Figure 4

chl1 cells show reduced association of the cohesin subunit Scc1p with CEN3 and Chl1p is required to maintain cohesion after S-phase. A. ChIP assay for detecting association of Scc1p at centromeres in US3335 (wild-type), US3335Dchl1 (chl1) and US3335Δsir3 (sir3) cells. Cells from all the three strains were grown to mid-log phase and fixed in formaldehyde for 2 h before chromatin isolation. + refers to "plus antibody", - refers to "no antibody" and SM refers to "starting material". PCR with CEN3 specific primers gave a 249 bp product. B. Quantification of the enrichment of the CEN3 PCR product over control levels in chl1 and sir3 mutants, relative to that in the wild-type. Averages and standard deviations are from three independent experiments performed as described above. C and D. Chl1p is required for the maintenance of cohesion in both S- and G2 phases. US3329 (wild-type), US3329Δchl1 (chl1) and SL25 (scc1-73) cells were arrested by alpha-factor in G1 at 25°C for 2 hours, washed and released in fresh YEPD containing nocodazole (15 μg/ml). After a further growth at 25°C for twenty minutes, the cultures were shifted to 35°C (0 min). CENV-GFP dot separation was monitored for 150 minutes after the temperature shift. (C) DNA content of the cells measured by flow cytometry. Arrows indicate G1 and G2 DNA contents. (D) Graph represents percentage of cells with 2 GFP signals (separated dots). 100-150 cells were analyzed in each case.