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`LCY Biotechnology Holding, Inc.
`Ex. 1056
`Page 1 of 30
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`LCY Biotechnology Holding, Inc.
`Ex. 1056
`Page 2 of 30
`
`

`

`Molecular Biology
`
`Fifth Edition
`
`Robert F. Weaver
`University of Kansas
`
`(cid:127) Succeed"
`
`onneet
`Learn
`
`}
`
`LCY Biotechnology Holding, Inc.
`Ex. 1056
`Page 3 of 30
`
`

`

`The McGrow-Hill Companies
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`(cid:127) Connect
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`Learn
`Succeed'"
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`MOI.ECULAk KIOLOGY, HHH EDITION
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`1. Molecular biology. I. Title.
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`LCY Biotechnology Holding, Inc.
`Ex. 1056
`Page 4 of 30
`
`

`

`BRIEF CONTENTS
`
`JV
`
`About the Author
`Preface xiii
`Acknowledgments xvu
`Guidr to Experimental Techniques in
`Molecular Biology xix
`
`PART I
`Introduction
`1 A Brief History 1
`2 The Molecular Nature of Genes 12
`3 An lntroduction to Gene Function 30
`
`PART 11
`Methods in Molecular Biology
`4 Molecular Cloning Methods 49
`5 Molecular Tools for Studying Genes and Gene
`Activity 75
`
`PART Ill
`Transcription in Bacteria
`6 The Mechanism of Transcription in Bacteria 121
`7 Operons: J-iinc Control of Bacterial
`Transcription 167
`8 Major Shifts in Bacterial Transcription 196
`9 DNA-Protein Interactions in Bacteria 222
`
`PART IV
`Transcription in Eukaryotes
`10 Eukaryotic RNA Polymerases and
`Their Promoters 244
`11 General Transcription Factors
`in Eukaryotes 2 73
`12 Transcription Activators in Eukaryotes 314
`13 Chromatin Structure and Its Effects
`on Transcription 355
`
`PART V
`Post-Transcriptional Events
`14 RNA Processing I: Splicing 394
`15 RNA Processing II: Capping and Polyadenylation 436
`16 Other RNA Processing Events and Post-Transcriptional
`Control of Gene Expression 4 71
`
`PART VI
`Translation
`17 The Mechanism of Translation I: Initiation 522
`18 The Mechanism of Translation II: Elongation
`and Termination 560
`19 Ribosomes and Transfer RNA 601
`
`PART VII
`DNA Replication, Recombination,
`and Transposition
`20 DNA Replication, Damage, and Repair 636
`21 DNA Replication 11: Detailed Mechanism 677
`22 Homologous Recombination 709
`23 Transposition 732
`
`PART VIII
`Genomes
`24 Introduction to Genomics: DNA Sequencing on
`a Genomic Scale 759
`25 Genomics II: Functional Genomics, Proteomics,
`and Bioinformatics 789
`
`Glossary 827
`Index 856
`
`V
`
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`
`

`

`iv
`
`About the Author
`Preface xiii
`Acknowledgments xvii
`Guide to Experimental Techniques
`in Molecular Biology xix
`
`PART I
`Introduction
`
`CHAPTER 1
`A Brief History 1
`1. l
`Transmission Genetics 2
`Mendel's Laws o f Tnheritancc 2
`The Chromosome Tht:ory o f Inhcrir.im:e 3
`Genetic ltecombination and Mapping 4
`Physical Evidence for Recombination 5
`1.2 Molecular Generics 5
`Th.e Discovery of DNA S
`The Relationship Between Genes and Proreim
`Activities of Genes 7
`1.3 The Three Domains of Life 9
`
`6
`
`C HAPTER 2
`The Molecular Nature of Genes 12
`2.1
`The Nature of Genetic Material 13
`Transformation in Ha<.:tcria 13
`The Chemical Nature of Polynuclcorides 15
`2.2 DNA Structure 18
`Experimental Backgro und 19
`T h~ Douhle Helix 19
`2.3 Genes Made of RNA 22
`2.4
`Physical Chemistry of Nucleic Adds 23
`A Variety of DNA Structurt:s 23
`DN,\s of Various Si1cs a nd Shapes 27
`
`vi
`
`C H A PT ER 3
`An Introduction to Gene Function 30
`3.1
`Storing Information 31
`Overview of Gene Expression 31
`Prnrein StJUCture 31
`Prott:in Function 35
`Discovery of Messenger RNA 37
`T ranscription 39
`Translation 40
`3 .2 Replication 45
`3.3 Mutations 45
`Sickle Cell Disease 45
`
`PART II
`Methods of Molecular Biology
`
`CHA P TER 4
`Molecular C]oning Methods 49
`4.1 Gene Cloning 50
`T he Role of Restriction Endonucleascs 50
`Vectors 53
`Idemifying a Speci fic Clone with a
`Specific Probe 58
`cDNJ\ Cloning 60
`Rapid Amplification of cDNA Ends 61
`4.2 The Polymerase Chain Reaction 62
`Standard PCR 62
`Box 4.1 Jurassic Park: More than a Fantasy? 63
`Using Rcverst: Transcriptase PCR (RT-PCR)
`in cDNA Cloning 64
`Real-Time PCR 64
`4.3 Methods of Expressing Cloned Genes 65
`Expression Vectors 65
`Other F.ukarymic Vectors 71
`Using t he Ti Plas mid to Trans fer Gene~
`to Plants 71
`
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`

