Molecular cloning and expression analysis of tumor necrosis factor α from a marine fish reveal its constitutive expression and ubiquitous nature

The tumor necrosis factor alpha (TNFalpha) gene from the marine fish, gilthead seabream (Sparus aurata L.), has been isolated by RT-PCR using degenerate primers designed against vertebrate TNFa conserved motifs and subsequent rapid amplification of cDNA ends (RACE). The TNFa cDNA consists of a 142 bp 5' untranslated region (5'UTR), a single open reading frame of 762 bp, which could code for a 253 amino acid protein, and a 476-bp 3'UTR. The protein sequence deduced from seabream TNFa gene shows a high degree of homology with the Japanese flounder TNFalpha (65.6% identity and 78.9% similarity) and, more important, it is more homologous to mammalian TNFalphas (41.1-48.6% similarity) than to TNFbetas (36.0-43.5% similarity). The prediction of a transmembrane domain between residues 37 and 54 of seabream TNFalpha and the presence of a conserved Thr-Leu sequence, which is associated with cleavage of the mouse TNFalpha molecule, suggest that seabream TNFalpha exists in two forms, a membrane-bound and a soluble form. RT-PCR shows that the seabream TNFalpha messenger was widely and constitutively accumulated. Lastly, stimuli known to up-regulate seabream IL-1beta, lipopolysaccharide and lymphocyte-derived macrophage-activating factor, failed to up-regulate TNFalpha in cultured macrophages. The putative role of three AU-rich endotoxin-responsive motifs (AREs) of seabream TNFalpha mRNA, found within two phylogenetically conserved protein binding regions, is discussed.