Caenorhibditus Elegans: Modern Biological Analysis of an Organism (eBook)

Front Cover 1
Methods in Cell Biology, Volume 48 4
Copyright Page 5
Contents 6
Contributors 14
Preface 18
Acknowledgments 22
Part I: Genetic and Culture Methods 24
Chapter 1. Basic Culture Methods 26
I. Life Cycle of Caenorltabditis elegans 27
II. Preparation of Petri Plates for Culturing Nematodes 30
III. Handling of Nematodes 33
IV. Decontamination and Preservation of Nematode Stocks 35
V. Preparation of Synchronized Cultures and Specific Stages 38
VI. Large-Scale Cultivation of Nematodes 39
VII. Growth of Radioactively Labeled Worms 46
VIII. Solutions 48
References 50
Chapter 2. Mutagenesis 54
I. Introduction 54
II. Development of the Germ Line 55
III. Mutagen Efficiency versus Mutagen Specificity 56
IV. The Dark Side of the Force 57
V. Chemical Mutagenesis 59
VI. Radiation Mutagenesis 66
VII. Spontaneous Mutagenesis 70
VIII. Mutator Strains 74
IX. Summary and Conclusions 76
References 77
Chapter 3. Reverse Genetics: From Gene Sequence to Mutant Worm 82
I. Introduction 82
II. Strategies 85
III. Isolation of Tc1 Alleles from a Mutant Library 86
IV. Analysis of Mutants 98
V. Isolation of Deletion Derivatives 99
VI. Concluding Remarks 101
References 101
Chapter 4. Genetic Mapping with Polymorphic Sequence-Tagged Sites 104
I. Introduction 104
II. Polymorphic Sequence-Tagged Site Markers 106
III. Summary of Mapping Strategy 106
IV. Results 108
V. How to Use the Method 110
VI. Conclusions and Prospects 117
References 118
Chapter 5. Genetic Dissection of Developmental Pathways 120
I. Epistasis Analysis 121
II. Epistasis Analysis of Switch Regulation Pathways 123
III. Extragenic Suppressors 137
IV. Prospects: Use of New Technologies for Pathway Analysis 142
V. Conclusion 143
References 143
Chapter 6. Mosaic Analysis 146
I. Questions Addressed by Mosaic Analysis 146
II. Generation of Mosaics by Spontaneous Mitotic Loss of Free Duplications 150
III. Fusing Two Unlinked Free Duplications 162
IV. Fusing a Free Duplication and an Extrachromosomal Transgenic Array 163
V. Using Extrachromosomal Arrays for Mosaic Analysis 164
VI. Other Methods for Generating Mosaics 165
VII. Conclusions 165
References 166
Chapter 7. Genetic Balancers 170
I. Introduction 171
II. Types of Balancers 174
III. Practical Considerations of Balancer Use 181
IV. The Balancer Map 192
V. A Field Guide to Balancers 192
References 205
PART II: Neurobiology 208
Chapter 8. Genetic Pharmacology: Interactions between Drugs and Gene Products in Caenorhabditis elegans 210
I. Introduction 210
II. Use of Drugs as a Method of Phenotypic Analysis 213
III. Use of Mutants to Study Mechanisms of Drug Action 216
IV. Use of Caenorhabditis elegans for Drug Discovery 223
References 224
Chapter 9. Methods of Studying Behavioral Plasticity in Caenorhabditis elegans 228
I. Introduction 228
II. Behavioral Plasticity Paradigms for Caenorhabditis elegans 232
III. Conclusion 244
References 245
Chapter 10. Laser Killing of Cells in Caenorhabditis elegans 248
I. Overview 248
II. Identifying Cells in Caenorhabditis elegans 250
III. Practical Aspects of Laser Ablation 257
IV. Experimental Design and Controls 269
V. Future Directions 271
References 272
Chapter 11. Electrophysiological Methods 274
I. Introduction 274
II. The Electropharyngeogram 275
III. Patch-Clamping 284
IV. Further Reading 291
References 291
PART III: Cell Biology and Molecular Biology 294
Chapter 12. Cell Biology of Nematode Sperm 296
I. General Introduction 296
II. Caenorhabditis elegans 297
III. Ascaris suum 311
IV. Solutions 318
V. Materials 322
References 322
Chapter 13. Blastomere Culture and Analysis 326
I. Introduction: Uses and Limitations of the System 326
II. Embryo Devitellinization and Blastomere Isolation 331
III. Culture Methods 335
IV. Drug Treatments 335
V. Radioactive Labeling 336
VI. Fixation and Cytochemistry 337
VII. Solutions and Culture Media 340
References 343
Chapter 14. Whole-Mount in Situ Hybridization for the Detection of RNA in Caenorhabditis elegans Embryos 346
I. Introduction 346
II. Materials 347
