MOLECULAR

BIOLOGY

°FTHE GENE SIXTH EDITIO N

James D . Watson

Tania A. Baker

Stephen P. Bell

Alexander Gan n

Michael Levine

Richard Losick

PART 1

CHEMISTRY AND GENETICS, 1

1 The Mendelian View of the World, 5

2 Nucleic Acids Convey Genetic Information, 1 93 The Importance of Weak Chemical Interactions, 4 34 The Importance of High-Energy Bonds, 5 7

5 Weak and Strong Bonds Determine Macromolecular Structure, 7 1

PART 2

MAINTENANCE OF THE GENOME, 9 5

6 The Structures of DNA and RNA, 10 1

7 Genome Structure, Chromatin, and the Nucleosome, 13 5

8 The Replication of DNA, 19 59 The Mutability and Repair of DNA, 25 7

10 Homologous Recombination at the Molecular Level, 28 311 Site-Specific Recombination and Transposition of DNA, 31 9

PART 3

EXPRESSION OF THE GENOME, 37 1

12 Mechanisms of Transcription, 37 713 RNA Splicing, 41 514 Translation, 45 7

15 The Genetic Code, 52 1

PART 4

REGULATION, 54 1

16 Transcriptional Regulation in Prokaryotes, 54 717 Transcriptional Regulation in Eukaryotes, 58 9

18 Regulatory RNAs, 63 319 Gene Regulation in Development and Evolution, 66 120 Genome Analysis and Systems Biology, 70 3

PART 5

METHODS, 73 3

20 Techniques of Molecular Biology, 73 9

21 Model Organisms, 78 3

Index, 819

PART 1

CHEMISTRY AND GENETICS, 1

CHAPTER 1 • The Mendelian View of the World, 5

Mendel's Discoveries, 6

Chromosome Mapping, 1 2The Principle of Independent Segregation, 6

The Origin of Genetic Variability through Mutations, 1 5ADM( 11) Cov(rPr) Box 1-1 Mendelian Laws, 6

Some Alleles Are neither Dominant nor Recessive, 8

Early Speculations about What Genes Are and Ho w

Principle of Independent Assortment, 8

They Act, 1 6

Chromosomal Theory of Heredity, 8

Preliminary Attempts to Find a Gene-Protei nRelationship, 1 6

Gene Linkage and Crossing Over, 9

SUMMARY, 1 7KEY EXPERIMENTS Box 1-2 Genes Are Linked to

BIBLIOGRAPHY, 1 8Chromosomes, 10

CHAPTER 2 • Nucleic Acids Convey Genetic Information, 1 9

Avery's Bombshell : DNA Can Carry Genetic

The Central Dogma, 3 2Specificity, 20

The Adaptor Hypothesis of Crick, 3 2

Viral Genes Are Also Nucleic Acids, 21

Discovery of Transfer RNA, 32

The Double Helix, 21

The Paradox of the Nonspecific-Appearing Ribosomes, 3 3

Finding the Polymerases That Make DNA, 23

Discovery of Messenger RNA (mRNA), 3 4

KEY EXPERIMENTS Box 2 1 Chargaff's Rules, 24

Enzymatic Synthesis of RNA upon DNA Templates, 35

Experimental Evidence Favors Strand Separation during

Establishing the Genetic Code, 3 6

DNA Replication, 25

Establishing the Direction of Protei n

The Genetic Information within DNA Is Conveyed by the

Synthesis, 3 8

Sequence of Its Four Nucleotide Building Blocks, 28

Start and Stop Signals Are Also Encoded within DNA, 39

KEY EXPERIMENTS Box 2-2, Evidence That Genes Control

The Era of Genomics, 3 9Amino Acid Sequences in Proteins, 29

DNA Cannot Be the Template That Directly Orders Amino

SUMMARY, 40

Acids during Protein Synthesis, 30

BIBLIOGRAPHY, 4 1

RNA Is Chemically Very Similar to DNA, 30

CHAPTER 3 • The Importance of Weak Chemical Interactions, 43

Characteristics of Chemical Bonds, 43

Some Ionic Bonds Are Hydrogen Bonds, 50Chemical Bonds Are Explainable in Quantum-Mechanical

Weak Interactions Demand Complementary Molecula rTerms, 44

Surfaces, 5 1

Chemical-Bond Formation Involves a Change in the Form of

Water Molecules Form Hydrogen Bonds, 5 1Energy, 45

Weak Bonds between Molecules in Aqueous Solutions, 5 1Equilibrium between Bond Making and Breaking, 45

Organic Molecules That Tend to Form Hydrogen Bonds Are

The Concept of Free Energy, 46

Water Soluble, 52

Key Is Exponentially Related to AG, 46

ADVANCED CONCEPTS Box 3-1 The Uniqueness of MolecularShapes and the Concept of Selective Stickiness, 5 3

Covalent Bonds Are Very Strong 46

Hydrophobic "Bonds" Stabilize Macromolecules, 5 4Weak Bonds in Biological Systems, 47

The Advantage of AG between 2 and 5 kcal/mole, 55

Weak Bonds Have Energies between 1 and 7 kcallmol, 47

Weak Bonds Attach Enzymes to Substrates, 55

Weak Bonds Are Constantly Made and Broken at

Weak Bonds Mediate Most Protein-DNA an dPhysiological Temperatures, 47

Protein-Protein Interactions, 55The Distinction between Polar and Nonpolar Molecules, 47

SUMMARY, 56van der Waals Forces, 48

BIBLIOGRAPHY, 56Hydrogen Bonds, 49

CHAPTER 4 • The Importance of High-Energy Bonds, 5 7

Molecules That Donate Energy Are Thermodynamically

Activation of Precursors in Group Transfer Reactions, 63Unstable, 57

ATP Versatility in Group Transfer, 64

Enzymes Lower Activation Energies in Biochemical

Activation of Amino Acids by Attachment of AMP, 6 5

Reactions, 59

Nucleic Acid Precursors Are Activated by the Presence o f0-0, 66

Free Energy in Biomolecules, 60 The Value of 0 -- 0 Release in Nucleic Acid Synthesis, 6 6High-Energy Bonds Hydrolyze with Large Negative AG, 60

0 -O Splits Characterize Most Biosynthetic Reactions, 6 7

High-Energy Bonds in Biosynthetic Reactions, 62

SUMMARY, 68Peptide Bonds Hydrolyze Spontaneously, 62

BIBLIOGRAPHY, 69Coupling of Negative with Positive AG, 6 3

CHAPTER 5 • Weak and Strong Bonds Determine Macromolecular Structure, 7 1

Higher-Order Structures Are Determined by Intra- and

Most Proteins Are Modular, Containing Two or Thre eIntermolecular Interactions, 71

Domains, 82DNA Can Form a Regular Helix, 71

Proteins Are Composed of a Surprisingly Small Number of

RNA Forms a Wide Variety of Structures, 73

Structural Motifs, 82

Chemical Features of Protein Building Blocks, 73

AI> L :' \. (

( r, (

I?

