The Bradysia hygida BhC4-1 gene is expressed in fourth instar larvae salivary glands. The mechanisms that control BhC4-1 expression in the salivary gland are conserved in transgenic Drosophila. The analysis of lines transformed with progressive deletions of the BhC4-1 promoter revealed gene expression in another organ, the ring gland. Functional studies identified a 67 bp (-253/-187) ring gland enhancer. To extend the characterization of this enhancer, five constructs (MUT 1-5), containing each a distinct 10 bp cluster of mutated sequences in the 67 bp fragment, were cloned upstream the Fbp1 mininal promoter, in the pCaSper-AUG-b-gal vector. At least five independent transgenic lines were obtained for each construct. Promoter activity was assayed employing a histochemical assay for b-galactosidase in embryos, first instar larvae (L1) and third instar larvae (L3). In embryos and L1 the mutations MUT4 and MUT5 abolish gene expression in the ring gland, whereas reporter gene expression is detected in the ring gland in lines transformed with either the MUT1, MUT2 or MUT3 constructs. In L3 the mutations MUT3, MUT4 and MUT5 abolish reporter gene expression in the ring gland, whereas the mutations MUT1 and MUT2 lead to an apparent decrease in the levels of reporter gene expression., when compared to the wild type activator. These results indicate that the sequences mutated in constructs MUT4 and MUT5 are essential for the gene activation in the ring gland. On the other hand, the sequences mutated in constructs MUT1 and MUT2 apparently promote correct levels of expression, without interfering with the developmental pattern of expression of the gene. The detection of reporter expression only in embryos and L1 in lines of the MUT3 series could be attributed to the fact the ring gland cells grow only by size, without proliferating. This could result in the dilution of the reporter protein in the ring gland cells of L3, rendering them negative for the histochemical assay employed. These functional data are further supported by a bioinformatics analysis which identified, in the Drosophila melanogaster genome, sequences identical to sequences contained in the ring gland enhancer. The majority (96%) of the identified D. melanogaster sequences align to a fragment located between positions (-231/-187), which comprises the regions mutated in constructs MUT3 (-222/-213), MUT4 (-210/-201) and MUT5 (-198-189).
Supported by FAPESP.