Figure 4.

Loss of Abf1 binding in abf1-1 yeast does not result in decreased RPS28A expression. (A) ChIP of FLAG-tagged wild-type or ts Abf1 after 1 h at 37°C. Immunoprecipitated and input DNA were amplified using primers spanning the Abf1 site in the RPS28A promoter (upper panel) and the PCR fragments visualized by gel electrophoresis followed by Southern blotting (middle panel). Lanes are labeled as containing PCR products amplified from input DNA (In), immunoprecipitated DNA (IP), or mock IP without antibody (No Ab). The lane labeled ‘−’ is empty. Quantified results from this and an independent ChIP experiment are depicted at the bottom; SDs (too small to be visible for the FLAG-abf1-1 sample) are indicated. (B) RNA was prepared from wild-type or abf1-1 ts yeast grown at 25°C or for 1 h at 37°C (upper panel), and also from yeast having the Abf1 binding site in the RPS28A promoter mutated (lower panel). RPS28A and PYK1 mRNA were reverse transcribed and amplified by PCR, electrophoresed and visualized by ethidium bromide staining. These experiments were performed at least three times with similar results.