Molecular Biology (eBook)

Front Cover 4
Molecular biology: Academic Cell Update 4
Copyright Page 5
Dedication 6
Preface 8
Introduction 9
Table of Contents 10
Chapter One Basic Genetics 20
Gregor Mendel Was the Father of Classical Genetics 21
Genes Determine Each Step in Biochemical Pathways 22
Mutants Result from Alterations in Genes 23
Phenotypes and Genotypes 24
Chromosomes Are Long, Thin Molecules ThatCarry Genes 25
Different Organisms may Have Different Numbers of Chromosomes 26
Dominant and Recessive Alleles 27
Partial Dominance, Co-Dominance, Penetrance and Modifier Genes 28
Genes from Both Parents Are Mixed by Sexual Reproduction 30
Sex Determination and Sex-Linked Characteristics 32
Neighboring Genes Are Linked during Inheritance 34
Recombination during Meiosis Ensures Genetic Diversity 35
Escherichia coli is a Model for Bacterial Genetics 36
Chapter Two Cells and Organisms 40
What Is Life? 41
Living Creatures Are Made of Cells 42
Essential Properties of a Living Cell 42
Prokaryotic Cells Lack a Nucleus 46
Eubacteria and Archaebacteria Are Genetically Distinct 47
Bacteria Were Used for Fundamental Studies of Cell Function 48
Escherichia coli (E. coli) Is a Model Bacterium 50
Where Are Bacteria Found in Nature? 51
Some Bacteria Cause Infectious Disease, but Most Are Beneficial 53
Eukaryotic Cells Are Sub-Divided into Compartments 53
The Diversity of Eukaryotes 55
Eukaryotes Possess Two Basic Cell Lineages 55
Organisms Are Classified 57
Some Widely Studied Organisms Serve as Models 59
Yeast Is a Widely Studied Single-Celled Eukaryote 59
A Roundworm and a Fly Are Model Multicellular Animals 60
Zebrafish are used to Study Vertebrate Development 61
Mouse and Man 63
Arabidopsis Serves as a Model for Plants 63
Haploidy, Diploidy and the Eukaryote Cell Cycle 64
Viruses Are Not Living Cells 65
Bacterial Viruses Infect Bacteria 66
Human Viral Diseases Are Common 67
A Variety of Subcellular Genetic Entities Exist 68
Chapter Three DNA, RNA and Protein 70
Nucleic Acid Molecules Carry Genetic Information 71
Chemical Structure of Nucleic Acids 71
DNA and RNA Each Have Four Bases 73
Nucleosides Are Bases Plus Sugars Nucleotides Are Nucleosides Plus Phosphate74
Double Stranded DNA Forms a Double Helix 75
Base Pairs are Held Together by Hydrogen Bonds 76
Complementary Strands Reveal the Secret of Heredity 78
Constituents of Chromosomes 79
The Central Dogma Outlines the Flow ofGenetic Information 82
Ribosomes Read the Genetic Code 84
The Genetic Code Dictates the Amino Acid Sequence of Proteins 86
Various Classes of RNA Have Different Functions 88
Proteins, Made of Amino Acids, Carry Out Many Cell Functions 89
The Structure of Proteins Has Four Levels of Organization 90
Proteins Vary in Their Biological Roles 92
Chapter Four Genes, Genomes and DNA 94
History of DNA as the Genetic Material 95
How Much Genetic Information Is Necessary to Maintain Life? 97
Non-Coding DNA 97
Coding DNA May Be Present within Non-coding DNA 99
Repeated Sequences Are a Feature of DNA in Higher Organisms 100
Satellite DNA Is Non-coding DNA in the Form of Tandem Repeats 102
Minisatellites and VNTRs 103
Origin of Selfish DNA and Junk DNA 103
Palindromes, Inverted Repeats and Stem and Loop Structures 105
Multiple A-Tracts Cause DNA to Bend 106
Supercoiling is Necessary for Packaging of Bacterial DNA 107
Topoisomerases and DNA Gyrase 108
Catenated and Knotted DNA Must Be Corrected 110
Local Supercoiling 110
Supercoiling Affects DNA Structure 110
Alternative Helical Structures of DNA Occur 111
Histones Package DNA in Eukaryotes 114
Further Levels of DNA Packaging in Eukaryotes 115
Melting Separates DNA Strands Cooling Anneals Them119
Chapter Five Cell Division and DNAReplication 122
Cell Division and ReproductionAre Not Always Identical 123
DNA Replication Is a Two-Stage Process Occurring at the Replication Fork 123
Supercoiling Causes Problems for Replication 124
