FIG. 7.

The php4⁺ gene is required for iron limitation-dependent down-regulation of pcl1⁺, sdh4⁺, and isa1⁺ transcripts. (A) Mid-logarithmic-phase cultures of isogenic strains FY435 (php4⁺) and AMY15 (php4Δ) were grown in the presence of FeCl₃ (0 and 100 μM) or under conditions of iron deprivation (250 μM Dip) at 30°C. Fifteen-milliliter samples were taken after 90 min of treatment. RNA was extracted from each sample and analyzed by RNase protection assays. mRNA steady-state levels of pcl1⁺, sdh4⁺, isa1⁺, and act1⁺ (indicated with arrows) were analyzed with respect to the php4⁺ allele status. As a positive control, php4Δ cells were also transformed with an integrating plasmid (int.) expressing S. pombe php4⁺ under the control of its own promoter and assayed for iron limitation-dependent repression of specific mRNAs (pcl1⁺, sdh4⁺, isa1⁺, and act1⁺) under the same conditions. (B) Graphic representation of quantification of three independent RNase protection assays, including the experiment shown in panel A.