Human mtDNA Sequences Research Articles

Evaluation of unmodifiedhuman cell-derived extracellular vesicle mitochondrial deoxyribonucleic acid-based biodistribution in rodents.

Recently, extracellular vesicles (EVs) have been developed as therapeutic targets for various diseases. Biodistribution is crucial for EVs intended for therapeutic purposes because it can determine the degree of on- and off-target effects. This study aimed to explore techniques to evaluate the biodistribution of unmodified EVs. We devised a novel quantitative polymerase chain reaction (qPCR)-based assay to detect unmodified EVs by targeting mitochondrial deoxyribonucleic acid (mtDNA), a constituent of EVs. We focused on specific mtDNA regions that exhibited homologous variations distinct from their rodent mtDNA counterparts to establish this analytical approach. Herein, we successfully designed primers and probes targeting human and rodent mtDNA sequences and developed a highly specific and sensitive qPCR method. Furthermore, the quantification range of EVs isolated from various cells differed based on the manufacturer and cell source. IRDye 800CW-labelled Expi293F EV mimetics were administered to the animals via the tail vein to compare the imaging test and mtDNA-qPCR results. The results obtained from imaging tests and mtDNA-qPCR to investigate EV biodistribution patterns revealed differences. The results revealed that our newly developed method effectively determined the biodistribution of unmodified EVs with high sensitivity and reproducibility.

Haplogroups determination from RNR1 and RNR2 regions of mitochondrial DNA

Mitochondrial DNA (mtDNA) has two regions, i.e. coding and control regions which are also known as hypervariation (HV) regions. Analysis of hypervariation regions or the entire human mtDNA genome is an important element that could be used during human evolutionary, forensic or phylogenetic studies. Although most human phylogenetics studies often use HV regions; the lack of information from those regions can complicate the process of haplogroup determination. Therefore, in this study, the human mtDNA coding regions such as MT-RNR1 and MT-RNR2 genes were used as an alternative to determine the haplogroups. A total of four primer pairs were designed based on MT-RNR1 and MT-RNR2 genes using the Primer Designing Tool software. The human mtDNA sequence from the National Center for Biotechnology Information (NCBI) database (accession number NC_012920.1) was used as a reference. These primers were used in the PCR amplification approach using DNA genomic samples isolated from human peripheral blood. Polymorphism analysis of MT-RNR1 and MT-RNR2 regions using the Mitomaster database was performed to determine haplogroups. The results indicated that three haplogroups were identified which are N11a, H2a and H1. In conclusion, the primers designed from mitochondrial RNR1 and RNR2 genes can be used for human haplogroup identification.

Human mitochondrial DNA diversity is compatible with the multiregional continuity theory of the origin of Homo sapiens

Confidence intervals for estimates of human mtDNA sequence diversity, chimpanzee-human mtDNA sequence divergence, and the time of splitting of the pongid-hominid lineages are presented. Consistent with all the data used in estimating the coalescence time for human mitochondrial lineages to a common ancestral mitochondrion is a range of dates from less than 79,000 years ago to more than 1,139,000 years ago. Consequently, the hypothesis that a migration of modern humans (Homo sapiens) out of Africa in the range of 140,000 to 280,000 years ago resulted in the complete replacement, without genetic interchange, of earlier Eurasian hominid populations (Homo erectus) is but one of several possible interpretations of the mtDNA data. The data are also compatible with the hypothesis, suggested earlier and supported by fossil evidence, of a single, more ancient expansion of the range of Homo erectus from Africa, followed by a gradual transition to Homo sapiens in Europe, Asia, and Africa.

Analysis of human mitochondrial genome co-occurrence networks of Asian population at varying altitudes

Networks have been established as an extremely powerful framework to understand and predict the behavior of many large-scale complex systems. We studied network motifs, the basic structural elements of networks, to describe the possible role of co-occurrence of genomic variations behind high altitude adaptation in the Asian human population. Mitochondrial DNA (mtDNA) variations have been acclaimed as one of the key players in understanding the biological mechanisms behind adaptation to extreme conditions. To explore the cumulative effects of variations in the mitochondrial genome with the variation in the altitude, we investigated human mt-DNA sequences from the NCBI database at different altitudes under the co-occurrence motifs framework. Analysis of the co-occurrence motifs using similarity clustering revealed a clear distinction between lower and higher altitude regions. In addition, the previously known high altitude markers 3394 and 7697 (which are definitive sites of haplogroup M9a1a1c1b) were found to co-occur within their own gene complexes indicating the impact of intra-genic constraint on co-evolution of nucleotides. Furthermore, an ancestral ‘RSRS50’ variant 10,398 was found to co-occur only at higher altitudes supporting the fact that a separate route of colonization at these altitudes might have taken place. Overall, our analysis revealed the presence of co-occurrence interactions specific to high altitude at a whole mitochondrial genome level. This study, combined with the classical haplogroups analysis is useful in understanding the role of co-occurrence of mitochondrial variations in high altitude adaptation.

