Schedule
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Lab 8 -pick mutants and restreak |
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Lab 9 -grid and mate mutants for complementation |
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Lab 10 -select for complementation analysis |
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Lab 11 -Complementation analysis scoring -beta-galactosidase assay |
Goal: To identify and characterize mutants that affect the expression of the HO gene.
Many thanks go to Marta Galova and Dr. Kim Nasymth for suppling the yeast strains used in this experiment.
Background
It may come as a surprize; organisms with sexual life cycles have a strong drive toward having sex. Yeast have sexual life cycles. There are two types of yeast life cycles: heterothallic and homothallic. In the hetrothallic life cycle, the mating type of haploid yeast is stable; at every mitotic division a haploid yeast cell of the same mating type is produced. The baker's yeast (Saccharomyces cerevisiae) has two haploid mating types a and a. Only an a and an a cell can mate to form an a/a diploid. In the homothallic life cycle, the mating type is not stable. If you start growing an a haploid yeast cell soon you have a colony of a/a diploids. This is because as the a or a cell divids it can switch mating type and produce a or a cells. This mating type switch is a recombination/gene conversion event that removes a information at the mating type locus and replaces it with a information. This recombination is dependent on the presence of the HO DNA endonuclease which makes a double strand DNA cut at the mating type locus. The HO gene encodes the HO endonuclease.
Below are the articles that we will be discussing in class.
Reference material
Modern Genetic Analysis 2nd edition
page 489
pages 187-188
pages 404-405
pages 251-252
Other material that you will find helpful.
A good review for Mating Type and HO regulation.
Simple overview of yeast genetic nomenclature published in Trends in Genetics.
Fred Sherman's Getting started with yeast.