Part D:Mutation Experiments

Spontaneous Mutation: Isolating "White" Mutants

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 ade2 strains, but it can be adapted for any of the haploid red adenine strains.

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: