Fig. 1.

TCF efficiently and specifically binds the dpp posterior spiracle enhancer. (A) Combinatorial signaling enhancer within the dppMX reporter. I. An alignment of dpp intron sequences from D. melanogaster, D. simulans, D. pseudoobscura and D. virilis is shown. The D. melanogaster sequence begins 796 nucleotides downstream of the dpp exon2 splice donor. Identities are shown as dots and gaps as dashes. The D. melanogaster sequence is 94% identical to D. pseudoobscura and 84% identical to D. virilis. The sequences removed in the ΔKX deletion mutant are shown. II. Both strands of the D. melanogaster sequence are shown with putative binding sites for TCF, Mad and Brk. (B) Gelshift with TCF-HMG domain protein. Lane 8 in red serves as a positive control for two sets of experiments run on the same gel. Lanes 1-8 demonstrate the efficiency of TCF enhancer binding to both sites (shift and supershift indicated with red arrows). Lanes 1, 3, 5, and 7 show the mobility of each oligo alone. Mobility of the WT oligo is significantly impeded in the presence of TCF (Lane 8). Under identical conditions reduced TCF binding to M1 (Lane 2), M2 (Lane 4), and M1+2 (Lane 6) is seen. Binding of M1+2 is significantly less than WT. Lanes 8-24 demonstrate the specificity of TCF enhancer binding. Lanes 9-24 reveal the affects of adding increasing amounts of cold competitor oligo (50X, 100X, 200X and 300X) to the WT reaction in Lane 8. Adding cold WT (Lanes 9-12) significantly reduces binding at 50X and essentially eliminates binding at 200X. Adding cold M1 (Lanes 13-16) shows a similar reduction at 50X. Slightly more binding is seen at 200X and 300X than in WT indicating that this oligo cannot compete as efficiently as WT. Adding cold M2 (Lanes 17-20) does not reduce binding as efficiently as WT or M1 - at 50X and 100X greater binding is seen. Adding cold M1+2 (Lanes 21-24) has only modest effects on binding at 50X or 100X. Even at 300X, TCF binding to the WT oligo is not eliminated with M1+2 (Lane 24).