignalingMoleculels Formedby Cleavage of an InactivePrecursor

ReceprorTyrosrneKrnases

679

L i g a n dB i n d i n gL e a d st o P h o s p h o r y l a t i oann d Activation of IntrinsicKinasein RTKs

680

Overexpression of HER2,a Receptor TyrosineKinase,Occursin Some Breast Cancers

680

s[ $f|

TGFFReceptors and the Direct

CytokinesInfluenceDevelopmentof Many Cell Types

G Protein-coupled Receptors That

sfl

I n s u l i na n d G l u c a g o nW o r k T o g e t h e rt o M a i n t a i na StableBlood GlucoseLevel

HedgehogSignalingRelievesRepressionof Target Genes

D o m a i n sA r e l m p o r t a n tf o r B i n d i n gS i g n a l Conserved TransductionProteinsto ActivatedReceptors Down-regulationof RTKSignalingOccursby Endocytosis and LysosomalDegradation

683

@

700

PathwaysThat InvolveSignal - l nduc ed 703 ProteinCleavage

o f R a sa n d MA P K i n ase @ Act i v a t i o n 584 Pathways

Degradationof an Inhibitor ProteinActivatesthe NF-rBTranscriptionFactors

703

Ras,a GTPaseSwitch Protein,CyclesBetweenActive and InactiveStates

68s

Ligand-ActivatedNotch ls CleavedTwice,Releasing a TranscriptionFactor

705

ReceptorTyrosineKinasesAre Linkedto Rasby Adapter Proteins

685

GeneticStudiesin Drosophilaldentified Key Proteinsin the Ras/MAP Signal-Transducing KinasePathway

685

Binding of SosProteinto InactiveRasCausesa ConformationalChangeThat ActivatesRas

587

SignalsPassfrom Activated Rasto a Cascadeof Protein Kinases

688

MAP KinaseRegulatesthe Activity of Many Transcription 690 Genes FactorsControlling Early-Response G Protein-CoupledReceptorsTransmitSignalsto MAP Kinasein YeastMating Pathways

691

ScaffoldProteinsSeparateMultiple MAP Kinase Pathwaysin EukaryoticCells

692

The Ras/MAPKinasePathwayCan InduceDiverse C e l l u l a rR e s p o n s e s

693

sf|

Phosphoinositides as signal Transducers

Phospholipase C" ls Activatedby SomeRTKsand CytokineReceptors

694

6e4

Recruitmentof Pl-3 Kinaseto Hormone-Stimulated ReceptorsLeadsto Synthesisof Phosphorylated Phosphatidyl inositols in the Plasma Accumulationof Pl 3-Phosphates Membrane Leadsto Activation of SeveralKinases 695 Activated Protein KinaseB InducesMany CellularResponses

696

The Pl-3KinasePathwayls NegativelyRegulated by PTENPhosphatase

s!|

698

Arrestin ActivatesSeveralKinaseCascades 698 GPCR-Bound Wnt SignalsTrigger Releaseof a Transcription Factorfrom CytosolicProteinComplex

RegulatedIntramembraneProteolysisof SREBP a TranscriptionFactorThat Acts to Releases Maintain Phospholipidand CholesterolLevels

1 7 C E L LO R G A N I Z A T I OANN D 713 M O V E M E N Tl : M I C R O F I L A M E N T S

@

and Actin Microfilaments Structures

715

Actin ls Ancient,Abundant, and Highly Conserved G-ActinMonomersAssembleinto Long, Helical F-ActinPolymers

717

F-ActinHasStructuraland FunctionalPolarity

718

@

Dynamicsof Actin Filaments

717

718

Actin Polymerizationin Vitro Proceedsin Three Steps 7 1 9 Actin FilamentsGrow Fasterat (+) EndsThan at 720 (-) Ends Actin FilamentTreadmillingls Acceleratedby P r o f i l i na n d C o f i l i n Providesa Reservoirof Actin for Thymosin-B4 Polymerization CappingProteinsBlockAssemblyand Disassembly a t A c t i n F i l a m e n tE n d s

Activationof GeneTranscription Cell-Surface by Seven-spanning 697 Receptors

CREBLinkscAMP and Protein KinaseA to Activation of GeneTranscription

CatalyzeCleavageof Many Matrix Metalloproteases 706 S i g n a l i n gP r o t e i n sf r o m t h e C e l lS u r f a c e InappropriateCleavageof Amyloid PrecursorProtein Can Leadto Alzheimer'sDisease

@

of Actin Filament Mechanisms AssemblY

F o r m i n sA s s e m b l eU n b r a n c h e dF i l a m e n t s The Arp2/3 ComplexNucleatesBranchedFilament Assembly lntracellularMovementsCan Be Poweredby Actin Polymerization

CONTENTS

721 722 722

723 723 724 726 xxix

ToxinsThat Perturbthe Pool of Actin Monomers Are Usefulfor StudyingActin Dynamics

726

ChemotacticGradientsInduceAltered Phosphoinositide LevelsBetweenthe Front and Backof a Cell 750

Cnsstc ExpenlvlrruT 17.1 Looking at Muscle

o r g a n i z a ti o no f A cti n -B a secellular d Structures 728

@

C r o s s - L i n k i nPgr o t e i n sO r g a n i z eA c t i n F i l a m e n t si n t o Bundlesor Networks

728

Adaptor ProteinsLink Actin Filamentsto Membranes

728

Myosins: Actin-Based Motor Proteins

fifl

Contraction

755

1 8 C E L LO R G A N I Z A T I OANN D M O V E M E N Tl l : M I C R O T U B U L E S AND INTERMEDIATE FILAM EN T S 757 [[

731

Micr otubuleStr uctur eand Or ganization

758

732

MicrotubuleWalls Are PolarizedStructuresBuilt f r o m a B - T u b u l i nD i m e r s

758

M y o s i n sM a k e U p a L a r g eF a m i l yo f MechanochemicalMotor proteins

733

MicrotubulesAre Assembledfrom MTOCsto GenerateDiverseOrganizations

760

C o n f o r m a t i o n aCl h a n g e si n t h e M y o s i nH e a d CoupleATPHydrolysisto Movement

736

Myosin HeadsTake DiscreteStepsAlong Actin Filaments

736

MyosinV Walks Hand Over Hand Down an Actin Filament

MicrotubulesAre DynamicStructuresDue to Kinetic Differencesat Their Ends

763

737

I n d i v i d u aM l i c r o t u b u l e sE x h i b i tD y n a m i cI n s t a bIi t y

763

LocaIized A s s e m b l ya n d " S e a r c h - a n d - C a p t u r e " H e l p O r g a n i z eM i c r o t u b u l e s

766

DrugsAffecting Tubulin PolymerizationAre Useful Experimentallyand to Treat Diseases

766

M y o s i n sH a v eH e a d ,N e c k ,a n d T a i l D o m a i n sw i t h DistinctFunctions

Myosin-poweredMovements

@

M y o s i nT h i c kF i l a m e n t sa n d A c t i n T h i n F i l a m e n t s i n S k e l e t aM l u s c l eS l i d eP a s tO n e A n o t h e r D u r i n gC o n t r a c t i o n

738

ftf|

738

[f!

S k e l e t aM l u s c l el s S t r u c t u r e db y S t a b i l i z i n ga n d ScaffoldingProteins

740

Contractionof SkeletalMusclels Regulatedby Ca2* a n d A c t i n - B i n d i n gP r o t e i n s

740

Actin and Myosin ll Form ContractileBundlesin N o n m u s c l eC e l l s

741 Myosin-DependentMechanismsRegulate C o n t r a c t i o ni n S m o o t hM u s c l ea n d N o n m u s c l eC e l l s 742 Myosin-V-Bound VesiclesAre CarriedAlong Actin Filaments 743

Microtubule Dynamics

762

Regulationof MicrotubuleStructure and Dynam ics 767

MicrotubulesAre Stabilizedby Side-and End-Binding Proteins

767

M i c r o t u b u l e sA r e D i s a s s e m b l ebdy E n d B i n d i n g and SeveringProteins

768

@

Kinesinsand Dyneins:Micr otubul eBasedMotor Proteins 769

Organellesin AxonsAre TransportedAlong Microtubulesin Both Directions

769

Kinesin-1PowersAnterogradeTransportof VesiclesDown Axons Toward the (+) End of Microtubules

770

745

KinesinsForm a LargeProtein Familywith Diverse Functions

771

747

Kinesin-l ls a Highly Processive Motor

772

C e l lM i g r a t i o nI n v o l v e st h e C o o r d i n a t eR e g u l a t i o n of Cdc42,Rac,and Rho

748

Dynein Motors TransportOrganellesTowardthe (-) E n do f M i c r o t u b u l e s

774

Migrating CellsAre Steeredby Chemotactic Molecules

750

Kinesinsand DyneinsCooperatein the Transport o f O r g a n e l l eT s hroughout he Cell

715

c e l l M i g r a t i o ns: i g n a l i n ga n d Chemotaxis

@

C e l lM i g r a t i o nC o o r d i n a t e F s o r c eG e n e r a t i o nw i t h C e l lA d h e s i o na n d M e m b r a n eR e c y c l i n g The SmallGTP-BindingProteinsCdc42,Rac,and Rho Control Actin Organization

XXX

.

CONTENTS

745

fifl

Ciliaand Flagella: Microtubule777 BasedSurfaceStructures

E u k a r y o t i cC i l i aa n d F l a g e l l aC o n t a i nL o n g D o u b l e t MicrotubulesBridged by Dynein Motors

777

r e a t i n gA r e P r o d u c e db y C i l i a r ya n d F l a g e l l a B C o n t r o l l e dS l i d i n go f O u t e r D o u b l e t Microtubules

778

IntraflagellarTransportMoves Material Up and D o w n C i l i aa n d F l a g e l l a

779

Defectsin IntraflagellarTransportCauseDisease by Affecting SensoryPrimaryCilia

780

Microfilamentsand MicrotubulesCooperateto TransportMelanosomes Cdc42Coordi nates M icrotubules and M icrofi laments D u r i n g C e l lM i g r a t i o n

1 9 I N T E G R A T I NCGE L L SI N T O TISSUES

$[

797

801

cell- celland cell- Matr ixAdhes i on: 803 An Overview

C e l l - A d h e s i oM n o l e c u l e sB i n dt o O n e A n o t h e r a n d t o I n t r a c e l l u l aPr r o t e i n s

803

782

805

C e n t r o s o m eD s u p l i c a t eE a r l yi n t h e C e l lC y c l ei n Preparationfor Mitosis

The ExtracellularMatrix Participatesin Adhesion, S i g n a l i n ga, n d O t h e r F u n c t i o n s

783

807

of The Mitotic SpindleContainsThree Classes Microtubules

The Evolutionof MultifacetedAdhesionMolecules Enabledthe Evolutionof DiverseAnimal Tissues

784

M i c r o t u b u l eD y n a m i c sI n c r e a s eDs r a m a t i c a l l y in Mitosis

784

$!|

Mitosis

MitosisCan Be Dividedinto Six Phases

781

l u r i n gM i t o s i s M i c r o t u b u l e sT r e a d m i l D

785

The KinetochoreCapturesand HelpsTransport Chromosomes

786

DuplicatedChromosomesAre Aligned by Motors a n d T r e a d m i l l i n gM i c r o t u b u l e s

788

to Polesby AnaphaseA MovesChromosomes Microtubule Shortening

789

AnaphaseB SeparatesPolesby the Combined A c t i o n o f K i n e s i n sa n d D y n e i n

789

s o n t r i b u t et o S D i n d l e A d d i t i o n a lM e c h a n i s mC Formation

789

CytokinesisSplitsthe DuplicatedCell in Two

789

e h e i r M i c r o t u b u l e sa n d P l a n tC e l l sR e o r g a n i z T B u i l da N e w C e l l W a l li n M i t o s i s

790

$fl

IntermediateFilaments

791

@

Junctio ns cell- celland celI- ECM and TheirAdhesionMolecules 808

, nd t p i c a l ,L a t e r a l a E p i t h e l i aC l e l l sH a v eD i s t i n c A BasalSurfaces

808

ThreeTypesof JunctionsMediate Many Cell-Cell and Cell-ECMInteractions

809

l l d h e s i o n si n A d h e r e n s C a d h e r i n sM e d i a t eC e l l - C e A J u n c t i o n sa n d D e s m o s o m e s

810

Tight JunctionsSealOff Body Cavitiesand Restrict D i f f u s i o no f M e m b r a n eC o m p o n e n t s

814

Adhesionsin EpithelialCells 816 IntegrinsMediateCelI-ECM s llow Small G a p J u n c t i o n sC o m p o s e do f C o n n e x i n A Moleculesto PassDirectlyBetweenAdjacent Cells 817

M atr ix l: The Extr acellular The BasalLamina

820

l a m i n aP r o v i d e sa F o u n d a t i o nf o r The BasaL Assemblyof Cellsinto Tissues

820 821

IntermediateFilamentsAre Assembledfrom S u b u n i tD i m e r s

792

M a t r i x P r o t e i n ,H e l p s L a m i n i n ,a M u l t i a d h e s i v e Cross-linkComponentsof the BasalLamina

Intermediate FilamentsProteins Are Expressed Manner in a Tissue-Specific

792

Type lV Collagenls a Major Structural Sheet-Forming 821 l amina C o m p o n e n to f t h e B a s a L

I n t e r m e d i a t eF i l a m e n t sA r e D y n a m i c

795

Defectsin Laminsand KeratinsCauseManv Diseases 795

[[

coordinationand cooperation between CytoskeletalElements 796

Proteins lntermediateFilament-Associated C o n t r i b u t et o C e l l u l a rO r g a n i z a t i o n

796

Components Perlecan,a Proteoglycan,Cross-links Receptors of the BasalLaminaand Cell-Surface

@The

Mra t r i xl l : Extracellula Connectiveand Other Tissues

s r e t h e M a j o r F i b r o u sP r o t e i n s F i b r i l l a rC o l l a g e n A in the ECMof ConnectiveTissues

CONTENTS

824

825 825

XXXI

F i b r i l l a rC o l l a g e nl s S e c r e t e da n d A s s e m b l e di n t o FibrilsOutsideof the Cell

926

ConnectionsBetweenthe ECMand Cytoskeleton Are Defectivein MuscularDystrophy

835

T y p eI a n d l l C o l l a g e n A s ssociate with Nonfibrillar Collagensto Form DiverseStructures

826

l g C A M sM e d i a t eC e l l - C e l l A d h e s i oi n N e u r o n a l and Other Tissues

836

LeukocyteMovement into Tissuesls Orchestrated by a Precisely Timed Sequenceof Adhesive Interactions

837

Proteoglycans and Their ConstituentGAGsPlay DiverseRolesin the ECM

827

HyaluronanResists Compression, Facilitates Cell Migration, and GivesCartilagelts Gel-likeProperties 829 FibronectinsInterconnectCellsand Matrix, Influencing Cell Shape,Differentiation,and Movement 830

AdhesiveInteractionsin Motile a n d N o n mo ti l eC e l l s

[[

I n t e g r i n sR e l a yS i g n a l sB e t w e e nC e l l sa n d T h e i r T h r e e - D i m e n s i o nE an l vironment

p[

PtantTissues

839

T h e P l a n tC e l lW a l l l s a L a m i n a t eo f C e l l u l o s eF i b r i l s in a Matrix of Glycoproteins

833 833

Regulationof Integrin-MediatedAdhesionand S i g n a l i n gC o n t r o l sC e l l M o v e m e n t

Looseningof the Cell Wall PermitsPlant Cell Growth

840

Plasmodesmata DirectlyConnectthe Cytosolsof A d j a c e n tC e l l si n H i g h e rP l a n t s Only a Few AdhesiveMoleculesHave Been ldentified in Plants

841

PartlV CellGrowth and Development 20 R E G U L AT IN T GH E E U K A R Y OT IC C E L LC Y C L E 847 Overviewof the CellCycle and lts Control

E[

849

The Cell Cyclels an Ordered Seriesof Events L e a d i n gt o C e l lR e p l i c a t i o n

849

RegulatedProtein Phosphorylationand DegradationControl Passage Through the Cell Cycle

849

DiverseExperimentalSystemsHave Been Usedto ldentify and lsolateCell-Cycle Control proteins

851

MPF Activity

853

Maturation-PromotingFactor(MpF)Stimulates Meiotic Maturation in Oocytesand Mitosis i n S o m a t i cC e l l s

854

Mitotic CyclinWas Firstldentified in EarlvSea U r c h i nE m b r y o s

856

CyclinB Levelsand KinaseActivity of MitosisPromoting Factor(MPF)ChangeTogetherin CyclingXenopusEgg Extracts Anaphase-Promoting Complex(APC/C)Controls Degradationof Mitotic Cyclinsand Exit from Mitosis

.

