John F. T. Spencer, Dorothy M. Spencer, I. J. Bruce

Yeast Genetics (eBook, PDF)

A Manual of Methods

• Format: PDF

Produktbeschreibung

The manual consists of two main sections. The first includes the essential, sometimes laborious, procedures for handling yeasts, for inducing mating and isolation of hybrids, for inducing sporulation and isolation of single-spore clones, with some details of tetrad analysis, and including techniques and ancillary equipment for use of the micromanipulator. There are also proce dures for induction of mutants by physical and chemical agents, and for iso lation of particular types of mutants, such as to temperature sensitivity, for increased frequency of mutations, for mutations in the mitochondrial genome, both to the petite colonie form and to resistance to antibiotics, for mutations in that part of the yeast genome controlling the glycolytic cycle, and numerous others. Mapping of mutations is discussed briefly, though this aspect of yeast genetics is probably one which should not be undertaken until the investigator has gained a certain amount of experience in the field. How ever, as is pointed out in the pertinent part of the manual, the task of mapping has been tremendouslysimplified by the availability from the Yeast Genetics Stock Center at the University of California at Berkeley of a set of auxo trophic strains designed to permit mapping of most unknown genes with a minimum number of crosses and tetrad analyses. The first section concludes with the description of methods for hybridization of yeasts by protoplast fusion, which has been described as the poor man's system for genetic engineering.

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Produktdetails

• Verlag: Springer Berlin Heidelberg
• Seitenzahl: 104
• Erscheinungstermin: 6. Dezember 2012
• Englisch
• ISBN-13: 9783642733567
• Artikelnr.: 53093965

Inhaltsangabe

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.Contents: Introduction.- "Classical" Yeast Genetics: Mating. Sporulation. Tetrad Analysis. Enzymes for Digestion of Yeast Ascus Walls. Ancillary Methods for Handling Clones Obtained by Ascus Dissection. Mutagenesis. Mapping and Fusion. Protoplast Formation and Fusion.- Methods Using Direct Manipulation of DNA and RNA: Separation of Large DNA Molecules, Greater than 25 Kbp, by Gel Electrophoresis. Isolation of Pure High-Molecular-Weight DNA from the Yeast Saccharomyces cerevisiae. Transformation of Yeast: Saccharomyces cerevisiae. Plasmid Isolation from Yeast. Rapid Isolation of DNA from Yeast. RNA Isolation.- Subject Index.