Fig. 2. Ubx binds to seven sites in a flight appendage-specific cis-regulatory element of sal. (A) DNase I footprinting of the sal 328 cis-regulatory element reveals three sites protected by Ubx homeodomain and is representative of footprinting of the entire sal 1.1 element. A G+A sequencing ladder is shown in the first lane, and DNase I digestions incubated with increasing concentrations of Ubx homeodomain from 0 to 90 ng, in three-fold increments, are shown in subsequent lanes. Sites numbered 5-7 are schematized to the right of the lanes and are represented by boxes; their orientation is indicated by the arrows. (B) A list of the sequences of the seven sites bound by Ubx in DNaseI footprinting assay showing 14 base pairs centered on the TAAT core sequence (highlighted in red). The numbers indicate the position of the sites within the 1.1 kb sal element. Below each Ubx binding site is the altered sequence (mut) that was introduced in the mutant sal elements to abolish the ability of Ubx to bind specifically to the site. The altered base pairs are highlighted in blue. (C) A schematic representation of the sal elements. The blue circles indicate Sd binding sites identified by Guss et al. (Guss et al., 2001), and red circles represent the seven Ubx binding sites identified by footprinting. We note that the sal 1.1 element contains other TAAT sites that were not footprinted by Ubx. (D) Gel shifts of oligos containing a Ubx consensus binding site (lanes 1-5), Ubx binding site 2 from the sal 1.1 element (lanes 6-10) or its mutant variant (lanes 11-15) using Ubx homeodomain protein indicate that the mutant variant of site 2 exhibits an approximate ten-fold decrease in affinity for Ubx. The open triangle indicates increasing concentrations of Ubx homeodomain, ranging from 0 to 30 ng in three-fold increments. The black arrowheads indicate the lane for each oligo in which binding of Ubx is closest to half-maximal.