Figure 6.—

Substantial silencing can occur at an HMRα locus without passage through S phase. (A) A diagram of the hybrid HMRα locus is shown. (B) Experimental outline. The scheme for this experiment is similar to that described in Figure 1, except that an HMRα sir3-8 strain (JRY32) was used. (C) DNA content. Samples for S phase arrest were withdrawn at the times indicated and their DNA content monitored by flow cytometry. (D) α1 expression at the HMRα locus. RNA was extracted at the indicated time points from both S phase-arrested and -released samples, subjected to RT–PCR, and quantified by real-time PCR. For either cell-cycle condition, the HMRα1 RNA level at the 0-hr time point, normalized to 18S rRNA, was set to 1.0. The average of two RNA measurements is shown. Also shown is the RNA level for cells grown at 23° overnight (labeled O/N).