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- Title
Molecular cloning, expression and characterization of ovine TNFa.
- Authors
Nash, A. D.; Barcham, G. J.; Brandon, M. R.; Andrews, A. E.
- Abstract
Tumour necrosis factor α (TNF α) is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. By hybridization with a human TNF α cDNA probe the corresponding ovine cDNA was isolated from a lipopolysaccharide (LPS) stimulated alveolar macrophage cDNA library. The sequence of the cDNA clone showed that ovine TNF α encodes a polypeptide of 234 amino acids that, based on analysis of human TNF α, is processed to a protein of 157 amino acids. The nucleotide and amino acid sequences showed a high degree of homology to eh equivalent human and mouse molecules. In a mammalian COS cell expression system the ovine cDNA was found to encode a protein which was able to lyse actinomycin-D treated WEHI-164 cells and induce COS cells to produce and secrete interleukin 6 (IL-6). Further experiments domnstrated the importance of sequences within the 3' untranslated region of the cDNA in determining the level of expression of ovine TNF α. Northern blot analysis was used to analyse the kinetics of induction of ovine TNF α mRNA in alveolar macrophages stimulated with a variety of mitogens. Addition of LPS increased mRNA encoding TNF α at 1 h and 5 but not 24 h post stimulation. In contrast, addition of phorbol myristic acid (PMA) led to increased TNF α mRNA at 5 h while the combination of PMA and ionomycin increased the level of specific mRNA detected at 1 h, 5 h and 24 h. From genomic analysis ovine TNF α appears to exist as a single copy.
- Subjects
MOLECULAR cloning; TUMOR necrosis factors; CYTOKINES; GLYCOPROTEINS; GENETIC engineering; MACROPHAGES
- Publication
Immunology & Cell Biology, 1991, Vol 69, Issue 4, p273
- ISSN
0818-9641
- Publication type
Article
- DOI
10.1038/icb.1991.38