Wilson Drosophila melanogaster: Practical Uses in Cell and Molecular Biology

1. Auflage 1995
ISBN: 978-0-08-085942-2
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 44, 755 Seiten, Web PDF

Reihe: Methods in Cell Biology

Drosophila melanogaster: Practical Uses in Cell and Molecular Biology is a compendium of mostly short technical chapters designed to provide state-of-the art methods to the broad community of cell biologists, and to put molecular and cell biological studies of flies into perspective. The book makes the baroque aspects of genetic nomenclature and procedure accessible to cell biologists. It also contains a wealth of technical information for beginning or advanced Drosophila workers. Chapters, written within a year of publication, make this topical volume a valuable laboratory guide today and an excellent general reference for the future.Key Features* Collection of ready-to-use, state-of-the art methods for modern cell biological and related research using Drosophila melanogaster* Accessible to both experienced Drosophila researchers and to others who wish to join in at the cutting edge of this system * Drosophila offers an easily managed life cycle, inexpensive lifestyle, extraordinarily manipulable molecular and classical genetics, now combined with powerful new cell biology techniques * Introduction and overview sections orient the user to the Drosophila literature and lore * Six full-color plates and over 100 figures and tables enhance the understanding of these cell biology techniques

Autoren/Hrsg.

Wilson, Leslie

Inhaltsverzeichnis

1;Front Cover;1
2;Drosophila melanogaster: Practical Uses in Cell and Molecular Biology;4
3;Copyright Page;5
4;Contents;6
5;Contributors;16
6;Preface: Why the Fly?;20
7;Part I: Basic Genetic Methodologies;22
7.1;Chapter 1. Sources of Information about the Fly: Where to Look It Up;24
7.1.1;I. Introduction;24
7.1.2;II. Technical References;25
7.1.3;III. Intellectual References;26
7.1.4;IV. Electronic References;28
7.1.5;V. Final Remarks;31
7.1.6;References;32
7.2;Chapter 2. Care and Feeding of Drosophila melanogaster;34
7.2.1;I. Introduction;35
7.2.2;II. Tools of the Trade;35
7.2.3;III. Room and Board;39
7.2.4;IV. Culturing Drosophila;40
7.2.5;V. Reading the Runes—Nomenclature;46
7.2.6;VI. Resources;50
7.2.7;VII. Summary;52
7.2.8;References;52
7.3;Chapter 3. Harnessing the Power of Drosophila Genetics;54
7.3.1;I. Introduction;55
7.3.2;II. How Do We Get Mutants from Cloned Genes or Clones from Mutated Genes?;56
7.3.3;III. How Do We Determine Which Stages of the Drosophila Life Cycle Are Affected by a Mutation?;67
7.3.4;IV. How Do We Analyze Mutants?;76
7.3.5;V. Using Mutants to Explore Processes and Pathways;85
7.3.6;VI. Conclusion;92
7.3.7;References;92
7.4;Chapter 4. From Clone to Mutant Gene;102
7.4.1;I. Introduction;102
7.4.2;II. Overview;106
7.4.3;III. Protocols;109
7.4.4;IV. Conclusion;113
7.4.5;References;114
8;Part II: Isolation and Culture Methods;116
8.1;Chapter 5. Raising Large Quantities of Drosophila for Biochemical Experiments;120
8.1.1;I. Introduction;120
8.1.2;II. The Drosophila Colony;121
8.1.3;III. Media Recipes;127
8.1.4;IV. Materials Used;129
8.1.5;References;129
8.2;Chapter 6. Isolation and Organ Culture of Imaginal Tissues;130
8.2.1;I. Perspectives and Applications;131
8.2.2;II. Mass Isolation of Imaginal Discs;132
8.2.3;III. In Vitro Culture of Imaginal Discs;138
8.2.4;IV. Analysis of Biosynthetic Activity;144
8.2.5;V. Isolation of Other Larval Organs;145
8.2.6;VI. Summary and Prospects;146
8.2.7;References;146
8.3;Chapter 7. Mass Isolation of Fly Tissues;150
8.3.1;I. Introduction;150
8.3.2;II. Method for Rearing Flies on Liquid Medium;151
