FIG 1.

Osmostress delays transcription initiation of G₁ cyclins. (A) G₁ cyclin expression is delayed upon osmostress. Wild-type cells were released from α-factor arrest into YPD medium (control) or 0.4 M NaCl. Samples were taken at the time points indicated for Northern blot analysis with radioactively labeled probes against CLN2, CLB5, and ENO1 (as a loading control). (B) Binding of RNA Pol II to G₁ cyclin promoters is delayed in response to osmostress. Wild-type cells were synchronized in G₁ with α-factor and released into either YPD medium (control) or 0.4 M NaCl. Chromatin-bound RNA Pol II was immunoprecipitated with an anti-Rpb1 monoclonal antibody (8WG16; Covance) at the times indicated. The precipitate was analyzed by real-time PCR with primers specific for the CLN2 (left panel) or CLB5 (right panel) promoter region. Graphs represent the averages ± the standard deviations from five independent experiments. (C) Osmostress inhibits transcriptional firing of G₁ cyclin promoters. A fluorescent reporter of CLN2 or CLB5 promoter activation was integrated into wild-type cells. Promoter-associated fluorescence of G₁-arrested cells (α-factor) or cells released for 40 min into YPD medium (control) or 0.4 M NaCl was analyzed by flow cytometry. A representative experiment with the CLN2 (left panel) and CLB5 (right panel) promoters is shown. Each overlaid histogram represents fluorescence distribution from 20,000 cells.