Richard G. Vogt - US grants

This proposal focuses on three classes of protein which control olfactory function: olfactory receptor proteins, odorant binding proteins, and odorant degrading enzymes. The last few years have seen the biochemical characterization of each of these components in a variety of animals ranging from rats and frogs to insects, lobsters and snails. We have ourselves amassed a substantial amount of biochemical and molecular biological information of olfactory processing in an insect model system: sex-pheromone reception/transduction in large moths. In previous work we have characterized candidate odorant receptor proteins; isolated, characterized and sequenced the cDNA of odorant binding proteins; and isolated and characterized odorant degrading enzymes. We now propose to isolate and characterize the nucleic acid sequences which encode these proteins, as well as those 5' transcription regulatory sequences which determine and modulate their expression. Nucleic acid and antibody probes will be generated to each of the previously identified proteins. These probes will be used to screen cDNA libraries which have been made from RNA isolated from olfactory tissue. In some cases enhanced cDNA libraries will be constructed using size fractionation and tissue specific subtraction techniques. Selected cDNA inserts will be screened by tissue specific RNA hybridization techniques and by physiological expression assays. Complementary DNA inserts thus selected will be used to isolate and characterize genomic DNA with special attention to the sequences of the 5' transcription regulatory sequences. By comparing the nucleic acid sequences encoding different species of related proteins from different species of related animals patterns will emerge enabling us to understand how tissue and sex specific coordination is accomplished in the nervous system.

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.