基因分子生物学 影印版PDF电子书下载

PART 1 CHEMISTRY AND GENETICS 1

1 The Mendelian View of the World 5

2 Nucleic Acids Convey Genetic Information 19

3 The Importance of Weak Chemical Interactions 43

4 The Importance of High-Energy Bonds 57

5 Weak and Strong Bonds Determine Macromolecular Structure 71

PART 2 MAINTENANCE OF THE GENOME 95

6 The Structures of DNA and RNA 101

7 Genome Structure,Chromatin,and the Nucleosome 135

8 The Replication of DNA 195

9 The Mutability and Repair of DNA 257

10 Homologous Recombination at the Molecular Level 283

11 Site-Specific Recombination and Transposition of DNA 319

PART 3 EXPRESSION OF THE GENOME 371

12 Mechanisms of Transcription 377

13 RNA Splicing 415

14 Translation 457

15 The Genetic Code 521

PART 4 REGULATION 541

16 Transcriptional Regulation in Prokaryotes 547

17 Transcriptional Regulation in Eukaryotes 589

18 Regulatory RNAs 633

19 Gene Regulation in Development and Evolution 661

20 Genome Analysis and Systems Biology 703

PART 5 METHODS 733

20 Techniques of Molecular Biology 739

21 Model Organisms 783

Index 819

PART 1 CHEMISTRY AND GENETICS 1

CHAPTER 1 The Mendelian View of the World 5

Mendel's Discoveries 6

The Principle of Independent Segregation 6

ADVANCED CONCEPTS Box 1-1 Mendelian Laws 6

Some Alleles Are neither Dominant nor Recessive 8

Principle of Independent Assortment 8

Chromosomal Theory of Heredity 8

Gene Linkage and Crossing Over 9

KEY EXPERIMENTS Box 1-2 Genes Are Linked to Chromosomes 10

Chromosome Mapping 12

The Origin of Genetic Variability through Mutations 15

Early Speculations about What Genes Are and How They Act 16

Preliminary Attempts to Find a Gene-Protein Relationship 16

SUMMARY 17

BIBLIOGRAPHY 18

CHAPTER 2 Nucleic Acids Convey Genetic Information 19

Avery's Bombshell:DNA Can Carry Genetic Specificity 20

Viral Genes Are Also Nucleic Acids 21

The Double Helix 21

Finding the Polymerases That Make DNA 23

KEY EXPERIMENTS Box 2-1 Chargaff's Rules 24

Experimental Evidence Favors Strand Separation during DNA Replication 25

The Genetic Information within DNA Is Conveyed by the Sequence of Its Four Nucleotide Building Blocks 28

KEY EXPERIMENTS Box2-2,Evidence That Genes Control Amino Acid Sequences in Proteins 29

DNA Cannot Be the Template That Directly Orders Amino Acids during Protein Synthesis 30

RNA Is Chemically Very Similar to DNA 30

The Central Dogma 32

The Adaptor Hypothesis of Crick 32

Discovery of Transfer RNA 32

The Paradox of the Nonspecific-Appearing Ribosomes 33

Discovery of Messenger RNA(mRNA) 34

Enzymatic Synthesis of RNA upon DNA Templates 35

Establishing the Genetic Code 36

Establishing the Direction of Protein Synthesis 38

Start and Stop Signals Are Also Encoded within DNA 39

The Era of Genomics 39

SUMMARY 40

BIBLIOGRAPHY 41

CHAPTER 3 The Importance of Weak Chemical Interactions 43

Characteristics of Chemical Bonds 43

Chemical Bonds Are Explainable in Quantum-Mechanical Terms 44

Chemical-Bond Formation Involves a Change in the Form of Energy 45

Equilibrium between Bond Making and Breaking 45

The Concept of Free Energy 46

Keq Is Exponentially Related to ΔG 46

Covalent Bonds Are Very Strong 46

Weak Bonds in Biological Systems 47

Weak Bonds Have Energies between 1 and 7 kcal/mol 47

Weak Bonds Are Constantly Made and Broken at Physiological Temperatures 47

The Distinction between Polar and Nonpolar Molecules 47

van der Waals Forces 48

Hydrogen Bonds 49

Some Ionic Bonds Are Hydrogen Bonds 50

Weak Interactions Demand Complementary Molecular Surfaces 51

Water Molecules Form Hydrogen Bonds 51

Weak Bonds between Molecules in Aqueous Solutions 51

Organic Molecules That Tend to Form Hydrogen Bonds Are Water Soluble 52

ADVANCED CONCEPTS Box 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness 53

Hydrophobic"Bonds"Stabilize Macromolecules 54

The Advantage of△G between 2 and 5 kcal/mole 55

Weak Bonds Attach Enzymes to Substrates 55

Weak Bonds Mediate Most Protein-DNA and Protein-Protein Interactions 55

SUMMARY 56

BIBLIOGRAPHY 56

CHAPTER 4 The Importance of High-Energy Bonds 57

Molecules That Donate Energy Are Thermodynamically Unstable 57

Enzymes Lower Activation Energies in Biochemical Reactions 59

Free Energy in Biomolecules 60

High-Energy Bonds Hydrolyze with Large Negative△G 60

High-Energy Bonds in Biosynthetic Reactions 62

Peptide Bonds Hydrolyze Spontaneously 62

Coupling of Negative with Positive△G 63

Activation of Precursors in Group Transfer Reactions 63

ATP Versatility in Group Transfer 64

Activation of Amino Acids by Attachment of AMP 65

Nucleic Acid Precursors Are Activated by the Presence of P～P 66

The Value of P～P Release in Nucleic Acid Synthesis 66

P～P Splits Characterize Most Biosynthetic Reactions 67

SUMMARY 68

BIBLIOGRAPHY 69

CHAPTER 5 Weak and Strong Bonds Determine Macromolecular Structure 71

Higher-Order Structures Are Determined by Intra-and Intermolecular Interactions 71

DNA Can Form a Regular Helix 71

RNA Forms a Wide Variety of Structures 73

Chemical Features of Protein Building Blocks 73

The Peptide Bond 75

There Are Four Levels of Protein Structure 75

αHelices andβSheets Are the Common Forms of Secondary Structure 76

TECHNIQUES Box 5-1 Determination of Protein Structure 78

The Specific Conformation of a Protein Results from Its Pattern of Hydrogen Bonds 80

αHelices Come ToGether to Form Coiled-Coils 80

Most Proteins Are Modular,Containing Two or Three Domains 82

Proteins Are Composed of a Surprisingly Small Number of Structural Motifs 82

ADVANCED CONCEPTS Box 5-2 Large Proteins Are Often Constructed of Several Smaller Polypeptide Chains 83

Different Protein Functions Arise from Various Domain Combinations 84

Weak Bonds Correctly Position Proteins along DNA and RNA Molecules 85

Proteins Scan along DNA to Locate a Specific DNA-Binding Site 87

Diverse Strategies for Protein Recognition of RNA 88

Allostery:Regulation of a Protein's Function by Changing Its Shape 90

The Structural Basis of Allosteric Regulation Is Known for Examples Involving Small Ligands,Protein-Protein Interactions,and Protein Modification 90

