Yeast Genetics - Bruce, I. J.

. .

Sommaire:
I. Classical Yeast Genetics.- 1. Mating.- a) Drop-Overlay Method.- Mating of Prototrophic Haploid Strains.- 2. Sporulation.- a) Spore Isolation.- i) Heat Killing of Vegetative Cells.- ii) Ether Killing of Vegetative Cells.- iii) Sporulation of Protoplasts and Bursting of Vegetative Cells.- iv) Separation of Asci and Vegetative Cells in Biphasic Systems.- v) Separation of Asci and Vegetative Cells on Renografin Density Gradients.- 3. Tetrad Analysis.- a) Operation of Micromanipulators.- b) Variants: Spore-Spore or Spore-Cell Pairing.- c) Detection of Post-Meiotic Segregation.- i) Procedure 1: Dissection on the Surface of an Open Plate.- ii) Procedure 2: Dissection on an Inverted Plate (Fogel-s Procedure).- 4. Enzymes for Digestion of Yeast Ascus Walls.- 5. Ancillary Methods for Handling Clones Obtained by Ascus Dissection.- a) Replica Plating.- i) Velvet Pad.- ii) Filter Paper.- b) Mating Type Determination by Cross-Stamping.- 6. Mutagenesis.- a) Ultraviolet and X-Irradiation.- i) Procedure 1: (Liquid Medium).- ii) Procedure 2: (Solid Medium).- iii) General Note on Mutagenesis.- iv) Special Note: Induction of Mitotic Recombination by Ultraviolet Irradiation.- b) Drying.- i) Freeze-Drying.- ii) Vacuum Drying.- c) Chemical Mutagens.- i) Nuclear Mutations.- ii) Mitochondrial Mutations.- d) Isolation of Particular Mutants or Classes of Mutants.- i) Mutator Mutants.- ii) Temperature-Sensitive Mutants.- iii) Isolation of kar Mutants (Defective in Nuclear Fusion).- iv) Cell Wall Mutants.- v) Antibiotic-Sensitive Mutants. Kamikaze Strains.- vi) PEP4 Mutants.- vii) Membranes. Fatty Acid and Inositol-Requiring Mutants.- viii) Mutants Auxotrophic for 2?-Deoxythymidine 5?-Monophosphate.- ix) Glycolytic Cycle Mutants.- x) Secretory Mutants, Transport Mutants, etc.- 7. Mapping and Fusion.- a) Tetrad Analysis.- b) Determination of Centromere Linkage.- c) Assignment of a Gene to a Particular Chromosome: Trisomic Analysis.- i) Known Chromosome is Disomic.- ii) Chromosome Bearing the Unmapped Gene is Disomic.- iii) Multiply Disomic Strains.- iv) Super-Triploid Method.- d) Mitotic Mapping Techniques.- i) Mitotic Crossing-Over.- ii) Mitotic Chromosome Loss.- e) Mapping by Chromosome Transfer.- f) Fine-Structure (Intragenic) Mapping.- g) Strategies for Mapping According to the Above Methods.- 8. Protoplast Formation and Fusion.- a) Regeneration in Solid Medium.- b) Regeneration in Liquid Medium.- II. Methods Using Direct Manipulation of DNA and RNA.- 1. Separation of Large DNA Molecules, Greater than 25 Kbp, by Gel Electrophoresis.- a) Preparation of Intact Chromosomal-Sized Yeast DNA Molecules.- b) Gel Preparation.- c) Size Standards.- d) Restriction Digests.- e) Conditions for Electrophoresis.- i) Loading the Gel.- ii) Running the Gel.- f) OFAGE.- g) FIGE.- 2. Isolation of Pure High-Molecular-Weight DNA from the Yeast Saccharomyces cerevisiae.- a) Protoplasting.- b) Protoplast Lysis and DNA Recovery.- 3. Transformation of Yeast: Saccharomyces cerevisiae.- a) Protoplast (Spheroplast) Transformation.- b) Intact Cell Transformation.- 4. Plasmid Isolation from Yeast.- 5. Rapid Isolation of DNA from Yeast.- 6. RNA Isolation.- a) With Glass Beads.- b) Without Glass Beads.