This book is designed for use as a manual of methods, to be used at the bench. Topics discussed and methods described include classical genetics as related to yeasts, such as mating, sporulation, isolation of hybrids, microdissection of asci or the isolation of single-spore clones, mapping of genes and the construction of new strains by protoplast fusion. There is a large section dealing with mutations in general, and with methods of isolating a number of important classes of mutants in particular. A second part deals with basic techniques in separation of chromosomes by electrophoresis, such…mehr
This book is designed for use as a manual of methods, to be used at the bench. Topics discussed and methods described include classical genetics as related to yeasts, such as mating, sporulation, isolation of hybrids, microdissection of asci or the isolation of single-spore clones, mapping of genes and the construction of new strains by protoplast fusion. There is a large section dealing with mutations in general, and with methods of isolating a number of important classes of mutants in particular. A second part deals with basic techniques in separation of chromosomes by electrophoresis, such as OFAGE, FIGE, and CHEF, and gives detailed protocols for the first two. This section further includes methods for isolation of high molecular weight DNA from yeast, isolation of RNA, and techniques for transformation of yeasts.
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.
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.
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