PRINCIPLES O F NUCLEAR STRUCTUR E AND FUNCTIO N. Peter R. Cook

Transcription

1 PRINCIPLES O F NUCLEAR STRUCTUR E AND FUNCTIO N Peter R. Cook

2

3 Preface xii i Acknowledgments xv 1. SOME PRINCIPLES 1 Overview of the Cell Nucleus 1 Box 1-1. Discovery of Cells, Nuclei, and DNA 2 A Sense of Scale 4 Box 1-2. Microscopy: Problems and Solutions 5 Box 1-3. Green Fluorescent Protein 7 Thermal Motion 9 Local Concentrations 1 0 Structures of Nucleic Acids 1 0 The DNA Double Helix 1 3 Box 1-4. Translation 1 8 The Length of DNA Molecules 2 1 Bending and Twisting DNA 2 1 Box 1-5. Gel Electrophoresis and Blotting 23 The Structure of RNA 26 Recognizing Specific DNA Sequences 2 8 Box 1-6. DNA: Protein Binding- "Gel-Shifts," "Footprinting," "ChIP" 3 1 DNA-Binding Proteins 32 Making Large Structures 34 Assembling Nuclei in Egg Extracts 3 6 Subcellular Localization 3 7 Tensegrity Architecture and Cellular Skeletons 3 8 Nuclear Position and Shape 38

4 Some Evolutionary Considerations 4 0 Genome Size 4 1 Box 1-7. The Amount of DNA in a Human Nucleus 4 2 Gene Number and Organization 4 4 Regulatory Networks: Redundant and Robust 47 Box 1-8. Protein:Protein Interactions - "Two-Hybrid," "FRET" 48 Box 1-9. Simulating Complex Control Circuits 49 Subcompartments and the Origin of Nuclei 5 0 Box The Three Primary Lineages of the Living World 53 Summary 5 5 References STRUCTURE 59 Overview of Nuclear Structure 59 Box 2-1. Preparing Metaphase Spreads 60 The Nuclear Membrane 6 1 The Nuclear Lamina 6 2 Nuclear Pores 62 Box 2-2. Intermediate Filaments 65 Box 2-3. Identifying Proteins in Large Structures by MALDI Mass Spectrometry 67 Importing Proteins of >60 kd 6 7 Box 2-4. Autoradiography 69 RNA Export 70 Is the Nuclear Membrane an Ion Barrier? 7 1 The Nucleolus 7 1 Packaging Chromatin During Interphase 7 3 Artifacts 7 3 Box 2-5. Isolating Nuclei 75 Box 2-6. Nuclear Matrices and Scaffolds 77 The Nucleosome 7 8 Box 2-7. Nucleoids 79 The Zig-zagging Nucleosomal String 8 4 Chromatin Loops 8 5 Chromatin "Clouds" 9 2 Box 2-8. Position-Effect Variegation in Drosophila 93 Chromosome Territories 9 4 Box 2-9. In Situ Hybridization 95 Nucleoskeletons and Nuclear Subcompartments 96 Chromosomes 98 Box Acute Promyelocytic Leukemia 99 Elements of Yeast Chromosomes 9 9 Telomeres 10 1 Chromosome Bands 102 Models for Chromosome Organization 102 Polytene Chromosomes 107

5 Summary 10 9 References REPLICATION 11 3 'Principles 113 Box 3-1. Bacterial DNA Polymerases 11 5 Tracking Versus Immobile DNA Polymerases 117 Replication Factories 12 1 The Mechanics of Synthesis at the Fork 124 Separating Parental Strands 124 Box 3-2. Topoisomerases and Anticancer Drugs 12 5 RNA Primers 12 6 The Asymmetric Fork 12 8 Proofreading 13 3 Replicating chromatin 13 5 The Initiation of Synthesis 135 Box 3-3. The Origin of Replication ofe. coli 136 Box 3-4. Two Methods for Mapping Origins 13 8 Simple Origins of SV40 Virus and Yeast 13 8 Complex Mammalian Origins 13 9 Role of Transcription During Initiation 14 2 Replicating Ends 14 3 Box 3-5. Telomerase in Aging and Cancer 14 7 Summary 14 8 References TRANSCRIPTION 15 1 Principles 15 1 Box 4-1. Mapping Transcription Units 153 Tracking Versus Immobile RNA Polymerases 15 6 Box 4-2. The RNA Polymerase of E. coli 157 The Untwining and Supercoiling Problems 160 Attached Polymerases Can Work In Vitro 164 Transcribing Chromatin 16 6 The Three Kinds of Eukaryotic RNA Polymerase 167 RNA Polymerase I 16 7 Box 4-3. Ribosome Synthesis 170 RNA Polymerase II and its Transcription Factors 17 1 Number and Activity of RNA Polymerases 17 5 Box 4-4. Transcription of Heat-Shock Loci 176 Box 4-5. Message/Protein-Profiles - "Microarrays," "SAGE," "Two - Hybrid" 177 Transcription Factories 17 9 Nucleolar Factories Containing Polymerase I 17 9 Extranucleolar Factories Containing Polymerases II and III 18 1 Dynamics 182

