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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.
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