C. elegans: a practical approach
Saved in:
| Other Authors: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Oxford
Oxford University Press
1999
|
| Series: | The practical approach series
213 |
| Subjects: | |
| Online Access: | Klappentext Inhaltsverzeichnis |
| Item Description: | Hier auch später erschienene, unveränderte Nachdrucke |
| Physical Description: | XXI, 281 S.,: graph. Darst. , Ill. |
| ISBN: | 0199637385 0199637393 |
Staff View
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| adam_text | R /V C T I C A L
P P R O A C H
E R I E S
Series Editor: B. D. Hames
This book provides all the information you need to start working on
Caenorhabditis elegans. It begins by giving basic background about the
organism: its natural history, anatomy, life cycle, development, and
evolution. The rest of the volume is an outstanding collection of
methods that describe how to use C. elegans in research. Topics covered
include: obtaining growing and maintaining C. elegans; the elegans
genome project, genome sequence database searching and analysis;
transformation of C. elegans; reverse genetic techniques such as gene
inactivation by RNA mediated interference and germ line gene
deletion; analysis of phenotypes by Nomarski, fluorescence, 4-
dimensional, and electron microscopy; C. elegans neurobiology;
approaches for studying gene expression; gene characterization;
procedures for producing biochemically useful preparations;
conventional genetics with procedures for selfing and crossing,
mutagenesis and mutant screening, characterization of mutants, gene
mapping, temperature-shift experiments and mosaic analysis.
C. elegans: A Practical Approach is a comprehensive source of data,
helpful advice and detailed protocols for all researchers who use this
organism in their research. If you intend to start working on C. elegans,
this is the practical guide you need.
UNIVERSITY PRESS
9
Contents
a
List of Contributors xvii
Abbreviations xix
1. Background on Caenorhabditis elegans 1
Ian A. Hope
1. Introduction 1
2. What is C. elegans? 2
Natural history 2
Anatomy 3
Life-cycle 6
Development 6
Evolutionary relationship 8
3. How can C. elegans be studied? 9
4. What biological subjects has C. elegans had a key role in? 12
5. Other books about C. elegans 13
References 13
2. The genome project and sequence homology
to other species 17
Mark Blaxter
1. The C. elegans genome project 17
2. The physical map 18
3. The expressed genome 20
4. The genome sequence 23
5. Sequence analysis and annotation 24
Annotation of coding segments 24
Other data on the sequence map 25
6. Searching the C. elegans databases for homologues of genes
of interest 26
Finding your homologue 27
7. Caveats on genome sequence data and gene predictions 32
8. Other nematode genome projects 32
References 33
Contents
3. C. elegans and the Web 39
Jean Thierry֊Mieg, Danielle Thierry-Mieg, Michel Potdevin, and
Lincoln Stein
1. Introduction 39
2. Starting from a sequence homology 40
Graphical view of the sequence and of the cDNAs 41
The genetic map 43
3. Starting from a direct BLAST search of the C. elegans data 46
4. Starting from ‘Text search/ 47
5. Other types of data 47
6. Other ways to look at the ACeDB data 48
7. Conclusions 49
Acknowledgements 49
References 49
4. Maintenance of C. elegans 51
Theresa Stiernagle
1. Introduction 51
2. Acquiring strains from the Caenorhabditis Genetics
Center (CGC) 51
The CGC 51
Requesting strains 52
Mailing strains 52
3. Preparation of growth media 53
Preparation of bacterial food source 53
Preparation of NGM Petri plates 54
Seeding NGM plates 55
4. Culturing C. elegans on Petri plates 55
Transferring worms grown on NGM plates 55
Transferring frequency 56
General stock maintenance tips 57
5. Growth of C. elegans in liquid medium 59
6. Cleaning contaminated C. elegans stocks 60
7. Obtaining synchronous cultures and staged animals 62