8.3.3;III. Methods for Isolating Specific Cells;152
8.3.4;IV. Isolation of Specific Stages of Oogenesis;158
8.3.5;V. Concluding Observations;161
8.3.6;VI. Solutions;161
8.3.7;References;162
8.4;Chapter 8. Preparation and Analysis of Pure Cell Populations from Drosophila;164
8.4.1;I. Introduction;164
8.4.2;II. Purifying Embryonic Cells by Fluorescence-Activated Cell Sorting;167
8.4.3;III. Culturing and Analysis of Purified Cells;176
8.4.4;IV. Conclusions;178
8.4.5;References;179
8.5;Chapter 9. Transformation Techniques for Drosophila Cell Lines;182
8.5.1;I. Introduction;182
8.5.2;II. Maintaining and Cloning Cells;184
8.5.3;III. Transforming Cells;187
8.5.4;References;197
9;Part III: Biochemical Preparation Methods;202
9.1;Chapter 10. Preparation of Drosophila Nuclei;206
9.1.1;I. Introduction;206
9.1.2;II. Dechorionation of Embryos;207
9.1.3;III. Disruption of Embryos;207
9.1.4;IV. Isolation of Nuclei;208
9.1.5;V. Nuclei from Other Developmental Stages;208
9.1.6;References;210
9.2;Chapter 11. Isolation and Characterization of RNA-Binding Proteins from Drosophila melanogaster;212
9.2.1;I. Introduction;213
9.2.2;II. Isolation of RNA-Binding Proteins by Affinity Chromatography;214
9.2.3;III. Characterization of Putative RNA-Binding Proteins;217
9.2.4;IV. Discussion;224
9.2.5;References;225
9.3;Chapter 12. Chromatin Assembly Extracts from Drosophila Embryos;228
9.3.1;I. General Introduction;229
9.3.2;II. Extract Preparation;230
9.3.3;III. Chromatin Assembly Reaction;233
9.3.4;IV. Analysis of Reconstituted Chromatin;235
9.3.5;V. Nucleosome Organization at Specific Sites;240
9.3.6;VI. Conclusions and Perspectives;242
9.3.7;References;243
9.4;Chapter 13. The Soluble Nuclear Fraction, a Highly Efficient Transcription Extract from Drosophila Embryos;246
9.4.1;I. Summary;246
9.4.2;II. Introduction;247
9.4.3;III. Preparation of the Soluble Nuclear Fraction;249
9.4.4;IV. In Vitro Transcription/Primer Extension Analysis;252
9.4.5;V. Notes on the Use of the Soluble Nuclear Fraction;254
9.4.6;References;255
9.5;Chapter 14. Basic Methods for Drosophila Muscle Biology;258
9.5.1;I. Introduction;259
9.5.2;II. Specific Methods;261
9.5.3;References;275
9.6;Chapter 15. Isolation of Cytoskeletal Proteins from Drosophila;280
9.6.1;I. Introduction;280
9.6.2;II. Affinity Methods for Isolation of Interacting Proteins;282
9.6.3;III. Characterization of Proteins Isolated by Affinity Methods;293
9.6.4;IV. Conclusions and Summary;296
9.6.5;References;297
9.7;Chapter 16. Isolation and Analysis of Microtubule Motor Proteins;300
9.7.1;I. Introduction;300
9.7.2;II. Isolation of Motor Proteins;302
9.7.3;III. Characterization of Microtubule Motors;305
9.7.4;IV. Conclusions and Summary;306
9.7.5;References;307
9.8;Chapter 17. Preparation and Analysis of Membranes and Membrane Proteins from Drosophila;310
9.8.1;I. Introduction;310
9.8.2;II. Immunoaffinty Purification of Drosophila Membrane Proteins;311
9.8.3;III. Isolation and Characterization of Cellular Membranes in Drosophila Embryos ;315
9.8.4;IV. Conclusion;320
9.8.5;References;321
9.9;Chapter 18. Preparation of Extracellular Matrix;324
9.9.1;I. Introduction;324
9.9.2;II. Isolation of ECM Proteins;326
9.9.3;III. Characterization of ECM Molecules;335
9.9.4;IV. Interactions of ECM Proteins with Cells in Vitro;340
9.9.5;V. Temporal and Spatial Expression of ECM Genes and Localization of ECM Products;341
9.9.6;VI. Miscellaneous Aspects and Conclusions;345
9.9.7;References;346
10;Part IV: Cytological Methods;350
10.1;Chapter 19. Looking at Polytene Chromosomes;354
10.1.1;I. The Beautiful Contig Maps of Drosophila;355
10.1.2;II. Sources of Polytene Chromosomes;356
10.1.3;III. Structure of Polytene Chromosomes;356
10.1.4;IV. Maps of Polytene Chromosomes;361
10.1.5;V. Equipment for Salivary Gland Preparations;362
10.1.6;VI. Protocols for Squash Preparations of Salivary Glands;363
10.1.7;VII. Nucleic Acid Probes for in Situ Hybridization;365
10.1.8;VIII. Protocols for in Situ Hybridization to Polytene Chromosomes;367
10.1.9;References;371
10.2;Chapter 20. Immunological Methods for Mapping Protein Distributions on Polytene Chromosomes;374
10.2.1;I. General Introduction;375
10.2.2;II. Mapping Known Chromosomal Components;376
10.2.3;III. Identification and Characterization of Unknown Chromosomal Components ;377
10.2.4;IV. Mapping Sites of Accumulation of Regulatory Proteins;377
10.2.5;V. Mapping Protein Domains;379
10.2.6;VI. Tests of Regulatory Interactions among Proteins of Related Function;379
10.2.7;VII. History of Antibody-Staining Methods for Polytene Chromosomes;380
10.2.8;VIII. Procedure for Enzyme-Linked Detection of Proteins on Polytene Chromosomes ;381
10.2.9;IX. Procedure for Fluorescent Labeling of Proteins on Polytene Chromosomes ;387
10.2.10;X. Double-Labeling of Proteins on Polytene Chromosomes Using Fluorescently Tagged Antibodies ;388
10.2.11;XI. Interpretations and Limitations;388
10.2.12;References;390
10.3;Chapter 21. Looking at Drosophila Mitotic Chromosomes;394
10.3.1;I. Introduction;395
10.3.2;II. Preparation of Larval Neuroblast Chromosomes;396
10.3.3;III. Chromosome-Banding Techniques;400
10.3.4;IV. In Situ Hybridization;406
10.3.5;V. Summary and Conclusions;412
10.3.6;References;413
10.4;Chapter 22. Looking at Diploid Interphase Chromosomes;418
10.4.1;I. Introduction;418
10.4.2;II. Two-Color Fluorescence in Situ Hybridization to Single-Copy DNA Sequences in Diploid Nuclei;421
10.4.3;III. Summary and Future Directions;433
10.4.4;References;433
10.5;Chapter 23. Electron Microscopy and EM Immunocytochemistry;436
10.5.1;I. Introduction;437
10.5.2;II. Criteria for Judging Ultrastructure Quality;438
10.5.3;III. Aim and Scope of This Chapter;443
10.5.4;IV. Embryos;443
10.5.5;V. Stages Other Than Embryos;453
10.5.6;VI. Immunolabeling;455
10.5.7;Appendix: Vendor Information;466
10.5.8;References;467
10.6;Chapter 24. Imaging Neuronal Subsets and Other Cell Types in Whole-Mount Drosophila Embryos and Larvae Using Antibody Probes;470
10.6.1;I. Introduction;471
10.6.2;II. Reagents Used to Visualize Specific Tissues and Subsets of Neurons;472
10.6.3;III. Fixation, Methanol Devitellinization, Storage, and Rehydration Protocols ;480
10.6.4;IV. Primary and Secondary Antibody Incubation Procedure;488
10.6.5;V. Histochemical Development Reactions;490
10.6.6;VI. Labeling with Multiple Primary Antibodies;492
10.6.7;VII. Rapid Staining Procedure;497
10.6.8;VIII. Clearing Embryos;498
10.6.9;IX. Dissecting and Mounting Stained Embryos;500
10.6.10;X. Hints for Photographing Histochemically Stained Specimens;502
10.6.11;XI. Hints for Looking at Mutants That Affect Central Nervous System Development ;503
10.6.12;XII. Trouble Shooting;505
10.6.13;XIII. Solutions;507
10.6.14;Appendix: Suppliers;511
10.6.15;References;512
10.7;Chapter 25. Immunofluorescence Analysis of the Cytoskeleton during Oogenesis and Early Embryogenesis;516
10.7.1;I. Introduction;516
10.7.2;II. The Cytoskeleton during Oogenesis;517
10.7.3;III. The Cytoskeleton during Early Embryogenesis;521
10.7.4;IV. Labeling Cytoskeletal Elements in Fixed Oocytes and Embryos;527
10.7.5;References;530
10.8;Chapter 26. High-Resolution Microscopic Methods for the Analysis of Cellular Movements in Drosophila Embryos;534
10.8.1;I. Introduction;534
10.8.2;II. Optimization of Resolution and Contrast;535
10.8.3;III. Thick, Living Specimens, High NA, and Spherical Aberration;539
