Figure 3.
Analysis of the association of Gal4p, Gal80p, and Gal3p with the UASGAL by chromatin immunoprecipitation. Yeast cells were grown to early-log phase in medium containing 2% glycerol, 3% lactic acid, and 0.05% glucose (-galactose, uninduced) and induced with 2% galactose for 20 min (+galactose, induced). The 344-bp PCR product (UASGAL) corresponds to the region extending from −600 to −257 relative to the +1 of the GAL1 gene. The 287-bp PCR product (interg. ctrl.) corresponds to an intergenic region located 5 kb downstream from the GAL1 promoter; it served as a control for background binding in the immunoprecipitation. The ratio between the 344-bp and 287-bp PCR products (fold enrichment) is calculated to normalize results and assess the promoter occupancy. (A) Gal4p and Gal80p occupancy at UASGAL sites in uninduced and induced cells. The results from twofold dilution of the template DNA used for the PCR reactions demonstrated that the reaction was within the linear range. Fold enrichments are indicated (enrichment). (B) Gal80p and Gal3p association with the UASGAL region evaluted before and after galactose induction. (C) Quantitation of the promoter occupancy by Gal4p, Gal80p, and Gal3p in uninduced and induced cells. Values are means (±SD) of at least five experiments carried out with two sets of independently prepared chromatin samples.