PHOTORECEPTION AND TRANSDUCTIONS IN VISION

我们研究的目的是检查分子基础 光感受器细胞中的光接收和光传导。在这五年里 在这项计划中，我们建议进行实验，以实现以下两个具体目标： 1.重组DNA片段的体外表达和诱变 编码分子光感受器视紫红质将用于检测 它的结构和活性。在大肠杆菌和哺乳动物细胞中的表达 是提议的。视紫红质和相应的突变受体将 检查结构折叠的特性，吸收的调节 胞质结构域与其相互作用的波长和功能 CGMP级联的组成成分，例如G蛋白、视蛋白激酶和 48K蛋白。 2.两种光感受器的分离和鉴定技术 质膜大分子、Na~+/Ca~(++)交换器和 CGMP/光依赖离子通道，控制细胞信号转导 概述。我们建议对基因和信使核糖核酸进行鉴定和表征 对每个分子进行编码，以确定预测的蛋白质结构 从获得的遗传信息中获得，并最终在体外使用 表达和诱变以研究其结构和功能 活动。 我们的长期研究目标是开发重组DNA技术 补充传统的生化分离/鉴定方法 用于研究视觉传导中的蛋白质功能。如AIM中所述 这样的技术将使我们能够研究对 视觉功能，但每个细胞只有几个拷贝。此外，我们 希望启动研究，使我们能够研究 视网膜中编码视觉转导蛋白的基因。这些研究 提供有关光感受器中大分子的基本信息 以某种方式受到视网膜色素变性基因缺陷的影响 视网膜母细胞瘤。

The objective of our research is to examine the molecular basis of photoreception and transduction in photoreceptor cells. In this five year plan, we propose experiments addressing the following two specific aims: 1. In vitro expression and mutagenesis of recombinant DNA fragments encoding the molecular photoreceptor rhodopsin will be applied to examine its structure and activity. Expression in both E. coli and mammalian cells is proposed. Rhodopsin and the corresponding mutant receptors will be examined for properties of structural folding, regulation of absorption wavelength, and functional interactions of the cytoplasmic domains with components of the cGMP cascade, e.g. G-protein, opsin kinase, and 48K-protein. 2. Techniques for isolation and characterization of two photoreceptor plasma membrane macromolecules, the Na+/Ca++ exchanger and the cGMP/light-dependent ion channel, that control cell signaling are outlined. We propose to identify and characterize the genes and mRNA encoding each of the molecules, to identify the predicted protein structure from genetic information obtained, and eventually to use in vitro expression and mutagenesis to study their structure and functional activities. Our long term research objective is to develop recombinant DNA techniques to supplement traditional biochemical isolation/characterization methods for study of proteins functional in visual transduction. As noted in aim 2, such techniques would enable us to study proteins that are important for visual function but present in only few copies per cell. In addition, we wish to initiate studies that will enable us to study the expression of genes encoding visual transduction proteins in the retina. These studies contribute basic information about macromolecules in photoreceptors that are somehow affected by genetic defects in retinitis pigmentosa or retinoblastomas.