Detection and quantitation by competitive PCR of an age-associated increase in a 4.8-kb deletion in rat mitochondrial DNA

Mutation Research/DNAging Pub Date : 1994-08-01 DOI:10.1016/0921-8734(94)90009-4

Wade Edris , Bradford Burgett , O. Colin Stine , Charles R. Filburn

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引用次数: 78

Abstract

Recent studies on human tissues have shown that the guantity of partially deleted mitochondrial DNA (mtDNA) increases with age. In this study, mtDNAs from the livers of young adult and old Wistar rats were analyzed by PCR. Evidence for partially deleted mtDNAs was found, with a 4834-bp deletion present in all animals and most easily detected in samples from senescent rats. The deletion breakpoint occurs at a 16-bp direct repeat present in the cytochrome oxidase I and ATPase 6 genes. This deletion in rats is similar in size and location to the 5.0-kb deletion observed in human mtDNA. The proportion of rat mtDNA with this 4.8-kb deletion was quantitated by a competitive PCR assay. The ratio of partially deleted mtDNA/ total mtDNA in liver mtDNA from individual 6 month old rats ranged from 5 × 10⁻⁶ to 3 × 10⁻⁵, while the ratio in 24 month old rats ranged from 8 × 10⁻⁴ 5 × 10⁻³, with a mean 100-fold increase with age. These increases are in the range observed for human mtDNA during aging. Thus senescent rats can be used as a model to study this type of mitochondrial DNA damage in aging. The method and reagents described should prove useful of the mechanism(s) underlying deletions, their significance to the aging process, and testing of various compounds or interventions for their ability to slow the process.

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通过竞争性PCR检测和定量大鼠线粒体DNA中4.8 kb缺失的年龄相关增加

最近对人体组织的研究表明，部分缺失的线粒体DNA (mtDNA)的数量随着年龄的增长而增加。本研究采用PCR方法对青壮年和老年Wistar大鼠肝脏的mtdna进行了分析。发现了部分缺失mtdna的证据，在所有动物中都有4834 bp的缺失，最容易在衰老大鼠的样本中检测到。缺失断点发生在细胞色素氧化酶I和atp酶6基因中直接重复的16bp处。在大鼠中，这种缺失在大小和位置上与在人类mtDNA中观察到的5.0 kb缺失相似。大鼠mtDNA中4.8 kb缺失的比例通过竞争性PCR测定进行定量分析。6月龄大鼠肝脏mtDNA部分缺失与总mtDNA之比为5 × 10−6 ~ 3 × 10−5,24月龄大鼠肝脏mtDNA部分缺失与总mtDNA之比为8 × 10−4 ~ 5 × 10−3，随年龄增长平均增加100倍。这些增加是在衰老过程中观察到的人类mtDNA的范围内。因此，衰老大鼠可以作为研究衰老过程中线粒体DNA损伤的模型。所描述的方法和试剂应证明对缺失的机制有用，它们对衰老过程的重要性，以及测试各种化合物或干预措施以减缓这一过程的能力。

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Mutation Research/DNAging

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