Heteroplasmy incidence in mitochondrial DNA

Mitochondrial DNA In humans, mitochondrial DNA (mtDNA) is predominantly maternally inherited. mtDNA is under selection to prevent heteroplasmy—the transmission of multiple genetic variants into the next generation. Wei et al. explored human mtDNA sequences to determine mtDNA genome structure, selection, and transmission. Whole-genome sequencing revealed that about 45% of individuals carry heteroplasmic mtDNA sequences at levels greater than 1% of their total mtDNA. Furthermore, studies of more than 1500 mother-offspring pairs indicated that the female line selected which mtDNA variants were passed on to children. This effect was influenced by the mother's nuclear genetic background. Thus, mtDNA is under selection at specific loci in the human germ line. Science , this issue p. [eaau6520][1] [1]: /lookup/doi/10.1126/science.aau6520

Germline selection shapes human mitochondrial DNA diversity.

Approximately 2.4% of the human mitochondrial DNA (mtDNA) genome exhibits common homoplasmic genetic variation. We analyzed 12,975 whole-genome sequences to show that 45.1% of individuals from 1526 mother-offspring pairs harbor a mixed population of mtDNA (heteroplasmy), but the propensity for maternal transmission differs across the mitochondrial genome. Over one generation, we observed selection both for and against variants in specific genomic regions; known variants were more likely to be transmitted than previously unknown variants. However, new heteroplasmies were more likely to match the nuclear genetic ancestry as opposed to the ancestry of the mitochondrial genome on which the mutations occurred, validating our findings in 40,325 individuals. Thus, human mtDNA at the population level is shaped by selective forces within the female germ line under nuclear genetic control, which ensures consistency between the two independent genetic lineages.

A Dawson-like clustering of human mitochondrial DNA sequences based on protein coding region

In the present paper, our main goal is focused in developing fast algorithms for human mtDNA sequence analyses, requiring minimum and explicit assumptions on mutation models and evolutionary pathways. We propose a new approach based on a construction of Dawson, a technique based on the ordering of the variable sites.In this approach, the first step corresponds to the computation of the order of the positions according to their capacity to separate the sequences into dichotomous groups. Aiming to avoid or at least to minimize the consideration of ambiguous evolutionary events such as insertions/deletions and recurrence, which cause well-known alignment problems, in the present study we only work with the protein coding sequence, the clearly more stable region in human mitochondrial genomes. This method was tested in a small set of 99 human mtDNA comprising representatives of all major haplogroups. The developed approach showed to be a choice to automate the clustering of human mtDNA sequences into broad groups, the output being in agreement with the canonical classification into macro-haplogroups deposited in the Phylotree database.

Identification and characterization of the novel m.8305C>T MTTK and m.4440G>A MTTM gene mutations causing mitochondrial myopathies

We report on two novel mtDNA mutations in patients affected with mitochondrial myopathy. The first patient, a 44-year-old woman, had bilateral eyelid ptosis and the m.8305C>T mutation in the MTTK gene. The second patient, a 56-year-old man, had four-limb muscle weakness and the MTTM gene m.4440G>A mutation. Muscle biopsies in both patients showed ragged red fibers and numerous COX-negative fibers as well as a combined defect of complex I, III and IV activities. The two mutations were heteroplasmic and detected only in muscle tissue, with a higher mutation load in COX-negative fibers. Additionally, both mutations occurred in highly conserved mt-tRNA sites, and were not found by an in silico search in 30,589 human mtDNA sequences. Our report further expands the mutational and phenotypic spectrum of diseases associated with mutations in mitochondrial tRNA genes and reinforces the notion that mutations in mitochondrial tRNAs represent hot spots for mitochondrial myopathies in adults.

MtDNA diversity in human populations highlights the merit of haplotype matching in gene therapies.

