Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus

We cloned a cDNA and the gene for Japanese flounder TNF, The TNF cDNA consi sted of 1217 bp, which encoded 225 amino acid residues. The identities betw een Japanese flounder TNF and members of the mammalian TNF family were simi lar to 22-30%. The positions of cysteine residues that are important for di sulfide bonds were conserved with respect to those in mammalian TNF-alpha. The Japanese flounder TNF gene has a length of similar to 2 kbp and consist s of four exons and three introns, The positions of the exon-intron junctio n positions of Japanese flounder TNF gene are similar to those of human TNF -alpha. However, the length of the first intron of Japanese flounder is muc h shorter than that of the human TNF-alpha gene. There are simple CA or AT dinucleotide repeats in the 5'-upstream and 3'-downstream regions of the Ja panese flounder TNF gene. Southern blot hybridization indicted that Japanes e flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-alpha than mammalian lymphotoxin-alpha, with respect to its gene struct ure, length of amino acid sequence, number and position of cysteine residue s, and regulation of gene expression.