7 Genetic Analyses

7.1 Overviews with Examples

There are numerous approaches for the isolation and characterization of mutations in yeast.
Generally, a haploid strain is treated with a mutagen, such as ethylmethanesulfonate, and the
desired mutants are detected by any one of a number of procedures. For example, if Yfg-
(Your Favorite Gene) represents an auxotrophic requirement, such as arginine, or
temperature-sensitive mutants unable to grow at 37°C, the mutants could be scored by replica
plating. Once identified, the Yfg- mutants could be analyzed by a variety of genetic and
molecular methods. Three major methods, complementation, meiotic analysis and molecular
cloning are illustrated in Figure 7.1.

Genetic complementation is carried out by crossing the Yfg- MATa mutant to each of the
tester strains MATα yfg1, MATα yfg2, etc., as well as the normal control strain MATα .
These yfg1, yfg2, etc., are previously defined mutations causing the same phenotype. The
diploid crosses are isolated and the Yfg trait is scored. The Yfg+ phenotype in the
heterozygous control cross establishes that the Yfg- mutation is recessive. The Yfg- phenotype
in MATα yfg1 cross, and the Yfg+ phenotype in the MATα yfg2, MATα yfg3, etc., crosses
reveals that the original Yfg- mutant contains a yfg1 mutation.

Meiotic analysis can be used to determine if a mutation is an alteration at a single genetic

locus and to determine genetic linkage of the mutation both to its centromere and to other
markers in the cross. As illustrated in Figure 7.1, the MATa yfg1 mutant is crossed to a normal
MATα strain. The diploid is isolated and sporulated. Typically, sporulated cultures contain
the desired asci with four spores, as well as unsporulated diploid cells and rare asci with less
than four spores. The sporulated culture is treated with snail extract which contains an enzyme
that dissolves the ascus sac, but leaves the four spores of each tetrad adhering to each other. A
portion of the treated sporulated culture is gently transferred to the surface of a petri plate or
an agar slab. The four spores of each cluster are separated with a microneedle controlled by a
micromanipulator. After separation of the desired number of tetrads, the ascospores are
allowed to germinate and form colonies on complete medium. The haploid segregants can
then be scored for the Yfg+ and Yfg- phenotypes. Because the four spores from each tetrad are
the product of a single meiotic event, a 2:2 segregation of the Yfg+:Yfg- phenotypes is
indicative of a single gene. If other markers are present in the cross, genetic linkage of the
yfg1 mutation to the other markers or to the centromere of its chromosome could be revealed
from the segregation patterns.

The molecular characterization of the yfg1 mutation can be carried out by cloning the wild-
type YFG1+ gene by complementation, as illustrated in Figure 7.1 and described below
(Section 11.1 Cloning by Complementation).