Plant Molecular Biology

, Volume 16, Issue 1, pp 59–70 | Cite as

Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays

  • Miguel Carballo
  • Ramon Giné
  • Mireia Santos
  • Pere Puigdomènech
Article
Received:
Accepted:

Abstract

We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits cukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.

Key words

abscisic acid auxin topoisomerases I and II Zea mays embryos 
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    AckermanP, GloverCVC, OsheroffN: Phosphorylation of DNA topoisomerase II by casein kinase II: Modulation of eukaryotic topoisomerase II activity in vitro. Proc Natl Acad Sci USA 82: 3164–3168 (1985).Google Scholar
  2. 2.
    AckermanP, GloverCVC, OsheroffH: Phosphorylation of DNA topoisomerase II in vivo in total homogenates of Drosophila Kc cells. J Biol Chem 263: 12653–12660 (1988).Google Scholar
  3. 3.
    BradfordMM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).Google Scholar
  4. 4.
    CarballoM, PuigdoménechP, PalauJ: DNA and histone H1 interact with different domains of HMG1 and 2 proteins. EMBO J 2: 1759–1764 (1983).Google Scholar
  5. 5.
    ChampouxJ, AronoffR: The effects of camptothecin on the reaction and specificity of the wheat germ type I topoisomerase. J Biol Chem 264: 1010–1015 (1989).Google Scholar
  6. 6.
    ChenGL, YangL, RoweCT, HalliganBD, TeweyMK, LiuLF: Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259: 13560–13566 (1984).Google Scholar
  7. 7.
    DaviesSM, RobsonCN, DaviesSL, HicksonID: Nuclear topoisomerase II levels correlate with the sensitivity of mammalian cells to intercalating agents and epipodophyllotoxins. J Biol Chem 263: 17724–17729 (1988).Google Scholar
  8. 8.
    DurbanE, MillsSS, RollD, BuschH: Phosphorylation of purified Novikoff Hepatoma Topoisomerase I. Biochem Biophys Res Commun 111: 897–905 (1983).Google Scholar
  9. 9.
    DurbanE, RollD, BecknerG, BuschH: Purification and characterization of a nuclear DNA-binding phosphoprotein in fetal and tumor tissues. Cancer Res 41: 537–545 (1981).Google Scholar
  10. 10.
    DynanWS, JendrisakSS, HagerDA, BurgesRR: Purification and characterization of wheat germ topoisomerase I (nicking-closing enzyme). J Biol Chem 256: 5860–5865 (1981).Google Scholar
  11. 11.
    EcheverriaM, MartinMT, RicardB, LitvakS: A DNA topoisomerase type I from wheat embryo mitochondria. Plant Mol Biol 6: 417–427 (1986).Google Scholar
  12. 12.
    EttlingerC, LehleL: Auxin induces rapid changes in phosphatidylinositol metabolites. Nature 331: 176–178 (1988).Google Scholar
  13. 13.
    FukataH, OhgamiK, FukasuraH: Isolation and characterization of DNA topoisomerase II from cauliflower inflorescences. Plant Mol Biol 6: 137–144 (1986).Google Scholar
  14. 14.
    GreenCE, PillipsRL: Plant regeneration from tissue cultures of maize. Crop Sci 15: 417–421 (1975).Google Scholar
  15. 15.
    HsiangYH, HertzbergR, HechtS, LiuLF: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260: 14873–14878 (1985).Google Scholar
  16. 16.
    HsiehTS: Purification and properties of type II DNA topoisomerase from embryos of Drosophila melanogaster. Methods Enzymol 100: 161–170 (1983).Google Scholar
  17. 17.
    HsiehT, GrutlagD: ATP-dependent DNA topoisomerase from Drosophila melanogaster reversibly catenates duplex DNA rings. Cell 21: 115–125 (1980).Google Scholar
  18. 18.
    LaemmliUK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).Google Scholar
  19. 19.
    LamE, ChuaNH: Chloroplast DNA gyrase and in vitro regulation of transcription by template topology and novobiocin. Plant Mol Biol 8: 415–424 (1987).Google Scholar
  20. 20.
    LiuLF: HeLa topoisomerase I. Methods Enzymol 100: 133–137 (1983).Google Scholar
  21. 21.
    ManiatisT, FrischE, SambrookJ: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).Google Scholar
  22. 22.
    MorriseySH: Silver stain for proteins in polyacrylamide gels: a modified produce with enhanced uniform sensitivity. Anal Biochem 117: 307–310 (1981).Google Scholar
  23. 23.
    NelsonGW, ChoRK, HsiangYH, LiuLF, CoffeyD: Growth-related elevations of DNA topoisomerase II levels found in Dunning R3327 rat prostatic adenocarcinomas. Cancer Res 47: 3246–3250 (1987).Google Scholar
  24. 24.
    NielsenBL, TewariKK: Pea chloroplast topoisomerase. I: Purification, characterization and role in replication. Plant Mol Biol 11: 3–14 (1988).Google Scholar
  25. 25.
    NishizukaY: The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature 308: 693–698 (1984).Google Scholar
  26. 26.
    OsheroffN, SheltonER, BrutlagDL: DNA topoisomerase II from Drosophila melanogaster: Relaxation of supercoil DNA. J Biol Chem 258: 9536–9543 (1983).Google Scholar
  27. 27.
    RottmannM, SchröderHC, GramazowM, RenneisenK, KurelecB, DornA, FrieseU, MüllerWEG: Specific phoshorylation of proteins in pore complex-laminal from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. EMBO J, 6: 3939–3944 (1987).Google Scholar
  28. 28.
    SahyounN, WolfM, BestermanJ, HsiehTS, SanderM, LeVineIIIH, ChangKJ, CuatrecasasP: Protein kinase C phosphorylates topoisomerase II: Topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. Proc Natl Acad Sci USA 83: 1603–1607 (1986).Google Scholar
  29. 29.
    SantosMA, TornéJM, BlancoJL: Methods of obtaining maize totipotent tissues in seedling segments culture. Plant Sci Lett 33: 309–315 (1984).Google Scholar
  30. 30.
    SiedleckiJ, ZimmermannW, WeissbachA: Characterization of a prokaryotic topoisomerase I activity in chloroplast extracts from spinach. Nucleic Acids Res 11: 1523–1536 (1983).Google Scholar
  31. 31.
    TornéJM, SantosMA, BlancoJL: Methods of obtaining maize totipotent tissues; atrophic tissue culture. Plant Sci Lett 33: 317–325 (1984).Google Scholar
  32. 32.
    VasilIK: Progress in regeneration and genetic manipulation of cereal crops. Bio/technology 6: 397–402 (1988).Google Scholar
  33. 33.
    WangJC: DNA topoisomerases. Annu Rev Biochem 54: 665–697 (1985).Google Scholar

Copyright information

© Kluwer Academic Publishers 1991

Authors and Affiliations

  • Miguel Carballo
    • 1
  • Ramon Giné
    • 1
  • Mireia Santos
    • 2
  • Pere Puigdomènech
    • 1
  1. 1.Departmento de Genética Molecular, Centro de Investigación y DesarrolloCSICBarcelonaSpain
  2. 2.Departamento de Biología Molecular y Agrobiología, Centro de Investigación y DesarrolloCSICBarcelonaSpain

Personalised recommendations