Verification of novel Gal4 targets. (A) ChIP assay for Gal4 binding. Chromatin was cross-linked to protein by treatment with formaldehyde, and Gal4 was tagged with the 18-myc epitope, which was precipitated with anti-myc antibody. The precipitated DNA was released from the protein and detected by PCR as described in the Methods, using primers specific for sequences upstream of the indicated putative Gal4 targets (query promoter) and primers specific for the GAL4 promoter (control promoter) that amplify a 150-bp fragment. (B) RT-PCR analysis compared the expression of novel Gal4 target genes in wild-type FM393 cells vs. gal4Δ cells grown on different carbon sources. Cells were grown to saturation in YPD and then diluted 100 times in fresh 2% glucose, 2% galactose plus 5% glycerol, or 5% glycerol. Cells were harvested once they reached mid-log phase (OD600 = 1.5–2.0), total RNA was prepared, and RT-PCR was performed on the indicated targets. Control reactions lacking reverse transcriptase produce no PCR products (data not shown).
