Molecular Mechanisms Underlying the Regulation of the Drosophila E93 Gene by Ecdysone and betaFTZ-F1
Steroid hormones play a critical role in the regulation of physiological and developmental processes in eukaryotes. Drosophila melanogaster is an ideal system for the study of steroid hormone action because it has only one known physiologically active steroid hormone: 20-hydroxyecdysone. During metamorphosis, ecdysone directs morphogenesis as well as programmed cell death. It regulates the expression of a set of genes called the early genes that, in turn, regulate the transcription of other genes important in metamorphosis called the late genes. In larval salivary glands, betaFTZ-F1, a nuclear hormone receptor, provides E93, an early gene, with the competence to respond to the high ecdysone titer in the late prepupal stage. Preliminary studies have indicated that betaFTZ-F1 binds to the 3 -end of the first intron of E93. This project delves into the molecular mechanisms by which betaFTZ-F1 and ecdysone regulate the expression of the E93 gene in Drosophila. It aims to map betaFTZ-F1 binding site(s) in E93. In order to achieve this goal, the betaFTZ-F1 open reading frame was cloned into a pET42a vector. The recombinant plasmid was used to transform competent bacterial expression cells. Protein expression was successfully induced in these bacterial cells and a purer form of betaFTZ-F1 was obtained. Gel-shift assays with betaFTZ-F1 and a consensus betaFTZ-F1 binding sequence was carried out. Gel-shift assays with betaFTZ-F1 and E93 are to be conducted in the near future to determine the exact DNA sequence of the betaFTZ-F1 binding site(s) in E93.