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PART 1 CHEMISTRY AND GENETICS1

1 The Mendelian View of the World5

2 Nucleic Acids Convey Genetic Information19

3 The Importance of Weak Chemical Interactions43

4 The Importance of High-Energy Bonds57

5 Weak and Strong Bonds Determine Macromolecular Structure71

PART 2 MAINTENANCE OF THE GENOME95

6 The Structures of DNA and RNA101

7 Genome Structure,Chromatin,and the Nucleosome135

8 The Replication of DNA195

9 The Mutability and Repair of DNA257

10 Homologous Recombination at the Molecular Level283

11 Site-Specific Recombination and Transposition of DNA319

PART 3 EXPRESSION OF THE GENOME371

12 Mechanisms of Transcription377

13 RNA Splicing415

14 Translation457

15 The Genetic Code521

PART 4 REGULATION541

16 Transcriptional Regulation in Prokaryotes547

17 Transcriptional Regulation in Eukaryotes589

18 Regulatory RNAs633

19 Gene Regulation in Development and Evolution661

20 Genome Analysis and Systems Biology703

PART 5 METHODS733

20 Techniques of Molecular Biology739

21 Model Organisms783

Index819

PART 1 CHEMISTRY AND GENETICS1

CHAPTER 1 The Mendelian View of the World5

Mendel's Discoveries6

The Principle of Independent Segregation6

ADVANCED CONCEPTS Box 1-1 Mendelian Laws6

Some Alleles Are neither Dominant nor Recessive8

Principle of Independent Assortment8

Chromosomal Theory of Heredity8

Gene Linkage and Crossing Over9

KEY EXPERIMENTS Box 1-2 Genes Are Linked to Chromosomes10

Chromosome Mapping12

The Origin of Genetic Variability through Mutations15

Early Speculations about What Genes Are and How They Act16

Preliminary Attempts to Find a Gene-Protein Relationship16

SUMMARY17

BIBLIOGRAPHY18

CHAPTER 2 Nucleic Acids Convey Genetic Information19

Avery's Bombshell:DNA Can Carry Genetic Specificity20

Viral Genes Are Also Nucleic Acids21

The Double Helix21

Finding the Polymerases That Make DNA23

KEY EXPERIMENTS Box 2-1 Chargaff's Rules24

Experimental Evidence Favors Strand Separation during DNA Replication25

The Genetic Information within DNA Is Conveyed by the Sequence of Its Four Nucleotide Building Blocks28

KEY EXPERIMENTS Box2-2,Evidence That Genes Control Amino Acid Sequences in Proteins29

DNA Cannot Be the Template That Directly Orders Amino Acids during Protein Synthesis30

RNA Is Chemically Very Similar to DNA30

The Central Dogma32

The Adaptor Hypothesis of Crick32

Discovery of Transfer RNA32

The Paradox of the Nonspecific-Appearing Ribosomes33

Discovery of Messenger RNA(mRNA)34

Enzymatic Synthesis of RNA upon DNA Templates35

Establishing the Genetic Code36

Establishing the Direction of Protein Synthesis38

Start and Stop Signals Are Also Encoded within DNA39

The Era of Genomics39

SUMMARY40

BIBLIOGRAPHY41

CHAPTER 3 The Importance of Weak Chemical Interactions43

Characteristics of Chemical Bonds43

Chemical Bonds Are Explainable in Quantum-Mechanical Terms44

Chemical-Bond Formation Involves a Change in the Form of Energy45

Equilibrium between Bond Making and Breaking45

The Concept of Free Energy46

Keq Is Exponentially Related to ΔG46

Covalent Bonds Are Very Strong46

Weak Bonds in Biological Systems47

Weak Bonds Have Energies between 1 and 7 kcal/mol47

Weak Bonds Are Constantly Made and Broken at Physiological Temperatures47

The Distinction between Polar and Nonpolar Molecules47

van der Waals Forces48

Hydrogen Bonds49

Some Ionic Bonds Are Hydrogen Bonds50

Weak Interactions Demand Complementary Molecular Surfaces51

Water Molecules Form Hydrogen Bonds51

Weak Bonds between Molecules in Aqueous Solutions51

Organic Molecules That Tend to Form Hydrogen Bonds Are Water Soluble52

ADVANCED CONCEPTS Box 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness53

Hydrophobic"Bonds"Stabilize Macromolecules54

The Advantage of△G between 2 and 5 kcal/mole55

Weak Bonds Attach Enzymes to Substrates55

Weak Bonds Mediate Most Protein-DNA and Protein-Protein Interactions55

SUMMARY56

BIBLIOGRAPHY56

CHAPTER 4 The Importance of High-Energy Bonds57

Molecules That Donate Energy Are Thermodynamically Unstable57

Enzymes Lower Activation Energies in Biochemical Reactions59

Free Energy in Biomolecules60

High-Energy Bonds Hydrolyze with Large Negative△G60

High-Energy Bonds in Biosynthetic Reactions62

Peptide Bonds Hydrolyze Spontaneously62

Coupling of Negative with Positive△G63

Activation of Precursors in Group Transfer Reactions63

ATP Versatility in Group Transfer64

Activation of Amino Acids by Attachment of AMP65

Nucleic Acid Precursors Are Activated by the Presence of P～P66

The Value of P～P Release in Nucleic Acid Synthesis66

P～P Splits Characterize Most Biosynthetic Reactions67

SUMMARY68

BIBLIOGRAPHY69

CHAPTER 5 Weak and Strong Bonds Determine Macromolecular Structure71

Higher-Order Structures Are Determined by Intra-and Intermolecular Interactions71

DNA Can Form a Regular Helix71

RNA Forms a Wide Variety of Structures73

Chemical Features of Protein Building Blocks73

The Peptide Bond75

There Are Four Levels of Protein Structure75

αHelices andβSheets Are the Common Forms of Secondary Structure76

TECHNIQUES Box 5-1 Determination of Protein Structure78

The Specific Conformation of a Protein Results from Its Pattern of Hydrogen Bonds80

αHelices Come ToGether to Form Coiled-Coils80

Most Proteins Are Modular,Containing Two or Three Domains82

Proteins Are Composed of a Surprisingly Small Number of Structural Motifs82

ADVANCED CONCEPTS Box 5-2 Large Proteins Are Often Constructed of Several Smaller Polypeptide Chains83

Different Protein Functions Arise from Various Domain Combinations84

Weak Bonds Correctly Position Proteins along DNA and RNA Molecules85

Proteins Scan along DNA to Locate a Specific DNA-Binding Site87

Diverse Strategies for Protein Recognition of RNA88

Allostery:Regulation of a Protein's Function by Changing Its Shape90

The Structural Basis of Allosteric Regulation Is Known for Examples Involving Small Ligands,Protein-Protein Interactions,and Protein Modification90

