The three homologous Odorant Binding Proteins (OBPs) of the moth Manduca sexta are uniquely expressed in the olfactory neuronal epithelium; the respective OBPs are expressed in different domains of the neuronal epithelium and OBP expression is induced by a decline in the insect steroid hormone 20-hydroxy ecdysone. Evidence for differential expression is from biochemical and histological studies. Evidence for steroid regulation is from tissue culture studies. Complementary DNAs of all three OBPs have been cloned and sequenced and are in hand. The experiments proposed in this application examine the regulatory mechanisms that account for differential expression and steroid regulation of the OBP genes. Development of the olfactory organ occurs in a progressive manner to form a tissue with at least three levels of spatial organization: the olfactory organ contains domains of neuronal and non-neuronal epithelium; the neuronal epithelium is subdivided into domains containing distinct subsets of olfactory neurons; neurons of the same phenotype are evenly distributed within their domain but are thought to converge onto a phenotypically defined site in the brain. This organization is observed in both rat and insect; the mechanisms underlying this organization may be highly conserved among different animal groups. The expression of the M. sexta OBPs reflects this organization. The M. sexta OBPs represent simple and accessible tools for identifying and mechanisms controlling spatially and temporally regulated gene expression during development of the olfactory organ and the olfactory neuronal epithelium.
Four specific aims are proposed: (l) histological analysis of the cellular and subcellular distributions of the three M. sexta OBPs; (2) isolation, sequencing and characterization of the three OBP genes from a M. sexta genomic library that is in hand; (3) identification of possible DNA regulatory sequences in the OBP genes that are responsible for differential expression; (4) identification of the molecular level of steroid regulation of OBP expression and identification of possible DNA regulatory sequences sensitive to steroid.
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