Skip to main content
SpringerLink
Log in

Investigation of plant organellar DNAs by pulsed-field gel electrophoresis

  • Original Paper
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Mitochondrial (mt) DNAs from several higher-plant species (Arabidopsis thaliana, Beta vulgaris, Brassica hirta, Chenopodium album, Oenothera berteriana, Zea mays) were separated by pulsed-field gel electrophoresis (PFGE). Hybridization of the separated DNA with mtDNA-specific probes revealed an identical distribution of mtDNA sequences in all cases: part of the DNA formed a smear of linear molecules migrating into the gel, the rest remained in the well. Hybridization signals in the compression zone of the gels disappeared after RNase or alkaline treatment. It was shown that the linear molecules are not products of unspecific degradation by nucleases. All plastid (pt) DNA from leaves of Nicotiana tabacum remained in the well after PFGE. Separation of linear monomers and oligomers of the chloroplast chromosomes of N. tabacum was achieved by mild DNase treatment of the well-bound DNA. DNase treatment of well-bound mtDNA, however, generated a smear of linear molecules. PtDNA from cultured cells of C. album was found after PFGE to be partly well-bound, and partly separated into linear molecules with sizes of monomeric and oligomeric chromosomes. The ease with which it was possible to detect large linear molecules of plastid DNA indicates that shearing forces alone can not explain the smear of linear molecules obtained after PFGE of mtDNA. The results are discussed in relation to the structural organization of the mt genome of higher plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Albring M, Griffith J, Attardi G (1977) Association of a protein of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication. Proc Natl Acad Sci USA 74:1348–1352

    Google Scholar 

  • André CP, Walbot V (1995) Pulsed-field gel mapping of maize mitochondrial chromosomes. Mol Gen Genet 247:255–263

    Google Scholar 

  • André CP, Levy AA, Walbot V (1992) Small repeated sequences and the structure of plant mitochondrial genomes. Trends Genet 8:128–132

    Google Scholar 

  • Bendich AJ (1993) Reaching for the ring: the study of mitochondrial genome structure. Curr Genet 24:279–290

    Google Scholar 

  • Bendich AJ, Smith SB (1990) Moving pictures and pulsed-field gel electrophoresis show linear DNA molecules from chloroplasts and mitochondria. Curr Genet 17:421–425

    Google Scholar 

  • Bendich AJ, Loretz CJ, Monnat RJ Jr (1993) The structure of the plant mitochondrial genome. In: Brennicke A, Kück U (eds) Plant mitochondria. Verlag Chemie, Weinheim, pp 171–180

    Google Scholar 

  • Beverley SM (1988) Characterization of the ‘unusual’ mobility of large circular DNAs in pulsed-field gradient electrophoresis. Nucleic Acids Res 16:925–939

    Google Scholar 

  • Beyermann B, Nürnberg P, Weihe A, Meixner M, Epplen JT, Börner T (1992) Fingerprinting plant genomes with oligonucleotide probes specific for simple repetitive DNA sequences. Theor Appl Genet 83:691–694

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Borst P, Hoeijmakers JHJ (1979) Kinetoplast DNA. Plasmid 2:20–40

    Google Scholar 

  • Brears T, Lonsdale DM (1988) The sugar beet mitochondrial genome: a complex organization generated by homologous recombination. Mol Gen Genet 214:514–522

    Google Scholar 

  • Brennicke A (1980) Mitochondrial DNA from Oenothera berteriana: purification and properties. Plant Physiol 65:1207–1210

    Google Scholar 

  • Brennicke A, Clayton DA (1981) Nucleotide assignment of alkalisensitive sites in mouse mitochondrial DNA. J Biol Chem 256:10613–10617

    Google Scholar 

  • Brennicke A, Blanz P (1982) Circular mitochondrial DNA species from Oenothera with unique sequences. Mol Gen Genet 187:461–466

    Google Scholar 

  • Deng X-W, Wing RA, Gruissem W (1989) The chloroplast genome exists in multimeric forms. Proc Natl Acad Sci USA 86:4156–4160

    Google Scholar 

  • Dörfel P, Weihe A, Knösche R, Börner T (1989) Mitochondrial DNA of Chenopodium album (L.): a comparison of leaves and suspension cultures. Curr Genet 16:375–380

