DROSOPHILA PARA LOCUS--GENETIC/MOLECULAR STUDIES

信息在神经系统中以以下形式编码和传输 电脉冲或动作电位。神经元的特征是一种 可电刺激的薄膜，使它们能够接收、处理和 传递这些冲动。跨膜蛋白，称为离子通道，形成 电压敏感，离子特定的孔在这些电池中，并执行密钥 通过调节特定离子通量在神经信号传递中的作用 穿过薄膜。尤其是钠通道表现为中枢 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、功能和调控。长期目标 在此提出的工作的目的是阐明其功能和调节 果蝇钠离子通道的遗传和分子结合研究 接近。我们将集中在副基因座上，它的突变导致 致命性或温度敏感型瘫痪与闭塞相关 神经传导。我们已经克隆了对位基因，测定了序列 并证明了PARA是一个钠通道 结构基因。可能存在其他可能的钠通道基因座 果蝇除了Para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，并可能为其服务 生理上不同的功能。要了解其功能和 我们现在建议对果蝇神经系统中对映体的调节 鉴定其表达及其基因产物的分布，并 检查PARA和其他两个基因座的突变引起的扰动 (NAP和TIP-E)明显干扰表情或功能或 第。段。为了实现这些目标，我们将使用Northern印迹和核酸酶S1 用实验测定卵泡刺激素的丰度和发育状况 野生型的文字记录。参照物的正态空间分布 转录本将通过组织原位杂交进行表征。Para- 将产生针对合成肽或对内酰胺的特定抗体 融合蛋白，并用于免疫定位副蛋白在 神经系统，并在蛋白质印迹上进行鉴定。为了澄清 Para、NAP和TIP-E突变的表型效应，类似的实验 将在这些突变体上进行，以确定它们对 转录本或蛋白质的结构、丰度或分布。 最后，我们将使用 混合基因组/cdna融合基因，为未来的实验铺平道路 将利用定点突变对帕金森病进行更详细的研究 在体内的作用和调节。因为PARA是唯一的钠通道 任何在原位突变的有机体中的结构基因，它提供了 获得对分子机制的新见解的独特机会 体内钠离子通道功能、表达和调控的研究。因为一个 已知许多人类神经遗传性疾病与 离子通道函数中的微扰，我们获得的信息 可能对理解和治疗该病有重要意义 这些障碍。

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.