Not All Regulation of Proteins Is Mediated by Allosteric Events93

SUMMARY93

BIBLIOGRAPHY94

PART 2 MAINTENANCE OF THE GENOME95

CHAPTER 6 The Structures of DNA and RNA101

DNA Structure102

DNA Is Composed of Polynucleotide Chains102

Each Base Has Its Preferred Tautomeric Form104

The Two Strands of the Double Helix Are Held Together by Base Pairing in an Antiparallel Orientation105

The Two Chains of the Double Helix Have Complementary Sequences106

Hydrogen Bonding Is Important for the Specificity of Base Pairing106

Bases Can Hip Out from the Double Helix107

DNA Is Usually a Right-Handed Double Helix107

The Double Helix Has Minor and Major Grooves108

KEY EXPERIMENTS Box 6-1 DNA Has 10.5 Base Pairs per Turn of the Helix in Solution:The Mica Experiment108

The Major Groove Is Rich in Chemical Information109

The Double Helix Exists in Multiple Conformations110

KEY EXPERIMENTS Box 6-2 How Spots on an X-ray Film Reveal the Structure of DNA112

DNA Can Sometimes Form a Left-Handed Helix113

DNA Strands Can Separate(Denature)and Reassociate113

Some DNA Molecules Are Circles116

DNA Topology117

Linking Number Is an Invariant Topological Property of Covalently Closed,Circular DNA117

Linking Number Is Composed of Twist and Writhe117

Lk0 Is the Linking Number of Fully Relaxed cccDNA under Physiological Conditions119

DNA in Cells Is Negatively Supercoiled120

Nucleosomes Introduce Negative Supercoiling in Eukaryotes120

Topoisomerases Can Relax Supercoiled DNA121

Prokaryotes Have a Special Topoisomerase That Introduces Supercoils into DNA121

Topoisomerases Also Unknot and DisentanGle DNA Molecules121

Topoisomerases Use a Covalent Protein-DNA Linkage to Cleave and Rejoin DNA Strands123

Topoisomerases Form an Enzyme Bridge and Pass DNA Segments through Each Other123

DNA Topoisomers Can Be Separated by Electrophoresis125

Ethidium Ions Cause DNA to Unwind126

RNA Structure127

RNA Contains Ribose and Uracil and Is Usually Single-Stranded127

RNA Chains Fold Back on Themselves to Form Local Regions of Double Helix Similar to A-Form DNA127

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 Rings128

RNA Can Fold Up into Complex Tertiary Structures129

Some RNAs Are Enzymes130

The Hammerhead Ribozyme Cleaves RNA by the Formation of a 2',3'Cyclic Phosphate131

Did Life Evolve from an RNA World?132

SUMMARY132

BIBLIOGRAPHY133

CHAPTER 7 Genome Structure,Chromatin,and the Nucleosome135

Genome Sequence and Chromosome Diversity136

Chromosomes Can Be Cirular or Linear136

Every Cell Maintains a Characteristic Number of Chromosomes137

Genome Size Is Related to the Complexity of the Organism139

The E.coli Genome Is Composed Almost Entirely of Genes140

More Complex Organisms Have Decreased Gene Density140

Genes Make Up Only a Small Proportion of the Eukaryotic Chromosomal DNA141

The Majority of Human Intergenic Sequences Are Composed of Repetitive DNA143

Chromosome Duplication and Segregation144

Eukaryotic Chromosomes Require Centromeres,Telomeres,and Origins of Replication to Be Maintained during Cell Division144

Eukaryotic Chromosome Duplication and Segregation Occur in Separate Phases of the Cell Cycle147

Chromosome Structure Changes as Eukaryotic Cells Divide149

Sister-Chromatid Cohesion and Chromosome Condensation Are Mediated by SMC Proteins150

Mitosis Maintains the Parental Chromosome Number152

During Gap Phases,Cells Prepare for the Next Cell Cycle Stage and Check That the Previous Stage Is Completed Correctly152

Meiosis Reduces the Parental Chromosome Number154

Different Levels of Chromosome Structure Can Be Observed by Microscopy156

The Nucleosome157

Nucleosomes Are the Building Blocks of Chromosomes157

KEY EXPERIMENTS Box 7-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome158

Histones Are Small,Positively Charged Proteins159

The Atomic Structure of the Nucleosome160

Histones Bind Characteristic Regions of DNA within the Nucleosome162

Many DNA Sequence-lndependent Contacts Mediate the Interaction between the Core Histones and DNA162

The Histone Amino-Terminal Tails Stabilize DNA Wrapping around the Octamer165

Wrapping of the DNA around the Histone Protein Core Stores Negative Superhelicity166

KEY EXPERIMENTS Box 7-2 Nucleosomes and Superhelical Density166

Higher-Order Chromatin Structure169

Heterochromatin and Euchromatin169

Histone H1 Binds to the Linker DNA between Nucleosomes169

Nucleosome Arrays Can Form More Complex Structures:The 30-nm Fiber170

The Histone Amino-Terminal Tails Are Required for the Formation of the 30-nm Fiber172

Further Compaction of DNA Involves Large Loops of Nucleosomal DNA172

Histone Variants Alter Nucleosome Function174

Regulation of Chromatin Structure174

The Interaction of DNA with the Histone Octamer Is Dynamic174

Nucleosome-Remodeling Complexes Facilitate Nucleosome Movement175

Some Nucleosomes Are Found in Specific Positions:Nucleosome Positioning179

KEY EXPERIMENTS Box 7-3 Determining Nucleosome Position in the Cell180

Modification of the Amino-Terminal Tails of the Histones Alters Chromatin Accessibility182

Protein Domains in Nucleosome-Remodeling and-Modifying Complexes Recognize Modified Histones184

Specific Enzymes Are Responsible for Histone Modification185

Nucleosome Modification and Remodeling Work Together to Increase DNA Accessibility186

Nucleosome Assembly187

Nucleosomes Are Assembled Immediately after DNA Replication187

Assembly of Nucleosomes Requires Histone"Chaperones"189

SUMMARY192

BIBLIOGRAPHy193

CHAPTER 8 The Replication of DNA195

The Chemistry of DNA Synthesis196

DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template Junction196

DNA Is Synthesized by Extendingthe 3'End of the Primer197

Hydrolysis of Pyrophosphate Is the Driving Force for DNA Synthesis198

The Mechanism of DNA Polymerase198

DNA Polymerases Use a Single Active Site to Catalyze DNA Synthesis198

TECHNIQUES Box 8-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis200

DNA Polymerases Resemble a Hand That Gripsthe Primer:Template Junction202

MEDICAL CONNECTIONS Box 8-2 Anticancer and Antiviral Agents Target DNA Replication203

