Morais R, Desjardins P, Turmel C, Zinkewich-Péotti K
Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
In Vitro Cell Dev Biol. 1988 Jul;24(7):649-58. doi: 10.1007/BF02623602.
Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.
鹌鹑和鸡细胞群体用溴化乙锭（一种线粒体DNA复制抑制剂）处理。长期接触该药物后，将细胞群体转移至不含溴化乙锭（EtdBr）的培养基中并进行克隆。克隆的HCF7（鹌鹑）和DUS - 3（鸡）传代培养一年多后进行特性分析。对从这些细胞中提取的总细胞DNA进行分析，用克隆的线粒体DNA片段对经HindIII或AvaI消化的总细胞DNA进行Southern印迹杂交，未发现特征性的线粒体DNA分子。在提取总细胞DNA之前，将少量亲代细胞与HCF7细胞和DUS - 3细胞混合进行重建实验，进一步强化了药物处理细胞缺乏线粒体DNA分子这一观点。发现这些细胞群体的增殖速度与其各自亲代相比适度降低，对尿苷营养缺陷，并且对EtdBr和氯霉素的生长抑制作用具有稳定抗性。在超微结构水平上，线粒体显著增大，细胞器内嵴的数量严重减少且嵴的取向丧失。形态计量分析显示线粒体轮廓面积增加了四倍，而线粒体轮廓数量减少了两倍。生化参数分析表明，这些细胞的生长伴随着缺乏功能性呼吸链的线粒体。线粒体酶二氢乳清酸脱氢酶的活性降低了95%，这可能是导致尿苷营养缺陷的原因。
