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. 2007 Dec 13;27(1):111–121. doi: 10.1038/sj.emboj.7601955

Figure 1.

Figure 1

The loading machinery is required for sustained cohesin binding to chromatin in G1. Cells bearing thermosensitive mutations in genes encoding the cohesin-loading complex mis4 and ssl3 were arrested in G1 at permissive temperature by overexpressing a C-terminal Res1 fragment, under the control of the inducible nmt1 promoter (Ayte et al, 1995). Cells were then shifted to 37°C for 2 h to inactivate cohesin loading. (A) Rad21-HA chromatin association was monitored on chromosome spreads by immunofluorescence at 25°C and after shift to 37°C. DNA was stained with 4′-6-diamidino-2-phenylindole (DAPI). Scale bar, 5 μm. (B) Quantification of Rad21-HA fluorescence intensity. A total of 50–100 nuclei were analyzed for each sample. The error bars show the confidence interval of the mean with α=0.05. (C) DNA content analysis and septation index (SI) show that cells remained arrested in G1 throughout the course of the experiment.