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?
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Last updated Friday August 19 2005