Does mitochondrial DNA (mtDNA) diversity in modern human populations potentially pose a challenge, via mtDNA segregation, to mitochondrial replacement therapies? The magnitude of mtDNA diversity in modern human populations is as high as in mammalian model systems where strong mtDNA segregation is observed; consideration of haplotype pairs and/or haplotype matching can help avoid these potentially deleterious effects. In mammalian models, substantial proliferative differences are observed between different mtDNA haplotypes in cellular admixtures, with larger proliferative differences arising from more diverse haplotype pairings. If maternal mtDNA is 'carried over' in human gene therapies, these proliferative differences could lead to its amplification in the resulting offspring, potentially leading to manifestation of the disease that the therapy was designed to avoid-but existing studies have not investigated whether mtDNA diversity in modern human populations is sufficient to permit significant amplification. This theoretical study used over 7500 human mtDNA sequences from The National Center for Biotechnology Information (NCBI), a range of international and British mtDNA surveys, and 2011 census data. A stochastic simulation approach was used to model random haplotype pairings from within different regions. In total, 1000 simulated pairings were analysed using the basic local alignment search tool (BLAST) for each region. Previous data from mouse models were used to estimate proliferative differences. Even within the same haplogroup, differences of around 20-80 single-nucleotide polymorphisms (SNPs) are common between mtDNAs admixed in random pairings. These values are sufficient to lead to substantial segregation in mouse models over an organismal lifetime, even given low starting heteroplasmy, inducing increases from 5% to 35% over 1 year. Substantial population mixing in modern UK cities increases the expected genetic differences. Hence, the likely genetic differences between humans randomly sampled from a population may well allow substantial amplification of a disease-carrying mtDNA haplotype over the timescale of a human lifetime. We report ranges and mean differences for all statistics to quantify uncertainty in our results. The mapping from mouse and other mammalian models to the human system is challenging, as timescales and mechanisms may differ. Reporting biases in NCBI mtDNA data, if present, may affect the statistics we compute. We discuss the robustness of our findings in the light of these concerns. Matching the mtDNA haplotypes of the mother and third-party donor in mitochondrial replacement therapies is supported as a means of ameliorating the potentially deleterious results of human mtDNA diversity. We present a chart of expected SNP differences between mtDNA haplogroups, allowing the selection of optimal partners for therapies. N/A STUDY FUNDING/COMPETING INTERESTS: The authors report no external funding sources or conflicts of interest.

Abstract 4322: The landscape of mitochondrial DNA mutations in human cancer

Abstract Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of human cancers. Although mitochondria in cancer cells have important roles in energy metabolism and survival, the origins and impact of somatic mutations in mitochondrial DNA (mtDNA) are unclear. Here, we provide a comprehensive analysis of somatic alterations in cancer mitochondria from 1,675 genome sequences across 31 tumor types. We identified 1,907 somatic substitutions which were mainly C:G>T:A and T:A>C:G. These mutations exhibited dramatic replicative strand bias, being almost all C>T and A>G on the mitochondrial heavy strand. This strand-asymmetric mitochondrial mutational signature is different from those found in nuclear cancer genomes but is similar to processes shaping human mtDNA sequence over evolutionary time. The number of mtDNA mutations showed considerable heterogeneity across tumor types, being significantly higher in tumors from gastrointestinal and hepatobiliary systems. With respect to selection, observed missense mutations were nearly neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation showed evidence of negative selection and were more frequently heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria, and is fundamentally linked to mtDNA replication. Citation Format: Young Seok Ju, Ludmil Alexandrov, Moritz Gerstung, Inigo Martincorena, ICGC Breast Cancer Group; ICGC Chronic Myeloid Disorders Group; ICGC Prostate Cancer Group, Mike Stratton, Peter J. Campbell. The landscape of mitochondrial DNA mutations in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2014-4322

Transcription factors bind negatively selected sites within human mtDNA genes.

