Strand Of mtDNA Research Articles

The simultaneous detection of mitochondrial DNA damage from sun-exposed skin of three whale species and its association with UV-induced microscopic lesions and apoptosis

Due to life history and physiological constraints, cetaceans (whales) are unable to avoid prolonged exposure to external environmental insults, such as solar ultraviolet radiation (UV). The majority of studies on the effects of UV on skin are restricted to humans and laboratory animals, but it is important to develop tools to understand the effects of UV damage on large mammals such as whales, as these animals are long-lived and widely distributed, and can reflect the effects of UV across a large geographical range. We and others have used mitochondrial DNA (mtDNA) as a reliable marker of UV-induced damage particularly in human skin. UV-induced mtDNA strand breaks or lesions accumulate throughout the lifespan of an individual, thus constituting an excellent biomarker for cumulative exposure. Based on our previous studies in human skin, we have developed for the first time in the literature a quantitative real-time PCR methodology to detect and quantify mtDNA lesions in skin from sun-blistered whales. Furthermore the methodology allows for simultaneous detection of mtDNA damage in different species. Therefore using 44 epidermal mtDNA samples collected from 15 blue whales, 10 fin whales, and 19 sperm whales from the Gulf of California, Mexico, we quantified damage across 4.3kilobases, a large region of the ~16,400 base pair whale mitochondrial genome. The results show a range of mtDNA damage in the skin of the three different whale species. This previously unreported observation was correlated with apoptotic damage and microscopic lesions, both of which are markers of UV-induced damage. As is the case in human studies, this suggests the potential use of mtDNA as a biomarker for measuring the effect of cumulative UV exposure in whales and may provide a platform to help understand the effects of changing global environmental conditions.

MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation

Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no invivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease ofde novo transcription manifested as reduced 7SRNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.

The Mitochondrial RNA-Binding Protein GRSF1 Localizes to RNA Granules and Is Required for Posttranscriptional Mitochondrial Gene Expression

RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression.

Mitochondrial dysfunction in osteoarthritis is associated with down‐regulation of superoxide dismutase 2

Superoxide dismutase 2 (SOD2) is down- regulated in osteoarthritis (OA). This study was undertaken to investigate the functional effects of this down-regulation in the context of oxidative damage and mitochondrial dysfunction. Lipid peroxidation in articular cartilage from OA patients and from lesion-free control subjects with femoral neck fracture was assessed by measuring malondialdehyde levels using the thiobarbituric acid reactive substances assay. Long-range polymerase chain reaction amplification and a mitochondrial DNA (mtDNA) strand break assay were used to investigate the presence of somatic large-scale mtDNA rearrangements in cartilage. Microscale oxygraphy was used to explore possible changes in mitochondrial respiratory activity between OA and control chondrocytes. RNA interference was used to determine the effects of SOD2 depletion on lipid peroxidation, mtDNA damage, and mitochondrial respiration. OA cartilage had higher levels of lipid peroxidation compared to control cartilage, and lipid peroxidation was similarly elevated in SOD2-depleted chondrocytes. SOD2 depletion led to a significant increase in mtDNA strand breaks in chondrocytes, but there was no notable difference in the level of strand breaks between OA and control chondrocytes. Furthermore, only very low levels of somatic, large-scale mtDNA rearrangements were identified in OA cartilage. OA chondrocytes showed less spare respiratory capacity (SRC) and higher proton leak compared to control chondrocytes. SOD2-depleted chondrocytes also showed less SRC and higher proton leak. This is the first study to analyze the effects of SOD2 depletion in human articular chondrocytes in terms of changes to oxidation and mitochondrial function. The findings indicate that SOD2 depletion in chondrocytes leads to oxidative damage and mitochondrial dysfunction, suggesting that SOD2 down-regulation is a potential contributor to the pathogenesis of OA.

Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis.

The Human Mitochondrial Transcriptome

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com).

