In the wild the haploid stage is usually brief. Yeast spores often mate shortly after they germinate, sometimes while they are still within the ascus.
Consider the cross:
a ade1 ADE2 X ` ADE1 ade2
Each complete, four-spored ascus produced from this cross should contain two spores of each mating type. The ade1 and ade2 alleles and their normal counterparts should segregate independently from each other and from the mating type alleles. Table I summarizes the types of spores expected, and their expected frequencies.
Genotypes of Spores Expected From the Cross: a ade1 ADE2 X ` ADE1 ade2
| Mating-type a spores | Mating-type ` spores |
|---|---|
| ade1 ADE2 | ade1 ADE2 |
| ADE1 ade2 | ADE1 ade2 |
| ade1 ade2 | ade1 ade2 |
| ADE1 ADE2 | ADE1 ADE2 |
When placed on growth medium, the spores in each ascus should mate, two-by-two, forming a random population of diploids.
Table II summarizes the distribution of
diploids expected from random matings;
however, in this case each colony will grow
from two diploid cells. Table III shows the
phenotypes and distribution of spores in the
asci expected from the cross. From these
genotypes you can predict the diploids that
would be expected if all the possible
matings occurred (see Table IV). There
should be three types of colonies:
1) those in
which both diploids are positive (cream-colored, adenine-independent),
2) those in
which both diploids are negative (red,
adenine- requiring), and
3) those in which
one diploid of each type occurs. The colony
types should occur in the ratio of 10:1:7.
This analysis is complicated by two factors. First, each colony that grows from an ascus in which two pairs of spores mate with each other will contain two different kinds of diploid cells. Second, in some asci only one, two, or three spores will have developed. Therefore some colonies will contain only haploid cells, some will contain only diploids, and others will contain a mixture of haploids and diploids.
Clearly, this is not a very convenient experiment for studying segregation, but by analyzing some colonies obtained this way, you can see how the life cycle progresses if the cells are left to do their own thing.
Experiment:
In this experiment you will
1) streak out
some intact asci
2) pick colonies and
3)
analyze the colonies for color and adenine
requirement.
Genotypes of Diploids Expected From All
Possible Matings of Spores from the Cross:
a ade1 ADE2 X ` ADE1 ade2
Mating type a spores:
| Mating type ` | ade1 ADE2 | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 | |
|---|---|---|---|---|---|
| ade1 ADE2 | ade1 ADE2 | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 | |
| ade1 ADE2 | ade1 ADE2 | ade1 ADE2 | ade1 ADE2 | ||
| (-) | (+) | (-) | (+) | ||
| ADE1 ade2 | ade1 ADE2 | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 | |
| ADE1 ade2 | ADE1 ade2 | ADE1 ade2 | ADE1 ade2 | ||
| (+) | (-) | (-) | (+) | ||
| ade1 ade2 | ade1 ADE2 | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 | |
| ade1 ade2 | ade1 ade2 | ade1 ade2 | ade1 ade2 | ||
| (-) | (-) | (-) | (+) | ||
| ADE1 ADE2 | ade1 ADE2 | ADE1 ade2 | ade1 ade2 | ADE1 ADE2 | |
| ADE1 ADE2 | ADE1 ADE2 | ADE1 ADE2 | ADE1 ADE2 | ||
| (+) | (+) | (+) | (+) |
Expected Asci From the Cross:
a ade1 ADE2 X ` ADE1 ade2
| a ade1 ADE2 | a ade1 ADE2 | a ade1 ade2 |
| a ade1 ADE2 | a ade1 ade2 | a ade1 ade2 |
| ` ADE1 ade2 | ` ADE1 ADE2 | ` ADE1 ADE2 |
| ` ADE1 ade2 | ` ADE1 ade2 | ` ADE1 ADE2 |
| 2:0 | 2:0/1:1 | 2:0 |
| 1 | 4 | 1 |
| a ade1 ADE2 | a ade1 ADE2 | a ade1 ade2 |
| ` ade1 ADE2 | ` ade1 ade2 | ` ade1 ade2 |
| a ADE1 ade2 | a ADE1 ADE2 | a ADE1 ADE2 |
| ` ADE1 ade2 | ` ADE1 ade2 | ` ADE1 ADE2 |
| 2:0/0:2 | 1:1/2:0 | 1:1/2:0 |
| 1 | 4 | 1 |
| ` ade1 ADE2 | ` ade1 ADE2 | ` ade1 ade2 |
| a ade1 ADE2 | a ade1 ade2 | a ade1 ade2 |
| ` ADE1 ade2 | ` ADE1 ADE2 | ` ADE1 ADE2 |
| a ADE1 ade2 | a ADE1 ade2 | a ADE1 ADE2 |
| 2:0/0:2 | 1:1/2:0 | 1:1/2:0 |
| 1 | 4 | 1 |
| ` ade1 ADE2 | ` ade1 ADE2 | ` ade1 ade2 |
| a ade1 ADE2 | a ade1 ade2 | a ade1 ade2 |
| a ADE1 ade2 | a ADE1 ADE2 | a ADE1 ADE2 |
| ` ADE1 ade2 | ` ADE1 ade2 | ` ADE1 ADE2 |
| 2:0/0:2 | 1:1/2:0 | 1:1/2:0 |
| 1 | 4 | 1 |
| a ade1 ADE2 | a ade1 ADE2 | a ade1 ade2 |
| ` ade1 ADE2 | ` ade1 ade2 | ` ade1 ade2 |
| ` ADE1 ade2 | ` ADE1 ADE2 | ` ADE1 ADE2 |
| a ADE1 ade2 a ADE1 ade2 | a ADE1 ADE2 | |
| 2:0/0:2 | 1:1/2:0 | 1:1/2:0 |
| 1 | 4 | 1 |
| ` ade1 ADE2 | ` ade1 ADE2 | ` ade1 ade2 |
| ` ade1 ADE2 | ` ade1 ade2 | ` ade1 ade2 |
| a ADE1 ade2 | a ADE1 ADE2 | a ADE1 ADE2 |
| a ADE1 ade2 | a ADE1 ade2 | a ADE1 ADE2 |
| 2:0 | 2:0/1:1 | 2:0 |
| 1 | 4 | 1 |
Table IV
Colony Phenotypes Expected From Matings of Spores Within Asci
1st Day: 15 min Streak out asci
4th Day: 30 min Record colony
color and plate
colonies onto
MV medium
5th Day: 15 min Record and analyze data
Materials:
Procedure: Time Line: 1st Day: 1. Streak out the sporulation mixture on YED medium to obtain isolated colonies.
Time Line: 4th Day:
1. Examine the isolated colonies for
cream-colored, red, and red/cream
phenotypes.
2. Compare the frequencies with those
predicted.
3. Pick several of each color and spot
them onto a MV plate to see if they
require adenine. Be sure to mark the
plate so you know the color of each
colony you test.
Time Line: 5th Day:
1. Examine the spots on the MV plate to
see if all the red colonies required
adenine and all the cream-colored ones
did not.
2. Compare the frequencies with those
predicted. Comments:
This experiment and its complicated analysis illustrates why
yeast geneticists isolate the spores from the asci and analyze
them as haploids.(Teacher
Tips)
You may be able to completely skip this procedure if you have a supply of asci from some previous work. (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.
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. Presporulate the diploids by transferring a small amount of the diploid culture to a fresh YED plate. Incubate the plate overnight.
4. 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.
Rather than having the whole class make the cross (HA1 X HB2) suggested for 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.
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Last updated Wednesday, 04-Dec-2002 14:59:16 CST