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Cee ey Molecular Biology David P. Clark _ Zn es | Table of Contents CHAPTER 1 CHAPTER 2 ‘CHAPTER 3 CHAPTER 4 CHAPTER 5 CHAPTER 6 (CHAPTER 7 ‘CHAPTER 8 CHAPTER 9 ‘CHAPTER 10 CHAPTER 11 CHAPTER 12 CHAPTER 13 CHAPTER 14 CHAPTER 15 CHAPTER 16 ‘CHAPTER 17 CHAPTER 18 (CHAPTER 19 CHAPTER 20 CHAPTER 21 CHAPTER 22 CHAPTER 23 CHAPTER 24 ‘CHAPTER 25 CHAPTER 26 Basic Geneties 1 Cells and Organisms 21 DNA, RNA and Protein 51 Genes, Genomes and DNA 75 Cell Division and DNA Replication 103 ‘Transcription of Genes 132 Protein Structure and Function 154 Protein Synthesis 197 Regulation of ‘Transcription in Prokaryotes 234 Regulation of Transcription in Eukaryotes 262 Regulation at the RNA Level 281 Processing of RNA 302 Mutations 333 Recombination and Repair 368 Mobile DNA 396 Plasmids 425 Viruses 453 Bacterial Genetics 484 Diversity of Lower Eukaryotes 508 Molecular Evolution 533 Nucleic Acids: Isolation, Purification, Detection, and Hybridization 567 Recombinant DNA Technology 599 The Polymerase Chain Reaction 634 Genomics and DNA Sequencing 662 Analysis of Gene Expression 693 Proteomics: The Global Analysis of Proteins 717 Glossary 745 Index 771 Detailed Contents CHAPTER 1 Basic Genetics Gregor Mendel Was the Father of Classical Gen Genes Determine Each Step in Biochemical Pathways Mutants Result from Alterations in Genes Phenotypes and Genotypes Chromosomes Are Long, Thin Molecules ‘That Carry Genes Different Organisms may Have Different Numbers of Chromosomes Dominant and Recessive Alleles Partial Dominance, Co-Dominance, Penetrance and Modifier Genes Genes from Both Parents Are Mixed by Sexual Reproduction Sex Determination and Sex-Linked Characteristics Neighboring Genes Are Linked during Inheritance Recombination during Meiosis Ensures Genetic Diversity Escherichia coli Is a Model for Bacterial Genetics CHAPTER 2 Cells and Organisms What Is Life? Living Creatures Are Made of Cells Essential Properties of a Living Cell Prokaryotie C ls Lack @ Nucleus Eubacteria and Archaebacteria Are Genetically Distinct Bacteria Were Used for Fundamental Studies of Cell Function Escherichia coli (E.coli) Is a Model Bacterium Where Are Bacteria Found in Nature? Some Bacteria Cause Infectious Disease, but Most Are Benefic Eukaryotic Cells Are Sub-Divided into Compartments, ‘The Diversity of Eukaryotes Eukaryotes Possess Two Basic Cell Lineages Organisms Are Classified 28 29 a1 2 34 4 36 36 38 Some Widely Studied Organisms Serve as Models Yeast Is a Widely Studied Single-Celled Eukaryote A Roundworm and a Fly are Model Muhticellular Animals Zebralish are used to Study Vertebrate Development Mouse and Man Arabidopsis Serves as a Model for Plants Haploidy, Diploidy and the Eukaryote Cell Cycle Viruses Are Not Living Cells Bacterial Viruses Infect Bacteria Human Viral Diseases Are Common, A Variety of Subcellular Genetic Entities Exist CHAPTER 3 DNA, RNA and Protein Nucleic Acid Molecules Carry Genetic Information Chemical Structure of Nucleic Acids DNA and RNA Each Have Four Bases Nucleosides Are Bases Plus Sugars; Nucleotides Are Nucleosides Plus Phosphate Double Stranded DNA Forms a Double H. Base Pairs are Held Together by Hydrogen Bonds Complementary Strands Reveal the Secret of Heredity Constituents of Chromosomes ‘The Central Dogma Outlines the Flow of Genetic Information Ribosomes Read the Genetic Code ‘The Genetic Code Dictates the Amino Acid Sequence of Proteins Various Classes of RNA Have Different Punetions Proteins, Made of Amino Acids, Carry Out Many Cell Funetions ‘The Structure of Proteins Has Four Levels of Organization Proteins Vary in Their Biological Roles 