TOPOGRAPHICAL ANALYSIS OF RHODOPSIN

描述（改编自申请人摘要）： 建议的研究是确定功能的意义，催化和 结构上，C-末端棕榈酰化和N-末端糖基化 脊椎动物视紫红质，视杆细胞色素。 聚合酶链反应 将用于通过定点诱变修饰视蛋白 糖基化和棕榈酰化位点和突变体视蛋白将被表达 在转基因小鼠中。 突变体视蛋白还将含有三个氨基酸， 牛特异性C-末端表位“标签”，其与 表位特异性单克隆抗牛视蛋白抗体，将允许 转基因“bouse”视蛋白的鉴定和定位 正常小鼠视蛋白的背景，使用常规蛋白质印迹法 和免疫细胞化学方法。 将进行初步实验， 测定“bouse”视蛋白（不含修饰的 棕榈酰化和糖基化位点），其本身不产生 结构和功能的变化。 这项建议旨在测试 假设视紫红质棕榈酰化参与失活 光活化视紫红质 它还旨在检验以下假设： 视紫红质的N-末端糖基化受损足以引起 外节的形态发生缺陷。 这些功能将 结合生物化学方法，光学显微镜 放射自显影、光镜和电镜以及免疫细胞化学。 将使用视网膜电图和吸管单细胞记录 以确定转基因表达对光转导的影响， 视网膜的整体电生理能力。

DESCRIPTION (Adapted from applicant's abstract): The purpose of the proposed research is to determine the functional significance, catalytic and structural, of C-terminal palmitoylation and N-terminal glycosylation of vertebrate rhodopsin, the rod visual pigment. Polymerase chain reaction will be utilized to modify, by site-directed mutagenesis, the opsin glycosylation and palmitoylation sites and mutant opsins will be expressed in transgenic mice. The mutant opsins will also contain a three amino acid, bovine-specific C-terminal epitope "tag" which, in conjunction with an epitope-specific monoclonal anti-bovine opsin antibody, will permit identification and localization of the transgenic "bouse" opsin against the background of the normal mouse opsin, using conventional Western blotting and immunocytochemical methods. Initial experiments will be performed to determine the level of expression of "bouse" opsin (not containing modified palmitoylation and glycosylation sites) that does not by itself produce structural and functional alternations. This proposal aims at testing the hypothesis that palmitoylation of rhodopsin is involved in the deactivation of photoactivated rhodopsin. It also aims at testing the hypothesis that impaired N-terminus glycosylation of rhodopsin is sufficient to cause defective morphogenesis of the outer segment. These features will be studied using a combination of biochemical methods, light microscopic autoradiography, and light and electron microscopy and immunocytochemistry. Electroretinography and suction pipette single cell recording will be used to determine the effects of transgene expression on phototransduction and overall electrophysiological competence of the retina.