Not All Regulation of Proteins Is Mediated by Allosteric Events 93

SUMMARY 93

BIBLIOGRAPHY 94

PART 2 MAINTENANCE OF THE GENOME 95

CHAPTER 6 The Structures of DNA and RNA 101

DNA Structure 102

DNA Is Composed of Polynucleotide Chains 102

Each Base Has Its Preferred Tautomeric Form 104

The Two Strands of the Double Helix Are Held Together by Base Pairing in an Antiparallel Orientation 105

The Two Chains of the Double Helix Have Complementary Sequences 106

Hydrogen Bonding Is Important for the Specificity of Base Pairing 106

Bases Can Hip Out from the Double Helix 107

DNA Is Usually a Right-Handed Double Helix 107

The Double Helix Has Minor and Major Grooves 108

KEY EXPERIMENTS Box 6-1 DNA Has 10.5 Base Pairs per Turn of the Helix in Solution:The Mica Experiment 108

The Major Groove Is Rich in Chemical Information 109

The Double Helix Exists in Multiple Conformations 110

KEY EXPERIMENTS Box 6-2 How Spots on an X-ray Film Reveal the Structure of DNA 112

DNA Can Sometimes Form a Left-Handed Helix 113

DNA Strands Can Separate(Denature)and Reassociate 113

Some DNA Molecules Are Circles 116

DNA Topology 117

Linking Number Is an Invariant Topological Property of Covalently Closed,Circular DNA 117

Linking Number Is Composed of Twist and Writhe 117

Lk0 Is the Linking Number of Fully Relaxed cccDNA under Physiological Conditions 119

DNA in Cells Is Negatively Supercoiled 120

Nucleosomes Introduce Negative Supercoiling in Eukaryotes 120

Topoisomerases Can Relax Supercoiled DNA 121

Prokaryotes Have a Special Topoisomerase That Introduces Supercoils into DNA 121

Topoisomerases Also Unknot and DisentanGle DNA Molecules 121

Topoisomerases Use a Covalent Protein-DNA Linkage to Cleave and Rejoin DNA Strands 123

Topoisomerases Form an Enzyme Bridge and Pass DNA Segments through Each Other 123

DNA Topoisomers Can Be Separated by Electrophoresis 125

Ethidium Ions Cause DNA to Unwind 126

RNA Structure 127

RNA Contains Ribose and Uracil and Is Usually Single-Stranded 127

RNA Chains Fold Back on Themselves to Form Local Regions of Double Helix Similar to A-Form DNA 127

KEY EXPERIMENTS BOX 6-3 Proving that DNA Has a Helical Periodicity of about 10.5 Base Pairs per Turn from the Topological Properties of DNA Rings 128

RNA Can Fold Up into Complex Tertiary Structures 129

Some RNAs Are Enzymes 130

The Hammerhead Ribozyme Cleaves RNA by the Formation of a 2',3'Cyclic Phosphate 131

Did Life Evolve from an RNA World? 132

SUMMARY 132

BIBLIOGRAPHY 133

CHAPTER 7 Genome Structure,Chromatin,and the Nucleosome 135

Genome Sequence and Chromosome Diversity 136

Chromosomes Can Be Cirular or Linear 136

Every Cell Maintains a Characteristic Number of Chromosomes 137

Genome Size Is Related to the Complexity of the Organism 139

The E.coli Genome Is Composed Almost Entirely of Genes 140

More Complex Organisms Have Decreased Gene Density 140

Genes Make Up Only a Small Proportion of the Eukaryotic Chromosomal DNA 141

The Majority of Human Intergenic Sequences Are Composed of Repetitive DNA 143

Chromosome Duplication and Segregation 144

Eukaryotic Chromosomes Require Centromeres,Telomeres,and Origins of Replication to Be Maintained during Cell Division 144

Eukaryotic Chromosome Duplication and Segregation Occur in Separate Phases of the Cell Cycle 147

Chromosome Structure Changes as Eukaryotic Cells Divide 149

Sister-Chromatid Cohesion and Chromosome Condensation Are Mediated by SMC Proteins 150

Mitosis Maintains the Parental Chromosome Number 152

During Gap Phases,Cells Prepare for the Next Cell Cycle Stage and Check That the Previous Stage Is Completed Correctly 152

Meiosis Reduces the Parental Chromosome Number 154

Different Levels of Chromosome Structure Can Be Observed by Microscopy 156

The Nucleosome 157

Nucleosomes Are the Building Blocks of Chromosomes 157

KEY EXPERIMENTS Box 7-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome 158

Histones Are Small,Positively Charged Proteins 159

The Atomic Structure of the Nucleosome 160

Histones Bind Characteristic Regions of DNA within the Nucleosome 162

Many DNA Sequence-lndependent Contacts Mediate the Interaction between the Core Histones and DNA 162

The Histone Amino-Terminal Tails Stabilize DNA Wrapping around the Octamer 165

Wrapping of the DNA around the Histone Protein Core Stores Negative Superhelicity 166

KEY EXPERIMENTS Box 7-2 Nucleosomes and Superhelical Density 166

Higher-Order Chromatin Structure 169

Heterochromatin and Euchromatin 169

Histone H1 Binds to the Linker DNA between Nucleosomes 169

Nucleosome Arrays Can Form More Complex Structures:The 30-nm Fiber 170

The Histone Amino-Terminal Tails Are Required for the Formation of the 30-nm Fiber 172

Further Compaction of DNA Involves Large Loops of Nucleosomal DNA 172

Histone Variants Alter Nucleosome Function 174

Regulation of Chromatin Structure 174

The Interaction of DNA with the Histone Octamer Is Dynamic 174

Nucleosome-Remodeling Complexes Facilitate Nucleosome Movement 175

Some Nucleosomes Are Found in Specific Positions:Nucleosome Positioning 179

KEY EXPERIMENTS Box 7-3 Determining Nucleosome Position in the Cell 180

Modification of the Amino-Terminal Tails of the Histones Alters Chromatin Accessibility 182

Protein Domains in Nucleosome-Remodeling and-Modifying Complexes Recognize Modified Histones 184

Specific Enzymes Are Responsible for Histone Modification 185

Nucleosome Modification and Remodeling Work Together to Increase DNA Accessibility 186

Nucleosome Assembly 187

Nucleosomes Are Assembled Immediately after DNA Replication 187

Assembly of Nucleosomes Requires Histone"Chaperones" 189

SUMMARY 192

BIBLIOGRAPHy 193

CHAPTER 8 The Replication of DNA 195

The Chemistry of DNA Synthesis 196

DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template Junction 196

DNA Is Synthesized by Extendingthe 3'End of the Primer 197

Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis 198

The Mechanism of DNA Polymerase 198

DNA Polymerases Use a Single Active Site to Catalyze DNA Synthesis 198

TECHNIQUES Box 8-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis 200

DNA Polymerases Resemble a Hand That Gripsthe Primer:Template Junction 202

MEDICAL CONNECTIONS Box 8-2 Anticancer and Antiviral Agents Target DNA Replication 203