Strand Separation Precedes DNA Synthesis 126
Properties of DNA Polymerase 126
Polymerization of Nucleotides 128
Supplying the Precursors for DNA Synthesis 128
DNA Polymerase Elongates DNA Strands 130
The Complete Replication Fork Is Complex 131
Discontinuous Synthesis of DNA Requires a Primosome 133
Completing the Lagging Strand 135
Chromosome Replication Initiates at oriC 137
DNA Methylation and Attachment to the Membrane Control Initiation of Replication 139
Chromosome Replication Terminates at terC 140
Disentangling the Daughter Chromosomes 141
Cell Division in Bacteria Occurs after Replication of Chromosomes 143
How Long Does It Take for Bacteria to Replicate? 143
The Concept of the Replicon 144
Replicating Linear DNA in Eukaryotes 145
Eukaryotic Chromosomes Have Multiple Origins 148
Synthesis of Eukaryotic DNA 149
Cell Division in Higher Organisms 149
Chapter Six Transcription of Genes 151
Genes are Expressed by Making RNA 152
Short Segments of the Chromosome Are Turned into Messages 153
Terminology: Cistrons, Coding Sequences and Open Reading Frames 153
How Is the Beginning of a Gene Recognized? 154
Manufacturing the Message 156
RNA Polymerase Knows Where to Stop 157
How Does the Cell Know Which Genes to Turn On? 159
What Activates the Activator? 160
Negative Regulation Results from the Action ofRepressors 162
Many Regulator Proteins Bind Small Molecules and Change Shape 163
Transcription in Eukaryotes Is More Complex 164
Transcription of rRNA and tRNA in Eukaryotes 165
Transcription of Protein-Encoding Genes in Eukaryotes 167
Upstream Elements Increase the Efficiency of RNA Polymerase II Binding 170
Enhancers Control Transcription at a Distance 171
Chapter Seven Protein Structure and Function 173
Proteins Are Formed from Amino Acids 174
Formation of Polypeptide Chains 174
Twenty Amino Acids Form Biological Polypeptides 174
Amino Acids Show Asymmetry around the Alpha-carbon 177
The Structure of Proteins Reflects Four Levels of Organization 179
The Secondary Structure of Proteins Relies on Hydrogen Bonds 179
The Tertiary Structure of Proteins 182
A Variety of Forces Maintain the 3-D Structure of Proteins 184
Cysteine Forms Disulfide Bonds 185
Multiple Folding Domains in Larger Proteins 185
Quaternary Structure of Proteins 186
Higher Level Assemblies and Self-Assembly 188
Cofactors and Metal Ions Are Often Associated with Proteins 188
Nucleoproteins, Lipoproteins and Glycoproteins Are Conjugated Proteins 191
Proteins Serve Numerous Cellular Functions 193
Protein Machines 196
Enzymes Catalyze Metabolic Reactions 196
Enzymes Have Varying Specificities 198
Lock and Key and Induced Fit Models Describe Substrate Binding 200
Enzymes Are Named and Classified According to the Substrate 200
Enzymes Act by Lowering the Energy of Activation 201
The Rate of Enzyme Reactions 203
Substrate Analogs and Enzyme Inhibitors Act at the Active Site 203
Enzymes May Be Directly Regulated 206
Allosteric Enzymes Are Affected by Signal Molecules 206
Enzymes May Be Controlled by Chemical Modification 208
Binding of Proteins to DNA Occurs in Several Different Ways 209
Denaturation of Proteins 213
Chapter Eight Protein Synthesis 216
Protein Synthesis Follows a Plan 217
Proteins Are Gene Products 217
Decoding the Genetic Code 218
Transfer RNA Forms a Flat Cloverleaf Shape and a Folded “L” Shape 219
Modified Bases Are Present in Transfer RNA 220
Some tRNA Molecules Read More Than One Codon 221
Charging the tRNA with the Amino Acid 223
The Ribosome: The Cell’s Decoding Machine 223
Three Possible Reading Frames Exist 227
The Start Codon Is Chosen 229
The Initiation Complexes Must Be Assembled 230
The tRNA Occupies Three Sites During Elongation of the Polypeptide 230
Termination of Protein Synthesis Requires Release Factors 232
Several Ribosomes Usually Read the Same Message at Once 233
Bacterial Messenger RNA Can Code for Several Proteins 234