6 Processing and Transport of Polymerase II Transcripts 18 4 Capping the 5' End 184 Box 4-6. Analyzing Caps 185 Polyadenylating the 3' End 186 Splicing the Middle 18 7 Box 4-7. Autoimmune Antibodies 188 Packaging Transcripts into Ribonucleoprotein Particles 19 2 Fidelity and Quality Control 19 2 The Organization of Processing 195 Transport to the Cytoplasm 195 Summary 197 References 198 i 5. REPAIR 20 1 The Need for Repair of Damage 20 1 Common Damaging Agents and Lesions 202 Box 5-1. Consequences of Deamination of C and 5-MeC 204 Some Experimental Approaches 20 5 Box 5-2. Repair Defects and Human Disease 206 Types of Repair 209 Direct Repair 21 1 Base-Excision Repair 21 1 Nucleotide-Excision Repair 21 2 Mismatch Repair 21 3 Other Repair Mechanisms 214 Box 5-3. Damage Response Systems in Bacteria 21 6 Accuracy in Gap Filling 21 7 Sites of Repair 21 7 Transcription and Repair 21 8 Some Consequences of Inefficient Repair 220 Summary 222 References REGULATION OF GENE EXPRESSION 225 Simple Regulatory Circuits in Bacteria and Yeast 22 5 Box 6-1. Positive and Negative Control in the lac Operon 22 7 Box 6-2. A Complex Circuit Involving the Lambda Repressor 22 8 Principles of Eukaryotic Gene Regulation 23 1 Most Cells in an Organism Contain the Same DNA 23 2 Box 6-3. Sequence Changes in Antibody Genes 234 Different Levels of Control 23 5 Box 6-4. Gene Amplification 236 Box 6-5. Inheritance of Methylated Sequences in DNA 23 7 Box 6-6. Alternative Splicing and Polyadenylation 23 9 Inheriting the Differentiated State Through Mitosis 24 0 Box 6-7. Co-suppression and RNA Interference 241

7 Differential Expression Can Require Continuous Regulation 24 3 Box 6-8. Identification of MyoD, a Myogenic Transactivator 244 Relative Expression Levels in Different Cells 24 4 Box 6-9. Designing a Eukaryotic Repressor 245 Regulation at the Level of the Nucleosome 24 6 Nucleosome Positioning and Modification 24 9 Chromatin Remodeling 25 0 Regulation at the Level of the Loop 25 1 Box Defining DNA Motifs That Regulate Transcription 252 Heterochromatin 25 3 Silencing in Yeast 25 5 DNA Methylation in Vertebrates 25 7 Polycomb Proteins of Drosophila 257 Establishing and Inheriting Patterns of Expression 25 8 Example: Inheriting Activity of rdna Genes 25 8 Example: Anterior-Posterior Patterning in Drosophila Eggs 25 9 Box Maternal-Effect Genes in Drosophila 261 Example: Commitment of Hematopoietic Stem Cells 262 Summary 263 References THE CELL CYCLE 267 Overview 26 7 Mitosis 26 9 Box 7-1. Synchronizing Mammalian Cells 270 The Centrosome Cycle 27 2 Box 7-2. Microtubules 274 Spindles, Centromeres and Kinetochores 27 7 Box 7-3. Different Kinds of Centromeres 27 9 Microtubule-Based Motors Drive Movement 28 0 Cytokinesis 28 1 Regulation of the Cell Cycle 28 3 Box 7-4. Glycogen Phosphorylase and Protein Kinases 285 Role of Trans-acting Factors 28 6 Frog Embryos : MPF and Cyclins 28 6 Yeasts : cdc Mutants, START, and ORC 28 9 Box 7-5. The Life Cycles of Two Yeasts 290 The G2 Checkpoint in Fission Yeast 29 4 The Anaphase-Promoting Complex 294 Some Other Checkpoints 295 Growth Factors 29 6 Size Control 298 Deranged Cycles and Cancer 29 9 Box 7-6. The Genetic Basis of Cancer 300 Box 7-7. Oncogenes 302 Genes Controlling Proliferation 303

8 Box 7-8. Tumor-Suppressor Genes 304 T antigen, p53, and Rb 305 Box 7-9. p53 and Human Tumors 307 Box Hereditary Colorectal Cancer 31 1 Cancer Therapy and Checkpoints 31 2 Apoptosis 31 2 The Genetic Basis of Apoptosis 31 3 Box Lineage Analysis in Caenorhabditis elegans 314 Box Caspases 31 5 Activating the Suicide Machinery 31 5 Box Death Receptors 31 8 Summary 31 8 References MEIOSIS AND RECOMBINATION 323 Overview 323 Meiosis 324 The Synaptonemal Complex 327 Chiasmata and Chromosome Segregation 327 The Production of Gametes 329 Recombination 329 Principles Involved in Strand Exchange 33 1 Gene Conversion 33 3 Box 8-1. RecBCD, RecA, and General Recombination 334 General Recombination in Eukaryotes 33 6 Box 8-2. The ARG4 Recombination Site in Budding Yeast 336 Chromosome Pairing 33 8 Summary 34 1 References 34 2 Appendix I : Some Websites 345 Index 347