Essentials of Molecular Biology

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Cover Contents Preface Acknowledgements Chapter 1: Nucleic Acids Introduction DNA and RNA as Genetic Materials DNA DNA RNA Structure of Nucleic Acids The Chemical Nature of DNA and RNA Keto-Enol Tautomerism of the Nitrogenous Bases Nucleosides Pyrimidines Exist Entirely in the Anti Conformation Chargaff Rule Nucleotides The Primary Structure of DNA Watson and Crick Model of DNA – Secondary Structure of DNA The DNA Grooves DNA Conformations Triple Stranded DNA Cruciforms or Holliday Junction Tertiary Structure Properties of the DNA Physical Properties of the DNA Chemical Stability of Nucleic Acids DNA Topology Linking, Twisting and Writhing Topoisomerases Types of RNA Types of DNA The Chromosomal DNA The Autosomal DNA Y-DNA Mitochondrial DNA (mtDNA) Chloroplast DNA Plasmid DNA DNA as Vehicle of Inheritance Griffith Transformation Oswald Avery, Colin Mac Leod and Maclyn McCarty Experiment Disadvantages of the Experiment Hershey and Chase Experiment Summary Review Questions References Chapter 2: Gene Organization Introduction Genes Non-coding DNA Repetitive DNA Sequences Satellite DNAs Cot Value – Renaturation Kinetics of DNA Chromatin Chromosomes Prokaryotic Gene Organization Gene Organizationin Eukaryotes Summary Review Questions References Chapter 3: DNA Replication Introduction Chemistry of DNA Synthesis Modes of DNA Replication Semi-Conservative Mode of Replication/Meselson and Stahl Experiment Enzymes of Replication Prokaryotic DNA Polymerases Eukaryotic DNA Polymerases Helicases Primases Single-Strand Binding Proteins Ligases Topoisomerases Models of Replication Replication Rolling Circle Replication Bacteriophage X174 Replicates by Rolling Circle Replication D-loop Replication Prokaryotic Replication Origin of Replication Initiation of DNA Replication Fidelity of DNA Replication Eukaryotic Replication Cell Cycle Control of DNA Replication Eukaryotic Replication Origins Initiation of DNA Replication Elongation Licensing of DNA Replication Termination of Replication Telomeres and Telomerases Inhibitors of Replication Inhibitors of Nucleotide Biosynthesis Inhibitors of Purine Biosynthesis Analogues of Purine and Pyrimidine Bases Inhibitors of Folate Synthesis Inhibitors of Deoxynucleotide Synthesis Catabolite Analogs Inhibitors that Modify DNA Inhibitors that Affects Enzyme of Replication Summary Review Questions References Chapter 4: Transcription Introduction Differences Between Replication and Transcription Chemistry of Transcription RNA Polymerase of Prokaryotes The Sigma Subunit Promoters Promoter Types Prokaryotic Transcription Binding of RNA Polymerase DNA Foot Printing Transcriptional Events Termination Eukaryotic RNA Polymerases RNA Polymerase-I RNA Polymerase-II RNA Polymerase-III Template-independent RNA Polymerases Mechanism of Action of RNA Pol-II Transcriptional Events of Eukaryotic RNA Class-I Transcriptional Units Class-II Transcription Units Class-III Transcriptional Unit Transcription of tRNA Gene Transcription of 5S rRNA Gene Post-Transcriptional Modifications m RNA Processing RNA Editing RNA Editing of apoB mRNA Processing of rRNA Processing of tRNA Inhibitors of RNA Metabolism Inhibitors Acting by Binding to DNA Inhibitors Acting by Bindingto RNA Polymerase Inhibitors Acting by Bindingto Growing RNA Chain Reverse Transcription Reverse Transcriptase Steps Involved in Reverse Transcription Summary Review Questions References Chapter 5: Translation Introduction Genetic Code Codons Deciphering the Genetic Code Nirenberg and Khorana Experiment Characteristic Features of the Genetic Code The Mitochondrial Genetic Code Wobble Hypothesis Codon–anticodon Interactions Wobble Hypothesis Ribosome Structure Prokaryotic Ribosome—70S Ribosome Self-assembly of Ribosomes Eukaryotic Ribosome—80S Ribosome Protein Synthesis in Prokaryotes Protein Synthesis in Eukaryotes Post-Translational Modifications Summary Review Questions References Chapter 6: Protein Sorting Introduction Signal Sequences Translocation of Secretory Proteins Across the ER The Signal Sequences of Secretory Proteins to ER The Signal Recognition Particle SRP Receptor Translocation into the ER Lumen Insertion of Proteins into the ER Membrane GPI-anchored Proteins Protein Modifications in the ER Protein Glycosylation Disulphide Bond Formation The Role of N-linked Glycosylation in ER Protein Folding Protein Targeting to Mitochondria and Chloroplast Transport of Proteins to Mitochondria Transport of Proteins to Chloroplast Protein Targeting to the Nucleus Nuclear Localization Signals Transport of Proteins into the Nucleus Transport of Proteins Out of the Nucleus Transport