MOLECULAR NEUROBIOLOGY OF OLFACTORY PROTEINS

这一建议集中在三类控制蛋白质的 嗅觉功能：嗅觉受体蛋白、气味结合 蛋白质和气味降解酶。过去的几年里 看到了每一种成分的生化特征 在从老鼠、青蛙到昆虫的各种动物中， 龙虾和蜗牛。我们自己已经积累了大量的 生物化学和分子生物学信息量 昆虫模型系统中的嗅觉处理：性信息素 在大蛾中的接收/转导。在以前的工作中，我们有 鉴定的候选气味受体蛋白；分离的， 对气味结合蛋白基因进行了鉴定和测序； 并分离和鉴定了气味降解酶。 我们现在提议分离和鉴定核酸 编码这些蛋白质的序列，以及那些5‘ 决定和调节转录调控序列 他们的表情。核酸和抗体探针将被 产生于每一种先前识别的蛋白质。这些 将使用探针来筛选已制作好的cDNA文库 从嗅觉组织中提取的RNA。在某些情况下增强 将使用大小分级和 组织特异性减影技术。选定的c DNA插入片段将 通过组织特异性RNA杂交技术和 生理表达分析。互补DNA插入 所选择的将用于分离和鉴定基因组DNA 对5‘转录序列的特别关注 调控序列。通过比较核酸序列 编码不同物种的不同来源的相关蛋白 相关动物的物种模式将会出现，使我们能够 了解组织和性别特异性协调是如何实现的 在神经系统中。

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.