The mutation rate in the human mtDNA control region

The mutation rate of the mitochondrial control region has been widely used to calibrate human population history. However, estimates of the mutation rate in this region have spanned two orders of magnitude. To readdress this rate, we sequenced the mtDNA control region in 272 individuals, who were related by a total of 705 mtDNA transmission events, from 26 large Icelandic pedigrees. Three base substitutions were observed, and the mutation rate across the two hypervariable regions was estimated to be 3/705 =.0043 per generation (95% confidence interval [CI].00088-.013), or.32/site/1 million years (95% CI.065-.97). This study is substantially larger than others published, which have directly assessed mtDNA mutation rates on the basis of pedigrees, and the estimated mutation rate is intermediate among those derived from pedigree-based studies. Our estimated rate remains higher than those based on phylogenetic comparisons. We discuss possible reasons for-and consequences of-this discrepancy. The present study also provides information on rates of insertion/deletion mutations, rates of heteroplasmy, and the reliability of maternal links in the Icelandic genealogy database.     

Premature aging is associated with higher levels of 8‐oxoguanine and increased DNA damage in the Polg mutator mouse

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg ^D257A/D257A mutator mouse (Polg ^mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg ^mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg ^mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.     

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.     

Compilation of human mtDNA control region sequences.

This paper describes the organisation of a database for human mitochondrial control-region sequences. The data are divided into three ASCII files that contain aligned sequences from the hypervariable region I (HVRI), from the hypervariable region II (HVRII), and the available information about the individuals, from whom the sequences stem. The current collection comprises 4079 HVRI and 969 HVRII sequences. From 728 individuals sequences of both HVRI and HVRII are available. For easy access, the collection is made available to the scientific community via World Wide Web at URL http://www.zi.biologie.uni-muenchen.de/[symbol: see text]meyers/mtdna.html     

Hypervariable sites in the mtDNA control region are mutational hotspots

Hypervariable sites in human mtDNA are readily identified in evolutionary studies and are usually assumed to represent mutational hotspots. Recently, an alternative hypothesis was proposed that holds that hypervariable sites may instead reflect ancient mtDNA mutations that have been "shuffled" among different lineages via recombination. These hypotheses can be tested by examining the evolutionary rates for sites at which new mtDNA mutations are observed; if hypervariable sites are mutational hotspots, then newly arisen mtDNA mutations should occur preferentially at hypervariable sites. Results of this study show that both germline and somatic mtDNA mutations occur preferentially at hypervariable sites, which supports the view that hypervariable sites are indeed mutational hotspots.     

Heteroplasmic point mutations in the human mtDNA control region.

As part of an investigation of the fixation mechanisms of mtDNA mutations in humans, we sequenced the first hypervariable segment of the control region in 180 twin pairs and found evidence of site heteroplasmy in 4 pairs. Significant levels of two mitochondrial haplotypes differing by a single point mutation were found in two MZ pairs, and within each pair, both members had similar levels of heteroplasmy. Two DZ pairs were found in which the predominant mitochondrial haplotype differed within the pair. We measured proportions of mitochondrial haplotypes within two twin pairs and their maternal relatives, using primer extension. In both maternal lineages, most family members were heteroplasmic, and the proportions of each genotype varied widely in different individuals. We used the changes in haplotype proportions within mother-offspring pairs to calculate the size range of potential bottlenecks in mitochondrial numbers occurring during development of the offspring. In most individuals, the most likely effective bottleneck sizes ranged from 3 to 20 segregating units, though in two individuals a small bottleneck was very unlikely and there was no upper limit on its possible size. We also used the data from this study, together with unpublished data from other populations, to estimate the frequency of site heteroplasmy in normal human populations. From this, we calculated that the rate of mutation and fixation in the first hypervariable segment of the human mtDNA control region is between 1.2 x 10(-6) and 2.7 x 10(-5) per site per generation. This range is in good agreement with published estimates calculated by other methods.     

