Rapid and random turnover of mitochondrial DNA in rat hepatocytes of primary culture
| Autor: | Yoichiro Kai, Mayumi Okamoto, Chihiro Takamatsu, Shosuke Takahashi, Kazuo Irita, Kentaro Tokuda |
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| Rok vydání: | 2006 |
| Předmět: |
DNA Replication
Male Mitochondrial DNA Bromouracil Time Factors Mitochondria Liver Biology DNA Mitochondrial Rats Sprague-Dawley chemistry.chemical_compound Animals Southwestern blot Molecular Biology Cells Cultured Southern blot Cell Biology Cell cycle Pulse-chase analysis Molecular biology Nuclear DNA Rats chemistry Turnover Hepatocytes Molecular Medicine Ethidium bromide |
| Zdroj: | Mitochondrion. 6(6) |
| ISSN: | 1567-7249 |
| Popis: | It is known that mitochondrial DNA (mtDNA) replication is independent of the cell cycle. Even in post-mitotic cells in which nuclear DNA replication has ceased, mtDNA is believed to still be replicating. Here, we investigated the turnover rate of mtDNA in primary rat hepatocytes, which are quiescent cells. Southwestern blot analysis using 5-bromo-2′-deoxyuridine (BrdU) was employed to estimate the activity of full-length mtDNA replication and to determine efficient doses of replication inhibitors. Southern blot analysis showed that a two-day treatment with 20 mM 2′,3′-dideoxycytidine and 0.2 μg/ml ethidium bromide caused a 37% reduction in the amount of mtDNA, indicating that the hepatocytes had a considerably high rate of turnover of mtDNA. Further, pulse-chase analysis using Southwestern analysis showed that the amount of newly synthesized mtDNA labeled with BrdU declined to 60% of the basal level within two days. Because the rate of reduction of the new mtDNA was very similar to the overall turnover rate described above, it appears that degrading mtDNA molecules were randomly chosen. Thus, we demonstrated that there is highly active and random turnover of mtDNA in hepatocytes. |
| Databáze: | OpenAIRE |
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