Transcription of mitochondrial DNA (mtDNA)-encoded genes is thought to be regulated by a handful of dedicated transcription factors (TFs), suggesting that mtDNA genes are separately regulated from the nucleus. However, several TFs, with known nuclear activities, were found to bind mtDNA and regulate mitochondrial transcription. Additionally, mtDNA transcriptional regulatory elements, which were proved important in vitro, were harbored by a deletion that normally segregated among healthy individuals. Hence, mtDNA transcriptional regulation is more complex than once thought. Here, by analyzing ENCODE chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified strong binding sites of three bona fide nuclear TFs (c-Jun, Jun-D, and CEBPb) within human mtDNA protein-coding genes. We validated the binding of two TFs by ChIP-quantitative polymerase chain reaction (c-Jun and Jun-D) and showed their mitochondrial localization by electron microscopy and subcellular fractionation. As a step toward investigating the functionality of these TF-binding sites (TFBS), we assessed signatures of selection. By analyzing 9,868 human mtDNA sequences encompassing all major global populations, we recorded genetic variants in tips and nodes of mtDNA phylogeny within the TFBS. We next calculated the effects of variants on binding motif prediction scores. Finally, the mtDNA variation pattern in predicted TFBS, occurring within ChIP-seq negative-binding sites, was compared with ChIP-seq positive-TFBS (CPR). Motifs within CPRs of c-Jun, Jun-D, and CEBPb harbored either only tip variants or their nodal variants retained high motif prediction scores. This reflects negative selection within mtDNA CPRs, thus supporting their functionality. Hence, human mtDNA-coding sequences may have dual roles, namely coding for genes yet possibly also possessing regulatory potential.

Association of G-quadruplex forming sequences with human mtDNA deletion breakpoints.

BackgroundMitochondrial DNA (mtDNA) deletions cause disease and accumulate during aging, yet our understanding of the molecular mechanisms underlying their formation remains rudimentary. Guanine-quadruplex (GQ) DNA structures are associated with nuclear DNA instability in cancer; recent evidence indicates they can also form in mitochondrial nucleic acids, suggesting that these non-B DNA structures could be associated with mtDNA deletions. Currently, the multiple types of GQ sequences and their association with human mtDNA stability are unknown.ResultsHere, we show an association between human mtDNA deletion breakpoint locations (sites where DNA ends rejoin after deletion of a section) and sequences with G-quadruplex forming potential (QFP), and establish the ability of selected sequences to form GQ in vitro. QFP contain four runs of either two or three consecutive guanines (2G and 3G, respectively), and we identified four types of QFP for subsequent analysis: intrastrand 2G, intrastrand 3G, duplex derived interstrand (ddi) 2G, and ddi 3G QFP sequences. We analyzed the position of each motif set relative to either 5' or 3' unique mtDNA deletion breakpoints, and found that intrastrand QFP sequences, but not ddi QFP sequences, showed significant association with mtDNA deletion breakpoint locations. Moreover, a large proportion of these QFP sequences occur at smaller distances to breakpoints relative to distribution-matched controls. The positive association of 2G QFP sequences persisted when breakpoints were divided into clinical subgroups. We tested in vitro GQ formation of representative mtDNA sequences containing these 2G QFP sequences and detected robust GQ structures by UV–VIS and CD spectroscopy. Notably, the most frequent deletion breakpoints, including those of the "common deletion", are bounded by 2G QFP sequence motifs.ConclusionsThe potential for GQ to influence mitochondrial genome stability supports a high-priority investigation of these structures and their regulation in normal and pathological mitochondrial biology. These findings emphasize the potential importance of helicases that subsequently resolve GQ to maintain the stability of the mitochondrial genome.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-677) contains supplementary material, which is available to authorized users.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

Hominin evolution and gene flow in the Pleistocene Africa

Africa demonstrates a complex process of the hominin evolution with a series of adaptive radiations during several millions of years that led to diverse morphological forms. Recently, Hammer et al. (2011) and Harvati et al. (2011) provided integrated morphological and genetic evidence of interbreeding between modern humans and unknown archaic hominins in Africa as recently as 35,000 years ago. However, a genetic evidence of hybridization between hominin lineages during the Lower and Middle Pleistocene epochs is unknown and the direct retrieval of DNA from extinct lineages of African hominins remains elusive. The availability of both nuclear and mitochondrial genome sequences from modern humans, Neanderthals, and Denisovans allows collecting nuclear DNA sequences of mitochondrial origin (numts) inserted into the nuclear genome of the ancestral hominin lineages and drawing conclusions about the hominin evolution in the remote past. The mtDNA and numt analysis uncovered a deep division of mtDNA lineages that existed in African hominins in the Middle Pleistocene. The first cluster included the human and Neanderthal-like mtDNA sequences while the second consisted of DNA sequences that are known today as mtAncestor-1, a nuclear fossil of the mtDNA, and the Denisova mtDNA isolated from a bone and a tooth found in southern Siberia. The two groups initially diverged 610,000-1,110,000 years ago. Approximately 220,000 years after the primary split, the Denisova - mtAncestor-1 mtDNA lineages mixed with the mtDNA pool of an ancestral population of Neanderthals and modern humans. This admixture after the profound division is demonstrated by the transposition of the Denisova-like mtDNA sequence into the nuclear genome of an ancestor of Neanderthals and modern humans. This finding suggests the matrilineal genetic structure among the Middle Pleistocene hominins as well as the existence of gene flow between African hominin lineages. Through paleogenomic analyses, it is impossible to exclude the theory that population structure and gene flow in African hominins influenced the admixture pattern observed in the nuclear genomes of non-Africans.

