Fundamental molecular biology

Preface, xviii 1 The beginnings of molecular biology, 1 1.1 Introduction 1.2 Historical perspective Insights into heredity from round and wrinkled peas: Mendelian genetics Insights into the nature of hereditary material: the transforming principle is DNA Creativity in approach leads to the one gene–one enzyme hypothesis The importance of technological advances: the Hershey–Chase experiment A model for the structure of DNA: the DNA double helix Chapter summary Analytical questions Suggestions for further reading 2 The structure of DNA, 13 2.1 Introduction 2.2 Primary structure: the components of nucleic acids Five-carbon sugars Nitrogenous bases The phosphate functional group Nucleosides and nucleotides 2.3 Significance of 5′ and 3′ 2.4 Nomenclature of nucleotides 2.5 The length of RNA and DNA 2.6 Secondary structure of DNA Hydrogen bonds form between the bases Base stacking provides chemical stability to the DNA double helix Structure of the Watson–Crick DNA double helix Distinguishing between features of alternative double-helical structures DNA can undergo reversible strand separation 2.7 Unusual DNA secondary structures Slipped structures Cruciform structures Triple helix DNA Disease box 2.1 Friedreich’s ataxia and triple helix DNA 2.8 Tertiary structure of DNA Supercoiling of DNA Topoisomerases relax supercoiled DNA What is the significance of supercoiling in vivo? Disease box 2.2 Topoisomerase-targeted anticancer drugs Chapter summary Analytical questions Suggestions for further reading 3 Genome organization: from nucleotides to chromatin, 37 3.1 Introduction 3.2 Eukaryotic genome v Chromatin structure: historical perspective Histones Nucleosomes Beads-on-a-string: the 10 nm fiber The 30 nm fiber Loop domains Metaphase chromosomes Alternative chromatin structures 3.3 Bacterial genome 3.4 Plasmids 3.5 Bacteriophages and mammalian DNA viruses Bacteriophages Mammalian DNA viruses 3.6 Organelle genomes: chloroplasts and mitochondria Chloroplast DNA (cpDNA) Mitochondrial DNA (mtDNA) Disease box 3.1 Mitochondrial DNA and disease 3.7 RNA-based genomes Eukaryotic RNA viruses Retroviruses Viroids Other subviral pathogens Disease box 3.2 Avian flu Chapter summary Analytical questions Suggestions for further reading 4 The versatility of RNA, 54 4.1 Introduction 4.2 Secondary structure of RNA Secondary structure motifs in RNA Base-paired RNA adopts an A-type double helix RNA helices often contain noncanonical base pairs 4.3 Tertiary structure of RNA tRNA structure: important insights into RNA structural motifs Common tertiary structure motifs in RNA 4.4 Kinetics of RNA folding 4.5 RNA is involved in a wide range of cellular processes 4.6 Historical perspective: the discovery of RNA catalysis Tetrahymena group I intron ribozyme RNase P ribozyme Focus box 4.1: The RNA world 4.7 Ribozymes catalyze a variety of chemical reactions Mode of ribozyme action Large ribozymes Small ribozymes Chapter summary Analytical questions Suggestions for further reading vi Contents 5 From gene to protein, 79 5.1 Introduction 5.2 The central dogma 5.3 The genetic code Translating the genetic code The 21st and 22nd genetically encoded amino acids Role of modified nucleotides in decoding Implications of codon bias for molecular biologists 5.4 Protein structure Primary structure Secondary structure Tertiary structure Quaternary structure Size and complexity of proteins Proteins contain multiple functional domains Prediction of protein structure 5.5 Protein function Enzymes are biological catalysts Regulation of protein activity by post-translational modifications Allosteric regulation of protein activity Cyclin-dependent kinase activation Macromolecular assemblages 5.6 Protein folding and misfolding Molecular chaperones Ubiquitin-mediated protein degradation Protein misfolding diseases Disease box 5.1 Prions Chapter summary Analytical questions Suggestions for further reading 6 DNA replication and telomere maintenance, 108 6.1 Introduction 6.2 Historical perspective Insight into the mode of DNA replication: the Meselson–Stahl experiment Insight into the mode of DNA replication: visualization of replicating bacterial DNA 6.3 DNA synthesis occurs from 5′ → 3′ 6.4 DNA polymerases are the enzymes that catalyze DNA synthesis Focus box 6.1 Bacterial