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 à> ade2 adeX, then the diploid will still be cream-colored and adenine-dependent because both genes are homozygous for recessive alleles.
ade2 adeX x ade2 adeX
ade2/ade2 adeX/ adeX
On the other hand, if you cross it to a mutant that is à> ade2 ADEX adeY, where adeY is a mutation in a different gene than adeX, then the two mutant strains will complement each other.
ade2 adeX ADEY ade2 ADEX adeY
(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
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 à> 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.
1. 1st Day: Set up fresh cultures of the
mutants by spotting them on a YED
A convenient arrangement is to put up to eight
mating-type a strains in a row across the top of a
plate and up to eight mating-type à> strains in a
column down one side of the same plate.
2. 2nd Day: At the intersections of the
rows and columns, make all the
possible mating mixtures.
Be sure to take a new toothpick after each mixture.
Incubate the plates overnight.
3. 3rd Day:
Examine the mating mixtures for development of red diploid clones in the cream-colored mixtures.
Group your unknown mutants into complementation groups based on whether or not they complement each other.
All strains that fail to complement are placed in the same group. (In these crosses if the mating mixture stays cream-colored the unknowns have failed to complement each other)
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Last updated Friday August 19 2005