Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Apr 1;291(Pt 1):315–322. doi: 10.1042/bj2910315

The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells.

W D Rees 1, S M Hay 1
PMCID: PMC1132519  PMID: 8385938

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

PDF
319

Images in this article

320Figure 5
on p.320
321Figure 7
on p.321

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. FLAVIN M., SLAUGHTER C. Threonine synthetase mechanism: studies with isotopic hydrogen. J Biol Chem. 1960 Apr;235:1112–1118. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES