Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-alpha

Kerry J Laing, Tiehui Wang, Jun Zou, Jason Holland, Suhee Hong, Niels Bols, Ikuo Hirono, Takashi Aoki, Christopher J Secombes

Research output: Contribution to journalArticle

213 Citations (Scopus)

Abstract

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)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.
Original languageEnglish
Pages (from-to)1315-1322
Number of pages8
JournalEuropean Journal of Biochemistry
Volume268
Issue number5
DOIs
Publication statusPublished - Mar 2001

Keywords

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • Exons
  • Gene Expression Profiling
  • Gene Library
  • Gills
  • Glycosylation
  • Humans
  • Interleukin-1
  • Introns
  • Kidney
  • Lipopolysaccharides
  • Macrophages
  • Molecular Sequence Data
  • Oncorhynchus
  • Open Reading Frames
  • RNA, Messenger
  • Sequence Alignment
  • Transcriptional Activation
  • Tumor Necrosis Factor-alpha
  • Gene structure
  • mRNA expression
  • Rainbow Trout
  • Tumour Necrosis
  • Growth-Factor-Beta
  • Messenger-RNA
  • Molecular-Cloning
  • TNF-alpha
  • Sequence
  • Interleukin-1-Beta
  • Receptor
  • Region
  • Genes
  • Chemokine

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