Heteroplasmy of the human mtDNA control region remains constant during life

In a longitudinal, retrospective study, we monitored the level of heteroplasmy at nucleotide position (nt) 309 and nt 16189 of the control region of human mtDNA. As a unique source of DNA, we analyzed multiple cervical-cell samples collected, during 1 or 2 decades, from four women with heteroplasmy at either nt 309 or nt 16189. According to accurate, quantitative analysis by solid-phase minisequencing, the level of heteroplasmy remained stable in the cervical-cell samples from all four women during the time studied. We also analyzed autopsy samples from several different tissues, all containing nt 309 in heteroplasmic form, of one of the women, who was deceased. On the basis of our results, heteroplasmy in the control region of mtDNA seems to be inherited and is not the result of somatic age-related accumulation.     

鼬科动物线粒体DNA控制区结构分析

利用PCR技术获得紫貂(Martes zibellina)和黄喉貂(Martes flavigula)线粒体DNA控制区全序列,并结合从GenBank中下载的9种鼬科动物相应序列,用ClustalX排序后对控制区结构进行分析,识别出延长终止序列区、中央区和保守序列区3个区域,指出了一个终止相关序列ETAS1及8个保守序列(CSB-F、E、D、C、B、1、2和3),并给出了序列通式,在CSB1和CSB2之间发现不同形式的短重复序列.此外,以狼为外类群,应用邻接法构建鼬科线粒体控制区全序列的系统进化树,结果表明:臭鼬亚科最先从鼬科中分化出来,随后剩余类群分为两大支系,即貂属种类与貂熊聚为一支,并与獾亚科的狗獾形成姐妹群;另一支为水獭亚科的物种与鼬属的林鼬形成姐妹群,再与虎鼬聚在一起,狗獾与貂属的紫貂亲缘关系最近,水獭亚科与鼬属亲缘关系最近.     

Powerful mutator activity of the polA1 mutation within the histidine region of Escherichia coli K-12.

We examined 122 spontaneous histidine auxotrophs accumulated in overnight cultures of polA1 strains of Escherichia coli K-12 at approximate frequencies of 10(-3). One hundred and thirteen appeared to be minus frameshifts, and nine appeared to be deletions. Of the frameshift mutations, 109 affected the hisC gene, and 4 affected genes hisD, hisH, hisA, and hisI. The lack of base substitutions supported the idea that polymerase-defective polA is a minus frameshift- and deletion-type mutator. Contrary to a previous report, we did not observe superior growth of PolA auxotrophs over their prototrophic progenitors (15 auxotrophs tested). We conclude that the polA1 mutation exerts a powerful mutator activity in this specific genetic context.     

Persistent heteroplasmy of a mutation in the human mtDNA control region: hypermutation as an apparent consequence of simple-repeat expansion/contraction

In the genealogical and phylogenetic analyses that are reported here, we obtained evidence for an unusual pattern of mutation/reversion in the human mitochondrial genome. The cumulative results indicate that, when there is a T-->C polymorphism at nt 16189 and a C-->T substitution at nt 16192, there is an extremely high rate of reversion (hypermutation) at the latter site. The apparent reversion rate is sufficiently high that there is persistent heteroplasmy at nt 16192 in maternal lineages and at the phylogenetic level, a situation that is similar to that observed for the rapid expansion/contraction of simple repeats within the control region. This is the first specific instance in which the mutation frequency at one site in the D-loop is markedly influenced by the local sequence "context." The 16189 T-->C polymorphism lengthens a (C:G)n simple repeat, which then undergoes expansion and contraction, probably through replication slippage. This proclivity toward expansion/contraction is more pronounced when there is a C residue, rather than a T, at nt 16192. The high T-->C reversion frequency at nt 16192 apparently is the result of polymerase misincorporation or slippage during replication, the same mechanism that also causes the expansion/contraction of this simple-repeat sequence. In addition to the first analysis of this mitochondrial hypermutation process, these results also yield mechanistic insights into the expansion/contraction of simple-repeat sequences in mtDNA.     