>-' Large Proteins Are Often

The Peptide Bond, 75

Constructed of Several Smaller Polypeptid e

There Are Four Levels of Protein Structure, 75

Chains, 83

a Helices and ß Sheets Are the Common Forms of

Different Protein Functions Arise from Various Domai n

Secondary Structure, 76

Combinations, 8 4

TECHNIQUES Box 5-1 Determination of Protein

Weak Bonds Correctly Position Proteins along DNA an d

Structure, 78

RNA Molecules, 85Proteins Scan along DNA to Locate a Specific DNA-Bind ing

The Specific Conformation of a Protein Results from Its

Site, 8 7Pattern of Hydrogen Bonds, 80

a Helices Come Together to Form Coiled-Coils, 80

Diverse Strategies for Protein Recognition of RNA, 88

Allostery : Regulation of a Protein's Function by Changing

Not All Regulation of Proteins Is Mediated byAllosteric

Its Shape, 90

Events, 93

The Structural Basis of Allosteric Regulation Is Known for

SUMMARY, 93Examples Involving Small Ligands, Protein-Protein

BIBLIOGRAPHY, 94Interactions, and Protein Modification, 9 0

PART 2

MAINTENANCE OF THE GENOME, 95

CHAPTER 6 • The Structures of DNA and RNA, 10 1

DNA Structure,1 02

Nucleosomes Introduce Negative Supercoiling i n

DNA Is Composed of Polynucleotide Chains, 102

Eukaryotes, 12 0

Each Base Has Its Preferred Tautomeric Form, 104

Topoisomerases Can Relax Supercoiled DNA, 72 1

The Two Strands of the Double Helix Are Held Together by

Prokaryotes Have a Special Topoisomerase That Introduce s

Base Pairing in an Antiparallel Orientation, 105

Supercoils into DNA, 12 1

The Two Chains of the Double Helix Have Complementary Topoisomerases Also Unknot and Disentangle DNA

Sequences, 106

Molecules, 12 1

Hydrogen Bonding Is Important for the Specificity of Base

Topoisomerases Use a Covalent Protein-DNA Linkage to

Pairing 106

Cleave and Rejoin DNA Strands, 12 3

Bases Can Flip Out from the Double Helix, 107

Topoisomerases Form an Enzyme Bridge and Pass DNASegments through Each Other, 123

DNA Is Usually a Right-Handed Double Helix, 107DNA Topoisomers Can Be Separated by Electrophoresis, 12 5

The Double Helix Has Minor and Major Grooves, 108

Ethidium Ions Cause DNA to Unwind, 126KEY EXPERIMENTS Box 6-1 DNA Has 10.5 Base Pairs per Tur n

of the Helix in Solution : The Mica Experiment, 108

RNA Structure, 12 7The Major Groove Is Rich in Chemical Information, 109

RNA Contains Ribose and Uracil and Is Usually Single-The Double Helix Exists in Multiple Conformations, 110

Stranded, 12 7

KEY EXPERIMENTS Box 6-2 How Spots on an X-ray Film Reveal

RNA Chains Fold Back on Themselves to Form Local Regionsthe Structure of DNA, 112

of Double Helix Similar to A-Form DNA, 12 7

DNA Can Sometimes Form a Left-Handed Helix, 113

KEY EXPERIMENTS Box 6-3 Proving that DNA Has a Helica l

DNA Strands Can Separate (Denature) and Reassociate, 113

Periodicity of about 10.5 Base Pairs per Turn from the

Some DNA Molecules Are Circles, 116

Topological Properties of DNA Rings, 12 8

RNA Can Fold Up into Complex Tertiary Structures, 12 9DNA Topology, 117

Some RNAs Are Enzymes, 13 0Linking Number Is an Invariant Topological Property of

The Hammerhead Ribozyme Cleaves RNA by the Formatio nCovalently Closed, Circular DNA, 117

of a 2', 3' Cyclic Phosphate, 13 1Linking Number Is Composed of Twist and Writhe, 117

Did Life Evolve from an RNA World?, 132Lk° Is the Linking Number of Fully Relaxed cccDNA under

SUMMARY, 132Physiological Conditions, 11 9

DNA in Cells Is Negatively Supercoiled, 120

BIBLIOGRAPHY, 133

CHAPTER 7 • Genome Structure, Chromatin, and the Nucleosome, 13 5

Genome Sequence and Chromosome Diversity, 136

The E . coli Genome Is Composed Almost Entirely o f

Chromosomes Can Be Circular or Linear, 136

Genes, 14 0

Every Cell Maintains a Characteristic Number of

More Complex Organisms Have Decreased Gene Density,

Chromosomes, 137

1 `w

Genome Size Is Related to the Complexity of the

Genes Make Up Only a Small Proportion of the Eukaryoti c

Organism, 139

Chromosomal DNA, 141

The Majority of Human Intergenic Sequences Are Composed

Higher-Order Chromatin Structure, 16 9of Repetitive DNA, 143

Heterochromatin and Euchromatin, 16 9

Chromosome Duplication and Segregation, 144

Histone H1 Binds to the Linker DNA between Nucleosomes,

Eukaryotic Chromosomes Require Centromeres, Telomeres,

169

and Origins of Replication to Be Maintained during Cell

Nucleosome Arrays Can Form More Complex Structures :Division, 144

The 30-nm Fiber, 170

Eukaryotic Chromosome Duplication and Segregation Occur

The Histone Amino-Terminal Tails Are Required for th ein Separate Phases of the Cell Cycle, 147

Formation of the 30-nm Fiber, 172

Chromosome Structure Changes as Eukaryotic Cells

Further Compaction of DNA involves Large Loops ofDivide, 149

Nucleosomal DNA, 172

Sister-Chromatid Cohesion and Chromosome Condensation

Histone Variants Alter Nucleosome Function, 174Are Mediated by SMC Proteins, 150

Regulation of Chromatin Structure, 17 4Mitosis Maintains the Parental Chromosome Number, 152

The Interaction of DNA with the Histone Octamer I sDuring Gap Phases, Cells Prepare for the Next Cell Cycle

Dynamic, 174Stage and Check That the Previous Stage Is CompletedCorrectly, 152

Nucleosome-Remodeling Complexes Facilitate Nucleosom e

Meiosis Reduces the Parental Chromosome Number, 154

Movement, 175Some Nucleosomes Are Found in Specific Positions :

Different Levels of Chromosome Structure Can Be Observed

Nucleosome Positioning, 17 9by Microscopy, 156

KEY EXPERIMENTS Box 7-3 Determining Nucleosome Positio n

The Nucleosome, 157

in the Cell, 180

Nucleosomes Are the Building Blocks of Chromosomes, 157

Modification of the Amino-Terminal Tails of the Histone s

KEY EXPERIMENTS Box 7-1 Micrococcal Nuclease and the

Alters Chromatin Accessibility, 182

DNA Associated with the Nucleosome, 158

Protein Domains in Nucleosome-Remodeling an d

Histones Are Small, Positively Charged Proteins, 159

-Modifying Complexes Recognize Modified Histones, 18 4

The Atomic Structure of the Nucleosome, 160

Specific Enzymes Are Responsible for Histone Modification ,185

Histones Bind Characteristic Regions of DNA within the

Nucleosome Modification and Remodeling Work TogetherNucleosome, 162

Many DNA Sequence-Independent Contacts Mediate

to Increase DNA Accessibility, 18 6

the Interaction between the Core Histones and

Nucleosome Assembly, 187DNA, 162

Nucleosomes Are Assembled Immediately after DNAThe Histone Amino-Terminal Tails Stabilize DNA Wrapping

Replication, 18 7around the Octamer, 165

Assembly of Nucleosomes Requires Histone "Chaperones, "Wrapping of the DNA around the Histone Protein Core