DNA Polymerases Are Processive Enzymes 207

Exonucleases Proofread Newly Synthesized DNA 208

The Replication Fork 209

Both Strands of DNA Are Synthesized Togetherat the Replication Fork 209

The Initiation of a New Strand of DNA Requires an RNA Primer 210

RNA Primers Must Be Removed to Complete DNA Replication 211

DNA Helicases Unwind the Double Helix in Advance of the Replication Fork 211

TECHNIQUES Box8-3 Determining the Polarity of a DNA Helicase 212

DNA Helicase Pulls Single-Stranded DNA through a Central Protein Pore 214

Single-Stranded DNA-Binding Proteins Stabilize ssDNA prior to Replication 215

Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork 216

Replication Fork Enzymes Extend the Range of DNA Polymerase Substrates 217

The Specialization of DNA Polymerases 218

DNA Polymerases Are Specialized for Different Roles in the Cell 218

Sliding Clamps Dramatically Increase DNA Polymerase Processivity 219

Sliding Clamps Are Opened and Placed on DNA by Clamp Loaders 222

ADVANCED CONCEPTS Box 8-4 ATP Control of Protein Function:Loading a Sliding Clamp 223

DNA Synthesis at the Replication Fork 225

Interactions between Replication Fork Proteins Form the E.coli Replisome 228

Initiation of DNA Replication 230

Specific Genomic DNA Sequences Direct the Initiation of DNA Replication 230

The Replicon Model of Replication Initiation 230

Replicator Sequences Include Initiator Binding Sites and Easily Unwound DNA 231

KEY EXPERIMENTS Box 8-5 The Identification of OriGins of Replication and Replicators 232

Binding and Unwinding:Origin Selection and Activation by the Initiator Protein 235

Protein-Protein and Protein-DNA Interactions Direct the Initiation Process 235

ADVANCED CONCEPTS Box 8-6 The Replication Factory Hypothesis 237

Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle 239

Prereplicative Complex Formation Is the First Step in the Initiation of Replication in Eukaryotes 240

Pre-RC Formation and Activation Are Regulated to Allow Only a Single Round of Replication during Each Cell Cycle 241

Similarities between Eukaryotic and Prokaryotic DNA Replication Initiation 244

ADVANCED CONCEPTS Box 8-7 E.coli DNA Replication Is Regulated by DnaA·ATP Levels and SeqA 244

Finishing Replication 246

TypeⅡTopoisomerases Are Required to Separate Daughter DNA Molecules 246

Lagging-Strand Synthesis Is Unable to Copy the Extreme Ends of Linear Chromosomes 247

Telomerase Is a Novel DNA Polymerase That Does Not Require an Exogenous Template 248

Telomerase Solves the End Replication Problem by Extending the 3'End of the Chromosome 250

MEDICAL CONNECTIONS Box 8-8 Aging Cancer and the Telomere Hypothesis 251

Telomere-Binding Proteins Regulate Telomerase Activity and Telomere Length 252

Telomere-Binding Proteins Protect Chromosome Ends 253

SUMMARY 255

BIBLIOGRAPHY 256

CHAPTER 9 The Mutability and Repair of DNA 257

Replication Errors and Their Repair 258

The Nature of Mutations 258

Some Replication Errors Escape Proofreading 259

MEDICAL CONNECTIONS Box 9-1 Expansion of Triple Repeats Causes Disease 259

Mismatch Repair Removes Errors That Escape Proofreading 260

DNA Damage 265

DNA Undergoes Damage Spontaneously from Hydrolysis and Deamination 265

DNA Is Damaged by Alkylation,Oxidation,and Radiation 265

MEDICAL CONNECTIONS Box 9-2 The Ames Test 266

Mutations Are Also Caused by Base Analogs and Intercalating Agents 268

Repair of DNA Damage 269

Direct Reversal of DNA Damage 270

Base Excision Repair Enzymes Remove Damaged Bases by a Base-Flipping Mechanism 270

Nucleotide Excision Repair Enzymes Cleave Damaged DNA 273

on Either Side of the Lesion 273

Recombination Repairs DNA Breaks by Retrieving Sequence Information from Undamaged DNA 275

DSBs in DNA Are Also Repaired by Direct Joining of Broken Ends 275

MEDICAL CONNECTIONS Box 9-3 Nonhomologous End Joining 276

Translesion DNA Synthesis Enables Replication to Proceed across DNA Damage 278

ADVANCED CONCEPTS Box 9-4 The Y Family of DNA Polymerases 280

SUMMARY 281

BIBLIOGRAPHY 282

CHAPTER 10 Homologous Recombination at the Molecular Level 283

DNA Breaks Are Common and Initiate Recombination 284

Models for Homologous Recombination 284

Strand Invasion Is a Key Early Step in Homologous Recombination 286

Resolving Holliday Junctions Is a Key Step to Finishing Genetic Exchange 288

The Double-Strand Break-Repair Model Describes Many Recombination Events 288

Homologous Recombination Protein Machines 291

ADVANCED CONCEPTS Box 10-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions 292

The RecBCD Helicase/Nuclease Processes Broken DNA Molecules for Recombination 293

Chi Sites Control RecBCD 296

RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion 297

Newly Base-Paired Partners Are Established within the RecA Filament 299

RecA Homologs Are Present in All Organisms 301

The RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch Migration 301

RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination 302

Homologous Recombination in Eukaryotes 303

Homologous Recombination Has Additional Functions in Eukaryotes 303

Homologous Recombination Is Required for Chromosome Segregation during Meiosis 304

Programmed Generation of Double-Stranded DNA Breaks Occurs during Meiosis 305

MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA-like Strand-Exchange Proteins 307

Dmcl Is a RecA-like Protein That Specifically Functions in Meiotic Recombination 308

Many Proteins Function Together to Promote Meiotic Recombination 308

MEDICAL CONNECTIONS Box 10-2 The Product of the Tumor 309

Suppressor Gene BRCA2 Interacts With Rad51 Protein and Controls Genome Stability 309

Mating-Type Switching 310

Mating-Type Switching Is Initiated by a Site-Specific Double-Strand Break 311

Mating-Type Switching Is a Gene Conversion Event and Not Associated with Crossing Over 312

Genetic Consequences of the Mechanism of Homologous Recombination 314

One Cause of Gene Conversion Is DNA Repairduring Recombination 315

SUMMARY 316

BIBLIOGRAPHY 317

CHAPTER 11 Site-Specific Recombination and Transposition of DNA 319

Conservative Site-Specific Recombination 320

Site-Specific Recombination Occurs at Specific DNA Sequences in the Target DNA 320

Site-Specific Recombinases Cleave and Rejoin DNA Using a Covalent Protein-DNA Intermediate 322

Serine Recombinases Introduce Double-Strand Breaks in DNA and Then Swap Strands to Promote Recombination 324

Structure of the Serine Recombinase-DNA Complex Indicates That Subunits Rotate to Achieve Strand Exchange 325

Tyrosine Recombinases Break and Rejoin One Pair of DNA Strands at a Time 326

Structures of Tyrosine Recombinases Bound to DNA Reveal the Mechanism of DNA Exchange 327

MEDICAL CONNECTIONS Box 11-1 Application of Site-Specific Recombination to Genetic Engineering 327

