Fig. 6. Identification of functional Krox20-binding sites in element A. (A) Alignments of chick and mouse nucleotide sequences of element A showing the presence of seven conserved putative Krox20-binding sites (boxed). Conserved residues are indicated with a dash in the mouse sequence. The mutations introduced by site-directed mutagenesis are indicated above each box. (B,E) Bandshift analysis of the wild-type chick element A (B), and a derivative carrying mutations in the seven Krox20-binding sites (E) using extracts from control (c) or Krox20-expressing bacteria. The positions of specific complexes are indicated with brackets. Specific complexes were identified by the addition of oligonucleotides carrying a high-affinity Krox20-binding site (wt) or a mutated version unable to bind the protein (mt). (C,D,F,G) Chick embryos analysed by X-gal staining after co-electroporation with constructs containing the wild-type (C,D) or mutant versions (F,G) of chick element A driving the ß-globin promoter-lacZ reporter together with the empty expression vector (C,F) or the Krox20 expression vector (D,G). FP, free probe; r, rhombomere; ov, otic vesicle.