40 40 4L a2 44 44 45 46 47 4B 49 60 6B 68 or ) 70 a B CHAPTER 4 Genes, Genomes and DNA History of DNA as the Genetic Material How Much Genetie Information Is Necessary to Maintain Life? Non-Coding DNA Coding DNA May Be Present within Non-coding DNA Repeated Sequences Are a Feature of DNA in Higher Organisms Satellite DNA Is Non-coding DNA in the Form of Tandem Repeats Minisatellites and VNTRs Origin of Selfish DNA and Junk DNA Palindromes, Inverted Repeats and Stem and Loop Struetures, Multiple A-Tracts Cause DNA to Bend Supercoiling is Necessary for Packaging of Bacterial DNA ‘Topoisomerases and DNA Gyrase Catenated and Knotted DNA Must Be Corrected Local Supereoiling Supercoiling Affect Alternative Helical Structures of DNA Oveur Histones Package DNA in Eukaryotes Further Levels of DNA Packaging in Eukaryotes Melting Separates DNA Strands; Cooling Anneals Them DNA Structure CHAPTER 5 Cell Division and DNA Replication Cell Division and Reproduction Are Not Always, Identi DNA Replication Is a Two-Stage Process Occurring at the Replication Fork Supercoiling Causes Problems for Replication. ‘Strand Separation Precedes DNA Synthesis Properties of DNA Polymerase Polymerization of Nucleotides Supplying the Precursors for DNA Synthesis, DNA Polymerase Blongates DNA Strands ‘The Complete Replication Fork Is Complex iscontinuous Synthesis of DNA Requires a Primosome Completing the Lagging Strand 76 8 78 SR EB SE 2 96 100 103 104 104 105 107 107 109 109 m4 m2 14 116 Detailed Coens xi Chromosome Replication Initiates at oriC DNA Methylation and Attachment to the Membrane Control Initiation of Replication Chromosome Replication Terminates at rerC Disentangling the Daughter Chromosomes Cell Division in Bacteria Ovcurs alter Replication of Chromosomes How Long Does It Take for Bacteria to Replicate? The Concept of the Replicon Replicating Linear DNA in Eukaryotes Eukaryotic Chromosomes Have Multiple Origins Synthesis of Eukuryotie DNA Cell Division in Higher Organisms CHAPTER 6 Transcription of Genes Genes are Expressed by Making RNA Short Segments of the Chromosome Are “Turned into Messages ‘Terminology: Cistrons, Coding Sequences and Open Reading Frames How Is the Beginning of a Gene Recognized? Manufacturing the Message RNA Polymerase Knows Where t0 Stop How Does the Cell Know Which Genes to Turn On? What Activates the Activator? Negative Regulation Results from the Action of Repressors Many Regulator Proteins Bind Small Molecules and Change Shape ‘Transcription in Eukaryotes Is More Complex: ‘Transcription of FRNA and (RNA in Bukaryotes ‘Transcription of Protein-Encoding Genes in Eukaryotes Upstream Elements Increase the Efficieney of RNA Polymerase II Binding Enhancers Control Transeription at a Distance CHAPTER 7 Protein Structure and Function Proteins Are Formed from Amino Acids Formation of Polypeptide Chains ‘Twenty Amino Acids Form Biologi Polypeptides Amino Acids Show Asymmetry around the Alpha-carbon 118 120 121 122 124 124 125 126 129 130 130 132 133 134 134 135 137 138 140 141 143 144 145, 146 148 Is} 152 154 155 155 158 xii Detailed Contents ‘The Structure of Proteins Reflects Four Levels. of Organization The Secondary Structure of Proteins Relies on Hydrogen Bonds The Tertiary Structure of Proteins A Variety of Forces Maintain the 3-D Structure of Proteins Cysteine Forms Disulfide Bonds Multiple Folding Domains in Larger Proteins Quaternary Structure of Proteins Higher Level Assemblies and Self-Assembly Cofactors and Metal Ions Are Often Associated with Proteins Nueleoproteins, Lipoproteins and Glycoproteins ‘Are Conjugated Proteins Proteins Serve Numerous Cellular Functions Protein Machines