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. 1985 May;5(5):1163–1169. doi: 10.1128/mcb.5.5.1163

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

P Desjardins, E Frost, R Morais
PMCID: PMC366835  PMID: 2987677

Abstract

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bibor-Hardy V., Kessous A., Simard R. Presence of herpes simplex virus type 2 DNA in hamster cells oncogenically transformed by ultraviolet-inactivated virus. Can J Biochem. 1979 Jun;57(6):867–872. doi: 10.1139/o79-106. [DOI] [PubMed] [Google Scholar]
  2. Bogenhagen D., Clayton D. A. The number of mitochondrial deoxyribonucleic acid genomes in mouse L and human HeLa cells. Quantitative isolation of mitochondrial deoxyribonucleic acid. J Biol Chem. 1974 Dec 25;249(24):7991–7995. [PubMed] [Google Scholar]
  3. Borst P., Kroon A. M. Mitochondrial DNA: physicochemical properties, replication, and genetic function. Int Rev Cytol. 1969;26:107–190. doi: 10.1016/s0074-7696(08)61635-6. [DOI] [PubMed] [Google Scholar]
  4. Borst P., Ruttenberg J. C., Kroon A. M. Mitochondrial DNA. I. Preparation and properties of mitochondrial DNA from chick liver. Biochim Biophys Acta. 1967 Nov 21;149(1):140–155. doi: 10.1016/0005-2787(67)90697-1. [DOI] [PubMed] [Google Scholar]
  5. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  6. Criddle R. S., Schatz G. Promitochondria of anaerobically grown yeast. I. Isolation and biochemical properties. Biochemistry. 1969 Jan;8(1):322–334. doi: 10.1021/bi00829a045. [DOI] [PubMed] [Google Scholar]
  7. D'Agostino M. A., Nass M. M. Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses. J Cell Biol. 1976 Dec;71(3):781–794. doi: 10.1083/jcb.71.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  9. Goldring E. S., Grossman L. I., Krupnick D., Cryer D. R., Marmur J. The petite mutation in yeast. Loss of mitochondrial deoxyribonucleic acid during induction of petites with ethidium bromide. J Mol Biol. 1970 Sep 14;52(2):323–335. doi: 10.1016/0022-2836(70)90033-1. [DOI] [PubMed] [Google Scholar]
  10. Grégoire M., Morais R., Quilliam M. A., Gravel D. On auxotrophy for pyrimidines of respiration-deficient chick embryo cells. Eur J Biochem. 1984 Jul 2;142(1):49–55. doi: 10.1111/j.1432-1033.1984.tb08249.x. [DOI] [PubMed] [Google Scholar]
  11. King M. E., Godman G. C., King D. W. Respiratory enzymes and mitochondrial morphology of HeLa and L cells treated with chloramphenicol and ethidium bromide. J Cell Biol. 1972 Apr;53(1):127–142. doi: 10.1083/jcb.53.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Labbe-Bois R., Rytka J., Litwinska J., Bilinski T. Analysis of heme biosynthesis in catalase and cytochrome deficient yeast mutants. Mol Gen Genet. 1977 Nov 14;156(2):177–183. doi: 10.1007/BF00283490. [DOI] [PubMed] [Google Scholar]
  13. Leblond-Larouche L., Dupuis C., Morais R. Poly(A) hydrolase of chick-embryo fibroblasts. Eur J Biochem. 1976 Jun 1;65(2):423–430. doi: 10.1111/j.1432-1033.1976.tb10357.x. [DOI] [PubMed] [Google Scholar]
  14. Leibowitz R. D. The effect of ethidium bromide on mitochondrial DNA synthesis and mitochondrial DNA structure in HeLa cells. J Cell Biol. 1971 Oct;51(1):116–122. doi: 10.1083/jcb.51.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. MASTER R. W. POSSIBLE SYNTHESIS OF POLYRIBONUCLEOTIDES OF KNOWN BASE-TRIPLET SEQUENCES. Nature. 1965 Apr 3;206:93–93. doi: 10.1038/206093b0. [DOI] [PubMed] [Google Scholar]
  16. Morais R., Giguère L. On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol. J Cell Physiol. 1979 Oct;101(1):77–88. doi: 10.1002/jcp.1041010110. [DOI] [PubMed] [Google Scholar]
