Basic Molecular Biology of Mitochondrial Replication

Scheffler, Immo E.

doi:10.1002/9780470372531.ch2

Cited by 4 publications

(7 citation statements)

References 178 publications

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“…This is because both mtDNA and the proteins they encode have low turnover rates. 3 For instance, the half-life of mtDNA from rat liver is ~10 days, 8 and the half-life of the 3 mtDNA-encoded subunits of Complex IV from the rat hepatoma cell line, H4-II-E, is ~4 days. 9 Moreover, the proteins involved in oxidative phosphorylation have to fall below a critical threshold in order for mitochondrial ATP synthesis to be impaired, the threshold varying for each cell type.…”

Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

“…9 Moreover, the proteins involved in oxidative phosphorylation have to fall below a critical threshold in order for mitochondrial ATP synthesis to be impaired, the threshold varying for each cell type. 3 Hence, compounds that alter mtDNA-encoded protein levels have usually been detected by Western blotting 6 or lateral-flow dipstick immunoassays 10 of cells grown in the compound of interest for several days, but neither of these techniques is amenable to high-throughput screening (HTS). To address this problem, we have developed a high-content screening (HCS) assay that identifies compounds that affect mtDNA-encoded protein levels in adherent eukaryotic cells.…”

Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

“…Structurally complex, mitochondria contain several hundred copies of circular DNA (mitochondrial DNA), ribosomes, 22 types of tRNA, and more than 1000 types of proteins. 3 Thirteen polypeptides, all subunits of proteins involved in oxidative phosphorylation, are encoded by mtDNA and synthesized on mitochondrial ribosomes. All other proteins located within mitochondria are nuclear DNA encoded, synthesized on cytosolic ribosomes, and subsequently imported to the mitochondria.…”

Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

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High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

et al. 2010

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Compounds that interfere with the synthesis of either mitochondrial DNA or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial adenosine triphosphate (ATP) production. Toxicity caused by these compounds is seldom identified in 24-to 72-h cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. To address this problem, the authors developed a 96-well format, high-content screening (HCS) assay that measures, in eukaryotic cells, the level of Complex IV-subunit 1, an mtDNA-encoded protein synthesized on mitochondrial ribosomes, and the level of Complex V-α subunit, a nuclear DNAencoded protein synthesized on cytosolic ribosomes. The effect of several antibiotics and antiretrovirals on these 2 proteins was assessed, in transformed human liver epithelial cells, 6 days after compound treatment. The results confirmed effects of drugs known to reduce mtDNA-encoded protein levels and also revealed novel information showing that several fluoroquinolones and a macrolide, josamycin, impaired expression of mtDNA-encoded proteins. The HCS assay was robust with an average Z′ factor of 0.62. The assay enables large-scale screening of compounds to identify those that potentially affect mtDNA-encoded protein levels and can be implemented within a screening paradigm to minimize compound attrition. (Journal of Biomolecular Screening 2010:937-948)

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Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

Section: Oxicity Is a Major Reason For Compound Attritionmentioning

confidence: 99%

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High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

et al. 2010

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“…Furthermore, they are necessary for fatty acid oxidation, steroid synthesis, heme synthesis, and apoptosis (Scheffler, 2008). Mitochondria are unique in that they contain several copies of DNA, referred to as mitochondrial DNA (mtDNA), a DNA-replicating enzyme known as DNA polymerase γ , their own ribosomes, 22 types of tRNA, and more than 1000 types of proteins (Scheffler, 2008). Thirteen mitochondrial proteins are encoded by mtDNA and are synthesized on mitochondrial ribosomes.…”

Section: Introductionmentioning

confidence: 99%

High‐Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA‐Encoded Protein Levels in Eukaryotic Cells

Nadanaciva

Murray²,

Wilson³

et al. 2011

CP Toxicology

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Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.

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“…The human mitochondrial genome is 16,569bp long, contains 37 genes, encoding 13 proteins and 24 transfer RNA and ribosomal RNAs crucial to mitochondrial function. The remaining mitochondrial proteins which are required to make functional mitochondria are coded for and transcribed from the nuclear genome, with resultant transcripts being translated into proteins at cytosolic ribosomes and transported into mitochondria for assembly (Scheffler, 2008). Mitochondria contain >1000 different proteins, some of which show tissue specific profiles (Johnson et al, 2007, Smith et al, 2012.…”

mentioning

confidence: 99%

Mitochondrial DNA as a Potential Translational Biomarker of Mitochondrial Dysfunction in Drug‐Induced Toxicity Studies

Malik

2018

Mitochondrial Dysfunction Caused by Drugs and Environmental Toxicants

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Basic Molecular Biology of Mitochondrial Replication

Cited by 4 publications

References 178 publications

High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

High‐Throughput Assays for Assessing Mitochondrial Dysfunction Caused by Compounds that Impair mtDNA‐Encoded Protein Levels in Eukaryotic Cells

Mitochondrial DNA as a Potential Translational Biomarker of Mitochondrial Dysfunction in Drug‐Induced Toxicity Studies

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