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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK039690-01
Application #
3239579
Study Section
Diabetes and Digestive and Kidney Diseases Special Grants Review Committee (DDK)
Project Start
1988-01-01
Project End
1990-12-31
Budget Start
1988-01-01
Budget End
1988-12-31
Support Year
1
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Wilkinson, D J; Strong, T V; Mansoura, M K et al. (1997) CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. Am J Physiol 273:L127-33
Strong, T V; Boehm, K; Collins, F S (1994) Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization. J Clin Invest 93:347-54
Cohn, J A; Strong, T V; Picciotto, M R et al. (1993) Localization of the cystic fibrosis transmembrane conductance regulator in human bile duct epithelial cells. Gastroenterology 105:1857-64
Strong, T V; Wilkinson, D J; Mansoura, M K et al. (1993) Expression of an abundant alternatively spliced form of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is not associated with a cAMP-activated chloride conductance. Hum Mol Genet 2:225-30
Sferra, T J; Collins, F S (1993) The molecular biology of cystic fibrosis. Annu Rev Med 44:133-44
Smit, L S; Nasr, S Z; Iannuzzi, M C et al. (1993) An African-American cystic fibrosis patient homozygous for a novel frameshift mutation associated with reduced CFTR mRNA levels. Hum Mutat 2:148-51
Smit, L S; Wilkinson, D J; Mansoura, M K et al. (1993) Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 90:9963-7
Koh, J; Sferra, T J; Collins, F S (1993) Characterization of the cystic fibrosis transmembrane conductance regulator promoter region. Chromatin context and tissue-specificity. J Biol Chem 268:15912-21
Collins, F S (1992) Cystic fibrosis: molecular biology and therapeutic implications. Science 256:774-9
Strong, T V; Smit, L S; Nasr, S et al. (1992) Characterization of an intron 12 splice donor mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Hum Mutat 1:380-7

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