MOLECULAR STUDIES OF RETINAL DEGENERATION IN DROSOPHILA

描述(改编自申请者的摘要)：该项目旨在 以果蝇为模型研究遗传的分子基础 视网膜疾病。第一个具体目标是进行表型和 W108突变体致病基因的分子特征 表型。对这种突变进行了鉴定和部分表征。 在前一个授权期内。申请者的高水平 之所以对这种基因感兴趣，是因为该突变体表现出两种相同的表型 与视紫红质运输和视网膜退化有关的基因。 这些表型是内质网(ER)在 光感受器和持续反应的减少 视网膜电流图。表型分析将扩展到 检测是否需要通过内质网运输的蛋白质的表达 会受到突变的影响，如果表型变得更严重 当这种突变与其他影响 贩运组件。 确定W108突变体作用的主要努力将是 克隆这个基因。基因克隆将利用转座子标签 在基因中。基因转录本将在 克隆测序并测序。这些信息将被用于 确定该蛋白质是否与已知蛋白质有相同的基序，因此， 可以允许一个人推断它在细胞中的功能。抗体将会是 根据蛋白质产生，然后用来推断 程序进行基因表达，并确定相关蛋白质是否 在脊椎动物的光感受器中表达。 第二个具体目标涉及额外基因的特征。 参与了细胞内的运输。申请者使用了创新的 从完整视紫红质中生化纯化标记形式视紫红质的技术 细胞，并计划利用这一点来寻找其他与 视紫红质。她已经证明了NINA亲环素将 与视紫红质配伍，并将使用柱层析进行鉴定 其他相关蛋白。这些蛋白质将被鉴定并 特征化，然后产生抗体或肽序列 这种蛋白质将被用来克隆相应的基因。变种人会 在这些基因中寻找视紫红质在突变动物中的命运 将会被调查。

DESCRIPTION (Adapted from applicant's abstract): The project seeks to use Drosophila as a model for studying the molecular basis of inherited retinal diseases. The first specific aim is to conduct a phenotypic and molecular characterization of the gene responsible for the W108 mutant phenotype. This mutation was identified and partially characterized during the previous granting period. The applicant's high level of interest in this gene is because the mutant shows two phenotypes shared with genes involved in rhodopsin transport and retinal degeneration. These phenotypes are the accumulation of endoplasmic reticulum (ER) in the photoreceptors, and the reduction of the sustained response of the electroretinogram. The phenotypic analysis will be extended by examining if expression of proteins requiring transport through the ER are affected by the mutation, and if the phenotype becomes more severe when this mutation is combined with other mutations affecting trafficking components. The major effort to determine the role of the W108 mutant will be to clone this gene. Gene cloning will take advantage of the transposon tag within the gene. The gene transcript will be identified within the cloned sequence and sequenced. This information will be used to determine if the protein shares motifs with known proteins and, hence, may allow one to deduce its function in the cell. Antibodies will be generated against the protein and subsequently used to deduce the program for gene expression and determine whether related proteins are expressed in vertebrate photoreceptors. The second specific aim concerns the characterization of additional genes involved in intracellular transport. The applicant has used innovative techniques to biochemically purify a tagged form of rhodopsin from intact cells and plans to use this to find other proteins that copurify with rhodopsin. She has already shown that the ninaA cyclophilin will copurify with rhodopsin, and will use column chromatography to identify other associated proteins. These proteins will be identified and characterized, and then antibodies or peptide sequences generated from the protein will be used to clone the corresponding genes. Mutants will be sought in these genes and the fate of rhodopsin in mutant animals will be investigated.