Analysis of putativetrans-acting regulators of thes15 chorion gene in Drosophila melanogaster.

摘要

n the laboratory of Dr. Fotis Kafatos at Harvard University, we are addressing the question of tissue and temporal specific gene regulation, using as our model system the chorion gene family in Drosophila melanogaster. My particular interests include isolating and studying trans- acting regulators of the late chorion gene s15. Proteins that bind in vitro to functionally relevant portions of the s15 promoter of D. melanogaster have been isolated from embryonic and ovarian cDNA libraries using the method of Vinson et al. (Genes Dev. 2: 801-806). These putative trans- acting factors, termed CF (chorion factor) 1, CF2, and CF3, have been characterized by sequencing, footprinting and Northern analysis and all have been found to be legitimate candidates for true regulators of s15. CF1 and CF2 are putative activators of s15. CF1 in particular is a member of the nuclear hormone receptor family and binds in vitro to the first half site of an imperfect palindrome with sequence similarity to vertebrate hormone response elements. CF3 on the other hand, is a putative repressor of s15 in non-ovarian tissues.;A functional test was devised that involved the generation and analysis of genetic mosaic follicles in flies that lack the CF1 gene and which have an s15 promoter-lac Z fusion as the reporter gene. The mosaic follicles are generated in Drosophila by an approach exploiting the FLP recombinase system of yeast to produce mitotic clones of varying size in a convenient manner (Cell 59: 499-509). CF1 mutant flies were rescued with a construct containing an 8.0 kb genomic fragment that had been shown to give complete male rescue for the CF1 null mutation (ultraspiracle; Nature 347: 298-301), adjacent to a nuclear