Biology Reference
In-Depth Information
FIGURE 22.3
Yeast tetrad dissection and spore viability analysis. (A) Diamond-shaped tetrad (arrow)
consisting of four haploid spores encapsulated by the ascus. (B) Cells heterozygous for a
TUB2 mutation linked to uracil prototrophy (tub2-URA3) were sporulated, Zymolyase-
treated, and spread across a dissection plate (asterisk). Four tetrads (1-4) were dissected
and the individual spores placed in positions A-D. All spores grew on YPD demonstrating the
mutation is nonlethal. Replica-plating onto SC-Ura revealed two spores contained the
mutated
tub2-URA3
allele (growth) and two contained the wild-type allele (no growth).
Assessing spore viability in independently created diploid mutants or elevated chro-
mosome missegregation rates in the surviving mutant spores can help discriminate
between these possibilities.
22.3.1
Sporulation
Grow yeast strains overnight on YPD agar plates and then transfer onto sporulation
plates as a patch using a sterile toothpick. Verify sporulation progress after 3-4 days
by picking a small amount of cells off the plate and scoring for the presence of asci.
The appearance of dyads/triads is usually a positive indicator of sporulation progress.
Formation of mature tetrads can take approximately a week depending on the genetic
background of the yeast strain. Proceed with dissection when sufficient tetrads have
formed.
Note I
: Dissection is facilitated when sporulation efficiency is
20%. Dissection
is possible, albeit more challenging, when tetrad frequency is lower.
Note II
: Tetrads can be stored at 4
C for an extended period of time.
>
22.3.2
Microdissection of haploid spores
Each complete ascus encapsulates four spores in what typically appears as a pyra-
midal or diamond-shaped structure (
Fig. 22.3
A). To isolate spores, the ascus is
digested with Zymolyase. Pick up a small amount of sporulated culture using a tooth-
pick or pipette tip and resuspend in 40
m
l of filtered 0.8 M KCl, 25 mM Tris, pH 7.5
buffer, containing 0.4 mg/ml Zymolyase. Incubate 10 min in a 37
C waterbath and
then dilute with 400
m
l YPD to inhibit the reaction. Mark a line through the center of
