Abstract
The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40–50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kbHind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells ofEscherichia coli, and colonies were recovered that carried pGEM withHind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transformE. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning intoE. coli.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ballas N, Zakai N, Friedberg D, Loyter A: Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts. Plant Mol Biol 11: 517–527 (1988).
Bates GW, Piastuch W, Riggs CD, Rabussay D: Electroporation for DNA delivery to plant protoplasts. Plant Cell Tissue Organ Cult 12: 213–218 (1988).
Czernilofsky AP, Hain R, Herrera-Estrella L, Lörz H, Goyvaerts E, Baker BJ, Schell J: Fate of selectable marker DNA integrated into the genome ofNicotiana tabacum. DNA 5: 101–113 (1986).
Damm B, Schmidt R, Willmitzer L: Effecient transformation ofArabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet 217: 6–12 (1989).
Deshayes A, Herrera-Estrella L, Caboche M: Liposome-mediated transformation of tobacco mesophyll protoplasts by aEscherichia coli plasmid. EMBO J 4: 2731–2737 (1985).
Dron M, Clouse SD, Dixon RA, Lawton MA, Lamb CJ: Gluathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts. Proc Natl Acad Sci USA 85: 6738–6742 (1988).
Feinberg AP, Vogelstein B: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Fromm M, Taylor LP, Walbot V: Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acid Sci USA 82: 5824–5828 (1985).
Fromm ME, Taylor LP, Walbot V: Stable transformation of maize after gene transfer by electroporation. Nature 319: 791–793 (1986).
Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2: 1044–1051 (1982).
Junker B, Zimny J, Luhrs R, Lörz H: Transient expression of chimaeric genes in dividing and non-dividing cereal protoplasts after PEG-induced DNA uptake. Plant Cell Rep 6: 329–332 (1987).
Krens FA, Mans RMW, van Slogteren TMS, Hoge JHC, Wullems GJ, Schilperoort RA: Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences. Plant Mol Biol 5: 223–234 (1985).
Lyznik LA, Ryan RD, Ritchie SW, Hodges TK: Stable co-transformation of maize protoplasts with gusA and neo genes. Plant Mol Biol 13: 151–161 (1989).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
McMahon AP, Novak TJ, Britten RJ, Davidson EH: Inducible expression of a cloned heat shock fusion gene in sea urchin embryos. Proc Natl Acad Sci USA 81: 7490–7494 (1984).
Meyer P, Kartzke S, Niedenhof I, Heidmann I, Bussmann K, Saedler H: A genomic DNA segment fromPetunia hybrida leads to increased transformation frequencies and simple integration patterns. Proc Natl Acad Sci USA 85: 8568–8572 (1988).
Miller CK, Temin HM: High-efficiency ligation and recombination of DNA fragments by vertebrate cells. Science 220: 606–609 (1983).
Nea LJ, Bates GW: Factors affecting protoplast electrofusion efficiency. Plant Cell Rep 6: 337–340 (1987).
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F: Hybrid genes in the analysis of transformation conditions. Plant Mol Biol 8: 363–373 (1987).
Paszkowski J, Shillito RD, Saul M, Mandak V, Hohn T, Hohn B, Potrykus I: Direct gene transfer to plants. EMBO J 3: 2717–2722 (1984).
Peerbolte R, Krens FA, Mans RMW, Floor M, Hoge JHC, Wullems GJ, Schilperoort RA: Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences. Plant Mol Biol 5: 235–246 (1985).
Riggs CD, Bates GW: Stable transformation of tobacco by electroporation: Evidence for plasmid concatenation. Proc Natl Acad Sci USA 83: 5602–5606 (1986).
Saxena PK, Fowke LC, King J: An efficient procedure for isolation of nuclei from plant protoplasts. Protoplasma 128: 184–189 (1985).
Schocher RJ, Shillito RD, Saul MW, Paszkowski J, Potrykus I: Co-transformation of unlinked foreign genes into plants by direct gene transfer. Bio/Technology 4: 1093–1096 (1986).
Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I: High efficiency direct gene transfer of plants. Bio/Technology 3: 1099–1103 (1985).
Shimamoto K, Terada R, Izawa T, Fujimoto H: Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338: 274–276 (1989).
Wake CT, Gudewicz T, Porter T, White A, Wilson JH: How damaged is the biologically active subpopulation of transfected DNA? Mol Cell Biol 4: 387–398 (1984).
Werr W, Lörz H: Transient gene expression in a Gramineae cell line. Mol Gen Genet 202: 471–475 (1986).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Bates, G.W., Carle, S.A. & Piastuch, W.C. Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization. Plant Mol Biol 14, 899–908 (1990). https://doi.org/10.1007/BF00019388
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00019388