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Plant Biol (Stuttg) 2003; 5(4): 383-392
DOI: 10.1055/s-2003-42709
Original Paper

Georg Thieme Verlag Stuttgart · New York

Daucus carota Cells Contain Specific DNA Methyltransferase Inhibitors that Interfere with Somatic Embryogenesis

G. Pedrali-Noy* 1 , G. Bernacchia* 2 , M. do Rosario Alvelos 3 , R. Cella 3 *equally contributed
  • 1Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Pavia, Italy
  • 2Dipartimento di Biologia, Università di Ferrara, Ferrara, Italy
  • 3Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy
Further Information

Publication History

Publication Date:
02 October 2003 (online)

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Abstract

Results reported in this paper show that carrot cells contain a thermostable inhibiting activity for cytosine-5-DNA methyltransferase that, upon filtration chromatography, can be resolved into three major peaks. Inhibiting activity was found in all plant species tested, though at a concentration lower than in carrot. These inhibiting activities differ in size, sensitivity to various hydrolytic treatments, specificity for DNA METases of eukaryotic and bacterial origin and kinetics of inhibition. Results of chemical analyses indicate that the inhibitors differ from lipidic inhibitors described in Escherichia coli and Streptomyces sp. and, given their sensitivity to proteinase K, appear to have a proteinaceous nature. The addition of these inhibitors (Sephadex G25 peak II and peak III) to actively growing suspension rice cells reduced the rate of in vivo DNA methylation without interfering with DNA synthesis. Peak II also induced a general demethylation effect in carrot cell suspension, even if weaker than that caused by 5-azacytidine. Interestingly, inhibitors suppressed carrot embryogenesis but did not prevent undifferentiated cell proliferation of suspension cultures.

Key words

DNA methylation - heat stable fraction - inhibition kinetics - suspension cultures - carrot and rice cells

