GENETIC DISSECTION--SYNAPTIC TRANSMISSION IN DROSOPHILA

基因解剖——果蝇的突触传递

• 批准号: 2184068
• 负责人: Michael J Stern
• 金额: $ 13.11万
• 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/2184068
• 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; nucleic acid sequence; 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.

一个对神经系统功能很重要的过程是突触 传输，神经元相互通信的过程以及 与目标肌肉细胞。 神经元离子通道在 控制此过程。 对机制的更完整理解 可以调节突触传播的情况需要识别 离子通道结构和调节组件。 但是很多 这些成分尚未抵抗分子表征。 这 这项工作的长期目标是在 果蝇识别和表征这些成分。 与遗传 方法论，确定调节突触传播的基因 通过突变。 因为任何基因都可以突变，因此任何蛋白质都可以是 通过突变识别，不论丰度如何，与以前的同源性 表征蛋白质甚至存在的先验知识。 因此 方法提供了一种独特的方式，可以在功能上识别新颖的类别 重要分子无法通过其他方式访问。 一旦确定， 这些基因在控制突触传播中的作用 使用电生理测定，最后将基因克隆，然后 测序使能够在分子上研究编码的产物 等级。 我以前在三个相互作用的新基因中鉴定了突变 与A型钾的结构基因相比行为上 渠道。 这些新突变体的电生理分析表明 每种表现出在幼虫神经肌肉的异常突触传播 由于运动神经元异常的兴奋性，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。 缺乏每个基因的苍蝇的表型， 以及每个基因的过表达，还将确定。 可能的 基因之间的协同相互作用将通过施工和 分析双突变体。 每个基因对神经终末的影响 将确定结构和电生理特性。 到 促进这些基因的克隆，P元素和X射线的诱变 将执行。 与这些cDNA的分离和序列分析 基因将提供有关基因产物功能的线索和 提供进一步研究的材料。 这些基因可能编码离子通道 亚基或调节分子，例如蛋白激酶，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物可能存在，并且可能是 涉及神经或神经肌肉系统的遗传性疾病。 另外，由于需要钾和钙通道功能 对于非神经过程，例如控制血压，胰岛素 释放和T淋巴细胞的激活，这些人类同源物可能 在这些过程的遗传性疾病中也有缺陷。 因此，我期望对离子通道结构和调节的研究 在果蝇中，将具有一般的医学意义。 将来，这个 遗传方法将进一步识别和分析其他 控制突触传播的重要过程的组件。