HvrBase: compilation of mtDNA control region sequences from primates.

HvrBase is a compilation of human and ape mtDNA control region sequences. Sequences and related information on individuals, such as from where the sequences were obtained, is stored in three ASCII files as described previously. Moreover, the collection is also available as Mac/PC database application with a graphical user interface. It can be accessed through the WWW at URL http://www.eva.mpg.de/hvrbase. The current collection comprises 5846 human sequences from hypervariable region I (HVRI) and 2302 human sequences from hypervariable region II (HVRII). From apes, 295 HVRI sequences and 13 HVRII sequences are available.     

Marmoset phylogenetics, conservation perspectives, and evolution of the mtDNA control region

Marmosets (genus Callithrix) are a diverse group of platyrrhine primates with 13-15 purported taxa, many of them considered endangered. Morphological analyses constitute most of the basis for recognition of these forms as distinct taxa. The purpose of this study was to provide a molecular view, based on mitochondrial control region sequences, of the evolutionary history of the marmosets, concomitant with a molecular phylogenetic perspective on species diversity within the group. An additional purpose was to provide the first comparative examination of a complete New World monkey control region sequence with those of other mammals. The phylogenetic analyses provide convincing support for a split between the Atlantic forest and Amazonian marmosets, with the inclusion of the pygmy marmoset (Cebuella pygmaea) at the base of the Amazonian clade. The earliest branch of the Atlantic forest group was C. aurita. In the Amazonian group, the analyses do not support the recognition of C. humeralifer and the recently described C mauesi as distinct taxa. They do, however, support a clear distinction between C. argentata and a strongly supported mixed clade of C. humeralifer and C. mauesi. In the Atlantic forest group, the phylogenetic tree suggests mixing between C. penicillata, C. kuhli, and possibly C. jacchus. Most of the sequence features characteristic of other mammal control regions were also evident in marmosets, with the exception that conserved sequence blocks (CSBs) 2 and 3 were not clearly identifiable. Tandem repeat units often associated with heteroplasmy in a variety of other mammals were not evident in the marmoset sequences.      

Point mutations of the mtDNA control region in normal and neurodegenerative human brains

Recent observations in cultured human fibroblasts suggest that the accumulation of point mutations in the noncoding control region of mtDNA may be important in human aging. We studied the mtDNA control region in brain tissue from 31 normal elderly individuals, from 35 individuals who had Alzheimer disease, and from 47 individuals who had dementia with Lewy bodies. We found no evidence that these somatic mtDNA point mutations accumulate either in the brains of normal elderly individuals or in the brains of individuals with neurodegenerative disease.     

Length heteroplasmy in the first hypervariable segment of the human mtDNA control region.

The first hypervariable segment of the human mtDNA control region contains a homopolymeric tract of cytosines between nt 16184 and 16193, interrupted at position 16189 by a thymine, according to the Cambridge reference sequence. A variant commonly found in population screening is a T-to-C transition at nt 16189, resulting in an uninterrupted homopolymeric tract. Direct sequencing of individuals with this variant produces a characteristic blurred sequence in nucleotides beyond the tract. Sequencing clones from these individuals revealed that this is caused by high levels of length heteroplasmy in the homopolymeric tract and low levels of length heteroplasmy in the four adenines following the tract. We have developed a rapid method involving densitometry of sequencing gels to quantify the relative proportions of different length variants present in an individual. We have used this to study the proportions of length variants in individuals from three twin pairs and two maternal lineages. While unrelated individuals usually have different proportions of length variants, all maternally related individuals studied have the same proportions, even if they are only distantly related. It is not obvious how identical heteroplasmic profiles are maintained in maternally related individuals, but some possible mechanisms are suggested.     

The mutator property of uvr502 mutation affecting UV sensitivity of Escherichia coli

Summary The frequency of direct and reverse mutations at several chromosomal loci increased in UV sensitiveuvr502 strains ofE. coli. Both UV sensitivity and mutator (Mut⁻) phenotype are due to the singleuvr502 mutation. Inuvr ⁺/uvr502 merodiploid the Mut⁻ phenotype is recessive.     

