| First Author | Wedlock DN | Year | 1996 |
| Journal | Immunol Cell Biol | Volume | 74 |
| Issue | 2 | Pages | 151-8 |
| PubMed ID | 8724002 | Mgi Jnum | J:33055 |
| Mgi Id | MGI:80542 | Doi | 10.1038/icb.1996.20 |
| Citation | Wedlock DN, et al. (1996) Molecular cloning and characterization of tumor necrosis factor alpha (TNF-alpha) from the Australian common brushtail possum, Trichosurus vulpecula. Immunol Cell Biol 74(2):151-8 |
| abstractText | 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. |
