MOLECULAR GENETICS OF CYSTIC FIBROSIS

囊性纤维化的分子遗传学

• 批准号: 3233191
• 负责人: LAP-CHEE TSUI
• 金额: $ 9.46万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233191
• 关键词: SDS polyacrylamide gel electrophoresis; complementary DNA; cystic fibrosis; developmental genetics; diagnosis design /evaluation; endonuclease; female; gene expression; gene mutation; genetic disorder diagnosis; genetic manipulation; genetic markers; genetic polymorphism; genetically modified animals; human genetic material tag; human population genetics; human subject; laboratory mouse; linkage mapping; molecular cloning; molecular genetics; molecular pathology; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; reporter genes; thin layer chromatography; tissue /cell culture

Cystic fibrosis (CF) is the most common severe autosomal recessive disorder in the Caucasian population. In North America, approximately 1 in 2,500 live-births is affected with CF, which is characterized by chronic obstructive lung disease, pancreatic insufficiency, abnormalities of electrolyte, fluid and macromolecule secretion of exocrine glands. The basic biochemical defect is unknown. The identification of the CF gene has provided an opportunity to understand the defect and the pathophysiology of the disease at the molecular level, which will allow the development of rational therapy. On the basis of DNA sequence analysis, the CF gene product (CFTR) is predicted to be a transmembrane protein with 2 ATP-binding domains. Genetic analysis shows that approximately 70% of the CF chromosomes suffer a 3 base pair deletion which corresponds to a single amino acid deletion at position 508 of CFTR. Five specific aims are proposed in this application: (1) Additional mutations in the CF gene will be identified in order to map the functional domains of CFTR; simple detection procedures will be developed for each mutation; a number of mutations have already been identified for the remaining 30% of CF chromosomes. (2) Mutations and "epitope tags" will be introduced into selected regions of CFTR to map its functional domains and topology in relation to the plasma membrane in mammalian cells. (3) The effect on the biosynthesis of CFTR of some of the naturally occurring mutations, especially the nonsense and frameshift mutations that have been detected, will be studied to investigate if true "null" mutations exist in CF. (4) In order to understand the factors governing CF gene expression, the sequence elements responsible for basal promoter activity and tissue-specificity for CFTR will be determined by conventional techniques with the use of reporter gene constructs (CAT); transgenic mice carrying the CF gene promoter and a reporter gene (E. coli lacZ) will also be constructed to study developmental regulation of CFTR. (5) DNA sequences that have been detected and shown in preliminary studies, to be closely related to CFTR in the human genome will be isolated and characterized, with respect to their chromosomal location, expression pattern, and their structural and functional relationships with CFTR. The latter information may provide important insight into the evolution function of the CF gene and other CFTR-like genes. These studies, conducted in parallel with 2 other separately funded approaches (use of yeast and mouse models), constitute a comprehensive research program in elucidating the basic defect in CF.

囊性纤维化（CF）是最常见的严重常染色体隐性疾病 在高加索人口中。在北美，大约有2500分之一 Live-births受CF的影响，CF的特征是慢性 阻塞性肺部疾病，胰腺功能不全，异常 外分泌腺的电解质，液体和大分子分泌。这 基本的生化缺陷尚不清楚。 CF基因的识别具有 提供了了解缺陷和病理生理学的机会 分子水平的疾病，这将允许发展 理性疗法。根据DNA序列分析，CF基因 产品（CFTR）被预测为跨膜蛋白，2 ATP结合域。遗传分析表明，约有70％ CF染色体患有3个基对删除，对应于一个 CFTR的位置508处的氨基酸缺失。五个具体目标是 在此应用中提出的：（1）CF基因中的其他突变将 为了映射CFTR的功能域；简单的 每个突变都将制定检测程序；许多 已经确定了其余30％CF的突变 染色体。 （2）将引入突变和“表位标签” CFTR的选定区域以绘制其功能域和拓扑 与哺乳动物细胞中的质膜有关。 （3）对 某些天然发生的突变的CFTR的生物合成， 特别是检测到的胡说八道和移码突变 将研究以研究CF中是否存在真正的“零”突变。 （4） 为了了解有关CF基因表达的因素， 负责基础启动子活性和 CFTR的组织特异性将由常规技术确定 使用记者基因构建体（CAT）；转基因小鼠携带 CF基因启动子和报告基因（大肠杆菌LACZ）也将是 构建以研究CFTR的发展调节。 （5）DNA序列 在初步研究中已检测到并显示了 与人类基因组中的CFTR有关 关于它们的染色体位置，表达方式及其 与CFTR的结构和功能关系。后者的信息 可以提供有关CF基因的演化函数的重要洞察力 和其他类似CFTR的基因。这些研究与2平行进行 其他单独资助的方法（使用酵母和小鼠模型）， 构成阐明基本缺陷的全面研究计划 在CF中。