PURIFICATION OF ALL FORMS OF HELA-CELL MITOCHONDRIAL-DNA AND ASSESSMENT OF DAMAGE TO IT CAUSED BY HYDROGEN-PEROXIDE TREATMENT OF MITOCHONDRIA OR CELLS
PURIFICATION OF ALL FORMS OF HELA-CELL MITOCHONDRIAL-DNA AND ASSESSMENT OF DAMAGE TO IT CAUSED BY HYDROGEN-PEROXIDE TREATMENT OF MITOCHONDRIA OR CELLS
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DOI：
10.1074/jbc.270.14.7950
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发表时间：
1995-04-07
影响因子：
4.8
通讯作者：
LINN, S
中科院分区：
文献类型：
--
作者：
HIGUCHI, Y;LINN, S
本文设计了一种线粒体DNA的纯化方案，该方案能最大限度地提高所有形式DNA的产量，同时使分离过程中对DNA的损伤最小化。用DNase I处理完整的线粒体，去除核DNA，避免苯酚，在不存在溴化乙锭的情况下通过CsCl密度梯度分离，随后通过斑点印迹的Southern杂交检测，最大限度地减少了DNA损伤。通过琼脂糖凝胶电泳和电子显微镜观察，获得了四种不同的无明显核DNA的mtDNA形式：闭合环状（I）、开放环状（II）、线性（III）和大的多聚体复合物（C）。使用这种方法，从用H2 O2处理的全细胞或完整线粒体中获得mtDNA。在37 ° C处理后观察到显著的片段化，但在0 ° C没有，并且当处理全细胞时观察到比分离的线粒体更多的损伤。在所有病例中均观察到极低水平的8-羟基脱氧鸟苷。然而，在剂量的H2 O2，这只是致命的，既没有增加DNA损伤，也没有失活的细胞色素c氧化酶观察。
A purification scheme for mitochondrial DNA (mtDNA) ws designed which maximized the yield of all forms of the DNA while minimizing damage to the DNA during its isolation. Treatment of intact mitochondria with DNase I removed nuclear DNA and the avoidance of phenol and the isolation by CsCl density gradients in the absence of ethidium bromide and subsequent detection by southern hybridization of dot-blots minimized DNA damage. Four different mtDNA forms free of apparent nuclear DNA were obtained: closed circular (I), open circular (II), linear (III), and a large multimer complex (C) which were characterized by agarose gel electrophoresis and electron microscopy. Using this procedure, mtDNA was obtained from both whole cells or intact mitochondria treated with H2O2. Significant fragmentation was observed after treatment at 37 degrees C, but not at 0 degrees C, and more damage was observed when treating whole cells than isolated mitochondria. Very low levels of 8-hydroxydeoxyguanosine were observed in all cases. However, at doses of H2O2 which were just lethal, neither increased DNA damage nor inactivation of cytochrome c oxidase was observed.