Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

Breen, Gail A. M.

doi:10.1128/mcb.2.7.772

Cited by 7 publications

(2 citation statements)

References 54 publications

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“…Importantly, oligomycin concentrations of 0.05 and 0.1 ng/ml in medium containing glucose did not affect the growth rate of the cell lines (not shown) and, in this medium, an oligomycin concentration of at least 1 ng/ml was necessary to kill both cell lines (not shown). These results are similar to those reported previously on hamster cells (15).…”

Section: T8993gsupporting

confidence: 93%

“…those containing a lower amount of mutant mtDNA), despite the fact that cells harboring this mutation are deficient in oxidative phosphorylation (5,6,16). We reasoned that a mitochondrial inhibitor such as oligomycin might be such a selective agent, because of its known interaction with the ATPase 6 subunit of complex V (8,9,15). Oligomycin belongs to a family of antibiotics produced by Streptomyces diastatochromogenes and is toxic to both eucaryotic and procaryotic organisms.…”

Section: Discussionmentioning

confidence: 99%

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Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Manfredi¹,

Gupta²,

Vazquez-Memije³

et al. 1999

Journal of Biological Chemistry

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A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F 0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.A T 3 G transversion at human mtDNA 1 position 8993 (1) is associated with a multisystemic syndrome characterized clinically by neuropathy, ataxia, and retinitis pigmentosa (NARP) (2), and with a maternally inherited form of Leigh's syndrome (MILS) (3, 4), a fatal encephalopathy of infancy. The mutation results in the conversion of leucine to arginine at amino acid 156 in ATPase 6, one of the two mtDNAencoded subunits of the F 0 portion of the mitochondrial ATPase complex (complex V). It has been reported that very high levels of mutant mtDNA are required for the clinical phenotype to be expressed (3), suggesting that this mutation is "recessive" in nature.Cultured cells harboring high levels (Ͼ95%) of the T8993G mutation have decreased ATP synthase activity (5, 6), implying that a defect in oxidative ATP production is the most likely cause of both disorders. Nevertheless, cultured fibroblasts from NARP and MILS patients do not display any growth abnormalities under standard culture conditions, despite often being homoplasmic for the mutation (i.e. containing 100% mutant mtDNA). Nevertheless, we hypothesized that we could identify culture conditions that would not only allow for selection against mutant cells, but would also affect the intracellular proportion of mutant mtDNAs.We took advantage of two factors that eventually allowed us to develop such a system. First, fibroblasts from patients with diverse mitochondrial disorders grow poorly when glucose is replaced by galactose as the sole carbon source (7). Second, resistance to the antibiotic oligomycin, an inhibitor of mitochondrial function, is associated with at least two mutations in the yeast ATPase 6 gene (8) and with at least one mutation in the hamster ATPas...

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Section: T8993gsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Manfredi¹,

Gupta²,

Vazquez-Memije³

et al. 1999

Journal of Biological Chemistry

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Two cytoplasmically inherited oligomycin-resistant Chinese hamster cell lines exhibit an altered mitochondrial translation product

Shew

Breen

1985

Somat Cell Mol Genet

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Mitochondria from two different cytoplasmically inherited oligomycin-resistant Chinese hamster ovary cell lines synthesize an altered polypeptide compared to mitochondria from wild-type cells. For example, mitochondria from both oligomycin-resistant cell lines synthesize a polypeptide with a molecular weight of approximately 20,500, which is present in very low amounts in wild-type cells. In contrast, mitochondria from wild-type cells synthesize a polypeptide with a molecular weight of approximately 19,500, which is present in very low amounts in one of the oligomycin-resistant mutants and in reduced amounts in the other mutant. The gene which encodes this altered polypeptide is cytoplasmically transferred together with the oligomycin-resistant phenotype. This is the first example in mammalian cells where an altered mitochondrial gene product is shown to be associated with the cytoplasmic transfer of oligomycin resistance.

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Altered form of subunit 6 of mitochondrial ATP synthase complex in oligomycin-resistant mutants of Chinese hamster ovary cells

Holmans

Breen

1987

Somat Cell Mol Genet

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Using an antiserum generated against a synthetic peptide predicted from the DNA sequence of the ATPase 6 gene of the mitochondrial DNA, we demonstrate that mitochondria from two oligomycin-resistant Chinese hamster ovary cell lines with a defined mutation in the ATPase 6 gene synthesize an altered ATPase 6 gene product. This altered gene product migrates in sodium dodecyl sulfate-polyacrylamide gels as if it has a molecular mass that is larger by 1000 daltons than the wild-type ATPase 6 gene product. We also demonstrate that mitochondria from four other independently isolated oligomycin-resistant Chinese hamster ovary mutant cell lines contain a similar altered ATPase 6 gene product. These results suggest that all six oligomycin-resistant cell lines have a similar mutation in the ATPase 6 gene of the mitochondrial DNA that encodes subunit 6 of the ATP synthase complex.

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Genetics of the mammalian oxidative phosphorylation system: characterization of a new oligomycin-resistant Chinese hamster ovary cell line.

Cited by 7 publications

References 54 publications

Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Oligomycin Induces a Decrease in the Cellular Content of a Pathogenic Mutation in the Human Mitochondrial ATPase 6 Gene

Two cytoplasmically inherited oligomycin-resistant Chinese hamster cell lines exhibit an altered mitochondrial translation product

Altered form of subunit 6 of mitochondrial ATP synthase complex in oligomycin-resistant mutants of Chinese hamster ovary cells

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