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. 1982 Jan;79(1):86–90. doi: 10.1073/pnas.79.1.86

Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens

Norimoto Murai 1, John D Kemp 1
PMCID: PMC345666  PMID: 16593142

Abstract

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of agrobacterium tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of Mr 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five 32P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the Mr 40,000 polypeptide and was not detected in the habituated callus.

Keywords: recombinant plasmid, hybridization, mRNA size, in vitro translation, immunoprecipitation

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Selected References

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

  1. Adams S. L., Alwine J. C., de Crombrugghe B., Pastan I. Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts. J Biol Chem. 1979 Jun 25;254(12):4935–4938. [PubMed] [Google Scholar]
  2. BRAUN A. C. The activation of two growth-substance systems accompanying the conversion of normal to tumor cells in crown gall. Cancer Res. 1956 Jan;16(1):53–56. [PubMed] [Google Scholar]
  3. Bantle J. A., Maxwell I. H., Hahn W. E. Specificity of oligo (dT)-cellulose chromatography in the isolation of polyadenylated RNA. Anal Biochem. 1976 May 7;72:413–427. doi: 10.1016/0003-2697(76)90549-2. [DOI] [PubMed] [Google Scholar]
  4. Bomhoff G., Klapwijk P. M., Kester H. C., Schilperoort R. A., Hernalsteens J. P., Schell J. Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1976 May 7;145(2):177–181. doi: 10.1007/BF00269591. [DOI] [PubMed] [Google Scholar]
  5. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  6. Chilton M. D., Drummond M. H., Merio D. J., Sciaky D., Montoya A. L., Gordon M. P., Nester E. W. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell. 1977 Jun;11(2):263–271. doi: 10.1016/0092-8674(77)90043-5. [DOI] [PubMed] [Google Scholar]
  7. Chilton M. D., Saiki R. K., Yadav N., Gordon M. P., Quetier F. T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4060–4064. doi: 10.1073/pnas.77.7.4060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gurley W. B., Kemp J. D., Albert M. J., Sutton D. W., Callis J. Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2828–2832. doi: 10.1073/pnas.76.6.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hack E., Kemp J. D. Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase. Plant Physiol. 1980 May;65(5):949–955. doi: 10.1104/pp.65.5.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Humphreys G. O., Willshaw G. A., Anderson E. S. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. doi: 10.1016/0005-2787(75)90318-4. [DOI] [PubMed] [Google Scholar]
  12. Kemp J. D. In Vivo Synthesis of Crown Gall-specific Agrobacterium tumefaciens-directed Derivatives of Basic Amino Acids. Plant Physiol. 1978 Jul;62(1):26–30. doi: 10.1104/pp.62.1.26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kemp J. D., Sutton D. W., Hack E. Purification and characterization of the crown gall specific enzyme nopaline synthase. Biochemistry. 1979 Aug 21;18(17):3755–3760. doi: 10.1021/bi00584a017. [DOI] [PubMed] [Google Scholar]
  14. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  15. Koekman B. P., Ooms G., Klapwijk P. M., Schilperoort R. A. Genetic map of an octopine TI-plasmid. Plasmid. 1979 Jul;2(3):347–357. doi: 10.1016/0147-619x(79)90018-0. [DOI] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. MENAGE A., MOREL G. SUR LA PR'ESENCE D'OCTOPINE DANS LES TISSUS DE CROWN-GALL. C R Hebd Seances Acad Sci. 1964 Dec 21;259:4795–4796. [PubMed] [Google Scholar]
  18. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McPherson J. C., Nester E. W., Gordon M. P. Proteins encoded by Agrobacterium tumefaciens Ti plasmid DNA (T-DNA) in crown gall tumors. Proc Natl Acad Sci U S A. 1980 May;77(5):2666–2670. doi: 10.1073/pnas.77.5.2666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Merlo D. J., Kemp J. D. Attempts to Detect Agrobacterium tumefaciens DNA in Crown-Gall Tumor Tissue. Plant Physiol. 1976 Jul;58(1):100–106. doi: 10.1104/pp.58.1.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Noyes B. E., Stark G. R. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell. 1975 Jul;5(3):301–310. doi: 10.1016/0092-8674(75)90105-1. [DOI] [PubMed] [Google Scholar]
  22. Park W. D., Lewis E. D., Rubenstein I. Heterogeneity of zein mRNA and protein in maize. Plant Physiol. 1980 Jan;65(1):98–106. doi: 10.1104/pp.65.1.98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  24. Scheele G., Blackburn P. Role of mammalian RNase inhibitor in cell-free protein synthesis. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4898–4902. doi: 10.1073/pnas.76.10.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith E. F., Townsend C. O. A PLANT-TUMOR OF BACTERIAL ORIGIN. Science. 1907 Apr 26;25(643):671–673. doi: 10.1126/science.25.643.671. [DOI] [PubMed] [Google Scholar]
  26. Sun S. M., Buchbinder B. U., Hall T. C. Cell-free Synthesis of the Major Storage Protein of the Bean, Phaseolus vulgaris L. Plant Physiol. 1975 Dec;56(6):780–785. doi: 10.1104/pp.56.6.780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomashow M. F., Nutter R., Montoya A. L., Gordon M. P., Nester E. W. Integration and organization of Ti plasmid sequences in crown gall tumors. Cell. 1980 Mar;19(3):729–739. doi: 10.1016/s0092-8674(80)80049-3. [DOI] [PubMed] [Google Scholar]
  29. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zaenen I., Van Larebeke N., Van Montagu M., Schell J. Supercoiled circular DNA in crown-gall inducing Agrobacterium strains. J Mol Biol. 1974 Jun 15;86(1):109–127. doi: 10.1016/s0022-2836(74)80011-2. [DOI] [PubMed] [Google Scholar]

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