DNA Polymerases Are Processive Enzymes207

Exonucleases Proofread Newly Synthesized DNA208

The Replication Fork209

Both Strands of DNA Are Synthesized Togetherat the Replication Fork209

The Initiation of a New Strand of DNA Requires an RNA Primer210

RNA Primers Must Be Removed to Complete DNA Replication211

DNA Helicases Unwind the Double Helix in Advance of the Replication Fork211

TECHNIQUES Box8-3 Determining the Polarity of a DNA Helicase212

DNA Helicase Pulls Single-Stranded DNA through a Central Protein Pore214

Single-Stranded DNA-Binding Proteins Stabilize ssDNA prior to Replication215

Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork216

Replication Fork Enzymes Extend the Range of DNA Polymerase Substrates217

The Specialization of DNA Polymerases218

DNA Polymerases Are Specialized for Different Roles in the Cell218

Sliding Clamps Dramatically Increase DNA Polymerase Processivity219

Sliding Clamps Are Opened and Placed on DNA by Clamp Loaders222

ADVANCED CONCEPTS Box 8-4 ATP Control of Protein Function:Loading a Sliding Clamp223

DNA Synthesis at the Replication Fork225

Interactions between Replication Fork Proteins Form the E.coli Replisome228

Initiation of DNA Replication230

Specific Genomic DNA Sequences Direct the Initiation of DNA Replication230

The Replicon Model of Replication Initiation230

Replicator Sequences Include Initiator Binding Sites and Easily Unwound DNA231

KEY EXPERIMENTS Box 8-5 The Identification of OriGins of Replication and Replicators232

Binding and Unwinding:Origin Selection and Activation by the Initiator Protein235

Protein-Protein and Protein-DNA Interactions Direct the Initiation Process235

ADVANCED CONCEPTS Box 8-6 The Replication Factory Hypothesis237

Eukaryotic Chromosomes Are Replicated Exactly Once per Cell Cycle239

Prereplicative Complex Formation Is the First Step in the Initiation of Replication in Eukaryotes240

Pre-RC Formation and Activation Are Regulated to Allow Only a Single Round of Replication during Each Cell Cycle241

Similarities between Eukaryotic and Prokaryotic DNA Replication Initiation244

ADVANCED CONCEPTS Box 8-7 E.coli DNA Replication Is Regulated by DnaA·ATP Levels and SeqA244

Finishing Replication246

TypeⅡTopoisomerases Are Required to Separate Daughter DNA Molecules246

Lagging-Strand Synthesis Is Unable to Copy the Extreme Ends of Linear Chromosomes247

Telomerase Is a Novel DNA Polymerase That Does Not Require an Exogenous Template248

Telomerase Solves the End Replication Problem by Extending the 3'End of the Chromosome250

MEDICAL CONNECTIONS Box 8-8 Aging Cancer and the Telomere Hypothesis251

Telomere-Binding Proteins Regulate Telomerase Activity and Telomere Length252

Telomere-Binding Proteins Protect Chromosome Ends253

SUMMARY255

BIBLIOGRAPHY256

CHAPTER 9 The Mutability and Repair of DNA257

Replication Errors and Their Repair258

The Nature of Mutations258

Some Replication Errors Escape Proofreading259

MEDICAL CONNECTIONS Box 9-1 Expansion of Triple Repeats Causes Disease259

Mismatch Repair Removes Errors That Escape Proofreading260

DNA Damage265

DNA Undergoes Damage Spontaneously from Hydrolysis and Deamination265

DNA Is Damaged by Alkylation,Oxidation,and Radiation265

MEDICAL CONNECTIONS Box 9-2 The Ames Test266

Mutations Are Also Caused by Base Analogs and Intercalating Agents268

Repair of DNA Damage269

Direct Reversal of DNA Damage270

Base Excision Repair Enzymes Remove Damaged Bases by a Base-Flipping Mechanism270

Nucleotide Excision Repair Enzymes Cleave Damaged DNA273

on Either Side of the Lesion273

Recombination Repairs DNA Breaks by Retrieving Sequence Information from Undamaged DNA275

DSBs in DNA Are Also Repaired by Direct Joining of Broken Ends275

MEDICAL CONNECTIONS Box 9-3 Nonhomologous End Joining276

Translesion DNA Synthesis Enables Replication to Proceed across DNA Damage278

ADVANCED CONCEPTS Box 9-4 The Y Family of DNA Polymerases280

SUMMARY281

BIBLIOGRAPHY282

CHAPTER 10 Homologous Recombination at the Molecular Level283

DNA Breaks Are Common and Initiate Recombination284

Models for Homologous Recombination284

Strand Invasion Is a Key Early Step in Homologous Recombination286

Resolving Holliday Junctions Is a Key Step to Finishing Genetic Exchange288

The Double-Strand Break-Repair Model Describes Many Recombination Events288

Homologous Recombination Protein Machines291

ADVANCED CONCEPTS Box 10-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions292

The RecBCD Helicase/Nuclease Processes Broken DNA Molecules for Recombination293

Chi Sites Control RecBCD296

RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion297

Newly Base-Paired Partners Are Established within the RecA Filament299

RecA Homologs Are Present in All Organisms301

The RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch Migration301

RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination302

Homologous Recombination in Eukaryotes303

Homologous Recombination Has Additional Functions in Eukaryotes303

Homologous Recombination Is Required for Chromosome Segregation during Meiosis304

Programmed Generation of Double-Stranded DNA Breaks Occurs during Meiosis305

MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA-like Strand-Exchange Proteins307

Dmcl Is a RecA-like Protein That Specifically Functions in Meiotic Recombination308

Many Proteins Function Together to Promote Meiotic Recombination308

MEDICAL CONNECTIONS Box 10-2 The Product of the Tumor309

Suppressor Gene BRCA2 Interacts With Rad51 Protein and Controls Genome Stability309

Mating-Type Switching310

Mating-Type Switching Is Initiated by a Site-Specific Double-Strand Break311

Mating-Type Switching Is a Gene Conversion Event and Not Associated with Crossing Over312

Genetic Consequences of the Mechanism of Homologous Recombination314

One Cause of Gene Conversion Is DNA Repairduring Recombination315

SUMMARY316

BIBLIOGRAPHY317

CHAPTER 11 Site-Specific Recombination and Transposition of DNA319

Conservative Site-Specific Recombination320

Site-Specific Recombination Occurs at Specific DNA Sequences in the Target DNA320

Site-Specific Recombinases Cleave and Rejoin DNA Using a Covalent Protein-DNA Intermediate322

Serine Recombinases Introduce Double-Strand Breaks in DNA and Then Swap Strands to Promote Recombination324

Structure of the Serine Recombinase-DNA Complex Indicates That Subunits Rotate to Achieve Strand Exchange325

Tyrosine Recombinases Break and Rejoin One Pair of DNA Strands at a Time326

Structures of Tyrosine Recombinases Bound to DNA Reveal the Mechanism of DNA Exchange327

MEDICAL CONNECTIONS Box 11-1 Application of Site-Specific Recombination to Genetic Engineering327

Biological Roles of Site-Specific Recombination328

λIntegrase Promotes the Integration and Excision of a Viral Genome into the Host-Cell Chromosome329

BacteriophageλExcision Requires a New DNA-Bending Protein331

The Hin Recombinase Inverts a Segment of DNA Allowing Expression of Alternative Genes331