`CHAPTER 5
`Molecular Tools for Studying Genes
`and Gene Activity 7 5
`5.1 Molecular Separations 76
`Gel Electrophoresis 76
`Two-Dimensional Gel Electrophoresis 79
`Ion-Exchange Chromatography 80
`Gel Filtrarion Chromatography 80
`Affinity Chromatography 81
`5 .2 Labeled Tracers 82
`Autoradiography 82
`Phosphorimaging 83
`Liquid Scintillation Counting 84
`Nonradioactive Tracers 84
`5.3 Using Nucleic Acid Hybridization 85
`Southern Blots: Identifying Specific DNA Fragments 85
`DNA Fingerprinting and DNA Typing 86
`Forensic Uses of DNA Fingerprinting and
`DNA Typing 87
`In Situ Hybridization: Locating Genes in
`Chromosomes 8 8
`Immunoblots (Western Blots) 89
`5 .4 DNA Sequencing and Physical Mapping 89
`The Sanger Chain-Termination
`Sequencing Method 90
`Automated DNA Sequencing 91
`High-Throughput Sequencing 93
`Restriction Mapping 95
`Protein Engineering with Cloned Genes:
`Site-Directed Mutagenesis 97
`5 .6 Mapping and Quantifying Transcripts 99
`Northern Blots 99
`S1 Mapping 100
`Primer Extension 102
`Run-Off Transcription and G-Less
`Cassette Transcription 103
`5.7 Measuring Transcription Rates in Vivo 104
`Nuclear Run-On Transcription 104
`Reporter Gene Transcription 105
`Measuring Protein Accumulation in Vivo
`Assaying DNA-Protein Interactions
`Filter Binding 108
`Gel Mobility Shift 109
`DNase Footprinting 109
`DMS Footprinting and Other Footprinting
`Methods 109
`Chromatin Immunoprecipitation (ChIP) 112
`5.9 Assaying Protein-Protein Interactions 112
`
`5 .5
`
`5.8
`
`Contents
`
`vii
`
`5.10 Finding RNA Sequences That Interact
`with Other Molecules 114
`SELEX 114
`Functional SELEX 114
`5 .11 Knockouts and Transgenics 115
`Knockout Mice 115
`Transgenic Mice 115
`
`PA RT 111
`Transcription in Bacteria
`
`6.2
`
`CHAPTER 6
`The Mechanism of Transcription
`in Bacteria 121
`6.1 RNA Polymerase Structure 122
`Sigma (er) as a Specificity Factor 122
`Promoters 123
`Binding of RNA Polymerase to Promoters 123
`Promoter Structure 125
`6.3 Transcription Initiation 126
`Sigma Stimulates Transcription Initiation 127
`Reuse of er 128
`The Stochastic a-Cycle Model 129
`Local DNA Melting at the Promoter 132
`Promoter Clearance 134
`Structure and Function of rr 139
`The Role of the o.-Subunit in UP Element
`Recognition 142
`6.4 Elongation 144
`Core Polymerase Functions in Elongation 144
`Structure of the Elongation Complex 146
`6.5 Termination of Transcription 156
`Rho-Independent Termination 156
`Rho-Dependent Termination 159
`
`CHAPTER 7
`Operons: Fine Control of Bacterial
`Transcription 167
`The lac Operon 168
`7.1
`Negative Control of the lac Operon 169
`Discovery of the Operon 169
`Repressor-Operator Interactions 173
`The Mechanism of Repression 174
`Positive Control of the lac Operon 177
`The Mechanism of CAP Action 178
`
`106
`108
`
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`viii
`
`Contents
`
`7 3
`
`7 .2 The ara Operon 182
`The ara Operon Repression Loop 183
`Evidence for the am Operon Repression Loop 183
`Autoregulation of araC 185
`The trp Operon 186
`Tryptophan's Role in Negative Control of the
`tip Opernn 186
`Control of the tip Operon by Attenuation 187
`Defeating Atrcnuation 188
`7.4 Riboswitchcs 190
`
`9.4 DNA-Rinding Proteins: Action at
`a Distance 237
`The ~al Operon 237
`Duplicated A Operators 237
`Enhancers 238
`
`PART IV
`Transcription in Eukaryotes
`
`CHAPTER 8
`Major Shifts in Bacterial
`Transcription 196
`8.1
`Sigma Factor Switching 197
`1.97
`Phage Infection
`Sporulation 199
`Gene.~ with Mulciple Promoters 201
`Other <r Switches 201
`Anti-<r-factors 202
`8.2 The RNA Polymerase Encoded in
`Phage T7 202
`Infection of E. coli by Phage A 203
`Lyric Rerro<lucrion of Phage >.. 204
`Establishing Lysogeny 211
`Autoregulation of the d Gene During
`Lysogeny 212
`Determining the Fate of a A Infection: [ ,ysis or
`Lysogeny 217
`Lysogcn Induction 218
`
`8.3
`
`CHAPTER 9
`DNA-Protein Interactions
`in Bacteria 222
`9.1 The A Family of Repressors 223
`Probing Binding Specificity by Site(cid:173)
`Directed Mutagcnesis 223
`Box 9.1 X-Ray Crystallography 224
`High-Resolution Analysis of A Repressor-Operaror
`hitcracrions 229
`l ligh-Resolution Analysi~ of Phage 4.)4
`Repressor-Operator lntern~tions 232
`9.2 The trp Repressor 234
`The Role of Tryptophan 234
`9 .J General Considerations on
`Protein-DNA Interactions 235
`Hydrogen F\onding Capabilities of the Four
`Different Base Pairs 235
`The Importance of Multimeric DNA-Binding Proteins 236
`
`CHAPTER 10
`Eukaryotic RNA Polymerases
`and Their Promoters 244
`10.1 Multiple Forms of Eukaryotic
`RNA Polymerase 245
`Separation of the Three Nudear Polymerases 245
`The Roles of the Three RNA Polymerases 246
`RNA Polymerase Sulmnit Structures 248
`Promoters 259
`Class 11 Promoters 259
`Class l Promoters 263
`Class Ill Promoters 264
`Enhancers and Silencers 267
`Enhancers 267
`Silencers 269
`
`10.2
`
`10.3
`
`CHAPTER 11
`General Transcription Factors
`in Eukaryotes 2 73
`11.1
`Class II Factors 274
`The Class 11 l'reinitiation Complex 274
`Structure and Function ofTfllD 276
`Structure anti Function of TFJIB 286
`Structun; and Function ofTf-IIH 288
`The Mediator Complex and the
`RNA Polymerase n Holoenzyme 295
`Elongation factors 296
`11.2 Class I Factors 299
`The Corc-Bindini-; factor 299
`The UPE-Bintling Factor 300
`Structure and Function of SU 301
`11.3 Class JI[ Factors J03
`TFIHA 303
`TFIIIB and C 304
`The Role of TBP 307
`
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`