189Stores Negative Superhelicity, 166

SUMMARY, 192KEY EXPERIMENTS Box 7-2 Nucleosomes and Superhelica l

Density, 166

BIBLIOGRAPHY, 193

CHAPTER 8 • The Replication of DNA, 19 5

The Chemistry of DNA Synthesis, 196

Primer:Template Junction, 202DNA Synthesis Requires Deoxynucleoside Triphosphates

MEDICAL CONNECTIONS Box 8-2 Anticancer and Antivira land a Primer:Template Junction, 196

Agents Target DNA Replication, 20 3DNA Is Synthesized by Extending the 3 ' End of the

DNA Polymerases Are Processive Enzymes, 20 7Primer, 197

Exonucleases Proofread Newly Synthesized DNA, 20 8Hydrolysis of Pyrophosphate Is the Driving Force for DNA

The Replication Fork, 20 9Synthesis, 198Both Strands of DNA Are Synthesized Together at th e

The Mechanism of DNA Polymerase, 198

Replication Fork, 209DNA Polymerases Use a Single Active Site to Catalyze DNA

The Initiation of a New Strand of DNA Requires an RNASynthesis, 198

Primer, 210TECHNIQUES Box 8-1 Incorporation Assays Can Be

RNA Primers Must Be Removed to Complete DNAUsed to Measure Nucleic Acid and Protein

Replication, 21 1Synthesis, 200

DNA Helicases Unwind the Double Helix in Advance of th eDNA Polymerases Resemble a Hand That Grips the

Replication Fork, 211

TECHNIQUES Box 8-3 Determining the Polarity of a DNA

Protein-Protein and Protein-DNA Interactions Direct th eHelicase, 212

Initiation Process, 235

DNA Helicase Pulls Single-Stranded DNA through a Central

Al .( /I) (()\( /PT.s Box 8-6 The Replication FactoryProtein Pore, 214

Hypothesis, 23 7

Single-Stranded DNA-Binding Proteins Stabilize ssDNA prior

Eukaryotic Chromosomes Are Replicated Exactly Once pe rto Replication, 215

Cell Cycle, 23 9

Topoisomerases Remove Supercoils Produced by DNA

Prereplicative Complex Formation Is the First Step in th eUnwinding at the Replication Fork, 216

Initiation of Replication in Eukaryotes, 24 0

Replication Fork Enzymes Extend the Range of DNA

Pre-RC Formation and Activation Are Regulated to AllowPolymerase Substrates, 217

Only a Single Round of Replication during Each Cel lCycle, 24 1

The Specialization of DNA Polymerases, 218

Similarities between Eukaryotic and Prokaryotic DNADNA Polymerases Are Specialized for Different Roles in the

Replication Initiation, 24 4Cell, 218

gill qv(II ( O,vr i'l :, S( 11 li-7 E . colt DNA Replication IsSliding Clamps Dramatically Increase DNA Polymerase

Regulated by DnaA•ATP Levels and SeqA, 24 4Processivity, 219

Sliding Clamps Are Opened and Placed on DNA by Clamp

Finishing Replication, 246

Loaders, 222

Type II Topoisomerases Are Required to Separate Daughter

AEA' ;\'CfD CC)NCFPln Box 8-4 ATP Control of Protein

DNA Molecules, 24 6

Function : Loading a Sliding Clamp, 223

I.9gging-Strand Synthesis Is Unable to Copy the Extreme Ends

DNA Synthesis at the Replication Fork, 225

of Linear Chromosomes, 24 7

Telomerase Is a Novel DNA Polymerase That Does No tInteractions between Replication Fork Proteins Form the E .

Require an Exogenous Template, 248colt Replisome, 228

Telomerase Solves the End Replication Problem by Extendin g

Initiation of DNA Replication, 230

the 3' End of the Chromosome, 25 0

Specific Genomic DNA Sequences Direct the Initiation of

MEDICAL CONNECTIONS Box 8-8 Aging, Cancer, and the

DNA Replication, 230

Telomere Hypothesis, 25 1

The Replicon Model of Replication Initiation, 230

Telomere-Binding Proteins Regulate Telomerase Activity andTelomere Length, 252

Replicator Sequences Include Initiator Binding Sites andEasily Unwound DNA, 231

Telomere-Binding Proteins Protect Chromosome

KEY EXPERIMENTS Box 8-5 The Identification of Origins of

Ends, 253

Replication and Replicators, 232

SUMMARY, 255

Binding and Unwinding : Origin Selection and Activation

BIBLIOGRAPHY, 256

by the Initiator Protein, 235

CHAPTER 9 • The Mutability and Repair of DNA, 25 7

Replication Errors and Their Repair, 258

Direct Reversal of DNA Damage, 270

The Nature of Mutations, 258

Base Excision Repair Enzymes Remove Damaged Bases by aSome Replication Errors Escape Proofreading, 259

Base Flipping Mechanism, 270

MEDICAL CONNECTIONS Box 9-1 Expansion of Triple Repeats

Nucleotide Excision Repair Enzymes Cleave Damaged DN ACauses Disease, 259

on Either Side of the Lesion, 273

Mismatch Repair Removes Errors That Escape

Recombination Repairs DNA Breaks by Retrieving SequenceProofreading, 260

Information from Undamaged DNA, 275

DSBs in DNA Are Also Repaired by Direct Joining of Broke nDNA Damage, 265

Ends, 275DNA Undergoes Damage Spontaneously from Hydrolysis

MEDICAL CONNECTIONS Box 9-3 Nonhomologous En dand Deamination, 265

Joining, 276DNA Is Damaged by Alkylation, Oxidation, and

Translesion DNA Synthesis Enables Replication to ProceedRadiation, 265

across DNA Damage, 278MEDICAL CONNECTIONS Box 9-2 The Ames T e s t , 266

vx is n ; ' o'r 13rn. 9-4 The Y Family of DNAMutations Are Also Caused by Base Analogs and

Polymerases, 280Intercalating Agents, 268

SUMMARY, 28 1

Repair of DNA Damage, 269

BIBLIOGRAPHY, 282

CHAPTER 10 • Homologous Recombination at the Molecular Level, 283

DNA Breaks Are Common and Initiate Recombination, 284

Homologous Recombination Is Required for Chromosom eModels for Homologous Recombination, 284

Segregation during Meiosis, 304

Strand Invasion Is a Key Early Step in Homologous

Programmed Generation of Double-Stranded DNA Break s

Recombination, 286

Occurs during Meiosis, 305

Resolving Holliday Junctions Is a Key Step to Finishing

MRX Protein Processes the Cleaved DNA Ends fo r

Genetic Exchange, 288

Assembly of the RecA-like Strand-Exchange

The Double-Strand Break-Repair Model Describes Many

Proteins, 30 7

Recombination Events, 288

Dmc1 Is a RecA-like Protein That Specifically Functions inMeiotic Recombination, 308

Homologous Recombination Protein Machines, 291

Many Proteins Function Together to Promote Meioti cc

hrr 10-1 How to Resolve a

Recombination, 308

Recombination Intermediate with Two Holliday

MEDICAL CONNECTIONS Box 10-2 The Product of the TumorJunctions, 292

Suppressor Gene BRCA2 Interacts with Rad5I ProteinThe RecBCD Helicase/Nuclease Processes Broken DNA

and Controls Genome Stability, 309

Molecules for Recombination, 293

Chi Sites Control RecBCD, 296

Mating Type Switching, 31 0

RecA Protein Assembles on Single Stranded DNA and

Mating-Type Switching Is Initiated by a Site-Specific Double -

Promotes Strand Invasion, 297

Strand Break, 31 1

Mating-Type Switching Is a Gene Conversion Event and No tNewly Base-Paired Partners Are Established within the RecA