CONTENTS

8s6

8s8

Cyclin-Dependent KinaseRegulation Dur ingMitosis 8s9

MPFComponentsAre ConservedBetween Lower and Higher Eukaryotes

860

Phosphorylationof the CDKSubunit Regulatesthe KinaseActivity of MPF

851

ConformationalChangesInducedby Cyclin Binding and PhosphorylationIncrease MPFActivity

862

@

Controlof Mitosisby Cyclinsand

@

![fl

MolecularMechanisms for Regulating Mitotic Events

864

Phosphorylationof NuclearLaminsand Other ProteinsPromotesEarlyMitotic Events

864

U n l i n k i n go f S i s t e rC h r o m a t i d sI n i t i a t e sA n a p h a s e

867

ChromosomeDecondensationand Reassembly of the NuclearEnvelopeDependon Dephosphorylation of MPFSubstrates

870

!!f|

Cyclin-cDK and Ubiquitin-protein Ligase Control of S phase

872

A Cyclin-Dependent Kinase(CDK)ls Criticalfor S-PhaseEntry in S. cerevisiae

872

Three G1CyclinsAssociatewith 5. cerevrsrae CDK to Form S-Phase-Promoting Factors

874

Degradationof the S-Phase Inhibitor TriggersDNA Replication Multiple CyclinsRegulatethe KinaseActivity of 5. cerevisiaeCDK During Different Cell-CyclePhases 877 Replication a t E a c hO r i g i n l s I n i t i a t e dO n l y O n c e During the Cell Cycle

!fil

877 |ffirhe

cell-cyclecontrolin Mammalian Cells

2 1 C E L LB I R T H L, I N E A G EA, N D DEATH

879

90s

Birthof cells:Stemcells, 905

N i c h e s ,a n d L i n e a g e

Stem CellsGive Riseto Both Stem Cellsand DifferentiatingCells

906

RestrictedDuring Cell FatesAre Progressively Development

907

The CompleteCell Lineageof C. elegansls Known

908

M a m m a l i a nR e s t r i c t i o n P o i n t l s A n a l o g o u st o STARTin Yeast Cells

880

Multiple CDKsand CyclinsRegulatePassage of M a m m a l i a nC e l l sT h r o u g ht h e C e l lC y c l e

881

HeterochronicMutants ProvideCluesAbout Control of Cell Lineage

909

RegulatedExpression of Two Classes of Genes R e t u r n sG eM a m m a l i a nC e l l st o t h e C e l lC y c l e

881

Cultured EmbryonicStem CellsCan Differentiateinto V a r i o u sC e l lT y p e s

911

Passage Through the RestrictionPoint Dependson Phosphorylationof the Tumor-Suppressor Rb Protein 882

Adult Stem Cellsfor Different Animal TissuesOccupy 912 S u s t a i n i n gN i c h e s

C y c l i nA l s R e q u i r e df o r D N A S y n t h e s iasn d C D K 1 for Entry into Mitosis

883

MeristemsAre Nichesfor Stem Cellsin Postnatal Plants

Two Typesof Cyclin-CDKInhibitorsContributeto Cell-Cycle Control in Mammals

883

@

!![

in cell-Cycle Checkpoints Regulation

The Presenceof UnreplicatedDNA PreventsEntry into Mitosis lmproper Assemblyof the Mitotic SpindlePrevents the Initiation of Anaphase ProperSegregationof Daughter Chromosomesls Monitored by the Mitotic Exit Network

884 888

92O

cell-Typespecificationin Yeast

921

Mati ng-TypeTranscription FactorsSpecify CellTypes

922

MCMl and a1-MCM1ComplexesActivate Gene Transcription

923

a2-MCMI and cr2-a1ComplexesRepressTranscription 923 888

PheromonesInduceMating of ct and a Cellsto G e n e r a t ea T h i r d C e l l T Y P e

923

889

Arrest of Cellswith DamagedDNA Depends Cell-Cycle 891 on Tumor Suppressors

!!|

and Differentiation Specification 924 of Muscle

EmbryonicSomitesGive Riseto Myoblasts

925

MyogenicGenesWere Firstldentified in Studieswith Cultured Fibroblasts

925

Two Classesof Regulatory FactorsAct in Concert to Guide Productionof MuscleCells

926

CohesinSubunit Recombinationand a Meiosis-Specific for the SpecializedChromosome Are Necessary 895 Segregationin Meiosis|

Differentiationof Myoblastsls Under Positiveand NegativeControl

927

Cell-CellSignalsAre Crucialfor Determinationand Migration of Myoblasts

928

SpecialPropertiesof Rec8Regulatelts Cleavagein M e i o s i sI a n d l l

896

bHLH RegulatoryProteinsFunctionin Creationof Other Tissues

929

The Monopolin ComplexCo-orientsSister Kinetochoresin Meiosis|

898

Tensionon SpindleMicrotubulesContributesto ProperSpindleAttachment

898

!![

Meiosis:A specialTypeof cell Division

Key FeaturesDistinguishMeiosisfrom Mitosis

892 892

Protein of G1Cyclinsand a Meiosis-Specific Repression 895 KinasePromote Premeiotic5 Phase

Emerging 20.1 cellBiology Cnsstc ExpenlueruT from the Sea:The Discoveryof Cyclins

903

@

Regulationof Asymmetric CellDivision

930

YeastMating-TypeSwitchingDependsupon AsymmetricCell Division

CONTENTS

930 '

xxxiii

ProteinsThat RegulateAsymmetryAre Localizedat OppositeEndsof Dividing Neuroblastsin Drosophila 9 3 1

cell Deathand tts Regulation

!@

936

ProgrammedCell Death OccursThrough Apoptosis Neurotrophinspromote Survivalof Neurons

937

A Cascadeof CaspaseProteinsFunctionsin One Apoptotic Pathway

938

Pro-ApoptoticRegulatorsPermit CaspaseActivation in the Absenceof TrophicFactors

941

SomeTrophicFactorsInduceInactivationof a Pro-ApoptoticRegulator

942

Tumor NecrosisFactorand RelatedDeath Signals PromoteCell Murder by Activating Caspases

943

937

2 2 T H EM O L E C U L ACRE L LB I O L O G Y O FD E V E L O P M E N T 949

Controlof Body Segmentation: Themesand Variationsin Insects and Vertebrates 969 Early Drosophila Development ls an Exercisein Speed 970 TranscriptionalControl Specifiesthe Embryo's Anterior and Posterior

971

TranslationInhibitorsReinforceAnterior-Posterior Patterning

973

InsectSegmentationls Controlledby a Cascade of Transcription Factors

974

VertebrateSegmentationls Controlledby Cyclical Expression of RegulatoryGenes

977

DifferencesBetweenSegmentsAre Controlledby Hox Genes

978

Hox-GeneExpressionls Maintained by a Variety of Mechanisms

982

Flower DevelopmentRequiresSpatiallyRegulated Productionof TranscriptionFactors

983

!![ H i g h l i g h tso f D e ve l o p me n t

@

DevelopmentProgresses from Egg and Sperm to an EarlyEmbryo

As the EmbryoDevelops,Cell LayersBecomeTissues and Organs 951 GenesThat RegulateDevelopmentAre at the Heart of Evolution

Neural Development

985

N e u r u l a t i o nB e g i n sF o r m a t i o no f t h e B r a i na n d SpinalCord

986

950 9s0

952

cell-TypeSpecification in Early

SignalGradientsand TranscriptionFactorsSpecifyCell Typesin the NeuralTube and Somites 987 Most Neuronsin the BrainArise in the Innermost NeuralTube and Migrate Outward

988

LateralInhibition Mediated by Notch Signaling CausesEarlyNeural Cellsto BecomeDifferent

988

Gametogenesis and Fertilization g53

@

Growth and Patterningof Limbs 990

G e r m - l i n eC e l l sA r e A l l T h a t W e I n h e r i t

953

!![

FertilizationUnifiesthe Genome

955

Hox GenesDeterminethe Right Placesfor Limbsto Grow

990

958

Limb DevelopmentDependson Integration o f M u l t i p l e E x t r a c e l l u l aSri g n a lG r a d i e n t s

991

958

Hox GenesAlso Control Fine Patterning of Limb Structures

992

So Fa[ So Good

994

Genomiclmprinting ControlsGene Activation Accordingto Maternal or PaternalChromosome Origin Too Much of a Good Thing: The X Chromosomels Regulatedby DosageCompensation

cell Diversityand patterning

!f,

in EarlyVertebrateEmbryos

CleavageLeadsto the FirstDifferentiationEvents The Genomesof Most SomaticCellsAre Complete

Cussrc ExprRlueruT 22.1 UsingLethal

961 961

SignalGradientsMay InduceDifferent Cell Fates

963

SignalAntagonistsInfluenceCell Fatesand Tissue Induction

965

A Cascadeof SignalsDistinguishes Left from Right

956

.

coNTENTs

999

960

GastrulationCreatesMultiple TissueLayers,Which BecomePolarized

xxxiv

Mutationsto Study Development

959

23 NERVE CELLS !fl

Neurons andGlia:Building Blocksof the NervousSystem

1001

lOOz

Information FlowsThrough Neuronsfrom Dendrites to Axons 1003

NXXX

.

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u o ll) u n l p u e a r n lr n r ls :su lln q o l6 0 u nuul

SpecificMutations TransformCulturedCells into Tumor Cells

1113

A Multi-hit Model of CancerInduction ls Supported by SeveralLinesof Evidence

1114

Successive OncogenicMutations Can Be Traced in Colon Cancers

1116

PatternsCan DNA MicroarrayAnalysisof Expression RevealSubtle DifferencesBetweenTumor Cells

1116

@ Th e

G e ne ti cB a si so f ca n ce r

1119

Gai n-of-FunctionM utationsConvertProto-oncogenes 1119 into Oncogenes VirusesContainOncogenesor Cancer-Causing Activate CellularProto-oncogenes

1121

Mutations in Tumor-Suppressor Loss-of-Function GenesAre Oncogenic

1123

Genes Inherited Mutations in Tumor-Suppressor lncreaseCancerRisk

1123

Aberrationsin SignalingPathwaysThat Control DevelopmentAre Associatedwith Many Cancers 1124

!fl

oncogenicMutationsin GrowthPromoting Proteins

1127

OncogenicReceptorsCan Promote Proliferationin the Absenceof External Growth Factors

1127

Viral Activators of Growth-Factor ReceptorsAct as Oncoproteins

1128

Many OncogenesEncodeConstitutivelyActive Proteins Sig nal-Transduction

1129

InappropriateProductionof NuclearTranscription FactorsCan InduceTransformation

11 3 0

M o l e c u l a rC e l lB i o l o g yl s C h a n g i n gH o w C a n c e r ls Treated

1132

MutationsCausingLossof and Gr owth- lnhibiting Controls Cell-Cycle

1134

from MutationsThat Promote UnregulatedPassage Gr to 5 PhaseAre Oncogenic

1134

MutationsAffecting ChromatinLoss-of-Function RemodelingProteinsContributeto Tumors

1135

Lossof p53 Abolishesthe DNA-DamageCheckpoint 1136 Apoptotic GenesCan Functionas Proto-oncogenes 1137 Genes or Tumor-Suppressor CheckpointsOften Leadsto Failureof Cell-Cycle T u m o rC e l l s i n Aneuploidy

1138

and CaretakerGenes Carcinogens 11 3 9 in Cancer CarcinogensInduceCancerby DamagingDNA

11 3 9

SomeCarcinogensHave Been Linkedto Specific Cancers

1139

Lossof DNA-RepairSystemsCan Leadto Cancer

1141

Contributesto TelomeraseExpression lmmortalizationof CancerCells

1143

GLOSSARY

G-1 l-1

INDEX

CONTENTS

.

