NEUROGENETICS OF SODIUM CHANNEL GENES IN DROSOPHILA

果蝇钠通道基因的神经遗传学

• 批准号: 2181787
• 负责人: BARRY S GANETZKY
• 金额: $ 18.68万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181787
• 关键词: Drosophilidae; complementary DNA; fusion gene; gene induction /repression; gene mutation; genetic regulatory element; in situ hybridization; neurons; nucleic acid probes; nucleic acid sequence; open reading frames; plasmids; polymerase chain reaction; protein structure function; reporter genes; single strand conformation polymorphism; sodium channel; structural genes; tissue /cell culture; transposon /insertion element

DESCRIPTION: Na channels perform the central role in the generation and propagation of action potentials, which are the primary form of electrical signalling in the nervous system. The long term objective of the proposed work is to elucidate the molecular mechanisms that underlie neural signalling. Here, the investigator focuses on analysis of the structure, function, and regulation of two Na channel structural genes in Drosophila, called para and Dsc. These Na channel genes comprise a small family whose members appear to be differentially utilized and could subserve physiologically distinct functions. The goals are to understand the respective functions of these two Na channel genes in the Drosophila nervous system, the basis of their differential expression, and the phenotypic consequences of mutations. The investigator will approach these goals using a combination of genetic, molecular, and electrophysiological techniques. To characterize Dsc, cDNAs representing the entire open reading frame will be isolated and sequenced; null mutations will be generated by imprecise excision of P element insertions within the gene; and behavioral and electrophysiological phenotypes of these mutants alone and in various double mutant combinations will be characterized. To characterize further the spatial and developmental expression of para and Dsc and to elucidate some of the regulatory mechanisms governing their expression, the investigator will: raise para- and Dsc-specific antisera for immunolocalization of the corresponding channel polypeptides; examine the cellular distribution of the large array of para splice forms using exon- specific probes for in situ hybridization, splice-dependent reporter constructs, or single-cell PCR analysis; and identify the upstream transcriptional regulatory sequences of para for use in germline transformation experiments. To explore structure-function relationships of para and Dsc, the investigator will determine the exact lesion in a large collection of in vivo-generated para mutations using a sensitive SSCP technique in combination with sequence analysis. Electrophysiological recording of Na currents in cultured Drosophila neurons and in heterologous expression systems will be used to compare the properties of channels encoded by wild-type and mutant alleles of para and Dsc as well as by the different splice variants of para. Finally, the investigator proposes to use a PCR-based strategy to search for additional Na channel genes in Drosophila that will be targeted for mutagenesis and phenotypic analysis. Because para and Dsc are the only neuronal Na channel genes in any organism that have been mutated in situ, these genes provide unique opportunities to obtain novel insights into the molecular mechanisms of Na channel expression, function, and regulation, in vivo. A number of human hereditary neuromuscular diseases are known to be associated with perturbation in the structure or function of ion channels, including Na channels. Therefore, the results obtained from these studies could have direct significance for the understanding and possible treatment of these disorders.

描述：Na通道在生成和 传播的动作电位，这是主要形式的 神经系统中的电信号 长期目标 这项工作的目的是阐明 是神经信号的基础 在这里，调查员着重分析 两个Na通道结构的结构、功能和调节 果蝇中的基因，叫做帕拉和Dsc。 这些钠通道基因 组成了一个小家庭，其成员似乎 利用，并可以辅助生理上不同的功能。 的 目的是了解这两种Na通道各自的功能 果蝇神经系统中的基因，其差异的基础 表达和突变的表型后果。 的 研究人员将通过遗传学、基因组学和生物学方法相结合， 分子和电生理技术。 为了表征Dsc， 分离代表整个开放阅读框的cDNA， 测序;无效突变将通过不精确切除P 基因内的元件插入;以及行为和 这些突变体的电生理表型单独和在各种 将表征双突变体组合。 表征 促进帕拉和Dsc的空间和发育表达， 阐明了一些控制其表达的调节机制， 研究者将：产生帕拉和Dsc特异性抗血清， 相应通道多肽的免疫定位;检查 使用外显子的大量帕拉剪接形式的细胞分布， 用于原位杂交的特异性探针，剪接依赖性报告基因 构建体，或单细胞PCR分析;并确定上游 用于种系的帕拉转录调节序列 转化实验 探索结构-功能关系 在帕拉和Dsc中，研究者将确定 使用敏感的 SSCP技术结合序列分析。 培养果蝇钠电流的电生理记录 神经元和异源表达系统将用于比较 由野生型和突变等位基因编码的通道的性质， 帕拉和Dsc以及para的不同剪接变体。 最后，研究者建议使用基于PCR的策略来搜索 果蝇中的其他Na通道基因， 诱变和表型分析。 因为帕拉和Dsc是唯一 任何生物体中的神经元Na通道基因， 原位，这些基因提供了独特的机会，以获得新的见解 钠通道表达、功能和 调节，在体内。 一些人类遗传性神经肌肉疾病 已知与结构中的扰动有关， 离子通道的功能，包括Na通道。 因此，结果 从这些研究中获得的信息可能对 了解和可能的治疗这些疾病。