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遗传疾病 在白人人口中。在北美，大约每 2,500 人中就有 1 人 活产受到 CF 的影响，其特点是慢性 阻塞性肺病、胰腺功能不全、 外分泌腺的电解质、液体和大分子分泌。这 基本生化缺陷尚不清楚。 CF基因的鉴定 提供了了解缺陷和病理生理学的机会 该疾病在分子水平上的发展，这将使得 理性治疗。根据DNA序列分析，CF基因 产物（CFTR）预计是一种跨膜蛋白，具有 2 ATP 结合域。遗传分析显示，大约 70% CF 染色体有 3 个碱基对缺失，对应于单个 CFTR第508位氨基酸缺失。五个具体目标是 本申请中提出：（1）CF基因中的额外突变将 被识别以绘制 CFTR 的功能域；简单的 将为每个突变制定检测程序；一些 其余 30% 的 CF 的突变已被确定 染色体。 (2) 突变和“表位标签”将被引入 CFTR 的选定区域来映射其功能域和拓扑 与哺乳动物细胞质膜的关系。 (3) 对系统的影响 一些自然发生的突变的 CFTR 的生物合成， 尤其是已经检测到的无义突变和移码突变， 将进行研究以调查 CF 中是否存在真正的“无效”突变。 (4) 为了了解控制 CF 基因表达的因素， 负责基础启动子活性的序列元件和 CFTR 的组织特异性将通过常规技术确定 使用报告基因构建体（CAT）；转基因小鼠携带 CF 基因启动子和报告基因（大肠杆菌 lacZ）也将被 构建用于研究 CFTR 的发育调节。 (5)DNA序列 已在初步研究中检测到并显示，需要密切关注 人类基因组中与 CFTR 相关的部分将被分离和表征， 关于它们的染色体位置、表达模式及其 与 CFTR 的结构和功能关系。后者的信息 可能为 CF 基因的进化功能提供重要的见解 和其他 CFTR 样基因。这些研究与 2 项研究同时进行 其他单独资助的方法（使用酵母和小鼠模型）， 制定一个全面的研究计划来阐明基本缺陷 在CF中。