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'sJaJrloJpue seqeue 'so;n1lnl1s to sarloru ur eesuef a.rl sE Sur8ueqc-lseJe^eq pue dlprder e^ou aruos '(1-1 arn8r4) sadeqs pup sezrs lo f,lBrrcr, Surzeute ue ur otuoJ sllaJ

slla) Io f1r;euourruo)pue Alrsla^lqaql lU 's11arSurdpnts sdp.^a snorrel ar{l ruo{ urpel uef, to aA\ let{,ryrpup (suorlf,unJIeJrtrrr pup sluenlrlsuoJ Jrsgq Jroql 's11ec dlrs.rallp aqt Surcnporrurdq rerdeqr anSolord srql ;o ur truls elrN'slleJ rltpJp pue 'ayr1'qrrrg aqr 1o ,{lors petJceJ Jo -ulnru oqt 3ur,teo,ndgenper8 'splaryasegt to IIE r.uoryu,ry\pJp saqreo.rddelpluorurrodxapue slq8rsureqrrJsep11r,ue,tt 'sral -deqc SurrnolloJer{r uI 'uorletuour.radxa;o a1d1spue srseqd ',(8o1orq -rua u^\o slr spq splort eseql Jo qre1 leluarudolo,r -ep pue'eruerf,s relndruor tSolorsdqd'slrlaue8 1(docsonrur iiSoyorqrplnf,oloru'sorsdqdorq'dtlsruaqcorqreqtoSol sBur.rq (qcr.r sr lSolorq r lerll erualrs a,rrlBr8elut [e] JEInJOI6W 'puodsa; o1 sdem eleudordde tsotu oqt uo aprrep daqr moq pue sllor q8no.rqrs^{olJuorler.uro;ur^/rl,oq lnoqe dlrelnrrued 'peureal eg ol sureruoJlunotup asuerururue '11115 'Joqlo qJEe aJuenlturpu€ qrnot dagr ,vroq'urEluoJ daqt sernlcnrls tEr{na 's11ac sluouodruor ar{t tnoqp Etep.ry\au;ouorsoldxaue aJe! 1o o.tr 'suado d.rntuec tsrrJ-.{tue,v\teqr sV 'aJII Jo lrun Ietueruep 'aJIIyo sartradord>lreurller{ -unt oql sr IIar eqr tpr{t SuneJrpul aqt IIe trqrqxo susrueS.rorulnlloJrun aldurrsua,lg '1ac epurs e Jo lsrsuof, sulsrue8rodueru 1nq 'salnlf,n.rlsxalduror olur pezrve8rc sllel Jo suor1lrrtro suorllq ureluor srusrueSrore1 -NIIE]qFI.U

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(b) Eukaryoticcell

( a ) P r o k a r y o t icce l l P e r i p l a s m iscp a c e a n d c e l lw a l l

Golgi vesicles

Lysosome

O u t e rm e m b r a n e

I n n e r( p l a s m a ) membrane

Nucleoid lu'btrm I

Nucleoid

N u c l e a rm e m b r a n e P l a s m am e m b r a n e Golgi vesicles Mitochondrion Peroxisome Lysosome

I n n e r( p l a s m a )m e m b r a n e

Periplasmicspace Outer membrane R o u g he n d o p l a s m i c reticulum

1-2 Prokaryotic cellshavea simplerinternal A FIGURE micrograph of a organizationthan eukaryoticcells.(a)Electron coli a commonintestinal bacteriumThe thin sectionof Escherichia withina of the bacterial DNA,is notenclosed nucleoid, consisting by two membraneE coli andsomeotherbacterra aresurrounded spaceThethincellwallis membranes separated bythe periplasmic (b)Electron mrcrograph of a plasma adjacent to the innermembrane antibodies Onlya single cell,a typeof whitebloodcellthatsecretes (theplasma membrane membrane) surrounds thecell,butthe manymembrane-limited compartments, or interior contains Thedefining of eukaryotic cellsis organelles. characteristic

whichis DNAwithina definednucleus, of thecellular segregation is membrane Theouternuclear by a doublemembrane bounded for factory a reticulum, withthe roughendoplasmic continuous process andmodifyproteins, proteinsGolgivesicles assembling to digestcellmaterials generate lysosomes energy, mitochondria and process usingoxygen, molecules them,peroxisomes recycle them to the surfaceto release carrycellmaterials vesicles secretory E M u r r a y . P a r t ( b ) fromPC G B u r d e t t a n d R D J lPart(a)courtesyofl lJltrastructure: A Functional 1993,CellandTissue Cross andK L Mercer, andCompanyl W H Freeman Perspective,

and 40 miles up in the atmosphere;they are quite adaptable! The carbon stored in bacteria is nearly as much as the carbon stored in plants. Eukaryotic cells, unlike prokaryotic cells, contain a defined membrane-bound nucleus and extensive internal membranes that enclose other compartments called organelles (Figure 1-2b). The region of the cell lying betweenthe plasma membrane and the nucleus is the cytoplasm, comprising the cytosol (aqueousphase)and the organelles.Eukaryotescompriseall membersof the plant and animal kingdoms,including

the fungi, which exist in both multicellular forms (molds) and unicellular forms (yeasts),and the protozoans (proto, primitive zoan, animal), which are exclusively unicellular. Eukaryotic cells are commonly about 10-100 pm across' generally much larger than bacteria. A typical human fibroblast, a connective tissuecell, might be about 15 p,m acrosswith a volume and dry weight some thousandsof times those of an E. coli bacterial cell. An ameba, a single-celledprotozoan, can be more than 0.5 mm long. An ostrich egg beginsas a singlecell that is evenlarger and is easilyvisible to the naked eye. THE DIVERSITA Y N D C O M M O N A L I T YO F C E L L S

All cells are thought to have evolved from a common progenitor becausethe structures and moleculesin all cells have so many similarities. In recent years, detailed analysis of the DNA sequencesfrom a variety of prokaryotic organisms has revealed two distinct types: the bacteria and the archaea. Working on rhe assumption that organisms with more similar genesevolvedfrom a common progenitor more recently than those with more dissimilar genes,researchers have developed the evolutionary lineage tree shown in Figure 1-3. According to this tree, the archaeaand the eukaryotes diverged from bacteria billions of years ago before they diverged from each other. In addition to DNA sequence distinctions that define the three groups of organisms,

Animals

Plants Fungi

Ciliates

Euglena

Microsporidia

EUKARYOTA S I i m em o l d s Diplomonads (Giardia lamblia)

EUBACTERIA E. coli

Sulfolobus ARCHAEA B. subtilus

Thermococcus

Thermotoga

Methanobacteriu m Halococcus

Flavobacteria G r e e ns u l f u r bacteria

Halobacterium Methanococcus jannaschii

Borrelia burgdorferi

P r e s u m e dc o m m o n p r o g e n i t o r of all extant organisms P r e s u m e dc o m m o n p r o g e n i t o r of archaebacteria and eukaryotes

A FIGURE 1-3 All organismsfrom simplebacteriato complex mammalsprobablyevolvedfrom a common,single-celled progenitor.Thisfamilytreedepicts theevolutionary relations among thethreemajorlineages of organisms. Thestructure of thetreewas initially ascertained frommorphological criteria: creatures that look alikewereput closetogetherMorerecently the sequences of DNA andproteins havebeenexamined asa moreinformation-rich criterion for assigning relationships Thegreater thesimilarities in thesemacromolecular sequences, the moreclosely related organisms arethoughtto be.Thetreesbasedon morphological comparisons andthefossilrecordgenerally agreewellwith thosebasedon molecular data.Althoughallorganisms in the eubacterial and archaean lineages areprokaryotes, archaea aremoresimilar to eukaryotes ("true"bacteria) thanto eubacteria in somerespects For instance, archaean andeukaryotic genomes encodehomologous histone proteins, whichassociate with DNA;in contrast, bacteria lack histonesLikewise, the RNAandproteincomponents of archaean ribosomes aremorelikethosein eukaryotes thanthosein bacteria. C H A P T E RI

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archaeacell membraneshave chemical properties that differ dramatically from those of bacteria and eukaryotes. Many archaeansgrow in unusual,often extreme,environments that may resemblethe ancient conditions that existed when life first appeared on earth. For instance, halophiles ("salt loving") require high concentrationsof salt to survive, and thermoacidophiles("heat and acid loving") grow in hot (80' C) sulfur springs,where a pH of lessthan 2 is common. Still other archaeanslive in oxygen-free milieus and generate methane(CH+) by combining water with carbon dioxide.

U n i c e l l u l aO r r g a n i s m sH e l p a n d H u r t U s Bacteria and archaea, the most abundant single-celledorganisms,are commonly 1-2 pm in size. Despite their small size and simple architecture,they are remarkable biochemical factories,converting simple chemicalsinto complex biological molecules.Bacteria are critical to the earth's ecology, but some cause major diseases:bubonic plague (Black Death) from Yersiniapestis,strep throat from Streptomyces, tuberculosis from Mycobacterium tubercwlosis, anthrax from Bacillus anthracis, cholera from Vibrio cholerae, and food poisoning from certain types of E. coli and Salmonella. Humans are walking repositories of bacteria, as are all plants and animals.'We provide food and shelter for a staggering number of "bugs," with the greatestconcentration in our intestines.In return for the food and shelter that allow them to reproduce, bacteria help us digest our food. One common gut bacterium,E. coli, is also a favorite experimental organism. In responseto signalsfrom bacteria such as E. coli, the intestinal cells form appropriate shapesto provide a niche where bacteria can live, thus facilitating proper digestion by the combined efforts of the bacterial and the intestinal cells. Conversely,exposure to intestinal cells changes the properties of the bacteria so that they participare more effectivelyin human digestion. Such communication and responseis a common feature of cells. The normal, peacefulmutualism of humans and bacteria is sometimesviolated by one or both parties. When bacteria begin to grow where they are dangerousto us (e.g.,in the bloodstream or in a wound), the cells of our immune system fight back, neutralizingor devouring the intruders. Powerful antibiotic medicines,which selectivelypoison prokaryotic cells, provide rapid assistanceto our relatively slow-developingimmune response.Understanding the molecular biology of bacterial cells leads to an understanding of how bacteria are normally poisonedby antibiotics,how they becomeresistantto antibiotics, and what processesor structurespresent in bacterial but not human cells might be usefully targeted by new drugs. Like bacteria) protozoa are usually beneficial members of the food chain. They play key roles in the fertility of soil, controlling bacterial populations and excreting nitrogenous and phosphatecompounds,and are key playersin wastetreatment systems-both natural and man-made.These unicellular eukaryotesare also critical parts of marine ecosystems, consuming large quantities of phytoplankton and harboring photosynthetic algae, which use sunlight to produce biologically usefulenergyforms and small fuel molecules.

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FfGURE 1-4 Plasmodiumorganisms,the parasitesthat cause malaria,are single-celledprotozoanswith a remarkablelife cycle.ManyPlasmodium species areknown,andtheycaninfecta The hosts. varrety of animals, cycling betweeninsect andvertebrate fourspecies thatcausemalaria in humansundergo several dramatic (a)Diagram withintheirhumanandmosquito hosts. transformations of the lifecycleSporozoites entera humanhostwhenan infected Anopheles mosquitobitesa personfl Theymigrateto the liver, intothe wheretheydevelop intomerozoites, whicharereleased fromsporozoites, sothis bloodE. Merozortes differsubstantially "to transform" (Greek, isa metamorphosis or "many transformation merozoites invaderedbloodcells(RBCs) and shapes")Circulating producedby somePlasmodium reproduce withinthem B Proteins RBCs, causing the cellsto species moveto the surface of infected infected RBCs adhere to thewallsof bloodvessels. Thrsprevents fromcirculating to thespleen, wherecellsof the immunesystem theyharbor woulddestroy the RBCs andthe Plasmodium organisms in RBCs for a periodof time Aftergrowingandreproducing suddenly characteristic of eachPlasmodium species, the merozoites

from largenumbersof infectedcells4. lt burstforth in synchrony chillsthatare isthiseventthatbringson thefeversandshaking of malariaSomeof the released symptoms thewell-known a cycleof production creating RBCs, infectadditional merozoites intomaleand develop somemerozoites Eventually, andinfection. cells, These 5, anothermetamorphosis. femalegametocytes cannot whichcontainhalftheusualnumberof chromosomes, in bloodIo an Anopheles theyaretransferred for longunless survive gametocytes are the stomach, mosquito s In the mosquito. yetanother intospermor eggs(gametes), transformed flagellaon of longhairlike markedby development metamorphosis zygotesZ, of spermandeggsgenerates the sperm6 Fusion wallandgrowinto intothecellsof thestomach whichimplant of Rupture sporozoites for producing factories essentially oocysts, of sporozoites E; thesemigrateto the thousands an oocystreleases glands, settingthe stagefor infectionof anotherhuman salivary and of matureoocysts micrograph electron host.(b)Scanning stomach of sudace the external abut Oocysts sporozoites. emerging them that protects withina membrane wallcellsandareencased (b)courtesy of R E Sinden ] fromthe hostimmunesystem. lPart

However, some protozoa do give us grief: Entamoeba histolytica causesdysentery;Trichomonas uaginalis,vaginitis; and Trypanosoma brucei, sleeping sickness.Each year the deadliest of the protozoa, Plasmodium falciparum and related species,is the cause of more than 300 million new casesof malaria, a diseasethat kills 1.5 to 3 million people annually. These protozoans inhabit mammals and mosquitoes alternately,changing their morphology and behavior in responseto signalsin each of theseenvironments.They also recognize receptors on the surfacesof the cells they infect. The complex life cycle of Plasmodium dramatically illustrates how a singlecell can adapt to each new challenge it encounters(Figure 1-4). All of the transformations in cell type that occur during the Plasmodium Iife cycle are gov-

erned by instructions encodedin the geneticmaterial of this parasite and triggered by environmental inputs. The other group of single-celledeukaryotes, the yeasts, also have their good and bad points with respect to humans. as do their multicellular cousins, the molds. Yeasts and molds, which collectively constitute the fungi, have an important ecological role in breaking down plant and animal remains for reuse.They also make numerous antibiotics and are used in the manufacture of bread, beer,wine, and cheese.Not so pleasant are fungal diseases'which range from relatively innocuous skin infections such as jock itch and athlete's foot to life-threatening Pneumocystis carinii pneumonia, a common cause of death among AIDS patients. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

VirusesAre the Ultimate Parasites

opportunity. Viral infections can be devastatingly destructive, causing cells to break open and tissuesto fall apart. However, many methods for manipulating cells depend on using virusesto convey geneticmaterial into cells.To do this, the portion of the viral genetic material that is potentially harmful is replaced with other genetic material, including human genes.The altered viruses, or vectors, still can enter cellstoting the introduced geneswith them (Chapter 9). One day, diseasescausedby defectivegenesmay be treated by using viral vectors to introduce a normal copy of a defective gene into patients. Current research is dedicated to overcoming the considerableobstaclesto this approach, such as getting the introduced genesto work at the right placesand trmes.

