A clue to the basic defect in cystic fibrosis from cloning the CF antigen gene

The metabolic basis of the autosomal recessive disease cystic fibrosis (CF) remains unidentified. Elevated levels of a serum protein in CF homozygotes and obligate heterozygotes have been described1. As heterozygotes are clinically unaffected, any consistently observed abnormality in these individuals is a likely pointer to the aetiology of the disease. The gene for this serum protein, called cystic fibrosis (CF) antigen, has been mapped to chromosome 1 (ref. 2). It is not the gene that is mutant in CF because this has since been assigned to chromosome 7 by cosegregation of the disease with closely linked DNA markers in CF families3–5. CF antigen is a product of normal and leukaemic granulocytes2 and is inducible in the promyelocytic cell line HL60 (M.N., J.D., C. Hayward, F. Northrop, D J.H.B., J. Walker, V. van H. and D.S.S., manuscript in preparation). We have isolated cDNA clones for this protein from a library constructed with messenger RNA from chronic myeloid leukaemia (CML) cells. The complete nucleotide sequence was obtained from the cDNA clone and by primer extension of mRNA. We have confirmed that the gene encoding CF antigen is on chromosome 1 and have localized it to a particular region. RNA blot analysis shows a 550-bp major transcript in CML cells and in induced HL60. The ammo-acid sequence predicted from the nucleotide sequence shows significant homology with intestinal and brain calcium-binding proteins. Abnormal accumulation of such a protein in CF is a clue which must be pursued now that evidence is gathering that the basic defect in CF is in pathways controlling chloride channel activity6,7.

[1]  T. Isobe,et al.  The Amino-Acid Sequence of the α Subunit in Bovine Brain S-100a Protein , 1981 .

[2]  C. Hayward,et al.  Monoclonal antibodies to cystic fibrosis antigen. , 1986, Journal of immunological methods.

[3]  M. Baggiolini,et al.  Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration , 1986, Nature.

[4]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[5]  C. Hayward,et al.  Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis , 1982, Clinical genetics.

[6]  V. Hruby,et al.  Activation of two signal-transduction systems in hepatocytes by glucagon , 1986, Nature.

[7]  C. Hayward,et al.  The Genetical Society Abstracts of Papers presented at the Two Hundred and Second Meeting of the Society on the 1st, 2nd and 3rd April 1985 at the University of Edinburgh , 1985, Heredity.

[8]  C. Fullmer,et al.  The amino acid sequence of bovine intestinal calcium-binding protein. , 1981, The Journal of biological chemistry.

[9]  K. Sato,et al.  Defective beta adrenergic response of cystic fibrosis sweat glands in vivo and in vitro. , 1984, The Journal of clinical investigation.

[10]  L. Kaczmarek,et al.  Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein. , 1986, The Journal of biological chemistry.

[11]  A. Hagemeijer,et al.  Expression of human myeloid-associated surface antigens in human-mouse myeloid cell hybrids. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[12]  F. Gros,et al.  Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. , 1981, The Journal of biological chemistry.

[13]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[14]  Peter J. Scambler,et al.  Localization of cystic fibrosis locus to human chromosome 7cen–q22 , 1985, Nature.

[15]  D. Porteous,et al.  Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Case.  Chloride ions and cystic fibrosis , 1986, Nature.

[17]  G. Rovera,et al.  Induction of differentiation of human myeloid leukemias: surface changes probed with monoclonal antibodies. , 1983, Blood.

[18]  P. Goodfellow,et al.  A human cell‐surface antigen defined by a monoclonal antibody and controlled by a gene on human chromosome 1 , 1985, Annals of human genetics.

[19]  George,et al.  Surface IgM-kappa specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. , 1968, Cancer research.

[20]  S. Collins,et al.  Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[21]  C. Wood,et al.  Impaired beta adrenergic receptor binding and function in cystic fibrosis neutrophils. , 1981, The Journal of clinical investigation.

[22]  D. Marshak,et al.  Calcium-binding proteins and the molecular basis of calcium action. , 1982, International review of cytology.

[23]  M. Houslay,et al.  Pleiotypic responses: regulation by programmable messengers or by multiple receptors? , 1986 .

[24]  J. Glenney,et al.  Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[25]  R. Kretsinger.  CRYSTALLOGRAPHIC STUDIES OF CALMODULIN AND HOMOLOGS , 1980, Annals of the New York Academy of Sciences.

[26]  V. Gerke,et al.  The regulatory chain in the p36‐kd substrate complex of viral tyrosine‐specific protein kinases is related in sequence to the S‐100 protein of glial cells. , 1985, The EMBO journal.

[27]  W. D. Benton,et al.  Screening lambdagt recombinant clones by hybridization to single plaques in situ. , 1977, Science.

[28]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Michael J. Berridge,et al.  Inositol trisphosphate, a novel second messenger in cellular signal transduction , 1984, Nature.

[30]  T. Boat,et al.  Beta adrenergic receptors in lymphocytes and granulocytes from patients with cystic fibrosis. , 1983, The Journal of clinical investigation.

[31]  P. Leder,et al.  Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Ivan Damjanov,et al.  Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line , 1979, Nature.

[33]  B. Hoffman,et al.  A simple and very efficient method for generating cDNA libraries. , 1983, Gene.

[34]  Michael Dean,et al.  A closely linked genetic marker for cystic fibrosis , 1985, Nature.

[35]  D. Glover.  DNA cloning : a practical approach , 1985 .

[36]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[37]  M. Welsh,et al.  Chloride and potassium channels in cystic fibrosis airway epithelia , 1986, Nature.

[38]  L. Tsui,et al.  A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7 , 1985, Nature.

[39]  R. Shoemaker,et al.  Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. , 1986, Science.