Associated with Crossing Over, 312Filament, 299

RecA Homologs Are Present in All Organisms, 301

Genetic Consequences of the Mechanism o f

The RuvAB Complex Specifically Recognizes Holliday

Homologous Recombination, 31 4Junctions and Promotes Branch Migration, 301

One Cause of Gene Conversion Is DNA Repair during

RuvC Cleaves Specific DNA Strands at the Holliday Junction

Recombination, 315

to Finish Recombination, 302

SUMMARY, 31 6Homologous Recombination in Eukaryotes, 303

BIBLIOGRAPHY, 31 7Homologous Recombination Has Additional Functions i n

Eukaryotes, 303

CHAPTER 11 • Site-Specific Recombination and Transposition of DNA, 31 9

Conservative Site-Specific Recombination, 320

The Hin Recombinase Inverts a Segment of DNA Allowing

Site-Specific Recombination Occurs at Specific DNA

Expression of Alternative Genes, 33 1

Sequences in the Target DNA, 320

Hin Recombination Requires a DNA Enhancer, 332

Site-Specific Recombinases Cleave and Rejoin DNA Using a

Recombinases Convert Multimeric Circular DNA Molecule sCovalent Protein-DNA Intermediate, 322

into Monomers, 333

Serine Recombinases Introduce Double-Strand Breaks in DNA

There Are Other Mechanisms to Direct Recombination t oand Then Swap Strands to Promote Recombination, 324

Specific Segments of DNA, 334

Structure of the Serine Recombinase-DNA Complex

Transposition, 33 4Indicates That Subunits Rotate to Achieve Stran dExchange, 325

Some Genetic Elements Move to New Chromosoma l

Tyrosine Recombinases Break and Rejoin One Pair of DNA

Locations by Transposition, 334

Strands at a Time, 326

ADVANCED CONCEPTS Box 11-2 The Xer Recombinase

Structures of Tyrosine Recombinases Bound to DNA Reveal

Catalyzes the Monomerization of Bacteria l

the Mechanism of DNA Exchange, 327

Chromosomes and of Many Bacterial Plasmids, 335

There Are Three Principal Classes of TransposableMEDICAL . CONNECTIONS Box 11-1 Application of Site-Specific Elements, 338Recombination to Genetic Engineering, 327

DNA Transposons Carry a Transposase Gene, Flanked byBiological Roles of Site-Specific Recombination, 328

Recombination Sites, 339

? Integrase Promotes the Integration and Excision of a Viral

Transposons Exist as Both Autonomous an dGenome into the Host-Cell Chromosome, 329

Nonautonomous Elements, 339

Bacteriophage 7l Excision Requires a New DNA-Bending

Virus-like Retrotransposons and RetrovirusesProtein, 331

Carry Terminal Repeat Sequences and Two Genes

Important for Recombination, 340

KEY EXPERIMENTS Box 11-4 Maize Elements and th e

Poly-A Retrotransposons Look Like Genes, 340

Discovery of Transposons, 356

DNA Transposition by a Cut-and-Paste Mechanism, 340

Tn 10 Transposition Is Coupled to Cellular DNA Replication ,

The Intermediate in Cut-and-Paste Transposition Is Finished

358

by Gap Repair, 342

Phage Mu Is an Extremely Robust Transposon, 35 9

There Are Multiple Mechanisms for Cleaving the

Mu Uses Target Immunity to Avoid Transposing into Its Ow n

Nontransferred Strand during DNA Transposition, 343

DNA, 359

DNA Transposition by a Rep/icative Mechanism, 345

\ ' ! ' ? N 'l t l'TS Box 11-5 Mechanism of Transposition

Virus-like Retrotransposons and Retroviruses Move Using an

Target Immunity, 36 1

RNA Intermediate, 347

Tc1/mariner Elements Are Extremely Successful DNA

Bo.\ 11-3 The Pathway of Retroviral

Elements in Eukaryotes, 362

cDNA Formation, 349

Yeast Ty Elements Transpose into Safe Havens in th e

DNA Transposases and Retroviral lntegrases Are Members of

Genome, 362

a Protein Superfamily, 351

LINEs Promote Their Own Transposition and Even Transpos e

Poly-A Retrotransposons Move by a "Reverse Splicing"

Cellular RNAs, 36 3

Mechanism, 352

V(D)J Recombination, 36 5

Examples of Transposable Elements and Their

The Early Events in V(D)J Recombination Occur by a

Regulation, 354

Mechanism Similar to Transposon Excision, 36 7

IS4-Family Transposons Are Compact Elements with Multiple

SUMMARY, 369Mechanisms for Copy Number Control, 355

BIBLIOGRAPHY, 369

PART 3

EXPRESSION OF THE GENOME, 37 1

C H APT E R 12 • Mechanisms of Transcription, 37 7

RNA Polymerases and the Transcription Cycle, 378

a!nAwoI ) .,\(1!'r, Box 12-2 The Single-Subunit RNA

RNA Polymerases Come in Different Forms but Share Many

Polymerases, 393

Features, 378

RNA Polymerase Can Become Arrested and Nee d

Transcription by RNA Polymerase Proceeds in a Series of

Removing, 394

Steps, 380

Transcription Is Terminated by Signals within the RN A

Transcription Initiation Involves Three Defined Steps, 382

Sequence, 39 4

The Transcription Cycle in Bacteria, 383

Transcription in Eukaryotes, 396

Bacterial Promoters Vary in Strength and Sequence but Have

RNA Polymerase II Core Promoters Are Made Up of

Certain Defining Features, 383

Combinations of Four Different Sequence Elements, 39 7

The a Factor Mediates Binding of Polymerase to the

RNA Polymerase Il Forms a Preinitiation Complex with

Promoter, 384

General Transcription Factors at the Promoter, 39 8

Transition to the Open Complex Involves Structural

Promoter Escape Requires Phosphorylation of the

Changes in RNA Polymerase and in the Promoter

Polymerase ''Tail," 39 8

DNA, 386

TBP Binds to and Distorts DNA Using a I Sheet Inserted into

TECHNIQUES Box 12-1 Consensus Sequences, 388

the Minor Groove, 400

Transcription Is Initiated by RNA Polymerase without the

The Other General Transcription Factors Also Have Specifi c

Need for a Primer, 388

Roles in Initiation, 40 1

During Initial Transcription, RNA Polymerase Remains

In Vivo, Transcription Initiation Requires Additional Proteins ,Stationary and Pulls Downstream DNA into Itself, 389

Including the Mediator Complex, 402

Promoter Escape Involves Breaking Polymerase-Promoter

Mediator Consists of Many Subunits, Some Conserved fro m

Interactions and Polymerase Core-a Interactions, 390

Yeast to Human, 403

The Elongating Polymerase Is a Processive Machine That

A New Set of Factors Stimulate Pol II Elongation and RNA

Synthesizes and Proofreads RNA, 391

Proofreading, 404

Elongating RNA Polymerase Must Deal with Histones in Its

RNA Pol I and Pol III Recognize Distinct Promoters, UsingPath, 405

Distinct Sets of Transcription Factors, but Still Requir eElongating Polymerase Is Associated with a New Set of

