Fig. 2. (A) Identification of Engrailed-binding consensus sequence. The YAATYANB consensus was deduced from sequence analysis of 107 selected clones, as defined in the Material and Methods. For each position, the ratio of A, C G or T is indicated. (B) Gel shift assay was performed on a pentamer of the motif CAATTAGC, the sequence of which is shown below the gel. Labeled DNA fragment was analyzed in the absence (-) or in the presence of different amounts of Engrailed protein: 1=2x10^-10 M; 2=3x10^-10 M; 3=5x10^-10 M; 4=10^-9 M. Competition experiments were performed in the presence of 5x10^-10 M En protein (+) and in the presence of 300-fold excess of different DNAs: D2, polyhomeotic D2 fragment, corresponding to a specific Engrailed-binding fragment (Serrano et al., 1995); C, double strand monomer `CAATTAGC'; N, polyhomeotic N fragment that does not bind Engrailed specifically in vitro (Serrano et al., 1995); Cm, double strand mutated monomer `CAGCCGGC'. Supershift assays were performed in the presence of 5x10^-10 M En protein (+) and of 4F11 monoclonal anti-Engrailed antibody. F indicates free DNA. The asterisks indicate the Engrailed protein-DNA complexes.