NEUROGENETICS OF SODIUM CHANNEL GENES IN DROSOPHILA

果蝇钠通道基因的神经遗传学

• 批准号: 2392098
• 负责人: BARRY S GANETZKY
• 金额: $ 16.59万
• 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/2392098
• 关键词: Drosophilidae; complementary DNA; fusion gene; gene induction /repression; gene mutation; genetic regulatory element; in situ hybridization; neurogenetics; 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 动作电位的传播，这是动作电位的主要形式 神经系统中的电信号。 长期目标 拟议工作的目的是阐明分子机制 是神经信号传导的基础。 在这里，研究者重点分析 两个Na通道结构的结构、功能和调控 果蝇中的基因，称为 para 和 Dsc。 这些Na通道基因 由一个小家庭组成，其成员似乎有所不同 利用并可以促进生理上不同的功能。 这 目标是了解这两个Na通道各自的功能 果蝇神经系统中的基因，其差异的基础 表达以及突变的表型后果。 这 研究人员将结合遗传、 分子和电生理学技术。 为了表征 Dsc， 代表整个开放阅读框的 cDNA 将被分离并 测序； P的不精确切除将产生无效突变 基因内的元件插入；以及行为和 这些突变体单独和各种的电生理表型 双突变体组合将被表征。 表征 进一步增强 para 和 Dsc 的空间和发展表达，并 阐明一些控制其表达的调节机制， 研究者将： 产生 para 和 Dsc 特异性抗血清 相应通道多肽的免疫定位；检查 使用外显子对大量对剪接形式的细胞分布 用于原位杂交、剪接依赖性报告基因的特异性探针 构建体或单细胞 PCR 分析；并确定上游 用于种系的para转录调控序列 转化实验。 探索结构与功能的关系 para 和 Dsc 中，研究者将确定 a 中的确切病变 使用敏感的方法收集大量体内产生的副突变 SSCP 技术与序列分析相结合。 培养果蝇中Na电流的电生理记录 神经元和异源表达系统将用于比较 野生型和突变型等位基因编码的通道的特性 para 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 果蝇中额外的 Na 通道基因将被靶向 诱变和表型分析。 因为 para 和 Dsc 是唯一的 任何生物体中的神经元Na通道基因已发生突变 在原位，这些基因提供了获得新见解的独特机会 Na通道表达、功能和的分子机制 调节，体内。 多种人类遗传性神经肌肉疾病 已知与结构中的扰动有关或 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结果可能对 对这些疾病的了解和可能的治疗。