  17. Morais R., Gregoire M., Jeannotte L., Gravel D. Chick embryo cells rendered respiration-deficient by chloramphenicol and ethidium bromide are auxotrophic for pyrimidines. Biochem Biophys Res Commun. 1980 May 14;94(1):71–77. doi: 10.1016/s0006-291x(80)80189-6. [DOI] [PubMed] [Google Scholar]
  18. Morais R. On the effect of inhibitors of mitochondrial macromolecular-synthesizing systems and respiration on the growth of cultured chick embryo cells. J Cell Physiol. 1980 Jun;103(3):455–466. doi: 10.1002/jcp.1041030311. [DOI] [PubMed] [Google Scholar]
  19. Nass M. M. Abnormal DNA patterns in animal mitochondria: ethidium bromide-induced breakdown of closed circular DNA and conditions leading to oligomer accumulation. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1926–1933. doi: 10.1073/pnas.67.4.1926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nass M. M. Differential effects of ethidium bromide on mitochondrial and nuclear DNA synthesis in vivo in cultured mammalian cells. Exp Cell Res. 1972 May;72(1):211–222. doi: 10.1016/0014-4827(72)90583-6. [DOI] [PubMed] [Google Scholar]
  21. Perlman P. S., Mahler H. R. Molecular consequences of ethidium bromide mutagenesis. Nat New Biol. 1971 May 5;231(18):12–16. [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Sharp P. A., Pettersson U., Sambrook J. Viral DNA in transformed cells. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J Mol Biol. 1974 Jul 15;86(4):709–726. doi: 10.1016/0022-2836(74)90348-9. [DOI] [PubMed] [Google Scholar]
  24. Shmookler Reis R. J., Goldstein S. Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation. J Biol Chem. 1983 Aug 10;258(15):9078–9085. [PubMed] [Google Scholar]
  25. Slonimski P. P., Perrodin G., Croft J. H. Ethidium bromide induced mutation of yeast mitochondria: complete transformation of cells into respiratory deficient non-chromosomal "petites". Biochem Biophys Res Commun. 1968 Feb 15;30(3):232–239. doi: 10.1016/0006-291x(68)90440-3. [DOI] [PubMed] [Google Scholar]
  26. Smith C. A., Jordan J. M., Vinograd J. In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture. J Mol Biol. 1971 Jul 28;59(2):255–272. doi: 10.1016/0022-2836(71)90050-7. [DOI] [PubMed] [Google Scholar]
  27. Soslau G., Nass M. M. Effects of ethidium bromide on the cytochrome content and ultrastructure of L cell mitochondria. J Cell Biol. 1971 Nov;51(21):514–524. doi: 10.1083/jcb.51.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  29. Storrie B., Attardi G. Expression of the mitochondrial genome in HeLa cells. 13. Effect of selective inhibition of cytoplasmic or mitochondrial protein synthesis on mitochondrial nucleic acid synthesis. J Mol Biol. 1972 Nov 14;71(2):177–199. doi: 10.1016/0022-2836(72)90345-2. [DOI] [PubMed] [Google Scholar]
  30. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wiseman A., Attardi G. Reversible tenfod reduction in mitochondria DNA content of human cells treated with ethidium bromide. Mol Gen Genet. 1978 Nov 16;167(1):51–63. doi: 10.1007/BF00270321. [DOI] [PubMed] [Google Scholar]
  32. Zylber E., Vesco C., Penman S. Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide. J Mol Biol. 1969 Aug 28;44(1):195–204. doi: 10.1016/0022-2836(69)90414-8. [DOI] [PubMed] [Google Scholar]
  33. de Lamirande G., Morais R., Blackstein M. Intracellular distribution of 5'-ribonuclease and 5'-phosphodiesterase in rat liver. Arch Biochem Biophys. 1967 Feb;118(2):347–351. doi: 10.1016/0003-9861(67)90359-1. [DOI] [PubMed] [Google Scholar]

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