Not all microscopic pathogensare cells. The other most familiar disease-causingorganisms are the viruses, which make use of the machinery inside the cellsthey infect to copy themselves.Virus-causeddiseasesare numerous and all too familiar: chickenpox, influenza, some types of pneumonia, polio, measles,rabies,hepatitis,the common cold, and many others. Smallpox, once a worldwide scourge,was eradicated by a decade-longglobal immunization effort beginning in the mid-1960s. Mral infections in plants (e.g.,dwarf mosaic virus in corn) have a major economic impact on crop production. Planting of virus-resistantvarieties, developed by traditional breeding methods and more recently by geneticengineeringtechniques,can reduce crop lossessignificantly. Most viruses have a rather limited host range, infecting certain bacteria,plants, or animals(Figure1-5). Becausevirusescannot grow or reproduce on their own, they are in this sensenot consideredto be alive. To survive. a virus must infect a host cell and take over its internal machinery to synthesizeviral proteins and in some casesreplicate the viral geneticmaterial. When newly made virusesare releasedby budding from the cell membrane or when the infected cell bursts, the cycle starts anew. Viruses are much smaller than cells,on the order of 100 nanometer (nm) in diameter; in comparison, bacterial cells are usually >1000 nm (1 nm : 10-e meters).A virus is typically composedof a protein coat that enclosesa core containing the genetic material, which carries the information for producing more viruses (Chapter 4). The coat protects a virus from the environment and allows it to stick to, or enter,specifichost cells. In some viruses, the protein coat is surrounded by an outer membrane-likeenvelope. The ability of viruses to rransport genetic material into cells and tissuesrepresentsa medical menaceand a medical

The most remarkable feature of organismsis their ability to reproduce.Biological reproduction, combined with continuing evolutionary selectionfor a highly functional body plan, is why today's horseshoecrabs look much as they did 300 million years ago, a time span during which entire mountain ranges have risen or fallen. The Teton Mountains in S7yoming,now about 14,000 feet high and still growing, did not exist a mere 10 million years ago. Yet horseshoecrabs, with a life span of about 19 years,have faithfully reproduced their ancient selvesmore than half a million times during that period. The common impression that biological structure is transient and geologicalstructure is stableis the exact opposite of the truth. Despite the limited duration of our individual lives, reproduction gives us a potential for immortality that a mountain or a rock does not have. Whereas some species have changed little over great periods of time, other organismshave changeddramatically

(a)T4 bacteriophage

(b)Tobaccomosaicvirus

C h a n g e si n C e l l sU n d e r l i eE v o l u t i o n

,

50nm

,

(c) Adenovirus

A FIGURE 1-5 Virusesmust infect a host cell to grow and reproduce.Theseelectronmicrographs illustrate someof the structural variety (a)T4 bacteriophage exhibited byviruses. (bracket) attaches to a bacterial cellviaa tailstructureViruses that infect bacteria arecalledbacteriophages, (b)Tobacco or simplyphages. mosarc viruscauses a mottlingof the leaves of infected tobacco C H A P T E R1

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plantsandstuntstherrgrowth.(c)Adenovirus causes eyeand respiratory tractrnfections in humans. Thisvirushasan outer membranous envelope fromwhichlongglycoprotein spikesprotrude [Part(a) from A Levine,1991, Viruses,ScientificAmericanLibrary,p 20 Part(b) courtesyof R C Valentine Part(c) courtesyof RobleyC Williams, Universityof Californial

during the same period. The changescame in responseto pressuresfrom the environment that causedincreasedsurvival of variant individuals. Both stasis and change are possible becausethe machinery of cells does an amazingly precisejob of copying geneticmaterial, yet its rare errors introduce some variation. If environmental conditions continue to select more or less the existing form, as in the caseof horseshoecrabs, the specieswill changelittle. If a new variant has a survival advantage, perhaps because conditions have changed, it may persist and replace the old form. Populations of bacteria exposedto antibiotics, for example, changetheir properties dramatically to escapeand live. They do this becauserare mutations, changesin the genetic material that allow antibiotic resistance,keep some cells alive while the cells without those mutations die. Most populations of any single specieshave a large repertoire of genetic alterations becausethere is a low but significant error rate in copying the genes.That error rate increasesin the presenceof radiation such as sunlight or certain chemical poisons. Current genomeproiects are exploring geneticvariation among humans. "The" human genome sequencethat has been determined already is just one version among billions. Understanding variation is essentialto learn how we respond differently to certain infections or drugs and to exploring how our geneticheritage combines with our experienceand learning to make each of us unique. Underlying the reproduction of organismsis the copying of cells, something that must be precise in order to control the size, shape, and organization of animals and to prevent unwanted growth, such as cancer.The cell is a machine that can copy itself, unlike viruses,which cannot do so on their own. As we will seein Chapters 20 and 21',the cell cyclefrom a single cell copying its own contents through division into two cells-is controlled by a seriesof elegant switches and cross-checking mechanisms. Reproducing cells accurately is a matter of life and death.

EvenSingleCellsCan Have Sex If genetic material was never shared or exchanged,each individual would be the beginning of a new clone of individuals, and the members of a clone would share most of the same genetic strengths and weaknesses.Sex is a processof mingling genetic variation from two individuals, creating new individuals with a combination of properties unlike either parent and that may be beneficial for survival and reproduction. Each chromosome except the sex chromosomes is representedtwice, one copy from the father and one from the mother. Since each pair of chromosomes trades piecesduring the formation of eggs and sperm, new combinations of genesare created and inherited together in the next generation-variation is accelerated. The other benefit of having two copiesof each chromosome is that a poorly functioning gene is backed up by the other copy. The common yeast used to make bread and beer, Saccharomycescereuisiae,appearsfairly frequently in this

Budding lS. cerevisi ael

1-6 The yeast Saccharomycescerevisraereproduces A FfGURE sexuallyand asexually'(a)Twocellsthat differin matingtype' calleda andcr,canmateto forman a/a cellIl. Thea anda cellsare theycontaina singlecopyof eachyeast meaning haploid, halfthe usualnumber'Matingyieldsa diploida/ctcell chromosome, Duringvegetative two containing copiesof eachchromosome. process an asexual by mitoticbudding, growth,diploidcellsmultiply a diploidcellsundergomeiosis, conditions, E Understarvation B. Rupture to formhaploidascospores typeof celldivision, special into whichcangerminate fourhaploidspores, releases of an ascus asexually E. (b)Scanning alsocanmultiply cells4. These haploid Aftereachbudbreaks of buddingyeastcells. micrograph electron buds free,a scaris left at the buddingsite,so the numberof previous (b) AbbeyA/isuals M bacteria are cells [Part canbe counted.Theorange lncl Unlimited, book becauseit has proven to be a great experimental organism. Like many other unicellular organisms' yeasts h"u. t*o mating types that are conceptually like the male and female gametes(eggsand sperm) of higher organisms'

ubiquitous. A N D C O M M O N A L I T YO F C E L L S THE DIVERSITY

.

7

We Developfrom a SingleCell In 1827, German physician Karl von Baer discovered that mammals grow from eggs that come from the mother's ovary. Fertilization of an egg by a sperm cell yields a zygote, a visually unimpressive cell 200 pm in diameter. Every human being begins as a zygote,which housesall the necessaryinstructions for building a human body containing about 100 trillion (1014)cells, an amazing feat. Development begins with the fertrlizedegg cell dividing into two, four, then eight cells, forming the very early embryo (Figure 1-7). Continued cell proliferation and then differentiation

Chapters 16 and 22. Making different kinds of cells-muscle, skin, bone, neuron, blood cells-is not enough to produce the human body. The cells must be properly arranged and organized into tissues,organs, and appendages.Our two hands have the same kinds of cells, yet their different arrangements-in a mirror image-are critical for function. In addition, many cells exhibit distinct functional and/or structural asymmetries, a property often called polarity. From such polarized cells arise asymmetric, polarized tissuessuch as the lining of the intestinesand structures such as hands and hearts. The features that make some cellspolarized and how they arise also are coveredin later chapters,including Chapter 21.

Video:EarlyEmbryonicDevelopment < FIGURE 1-7 The first few cell divisionsof a fertilized egg set the stagefor all subsequentdevelopment.A developing mouseembryois shownat (a)the two-cell,(b) four-cell, and(c)eight-cell stages. Theembryoissurrounded by supporting membranes. The corresponding stepsin human development occurduringthe firstfew daysafterfertilization. Researchers. IClaudeEdelmann/Photo I n cl

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Stem Cells,Fundamentalto FormingTissues and Organs,Offer MedicalOpportunities The biology of stem cells, cells that can give rise to specific cell types and tissues,has generated, great interest. 'We can 'Sfhen contrast stem cells to the simpler types of bacteria. a bacterial E. coli cell divides, both daughter cells are pretty much equivalent in content, size, and shape. In some other bacteria and in many casesof eukaryotic cell division, the two daughters differ in important ways. Although both will have the same genetic material, the cells may differ in size, shape, and contents. The cells may have different fates, that is, they may become different types of differentiated cell. A division that produces two different daughter cells is sometimes described as an asymmetric cell division. Stem-celldivisions are a specialcaseof asymmetric division. One of the two daughter cells is identical to the parent cell; the other follows a path of differentiation, such as becoming a blood cell. The parent cell, called a stem cell, can go on reproducing itself at every division, at each division also producing anorher blood cell. Mosr tissuesin our bodies form from stem cells. Blood, for example, is produced from stem cells that residein the bone marrow and continue to produce new blood cellsfor our entire lives. This is the basis of the often successfulbone marrow transplants that are used to treat cancer patients who have had their blood stem cells damaged by cancer treatments: what is being transplanted is stem cells.However, blood stem cells produce onlv more of themselvesand blood cells, not othir cell types. Thus eachtissuemust have its own stem cells,at leastduring the period of development when the tissue is formed. Stem cells for each tissue arise from even more capable stem cells that have the ability to form multiple stem cell types. The first stem cells are found in early embryos,where all the cells are capable of producing all cell types. In mammals the ultimate stem cell is the fertilized egg, which produces early embryo cells capable of forming all the tissuesof the body. This capability is illustrated by the formation of identical twins, which occur naturally when the mass of cells composing an early embryo divides into two parts, each of which develops and grows into an individual animal. This means that the cells cannor have divided up their embryo-forming duties prior to the time of embryo division. Each cell in an eight-cell-stagemouse embryo has the potential to give rise to any part of the entire animal. Cells with this capability are referred to as embryonic stem (ES)cells. As we will learn in Chapter 22, ES cells can be grown in the laboratory (cultured) and will develop into various types of differentiated cells under appropriate conditions. The ability to make and manipulate mammalian embryos in the laboratory has led to new medical opportunities as well as various social and ethical concerns.In vitro fertilization, for instance, has allowed many otherwise infertile couples to have children. One technique involves extractron of nuclei from defective sperm incapable of normally fertilizing an egg,injection of the nuclei into eggs,and implantation of the resulting fertilized eggsinto the mother.

In recent years, nuclei taken from cells of adult animals have been used to produce new animals. In this procedure' the nucleus is removed from a body cell (e.g., skin or blood cell) of a donor animal and introduced into an unfertilized mammalian egg that has beendeprived of its own nucleus.In a step that has now been done with mice, cows, sheep, mules, and some other animals, the egg with its donor nucleus is implanted into a foster mother. The ability of such a donor nucleusto direct the developmentof an entire animal shows that all the information required for life is retained in the nuclei of some adult cells. Sinceall the cells in an animal produced in this way have the genesof the single original donor cell, the new animal is a genetic clone of the donor (Figure 1-8), though the animals may differ anyway due to their distinct environments and experiences.Repeating the processcan give rise to many clones. Nuclei taken from ES cellswork especiallywell, whereasnuclei from other parts of the body at later times in life work far lesswell. The majority of embryos produced by this technique do not survive due to birth defects,so the donor nuclei may not have all the needed information or the nuclei may be damaged by the cloning process.Even those animals that are born alive have abnormalities,including acceleratedaging. The "rooting" of plants, in contrast, is a type of cloning that is readily accomplished by gardeners,farmers, and laboratory technicians. Scientific interest in the cloning of humans is very limited. Virtually all scientistsoppose it becauseof its high risk to the embryo (also, most people don't believe there is a

critical shortage of twins and triplets). Of much gteater scientific and medical interest is the ability to generatespecific cell types starting from embryonic or adult stem cells' This procedure, somatic cell nuclear transfer (SCNT)' produces cells that are grown in culture and never turned into

If the cells are produced using a donor nucleus from a patient, the properties of the cells may allow them to escaperejection by the patient's immune system,opening new possibilities for cell-transplanttherapies.

E

The Moleculesof a Cell

Molecular cell biologists explore how all the remarkable

ture and function.

Small MoleculesCarryEnergy,TransmitSignals, and Are Linkedinto Macromolecules Much of the cell's content is a watery soup flavored with small molecules (e.g., simple sugars' amino acids, vitamins) and ions (e.g.,sodium, chloride, calcium ions)' The locations conce.tirations of small molecules and ions within the "nd controlled by numerous proteins inserted in cellular are cell membranes. These pumps' transporters, and ion channels move nearly all small moleculesand ions into or out of the cell and its organelles(Chapter 11). One of the best-known small molecules is adenosine triphosphate (ATP), which storesreadily available chemical 'When ..r"rgy itt two of its chemical bonds (seeFigure 2-31)' cells"splitapart theseenergy-richbonds in AIP' the released ..r.rgy ."n b. harnessed to power an energy-requiring pro..tt such as muscle contraction or protein biosynthesis' 1-8 Fivegeneticallyidenticalclonedsheep.An early To obtain energy for making ATR cells break down food a FIGURE intofivegroupsof cellsandeachwas sheepembryowasdivided molecules.For instance, when sugar is degraded to carbon natural like the much mother, intoa surrogate implanted separately dioxide and water, the energy stored in the original chemical process of twinningAt an earlystagethecellsareableto adjustand bonds is releasedand much of it can be "captured" in ATP the cellsbecome laterin development forman entireanimal; (Chapter 72).Bacterial,plant, and animal cells can all make way andcanno longerdo so.An alternative progressively restricted ATP ty this process.In addition, plants and a few other orsingle-celled the nucleiof multiple isto replace to cloneanimals ganisms can harvest energy from sunlight to form ATP in with donornucleifromcellsof an adultsheep'Eachembryo embryos photosynthesis. ' was to the adultfromwhichthe nucleus identical will be genetically Other small moleculesact as signalsboth within and beto theseprocedures survive of embryos obtained.Lowpercentages tween cells; such signals direct numerous cellular activities on the givehealthy andthefull impactof the techniques animals, effect on our bodies of Library/Photo (Chapters iS uttd f e ;. fne powerful Photo Tompkinson/Science animalsis not yet known.lGeoff flooding of instantaneous the from comes a frightening event Incl Researchers, S F A CELL T H E M O L E C U L EO

O

9

the body with epinephrine, a small-moleculehormone that mobilizes the "fight-or-flight" response.The movements neededto fight or flee are triggered by nerve impulses that flow from the brain to our muscleswith the aid of neurotransmitters, another type of small-moleculesignal that we discussin Chapter23.

acids. Sugars,for example, are the monomers used to form polysaccharides. These macromolecules are critical structural components of plant cell walls and insect skeletons.A typical polysaccharideis a linear or branched chain of repeating identical sugar units. Such a chain carries information: the number of units. However, if the units arenot identical, then the order and type of units carry additional information. As we will seein Chapter 6, some polysaccharides exhibit the greaterinformational complexity associated with a linear code made up of different units assembledin a particular order. But this property is most typical of the two other types of biological macromolecules-proteins and nucleic acids.

