Bioenergetics pp 441-449 | Cite as

S1 Nuclease Analysis and Direct Sequencing of Deleted Mitochondrial DNA in Myopathic Patients: Role of Directly Repeated Sequences in Deletion

  • Masashi Tanaka
  • Wataru Sato
  • Kinji Ohno
  • Tomoko Yamamoto
  • Takayuki Ozawa


Heteroplasmy of the normal-sized and the deleted mitochondrial DNA (mtDNA) has been observed in mitochondrial myopathy. To investigate the mechanism of mtDNA deletion, we analyzed the crossover sequences of mtDNA in the skeletal muscles of five patients with mitochondrial myopathy. We localized the deleted region using the combination of polymerase chain reaction and S1 nuclease digestion. Then, we directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5′-ATCCCCA-3′ was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, a 13-bp sequence of 5′-ACCTCCCTC ACCA-3′ was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 3, a 3-bp sequence of 5′-CCT-3′ was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. In Patients 4 and 5 with multiple deletions, one of the mutant mitochondrial DNA was amplified. Direct sequencing of the mutant mtDNA revealed the same directly repeated sequence as in Patient 2. These directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.


Polymerase Chain Reaction Method Mitochondrial Myopathy ATPase Subunit Nuclease Digestion Heteroduplex Formation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. 1.
    Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.L., Coulson, A.R., Drouin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J.H., Staden, R., and Young, I.G. (1981) Nature 290, 457–465PubMedCrossRefGoogle Scholar
  2. 2.
    Chomyn, A., Mariottini, P., Cleeter, M.W.J., Ragan, C.I., Matsuno-Yagi, A., Hatefi, Y., Doolittle, R.F., and Attardi, G. (1985) Nature 314, 592–597PubMedCrossRefGoogle Scholar
  3. 3.
    Gyles, R.E., Blanc, H., Cann, H.M., and Wallace, D.C. (1980) Proc. Natl Acad. Sci. USA 77, 6715–6719.CrossRefGoogle Scholar
  4. 4.
    Neckelmann, N., Li, K., Wade, R.P., Sinister, R., and Wallace, D.C. (1980) Proc. Natl. Acad. Sci. USA 87, 7580–7584.Google Scholar
  5. 5.
    Linnane, A.W., Marzuki, S., Ozawa, T., and Tanaka, M. (1989) Lancet i, 642–645.CrossRefGoogle Scholar
  6. 6.
    Holt, I.J., Harding, A.E., and Morgan-Hughes, J.A. (1988) Nature 331, 717–719.PubMedCrossRefGoogle Scholar
  7. 7.
    Lestienne, P., and Ponsot, G. (1988) Lancet i, 885.CrossRefGoogle Scholar
  8. 8.
    Zeviani, M., Moraes, C.T., DiMauro, S., Nakase, H., Bonilla, E., Schon, E.A., and Rowland, L.P. (1988) Neurology 38, 1339–1346.PubMedGoogle Scholar
  9. 9.
    Ozawa, T., Yoneda, M., Tanaka, M., Ohno, K., Sato, W., Suzuki, H., Nishikimi, M., Yamamoto, M., Nonaka, I., and Horai, S. (1988) Biochem. Biophys. Res. Commun. 154, 1240–1247.PubMedCrossRefGoogle Scholar
  10. 10.
    Noer, A.S., Marzuki, S., Trounce, I., and Byrne, E. (1988) Lancet ii, 1253–1254.CrossRefGoogle Scholar
  11. 11.
    Rotig, A., Colonna, M., Blanche, S., S., Fischer, A., Le Deist, F., Frezal, J., Saudubray, L-M., and Munnich, A. (1988) Lancet ii, 567–568.CrossRefGoogle Scholar
  12. 12.
    Rotig, A., Colonna, M., Bonnefont, J.P., Blanche, S., Fischer, A., Saudubray, J.M., and Munnich, A. (1989) Lancet i, 902–903.CrossRefGoogle Scholar
  13. 13.
    Tanaka-Yamamoto, T., Tanaka, M., Ohno, K., Sato, W., Horai, S., and Ozawa, T. (1989) Biochim. Biophys. Acta 1009, 151–155.PubMedGoogle Scholar
  14. 14.
    Gyllensten, U.B., and Erlich, H.A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652–7656.PubMedCrossRefGoogle Scholar
  15. 15.
    Tanaka, M., Sato, W., Ohno, K., Yamamoto, T., and Ozawa, T. (1989) Biochem. Biophys. Res. Commun. 164, 156–163.PubMedCrossRefGoogle Scholar
  16. 16.
    Sato, W., Tanaka, M., Ohno, K., Yamamoto, T., Takada, G., and Ozawa, T. (1989) Biochem. Biophys. Res. Commun. 162, 664–672.PubMedCrossRefGoogle Scholar
  17. 17.
    Taber, S. (1987) Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J. G., Smith, J.A., and Struhl, K., eds) Vol. 1, 3.12.2, John Wiley & Sons, New York.Google Scholar
  18. 18.
    Perbel, B. (1988) Practical Guide to Molecular Cloning, pp. 636–640. John Wiley & Sons, New York.Google Scholar
  19. 19.
    Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463–5467.PubMedCrossRefGoogle Scholar
  20. 20.
    Schon, E.A., Rizzuto, R., Moraes, C.T., Nakase, H., Zeviani, M., and DiMauro, S. (1989) Science 244, 346–349.PubMedCrossRefGoogle Scholar
  21. 21.
    Attardi, G. (1981) Trends Biochem. Sci. 89, 100–103.CrossRefGoogle Scholar
  22. 22.
    Vnencak-Jones, C.L., Phillips, J.A.III, Chen, E.Y., and Seeburg, P.H. (1988) Proc. Natl. Acad. Sci. USA 85, 5615–5619.PubMedCrossRefGoogle Scholar
  23. 23.
    Zeviani, M., Servidei, S., Gellera, C., Bertini, E., DiMauro, S., and DiDonato, S. (1989) Nature 339, 309–311.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Masashi Tanaka
    • 1
  • Wataru Sato
    • 1
  • Kinji Ohno
    • 1
  • Tomoko Yamamoto
    • 1
  • Takayuki Ozawa
    • 1
  1. 1.Department of Biomedical Chemistry, Faculty of MedicineUniversity of NagoyaNagoya 466Japan

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