Figure 4.—

Telomere repression is not established in S phase. (A) Experimental design for G₁–G₂/M interval. Cultures of strain YSH505 were grown to early log phase in raffinose media. Levels of URA3 message were examined in one experimental and three control cultures. (i) Experimental culture. Following efficient arrest in G₁ phase galactose was added and cultures were incubated in the presence of α-factor for an additional 8 hr. Cultures were then washed to remove α-factor and resuspended in galactose media containing nocodazole. Cells were then incubated an additional 6 hr. (ii) No galactose control. This culture was treated exactly the same as the experimental culture, but was not induced with galactose. (iii) No nocodazole control. This control was treated the same as the experimental culture, except that following the wash step cells were released into galactose media without nocodazole. Data for this control are shown in Figure 4C. (iv) Cycling cells control. This culture was induced with galactose but not subjected to cell-cycle blocks. (B) G₁–G₂/M interval. RT-PCR was used to determine the levels of URA3 message of cultures grown according to the design described in A. All times listed are in hours following initial addition of galactose to the culture. In the experiment presented galactose was added when 96% of the culture consisted of unbudded cells; following 8 hr of galactose induction 97% of the culture was unbudded. After washing out α-factor and incubating 6 hr in nocodazole, 91% of the cells had large buds. For each lane the URA3/ACT1 ratio is shown, as determined by quantification of the bands and expressed relative to the uninduced (no galactose) control.(C) Establishment of repression following a G₁ block. A culture of YSH505 was blocked in G₁, induced with galactose for 8 hr, and then released from the G₁ block into galactose media and allowed to progress through the cell cycle (lanes 1 and 6). Additional data shown in this figure constitute a replication of the experiment shown in B.