GENETIC DISSECTIO OF SYNAPTIC TRANSMISSION IN DROSOPHILA

果蝇突触传递的基因剖析

• 批准号: 3306019
• 负责人: Michael J Stern
• 金额: $ 13.56万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306019
• 关键词: Drosophilidae; X ray; alleles; calcium; calcium channel; calcium flux; electrophysiology; gene deletion mutation; gene duplication; gene expression; gene interaction; genetic mapping; genetic regulation; in situ hybridization; membrane channels; molecular cloning; molecular genetics; motor neurons; mutant; neuromuscular junction; neuromuscular transmission; nontherapeutic iontophoresis; northern blottings; potassium channel; protein structure; southern blotting; structural genes; synapses; voltage /patch clamp

One process that is important for nervous system function is synaptic transmission, the process by which neurons communicate with each other and with target muscle cells. Neuronal ion channels play key roles in controlling this process. A more complete understanding of the mechanisms by which synaptic transmission can be regulated requires identification of the ion channel structural and regulatory components. However many of these components have as yet resisted molecular characterization. The long-term objective of this work is to use genetic methodology in Drosophila to identify and characterize these components. With genetic methodology, the genes that regulate synaptic transmission are identified by mutation. Because any gene can be mutated, any protein can be identified by mutation regardless of abundance, homology to previously characterized proteins or even prior knowledge of existence. Thus this approach provides a unique way identifying novel classes of functionally important molecules not accessible by other means. Once identified, the roles of these genes in controlling synaptic transmission are determined with electrophysiological assays, and finally the genes are cloned and sequenced which enables the encoded products to be studied at the molecular level. I previously identified mutations in three new genes that interact behaviorally with Shaker, the structural gene for the A type potassium channel. Electrophysiological analysis of these new mutants has shown that each exhibits aberrant synaptic transmission at the larval neuromuscular junction as a result of aberrant excitability of the motor neuron. In the present application, further functional and molecular characterization of these three genes is proposed. The phenotypes of flies lacking each gene, as well as overexpressing each gene, will be determined. Possible synergistic interactions among the genes will be tested by construction and analysis of double mutants. Effects of each gene on nerve terminal structure and electrophysiological properties will be determined. To facilitate cloning of these genes, mutagenesis with P-elements and X-rays will be performed. Isolation and sequence analysis of cDNAs from these genes will provide clues as to the function of the gene products and provide material for further studies. These genes might encode ion channel subunits or regulatory molecules such as protein kinases, G-proteins, or calcium binding proteins. Because such genes are well conserved in evolution, human homologues of these genes will likely exist and might be involved in hereditable disorders of the nervous or neuromuscular system. In addition, because potassium and calcium channel functions are required for non-neural processes such as the control of blood pressure, insulin release and the activation of T-lymphocytes, these human homologues might be defective in hereditable disorders of these processes as well. Therefore I expect that the study of ion channel structure and regulation in Drosophila will have general medical significance. In the future, this genetic approach will be used further to identify and analyze additional components that control the important process of synaptic transmission.

对神经系统功能很重要的一个过程是突触 传递，神经元相互交流的过程，以及 与目标肌肉细胞。神经元离子通道在血管紧张性疾病中起关键作用 控制着这个过程。对机制有了更全面的了解 通过什么来调节突触传递需要识别 离子通道的结构和调节组件。然而，许多人 到目前为止，这些组分还不能进行分子表征。这个 这项工作的长期目标是将遗传学方法学用于 来鉴定和表征这些成分。与基因 方法，确定了调节突触传递的基因 通过突变。因为任何基因都可以突变，任何蛋白质都可以 通过突变鉴定，而不考虑丰度，与以前的同源性 表征蛋白质，甚至预先知道存在的情况。因此，这就是 方法提供了一种独特的方式来识别功能上的新类别 重要的分子是无法通过其他方式获取的。一旦确定， 确定了这些基因在控制突触传递中的作用 通过电生理测试，最后克隆了这些基因并 测序，使编码产物能够在分子水平上进行研究 水平。我之前发现了三个相互作用的新基因的突变 A型钾的结构基因Shaker的行为 频道。对这些新突变体的电生理分析表明， 它们都在幼虫的神经肌肉部位表现出异常的突触传递。 由于运动神经元的异常兴奋性而导致的连接。在 目前的应用，进一步的功能和分子表征 提出了这三个基因。缺少每个基因的果蝇的表型， 以及每个基因的过度表达，都将被确定。可能的 基因之间的协同作用将通过构建和测试 双突变体的分析。各基因对神经末梢的影响 将确定其结构和电生理特性。至 方便克隆这些基因，用P-元件和X射线进行诱变 将会被执行。它们的cDNA的分离和序列分析 基因将提供关于基因产物功能的线索和 为进一步研究提供素材。这些基因可能编码离子通道 亚基或调节分子，如蛋白激酶、G蛋白或 钙结合蛋白。因为这样的基因在 进化，这些基因的人类同源基因很可能存在，也可能是 涉及神经或神经肌肉系统的遗传性疾病。 此外，由于钾和钙通道功能是必需的 用于非神经过程，如控制血压、胰岛素 T淋巴细胞的释放和激活，这些人类同源物可能 在这些过程的遗传性障碍中也有缺陷。 因此，我期望对离子通道结构和调节的研究 对果蝇的研究将具有普遍的医学意义。在未来，这是 将进一步使用遗传方法来识别和分析其他 控制突触传递的重要过程的组件。