The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following3H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA.
Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells.
The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis.
These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.
DNA synthesis inhibitors synchronization tobacco suspension cultures
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Blamire J, Flechtner VR, Sager R: Regulation of nuclear DNA replication by the chloroplast inChlamydomonas. Proc Natl Acad Sci USA 71:2867–2871, 1974.PubMedCrossRefGoogle Scholar
Boffey SA, Ellis JR, Sellden G, Leech RM: Chloroplast division and DNA synthesis in light-grown wheat leaves. Plant Physiol 64:502–505, 1979.PubMedCrossRefGoogle Scholar
Butterfass T: The continuity of plastids and the differentiation of plastid populations. In: Reinert J (ed) Results and problems in cell differentiation, Vol 10. Springer-Verlag, Berlin, Heidelberg, New York, 1980, pp 29–45.Google Scholar
Cashmore AR: Protein synthesis in plant leaf tissue. The sites of synthesis of the major proteins. J Biol Chem 251:2848–2853, 1976.PubMedGoogle Scholar
Denhardt D: A membrane filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23:641–646, 1966.PubMedCrossRefGoogle Scholar
Ellis RJ: Chloroplast protein synthesis: Principles and problems. In: Roodyn DB (ed) Subcellular Biochemistry, Vol 9. Plenum Publishing Corporation, 1983, pp 237–261.PubMedGoogle Scholar
Flechtner VR, Sager R: Ethidium bromide induced selective and reversible loss of chloroplast DNA. Nature New Bio 241:277–279, 1973.CrossRefGoogle Scholar
Fluhr R, Edelman M: Conservation of sequence arrangement among higher plant chloroplast DNAs: molecular cross hybridization among the Solanaceae and betweenNicotiana andSpinacea. Nucleic Acids Res 9:6841–6853, 1981.PubMedGoogle Scholar
Galli MG, Sala F: Aphidicolin as synchronizing agent in root tip meristems ofHaplopappus gracilis. Plant Cell Reports 2:156–159, 1983.CrossRefGoogle Scholar
Gillespie D, Spiegelman S: A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol 12:819–842, 1965.CrossRefGoogle Scholar
Goldring ES, Grossman LI, Krupnik D, Cryer DR, Marmur J: The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol 52:323–335, 1970.PubMedCrossRefGoogle Scholar
De Heij HT, Jochemsen AG, Willemsen PTJ, Groot GSP: Protein synthesis during chloroplast development inSpirodela oligorhiza. Eur J Biochem 138:161–168, 1984.PubMedCrossRefGoogle Scholar
Herrmann RG, Feierabend J: The presence of DNA in ribosome-deficient plastids of heat-bleached rye leaves. Eur J. Biochem 104:603–609, 1980.PubMedCrossRefGoogle Scholar
Lamppa GK, Bendich AJ: Changes in chloroplast DNA levels during development of pea(Pisum sativum). Plant Physiol 64:126–130, 1979.PubMedGoogle Scholar
Lurquin PF, Kleinhofs A: Effects of chloramphenicol on plant cells: Potential as a selectable marker for transformation studies. Biochem Biophys Res Commun 107:286–293, 1982.PubMedCrossRefGoogle Scholar
Misumi M, Weissbach A: The isolation and characterization of DNA polymeraseα from spinach. J Biol Chem 257:2323–2329, 1982.PubMedGoogle Scholar
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497, 1962.CrossRefGoogle Scholar
Murray MG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325, 1980.PubMedGoogle Scholar
Nass MMK: Abnormal DNA pattern in animal mitochondria: Ethidium bromide-induced breakdown of closed circular DNA and conditions leading to oligomer accumulation. Proc Natl Acad Sci USA 67:1926–1933, 1970.PubMedCrossRefGoogle Scholar
Sala F, Amileni AR, Parisi B, Spadari S: Aγ-like DNA polymerase in spinach chloroplasts. Eur J Biochem 112:211–217, 1980PubMedCrossRefGoogle Scholar
Sala F, Galli MG, Levi M, Burroni D, Parisi B, Pedrali-Noy G, Spadari S: Functional roles of the plantα-like andγ-like DNA polymerases. FEBS Letters 124:112–118, 1981.PubMedCrossRefGoogle Scholar
Sala F, Galli MG, Nielsen E, Magnien E, Devreux M, Pedrali-Noy G, Spadari S: Synchronization of nuclear DNA synthesis in culturedDaucus carota L. cells by aphidicolin. FEBS Letters 153:204–208, 1983.CrossRefGoogle Scholar
Sala F, Parisi B, Burroni D, Amileni AR, Pedrali-Noy G, Spadari S: Specific and reversible inhibition by aphidicolin of theα-like DNA polymerases of plant cells. FEBS Letters 117:93–98, 1980.PubMedCrossRefGoogle Scholar
Sala F, Sala C, Galli MG, Nielsen E, Pedrali-Noy G, Spadari S: Inactivation of aphidicolin by plant cells. Plant Cell Reports 2:265–268, 1983.CrossRefGoogle Scholar
Schmidt GW, Bartlett SG, Grossman AR, Cashmore AR, Chua N-H: Biosynthetic pathway of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex. J Cell Biol 9:468–478, 1981.CrossRefGoogle Scholar
Scott NS, Cain P, Possingham JV: Plastid DNA levels in albino and green leaves of the ‘albostrians’ mutant ofHordeum vulgare. Z. Pflanzenphysiol 108:187–191, 1982.Google Scholar
Scott NS, Possingham JV: Changes in chloroplast DNA levels during growth of spinach leaves. J Exp Botany 34:1756–1767, 1983.Google Scholar
Summers J: Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme fromHaemophilus aegypticus, endonuclease R.HaeIII. J Virol 15:946–953, 1975.PubMedGoogle Scholar
Tassi F, Restino FM, Ferrari C, Puglisi PP: Erythromycin as a tool for discriminatingin vivo between mitochondrial and chloroplastic protein synthesis inNicotiana sylvestris. Plant Sci Lett 29:215–225, 1983.CrossRefGoogle Scholar