MOLECULAR MECHANISM OF VISUAL EXCITATION

该项目的长期目标是了解分子过程 使用果蝇的基础感光功能。 使用 果蝇作为研究视觉功能的模型系统具有以下特点 优点：（1）有多种视觉缺陷突变体，（2） 先进的遗传技术是可用的，（3）在体内以及在体外 实验可以相对容易地进行，（4）果蝇表明， 与高等哺乳动物在基因的核苷酸序列和 蛋白质的氨基酸序列 因此，仔细研究 果蝇突变体系统可以给我们一个难得的机会来解剖， 了解生物化学和分子生物学中视觉途径的每一步 生物学术语 此外，从果蝇中获得的结果 研究可能适用于哺乳动物系统。 七类 在果蝇中发现了视网膜特异性多肽。 其中三 七类在体内经历光诱导的磷酸化。 的 累积的结果表明，这些视网膜特异性蛋白及其 光依赖性磷酸化可能参与视觉 流程. 其中一种磷蛋白是果蝇的主要种类 视紫红质 然而，另外两个还没有特征。 为了 表征这些分子的分子性质和功能特性 视网膜特异性磷蛋白，我们建议克隆基因编码 这些磷蛋白。 拟议项目采用多步骤方法 由三个主要协议代表。 首先，这两类 视网膜特异性磷蛋白将被纯化至接近同质， 二维凝胶和蛋白质将被注射到兔子体内， 以产生多克隆抗体。 第二，通过使用抗体， 将筛选果蝇基因组DNA的表达文库， 鉴定含有DNA插入片段（或其一部分）的阳性噬菌体 编码磷蛋白。 最后，将筛选cDNA文库， 获得与初始阳性克隆同源的序列。 的积极 将对cDNA克隆进行测序，以推导出 基因产物。 与此同时，它们的部分氨基酸序列 将测定磷蛋白以确认测序的 基因 这些努力将为我们提供一条线索， 这些视网膜特异性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.