Figure 3.—

Establishment of silencing at HML and HMR in a conditional sir3-8 strain. (A) The establishment of silencing is not observed at HMR at G1. Strain YSH829 was grown to log phase in low pH YPD. α-Factor was added to one-half of the culture to arrest cells in G1. Following G1 arrest, hydroxyurea was added; after an additional 20-min incubation, the culture was further divided and one-half of the arrested cells were shifted to the nonpermissive temperature (NPT, 37°) for 1 hr (lane 2). Cells were then shifted back to the permissive temperature (PT, 23°) and cells were harvested every 2 hr for 6 hr (lanes 4–6). The unblocked cycling cells that served as the control were treated in a similar manner. In independent control experiments, cells shifted from 37° to 23° exhibited full repression of HML and HMR within 4 hr (Figure S3). The lane 7 control culture was blocked in G1 and then washed to remove α-factor and hydroxyurea to allow resumption of cell cycle progress. The lane 8 control was blocked in G1 but maintained at 23° throughout the experiment. (B) The establishment of silencing is not observed at HMR at G2/M. Strain YSH829 was subject to the same experimental design as that described in panel A, except that nocodazole was used to block cells at G2/M. (C) The establishment of silencing is observed at HML at G1. α1 message was measured from RNA obtained from the same cell culture as described in A. (D) The establishment of silencing is not observed at HML at G2/M. α1 message was measured from RNA obtained from the same cell culture described in B. (E) Silencing at the HML locus does not require HMR expression. An experiment identical to that described in C was performed on strain YSH854 (sir3-8 Δmat∷HYG Δhmr∷NAT).