REVERSE GENETICS OF CF LOCUS USING CHROMOSOME JUMPING

囊性纤维化是最常见的常染色体隐性遗传 影响高加索人口的疾病，导致复发 肺部感染、胰腺功能不全和过早死亡。 受影响组织中顶端氯离子通道的缺陷 已经被描述过了，但最近的证据表明 Cf存在于环状AMP依赖的调节通路中 渠道，而不是在渠道本身。在缺少 在蛋白质水平上定义异常，我们提出了一系列 用“反向遗传学”鉴定Cf基因的实验。这 一种新的方法寻求从紧密相连的DNA 7号染色体上的标记(原癌基因MET和 匿名DNA片段pJ3.11)连接到Cf基因本身。去做 这是一种新的染色体跳跃技术，它允许 在单个克隆中遍历100千个碱基)或mroe的DNA 步骤，将被密集应用，以产生多个 额外的DNA探针，距离CF依次更近 吉恩。此外，一张完整的CF区域物理地图将 使用脉冲场凝胶电泳构建，这是 能够解析大小达10,000 kb的片段。CF卡 基因将通过以下方式定位于该区域：1)额外的连锁 分析，直到找到零重组的探针；2)寻找 用标准电泳法和脉冲场进行基因组缺失 Cf纯合子患者的凝胶电泳法；3)鉴定 该区域编码的正常胰腺转录本；4) 确定进化保守区。这 方法的组合，适用于任何单个基因 有紧密连锁标记的疾病，应该是 能够识别Cf基因及其转录本，从而还 定义它的蛋白质产品。这应该允许开发一种 Cf的载体试验，以及最终确定的生化 CF的异常，这反过来可能提示新的治疗方法 医疗模式。

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.