Transcription and Translation Are Coupled in Bacteria 235
Some Ribosomes Become Stalled and Are Rescued 236
Differences between Eukaryotic and Prokaryotic Protein Synthesis 237
Initiation of Protein Synthesis in Eukaryotes 237
Protein Synthesis Is Halted When Resources Are Scarce 240
A Signal Sequence Marks a Protein for Export from the Cell 240
Molecular Chaperones Oversee Protein Folding 243
Protein Synthesis Occurs in Mitochondria and Chloroplasts 244
Proteins Are Imported into Mitochondria and Chloroplasts by Translocases 245
Mistranslation Usually Results in Mistakesin Protein Synthesis 245
The Genetic Code Is Not “Universal” 246
Unusual Amino Acids are Made in Proteins by Post-Translational Modifications 246
Selenocysteine: The 21st Amino Acid 246
Pyrrolysine: The 22nd Amino Acid 247
Many Antibiotics Work by Inhibiting Protein Synthesis 249
Degradation of Proteins 250
Chapter Nine Regulation of Transcriptionin Prokaryotes 253
Gene Regulation Ensures a Physiological Response 254
Regulation at the Level of Transcription Involves Several Steps 255
Alternative Sigma Factors in Prokaryotes Recognize Different Sets of Genes 257
Heat Shock Sigma Factors in Prokaryotes Are Regulated by Temperature 257
Cascades of Alternative Sigma Factors Occurin Bacillus Spore Formation 258
Anti-sigma Factors Inactivate Sigma Anti-anti-sigma Factors Free It to Act261
Activators and Repressors Participate in Positive and Negative Regulation 262
The Operon Model of Gene Regulation 263
Some Proteins May Act as Both Repressors and Activators 265
Nature of the Signal Molecule 267
Activators and Repressors May Be Covalently Modified 271
Two-Component Regulatory Systems 272
Phosphorelay Systems 273
Specific versus Global Control 273
Crp Protein Is an Example of a Global Control Protein 274
Accessory Factors and Nucleoid Binding Proteins 275
Action at a Distance and DNA Looping 276
Anti-Termination as a Control Mechanism 277
Chapter Ten Regulation of Transcription in Eukaryotes 281
Transcriptional Regulation in Eukaryotes Is More Complex Than in Prokaryotes 282
Specific Transcription Factors Regulate Protein Encoding Genes 283
The Mediator Complex Transmits Information to RNA Polymerase 283
Enhancers and Insulator Sequences Segregate DNA Functionally 284
Matrix Attachment Regions Allow DNA Looping 287
Negative Regulation of Transcription Occurs in Eukaryotes 288
Heterochromatin Causes Difficulty for Access to DNA in Eukaryotes 289
Methylation of DNA in Eukaryotes Controls Gene Expression 292
Silencing of Genes Is Caused by DNA Methylation 294
Genetic Imprinting in Eukaryotes Has Its Basis in DNA Methylation Patterns 294
X-Chromosome Inactivation Occurs in Female XX Animals 296
Chapter Eleven Regulation at the RNA Level 300
Regulation at the Level of RNA 301
Binding of Proteins to mRNA Controls The Rate of Degradation 301
Some mRNA Molecules Must Be Cleaved Before Translation 302
Some Regulatory Proteins May Cause Translational Repression 303
Some Regulatory Proteins Can Activate Translation 306
Translation May Be Regulated by Antisense RNA 307
Regulation of Translation by Alterations to the Ribosome 309
RNA Interference (RNAi) 310
Amplification and Spread of RNAi 311
Experimental Administration of siRNA 312
PTGS in Plants and Quelling in Fungi 313
Micro RNA—A Class of Small Regulatory RNA 314
Premature Termination Causes Attenuation of RNA Transcription 316
Riboswitches—RNA Acting Directly as a Control Mechanism 318
Chapter Twelve Processing of RNA 321
RNA is Processed in Several Ways 322
Coding and Non-Coding RNA 323
Processing of Ribosomal and Transfer RNA 324
Eukaryotic Messenger RNA Contains a Cap and a Tail 324
Capping is the First Step in Maturation of mRNA 325
A Poly(A) Tail is Added to Eukaryotic mRNA 327
Introns are Removed from RNA by Splicing 329
Different Classes of Intron Show Different Splicing Mechanisms 333