Hin Recombination Requires a DNA Enhancer332

Recombinases Convert Multimeric Circular DNA Molecules into Monomers333

There Are Other Mechanisms to Direct Recombination to Specific Segments of DNA334

Transposition334

Some Genetic Elements Move to New Chromosomal Locations by Transposition334

ADVANCED CONCEPTS Box 11-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids335

There Are Three Principal Classes of Transposable Elements338

DNA Transposons Carry a Transposase Gene,Flanked by Recombination Sites339

Transposons Exist as Both Autonomous and Nonautonomous Elements339

Virus-like Retrotransposons and Retroviruses Carry Terminal Repeat Sequences and Two Genes Important for Recombination340

Poly-A Retrotransposons Look Like Genes340

DNA Transposition by a Cut-and-Paste Mechanism340

The Intermediate in Cut-and-Paste Transposition Is Finished by Gap Repair342

There Are Multiple Mechanisms for Cleaving the Nontransferred Strand during DNA Transposition343

DNA Transposition by a Replicative Mechanism345

Virus-like Retrotransposons and Retroviruses Move Using anRNA Intermediate347

ADVANCED CONCEPTS Box 11-3 The Pathway of Retroviral cDNA Formation349

DNA Transposases and Retroviral Integrases Are Members of a Protein Superfamily351

Poly-A Retrotransposons Move by a"Reverse Splicing" Mechanism352

Examples of Transposable Elements and Their Regulation354

IS4-Family Transposons Are Compact Elements with Multiple Mechanisms for Copy Number Control355

KEY EXPERIMENTS Box 11-4 Maize Elements and the Discovery of Transposons356

Tn10 Transposition Is Coupled to Cellular DNA Replication358

Phage Mu Is an Extremely Robust Transposon359

Mu Uses Target Immunity to Avoid Transposing into Its Own DNA359

ADVANCED CONCEPTS Box 11-5 Mechanism of Transposition Target lmmunity361

Tc1/mariner Elements Are Extremely Successful DNA Elements in Eukaryotes362

Yeast Ty Elements Transpose into Safe Havens in the Genome362

LINEs Promote Their Own Transposition and Even Transpose Cellular RNAs363

V(D)J Recombination365

The Early Events in V(D)J Recombination Occur by a Mechanism Similar to Transposon Excision367

SUMMARY369

BIBLIOGRAPHY369

PART 3 EXPRESSION OF THE GENOME371

CHAPTER 12 Mechanisms of Transcription377

RNA Polymerases and the Transcription Cycle378

RNA Polymerases Come in Different Forms but Share Many Features378

Transcription by RNA Polymerase Proceeds in a Series of Steps380

Transcription Initiation Involves Three Defined Steps382

The Transcription Cycle in Bacteria383

Bacterial Promoters Vary in Strength and Sequence but Have Certain Defining Features383

TheσFactor Mediates Binding of Polymerase to the Promoter384

Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA386

TECHNIQUES Box 12-1 Consensus Sequences388

Transcription Is Initiated by RNA Polymerase without the Need for a Primer388

During Initial Transcription,RNA Polymerase Remains Stationary and Pulls Downstream DNA into Itself389

Promoter Escape Involves Breaking Polymerase-Promoter390

Interactions and Polymerase Core-σInteractions390

The Elongating Polymerase Is a Processive Machine That Synthesizes and Proofreads RNA391

ADVANCED CONCEPTS Box 12-2 The Single-Subunit RNA Polymerases393

RNA Polymerase Can Become Arrested and Need Removing394

Transcription Is Terminated by Signals within the RNA Sequence394

Transcription in Eukaryotes396

RNA PolymeraseⅡCore Promoters Are Made Up of Combinations of Four Different Sequence Elements397

RNA PolymeraseⅡForms a Preinitiation Complex with General Transcription Factors at the Promoter398

Promoter Escape Requires Phosphorylation of the Polymerase"Tail"398

TBP Binds to and Distorts DNA Using aβSheet Inserted into the Minor Groove400

The Other General Transcription Factors Also Have Specific Roles in Initiation401

In Vivo,Transcription Initiation Requires Additional Proteins,Including the Mediator Complex402

Mediator Consists of Many Subunits,Some Conserved from Yeast to Human403

A New Set of Factors Stimulate PolⅡElongation and RNA Proofreading404

Elongating RNA Polymerase Must Deal with Histones in Its Path405

Elongating Polymerase Is Associated with a New Set of Protein Factors Required for Various Types of RNA Processing406

Transcription Termination Is Linked to RNA Destruction by a Highly Processive RNase410

Transcription by RNA PolymerasesⅠandⅢ410

RNA PolⅠand PolⅢRecognize Distinct Promoters,Using Distinct Sets of Transcription Factors,but Still Require TBP410

PolⅢPromoters Are Found Downstream of Transcription Start Site412

SUMMARY413

BIBLIOGRAPHY414

CHAPTER 13 RNA Splicing415

The Chemistry of RNA Splicing417

Sequences within the RNA Determine Where Splicing Occurs417

The Intron Is Removed in a Form Called a Lariat as the Flanking Exons Are Joined418

KEY EXPERIMENTS Box 13-1 Adenovirus and the Discovery of Splicing419

Exons from Different RNA Molecules Can Be Fused by trans-Splicing421

The Spliceosome Machinery422

RNA Splicing Is Carried Out by a Large Complex Called the Spliceosome422

Splicing Pathways424

Assembly,Rearrangements,and Catalysis within the Spliceosome:The Splicing Pathway424

Self-Splicing Introns Reveal That RNA Can Catalyze RNA Splicing426

GroupⅠIntrons Release a Linear Intron Rather Than a Lariat426

KEY EXPERIMENTS Box 13-2 Converting GroupⅠIntrons into Ribozymes428

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 snRNPs432

Alternative Splicing432

Single Genes Can Produce Multiple Products by Alternative Splicing432

Several Mechanisms Exist to Ensure Mutually Exclusive Splicing435

The Curious Case of the Drosophila Dscam Cene:Mutually Exclusive Splicing on a Grand Scale436

Mutually Exclusive Splicing of Dscam Exon 6 Cannot Be Accounted for by Any Standard Mechanism and Instead Uses a Novel Strategy437

Alternative Splicing Is Regulated by Activators and Repressors439

Regulation of Alternative Splicing Determines the Sex of Flies441

KEY EXPERIMENTS Box 13-3 Identification of Docking Site and Selector Sequences442

MEDICAL CONNECTIONS Box 13-4 Defects in Pre-mRNA Splicing Cause Human Disease445

Exon Shuffling446

Exons Are Shuffled by Recombination to Produce Genes Encoding New Proteins446

RNA Editing448

RNA Editing Is Another Way of Altering the Sequence of an mRNA448

Guide RNAs Direct the Insertion and Deletion of Uridines450

MEDICAL CONNECTIONS Box 13-5 Deaminases and HIV450

mRNA Transport452

Once Processed,mRNA Is Packaged and Exported from the Nucleus into the Cytoplasm for Translation452

