The red pigment in ade1 and ade2 strains results from oxidative metabolism of the intermediate, called AIR, in the AMP synthesis pathway. If AIR is not produced or if it is not acted on by oxidative metabolism the red pigment does not form.
Additional mutations in genes that control the earlier steps in the AMP pathway -- the genes required for the synthesis of the precursors of AIR -- will block the synthesis of AIR and prevent ade1 and ade2 mutants from developing the red color. (See Figure 1: Pathway for synthesis of AMP)
Close examination reveals that some of the "white" mutations arising in red strains are whiter that others. In contrast to those that are the cream color of normal yeast, some are very nearly pure white. These pure white mutants also form smaller colonies and are, therefore, called"petite colony" mutants, or just "petites" (Dujon 1981). Rather than being blocked in the adenine pathway like the cream-colored early AMP pathway mutants, they fail to make the red pigment because they lack oxidative metabolism (respiration). Most of them have some kind of defect in their mitochondria. Mitochondria are complicated organelles necessary for respiration. There are many genes that can mutate to produce the petite phenotype so petite mutants are very common. The petites get energy by fermenting certain types of sugars. It is possible to identify petites using a special type of growth medium containing only nonfermentable sugars.
Finally there is a small chance that an ade1 or ade2 mutation may revert back to its normal DNA sequence. Such an event would restore the wild-type cream-colored adenine -independent phenotype.
These three types of mutations occur spontaneously during the growth of a colony. Whenever a mutation occurs, all the progeny of that mutant cell will also be mutant. That is, mutants occur as clones in a colony. When the colony is one that normally turns red and the mutants are white or cream, they appear as white or cream spots in the red colony. We will describe the experiment using the a ade2 and à ade2 strains, but it can be adapted for any of the haploid red adenine strains.
Experiment:
In this experiment you will subculture
ade2 strains of both mating types. After two
weeks you will pick spontaneous "white"
mutant cells from the cultures and identify
them as early AMP pathway mutants,
adenine-independent mutants or petite
mutants.
Time Line:
1st Day: 10 min Streak red
strains for
single colonies
10th Day: 15 min Streak "white"
mutants for
single colonies
12th Day: 15 min Pick "white"
mutants
15th Day: 15 min Replica plate
16th Day: 30 min Record and
analyze results
Materials:
MV medium Petite medium
Growth Growth
(adenine independent (not a petite)
revertant)
No growth No growth
2. 10th Day: Using the pointed end of a
toothpick pick "white" spots from
colonies, try to get as few red cells as
possible.
Use a fresh sterile toothpick for each different
sample.
Streak the samples on separate YED plates and
then streak each sample out to isolate single
colonies. Use the flat ends of fresh sterile
toothpicks after the first streak.
Incubate the plate for two to three days.
3. 12th Day:Pick several "white" colonies
from each sample plate and make short
streaks on a YED plate.
Use a different sterile toothpick for each colony.
Incubate the plate overnight.
4. 15th Day:Replica plate the samples
from the YED plate to YED, MV, and
PETITE media.
Incubate the plate overnight.
5. 16th Day:Record and analyze your
results.(Teacher Tip )
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contents
Figure
Last updated Wednesday, 04-Dec-2002 14:58:28 CST