Griffiths Lyra M, Doudican Nicole A, Shadel Gerald S, Doetsch Paul W
Emory University School of Medicine, Atlanta, GA, USA.
Methods Mol Biol. 2009;554:267-86. doi: 10.1007/978-1-59745-521-3_17.
Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases such as cancer, diabetes, and Parkinson's disease. Mutations in mtDNA also accumulate with age and are therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the "petite induction" assay.
人类线粒体DNA（mtDNA）的突变与母系遗传的神经肌肉疾病有关，并且在诸如癌症、糖尿病和帕金森病等更常见的疾病中也有牵连。mtDNA中的突变也会随着年龄的增长而积累，因此被认为与衰老及与年龄相关的病理过程有关。mtDNA位于线粒体基质中，它编码了几种通过氧化磷酸化过程参与ATP生成的蛋白质，氧化磷酸化过程涉及高能电子通过电子传递链（ETC）的流动。由于mtDNA靠近ETC，它极易受到活性氧（ROS）介导的氧化损伤，如过氧化氢、超氧化物和羟基自由基，这些都是该系统不断产生的。因此，能够在正常生理条件下以及在环境或疾病相关应激期间测量氧化的mtDNA损伤非常重要。出芽酵母，酿酒酵母，是一种便于研究此类mtDNA氧化损伤的信息丰富的模型系统，因为它是一种单细胞真核兼性厌氧菌，其生存能力有条件地依赖于线粒体氧化磷酸化。在这里，我们描述了定量酿酒酵母中氧化mtDNA损伤和诱变的方法，其中几种方法可应用于开发哺乳动物细胞和组织中的类似检测方法。这些方法包括用红霉素抗性检测法测量mtDNA中发生的点突变数量，利用改良的Southern印迹检测法定量氧化DNA损伤的量，以及用“小菌落诱导”检测法测量mtDNA的完整性。
