Abstract
We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cossart P., Katinka M., Yaniv M. Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions. Nucleic Acids Res. 1981 Jan 24;9(2):339–347. doi: 10.1093/nar/9.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- FLAVIN M., SLAUGHTER C. Threonine synthetase mechanism: studies with isotopic hydrogen. J Biol Chem. 1960 Apr;235:1112–1118. [PubMed] [Google Scholar]
- Hartman S. C., Mulligan R. C. Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8047–8051. doi: 10.1073/pnas.85.21.8047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
- Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parsot C., Cossart P., Saint-Girons I., Cohen G. N. Nucleotide sequence of thrC and of the transcription termination region of the threonine operon in Escherichia coli K12. Nucleic Acids Res. 1983 Nov 11;11(21):7331–7345. doi: 10.1093/nar/11.21.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rees W. D., Flint H. J., Fuller M. F. A molecular biological approach to reducing dietary amino acid needs. Biotechnology (N Y) 1990 Jul;8(7):629–633. doi: 10.1038/nbt0790-629. [DOI] [PubMed] [Google Scholar]
- Rees W. D., Hay S. M., Flint H. J. Expression of Escherichia coli homoserine kinase in mouse 3T3 cells. Biochem J. 1992 Feb 1;281(Pt 3):865–870. doi: 10.1042/bj2810865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
- Stacey A., Schnieke A. SVpoly: a versatile mammalian expression vector. Nucleic Acids Res. 1990 May 11;18(9):2829–2829. doi: 10.1093/nar/18.9.2829. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Szczesiul M., Wampler D. E. Regulation of a metabolic system in vitro: synthesis of threonine from aspartic acid. Biochemistry. 1976 May 18;15(10):2236–2244. doi: 10.1021/bi00655a033. [DOI] [PubMed] [Google Scholar]
- Théze J., Kleidman L., St Girons I. Homoserine kinase from Escherichia coli K-12: properties, inhibition by L-threonine, and regulation of biosynthesis. J Bacteriol. 1974 May;118(2):577–581. doi: 10.1128/jb.118.2.577-581.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]