Figure 3.—

IREu functions as a positive element in IME1 promoter. (A) Alignment of IREu and IREd elements and the putative Msn2 and Sok2 binding sites. (B) Alignment of IREu and IREd in the three Saccharomyces sensu stricto strains. (C) RNA was isolated from 1 × 10⁷ cells/ml grown in either SD (shaded column) or SA (striped column) media (left panel). In addition, cells grown in SA to 1 × 10⁷ cells/ml were shifted to SPM, and at the indicated hours samples were taken to isolate RNA (right panel). Level of RNA was determined by qPCR. The relative level of IME1 RNA in comparison to RNA levels of SUM1 is given. The isogenic strains used were Y1631 (wild type, squares) and Y1795 (IME1–ΔIREu/IME1–ΔIREu, triangle). (D) Following 24 (open column) and 48 (solid column) hr of incubation in SPM, the percentage of asci was determined. (E) Samples for ChIP were taken from cells grown in either SD or SA media to 1 × 10⁷ cells/ml. Strain used was Y422 (wild-type diploid) carrying either pCDC28–3xHA–sok2 (YEp2562) or pCDC28–GST–MSN2 (YEp2536). The PCRs amplified IME1 IREu region or the nonspecific ARS305 loci. Detection of ARS305 DNA required a 10-fold increase in the level of the input DNA. C, control (a strain without tagged proteins); WCE, whole-cell extract (input); M, marker. (F) Samples for ChIP were taken from cells grown in SA media to 1 × 10⁷ cells/ml. Strains used were Y422 (wild-type diploid, lanes 1 and 2), Y1064 (wild-type haploid, lane 4), Y1171 (msn2Δ msn4Δ/msn2Δ msn4Δ diploid, lane 3), and Y1162 (sok2Δ haploid, lane 5). These strains carried on 2μ plasmid pADH1–GAL4(bd)–ime1(id) (YEp2780).