Advertisement

Somatic Cell and Molecular Genetics

, Volume 13, Issue 1, pp 67–76 | Cite as

Expression of mouse dihydrofolate reductase gene confers methotrexate resistance in transgenic petunia plants

  • David A. Eichholtz
  • Stephen G. Rogers
  • Robert B. Horsch
  • Harry J. Klee
  • Maria Hayford
  • Nancy L. Hoffmann
  • Sarah B. Braford
  • Cindy Fink
  • Jeff Flick
  • Keith M. O'Connell
  • Robert T. Fraley
Article

Abstract

Transgenic petunia plants containing an altered (Leu22→Arg22) mouse dihydrofolate reductase gene fused to the cauliflower mosiac virus 35S (CaMV 35S) promoter and nopaline synthase (nos) polyadenylation site were obtained by transforming petunia leaf disks with anAgrobacterium tumefaciens strain carrying the chimeric gene. Transformants were directly selected for and rooted on medium containing 1ΜM methotrexate (MTX). The chimeric gene was present in the regenerated plants at one to three copies and produced the expected 950-nucleotide-long transcript based on Southern and Northern hybridization analyses, respectively. Leaf pieces from the regenerated transgenic plants were able to form callus when cultured on medium containing 1 ΜM MTX and were able to incorporate32P into high-molecular-weight DNA in the presence of > 100 ΜM MTX, thus demonstrating that the chimeric mouse dhfr gene was fully functional and useful as a selectable marker in plant transformation experiments. To date, this is the first report of successful expression of a vertebrate gene in transformed plant cells.

Keywords

Methotrexate Leaf Disk Chimeric Gene Northern Hybridization Chimeric Mouse 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature cited