ProteinsGive CellsStructureand perform Most CellularTasks The varied, intricate structures of proteins enable them to carry out numerous functions. Cells string together Z0 dif_ ferent amino acids in a linear chain to foim a protein (see Figure 2-14).Proteins commonly range in length from 100

to 1000 amino acids, but some are much shorter and others longer. We obtain amino acids either by synthesizingthem from other moleculesor by breaking down proteins that we eat. The "essential" amino acids, from a dietary standpoint, are the eight that we cannot synthesizeand must obtain from food. Beans and corn together have all eight, making their combination particularly nutritious. Once a chain of amino acids is formed, it folds into a complex shape,conferring a distinctive three-dimensionalstructure and function on each protein (Figure1-9). Some proteins are similar to one another and therefore can be consideredmembers of a protein family. A few hundred such families have been identified. Most proreins are designedto work in particular places within a cell or to be releasedinto the extracellular (extra, "outside") space.Elaborate cellular pathways ensurethat proteins are transported to their proper intracellular (intra, "within") locations or secreted (Chapters 13 and14). Proteins can serveas structural componentsof a cell. for example,by forming an internal skeleton(Chapters tO, tZ, and 18). They can be sensorsthat changeshapeas remperature, ion concentrations, or other properties of the cell change. They can import and export substancesacross the plasmamembrane(Chapter11). They can be enzymes,causing chemicalreactionsto occur much more rapidly than they would without the aid of theseprotein catalysts(Chapter 3). They can bind to a specificgene,turning it on or off (Chapter 7). They can be extracellular signals,releasedfrom one cell to communicatewith other cells, or intracellular signals, carrying information within the cell (Chapters 15 and 16). They can be motors that move other moleculesaround, burning chemicalenergy (ATP) to do so (Chapters17 and 1g).

Insulin

Glutaminesynthetase Lrturamtne synthetase

Hemoglobin

FIGURE 1-9 Proteinsvary greatly in size,shape,and function.Thesemodels of thewater-accessible surface of some representative proteins aredrawnto a commonscaleandreveal the numerous projections andcrevices on thesurface. Eachprotern hasa definedthree-dimensional shape(conformation) thatisstabilized by numerous chemrcal interactions discussed in Chapters 2 and3. The illustrated proteins include (glutamine enzymes synthetase and

10

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L | F EB E G T N W S |TH CELLS

DNA molecule

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adenylatekinase),an antibody(immunoglobuiln), a hormone (insulin), and the bloods oxygencarrier(hemoglobin)Modelsof a segmentof the nucleicacid DNA and a smallregionof the lipid bilayerthat formscellularmembranes(seeSection1.3)demonstrate the relativewidth of thesestructures comparedwith that of tvpical proteins.[Courtesy of GarethWhite]

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'g rardeq3 ur lretepur uortElsuertpuB uorldrrrsuPrl lno d.rrel leqt stuauodruor IIer eql euruexe al1 'opof, f,rteu -aE les.ra.trundlreeu aql ot Surprorre aruanbasVNUIU oqr dq parelrrp rap.ro asne;d eqt ur sprre ourrue raglaSol s>1ur1 (uorlelsuB-rl SurrnC 'uorlelsueJl pue selqtuesseatuosoqrrar{l pell€r <sserord puocaseqt lno srrJrer'uralord pue VNU qtoq yo pasodruor eurqJprurelnJelou xalduoc ,{lsnourrouaue 'aruosogrraLIteJeH 'ruseldoldceql ol se,l.oruqcrq.u 'a1nca1ou (VNUru) y51A ra8uasserurelleruse otur possarordsr rrnpord 'sl1ar 'arelduer E se ruodre>1na u1 vNd IBrlrur aqr vNC Sursnureqr VNU p otur saprloe]f,nuJo a8e>1ur1 aql sez.{1etec 'au.,(zua 'VNC 'aserorudlod y51g e8rel pepuens-alqnop V eql Jo uorsre^ (VNU) prJp Jrelf,nuoqrrpepuerts-a13urs e otur pardor sr aua8 e yo uor8er Surpor eqt 'uorldrrcsuprl pellel 'rs-rr; '(II-I aqt uI arn8lg) suretordorur VNC ur uorlerurot -ur pepol aqt ol sorrasur sessecordo.la.lesn slla] lJeluor 'opuru sr uratord Jr{t sllal qlrr{.a uI puE uel{,{\ slorluof, teqt uo8al Ktolap8at E pue urelord e ;o acuanb -es prf,p ounue aqt sarpads rcq7 uot8at Surpoc e :strud orrrl ureluoJ dluorutuoc suralord 3ur1eu Jot suorlfnJlsur d.r.rer teqr saue8eql'000'SZ-000'02 lnoqe'suerunq lsaue8pues -noqt .,taale e^eq errolf,pq tso14 '3uo1 seprloallnu 000'00I or 000S a.re dlyecrddrqlrq^\ 'souo8 eql (strun leuorlrunt elerrslP olul poPI^lPsl VNq Jo uorlrod Surreaq-uotlEturolul oql 'puerts e 3uo1esoprloalJnuJo rapro reaurl Jrll 'aluenb -esslr ur seprser VNC dq perrrpr uorteruroturlrleueg or{J. 'gfleru lou oP ]Pql sPuprls ror{rodueu sureluotroslEuorl vNC -nlos oqt ue^a 'Surraddrzot peal 'r llr.{\ puerts dretuauralduroc rel{lo eqt Surureluocuortnlos e ur raded aql Suopos taded 'alduruxerod ;o aoarde o] Paqleue pue par;rrndsI puerls auo;t reqlo aql Sursnpue.rtseuo Surlcalaprot Intesndlauarlxa sruorl -enpuq;1.i:rl prf,e f,rolf,nur.lrns 'suorlrpuoo arnle.radtuetpue tles drz dlsnoauetuods11,udaqt 'pale.r rq8rr eqr ur raqtaSor 4ceq -edes a.le spuprts .,(reluerualduol teqt Suols os sr spuerls Jr o.,rueqr;o Surqrreru,{-ruluaualduol srr{J '(91-1 arn8r4)3 e qtr.&\peqlteru sr J qrea pue 1 e ser1puerls reqlo eqt 'y ue seq pupJls ouo Jeleror{,la.:uouJnrlsuof aydrursp spr{ x[eq elqnop 'puens rqr VNC r{leg to auoqlleq lerqeq eqr tuo4 lno Sunoal 'puerts -ord stred eseq eqt qtrlr E ur puo ol pua paurol VNCI are '9 pue 'J t'V peter^argqe'seprtoalcnuluora11rp rnoC

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a)eld pue ourrl lq6tu aql le surelord6ur4ey1ro; uolleturo+ulpapo) f.r.re1sp!)v )rel)nN '(solnralou 80I x S) utlle utotord IErnltnDS lu€punqE eqr or (salncalotu000'02) utalo.rdroldoc 'dleprm -a.r sutetord alrnb eqt uo.r; sarrB,r Surpurq-urlnsur erpr 'e8e.ra,reuo urolord tuere;trp Jo eruepunqe aql 'lce1 u1 1o addr qree Jo selnrelouruorlpu e ot osolf,suleluol IIOJE snqr isurelord ruere;Jrp000'0I rnoqe suleluor IIat re^II p leq] raprsuoJ taqrrny dels auo uollelnf,lef, srqt drrec oI '(Ez0I x ZO'9) punoduor lerlueqr due 1o ayourrad salnrelotu Jo requnu eql laqunu s,orpeSorrypue lq8ra,t uratord Ielol eqt trrorl sp IIer re^II .radsalncalou utarord 60I x 6'L lnoqe 'suratord f,rlofre>lna to reqrunu letor oqt elelnllel uer e,l.r '(1ou73) 00L'TS yo rq8rorr ;o lerrddr sr enlE^ srql Surunssy relnrelorrr p seq uratord lseef e8erarreeql '3 or_0I x L er{r 'tq8ta,trs(ller e to tuer sr ureto.rdrelnller rq8rarvr ;o I€tol -nd 97,,(laleturxo.rddpro; slunoJJe uralord erur5 '3 r_0I x g'g q8ra.uplno.vi aql'1uF E0'I Jo &lsuep 11ec e Sururns IIer -sy '(s;arqqlur ro) etunlol e seg 'aprse e*l o_0I x V't p srql '(1ac re,rr1) uo (tuc rurl eqnr E ,{1q3nor'11ac SI SI00'0) 'req ardcoledeqe se r1rns'11accrlodre>1ne lerrd& e a4et s.ta1 -unu srqt eterurtseoJ 'lasrr urelureru pue alerado ol spJJu IIar e selnrelotu urarord dueur .uoq >1serq8nu a^.\txaN 'dtlulJul 3uo1e pre,&ol 1a,'nsr sutetord algrssodIo reqrunu .ro'alqetsun'luele.trnba llleuorr eqt'elqElunorsrpasr,&\rar.{lo 'seruanb -lunJ dueru aq asaql Surunsse ua,tg p1no.l.r leqt Jo '3uo1 sprre -as uralord luereJJrp alqrssod oov17ere araqt ounue 00t rnoqe sr ureto.rd..1errddl,,E Jr lng 'ecue13lsrr; re alqrssodrursrueestI is>lserperre^ eseqlurotred or papaeu surolord ruereJtrpoqt IIE ruroJ splre ouIIuE 0Z uel ,{\oH

ur pegrrtsap are aruosrldarer{r Jo sluauodruol raqto dueru aql lpue;rs VNC € otur seprtoepnu Surlury ro; alqrsuodsa_r sr eserau,(1odVNq 'leur8r.roeql ot Iefrluepr ql€e ,serrlaq elqnop 1o ;red e sr eruortno eqI .(0I-I a.rn8rgaas)pue_us dreruetualduoJ 1y\eue olur saprloopnu elquesse ol aleldual e se qrea sasnpup seuosoruorql eqr ul vNC Ielrleq PuErls -elqnop spuertso.,vrtrqt seteredas,aruosrlde.r eqt (eurqrpru Jo uorlerrlda.ruralordrllnru a8rel e ,saprlrp 11ace arurl d.ra,rg 'pec ol 'JlpLlreqlo eq.l 'asaql !tuoy41o1 IlEq peJen og UEJ'seue8eqt;o JIeq snLlrpue '(71-1 ern8r4)sauosoruorqr seq uerunq Jo 'lpH 9t IIal lpur -rou dre,te 'rulads pue s33a uondarxe egr qtrl1 .VNC 1o to luerualduror eJrluastr sasr.rduorrusrue8roue ;o auroua8eq1 '(e7-1 a-rn8r1 aes) aql yo uor8arI€rtueJaqt ur ,saurt duetu 11ac 'sat1alncalorusrqr lq13ua1ur ralaurllru Jlesrluo >lrEqPePIoJ B tnoqE alnrelotu VNC relnrrrr a13ursB ur sJprseruorterurot 's11arrrtodrelord u1 'suretord -ut cttaue83qt to IIe ro tsoru urEtJaJqtr.4apelerJosseelnfelou VNC Jeeurl a13urse surel -uor aruosoruonll qleg '(9 ;erdeq3) saruosoruorrlf,se.{\ou{ J^\ soJnlJnrls JprlrrueJor.[l olur peplot dle,rrsuelxa ,snap -nu agl ur petef,ol sr sllJJ lrodrelna ul VNC er.[]Jo rsory

u o t s r ^ t ol l o ) 6 u u n c p o l e ) r l d o up u e saurosotuotq)olur pa6e{)ed sl oruouagoql 'paroldxa Surag ere uorteln8e.r 11rrs ,saue8 1o ed& srql Jo &rnbrqn pue sursrupq)au eql qSnoql II€ ro tsoru alelnSer deu sygg llerus seteturlsaaruos dg 'sldrrcsuerlauaS;o drrpqerspue uortrnpord ,uors Surtuln8er -sardxa aue8 uo tleJJef,rt€uerp p e^eq uel selnrelour VNU 'serurtrq8u aqr sllet le rq8rr aqr ur saua8tq8u or1tsale^ IIpruS -ItrE legt ualsds IortuoJ alrsrnbxaup ruroJ ol padoldruaa.re srolreJ uotldtnsue;l lueretlrp Jo sperpunq 'susruefiro xald -ruoJ ul 'seua8pereln8er aqr Suqcalasol dlpr;rceds Surpurq -VNC u,4o str Surtnquluoc uralord auo ueql JJou qlr.^A 'saxaldruor uleloJdrtlnu sE lJo,ln uouo sJolfet uortdr.rrsuerl ']as eqt ur aueSqrea rEau lsrxo rotret teqr roJ satrsSurpurq ;r seua8Jo las E eteln8er dlateurproor uer rotf,€J uortdr.rcs 'uorssarda;pue uortelrl -uert Jo addl auo 1o serdocaldrrlnyq '11ar -re eua8 e Jo drlclJlradseqt Suunsue ;o VNC aql ur rnl -lo e dlug .seprtoaltnu IIr.^aacuanbesrJf,nsqrpe 1o sardor.ra.a1 rnoJ Jo due aq uec uortrsodr1f,Ee erurs (9tS'gV1't) saruanb -as alqrssod o^eq uec s.rred espq 0I Sururetuor ylrlq 0rt '3uo1srred aseq y51q ;o ruaru8asy ZI-9 $oqe seouanbes t.roqs azruSoJerllr,l.r'uralord Surpurg-y51q e d1lecrdd1 'VNC s,ller e ut luaserdspuesnoqlJrll Jo rno seue8,ua1e tsnl 1o suor8a.rdroleln8ar aqr o1 dlyerluare;ardpurq ot elqp ere .{eql reql dlasnard os pedeqs are srotreJ uortdrrrsue{ '(1 retdeq3) saue8 relncrtred ;o uortdursuerl Surssard 'saqrlr,rs sE tJE pue -ar ;o SurtB,r.rlJe JerJlre VNC ot purq (srolreJuorldrrrsuprl qcrq,rrr pallEt suralordSurpurq-ypq uo spuadapdtr,rrlceaua8yo lortuoJ r{tns 'speeuluorrnr }eetu ol 'go surato.rd;o arrolrada.r rragl Surldepe ,(qareqr ro uo saue8 rrPeds Suturnt dq suortrpuof, ur se8ueqc.ro sleu8rs leuJetxe 'esrol aJrApue IeuJetxeot puodsa;ue) sllJJlueru taaoerory s11ecdaupr>1,{q peenpord tou er€ teqt suralord oruos ernp -ord s1larra,tq dq,rrr. s(teqJ 'sureto-rdaleu ot pesn pup (uo paurnt lo 'a,ttlf,eare saua8esoqtJo eruosdluo ad.{rIIac r{rea