Alternative Splicing Produces Multiple Forms of RNA 334
Inteins and Protein Splicing 337
Base Modification of rRNA Requires Guide RNA 341
RNA Editing Involves Altering the Base Sequence 343
Transport of RNA out of the Nucleus 346
Degradation of mRNA 346
Nonsense Mediated Decay of mRNA 347
Chapter Thirteen Mutations 352
Mutations Alter the DNA Sequence 353
The Major Types of Mutation 354
Base Substitution Mutations 355
Missense Mutations May Have Major or Minor Effects 355
Nonsense Mutations Cause Premature Polypeptide Chain Termination 357
Deletion Mutations Result in Shortened or Absent Proteins 359
Insertion Mutations Commonly Disrupt Existing Genes 360
Frameshift Mutations Sometimes Produce Abnormal Proteins 362
DNA Rearrangements Include Inversions,Translocations, and Duplications 362
Phase Variation Is Due to Reversible DNA Alterations 364
Silent Mutations Do Not Alter the Phenotype 365
Chemical Mutagens Damage DNA 367
Radiation Causes Mutations 369
Spontaneous Mutations Can Be Caused by DNA Polymerase Errors 370
Mutations Can Result from Mispairing and Recombination 372
Spontaneous Mutation Can Be the Result of Tautomerization 372
Spontaneous Mutation Can Be Caused by Inherent Chemical Instability 372
Mutations Occur More Frequently at Hot Spots 374
How Often Do Mutations Occur? 377
Reversions Are Genetic Alterations That Change the Phenotype Back to Wild-type 378
Reversion Can Occur by Compensatory Changes in Other Genes 380
Altered Decoding by Transfer RNA May Cause Suppression 381
Mutagenic Chemicals Can Be Detected by Reversion 382
Experimental Isolation of Mutations 383
In Vivo versus In Vitro Mutagenesis 384
Site-Directed Mutagenesis 385
Chapter Fourteen Recombination and Repair 387
Overview of Recombination 388
Molecular Basis of Homologous Recombination 389
Single-Strand Invasion and Chi Sites 390
Site-Specific Recombination 392
Recombination in Higher Organisms 395
Overview of DNA Repair 397
DNA Mismatch Repair System 398
General Excision Repair System 400
DNA Repair by Excision of Specific Bases 402
Specialized DNA Repair Mechanisms 403
Photoreactivation Cleaves Thymine Dimers 406
Transcriptional Coupling of Repair 406
Repair by Recombination 407
SOS Error Prone Repair in Bacteria 407
Repair in Eukaryotes 410
Double-Strand Repair in Eukaryotes 411
Gene Conversion 411
Chapter Fifteen Mobile DNA 415
Sub-Cellular Genetic Elements as Gene Creatures 416
Most Mobile DNA Consists of Transposable Elements 416
The Essential Parts of a Transposon 417
Insertion Sequences—the Simplest Transposons 419
Movement by Conservative Transposition 420
Complex Transposons Move by Replicative Transposition 421
Replicative and Conservative Transposition are Related 425
Composite Transposons 425
Transposition may Rearrange Host DNA 427
Transposons in Higher Life Forms 429
Retro-Elements Make an RNA Copy 431
Repetitive DNA of Mammals 433
Retro-Insertion of Host-Derived DNA 434
Retrons Encode Bacterial Reverse Transcriptase 435
The Multitude of Transposable Elements 436
Bacteriophage Mu is a Transposon 436
Conjugative Transposons 439
Integrons Collect Genes for Transposons 439
Junk DNA and Selfish DNA 441
Homing Introns 442
Chapter Sixteen Plasmids 444
Plasmids as Replicons 445
General Properties of Plasmids 446
Plasmid Families and Incompatibility 447
Occasional Plasmids are Linear or Made of RNA 447
Plasmid DNA Replicates by Two Alternative Methods 449
Control of Copy Number by Antisense RNA 451
Plasmid Addiction and Host Killing Functions 454
Many Plasmids Help their Host Cells 455
Antibiotic Resistance Plasmids 455
Mechanisms of Antibiotic Resistance 457
Resistance to Beta-Lactam Antibiotics 457
Resistance to Chloramphenicol 458
Resistance to Aminoglycosides 459
Resistance to Tetracycline 460
Resistance to Sulfonamides and Trimethoprim 461
Plasmids may Provide Aggressive Characters 461
Most Colicins Kill by One of Two Different Mechanisms 463