`

`12.2
`
`12.3
`
`J 2.4
`
`CHAPTER 12
`Transcription Activators
`in Eukaryotes 314
`12.1 Categories of Activators 315
`DNA-Binding Domains 315
`Transcription-Activating Domains 315
`Structures of the DNA-Binding
`Motifs of Activators 316
`Zinc Fingers 316
`The GAL4 Protein 318
`The Nuclear Receptors 319
`Homeo<lomains 320
`The bZIP an<l hHLH Domains 321
`Independence of the Domains
`of Activators 323
`Functions of Activators 324
`Recruitment ofTFIID 324
`Recruitment of the Holoenzyme 325
`Interaction Among Activators 328
`Dirnerization 328
`Action at a Distance 329
`Box 12.1 Genomic Imprinting 332
`Transcription Factories 334
`Complex Enhancers 336
`Architectural Transcription Factors 337
`Enhanccosomes 338
`Insulators 339
`12.6 Regulation of Transcription Factors 343
`Coactivators 344
`Activator Ubiquitylation 346
`Activator Sumoylarion 347
`Activator Acetylation 348
`Signal Transduction Pathways 348
`
`12.5
`
`CHAPTER 13
`Chromatin Structure and Its Effects
`on Transcription 355
`13.1 Chromatin Structure 356
`Histones 356
`Nucleosomes 357
`The 30-nm Fiber 360
`Higher-Order Chromatin Folding 362
`13.2 Chromatin Structure and Gene
`Activity 364
`The Effects of Histones on Transcription
`of Class II Genes 365
`
`Contents
`
`ix
`
`Nucleosome Positioning 367
`Histone Acerylation 3 72
`Histone Deacetylation 373
`Chromatin Remodeling 376
`Hcterochromatin and Silencing 383
`Nuclcosomes an<l Transcription
`Elongation 387
`
`PART V
`Post-Transcriptional Events
`
`CHAPTER 14
`RNA Processing I: Splicing 394
`14.1 Genes in Pieces 395
`Evidence for Split Genes 395
`RNA Splicing 396
`Splicing Signals 397
`Effect of Splicing on Gene Expression 398
`14.2 The Mechanism of Splicing of Nuclear
`mRNA Precursors 399
`A Branched Intermediate 399
`A Signal at the Branch 401
`Spliceosomcs 402
`Spliceosome Assembly and Function 411
`Commitment, Splice Site Selection, and Alternative
`Splicing 415
`Control of Splicing 425
`Self-Splicing RNAs 427
`Group l Incrons 42 7
`Group II Introns 430
`
`14.3
`
`CHAPTER 15
`RNA Processing II: Capping and
`Polyadenylation 436
`15.1 Capping 437
`Cap Structure 437
`Cap Synthesis 438
`Functions of Caps 440
`Polyadenylation 442
`Poly(A) 442
`Functions of Poly(A} 443
`llasic Mechanism of Polyadcnylation 445
`I'olyadenylation Signals 446
`Cleavage and Polyadcnylation of a Pre-mRNA 448
`Poly{A} Polymerase 454
`Turnover of Poly(A) 454
`
`15.2
`
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`

`x
`
`Contents
`
`15.3 Coordination of mRNA Processing Events 456
`Binding of the CTD of Rpbl co mRNA-Prncessing
`Proteins 457
`Changes in Association of RNA-Processing Proteins
`with the CTD Correlate with Changes in C'fl)
`Phosphorylation 458
`·
`A CTO Code? 460
`Coupling Transcription Termination with mRNA
`3 '-End Processing 461
`Mechanism of Termination 462
