MOLECULAR ANALYSIS OF PHOTOTRANSDUCTION IN DROSOPHILA

光传导级联反应由以下物质的光激发引发： 视紫红质和终止与极化的细胞由钠离子 在脊椎动物和无脊椎动物中。 在果蝇中，视网膜电图 (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。 已分离出几种ERG突变，并已显示与 在光传导中。 该提案描述了一种神经原性分析， 其中三种突变。 视网膜变性B（rdg B）阴性 与磷脂酶C或其酶促产物肌醇相互作用 1，4，5-三磷酸，这是级联反应中的第二信使， 激活钠通道产生梯度电位。 No对 瞬时A（nonA）影响感光细胞电位的转导 信号传递到板层中间神经元，而慢受体电位（slrp） 在去极化之后返回到基态时表现出缺陷 感光细胞的。 分离了56个果蝇视觉系统特异性cDNA克隆， 定位在rdgB和nonA或slrp附近的cDNA。 我会分离出相应的 基因组克隆，并将其转化为果蝇种系， 如果它们能够挽救突变的ERG表型， 克隆体含有野生型基因 这些基因将被 在分子水平上通过内含子/外显子作图，RNA 5'末端分析， 和测序。 推导的氨基酸序列将由DNA序列确定 并将用于搜索与已知功能蛋白质结构域的同源性 并通过跨膜区的疏水性图进行检查。 这些数据 应该能揭示级联反应中分子的潜在作用。 体外 诱变将被用于在推导的基因组中引入点突变。 功能区。 诱变基因将被引入果蝇， 将观察表型。 未观察到野生型ERG， 引入的基因将表明，诱变的结构域起着重要的作用， 在蛋白质功能中的作用。 此外，抗体将被提高到 检查蛋白质表达的时间和空间定位， 免疫电子显微镜将用于定位蛋白质的功能。 本文的目的是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，以及如何第二 信使受到调节，从而也调节反应。

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.