Figure 2.—

Silencing is not established at an HMLa locus without passage through S phase. (A) A diagram of the modified HML locus, HMLa, is shown. It contains the a1/a2 transcription unit from HMR instead of the usual HMLα1/α2 transcription unit, but flanked by the usual HML silencers. (B) Experimental outline. The scheme for this experiment is similar to that described in Figure 1, except that HMLa sir3-8 cells (JRY19) were used. (C) DNA content. Samples for S phase arrest were withdrawn at the time points indicated and their DNA content monitored by flow cytometry. (D) HMLa1 expression. 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. The HMLa1 RNA level at the 0-hr time point, normalized to either an 18S rRNA or the ACT1 internal control, was set to 1.0. The average of two independent experiments is shown. Also shown is the RNA level for cells grown at 23° overnight (labeled O/N).