Figure 4

Effect on the transcriptional activity of site-directed mutagenesis of the putative Sp1-binding site of the MsrB1 promoter. (A) the sequence spanning Sp1- binding site (Sp1E) contained in the construct P-203 WT was subjected to site-directed mutagenesis. The mutated construct was designed P-203 Sp1EMut. Luciferase reporter activity was assessed following transfection into MCF7 cells. Luciferase activity was inhibited by approximately 80% by mutation of Sp1E site. (B) the sequences spanning Sp1-binding sites (Sp1C and Sp1E) contained in the P-296 WT construct were subjected to site-directed mutagenesis. The constructs obtained were designated as P-296 Sp1CMut and P-296 Sp1EMut. In addition a double mutant containing both mutations was indicated as P-296 Sp1C/EMut. The mutated constructs were transiently expressed in MCF7 cells for luciferase assays. Luciferase activity was inhibited by approximately 30% by mutation of Sp1C site; 24% by mutation of Sp1E site and by approximately 76% by mutation of both Sp1 sites (Sp1C/Sp1E). The results are expressed as mean ± S.D. of at least five different experiments, in duplicate for each construct. Statistically significant differences compared to the appropriate WT construct are indicated by *P < 0.005 and **P < 0.002.