Skip to main content
SpringerLink
Log in

Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis

  • Papers
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

TheSalmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of theS. typhimurium andEscherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. Thein vitro andin vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. Thecys M gene ofS. typhimurium possessed a GUG initiation codon, like itsE. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteinein vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adra, C.N., Boer, P.H. and McBurney, M.W. (1987) Cloning and expression of the mousepgk-1 gene and the nucleotide sequence of its promoter.Gene 60, 65–74.

    Article  PubMed  CAS  Google Scholar 

  • Anderson, S. (1981) Shotgun sequencing using cloned DNasel-generated fragments.Nucl. Acids Res. 9, 3015–27.

    PubMed  CAS  Google Scholar 

  • Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–54.

    Article  PubMed  CAS  Google Scholar 

  • Bray, A.C. and Till, A.R. (1975) Metabolism of sulphur in the gastro-intestinal tract. In: McDonald, I.W. and Warner, A.C.I. (eds)Digestion and Metabolism in the Ruminant, pp. 243–60. New England: University of New England Press.

    Google Scholar 

  • Burnham, B.F. and Lascelles, J. (1963) Control of porphyrin biosynthesis through a negative-feedback mechanism: Studies with preparations of d-aminolaevulate synthetase and d-aminolaevulate dehydratase fromRhodopseudomonas spheroides.Biochem. J. 87, 462–72.

    PubMed  CAS  Google Scholar 

  • Byrne, C.R., Monroe, R.S., Ward, K.A. and Kredich, N.M. (1988) DNA sequences of thecys K regions ofSalmonella typhimurium andEscherichia coli and linkage of thecys K regions topts H.J. Bacteriol 170, 3150–7.

    PubMed  CAS  Google Scholar 

  • Cherest, H., Eichler, F. and de Robichon-Szulmajster, H. (1969) Genetic and regulatory aspects of methionine biosynthesis inSaccharomyces cerevisiae.J. Bacteriol 97, 327–36.

    Google Scholar 

  • Cook, P.F. and Wedding, R.T. (1978) Cysteine synthetase fromSalmonella typhimurium LT-2. Aggregation, kinetic behaviour and effect of modifiers.J. Biol. Chem. 253, 7874–9.

    PubMed  CAS  Google Scholar 

  • D'Andrea, R.J., Sivaprasad, A.V., Bawden, C.S., Kuczek, E.S., Whitbread, L.A. and Rogers, G.E. (1989) Isolation of microbial genes for cysteine synthesis and prospects for their use in increasing wool growth. In: Rogers, G.E., Reis, P.J., Ward, K.A. and Marshal, R.C. (eds).,The Biology of Wool and Hair, pp. 447–64. London: Chapman & Hall.

    Google Scholar 

  • Davies, R.W., Botstein, D. and Roth, J.R. (1980)Advanced Bacterial Genetics, Cold Spring Harbour, N.Y.: Cold Spring Harbour Press. pp. 201.

    Google Scholar 

  • DeNoto, F.M., Moore, D.D. and Goodman, H.W. (1981) Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.Nucl. Acids Res. 9, 3719–30.

    PubMed  CAS  Google Scholar 

  • Denk, D. and Bock, A. (1987) L-cysteine biosynthesis inEscherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cys E) gene from the wild-type and a cysteine-excreting mutant.J. Gen. Microbiol. 133, 515–25.

    PubMed  CAS  Google Scholar 

  • Doyle, P.T. and Moir, R.J. (1980) Sulphur and methionine metabolism in Sheep II. Quantitative estimates of sulphur metabolism in the sheep's stomach.Aust. J. Biol. Sci. 32, 65–76.

    Google Scholar 

  • Flavin, M. and Slaughter, C. (1965) Synthesis of the succinic ester of homoserine, a new intermediate in the bacterial biosynthesis of methionine.Biochemistry 4, 1370–5.

    Article  PubMed  CAS  Google Scholar 

  • Gillespie, J.M. (1983) The structural proteins of hair: isolation, characterization and regulation of biosynthesis. In: Goldsmith, L.A. (ed.),Biochemistry and Physiology of the Skin Vol. I, pp. 475–510. Oxford: Oxford University Press.

    Google Scholar 

  • Gold, L. and Stormo, G. (1987) Translational initiation. In: Neidhardt, F.C. (ed.),Escherichia coli andSalmonella typhimurium: Cellular and Molecular Biology, pp. 1302–7. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory.

    Google Scholar 

  • Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I. and Howard, B.H. (1982) The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.Proc. Natl Acad. Sci. USA 79, 6777–81.

