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.
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