REVERSE GENETICS OF CF LOCUS USING CHROMOSOME JUMPING

囊性纤维化（CF）是最常见的常染色体隐性遗传疾病， 影响高加索人群的疾病，导致复发性 肺部感染胰腺功能不全和早逝 受影响组织中顶端氯离子通道的缺陷， 但最近的证据表明， CF存在于环AMP依赖的调节途径中， 而不是在渠道本身。 在没有 在蛋白质水平上定义的异常，我们提出了一系列 通过“反向遗传学”鉴定CF基因的实验。 这 一种新的方法试图从紧密相连的DNA 7号染色体上的标志物（原癌基因met和 匿名DNA片段pJ3.11）与CF基因本身。 做 这项新的染色体跳跃技术， 100个酶的遍历）或在单个克隆中的DNA片段 步骤，将被集中应用，以产生多个 在与CF的连续更近距离处的附加DNA探针 基因 此外，CF区域的完整物理地图将 使用脉冲场凝胶电泳构建， 能够分辨大小达10，000 kb的片段。 的CF 基因将通过以下方式定位在该区域：1）额外的连锁 分析直到发现具有零重组的探针; 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）鉴定 在该区域编码的正常胰腺转录物; 4） 确定进化保护区。 这 方法的组合，其适用于任何单个基因 一个紧密连锁的标志物是可用的疾病，应该是 能够鉴定CF基因及其转录物，从而还 定义其蛋白质产物。 这将有助于开发一个 CF的携带者测试，以及最后定义的生化 CF异常，这反过来可能表明新的治疗方法 方式。

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.