BIOCHEMISTRY AND MOLECULAR BIOLOGICAL

该研究计划的总体和长期目标是 研究视网膜功能所必需的分子，并识别和 视网膜谷氨酸丰富蛋白（GARP）的表征， 先前在与棒共纯化的牛棒外节段中发现 cGMP磷酸二酯酶。 最近，GARP被证明是 与cGMP门控阳离子的第二亚基共价连接 牛视杆细胞光感受器通道，使其成为候选基因， 视网膜疾病 至少一种形式的Bardet-Biedl综合征，a 包括色素性视网膜炎在内的全身性发育障碍， 与16号染色体长臂相连 我们发现人类 garp基因定位于同一区域，这使我们认为， GARP的缺陷可能与这种疾病的病因有关。 到 为了研究这种可能性，将首先对garp基因进行表征， 在正常和Bardet-Biedl患者的DNA中使用异源双链体， 单链确认多态性分析。 任何点突变 将通过直接测序来研究这些试验所建议的 PCR片段。 结合基因特征研究， 将进行人GARP的免疫学表征， 确定这种蛋白质是否与类似的通道相关联， 对牛的态度。 最后，分析了GARP在 将使用核糖核酸酶研究新生和成年小鼠 保护试验和定量Western分析。 成功 这些实验的完成将提供有价值的见解， 严重形式的视网膜色素变性的分子基础，并提供 必要的信息，以调查GARP的功能中的作用， cGMP门控阳离子通道

The general and long term objectives of this research program are to investigate molecules essential to retinal function, and to identify and characterization of a retinal glutamic acid rich protein (garp) which was previously identified in bovine rod outer segments copurifying with rod cGMP phosphodiesterase. More recently, garp has been shown to be covalently linked with the second subunit of the cGMP-gated cation channel of bovine rod photoreceptors, making it a candidate gene for retinal disorders. At least one form of Bardet-Biedl syndrome, a systemic disorder of development that includes retinitis pigmentosa, has been linked to the long arm of chromosome 16. Our finding that the human garp gene is localized to the same region leads us to suggest that defects in garp may be involved in the etiology of this disease. To investigate this possibility, the garp gene will first be characterized in the DNA of normal and Bardet-Biedl patients using heteroduplex and single strand confirmation polymorphism analysis. Any point mutations suggested by these assays will be investigated by direct sequencing of PCR fragments. In conjunction with the gene characterization studies, an immunological characterization of human garp will be undertaken to determine if this protein associates with the channel in an analogous manner to bovine garp. Finally, the expression pattern of garp in neonatal and adult mice will be investigated using ribonuclease protection assay and quantitative Western analysis. Successful completion of these experiments will provide valuable insight into molecular basis of a severe form of retinitis pigmentosa, and provide the information necessary to investigate the role of garp in the function of the cGMP-gated cation channel.