MAB-3 binds the minimal 247 bp vit- 2 promoter in vitro in a gel-mobility shift assay (Fig. 5, lanes 1 and 2). To test whether MAB-3 binds the site identified by sequence analysis, we made two mutant promoters,
vit-2 M1 and
vit-2 M2, which have two and five nucleotide changes in the potential MAB-3 binding site, respectively. MAB-3 does not bind to either of the mutant promoters (Fig. 5, lanes 3 and 4), demonstrating that the site is required for MAB-3 binding in vitro.