The four meiotic products of a diploid yeast cell form into spores in a structure called an ascus. If the spores can be released from the ascus, then each spore can be grown as a pure haploid culture and its phenotype and genotype can be determined. Clearly, this provides much more information than one can obtain by letting the spores mate in the ascus. The ascus wall can be removed with an enzyme preparation from snails, glusulase, which is available from Sigma Biochemical Co. Then the spores can be isolated by streaking them out for single colonies. Examination of Table I shows that 75 percent of the spores should be red, adenine-requiring, and 25 percent should be white, adenine-independent.
Experiment: In this experiment you will free the spores from asci produced by the diploid (HA1 X HB2). You will then grow the spores into individual colonies and analyze each colony's phenotype and genotype.
Genotypes of Spores Expected
From the Cross:
a ade1 ADE2 X à ADE1 ade2
Mating type a: | ade1 ADE | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 |
Mating type à: | ade1 ADE | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 |
Each of the eight spore genotypes is expected to occur with the same frequency.
Phenotypes of Spores Expected From the
Cross:
75% pink and 25% cream-colored
Pink phenotype produced by ade1 ADE2 ADE1 ade2 ade1 ade2 in both mating types.
Cream-colored phenotype produced by ADE1 ADE2 in both mating types.
1st - 8th
Day: 30 min Getting Ready
9th Day: 50 min Digestion of
Asci, Plating
Cells and
11th Day: 20 min Counting Colonies
11th Day: 10 min Analysis of Cream-colored
15th Day: 30 min Colonies
Optional genotype analysis
15th Day: 20 min Subculture of Ascospore Colonies and Tester Strains
16th Day: 10 min Mating of Strains
17th Day: 50 min Observation and Plating of Mating Mixtures
18th Day: 50 min Observation of
MV Plates and
Analysis of Data
Materials:
Time Line: 1st Day-8th Day: 30 min
You may skip this procedure if you have asci from a previous work such as the Yeast Life Cycle experiment. ( See The Yeast Life Cycle for more information on mating and sporulating yeast)
1. Subculture the parent strains, HA1 and
HB2, overnight on a YED plate. Materials:
Time Line: 9th Day: 50 min
2. Pick a small amount of the sporulation
mixture with a sterile toothpick or an
inoculating loop and stir it into the
drop until the cells are suspended.
It should be slightly cloudy. (Alternatively, you
can do the above procedure in a small sterile test
tube.)
3. After 30 to 45 minutes examine a
small amount of the suspension using a
microscope.
Time Line: 11th Day: 20 min
2. Compare this result with the expected
phenotype frequency in Table I.
Materials:
Isolated cream-colored colonies
YEKAC plate
Sterile toothpicks
Time Line: 11th Day: 10 min
1. Transfer 5 - 10 isolated cream-colored
colonies to a YEKAC plate.
Time Line: 15th Day: 30 min
You can use the following formula to
determine the haploid fraction of your
sample.
Haploid fraction = # of haploid colonies /
total # of sample colonies
Teacher tips
Time Line:
15th Day: Subculturing Ascospore
Colonies and Tester Strains 20 min
Materials:
2. Also, make one-centimeter streaks of
the four tester strains across the top of
each YED plate.
Teacher tips
Technical Tip:
Time Shifting: Yeast cells follow a
clock that depends on temperature, so
you can easily control how fast they
grow and develop. If you can't look at
the mating mixture after three or four
hours, you or a friend (ask your teacher)
can put the plate in the refrigerator until
you have time.
Another way to slow the growth of the
yeast, is to put the plate in the
refrigerator as soon as you mix the cells
and then take it out four or five hours
before looking at the cells. If you leave
the plate in the refrigerator for one day,
then you need to add another day to
your experiment.
Mating of Strains
1. Place a spot of each of the four tester
strains beside a different spot of each
strain to make 32 pairs of spots.
Observation and Plating of Mating
Mixtures
1. Using a fresh toothpick for each sample
pick from each mating mixture of red
spore colonies and spot it on a MV
plate.
Use the same pattern to make it easy to keep track
of which spot is which.
This entire test can be done by replica
plating the plate with the mixtures to a plate
of MV.
Teacher tips
Observation of MV Plates and Analysis of
Data
1. Make a table of the test results
indicating growth or no growth.
Normally, each red spore strain should mate with
two of the testers, those of the opposite mating
type. The red spore strain should complement
with one of the testers it mates with unless it has
both the ade1 and ade2 recessive alleles.
Remember that the red spore strain being tested
carries the dominant allele of the mutation that it
complements, and has the opposite mating type to
the one it mates with.
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2. Use sterile toothpicks to transfer a
small amount of each parent strain to
separate spots close to each other on
the plate.
Stir the parent strains together using a third sterile
toothpick and then incubate the plate overnight.
3. Purify the diploid strain by transferring
a small amount of the mating mix to a
MV plate.
Incubate the plate overnight.
4. Presporulate the diploids by
transferring a small amount of the
diploid culture from the MV plate to a
fresh YED plate.
Incubate the plate overnight.
5. To sporulate the diploid cells transfer
several streaks of cells from the
presporulation plate to a YEKAC
plate.
Incubate the plate for 3-5 days.
Asci from (HA1XHB2)
Snail enzyme preparation
Sterile eye dropper
Waxed paper or small sterile test
tube
Inoculating loop or sterile toothpicks
Petri plates of YED medium
1. Take a piece of waxed paper from a
clean roll without touching the inside
surface and place it on the bench with
the inside surface up.
Place a drop of snail enzyme on it and cover it
with half of a sterile petri plate or a sterile beaker.
The individual spores should look like shiny
spheres.
If so, then streak out some of the suspension on
YED plates to isolate single colonies.(Teacher Tips)
1. Examine the isolated colonies and
count the number of red ones and the
number of cream-colored ones.
Incubate the plate for 4 to 5 days.
1. Make a wet mount slide of each
sample and use a microscope to look
for asci.
Draw and label a diagram of the plate showing
which colonies formed asci.
2. Count the number of haploid colonies
in your sample. Diploid cells sporulate
and form asci on YEKAC and haploid
cells do not.
Analysis of Genotypes
16th Day: Mating of Strains
20 min
17th Day: Observation and Plating of
Mating Mixtures
50 min
18thDay: Observation of MV Plates
and Analysis of Data
50 min
1. Use sterile toothpicks to pick a sample
of the isolated colonies for testing.
(Refer to your previous data on cream-colored colonies so that you pick only
haploid colonies)
Make streaks about one centimeter long in a
column down the side on a YED plate.
Place eight streaks in each column.
Mark the plates so you know which are red and
which are cream-colored. It will be simpler if the
red and cream-colored colonies are on separate
plates.
Time Line: 16th Day: 20 min
2. Then mix each pair of spots to make a
mating mixture.
3. Incubate the mixtures from red spore
colonies.
4. Make wet mount slides and examine the
mating mixtures from cream-colored
spore colonies under the microscope
after 3 to 6 hours.
The mixtures showing the characteristic peanut
and cloverleaf shaped zygotes are mating.
5. Make a diagram of your cream-colored
spore colony plate and label it showing
which mating mixtures are mating.
Time Line: 17th Day:50 min
2. Also transfer the red haploid strains
onto MV to see if the growth difference
between ade1 and ade2 holds for these
strains.
Time Line: 18th Day: 50 min
2. To determine the mating type of the
ade1 ade2 red spore strains (they do
not complement with any of the
testers) you will have to repeat the
microscopic examination mating test
that you used for the cream-colored
haploids.
Last updated Wednesday, 04-Dec-2002 14:59:16 CST