Part E: Analysis of Meiotic Products

Genetic Analysis of Free Ascospores

The four meiotic products of a diploid yeast cell form into spores in a structure called an ascus. If the spores can be released from the ascus, then each spore can be grown as a pure haploid culture and its phenotype and genotype can be determined. Clearly, this provides much more information than one can obtain by letting the spores mate in the ascus. The ascus wall can be removed with an enzyme preparation from snails, glusulase, which is available from Sigma Biochemical Co. Then the spores can be isolated by streaking them out for single colonies. Examination of Table I shows that 75 percent of the spores should be red, adenine-requiring, and 25 percent should be white, adenine-independent.

Experiment: In this experiment you will free the spores from asci produced by the diploid (HA1 X HB2). You will then grow the spores into individual colonies and analyze each colony's phenotype and genotype.

Table I :

Genotypes of Spores Expected From the Cross:
a ade1 ADE2 X ADE1 ade2

Mating type a: ade1 ADE ADE1 ade2 ade1 ade2 ADE1 ADE2
Mating type : ade1 ADE ADE1 ade2 ade1 ade2 ADE1 ADE2

Each of the eight spore genotypes is expected to occur with the same frequency.

Phenotypes of Spores Expected From the Cross:
75% pink and 25% cream-colored

Pink phenotype produced by ade1 ADE2 ADE1 ade2 ade1 ade2 in both mating types.

Cream-colored phenotype produced by ADE1 ADE2 in both mating types.

Time Line:

1st - 8th Day: 30 min Getting Ready
9th Day: 50 min Digestion of Asci, Plating Cells and
11th Day: 20 min Counting Colonies
11th Day: 10 min Analysis of Cream-colored 15th Day: 30 min Colonies

Optional genotype analysis
15th Day: 20 min Subculture of Ascospore Colonies and Tester Strains
16th Day: 10 min Mating of Strains
17th Day: 50 min Observation and Plating of Mating Mixtures
18th Day: 50 min Observation of MV Plates and Analysis of Data

You may skip this procedure if you have asci from a previous work such as the Yeast Life Cycle experiment. ( See The Yeast Life Cycle for more information on mating and sporulating yeast)

1. Subculture the parent strains, HA1 and HB2, overnight on a YED plate.
2. Use sterile toothpicks to transfer a small amount of each parent strain to separate spots close to each other on the plate.
Stir the parent strains together using a third sterile toothpick and then incubate the plate overnight.
3. Purify the diploid strain by transferring a small amount of the mating mix to a MV plate.
Incubate the plate overnight.
4. Presporulate the diploids by transferring a small amount of the diploid culture from the MV plate to a fresh YED plate.
Incubate the plate overnight.
5. To sporulate the diploid cells transfer several streaks of cells from the presporulation plate to a YEKAC plate.
Incubate the plate for 3-5 days.

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