MOLECULAR MECHANISM OF VISUAL EXCITATION

这个项目的长期目标是理解分子过程。 利用黑腹果蝇的潜在光感受器功能。它的用途 以果蝇为模型系统来研究视觉功能有以下几点 优点：(1)有多种视力缺陷突变体可供选择，(2) 先进的基因技术是可用的，(3)体内和体外 实验可以相对容易地进行，(4)果蝇表明 在基因的核苷酸序列上与高等哺乳动物的同源性 蛋白质的氨基酸序列。因此，仔细研究 果蝇突变系统可以给我们一个难得的机会来解剖和 在生物化学和分子水平上理解视觉通路的每一步 生物学术语。此外，从果蝇身上获得的结果 这项研究可能适用于哺乳动物系统。七个班 在果蝇体内发现了视网膜特异性多肽。其中三个 有七类基因在体内经历了光诱导的磷酸化。这个 累积的结果表明，这些视网膜特异的蛋白质和它们的 光依赖的磷酸化可能涉及视觉 流程。其中一种磷蛋白是果蝇的主要类别 视紫红质。然而，另外两个还没有被描述出来。为了 表征这些分子的性质和功能同一性 视网膜特异的磷酸蛋白，我们建议克隆编码基因 这些磷蛋白。拟议的项目采用多步骤方法。 由三个主要协议代表。首先，这两类人 视网膜特异的磷酸蛋白将被提纯到接近均一的水平 二维凝胶和蛋白质将注射到兔体内 以获得多克隆抗体。其次，通过使用抗体， 果蝇基因组DNA表达文库的筛选 鉴定含有DNA插入片段(或其中一部分)的阳性噬菌体 编码磷酸蛋白。最后，对文库进行筛选，以 获得与初始阳性克隆同源的序列。积极的一面 对克隆的cdna进行测序，推导出其氨基酸序列。 基因产物。同时，获得了这些基因的部分氨基酸序列 将测定磷蛋白以确认测序的身份 吉恩。这些努力将给我们一条线索来理解 这些视网膜特有的分子及其在视觉兴奋中的功能。

The long term goal of this project is to understand the molecular processes underlying photoreceptor function using Drosophila melanogaster. The use of Drosophila as a model system to study visual function has the following merits: (1) many kinds of vision-defective mutants are available, (2) advanced genetic techniques are available, (3) in vivo as well as in vitro experiments can be performed relatively easily, and (4) Drosophila shows homology to higher mammals in the nucleotide sequence of genes and in the amino acid sequence of proteins. Therefore, careful studies on the Drosophila mutant system could give us a rare opportunity to dissect and understand each step of the visual pathway in biochemical and molecular biological terms. Moreover, the results thus obtained from Drosophila research might be applicable to mammalian systems. Seven classes of retina-specific polypeptides have been found in Drosophila. Three of these seven classes undergo light-induced phosphorylation in vivo. The accumulated results indicate that these retina-specific proteins and their light-dependent phosphorylation are likely to be involved in visual processes. One of these phosphoproteins is the major class of Drosophila rhodopsin. The other two, however, are not yet characterized. In order to characterize the molecular nature and functional identity of these retina-specific phosphoproteins, we propose to clone the genes encoding these phosphoproteins. The proposed project employs multi-step approaches represented by three major protocols. First, these two classes of retina-specific phosphoproteins will be purified to near homogeneity on two-dimensional gels and the proteins will be injected into rabbits in order to raise polyclonal antibodies. Second, with use of the antibodies, an expression library of Drosophila genomic DNA will be screened in order to identify the positive phage containing the DNA insert (or a part of it) encoding the phosphoprotein. Finally, cDNA libraries will be screened to obtain sequences homologous to the initial positive clones. The positive cDNA clones will be sequenced in order to deduce the amino acid sequence of the gene product. At the same time a partial amino acid sequence of these phosphoproteins will be determined to confirm the identity of the sequenced gene. These efforts would give us a clue to understand the identity of these retina-specific molecules and their function in visual excitation.