`Role of Polyadenylation in mRNA Transport 466
`
`16.3
`
`16.5
`
`16.6
`
`CHAPTER 16
`Other RNA Processing Events and
`Post-Transcriptional Control of Gene
`Expression 471
`16.1 Ribosomal RNA Processing 472
`Eukaryotic rRNA Processing 472
`Bacteria\ rRNA Processing 474
`16.2 Transfer RNA Processing 475
`Cutting Apart Polycistronic Precursors 475
`Forming Mature 5'-Ends 475
`forming Mature 3'-Ends 476
`Trans-Splicing 477
`The Mechanism of Trans-Splicing 477
`16.4 RNA Editing 479
`Mechanism of Editing 479
`Editing hy Nucleotide Deamination 482
`Post-Transcriptional Control of Gene
`Expression: mUNA Stability 483
`Casein mRNA Stability 484
`Transfcrrin Receptor rnRNJ\ Stability 484
`Post-Transcriptional Control of Gene Expression:
`RNA Interference 488
`Mechanism of RN/\i 489
`Amplification of siRN/\ 494
`Role of the RN Ai Machinery in Heterochromarin
`Formation and Gene Silencing 495
`Piwi-Interacting RNAs and Transposon Control
`Post-Transcriptional Control of Gene
`Expression: MicroRNAs 502
`Silencing ofTra1Jslation hy miRN/\s 502
`Stimulation of Translation by rniRN/\s 507
`Translation Repression, mRNA Degradation,
`and P-Bodics S 10
`Processing Bodies 510
`Degradation of mRNAs in P-Bodies 511
`Relief of Repression in P-Bo<lies 514
`Other Small RNl\s 517
`
`16.7
`16.8
`
`16.9
`
`501
`
`PA RT VI
`Translation
`
`CHAPTER 17
`The Mechanism of Translation I:
`Initiation 522
`Initiation of Translation in Bacteria 523
`17.1
`tRNA Charging 523
`Dissociation of Ribosomes 523
`Formation of the 305 Initiation Complex 525
`Formation of the 705 initiation Complex 531
`Summary of initiation in Bacteria 533
`Initiation in Eukaryotes 533
`.s:n
`The Scanning Model of Initiation
`Eukaryotic fnitiacion Factors 537
`17.3 Control of Initiation 545
`Racterial Translational Control 545
`Eukaryotic Translational Control 548
`
`17.2
`
`CHAPTER 18
`The Mechanism of Translation II:
`Elongation and Termination 560
`18.1
`The Direction of Polypeptide Synthesis and
`of mRNA Translation 561
`18.2 The Genetic Code 562
`Nonoverlapping Codons 562
`No Gaps in the Co<le 563
`The Triplet Code 563
`Breaking the C()dc 564
`Unusual Base Pairs Between Co<lon and Anticodon 566
`The (Almost} Universal Code 567
`18.3 The Elongation Cycle 569
`Overview of Elongation 569
`A Three-Site Model of the Rihosomc 570
`Elongation Step I: Binding an Aminoacyl-tRNA to the
`A Site of the Ribosome S7J
`Elongation Step 2: Pepridc Bond Formation 577
`Elongation Step 3: Translocacion 5/lO
`G Proteins and Translation 582
`The Strncr.u res of Ef-Tu and fF-G 583
`Termination 584
`Termination Codons 584
`Stop Co<lon Suppression 5ll6
`Release Factors 5 86
`Dealing with Aberrant Termination 588
`Use of Stop Codons to lnsert Unusual Amino Acids 593
`
`18.4
`
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`

