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.
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