In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture

Smith, Charles A.; Jordan, John M.; Vinograd, Jerome

doi:10.1016/0022-2836(71)90050-7

Cited by 117 publications

(27 citation statements)

References 13 publications

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“…Although a morphologic analysis has been applied to similar problems (12), we chose not to attempt this method because of difficulties relative to the presence of mitochondrial DNA. Thus, mitochondrial DNA, exactly twice the size of Shope DNA (16), exists as monomeric and dimeric circles as well as interlocked dimers and higher oligomers (16)(17)(18). Furthermore, this DNA would be present at a concentration at least 1 order of magnitude higher than Shope DNA in supercoiled DNA fractions isolated from tumor cells.…”

Section: Discussionmentioning

confidence: 99%

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

Wettstein¹,

Stevens²

1982

Proc. Natl. Acad. Sci. U.S.A.

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BiochemistryVariable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some (virus-induced Communicated by P. D. Boyer, October 22, 1981 ABSTRACT The state ofrabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electiophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and SI nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of-increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA-was linear and comigrated with cellular DNA. Integration ofat least some ofthese sequences was suggested by the detection ofviral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.When applied to scarified skin on the domestic rabbit, Shope papilloma virus induces papillomas, and carcinomas later appear at the same site (1, 2). Although few, if any, virions are present in either tumor, we recently determined that multiple copies of the viral genome are present in every neoplastic cell (3). Preliminary experiments also showed that, in carcinomas, most viral sequences are present in molecules larger then unit length viral DNA, although free episomes consisting of form I and form II DNA are present. In addition, most of the DNA in the larger molecules is converted to unit length linear molecules when digested with a restriction enzyme that cleaves the viral genome once (4).We show here that, in all tumors on domestic rabbits, a large fraction of the viral DNA persists in a free episomal form. In papillomas, form .1 and form II viral DNA are the most common. In carcinomas, larger free episomes predominate, and molecules consisting of multiple genomic equivalents of viral DNA (dimers through hexamers) have been resolved. In some tumors (both papillomas and carcinomas), a fraction of the viral sequences comigrates with cellular DNA; and in one tumor, cellular-viral DNA junction pieces were identified after restriction endonuclease digestion.MATERIALS AND METHODS Animals and Virus. The source of animals and virus and the mode of infection were as described (2).Isolation and Purification of Tumor DNA. High molecular weight DNA was extracted from tumors by the method of Blin and Stafford (5) with some modifications (4), and supercoiled DNA was separated from nicked and linear DNA by density equilibrium centrifugation in CsCl (density = 1.56 g/ml) containingO.01'M Tris HCl (pH 8.0), 1 mM EDTA, and propidium iodide at 100 Ag/ml. After centrifugation for 40 hr at 35,000 rpm and 200C ...

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Section: Discussionmentioning

confidence: 99%

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

Wettstein¹,

Stevens²

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…Two milliliters of 0.01 M Tris-hydrochloride (pH 7.2)-i mM EDTA-0.5 M sucrose was then added to bring the sucrose molarity to 0.25. Cells were fractionated by differential centrifugation as previously described (17), and the mitochondrial fraction was further purified on a sucrose step gradient (30). Radioactivity was determined on samples of total homogenate and purified mitochondria as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

“…On the other hand, other than decreasing the number of mtDNA copies per cell, EtdBr might also mutate mtDNA in CEF so that the expression of some or of all the genes is prevented upon the removal of the drug from the culture medium. Although there is no direct evidence in the literature to support the latter possibility, the drug is known to induce a structural alteration in covalently closed mtDNA of vertebrate cells, consisting in part of an increased degree of supercoiling (23,30) and possibly a breakage of circular DNA without reclosing as observed by electron microscopy (23). These changes appear to be reversible when the cells are transferred to EtdBr-free medium.…”

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confidence: 99%

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

Desjardins¹,

Frost²,

Morais³

1985

Mol. Cell. Biol.

