title: Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula.
abstract: Immune responses in the Australian common brushtail possum (Trichosurus vulpecula) and in particular the role of cytokines are poorly understood. We have undertaken to isolate cytokine genes using reverse transcriptase-polymerase chain reaction (RT-PCR) and in this study describe the molecular cloning of TNF-alpha. Primers were designed from consensus sequences at the N-terminus end of eutherian mammalian TNF-alpha and the possum cDNA, derived from spleen RNA, identified by RT-PCR. The complete cDNA encoding possum TNF-alpha was amplified from lymphocyte RNA by 5' and 3' rapid amplification of cDNA ends (RACE). The nucleotide sequence of the protein coding region of this cDNA shared 66-69% identity with other mammalian TNF-alpha genes. The predicted protein of 233 amino acids shared 56-58% identity with eutherian mammalian TNF-alpha was expressed in both Saccharomyces cerevisiae and Escherichia coli by constructing expression plasmid derivatives of the vectors pYES2 and pGEX-2T respectively. Cell extracts prepared from transformants and the purified GST/TNF-alpha fusion protein exhibited cytotoxic activity on the TNF-alpha-sensitive murine fibroblast L929 cells and stimulated proliferation of possum thymocyte cells. The induction of possum TNF-alpha mRNA in alveolar macrophages was analysed by RT-PCR using possum-specific TNF-alpha primers. Macrophages cultured in the presence of LPS showed enhanced transcription of TNF-alpha mRNA. This is the first report of the cloning and sequence analysis of the cDNA encoding a marsupial cytokine gene.
authors.affiliation: AgResearch, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
authors.forename: D N
authors.initials: DN
authors.lastname: Wedlock
authors.forename: F E
authors.initials: FE
authors.lastname: Aldwell
authors.forename: B M
authors.initials: BM
authors.lastname: Buddle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001315:Australia; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D016676:Macrophages, Alveolar / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D009893:Opossums / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha