Cloning of a complementary DNA coding for the 100-kD antigenic protein of the PM-Scl autoantigen.
Anti-PM-Scl antibodies are associated with polymyositis-scleroderma overlap or either disease alone. Among sera from 39 patients with anti-PM-Scl, 23 recognized the 100-kD band in immunoblot against HeLa cell extract, 16 of which also stained the 70-kD band. A human thymocyte lambda gt11 cDNA expression library was screened with anti-PM-Scl serum, and two clones were identified whose products reacted with 33 and 37 of 39 anti-PM-Scl sera, respectively, but none of 26 negative control sera. Affinity-purified antibody reacting specifically with plaques of the clone stained the 100-kD band on immunoblot, reacted with nucleoli of HEp-2 cells, and immunoprecipitated the PM-Scl protein complex. Partial sequences of both inserts were identical. One insert was fully sequenced, and additional 5' and 3' sequence was obtained using a gene-specific primer to form a cDNA with HeLa cell RNA as template followed by PCR. The complete nucleotide sequence included 2,739-bp coding for a predicted full-length protein of 98,088 D. There was no homology with the PM-Scl 75-kD protein and no significant homology with other proteins. A mixed-charge cluster was identified, with 22 charged amino acids of 37. In conclusion, the full-length cDNA sequence was determined coding for the PM-Scl 100-kD protein, the most commonly antigenic protein of the PM-Scl complex.
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Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Frank
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MB
O'Brien
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Targoff
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IN
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000235:genetics*; D001327:Autoimmune Diseases / Q000276:immunology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; D006801:Humans; D008969:Molecular Sequence Data; D009135:Muscular Diseases / Q000276:immunology*; D009687:Nuclear Proteins / Q000235:genetics* / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D015183:Restriction Mapping; D012595:Scleroderma, Systemic / Q000276:immunology*
D001324:Autoantigens; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C077044:EXOSC10 protein, human