SUMMARY454

BIBLIOGRAPHY455

CHAPTER 14 Translation457

Messenger RNA458

Polypeptide Chains Are Specified by Open Reading Frames458

Prokaryotic mRNAs Have a Ribosome-Binding Site That Recruits the Translational Machinery459

Eukaryotic mRNAs Are Modified at Their 5'and 3'Ends to Facilitate Translation460

Transfer RNA461

tRNAs Are Adaptors between Codons and Amino Acids461

ADVANCED CONCEPTS Box 14-1 CCA-Adding Enzymes:Synthesizing RNA without a Template462

tRNAs Share a Common Secondary Structure That Resembles a Cloverleaf462

tRNAs Have an L-shaped Three-Dimensional Structure463

Attachment of Amino Acids to tRNA464

tRNAs Are Charged by the Attachment of an Amino Acid to the 3'-Terminal Adenosine Nucleotide via a High-Energy Acyl Linkage464

Aminoacyl-tRNA Synthetases Charge tRNAs in Two Steps464

Each Aminoacyl-tRNA Synthetase Attaches a Single Amino Acidto One or More tRNAs466

tRNA Synthetases Recognize Unique Structural Features of Cognate tRNAs466

Aminoacyl-tRNA Formation Is Very Accurate468

Some Aminoacyl-tRNA Synthetases Use an Editing468

Pocket to Charge tRNAs with High Accuracy468

The Ribosome Is Unable to Discriminate between469

Correctly and Incorrectly Charged tRNAs469

The Ribosome469

ADVANCED CONCEPTS Box 14-2 Selenocysteine470

The Ribosome Is Composed of a Large and a Small Subunit471

The Large and Small Subunits Undergo Association and Dissociation during Each Cycle of Translation472

New Amino Acids Are Attached to the Carboxyl Terminus of the Growing Polypeptide Chain474

Peptide Bonds Are Formed by Transfer of the Growing Polypeptide Chain from One tRNA to Another474

Ribosomal RNAs Are Both Structural and Catalytic Determinants of the Ribosome475

The Ribosome Has Thtee Binding Sites for tRNA475

Channels through the Ribosome Allow the mRNA and Growing Polypeptide to Enter and/or Exit the Ribosome476

Initiation of Translation479

Prokaryotic mRNAs Are Initially Recruited to the Small480

Subunit by Base Pairing to rRNA480

A Specialized tRNA Charged with a Modified Methionine Binds Directly to the Prokaryotic Small Subunit480

Three Initiation Factors Direct the Assembly of an Initiation Complex That Contains mRNA and the Initiator tRNA481

Eukaryotic Ribosomes Are Recruited to the mRNA by the 5'Cap482

The Start Codon Is Foundby Scanning Downstream from the 5'End of the mRNA483

ADVANCED CONCEPTS Box 14-3 uORFs and IRESs:Exceptions That Prove the Rule485

Translation Initiation Factors Hold Eukaryotic mRNAs in Circles487

Translation Elongation487

Aminoacyl-tRNAs Are Delivered to the A Site by Elongation Factor EF-Tu488

The Ribosome Uses Multiple Mechanisms to Select against Incorrect Aminoacyl-tRNAs488

The Ribosome Is a Ribozyme491

Peptide Bond Formation and the Elongation Factor EF-G Drive Translocation of the tRNAs and the mRNA492

EF-G Drives Translocation by Displacing the tRNA Bound to the A Site494

EF-Tu-GDP and EF-G-GDP Must Exchange GDP for GTP prior to Participating in a New Round of Elongation495

A Cycle of Peptide Bond Formation Consumes Two Molecules of GTP and One Molecule of ATP495

Termination of Translation496

Release Factors Terminate Translation in Response to Stop Codons496

Short Regions of ClassⅠRelease Factors Recognize Stop Codons and Trigger Release of the Peptidyl Chain496

ADVANCED CONCEPTS Box 14-4 GTP-Binding Proteins,Conformational Switching,and the Fidelity and Ordering of the Events of Translation498

GDP/GTP Exchange and GTP Hydrolysis Control the Function of the ClassⅡRelease Factor499

The Ribosome Recycling Factor Mimics a tRNA500

MEDICAL CONNECTIONS Box 14-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation502

Regulation of Translation503

Protein or RNA Binding Near the Ribosome-Binding Site Negatively Regulates Bacterial Translation Initiation504

Regulation of Prokaryotic Translation:Ribosomal Proteins Are Translational Repressors of Their Own Synthesis505

Global Regulators of Eukaryotic Translation Target Key Factors Required for mRNA Recognition and Initator tRNA Ribosome Binding508

Spatial Control of Translation by mRNA-Specific 4E-BPs510

An Iron-Regulated,RNA-Binding Protein Controls Translation of Ferritin511

Translation of thet Yeast Transcriptional Activator Gcn4 Is Controlled by Short Upstream ORFs and Ternary Complex Abundance512

Translation-Dependent Regulation of mRNA and Protein Stability514

The SsrA RNA Rescues Ribosomes That Translate Broken mRNAs514

Eukaryotic Cells Degrade mRNAs That Are Incomplete or Have Premature Stop Codons516

SUMMARY518

BIBLIOGRAPHY519

CHAPTER 15 The Genetic Code521

The Code Is Degenerate521

Perceiving Order in the Makeup of the Code522

Wobble in the Anticodon523

Three Codons Direct Chain Termination525

How the Code Was Cracked525

Stimulation of Amino Acid Incorporation by Synthetic mRNAs526

Poly-U Codes for Polyphenylalanine527

Mixed Copolymers Allowed Additional Codon Assignments527

Transfer RNA Binding to Defined Trinucleotide Codons528

Codon Assignments from Repeating Copolymers529

Three Rules Govern the Genetic Code530

Three Kinds of Point Mutations Alter the Genetic Code531

Genetic Proof That the Code Is Readin Units of Three532

Suppressor Mutations Can Reside in the Same or a Different Gene532

Intergenic Suppression Involves Mutant tRNAs533

Nonsense Suppressors Also Read Normal Termination Signals535

Proving the Validity of the Genetic Code535

The Code Is Nearly Universal536

SUMMARY538

BIBLIOGRAPHY538

PART 4 REGULATION541

CHAPTER 16 Transcriptional Regulation in Prokaryotes547

Principles of Transcriptional Regulation547

Gene Expression Is Controlled by Regulatory Proteins547

Most Activators and Repressors Act at the Level of Transcription Initiation548

Many Promoters Are Regulated by Activators That Help RNA Polymerase Bind DNA and by Repressors That Block That Binding548

Some Activators and Repressors Work by Allostery and Regulate Steps in Transcriptional Initiation after RNA Polymerase Binding550

Action at a Distance and DNA Looping551

Cooperative Binding and Allostery Have Many Roles in Gene Regulation552

Antitermination and Beyond:Not All of Gene Regulation Targets Transcription Initiation552

