Biology Reference
In-Depth Information
With a single sterile velvet towel, replica-plate the colonies on a tetrad dissection
plate onto two YPD plates. Using clean velvets, now replica-plate a lawn of
MAT
a
tester cells onto the first YPD plate. Repeat with a lawn of
MAT
testers on the sec-
ond YPD plate. Incubate the YPD plates for 8-24 h at 30
C to allow conjugation,
then replica-plate each onto minimal media (YNB), and grow overnight. Only newly
formed diploids with complimented markers will grow on minimal media. Growth
after mating with
MAT
a tester cells indicates that the spore in the corresponding po-
sition is
MAT
a
testers denotes
a
MAT
a spore. A properly dissected, valid tetrad will generate two
MAT
a and two
MAT
haploid. Conversely, growth after mating with
MAT
a
a
spores. If this is not the case, the tetrad should not be considered for analysis.
a
Note
: To prepare mating-type tester lawns, spread
l saturated culture onto
YPD plates, incubate overnight at 30
C, and store in a sealed container at 4
Cup
to several months.
200
m
22.4
ASSESSING MICROTUBULE STABILITY BY DRUG
SENSITIVITY
Challenging yeast cells with the microtubule destabilizing drug benomyl is a com-
mon method to assess changes in microtubule stability
in vivo
. Benomyl supersen-
sitivity is indicative of less stable microtubules while improved resistance implies
increased stability. For example, the
-tubulin C354S substitution resulted in robust
benomyl resistance. The increased microtubule stability was corroborated by direct
visualization of green fluorescent protein (GFP)-labeled microtubule dynamics and
confirmed to be independent of any potential changes to benomyl binding and/or reg-
ulatory proteins by purifying the mutated tubulin and measuring microtubule dynam-
ics
in vitro
(
Gupta et al., 2002
). Overall, the benomyl plate assay is representative of
changes in microtubule stability
in vivo
.
b
22.4.1
Benomyl plate assay
Prepare 5-ml overnight cultures in YPD. The following day, dilute 1:50 in fresh me-
dia and grow to log phase (4-6 h). Measure optical density of the cultures at 600 nm
(OD600) and equalize cell concentration by adjusting volumes with YPD. In a 96-
well plate, prepare logarithmic serial dilutions of each culture in YPD. With a multi-
channel pipette or a “frogger,” spot 3
l of each dilution onto the assay plates. We
typically use plates with 0, 4, 6, 9, 12, 15, 18, and 21
m
g/ml benomyl. Incubate plates
at 24
C for 72 h. A larger dynamic readout is consistently obtained by conducting
this assay at 24
C rather than 30
C.
Drug sensitivity is scored as the concentration that inhibits cell growth. Spots
where high-density cultures were deposited often have minor but visible growth
at high drug concentrations. Growth inhibition becomes more obvious where low-
density cultures were spotted and individual colonies are dispersed.
m
