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.
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.
Mimori
T
T
Department of Medicine, Keio University School of Medicine, Tokyo, Japan.
Ohosone
Y
Y
Hama
N
N
Suwa
A
A
Akizuki
M
M
Homma
M
M
Griffith
A J
AJ
Hardin
J A
JA
D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology* / Q000302:isolation & purification; D001327:Autoimmune Diseases / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D015723:Gene Library; D006801:Humans; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009220:Myositis / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012595:Scleroderma, Systemic / Q000235:genetics* / Q000276:immunology; D013154:Spleen / Q000276:immunology; D013577:Syndrome
D034961:Antigens, Nuclear; D000954:Antigens, Surface; D001323:Autoantibodies; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen