Elsevier S & T

Tetrahymena Thermophila

Wilson, Leslie

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Table of contents

Cover
Copyright Pageiv
Contentsv
Contributorsxiii
Prefacexvii
Part I: Overview1
Chapter 1. Laboratory and Evolutionary History of Tetrahymena thermophila3
Summary4
I. Characters in Search of a Script4
II. Paramecium Precursors8
III. Tetrahymena Breeding Studies11
IV. Perils of Passage16
V. Doing Genetics by Evolution18
References22
Chapter 2. Cell Biology of Tetrahymena thermophila27
I. Introduction28
II. Membranes and Membrane-Bounded Compartments35
III. The Cytoskeleton and Membrane Skeleton52
IV. Dynamics of the Cell Cycle68
V. Spacial Patterning79
VI. Starvation-Mediated Development88
VII. Concluding Remarks99
References103
Chapter 3. Tetrahymena Genetics: Two Nuclei Are Better Than One127
I. Asexual and Sexual Reproduction128
II. Nuclear Dualism134
III. Special Features of Tetrahymena Genetics150
IV. DNA-Mediated Transformation163
V. Summary and Prospects173
References176
Part II: Methods187
Chapter 4. Tetrahymena as a Laboratory Organism: Useful Strains, Cell Culture, and Cell Line Mainten189
I. Introduction190
II. Useful Tetrahymena Strains190
III. Culture Media194
IV. Culturing Tetrahymena Cells199
V. Cell Line Storage202
References208
Chapter 5. Long-Term Storage213
I. Introduction213
II. Supplies214
III. Preparation of Cells before Freezing215
IV. Freezing216
V. Thawing and Recovery216
VI. Conclusion217
References217
Chapter 6. Genetic Crosses: Setting Up Crosses, Testing Progeny, and Isolating Phenotypic Assortants219
I. Introduction219
II. Methods for Mating Cells220
III. How to Isolate and/or Select Progeny from a Cross222
IV. Mating Type Tests and Sexual Immaturity Tests225
V. Isolating Assortants227
References228
Chapter 7. Methods for Genetic Analysis229
I. Introduction229
II. Reagents230
III. Equipment231
IV. Strains234
V. Strategies235
References240
Chapter 8. Isolation of Micronuclear and Macronuclear DNA241
I. Introduction242
II. Cell Culture243
III. Percoll Method for Isolation of Nuclei244
IV. Isolation of High Molecular Weight DNA from Nuclei247
V. Solutions249
References252
Chapter 9. Genetically Sorting a Collection of Tetrahymena Mutants253
I. Introduction253
II. Mapping the Mutations to Chromosome Arms255
III. Genetic Complementation Tests255
References263
Chapter 10. Genetically Mapping New Mutants and Cloned Genes265
I. Introduction265
II. Genetically Mapping a Mutation266
III. Genetically Mapping a Cloned Gene or Sequence278
References279
Chapter 11. Selection of Motility Mutants281
I. Introduction281
II. Materials282
III. Special Procedures and Equipment283
IV. Strains283
V. Inducing and Screening for Temperature-Sensitive Mutations Affecting Cilia Regeneration283
VI. Mutagenesis by Targeted Gene Knockout286
References289
Chapter 12. Nuclear and Cytoskeletal Fluorescence Microscopy Techniques291
I. Introduction291
II. DAPI: A Fluorescent Nuclear Stain292
III. Immunofluorescence Labeling for Conventional Fluorescence Microscopy299
IV. Immunofluorescence Labeling for Confocal Fluorescence Microscopy305
References310
Chapter 13. Nuclear and Cortical Histology for Brightfield Microscopy313
I. Introduction313
II. Silver Staining314
III. Protargol Staining317
IV. Giemsa Staining319
References322
Chapter 14. Fixation of Tetrahymena Cells for Electron Microscopy323
I. Introduction323
II. Cell Culture324
III. Fixation324
IV. Critical Point Drying for SEM327
V. Embedding for TEM328
VI. Staining Sections (TEM)330
References331
Chapter 15. Immunoelectron Microscopy of Tetrahymena333
I. Introduction334
II. General Considerations334
III. Postembedding Labeling336
IV. Double Labeling with the Postembedding Technique338
V. Pre-embedding Labeling340
VI. Controls for Immunogold Labeling340
VII. Quantitative Analysis of Colloidal Gold Labeling341
VIII. Troubleshooting: Problems and Recommended Remedies342
References342
Part III: Cell Biology345
Chapter 16. Regulated Protein Secretion in Tetrahymena thermophila347
I. Introduction347
II. Isolation of the Released Contents of Dense-Core Secretory Vesicles (Mucocysts)350
III. Stimulation of Cells to Test Exocytic Capability or to Study Postexocytic Events354
