In recent years it has become obvious that multigene families are important features of eukaryotic genomes. Gene families are especially important for development, since their members frequently diversify after reduplication, and come to be expressed in different cells or disparate times during development. Diversified members are favorable for analysis of the molecular mechanisms that regulate gene expression in development, and of the evolution of these mechanisms. Such studies are clearly health related, insofar as correctly regulated gene expression is fundamental to proper development and health. The families of chorion (eggshell) genes of fruitflies and silkmoths exhibit a high degree of tissue, temporal and spatial regulation during development and high conservation of regulatory mechanisms during evolution, in the face of considerable structural diversification. Taking advantage of recent progress in characterizing chorion cis-regulatory elements and putative trans-acting (CF) factors, we intend to study regulatory protein-DNA interactions in an evolutionary context, using the moth A/B.L12 and the fly s15 promoters. The specific objectives are: 1. To map on the A/B.L12 promoter binding sites for fly CF factors. 2. To verify which CF components are indeed regulatory in vivo, using a combination of cell culture assays and detection of CF factors on the promoter in vivo through immunoprecipitation of chromatin. 3. To clone and characterize moth cognates of these functionally important factors. 4. To study in detail the DNA recognition properties and mechanisms of selected functionally important CF factors, from both Drosophila and the moth, with a view towards understanding the paradox of evolutionarily conserved chorion gene regulation, in the face of promoter sequence diversification.
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