s r r r ) H r M s N r g r sl j r ' r I

t u:ravul

zl

ur tnq 'seua3ueunq to tes IInJ aql uretuor serpogrno ur sllar eL[] IIE dl.reau'aruetsur roC 'paquf,suurl aq UEJ seua8rraql eJeq.4 pue uoq,lt IoJluoc ol sde,,vreleq s{usrup8ro 11y ' u r a t o r do t u l s J l n f , 'salncalotuy51a re8rel -elou VNUIU to uouplsuerr eqt pue (seLuosoluorqr Jo drlllqers eql Jo uollrunJ Pue erntf,nrts '3uo1 sepnoepnu 'sVNU aqr aleln8a.rd11err;roads 002-02 'dr{lqers pue Surssecord VNU pue ornl Ilprus sas€f,dueru u1 -fnrts eruosoruoJqlSurpnycurtlrrrrlre eua8 slf,odseduetu 1o Suneln8e.rur alor tuet.rodurr flqelreua; e Aeld ol puno' ueaq osle ser{VNU 'flruareg razrsaqludsuratord pue repear VNUIU luarrr]'a pue asrcard dlqe>1reua.rp a{pru ol surel -ord 69 ueqt eroril qlrrr,rdn rueat leqt sureqf,VNU JnoJ seq euosoqrr agt (oldurexa roC 'aurLlleru rplnlelou e Surplrnq ro; {ro^\euer; E se enres uer y51g 'useldoldc ol snolf,nu ruoJJ uorleruro;ur Surr.ra;supJlur aloJ slr ol uortrppe uI

ureqlrpourle olurspr)eouru.re 6ur1ur1 {1;erruaqr {q ureloLd e puearuenbes slrpear]eql sourosoqu {q punoqsr}r oloqm alqrlosse 'useldoyb y11y (VNUul) eq] o1 sa^oLr; rebuesseu ernleur oql 'llo) p ul :E de15saruanbas 6urporuou oAoLUar o1pessa:ord rrloi{re>1ne sr aq1: E dals eleldua]e sespuer]s ldursuerl VNCaq],o auo pepuerls-a16urs 6ursn]drnsuert e olurseprloapnu 6ur>1ur1 VNUU-ard esereuri;od aq_La]rsuelsoq] 'uorle)ol>rloeds VNc oq] 6uo;esenou.r 'VNC ue,Louorldursuerl sur6aq asereu{1od e ]e auebpale^r])e VNU aq]o] punoqxelduoruorlpr]rur ure1o.rdr1lnu e 1o{lquasse6urnnollo3 : g da15uJeq]ole^r]lepuelorluo),{eq}seue6:r;r:edsaq11o ,lo1eln6er aql o1purqsrolreluorldursue{:It da15'ssarord suor6el delsrllnu e {q surelold;o saruenbaspr)eourue oq} o}ul pouo^uo)s! vNc u! uollewrolu!papo)aqM-! lun9lJ v ureq3prce ouil..uv I 1 I I

v l g 1 o u o r o e ro u r p o c u o N V r u U ; ou o t 6 a l6 u t P o c - u t a 1 o l 3 vNc Jo uorOorpaqrrcsuerluoN -n2q2q2qa V N C + o u o r 0 o rp a q u 3 s u e {

uollolJcsue{

d uorleAllcv

can be "painted" for easy 1-12 Chromosomes FIGURE A normalhumanhas23 pairsof morphologically identification. fromthe onememberof eachpairis inherited chromosomes; distinct motherandthe othermemberfromthefather(Left)A chromosome whenthe froma humanbodycellmidwaythroughmitosis, spread wastreatedwith Thispreparation arefullycondensed chromosomes reaqents thatalloweachof the 22 pairs staininq fluorescent-labeled

Chapter 4. The molecular design of DNA and the remarkable properties of the replisome ensure rapid, highly accurate copying. Many DNA polymerasemoleculeswork in concert, each one copying part of a chromosome.The entire genome of fruit flies, about 1.2 x 10o nucleotideslong, can be copied in three minutes! Becauseof the accuracyof DNA replication, nearly all the cells in our bodies carry the same genetic instructions, and we can inherit Mom's brown hair and Dad's blue eyes. A rather dramatic example of genecontrol involves inac'Women tivation of an entire chromosomein human females. have two X chromosomes,whereas men have one X chromosome and one Y chromosome, which has different genes than the X chromosome. Yet the genes on the X chromosome must, for the most part, be equally active in female cells(XX) and male cells(XY). To achievethis balance'one of the X chromosomesin female cells is chemically modified and condensedinto a very small mass called a Barr body' which is inactive and never transcribed. Surprisingly,we inherit a small amount of geneticmaterial entirely and uniquely from our mothers. This is the circular DNA present in mitochondria' the organelles in eukaryotic cells that synthesizeAIP using the energy released by the breakdown of nutrients. Mitochondria contain multiple copies of their own DNA genomes,which code for some of the mitochondrialproteins(Chapter6). Becauseeachhuman inherits mitochondrial DNA only from his or her mother (it comes with the egg but not the sperm), the

colorwhen to appearin a different andtheX andY chromosomes of multiplex Thistechnique microscope viewedin a fluorescence (M-FISH) iscalled sometimes in situhybridization fluorescence (RiSht) fromthe (Chapter Chromosomes 6) painting chromosome orderof size, in pairsrndescending preparation on the leftarranged of X andY chromosomes Thepresence an arraycalleda karyotype. of M R Speicher] asmale [Courtesy the sexof the individual identifies

distinctive features of a particular mitochondrial DNA can be used to trace maternal history. Chloroplasts, the organelles that carry out photosynthesisin plants' also have Ih.i, o*tt circular genomes'Both mitochondria and chloroolasts are believedto be derived from endosymbionts,bacteii" th"t took up residenceinside eukaryotic cells in a mutually beneficialpartnership.The mitochondrial and chloroplast circular DNAs appeatto have originated as bacterial genomes' which also are usually circular,though the organellegenomes have lost most of the bacterialgenes.

Mutations May Be Good, Bad,or Indifferent Mistakes occasionallydo occur spontaneouslyduring DNA replication, causing changesin the sequenceof nucleotides' Soch ch"ng.s, or mutations, also can arise from radiation that causesdamageto the nucleotidechain or from chemical poisons, such as those in cigarette smoke, that lead to errors iuring the DNA-copying process (Chapter 25)' Mutations .o-.1., various forms: a simple swap of one nucleotide for another; the deletion, insertion, or inversion of one to millions of nucleotides in the DNA of one chromosome; and translocation of a stretch of DNA from one chromosome to another. In sexually reproducing animals such as ourselves,mutations can be inherited only if they are presentin cellsthat potentially contribute to the formation of offspring' Such germJine cells include eggs'sperm' and their precursor cells' T H E M O L E C U L EOSF A C E L L

O

13

Body cells that do not contribute to offspring are called somatic cells. Mutations that occur in these cells never are inherited, although they may contribute to the onset of cancer. Plants have a less distinct division between somatic and germ-line cells, since many plant cells can function in both capacltres. Mutated genesthat encode altered proteins or that cannot be controlled properly ."u.. .rurn..ous inherited diseases.For example, sickle-celldiseaseis atributable to a single nucleotide substitution in the hemoglobin gene, which encodesthe protein that carries oxygen in red blood cells. The single amino acid changecausedby the sickle cell muta-

Sequencingof the human genome has shown that a

transcription factors typically are only 70-12 nucleotides long, a few single-nucleotidemutations might convert a nonfunctional bit of DNA into a functional protein-binding regulatory site. Much of the nonessentialDNA in both eukaryotesand prokaryotes consistsof highly repeatedsequencesthat can move from one place in the genome to another. These mobile DNA elementscan jump (transpose)inro genes,most commonly damaging but sometimes activating them. Jumping generallyoccursrarely enoughto avoid endangering the host organism. Mobile elements,which *... di.coveredfirst in plants, are responsiblefor leaf color variegation and the diverse beautiful color patterns of Indian corn kernels.By jumping in and out of ge.resthat control prgmentationas plant developmentprogresses,the mobile elemenrsgive rise to elaboratecolorid putt...rr. Mobile elements were later found in bacteria, in which they often carry and, unfortunatelS disseminategenesfor antibiotic resrstance. Now we understand that mobile elements have multi_ plied and slowly accumulatedin genomesover evolutionary time, becoming a universal property of genomesin preseni_ day_organisms. They account for an astounding45 percent of the human genome. Some of our own mobile DNA ele_ ments are copies-often highly mutated and damaged_of genomesfrom viruses that spend part of their life iycle as DNA segmentsinserted into host-cell DNA. Thus we carry in our chromosomes the genetic residues of infections ac_ quired by our ancestors.Once viewedonly as molecularpar_ asites,mobile DNA elementsare now thoueht to have ion_ tnbuted significantly to the evolution of higher organisms (Chapter6). '14

.

cHAprE1 R | L | F EB E G t Nwsl r H c E L L s

IE

TheWorkof Cetts

In essence,any cell is simply a compartment with a watery interior that is separatedfrom the external environment by a surfacemembrane (the plasma membrane) that preventsthe free flow of moleculesin and out. In addition, as we've noted, eukaryotic cells have extensive internal membranes that further subdivide the cell into various comparrmenrs, the organelles.Each compartment has contents and properties, such as specializedproteins or a certain pH, suited to its job. The plasma membrane and other cellular membranes are composedprimarily of two layers of phospholipid molecules. These bipartite moleculeshave a "water-loving" (hydrophilic) end and a "water-hating" (hydrophobic) end. The two phospholipid layersof a membrane are orienred with all the hydrophilic ends directed toward the inner and outer surfacesand the hydrophobic ends buried within the interior (Figure1-13). Smalleramounts of other lipids, such as cholesterol, and many kinds of proteins are inserted into the phospholipid framework. The lipid moleculesand someproteins can float sidewisein the plane of the membrane,giving membranes a fluid character. This fluidity allows cells to change shape and even move. However, the attachment of some membrane proteins to other molecules inside or outside the cell restricts their lateral movement. We will learn more about membranes and how molecules cross them in Chapters10 and 11. The cytosol and the internal spacesof organellesdiffer from each other and from the cell exterior in terms of aciditg ionic composition, and protein contents. For example, the composition of saltsinside the cell is often drastically different from what is outside. Becauseof these different ,,microclimates," each cell compartment has its own assigned

Cholesterol

Water-seeking h e a dg r o u p

Water

FIGURE 1-13Thewatery interiorof cellsis surroundedby the plasmamembrane,a two-layeredshell of phospholipids. Thephospholipid molecules areoriented with theirfattyacylchains (blacksquiggly lines) facinginwardandtheirwater-seeking head groups(whitespheres) facingoutward.Thusbothsidesof the membrane arelinedby headgroups, mainlycharged phosphates, adjacent to thewateryspaces tnside andoutside thecell.All biological membranes havethesamebasicphospholipid bilayer structureCholesterol (red)andvarious (notshown)are proteins embedded in the bilayer. Theinteriorspaceisactually muchlarger relative to thevolumeof the plasma membrane depicted here.

tasks in the overall work of the cell (Chapters 10, L2, and 13). The unique functions and microclimates of the various cell compartm€nts are due largely to the proteins that reside in their membranesor interior. 'We can think of the entire cell compartment as a factory dedicatedto sustainingthe well-being of the cell. Much cellular work is performed by molecular machines, some housed in the cytosol, some attached to the cytoskeleton' and some in various organelles.Here we quickly review the major tasks that cells carry out in their pursuit of the good life.

Cells needto break down worn-out or obsoleteparts into small molecules that can be discarded or recycled' This housekeepingtask is assignedlargely to lysosomes,organelles crammedwith degradativeenzymes.The interior of a lysosome has a pH of about 5.0' roughly 100 times more acidic than that of the surrounding cytosol. This aids in the breakdown of materials by lysosomal enzymes' which are specially

CellsBuild and DegradeNumerous Moleculesand Structures As chemical factories,cells produce an enormous number of complex molecules from simple chemical building blocks. All of this syntheticwork is powered by chemical energy extracted primarily from sugarsand fats or sunlight, in the case of plant cells, and stored primarily in ATP, the universal "currency" of chemical energy (Figure 1-14). In animal and plant cells, most ATP is produced by large molecular machines located in two organelles,mitochondria and chloroplasts. Similar machines for generating ATP are located in the plasma membrane of bacterial cells. Both mitochondria and chloroplasts are thought to have originated as bacteria that took up residenceinside eukaryotic cells and then became welcome collaborators (Chapter 12). Directly or indirectlS all of our food is created by plant cells using sunlight to build complex macromolecules during photosynthesis. Even underground oil suppliesare derived from the decay of plant material.

llll+ overviewAnimation:BiologicalE

Most of the structural and functional properties of cells de-

cell and most membrane proteins, however' are made on ribosomes associatedwith the endoplasmic reticulum (ER)' This organelleproduces,processes'and ships out both proteins and lipids. Prolein chains produced on the ER move to the Golgi comple*, where they are further modified before being for*ari.d to their final destinations. Proteins that travel in this way contain short sequencesof amino acids or attached sugar ch"itts (oligosaccharides)that serve as addressesfor directing them to their correct destinations. These addresseswork becausethey arerecognizedand bound by other proteins that do the sorting and shipping in various cell compartments'

lnterconversions or Light (photosynthesis) compoundswith high potentialenergY(resPiration )

Synthesisof cellularmacromolecules(DNA, RNA, proteins, polysaccharides)

Synthesisof other cellularconstituents (suchas membrane phospholipidsand certainrequired metabolites)

Cellularmovements, includingmusclecontraction,crawling movements of entire cells, and movement of chromosomesduring mitosis

A FIGURE1-14 ATP is the most common molecule used by cells to capture and transfer energy. ATPis formed from ADP and in plantsand by the inorganicphosphate(Pi)by photosynthesis

Transportof moleculesagainst a concentration

gradient

Generationof an electricpotential acrossa memDrane

Heat

for nerve 1fi..:.,;t;,",

by andfatsin mostcells.Theenergyreleased of sugars breakdown processes (hydrolysis) of PifromATPdrivesmanycellular thesplitting

THEWORK OF CELLS

15

lntermediate f ilaments

Microtubules

FIGURE 1-15 The three types of cytoskeletalfilamentshave characteristic distributionswithin cells.Threeviewsof the same cell.A cultured fibroblast wastreatedwith threedifferent antibodv preparations. Eachantibody bindsspecifically to the protein monomers formingonetypeof filamentandischemically linkedto a differently colored fluorescent dye(green, blue,or red).Visualization