Biological Roles of Site-Specific Recombination 328

λIntegrase Promotes the Integration and Excision of a Viral Genome into the Host-Cell Chromosome 329

BacteriophageλExcision Requires a New DNA-Bending Protein 331

The Hin Recombinase Inverts a Segment of DNA Allowing Expression of Alternative Genes 331

Hin Recombination Requires a DNA Enhancer 332

Recombinases Convert Multimeric Circular DNA Molecules into Monomers 333

There Are Other Mechanisms to Direct Recombination to Specific Segments of DNA 334

Transposition 334

Some Genetic Elements Move to New Chromosomal Locations by Transposition 334

ADVANCED CONCEPTS Box 11-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids 335

There Are Three Principal Classes of Transposable Elements 338

DNA Transposons Carry a Transposase Gene,Flanked by Recombination Sites 339

Transposons Exist as Both Autonomous and Nonautonomous Elements 339

Virus-like Retrotransposons and Retroviruses Carry Terminal Repeat Sequences and Two Genes Important for Recombination 340

Poly-A Retrotransposons Look Like Genes 340

DNA Transposition by a Cut-and-Paste Mechanism 340

The Intermediate in Cut-and-Paste Transposition Is Finished by Gap Repair 342

There Are Multiple Mechanisms for Cleaving the Nontransferred Strand during DNA Transposition 343

DNA Transposition by a Replicative Mechanism 345

Virus-like Retrotransposons and Retroviruses Move Using anRNA Intermediate 347

ADVANCED CONCEPTS Box 11-3 The Pathway of Retroviral cDNA Formation 349

DNA Transposases and Retroviral Integrases Are Members of a Protein Superfamily 351

Poly-A Retrotransposons Move by a"Reverse Splicing" Mechanism 352

Examples of Transposable Elements and Their Regulation 354

IS4-Family Transposons Are Compact Elements with Multiple Mechanisms for Copy Number Control 355

KEY EXPERIMENTS Box 11-4 Maize Elements and the Discovery of Transposons 356

Tn10 Transposition Is Coupled to Cellular DNA Replication 358

Phage Mu Is an Extremely Robust Transposon 359

Mu Uses Target Immunity to Avoid Transposing into Its Own DNA 359

ADVANCED CONCEPTS Box 11-5 Mechanism of Transposition Target lmmunity 361

Tc1/mariner Elements Are Extremely Successful DNA Elements in Eukaryotes 362

Yeast Ty Elements Transpose into Safe Havens in the Genome 362

LINEs Promote Their Own Transposition and Even Transpose Cellular RNAs 363

V(D)J Recombination 365

The Early Events in V(D)J Recombination Occur by a Mechanism Similar to Transposon Excision 367

SUMMARY 369

BIBLIOGRAPHY 369

PART 3 EXPRESSION OF THE GENOME 371

CHAPTER 12 Mechanisms of Transcription 377

RNA Polymerases and the Transcription Cycle 378

RNA Polymerases Come in Different Forms but Share Many Features 378

Transcription by RNA Polymerase Proceeds in a Series of Steps 380

Transcription Initiation Involves Three Defined Steps 382

The Transcription Cycle in Bacteria 383

Bacterial Promoters Vary in Strength and Sequence but Have Certain Defining Features 383

TheσFactor Mediates Binding of Polymerase to the Promoter 384

Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA 386

TECHNIQUES Box 12-1 Consensus Sequences 388

Transcription Is Initiated by RNA Polymerase without the Need for a Primer 388

During Initial Transcription,RNA Polymerase Remains Stationary and Pulls Downstream DNA into Itself 389

Promoter Escape Involves Breaking Polymerase-Promoter 390

Interactions and Polymerase Core-σInteractions 390

The Elongating Polymerase Is a Processive Machine That Synthesizes and Proofreads RNA 391

ADVANCED CONCEPTS Box 12-2 The Single-Subunit RNA Polymerases 393

RNA Polymerase Can Become Arrested and Need Removing 394

Transcription Is Terminated by Signals within the RNA Sequence 394

Transcription in Eukaryotes 396

RNA PolymeraseⅡCore Promoters Are Made Up of Combinations of Four Different Sequence Elements 397

RNA PolymeraseⅡForms a Preinitiation Complex with General Transcription Factors at the Promoter 398

Promoter Escape Requires Phosphorylation of the Polymerase"Tail" 398

TBP Binds to and Distorts DNA Using aβSheet Inserted into the Minor Groove 400

The Other General Transcription Factors Also Have Specific Roles in Initiation 401

In Vivo,Transcription Initiation Requires Additional Proteins,Including the Mediator Complex 402

Mediator Consists of Many Subunits,Some Conserved from Yeast to Human 403

A New Set of Factors Stimulate PolⅡElongation and RNA Proofreading 404

Elongating RNA Polymerase Must Deal with Histones in Its Path 405

Elongating Polymerase Is Associated with a New Set of Protein Factors Required for Various Types of RNA Processing 406

Transcription Termination Is Linked to RNA Destruction by a Highly Processive RNase 410

Transcription by RNA PolymerasesⅠandⅢ 410

RNA PolⅠand PolⅢRecognize Distinct Promoters,Using Distinct Sets of Transcription Factors,but Still Require TBP 410

PolⅢPromoters Are Found Downstream of Transcription Start Site 412

SUMMARY 413

BIBLIOGRAPHY 414

CHAPTER 13 RNA Splicing 415

The Chemistry of RNA Splicing 417

Sequences within the RNA Determine Where Splicing Occurs 417

The Intron Is Removed in a Form Called a Lariat as the Flanking Exons Are Joined 418

KEY EXPERIMENTS Box 13-1 Adenovirus and the Discovery of Splicing 419

Exons from Different RNA Molecules Can Be Fused by trans-Splicing 421

The Spliceosome Machinery 422

RNA Splicing Is Carried Out by a Large Complex Called the Spliceosome 422

Splicing Pathways 424

Assembly,Rearrangements,and Catalysis within the Spliceosome:The Splicing Pathway 424

Self-Splicing Introns Reveal That RNA Can Catalyze RNA Splicing 426

GroupⅠIntrons Release a Linear Intron Rather Than a Lariat 426

KEY EXPERIMENTS Box 13-2 Converting GroupⅠIntrons into Ribozymes 428

How Does the Spliceosome Find the Splice Sites Reliably? 430

A Small Group of Introns Are Spliced by an Alternative Spliceosome Composed of a Different Set of snRNPs 432

Alternative Splicing 432

Single Genes Can Produce Multiple Products by Alternative Splicing 432

Several Mechanisms Exist to Ensure Mutually Exclusive Splicing 435

The Curious Case of the Drosophila Dscam Cene:Mutually Exclusive Splicing on a Grand Scale 436

Mutually Exclusive Splicing of Dscam Exon 6 Cannot Be Accounted for by Any Standard Mechanism and Instead Uses a Novel Strategy 437

Alternative Splicing Is Regulated by Activators and Repressors 439

Regulation of Alternative Splicing Determines the Sex of Flies 441

KEY EXPERIMENTS Box 13-3 Identification of Docking Site and Selector Sequences 442

MEDICAL CONNECTIONS Box 13-4 Defects in Pre-mRNA Splicing Cause Human Disease 445