References

  • 1 Adams R. L. P.. Eukaryotic DNA methyltransferase-structure and function.  BioEssays. (1995);  17 139-145
    CrossrefPubMedGoogle Scholar
  • 2 Adams R. L. P., Burdon R. H.. Molecular biology of DNA methylation. New York; Springer Verlag (1985)
    Google Scholar
  • 3 Balestrazzi A., Bernacchia G., Cella R., Ferretti L., Sora S.. Preparation of high molecular weight plant DNA and its use for artificial chromosome construction.  Plant Cell Reports. (1991);  10 315-320
    PubMedGoogle Scholar
  • 4 Banks J. A., Masson P., Federoff N.. Molecular mechanism in the developmental regulation of the maize suppressor-mutator transposable element.  Genes Dev.. (1988);  2 1364-1380
    PubMedGoogle Scholar
  • 5 Barbes C., Sanchez J., Yerba M. J., Robert-Gero M., Hardsson C.. Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria.  FEMS Microbiol. Lett.. (1990);  69 239-244
    CrossrefPubMedGoogle Scholar
  • 6 Bernacchia G., Primo A., Giorgetti L., Pitto L., Cella R.. Carrot DNA-methyltransferase is encoded by two classes of genes with differing patterns of expression.  Plant J.. (1998);  13 317-330
    CrossrefPubMedGoogle Scholar
  • 7 Bohlen P., Stein S., Dairman W., Udenfriend S.. Fluorometric assay of proteins in the nanogram range.  Arch. Biochem. Biophys.. (1973);  155 213-220
    PubMedGoogle Scholar
  • 8 Bolden A., Ward C., Siedlecki J. A., Weissbach A.. DNA methylation: Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides.  J. Biol. Chem.. (1984);  259 12437-12443
    PubMedGoogle Scholar
  • 9 Burn J. A., Bagnall D. J., Metzger J. D., Dennis E. S., Peacock W. J.. DNA methylation, vernalization, and the initiation of flowering.  Proc. Natl. Acad. Sci. USA. (1993);  90 287-291
    PubMedGoogle Scholar
  • 10 Cedar H., Verdine G. L.. Gene expression: The amazing demethylase.  Nature. (1999);  397 568-569
    CrossrefPubMedGoogle Scholar
  • 11 Christman J. K.. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy.  Oncogene. (2002);  21 5483-5495
    CrossrefPubMedGoogle Scholar
  • 12 Colot V., Rossignol J.-L.. Eukaryotic DNA methylation as an evolutionary device.  BioEssays. (1999);  21 402-411
    CrossrefPubMedGoogle Scholar
  • 13 Doyle J. J., Doyle J. L.. Isolation of plant DNA from fresh tissue.  Focus. (1990);  12 13-15
    PubMedGoogle Scholar
  • 14 Falaschi A., Kornberg A.. A lipopolysaccharide inhibitor of a DNA methyl transferase.  Proc. Natl. Acad. Sci. USA. (1965);  54 1713-1720
    PubMedGoogle Scholar
  • 15 Fedoroff N. V., Banks J. A.. Is the suppressor-mutator element controlled by a basic development regulatory mechanism?.  Genetics. (1988);  120 559-577
    PubMedGoogle Scholar
  • 16 Finnegan E. J., Dennis E. S.. Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana. .  Nucl. Acids Res.. (1994);  21 2383-2388
    PubMedGoogle Scholar
  • 17 Finnegan E. J., Kovac K. A.. Plant DNA methyltransferases.  Plant Mol. Biol.. (2000);  43 189-201
    CrossrefPubMedGoogle Scholar
  • 18 Finnegan E. J., Genger R. K., Peacock W. J., Dennis E. S.. DNA methylation in plants.  Ann. Rev. Plant Physiol. Plant Mol. Biol.. (1998);  49 223-247
    PubMedGoogle Scholar
  • 19 Finnegan E. J., Peacock W. J., Dennis E. S.. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.  Proc. Natl. Acad. Sci. USA. (1996);  93 8449-8454
    CrossrefPubMedGoogle Scholar
  • 20 Fojtova M., Kovarik A., Votruba I., Holy A.. Evaluation of the impact of S-adenosylhomocysteine metabolic pools on cytosine methylation of the tobacco genome.  Eur. J. Biochem.. (1998);  252 347-352
    CrossrefPubMedGoogle Scholar
  • 21 Galaud J. P., Gaspar T., Boyer N.. Inhibition of internode growth due to mechanical stress in Bryonia dioica: relationship between changes in DNA methylation and ethylene metabolism.  Physiol. Plant.. (1993);  87 25-30
    CrossrefPubMedGoogle Scholar
  • 22 Giordano M., Mattachini M. E., Cella R., Pedrali-Noy G.. Purification and properties of a novel DNA methyltransferase from cultured rice cells.  Biochem. Biophys. Res. Commun.. (1991);  177 711-719
    CrossrefPubMedGoogle Scholar
  • 23 Gruenbaum Y., Naveh-Many T., Cedar H., Razin A.. Sequence specificity of methylation in higher plant DNA.  Nature. (1981);  292 860-862
    CrossrefPubMedGoogle Scholar
  • 24 Jones P. A.. Altering gene expression with 5-azacytidine.  Cell. (1985);  40 485-486
    CrossrefPubMedGoogle Scholar
  • 25 Jones P. A.. The DNA methylation paradox.  Trends Genet.. (1999);  15 3437
    PubMedGoogle Scholar