Enzymes Catalyze Metabolic Reactions Enzymes Have Varying Specificities Lock and Key and Induced Fit Models Describe Substrate Binding Enzymes Are Named and Classified According to the Substrate Enzymes Act by Lowering the Energy of Activation ‘The Rate of Enzyme Reactions Substrate Analogs and Enzyme Inhibitors Act at the Active Site Enzymes May Be Directly Regulated Allosteric Enzymes Are Affected by Signal Molecules Enzymes May Be Controlled by Chemical Modification Binding of Proteins to DNA Occurs in Several Different Ways Denaturation of Proteins CHAPTER 8 Protein Synthesis Protein Synthesis Follows a Plan Proteins Are Gene Products Decoding the Genetic Code Transfer RNA Forms a Flat Cloverleaf Shape and a Folded “L” Shape Modified Bases Are Present in Transfer RNA Some tRNA Molecules Read More Than ‘One Codon 160 160 163 165 166 166 167 169 169 in 174 7 7 179 18 181 182 184 Ist 187 Is7 189 190 194 197 198 198, 199 200 201 202 Charging the tRNA with the Amino Acid ‘The Ribosome:The Cell’s Decoding Machine ‘Three Possible Reading Frames Exist ‘The Start Codon Is Chosen ‘The Initiation Complexes Must Be Assembled The (RNA Occupies Three Sites During Elongation of the Polypeptide ‘Termination of Protein Synthesis Requires Release Factors Several Ribosomes Message at Once Bacterial Messenger RNA Can Code for ‘Several Proteins Usually Read the Same ‘Transcription and Translation Are Coupled in Bacteria Some Ribosomes Become Stalled and Are Rescued Dilferences between Eukaryotic and Prokaryotic Protein Synthesis Initiation of Protein Synthesis in Bukaryotes Protein Synthesis Is Halted When Resources Are Scarce A Signal Sequence Marks a Protein for Export from the Cell Molecular Chaperones Oversee Protein Folding Protein Synthesis Occurs in Mitochondria and Chloroplas Proteins Are Imported into Mitochondria and Chloroplasts by Transtocases Mistranslation Usually Results in Mistakes in Protein Synthesis The € Unusual Amino Acids are Made in Proteins by Post-Translational Modifications “ode Is Not “Universal” netic Selenocysteine: The 21st Amino Acid Pyrrolysine: The 22nd Amino Acid Many Antibiotics Work by Inhibiting Protein Synthesis Degradation of Proteins CHAPTER 9 Regulation of Transcription in 204 204 208 210 au au 23 aud ais 218. a8. 221 201 204 205 226 226 207 an 27 28 230 231 Prokaryotes Regulation at the Level of Transcription Involves Several Steps Alternative Sigma Faetors in Prokaryotes Recognize Different Sets of Genes Heat Shock Sigma Factors in Prokaryotes Are Regulated by Temperature Cascades of Alternative Sigma Factors Occur in Bacillus Spore Formation sigma Factors Inactivate Sigma; i-sigma Factors Free Tt to Aet Activators and Repressors Participate in Positive and Negative Regulation ‘The Operon Model of Gene Regulation Some Proteins May Act as Both Repressors and Activators Nature of the Signal Molecule Activators and Repressors May Be Covalently Modified ‘Two-Component Regulatory Systems Phosphorelay Systems Specific Versus Global Control Crp Protein Is an Example of a Global Control Protein Accessory Factors and Nucleoid Binding Proteins Action at a Distance and DNA Looping Anti-termination as a Control Mechanism CHAPTER 10 Regulation of ‘Transcription in Eukaryotes ‘Transcriptional Regulation in Eukaryotes Is More Complex Than in Prokaryotes Specific Transcription Factors Regulate Protein Encoding Genes ‘The Mediator Complex Transmits Information to RNA Polymerase Enhancers and Insulator Sequences Segregate DNA Functionally Matrix Attachment Regions Allow DNA Looping Negative Regulation of Transeription Occurs in Bukaryotes Heterochromatin Causes Difficulty for Access t DNA in Eukaryotes Methylation of DNA in Eukaryotes Controls Gene Expression Silencing of Genes Is Caused by DNA Methylation 238 238 20 242 243 244 262 263 268. 