TOPOGRAPHICAL ANALYSIS OF RHODOPSIN

说明(改编自申请人的摘要)： 拟议的研究是确定其功能意义、催化和 C-末端棕榈酰化和N-末端糖基化的结构 脊椎动物视紫红质，视杆状色素。聚合酶链式反应 将被用来通过定点突变来修饰视蛋白 糖基化和棕榈酸化位点和突变的视蛋白将被表达 在转基因小鼠身上。突变的视蛋白还将包含三个氨基酸， 牛特异的C末端表位“标签”，与一种 表位特异性的抗牛视蛋白单抗，将允许 抗白粉病转基因“bouse”opsin的鉴定与定位 正常小鼠视蛋白的背景，使用常规的Western blotting 和免疫细胞化学方法。将进行初步实验以 测定“bouse”视蛋白的表达水平(不含修饰 棕榈酰化和糖基化位点)本身不会产生 结构和功能的交替。这项提案旨在测试 视紫红质棕榈酰化参与失活的假说 光激活的视紫红质。它还旨在检验这样的假设 视紫红质N末端糖基化受损足以导致 外节的形态发生有缺陷。这些功能将是 结合生物化学方法进行研究，光学显微镜 放射自显影、光镜、电子显微镜和免疫细胞化学。 将使用视网膜电描记术和吸管单细胞记录 目的：研究转基因表达对光转化的影响。 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.