FIG. 4.

Multiple Rgt1 binding sites mediate synergistic repression. (A) Three different segments of the HXT3 promoter were fused to a HIS3-lacZ reporter gene (pBM2832) and introduced into yeast wild-type (WT) (YM4127) and Δrgt1 (YM4509) strains. Expression of the reporter gene was measured in cells grown on minimal medium containing 2% galactose (Gal) to mid-log phase and then switched to glucose (4%) (Glu) for 1.5 h. (B) Single or multiple copies of Rgt1 sites in cluster I were inserted in the reporter plasmid (pBM2832) assayed for the function of Rgt1 binding sites as described for panel A. These Rgt1 binding sites were generated by annealing complementary oligonucleotides containing one or two Rgt1 sites or by amplifying more than three Rgt1 sites. (C) Mutations in Rgt1 binding sites were generated by recombinational gap repair. Repression is the ratio of β-galactosidase activity in the WT (YM4127) to that in Δrgt1 (YM4509). Arrows (B and C), locations and orientations of Rgt1 binding sites.