269 20 Derailed Comets Genetic Imprinting in Eukaryotes Has tts Basis in DNA Methylation Patterns X-chromosome Inactivation Occurs in Female XX Animals: CHAPTER 11 Regulation at the RNA Level Regulation at the Level of RNA Binding of Proteins to mRNA Controls ‘The Rate of Degradation Some mRNA Molecules Must Be Cleaved Before Translation Some Regulatory Proteins May Cause ‘Translational Repression Some Regulatory Proteins Can Activ: ‘Translation ‘Translation May Be Regulated by Antisense RNA Regulation of Translation by Alterations to the Ribosome RNA Interference (RNAi) Amplification and Spread of RNAi Experimental Administration of SRNA PTGS in Plants and Quelling in Fungi Micro RNA—A Class of Small Regulatory RNA. Premature Termination Causes Attenuation of RNA Transcription Riboswitches—RNA Acting Directly as a Control Mechanism CHAPTER 12 Processing of RNA RNA js Processed in Several Ways Coding and Noa-Coding RNA. Processing of Ribosomal and Transfer RNA Eukaryotic Messenger RNA Contains a Cap and Tail Capping is the First Step in Maturation of mRNA, A Poly(A) Tail is Added to Eukaryotic mRNA Introns are Removed from RNA by Splicing Different Classi Splicing Me Aliernative Splicing Produces Multiple Forms of RNA of Intron Show Different 25 am 281 282 283, 284 287 288 290 201 202 293, 204 207 299 305 306 308, 310 314 315 xiv Detailed Contens Inteins and Protein Splicing Modification of rRNA Requires Guide RNA Editing Involves Altering the Base Sequence Transport of RNA out of the Nucleus Degradation of mRNA Nonsense Mediated Decay of mRNA CHAPTER 13 Mutations Mutations Alter the DNA Sequence ‘The Major Types of Mutation Base Substitution Mutations Missense Mutations May Have Major or Minor Eficets Nonsense Mutations Cause Premature Polypeptide Chain Termination Deletion Mutations Result in Shortened or Absent Proteins Insertion Mutations Commonly Disrupt Existing Genes Frameshift Mutations Sometimes Produce Abnormal Proteins. DNA Rearrangements Include Inversions, 1 Duplications Phase Variation Is Due to Reversible DNA Alterations Translocation: Silent Mutations Do Not Alter the Phenotype Chemical Mutagens Damage DNA Radiation Causes Mutations Spontaneous Mutations Can Be C DNA Polymerase Errors Mutations Can Result from Mispairing and Recombination Spontaneous Mutation Can Be the Result of Tautomerization used by Spontancous Mutation Can Be Caused by Inherent Chemical Instability Mutations Occur More Frequently at Hot Spots How Often Do Mutations Occur? Reversions Are Genetic Alterations That Change the Phenotype Back to Wild-type Reversion Can Occur by Compensatory Changes in Other Genes Altered Decoding by Transfer RNA May Cause Suppression Mutagenic Chemicals Can Be Detected by Reversion 324 327 307 328, 333 34 335 336 336 28 340 3a 343 353 353 353 355 358, 359 361 362 363 Experimental Isolation of Mutations In Vivo versus In Vitro Mi Site-Directed Mutagenesis CHAPTER 14 Recombination and Repair Overview of Recombination Molecular Basis of Homologous Recombination Single-Strand Invasion and Chi Sites Site-Specific Recombination Recombination in Higher Organisms Overview of DNA Repair DNA Mismatch Repair § General Excision Repair Sy DNA Repair by Excision of Specific Bases Specialized DNA Repair Mechanisms Photoreactivation Cleaves Thymine Dimers ‘Transcriptional Coupling of Repair Repair by Recombination SOS Error Prone Repair in Bacteria Repair Double-Strand Repair in Eukaryotes Gene Conversion tem stem in Bukaryotes CHAPTER 15 