Molecular and cellular biology of filamentous fungi: a practical approach
This volume brings together detailed practical guidance from experienced researchers using genetic, genomic, cellular and biochemical methods, to attempt to determine the functions of genes and how they contribute to the biology of fungi.
Saved in:
| Format: | Book |
|---|---|
| Language: | English |
| Published: |
Oxford [u.a.]
Oxford Univ. Pr.
2001
|
| Edition: | 1. publ. |
| Series: | The practical approach series
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Summary: | This volume brings together detailed practical guidance from experienced researchers using genetic, genomic, cellular and biochemical methods, to attempt to determine the functions of genes and how they contribute to the biology of fungi. |
| Item Description: | Reprinted 2007 |
| Physical Description: | XX, 267 S. Ill., graph. Darst. |
| ISBN: | 0199638381 0199638373 |
Staff View
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Record in the Search Index
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| adam_text | Molecular and Cellular
Biology of Filamentous
Fungi
U |Q)
Edited by
Nick Talbot
University of Exeter, School of Biological
Sciences, Washington Singer Laboratories,
Perry Road, Exeter EX4 4QG, UK
OXTORD
UNIVERSITY PRESS
© 2008 AGI-Information Management Consultants
May be used for personal purporses only or by
libraries associated to dandelon com network
Contents
List of protocols page xv
Abbreviations xix
1 Identification and genotypic characterization of filamentous fungi i
Michele D Piercey-Normore and Keith N Egger
1 Introduction 1
2 A synopsis of genome variation 1
3 Determining your objectives 4
4 Techniques for genome analysis 4
PCR amplification 4
Analysing variation at a single locus 7
Analysing variation at multiple loci 9
Analysis of environmental samples 11
Analysing expressed genes 12
5 Assumptions 13
6 Choosing the best strategy 15 *
Distinguishing individuals or populations 15
Distinguishing closely related species 17
Higher taxa 18
7 Databases and WWW resources 19
References 20
2 Nucleic acid isolation and analysis 23
Nicholas] Talbot
1 Introduction 23
2 Isolation of genomic DNA 23
Genomic DNA extraction using cetyltrimethylammonium bromide (CTAB) 23
Isolation of high molecular weight DNA 26
3 Isolation of total RNA from filamentous fungi 28
Manipulation of RNA 30
Acknowledgements 31
References 31
IX
CONTENTS
3 DNA-mediated transformation of fungi 33
Paul Bowyer
1 Introduction 33
2 Selectable markers and vectors for transformation 33
Hygromycin B resistance 35
Phleomycin resistance 35
Bialophos resistance 35
Sulfonylurea resistance 35
Benomyl resistance 36
Acetamidase (AmdS) 36
3 Quality of DNA required for transformation 36
4 Entry of DNA into fungi 36
Protoplast formation 36
5 REMI transformation 37
6 Agrobacterium-mediated transformation of fungi 39
7 Particle bombardment 41
8 Transformation of fungi by electroporation 42
9 Fate of transforming DNA 43
Integration/replication of transforming DNA 43
References 45
4 Genetic analysis of ascomycete fungi 47
Steven D Harris
1 Introduction 47
2 Identification and isolation of mutants 47
Important considerations 47
Chemical mutagenesis 48
UV mutagenesis 49
Recovery of mutants 50
Types of mutants 50
3 Classical genetic analysis of mutants 52
Setting up crosses 52
Random ascospore analysis 53
Octad analysis 55
Parasexual genetics in A nidulans 55
References 58
5 Genetic analysis of basidiomycete fungi 59
Piers] Walser, Marcel Hollenstein, Michaela] Klaus, and Ursula Kiies
1 Introduction 59
Life cycles 59
Breeding systems 60
Mating types 61
2 Strain isolation 61
Isolation of haploid strains from dikaryons through meiosis 61
Isolation of haploid strains through dedikaryotization of dikaiyotic mycelium 63
CONTENTS
Mating for dikaryon formation 64
Isolation of strains of special genetic condition: forced heterokaryons, diploid
strains, solo-pathogenic strains, and AmutBmut homokaryons 65
Preservation and maintenance of cultures 66
3 Genetic complementation 67
4 Genetic mapping 68
Genetic mapping by sexual recombination 68
Electrophoretic karyotyping and RFLP analysis 69
Tetrad analysis 71
Gene mapping by parasexual recombination 72
5 Natural mutants 73
