Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts

• Published in: American journal of human genetics 1993-12, Vol.53 (6), p.1206-1216
• Main Authors: STEPHEN MEYN, M, LU-KUO, J. M, HERZING, L. B. K
• Format: Article
• Language: English
• Subjects: ataxia telangiectasia; Ataxia Telangiectasia - genetics; Ataxia Telangiectasia - pathology; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; Blotting, Northern; Blotting, Southern; cDNA; Cell Line, Transformed; Cell Survival - drug effects; Cell Survival - radiation effects; Chromosome Mapping; CHROMOSOMES; Chromosomes, Human, Pair 17; CLONING; Cloning, Molecular; complementation; damage; defects; DISEASES; DNA; DNA - isolation & purification; DNA HYBRIDIZATION; DNA-CLONING; Fibroblasts - drug effects; Fibroblasts - radiation effects; Fibroblasts - ultrastructure; Gene Library; GENE MUTATIONS; GENES; Genetic Complementation Test; GENETIC MAPPING; HUMAN CHROMOSOME 17; HUMAN CHROMOSOMES; Humans; HYBRIDIZATION; IMMUNE SYSTEM DISEASES; Immunodeficiencies; Immunodeficiencies. Immunoglobulinopathies; Immunopathology; isolation; Lesch-Nyhan Syndrome - pathology; man; MAPPING; Medical sciences; mutagens; MUTATIONS 550400 > Genetics; NERVOUS SYSTEM DISEASES; Phenotype; phenotypes; RADIOSENSITIVITY; RNA - analysis; sensitivity; Sequence Analysis, DNA; Streptonigrin - pharmacology; Transfection; Transformation, Genetic
• ISSN: 0002-9297; 1537-6605

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.