Plant Molecular Biology

, Volume 62, Issue 4–5, pp 611–621 | Cite as

Linear molecules of tobacco ptDNA end at known replication origins and additional loci

  • Lars B. Scharff
  • Hans-Ulrich KoopEmail author


Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with ‘single cutter’ restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and—less pronouncedly—with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and␣might therefore represent additional origins of replication.


Replication Chloroplast PFGE Tobacco Origin of replication Plastome 



Inverted repeat in the plastome


Large single copy region in the plastome


Origin of replication


Pulsed-field gel electrophoresis


Plastid DNA


Small single copy region in the plastome


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The authors gratefully acknowledge the expert technical assistance of Stefan Kirchner, the suggestion of Arnold Bendich and Delaine Oldenburg to use pre-electrophoresis and the funding of this work by a grant of the Deutsche Forschungsgemeinschaft (Ko 632/14–1,2).


  1. Asai T, Bates DB, Boye E, Kogoma T (1998) Are minichromosomes valid model systems for DNA replication control? Lessons learned from Escherichia coli. Mol Microbiol 29(3):671–675PubMedCrossRefGoogle Scholar
  2. Backert S Dörfel P, Börner T (1995) Investigation of plant organellar DNAs by pulsed-field gel electrophoresis. Curr Genet 28(4):390–399CrossRefGoogle Scholar
  3. Bitinaite J, Schildkraut I (2002) Self-generated DNA termini relax the specificity of SgrAI restriction endonuclease. Proc Natl Acad Sci USA 99(3):1164–1169PubMedCrossRefGoogle Scholar
  4. Buchanan BB, Gruissem W, Jones RL (2000) Biochemistry & molecular biology of plants. American Society of Plant Phsysiologist, Rockville, p 284Google Scholar
  5. Carrillo N, Bogorad L (1988) Chloroplast DNA replication in vitro: site-specific initiation from preferred templates. Nucleic Acids Res 16(12):5603–5620PubMedGoogle Scholar
  6. Chiu WL, Sears BB (1992) Electron microscopic localization of replication origins in Oenothera chloroplast DNA. Mol Gen Genet 232(1):33–39PubMedCrossRefGoogle Scholar
  7. Dovzhenko A, Bergen U, Koop HU (1998) Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma 204:114–118CrossRefGoogle Scholar
  8. Drescher A, Ruf S, Calsa T Jr, Carrer H, Bock R (2000) The two␣largest chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J 22(2):97–104PubMedCrossRefGoogle Scholar
  9. Fujie M, Kuroiwa H, Kawano S, Mutoh S, Kuroiwa T (1994) Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L. Planta 194(3):395–405CrossRefGoogle Scholar
  10. Hedrick LA, Heinhorst S, White MA, Cannon GC (1993) Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis. Plant Mol Biol 23(4):779–792PubMedCrossRefGoogle Scholar
  11. Hornung S, Fulgosi H, Dörfel P, Herrmann RG (1996) Sequence variation in the putative replication origins of the five genetically distinct basic Euoenothera plastid chromosomes (plastomes). Mol Gen Genet 251(5):609–612PubMedGoogle Scholar
  12. Holt IJ, Lorimer HE, Jacobs HT (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100(5):515–524PubMedCrossRefGoogle Scholar
  13. Jeong SY, Rose A, Meier I (2003) MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure. Nucleic Acids Res 31(17):5175–5185PubMedCrossRefGoogle Scholar
  14. Johnson DA, Hattori J (1996) Analysis of a hotspot for deletion formation within the intron of the chloroplast trnI gene. Genome 39(5):999–1005PubMedGoogle Scholar
  15. Kolodner RD, Tewari KK (1972) Molecular size and conformation of chloroplast deoxyribonucleic acid from pea leaves. J Biol Chem 247(19):6355–6364PubMedGoogle Scholar
  16. Kolodner RD, Tewari KK (1975) Chloroplast DNA from higher plants replicates by both the Cairns and the rolling circle mechanism. Nature 256(5520):708–711PubMedCrossRefGoogle Scholar
  17. Kolodner RD, Tewari KK (1979) Inverted repeats in chloroplast DNA from higher plants. Proc Natl Acad Sci USA 76(1):41–45PubMedCrossRefGoogle Scholar
  18. Kreuzer KN (2000) Recombination-dependent DNA replication in phage T4. Trends Biochem Sci 25(4):165–173PubMedCrossRefGoogle Scholar
  19. Kunnimalaiyaan M, Shi F, Nielsen BL (1997) Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene. J Mol Biol 268(2):273– 283PubMedCrossRefGoogle Scholar
  20. Kunnimalaiyaan M, Nielsen BL (1997) Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA. Nucleic Acids Res 25(18):3681–3686PubMedCrossRefGoogle Scholar
  21. Lawrence ME, Possingham JV (1986) Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry. J Histochem Cytochem 34(6):761–768PubMedGoogle Scholar
