The goal of the proposed research is the elucidation of mechanisms determining the tissue and temporal specificity of transcriptional activation. The model system to be used is insect choriogenesis, a process involving stringent development controls on the expression of a number of related genes, each of which is differentially regulated at eh transcriptional level. The studies will focus on the molecular biology of DNA-protein interactions regulating the expression of chorion genes and the analysis of the extent of conservation of such regulatory interactions between species.
Specific aims will be: Localization and functional analysis of silkmoth chorion gene cis- regulatory elements. Regions on chorion gene promoters recognized in vitro by Drosophila follicular cell nuclear proteins and cloned Drosophila transcription factors will be identified through DNase I protection and methylation interference studies. These regions will be the targets of in vitro mutagenesis and the effect of these mutations on the tissue and temporal specificity of promoter activation will be analyzed in transgenic Drosophila. Cloning and characterization of silkmoth genes regulating chorion gene expression. Silkmoth follicular cell cDNA expression libraries will be screened for binding to synthetic oligonucleotides representing the recognition sites of follicular nuclear proteins. In addition, cDNAs of Drosophila transcription factors associated with chorion gene transcription will be used in sequence homology screening. Clones representing cDNAs of putative regulatory genes will be structurally characterized and used for the production of proteins in bacterial or tissue culture expression systems. Reconstruction of transcriptional control interactions in cell-free systems. Transcriptionally active nuclear extracts derived from follicles and tissue culture cell lines will be supplemented with Drosophila and silkmoth putative regulatory proteins and the effect on the in vitro transcription of chorion and non-chorion gene promoters will be analyzed. The interspecies supplementation studies will be especially revealing on the extent of functional conservation in transcriptional regulatory interactions. The above proposed studies will provide information on molecular mechanisms involved in transcriptional regulation of a class of genes under stringent developmental control. Interspecies conservation of regulatory interactions, such as observed in this insect system, are also evident in mammalian systems, although extensively characterized in only a few cases. Basic information on the extent of conservation of transcriptional regulatory interactions between species could be relevant in the development of animal models for human disease.
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