Figure 4.

Wild-type p53 directly activates the bbc3 promoter. (A) Schematic of the bbc3 gene structure, and cloned segments of the candidate bbc3 promoter region. Restriction sites for BamHI, PstI, and EcoRI are indicated by B, P, and R, respectively. The putative p53 binding sites in the human and mouse bbc3 promoter regions are shown, together with the consensus p53 binding site, and substitution mutations introduced into the human site. (B) Transactivation of the bbc3 promoter by wild-type p53. The bbc3 promoter region fragments shown in A were cloned into a luciferase reporter plasmid and cotransfected with plasmids expressing either wild-type p53 (wt) or mutant p53 (mut) into Saos-2 cells. Luciferase activity (measured in triplicate) was normalized to an internal transfection efficiency control (β-galactosidase). (C) Mutation of the p53 binding site abolishes transactivation of the bbc3 promoter by p53. Transfections were performed as in B, with bbc3 promoter reporter plasmids containing an intact (pGL3/0.9) or mutated (pGL3/0.9mut) p53 binding site, in the presence or absence of wild-type p53.