  1. 1.
    Fraley, R.T., Rogers, S.G., Horsch, R.B., Sanders, P.R., Flick, J.S., Adams, S.P., Bittner, M.L., Brand, L.A., Fink, C.L., Fry, J.S., Galluppi, G.R., Goldberg, S.B., Hoffmann, N.L., and Woo, S.C. (1983).Proc. Natl. Acad. Sci. U.S.A. 80:4803–4807.PubMedGoogle Scholar
  2. 2.
    Herrera-Estrella, J., DeBlock, M., Messens, E., Hernalsteens, J.-P., Van Montagu, M., and Schell, J. (1983).EMBO J. 2:987–995.PubMedGoogle Scholar
  3. 3.
    Bevan, M., Flavell, R., and Chilton, M.-D. (1983).Nature 304:184–187.CrossRefGoogle Scholar
  4. 4.
    Colbere-Garapin, F., Horodniceanu, F., Kourilsky, P., and Garapin, A.-C. (1981).J. Mol. Biol. 150:1–14.CrossRefPubMedGoogle Scholar
  5. 5.
    Mulligan, R.C., and Berg, P. (1981).Proc. Natl. Acad. Sci. U.S.A. 78:2072–2076.PubMedGoogle Scholar
  6. 6.
    Southern, P.J., and Berg, P. (1982).J. Mol. Appl. Gen. 1:327–341.Google Scholar
  7. 7.
    Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y., and Axel, R. (1977).Cell 11:223–232.CrossRefPubMedGoogle Scholar
  8. 8.
    Wigler, M., Sweet, R., Sim, G.K., Wold, B., Pellicer, A., Lacy, E., Maniatis, T., Silverstein, S., and Axel, R. (1979).Cell 16:777–785.CrossRefPubMedGoogle Scholar
  9. 9.
    Subramani, S., Mulligan, R., and Berg, P. (1981).Mol. Cell. Biol. 1:854–864.PubMedGoogle Scholar
  10. 10.
    Simonsen, C.C., and Levinson, A.D. (1983).Proc. Natl. Acad. Sci. U.S.A. 80:2495–2499.PubMedGoogle Scholar
  11. 11.
    DeBlock, M., Herrera-Estrella, L., Van Montagu, M., Schell, J., and Zambryski, P. (1984).EMBO J. 3:1681–1689.Google Scholar
  12. 12.
    Brisson, N., Paszkowski, J., Penswick, J.R., Gronenborn, B., Potrykus, I., and Kohn, T. (1984).Nature 310:511–514.Google Scholar
  13. 13.
    Haber, D.A., Beverley, S.M., Kiely, M.L., and Schimke, R.T. (1981).J. Biol. Chem. 256:9501–9510.PubMedGoogle Scholar
  14. 14.
    Nunberg, J., Kaufman, R., Chang, A., Cohen, S., and Schimke, R. (1980).Cell 19:355–364.CrossRefPubMedGoogle Scholar
  15. 15.
    Vieira, J., and Messing, J. (1982).Gene 19:259–268.CrossRefPubMedGoogle Scholar
  16. 16.
    Zoller, M., and Smith, M. (1982).Nucleic Acids Res. 10:6487–6500.PubMedGoogle Scholar
  17. 17.
    Guilley, H., Dudley, R.K., Jonard, G., Balazs, E., and Richards, K.E. (1982).Cell 30:763–773.CrossRefPubMedGoogle Scholar
  18. 18.
    Gardner, R., Howarth, A., Hahn, P., Brown-Luedi, M., Shepherd, R., and Messing, J. (1981).Nucleic Acids Res. 9:2871–2888.PubMedGoogle Scholar
  19. 19.
    Rogers, S., O'Connell, K., Horsch, R., and Fraley, R. (1985). InBiotechnology in Plant Science, (eds.) Day, P., Zaitlin, M., and Hollander, A. (Academic Press, Orlando), pp. 219–226.Google Scholar
  20. 20.
    Schneidhauser, T.J., and Helinski, D.R. (1985).J. Bacteriol. 164:446–455.Google Scholar
  21. 21.
    Fraley, R.T., Rogers, S.G., Horsch, R.B., Eichholtz, D.A., Flick, J.S., Fink, C.L., Hoffmann, N.L., and Sanders, P.R. (1985).Bio/Technology 3:629–635.CrossRefGoogle Scholar
  22. 22.
    Sutcliffe, J.G. (1979).Cold Spring Harbor Symp. 43:77–90.Google Scholar
  23. 23.
    DeGreve, H., Decraemer, H., Seurinck, J., Van Montagu, M., and Schell, J. (1981).Plasmid 6:235–248.CrossRefGoogle Scholar
  24. 24.
    Barker, R.F., Idler, K.B., Thompson, D.V., and Kemp, J.D. (1983).Plant Mol. Biol. 2:335–350.CrossRefGoogle Scholar
  25. 25.
    Horsch, R.B., Fry, J.E., Hoffmann, N.L., Wallroth, M., Eichholtz, D.A., Rogers, S.G., and Fraley, R.T. (1985).Science 227:1229–1231.Google Scholar
  26. 26.
    Horsch, R.B., and Klee, H.J. (1986).Proc. Natl. Acad. Sci. U.S.A. 83:4428–4432.PubMedGoogle Scholar
  27. 27.
    Southern, E. (1985).J. Mol. Biol. 98:503–517.Google Scholar
  28. 28.
    Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). InMolecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), pp. 200–201.Google Scholar
  29. 29.
    Bevan, M.W., Mason, S.E., and Goelet, P. (1985).EMBO J. 4:1921–1926.PubMedGoogle Scholar
  30. 30.
    Kornberg, A. (1974).DNA Replication, (W.H. Freeman and Co., San Francisco), p. 422.Google Scholar
  31. 31.
    Waldron, C., Murphy, E.B., Roberts, J.L., Gustafson, G.D., Armour, S.L., and Malcolm, S.K. (1985).Plant Mol. Biol. 5:103–108.CrossRefGoogle Scholar
  32. 32.
    Van den Elzen, P.J.M., Townsend, J., Lee, K.Y., and Bedbrook, J.R. (1985).Plant Mol. Biol. 5:299–302.CrossRefGoogle Scholar
  33. 33.
    Pattishall, K.H., Acar, J., Burchall, J.J., Goldstein, F.W., and Harvey, R.J. (1977).J. Biol. Chem. 252:2319–2323.PubMedGoogle Scholar
  34. 34.
    Caplan, A., Herrera-Estrella, L., Inze, D., Van Haute, E., Van Montague, M., Schell, J., and Zambryski, P. (1983).Science 222:815–821.Google Scholar
  35. 35.
    Koncz, C., Kreuzaler, F., Kalman, Zs., and Schell, J. (1984).EMBO J. 3:1029–1037.PubMedGoogle Scholar
  36. 36.
    Odell, J.T., Nagy, F., and Chua, N.-H. (1985).Nature 313:810–812.CrossRefPubMedGoogle Scholar
  37. 37.
    Dennis, E., and Berg, P. (1985).Nucleic Acids Res. 13:7945–7957.PubMedGoogle Scholar
  38. 38.
    Nagy, F., Morelli, G., Fraley, R.T., Rogers, S.G., and Chua, N.-H. (1985).EMBO J. 4:3063–3068.PubMedGoogle Scholar
  39. 39.
    Jones, J.D.G., Dunsmuir, P., and Bedbrook, J. (1985).EMBO J. 4:2411–2418.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1987

Authors and Affiliations

  • David A. Eichholtz
    • 1
  • Stephen G. Rogers
    • 1
  • Robert B. Horsch
    • 1
  • Harry J. Klee
    • 1
  • Maria Hayford
    • 1
  • Nancy L. Hoffmann
    • 1
  • Sarah B. Braford
    • 1
  • Cindy Fink
    • 1
  • Jeff Flick
    • 1
  • Keith M. O'Connell
    • 1
  • Robert T. Fraley
    • 1
  1. 1.Monsanto CompanySt. Louis

Personalised recommendations