Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor‐α

• DOI: 10.1046/j.1432-1327.2001.01996.x
• Publication Date: 2003-03-11T18:25:13Z
• Authors: Kerry J. Laing; Tiehui Wang; Jun Zou; Jason Holland; Suhee Hong; Niels Bols; Ikuo Hirono; Takashi Aoki; Christopher J. Se...
• Keywords: Gills; Lipopolysaccharides; Glycosylation; Base Sequence; Oncorhynchus; Gene Expression Profiling; Macrophages; Molecular Sequence Data; Exons; Kidney; Introns; Cell Line; Open Reading Frames; 03 medical and health sciences; 0302 clinical medicine; Animals; Humans; Amino Acid Sequence; RNA, Messenger; Cloning, Molecular; Gene Library; Interleukin-1

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino‐acid putative peptide, with a 5′ untranslated region (UTR) of 140 bp and a 3′ UTR of 506 bp. Two potential N‐linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT‐PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up‐regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)‐1β. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL‐1β, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF‐α is discussed.