8. Freezing and recovery of C. elegans stocks 64
Acknowledgements 67
References 67
X
Contents
5. Transformation 69
Y is hi Jin
1. Introduction 69
2. The C. elegans germline 70
3. Equipment for transformation 71
4. Procedures for transformation 72
Preparing DNA samples for transformation 73
Needle preparation and sample loading 79
Mounting worms 80
Injections 82
Recovery of inj ected worms 84
Identification of transformants and establishing lines 84
Troubleshooting 85
5. Fate of injected DNA 87
6. Integration of extrachromosomal arrays 88
7. Transformation markers 90
Wild-type or mutant genes as co-injection markers 90
Reporter constructs as co-injection markers 92
8. Gene cloning by germline transformation using the
wild-type DNA 93
Acknowledgements 95
References 95
6. Reverse genetics 97
Robert J. Barstead
1. Introduction 97
2. Epigenetic gene inactivation 97
RNA-mediated gene inactivation (RNAi) 97
3. Mutational germline gene inactivation 99
Phenotype-dependent mutational screens 99
Phenotype-independent mutational screens 100
4. Analysis of phenotypes 116
General considerations 116
References 118
xi
Contents
7. Microscopy 119
Ralf Schnabel
1. Introduction 119
2. Stereomicroscopy 120
3. Light microscopy 120
Acquiring a new microscope 120
4. Nomarski microscopy 125
Mounting embryos or worms 125
Setting up the microscope 126
Analysing worms 129
5. 4-dimensional microscopy 131
What is 4D-microscopy useful for? 132
Technology 133
Lineaging with a 4D-record 134
6. Confocal microscopy 136
7. Documentation 137
Photography 137
Collecting and processing digital pictures 139
8. Electron microscopy 140
References 141
8. Neurobiology 143
James H. Thomas and Shawn Lockery
1. Introduction 143
2. Structure 144
Overview 144
Sensory anatomy 145
Motor anatomy 145
Synapses 146
Tools for analysing neuron structure 147
3. Genes 147
Neurotransmitters 147
Neuropeptides 148
Voltage-gated ion channels 150
Transmitter systems and ion channels as nuts and bolts 150
4. Behaviours 152
Locomotion 152
Egg-laying 156
Defecation 158
XII
Contents
Pharyngeal pumping 159
Chemotaxis 160
Chemical avoidance 162
Thermotaxis 163
Mechanosensory responses 165
Dauer formation 166
Food responses 167
Male mating 167
Integrative functions 168
5. Pharmacology 168
Overview of useful drugs 168
Egg-laying pharmacology 170
Why are pharmacological targets so conserved? 170
6. Electrophysiology 171
Electrophysiological methods 171
Neurons 173
Muscles 175
Chemical synapses 175
Electrical synapses 175
7. Future research 176
References 176
9. Gene expression patterns isi
Andrew Mounsey, Laurent Molin, and Ian A. Hope
1. Introduction 181
2. Reporter genes 181
Selection of an expression vector 182
Designing a reporter gene fusion 183
Staining for lacZ expression 184
Examination of GFP expression 187
Interpretation of expression patterns 189
3· Localization of RNAs in C. elegans embryos by whole-mount,
in situ hybridization 189
Preparation of digoxigenin-labelled RNA probes 190
Preparation of the embryos 191
Pre-hybridization and hybridization of the embryos 192
Immunochemical detection of the probe 194
4. Immunomicroscopy 195
5. Conclusion 198
References 198
xiii
Contents
10. Molecular biology 201
Iain L. Johnstone
1. Introduction 201
2. The raw material—worms 201
External contamination 202
Internal contamination 202
Factors influencing gene expression 203
3. DNA methods 205
General considerations 205
Southern analysis 208
Genomic PCR analysis 209
4. RNA methods 209
General considerations on worm gene expression 209
RNA purification methods 212
Northern blot analysis 215
Semi-quantitative rtPCR 215
Transcript analysis 220
5. Protein detection 223
Simple, whole animal lysates 223
Protein extraction methods 223
References 225
11. Biochemistry of C.elegans
Paul E. Mains and James D. McGhee
1. Introduction 227
2. Large-scale growth of mixed-stage C. elegans on solid media 227