Bacteria are Immune to their own Colicins 464
Colicin Synthesis and Release 465
Virulence Plasmids 465
Ti-Plasmids are Transferred from Bacteria to Plants 466
The 2-Micron Plasmid of Yeast 469
Certain DNA Molecules may Behave as Viruses or Plasmids 470
Chapter Seventeen Viruses 443
Viruses are Infectious Packages of Genetic Information 473
Life Cycle of a Virus 474
Bacterial Viruses are Known as Bacteriophage 477
Lysogeny or Latency by Integration 479
The Great Diversity of Viruses 481
Small Single-Stranded DNA Viruses of Bacteria 482
Complex Bacterial Viruses with Double Stranded DNA 484
DNA Viruses of Higher Organisms 485
Viruses with RNA Genomes Have Very Few Genes 486
Bacterial RNA Viruses 488
Double Stranded RNA Viruses of Animals 488
Positive-Stranded RNA Viruses Make Polyproteins 488
Strategy of Negative-Strand RNA Viruses 489
Plant RNA Viruses 489
Retroviruses Use both RNA and DNA 491
Genome of the Retrovirus 496
Subviral Infectious Agents 496
Satellite Viruses 498
Viroids are Naked Molecules of Infectious RNA 499
Prions are Infectious Proteins 500
Chapter Eighteen Bacterial Genetics 503
Reproduction versus Gene Transfer 504
Fate of the Incoming DNA after Uptake 504
Transformation is Gene Transfer by Naked DNA 506
Transformation as Proof that DNA is the Genetic Material 507
Transformation in Nature 510
Gene Transfer by Virus—Transduction 512
Generalized Transduction 512
Specialized Transduction 513
Transfer of Plasmids between Bacteria 514
Transfer of Chromosomal Genes Requires Plasmid Integration 515
Gene Transfer among Gram-Positive Bacteria 520
Archaebacterial Genetics 523
Whole Genome Sequencing 525
Chapter Nineteen Diversity of Lower Eukaryotes 527
Origin of the Eukaryotes by Symbiosis 528
The Genomes of Mitochondria and Chloroplasts 529
Primary and Secondary Endosymbiosis 530
Is Malaria Really a Plant? 531
Symbiosis: Parasitism versus Mutualism 534
Bacterial Endosymbionts of Killer Paramecium 534
Is Buchnera an Organelle or a Bacterium? 536
Ciliates have Two Types of Nucleus 536
Trypanosomes Vary Surface Proteins to Outwit the Immune System 539
Mating Type Determination in Yeast 544
Multi-Cellular Organisms and Homeobox Genes 549
Chapter Twenty Molecular Evolution 552
Getting Started—Formation of the Earth 553
The Early Atmosphere 553
Oparin’s Theory of the Origin of Life 554
The Miller Experiment 555
Polymerization of Monomers to Give Macromolecules 557
Enzyme Activities of Random Proteinoids 558
Origin of Informational Macromolecules 559
Ribozymes and the RNA World 559
The First Cells 561
The Autotrophic Theory of the Origin of Metabolism 563
Evolution of DNA, RNA and Protein Sequences 564
Creating New Genes by Duplication 566
Paralogous and Orthologous Sequences 568
Creating New Genes by Shuffling 569
Different Proteins Evolve at Very Different Rates 569
Molecular Clocks to Track Evolution 571
Ribosomal RNA—A Slowly Ticking Clock 571
The Archaebacteria versus the Eubacteria 573
DNA Sequencing and Biological Classification 574
Mitochondrial DNA—A Rapidly Ticking Clock 578
The African Eve Hypothesis 579
Ancient DNA from Extinct Animals 581
Evolving Sideways: Horizontal Gene Transfer 583
Problems in Estimating Horizontal Gene Transfer 584
Chapter Twenty-one Nucleic Acids: Isolation,Purification, Detection, and Hybridization 502
Isolation of DNA 587
Purification of DNA 587
Removal of Unwanted RNA 588
Gel Electrophoresis of DNA 589
Pulsed Field Gel Electrophoresis 591
Denaturing Gradient Gel Electrophoresis 592
Chemical Synthesis of DNA 593
Chemical Synthesis of Complete Genes 599
Peptide Nucleic Acid 599
Measuring the Concentration of DNA and RNA withUltraviolet Light 601
Radioactive Labeling of Nucleic Acids 602
Detection of Radio-Labeled DNA 602
Fluorescence in the Detection of DNA and RNA 604
Chemical Tagging with Biotin or Digoxigenin 606
The Electron Microscope 607
Hybridization of DNA and RNA 609
