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Fig. 2. Assay for zygote formation. (A) Mating pair formation. Cultures of opposite mating type of mdy2 mutant (MATa and MAT{alpha}) and wild-type strains were grown to log phase. Equal numbers of cells (3x106) of each mating type were mixed with each other. The cell pellets were then transferred onto a NC membrane on an YPD plate and incubated at 30°C. Samples were taken after various times and the numbers of mating pairs in samples of at least 800 cells were counted. The percentage of mating pairs at each time point is shown. (B) Zygote formation. The same samples were used as in A and the nuclei were stained with DAPI, and the numbers of zygotes was counted. The percentage zygote formation is shown. (C) Classification of mating pairs. Cultures of mdy2 mutant (MATa and MAT{alpha}) and wild-type strains of opposite mating type were grown to log phase. Equal numbers (3x106 cells) of each mating type were mixed, pelleted, transferred onto a NC membrane on an YPD plate and incubated at 30°C. Samples were taken after 3.5 hours incubation and stained with DAPI to identify zygotes and follow the course of karyogamy. The percentage of cells in the different stages of zygote formation was determined in samples containing at least 200 zygotes.





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