Exon Shuffling 446

Exons Are Shuffled by Recombination to Produce Genes Encoding New Proteins 446

RNA Editing 448

RNA Editing Is Another Way of Altering the Sequence of an mRNA 448

Guide RNAs Direct the Insertion and Deletion of Uridines 450

MEDICAL CONNECTIONS Box 13-5 Deaminases and HIV 450

mRNA Transport 452

Once Processed,mRNA Is Packaged and Exported from the Nucleus into the Cytoplasm for Translation 452

SUMMARY 454

BIBLIOGRAPHY 455

CHAPTER 14 Translation 457

Messenger RNA 458

Polypeptide Chains Are Specified by Open Reading Frames 458

Prokaryotic mRNAs Have a Ribosome-Binding Site That Recruits the Translational Machinery 459

Eukaryotic mRNAs Are Modified at Their 5'and 3'Ends to Facilitate Translation 460

Transfer RNA 461

tRNAs Are Adaptors between Codons and Amino Acids 461

ADVANCED CONCEPTS Box 14-1 CCA-Adding Enzymes:Synthesizing RNA without a Template 462

tRNAs Share a Common Secondary Structure That Resembles a Cloverleaf 462

tRNAs Have an L-shaped Three-Dimensional Structure 463

Attachment of Amino Acids to tRNA 464

tRNAs Are Charged by the Attachment of an Amino Acid to the 3'-Terminal Adenosine Nucleotide via a High-Energy Acyl Linkage 464

Aminoacyl-tRNA Synthetases Charge tRNAs in Two Steps 464

Each Aminoacyl-tRNA Synthetase Attaches a Single Amino Acidto One or More tRNAs 466

tRNA Synthetases Recognize Unique Structural Features of Cognate tRNAs 466

Aminoacyl-tRNA Formation Is Very Accurate 468

Some Aminoacyl-tRNA Synthetases Use an Editing 468

Pocket to Charge tRNAs with High Accuracy 468

The Ribosome Is Unable to Discriminate between 469

Correctly and Incorrectly Charged tRNAs 469

The Ribosome 469

ADVANCED CONCEPTS Box 14-2 Selenocysteine 470

The Ribosome Is Composed of a Large and a Small Subunit 471

The Large and Small Subunits Undergo Association and Dissociation during Each Cycle of Translation 472

New Amino Acids Are Attached to the Carboxyl Terminus of the Growing Polypeptide Chain 474

Peptide Bonds Are Formed by Transfer of the Growing Polypeptide Chain from One tRNA to Another 474

Ribosomal RNAs Are Both Structural and Catalytic Determinants of the Ribosome 475

The Ribosome Has Thtee Binding Sites for tRNA 475

Channels through the Ribosome Allow the mRNA and Growing Polypeptide to Enter and/or Exit the Ribosome 476

Initiation of Translation 479

Prokaryotic mRNAs Are Initially Recruited to the Small 480

Subunit by Base Pairing to rRNA 480

A Specialized tRNA Charged with a Modified Methionine Binds Directly to the Prokaryotic Small Subunit 480

Three Initiation Factors Direct the Assembly of an Initiation Complex That Contains mRNA and the Initiator tRNA 481

Eukaryotic Ribosomes Are Recruited to the mRNA by the 5'Cap 482

The Start Codon Is Foundby Scanning Downstream from the 5'End of the mRNA 483

ADVANCED CONCEPTS Box 14-3 uORFs and IRESs:Exceptions That Prove the Rule 485

Translation Initiation Factors Hold Eukaryotic mRNAs in Circles 487

Translation Elongation 487

Aminoacyl-tRNAs Are Delivered to the A Site by Elongation Factor EF-Tu 488

The Ribosome Uses Multiple Mechanisms to Select against Incorrect Aminoacyl-tRNAs 488

The Ribosome Is a Ribozyme 491

Peptide Bond Formation and the Elongation Factor EF-G Drive Translocation of the tRNAs and the mRNA 492

EF-G Drives Translocation by Displacing the tRNA Bound to the A Site 494

EF-Tu-GDP and EF-G-GDP Must Exchange GDP for GTP prior to Participating in a New Round of Elongation 495

A Cycle of Peptide Bond Formation Consumes Two Molecules of GTP and One Molecule of ATP 495

Termination of Translation 496

Release Factors Terminate Translation in Response to Stop Codons 496

Short Regions of ClassⅠRelease Factors Recognize Stop Codons and Trigger Release of the Peptidyl Chain 496

ADVANCED CONCEPTS Box 14-4 GTP-Binding Proteins,Conformational Switching,and the Fidelity and Ordering of the Events of Translation 498

GDP/GTP Exchange and GTP Hydrolysis Control the Function of the ClassⅡRelease Factor 499

The Ribosome Recycling Factor Mimics a tRNA 500

MEDICAL CONNECTIONS Box 14-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation 502

Regulation of Translation 503

Protein or RNA Binding Near the Ribosome-Binding Site Negatively Regulates Bacterial Translation Initiation 504

Regulation of Prokaryotic Translation:Ribosomal Proteins Are Translational Repressors of Their Own Synthesis 505

Global Regulators of Eukaryotic Translation Target Key Factors Required for mRNA Recognition and Initator tRNA Ribosome Binding 508

Spatial Control of Translation by mRNA-Specific 4E-BPs 510

An Iron-Regulated,RNA-Binding Protein Controls Translation of Ferritin 511

Translation of thet Yeast Transcriptional Activator Gcn4 Is Controlled by Short Upstream ORFs and Ternary Complex Abundance 512

Translation-Dependent Regulation of mRNA and Protein Stability 514

The SsrA RNA Rescues Ribosomes That Translate Broken mRNAs 514

Eukaryotic Cells Degrade mRNAs That Are Incomplete or Have Premature Stop Codons 516

SUMMARY 518

BIBLIOGRAPHY 519

CHAPTER 15 The Genetic Code 521

The Code Is Degenerate 521

Perceiving Order in the Makeup of the Code 522

Wobble in the Anticodon 523

Three Codons Direct Chain Termination 525

How the Code Was Cracked 525

Stimulation of Amino Acid Incorporation by Synthetic mRNAs 526

Poly-U Codes for Polyphenylalanine 527

Mixed Copolymers Allowed Additional Codon Assignments 527

Transfer RNA Binding to Defined Trinucleotide Codons 528

Codon Assignments from Repeating Copolymers 529

Three Rules Govern the Genetic Code 530

Three Kinds of Point Mutations Alter the Genetic Code 531

Genetic Proof That the Code Is Readin Units of Three 532

Suppressor Mutations Can Reside in the Same or a Different Gene 532

Intergenic Suppression Involves Mutant tRNAs 533

Nonsense Suppressors Also Read Normal Termination Signals 535

Proving the Validity of the Genetic Code 535

The Code Is Nearly Universal 536

SUMMARY 538

BIBLIOGRAPHY 538

PART 4 REGULATION 541

CHAPTER 16 Transcriptional Regulation in Prokaryotes 547

Principles of Transcriptional Regulation 547

Gene Expression Is Controlled by Regulatory Proteins 547

Most Activators and Repressors Act at the Level of Transcription Initiation 548

Many Promoters Are Regulated by Activators That Help RNA Polymerase Bind DNA and by Repressors That Block That Binding 548

