Baumer A, Zhang C, Linnane A W, Nagley P
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Am J Hum Genet. 1994 Apr;54(4):618-30.
Deletions in mtDNA accumulate during the human aging process, arising from either intramolecular illegitimate recombination or strand slippage during replication, which results in subgenomic mtDNA molecules. We identify here two classes of mtDNA deletions--class A deletions, which are homogeneous at the breakpoints, with all subgenomic molecules therefore being identical in size, and class B deletions, which arise from a less stringent process that gives rise to heterogeneity at the breakpoints, with the subgenomic molecules being of slightly different sizes. A novel approach is described that offers a global overview of the populations of different deletions in individual tissues. It is based on PCR cycle-sequencing reactions that are carried out directly on mtDNA segments, amplified by PCR from total cellular DNA. The results show a clear size homogeneity of the subgenomic mtDNA molecules representative of class A, which carry a commonly detected 4,977-bp deletion occurring at a pair of 13-bp directly repeated sequences. In this case, precisely one copy of the repeat is retained in the subgenomic molecules. We then describe a class B situation comprising a family of at least nine closely related 8.04-kb deletions involving the same pair of 5-bp direct repeats. In this situation, the breakpoints differ at the base-pair level (8,037-8,048-bp deletions); the subgenomic molecules retain > 1 copy, 1 copy, or < 1 copy of the 5-bp repeat. In different tissues from either the same individual or among different individuals, there is a widely variable occurrence of particular deletions in the subgenomic mtDNA population within this class B set. Class B deletions offer a new approach for studying the accumulation of mtDNA deletions, thereby providing insight into the independent somatic origin of mutated mtDNA molecules, both in aging and in mitochondrial diseases. We also report a convenient method for ascertaining whether a given PCR product results from the amplification of a subgenomic mtDNA template, on the basis of the selective degradation of full-length mtDNA molecules prior to PCR.
线粒体DNA（mtDNA）缺失在人类衰老过程中会不断积累，其产生原因是分子内非法重组或复制过程中的链滑动，这会导致亚基因组mtDNA分子的出现。我们在此识别出两类mtDNA缺失——A类缺失，其断点处是均匀一致的，因此所有亚基因组分子大小相同；B类缺失，它源于一个不那么严格的过程，导致断点处存在异质性，亚基因组分子大小略有不同。本文描述了一种新方法，可全面概述个体组织中不同缺失的群体情况。该方法基于直接在mtDNA片段上进行的PCR循环测序反应，这些片段是通过PCR从总细胞DNA中扩增得到的。结果显示，代表A类的亚基因组mtDNA分子具有明显的大小同质性，这类分子携带一个常见的4977 bp缺失，该缺失发生在一对13 bp的直接重复序列处。在这种情况下，亚基因组分子中恰好保留了一个重复序列拷贝。然后我们描述了一种B类情况，它包含一个至少由九个密切相关的8.04 kb缺失组成的家族，这些缺失涉及同一对5 bp的直接重复序列。在这种情况下，断点在碱基对水平上有所不同（8037 - 8048 bp缺失）；亚基因组分子保留了5 bp重复序列的>1个拷贝、1个拷贝或<1个拷贝。在同一个体的不同组织或不同个体之间，B类亚基因组mtDNA群体中特定缺失的出现情况差异很大。B类缺失为研究mtDNA缺失的积累提供了一种新方法，从而有助于深入了解衰老和线粒体疾病中突变mtDNA分子的独立体细胞起源。我们还报告了一种便捷方法，可在PCR之前基于全长mtDNA分子的选择性降解来确定给定的PCR产物是否来自亚基因组mtDNA模板的扩增。
