Molecular cloning and expression analysis of tumor necrosis factor alpha from a marine fish reveal its constitutive expression and ubiquitous nature.
The tumor necrosis factor alpha ( TNF alpha) gene from the marine fish, gilthead seabream (Sparus aurata L.), has been isolated by RT-PCR using degenerate primers designed against vertebrate TNF alpha conserved motifs and subsequent rapid amplification of cDNA ends (RACE). The TNF alpha 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 TNF alpha gene shows a high degree of homology with the Japanese flounder TNF alpha (65.6% identity and 78.9% similarity) and, more important, it is more homologous to mammalian TNF alphas (41.1-48.6% similarity) than to TNF betas (36.0-43.5% similarity). The prediction of a transmembrane domain between residues 37 and 54 of seabream TNF alpha and the presence of a conserved Thr-Leu sequence, which is associated with cleavage of the mouse TNF alpha molecule, suggest that seabream TNF alpha exists in two forms, a membrane-bound and a soluble form. RT-PCR shows that the seabream TNF alpha messenger was widely and constitutively accumulated. Lastly, stimuli known to up-regulate seabream IL-1 beta, lipopolysaccharide and lymphocyte-derived macrophage-activating factor, failed to up-regulate TNF alpha in cultured macrophages. The putative role of three AU-rich endotoxin-responsive motifs (AREs) of seabream TNF alpha mRNA, found within two phylogenetically conserved protein binding regions, is discussed.
García-Castillo
Jesús
J
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.
Pelegrín
Pablo
P
Mulero
Victoriano
V
Meseguer
José
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis; D021541:Sea Bream / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D016415:Sequence Alignment; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*
D029941:Fish Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha