STUDIES OF VISUAL CYCLE PROTEINS

拟议研究的主要重点是表征 编码细胞视黄醇结合蛋白的人基因 （CRALBP），并鉴定其内的功能结构域。 CRALBP的三维结构。 CRALBP可能是一个 视觉周期的功能组件，也许可以作为一个 底物载体和/或立体选择性试剂。 CRALBP携带作为 内源性配体类维生素A仅已知在 视觉，即11-顺式-视黄醇和11-顺式-视黄醇醛， 在对光有反应的组织（视网膜和松果体）中发现。 将适当注意克隆和测序 人CRALBP基因的分析和鉴定 负责组织特异性表达的调节区。 一 此外，政府亦会共同努力， 负责类维生素A的结合和与视网膜的相互作用 色素上皮视黄醇脱氢酶和视黄酯 合成酶 蛋白质的细胞定位将是 通过原位技术证实， 进行人类基因测定和临床连锁研究。 的 CRALBP可能参与遗传性视网膜疾病， 因为色素性视网膜炎是主要关注的。 长远目标 是通过基因/蛋白质工程研究来定义 蛋白质的结构特征，负责其 立体选择性高、光敏性低。 该基因的调控区将通过初级RPE表征 和Muller细胞培养转染CAT测定。 CRALBP mRNA 将在RPE和Muller细胞培养物中测量水平， 用各种类固醇激素、生长因子和 类维生素A。 整个蛋白质将在一个 有限数量的肽（16-30个残基/肽），通过自动 固相方法和抗CRALBP肽抗体的产生， 家兔 功能结构域将通过筛选 功能阻断抗体和肽以及有限的 蛋白水解 拟议研究的综合结果将 提供了一个精确的分子框架， CRALBP的正常功能及其可能引起的视觉障碍 可以用具体的术语来回答。

The major emphasis of the proposed research is to characterize the human gene encoding the cellular retinaldehyde-binding protein (CRALBP) from retina and to identify functional domains within the three-dimensional structure of CRALBP. CRALBP could be a functional component of the visual cycle, perhaps serving as a substrate carrier and/or stereoselective agent. CRALBP carries as endogenous ligands retinoids that are only known to function in vision, namely 11-cis-retinol and 11-cis-retinaldehyde and has only been found in tissues that respond to light (retina and pineal). Appropriate attention will be given to cloning and sequence analysis of the human CRALBP gene and to identification of regulatory regions responsible for tissue-specific expression. A concerted effort will also be made to identify the domains responsible for retinoid binding and interaction with retinal pigment epithelium (RPE) retinol dehydrogenase and retinyl ester synthase. The cellular localization of the protein will be confirmed by in situ techniques, the chromosomal location of the human gene determined and clinical linkage studies pursued. The possible involvement of CRALBP in hereditary retinal diseases such as retinitis pigmentosa is of major interest. A long-range goal is to define through genetic/protein engineering studies the structural features of the protein that are responsible for its high stereoselectivity and low photosensitivity. Regulatory regions of the gene will be characterized by primary RPE and Muller cell culture transfection CAT assays. CRALBP mRNA levels will be measured in RPE and Muller cell culture following treatment with various steroid hormones, growth factors and retinoids. The whole protein will be chemically synthesized in a limited number of peptides (16-30 residues/peptide) by automatic solid phase methods and anti-CRALBP peptide antibodies raised in rabbits. Functional domains will be probed by screening for function-blocking antibodies and peptides and by limited proteolysis. The combined results from the proposed research will provide a precise molecular framework in which questions concerning the normal function of CRALBP and the visual disorders it may be associated with can be answered in specific terms.