ade2 adeX, then the diploid will
still be cream-colored and
adenine-dependent because both genes are
homozygous for recessive alleles.
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.
Materials:
ade2 strains. (See Spontaneous Mutation:
Isolation of White Mutants and Ultraviolet
Lethality and Mutation in Yeast)
1. 1st Day: Set up fresh cultures of the
mutants by spotting them on a YED
plate.
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 strains in a
column down one side of the same plate.
Incubate overnight.
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