Some Activators and Repressors Work by Allostery and Regulate Steps in Transcriptional Initiation after RNA Polymerase Binding 550

Action at a Distance and DNA Looping 551

Cooperative Binding and Allostery Have Many Roles in Gene Regulation 552

Antitermination and Beyond:Not All of Gene Regulation Targets Transcription Initiation 552

Regulation of Transcription Initiation:Examples from Prokaryotes 553

An Activator and a Repressor Together Controlthe Iac Genes 553

CAPand Lac Repressor Have Opposing Effects on RNA Polymerase Binding to the Iac Promoter 554

CAP Has Separate Activating and DNA-Binding Surfaces 555

CAP and Lac Repressor Bind DNA Using a Common Structural Motif 556

EDY EXPERIMENTS Box 16-1 Activator Bypass Experiments 557

The Activities of Lac Repressor and CAP Are Controlled Allosterically by Their Signals 559

Combinatorial Control:CAP Controls Other Genes As Well 560

KEY EXPERIMENTS Box 16-2 Jacob,Monod,and the Ideas Behind Gene Regulation 561

AlternativeσFactors Direct RNA Polymerase to Alternative Sets of Promoters 563

NtrC and MerR:Transcriptional Activators That Work by Allostery Rather than by Recruitment 564

NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene 564

MerR Activates Transcription by Twisting Promoter DNA 565

Some Repressors Hold RNA Polymerase at the Promoter Rather than Excluding It 566

AraC and Control of the araBAD Operon by Antiactivation 567

The Case of Bacteriophageλ:Layers of Regulation 568

Alternative Patterns of Gene Expression Control Lytic and Lysogenic Growth 569

Regulatory Proteins and Their Binding Sites 570

λRepressor Binds to Operator Sites Cooperatively 571

ADVANCED CONCEPTS Box 16-3 Concentration,Affinity,and Cooperative Binding 572

Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth 573

Lysogenic Induction Requires Proteolytic Cleavage ofλRepressor 574

Negative Autoregulation of Repressor Requires Long-Distance Interactions and a Large DNA Loop 575

Another Activator,λCll,Controls the Decision between Lytic and Lysogenic Growth upon Infection of a New Host 577

The Number of Phage Particles Infecting a Given Cell Affects Whether the Infection Proceeds Lytically or Lysogenically 578

Growth Conditions of E.coli Control the Stability of Cll Protein and thus the Lytic/Lysogenic Choice 578

KEY EXPERIMENTS Box 16-4 Evolution of theλSwitch 579

KEY EXPERIMENTS Box 16-5 Genetic Approaches That Identified Genes Involved in the Lytic/Lysogenic Choice 581

Transcriptional Antitermination inλDevelopment 582

Retroregulation:An Interplay of Controls on RNA Synthesis and Stability Determines int Gene Expression 584

SUMMARY 585

BIBLIOGRAPHY 586

CHAPTER 17 Transcriptional Regulation in Eukaryotes 589

Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals 591

Activators Have Separate DNA-Binding and Activating Functions 591

Eukaryotic Regulators Use a Range of DNA-Binding Domains,but DNA Recognition Involves the Same Principles as Found in Bacteria 593

TECHNIQUES Box 17-1 The Two-Hybrid Assay 594

Activating Regions Are Not Well-Defined Structures 596

Recruitment of Protein Complexes to Genes by Eukaryotic Activators 597

Activators Recruit the Transcriptional Machinery to the Gene 597

Activators Also Recruit Nucleosome Modiffers That Help the Transcriptional Machinery Bind at the Promoter or Initiate Transcription 598

Activators Recruit an Additional Factor Needed for Efficient Initiation or Elongation at Some Promoters 600

Action at a Distance:Loops and Insulators 601

Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions 603

KEY EXPERIMENTS Box 17-2 Long-Distance Interactions on the Same and Different Chromosomes 604

Signal Integration and Combinatorial Control 605

Activators Work Synergistically to Integrate Signals 605

SignalIntegration:The HO Gene Is Controlledby Two Regulators—One Recruits Nucleosome Moditiers and the Other Recruits Mediator 607

Signal Integration:Cooperative Binding of Activators at theHumanβ-Interferon Gene 608

Combinatorial Control Lies at the Heart of the Complexity and Diversity of Eukaryotes 610

Combinatorial Control of the Mating-Type Genes from S.cerevisiae 611

KEY EXPERIMENTS Box 17-3 Evolvability of a Regulatory Circuit 612

Transcriptional Repressors 613

Signal Transduction and the Control of Transcriptional Regulators 615

Signals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways 615

Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways 617

Activators and Repressors Sometimes Come in Pieces 619

Gene"Silencing"by Modification of Histones and DNA 620

Silencing in Yeast Is Mediated by Deacetylation and Methylation of Histones 621

In Drosophila,HP1 Recognizes Methylated Histones and Condenses Chromatin 622

ADVANCED CONCEPTS Box 17-4 Is There a Histone Code? 623

DNA Methylation Is Associated with Silenced Genes in Mammalian Cells 624

MEDICAL CONNECTIONS Box 17-5 Transcriptional Repression and Human Disease 626

Epigenetic Gene Regulation 626

Some States of Gene Expression Are Inherited through Cell Division Even When the Initiating Signal Is No Longer Present 627

MEDICAL CONNECTIONS Box 17-6 Using Transcription Factors to Reprogram Somatic Cells into Embryonic Stem Cells 629

SUMMARY 630

BIBLIOGRAPHY 631

CHAPTER 18 Regulatory RNAs 633

Regulation by RNAs in Bacteria 633

Riboswitches Reside within the Transcripts of Genes 635

Whose Expression They Control through Changes in Secondary Structure 635

ADVANCED CONCEPTS Box 18-1 Amino Acid Biosynthetic Operons Are Controlled by Attenuation 639

RNA Interference Is a Major Regulatory Mechanism in Eukaryotes 641

Short RNAs That Silenee Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in Three Different Ways 641

Synthesis and Function of miRNA Molecules 643

miRNAs Have a Characteristic Structure That Assists in Identifying Them and Their Target Genes 643

An Active miRNA Is Generated through a Two-Step Nucleolytic Processing 645

Dicer Is the Second RNA-Cleaving Enzyme Involved in miRNA Production 646

Incorporation of a Guide Strand RNA into RISC Makes the Mature Complex That Is Ready to Silence Gene Expression 647

siRNAs Are Regulatory RNAs Generated from Long Double-Stranded RNAs 649

Small RNAs Can Transcriptionally Silence Genes by Directing Chromatin Modification 649

KEY EXPERIMENTS Box 18-2 History of miRNAs and RNAi 650

The Evolution and Exploitation of RNAi 652

Did RNAi EvolveAs an Immune System? 652

RNAi Has Become a Powerful Tool for Manipulating Gene Expression 654

MEDICAL CONNECTIONS Box 18-3 RNAi and Human Disease 656

Regulatory RNAs and X-inactivation 657

X-inactivation Creates Mosaic Individuals 657

Xist Is an RNA Regulator That Inactivates a Single X Chromosome in Female Mammals 657