6 Classical mutagenesis 73
UV mutagenesis 74
Chemical mutagenesis 75
Mutant selection 76
7 Insertional mutagenesis 79
Transposon mutagenesis 79
REMI mutagenesis 79
Acknowledgements 87
References 87
Appendix 1 Standard basidiomycete media 89
6 Genome analysis in filamentous fungi 91
Mark L Farman
1 Introduction 91
2 Genetic mapping 91
Whole genome mapping versus targeted mapping 92
Genetic markers and genome analysis 92
Other types of markers 95
3 Electrophoretic karyotyping 96
Preparation of intact chromosomal DNA 97
CHEFelectrophoresis 98
4 Physical mapping 99
Procedures for physical mapping in filamentous fungi 99
RecA-assisted Achilles cleavage (RecA-AC) 99
5 Construction and screening of genomic DNA libraries 101
Bacterial artificial chromosome (BAC) libraries 103
Picking colonies and library replication 106
Arraying library clones on hybridization membranes 108
Pooling library clones 109
Library storage 110
6 Screening libraries 110
Screening libraries by hybridization 110
Recovery of clones from frozen library stocks 111
7 Chromosome walking 112
Synthesis of probes from insert ends 112
Determining the correct direction of a walk 114
Monitoring progress 114
XI
CONTENTS
Overcoming obstacles to walking 115
Identification of clones containing a target gene 115
8 Resources for fungal genome analysis 116
References 116
7 Cell biology of the duplication cycle in fungi 119
Michelle Momany
1 Introduction 119
2 Staining nuclei and cell walls 120
3 Indirect immunofluorescence 122
Commercially available antibodies 122
Raising antibodies 123
References 124
8 Investigations of Oomycete cell biology 127
Adrienne R Hardham
1 Introduction 127
Oomycete phylogeny 127
General biology and life cycle of Oomycetes 128
2 Observations of living cells 130
Assessment of hyphal health 130
Video microscopy of living cells 131
Introduction of fluorescent probes into living cells 131
3 Investigations of cell ultrastructure 134
The flagellar apparatus of Oomycete zoospores 134
CryoSEM: scanning electron microscopy of frozen, hydrated cells 135
Chemical fixation and processing for transmission electron microscopy 135
Cryofixation and freeze-substitution for transmission electron microscopy 138
4 Immunolabelling of Oomycete cells 140
Fixation 140
Cryosectioning for immunofluorescence microscopy 142
Methacrylate embedding 145
Immunolabelling for light microscopy 149
Post-embedding immunogold labelling 152
Acknowledgements 154
References 154
9 Signal transduction in filamentous fungi: regulation of protein
kinases 157
XiangS Ye, LipingWu, and Stephen A Osmani
1 Introduction 157
Scope of the chapter 158
2 Conserved properties of protein kinases 158
3 Protein extraction: importance of protecting state of phosphorylation 159
Xll
CONTENTS
4 Protein immunoprecipitation 162
Epitope tagging using the Quick Change mutagenesis kit 163
Heterologous expression and antibody production 164
5 Gel-shift assay for detection of protein phosphorylation state 167
6 Protein kinase assay and substrate specificity 169
7 Ordering a signal transduction pathway 172
Acknowledgements 173
References 173
10 Electron microscopy of filamentous fungi 175
Richard] O Connell and Raffaella Carzaniga
1 Introduction 175
2 Conventional preparation techniques for TEM 175
Chemical fixation and dehydration 176
Resin embedding 178
Sectioning 180
Section contrasting 182
3 Low temperature preparation techniques for TEM 184
Cryofixation 186
Freeze-substitution 190
Artefacts of cryofixation and freeze-substitution 192
4 Cytochemical techniques 193
PATAg staining of carbohydrates 193
Immunogold labelling 194
Lectin-gold cytochemistry 199
Enzyme-gold cytochemistry 200
5 Shadowing and replication techniques 201
Freeze-fracture 201
Surface replication 203
6 Scanning electron microscopy 204
Ambient temperature SEM 204
Low temperature SEM 205
7 Safety 206
Acknowledgements 206
References 207
1 1 Biochemical methods for filamentous fungi 209
Hans von Db hren
1 Introduction 209
2 Preparation of extracts 210
Preparation of mycelium 210
Extraction of mycelium 211
3 Preparation of cellular fractions 212
Plasma membranes 212
Organelles 213
Xlll
CONTENTS
4 Protein analysis 218
Protein survey 218
Detection of specific proteins 218
Protein isolation and purification 219
