REVERSE GENETICS OF CF LOCUS USING CHROMOSOME JUMPING

利用染色体跳跃的 CF 基因座的反向遗传学

• 批准号: 3239581
• 负责人: Francis S. Collins
• 金额: $ 18.21万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-01-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3239581
• 关键词: autosomal recessive trait; chloride channels; chromosome disorders; cyclic AMP; cystic fibrosis; developmental genetics; gel electrophoresis; gene deletion mutation; genetic disorder; genetic mapping; genetic markers; genetic transcription; homozygote; linkage mapping; molecular pathology; nucleic acid probes; oncogenes

Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting the Caucasian population, leading to recurrent pulmonary infections, pancreatic insufficiency, and early death. A defect in the apical chloride ion channel in affected tissues has been described, but recent evidence suggests that the defect in CF lies in a cyclic AMP dependent regulation pathway of this channel, rather than in the channel itself. In the absence of a defined abnormality at the protein level, we propose a series of experiments to identify the CF gene by "reverse genetics". This novel approach seeks to move from the closely linked DNA markers on chromosome 7 (the proto oncogene met and the anonymous DNA fragment pJ3.11) to the CF gene itself. To do this, the new technique of chromosome jumping, which allows the traversal of 100 kilobases) or mroe of DNA in a single cloning step, will be intensively applied in order to generate multiple additional DNA probes at successively closer distances to the CF gene. In addition, a complete physical map of the CF region will be constructed using pulsed field gel electrophoresis, which is capable of resolving fragments up to 10,000 kb in size. The CF gene will be localized in this region by: 1) additional linkage analysis until probes with zero recombination are found; 2) looking for genomic deletions by standard electrophoresis and pulsed field gel electrophoresis in patients homozygous for CF; 3) identifying normal pancreatic transcripts coded for in this region; 4) identifying regions of evolutionary conservation. This combination of approaches, which is applicable to any single gene disorder for which a closely linked marker is available, should be capable of identifying the CF gene and its transcript, thereby also defining its protein product. This should allow development of a carrier test for CF, as well as at last defining the biochemical abnormality in CF, which in turn may suggest new therapeutic modalities.

囊性纤维化（CF）是最常见的常染色体隐性遗传病 影响白种人的疾病，导致复发 肺部感染、胰腺功能不全和过早死亡。 受影响组织的顶端氯离子通道存在缺陷 已被描述，但最近的证据表明，该缺陷 CF 位于这一循环 AMP 依赖性调节途径中 渠道，而不是渠道本身。 在没有一个 在蛋白质水平上定义异常，我们提出了一系列 通过“反向遗传学”鉴定CF基因的实验。 这 新方法试图摆脱紧密相连的DNA 7 号染色体上的标记（原癌基因 met 和 匿名 DNA 片段 pJ3.11) 到 CF 基因本身。 要做的事 这是染色体跳跃的新技术，它允许 在单次克隆中遍历 100 kilobase) 或 mroe DNA 步骤，将被集中应用，以产生多个 与 CF 的距离逐渐靠近的附加 DNA 探针 基因。 此外，CF区域的完整物理图将 采用脉冲场凝胶电泳构建， 能够解析最大 10,000 kb 的片段。 CF 基因将通过以下方式定位在该区域：1）额外的连锁 分析直至找到重组为零的探针； 2）寻找 通过标准电泳和脉冲场进行基因组缺失 CF 纯合子患者的凝胶电泳； 3）识别 该区域编码的正常胰腺转录本； 4） 识别进化保护区域。 这 方法组合，适用于任何单个基因 对于有密切相关标记可用的疾病，应该 能够识别CF基因及其转录本，从而也 定义其蛋白质产品。 这应该允许开发一个 CF 的携带者测试，以及最后定义生化 CF 异常，这反过来可能会提示新的治疗方法 方式。