A n i m a l C e l l sP r o d u c eT h e i r O w n E x t e r n a l E n v i r o n m e nat n d G l u e s The simplest multicellular animals are sinsle cells embedded in a jelly of proteins and polysaccharides."ll.d th. extracellular matrix. Cells themselvesproduce and secretethesematerials, thus creating their own immediate environment (Chapter 19). Collagen, the single most abundant protein in the animal kingdom, is a major component of the extracellular matrix in most tissues.In animals, the extracellularmatrix cushions and lubricates cells. A specialized,especially tough matrix, the basal lamina, forms a supporting layer un_ derlying sheetlikecell layers and helps pr.u"rrt the cells from rlppmg apart. The cells in animal tissuesare ..glued" togerher by cell_ adhesionmolecules(CAMs) embeddedin their surfacemem_

Microfilaments

of the stainedcellin a fluorescence microscope reveals the location of filaments boundto a particular preparation dye-antibody In this case,intermediate filaments green;microtubules, arestained blue; and microfilaments, red.All threefibersystems contributeto the shapeandmovements of cells.lcourtesy of V Small ]

animal cells (Figure 1-15). The cytoskeleton prevents the plasma membrane of animal cells from relaxing into a sphere(Chapter 10); it also functions in cell locomotion and the intracellular transport of vesicles,chromosomes, and macromolecules(Chapters 17 and 18). The cytoskeletoncan be linked through the cell surfaceto the extracellular matrix or to the cytoskeletonof other cells,thus helping to form tissues(Chapter 19). All cytoskeletal filaments are long polymers of protein subunits. Elaborate systemsregulatethe assembly dirursembly of the cytoskeleton,thereby controlling cell".rd shape.In some cells the cytoskeletonis relatively stable, but in others it changesshapecontinuously. Shrinkageof the cytoskeleton in some parts of the cell and its growrh in other parts can produce coordinated changesin shape that result in cell locomotion. For instance,a cell can send out an extensionthat attachesto a surfaceor to other cellsand then retract the cell body from the other end. As this processcontinues due to coordinated changesin the cytoskeleton,the cell moves forward. Cells can move at rates on the order of 20 pm./second.

C e l l sC h a n g eS h a p ea n d M o v e

largement but not movement of cells from one position to another.

Cells changeshapeand move becausetheir internal skeleton. the cytoskeleton,exerts forces on the rest of the cell and its

CellsSenseand SendInformation

attachments- Three types of protein filaments, organized into networks and bundles, form the cytoskeleton within

A living cell continuously monitors its surroundings and adjusts its own activities and composition accordingly. Cells also communicate by deliberatelysendingsignalsthat can be receivedand interpreted by other cells. Suchsignalsare com_ mon not only within an individual organism but also be_ tween organisms.For instance,the odor of a pear signals a food source to us and other animals; consumption of the

16

.

c H A p r E1R | L I F E B E G t Nwst r H c E L L s

pear by an animal aids in distributing the pear'sseeds.Everyone benefits! The signals employed by cells include simple small chemicals, gases, proteins, light, and mechanical movements. Cells possessnumerous receptor proteins for detecting signals and elaborate pathways for transmitting them within the cell to evoke a response.At any time, a cell may be able to senseonly some of the signalsaround it, and how a cell responds to a signal may change with time. In some cases,receivingone signal primes a cell to respond to a subsequentdifferent signal in a particular way. Both changesin the environment (e.g.,an increaseor decreasein a particular nutrient or the light level) and signals receivedfrom other cells representexternal information that cells must process.The most rapid responsesto such signals generally involve changesin the location or activity of preexisting proteins. For instance, soon after you eat a carbohydrate-rich meal, glucosepours into your bloodstream.The rise in blood glucose is sensedby B cells in the pancreas, which respond by releasingtheir stored supply of the protein hormone insulin. The circulating insulin signal causesglucose transporters in the cytoplasm of fat and muscle cells to move to the cell surface,where they begin importing glucose. Meanwhile, liver cellsalso are furiously taking in glucosevia a different glucosetransporter.In both liver and musclecells, an intracellular signalingpathway triggeredby binding of insulin to cell-surfacereceptorsactivatesa key enzymeneeded to make glycogen,a large glucosepolymer (Figure 1-16a). The net result of thesecell responsesis that your blood glucoselevel falls and extra glucoseis stored as glycogen,which your cells can use as a glucosesource when you skip a meal to cram for a test. The ability of cells to send and respond to signalsis crucial to development. Many developmentallyimportant signals are secretedproteins produced by specific cells at specific times and places in a developing organism. Often a receiving cell integratesmultiple signals in deciding how to behave,for example, to differentiate into a particular tissue type, to extend a process,to die, to send back a confirming s i g n a l( y e s ,I ' m h e r e ! ) ,o r t o m i g r a t e . The functions of about half the proteins in humans, roundworms, yeast, and severalother eukaryotic organisms have beenpredicted basedon analysesof genomic sequences (Chapter6). Suchanalyseshave revealedthat at least10-15 percent of the proteins in eukaryotesfunction as secretedextracellular signals, signal receptors, or intracellular signaltransduction proteins, which pass along a signal through a seriesof steps culminating in a particular cellular response (e.g., increased glycogen synthesis).Clearly, signaling and signal transduction are maior activities of cells.

CellsRegulateTheir Gene Expressionto Meet C h a n g i n gN e e d s In addition to modulating the activities of existing proteins, cells often respond to changing circumstancesand to signals from other cells by altering the amount or types of proteins they contain. Gene expression,the overall processof selectively reading and using genetic information, is commonly

(a)

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lncreasedtranscription of specificgenes

1-16 Externalsignalscommonlycausea changein FIGURE the activity of preexistingproteinsor in the amountsand or of a hormone typesof proteinsthat cellsproduce.(a)Binding cantriggeran receptors to itsspecific molecule othersignaling of a the activity or decreases pathway that increases intracellular in to receptors bindingof insulin protein. Forexample, preexisting of cellsleadsto activation of liverandmuscle membrane the plasma from of glycogen in thesynthesis a keyenzyme glycogen synthase, within arelocated for steroidhormones glucose(b)Thereceptors complexes hormone-receptor The surface the cell cells,noton to increased tarqetgenes,leading of specific transcription activate that bindto proteins. Manysignals production of the encoded pathways, to alsoact,by morecomplex on the cellsurface receptors geneexPression modulate

controlled at the level of transcription, the first step in the production of proteins. In this way cells can produce a pariicular mRNA only when the encoded protein is needed' thus minimizing wasted energy. Producing an mRNA is, however,only the first in a chain of regulatedeventsthat together determine whether an active protein product is produced from a particular gene. Transcriptional control of geneexpressionwas first decisively demonstratedin the responseof the gut bacterium E. coli to different sugar sources.E. coli cells prefer glucose as a sugar source,but they can survive on lactosein a pinch' These bacteria use both a DNA-binding repressor protein and a DNA-binding actiuator protein to change the rate of transcription of three genesneededto metabolize lactosedepending on the relative amounts of glucoseand lactosepresint (Chapter 4). Such dual positiveinegativecontrol of gene exoression fine-tunes the bacterial cell's enzymatic equip-..rt fo. the job at hand. Like bacterial cells, unicellular eukaryotes may be subjected to widely varying environmental conditions that require extensivechangesin cellular structuresand function' T H EW O R K O F C E L L S

17

For instance,in starvation conditions yeastcellsstop growing and form dormant spores (seeFigure 1-6). In multicellular organisms, however, the environment around most cells is relatively constant. The major purpose of gene control in us and in other complex organismsis to tailor the properties of various cell types to the benefit of the entire animal or plant. Control of gene activity in eukaryotic cells usually involves a balance between the actions of transcriptional activators and repressors.Binding of activators to specificDNA regulatory sequencescalled enhancers turns on transcription, and binding of repressorsto other regulatory sequences called silencersturns off transcription. In Chapters 7 and 8, we take a closelook at transcriptional activators and repressors and how they operate, as well as other mechanismsfor controlling gene expression.In an extreme case,expression of a particular gene could occur only in part of the brain, only during eveninghours, only during a certain stageof development, only after a large meal, and so forth. Many external signals modify the activity of transcriptional activators and repressorsthat control specific genes. For example, lipid-soluble steroid hormones, such as estrogen and testosterone,can diffuse across the plasma membrane and bind to their specific receptors located in the cytoplasm or nucleus(Figure 1-16b).Hormone binding changes

Overview Animation: Life Cycle of a Cell {lltt Nondividing cells

o

Resting c el l s

R N Aa n d protein synthesis G2

A FIGURE 1-17 Duringgrowth, eukaryoticcellscontinually progressthrough the four stagesof the cell cycle, generatingnew daughtercells.In mostproliferating cells,the four phases of the cellcycleproceedsuccessively, takingfrom 10-20 hoursdepending on celltypeanddevelopmental state Duringinterphase, whichconsists of the G,,,S,andG2phases, the cellroughly doubles itsmassReplication of DNAdurinqthe S phaseleaves the cellwith four copiesof eachtypeof chromosome Inthe mitotic(M)phase, thechromosomes are evenlypartitioned to two daughter cells,andthecytoplasm divides roughlyin halfin mostcases. Undercertainconditions suchasstarvation or whena tissuehasreached itsfinalsize,cells willstopcycling andremainin a waitingstatecalledGs Mostcells in Gscanreenter thecycleif conditions chanqe.

18

C H A P T E R1

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L t F EB E G T N W S |TH CELLS

the shape of the receptor so that it can bind to specific enhancer sequencesin the DNA, thus turning the receptor into a transcriptional activator. By this rather simple signaltransduction pathway, steroid hormones cause cells to change which genes they transcribe (Chapter 7). Since steroid hormones can circulate in the bloodstream, they can affect the properties of many or all cells in a temporally coordinated manner. Binding of many other hormones and of growth factors to receptors on the cell surface triggers different signal-transduction pathways that also lead to changesin the transcription of specific genes (Chapters 15 and 16). Although thesepathways involve multiple components and are more complicated than those transducing steroid hormone signals,the generalidea is the same.

CellsGrow and Divide As we have discussed,reproduction is at the heart of biology; rocks don't do it. The reproduction of organisms depends on the reproduction of cells. The simplest type of reproduction entails the division of a "parent" cell into two "daughter" cells.This occurs as part of the cell cycle,a series of eventsthat preparesa cell to divide followed by the actual division process, called mitosis. The eukaryotic cell cycle commonly is representedas four stages(Figure 1-17). The chromosomes and the DNA they carry are copied during the S (synthesis)phase.The replicatedchromosomesseparare during the M (mitotic) phase,with each daughter cell getting a copy of each chromosome during cell division. The M and S phasesare separatedby two gap stages,the G1 phase and G2 phase, during which mRNAs and proteins are made. In single-celledorganisms,both daughtercellsoften (though not always) resemblethe parent cell. In multicellular organisms, stem cells can give rise to two different cells, one that resembles the parent cell and one that does not. Such asymmetric cell division is critical to the generationof different cell types in the body (Chapter21). During growth the cell cycle operatescontinuously with newly formed daughter cells immediately embarking on their own path ro mitosis. Under optimal conditions bacteria can divide to form two daughter cells once every 30 minutes. At this rate, in an hour one cell becomesfour: in a day one cell becomesmore than 101a, which if dried would weigh about 25 grams. Under normal circumstances, however, growth cannot continue at this rate becausethe food supply becomeslimiting. Most eukaryotic cells take considerably longer than bacterial cells to grow and divide. Moreover, the cell cycle in adult plants and animalsnormally is highly regulated(Chapter20). This tight control prevents imbalanced, excessivegrowth of tissueswhile ensuring that worn-out or damaged cells are replaced and that additional cells are formed in responseto new circumstancesor developmental needs.For instance, the proliferation of red blood cellsincreasessubstantiallywhen a person ascendsto a higher altitude and needs more capacity to capture oxygen. Somehighly specializedcells in adult animals, such as nervecellsand striatedmusclecells,rarely divide, if at all. The fundamental defect in cancer is loss of the ability to

control the growth and division of cells.In Chapter 25, we examine the molecular and cellular eventsthat lead to inappropriate, uncontrolled proliferation of cells. Mitosis is an asexual process since the daughter cells carry the exact samegeneticinformation as the parental cell. ln sexwalreproduction, fusion of two cells produces a third cell that contains genetic information from each parental cell. Sincesuch fusions would causean ever-increasingnumber of chromosomes, sexual reproductive cycles employ a special type of cell division, called meiosis,that reducesthe number of chromosomesin preparation for fusion (seeFigure 5-3). Cells with a full set of chromosomes are called diploid cells. During meiosis, a diploid cell replicates its chromosomes as usual for mitosis but then divides twice without copying the chromosomesin between. Each of the resulting four daughter cells, which have only half the full number of chromosomes,is said to be haploid. Sexual reproduction occurs in animals and plants and even in unicellular organismssuch as yeasts(seeFigure 1-5). Animals spendconsiderabletime and energygeneratingeggs and sperm, the haploid cells, called gametes,which are used for sexualreproduction. A human femalewill produce about half a million eggsin a lifetime, all of thesecells forming before she is born; a young human male produces about 100 million sperm each day. Gametes are formed from diploid precursor germ-line cells,which in humans contain 46 chromosomes.In humans the X and Y chromosomesare called sex chromosomes becausethey determine whether an individual is male or female. In human diploid cells, the 44 remaining chromosomes,called autosomes,occur as pairs of 22 different kinds. Through meiosis,a man produces sperm that have 22 chromosomes plus either an X or a Y, and a woman produces ova (unfertilized eggs) with 22 chromosomesplus an X. Fusion of an egg and sperm (fertilization) yields a fertilized egg,the zygote,with46 chromosomes,one pair of each of the 22 kinds and a pair of Xs in femalesor an X and a Y in males(Figure1-18). Errors during meiosiscan lead to disorders resulting from an abnormal number of chromosomes.These include Down's syndrome, caused by an extra chromosome 21, and Klinefelter's syndrome, causedby an extra X chromosome.

