Cloning and Expression of Human Lymphotoxin mRNA Derived from a Human T Cell Hybridoma

We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells. The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1986, Vol.100 (3), p.727-733
Main Authors: KOBAYASHI, Yoshiro, MIYAMOTO, Daisei, ASADA, Makoto, OBINATA, Masuo, OSAWA, Toshiaki
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Biological and medical sciences
Biotechnology
cDNA
cloning
Cloning, Molecular
DNA - analysis
DNA - genetics
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
gene expression
genes
Genetic engineering
Genetic technics
Humans
hybridoma
Hybridomas
lymphocytes T
lymphotoxin
Lymphotoxin-alpha - biosynthesis
Lymphotoxin-alpha - genetics
man
Methods. Procedures. Technologies
Nucleic Acid Hybridization
nucleotide sequence
Plasmids
RNA, Messenger - analysis
RNA, Messenger - genetics
T-Lymphocytes
transformation
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Summary:We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells. The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene. Four stretches, containing 14 nucleotides in total, were different among the three sequences. The differences included one base change from C to A in the coding region. Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E. coli expression plasmid (pLT13tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT. Both plasmids were found to synthesize active LT molecules after transfection into JM1O5 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum. Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity. The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.
ISSN:0021-924X
1756-2651
DOI:10.1093/oxfordjournals.jbchem.a121765