3. Production of dauer larvae 231
4. Production of C. elegans adults and embryos 232
5. Production of different stages of the C. elegans life-cycle 234
6. Production of embryos blocked in mid-proliferation phase
by growth on fluorodeoxyuridine (FUdR) 234
7. Production of unfertilized oocytes 236
8. Methods of worm disruption 238
9. Preparation of nuclei and nuclear extracts from embryos
and oocytes 239
10. The use of nuclear extracts to investigate DNA—protein
interactions 241
xiv
Contents
11. Manipulation of the living C. elegans embryo 242
Blastomere culture 242
Laser ablation 243
12. Conventional genetics 245
Jonathan Hodgkin
1. Introduction 245
2. Reproduction, maintenance, and scoring 246
Selfing 246
Crossing 246
Source of males for crossing 247
Maintenance of lethal or sterile mutations with genetic balancers 248
Scoring crosses 249
Analysing crosses 249
3. Mutagenesis and screening 251
Mutagenesis 251
Mutant screens and selections; clonal screens 255
Isolation of lethal mutations 256
4. Cleaning and characterizing mutants 257
Mutant isolation 257
Mutant characterization 258
Temperature-shift analysis 260
Mosaic analysis 260
5. Genetic mapping procedures 262
Assignment of linkage 263
Standard 2-factor mapping 263
3-factor and multipoint crosses for ordering genes 264
Mapping by means of deficiencies and duplications 265
STS mapping by means of PCR 265
PCR mapping of deficiencies 268
Molecular cloning of mapped genes 269
References 269
List of suppliers 271
Index 274
XV
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| discipline | Biologie |
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| spelling | C. elegans a practical approach edited by Ian A. Hope, School of Biology University of Leeds Caenorhabditis elegans Oxford Oxford University Press 1999 XXI, 281 S.,: graph. Darst. , Ill. txt rdacontent n rdamedia nc rdacarrier The practical approach series 213 Hier auch später erschienene, unveränderte Nachdrucke Caenorhabditis elegans Genetics Caenorhabditis elegans genetics Genetik (DE-588)4071711-2 gnd rswk-swf Caenorhabditis elegans (DE-588)4147127-1 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content Caenorhabditis elegans (DE-588)4147127-1 s Genetik (DE-588)4071711-2 s DE-604 Hope, Ian A. edt The practical approach series 213 (DE-604)BV007921321 213 Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010278286&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Klappentext Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010278286&sequence=000002&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | C. elegans a practical approach The practical approach series Caenorhabditis elegans Caenorhabditis elegans Genetics Caenorhabditis elegans genetics Genetik (DE-588)4071711-2 gnd Caenorhabditis elegans (DE-588)4147127-1 gnd |
| subject_GND | (DE-588)4071711-2 (DE-588)4147127-1 (DE-588)4143413-4 |
| title | C. elegans a practical approach |
| title_alt | Caenorhabditis elegans |
| title_auth | C. elegans a practical approach |
| title_exact_search | C. elegans a practical approach |
| title_full | C. elegans a practical approach edited by Ian A. Hope, School of Biology University of Leeds |
| title_fullStr | C. elegans a practical approach edited by Ian A. Hope, School of Biology University of Leeds |
| title_full_unstemmed | C. elegans a practical approach edited by Ian A. Hope, School of Biology University of Leeds |
| title_short | C. elegans |
| title_sort | c elegans a practical approach |
| title_sub | a practical approach |
| topic | Caenorhabditis elegans Caenorhabditis elegans Genetics Caenorhabditis elegans genetics Genetik (DE-588)4071711-2 gnd Caenorhabditis elegans (DE-588)4147127-1 gnd |
| topic_facet | Caenorhabditis elegans Caenorhabditis elegans Genetics Caenorhabditis elegans genetics Genetik Aufsatzsammlung |
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