FIG. 3.

Bas1-Myc₁₃ binds to ADE5,7 promoters carrying a single binding site in vivo. (A) Schematic representation of the ADE5,7 promoter and binding sites for Bas1 and Pho2. The Bas1 binding sites are represented by arrows: the distal site (white arrow) is located at −217, and the proximal site (gray arrow) is located at position −184. The Pho2 binding site (black bar) is located at −169. (B) Northern analysis was performed on strains with the wild-type or mutant ADE5,7 promoters at the native locus. Strain RR376 carries the wild-type promoter (WT), strain RR405 carries the proximal Bas1 binding site, and strain RR406 carries the distal Bas1 binding site. RNA was prepared from strains that had been grown in SC medium containing adenine (+ade) and lacking adenine (−ade) and blotted to membranes. Radiolabeled probes recognizing the ADE5,7 and ACT1 genes were hybridized, and the amount of label bound was quantified using a phosphorimager. The levels were normalized to ACT1 and are graphically represented in arbitrary units. Repressed levels, black bars; derepressed levels, gray bars. (C) ChIP assays were performed on the same three isogenic strains as described in panel B that had been grown in SC medium containing (+ade) and lacking (−ade) adenine. PCR was performed with ADE5,7- and POL1-specific primers. ChIP values and the ratio were calculated as described in Table 3 and are listed above the schematic of the promoter. The ratios were calculated as 2.6 for wild type, 2.2 for the proximal site alone, and 0.8 for the distal site alone. I, input DNA.