HUMAN RETINAL HOMOLOGS OF DROSOPHILA EYE GENES

果蝇眼睛基因的人类视网膜同源物

• 批准号: 2162882
• 负责人: SEYMOUR BENZER
• 金额: $ 22.45万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2162882
• 关键词: Drosophilidae; alternatives to animals in research; cell differentiation; cell growth regulation; complementary DNA; computer assisted sequence analysis; disease /disorder model; fusion gene; gene expression; gene mutation; gene therapy; genetic mapping; genetic promoter element; germ cells; human genetic material tag; human tissue; laboratory mouse; laboratory rabbit; molecular cloning; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phenotype; polymerase chain reaction; protein structure function; retina; retina disorder; retinoblastoma; rhodopsin; structural genes; tissue /cell culture; visual phototransduction; western blottings

Humans and flies display a wide variety of genetic defects affecting the eye. Our goal is to elucidate the functions of genes expressed in the human retina by using Drosophila as a model system. We will isolate human genes with homology to Drosophila eye genes, and Drosophila homologs of human genes involved in retinal disease, and use them for functional studies in Drosophila. A set of previously isolated eye-specific Drosophila cDNAs will be used to isolate potential functional human retinal cDNA homologs. The Drosophila cDNAs include several with known sequence homology to human eye genes, such as arrestin and rhodopsin, as well as others of novel sequence. Mutations will be induced in the fly genes corresponding to the Drosophila eye cDNAs, and the resultant phenotypes characterized for anatomical and functional defects in the retina and visual transduction pathways. We have also identified other Drosophila genes based on striking defects in retinal differentiation, or expression patterns of interest to visual system development or function. These genes are currently being cloned in our laboratory, and will also be used to isolate candidate human homologs. To test whether a putative human homolog expresses a similar function in the fly, we win attempt to rescue the fly mutant phenotype with the human cDNA. This will be done by constructing hybrid genes, in which Drosophila promoter sequences are linked to the protein-encoding regions of the human homologs. These constructs will be tested for gene and protein expression in Drosophila tissue culture cells, then introduced into the Drosophila germ line by P element-mediated transformation, and analyzed for expression in the fly. Partial or full rescue of the phenotype will be evidence for conservation of function. This methodology will be tested by using human rhodopsin to rescue the phenotype of the Drosophila rhodopsin mutant, hinae. We will also search for Drosophila homologs of the human retinoblastoma gene, for which a clone is available. Drosophila homologs of the gene will be searched for by low stringency hybridization and PCR amplification of regions of the gene conserved through vertebrate evolution. The function will be investigated by the molecular and genetic methodologies available in Drosophila.

人类和苍蝇表现出各种影响的遗传缺陷 眼睛。 我们的目标是阐明在 人类视网膜通过使用果蝇作为模型系统。 我们将孤立人类 与果蝇眼基因同源的基因和果蝇同源物的基因 人类基因参与视网膜疾病，并将其用于功能 果蝇研究。 一组先前隔离的眼睛特异性果蝇cDNA将用于 分离潜在的功能性人视网膜cDNA同源物。 果蝇 cDNA包括与人眼基因具有已知序列同源性的几种，这样的 作为抑制素和视紫红质，以及其他新序列。 突变 将在对应于果蝇眼cDNA的蝇基因中诱导 以及用于解剖学和功能的表征的结果表型 视网膜和视觉转导途径中的缺陷。 我们也有 基于视网膜中的醒目缺陷确定了其他果蝇基因 视觉系统感兴趣的分化或表达模式 开发或功能。 这些基因目前被克隆在我们的 实验室，还将用于隔离候选人类同源物。 测试假定的人类同源物在 苍蝇，我们赢得了与人类一起营救蝇突变表型的尝试 cDNA。 这将通过构建杂种基因来完成，其中果蝇中 启动子序列与人类的蛋白质编码区域有关 同源物。 这些结构将用于基因和蛋白质表达 在果蝇组织培养细胞中，然后引入果蝇中 通过P元素介导的转化生殖系，并分析表达 苍蝇。 表型的部分或完全营救将是证据 功能保护。 该方法将通过人类进行测试 Rhodopsin挽救果蝇Rhopopsin突变体的表型， Hinae。 我们还将搜索人类的果蝇同源物 视网膜母细胞瘤基因，为此克隆。 果蝇同源物 该基因将通过低严格杂交和PCR搜索 通过脊椎动物保存的基因区域的扩增 进化。 该功能将通过分子和遗传来研究 果蝇可用的方法。