TBP, 41 0Protein Factors Required for Various Types of RNA

Pol Ill Promoters Are Found Downstream of TranscriptionProcessing, 406

Start Site, 412Transcription Termination Is Linked to RNA Destruction by a

SUMMARY, 41 3Highly Processive RNase, 410BIBLIOGRAPHY, 41 4

Transcription by RNA Polymerases I and III, 41 0

CHAPTER 13 • RNA Splicing, 41 5

The Chemistry of RNA Splicing, 417

Several Mechanisms Exist to Ensure Mutually ExclusiveSequences within the RNA Determine Where Splicing

Splicing, 43 5Occurs, 417

The Curious Case of the Drosophila Dscam Gene : MutuallyThe Intron Is Removed in a Form Called a Lariat as the

Exclusive Splicing on a Grand Scale, 43 6Flanking Exons Are Joined, 418

Mutually Exclusive Splicing of Dscam Exon 6 Cannot B eKEY EXPERIMENTS Box 13-1 Adenovirus and the Discovery

Accounted for by Any Standard Mechanism and Instea dof Splicing, 419

Uses a Novel Strategy, 43 7

Exons from Different RNA Molecules Can Be Fused by

Alternative Splicing Is Regulated by Activators an dtrans-Splicing, 421

Repressors, 43 9Regulation of Alternative Splicing Determines the Sex

The Spliceosome Machinery, 422

of Flies, 44 1RNA Splicing Is Carried Out by a Large Complex Called

KEY EXPERIMENTS Box 13-3 Identification of Docking Site andthe Spliceosome, 422

Selector Sequences, 442

Splicing Pathways, 424

MEDICAL CONNECTIONS Box 13-4 Defects in Pre-mRNA

Assembly, Rearrangements, and Catalysis within the

Splicing Cause Human Disease, 44 5

Spliceosome: The Splicing Pathway, 424

Exon Shuffling, 446Self-Splicing Introns Reveal That RNA Can Catalyze RNA

Exons Are Shuffled by Recombination to Produce GenesSplicing, 426

Encoding New Proteins, 44 6Group I Introns Release a Linear Intron Rather Than a

Lariat, 426

RNA Editing, 448

KEY EXPERIMENTS Box 13-2 Converting Group I Introns into

RNA Editing Is Another Way of Altering the Sequence of a n

Ribozymes, 428

mRNA, 44 8

How Does the Spliceosome Find the Splice Sites

Guide RNAs Direct the Insertion and Deletion of Uridines, 45 0

Reliably?, 430

MEDICAL CONNECTIONS Box 13-5 Deaminases and HIV4450

A Small Group of Introns Are Spliced by an Alternative

mRNA Transport, 452Spliceosome Composed of a Different Set of snRNPs,432

Once Processed, mRNA Is Packaged and Exported from th eNucleus into the Cytoplasm for Translation, 45 2

Alternative Splicing, 432

SUMMARY, 454Single Genes Can Produce Multiple Products by Alternative

Splicing 432

BIBLIOGRAPHY, 45 5

CHAPTER 14 • Translation, 45 7

Messenger RNA, 458

Transfer RNA, 46 1

Polypeptide Chains Are Specified by Open Reading

tRNAs Are Adaptors between Codons and AminoFrames, 458

Acids, 46 1Prokaryotic mRNAs Have a Ribosome-Binding Site That

ADVANCED CONCEPTS Box 14-1 CCA-Adding Enzymes :Recruits the Translational Machinery, 459

Synthesizing RNA without a Template, 462

Eukaryotic mRNAs Are Modified at Their 5 ' and 3 ' Ends to

tRNAs Share a Common Secondary Structure Tha tFacilitate Translation, 460

Resembles a Cloverleaf, 462

tRNAs Have an L-shaped Three-Dimensional

The Ribosome Uses Multiple Mechanisms to Select agains tStructure, 463

Incorrect Aminoacyl-tRNAs, 488

Attachment of Amino Acids to tRNA, 464

The Ribosome Is a Ribozyme, 49 1

tRNAs Are Charged by the Attachment of an Amino

Peptide Bond Formation and the Elongation Facto r

Acid to the 3'-Terminal Adenosine Nucleotide via

EF G Drive Translocation of the tRNAs and the

a High-Energy Acyl Linkage, 464

mRNA, 492

Aminoacyl tRNA Synthetases Charge tRNAs in Two

EF-G Drives Translocation by Displacing the tRNA Bound to

Steps, 464

the A Site, 494

Each Aminoacyl-tRNA Synthetase Attaches a Single Amino

EF-Tu-GDP and EF-G-GDP Must Exchange GDP fo r

Acid to One or More tRNAs, 466

GTP prior to Participating in a New Round ofElongation, 495

tRNA Synthetases Recognize Unique Structural Features of

A Cycle of Peptide Bond Formation ConsumesCognate tRNAs, 466Two Molecules of GTP and One Molecule o f

Aminoacyl-tRNA Formation Is Very Accurate, 468

ATP, 49 5Some Aminoacyl-tRNA Synthetases Use an Editin g

Pocket to Charge tRNAs with High Accuracy, 468

Termination of Translation, 496

The Ribosome Is Unable to Discriminate between

Release Factors Terminate Translation in Response to Stop

Correctly and Incorrectly Charged tRNAs, 469

Codons, 496

Short Regions of Class I Release Factors RecognizeThe Ribosome, 469

Stop Codons and Trigger Release of the PeptidylADVANCED CONCEPTS Box 14-2 Selenocysteine, 470

Chain, 496

The Ribosome Is Composed of a Large and a Small

11)V AN( I U r O y(I rr' hO 14-4 GTP-Binding Proteins,Subunit, 471

Conformational Switching, and the Fidelity an dThe Large and Small Subunits Undergo Association

Ordering of the Events of Translation, 498and Dissociation during Each Cycle of

GDP/GTP Exchange and GTP Hydrolysis Control theTranslation, 472

Function of the Class 11 Release Factor, 49 9New Amino Acids Are Attached to the Carboxyl

The Ribosome Recycling Factor Mimics a tRNA, 50 0Terminus of the Growing Polypeptide Chain, 474

MEDICAL CONNECTIONS Box 14-5 Antibiotics Arres tPeptide Bonds Are Formed by Transfer of the Growing

Cell Division by Blocking Specific Steps inPolypeptide Chain from One tRNA to Another, 474

Translation, 50 2

Ribosomal RNAs Are Both Structural and Catalytic

Regulation of Translation, 50 3Determinants of the Ribosome, 47 5

The Ribosome Has Three Binding Sites for tRNA, 475

Protein or RNA Binding Near the Ribosome-Bindin gSite Negatively Regulates Bacterial Translatio n