Mobile DNA Sub-Cellular G Creatures Most Mobile DNA Consists of Transposable Elements ments as Gene netic ‘The Essential Parts of a Transposon Insertion Sequences—the Simplest Transposons. Movement by Conservative Transposi Complex Transposons Move by Replicative ‘Transposition Replicative and Conservative Transposition are Related Composite Transposons ‘Transposition may Rearrange Host DNA ‘Transposons in Higher Life Forms Retro-Elements Make an RNA Copy Repetitive DNA of Mamm: Retro-Insertion of Host-Derived DNA. Retrons Encode Bacterial Reverse Transcriptase The Multitude of Transposable Elements 364 365 306 368 369 370 a7 373 376 378. 379 381 383 384 387 387 388 388 301 392 302 396 307 397 398 400 401 402 406 406 408 10 412 44 41s 416 a7 Bacteriophage Mu is a Transposon Conjugative Transposons Integrons Collect Genes for Transposons Junk DNA and Selfish DNA ‘Homing Introns CHAPTER 16 Plasmids Plasmids as Replicons General Properties of Plasmids Plasmid Families and Incompatibility Occasional Plasmids are Linear or Made of RNA Plasmid DNA Replicates by Two Alternative Methods Control of Copy Number by Antisense RNA Plasmid Addiction and Host Killing Functions Many Plasmids Help their Host Cells Antibiotic Res Mechanism of Antibiotic Resistance Resistance to Beta-Lactam Antibiotics Resistance to Chloramphenicol Resistance to Aminogly Resistance to Tetracycline Resistance to Sulfonamides and Trimethoprim Plasmids may Provide Aggee Most Colicins Kill by One of Two Different Mechanisms Bacteria are Immune to their own Colicins tance Plasmids vosides ve Characters Colicin Synthesis and Release Virulence Plasmids ‘Ti-Plasmids are Transferred from Bacteria to Plants The 2 Micron Plasmid of Yeast Certain DNA Molecules may Behave as ‘Viruses ot Plasmids CHAPTER 17 Viruses Viruses are Infectious Packages of Genetic Information Life Bacterial Viruses are Known as Bacteriophage Lysogeny or Lateney by Integration ‘The Great Diversity of Viruses Small Single-Stranded DNA Viruses of Bacteria Complex Bacterial Viruses with Double ‘Stranded DNA, cle of a Virus 4n7 20 20 22 23 425 26 27 ps 442 445, 446, “7 450 451 453 454 455 458, 460) 462. 463 Dezailed Contents x¥ DNA Viruses of Higher Organisms Viruses with RNA Genomes Have Very Few Genes Bacterial RNA Viruses Double Stranded RNA Viruses of Animals Positive-Stranded RNA Viruses Make Polyproteins Strategy of Negative-Strand RNA Viruses Plant RNA Viruses Retroviruses Use both RNA and DNA Genome of the Retrovirus Subviral Infectious Agents Satellite Viruses Viroids are Naked Molecules of Infectious RNA Prions are Infectious Proteins CHAPTER 18 Bacterial Genetics Reproduction versus Gene Transfer Fate of the Incoming DNA after Uptake is Gene Transfer by Naked DNA Transformation as Proof that DNA is the Genetic Material ‘Transformation in Nature Gene Transfer by Virus—Transduction Generalized Transduction Specialized Transduction ‘Transfer of Plasmids between Bacteria ‘Transfer of Chromosomal Genes Requires Plasmid Integration Gene Transfer among Gram-Positive Bacteria ‘Transform: Archacbacterial Genetics Whole Genome Sequencing CHAPTER 19 Diversity of Lower Eukaryotes Origin of the Eukaryotes by Symbiosis The Genomes of Mitochondria and Chloroplasts, Primary and Secondary Endosymbiosis Is Malaria Really a Plant? Symbiosis: Parasitism versus Mutualism Bacerial Endosymbionts of Killer Paramecium s Buchnera an Organelle or a Bacterium’? Ciliates have Two Types of Nucleus ‘Trypanosomes Vary Surface Proteins to Outwit the Immune System 466 467 469 409 469 70 470 472 471 amt 479 480 481 485 485 487 488 41 493 493 494 495; 496 S01 S04 506 509 510 su 512 sis sis si7 su