Fig. 6. Six3 binds to its own promoter. (A) The Six3 promoter region (GenBank Accession Number, AD487887). The putative Six3 recognition sequences identified in that genomic fragment are labeled I, II and III. (B) Bacterially expressed Six3 protein (full length) and 32P-labeled DNA probes I, II and III were subjected to an electrophoretic mobility shift assay (EMSA). The upper band containing the Six3-bound DNA fragment is indicated (arrow). The binding specificity was determined by the ability of the complex to be competed by nonradioactive wild-type oligonucleotides (lanes 3, 7 and 11), but not by nonradioactive mutated oligonucleotides (lanes 4, 8 and 12). The lower band seen in lanes 2 and 4 (arrowhead) probably represents truncated form of Six3 protein bound to the probe. This band was also efficiently competed by the cold wild-type probe but not by the mutated form. (C) The sense strand sequences of the oligonucleotide probes I, II and III used in the EMSA are represented. The core sequence motif ATTA of probes I, II and III was mutated into AGCA in the mutated oligonucleotide probes mut1, mut2 and mut3.
