Kai Yoichiro, Takamatsu Chihiro, Tokuda Kentaro, Okamoto Mayumi, Irita Kazuo, Takahashi Shosuke
Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Mitochondrion. 2006 Dec;6(6):299-304. doi: 10.1016/j.mito.2006.10.002. Epub 2006 Oct 17.
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 20mM 2',3'-dideoxycytidine and 0.2mug/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.
已知线粒体DNA（mtDNA）复制独立于细胞周期。即使在核DNA复制已经停止的有丝分裂后细胞中，mtDNA仍被认为在进行复制。在此，我们研究了原代大鼠肝细胞（一种静止细胞）中mtDNA的更新率。使用5-溴-2'-脱氧尿苷（BrdU）的蛋白质印迹分析被用于估计全长mtDNA复制的活性并确定复制抑制剂的有效剂量。Southern印迹分析表明，用20mM 2'，3'-二脱氧胞苷和0.2μg/ml溴化乙锭处理两天导致mtDNA量减少37%，这表明肝细胞中mtDNA的更新率相当高。此外，使用蛋白质印迹分析的脉冲追踪分析表明，用BrdU标记的新合成mtDNA量在两天内降至基础水平的60%。由于新mtDNA的减少速率与上述总体更新率非常相似，似乎降解的mtDNA分子是被随机选择的。因此，我们证明了肝细胞中存在高度活跃且随机的mtDNA更新。