`

`18.5
`
`Po.~ttranslation 593
`Folding Nascent Proteins 594
`Release of Ribosomes from mRNA 595
`
`CHAPTER 19
`Ribosomes and Transfer RNA 601
`19.1 Ribosomes 602
`Fine Structure of the 70S Ribosome 602
`Ribosome Composition 605
`Fine Structure of the 30S Subunit 606
`Fine Structure of the 50S Subunit 612
`Ribosome Structure and the Mechanism of Translation 616
`Polysomes 621
`19.2 Transfer RNA 623
`Th.e Discovery of rRNA 623
`tRNA Structure 623
`Recognition of tRNAs hy Aminoacyl-tRNA Synthetase:
`The Second Genetic Code 626
`Proofreading and Editing hy Aminoacyl-tRNA
`Syntherases 630
`
`PART VI I
`DNA Replication, Recombination,
`and Transposition
`
`CHAPTER 20
`DNA Replication, Damage,
`and Repair 636
`20.1 General Features of DNA Replication 637
`Semiconscrva tive Replication 63 7
`At Least Scmidiscontinuous Replication 639
`Priming of DNA Synthesis 641
`Bidirectional Replication 642
`Rolling Circle Replication 645
`20.2 Enzymology of DNA Replication 646
`Three DNA Polymerases in E. coli 646
`Fidelity of Replication 649
`Multiple Eukaryotic DNA Polymerases 650
`Strand Separation 651
`Singlt:-Strand DNA-Binding Protein~ 651
`Topoisomerases 653
`20.3 DNA Damage and Repair 656
`Damage Caused by A!kylation of Bases 657
`Damage Caused hy Ultraviolet Radiation 658
`Damage Caused hy Gamma and X-Rays 658
`Directly Undoing DNA Damage 659
`
`Contents
`
`x,
`
`Excision Repair 660
`Douhle-Strand Break Repair in Eukaryotes 665
`Mismatd1 Repair 667
`Failmc of Mismatch Repair in Humans 668
`Coping with DNA Damage Without Repairing It 668
`
`CHAPTER 21
`DNA Replication II:
`Detailed Mechanism 677
`21.1
`Initiation 678
`Priming in E.coli 678
`Priming in Eukaryotes 679
`21.2 Elongation 683
`Speed of Replication 683
`The Pol HI Holoenzynie and Processivity
`of Replication 683
`21.3 Termination 694
`Decatenation: Disentangling Da11ghter DNAs 694
`Termination in Eukaryotes 695
`Box 21.1 Telomeres, the Hayflick Limit, and
`Cancer 699
`Telomere Structllre and Telomere-Binding Proteins in
`Lower Eukaryotes 702
`
`CHAPTER 22
`Homologous Recombination 709
`22.1 The RccBCD Pathway for Homologous
`Recombination 710
`22.2 Experimental Support for the RecBCD
`Pathway 712
`RecA 712
`RecBCD 715
`RuvA and RuvB 717
`RuvC 719
`22.3 Meiotic Recombination 721
`The Mechanism of Meiotic Recombination:
`Overview 721
`The Double-Stranded DNA Break 722
`Creation of Single-Stranded Ends at DSBs 728
`22.4 Gene Conversion 728
`
`CHAPTER 23
`Transposition 732
`23.1 Bacterial Transposons 733
`Discovery of Bacterial Transposons 733
`Insertion Sequences: Tl1e Simplest Bacterial
`Transposons 733
`
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`xii
`
`Contents
`
`23.2
`
`More Complex Transposons 734
`Mechanisms of Transposition 734
`Eukaryotic Transposoos 737
`The Fir.st Examples of Transposable Elements:
`TJs an<l Ac of Maize 737
`P Elements 739
`23.3 Rearrangement of lmmunoglohulin Genes 740
`Recombination Signals 742
`The Recornbioase 743
`Mec hanism ofV(D)J Recombination 743
`23.4 Retrotransposons 745
`Retroviruses 745
`Retrotra nsposons 749
`
`PART VIII
`Genomes
`
`CHAPTER 24
`Introduction to Genomics: DNA
`Sequencing on a Genomic Scale 759
`Positional Cloning: An Introduction
`24. l
`to Genomic.<; 760
`Classical Tools of Positional Cloning 760
`I<lenrifying the Gene Mutated in a Human Disease 762
`24.2 Techniques in Genomic Sequencing 765
`The Human Genome Projea 767
`Vectors for Large-Scale Genome Projects 769
`The Clone-by-Clone Strategy 770
`
`24.3
`
`Shotgun Sequencing 773
`Sequencing Standards 774
`Studying and Comparing Genomic Sequences 774
`The Uuman Genome 774
`Personal Gcnomics 779
`O ther Verrebrate Genomes 779
`The Minimal Genome 782
`The Barco<le of Life 784
`
`CHA P TER 25
`Genomics II: Functional Genomics,
`Protcomics, and Bioinformatics 789
`25 .1
`Functional Gcnomics: Gene Expression
`on a Genomic Scale 790
`Transcripromics 790
`Genomic functional Profiling 799
`Single-N ucleotide Polymorphisms:
`Pharniacogenomics 81 0
`Proteomics 812
`Protein Separations 8 12
`Protein Analysis 813
`Quanti ra rive Protcomics 814
`Protein Jnteractions s ·16
`25.3 Bioinformatics 820
`finding Regulatory Motifs in Mammalian
`Genomes 820
`Using the Databases Yourself 822
`
`25 .2
`
`Glossary 82 7
`Index 856
`
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`