IV. Isolation of Intact Secretory Vesicles357
References360
Chapter 17. Electrophysiology of Tetrahymena363
I. Introduction363
II. Materials and Methods367
III. Commentary and Summary374
References392
Chapter 18. Isolation and Characterization of in Vivo Modified Histones and an Activity Gel Assay fo379
I. Introduction379
II. Methods382
References392
Chapter 19. Tetrahymena Telomerase Activity, Purification, and Reconstitution395
I. Introduction396
II. Materials and Methods400
III. Commentary and Summary409
References412
Chapter 20. Studying the Telomerase RNA in Tetrahymena417
I. Introduction417
II. General Strategy and Overview419
III. Basic Methods420
References431
Chapter 21. Isolation and Characterization of 22S Outer Arm Dynein from Tetrahymena Cilia433
I. Introduction433
II. Methods435
III. Summary438
References439
Chapter 22. Preparation of Cytoskeletal Fractions from Tetrahymena thermophila441
I. Introduction441
II. Methods442
References446
Chapter 23. Immunoprecipitation Procedures449
I. Introduction449
II. Method450
References453
Chapter 24. Tetrahymena Calcium-Binding Proteins, TCBP-25 and TCBP-23455
I. Introduction455
II. Methods456
References465
Part IV: Manipulating Genes467
Chapter 25. Microinjection of Tetrahymena thermophila469
I. Introduction469
II. The Technique of Microinjection472
III. Concluding Remarks482
References483
Chapter 26. Transient and Stable DNA Transformation of Tetrahymena thermophila by Electroporation485
I. Introduction486
II. Considerations487
III. Preparing Plasmid DNA for Electrotransformation488
IV. General Protocol for Conjugant Electrotransformation489
V. Transformation with Integrating Vectors: Gene Replacements and Gene Knockouts492
VI. Transformation with Autonomously Replicating Vectors495
References498
Chapter 27. Biolistic Transformation of Macro- and Micronuclei501
I. Introduction501
II. Cells502
III. Transformation504
References511
Chapter 28. Knockout Heterokaryons Enable Facile Mutagenic Analysis of Essential Genes in Tetrahymen513
I. Introduction514
II. Strategy515
III. Methods for Creating Knockout Heterokaryons524
References530
Chapter 29. Creation and Use of Antisense Ribosomes in Tetrahymena thermophila533
I. Introduction533
II. Using Antisense Ribosomes in Tetrahymena thermophila534
III. Considerations Particular to Antisense Ribosomes538
IV. Evaluation and Applications of the Antisense Ribosome System544
References545
Chapter 30. Protein Tagging in Tetrahymena549
I. Introduction549
II. Strategies for Gene Tagging in Tetrahymena551
III. Methods557
References557
Appendix I. Genetic Nomenclature Rules for Tetrahymena thermophila561
Appendix II. Codon Usage in Tetrahymena thermophila565
Index569
Volumes in Series575

Book details

Vendor Elsevier S & T
SKU 9780125441643
ISBN-13 9780080573625
Author Wilson, Leslie
Category Science
Subject Developmental Biology

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Tetrahymena thermophila is emerging as a powerful experimental system for functional genetics. The ciliated protozoan offers numerous advantages, not the least of which is the ability to eliminate any specific gene of interest and then to evaluate the effect of that mutation on the living cell. Past investigations with T. thermophila have yielded several key discoveries, including dynein, catalytic RNA, and telomerase. This volume is a comprehensive resource for using Tetrahymena in the laboratory and is aimed at persons already experienced, as well as newcomers to the organism. It covers both the biological background and essential protocols for investigators rapidly turning to Tetrahymena as the experimental system of choice.

Key Features
* Contains both theoretical and practical issues in 30 chapters contributed by the world authorities on Tetrahymena
* Indispensible for both the novice and the experienced researcher
* Overviews the history, cell biology, and genetics of the organism
* Describes essential protocols on the growth of cells, genetic techniques, and how to look at the cells with the microscope
* Illustrates how the methods can be applied to solve various cell biological problems
* Reviews recently developed strategies for altering gene expression

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