Part D:Mutation Experiments

Testing Early AMP Pathway Mutants for Allelism

There are five different genes in the earlier part of the adenine pathway in which mutations can occur and block pigment formation (ade4 through ade8). If you isolate a number of early AMP pathway mutants from red strains they will carry mutations in several different loci. Furthermore, if some of these mutants are of each mating type, it is possible to determine which mutations are in the same gene by doing complementation tests. If two mutations are in the same gene we say they are alleles, so this is called an allelism test. If you cross a mutant that is a ade2 adeX by one that is ade2 adeX, then the diploid will still be cream-colored and adenine-dependent because both genes are homozygous for recessive alleles.

Haploids crossed:
ade2 adeX x ade2 adeX

Diploid produced:
ade2/ade2 adeX/ adeX

On the other hand, if you cross it to a mutant that is ade2 ADEX adeY, where adeY is a mutation in a different gene than adeX, then the two mutant strains will complement each other.

Haploids crossed:
ade2 adeX ADEY ade2 ADEX adeY

Diploid produced:
(ade2/ade2 adeX/ADEX ADEY/adey)

The resulting diploid will be red and adenine requiring because it is homozygous for ade2 but heterozygous for the other adenine genes. Experiment:
In this investigation you will collect cream-colored mutants that carry the ade2 gene and then cross all of the a mating types in every combination with the mating types. You will use your data to group the cream-colored mutants into complementation groups. All of the members of a single complementation group are mutant in the same gene.

Time Line:

1st Day: 15 min Subculture mutant strains
2nd Day: 15 min Make mating mixtures
3rd Day: 30 min Record and analyze data