Rather than having the whole class make the cross (HA1 X HB2)
suggested in this experiment you may wish to have several
students perform the Getting Ready steps as a class
demonstration. This will offer the opportunity for the whole
class to review the steps of the yeast sexual life cycle. One
plate of asci will supply enough ascospores for the entire class
to do this spore analysis experiment.
Getting Ready:
1st Day - 8th Day: 30 min
It takes less than 10 minutes to perform each of the yeast
transfers in these Getting Ready steps. You will need to prepare
the agar plates in advance.
1. To avoid contamination it's usually best to limit student access to your master collection of yeast strains. You may wish to subculture the strains on one plate of YED and then use that plate as the source of strains for student work.
3. The diploid cells will be cream-colored because each cell has at least one functional copy of each adenine gene. (See Genetics of Baker's Yeast in this manual) HA1 (ade1 ADE2); HB2 (ADE1 ade2); HA1 X HB2 (ade1/ADE1 ADE2/ade2)
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This is sufficient to test whether the
overall segregation
frequency is as expected. (75% pink, 25% cream-colored) Chances
are, however, there will be a large excess of cream-colored,
adenine-independent colonies. This is because the sample will
include some unsporulated diploids. If the sporulation step
doesn't work well the cream-colored diploid colonies could
account for most of the isolated cream-colored colonies that you
count. Longer digestion in snail enzyme will kill some of the
diploids, but if you go too long it will kill spores, too. To
get a better estimation of the segregation frequency you must
determine what fraction of your isolated cream-colored colonies
are haploid.
Digestion of Asci, Plating Cells and Counting Colonies:
9th Day:
2. Toothpicks are sterile from the box. If you use an
inoculating loop you will need to supply a flame to sterilize
the loop each time it is used. (See Laboratory Methods,
Using Toothpicks and Inoculating Loops)
3. Use a sterile loop to transfer a small amount of the
suspension to a microscope slide. You may need to add a
small amount of water to the yeast suspension before you add
the coverslip. (See Laboratory Methods Using Toothpicks and
Inoculating Loops)
Five to six single colony isolation plates should be enough
for each class if the ascus digestion works well. You may wish
to have each student make an isolation plate to practice the
technique and to increase the number of haploid colonies
available for analysis. (See Laboratory Methods, Isolating
Single Colonies)
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Comment:
You can now go back to your original colony count data from Day
10. Multiply the original cream-colored colony count by the
haploid fraction that you just determined to get a more accurate
number of cream-colored haploid colonies.
"corrected" cream-colored haploid count = original cream-colored
count * haploid fraction
When you use the "corrected" cream-colored colony count and the
original pink colony count does your phenotype segregation
frequency equal the expected 75% pink and 25% cream-colored?
When you pool the class data does the phenotype segregation
frequency come closer to the expected frequency of 75% to 25% ?
If there is a reasonable number of red colonies, indicating that
the sporulation worked (remember that theoretically 75 percent
could be red), then it is instructive to determine the mating
type and adenine genotype of some of the spores.
Analysis of Cream-colored Colonies:
11th Day:
15th Day:
2. Haploid fraction
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Determining the genotypes of ascospore colonies:
The possible genotypes of colonies from the
digested
sporulation mixture are the parental diploid genotype and the
eight possible haploid genotypes in Table I. Any red colony must
be haploid, but the cream-colored ones could be either haploid or
diploid. The red spore strains can be tested for ade1 or ade2 at
the same time that they are tested for mating type. This can be
done by using the mating and complementation test using four
tester strains: a ade1, ` ade1, a ade2, and ` ade2.
Each red
spore strain is crossed with each of the four testers and then
tested for mating by complementation on MV. Neither the red
spore strain nor the tester strains can grow on MV. If they mate
and complement the resulting diploid will be able to grow on MV.
Each should mate and complement with no more than one tester; a
double ade1 ade2 mutant will not complement with either. Its
mating type, and the mating types of the cream-colored,
adenine-independent spores, can only $
in the mating mixture.
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Mating of Strains
2. Remember to use a fresh sterile toothpick for each of the
mating mixtures when you stir each mixture together.
3. The mixtures from red spore colonies will be transferred to
MV.
4. You may wish to use a video microscope to show the class asci
so they will know for sure what to look for in their slides.
5. This test determines the mating type. The cream color of the
haploid strains indicates the ADE1 ADE2 adenine independent
genotype. If you wish to confirm the genotype you may wish
to plate the haploid strains on MV medium.
Comments: This completes a total analysis of the
segregation of three
pairs of heterozygous markers. The results can be compared
statistically with the expected results by using the Chi-square
test. For this to be valid it is important that the sample of
spores not be biased for or against red colonies. The best way
is to test all the colonies that can be picked from each plate.
It may be useful to organize a group effort and pool the results.
1. The snail enzyme used in this experiment is Glusulase, a
-glucuronidase/sulfatase prepara$
of snails, Helix pomatia. A crude solution is adequate.
Sigma Chemical Co. is one possible supplier. It is usually
best to dilute the crude solution with sterile water. A one
2.to twenty dilution usually works well. (1 part glusulase in
19 parts sterile water) Because the activity of these crude
enzyme solutions may vary you may wish to experiment with
several different dilutions. Store the unused solutions in
the refrigerator.
1. Make a 2 to 3 centimeter streak from each
colony. You may
wish to number each source colony on the
bottom of the source
plate and then record those numbers on the
YEKAC plate. You
will then be able to go back to the source
plate when you
identify which cream-colored colonies are
haploid. (See The
Yeast Life Cycle section of more
information on sporulating
cells)
1. (See Laboratory Methods Microscopic
Examination of Yeast and
the Yeast Life Cycle section)
If the original sporulation frequency was low
it is possible
for an individual student to get 0 haploids in their sample of 5
to 10 cream-colored colonies.
:
1. Be sure to use a fresh sterile toothpick each time you start
working with a new strain.
Last updated Wednesday, 04-Dec-2002 15:09:54 CST