Figure 3.—

Silencing is not established at a hybrid HML-Pa locus without passage through S phase. (A) A diagram of the hybrid HML-Pa locus is shown. It expresses the α1 protein from the a1 promoter instead of the usual α1 promoter. (B) Experimental outline. An HML-Pa sir3-8 strain (JRY27) was treated with αF at 23° for 2.5 hr, then shifted to 37° in the presence of αF and HU for 2 hr to synchronize cells in early S phase. The culture was then shifted back to 23°, either with HU for S phase arrest or released into fresh YPD to allow for cell-cycle progression. (C) DNA content. Samples for S phase arrest were withdrawn at the times indicated and their DNA content monitored by flow cytometry. (D) HMLα1 expression at the HML-Pa 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 HMLα1 RNA level at the 0-hr time point, normalized to 18S rRNA, was set to 1.0. The average of two independent experiments is shown.