Figure 2.

Analysis of DNA-protein complexes binding to the secretin gene E box. (A) Three complexes denoted 1, 2, and 3 were generated from HIT cell nuclear extracts in gel shift assays. Competitor fragments consisting of the wild-type sequence (WT), a similar fragment with a double-point mutation in the E box (mE2), the core E-box sequence without upstream sequence (E), or 4-bp transversion mutant (m5) were used to determine the binding specificity for each complex. The three lanes shown for each competitor contained a 25-, 50-, or 100-fold mol excess, respectively. (B) Methylation interference analysis of complex 1 binding. Piperidine cleavage patterns of bound (B) and free (F) methylated sense and antisense strands are shown for DNA-protein-binding reactions. Arrowheads denote fragments not present in complex 1. Brackets denote the position of the E box. (C) The corresponding sequence for B with putative contact points necessary for binding indicated by ∗. The E-box sequence is highlighted.