    Article  PubMed  CAS  Google Scholar 

  • Gough, N.M., Metcalf, D., Gough, J., Grail, D. and Dunn, A.R. (1985) Structure and expression of the mRNA for granulocyte-macrophage colony stimulating factor.EMBO J. 4, 645–53.

    PubMed  CAS  Google Scholar 

  • Greenstein, J.P. and Winitz, M. (1967) In:Chemistry of the Amino Acids, pp. 1053 and 2230. NY and London: John Wiley and Sons, Inc.

    Google Scholar 

  • Hartzell, S.W., Byrne, B.J. and Subramanian, K.N. (1984) Mapping of the late promoter of simian virus 40.Proc. Natl Acad. Sci. USA 81, 23–7.

    Article  PubMed  CAS  Google Scholar 

  • Hulanicka, M.D. and Kredich, N.M. (1976) A mutation affecting expression of the gene coding for serine transacetylase inSalmonella typhimurium.Mol. Gen. Genet. 148, 143–8.

    Article  PubMed  CAS  Google Scholar 

  • Hulanicka, M.D., Garrett, C., Jagura-Burdzy, G. and Kredich, N.M. (1986) Cloning and characterization of thecys AMK region ofSalmonella typhimurium.J. Bacteriol. 168, 322–7.

    PubMed  CAS  Google Scholar 

  • Hulanicka, M.D., Kredich, N.M. and Treiman, D.M. (1974) The structural gene for O-acetylserine sylphydrylase A inSalmonella typhimurium.J. Biol. Chem.,249, 867–72.

    PubMed  CAS  Google Scholar 

  • Jenkins, B.J. (1990) Characterization of the transcription of cysteine biosynthesis genes in mammalian cells.Honours Thesis, Department of Biochemistry, University of Adelaide, Adelaide, Australia.

    Google Scholar 

  • Jones-Mortimer, M.C. (1968) Positive control of sulphate inEscherichia coli: the nature of the pleiotropic cysteineless mutants ofE. coli K12.Biochem. J. 110, 597–602.

    PubMed  CAS  Google Scholar 

  • Kozak, M. (1984) Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.Nucl. Acids Res. 12, 857–72.

    PubMed  CAS  Google Scholar 

  • Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that mediates translation by eukaryotic ribosomes.Cell 44, 283–92.

    Article  PubMed  CAS  Google Scholar 

  • Kredich, N.M. (1987) Biosynthesis of cysteine. In: Neidhardt F.C. (ed.), Escherichia coliand Salmonella typhimurium:Cellular and molecular biology, pp. 419–28. New York: Cold Spring Harbour.

    Google Scholar 

  • Kredich, N.M. and Tomkins, G.M. (1966) The enzymatic synthesis of L-cysteine inEscherichia coli andSalmonella typhimurium.J. Biol Chem. 241, 4955–65.

    PubMed  CAS  Google Scholar 

  • Kredich, N.M., Becker, M.A. and Tomkins, G.M. (1969) Purification and characterization of cysteine synthetase, a bifunctional protein complex, fromSalmonella typhimurium.J. Biol. Chem. 244, 2428–39.

    PubMed  CAS  Google Scholar 

  • Krieg, P.A. and Melton, D.A. (1987)In vitro RNA synthesis with SP6 RNA polymerase.Methods Enzymol. 155, 397–415.

    PubMed  CAS  Google Scholar 

  • Layne, E. (1957) Spectrophotometric and turbidimetric methods for measuring proteins.Methods Enzymol. 3, 447–54.

    Google Scholar 

  • Maurer, R., Osmond, B.C., Shekhtman, E., Wong, A. and Botstein, D. (1984) Functional interchangeability of DNA replication genes inSalmonella typhimurium andEscherichia coli demonstrated by a general complementation procedure.Genetics 108, 1–23.

    PubMed  CAS  Google Scholar 

  • McKnight, S.L., Gavis, E.R., Kingsbury, R. and Axel, R. (1982) Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region.Cell 25, 385–98.

    Article  Google Scholar 

  • Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficientin vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.Nucleic Acids Res. 12, 7035–56

    PubMed  CAS  Google Scholar 

  • Messing, J. (1983) New M13 vectors for cloning.Methods Enzymol. 101, 20–78.

    Article  PubMed  CAS  Google Scholar 

  • Monroe, R.S. and Kredich, N.M. (1988) Isolation ofSalmonella typhimurium cys genes by transduction with a library of recombinant plasmids packaged in bacteriophage P22HT capsids.J. Bacteriol. 170, 42–7.