5 Metabolite identification 223
Direct determination of metabolites 224
Metabolite extraction 224
Isotope labelling of metabolites 225
6 Enzyme analysis 225
References 225
12 Immunological methods for fungi 227
Christopher R Thornton
1 Introduction 227
2 Properties of a molecule that make it immunogenic 228
Selection of fungal material for immunization 229
Preparation of immunogens 230
3 Raising polyclonal antisera 232
Which species/strain and how many animals? 233
Immunization procedures, determination of immune response by test bleeds,
and boosters 233
Serum preparation, purification, storage 234
Titre determination 236
4 Raising monoclonal antibodies 240
Setting up a tissue culture facility 240
Immunization procedures, the immune response, and titre determination 240
Production of murine hybridomas 241
Screening procedure for selection of hybridomas 243
Subcloning, expansion, and routine culture of hybridomas and long-term
storage of mAb supernatants 245
Long-term storage of hybridoma cell lines 247
Determination of immunoglobulin class/subclass 249
5 Characterization of protein and carbohydrate epitopes 250
Periodate oxidation 250
Protease digestion using pronase and trypsin 251
Heating 251
Mild alkaline hydrolysis (p-elimination) 251
6 Other commonly used immunological assays 253
7 Availability and commercial development of antibodies 255
References 256
A l List of suppliers 259
Index 265
XIV
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| spelling | Molecular and cellular biology of filamentous fungi a practical approach ed. by Nick Talbot 1. publ. Oxford [u.a.] Oxford Univ. Pr. 2001 XX, 267 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series Reprinted 2007 This volume brings together detailed practical guidance from experienced researchers using genetic, genomic, cellular and biochemical methods, to attempt to determine the functions of genes and how they contribute to the biology of fungi. Biologie moléculaire fongique Champignons filamenteux - Cytologie Filamentous fungi Cytology Fungal molecular biology Methode (DE-588)4038971-6 gnd rswk-swf Molekularbiologie (DE-588)4039983-7 gnd rswk-swf Filamentöse Pilze (DE-588)1105438414 gnd rswk-swf Cytologie (DE-588)4070177-3 gnd rswk-swf Filamentöse Pilze (DE-588)1105438414 s Molekularbiologie (DE-588)4039983-7 s Methode (DE-588)4038971-6 s DE-604 Cytologie (DE-588)4070177-3 s Talbot, Nick Sonstige oth HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009620074&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Molecular and cellular biology of filamentous fungi a practical approach Biologie moléculaire fongique Champignons filamenteux - Cytologie Filamentous fungi Cytology Fungal molecular biology Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd Filamentöse Pilze (DE-588)1105438414 gnd Cytologie (DE-588)4070177-3 gnd |
| subject_GND | (DE-588)4038971-6 (DE-588)4039983-7 (DE-588)1105438414 (DE-588)4070177-3 |
| title | Molecular and cellular biology of filamentous fungi a practical approach |
| title_auth | Molecular and cellular biology of filamentous fungi a practical approach |
| title_exact_search | Molecular and cellular biology of filamentous fungi a practical approach |
| title_full | Molecular and cellular biology of filamentous fungi a practical approach ed. by Nick Talbot |
| title_fullStr | Molecular and cellular biology of filamentous fungi a practical approach ed. by Nick Talbot |
| title_full_unstemmed | Molecular and cellular biology of filamentous fungi a practical approach ed. by Nick Talbot |
| title_short | Molecular and cellular biology of filamentous fungi |
| title_sort | molecular and cellular biology of filamentous fungi a practical approach |
| title_sub | a practical approach |
| topic | Biologie moléculaire fongique Champignons filamenteux - Cytologie Filamentous fungi Cytology Fungal molecular biology Methode (DE-588)4038971-6 gnd Molekularbiologie (DE-588)4039983-7 gnd Filamentöse Pilze (DE-588)1105438414 gnd Cytologie (DE-588)4070177-3 gnd |
| topic_facet | Biologie moléculaire fongique Champignons filamenteux - Cytologie Filamentous fungi Cytology Fungal molecular biology Methode Molekularbiologie Filamentöse Pilze Cytologie |
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