    Google Scholar 

  • Dörfel P, Weihe R, Dolferus R, Börner T (1991) DNA sequence of a mitochondrial plasmid from Chenopodium album. Plant Mol Biol 17:155–156

    Google Scholar 

  • Echeverria M, Robert D, Carde JP, Litvak S (1991) Isolation from wheat mitochondria of a membrane-associated high-molecular-weight complex involved in DNA synthesis. Plant Mol Biol 16:301–315

    Google Scholar 

  • Eckhardt T (1978) A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid 1:584–588

    Google Scholar 

  • Gwynn B, Dewey RE, Sederoff RR, Timothy DH, Levings III CS (1987) Sequence of the 18s–5s ribosomal gene region and the cytochrome oxidase II gene from mtDNA of Zea diploperennis. Theor Appl Genet 74:781–788

    Google Scholar 

  • Hiratsuka J, Shimada H, Whittier RF, Ishibashi T, Sakamoto M, Mori M, Kondo C, Honji Y, Sun C-R, Meng B-Y, Li Y-Q, Kanno A, Nishizawa Y, Hirai A, Shinozaki K, Sugiura M (1989) The complete sequence of the rice (Oryza sativa) chloroplast genome: intermolecular recombination between distinct tRNA genes accounts for a major plastid DNA inversion during the evolution of the cereals. Mol Gen Genet 217:185–194

    Google Scholar 

  • Kawano S, Kuroiwa T (1985) Isolation and characterization of a membrane-DNA complex in the mitochondria of Physarum polycephalum. Exp Cell Res 161:460–472

    Google Scholar 

  • Kenwrick S, Pattersohn M, Speer A, Fischbeck K, Davies K (1987) Molecular analysis of the Duchenne muscular dystrophy region using pulsed-field gel electrophoresis. Cell 48:351–357

    Google Scholar 

  • Klein RR, Mullet JE (1986) Regulation of chloroplast-encoded chlorophyll-binding protein translation during higher-plant chloroplasts biogenesis. J Biol Chem 261:11138–11145

    Google Scholar 

  • Knösche R, Günther G (1988) A cell-division cycle in suspension cultures from Chenopodium album with unspecific arrest at G1 and G2 phase under stationary growth conditions. Biol Zentralbl 107:653–661

    Google Scholar 

  • Leaver CJ, Hack E, Forde BG (1983) Protein synthesis by isolated plant mitochondria. Methods Enzymol 97:476–484

    Google Scholar 

  • Levenne SD, Zimm BH (1987) Separation of open-circular DNA using pulsed-field electrophoresis. Proc Natl Acad Sci USA 84:4054–4057

    Google Scholar 

  • Levy AA, André CP, Walbot V (1991) Analysis of a 120-kilobase mitochondrial chromosome in maize. Genetics 128:417–424

    Google Scholar 

  • Lonsdale DM, Jones IG (1978) Localization of the ribonucleotide sites in rat liver mitochondrial deoxyribonucleic acid. Biochem J 169:79–85

    Google Scholar 

  • Lonsdale DM, Hodge TP, Fauron CM-R (1984) The physical map and organization of the mitochondrial genome from the fertile cytoplasm of maize. Nucleic Acids Res 12:9249–9261

    Google Scholar 

  • Maleszka R, Skelly PJ, Clark-Walker GD (1991) Rolling-circle replication of DNA in yeast mitochondria. EMBO J 10:3923–3929

    Google Scholar 

  • Manna F, Brennicke A (1985) Primary and secondary structure of 26 ribosomal RNA of Oenothera berteriana. Curr Genet 9:505–515

    Google Scholar 

  • Margolin K, Doda JN, Clayton DA (1981) Mechanism of mitochondrial DNA replication in mouse L-cells: localization of alkali sensitive sites at the two origins of replication. Plasmid 6:332–341

    Google Scholar 

  • Meißner K, Dörfel P, Börner T (1992) Topoisomerase activity in mitochondrial lysates of a higher plant (Chenopodium album L.) Biochem Int 27:1119–1125

    Google Scholar 

  • Meißner K, Heinhorst S, Cannon GC, Börner T (1993) Purification and characterization of a γ-like DNA polymerase from Chenopodium album L. Nucleic Acids Res 21:4893–4899