DNA polymerases 6.5 Semidiscontinuous DNA replication Leading strand synthesis is continuous Lagging strand synthesis is discontinuous 6.6 Nuclear DNA replication in eukaryotic cells Replication factories Histone removal at the origins of replication Prereplication complex formation at the origins of replication Replication licensing: DNA only replicates once per cell cycle Duplex unwinding at replication forks RNA priming of leading strand and lagging strand DNA synthesis Contents vii Polymerase switching Elongation of leading strands and lagging strands Proofreading Maturation of nascent DNA strands Termination Histone deposition Focus box 6.2 The naming of genes involved in DNA replication Disease box 6.1 Systemic lupus erythematosus and PCNA 6.7 Replication of organelle DNA Models for mtDNA replication Replication of cpDNA Disease box 6.2 RNase MRP and cartilage-hair hypoplasia 6.8 Rolling circle replication 6.9 Telomere maintenance: the role of telomerase in DNA replication, aging, and cancer Telomeres Solution to the end replication problem Maintenance of telomeres by telomerase Other modes of telomere maintenance Regulation of telomerase activity Telomerase, aging, and cancer Disease box 6.3 Dyskeratosis congenita: loss of telomerase function Chapter summary Analytical questions Suggestions for further reading 7 DNA repair and recombination, 152 7.1 Introduction 7.2 Types of mutations and their phenotypic consequences Transitions and transversions can lead to silent, missense, or nonsense mutations Insertions or deletions can cause frameshift mutations Expansion of trinucleotide repeats leads to genetic instability 7.3 General classes of DNA damage Single base changes Structural distortion DNA backbone damage Cellular response to DNA damage 7.4 Lesion bypass 7.5 Direct reversal of DNA damage 7.6 Repair of single base changes and structural distortions by removal of DNA damage Base excision repair Mismatch repair Nucleotide excision repair Disease box 7.1 Hereditary nonpolyposis colorectal cancer: a defect in mismatch repair 7.7 Double-strand break repair by removal of DNA damage Homologous recombination Nonhomologous end-joining Disease box 7.2 Xeroderma pigmentosum and related disorders: defects in nucleotide excision repair Disease box 7.3 Hereditary breast cancer syndromes: mutations in BRCA1 and BRCA2 viii Contents Chapter summary Analytical questions Suggestions for further reading 8 Recombinant DNA technology and molecular cloning, 180 8.1 Introduction 8.2 Historical perspective Insights from bacteriophage lambda (l) cohesive sites Insights from bacterial restriction and modification systems The first cloning experiments 8.3 Cutting and joining DNA Major classes of restriction endonucleases Restriction endonuclease nomenclature Recognition sequences for type II restriction endonucleases DNA ligase Focus box 8.1 Fear of recombinant DNA molecules 8.4 Molecular cloning Vector DNA Choice of vector is dependent on insert size and application Plasmid DNA as a vector Bacteriophage lambda (l) as a vector Artificial chromosome vectors Sources of DNA for cloning Focus box 8.2 EcoRI: kinking and cutting DNA Tool box 8.1 Liquid chromatography 8.5 Constructing DNA libraries Genomic library cDNA library 8.6 Probes Heterologous probes Homologous probes Tool box 8.2 Complementary DNA (cDNA) synthesis Tool box 8.3 Polymerase chain reaction (PCR) Tool box 8.4 Radioactive and nonradioactive labeling methods Tool box 8.5 Nucleic acid labeling 8.7 Library screening Transfer of colonies to a DNA-binding membrane Colony hybridization Detection of positive colonies 8.8 Expression libraries 8.9 Restriction mapping 8.10 Restriction fragment length polymorphism (RFLP) RFLPs can serve as markers of genetic diseases Tool box 8.6 Electrophoresis Tool box 8.7 Southern blot Disease box 8.1 PCR-RFLP assay for maple syrup urine disease 8.11 DNA sequencing Manual DNA sequencing by the Sanger “dideoxy” DNA method Automated DNA sequencing Contents ix Chapter summary Analytical questions Suggestions for further reading 9 Tools for analyzing gene expression, 232 9.1 Introduction 9.2 Transient and stable transfection assays 9.3 Reporter genes Commonly used reporter genes Analysis of gene regulation Purification and detection tags: fusion proteins Tool box 9.1 