FIG. 3.
Sum1 and Ndt80 compete for binding to the SMK1 MSE in vivo. (A) Sum1-dependent repression of lacZ reporter expression in the presence and absence of Ndt80. Reporter plasmids containing wild-type (lanes 1 and 3) or A12T mutant (lanes 2 and 4) SMK1 MSE were cotransformed with a SUM1-myc plasmid into sum1Δ strains. One host strain had an integrated copy of a GAL-NDT80 expression construct (MPY4, lanes 3 and 4), and the other did not (JXY3, lanes 1 and 2). Cells were grown in galactose, and the expression levels of the reporter were measured using β-galactosidase liquid assays. The repression of each reporter construct was calculated by comparison with a reporter lacking an MSE in the same strain background. (B) Western blot of Sum1-myc from lysates containing wild-type (lanes 1 to 3) and A12T mutant (lanes 4 to 6) MSE reporters. Lysates in lanes 2, 3, 5, and 6 were from strains that contained the SUM1-myc-tagged gene expressed from the SUM1 promoter. Lanes 1 and 4 are lysates from strains containing an empty vector, pRS415. Lanes 2 and 5 were lysates from strain JXY3, lacking the GAL-NDT80 construct, and lanes 3 and 6 are lysates from strain MPY4, which contains the GAL-NDT80 construct.