Diploid (2n)

Haploid(1nl

(",,o )

One type of female gamete

Two types of male gamete

Diploid (2n) Female zygote

Male zygote

meiosis 1-18 Dad madeyou a boy or girl. In animals, FIGURE Themale cellsformseggsandsperm(gametes). of diploidprecursor the sexof the parentproduces two typesof spermanddetermines sex chromosomes; Y are the here, X and asshown zygoteIn humans, fromthe maleparentto a Y chromosome the zygotemustreceive (non-sex chromosomes). developintoa male A : autosomes bing" between the fingers; the cells in the webbing subsequently die in an orderly and precisepattern that leavesthe fingers and thumb free to play the piano. Nerve cells in the brain soon die if they do not make proper or useful electrical

CellsDie from AggravatedAssaultor an lnternal Program 'lfhen

cells in multicellular organismsare badly damagedor infectedwith a virus, they die. Cell death resulting from such a traumatic event is messy and often releasespotentially toxic cell constituents that can damage surrounding cells. Cells also may die when they fail to receivea life-maintaining signal or when they receive a death signal. In this type of programmed cell death, called apoptosis, a dying cell actually produces proteins necessaryfor self-destruction.Death by apoptosis avoids the releaseof potentially toxic cell constituents(Figure1-19). Programmed cell death is critical to the proper development and functioning of our bodies (Chapter 21). During fetal life, for instance,our hands initially developwith "web-

1-19ApoPtoticcellsbreakapartwithout spewing FIGURE forth cell constituentsthat might harm neighboringcells. looklikethe cellon the left Cells Whitebloodcellsnormally likethe cellon the programmed celldeath(apoptosis), undergoing The arereleased blebsthateventually surface right,formnumerous is growth Apoptosis signals. it lackscertain cellisdyingbecause cellswherethey cells,to remove virus-infected to eliminate important (likethewebbingthatdisappears asfingersdevelop), arenot needed cellsthatwouldreactwith ourown immunesystem andto destroy Inc] Unlimited, Murtil/isuals bodies[Gopal THE WORK OF CELLS

19

connectionswith other cells. Some developinglymphocytes, the immune-systemcells intended to recognizeforeign proteins and polysaccharides,have the ability to reac against our own tissues.Such self-reactivelymphocytes becomeprogrammed to die before they fully marure. If these cells are not weededout before reaching maturity, they can causeautoimmune diseases,in which our immune system destroys the very tissuesit is meant to protect.

IE

InvestigatingCellsand TheirParts

To build an integrared understanding of how the various molecular components that underlie cellular functions work together in a living cell, we must draw on various perspectives. Here, we look at how five disciplines-cell biology, biochemistry and biophysics,genetics,genomics,and developmental biology-can conrribure to our knowledge of cell structure and function. The experimental approachesof each field probe the cell's inner workings in different ways, allowing us to ask different types of questions about cells and what they do. Cell division provides a good example to illustrate the role of different perspectivesin analyzing a complex cellular process.Although we discussthe different disciplinesseparatelyfor clarity, in practice most biologists use multiple approachesin concert. This is part of the fun of

Nanometers

The goal of cell biologists is to understandhow a cell is able to control its own shape and surface properties, transport materials to the right locations, copy itself, and receiveand send signals.Cell biologists use severaltypes of microscopy to observecells, while at the same time labeling specificcell components and altering them to seewhat happens.Analysesare generally done at the micrometer scale. Actual observation of cells awaited development of the first, crude microscopes in the early 1600s. A compound

Meters

Assemblies Macromolecules

Glucose

C-C bond

Cell Biology Revealsthe Size,Shape,Location, and Movementsof Cell Components

Millimeters

Micrometers

Small molecules Atoms

cell biologg putting together genetics with microscopy or enzymology with development. The realm of biology rangesin scalemore than a billionfold (Figure 1-20). Beyond that, it's ecology and earth science at the "macro" end, chemistry and physics at the "micro" end. The visible plants and animals that surround us are measuredin meters (100-102 m). By looking closely,we can seea biologicalworld of millimeters(1 mm : 10-3 m) and even tenths of millimeters (10-a m). Setting aside oddities like chicken eggs, most cells are 1-100 micrometers (1 pm : 10-5 m) long and thus clearlyvisibleonly when magnified. To seethe structureswithin cells, we must go farther down the sizescaleto 10-100 nanometers(1 nm : 10 v m).

Hemoglobin

M u l t i c e l l u l aor r g a n i s m s

Cells

Ribosome

C. elegans

Bacterium

Mitochondrion

Newborn human

Bumblebee

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10sm

1 0 ' 8m

'1 0-7m

0.1nm

1 nm

10nm

1 0 0n m

10'6m 1 pm

1 0 - 5m 10pm

FIGURE 1-20 Biologistsare interestedin objectsrangingin sizefrom smallmolecules to the tallesttrees.A sampling of biological objects aligned on a logarithmic scale(a)TheDNAdouble helixhasa diameter of about2 nm (b)Eight-cell-stage numan embryothreedaysafterfertilization, about200 rr"macross(c)A wolf

20

CHAPTER 1

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L I F EB E G I N S WITHCELLS

1 0 - am 1 0 0u m

1 0 - 3m

1 0 2m

1 0 ' 1m

1 mm

1 0m m

100mm

1 0 0m 1m

spider, about15 mm across(d)Emperor penguins areabout1 m tall. [Part(a)Will andDeniMclntyrePart(b)YorgasNikas/photo Researchers, Inc Part(c)GaryGauglerl/isuals Unlimited, Inc Part(d)HughS Rosefuisuals Unlimited. lncl

microscope,the most useful type of light microscope,has two lenses.The total magnifying power is the product of the magnificationby each lens. As better lenseswere invented, the magnifying power and the ability to distinguish closely spacedobjects, the resolution, increasedgreatly. Modern cclmpoundmicroscopesmagnify the view about a thousandfold, so that a bacterium 1 micrometer (1 p-) long looks like it's a millimeterlong. Objectsabout 0.2 pm apart can be discernedin theseinstruments. Microscopy is most powerful when particular components of the cell are stained or labeled specifically,enabling them to be easilyseenand locatedwithin the cell. A simple exampleis stainingwith dyesthat bind specificallyto DNA to visualize the chromosomes. Specific proteins can be detected by harnessingthe binding specificityof antibodies,the proteins whose normal task is to help defend animals against infection and foreign substances.In general,eachtype of antibody binds to one protein or large polysaccharideand no other (Chapter 3). Purified antibodies can be chemically linked to a fluorescent molecule, which permits their detection in a specialfluorescencemicroscope(Chapter 3). lf a cell or tissueis treated with a detergentthat partially dissolvescell membranes,fluorescentantibodiescan drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope,the bound fluorescent antibodies identify the location of the target protein (see F i g u r e1 - 1 5 ) . Betrer still is pinpointing proteins in living cells with intact membranes. One way of doing this is to introduce an engineeredgenethat codesfor a hybrid protein: part of the hybrid protein is the cellular protein of interest;the other part is a protein that fluoresceswhen struck by ultraviolet light. A common fluorescentprotein used for this purpose is green fluorescent protein (GFP), a natural protein that makes some iellyfish colorful and fluorescent. GFP "tagging" could reveal,for instance,that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes.In this case,first the endoplasmicreticulum and later the lysosomeswould glow in the dark. Chromosomesare visible in the light microscopeonly during mitosis,when they becomehighly condensed.The extraordinary behavior of chromosomesduring mitosis first was discoveredusing the improved compound microscopes of the late 1800s.About halfway through mitosis,the replicatedchromosomesbeginto move apart. Microtubules,one of the three types of cytoskeletalfilaments, participate in this movementof chromosomesduring mitosis.Fluorescenttagging of tubulin, the protein subunitthat polymerizesto form microtubules, reveals structural details of cell division that otherwisecould not be seenand allows observationof chromosomemovement(Figure1-21). Electron miqroscopesuse a focusedbeam of electronsinstead of a beam of light. In transmission electron microscopy, specimensare cut into very thin sections and placedunder a high vacuum,precludingexaminationof living cells. The resolution of transmission electron micro-

llll|

5e6nsAnimation:Mitosis

A FIGURE1-21 During the later stages of mitosis, microtubules (red) pull the replicated chromosomes(black) toward the ends of a dividing cell. Thisplantcell is stained and with a DNA-bindingdye (ethidium)to revealchromosomes antibodiesspecificfor tubulinto reveal with f luorescent-tagged microtubulesAt this stagein mitosis,the two copiesof each replicatedchromosome(calledchromatids)haveseparatedand are of AndrewBajer ] movinqawavf rom eachother [Courtesy

scopes,about 0.1 nm, permits fine structural details to be distinguished,and their powerful magnification would make a 1-pm-long bacterialcell look like a soccerball. Most of the organelles in eukaryotic cells and the double-layered structure of the plasma membrane were first observedwith electron microscopes(Chapter 9). \fith new specializedelectron microscopy techniques,three-dimensionalmodels of organellesand large protein complexes can be constructed from multiple images.But to obtain a more detailed look at the individual macromoleculeswithin cells, we must turn to techniqueswithin the purview of biochemistry and biophysics.

s e v e a tl h e B i o c h e m i s t r ay n d B i o p h y s i c R M o l e c u l a rS t r u c t u r ea n d C h e m i s t r yo f P u r i f i e d Cell Constituents Biochemistsextract the contentsof cellsand separatethe constituents based on differencesin their chemical or physical properties,a processcalledfractionation. The attention to individual moleculesmeans operating at the nanometer scale. Of particular interest are proteins, the workhorses of many A typical fractionation schemeinvolvesuse cellular processes. of various separationtechniquesin a sequentialfashion.These separationtechniquescommonly are basedon differencesin the size of moleculesor the electric charge on their surface (Chapter 3). To purify a particular protein of interest, a purification scheme is designed so that each step yields a

I N V E S T I G A T I NC GE L L SA N D T H E I RP A R T S

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FIGURE 1-22 Biochemicalpurificationof a protein from a cell extract often requiresseveralseparationtechniques.The purificationcanbe followedby gelelectrophoresis of thestarting proteinmixture andthefractions obtained fromeachpurification step Inthisprocedure, a sample isapplied to wellsin thetop of a gelatin-like slabandan electric fieldisappliedInthe presence of appropriate saltanddetergent concentrations, the proteins move throughthefibersof the geltowardtheanode,with largerproteins movingmoreslowlythroughthe gelthansmaller ones(seeFigure 3-35)Whenthegelisstained, proteins separated arevisible as distinctbandswhoseintensities areroughlyproportional to the proteinconcentration Shownhereareschematic depictions of oels for thestarting (lane1)andsamples mixture of proteins takenaiter eachof several purification stepsIn the firststep,saltf ractionation, proteinsthat precipitated with a certainamountof saltwereredissolved; electrophoresis (lane2) showsthatit of thissample contains fewerproteins thantheoriginal mixtureThesample then wassubjected in succession to threetypesof column chromatography proteins thatseparate by electrical charge, size,or bindingaffinityfor a particular (seeFigure smallmolecule 3-37).The finalpreparation isquitepure,ascanbe seenfromtheappearance of justoneproteinbandin lane5 [AfterJBergetal,2o02,Biochemistry W H Freeman andCompany, p 87l preparation with fewer and fewer contaminating proteins, until finally only the protein of interestremains (Figure 1-22). The initial purification of a protein of interest from a cell extract often is a tedious, time-consumingtask. Once a small amount of purified protein is obtained, antibodies to it can be produced by methods discussedin Chapter 19. For a biochemist, antibodies are near-perfecttools for isolating larger amounts of a protein of interest for further analysis.In effect, antibodies can "pluck out" the protein they specifically recognizeand bind from a semipure sample containing numerous different proteins. An increasinglycommon alterna-

22

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L I F EB E G I N SW I T H C E L L S

tive is to engineera gene that encodesa protein of interest with a small attached protein "tag," which can be used to pull out the protein from whole cell extracts. Purification of a protein is a necessaryprelude to studies on how it catalyzesa chemical reaction or carries out other functions and how its activiry is regulated. Some enzymesare made of multiple protein chains (subunits) with one chain catalyzing a chemical reaction and other chains regulating when and where that reaction occurs. The molecular machines that perform many critical cell processesconstitute even larger assembliesof proteins. By separating the individual proteins composing such assemblies,their individual catalytic or other activitiescan be assessed. For example,purification and study of the activity of the individual proteins composing the DNA replication machine provided clues about how they work together to replicate DNA during cell division (Chapter 4). The folded, three-dimensionalstructure, or conformation, of a protein is vital to its function. To understand the relation between the function of a protein and its form, we need to know both what it does and its detailed structure. The most widely used method for determining the complex structures of proteins, DNA, and RNA is x-ray crystallography, one of the powerful tools of biophysics.Computer-assistedanalysisof the data often permits the location of every atom in a large, complex molecule to be determined. The double-helix structure of DNA, which is key to its role in herediry was first proposed basedon x-ray crystallographic studies.Throughout this book you will encounter numerous examplesof protein structuresas we zero in on how proteins work.

GeneticsRevealsthe Consequences of DamagedGenes Biochemical and crystallographic studies can tell us much about an individual protein, but they cannot prove that it is required for cell division or any other cell process.The importance of a protein is demonstratedmost firmly if a mutation that prevents its synthesis or makes it nonfunctional adverselyaffects the processunder study. !(/e define the genotype of an organism as its composition of genes;the term also is commonly used in referenceto different versionsof a singlegeneor a small number of genes of interest in an individual organism. A diploid organism generally carries two versions (alleles)of each gene, one derived from each parent. There are important exceptions, such as the geneson the X and Y chromosomesin males of some species,including our own. The phenotype is the visible outcome of a gene's action, such as blue eyes versus brown eyesor the shapesof peas.In the early days of genetics, the location and chemical identity of genes were unknown; only the observablecharacteristics,the phenotypes, could be followed. The concept that genesare like "beads" on a long "string," the chromosome,was p,roposedearly in the 1900s basedon geneticwork with the fruit fly Drosophila. In the classicalgeneticsapproach, mutants are isolated that lack the ability to do something a normal organism can do. Often large genetic "screens" are done to look for many different mutant individuals (e.g., fruit flies, yeast cells) that are

unable to complete a certain process,such as cell division or muscle formation. In experimental organismsor cultured cells, mutations usually are produced by treatment with a mutagen, a chemical or physical agent that promotes mutations in a largely random fashion.But how can we isolateand maintain mutant organismsor cellsthat are defectivein some process, such as cell division, that is necessaryfor survival?One way is to look for organisms with a temperature-sensitivemutation. Thesemutants are able to grow at one temperature,the permissiue temperature, but not at anothe5 usually higher temperature, the nonpermissiuetemperature.Normal cells can grow at either temperature. In most cases,a temperature-sensitivemutant produces an altered protein that works at the permissive temperaturebut unfolds and is nonfunctional at the nonpermissive temperature. Temperature-sensitive screensare readily done with viruses,bacteria, yeast,roundworms, and fruit flies. By analyzing the effectsof numerous different temperaturesensitivemutations that altered cell division, geneticistsdiscoveredall the genesnecessaryfor cell division without knowing anything, initially, about which proteins they encode or how theseproteinsparticipatein the process.The greatpower of geneticsis to reveal the existenceand relevanceof proteins without pr