Chromosome spreads and immunofluorescence were performed as described by Sym et al. (1993) (link). Except as noted, spreads were prepared 15 h after transfer to sporulation medium. Anti-Red1 and anti-Hop1 antibodies were used at a 1:100 dilution. The rabbit anti-Zip1 antibody was diluted 1:100, and the mouse anti-Zip1 antibody (from P. Moens) was diluted 1:1,000. The mouse anti-nucleolus antibody (monoclonal antibody 2.3b; from C. Copeland, H. Friedman, J. Woolford, and M. Snyder) was diluted 1:5. The rabbit anti-Histone2B antibody (from A. Carmen and M. Grunstein) was used at 1:500. Rabbit antibodies were detected with goat anti–rabbit antibodies conjugated to Texas red (Jackson ImmunoResearch Labs, West Grove, PA) diluted 1:200. Mouse antibodies were detected with goat anti–mouse antibodies conjugated to FITC (Jackson ImmunoResearch) diluted 1:200. After antibody staining, spreads were stained with 1 μg/ml 4′-6-diamidino-2-phenylindole (DAPI) to visualize the DNA. A Leitz DMRB microscope equipped with fluorescence and a PL APO 100× objective was used to observe antibody-stained preparations. Images were captured using a Photometrics Imagepoint CCD camera.
Time course analysis of meiotic prophase progression was carried out as follows. Strain BR2495 was grown to saturation overnight at 30°C in rich medium (YPAD; Sherman et al. [1986]). The culture was diluted 1:1 into fresh YPAD and incubated with shaking for 8 h at 30°C. Cells from 10 ml of culture were then washed once with H2O and resuspended in 100 ml of 2% KAc and placed in a one-liter flask and shaken at 250 rpm at 30°C. At 1-h intervals, starting 10 h after transfer to 2% KAc, 10 ml of culture were removed and used to prepare spread chromosomes on each of the four slides. The spreading procedure required 40 min after the removal of cells from KAc. The times given in the text and figure legends indicate when the spreading procedure was initiated. At each time point, two slides were used for double labeling Red1 and Zip1; one was used for double labeling Red1 and Hop1, and one was used for double labeling Hop1 and Zip1.
DNase I digestion of meiotic spreads was carried out as follows. Wildtype spreads from BR2495 were prepared 15 h after transfer to KAc. Spreads were then incubated for 1 h at 37°C in 100 U/ml DNase I (Boehringer Mannheim ) in 50 mM Tris (pH 7.5), 10 mM MnCl2, and 50 μg/ml BSA. Control spreads were incubated in buffer lacking DNase I. Subsequent antibody incubations were performed as described above.
Time course analysis of meiotic prophase progression was carried out as follows. Strain BR2495 was grown to saturation overnight at 30°C in rich medium (YPAD; Sherman et al. [1986]). The culture was diluted 1:1 into fresh YPAD and incubated with shaking for 8 h at 30°C. Cells from 10 ml of culture were then washed once with H2O and resuspended in 100 ml of 2% KAc and placed in a one-liter flask and shaken at 250 rpm at 30°C. At 1-h intervals, starting 10 h after transfer to 2% KAc, 10 ml of culture were removed and used to prepare spread chromosomes on each of the four slides. The spreading procedure required 40 min after the removal of cells from KAc. The times given in the text and figure legends indicate when the spreading procedure was initiated. At each time point, two slides were used for double labeling Red1 and Zip1; one was used for double labeling Red1 and Hop1, and one was used for double labeling Hop1 and Zip1.
DNase I digestion of meiotic spreads was carried out as follows. Wildtype spreads from BR2495 were prepared 15 h after transfer to KAc. Spreads were then incubated for 1 h at 37°C in 100 U/ml DNase I (