Update to Eller et al.'s “Local Extinction and Recolonization, Species Effective Population Size, and Modem Human Origins” (2004)

Humans have about the level of genetic diversity that you would expect from a Wright-Fisher population model with 10,000 individuals. That sentence opens a labyrinth of interpretation. Why do we have the genetic variation that we do? Is 10,000 the number of our ancestors, or are we inbred for other reasons? Haigh and Maynard Smith, writing in 1972 about /?-globin polymorphism, first proposed a population bottleneck. At the time, few human biologists recognized that such a hypothesis would cut the Neanderthals and other archaic humans from our ancestry. A relationship between population size and protein diversity across a large sample of animal species was documented by Soule (1976). Nei and Graur (1984) showed low protein polymorphism in dozens of species, from wolves to flies, suggesting that Pleistocene bottlenecks may have affected most species. When human mtDNA sequence data showed low worldwide nucleotide diversity in humans (Cann et al. 1987), a population bottleneck entered the human genetics mainstream. But long before this, the interpretation of polymorphisms became a focus of the antagonists in the neutralist/selectionist controversy (Gillespie 1991). Neutral evolution in a Wright-Fisher population model is algebraically simple, but natural populations depart from the assumptions of this model in many ways. Some population structures radically increase the chance of inbreeding, thereby lowering genetic variation even in large populations. Motoo Kimura and his students explored the consequences of many such models. One is the subject of our 2004 paper (Eller et al. 2004). Maruyama and Kimura (1980) initially developed the model of extinction and recolonization, showing that the right combination of parameters could generate strong inbreeding. The model was interesting because of its application to a famous model species: E. coli. This species may be a ponderous swarm of quadrillions of cells, but it behaves genetically like a nimble population of a few billion. When its colonic hosts die, they take whole communities of gut flora with them.

Update to Eller et al.'s "Local Extinction and Recolonization, Species Effective Population Size, and Modern Human Origins" (2004)

