Techniques in Molecular and cellular Biology

Contents......Page 6
Introduction......Page 10
Section I Saccharomyces cerevisiae as a Genetic Research Organism......Page 14
Culture Conditions......Page 16
Mating Type, Mating, and the Sexual Life Cycle......Page 18
Genetic Nomenclature......Page 20
Genetic Crosses and Linkage Analysis......Page 21
Single Gene Cross......Page 22
Two Gene Cross......Page 23
Classes of Saccharomyces Cloning Plasmid Vectors......Page 25
YIp Plasmid......Page 26
YRp Plasmid......Page 27
YAC Plasmid......Page 28
Gene Disruption/Deletion in Saccharomyces (One-Step Gene Replacement)......Page 29
Gap Repair......Page 30
Reporter and Other Types of Fusion Gene......Page 31
References and Further Reading......Page 33
Preparation of the Cell Extract......Page 36
Equilibrium Density Gradient Centrifugation......Page 37
Fluorescence Microscropy, Immunofluorescence, and GFP......Page 39
Nomarski Interference Microscropy......Page 43
Electron Microscropy......Page 44
Protein Extraction and Purification......Page 45
Western Analysis......Page 48
Epitope-Tagging and Immunodetection of Epitope-Tagged Proteins......Page 50
Immunoprecipitation......Page 52
Metal Chelate Affinity Purification......Page 53
References and Further Reading......Page 54
Cell Shape and Growth Patterns......Page 56
Cell Wall, Cell Surface Morphology, and Morphological Variation......Page 57
Bud Scars, Birth Scars, and Budding Patterns......Page 58
Bud Site Selection and Polarized Cell Growth......Page 60
Nucleus......Page 64
Cytoskeleton......Page 65
Actin Cytoskeleton......Page 66
Microtubule Morphology in Cell Division and Mating......Page 68
Endoplasmic Reticulum......Page 70
Vacuole......Page 72
Membrane Trafficking......Page 73
Peroxisome......Page 74
References and Further Reading......Page 75
Section II Techniques of Genetic Analysis......Page 78
4 Mutant Hunts—To Select or to Screen (Perhaps Even by Brute Force)......Page 80
5 Complementation Analysis: How Many Genes are Involved?......Page 86
References......Page 90
Overview......Page 92
Epistasis Analysis of a Substrate-Dependent Pathway......Page 94
Epistasis Analysis of a Switch Regulatory Pathway......Page 95
References and Further Reading......Page 97
Preparation of the Library......Page 98
Cloning by Complementation......Page 99
Positional Cloning......Page 100
Analysis of Multiple Mutant Alleles......Page 102
Reference......Page 103
Intragenic Suppression......Page 104
Intergenic Suppression......Page 105
By-Pass Suppression......Page 106
Allele-Specific Suppression......Page 107
Suppression by Epistasis......Page 108
Overexpression Suppression......Page 109
References and Further Reading......Page 110
Mechanisms of Enhancement......Page 112
Synthetic Enhancement......Page 113
Conditional Lethal Mutations for the Isolation of Enhancer Mutations......Page 114
Further Reading......Page 115
Two-Hybrid Analysis......Page 116
One-Hybrid and Three-Hybrid Analysis......Page 118
References and Further Reading......Page 119
Reverse Genetics......Page 120
Dominant Negative Mutations......Page 122
References and Further Reading......Page 124
12 Genomic Analysis......Page 126
Databases......Page 127
DNA Microarray Analysis......Page 128
Genome-Wide Two-Hybrid Screens......Page 129
Transposon Mutagenesis......Page 130
References and Further Reading......Page 131
Section III Case Studies from the Saccharomyces Genetic Literature......Page 134
Case Study I Glucose Sensing and Signaling Mechanisms in Saccharomyces......Page 136
Case Study II Secretion, Exocytosis, and Vesicle Trafficking in Saccharomyces......Page 156
Case Study III The Cell Division Cycle of Saccharomyces......Page 186
Case Study IV Mating-type Pheromone Response Pathway of Saccharomyces......Page 218
C......Page 248
E......Page 249
H......Page 250
M......Page 251
P......Page 252
T......Page 253
Y......Page 254