ANALYSES OF GCAP IN NORMAL AND RD MUTANT RETINA

正常和 RD 突变视网膜中的 GCAP 分析

• 批准号: 2391754
• 负责人: SUSAN Lynn SEMPLE-ROWLAND
• 金额: $ 17.53万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391754
• 关键词: autoradiography; chickens; complementary DNA; cone cell; gene expression; gene mutation; guanylate cyclase; immunocytochemistry; introns; northern blottings; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phenotype; polymerase chain reaction; retina; retina degeneration; southern blotting; visual photoreceptor; western blottings

Our long-range goal is to understand the biochemical processes within photoreceptor cells, that if disrupted, lead to cell dysfunction and degeneration. The retinal degeneration (rd) chicken, the only animal model of inherited retinal disease that possesses a cone-dominant retina, is the focus of the proposed studies. Retinas of chicks homozygous for the rd mutation are fully differentiated and ultrastructurally indistinguishable from normal chick retina at hatch but do not produce scotopic or photopic ERG responses following light stimulation. Within 7- 10 days of hatch, the photoreceptors begin to degenerate. The molecular defect underlying this phenotype is not known; however, levels of cyclic GMP in the photoreceptor cells of the mutant retina are significantly reduced prior to their degeneration suggesting that the rd gene may encode a protein directly involved in photoreceptor cGMP metabolism. Mammalian photoreceptors express at least two guanylate cyclase activating proteins (GCAPs), GCAP1 and GCAP2 (p24). Western blot analyses show that normal chick retina contains a third GCAP protein that is antigenically similar to GCAP1. Western blot analyses of this protein in the rd mutant retina shows that it is not expressed in this retina. The aims of the proposed studies are (1) to clone and characterize the GCAP1 variant present in normal chicken retina that is not expressed in the mutant retina and (2) to identify possible mutations in the GCAP1 candidate gene that would disable expression of this variant in the mutant retina. GCAP cDNAs will be amplified from first-strand cDNA using the PCR and/or isolated from our normal and mutant chick retina cDNA expression libraries using antibody and cDNA probes. The clones will be analyzed by sequencing, northern and Southern blot. Anti-peptide antibodies will be generated to allow western blot and immunocytochemical analyses of the proteins encoded by these clones. Mutations found within the GCAP candidate cDNAs will be verified through gene analyses, and pedigree analyses will be carried out to confirm association of the mutation with the rd mutant phenotype. Eventually, mutant GCAP will be expressed in vitro and tested for its competence to activate guanylate cyclase. The results of these studies will improve our understanding of the cGMP metabolism and how a defect in cGMP synthesis leads to retinal disease.

我们的长期目标是了解 感光细胞，如果破坏，导致细胞功能障碍， 退化 视网膜变性（rd）鸡是唯一的动物 一种遗传性视网膜疾病模型，具有视锥细胞占优势的视网膜， 这是拟议研究的重点。 纯合子的小鸡视网膜 RD突变是完全分化，且超微结构 在孵化时与正常小鸡视网膜没有区别，但不产生 光刺激后的暗视或明视ERG反应。 在7- 孵化10天后，光感受器开始退化。 分子 这种表型的潜在缺陷尚不清楚;然而， 突变型视网膜感光细胞中GMP的表达显著降低， 在它们变性之前减少，这表明RD基因可能编码 一种直接参与光感受器cGMP代谢的蛋白质。 哺乳动物 光感受器表达至少两种鸟苷酸环化酶激活蛋白 （GCAP）、GCAP 1和GCAP 2（p24）。 蛋白质印迹分析显示正常 鸡视网膜含有第三种抗原相似的GCAP蛋白 GCAP1。 rd突变型视网膜中该蛋白的westernblot分析 显示它在视网膜中没有表达。 本研究的目的是：（1）克隆和表征 正常鸡视网膜中存在的GCAP 1变体，在正常鸡视网膜中不表达。 突变的视网膜和（2）确定GCAP 1中可能的突变 在突变体中使该变体不能表达的候选基因 视网膜。 使用PCR从第一链cDNA扩增GCAP cDNA 和/或分离自我们的正常和突变的鸡视网膜cDNA表达 使用抗体和cDNA探针的文库。 将通过以下方法分析克隆 测序、北方和南方印迹。 抗肽抗体将是 产生的蛋白质印迹和免疫细胞化学分析， 由这些克隆体编码的蛋白质。 GCAP中发现的突变 候选cDNA将通过基因分析和系谱分析进行验证。 将进行分析，以确认突变与 rd突变体表型。 最终，突变的GCAP将在 体外并测试其激活鸟苷酸环化酶的能力。 的 这些研究的结果将提高我们对cGMP的理解 以及cGMP合成缺陷如何导致视网膜疾病。