Production of recombinant proteins 9.4 In vitro mutagenesis Tool box 9.2 Fluorescence, confocal, and multiphoton microscopy 9.5 Analysis at the level of gene transcription: RNA expression and localization Northern blot In situ hybridization RNase protection assay (RPA) Reverse transcription-PCR (RT-PCR) 9.6 Analysis at the level of translation: protein expression and localization Western blot In situ analysis Enzyme-linked immunosorbent assay (ELISA) Tool box 9.3 Protein gel electrophoresis Tool box 9.4 Antibody production 9.7 Antisense technology Antisense oligonucleotides RNA interference (RNAi) 9.8 Analysis of DNA–protein interactions Electrophoretic mobility shift assay (EMSA) DNase I footprinting Chromatin immunoprecipitation (ChIP) assay Disease box 9.1 RNAi therapies 9.9 Analysis of protein–protein interactions Pull-down assay Yeast two-hybrid assay Coimmunoprecipitation assay Fluorescence resonance energy transfer (FRET) 9.10 Structural analysis of proteins X-ray crystallography Nuclear magnetic resonance (NMR) spectroscopy Cryoelectron microscopy Atomic force microscopy (AFM) 9.11 Model organisms Yeast: Saccharomyces cerevisiae and Schizosaccharomyces pombe Worm: Caenorhabditis elegans Fly: Drosophila melanogaster Fish: Danio rerio Plant: Arabidopsis thaliana Mouse: Mus musculus x Contents Frog: Xenopus laevis and Xenopus tropicalis Chapter summary Analytical questions Suggestions for further reading 10 Transcription in prokaryotes, 278 10.1 Introduction 10.2 Transcription and translation are coupled in bacteria 10.3 Mechanism of transcription Bacterial promoter structure Structure of bacterial RNA polymerase Stages of transcription Proofreading Direction of transcription around the E. coli chromosome Focus box 10.1 Which moves – the RNA polymerase or the DNA? 10.4 Historical perspective: the Jacob–Monod operon model of gene regulation The operon model led to the discovery of mRNA Characterization of the Lac repressor 10.5 Lactose (lac) operon regulation Lac operon induction Basal transcription of the lac operon Regulation of the lac operon by Rho The lac promoter and lacZ structural gene are widely used in molecular biology research 10.6 Mode of action of transcriptional regulators Cooperative binding of proteins to DNA Allosteric modifications and DNA binding DNA looping 10.7 Control of gene expression by RNA Differential folding of RNA: transcriptional attenuation of the tryptophan operon Riboswitches Riboswitch ribozymes Chapter summary Analytical questions Suggestions for further reading 11 Transcription in eukaryotes, 312 11.1 Introduction 11.2 Overview of transcriptional regulation 11.3 Protein-coding gene regulatory elements Structure and function of promoter elements Structure and function of long-range regulatory elements Focus box 11.1 Position effect and long-range regulatory elements Disease box 11.1 Hispanic thalassemia and DNase I hypersensitive sites Focus box 11.2 Is there a nuclear matrix? Focus box 11.3 Chromosomal territories and transcription factories 11.4 General (basal) transcription machinery Components of the general transcription machinery Structure of RNA polymerase II General transcription factors and preinitiation complex formation Mediator: a molecular bridge Contents xi 11.5 Transcription factors Transcription factors mediate gene-specific transcriptional activation or repression Transcription factors are modular proteins DNA-binding domain motifs Transactivation domain Dimerization domain Focus box 11.4 Homeoboxes and homeodomains Disease box 11.2 Greig cephalopolysyndactyly syndrome and Sonic hedgehog signaling Disease box 11.3 Defective histone acetyltransferases in Rubinstein–Taybi syndrome 11.6 Transcriptional coactivators and corepressors Chromatin modification complexes Linker histone variants Chromatin remodeling complexes Focus box 11.5 Is there a histone code? 11.7 Transcription complex assembly: the enhanceosome model versus the “hit and run” model Order of recruitment of various proteins that regulate transcription Enhanceosome model Hit and run model Merging of models 11.8 Mechanism of RNA polymerase II transcription Promoter clearance Elongation: polymerization of RNA Proofreading and backtracking Transcription elongation through the nucleosomal barrier Disease box 11.4 Defects in Elongator