SUMMARY 659

BIBLIOGRAPHY 660

CHAPTER 19 Gene Regulation in Development and Evolution 661

TECHNIQUES Box 19-1 Microarray Assays:Theory and Practice 662

Three Strategies by Which Cells Are Instructed to Express Specific Sets of Genes during Development 663

Some mRNAs Become Localized within Eggs and Embryos because of an Intrinsic Polarity in the Cytoskeleton 663

Cell-to-Cell Contact and Secreted Cell-Signaling Molecules Both Elicit Changes in Gene Expression in Neighboring Cells 664

Gradients of Secreted Signaling Molecules Can Instruct Cells to Follow Different Pathways of Development Based on Their Location 665

Examples of the Three Strategies for Establishing Differential Gene Expression 666

The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO Gene 666

ADVANCED CONCEPTS Box 19-2 Review of Cytoskeleton:Asymmetry and Growth 669

A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo 670

ADVANCED CONCEPTS Box 19-3 Overview of Ciona Development 671

Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium,Bacillus subtilis 672

A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect Central Nervous System 673

A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube 674

The Molecular Biology of Drosophila Embryogenesis 676

An Overview of Drosophila Embryogenesis 676

ADVANCED CONCEPTS Box 19-4 Overview of Drosophila Development 677

A Morphogen Gradient Controls Dorsoventral Patterning of the Drosophila Embryo 679

Segmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Egg 682

Bicoid and Nanos Regulate hunchback 683

KEY EXPERIMENTS Box 19-5 The Role of Activator Synergy in Development 684

MEDICAL CONNECTIONS Box 19-6 Stem Cells 686

The Gradient of Hunchback Repressor Establishes Different Limits of Gap Gene Expression 687

Hunchback and Gap Proteins Produce Segmentation Stripes of Gene Expression 688

Gap Repressor Gradients Produce Many Stripes of Gene Expression 689

KEY EXPERIMENTS Box 19-7 cis-Regulatory Sequences in Animal Development and Evolution 690

Short-Range Transcriptional Repressors Permit Different Enhancers to Work Independently of One Another within the Complex eve Regulatory Region 692

Homeotic Genes:An Important Class of Developmental Regulators 693

Changes in Homeotic Gene Expression Are Responsible for Arthropod Diversity 695

Arthropods Are Remarkably Diverse 695

Changesin Ubx Expression Explain Modification of Limbs among the Crustaceans 695

ADVANCED CONCEPTS Box 19-8 Homeotic Genes of Drosophila AreOrganized in Special Chromosome Clusters 696

Why Insects Lack Abdominal Limbs 698

Modification of Flight Limbs Might Arise from the Evolution of Regulatory DNA Sequences 699

SUMMARY 701

BIBLIOGRAPHY 702

CHAPTER 20 Genome Analysis and Systems Biology 703

Genomics Overview 703

Bioinformatics Tools Facilitate the Genome-wide Identification of Protein-Coding Genes 703

Whole-Genome Tiling Arrays Are Used to Visualize the Transcriptome 704

Regulatory DNA Sequences Can Be Identified by Using Specialized Alignment Tools 706

The ChIP-Chip Assay Is the Best Method forIdentifying Enhancers 708

TECHNIQUES Box 20-1 Bioinformatics Methods for the Identification of Complex Enhancers 708

Diverse Animals Contain Remarkably Similar Sets of Genes 711

Many Animals Contain Anomalous Genes 712

Synteny Is Evolutionarily Ancient 713

Deep Sequencing Is Being Used to Explore Human Origins 715

Systems Biology 715

Transcription Circuits Consist of Nodes and Edges 716

Negative Autoregulation Dampens Noise and Allows a Rapid Response Time 717

Gene Expression Is Noisy 718

Positive Autoregulation Delays Gene Expression 720

Some Regulatory Circuits Lock in Alternative Stable States 720

Feed-Forward Loops Are Three-Node Networks with Beneficial Properties 722

KEY EXPERIMENTS Box 20-2 Bistability and Hysteresis 722

Feed-Forward Loops Are Used in Development 725

Some Circuits Generate Oscillating Patterns of Gene Expression 727

Synthetic Circuits Mimic Some of the Features of Natural Regulatory Networks 729

Prospects 730

SUMMARY 730

BIBLIOGRAPHY 731

PART 5 METHODS 733

CHAPTER 21 Techniques of Molecular Biology 739

Nucleic Acids 740

Electrophoresis through a Gel Separates DNA and RNA Molecules according to Size 740

Restriction Endonucleases Cleave DNA Molecules at Particular Sites 742

DNA Hybridization Can Be Used to Identify Specific DNA Molecules 743

Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs 744

Isolation of Specific Segments of DNA 746

DNA Cloning 746

Cloning DNA in Plasmid Vectors 746

Vector DNA Can Be Introduced into Host Organisms by Transformation 748

Libraries of DNA Molecules Can Be Created by Cloning 748

Hybridization Can Be Used to Identify a Specific Clone in a DNA Library 749

Chemically Synthesized Oligonucleotides 750

The Polymerase Chain Reaction Amplifies DNAs by Repeated Rounds of DNA Replication in Vitro 751

TECHNIQUES Box 21-1 Forensics and the Polymerase Chain Reaction 753

Nested Sets of DNA Fragments Reveal Nucleotide Sequences 753

KEY EXPERIMENTS Box 21-2 Sequenators Are Used for High-Throughput Sequencing 757

Shotgun Sequencing a Bacterial Genome 757

The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences 758

The Paired-End Strategy Permits the Assembly of Large-Genome Scaffolds 760

The$1000 Human Genome Is within Reach 762

Proteins 764

Specific Proteins Can Be Purified from Cell Extracts 764

Purification of a Protein Requires a Specific Assay 764

Preparation of Cell Extracts Containing Active Proteins 765

Proteins Can Be Separated from One Another Using Column Chromatography 765

Affinity Chromatography Can Facilitate More Rapid Protein Purification 767

Separation of Proteins on Polyacrylamide Gels 768

Antibodies Are Used to Visualize Electrophoretically Separated Proteins 769

Protein Molecules Can Be Directly Sequenced 769

Proteomics 771

Combining Liquid Chromatography With Mass Spectrometry Identifes Individual Proteins within a Complex Extract 771

Proteome Comparisons Identify Important Differences beween Cells 773

Mass Spectrometry Can Also Monitor Protein Modification States 773

Protein-Protein Interactions Can Yield Information about Protein Function 774

Nucleic Acid-Protein Interactions 775

The Electrophoretic Mobility of DNA Is Altered by Protein Binding 776

DNA-Bound Protein Protects the DNA from Nucleases and Chemical Modification 777

Chromatin Immunoprecipitation Can Detect Protein Association with DNA in the Cell 778

In Vitro Selection Can Be Used to Identify a Protein's DNA-or RNA-Binding Site 780

