Fig. 5. The activity of a Ubx protein lacking the YPWM Exd-interaction motif. (A) Electromobility shift analysis of an oligonucleotide probe bearing a consensus composite Ubx/Exd binding site using wild-type and YPWM-YAAA mutant Ubx proteins with and without Exd. Exd alone does not bind the oligo (lane 2). Together with Exd, wild-type Ubx protein binds with higher affinity than Ubx alone (compare lanes 3 and 4). Surprisingly, the Ubx YPWM-YAAA mutant protein also exhibits an increased binding affinity for a composite Ubx/Exd binding site when complexed with Exd (compare lanes 5 and 6), although not as great an increase as exhibited by wild-type Ubx and Exd (20% less) (compare lanes 4 and 6). The Ubx YAAA mutant protein alone exhibits a slight increase in binding to the probe than the wild-type protein (1.5-fold) (compare lanes 3 and 5). The closed arrowhead indicates the position of shifts due to Ubx/Exd complexes, the open arrowhead indicates Ubx shifts, and the arrow indicates the position of free probe. (B-D) Dark-field photomicrographs of cuticle preparations showing ventral denticle belts in the third thoracic segment (T3) and the first and second abdominal segments (A1 and A2, respectively), from left to right. Segmental identities are indicated next to each of the three denticle belts in each panel. (E-G) Confocal photomicrographs of the three thoracic segments in embryos carrying a ß-galactosidase reporter trans-gene driven by the Dll embryonic limb enhancer and stained with anti-ß-galactosidase antibody (green). (H-J) Confocal photomicrographs of embryos stained for Ubx protein (green). White arrowheads indicate the boundary between A1 and T3. Ectopic expression of the Ubx proteins is driven by arm¹¹-Gal4. In all images, anterior is to the left and ventral is down. (B) In wild-type larvae, the T3, A1 and A2 denticle belts each have distinct morphologies. The T3 denticle belt comprises two rows of small hairs, the A1 denticle belt comprises four rows of larger hairs, and the A2 denticle belt comprises six rows arranged in a trapezoidal shape. (C) The ectopic expression of wild-type Ubx protein transforms T3 segmental identity to that of A1. Thus, wild-type Ubx specifies A1 segmental identity. (D) Ectopically expressing the Ubx YAAA mutant protein induces segmental identity transformations to A2 in the T3 and A1 segments. Therefore, the Ubx YAAA mutant protein specifies A2 segmental identity, a phenotype consistent with the inability of this protein to physically interact with Exd. (E) The Dll304 embryonic limb enhancer drives reporter gene expression in the three limb primordia in wild-type embryos. (F) Ecoptic expression of Ubx strongly represses the Dll304 enhancer. (G) Ectopic expression of the Ubx YAAA mutant protein similarly represses the Dll304 enhancer, indicating that an interaction between Ubx and Exd is not required to repress an embryonic target gene. (H) In a wild-type embryo, the anterior boundary of Ubx expression is posterior T2. (I) Ectopic expression of wild-type UbxIa protein occurs anterior to its normal anterior boundary in thoracic and head segments. (J) The Ubx YAAA protein is ectopically expressed at levels similar to ectopic UbxIa (I).