The evolution of low mutation rates in experimental mutator populations of Saccharomyces cerevisiae

Mutation is the source of both beneficial adaptive variation and deleterious genetic load, fueling the opposing selective forces than shape mutation rate evolution. This dichotomy is well illustrated by the evolution of the mutator phenotype, a genome-wide 10- to 100-fold increase in mutation rate. This phenotype has often been observed in clonally expanding populations exposed to novel or frequently changing conditions. Although studies of both experimental and natural populations have shed light on the evolutionary forces that lead to the spread of the mutator allele through a population, significant gaps in our understanding of mutator evolution remain. Here we use an experimental evolution approach to investigate the conditions required for the evolution of a reduction in mutation rate and the mechanisms by which populations tolerate the accumulation of deleterious mutations. We find that after ～6,700 generations, four out of eight experimental mutator lines had evolved a decreased mutation rate. We provide evidence that the accumulation of deleterious mutations leads to selection for reduced mutation rate clones in populations of mutators. Finally, we test the long-term consequences of the mutator phenotype, finding that mutator lines follow different evolutionary trajectories, some of which lead to drug resistance.     

Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA

Autosomal dominant and/or recessive progressive external ophthalmoplegia (ad/arPEO) is associated with mtDNA mutagenesis. It can be caused by mutations in three nuclear genes, encoding the adenine nucleotide translocator 1, the mitochondrial helicase Twinkle or DNA polymerase γ (POLG). How mutations in these genes result in progressive accumulation of multiple mtDNA deletions in post- mitotic tissues is still unclear. A recent hypothesis suggested that mtDNA replication infidelity could promote slipped mispairing, thereby stimulating deletion formation. This hypothesis predicts that mtDNA of ad/arPEO patients will contain frequent mutations throughout; in fact, our analysis of muscle from ad/arPEO patients revealed an age-dependent, enhanced accumulation of point mutations in addition to deletions, but specifically in the mtDNA control region. Both deleted and non-deleted mtDNA molecules showed increased point mutation levels, as did mtDNAs of patients with a single mtDNA deletion, suggesting that point mutations do not cause multiple deletions. Deletion breakpoint analysis showed frequent breakpoints around homopolymeric runs, which could be a signature of replication stalling. Therefore, we propose replication stalling as the principal cause of deletion formation.     

Tandemly repeated sequences in mtDNA control region of whitefish, Coregonus lavaretus.

Length variation of the mitochondrial DNA control region was observed with PCR amplification of a sample of 138 whitefish (Coregonus lavaretus). Nucleotide sequences of representative PCR products showed that the variation was due to the presence of an approximately 100-bp motif tandemly repeated two, three, or five times in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA. This is the first report on the tandem array composed of long repeat units in mitochondrial DNA of salmonids.     

Phylogeography of the mallard Anas platyrhynchos from Eurasia inferred from sequencing of the mtDNA control region

Phylogenetic relationships, demographic history, and geographic distribution of the mtDNA haplotypes of the mallard Anas platyrhynchos were examined in three populations, Indian, Northern European, and Far Eastern. Two divergent halotype groups, A and B, were found in the Far Eastern population, while haplotypes identified in Northern European and Far Eastern populations were exclusively of the A group. The presence of B group haplotypes in the Far Eastern population can be explained either in terms of hybridization of the mallard with spot-billed duck Anas zonorhyncha at the south of the Russian Far East, or by the mtDNA paraphyly in mallards. In general, mallards from Eurasia were characterized by low genetic population differentiation along with slightly expressed phylogeographic structure. The most differentiated was the population from India (??_st = 0.076?0.077), while the difference between Northern European and Far Eastern populations was extremely low (??_st = 0.0029). Differentiation of Anas platyrhynchos Indian population was determined by the fact that a part of the population, inhabiting southern and eastern coasts of the Hindustan Peninsula, was resident.