BIBLIOGRAPHY 782

CHAPTER 22 Model Organisms 783

Bacteriophage 784

Assays of Phage Growth 786

The Single-Step Growth Curve 787

Phage Crosses and Complementation Tests 787

Transduction and Recombinant DNA 788

Bacteria 789

Assays of Bacterial Growth 789

Bacteria Exchange DNA by Sexual Conjugation,Phage-Mediated Transduction,and DNA-Mediated Transformation 790

Bacterial Plasmids Can Be Usedas Cloning Vectors 791

Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions 791

Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Technology,Whole-Genome Sequencing,and Transcriptional Profiling 793

Biochemical Analysis Is Especially Powerful in Simple Cells with Well-Developed Tools of Traditional and Molecular Genetics 793

Bacteria Are Accessible to Cytological Analysis 793

Phage and Bacteria Told Us Most of the Fundamental Things about the Gene 794

Baker's Yeast,Saccharomyces cerevisiae 795

The Existence of Haploid and Diploid Cells Facilitate Genetic Analysis of S.cerevisiae 795

Generating Precise Mutations in Yeast Is Easy 796

S.cerevisiae Has a Small,Well-Characterized Genome 796

S.cerevisiae Cells Change Shape as They Grow 797

Arabidopsis 798

Arabidopsis Has a Fast Life Cycle with Haploid and Diploid Phases 798

Arabidopsis Is Easily Transformed for Reverse Genetics 799

Arabidopsis Has a Small Genome That Is Readily Manipulated 800

Epigenetics 801

Plants Respond to the Environment 801

Development and Pattern Formation 802

The Nematode Worm,Caenorhabditis elegans 802

C.elegans Has a Very Rapid Life Cycle 803

C.elegans Is Composed of Relatively Few,Well-Studied Cell Lineages 804

The Cell Death Pathway Was Discovered in C.elegans 805

RNAi Was Discovered in C.elegans 805

The Fruit Fly,Drosophila melanogaster 806

Drosophila Has a Rapid Life Cycle 806

The First Genome Maps Were Produced in Drosophila 807

Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Hies 809

The Yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics 809

It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA 810

The House Mouse,Mus musculus 812

Mouse Embryonic Development Depends on Stem Cells 813

It Is Easy to Introduce Foreign DNA into the Mouse Embryo 813

Homologous Recombination Permits the Selective Ablation of Individual Genes 814

Mice Exhibit Epigenetic Inheritance 816

BIBLIOGRAPHY 818

Index 819

Advanced Concepts 6

Box 1-1 Mendelian Laws 6

Box 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness 53

Box 5-2 Large Proteins Are Often Constructed of Several Smaller Polypeptide Chains 83

Box 8-4 ATP Control of Protein Function:Loading a Sliding Clamp 223

Box 8-6 The Replication Factory Hypothesis 237

Box 8-7 E.coli DNA Replication Is Regulated by DnaA·ATP Levels and SeqA 244

Box 9-4 The Y Family of DNA Polymerases 280

Box 10-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions 292

Box 11-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids 335

Box 11-3 The Pathway of Retroviral cDNA Formation 349

Box 11-5 Mechanism of Transposition Target lmmunity 361

Box 12-2 The Single-Subunit RNA Polymerases 393

Box 14-1 CCA-Adding Enzymes:Synthesizing RNA without a Template 462

Box 14-2 Selenocysteine 470

Box 14-3 uORFs and IRESs:Exceptions That Prove the Rule 485

Box 14-4 GTP-Binding Proteins,Conformational Switching,and the Fidelity and Ordering of the Events of Translation 498

Box 16-3 Concentration,Affinity,and Cooperative Binding 572

Box 17-4 Is There a Histone Code? 623

Box 18-1 Amino Acid Biosynthetic Operons Are Controlled by Attenuation 639

Box 19-2 Review of Cytoskeleton:Asymmetry and Growth 669

Box 19-3 Overview of Ciona Development 671

Box 19-4 Overview of Drosophila Development 677

Box 19-8 Homeotic Genes of Drosophila Are Organized in Special Chromosome Clusters 696

Key Experiments 10

Box 1-2 Genes Are Linked to Chromosomes 10

Box 2-1 Chargaff's Rules 24

Box 2-2 Evidence That Genes Control Amino Acid Sequences in Proteins 29

Box 6-1 DNA Has 10.5 Base Pairs per Turn of the Helix in Solution:The Mica Experiment 108

Box 6-2 How Spots on an X-ray Film Reveal the Structure of DNA 112

Box 6-3 Proving that DNA Has a Helical Periodicity of about 10.5 Base Pairs per Turn from the Topological Properties of DNA Rings 128

Box 7-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome 158

Box 7-2 Nucleosomes and Superhelical Density 166

Box 7-3 Determining Nucleosome Position in the Cell 180

Box 8-5 The Identification of Origins of Replication and Replicators 232

Box 11-4 Maize Elements and the Discovery of Transposons 356

Box 13-1 Adenovirus and the Discovery of Splicing 419

Box 13-2 Converting GroupⅠIntrons into Ribozymes 428

Box 13-3 Identification of Docking Site and Selector Sequences 442

Box 16-1 Activator Bypass Experiments 557

Box 16-2 Jacob,Monod,and the Ideas Behind Cene Regulation 561

Box 16-4 Evolution of theλSwitch 579

Box 16-5 Genetic Approaches That Identiffed Genes Involved in the Lytic/Lysogenic Choice 581

Box 17-2 Long-Distance Interactions on the Same and Different Chromosomes 604

Box 17-3 Evolvability of a Regulatory Circuit 612

Box 18-2 History of miRNAs and RNAi 650

Box 19-5 The Role of Activator Synergy in Development 684

Box 19-7 cis-Regulatory Sequences in Animal Development and Evolution 690

Box 20-2 Bistability and Hysteresis 722

Box 21-2 Sequenators Are Used for High-Throughput Sequencing 757

Medical Connections 203

Box 8-2 Anticancer and Antiviral Agents Target DNA Replication 203

Box 8-8 Aging,Cancer,and the Telomere Hypothesis 251

Box 9-1 Expansion of Triple Repeats Causes Disease 259

Box 9-2 The Ames Test 266

Box 9-3 Nonhomologous End Joining 276

Box 10-2 The Product of the Tumor Suppressor Gene BRCA2 Interacts with Rad51 Protein and Controls Genome Stability 309

Box 11-1 Application of Site-Specific Recombination to Genetic Engineering 327

Box 13-4 Defects in Pre-mRNA Splicing Cause Human Disease 445

Box 13-5 Deaminases and HIV 450

Box 14-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation 502

Box 17-5 Transcriptional Repression and Human Disease 626

Box 17-6 Using Transcription Factors to Reprogram Somatic Cells into Embryonic Stem Cells 629

Box 18-3 RNAi and Human Disease 656

Box 19-6 Stem Cells 686

Techniques 78

Box 5-1 Determination of Protein Structure 78

Box 8-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis 200

Box 8-3 Determining the Polarity of a DNA Helicase 212

Box 12-1 Consensus Sequences 388

Box 17-1 The Two-Hybrid Assay 594

Box 19-1 Microarray Assays:Theory and Practice 662

Box 20-1 Bioinformatics Methods for the Identification of Complex Enhancers 708

Box 21-1 Forensics and the Polyrnerase Chain Reaction 753