Contents

The Scar Solution Natural Scar Removal

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Introduction ix

Section I Saccharomyces cerevisiae as a Genetic Research Organism 1

1 Saccharomyces cerevisiae as a Genetic Model Organism 3

Overview 3

Culture Conditions 3

The Mitotic Life Cycle 5

Mating Type, Mating, and the Sexual Life Cycle 5

Saccharomyces Genome and Nomenclature 7

Genome Sequence 7

Genetic Nomenclature 7

Phenotype Nomenclature 8

Strain Nomenclature 8

Protein Nomenclature 8

Genetic Crosses and Linkage Analysis 8

Single Gene Cross 9

Two Gene Cross 10

Classes of Saccharomyces Cloning Plasmid Vectors 12

Yip Plasmid 13

YRp Plasmid 14

YEp Plasmid 15

YCp Plasmid 15

YAC Plasmid 15

Libraries 16

Gene Disruption/Deletion in Saccharomyces (One-Step Gene

Replacement) 16

Gap Repair 17

Reporter and Other Types of Fusion Gene 18

Expression Vectors 20

References and Further Reading 20

2 Techniques in Cell and Molecular Biology 23

Cell Fractionation 23

Preparation of the Cell Extract 23

Differential-Velocity Centrifugation 24

Equilibrium Density Gradient Centrifugation 24

Microscropy Techniques 26

Fluorescence Microscropy, Immunofluorescence, and GFP 26

Confocal Scanning Microscropy 30

Nomarski Interference Microscropy 30

Electron Microscropy 31

Flow Cytometry 32

Protein Extraction and Purification 32

Western Analysis 35

Epitope-Tagging and Immunodetection of Epitope-Tagged Proteins 37

Hemagglutinin (HA) Epitope 39

FLAG Epitope 39

Myc Epitope 39

Immunoprecipitation and Related Methods 39

Immunoprecipitation 39

Metal Chelate Affinity Purification 40

GST-Tagged and MalB-Tagged Proteins 41

References and Further Reading 41

3 Saccharomyces Cell Structure 43

Cell Shape and Growth Patterns 43

Cell Wall, Cell Surface Morphology, and Morphological Variation 44

Cell Wall Composition and Synthesis 45

Bud Scars, Birth Scars, and Budding Patterns 45

Schmoo Formation and Mating 47

Bud Site Selection and Polarized Cell Growth 47

Spore Formation 51

Nucleus 51

Nuclear Envelope 52

Spindle Pole Body 52

Cytoskeleton 52

Actin Cytoskeleton 53

Microtubule Cytoskeleton 55

Microtubule Morphology in Cell Division and Mating 55 Plasma Membrane, Endoplasmic Reticulum, Golgi Complex, Vacuole, and Membrane Trafficking 57

Endoplasmic Reticulum 57

Golgi Complex 59

Vacuole 59

Membrane Trafficking 60

Mitochondrion 61

Peroxisome 61

References and Further Reading 62

Section II Techniques of Genetic Analysis 65

4 Mutant Hunts—To Select or to Screen (Perhaps Even by Brute Force) 67

5 Complementation Analysis: How Many Genes are Involved? 73

References 77

6 Epistasis Analysis 79

Overview 79

Epistasis Analysis of a Substrate-Dependent Pathway 81

Epistasis Analysis of a Switch Regulatory Pathway 82

Epistasis Group 84

References and Further Reading 84

7 Gene Isolation and Analysis of Multiple Mutant Alleles 85

Preparation of the Library 85

Cloning by Complementation 86

Positional Cloning 87

Cloning by Sequence Homology 89

Analysis of Multiple Mutant Alleles 89

Reference 90

8 Suppression Analysis 91

Overview 91

Intragenic Suppression 91

Intergenic Suppression 92

By-Pass Suppression 93

Allele-Specific Suppression 94

Suppression by Epistasis 95

Overexpression Suppression 96

By-Pass Suppression by Overexpression 97

Allele-Specific Suppression by Overexpression 97

Overexpression Suppression by Epistasis 97

References and Further Reading 97

9 Enhancement and Synthetic Phenotypes 99

Overview 99

Mechanisms of Enhancement 99

Synthetic Enhancement 100 Conditional Lethal Mutations for the Isolation of Enhancer

Mutations 101

Genetic Interaction 102

Further Reading 102

10 Two-Hybrid Analysis 103

Two-Hybrid Analysis 103

One-Hybrid and Three-Hybrid Analysis 105

References and Further Reading 106

11 Advanced Concepts in Molecular Genetic Analysis 107

Reverse Genetics 107

Cold-Sensitive Conditional Mutations 109

Dominant Negative Mutations 109

Charged-Cluster to Alanine Scanning Mutagenesis 111

References and Further Reading 111

12 Genomic Analysis 113

Databases 114

Biochemical Genomic Analysis 115

DNA Microarray Analysis 115

Genome-Wide Two-Hybrid Screens 116

Genome-Wide Generation of Null Mutations 117

Gene Disruption Strains 117

Transposon Mutagenesis 117

References and Further Reading 118

Section III Case Studies from the Saccharomyces Genetic Literature 121

Case Study I Glucose Sensing and Signaling Mechanisms in

Saccharomyces 123

Case Study II Secretion, Exocytosis, and Vesicle Trafficking in

Saccharomyces 143

Case Study III The Cell Division Cycle of Saccharomyces 173

Case Study IV Mating-type Pheromone Response Pathway of

Saccharomyces 205

Index 235

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