Channels through the Ribosome Allow the mRNA and

Initiation, 504Growing Polypeptide to Enter and/or Exit the

Regulation of Prokaryotic Translation : Ribosoma lRibosome, 476

Proteins Are Translational Repressors of Their Own

Initiation of Translation, 479

Synthesis, 505

Prokaryotic mRNAs Are Initially Recruited to the Small

Global Regulators of Eukaryotic Translation Target Key

Subunit by Base Pairing to rRNA, 480

Factors Required for mRNA Recognition and Initato r

A Specialized tRNA Charged with a Modified

tRNA Ribosome Binding 508

Methionine Binds Directly to the Prokaryotic

Spatial Control of Translation by mRNA-SpecificSmall Subunit, 480

4E-BPs, 51 0

Three Initiation Factors Direct the Assembly of an

An Iron-Regulated RNA-Binding Protein Control s

Initiation Complex That Contains mRNA and the

Translation of Ferritin, 51 1

Initiator tRNA, 481

Translation of thet Yeast Transcriptional Activator Gcn4 I s

Eukaryotic Ribosomes Are Recruited to the mRNA by the

Controlled by Short Upstream ORFs and Ternary

5' Cap, 482

Complex Abundance, 51 2

The Start Codon Is Found by Scanning Downstream from

Translation-Dependent Regulation of mRNA and Protei nthe 5' End of the mRNA, 483

Stability, 51 4ADVANCED CC?\CEPTS Box 14- uORFs and IRESs : Exceptions

The SsrA RNA Rescues Ribosomes That Translate Broke nThat Prove the Rule, 485

mRNAs, 51 4Translation Initiation Factors Hold Eukaryotic mRNAs in

Eukaryotic Cells Degrade mRNAs That Are Incomplete o rCircles, 487

Have Premature Stop Codons, 51 6

Translation Elongation, 487

SUMMARY, 51 8Aminoacyl-tRNAs Are Delivered to the A Site by Elongation

BIBLIOGRAPHY, 51 9Factor EF-Tu, 488

CHAPTER 15 • The Genetic Code, 52 1

The Code Is Degenerate, 521

Three Kinds of Point Mutations Alter the Genetic Code, 53 1

Perceiving Order in the Makeup of the Code, 522

Genetic Proof That the Code Is Read in Units of Three, 532

Wobble in the Anticodon, 523

Suppressor Mutations Can Reside in the Same or aThree Codons Direct Chain Termination, 525

Different Gene, 532How the Code Was Cracked, 525

Intergenic Suppression Involves Mutant tRNAs, 533Stimulation of Amino Acid Incorporation by Synthetic

Nonsense Suppressors Also Read Normal Terminatio nmRNAs, 526

Signals, 535Poly-U Codes for Polyphenylalanine, 527

Proving the Validity of the Genetic Code, 535Mixed Copolymers Allowed Additional Codon Assignments ,

527

The Code Is Nearly Universal, 536

Transfer RNA Binding to Defined Trinucleotide Codons, 528

SUMMARY, 538Codon Assignments from Repeating Copolymers, 529

BIBLIOGRAPHY, 538

Three Rules Govern the Genetic Code, 530

PART 4

REGULATION, 54 1

C H A P T E R 16 • Transcriptional Regulation in Prokaryotes, 54 7

Principles of Transcriptional Regulation, 547

Combinatorial Control: CAP Controls Other Genes As Well,

Gene Expression Is Controlled by Regulatory

560

Proteins, 547

KEY EXPERIMENTS Box 16-2 Jacob, Monod, and the Ideas

Most Activators and Repressors Act at the Level of

Behind Gene Regulation, 56 1

Transcription Initiation, 548

Alternative 6 Factors Direct RNA Polymerase to Alternative

Many Promoters Are Regulated by Activators That Help RNA

Sets of Promoters, 563

Polymerase Bind DNA and by Repressors That Block That

NtrC and MerR: Transcriptional Activators That Work by

Binding, 548

Allostery Rather than by Recruitment, 564

Some Activators and Repressors Work by Allostery and

NtrC Has ATPase Activity and Works from DNA Sites Fa r

Regulate Steps in Transcriptional Initiation after RNA

from the Gene, 564Polymerase Binding, 550

MerR Activates Transcription by Twisting Promoter

Action at a Distance and DNA Looping, 551

DNA, 565

Cooperative Binding and Allostery Have Many Roles in Gene

Some Repressors Hold RNA Polymerase at the Promoter

Regulation, 552

Rather than Excluding It, 566

Antitermination and Beyond: Not All of Gene Regulation

AraC and Control of the araBAD Operon by Antiactivation, 567

Targets Transcription Initiation, 552The Case of Bacteriophage X : Layers of Regulation, 568

Regulation of Transcription Initiation : Examples from

Alternative Patterns of Gene Expression Control Lytic an d

Prokaryotes, 553

Lysogenic Growth, 569

An Activator and a Repressor Together Control the lac

Regulatory Proteins and Their Binding Sites, 570

Genes, 553

k. Repressor Binds to Operator Sites Cooperatively, 57 1

CAP and Lac Repressor Have Opposing Effects on RNA

AIA,xv (~) iB » 16-S Concentration, Affinity, andPolymerase Binding to the lac Promoter, 554

Cooperative Binding, 572CAP Has Separate Activating and DNA-Binding

Repressor and Cro Bind in Different Patterns to Control LyticSurfaces, 555

and Lysogenic Growth, 573CAP and Lac Repressor Bind DNA Using a Common

Lysogenic Induction Requires Proteolytic Cleavage of AStructural Motif, 556

Repressor, 574KEY EXPERIMENTS Box 16-1 Activator Bypass Experiments, 557

Negative Autoregulation of Repressor Requires Long-

The Activities of Lac Repressor and CAP Are Controlled

Distance Interactions and a Large DNA Loop, 575

Allosterically by Their Signals, 559

Another Activator. A Cll. Controls the Decision between

Lytic and Lysogenic Growth upon Infection of a New

KEY EXPERIMENTS Box 16-5 Genetic Approaches That IdentifiedHost, 577

Genes Involved in the LyticlLysogenic Choice, 58 1

The Number of Phage Particles Infecting a Given Cell

Transcriptional Antitermination in Development, 58 2Affects Whether the Infection Proceeds Lytically or

Retroregulation : An Interplay of Controls on RNA SynthesisLysogenically, 578

and Stability Determines int Gene Expression, 584Growth Conditions of E . coil Control the Stability of CI

SUMMARY, 585Protein and thus the Lytic/Lysogenic Choice, 578

KEY EXPERIMENTS Box 16-4 Evolution of the X Switch, 579

BIBLIOGRAPHY, 586

CHAPTER 17 • Transcriptional Regulation in Eukaryotes, 589

Conserved Mechanisms of Transcriptional Regulation

KEY EXPERIMENTS Box 17-3 Evolvability of a Regulatoryfrom Yeast to Mammals, 591

Circuit, 612

Activators Have Separate DNA-Binding and Activating

Transcriptional Repressors, 61 3Functions, 59 1

Eukaryotic Regulators Use a Range of DNA-Binding

Signal Transduction and the Control of Transcriptiona l

Domains, but DNA Recognition Involves the Same

Regulators, 61 5

Principles as Found in Bacteria, 593

Signals Are Often Communicated to Transcriptiona l

TECHNIQUES Box 17-1 The Two-Hybrid Assay, 594

Regulators through Signal Transduction

Activating Regions Are Not Well-Defined Structures, 596

Pathways, 61 5

Signals Control the Activities of Eukaryotic Transcriptiona lRecruitment of Protein Complexes to Genes by

Regulators in a Variety of Ways, 61 7Eukaryotic Activators, 597

Activators and Repressors Sometimes Come in Pieces, 61 9Activators Recruit the Transcriptional Machinery to th e