and Sorting of Proteins to the Golgi Apparatus Transport of Proteins to the Lysosomes Receptor-Mediated Endocytosis and Sorting of Internalized Proteins Inhibitors of Protein Synthesis Streptomycin Puromycin Diphtheria Toxin Ricin Summary Review Questions References Chapter 7: Gene Regulation Introduction Regulation of Gene Expression in Prokaryotes Induction and Repression Types of Operons Lac Operon The Lactose Operon The Repressor The Operator The structural Genes Regulation of Lactose Operon Negative Regulation of Lactose Operon Catabolite Repression Positive Regulation of Lac Operon Positive Regulation by cAMP Gratuitous Inducers Tryptophan Operon and Attenuation Structural Genes Regulatory Gene Operator Co-repressor Attenuation Regulation of Eukaryotic Gene Expression Regulation of the Chromatin Structure Chromatin Remodelling Nucleosome-Remodelling Factors Histone Modifications DNA Methylation Regulation of Gene Expression at Transcription Level Cis-Acting Regulatory Sequences: Promoters and Enhancers Transcriptional Regulatory Proteins Eukaryotic Repressors RNA Interference MicroRNAs (miRNAs) The DNA-Binding Domains of the Regulatory Proteins Involved in Transcriptional Control Regulatory Proteins also have Protein–Protein Interaction Domain Leucine Zipper HLH Domain Regulation of Gene Expression by Hormone Action At the Level of Post-transcriptional Modification By Controlling the Stability of mRNA Molecule At Translational Level At Post-translational Level By Hormone Action Summary Review Questions References Chapter 8: Gene Mutation and DNA Repair Introduction Types of DNA Damages Endogenous DNA Damages Exogenous DNA Damages DNA Damages and Mutations Mutations and Types Classification of Mutations Physical and Chemical Mutagens Chemical Mutagens Physical Mutagens Biological Agents of Mutation Test for Mutagenicity Chromosomal Aberrations Chromosomal Numerical Changes Polyploidy Repair of DNA Direct Repair Systems Excision Repair Systems Base Excision Repair Nucleotide Excision Repair Mismatch Repair Recombination Repair SOS Repair/Error-Prone Repair Oncogenes Summary Review Questions References Chapter 9: Recombinant DNA Technology Introduction DNA Isolation and Sequencing Tools of Recombinant DNA Technology Restriction Endonucleases Types of Restriction Endonucleases Nomenclature of Restriction Endonuclease Type II Restriction Endonucleases Producing rDNA by Sticky-End Ligation Producing rDNA by Blunt-End Ligation Homopolymer Tailing Other Enzymes Used in rDNA Technology S1 Nuclease DNA Pol-I, Klenow Fragment Alkaline Phosphatase Reverse Transcriptase Deoxyribonuclease I (DNAse I) Cloning Vectors Types of Cloning Vectors Cosmid Vectors Shuttle Vectors Yeast Episomal Plasmids YAC Vectors Vectors for Plants Genes of Ti Plasmid and their Function Nucleic Acid Hybridization and Probes Nick Translation End Labelling or End Filling Non-radioactive Labelling Fluorescein Labelling Enzyme Labels Advantages of non-radioactive Labels In Situ Hybridization Fluorescent in Situ Hybridization (FISH) Molecular Cloning Preparation of Vector DNA Preparation of Target DNA Construction of rDNA Transport into the Host Cell (Transfection) Selection of Transformed Cells Construction of C-DNA and Genomic Libraries Genomic Library Applications of Recombinant DNA Technology In Pharmaceutical Industry Construction of Industrially Important Bacteria Summary Review Questions References Chapter 10: Epigenetics Introduction Heterochromatin and Histone Interactions Telomeric Silencing Polycomb and Trithorax CpG Islands Genomic Imprinting Epigenetic Effects and Inheritance The Epigenetic Inheritance of X Chromosomes Prions Summary Review Questions References Chapter 11: Genomics and Proteomics Introduction Genomics Classification of Genomics Structural Genomics Genome Mapping Genome Sequencing Genome Sequence Assembly Genome Annotation GenBank Gene Ontology Functional Genomics Expressed Sequence Tags (ESTs) Serial Analysis of Gene Expression (SAGE) Microarray or Gene Chip Applications of Microarray Technology Proteomics Classification of Proteomics Expression Proteomics Structural Proteomics Functional Proteomics Proteomics Tools Proteomics and Drug Discovery Summary Review Questions References Chapter 12: Techniques in Molecular Biology Introduction Gel Electrophoresis DNA Electrophoresis Protein Electrophoresis Blotting Techniques Southern Blotting Northern Blotting Western Blotting Restriction Digestion and Ligation Ligation Random Amplification of Polymorphic DNA Applications of RAPD Analysis Polymerase Chain Reaction The PCR Reaction Components PCR Thermocycling Types of PCR Summary Review Questions References Colour Illustrations Index