MOLECULAR ANALYSIS OF PHOTOTRANSDUCTION IN DROSOPHILA

光传导级联是由红光激发而来的。 N 并以钠离子在电池中的极化终止 脊椎动物和无脊椎动物。果蝇的视网膜电信号(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。这项建议描述了对三叉神经痛的神经源性分析 E 在这些突变中。视网膜退行性变B(RdgB)与 磷脂酶C或其酶产物1，4，5-三磷酸肌醇 是级联反应中的第二个信使，它激活钠通道以 产生分级势。暂态A上的NO(NONA)影响 光感受器电位信号向板层的转导 而慢感受器电位(SLRP)则显示出一种缺陷 光感受器去极化后回到基态 共分离到56个果蝇视觉系统特异性cDNA克隆，其中2个为CDN S 已映射到rdgB和NONA或SLRP附近。我会分离出相应的基因组 克隆并将它们转化为果蝇种系，以检查它们是否 E 能够挽救突变的ERG表型，证明克隆 含有野生型基因。然后对这些基因进行分析 分子水平通过内含子/外显子作图、RNA5‘末端分析和测序 推导的氨基酸序列将从DNA序列和 将用于搜索已知功能蛋白结构域的同源性 通过跨膜区的疏水性图进行检查。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。未能观察到野生型视网膜电图 引入的基因将表明诱变结构域起着至关重要的作用 在蛋白质的功能上。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，如光感受器去极化，以及第二信使如何 S 被调节，从而也调节反应。

The phototransduction cascade initiates with the photoexcitation of rhodops n 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 thr e of these mutations. Retinal degeneration B(rdgB) negatively interacts with phospholipase C or its enzymatic product, inositol 1,4,5-triphosphate, whic 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 cDN s mapped near rdgB and nonA or slrp. I will isolate the corresponding genomi clones and transform them into the Drosophila germline to examine if they a e 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 an 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 th 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 messenge s are regulated, thereby also regulating the response.