Part D:Mutation Experiments

Analysis of "White" Mutants of Red Strains

How can we test the hypothesis that the cream-colored clonies that occur in red colonies are the result of a second mutation in the early part of the AMP synthesis pathway? If the hypothesis is correct, then we should be able to demonstrate that the original red adenine mutation is still there. This could be done by restoring the function of the hypothetical second mutant gene by complementation. If our cream-colored, adenine-requiring mutant has the genotype a ade2 adeX, where adeX represents the new mutation in one of the early genes, then when we cross that mutant with the ade2 ADEX strain we will get a diploid that is homozygous for ade2/ade2 and heterozygous for ADEX/adeX. This diploid should have the phenotype of the original red haploid colonies, red and adenine-requiring. When we cross the mutant with the ade1 ADE2 ADEX strain we get a diploid that is heterozygous at all three loci and is cream-colored, adenine-independent. (ade1/ADE1 ADE2/ade2 ADEX/adeX)

Experiment:
In this investigation you will cross two tester strains with mutant strains that are presumed to be early AMP synthesis pathway mutants that carry the ade2 allele. One of the tester strains carries ade1 and the other carries ade2. You will use the phenotype of the resulting diploid cells to help determine the genotype of the presumed early AMP pathway mutants.

Time Line: 1st Day: 15 min Subculture testers and mutant strains
2nd Day:: 15 min Make mating mixtures
4th Day: 45 min Record color, replica plate to MV and set up diploid test
5th Day: 15 min Record results from MV plate
8th Day: 50 min Record diploid test and analyze results

Materials:

Procedure: Isolate a number of early AMP synthesis pathway mutants from HA2 (a ade2) (See Spontaneous Mutation: Isolation of "White" Mutants)

1. 1st Day: Set up fresh cultures of the mutants by using toothpicks to make short streaks of cells on a YED plate.
A convenient arrangement is to put up to eight mating-type a strains in a row across the top of a plate and the mating-type tester strains in a column down one side of the same plate.
Incubate the plate overnight.

2. 2nd Day: At the intersections of the rows and columns, make all the possible mating mixtures.
Be sure to take a new toothpick after each mixture.
Incubate the plates overnight.

3. 4th Day: Record colony color and analyze your results. What does it mean if the cross with HB1 is cream-colored and the same mutant crossed with HB2 is red? 4. Use sterile toothpicks to make two replicas of the mating plate, one replica on MV and the other replica on YEKAC. 5. 5th Day: Record and analyze the results from the MV plate.
For these mutants growth on MV indicates adenine independence and no growth indicates an adenine requirement.

6. 8th Day: Use a microscope to examine each mating mixture for asci.
Which mixtures contained diploid cells?

7. Your original hypothesis stated that the early AMP pathway mutants also contained the ade2 allele. Does your data support this hypothesis?
( Teacher Tips )

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Last updated Wednesday, 04-Dec-2002 20:56:44 UTC