III. Procedure 350
IV. Double Labeling 356
V. Interpretation 357
VI. Troubleshooting 359
References 359
Chapter 15. Fluorescence in Situ Hybridization for the Detection of DNA and RNA 362
I. Introduction 363
II. Nucleotides and Stains Used for Fluorescence in Situ Hybridization 364
III. Sources of Target Material for Hybridization 364
IV. Applications 367
V. Probe Labeling 372
VI. Preparation of Material for Hybridization 380
VII. Visualization of the Site of Hybridization 387
References 388
Chapter 16. Immunofluorescence Microscopy 390
I. Introduction 390
II. Preparing Specimens for Immunolocalization Studies 392
III. Preparation of Antibodies to Nematode Antigens 405
IV. Microscopy: Necessary Equipment and Photomicrography Tips 411
V. Concluding Remarks 413
VI. Recipes 416
References 417
Chapter 17. Electron Microscopy and Three-Dimensional Image Reconstruction 422
I. Introduction 423
II. Fixation and Embedding for Electron Microscopy 424
III. Sectioning Methods 428
IV. Basic Electron Microscopy of Caenorhabditis elegans 439
V. Three-Dimensional Reconstruction Methods 442
VI. Other Electron Microscopy Methods 447
VII. Equipment 455
VIII. Materials 457
References 461
Chapter 18. Proteins and Protein Assemblies 464
I. Introduction: Approaches to Subcellular Fractionation and Biochemical Purification 464
II. Inhibition of Proteolysis and Proteases 465
III. Fractionation and Isolation of Cell Constituents 469
IV. Additional Purification Techniques 473
V. A Model Protein: Myosin 474
VI. A Model Assembly: The Thick Filament 475
References 476
Chapter 19. DNA Transformation 478
I. Introduction 479
II. Microinjection Techniques for Caenorhabditis elegans 479
III. Identification and Inheritance Properties of Transformed Animals 489
IV. Assays for Function of Native Caenorhabditis elegans Genes 495
V. Use of Chimeric "Reporter" Constructs to Analyze Gene Expression 498
VI. Directed Expression of Coding Regions and Antisense RNAs 500
VII. Possible Sources for Inefficient or Inappropriate Expression 503
VIII. Other Applications of Transformation Technology 506
References 507
Chapter 20. Transcription and Translation 510
I. Introduction 510
II. Transcription 511
III. Post-transcriptional Regulation 526
IV. Translation 529
References 532
Chapter 21. Techniques for Analyzing Transcription and Translation 540
I. Introduction 540
II. Worm Growth for RNA Preparations 541
III. RNA Preparation 542
IV. Northern Blot Analysis 543
V. Hybridizations 545
VI. RNase Protection 546
VII. S1 Mapping 547
VIII. Primer Extension 548
IX. In Situ Hybridization 549
X. ß-Galactosidase Reporters 549
XI. Antisense Inhibition 549
XII. Reverse Transcriptase Polymerase Chain Reactions 550
XIII. Nuclear Extracts 553
XIV. Gel Mobility Shift Assays 553
References 554
PART IV: Genomics and Informatics 558
Chapter 22. The Physical Map of the Caenorhabditis elegans Genome 560
I. Introduction 561
II. Databases 561
III. Interpretation of the Map 563
IV. Central Resources 568
V. Protocols 570
VI. General Recipes 575
References 576
Chapter 23. Genomic DNA Sequencing Methods 578
I. Introduction 578
II. Methods 579
III. Summary 595
References 596
Chapter 24. Large-Scale Complementary DNA Sequencing Methods 598
I. Introduction 598
II. Methods 600
III. Summary 608
References 609
Chapter 25. ACeDB and Macace 610
I. Introduction 611
II. System Information and Requirements 611
III. Basic Use of ACeDB: Browsing and Searching 614
IV. Caenorhabditis elegans Data and Map Displays 619
V. Concepts 625
VI. Using ACeDB to Store One's Own Data 628
VII. Conclusion 631
References 631
Chapter 26. The Worm Community System, Release 2.0 (WCSr2) 634
I. Introduction 635
II. System Information and Requirements 635
III. Concepts and Conventions 638
IV. Browsing and Searching 640
V. Sharing Data 646
VI. Discussion 649
VII. The Future 651
References 652
Appendix A: Additional Information and Resources 654
Appendix B: Caenorhabditis Genetics Center—Laboratory Designations 658
Appendix C: Gene Names and Descriptions 662
Index 668
Contents of Previous Volumes 682