Figure 2

The structure of the cropped locus and its gene products. (A) The structure of the crp locus. The positions of P[lacZ, w] insertions (inverted triangles) in crp mutants are shown. The boxes at the bottom show the exons and the black boxes indicate the coding region. (B) Schematics of the primary structure of the Cropped and human AP-4 (hAP-4) proteins. The bHLH DNA-binding and dimerization domain (red), two leucine zippers (LZ, blue and green), and a region containing a conserved motif LETIV (yellow) are shown. The percent sequence identity between Cropped and hAP-4 is shown for the regions indicated. The positions of the in-frame initiator codons corresponding to the long (L) and short (S) Cropped protein isoforms are shown. The mutation of crp ^35Fd2 is indicated. (C) An alignment of the primary sequences of the 4 conserved domains of Cropped from D. melanogaster, D. virilis, D. willistoni, Apis mellifera, mouse AP-4, and hAP-4 is shown. The consensus sequence (Cons) is shown on the bottom. The bracket indicates the basic DNA binding region, which is deleted in DN-Cropped. (D) Immunoblots of equivalent amounts of protein extracts from crp ⁺ (crp ^35Fd2/+) and crp ⁻ (crp ^35Fd2/crp ^35Fd2) first instar larvae using anti-Cropped antiserum. The 85-kD band corresponds to the long Cropped isoform (L), and the 78-kD band corresponds to the short isoform (S). The lower molecular band (asterisk) is variable and may also be a cross-reacting species. (E) Immunoblots, as above, of protein extracts from control (hs-Gal4 alone) and UAS-cropped ^Short; hs-Gal4 adult flies subjected to heat-shock at 35°C for 1 hr to induce transgene expression. (F) An immunoblot of extracts of E. coli transformed with an inducible T7-cropped(short) vector before (−) and after (+) induction with IPTG. The Cropped (short) protein migrates at the same position as the short isoform in Drosophila cell extract.