Southern, Northern, and Western Blotting 611
Zoo Blotting 614
Fluorescence in Situ Hybridization (FISH) 614
Molecular Beacons 617
Chapter Twenty-Two Recombinant DNA Technology 618
Introduction 619
Nucleases Cut Nucleic Acids 619
Restriction and Modification of DNA 619
Recognition of DNA by Restriction Endonucleases 620
Naming of Restriction Enzymes 620
Cutting of DNA by Restriction Enzymes 621
DNA Fragments are Joined by DNA Ligase 622
Making a Restriction Map 623
Restriction Fragment Length Polymorphisms (RFLPs) 626
Properties of Cloning Vectors 627
Multicopy Plasmid Vectors 629
Inserting Genes into Vectors 629
Detecting Insertions in Vectors 631
Moving Genes between Organisms: Shuttle Vectors 634
Bacteriophage Lambda Vectors 635
Cosmid Vectors 636
Yeast Artificial Chromosomes 639
Bacterial and P1 Artificial Chromosomes 639
A DNA Library Is a Collection of Genesfrom One Organism 640
Screening a Library by Hybridization 642
Screening a Library by Immunological Procedures 642
Cloning Complementary DNA Avoids Introns 643
Chromosome Walking 645
Cloning by Subtractive Hybridization 647
Expression Vectors 650
Chapter Twenty-Three The Polymerase Chain Reaction 653
Fundamentals of the Polymerase Chain Reaction 654
Cycling Through the PCR 657
Degenerate Primers 659
Inverse PCR 660
Adding Artificial Restriction Sites 661
TA Cloning by PCR 662
Randomly Amplified Polymorphic DNA (RAPD) 662
Reverse Transcriptase PCR 665
Differential Display PCR 666
Rapid Amplification of cDNA Ends (RACE) 668
PCR in Genetic Engineering 668
Directed Mutagenesis 670
Engineering Deletions and Insertions by PCR 670
Use of PCR in Medical Diagnosis 671
Environmental Analysis by PCR 672
Rescuing DNA from Extinct Life Forms by PCR 673
Realtime Fluorescent PCR 674
Inclusion of Molecular Beacons in PCR—Scorpion Primers 675
Chapter Twenty-Four Genomics and DNA Sequencing 652
Introduction to Genomics 682
DNA Sequencing—General Principle 682
The Chain Termination Method for Sequencing DNA 682
DNA Polymerases for Sequencing DNA 687
Producing Template DNA for Sequencing 687
Primer Walking along a Strand of DNA 689
Automated Sequencing 689
The Emergence of DNA Chip Technology 691
The Oligonucleotide Array Detector 691
Pyrosequencing 693
Nanopore Detectors for DNA 695
Large Scale Mapping with Sequence Tags 695
Mapping of Sequence Tagged Sites 696
Assembling Small Genomes by Shotgun Sequencing 699
Race for the Human Genome 699
Assembling a Genome from Large Cloned Contigs 702
Assembling a Genome by Directed Shotgun Sequencing 702
Survey of the Human Genome 702
Sequence Polymorphisms: SSLPs and SNPs 705
Gene Identification by Exon Trapping 707
Bioinformatics and Computer Analysis 709
Chapter Twenty-Five Analysis of Gene Expression 712
Introduction 713
Monitoring Gene Expression 713
Reporter Genes for Monitoring Gene Expression 713
Easily Assayable Enzymes as Reporters 715
Light Emission by Luciferase as a Reporter System 715
Green Fluorescent Protein as Reporter 718
Gene Fusions 718
Deletion Analysis of the Upstream Region 721
Locating Protein Binding Sites in the Upstream Region 721
Location of the Start of Transcription byPrimer Extension 725
Location of the Start of Transcription by S1 Nuclease 726
Transcriptome Analysis 728
DNA Microarrays for Gene Expression 728
Serial Analysis of Gene Expression (SAGE) 732
Chapter Twenty-Six Proteomics: The Global Analysis of Proteins 736
Introduction to Proteomics 737
Gel Electrophoresis of Proteins 738
Two Dimensional PAGE of Proteins 739
Western Blotting of Proteins 741
Mass Spectrometry for Protein Identification 741
Protein Tagging Systems 745
Full-Length Proteins Used as Fusion Tags 745
Self Cleavable Intein Tags 748
Selection by Phage Display 748
Protein Interactions: The Yeast Two-Hybrid System 751
Protein Interaction by Co-Immunoprecipitation 756
Protein Arrays 760
Metabolomics 760
Glossary 764
Index 790