The 100-kDa 2',5'-oligoadenylate synthetase catalyzing preferentially the synthesis of dimeric pppA2'p5'A molecules is composed of three homologous domains.
The 2-5A synthetases represent a family of proteins implicated in the mechanism of the antiviral action of interferon. When activated by double-stranded RNA, these proteins polymerize ATP into 2'-5'-linked oligomers with the general formula pppA(2'p5'A)n, n >/= 1. Three forms of human 2-5A synthetases have been described corresponding to proteins of 40/46 (p40/p46), 69/71 (p69/p71), and 100 kDa (p100). Here we describe the molecular cloning and characterization of p100. By screening a cDNA expression library with a specific p100 polyclonal antibody, we first isolated a 590-nucleotide cDNA fragment which was subsequently used to isolate the full-length 6365-nucleotide cDNA. This cDNA recognizes a distinct interferon-induced messenger RNA of 7 kilobases. It has an open reading frame encoding a protein of 1087 amino acids including the sequence of seven peptides obtained by microsequencing of the natural p100 protein, which was purified from interferon-treated human cells. p100 is composed of three adjacent domains, each homologous to the previously defined catalytic unit of 350 amino acids, which is present as one unit in p40/p46 and as two units in p69/p71. The recombinant p100 synthesized preferentially dimeric 2', 5'-oligoadenylate molecules and displayed parameters for maximum enzyme activity similar to the natural p100. These results confirm that the enzymatic activity of p100 is distinct compared with that of p40/p46 and p69/p71.
Rebouillat
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Unité de Virologie et Imunologie Cellulaire, ERS CNRS 572, Institut Pasteur, 75724 Paris Cédex 15, France.
Hovnanian
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A
Marié
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I
Hovanessian
A G
AG
D015088:2',5'-Oligoadenylate Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000096:biosynthesis*; D015152:Blotting, Northern; D002384:Catalysis; D018076:DNA, Complementary / Q000737:chemistry; D019281:Dimerization; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016898:Interferon-alpha / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009843:Oligoribonucleotides / Q000096:biosynthesis*; D016366:Open Reading Frames; D012330:RNA, Double-Stranded / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000227:Adenine Nucleotides; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D016898:Interferon-alpha; D009843:Oligoribonucleotides; D012330:RNA, Double-Stranded; D011994:Recombinant Proteins; C016964:adenosine triphosphate adenosine monophosphate adenosine monophosphate; D015088:2',5'-Oligoadenylate Synthetase