`C HAPTER
`
`2
`
`The Molecular Nature of Genes
`
`B efore we begin to study in detail
`
`the structure and activities of genes, and the
`experimental evidence underlying those
`concepts, we need a fuller outline of the
`adventure that lies before us. Thus, in this
`chapter and in Chapter 3, we will flesh out
`the brief history of molecular biology pre(cid:173)
`sented in Chapter 1 . In this chapter we will
`begin this task by considering the behavior
`of genes as molecules.
`
`Computer model of the DNA double helix.
`© Comstock lmagcs/Jupilcr RF
`
`LCY Biotechnology Holding, Inc.
`Ex. 1056
`Page 13 of 30
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`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`2.1 The Nature of Genetic Material
`
`13
`
`2.1 The Nature of Genetic
`Material
`
`The studies that eventually revealed the chemistry of
`gene began in Tiibingen, Germany, in 1869. There Friedrich
`Mie cher isolated nuclei from pus cells (white blo d ceH ·)
`in waste surgical bandages. He found that these nuclei
`contained a novel phosphorus-bearing substance that he
`named nuclein. Nuclein is mostly chromatin which is a
`''
`complex of deoxyribonucleic acid (DNA) and :chromoso-
`mal proteins.
`By the end of the nineteenth century, both DNA and
`ribonucleic acid (RNA) had been separated from the pro(cid:173)
`tein that clings to them in the cell. This allowed more de(cid:173)
`tailed chemical analysis of these nucleic acids. (Notice that
`the term nucleic acid and its derivatives, DNA and RNA,
`come directly from Miescher's term nuclein.) By the begin(cid:173)
`ning of the 1930s, P. Levene, W. Jacobs, and others had
`demonstrated that RNA is composed of a sugar (ribose)
`plus four nitrogen-containing bases, and that DNA con(cid:173)
`t~ins a different sugar (deoxyribose) plus four bases. They
`discovered that each base is coupled with a sugar-phosphate
`to form a nucleotide. We will return to the chemical struc(cid:173)
`tures of DNA and RNA later in this chapter. First, let us
`examine the evidence that genes are made of DNA.
`
`Transformation in Bacteria
`Frederick Griffith laid the foundation for the identification
`of DNA as the genetic material in 1928 with his experi(cid:173)
`ments on transformation in the bacterium pneumococcus,
`now known as Streptococcus pneumoniae. The wild-type
`organism is a spherical cell surrounded by a mucous coat
`called a ~apsule. The cells form large, glistening colonies,
`characterized as smooth (S) (Figure 2.la). These cells are
`:~ule~t, t~at is, ~apable of causing lethal infections upon
`m1ect1on mto mice. A certain mutant strain of S. pneu(cid:173)
`moniae has lost the ability to form a capsule. As a result, it
`grows as small, rough (R) colonies (Figure 2.lb). More im(cid:173)
`portantly, it is avirulent; because it has no protective coat, it
`1s engulfed by the host's white blood cells before it can pro(cid:173)
`liferate enough to do any damage.
`The key finding of Griffith's work was that heat-killed
`virulent colonies of S. pneumoniae could transform aviru(cid:173)
`lent cells to virulent ones. Neither the heat-killed virulent
`bacteria nor the live avirulent ones by themselves could
`cause a lethal infection. Together, however, they were
`deadly. Somehow the virulent trait passed from the dead
`cells to the live, avirulent ones. This transformation phe(cid:173)
`nomenon is illustrated in Figure 2.2. Transformation was
`not transient; the ability to make a capsule and therefore to
`kill host animals, once conferred on the avirulent bacteria
`was passed to their descendants as a heritable trait. In othe;
`words, the avirulent cells somehow gained the gene for
`
`,,. ..
`
`•
`
`#
`
`~
`
`~
`
`-.
`-
`
`(a)
`
`(b)
`
`Figure 2.1 Variants of Streptococcus pneumoniae: (a) The large,
`glossy colonies contain smooth (S) virulent bacteria; (b} the small,
`mottled colonies are composed of rough (R) avirulent bacteria.
`(Source: (a, b) Harriet Ephrussi-Taylor.)
`
