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. 1990 Mar;87(5):1777–1781. doi: 10.1073/pnas.87.5.1777

Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome.

T Mimori 1, Y Ohosone 1, N Hama 1, A Suwa 1, M Akizuki 1, M Homma 1, A J Griffith 1, J A Hardin 1
PMCID: PMC53566  PMID: 2308937

Abstract

Anti-Ku (p70/p80) autoantibodies in patients with scleroderma-polymyositis overlap syndrome recognize a 70-kDa/80-kDa protein heterodimer which binds to terminal regions of double-stranded DNA. In the present study, we isolated full-length cDNAs that encode the 80-kDa Ku subunit. Initial screening of a human spleen cDNA library with anti-Ku antibodies yielded a cDNA of 1.0 kilobase (kb) (termed K71) encoding a portion of the 80-kDa Ku polypeptide (identification based on immunological criteria). In RNA blots, this cDNA hybridized with two mRNAs of 3.4 and 2.6 kb. In rescreening of a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA with the K71 cDNA as a hybridization probe, three positive clones were isolated, and that bearing the longest insert (termed Ku80-6) was selected for further characterization. In vitro transcription and translation experiments produced an immunoprecipitable polypeptide which comigrated with the 80-kDa Ku subunit. The Ku80-6 cDNA proved to be 3304 nucleotides in length, with an additional poly(A) tail, closely approximating the size of the larger mRNA. It contains a single long open reading frame encoding 732 amino acids (Mr = 82,713). The putative polypeptide has a high content of acidic amino acids and a region with periodic repeat of leucine in every seventh position which may form the "leucine zipper" structure. In genomic DNA blots, probes derived from the opposite ends of cDNA Ku80-6 hybridized with several nonoverlapping restriction fragments from human leukocyte DNA, indicating that the gene encoding the 80-kDa Ku polypeptide is divided into several exons by intervening sequences.

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Selected References

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