MOLECULAR STUDIES OF RETINAL DEGENERATION IN DROSOPHILA

描述（改编自申请人的摘要）：该项目旨在 使用果蝇作为模型来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 负责W108突变体的基因的分子表征 表型。该突变被鉴定并部分表征 在上一个授予期间。申请人的高水平 对该基因的兴趣是因为突变体显示出两种共有的表型 与视紫红质运输和视网膜变性有关的基因。 这些表型是内质网（ER）的积累， 光感受器的持续反应的减少， 视网膜电图表型分析将通过以下方式进行扩展： 检查是否需要通过ER转运的蛋白质的表达 受到突变的影响，如果表型变得更严重， 当这种突变与其他突变结合时， 贩运的组成部分。 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 克隆序列并测序。此信息将用于 确定蛋白质是否与已知蛋白质共享基序，因此， 可以让我们推断出它在细胞中的功能。抗体将 产生的蛋白质，并随后用于推导 基因表达的程序，并确定相关蛋白质是否 在脊椎动物光感受器中表达。 第二个具体的目标涉及额外的基因的特性 参与细胞内运输。申请人使用了创新的 生物化学纯化标记形式的视紫红质的技术， 细胞，并计划利用这一点来寻找其他蛋白质， 视紫红质她已经证明了ninaA亲环蛋白 与视紫红质共同纯化，并将使用柱层析进行鉴定 其他相关蛋白质这些蛋白质将被识别， 特征化，然后由其产生抗体或肽序列， 该蛋白质将用于克隆相应的基因。突变株 在这些基因和突变动物中视紫红质的命运中寻找 将被调查。

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.