Gene, 597

Gene "Silencing" by Modification of Histones an d

Activators Also Recruit Nucleosome Modifiers That Help the

DNA, 620

Transcriptional Machinery Bind at the Promoter or

Silencing in Yeast Is Mediated by Deacetylation an dInitiate Transcription, 598

Methylation of Histones, 62 1

Activators Recruit an Additional Factor Needed for Efficient

In Drosophila, HP1 Recognizes Methylated Histones an d

Initiation or Elongation at Some Promoters, 600

Condenses Chromatin, 622

Action at a Distance : Loops and Insulators, 601

ADC \u II)

1'1 5 Box 17-4 Is There a Histon e

Appropriate Regulation ofSome Groups ofGenes Requires

Code?, 62 3

Locus Control Regions, 603

DNA Methylation Is Associated with Silenced Genes in

KEY EXPERIMENTS Box 17-2 Long-Distance Interactions on the

Mammalian Cells, 62 4

Same and Different Chromosomes, 604

MEDKÜI CY)\\r( nr,\S Box 17-5 Transcriptional Repressionand Human Disease, 626

Signal Integration and Combinatorial Control, 605

Activators Work Synergistically to Integrate Signals, 605

Epigenetic Gene Regulation, 626

Signal Integration: The HO Gene Is Controlled by Two

Some States of Gene Expression Are Inherited through CellDivision Even When the Initiating Signal Is No LongerRegulators-One Recruits Nucleosome Modifiers and the

Present, 62 7Other Recruits Mediator, 60 7

Signal Integration: Cooperative Binding ofActivators at the

MEDIG41 CONNECTIONS Box 17-6 Using Transcriptio nFactors to Reprogram Somatic Cells into EmbryonicHuman 8-Interferon Gene, 608

Stem Cells, 62 9Combinatorial Control Lies at the Heart of the Complexity

and Diversity of Eukaryotes, 610

SUMMARY, 630

Combinatorial Control of the Mating-Type Genes from S .

BIBLIOGRAPHY, 63 1cerevisiae, 61 1

CHAPTER 18 • Regulatory RNAs, 63 3

Regulation by RNAs in Bacteria, 633

in Secondary Structure, 635

Riboswitches Reside within the Transcripts of Genes

r n cO c r i' - Box 18-1 Amino Acid BiosyntheticWhose Expression They Control through Changes

Operons Are Controlled by Attenuation, 639

RNA Interference Is a Major Regulatory Mechanism in

Small RNAs Can Transcriptionally Silence Genes by DirectingEukaryotes, 641

Chromatin Modification, 64 9

Short RNAs That Silence Genes Are Produced from a Variety

KEY EXPERIMENTS Box 18-2 History of miRNAs and RNA1, 65 0of Sources and Direct the Silencing of Genes in Thre eDifferent Ways, 641

The Evolution and Exploitation of RNAi, 652Did RNAi Evolve As an Immune System?, 652

Synthesis and Function of miRNA Molecules, 643

RNAi Has Become a Powerful Tool for Manipulating Gen emiRNAs Have a Characteristic Structure That Assists in

Expression, 65 4Identifying Them and Their Target Genes, 643

MEDICAL CONNECTIONS Box 18-3 RNAi and HumanAn Active miRNA Is Generated through a Two-Step

Disease, 65 6Nucleolytic Processing, 645

Dicer Is the Second RNA-Cleaving Enzyme Involved in

Regulatory RNAs and X inactivation, 65 7miRNA Production, 646

X-inactivation Creates Mosaic Individuals, 65 7

Incorporation of a Guide Strand RNA into RISC Makes the

Xist Is an RNA Regulator That Inactivates a Single XMature Complex That Is Ready to Silence Gene

Chromosome in Female Mammals, 65 7Expression, 647

SUMMARY, 65 9siRNAs Are Regulatory RNAs Generated from Long Double-

BIBLIOGRAPHY, 660Stranded RNAs, 64 9

CHAPTER 19 • Gene Regulation in Development and Evolution, 66 1

TECHNIQUES Box 19-1 Microarray Assays : Theory and

A Morphogen Gradient Controls Dorsoventral Patterning o fPractice, 662

the Drosophila Embryo, 679

Three Strategies by Which Cells Are Instructed to

Segmentation Is Initiated by Localized RNAs at the Anterio r

Express Specific Sets of Genes during Development, 663

and Posterior Poles of the Unfertilized Egg, 682

Some mRNAs Become Localized within Eggs and Embryos

Bicoid and Nanos Regulate hunchback 683

because of an Intrinsic Polarity in the Cytoskeleton, 663

KEY EXPERIMENTS Box 19-5 The Role of Activator Synergy i n

Cell-to-Cell Contact and Secreted Cell-Signaling Molecules

Development, 684

Both Elicit Changes in Gene Expression in Neighboring

MEDICAL CONNECTIONS Box 19 6 Stem Cells, 68 6

Cells, 664

The Gradient of Hunchback Repressor Establishes Different

Gradients of Secreted Signaling Molecules Can Instruct Cells

Limits of Gap Gene Expression, 68 7

to Follow Different Pathways of Development Based on

Hunchback and Gap Proteins Produce Segmentation Stripes

Their Location, 665

of Gene Expression, 688

Gap Repressor Gradients Produce Many Stripes of GeneExamples of the Three Strategies for Establishing

Expression, 68 9Differential Gene Expression, 666

KEY EXPERIMENTS Box 19-7 cis-Regulatory Sequences inThe Localized Ash l Repressor Controls Mating Type in Yeast

Animal Development and Evolution, 690by Silencing the HO Gene, 666

Short-Range Transcriptional Repressors Permit Differen tADVANCED CONCEPfS Box 19-2 Review of Cytoskeleton :

Enhancers to Work Independently of One Another withi nAsymmetry and Growth, 669

the Complex eve Regulatory Region, 692

A Localized mRNA Initiates Muscle Differentiation in the Sea

Homeotic Genes: An Important Class of DevelopmentalSquirt Embryo, 670

Regulators, 69 3\LNANCED CONCEPTS Box 19-3 Overview of Cion a

Development, 671

Changes in Homeotic Gene Expression Are Responsible for

Arthropod Diversity, 695Cell-to-Cell Contact Elicits Differential Gene Expression in

Arthropods Are Remarkably Diverse, 69 5the Sporulating Bacterium, Bacillus subtilis, 672

A Skin-Nerve Regulatory Switch Is Controlled by Notch

Changes in Ubx Expression Explain Modification of Limb s

Signaling in the Insect Central Nervous System, 673

among the Crustaceans, 695

A Gradient of the Sonic Hedgehog Morphogen Controls the

ADVANCED CONCEPTS Box 19-8 Homeotic Genes of Drosophil a

Formation of Different Neurons in the Vertebrate Neural

Are Organized in Special Chromosome Clusters, 696

Tube, 674

Why Insects Lack Abdominal Limbs, 69 8

The Molecular Biology of Drosophila Embryogenesis, 676

Modification of Flight Limbs Might Arise from the Evolutio nof Regulatory DNA Sequences, 699