`virulence during transformation. This meant that the trans(cid:173)
`forming substance in the heat-killed bacteria was probably
`the gene for virulence itself. The missing piece of the puzzle
`was the chemical nature of the transforming substance.
`
`DNA: The Transforming Material Oswald Avery, Colin
`Macleod, and Maclyn McCarty supplied the missing
`piece in 1944. They used a transformation test similar to
`the one that Griffith had introduced, and they took pains
`to define the chemical nature of the transforming sub(cid:173)
`stance from virulent cells. First, they removed the protein
`from the extract with organic solvents and found that the
`extract still transformed. Next, they subjected it to diges(cid:173)
`tio~ with various enzymes. Trypsin and chymotrypsin,
`which destroy protein, had no effect on transformation.
`Neither did ribonuclease, which degrades RNA. These
`experiments ruled out protein or RNA as the transforming
`material. On the other hand, Avery and his coworkers
`found that the enzyme deoxyribonuclease (DNase), which
`breaks down DNA, destroyed the transforming ability of
`the virulent cell extract. These results suggested that the
`transforming substance was DNA.
`Direct physical-chemical analysi; supported the hypo(cid:173)
`thesis that the purified transforming substance was DNA.
`The analytical tools Avery and his colleagues used were
`the following:
`
`1. Ultracentrifugation They spun the transforming
`substance in an ultracentrifuge (a very high-speed
`
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`14
`
`Chapter 2 / Tl1e Molecular Nature of Genes
`
`Strain of
`Colony
`
`Strain of
`Colony
`
`Cell type
`
`Effect
`
`Cell type
`
`Elfect
`
`No capsule
`
`J
`"'~
`
`• II
`:,
`
`..
`
`Rough {A)
`
`Live R
`strain
`
`.. ti~
`
`l
`
`I,.
`
`,,
`
`•
`
`•
`
`Effoct
`
`Smooth (S)
`
`Live S
`strain
`
`(a)
`
`.//
`~
`
`h·'
`
`Heat-killed
`S strain
`
`/
`
`(c)
`
`(b)
`
`(d)
`
`Hti,-Sn-,
`S strain
`
`+ • I> ~ Effect
`I Live R stram •
`,.~1- 1/
`(cid:141)
`1
`r &
`
`Live S and R strains
`isolated from dead
`mo1Jse
`
`,
`
`• . ,
`
`Figure 2.2 Griffith's transformation experiments. (a) Virulent strain S S. pneumoniae bacteria kill their host;
`{b) avirulent strain R bacteria cannot infect successfully, so the mouse survives; (c} strain S bacteria that are
`heat-killed can no longer infect; (d) a mixture of strain R and heat-killed strain S bacteria kills the mouse. The
`killed virulent (S) bacteria have transformed the avirulent (R) bacteria to virulent (S).
`
`centrifuge) to estimate its size. The material with
`transforming activity sedimentec.l rapidly (moved
`rapidly toward the bottom of the centrifuge rube),
`suggesting a very high molecular weight, characteris(cid:173)
`tic of DNA.
`2. 1:,/ectrofJhoresis They placed the transforming
`substance in an electric field to see how rapidly it
`moved. The transforming activity had a relatively high
`mobility, also characteristic of DNA because of its
`high charge-to-mass ratio.
`3. Ultraviolet Absorption Spectrophotometry They
`placed a solution of the transforming substance in a
`spectrophotometer to see what kind of ultraviolet
`(UV) light it absorbed most strongly. Its absorption
`spectrum matched that of DNA. That is, the light it
`
`absorbed most strongly had a wavelength of about
`260 nanometers (nm), in contrast to protein, which
`absorbs maximally at 280 nm.
`4. Elementary Chemical Analysis This yielded an
`average nitrogen-to-phosphorus ratio of 1.67,
`about what one would expect. for ONA, which is
`rich in both elements, but vastly lower than the
`value expected for protein, which is rich in nitrogen
`but poor in phosphorus. Even a slight protein
`contamination would have raised the nitrogen-to(cid:173)
`phosphorus ratio.
`
`Further Confirmation These findings should have settled
`the issue of the nature of the gene, but they ha<l litrle imme(cid:173)
`diate effect. The mistaken notion, from early chemical
`
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`