    Google Scholar 

  • Oda K, Yamato K, Ohta E, Nakamura Y, Takemura M, Nozato N, Akashi K, Kanegae T, Ogura Y, Kohchi T, Ohyama K (1992) Gene organization deduced from the complete sequence of liverwort marchantia polymorpha mitochondrial DNA. J Mol Biol 223:1–7

    Google Scholar 

  • Narayanan KK, André CP, Yang J, Walbot V (1993) Organization of a 117-kb circular mitochondrial chromosome in IR36 rice. Curr Genet 23:248–254

    Google Scholar 

  • Palmer (1992) Chloroplast and mitochondrial genome evolution in land plants. In: Herrmann RG (eds) Cell organelles. Springer Verlag, Wien, pp 99–133

    Google Scholar 

  • Palmer JD, Shields CR (1984) Tripartite structure of the Brassica campestris mitochondrial genome. Nature 307:437–440

    Google Scholar 

  • Palmer JD, Herbon LA (1987) Unicircular structure of the Brassica hirta mitochondrial genome. Curr Genet 11:565–570

    Google Scholar 

  • Schuster W, Brennicke A (1985) TGE-termination codon in the apocytochrome b gene from Oenothera mitochondria. Curr Genet 9:157–163

    Google Scholar 

  • Schuster W, Brennicke A (1994) The plant mitochondrial genome: physical structure, information content, RNA editing, and gene migration to the nucleus. Annu Rev Plant Physiol Plant Mol Biol 45:61–78

    Google Scholar 

  • Schuster W, Hiesel B, Wiesinger B, Schobel W, Brennicke A (1988) Isolation and analysis of plant mitochondria and their genomes. In: Shaw CH (ed) Plant molecular biology — a practical approach, IRL Press, Oxford, pp 79–102

    Google Scholar 

  • Shapiro TA (1993) Kinetoplast DNA maxicircles: networks within networks. Proc Natl Acad Sci USA 90:7809–7813

    Google Scholar 

  • Shearman CW, Kalf GF (1977) DNA replication by a membrane-DNA complex from rat liver mitochondria. Arch Biochem Biophys 182:573–582

    Google Scholar 

  • Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki, 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 the tobacco chloroplast genome: its gene organization and expression. EMBO U 5:2043–2049

    Google Scholar 

  • Siemenroth A, Wollgiehn R, Neumann D, Börner T (1981) Synthesis of ribosomal RNA in ribosome-deficient plastids of the mutant ‘albostrians’ of Hordeum vulgare L. Planta 153:547–555

    Google Scholar 

  • Small ID, Suffolk R, Leaver CJ (1989) Evolution of plant mitochondrial genomes via substoichiometric intermediates. Cell 58:69–76

    Google Scholar 

  • Unseld M, Brandt P, Heinze B, Eckert-Ossenkopp U, Brennicke A (1993) The mitochondrial genome of Arabidopsis thaliana. In: Brennicke A, Kück U (eds) Plant mitochondria. Verlag Chemie, pp 137–143

  • Ward BL, Anderson RS, Bendich AJ (1981) The mitochondrial genome is large and variable in a family of plants (Cucurbitaceae). Curr Genet 12:55–67

    Google Scholar 

  • Wilson AJ, Choury PS (1984) An rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources. Plant Cell Rep 3:237–239

    Google Scholar 

  • Wolf G, Kück U (1993) Organization and coding capacity of mitochondrial genomes of algae. In: Brennicke A, Kück U (eds) Plant mitochondria. Verlag Chemie, Weinheim, pp 103–113

    Google Scholar 

Download references

Author information

Author notes

  1. Peter Dörfel

    Present address: Botanisches Institut, Ludwig-Maximilians-Universität München, Menzinger Strasse 67, D-80638, München, Germany

Authors and Affiliations

  1. Institut für Biologie, Humboldt-Universität zu Berlin, Invalidenstrasse 43, D-10115, Berlin, Germany

    Steffen Backert, Peter Dörfel & Thomas Börner

Authors
  1. Steffen Backert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Dörfel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Börner
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by H. Kössel

Rights and permissions

Reprints and permissions

About this article

Cite this article

Backert, S., Dörfel, P. & Börner, T. Investigation of plant organellar DNAs by pulsed-field gel electrophoresis. Curr Genet 28, 390–399 (1995). https://doi.org/10.1007/BF00326439

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00326439

Key words

Search

Navigation