MOLECULAR ANALYSIS OF PHOTOTRANSDUCTION IN DROSOPHILA

光传导级联是在光激发下启动的 视紫红质和终止细胞的极化由钠离子 在脊椎动物和无脊椎动物中。在果蝇身上，视网膜电信号 (ERG)测量整个视网膜的质量电梯度电位，因此 测量复眼的功能和结构完整性。 已经分离出几个ERG突变，并被证明与之有关 在光转导中。这项建议描述了一种神经性分析 这些突变中有三个。视网膜变性B(RdgB)阴性 与磷脂酶C或其酶产物肌醇相互作用 1，4，5-三磷酸，它是级联中的第二信使， 激活钠通道以产生渐变电位。不是的 瞬时A(NONA)影响光感受器电位的转导 向板层中间神经元传递信号，同时慢感受器电位(SLRP) 表现出退极化后返回基态的缺陷 光感受器的。 共分离到56个果蝇视觉系统特异的cDNA克隆，其中2个 定位于rdgB和NONA或SLRP附近的cDNA。我会分离出相应的 基因组克隆并转化果蝇种系进行检测 如果他们能够挽救突变的ERG表型，证明 克隆中含有野生型基因。然后这些基因将会是 通过内含子/外显子图谱、RNA 5‘末端分析在分子水平上进行分析， 和测序。 推导的氨基酸序列将从DNA序列中确定 并将用于搜索已知功能蛋白结构域的同源性 并通过跨膜区的疏水性图进行检测。这些数据 应该能揭示这些分子在级联中的潜在作用。离体 突变将被用来在推导出的 功能区。诱变基因将被引入苍蝇体内，并 将观察其表型。未能观察到野生型ERG与 引入的基因将表明，诱变的区域在 在蛋白质功能中的作用。此外，抗体将被提高到 检测蛋白质表达的时间和空间局部性 免疫电子显微镜将被用来定位蛋白质的功能。 这项工作的目的是为了更好地理解信号的机制 转导，特别是第二信使如何调节细胞 反应，如感光细胞去极化，以及如何第二次 信使受到管制，因此也调节了反应。

The phototransduction cascade initiates with the photoexcitation of rhodopsin and terminated with the polarization of the cell by sodium ions in vertebrates and invertebrates. In Drosophila, an electroretinogram (ERG) measures the mass electrical graded potential across the retina, thus measuring the functional and structural integrity of the compound eye. Several ERG mutations have been isolated and have been shown to be involved in phototransduction. This proposal describes a neurogenic analysis of three of these mutations. Retinal degeneration B(rdgB) negatively interacts with phospholipase C or its enzymatic product, inositol 1,4,5-triphosphate, which is a second messenger in the cascade which activates the sodium channel to produce the graded potential. No on transient A (nonA) affects the transduction of the photoreceptor potential signal to the laminal interneurons, while slow receptor potential (slrp) exhibits a defect in returning to the ground state after the depolarization of the photoreceptor. 56 Drosophila visual system-specific cDNA clones were isolated, and two cDNAs mapped near rdgB and nonA or slrp. I will isolate the corresponding genomic clones and transform them into the Drosophila germline to examine if they are capable of rescuing the mutant ERG phenotype, demonstrating that the clone contains the wild-type gene. These genes will then be analyzed at a molecular level by intron/exon mapping, RNA 5' end analysis, and sequencing. The deduced amino acid sequence will be determined from the DNA sequence and will be used to search for homology to known functional protein domains and examined by hydrophobicity plots for transmembrane regions. These data should reveal potential roles of the molecules in the cascade. In vitro mutagenesis will be used to introduce point mutations within the deduced functional regions. The mutagenized gene will be introduced into flies and the phenotype will be observed. Failure to observe a wild-type ERG with the introduced gene will suggest that the mutagenized domain plays a vital role in the protein's function. Additionally, antibodies will be raised to examine the temporal and spatial localization of protein expression and immuno-electron microscopy will be used to localize the protein's function. It is the goal of this work to better understand the mechanism of signal transduction, particularly how the second messengers regulate the cellular response, such as photoreceptor depolarization, and how the second messengers are regulated, thereby also regulating the response.