Structure–Function Analysis of Yeast Tubulin - Methods in Cell Biology - page 355

an YPD or appropriate selection plate. While holding the plate nearly vertical, place

l of Zymolyase-treated cells at the top and allow the drop to run across the
surface along the line. Let the plate dry

20 m

10 min, then, using a dissection microscope
with attached micromanipulator, move a complete tetrad to the first of four clearly
marked locations, separate the spores, and place one spore in each of the three
remaining positions ( Fig. 22.3 B). Repeat until a sufficient number of tetrads have
been dissected. Micromanipulators for yeast dissection typically have well-marked
grids clearly denoting the four positions for depositing the spores. Each plate can
accommodate

10-20 tetrads. Incubate at 30 C until the spores form colonies.
For a description of the microdissection technique, see Treco and Winston (2008) .

Note I : Handle Zymolyase-treated cells gently. Avoid vortexing or vigorous
pipetting.
Note II : The concentration of Zymolyase and duration of digestion required may
vary by strain, thus optimization is recommended for best results.
Note III : For statistical significance, we recommend assessing the viability of at
least 25 tetrads per mutation.

22.3.3 Genotype analysis of haploid spores
To determine the effects of a specific mutation, the phenotype of the haploid spores
must be correlated with genotype. In the protocols presented above, the mutated

a -or

b -tubulin is tightly linked to tryptophan or uracil prototrophy, respectively, and the

dissection plate should be replica-plated onto SC-Trp or SC-Ura selection media to
determine whether spores contain the mutated tubulin gene.
Common replica-plating devices consist of a ring that is used to spread a sterile
velvet towel over a Petri dish-sized circular pedestal. Gently press the dissection
plate onto the spread velvet towel. Subsequently, press the selection plate onto
the same towel and mark the orientation with respect to the dissection plate. As
an example, spores from a heterozygous -tubulin mutant diploid that grow on

SC-Ura plates inherited the mutant allele whereas spores that fail to grow contain
the wild-type allele ( Fig. 22.3 B).

b

Note : If haploid spores containing the mutant allele are viable, they should be
saved for analysis with respect to the wild-type sister spores.

22.3.4 Mating-type analysis
Interpretation of tetrad dissection results depends on the fidelity of meiotic segrega-
tion and the reliable separation of spores from complete tetrads. Tetrad dissection can
be verified by following 2

þ

segregation of independent heterozygous markers
and/or haploid mating type. Mating-type tester strains are prototrophic for common
laboratory markers and auxotrophic for an uncommon marker. Upon conjugation
with laboratory strains, the auxotrophic markers in both haploid strains are compli-
mented and the resultant diploid can grow on minimal media.

/2