Plant Molecular Biology

, Volume 24, Issue 3, pp 505–514 | Cite as

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

  • James D. Jones
  • Stephen C. Weller
  • Peter B. Goldsbrough
Research Articles
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Abstract

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 μg/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 μg/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.

Key words

co-amplification gene amplification kanamycin resistance nptII petunia cell cultures 
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References

  1. 1.
    Altenbuchner J, Cullum J: Amplification of cloned genes in Streptomyces. Bio/technology 5: 1328–1329 (1987).CrossRefGoogle Scholar
  2. 2.
    Barredo JL, Diez B, Alvarez E, Martin JF: Large amplification of a 35 kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum. Curr Genet 16: 453–459 (1989).PubMedGoogle Scholar
  3. 3.
    Beck E, Ludwig G, Auerswald EA, Reiss B, Schaller H: Nucleotide sequence and exact localization of the neomycin phosophotransferase gene from transposon Tn5. Gene 19: 327–336 (1982).CrossRefPubMedGoogle Scholar
  4. 4.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).CrossRefPubMedGoogle Scholar
  5. 5.
    Brown PC, Beverley SM, Schimke RT: Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines. Mol Cell Biol 1: 1077–1083 (1981).PubMedGoogle Scholar
  6. 6.
    Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation: version II. Plant Mol Biol Rep 1 (4): 19–21 (1983).Google Scholar
  7. 7.
    Donn G, Tischer E, Smith JA, Goodman HM: Herbicide-resistant alfalfa cells: an example of gene amplification in plants. J Mol Appl Genet 2: 621–635 (1984).PubMedGoogle Scholar
  8. 8.
    Dorai H, Moore GP: The effect of dihydrofolate reductase-mediated gene amplification on the expression of transfected immunoglobulin genes. J Immunol 139: 4232–4241 (1987).PubMedGoogle Scholar
  9. 9.
    Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).PubMedGoogle Scholar
  10. 10.
    Goldsbrough PB, Gelvin SB, Larkins BA: Expression of maize zein genes in transformed sunflower cells. Mol Gen Genet 202: 374–381 (1986).CrossRefGoogle Scholar
  11. 11.
    Goldsbrough PB, Hatch EM, Huang B, Kosinki WG, Dyer WE, Herrmann KM, Weller SC: Gene amplification in glyphosate tolerant tobacco cells. Plant Sci 72: 53–62 (1990).CrossRefGoogle Scholar
  12. 12.
    Gupta A, Whitton BA, Morby AP, Huckle JW, Robinson NJ: Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium. Proc R Soc Lond B 248: 273–281 (1992).PubMedGoogle Scholar
  13. 13.
    Harms CT, Armour SL, DiMaio JJ, Middlesteadt LA, Murray D, Negrotto DV, Thompson-Taylor H, Weymann K, Montoya AL, Shillito RD, Jen GC: Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene. Mol Gen Genet 233: 427–435 (1992).CrossRefPubMedGoogle Scholar
  14. 14.
    Hasegawa PM, Bressan RA, Handa AK: Growth characteristics of NaCl-selected and nonselected cells of Nicotiana tabacum L. Plant Cell Physiol 21: 1347–1355 (1980).Google Scholar
  15. 15.
    Hayes RJ, Petty ITD, Coutts RHA, Buck KW: Gene amplification and expression in plants by a replicating geminivirus vector. Nature 334: 179–182 (1988).CrossRefGoogle Scholar
  16. 16.
    Hendricks MB, Luchette CA, Banker MJ: Enhanced expression of an immunoglobulin-based vector in myeloma cells mediated by coamplification with a mutant dihydrofolate reductase gene. Bio/technology 7: 1271–1274 (1989).Google Scholar
  17. 17.
    Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).Google Scholar
  18. 18.
    Jones JD, Goldsbrough PB, Weller SC: Characterization of tobacco plantlets regenerated from glyphosate resistant cell cultures. Plant Physiol Suppl 99: 48 (1992).Google Scholar
  19. 19.
    Kaufman RJ: Selection and coamplification of heterologous genes in mammalian cells. Meth Enzymol 185: 537–566 (1990).PubMedGoogle Scholar
  20. 20.
    Kay R, Chan A, Daly M, McPherson J: Duplication of CAMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302 (1987).Google Scholar
  21. 21.
    Linn F, Heidmann I, Saedler H, Meyer P: Epigenetic changes in the expression of the maize A1 gene in Petunia hybrida: role of numbers of integrated gene copies and state of methylation. Mol Gen Genet 222: 329–336 (1990).PubMedGoogle Scholar
  22. 22.
    Mantias T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  23. 23.
    Mouches C, Pasteur N, Berge JB, Hyrien O, Raymond M, Vincent BRDJ, Silvestri MD, Georghiou GP: Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito. Science 233: 778–780 (1986).PubMedGoogle Scholar
  24. 24.
    Muller E, Brown PTH, Hartke S, Lorz H: DNA variation in tissue-culture-derived rice plants. Theor Appl Genet 80: 673–679 (1990).Google Scholar
  25. 25.
    Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–479 (1962).Google Scholar
  26. 26.
    Nakamori S, Ishida M, Takagi H, Ito K, Miwa K, Sano K: Improved L-threonine production by the amplification of the gene encoding homoserine dehydrogenase in Brevibacterium lactofermentum. Agric Biol Chem 51: 87–91 (1987).Google Scholar
  27. 27.
    Napoli C, Lemieux C, Jorgensen R: Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279–289 (1990).CrossRefPubMedGoogle Scholar
  28. 28.
    Shah DM, Horsch RB, Klee HS, Kishore GM, Winter JA, Tumer NE, Hironaka CM, Sanders PR, Gasser CS, Aykent S, Siegel NR, Rogers SG, Fraley RT: Engineering herbicide tolerance in transgenic plants. Science 233: 478–481 (1986).Google Scholar
  29. 29.
    Shyr YYJ, Hepburn AG, Widholm JM: Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells. Mol Gen Genet 232: 377–382 (1992).PubMedGoogle Scholar
  30. 30.
    Spradling AC, Mahowald AP: Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc Natl Acad Sci USA 77: 1096–1100 (1980).PubMedGoogle Scholar
  31. 31.
    Stark GR: Gene amplification. Annu Rev Biochem 53: 447–491 (1984).PubMedGoogle Scholar
  32. 32.
    Stark GR, Debatisse M, Giulotto E, Wahl GM: Recent progress in understanding mechanisms of mammalian DNA amplification. Cell 57: 901–908 (1989).PubMedGoogle Scholar
  33. 33.
    Suh H, Hepburn AG, Kriz AL, Widholm JM: Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells. Plant Mol Biol 22: 195–205 (1993).PubMedGoogle Scholar
  34. 34.
    Tartof KD: Redundant genes. Annu Rev Genet 9: 355–385 (1975).PubMedGoogle Scholar
  35. 35.
    Wang Y, Jones JD, Weller SC, Goldsbrough PB: Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells. Plant Mol Biol 17: 1127–1138 (1991).PubMedGoogle Scholar
  36. 36.
    Welch JW, Fogel S, Cathala G: Industrial yeasts display tandem gene iteration at the CUP1 region. Mol Cell Biol 3: 1353–1361 (1983).PubMedGoogle Scholar
  37. 37.
    Welch JW, Maloney DH, Fogel S: Unequal crossingover and gene conversion at the amplified CUP1 locus of yeast. Mol Gen Genet 222: 304–310 (1990).PubMedGoogle Scholar
  38. 38.
    Wernars K, Goosen T, Wennekes LMJ, Visser J, Bos CJ, van den Broek HWJ, van Gorcom RFM, van den Hondel CAMJJ, Pouwels PH: Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene. Curr Genet 9: 361–368 (1985).PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • James D. Jones
    • 1
  • Stephen C. Weller
    • 1
  • Peter B. Goldsbrough
    • 1
  1. 1.Department of HorticulturePurdue UniversityW. LafayetteUSA

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