Pds5 is necessary for meiotic recombination. (A) A physical assay of DSB formation and processing at the YCR047c/YCR048w locus. Yeast cells were induced to undergo synchronous meiosis, and DNA samples were extracted at the indicated time points. DSBs were detected by Southern blotting. Two prominent DSB sites at this location are depicted (DSB1 and DSB2). (B) Quantitative measurement of DSB formation in wild-type (WT) and P_CLB2PDS5 cells. The ratio of the intensity of DSB1 to that of the parental band is shown on the y axis. (C) Rad51 focus formation during meiosis. Yeast cells were induced to enter meiosis as in A. Yeast nuclear spreads were prepared for immunofluorescence. Rad51 was detected with an anti-Rad51 antibody and microtubules with an anti–α-tubulin antibody. Red, DNA stained by DAPI; green, Rad51; blue, microtubules. Bar, 4 µm. (D and E) Quantification of Rad51 foci in cells with aster microtubules and short bipolar spindles is shown. Note that Rad51 foci persist in P_CLB2PDS5 cells with a bipolar spindle. 20 cells from each strain were scored. (F) Cell viability was assayed by return to growth. (E) Yeast cells were induced to enter meiosis, and aliquots were withdrawn at the indicated times and plated on both YPD and Arg minus plates. Colony-forming units are defined as 1 at time 0. (G) Meiotic recombination at the ARG4 locus. Two heteroalleles of ARG4 (arg4-Bgl and arg4-Nsp) are present in the strains assayed. Recombination between the heteroalleles generates a wild-type ARG4 allele, which is detected as an Arg-positive colony. The ratio of colonies formed on Arg minus plates to those on YPD plates determines the recombination rate.