10.8.4;IV. Chambers for the Observation of Embryos;543
10.8.5;V. Handling the Embryos;546
10.8.6;VI. High-Fidelity Digital Electronic Imaging and Image Acquisition;547
10.8.7;VII. Dynamics of Image Capture;552
10.8.8;VIII. Digital Image Processing;553
10.8.9;IX. Hard Copy of Electronic Images;553
10.8.10;X. Summary and Prospectus;558
10.8.11;References;559
10.9;Chapter 27. The Use of Photoactivatable Reagents for the Study of Cell Lineage in Drosophila Embryogenesis;560
10.9.1;I. Introduction;560
10.9.2;II. Preparation of the Photoactivatable Lineage Tracer;562
10.9.3;III. Injection and Activation of the Photoactivatable Lineage Tracer;563
10.9.4;IV. Fixation of Embryos Carrying Marked Cells;566
10.9.5;V. Analysis of Living Embryos Carrying Marked Cells;568
10.9.6;VI. Summary;568
10.9.7;References;569
10.10;Chapter 28. Looking at Oogenesis;572
10.10.1;I. General Background;572
10.10.2;II. Gross Morphological Analysis;576
10.10.3;III. More Detailed Examination;582
10.10.4;IV. Protocols;584
10.10.5;References;587
11;Part V: Analysis of Gene Expression in Situ;590
11.1;Chapter 29. Methods for Quantitative Analysis of Transcription in Larvae and Prepupae;592
11.1.1;I. Introduction;592
11.1.2;II. Staging of Third Instar Larvae and Prepupae;593
11.1.3;III. Organ Culture;596
11.1.4;IV. RNA Isolation;597
11.1.5;References;600
11.2;Chapter 30. In Situ Hybridization to RNA;602
11.2.1;I. Introduction;603
11.2.2;II. Embryo Collection and Fixation;604
11.2.3;III. Hybridization;608
11.2.4;IV. Detection;610
11.2.5;V. Mounting Techniques;612
11.2.6;VI. Probe Preparation;614
11.2.7;VII. Double Labeling: RNA–Protein;617
11.2.8;VIII. Double Labeling: RNA–RNA;621
11.2.9;IX. Conclusions;626
11.2.10;References;626
11.3;Chapter 31. Looking at mRNA Splicing and Transport in Situ;628
11.3.1;I. Introduction;628
11.3.2;II. Protocols;629
11.3.3;III. Concluding Technical Remarks;638
11.3.4;IV. Concluding General Remarks;639
11.3.5;References;640
11.4;Chapter 32. EM Methods for Visualization of Genetic Activity from Disrupted Nuclei;642
11.4.1;I. Introduction;642
11.4.2;II. Methods;644
11.4.3;III. Summary and Conclusions;657
11.4.4;References;658
12;Part VI: Molecular and Classical Genetic Analysis;660
12.1;Chapter 33. Ectopic Expression in Drorophila;664
12.1.1;I. Introduction;664
12.1.2;II. The Heat-Shock Method;668
12.1.3;III. The GAL4 System;672
12.1.4;References;681
12.2;Chapter 34. Mosaic Analysis Using FLP Recombinase;684
12.2.1;I. Introduction;684
12.2.2;II. Mosaic Analysis;685
12.2.3;III. Genetic Screens;705
12.2.4;IV. Concluding Remarks;709
12.2.5;References;710
12.3;Chapter 35. Analysis of Cellular Adhesion in Cultured Cells;714
12.3.1;I. Introduction;714
12.3.2;II. Cell Transformation and Expression of Cell Adhesion Molecules;715
12.3.3;III. Analysis of Cellular Adhesion;721
12.3.4;IV. Concluding Remarks;725
12.3.5;References;726
12.4;Chapter 36. Using Inhibitors t o Study Embryogenesis;728
12.4.1;I. Introduction: Using Chemical Inhibitors to Study Development;729
12.4.2;II. Introduction of Substances into the Embryo;729
12.4.3;III. Summary of Drug Uses and Effects;738
12.4.4;IV. Concluding Remarks;741
12.4.5;References;742
12.5;Chapter 37. Chromophore-Assisted Laser Inactivation of Cellular Proteins;746
12.5.1;I. Introduction;747
12.5.2;II. Malachite Green Labeling of Antibodies;748
12.5.3;III. Laser Instrumentation and Setup;749
12.5.4;IV. CALI of Drosophila Embryos;751
12.5.5;V. Preparation for Phenotype Analysis;756
12.5.6;VI. Materials;757
12.5.7;VII. Micro-CALI Setup and Application;758
12.5.8;VIII. Limitations;761
12.5.9;IX. Concluding Remarks;762
12.5.10;References;762
13;Index;764
14;Volumes in Series;782