DROSOPHILA PARA LOCUS--GENETIC/MOLECULAR STUDIES

信息在神经系统中以 电脉冲或动作电位。 神经元的特征是 电兴奋膜，使他们能够接受，处理和 传递这些脉冲。 跨膜蛋白，称为离子通道， 电压敏感，离子特异性孔在这些细胞和执行的关键 通过调节特定离子的流量在神经信号传导中起作用 穿过细胞膜 钠离子通道特别是执行中央 在动作电位的产生和传播中的作用。 尽管 尽管最近取得了重要进展，但仍有许多关于 钠通道的结构、功能和调节。 长期目标 本文提出的工作是阐明功能和调节 果蝇钠离子通道的遗传和分子生物学研究 approach. 我们将集中讨论帕拉基因座， 致死性或温度敏感性麻痹与阻断 神经传导 我们已经克隆了帕拉基因座， 并证明帕拉是一种钠通道 结构基因 其他假定的钠通道位点的存在， 果蝇除了帕拉之外，还表明，与哺乳动物一样，这些基因包括 一个小家庭，其成员被不同地利用， 生理上不同的功能。 为了了解功能和 调节帕拉在果蝇神经系统，我们现在建议， 表征其表达及其基因产物的分布， 检查帕拉和其他两个基因座突变引起的扰动 (nap和tip-E），明显干扰表达或功能，或 段 为了实现这些目标，我们将使用北方印迹和核酸酶S1 实验，以确定帕拉丰度和发展概况的副 野生型的转录本。 帕拉的正态空间分布 转录物将通过组织原位杂交来表征。 帕拉，对位 将产生针对合成肽或para-lacZ的特异性抗体 融合蛋白，并用于免疫定位的副蛋白在 神经系统，并确定它的蛋白质印迹。 阐明本 帕拉、nap和tip-E突变的表型效应，类似实验 将对这些突变体进行研究，以确定它们对 结构、丰度或分布的帕拉转录物或蛋白质。 最后，我们将开发生殖系转化的帕拉通过使用一个 杂交基因组/cDNA融合基因，为未来的实验铺平道路， 将利用定点突变进行更详细的研究帕拉 在体内的功能和调节。 因为帕拉是唯一的钠离子通道 在任何生物体中的结构基因已经在原位突变，它提供了 一个独特的机会，以获得新的见解到分子机制 钠通道功能、表达和调节的研究。 由于 已知许多人类神经遗传性疾病与 离子通道功能的扰动，我们获得的信息 可能对理解和治疗 这些紊乱。

Information is encoded and transmitted in the nervous system in the form of electrical impulses or action potentials. Neurons are characterized by an electrically excitable membrane that enables them to receive, process and relay these impulses. Transmembrane proteins, called ion channels, form voltage-sensitive, ion-specific pores in these cells and carry out the key functions in nerve signalling by mediating the fluxes of particular ions across the membrane. Sodium channels in particular perform the central role in the generation and propagation of action potentials. Despite important recent advances, many unanswered questions remain concerning the structure, function and regulation of sodium channels. The long term goal of the work proposed here is to elucidate the function and regulation of sodium channels in Drosophila by use of a combined genetic and molecular approach. We will concentrate on the para locus, mutations of which cause lethality or temperature-sensitive paralysis correlated with a block in nerve conduction. We have cloned the para locus, determined the sequence of the encoded protein and demonstrated that para is a sodium channel structural gene. The existence of other putative sodium channel loci in Drosophila besides para suggests that, as in mammals, these genes comprise a small family whose members are differentially utilized and may subserve physiologically distinct functions. To understand the function and regulation of para in the Drosophila nervous system we now propose to characterize its expression and the distribution of its gene product and to examine the perturbations caused by mutations at para and two other loci (nap and tip-E) that apparently interfere with expression or function or para. To accomplish these goals we will use NOrthern blot and nuclease S1 experiments to determine the abundance and developmental profile of para transcripts in wild type. The normal spatial distribution of para transcripts will be characterized by tissue in situ hybridization. para- specific antibodies will be raised against synthetic peptides or para-lacZ fusion proteins and used for immunolocalization of the paraprotein in the nervous system and to identify it on Western blots. To elucidate the phenotypic effects of para, nap and tip-E mutations, similar experiments will be carried out on these mutants to determine their effect on the structure, abundance or distribution of the para transcript or protein. Finally, we will develop germline transformation for para by use of a hybrid genomic/cDNA fusion gene to pave the way for future experiments that will utilize site-directed mutagenesis for more detailed studies of para function and regulation in vivo. Because para is the only sodium channel structural gene in any organism that has been mutated in situ, it provides a unique opportunity to obtain novel insights into to molecular mechanisms of sodium channel function, expression and regulation in vivo. Since a number of human neurogenetic disease are known to be associated with perturbations in the function of ion channels, the information we obtain may have significant implications for the understanding and treatment of these disorders.