Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.
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.
Meyn
M S
MS
Department of Genetics, School of Medicine, Yale University, New Haven, CT 06510.
Lu-Kuo
J M
JM
Herzing
L B
LB
D001260:Ataxia Telangiectasia / Q000235:genetics* / Q000473:pathology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002874:Chromosome Mapping; D002886:Chromosomes, Human, Pair 17; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects / Q000648:ultrastructure*; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D007926:Lesch-Nyhan Syndrome / Q000473:pathology; D010641:Phenotype; D012313:RNA / Q000032:analysis; D017422:Sequence Analysis, DNA; D013308:Streptonigrin / Q000494:pharmacology; D014162:Transfection; D014170:Transformation, Genetic
D013308:Streptonigrin; D012313:RNA; D004247:DNA