Part E: Analysis of Meiotic Products

Genetic Analysis of Ascospores by a Complementation Test

Yeast geneticists isolate ascospores, either as random mixtures or by micromanipulation and then score the phenotypes for the mutant alleles segregating in the cross. These more complicated and time-consuming experiments are described in other sections of this book. A simpler method to determine the genotype of each phenotype is the complementation test.

You can use the complementation test to determine whether a red isolate is haploid or diploid, and if haploid, to determine its mating type. The ade1 strains provide an elegant way to do this. When an ade1 strain mates with a red ade2 strain, the resulting diploid will be cream-colored, because ade1 and ade2 are different genes and the dominant alleles will complement the deficiencies in the recessive alleles. By mixing each red segregant with each of the two tester strains HA1 (a ade1) and HB1 (` ade1) and observing the resulting pattern of colors, you can infer the unknown genotypes. If the cells in a mixture mate the mixture will turn cream-colored. If your unknown strain does not mate with either tester, it must be diploid. You can confirm this by sporulating it. If it mates with the a tester it must be an `, and if it mates with the ` tester it must be an a.

This experiment illustrates the basic strategy of yeast genetics. From the point of view of the red and cream-colored variations of the color trait, which correspond to the ade2 and ADE2 alleles, this is a simple monohybrid cross. However, when you consider the mating-type alleles, a and `, it is a dihybrid cross. You should find approximately equal numbers of mating-type a and mating-type ` spores, demonstrating independent assortment of the alleles of the ADE2 gene and the mating type alleles in your original monohybrid cross. The mating-type ` ade2 strains are new recombinant strains that have inherited the ade2 allele from one parent and the mating-type ` allele from the other. Of course, this is not a conventional "textbook" dihybrid cross, because the mating-type alleles do not have a simple dominant/recessive mode of expression.

Experiment:


In this experiment you will mix known tester strains to unknown yeast strains and then determine if the cells are haploid or diploid. If the cells are haploid you will determine the mating type and genotype of the red trait.

Time Line:

1st Day: 10 min Subculture strains
2nd Day: 10 min Making mating mixtures
4th Day: 30 min Record and analyze results

Figure
Materials:

Procedure: 1. 1st Day: Use sterile toothpicks to subculture the tester strains (across top of plate) and unknown strains (down side of plate). It is possible to get eight to ten strains down the side of a single plate.

2. 2nd Day: Mix each of the unknown strains with each of the two tester strains.

3. 3rd Day: Observe and record the colors of the mixtures. Use your data to determine the mating type of each unknown.
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