Molecular Analyses of Hermansky-Pudlak Syndrome Genes

赫曼斯基-普德拉克综合征基因的分子分析

• 批准号: 6860993
• 负责人: RICHARD T SWANK
• 金额: $ 32.65万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2006-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6860993
• 关键词: RNase protection assay; albinism; artificial chromosomes; autoradiography; complementary DNA; denaturing gradient gel electrophoresis; diagnosis design /evaluation; gene complementation; gene expression; gene interaction; gene mutation; genetic mapping; genetic regulation; human genetic material tag; immunofluorescence technique; immunoprecipitation; laboratory mouse; molecular cloning; northern blottings; nucleic acid sequence; platelet disorder; polymerase chain reaction; single strand conformation polymorphism; southern blotting; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): The proposed research is part of a comprehensive research effort to molecularly define genes of the mouse, which cause oculocutaneous albinism (OCA), and in particular, the multigenic subform of OCA termed Hermansky-Pudlak Syndrome (HPS). HPS is a genetically heterogeneous, recessively inherited disease, which causes visual defects, hemorrhaging and significantly shortened lifespan due to fibrotic lung disease. A long-term goal is to devise diagnostic and therapeutic strategies for HPS, which presently has no efficacious treatment. The second long-term goal is to understand the mechanism of action of genes responsible for the normal biosynthesis of specialized mammalian subcellular organelles such as melanosomes, platelet dense granules and lysosomes. Two mouse HPS mutants, ruby eye and ruby eye-2, are of special interest, not only because they accurately model HPS, but also because they are phenotypic mimic mutants. Their molecular characterizations are therefore expected to lead to identification of a common pathway and perhaps a common protein complex involved in the disease. The specific aims of this proposal are to: 1) complete the identification and partial characterization of the mouse ruby eye (ru) HPS gene; 2) identify and partially characterize the closely related ruby eye-2 (ru2) HPS gene; 3) isolate human homologues of the cloned ru and ru2 HPS genes and test for alterations of these genes in human kindreds; and 4) identify interactions between the products of the ru and ru2 genes. A multidisciplinary positional/candidate cloning approach will be used to accomplish these aims including construction of high-resolution genetic maps through the use of large interspecific mouse backcrosses and construction of high-resolution physical maps through identification of overlapping contigs of RPCI-23 bacterial artificial chromosomes (BACs). Critical BACs containing the gene of interest will be identified through insertion into recipient transgenic mutant mice. Transcripts within critical BACs will be identified by a combination of exon trapping, cDNA selection and complete BAC sequencing. Transcripts containing the ruby eye and ruby eye-2 mutations will be identified by qualitative and quantitative approaches including complete cDNA sequencing and ribonuclease protection assays, respectively. To identify patients with mutations in the corresponding human genes, the cDNA sequence of the human homologue of each gene and its expression level will be determined in normal individuals and in HPS patients with no mutations in known HPS genes. Each gene will be partially characterized by assays of transcript and protein tissue distribution together with subcellular localization measurements by immunofluorescence techniques. Interaction of the ruby eye and ruby eye-2 genes will be measured by a variety of phenotypic assays in mice bred to be doubly mutant for each gene together with direct tests for interaction of their protein products by co-immunoprecipitation techniques and yeast two-hybrid approaches.

描述（由申请人提供）：拟议的研究是 全面的研究工作，以分子定义小鼠的基因，这些基因 引起眼皮白化病（OCA），尤其是多基因型 OCA称为Hermansky-Pudlak综合征（HPS）。 HPS是遗传 异质性，隐性遗传性疾病，导致视觉缺陷， 由于纤维化肺部疾病而导致的出血和显着缩短的寿命。 一个长期目标是设计HPS的诊断和治疗策略， 目前没有有效的治疗方法。第二个长期目标是 了解负责正常的基因的作用机理 专业哺乳动物亚细胞细胞器的生物合成，例如 黑素体，血小板致密颗粒和溶酶体。两个小鼠HPS突变体，红宝石 Eye和Ruby Eye-2，特别感兴趣，不仅因为它们准确 HPS模型，也是因为它们是表型模仿突变体。他们的分子 因此，预计特征会导致识别共同 途径，也许是该疾病涉及的常见蛋白质复合物。这 该提案的具体目的是：1）完成身份证明和 小鼠Ruby Eye（RU）HPS基因的部分表征； 2）识别和 部分表征密切相关的红宝石眼2（RU2）HPS基因； 3） 分离克隆RU和RU2 HPS基因的人类同源物并测试 这些基因在人类品系中的改变； 4）确定互动 在RU和RU2基因的产物之间。多学科 位置/候选克隆方法将用于实现这些目标 包括通过大型建造高分辨率遗传图 种间鼠标反向交叉和高分辨率物理的构建 通过鉴定RPCI-23细菌的重叠重叠群的地图 人造染色体（BAC）。关键的BAC包含感兴趣的基因 将通过插入接受者转基因突变小鼠来识别。 临界BAC中的笔录将通过外显子的组合确定 捕获，选择cDNA和完整的BAC测序。包含成绩单 Ruby Eye和Ruby Eye-2突变将通过定性和 定量方法，包括完整的cDNA测序和核糖核酸酶 保护测定法。识别患有突变的患者 相应的人类基因，每个人的cDNA序列 基因及其表达水平将在正常个体和 在已知HPS基因中无突变的HPS患者。每个基因将部分是 以转录本和蛋白质组织分布共同的测定为特征 通过免疫荧光技术进行亚细胞定位测量。 Ruby Eye和Ruby Eye-2基因的相互作用将通过一种品种测量 在每个基因繁殖的小鼠中的表型测定 直接测试通过 共免疫沉淀技术和酵母两种杂交方法。