Accumulation of point mutations in mitochondrial DNA of aging mice

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Abstract

Mitochondrial DNA (mtDNA) exists in a highly genotoxic environment created by exposure to reactive oxygen species, somewhat deficient DNA repair, and the relatively low fidelity of polymerase gamma. Given the severity of the environment, it was anticipated that mutation accumulation in the mtDNA of aging animals should exceed that of nuclear genes by several orders of magnitude. We have analyzed fragments amplified from the D-loop region of mtDNA from 2 to 22-month-old mice. The amplified 432 bp fragments were cloned into plasmid vectors, and plasmid DNAs from individual clones were purified and sequenced. None of 110 fragments from young mice contained a mutation, while 9 of 87 clones originating from old animals contained base substitutions (chi square = 11.9, P<0.001). The estimated mutation frequency in mtDNA from old mice was 11.6±2.7 or 25.4±7.8 per 105 nucleotides (depending on assumptions of clonality), which exceeds existing estimates for mutation frequencies for nuclear genes by approximately 1000-fold. Our data suggest that at 22 months of age, which roughly corresponds to 3/4 of the mouse natural life span, most mtDNA molecules carry multiple point mutations.

Introduction

While it is firmly established that carcinogenesis is closely linked to an accumulation of mutations in a diverse group of nuclear genes [1], [2], [3], the connection between aging and somatic cell mutations is less clear. Mutations in nuclear genes do accumulate with time [4], [5], but they are probably not the sole determinants of aging. The spontaneous mutation frequency observed in selectively neutral genes does not exceed 10−4 per locus at the end of an animal’s lifespan [6]. In addition, few of the age-associated changes in gene expression, activity, and inducibility decline [7], [8], [9], [10], [11]. This suggests that the observed shifts in the activity of enzymes are likely adaptive in nature and that the functional capability of nuclear genes remains largely intact.

Further evidence that nuclear genes are not solely responsible for aging can be found in experiments on nuclear transfer and animal cloning [12], [13]. Although nuclear transfer using somatic cells from adult animals as nuclear donors often produces defective offspring, these offspring appear to age essentially at the same rate as control animals [14], despite the shorter length of telomere fragments [15]. Nuclear transfer from pre-quiescent fibroblasts into embryonic fibroblasts restores the in vitro doubling potential to the levels of untreated embryonic cells. In light of these findings, it seems probable that aging and the development of degenerative pathologies result from a combination of factors, including cytoplasmic elements.

The mitochondria are possible cytoplasmic determinants of aging. These organelles harbor a second cellular genome and perform the universally important functions of bioenergy production and apoptosis. Mitochondrial DNA (mtDNA) is located in close proximity to the oxidative phosphorylation machinery, and presumably is subjected to more oxidative damage than nuclear genes [16], [17], although a recent report indicates that this may not be necessarily the case [18]. A combination of elevated oxidative damage, possible deficiencies in mismatch and nucleotide excision repair [19], [20], an excess of direct repeats, and the asymmetrical replication of mtDNA, leaving a considerable portion of the H-strand displaced for an extended period of time [21], may result in accelerated mutation accumulation.

Given the possible role of mitochondria in aging, the analysis of spontaneous mutation accumulation in mtDNA is of interest. It has been shown that deletions in mtDNA are typically flanked by direct repeats, tend to accumulate with time [22], and are found in a majority of mtDNA molecules in advanced age [23]. Studies on accumulation of point mutations, however, have produced widely differing results depending on the methods employed [24], [25], [26], [27], [28], [29]. The analyses based on quantitative PCR techniques have shown that specific base substitutions may accumulate in a high portion of mtDNA, reaching mutation frequencies of up to 2–3% in aging rodents and humans [26]. On the other hand, mutation frequencies based on direct sequencing of cloned mtDNA (either digested or amplified) are much lower, and do not exceed 10−4 per nucleotide. In the present study, we employed a non-selective approach to measuring the frequency of point mutations and small deletions/insertions in the mtDNA of aging mice. We used high fidelity amplification of the D-loop fragment with subsequent cloning and sequencing of individual plasmids.

Section snippets

Mitochondrial DNA isolation

Mitochondrial DNA was extracted from the livers of five 2-month-old and three 22-month-old C57Bl/6N mice bred maintained at the National Center for Toxicological Research using the mtDNA extractor CT Kit (Wako BioProducts, Richmond, VA). The presence of mtDNA was verified by electrophoresis on 0.8% agarose gels containing 0.5 μg/ml ethidium bromide. Bands corresponding to full-length mtDNA were excised and extracted using the QIAEX® II Gel Extraction System (Qiagen, Valencia, CA).

PCR amplifications and cloning

PCR

Co-transfection with multiple vectors

A co-transfection assay was performed in duplicate using serial dilutions of the PCR-Blunt-II-TOPO (Kan®, Invitrogen) and BlueScript SK+ (Amp®, Stratagene) vectors, both of which contain pUC origins of replication. Aliquots of TOP10 bacteria (Invitrogen) were transformed with equimolar mixtures of the two vectors at a total concentration that yields 1200–1500 colonies, the number typically observed when cloning mtDNA PCR products. The transformed bacteria were plated onto LB agar plates

Discussion

Our approach for detecting mutations in mtDNA was based on the expectation of a relatively high frequency of point mutations in aging animals. In previous studies using quantitative PCR-based methods, the frequency of specific base substitutions in defined sequences significantly increased with age and ranged from mutation frequencies of 0.1–2.4% [26], [30]. Extrapolating these estimates to the entire mtDNA sequence (close to 5×104 possible base substitutions) suggests that every mtDNA molecule

Acknowledgements

The authors are indebted to Michelle Bishop and Lascelles Lyn-Cook for their technical assistance. This research was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between US Department of Energy and US Food and Drug Administration.

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