Update to Eller et al.'s "Local Extinction and Recolonization, Species Effective Population Size, and Modern Human Origins" (2004) John Hawks Keywords Hunter-Gatherers, Pleistocene, Genetic Variation, Inbreeding Humans have about the level of genetic diversity that you would expect from a Wright-Fisher population model with 10,000 individuals. That sentence opens a labyrinth of interpretation. Why do we have the genetic variation that we do? Is 10,000 the number of our ancestors, or are we inbred for other reasons? Haigh and Maynard Smith, writing in 1972 about β-globin polymorphism, first proposed a population bottleneck. At the time, few human biologists recognized that such a hypothesis would cut the Neanderthals and other archaic humans from our ancestry. A relationship between population size and protein diversity across a large sample of animal species was documented by Soulé (1976). Nei and Graur (1984) showed low protein polymorphism in dozens of species, from wolves to flies, suggesting that Pleistocene bottlenecks may have affected most species. When human mtDNA sequence data showed low worldwide nucleotide diversity in humans (Cann et al. 1987), a population bottleneck entered the human genetics mainstream. But long before this, the interpretation of polymorphisms became a focus of the antagonists in the neutralist/selectionist controversy (Gillespie 1991). Neutral evolution in a Wright-Fisher population model is algebraically simple, but natural populations depart from the assumptions of this model in many ways. Some population structures radically increase the chance of inbreeding, thereby lowering genetic variation even in large populations. Motoo Kimura and his students explored the consequences of many such models. One is the subject of our 2004 paper (Eller et al. 2004). Maruyama and Kimura (1980) initially developed the model of extinction and recolonization, showing that the right combination of parameters could generate strong inbreeding. The model was interesting because of its application to a famous model species: E. coli. This species may be a ponderous swarm of quadrillions of cells, but it behaves genetically like a nimble population of a few billion. When its colonic hosts die, they take whole communities of gut flora with them. [End Page 825] Of course, humans aren't bacteria. We set out to show whether Maruyama and Kimura's model could work in a hunter-gatherer population setting. Takahata (1994) had considered the scenario as a possible explanation for human variation, but it was clear that we needed to consider what parameter values could be realistic in the Pleistocene. Hunter-gatherer groups historically intermarry extensively. Groups sometimes disappear, but most of the individuals don't die—they just move in with their neighbors. These facts could have proven fatal to the model, which depends on a high rate of extinction and a low rate of gene flow to cause inbreeding. Still, we found a range of parameter values that might be realistic and that really cut down the genetic variation in simulation models. After five years, how does our study hold up? The biggest change in the succeeding interval has been a better appreciation of variation in our close relatives. In 2004, it was evident that the mtDNA variation of apes was many times higher than it was in humans, but autosomal variation was only starting to be understood. Bonobos have roughly the same variation as humans (Yu et al. 2003), chimpanzees and gorillas run around double, and orangutans have three times the human value or higher (Caswell et al. 2008; Fischer et al. 2006; Yu et al. 2003). It now seems possible that humans and apes were subject to similar demographic processes, with inbreeding amped up a bit in our ancestors and regional isolation higher in the ancestral populations of most apes. Recently Neanderthal genetic information, including multiple mtDNA sequences (Briggs et al. 2009) and nuclear genome analysis (Green et al. 2006; Noonan et al. 2006), has yielded empirical observations of genetic variation within ancient humans. Modern humans, Neanderthals, and the human-Neanderthal ancestral population all appear to have had humanlike levels of genetic variation (Premo and Hublin 2009). Explaining human genetic variation is no longer a problem of a single bottleneck; it requires a demographic model that can encompass Pleistocene humans and to a greater or lesser extent other...

The worm in the fruit of the mitochondrial DNA tree

The worm in the fruit of the mitochondrial DNA tree

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.

Distilling Artificial Recombinants from Large Sets of Complete mtDNA Genomes

BackgroundLarge-scale genome sequencing poses enormous problems to the logistics of laboratory work and data handling. When numerous fragments of different genomes are PCR amplified and sequenced in a laboratory, there is a high immanent risk of sample confusion. For genetic markers, such as mitochondrial DNA (mtDNA), which are free of natural recombination, single instances of sample mix-up involving different branches of the mtDNA phylogeny would give rise to reticulate patterns and should therefore be detectable.Methodology/Principal FindingsWe have developed a strategy for comparing new complete mtDNA genomes, one by one, to a current skeleton of the worldwide mtDNA phylogeny. The mutations distinguishing the reference sequence from a putative recombinant sequence can then be allocated to two or more different branches of this phylogenetic skeleton. Thus, one would search for two (or three) near-matches in the total mtDNA database that together best explain the variation seen in the recombinants. The evolutionary pathway from the mtDNA tree connecting this pair together with the recombinant then generate a grid-like median network, from which one can read off the exchanged segments.ConclusionsWe have applied this procedure to a large collection of complete human mtDNA sequences, where several recombinants could be distilled by our method. All these recombinant sequences were subsequently corrected by de novo experiments – fully concordant with the predictions from our data-analytical approach.

Recovery of DNA and pollen from New Zealand lake sediments

An undisturbed sediment core taken from Round Lake, Hawke's Bay, New Zealand, using a specially designed sterile sampler, has been dated by tephra analysis and radiocarbon and analysed for pollen, charcoal and DNA. It appears to cover the last ca. 3600 years. There are clear indications of Māori effects on vegetation from ca. 800 BP. DNA was extracted from the core and amplified using bacterial and a range of other PCR primers targeting human and other animal mtDNA sequences. Cloned bacterial DNA sequences from just above the Taupo tephra (dated to AD 217–247) produced 98% and 99% similarity matches with human faecal bacteria. The 98% match was to the human gut bacterial genus Prevotella. This genus can, however, be found in a range of animal (including insect) guts. Extracted DNA was matched closely to the New Zealand native fish species Gobiomorphus cotidianus. Further work is needed to establish unequivocally that no DNA movement through the profile is occurring, but these results suggest a new technique of potentially revolutionary importance for Quaternary research.