FIG. 4.

MBF-regulated genes are repressed in a stb1Δ swi4Δ double mutant. (A) Anti-Stb1 Western blot analysis of extracts prepared from a stb1Δ swi4Δ strain harboring a MET25pr-STB1 plasmid (BY1695; lanes 1 to 5), a wild-type (wt) strain (BY263; lane 6), and a stb1Δ strain (BY806; lane 7). Log-phase stb1Δ swi4Δ strain plus MET25pr-STB1 cultures were grown in the absence of methionine (lane 1) and subsequently grown in medium supplemented with 5 mM methionine (lanes 2 to 5). Wild-type and stb1Δ cultures were also grown in medium supplemented with 5 mM methionine (lanes 6 to 7). (B) A stb1Δ swi4Δ strain containing a MET25pr-STB1 construct (BY1695) was grown to mid-log phase in the absence of methionine. Cultures were then diluted and grown in the absence or presence of 5 mM methionine. cDNAs derived from cultures grown in the absence of methionine were labeled with Cy3 fluor, while cDNAs from cultures grown in 5 mM methionine were labeled with a Cy5 fluor. The results for a subset of genes whose expression levels changed at least threefold are shown. Green indicates methionine-dependent repression, while red indicates genes induced in the presence of 5 mM methionine. The reciprocal experiment was also performed (data not shown). (C) RNR1 is repressed in a stb1Δ swi4Δ double mutant. Wild-type (wt) (BY263), stb1Δ (BY806), swi4Δ (BY184), and stb1Δ swi4Δ (BY1695) mutants (containing a MET25pr-STB1 plasmid) were grown to mid-log phase in the absence of methionine (t = 0). Strains were subsequently grown in minimal medium containing 5 mM methionine. Aliquots were taken at 4- and 8-h time points. Total RNA was isolated from cells and probed with radiolabeled RNR1 and ACT1. The histogram depicts quantitation of the Northern blot. The RNR1 signal was quantified by phosphorimager analysis, and the values were normalized to the ACT1 loading control before plotting.