MOLECULAR ANALYSIS OF THE CYSTIC FIBROSIS LOCUS

我们的研究计划的长期目标是确定 囊性纤维化（CF）。 我们采取的方法 最初绕过了对疾病中特定缺陷的搜索。 相反，我们的目标是确定DNA标记（探针）密切相关， 在7 q31的疾病位点，然后使用这些标记， 寻找CF基因的参考点。 虽然一些 紧密连锁的DNA标记最近已经分离出来，所有 现有数据表明， 这些标记和CF基因座之间的联系 在本申请中，我们 建议从CF区域分离额外的DNA标记， 将它们用作探针， 电泳以产生跨越 假定的基因位点，从CF突变本身可以 最终被识别。 将克隆适合用作探针的基因组DNA片段， 从流式分选的7号染色体特异性文库中分离 由洛斯阿拉莫斯和劳伦斯利弗莫尔国家 Laboratories. 这些探针中的每一个都将用于杂交 从一组人-啮齿动物体细胞中分离的DNA的分析 每个都含有人类7号染色体材料的一个子集的杂种 跨越Q31区域。 目前共有13种DNA探针 在我们的收藏中。 据估计， 35-40个DNA探针将足以使CF区域饱和， 标记。 将鉴定限制性片段长度多态性， 在q31区域中的每种探针， 确定连锁关系 这些DNA标记和CF之间的联系 这些家庭已经被证明 在我们以前的研究中，在CF附近包含交叉点 基因座 这些DNA标记的相对顺序 与CF的连锁将进一步通过连锁不平衡来检验 分析和脉冲场凝胶电泳。 基于 结合遗传和物理图谱，将有可能启动 从最接近CF位点的点进行染色体步移， 系统地搜索在这些基因中表达的序列， 受影响的组织 这些实验将与其他正在进行的实验同时进行。 利用受影响的上皮细胞和组织的研究 在CF。 具体来说，上皮细胞cDNA文库正在被 构建并克隆对应于7 q31区域中的基因 正在被确定和测试， 疾病 还在试图建立永久性的合作框架 上皮细胞系，以开发用于 CF基因通过DNA转染的方式来纠正离子 这些细胞中的运输缺陷。 结合这些 各种分子遗传学方法，CF的基本缺陷将 解决。

The long term objective our research program is to define the basic defect in cystic fibrosis (CF). The approach we have taken initially bypasses the search for a specific defect in the disease. Instead, our aim is to identify DNA markers (probes) closely linked to the disease locus at 7q31 and to then use these markers as reference points to search for the CF gene. Although a number of tightly linked DNA marker have been isolated recently, all available data suggest that there is still a considerable distance between these markers and the CF locus. In this application, we propose to isolate additional DNA markers from the CF region and to use them as probes in combination with pulsed field gel electrophoresis to generate a physical (restriction) map spanning the putative gene locus, from which the CF mutation itself can eventually be identified. Cloned genomic DNA fragments suitable for use as probes will be isolated from the flow sorted chromosome 7-specific library constructed by the Los Alamos and Lawrence Livermore National Laboratories. Each of these probes will be used in hybridization analysis with DNA isolated from a set of human-rodent somatic cell hybrids each containing a subset of human chromosome 7 material spanning the q31 region. A total of 13 DNA probes are now available in our collection. It is estimated that an additional 35-40 DNA probes will be sufficient to saturate the CF region with markers. Restriction fragments length polymorphisms will be identified for each of the probes in the q31 region and studied in a small number of informative families to determine the linkage relationship between these DNA markers and CF. These families have been shown tin our previous studies to contain crossover points near the CF locus. The relative order for those DNA markers that are tightly linked to CF will be further examined by linkage disequilibrium analysis and pulsed field gel electrophoresis. Based on the combined genetic and physical map, it will be possible to initiate chromosome walking from points closet to the CF locus and systematically search for sequences that are expressed in the affected tissues. These experiments will be performed in parallel with other ongoing studies utilizing epithelial cells and tissues that are affected in CF. Specifically, epithelial cell cDNA libraries are being constructed and clones that correspond to genes in the 7q31 region are being identified and tested for their involvement in the disease. Attempts are also being made to establish permanent CF epithelial cell lines in order to develop a functional assay for the CF gene by means of DNA transfection to correct the ion transport defect in these cells. With a combination of these various molecular genetic approaches, the basic defect in CF will be resolved.