Part D:Mutation Experiments

Separating the White Adenine Mutants From the Red Ones

The final proof of the double mutant hypothesis is to isolate the new adeX mutation in a haploid that is ADE1 and ADE2. In other words, in a strain with only a single mutant gene. This is done by crossing the double mutant a ade2 adeX to the nonmutant strain that is ADE2 ADEX.

Unfortunately, this experiment is complicated because the ADE2 ADEX parent strain is adenine independent, and so we cannot select a diploid. Therefore, the best we can do is select a number of large, cream-colored colonies from the mating mixture, because diploids grow better than haploids, and test them for sporulation. When we find one that sporulates, we will know it was a diploid.

When the spores from this cream-colored, adenine-independent diploid are characterized four types of spores will be obtained in each mating type: ade2 adeX, ade2 ADEX, ADE2 ADEX, and ADE2 adeX. The first two are the parental types and the last two are recombinant types. The last one is the one we want. This will have a cream-colored, adenine-requiring phenotype.

Experiment:
In this investigation you will cross cream-colored mutants that carry the ade2 gene with wild-type ( ADE1 ADE2). Then you will sporulate the diploid cells, isolate the spores and determine their genotypes.

Time Line:

1st Day: 15 min Subculture mutant strains
2nd Day: 15 min Make mating mixtures
3rd Day: 15 min Streak for single colonies
8th Day: 15 min Plate cells on YEKAC
13th Day:` 50 min Check for asci, digest asci and plate for single colonies
15th Day: 10 min Pick colonies
16th Day: 15 min Replica plate
17th Day: 50 min Record and analyze results; Set up mating plates
18th Day: 15 min Make test cross
19th Day: 30 min Record and analyze results

Materials:

Procedure: Isolate a number of early AMP pathway mutants from HA2 ( a ade2 ). (See Spontaneous Mutation: Isolation of White Mutants and Ultraviolet Lethality and Mutation in Yeast)

1. 1st Day: Subculture one or more of the early adenine pathway mutants and HB0 on a YED plate.

2. 2nd Day: Use sterile toothpicks to make all possible mating mixtures on the subculture plate.
Because you can't select for the diploid, after three hours use a microscope to check to be sure mating was efficient.
It might also help to use about twice as much of the strain carrying ade2 as of the wild-type strain (HB0).
Incubate the plate overnight.

3. 3rd Day: Streak the mixtures out on YED to isolate single colonies.

4. 8th Day: Pick 20 to 30 of the largest colonies and spot them onto YEKAC sporulation medium. Incubate the plate at least five days at room temperature.
( Teacher Tips )

Technical Tip: You can cross the adeX strain that you have isolated with more known mutants to further characterize its identity. 5. 13th Day: Make wet mount slides of the spots on the sporulation plate and examine them using a microscope until you find one that contains asci.
Digest these asci with snail enzyme and streak the spores out for single colonies on YED medium. (See Analysis of Free Ascospores)

6. 15th Day: Pick 20-30 cream-colored colonies and plate them in a grid pattern on YED.
Incubate the plate overnight.

7. 16th Day: Use toothpicks or replica plating equipment to make a replica of the YED plate on MV medium.
Incubate the plate overnight.

8. 17th Day: Record and analyze your results. Which of the four expected spore types have you been able to identify so far?
Set up a test cross plate that crosses HA2 and HB2 with each of the possible ade2 adeX and adeX strains that you have identified. Incubate the plate overnight.

9. 18th Day: Use sterile toothpicks to make all possible crosses.
Incubate the plate overnight.

10. 19th Day: Record and analyze your results.

Teacher Tips

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