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Figure 2
Fkh2 binds to FLEX and FLEXL sequences in the putative promoter region of ste11+ and thereby induces ste11+ mRNA. (A) Schematic representation of the region upstream of the open reading frame (ORF) of ste11+ showing FLEX and FLEXL sequences. The major transcription initiation site of ste11+ is indicated by the arrow, and the regions targeted by primer sets in ChIP analysis are also shown. (B) An EMSA was performed with recombinant GST–Fkh2p(216–330) (or GST alone) and with FLEX1, FLEXL1, FLEXL2, FLEXL3, or TR (negative control) probes labeled with 32P. Competition was evaluated with excess amounts of unlabeled FLEX1, TR, or FLEXL1 oligonucleotides, and supershift analysis was performed with antibodies to GST, as indicated. The positions of shifted and supershifted bands are shown. (C) No tagged cells (no tag, HM6) and cells expressing GFP-tagged Fkh2p (Tag, HM5719) were grown to late log-phase, washed, and resuspended in medium without nitrogen. After incubation for 2 h at 30°C, cells were collected and analyzed by ChIP with antibodies to GFP and with the primer sets indicated in (A). Data are means s.e. *P<0.006 (Student's t-test). (D) No tagged cells (no tag, HM6) and cells expressing GFP-tagged Fkh2p (wt; HM5719 and ste11-dFLEX1: HM6124) were treated and analyzed as in (C) with primer set A. Samples were collected at 0 and 2 h after nitrogen withdrawal. Data are meanss.e. of values from three separate experiments. *P<0.007 (Student's t-test). (E) wt (HM6) or ste11-dFLEX1 (HM5832) cells were treated and analyzed for mating efficiency as in Figure 1A. (F) Total RNA was extracted from cells treated as in (E) and was subjected to northern blot analysis of ste11+ mRNA.
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