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