  • 26 Klaas M., Amasino R. M.. DNA methylation is reduced in DNase I-sensitive regions of plant chromatin.  Plant Physiol.. (1989);  91 451-454
    PubMedGoogle Scholar
  • 27 Leonhardt H., Page A. W., Weier H.-U., Bestor T. H.. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.  Cell. (1992);  71 865-873
    CrossrefPubMedGoogle Scholar
  • 28 Li E., Bestor T. H., Jaenisch R.. Targeted mutation of DNA methyltransferase gene results in embryonic lethality.  Cell. (1992);  69 915-926
    CrossrefPubMedGoogle Scholar
  • 29 Liu Y., Sun L., Jost J.-P.. In differentiating mouse myoblasts DNA methyltransferase is posttranscriptionally and posttranslationally regulated.  Nucl. Acids Res.. (1996);  24 2718-2722
    CrossrefPubMedGoogle Scholar
  • 30 LoSchiavo F., Pitto L., Giuliano G., Torti G., Nuti-Ronchi V., Marazziti D., Vergara R., Orselli S., Terzi M.. DNA methylation of embryogenic carrot cell cultures and its variation as caused by mutation, differentiation, hormones and hypomethylating drugs.  Theor. Appl. Genet.. (1989);  77 325-331
    CrossrefPubMedGoogle Scholar
  • 31 Lowry O. H., Rosebrough N. J., Farr A., Randall R. J.. Protein measurement with the Folin phenol reagent.  J. Biol. Chem.. (1951);  193 265-275
    PubMedGoogle Scholar
  • 32 Messeguer R., Ganal M. W., Steffens J. C., Tanksley S. D.. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA.  Plant Mol. Biol.. (1991);  16 753-770
    CrossrefPubMedGoogle Scholar
  • 33 Modrich P., Lehman I. R.. Enzymatic joining of polynucleotides. IX. A simple and rapid assay of polynucleotide joining (ligase) activity by measurement of circle formation from linear deoxyadenylate-deoxythymidylate copolymer.  J. Biol. Chem.. (1970);  245 3626-3636
    PubMedGoogle Scholar
  • 34 Nielsen E., Rollo F., Parisi B., Cella R., Sala F.. Genetic markers in cultured plant cells: different sensitivities to amethopterin, azetidine-2-carboxylic acid and hydroxyurea.  Plant Science Lett.. (1979);  15 113-125
    PubMedGoogle Scholar
  • 35 Palmgren G., Mattsson O., Okkels F. T.. Specific levels of DNA methylation in various tissues, cell lines, and cell lines of Daucus carota. .  Plant Physiol.. (1991);  95 174-178
    PubMedGoogle Scholar
  • 36 Pedrali-Noy G., Weissbach A.. Mammalian DNA methyltranferases prefer poly(dI-dC) as substrate.  J. Biol. Chem.. (1986);  261 7600-7602
    PubMedGoogle Scholar
  • 37 Podestà A., Castiglione M., Avanzi S., Montagnoli G.. Molecular geometry of antigen binding by a monoclonal antibody against 5-methylcytidine.  Int. J. Biochem.. (1993);  25 929-933
    CrossrefPubMedGoogle Scholar
  • 38 Pradhan S., Adams R. L. P.. Distinct CG and CNG DNA methylation in Pisum sativum. .  Plant J.. (1995);  7 471-481
    CrossrefPubMedGoogle Scholar
  • 39 Razin A., Kafri T.. DNA methylation from embryo to adult.  Prog. Nucl. Acid Res. Mol. Biol.. (1994);  48 53-81
    PubMedGoogle Scholar
  • 40 Ronemus J., Galbiati M., Ticknor C., Chen J., Dellaporta S. L.. Demethylation-induced developmental pleiotropy in Arabidopsis. .  Science. (1996);  273 654-656
    PubMedGoogle Scholar
  • 41 Smith S. S., Kaplan B. E., Sowers L. C., Newman E. M.. Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.  Proc. Natl. Acad. Sci. USA. (1992);  89 4744-4748
    PubMedGoogle Scholar
  • 42 Suzuki K., Nagao K., Tokunaga J., Katayama N., Uyeda M.. Inhibition of DNA methyltransferase by microbial inhibitors and fatty acids.  J. Enzyme Inhib.. (1996);  10 271-280
    PubMedGoogle Scholar
  • 43 Theiss G., Schleicher R., Schimpff-Weiland G., Follmann H.. DNA methylation in wheat. Purification and properties of DNA methyltransferase.  Eur. J. Biochem.. (1987);  167 89-96
    CrossrefPubMedGoogle Scholar
  • 44 Vanyushin B. F.. Replicative DNA methylation in animal and higher plants. Trautner, T. A., ed. Current Topics in Microbiology and Immunology, Vol. 108. Berlin; Springer Verlag (1984): 99-114
    Google Scholar
  • 45 Vergara R., Verde F., Pitto L., Lo Schiavo F., Terzi M.. Reversible variations in the methylation pattern of carrot DNA during somatic embryogenesis.  Plant Cell Rep.. (1990);  8 697-700
    CrossrefPubMedGoogle Scholar
  • 46 Yesufu H. M. I., Hanley A., Rinaldi A., Adams R. L. P.. DNA methylase from Pisum sativum. .  Biochem. J.. (1991);  273 469-475
    PubMedGoogle Scholar

G. Bernacchia

Dipartimento di Biologia
Università di Ferrara

Via Borsari 46

44100 Ferrara

Italy

Email: bhg@dns.unife.it

Section Editor: S. M. Wick

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