  22. Lilly JW, Havey MJ, Jackson SA, Jiang J (2001) Cytogenomic analyses reveal the structural plasticity of the chloroplast genome in higher plants. Plant Cell 13(2):245–254PubMedCrossRefGoogle Scholar
  23. Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P, Darnell J (1995) Molecular cell biology, 3rd edn. Scientific American Books, New York, p 830Google Scholar
  24. Lou JK, Wu M, Chang CH, Cuticchia AJ (1987) Localization of␣a r-protein gene within the chloroplast DNA replication origin of Chlamydomonas. Curr Genet 11(6–7):537–541PubMedCrossRefGoogle Scholar
  25. Lu Z, Kunnimalaiyaan M, Nielsen BL (1996) Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA. Plant Mol Biol 32(4):693–706PubMedCrossRefGoogle Scholar
  26. Lugo SK, Kunnimalaiyaan M, Singh NK, Nielsen BL (2004) Required sequence elements for chloroplast DNA replication activity in vitro and in electroporated chloroplasts. Plant Sci 166(1):151–161CrossRefGoogle Scholar
  27. Mühlbauer SK, Lössl A, Tzekova L, Zou Z, Koop HU (2002) Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation. Plant J 32(2):175–184PubMedCrossRefGoogle Scholar
  28. Newman SM, Harris EH, Johnson AM, Boynton JE, Gillham NW (1992) Nonrandom distribution of chloroplast recombination events in Chlamydomonas reinhardtii: evidence for a hotspot and an adjacent cold region. Genetics 132(2):413–429PubMedGoogle Scholar
  29. Oldenburg DJ, Bendich AJ (2001) Mitochondrial DNA from the liverwort Marchantia polymorpha: circularly permuted linear molecules, head-to-tail concatemers, and a 5′ protein. J Mol Biol 310(3):549–562PubMedCrossRefGoogle Scholar
  30. Oldenburg DJ, Bendich AJ (2004) Most chloroplast DNA of maize seedlings in linear molecules with defined ends and branched forms. J Mol Biol 335(4):953–970PubMedCrossRefGoogle Scholar
  31. Palmer JD (1983) Chloroplast DNA exists in two orientations. Nature 301:92–93CrossRefGoogle Scholar
  32. Sato N, Albrieux C, Joyard J, Douce R, Kuroiwa T (1993) Detection and characterization of a plastid envelope DNA-binding protein which may anchor plastid nucleoids. EMBO J 12(2):555–561PubMedGoogle Scholar
  33. Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torazawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete nucleotide sequence of tobacco chloroplast genome: its gene organization and expression. EMBO J 5:2043–2049PubMedGoogle Scholar
  34. Sugiura M, Shinozaki K, Zaita N, Kusuda M, Kumano M (1986) Clone bank of the tobacco (Nicotiana tabacum) chloroplast genome as a set of overlapping restriction endonuclease fragments: mapping of eleven ribosomal proteins. Plant Sci 44:211–216CrossRefGoogle Scholar
  35. Swiatek M, Greiner S, Kemp S, Drescher A, Koop H-U, Herrmann RG, Maier RM (2003) PCR analysis of pulsed field gel electrophoresis-purified plastid DNA, a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants. Curr Genet 43: 45–53PubMedGoogle Scholar
  36. Takeda Y, Hirokawa H, Nagata T (1992) The replication origin of proplastid DNA in cultured cells of tobacco. Mol Gen Genet 232(2):191–198PubMedGoogle Scholar
  37. Wang JC (1996) DNA topoisomerases. Annu Rev Biochem 65:635–692PubMedCrossRefGoogle Scholar
  38. Wang Y, Tamura K, Saitoh Y, Sato T, Hidaka S, Tsutsumi K (2002) Mapping major replication origins on the rice plastid DNA. Plant Biotech 19:27–35Google Scholar
  39. Wang Y, Saitoh Y, Sato T, Hidaka S, Tsutsumi K (2003) Comparison of plastid DNA replication in different cells and tissues of the rice plant. Plant Mol Biol 52(4):905–913PubMedCrossRefGoogle Scholar
  40. Weigel C, Messer W, Preiss S, Welzeck M, Boye E (2001) The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome. Mol Microbiol 40(2):498–507PubMedCrossRefGoogle Scholar
  41. Williamson DH, Preiser PR, Moore PW, McCready S, Strath M, Wilson RJ (2002) The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms. Mol Microbiol 45(2):533–542PubMedCrossRefGoogle Scholar
  42. Woelfle MA, Thompson RJ, Mosig G (1993) Roles of novobiocin-sensitive topoisomerases in chloroplast DNA replication in Chlamydomonas reinhardtii. Nucleic Acids Res 21(18):4231–4238PubMedGoogle Scholar
  43. Wycliffe P, Sitbon F, Wernersson J, Ezcurra I, Ellerstrom M, Rask L (2005) Continuous expression in tobacco leaves of a Brassica napus PEND homologue blocks differentiation of plastids and development of palisade cells. Plant J 44(1):1–15PubMedCrossRefGoogle Scholar
  44. Yap WY, Kreuzer KN (1991) Recombination hotspots in bacteriophage T4 are dependent on replication origins. Proc Natl Acad Sci USA 88(14):6043–6047PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  1. 1.Department of Biology I – BotanyUniversity of MunichMünchenGermany

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