156

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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 [3lHlthymidine 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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“…mtDNA was isolated from a DNasetreated and NaDodSO4-solubilized crude mitochondrial fraction by banding in a CsCl/ethidium bromide (EtdBr) gradient (4); the material in the upper and lower bands was pooled and pelleted, and the total mtDNA was separated from degraded nuclear DNA by centrifugation through a 2-step CsCI/EtdBr gradient (5 In some experiments, the mtDNA-protein complexes were crosslinked by treating the crude or cleared lysate with formaldehyde and glutaraldehyde, as described elsewheref.…”

Section: Methodsmentioning

confidence: 99%

Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication.

Albring¹,

Griffith²,

Attardi³

1977

Proc. Natl. Acad. Sci. U.S.A.

179

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Almost all (about 95%) of the mitochondrial DNA molecules released by Triton X-100 lysis of HeLa cell mitochondria in the presence of 0.15 M salt are associated with a single protein-containing structure varying in appearance between a 10-20 nm knob and a 100-500 nm membrane-like patch. Analysis by high resolution electron microscopy and by polyacrylamide gel electrophoresis after cleavage of mitochondrial DNA with the endonucleases EcoRI, HindIII, and Hpa II has shown that the protein structure is attached to the DNA in the region of the D-loop, and probably near the origin of mitochondrial DNA replication. The data strongly suggest that HeLa cell mitochondrial DNA is attached in vivo to the inner mitochondrial membrane at or near the origin of replication, and that a membrane fragment of variable size remains associated with the DNA during the isolation. After sodium dodecyl sulfate extraction of mitochondrial DNA, a small 5-10 nm protein is found at the same site on a fraction of the mitochondrial DNA molecules.The attachment of animal cell mitochondrial DNA (mtDNA) to the inner mitochondrial membrane has been surmised in the past on the basis of electron microscopy (EM) observations (1); however, nonspecific adsorption phenomena could not be excluded in these early observations because of the lack of suitable position markers. In the present work, by applying to mtDNA extracted under mild conditions EM techniques designed to visualize DNA-protein complexes, we have made observations that support the idea of a specific membrane attachment of mtDNA. In particular, we have found that almost all (about 95%) circular mtDNA molecules released by Triton X-100 lysis of mitochondria in the presence of low salt exhibit a single protein structure associated with them, which varies in appearance between a 10-20 nm knob and a 100-500 nm membrane-like patch. We also report on the unique location of the protein structure near the origin of mtDNA replication and on the occurrence of protein(s) resistant to sodium dodecyl sulfate (NaDodSO4) at the same site on a fraction of the mtDNA molecules. MATERIALS AND METHODSGrowth in Suspension and Subcellular Fractionation of HeLa Cells. These were carried out as previously described (2, 3).Isolation of mtDNA. mtDNA was isolated from a DNasetreated and NaDodSO4-solubilized crude mitochondrial fraction by banding in a CsCl/ethidium bromide (EtdBr) gradient (4); the material in the upper and lower bands was pooled and pelleted, and the total mtDNA was separated from degraded nuclear DNA by centrifugation through a 2-step CsCI/EtdBr gradient (5). NaCl/0.25% Triton X-100 (TENT), and centrifuged 17 hr at 40,000 rpm (2°) in an SW 41 rotor. The mtDNA-containing fractions were pooled and diluted with an equal volume of TENT; 9 ml portions of the diluted sample were then layered over 3 ml of saturated solution of CsCl in 0.01 M Tris, pH 8.0/0.01 M EDTA/0.02 or 0.2% sodium dodecyl-N-sarcosinate (Sarkosyl, Ciba-Geigy Corp.) and centrifuged in an SW 41 rotor at 39,000 rpm for 14 hr at 5...

show abstract

In vivo effects of intercalating drugs on the superhelix density of mitochondrial DNA isolated from human and mouse cells in culture

Cited by 117 publications

References 13 publications

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

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

Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication.

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