Regulation of Transcription Initiation:Examples from Prokaryotes553

An Activator and a Repressor Together Controlthe Iac Genes553

CAPand Lac Repressor Have Opposing Effects on RNA Polymerase Binding to the Iac Promoter554

CAP Has Separate Activating and DNA-Binding Surfaces555

CAP and Lac Repressor Bind DNA Using a Common Structural Motif556

EDY EXPERIMENTS Box 16-1 Activator Bypass Experiments557

The Activities of Lac Repressor and CAP Are Controlled Allosterically by Their Signals559

Combinatorial Control:CAP Controls Other Genes As Well560

KEY EXPERIMENTS Box 16-2 Jacob,Monod,and the Ideas Behind Gene Regulation561

AlternativeσFactors Direct RNA Polymerase to Alternative Sets of Promoters563

NtrC and MerR:Transcriptional Activators That Work by Allostery Rather than by Recruitment564

NtrC Has ATPase Activity and Works from DNA Sites Far from the Gene564

MerR Activates Transcription by Twisting Promoter DNA565

Some Repressors Hold RNA Polymerase at the Promoter Rather than Excluding It566

AraC and Control of the araBAD Operon by Antiactivation567

The Case of Bacteriophageλ:Layers of Regulation568

Alternative Patterns of Gene Expression Control Lytic and Lysogenic Growth569

Regulatory Proteins and Their Binding Sites570

λRepressor Binds to Operator Sites Cooperatively571

ADVANCED CONCEPTS Box 16-3 Concentration,Affinity,and Cooperative Binding572

Repressor and Cro Bind in Different Patterns to Control Lytic and Lysogenic Growth573

Lysogenic Induction Requires Proteolytic Cleavage ofλRepressor574

Negative Autoregulation of Repressor Requires Long-Distance Interactions and a Large DNA Loop575

Another Activator,λCll,Controls the Decision between Lytic and Lysogenic Growth upon Infection of a New Host577

The Number of Phage Particles Infecting a Given Cell Affects Whether the Infection Proceeds Lytically or Lysogenically578

Growth Conditions of E.coli Control the Stability of Cll Protein and thus the Lytic/Lysogenic Choice578

KEY EXPERIMENTS Box 16-4 Evolution of theλSwitch579

KEY EXPERIMENTS Box 16-5 Genetic Approaches That Identified Genes Involved in the Lytic/Lysogenic Choice581

Transcriptional Antitermination inλDevelopment582

Retroregulation:An Interplay of Controls on RNA Synthesis and Stability Determines int Gene Expression584

SUMMARY585

BIBLIOGRAPHY586

CHAPTER 17 Transcriptional Regulation in Eukaryotes589

Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals591

Activators Have Separate DNA-Binding and Activating Functions591

Eukaryotic Regulators Use a Range of DNA-Binding Domains,but DNA Recognition Involves the Same Principles as Found in Bacteria593

TECHNIQUES Box 17-1 The Two-Hybrid Assay594

Activating Regions Are Not Well-Defined Structures596

Recruitment of Protein Complexes to Genes by Eukaryotic Activators597

Activators Recruit the Transcriptional Machinery to the Gene597

Activators Also Recruit Nucleosome Modiffers That Help the Transcriptional Machinery Bind at the Promoter or Initiate Transcription598

Activators Recruit an Additional Factor Needed for Efficient Initiation or Elongation at Some Promoters600

Action at a Distance:Loops and Insulators601

Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions603

KEY EXPERIMENTS Box 17-2 Long-Distance Interactions on the Same and Different Chromosomes604

Signal Integration and Combinatorial Control605

Activators Work Synergistically to Integrate Signals605

SignalIntegration:The HO Gene Is Controlledby Two Regulators—One Recruits Nucleosome Moditiers and the Other Recruits Mediator607

Signal Integration:Cooperative Binding of Activators at theHumanβ-Interferon Gene608

Combinatorial Control Lies at the Heart of the Complexity and Diversity of Eukaryotes610

Combinatorial Control of the Mating-Type Genes from S.cerevisiae611

KEY EXPERIMENTS Box 17-3 Evolvability of a Regulatory Circuit612

Transcriptional Repressors613

Signal Transduction and the Control of Transcriptional Regulators615

Signals Are Often Communicated to Transcriptional Regulators through Signal Transduction Pathways615

Signals Control the Activities of Eukaryotic Transcriptional Regulators in a Variety of Ways617

Activators and Repressors Sometimes Come in Pieces619

Gene"Silencing"by Modification of Histones and DNA620

Silencing in Yeast Is Mediated by Deacetylation and Methylation of Histones621

In Drosophila,HP1 Recognizes Methylated Histones and Condenses Chromatin622

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

DNA Methylation Is Associated with Silenced Genes in Mammalian Cells624

MEDICAL CONNECTIONS Box 17-5 Transcriptional Repression and Human Disease626

Epigenetic Gene Regulation626

Some States of Gene Expression Are Inherited through Cell Division Even When the Initiating Signal Is No Longer Present627

MEDICAL CONNECTIONS Box 17-6 Using Transcription Factors to Reprogram Somatic Cells into Embryonic Stem Cells629

SUMMARY630

BIBLIOGRAPHY631

CHAPTER 18 Regulatory RNAs633

Regulation by RNAs in Bacteria633

Riboswitches Reside within the Transcripts of Genes635

Whose Expression They Control through Changes in Secondary Structure635

ADVANCED CONCEPTS Box 18-1 Amino Acid Biosynthetic Operons Are Controlled by Attenuation639

RNA Interference Is a Major Regulatory Mechanism in Eukaryotes641

Short RNAs That Silenee Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in Three Different Ways641

Synthesis and Function of miRNA Molecules643

miRNAs Have a Characteristic Structure That Assists in Identifying Them and Their Target Genes643

An Active miRNA Is Generated through a Two-Step Nucleolytic Processing645

Dicer Is the Second RNA-Cleaving Enzyme Involved in miRNA Production646

Incorporation of a Guide Strand RNA into RISC Makes the Mature Complex That Is Ready to Silence Gene Expression647

siRNAs Are Regulatory RNAs Generated from Long Double-Stranded RNAs649

Small RNAs Can Transcriptionally Silence Genes by Directing Chromatin Modification649

KEY EXPERIMENTS Box 18-2 History of miRNAs and RNAi650

The Evolution and Exploitation of RNAi652

Did RNAi EvolveAs an Immune System?652

RNAi Has Become a Powerful Tool for Manipulating Gene Expression654

MEDICAL CONNECTIONS Box 18-3 RNAi and Human Disease656

Regulatory RNAs and X-inactivation657

X-inactivation Creates Mosaic Individuals657

Xist Is an RNA Regulator That Inactivates a Single X Chromosome in Female Mammals657

SUMMARY659

BIBLIOGRAPHY660

CHAPTER 19 Gene Regulation in Development and Evolution661

TECHNIQUES Box 19-1 Microarray Assays:Theory and Practice662

Three Strategies by Which Cells Are Instructed to Express Specific Sets of Genes during Development663