`

`analyses, that DNA was a monotonous repeat of a four(cid:173)
`nucleotide sequence, such as ACTG-ACTG-ACTG, and so
`011 , persuaded many geneticists that it could not be the
`genetic materia l. Furthermor e, controversy persisted about
`possible protein conta minatkm in the transforming mate(cid:173)
`rial, whether transformation could be accomplished with
`other genes besides those governing R and S, and even
`•whether bacterial genes were like the genes of higher
`organisms.
`Yet, by 1953, when James Watson and Fra,ncis Crick
`published the double-helical model of DNA structure,
`most geneticists agreed that genes were made of DNA.
`What had changed? For one thing, Erwin Chargaff had
`shown in 1950 that the bases were not really found in
`equal proportions in DNA, as previous evidence had sug(cid:173)
`gested, and that the base composition of DNA varied
`from one species to another. In fact, this is exactly what
`one would expect for genes, which also vary from one
`species to another. Furthermore, Rollin Hotchkiss had
`refined and extended Avery's findings. He purified the
`transforming substance to the point where it contained
`only 0.02 % protein and showed that it could still change
`the genetic characteristics of bacterial cells. He went on
`to show that such highly purified DNA could transfer
`genetic traits other than R and S.
`Finally, in 1952, A.D. Hershey and Martha Chase per(cid:173)
`formed another experiment that added to the weight of
`evidence that genes were made of DNA. This experiment
`involved a bacteriophage (bacterial virus) called T2
`that infects the bacterium Escherichia coli (Figure 2.3).
`
`Figure 2.3 A false color transmission electron micrograph of T2
`phages infecting an E.coli cell. Phage particles at left and top
`appear ready to inject their DNA into the host cell. Another T2 phage
`has already infected the cell, however, and progeny phage particles
`are being assembled. The progeny phage heads are readily discernible
`as dark polygons inside the host cell. (Source: © Lee Simon/Photo
`Resear~hers, Inc.)
`
`2 .1 The Nature of Genetic Material
`
`15
`
`(The term bacteriophage is usually shortened to phage.)
`During infection, the phage genes enter the host cell and
`direct the synthesis of new phage particles. The phage is
`composed of protein and DNA only. The question is
`this: Do the genes