and familial dysautonomia 11.9 Nuclear import and export of proteins Karyopherins Nuclear localization sequences (NLSs) Nuclear export sequences (NESs) Nuclear import pathway Nuclear export pathway Focus box 11.6 The nuclear pore complex Focus box 11.7 Characterization of the first nuclear localization sequence 11.10 Regulated nuclear import and signal transduction pathways Regulated nuclear import of NF-kB Regulated nuclear import of the glucocorticoid receptor Chapter summary Analytical questions Suggestions for further reading 12 Epigenetics and monoallelic gene expression, 392 12.1 Introduction 12.2 Epigenetic markers Cytosine DNA methylation marks genes for silencing Stable maintenance of histone modifications Disease box 12.1 Cancer and epigenetics 12.3 Genomic imprinting Establishing and maintaining the imprint Mechanisms of monoallelic expression Genomic imprinting is essential for normal development xii Contents Origins of genomic imprinting Disease box 12.2 Fragile X mental retardation and aberrant DNA methylation Disease box 12.3 Genomic imprinting and neurodevelopmental disorders 12.4 X chromosome inactivation Random X chromosome inactivation in mammals Molecular mechanisms for stable maintenance of X chromosome inactivation Is there monoallelic expression of all X-linked genes? 12.5 Phenotypic consequences of transposable elements Historical perspective: Barbara McClintock’s discovery of mobile genetic elements in maize DNA transposons have a wide host range DNA transposons move by a “cut and paste” mechanism Retrotransposons move by a “copy and paste” mechanism Some LTR retrotransposons are active in the mammalian genome Non-LTR retrotransposons include LINEs and SINEs Tool box 12.1 Transposon tagging Disease box 12.4 Jumping genes and human disease 12.6 Epigenetic control of transposable elements Methylation of transposable elements Heterochromatin formation mediated by RNAi and RNA-directed DNA methylation 12.7 Allelic exclusion Yeast mating-type switching and silencing Antigen switching in trypanosomes V(D)J recombination and the adaptive immune response Disease box 12.5 Trypanosomiasis: human “sleeping sickness” Focus box 12.1 Did the V(D)J system evolve from a transposon? Chapter summary Analytical questions Suggestions for further reading 13 RNA processing and post-transcriptional gene regulation, 452 13.1 Introduction 13.2 RNA splicing: historical perspective and overview 13.3 Group I and group II self-splicing introns Group I introns require an external G cofactor for splicing Group II introns require an internal bulged A for splicing Mobile group I and II introns Focus box 13.1 Intron-encoded small nucleolar RNA and “inside-out” genes 13.4 Archael and nuclear transfer RNA introns Archael introns are spliced by an endoribonuclease Some nuclear tRNA genes contain an intron 13.5 Cotranscriptional processing of nuclear pre-mRNA Addition of the 5′-7-methylguanosine cap Termination and polyadenylation Splicing Disease box 13.1 Oculopharyngeal muscular dystrophy: trinucleotide repeat expansion in a poly(A)-binding protein gene Disease box 13.2 Spinal muscular atrophy: defects in snRNP biogenesis Disease box 13.3 Prp8 gene mutations cause retinitis pigmentosa 13.6 Alternative splicing Effects of alternative splicing on gene expression Contents xiii Regulation of alternative splicing Focus box 13.2 The DSCAM gene: extreme alternative splicing 13.7 Trans-splicing Discontinuous group II trans-splicing Spliced leader trans-splicing tRNA trans-splicing Focus box 13.3 Apoptosis 13.8 RNA editing RNA editing in trypanosomes RNA editing in mammals Disease box 13.4 Amyotrophic lateral sclerosis: a defect in RNA editing? 13.9 Base modification guided by small nucleolar RNA molecules 13.10 Post-transcriptional gene regulation by microRNA Historical perspective: the discovery of miRNA in Caenorhabditis elegans Processing of miRNAs miRNAs target mRNA for degradation and translational inhibition 13.11 RNA turnover in the nucleus and cytoplasm Nuclear exosomes and quality control Quality control and the formation of nuclear export-competent RNPs Cytoplasmic RNA turnover Chapter summary Analytical questions Suggestions for further reading 14 The mechanism of translation, 512 14.1 Introduction 14.2 Ribosome structure and assembly Structure of ribosomes The nucleolus Ribosome biogenesis Focus box 14.1 What is “S”? 