Some mRNAs Become Localized within Eggs and Embryos because of an Intrinsic Polarity in the Cytoskeleton663

Cell-to-Cell Contact and Secreted Cell-Signaling Molecules Both Elicit Changes in Gene Expression in Neighboring Cells664

Gradients of Secreted Signaling Molecules Can Instruct Cells to Follow Different Pathways of Development Based on Their Location665

Examples of the Three Strategies for Establishing Differential Gene Expression666

The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO Gene666

ADVANCED CONCEPTS Box 19-2 Review of Cytoskeleton:Asymmetry and Growth669

A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo670

ADVANCED CONCEPTS Box 19-3 Overview of Ciona Development671

Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium,Bacillus subtilis672

A Skin-Nerve Regulatory Switch Is Controlled by Notch Signaling in the Insect Central Nervous System673

A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube674

The Molecular Biology of Drosophila Embryogenesis676

An Overview of Drosophila Embryogenesis676

ADVANCED CONCEPTS Box 19-4 Overview of Drosophila Development677

A Morphogen Gradient Controls Dorsoventral Patterning of the Drosophila Embryo679

Segmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Egg682

Bicoid and Nanos Regulate hunchback683

KEY EXPERIMENTS Box 19-5 The Role of Activator Synergy in Development684

MEDICAL CONNECTIONS Box 19-6 Stem Cells686

The Gradient of Hunchback Repressor Establishes Different Limits of Gap Gene Expression687

Hunchback and Gap Proteins Produce Segmentation Stripes of Gene Expression688

Gap Repressor Gradients Produce Many Stripes of Gene Expression689

KEY EXPERIMENTS Box 19-7 cis-Regulatory Sequences in Animal Development and Evolution690

Short-Range Transcriptional Repressors Permit Different Enhancers to Work Independently of One Another within the Complex eve Regulatory Region692

Homeotic Genes:An Important Class of Developmental Regulators693

Changes in Homeotic Gene Expression Are Responsible for Arthropod Diversity695

Arthropods Are Remarkably Diverse695

Changesin Ubx Expression Explain Modification of Limbs among the Crustaceans695

ADVANCED CONCEPTS Box 19-8 Homeotic Genes of Drosophila AreOrganized in Special Chromosome Clusters696

Why Insects Lack Abdominal Limbs698

Modification of Flight Limbs Might Arise from the Evolution of Regulatory DNA Sequences699

SUMMARY701

BIBLIOGRAPHY702

CHAPTER 20 Genome Analysis and Systems Biology703

Genomics Overview703

Bioinformatics Tools Facilitate the Genome-wide Identification of Protein-Coding Genes703

Whole-Genome Tiling Arrays Are Used to Visualize the Transcriptome704

Regulatory DNA Sequences Can Be Identified by Using Specialized Alignment Tools706

The ChIP-Chip Assay Is the Best Method forIdentifying Enhancers708

TECHNIQUES Box 20-1 Bioinformatics Methods for the Identification of Complex Enhancers708

Diverse Animals Contain Remarkably Similar Sets of Genes711

Many Animals Contain Anomalous Genes712

Synteny Is Evolutionarily Ancient713

Deep Sequencing Is Being Used to Explore Human Origins715

Systems Biology715

Transcription Circuits Consist of Nodes and Edges716

Negative Autoregulation Dampens Noise and Allows a Rapid Response Time717

Gene Expression Is Noisy718

Positive Autoregulation Delays Gene Expression720

Some Regulatory Circuits Lock in Alternative Stable States720

Feed-Forward Loops Are Three-Node Networks with Beneficial Properties722

KEY EXPERIMENTS Box 20-2 Bistability and Hysteresis722

Feed-Forward Loops Are Used in Development725

Some Circuits Generate Oscillating Patterns of Gene Expression727

Synthetic Circuits Mimic Some of the Features of Natural Regulatory Networks729

Prospects730

SUMMARY730

BIBLIOGRAPHY731

PART 5 METHODS733

CHAPTER 21 Techniques of Molecular Biology739

Nucleic Acids740

Electrophoresis through a Gel Separates DNA and RNA Molecules according to Size740

Restriction Endonucleases Cleave DNA Molecules at Particular Sites742

DNA Hybridization Can Be Used to Identify Specific DNA Molecules743

Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs744

Isolation of Specific Segments of DNA746

DNA Cloning746

Cloning DNA in Plasmid Vectors746

Vector DNA Can Be Introduced into Host Organisms by Transformation748

Libraries of DNA Molecules Can Be Created by Cloning748

Hybridization Can Be Used to Identify a Specific Clone in a DNA Library749

Chemically Synthesized Oligonucleotides750

The Polymerase Chain Reaction Amplifies DNAs by Repeated Rounds of DNA Replication in Vitro751

TECHNIQUES Box 21-1 Forensics and the Polymerase Chain Reaction753

Nested Sets of DNA Fragments Reveal Nucleotide Sequences753

KEY EXPERIMENTS Box 21-2 Sequenators Are Used for High-Throughput Sequencing757

Shotgun Sequencing a Bacterial Genome757

The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences758

The Paired-End Strategy Permits the Assembly of Large-Genome Scaffolds760

The$1000 Human Genome Is within Reach762

Proteins764

Specific Proteins Can Be Purified from Cell Extracts764

Purification of a Protein Requires a Specific Assay764

Preparation of Cell Extracts Containing Active Proteins765

Proteins Can Be Separated from One Another Using Column Chromatography765

Affinity Chromatography Can Facilitate More Rapid Protein Purification767

Separation of Proteins on Polyacrylamide Gels768

Antibodies Are Used to Visualize Electrophoretically Separated Proteins769

Protein Molecules Can Be Directly Sequenced769

Proteomics771

Combining Liquid Chromatography With Mass Spectrometry Identifes Individual Proteins within a Complex Extract771

Proteome Comparisons Identify Important Differences beween Cells773

Mass Spectrometry Can Also Monitor Protein Modification States773

Protein-Protein Interactions Can Yield Information about Protein Function774

Nucleic Acid-Protein Interactions775

The Electrophoretic Mobility of DNA Is Altered by Protein Binding776

DNA-Bound Protein Protects the DNA from Nucleases and Chemical Modification777

Chromatin Immunoprecipitation Can Detect Protein Association with DNA in the Cell778

In Vitro Selection Can Be Used to Identify a Protein's DNA-or RNA-Binding Site780

BIBLIOGRAPHY782

CHAPTER 22 Model Organisms783

Bacteriophage784

Assays of Phage Growth786

The Single-Step Growth Curve787

Phage Crosses and Complementation Tests787

Transduction and Recombinant DNA788

Bacteria789

Assays of Bacterial Growth789

Bacteria Exchange DNA by Sexual Conjugation,Phage-Mediated Transduction,and DNA-Mediated Transformation790

Bacterial Plasmids Can Be Usedas Cloning Vectors791

Transposons Can Be Used to Generate Insertional Mutations and Gene and Operon Fusions791

Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Technology,Whole-Genome Sequencing,and Transcriptional Profiling793

