Fig. 6. In vitro analyses of the NCE identify two Krox20 binding sites. (A) Both the upper (left) and the lower (right) strand of the 247 bp fragment #17 (Fig. 3) were analysed for Krox20 binding sites in DNase I footprinting assays using extracts from control or Krox20-expressing bacteria. Two doses of DNase I were used (0.2 and 0.5 units). The G+A reaction is included for both strands. The position of the two Krox20 footprints and the range of sequence analysed are indicated for both strands. (B) Control and Krox20-expressing bacterial extracts were analysed in bandshift assays using either the wild-type (upper) or Krox20 binding site mutant (lower) fragment #17 as a probe. The volume of bacterial extract was varied between 0.5-2.0 µl. To identify specific complexes, unlabelled competitor oligonucleotides corresponding to a high affinity Krox20 binding site (wt) or a mutant version (mt) were included in the binding reaction at 50-200-fold molar excess (Sham et al., 1993). Specific complexes are indicated with brackets. The different mobility protein-DNA complexes are likely a result of the presence of two Krox20 binding sites, one of which is asymetrically localised (Fig. 5). FP, free probe.