ADE2 ADEX.
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:
ADE2 ADEX )
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.
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Last updated Friday August 19 2005