14.3 Aminoacyl-tRNA synthetases Aminoacyl-tRNA charging Proofreading activity of aminoacyl-tRNA synthetases 14.4 Initiation of translation Ternary complex formation and loading onto the 40S ribosomal subunit Loading the mRNA on the 40S ribosomal subunit Scanning and AUG recognition Joining of the 40S and 60S ribosomal subunits Tool box 14.1 Translation toeprinting assays Disease box 14.1 Eukaryotic initiation factor 2B and vanishing white matter 14.5 Elongation Decoding Peptide bond formation and translocation Peptidyl transferase activity Events in the ribosome tunnel 14.6 Termination 14.7 Translational and post-translational control Phosphorylation of eIF2a blocks ternary complex formation eIF2a phosphorylation is mediated by four distinct protein kinases xiv Contents Chapter summary Analytical questions Suggestions for further reading 15 Genetically modified organisms: use in basic and applied research, 545 15.1 Introduction 15.2 Transgenic mice How to make a transgenic mouse Inducible transgenic mice Focus box 15.1 Oncomouse patent 15.3 Gene-targeted mouse models Knockout mice Knockin mice Knockdown mice Conditional knockout and knockin mice Focus box 15.2 A mouse for every need 15.4 Other applications of transgenic animal technology Transgenic primates Transgenic livestock Gene pharming Focus box 15.3 Transgenic artwork: the GFP bunny 15.5 Cloning by nuclear transfer Genetic equivalence of somatic cell nuclei: frog cloning experiments Cloning of mammals by nuclear transfer “Breakthrough of the year”: the cloning of Dolly Method for cloning by nuclear transfer Source of mtDNA in clones Why is cloning by nuclear transfer inefficient? Applications of cloning by nuclear transfer Focus box 15.4 Genetically manipulated pets 15.6 Transgenic plants T-DNA-mediated gene delivery Electroporation and microballistics Focus box 15.5 Genetically modified crops: are you eating genetically engineered tomatoes? Chapter summary Analytical questions Suggestions for further reading 16 Genome analysis: DNA typing, genomics, and beyond, 581 16.1 Introduction 16.2 DNA typing DNA polymorphisms: the basis of DNA typing Minisatellite analysis Polymerase chain reaction-based analysis Short tandem repeat analysis Mitochondrial DNA analysis Y chromosome analysis Randomly amplified polymorphic DNA (RAPD) analysis Focus box 16.1 DNA profiles of marijuana Focus box 16.2 Nonhuman DNA typing Contents xv 16.3 Genomics and beyond What is bioinformatics? Genomics Proteomics The age of “omics” 16.4 The Human Genome Project Clone by clone genome assembly approach Whole-genome shotgun approach Rough drafts versus finished sequences 16.5 Other sequenced genomes What is a gene and how many are there in the human genome? Focus box 16.3 Comparative analysis of genomes: insights from pufferfish and chickens 16.6 High-throughput analysis of gene function DNA microarrays Protein arrays Mass spectrometry 16.7 Single nucleotide polymorphisms Focus box 16.4 The nucleolar proteome Disease box 16.1 Mapping disease-associated SNPs: Alzheimer’s disease Chapter summary Analytical questions Suggestions for further reading 17 Medical molecular biology, 618 17.1 Introduction 17.2 Molecular biology of cancer Activation of oncogenes Inactivation of tumor suppressor genes Inappropriate expression of microRNAs in cancer Chromosomal rearrangements and cancer Viruses and cancer Chemical carcinogenesis Focus box 17.1 How cancer cells metastasize: the role of Src Disease box 17.1 Knudson’s two-hit hypothesis and retinoblastoma Disease box 17.2 Cancer gene therapy: a “magic bullet?” Focus box 17.2 The discovery of p53 Disease box 17.3 Human papilloma virus (HPV) and cervical cancer 17.3 Gene therapy Vectors for somatic cell gene therapy Enhancement genetic engineering Gene therapy for inherited immunodeficiency syndromes Cystic fibrosis gene therapy HIV-1 gene therapy Focus box 17.3 Retroviral-mediated gene transfer: how to make a “safe vector” Focus box 17.4 The first gene therapy fatality Focus box 17.5 HIV-1 life cycle 17.4 Genes and human behavior Aggressive, impulsive, and violent behavior Schizophrenia susceptibility loci xvi Contents Chapter summary Analytical questions Suggestions for further reading Glossary, 668 Index, 711