Skip to main content
. 2017 Feb 28;206(1):497–512. doi: 10.1534/genetics.117.199703

Figure 5.

Figure 5

RAD54 functions after NDT80 induction to promote meiotic progression and the formation of viable spores. (A) MMS sensitivity. WT (NH144), rad54Δ (NH2136), RAD54-IN (NH2319), NDT80-IN (NH2127), NDT80-IN rad54Δ (NH2126), and NDT80-IN RAD54-IN (NH2185) diploids were grown to log phase in YPD. For one set of strains, ED was added to a final concentration of 1 μM for 2 hr prior to plating. Tenfold serial dilutions were spotted onto YP plates containing either 2% glu or gal with or without 0.04% MMS as indicated. (B) Meiotic progression in NDT80 diploids. WT, rad54∆, and RAD54-IN diploids were transferred to Spo medium and incubated at 30°. After 3 hr, the RAD54-IN culture was split with a final concentration of 1 μM ED added to one of the RAD54-IN cultures (indicated by black arrow). Meiotic progression was assayed using fluorescent microscopy of fixed DAPI-stained nuclei to determine the percentage of MI and MII cells. Two hundred cells were counted for each strain at each time point. The average values from three biological replicates are shown. Error bars indicate the SDs. (C) Meiotic progression in NDT80-IN diploids. ED was added at 7 hr (black arrow) and progression was monitored as in (B). (D) Spore viability. ED was added to 1 μM at the indicated timepoints. Lines indicate the P-values using a χ2 test. Average values are shown with error bars indicating the SDs. For the NDT80-IN strains, two independently constructed diploids using different SK1 parents exhibited similar results and therefore the data were combined. For each strain, the number of biological replicates: number of tetrads is: NH144 (3:120), (4:162), NH2319 −ED (7:185), NH2319 +ED at 3 hr (7:172), NH2127 + NH2232 (14:524), NH2126 + NH2240 (10:423), and NH2185 + NH2245 (10:318). (E) Distribution of viable spores in tetrads for WT, rad54∆, and RAD54-IN without and with ED from the dissections presented in (D). (F) Induction of Rad54 and Ndt80 in NDT80-IN diploids. The black arrows indicate addition of ED at 7 hr after transfer to Spo medium. This experiment was performed three times with similar results. ED, β-estradiol; gal, galactose; glu, glucose; MI, binucleate; MII, tetranucleate; MMS, methyl methanesulfonate; Spo, sporulation medium; WT, wild type.