PRINCIPLES O F NUCLEAR STRUCTUR E AND FUNCTIO N Peter R. Cook
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
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 1-10. The Three Primary Lineages of the Living World 53 Summary 5 5 References 5 6 2. 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 2-10. Acute Promyelocytic Leukemia 99 Elements of Yeast Chromosomes 9 9 Telomeres 10 1 Chromosome Bands 102 Models for Chromosome Organization 102 Polytene Chromosomes 107
Summary 10 9 References 10 9 3. 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 14 9 4. 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
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 22 3 6. 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
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 6-10. 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 6-11. Maternal-Effect Genes in Drosophila 261 Example: Commitment of Hematopoietic Stem Cells 262 Summary 263 References 264 7.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
Box 7-8. Tumor-Suppressor Genes 304 T antigen, p53, and Rb 305 Box 7-9. p53 and Human Tumors 307 Box 7-10. Hereditary Colorectal Cancer 31 1 Cancer Therapy and Checkpoints 31 2 Apoptosis 31 2 The Genetic Basis of Apoptosis 31 3 Box 7-11. Lineage Analysis in Caenorhabditis elegans 314 Box 7-12. Caspases 31 5 Activating the Suicide Machinery 31 5 Box 7-13. Death Receptors 31 8 Summary 31 8 References 31 9 8. 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