Figure 5.

pCUP-IME1 system can be combined with the pGAL-NDT80 system to improve synchrony of sporulation. (A) Scheme of experimental setup. The diploid cells harboring IME1 fused to CUP promoter (FW2444), NDT80 expressed from the GAL promoter together with Gal4 fused to the estrogen receptor (GAL4.ER pGAL-NDT80) (FW1541) or a strain expressing both (pCUP-IME1 and GAL4.ER pGAL-NDT80) (FW2795) were grown in YPD overnight. Cells harboring GAL4.ER GAL-NDT80 (FW1541) were transferred to presporulation medium (BYTA). Subsequently, cells were pelleted by centrifugation, washed with sterile water and resuspended to a final OD₆₀₀ of 2.5 in SPO; 50 µM copper (II) sulfate was added 2 hr after the cells were transferred to SPO, and 1 µM β-estradiol was added 6 hr after transfer to SPO. (B) Kinetics of meiotic divisions in strains, using procedures described in (A). Samples were taken at the indicated time points, fixed in ethanol, nuclei were stained with DAPI, and DAPI masses were counted. Cells that harbored two, three, or four DAPI masses were classified as cells undergoing meiosis I or meiosis II (% MI + MII). For each time point, at least 200 cells were counted. We also computed the time or period taken for 75% of the cells to complete meiotic divisions (see Materials and Methods for details). This number is displayed in brackets next to the legend, and represents the mean number of hours followed by the SEM of three independent experiments. (C) Similar to B except that the percentages of bi- (left panel), or tri- and tetra-nucleate (right panel) cells (n = 200 cells) of strains described in (A) were determined. *, time of IME1 induction; **, time of NDT80 induction.