Biochemical Analysis Is Especially Powerful in Simple Cells with Well-Developed Tools of Traditional and Molecular Genetics793

Bacteria Are Accessible to Cytological Analysis793

Phage and Bacteria Told Us Most of the Fundamental Things about the Gene794

Baker's Yeast,Saccharomyces cerevisiae795

The Existence of Haploid and Diploid Cells Facilitate Genetic Analysis of S.cerevisiae795

Generating Precise Mutations in Yeast Is Easy796

S.cerevisiae Has a Small,Well-Characterized Genome796

S.cerevisiae Cells Change Shape as They Grow797

Arabidopsis798

Arabidopsis Has a Fast Life Cycle with Haploid and Diploid Phases798

Arabidopsis Is Easily Transformed for Reverse Genetics799

Arabidopsis Has a Small Genome That Is Readily Manipulated800

Epigenetics801

Plants Respond to the Environment801

Development and Pattern Formation802

The Nematode Worm,Caenorhabditis elegans802

C.elegans Has a Very Rapid Life Cycle803

C.elegans Is Composed of Relatively Few,Well-Studied Cell Lineages804

The Cell Death Pathway Was Discovered in C.elegans805

RNAi Was Discovered in C.elegans805

The Fruit Fly,Drosophila melanogaster806

Drosophila Has a Rapid Life Cycle806

The First Genome Maps Were Produced in Drosophila807

Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Hies809

The Yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics809

It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA810

The House Mouse,Mus musculus812

Mouse Embryonic Development Depends on Stem Cells813

It Is Easy to Introduce Foreign DNA into the Mouse Embryo813

Homologous Recombination Permits the Selective Ablation of Individual Genes814

Mice Exhibit Epigenetic Inheritance816

BIBLIOGRAPHY818

Index819

Advanced Concepts6

Box 1-1 Mendelian Laws6

Box 3-1 The Uniqueness of Molecular Shapes and the Concept of Selective Stickiness53

Box 5-2 Large Proteins Are Often Constructed of Several Smaller Polypeptide Chains83

Box 8-4 ATP Control of Protein Function:Loading a Sliding Clamp223

Box 8-6 The Replication Factory Hypothesis237

Box 8-7 E.coli DNA Replication Is Regulated by DnaA·ATP Levels and SeqA244

Box 9-4 The Y Family of DNA Polymerases280

Box 10-1 How to Resolve a Recombination Intermediate with Two Holliday Junctions292

Box 11-2 The Xer Recombinase Catalyzes the Monomerization of Bacterial Chromosomes and of Many Bacterial Plasmids335

Box 11-3 The Pathway of Retroviral cDNA Formation349

Box 11-5 Mechanism of Transposition Target lmmunity361

Box 12-2 The Single-Subunit RNA Polymerases393

Box 14-1 CCA-Adding Enzymes:Synthesizing RNA without a Template462

Box 14-2 Selenocysteine470

Box 14-3 uORFs and IRESs:Exceptions That Prove the Rule485

Box 14-4 GTP-Binding Proteins,Conformational Switching,and the Fidelity and Ordering of the Events of Translation498

Box 16-3 Concentration,Affinity,and Cooperative Binding572

Box 17-4 Is There a Histone Code?623

Box 18-1 Amino Acid Biosynthetic Operons Are Controlled by Attenuation639

Box 19-2 Review of Cytoskeleton:Asymmetry and Growth669

Box 19-3 Overview of Ciona Development671

Box 19-4 Overview of Drosophila Development677

Box 19-8 Homeotic Genes of Drosophila Are Organized in Special Chromosome Clusters696

Key Experiments10

Box 1-2 Genes Are Linked to Chromosomes10

Box 2-1 Chargaff's Rules24

Box 2-2 Evidence That Genes Control Amino Acid Sequences in Proteins29

Box 6-1 DNA Has 10.5 Base Pairs per Turn of the Helix in Solution:The Mica Experiment108

Box 6-2 How Spots on an X-ray Film Reveal the Structure of DNA112

Box 6-3 Proving that DNA Has a Helical Periodicity of about 10.5 Base Pairs per Turn from the Topological Properties of DNA Rings128

Box 7-1 Micrococcal Nuclease and the DNA Associated with the Nucleosome158

Box 7-2 Nucleosomes and Superhelical Density166

Box 7-3 Determining Nucleosome Position in the Cell180

Box 8-5 The Identification of Origins of Replication and Replicators232

Box 11-4 Maize Elements and the Discovery of Transposons356

Box 13-1 Adenovirus and the Discovery of Splicing419

Box 13-2 Converting GroupⅠIntrons into Ribozymes428

Box 13-3 Identification of Docking Site and Selector Sequences442

Box 16-1 Activator Bypass Experiments557

Box 16-2 Jacob,Monod,and the Ideas Behind Cene Regulation561

Box 16-4 Evolution of theλSwitch579

Box 16-5 Genetic Approaches That Identiffed Genes Involved in the Lytic/Lysogenic Choice581

Box 17-2 Long-Distance Interactions on the Same and Different Chromosomes604

Box 17-3 Evolvability of a Regulatory Circuit612

Box 18-2 History of miRNAs and RNAi650

Box 19-5 The Role of Activator Synergy in Development684

Box 19-7 cis-Regulatory Sequences in Animal Development and Evolution690

Box 20-2 Bistability and Hysteresis722

Box 21-2 Sequenators Are Used for High-Throughput Sequencing757

Medical Connections203

Box 8-2 Anticancer and Antiviral Agents Target DNA Replication203

Box 8-8 Aging,Cancer,and the Telomere Hypothesis251

Box 9-1 Expansion of Triple Repeats Causes Disease259

Box 9-2 The Ames Test266

Box 9-3 Nonhomologous End Joining276

Box 10-2 The Product of the Tumor Suppressor Gene BRCA2 Interacts with Rad51 Protein and Controls Genome Stability309

Box 11-1 Application of Site-Specific Recombination to Genetic Engineering327

Box 13-4 Defects in Pre-mRNA Splicing Cause Human Disease445

Box 13-5 Deaminases and HIV450

Box 14-5 Antibiotics Arrest Cell Division by Blocking Specific Steps in Translation502

Box 17-5 Transcriptional Repression and Human Disease626

Box 17-6 Using Transcription Factors to Reprogram Somatic Cells into Embryonic Stem Cells629

Box 18-3 RNAi and Human Disease656

Box 19-6 Stem Cells686

Techniques78

Box 5-1 Determination of Protein Structure78

Box 8-1 Incorporation Assays Can Be Used to Measure Nucleic Acid and Protein Synthesis200

Box 8-3 Determining the Polarity of a DNA Helicase212

Box 12-1 Consensus Sequences388

Box 17-1 The Two-Hybrid Assay594

Box 19-1 Microarray Assays:Theory and Practice662

Box 20-1 Bioinformatics Methods for the Identification of Complex Enhancers708

Box 21-1 Forensics and the Polyrnerase Chain Reaction753