An Overview of Drosophila Embryogenesis, 676

v>In c()N( LPTti Box 19-4 Overview of Drosophila

SUMMARY, 70 1

Development, 677

BIBLIOGRAPHY, 702

CHAPTER 20 • Genome Analysis and Systems Biology, 70 3

Genomics Overview, 703

Negative Autoregulation Dampens Noise and Allows a Rapid

Bioinformatics Tools Facilitate the Genome-wide

Response Time, 71 7

Identification of Protein-Coding Genes, 703

Gene Expression Is Noisy, 718

Whole-Genome Tiling Arrays Are Used to Visualize the

Positive Autoregulation Delays Gene Expression, 720

Transcriptome, 704

Some Regulatory Circuits Lock in Alternative Stabl e

Regulatory DNA Sequences Can Be Identified by Using

States, 720Specialized Alignment Tools, 706

Feed-Forward Loops Are Three-Node Networks with

The ChIP-Chip Assay Is the Best Method for Identifying

Beneficial Properties, 722Enhancers, 708

KEY EXPERIMENTS Box 20-2 Bistability and Hysteresis, 722TECHNIQUES Box 20-1 Bioinformatics Methods for the

Feed-Forward Loops Are Used in Development, 725Identification of Complex Enhancers, 708

Some Circuits Generate Oscillating Patterns of Gen eDiverse Animals Contain Remarkably Similar Sets of

Expression, 72 7Genes, 711

Synthetic Circuits Mimic Some of the Features of NaturalMany Animals Contain Anomalous Genes, 712

Regulatory Networks, 729

Synteny Is Evolutionarily Ancient, 713

Prospects, 730Deep Sequencing Is Being Used to Explore Human

SUMMARY, 73 0Origins, 715

BIBLIOGRAPHY, 73 1Systems Biology, 71 5

Transcription Circuits Consist of Nodes and Edges, 716

PART 5

METHODS, 73 3

CHAPTER 21 • Techniques of Molecular Biology, 73 9

Nucleic Acids, 740

Nested Sets of DNA Fragments Reveal Nucleotid e

Electrophoresis through a Gel Separates DNA and RNA

Sequences, 753

Molecules according to Size, 740

KEY EXPERIMENTS Box 21-2 Sequenators Are Used for High -

Restriction Endonucleases Cleave DNA Molecules at

Throughput Sequencing, 75 7

Particular Sites, 742

Shotgun Sequencing a Bacterial Genome, 757

DNA Hybridization Can Be Used to Identify Specific DNA

The Shotgun Strategy Permits a Partial Assembly of Larg e

Molecules, 743

Genome Sequences, 758

Hybridization Probes Can Identify Electrophoretically

The Paired-End Strategy Permits the Assembly of Large-

Separated DNAs and RNAs, 744

Genome Scaffolds, 760

Isolation of Specific Segments of DNA, 746

The $1000 Human Genome Is within Reach, 762

DNA Cloning, 746

Proteins, 764Cloning DNA in Plasmid Vectors, 746

Specific Proteins Can Be Purified from Cell Extracts, 764Vector DNA Can Be Introduced into Host Organisms by

Purification of a Protein Requires a Specific Assay, 764Transformation, 748

Preparation of Cell Extracts Containing Active Proteins, 765Libraries of DNA Molecules Can Be Created by

Proteins Can Be Separated from One Another Using ColumnCloning, 748

Chromatography, 765Hybridization Can Be Used to Identify a Specific Clone in a

Affinity Chromatography Can Facilitate More Rapid ProteinDNA Library, 749

Purification, 767Chemically Synthesized Oligonucleotides, 750

Separation of Proteins on Polyacrylamide Gels, 768The Polymerase Chain Reaction Amplifies DNAs by Repeated

Antibodies Are Used to Visualize Electrophoreticall yRounds of DNA Replication in Vitro, 751

Separated Proteins, 769TECHNIQUES Box 21-1 Forensics and the Polymerase Chain

Protein Molecules Can Be Directly Sequenced, 769Reaction, 753

Proteomics, 771

Nucleic Acid-Protein Interactions, 77 5

Combining Liquid Chromatography with Mass

The Electrophoretic Mobility of DNA Is Altered by ProteinSpectrometry Identifies Individual Proteins

Binding, 776within a Complex Extract, 771

DNA-Bound Protein Protects the DNA from Nucleases an dProteome Comparisons Identify Important Differences

Chemical Modification, 77 7beween Cells, 773

Chromatin Immunoprecipitation Can Detect Protei nMass Spectrometry Can Also Monitor Protein Modification

Association with DNA in the Cell, 778States, 773

In Vitro Selection Can Be Used to Identify a Protein's DNA -Protein-Protein Interactions Can Yield Information about

or RNA-Binding Site, 78 0Protein Function, 774

BIBLIOGRAPHY, 782

CHAPTER 22 • Model Organisms, 783

Bacteriophage, 784

Arabidopsis Is Easily Transformed for Reverse Genetics, 799

Assays of Phage Growth, 786

Arabidopsis Has a Small Genome That Is ReadilyThe Single-Step Growth Curve, 787

Manipulated, 800

Phage Crosses and Complementation Tests, 787

Epigenetics, 80 1

Transduction and Recombinant DNA, 788

Plants Respond to the Environment, 80 1Development and Pattern Formation, 80 2

Bacteria, 78 9Assays of Bacterial Growth, 789

The Nematode Worm, Caenorhabditis elegans, 80 2

Bacteria Exchange DNA by Sexual Conjugation, Phage-

C . elegans Has a Very Rapid Life Cycle, 803Mediated Transduction, and DNA-Mediated

C. elegans Is Composed of Relatively Few, Well-Studied CellTransformation, 790

Lineages, 804Bacterial Plasmids Can Be Used as Cloning Vectors, 791

The Cell Death Pathway Was Discovered in C . elegans, 805

Transposons Can Be Used to Generate Insertional Mutations

RNA1 Was Discovered in C . elegans, 805and Gene and Operon Fusions, 79 1

Studies on the Molecular Biology of Bacteria Have Been

The Fruit Fly, Drosophila melanogaster, 806

Enhanced by Recombinant DNA Technology, Whole-

Drosophila Has a Rapid Life Cycle, 80 6Genome Sequencing, and Transcriptional Profiling, 793

The First Genome Maps Were Produced in Drosophila, 807

Biochemical Analysis Is Especially Powerful in Simple Cells

Genetic Mosaics Permit the Analysis of Lethal Genes in Adultwith Well-Developed Tools of Traditional and Molecular

Flies, 809Genetics, 793

The Yeast FLP Recombinase Permits the Efficient Productio nBacteria Are Accessible to Cytological Analysis, 793

of Genetic Mosaics, 809Phage and Bacteria Told Us Most of the Fundamental Things

It Is Easy to Create Transgenic Fruit Flies that Carry Foreignabout the Gene, 794

DNA, 810

Baker 's Yeast, Saccharomyces cerevisiae, 795

The House Mouse, Mus musculus, 81 2The Existence of Haploid and Diploid Cells Facilitate Genetic

Mouse Embryonic Development Depends on StemAnalysis of S . cerevisiae, 795

Cells, 81 3Generating Precise Mutations in Yeast Is Easy, 796

It Is Easy to Introduce Foreign DNA into the Mous eS . cerevisiae Has a Small, Well-Characterized Genome, 796

Embryo, 81 3

S . cerevisiae Cells Change Shape as They Grow, 797

Homologous Recombination Permits the Selective Ablatio nof Individual Genes, 81 4

Arabidopsis, 798

Mice Exhibit Epigenetic Inheritance, 81 6Arabidopsis Has a Fast Life Cycle with Haploid and Diploid

BIBLIOGRAPHY, 81 8Phases, 79 8

Index, 819