    PubMed  CAS  Google Scholar 

  • Nakamura, T., Kon, Y., Iwahashi, H. and Eguchi, Y. (1983) Evidence that thiosulfate assimilation bySalmonella typhimurium is catalyzed by cysteine synthase B.J. Bacteriol. 156, 656–62.

    PubMed  CAS  Google Scholar 

  • Nakamura, T., Iwahashi, H. and Eguchi, Y. (1984) Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase ofSalmonella typhimurium.J. Bacteriol. 158, 1122–7.

    PubMed  CAS  Google Scholar 

  • Nordstrom, J.L., Hall, S.L. and Kessler, M.M. (1985) Polyadenylation of sea urchin histone RNA sequences in transfected COS cells.Proc. Natl Acad. Sci. USA 82, 1094–8.

    Article  PubMed  CAS  Google Scholar 

  • Overbeek, P.A., Lai, S.P., Van Quill, K.R. and Westphal, H. (1986) Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences.Science 231, 1574–7.

    Article  PubMed  CAS  Google Scholar 

  • Pataki, G. (1967)Techniques of Thin-layer Chromatography, pp. 67, Ann Arbor and London: Humphrey Science Publishers Inc.

    Google Scholar 

  • Peabody, D.S. (1987) Translation initiation at an ACG triplet in mammalian cells.J. Biol. Chem. 262, 11847–51.

    PubMed  CAS  Google Scholar 

  • Peabody, D.S. (1989) Translation initiation at non-AUG triplets in mammalian cells.J. Biol. Chem. 264, 5031–5.

    PubMed  CAS  Google Scholar 

  • Rathjen, P.D., Toth, S., Willis, A., Heath, J.K. and Smith, A.G. (1990) Differentiation inhibiting activity is produced in matrix-associated and diffusable forms that are generated by alternate promoter usage.Cell,62, 1105–1114.

    Article  PubMed  CAS  Google Scholar 

  • Reis, P.J. (1979) Effects of amino acids on the growth and properties of wool. In: Black, J.L. and Reis, P.J. (eds.),Physiological and Environmental Limitations to Wool Growth, pp. 223–42. New England: University of New England Press.

    Google Scholar 

  • Rogers, G.E. (1990) Improvement of wool production through genetic engineering.Trends Biotech. 8, 6–11.

    Article  CAS  Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning, A laboratory manual. Second Ed., Cold Spring Harbour, N.Y: Cold Spring Harbour Press.

    Google Scholar 

  • Sanger, F.G., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl Acad. Sci. USA 74, 5463–7.

    Article  PubMed  CAS  Google Scholar 

  • Sirko, A., Hryniewicz, M., Hulanicka, D. and Bock, A. (1990) Sulphate and thiosulphate transport inEscherichia coli K-12: nucleotide sequence and expression of thecys TWAM gene cluster.J. Bacteriol. 172, 3351–7.

    PubMed  CAS  Google Scholar 

  • Todaro, G.J., Green, H. and Swift, M.R. (1966) Susceptibility of human diploid fibroblast strains to transformation by SV40 virus.Science 153, 1252–4.

    Article  PubMed  CAS  Google Scholar 

  • Tooze, J. (1981) Transformation by polyoma virus and SV40. In Tooze, J. (ed).Molecular Biology of Tumor Viruses, pp. 350–402. Cold Spring Harbour, New York: Cold Spring Harbour Laboratory.

    Google Scholar 

  • Vogel, H.J. and Bonner, D.M. (1956) Acetylornithinase ofEscherichia coli: partial purification and some properties.J. Biol. Chem,218, 97–106.

    PubMed  CAS  Google Scholar 

  • Yamamoto, T., deCombrugghe, B. and Pastan, I. (1980) Identification of a functional promoter in the long terminal repeat of Rous sarcoma virus.Cell 22, 787–97.

    Article  PubMed  CAS  Google Scholar 

  • Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.Gene 33, 103–19.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Elizabeth S. Kuczek

    Present address: Department of Microbiology and Immunology, University of California, 94143, San Francisco, CA, USA

Authors and Affiliations

  1. Biochemistry Department, The University of Adelaide, GPO Box 498, 5001, Adelaide, South Australia, Australia

    Arapaut V. Sivaprasad, Elizabeth S. Kuczek, C. Simon Bawden & George E. Rogers

Authors
  1. Arapaut V. Sivaprasad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth S. Kuczek
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C. Simon Bawden
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. George E. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sivaprasad, A.V., Kuczek, E.S., Bawden, C.S. et al. Coexpression of thecys E andcys M genes ofSalmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis. Transgenic Research 1, 79–92 (1992). https://doi.org/10.1007/BF02513025

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02513025

Keywords

Search

Navigation