Involvement of human ELAC2 gene product in 3' end processing of mitochondrial tRNAs

Accurate tRNA processing is crucial for human mitochondrial genome expression, but the mechanisms of mt-tRNA cleavage and the key enzymes involved in this process are poorly characterized. At least two activities are required for proper mt-tRNA maturation: RNase P cleaving precursor molecules at the 5' end and tRNase Z at the 3' end. In human mitochondria only RNase P has been identified so far. Using RT-PCR and northern blot analyses we found that silencing of the human ELAC2 gene results in impaired 3' end of mt-tRNAs. We demonstrate this for several mitochondrial tRNAs, encoded on both mtDNA strands, including tRNAVal, tRNALys, tRNAArg, tRNAGly, tRNALeu(UUR) and tRNAGlu. The silencing of the MRPP1 gene that encodes a subunit of mtRNase P resulted in inhibition of both 5' and 3' processing. We also demonstrate the double mitochondrial/nuclear localization of the ELAC2 protein using immunofluorescence. Our results indicate that ELAC2 functions as a tRNase Z in human mitochondria and suggest that mt-tRNase Z preferentially cleaves molecules already processed by the proteinaceous mtRNase P.

DNA methyltransferase 1, cytosine methylation, and cytosine hydroxymethylation in mammalian mitochondria

Mitochondrial DNA (mtDNA) has been reported to contain 5-methylcytosine (5mC) at CpG dinucleotides, as in the nuclear genome, but neither the mechanism generating mtDNA methylation nor its functional significance is known. We now report the presence of 5-hydroxymethylcytosine (5hmC) as well as 5mC in mammalian mtDNA, suggesting that previous studies underestimated the level of cytosine modification in this genome. DNA methyltransferase 1 (DNMT1) translocates to the mitochondria, driven by a mitochondrial targeting sequence located immediately upstream of the commonly accepted translational start site. This targeting sequence is conserved across mammals, and the encoded peptide directs a heterologous protein to the mitochondria. DNMT1 is the only member of the three known catalytically active DNA methyltransferases targeted to the mitochondrion. Mitochondrial DNMT1 (mtDNMT1) binds to mtDNA, proving the presence of mtDNMT1 in the mitochondrial matrix. mtDNMT1 expression is up-regulated by NRF1 and PGC1α, transcription factors that activate expression of nuclear-encoded mitochondrial genes in response to hypoxia, and by loss of p53, a tumor suppressor known to regulate mitochondrial metabolism. Altered mtDNMT1 expression asymmetrically affects expression of transcripts from the heavy and light strands of mtDNA. Hence, mtDNMT1 appears to be responsible for mtDNA cytosine methylation, from which 5hmC is presumed to be derived, and its expression is controlled by factors that regulate mitochondrial function.

Effects on mitochondrial transcription of manipulating mTERF protein levels in cultured human HEK293 cells

BackgroundBased on its activities in vitro, the mammalian mitochondrial transcription termination factor mTERF has been proposed to regulate mitochondrial transcription by favouring termination at its high-affinity binding immediately downstream of the rDNA segment of mitochondrial DNA, and initiation selectively at the PH1 site of the heavy-strand promoter. This defines an rDNA transcription unit distinct from the 'global' heavy-strand transcription unit initiating at PH2. However, evidence that the relative activities of the two heavy-strand transcription units are modulated by mTERF in vivo is thus far lacking.ResultsTo test this hypothesis, we engineered human HEK293-derived cells for over-expression or knockdown of mTERF, and measured the steady-state levels of transcripts belonging to different transcription units, namely tRNALeu(UUR) and ND1 mRNA for the PH2 transcription unit, and tRNAPhe plus 12S and 16S rRNA for the PH1 transcription unit. The relative levels of 16S rRNA and ND1 mRNA were the same under all conditions tested, although mTERF knockdown resulted in increased levels of transcripts of 12S rRNA. The amount of tRNAPhe relative to tRNALeu(UUR) was unaffected by mTERF over-expression, altered only slightly by mTERF knockdown, and was unchanged during recovery from ethidium bromide-induced depletion of mitochondrial RNA. mTERF overexpression or knockdown produced a substantial shift (3-5-fold) in the relative abundance of antisense transcripts either side of its high-affinity binding site.ConclusionsmTERF protein levels materially affect the amount of readthrough transcription on the antisense strand of mtDNA, whilst the effects on sense-strand transcripts are complex, and suggest the influence of compensatory mechanisms.

Development of a Model for the Teaching of Mitochondrial DNA

The Cellular Biology and Molecular Biology are fields of Science that use very abstract concepts, because they look into microscopic and molecular aspects of the nature. The process of teaching/learning of those disciplines requires didactic material, as an alternative approach for the students, to increase the chances of understanding these issues and to become an important tool in the synthesis of this knowledge. One of the methods that can be employed is the didactic models based on multimedia, because they allow an easy and fun interaction with these subjects. On this work was created a new educational model that represents the human mitochondrial DNA molecule, mtDNA, in its circular form, using the softwares Excel 2007 and PowerPoint 2007. The model was constructed in hypertext format, which allowed a quick and interactive access to the information contained in the genes found in the L and the H strands of mtDNA, and its function in the mitochondrial processes, like themechanism of energy production that occurs inside of the mitochondria by the coupling of electron transfer and ATP synthesis or still others uses like forensic identification.

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

The mitochondrial genome: Dynamics, mechanisms of repair, and a target in disease and therapy

The mitochondrial genome: Dynamics, mechanisms of repair, and a target in disease and therapy

Profound Length Heteroplasmic Alterations in Mitochondrial DNA Control Region From Primary AML Cells: Implication of Impaired Mitochondrial Replication and Demonstration of ‘vicious cycle' Hypothesis.

Abstract 3116Poster Board III-53Mitochondrial DNA (mtDNA) control region (displacement (D)-loop including HV1 and HV2) is a non-coding region of 1124 bp (nucleotide positions, np 16 024–576), which acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements (Blood 2004;103:4466-77). Importantly, mutations in the D-loop regulatory region might change mtDNA replication rate by modifying the binding affinity of significant trans-activating factors (Eur J Cancer 2004;40:2519-24). Thus, length heteroplasmic alterations of mtDNA control region may be related with mitochondrial dysfunction resulting in ‘vicious cycle' (Mol Med Today 2000;6:425-32). In an attempt to investigate profiling of mtDNA length heteroplasmic alterations in primary AML cells, we carried out a quantitative size-based PCR product separation by capillary electrophoresis (ABI 3130XL Genetic Analyzer and ABI Prism Genotyper version 3.1) using six targets (np 303-315 poly C, np 16184-16193 poly C, np 514-511 CA repeats, np 3566-3572 poly C, np 12385-12391 poly C and np 12418-12426 poly A). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen) and Rotor-Gene 3000 (Corbett Research). Forty-eight AML bone marrow samples were collected after receiving Institutional Review Board approval and informed consent. There were profound alterations of mtGI in 303 poly C, 16184 poly C and 514 CA repeats. The length heteroplasmy pattern of 303 poly C tract in the HV2 region disclosed mixture of 7C, 8C, 9C and 10C mtDNA types. In the HV2 region, length heteroplasmy in poly-C tract at np 303 - 309 exhibited 5 variant peak patterns: 7CT6C+8CT6C (50.0%), 8CT6C+9CT6C (14.0%), 8CT6C+ 9CT6C+ 10CT6C (10.4%), 9CT6C+10CT6C+11CT6C (8.3%) 9CT6C + 10CT6C + 11CT6C+12CT6C (2.1%). The length heteroplasmy pattern of 514-523 CA repeats in the HV2 region exhibited 2 variant peak patterns: CACACACACA (56.3%) and CACACACA (43.7%). In the HV1 region, length heteroplasmy in the poly-C tract at np 16184 - 16193 exhibited 9 variant peak patterns: 5CT4C+5CT3C (31.0%), 6CT4C+6CT3C (2.1%), 9C+10C+11C+12C (16.7%), 9C+10C+11C (2.1%), T4CT4C+5CT3C (4.2%), 9C+10C+11C+12C+13C (2.1%), 3CTC4C+5CT3C (2.1%), 10C+11C+12C+13C (4.2%), 8C+9C+10+11C (2.1%). Primary AML cells revealed decreased enzyme activity in respiratory chain complex I, II and III. AML cells had about a two-fold decrease in mtDNA copy number compared with normal blood mononuclear cells. Current study demonstrates that profound length heteroplasmic alterations in mtDNA control region of primary AML cells may lead to impairment of mitochondrial biogenesis (reduction of mtDNA copy number) and derangement of mitochondrial ATP synthesis. During this perturbation, mitochondria in primary AML cells might produce a large amount of reactive oxygen species, which causes the vicious cycle observed in chronic inflammatory diseases and cancers as well. DisclosuresNo relevant conflicts of interest to declare.

Comparative genomics of Drosophila mtDNA: Novel features of conservation and change across functional domains and lineages.

To gain insight on mitochondrial DNA (mtDNA) evolution, we assembled and analyzed the mitochondrial genomes of Drosophila erecta, D. ananassae, D. persimilis, D. willistoni, D. mojavensis, D. virilis and D. grimshawi together with the sequenced mtDNAs of the melanogaster subgroup. Genomic comparisons across the well-defined Drosophila phylogeny impart power for detecting conserved mtDNA regions that maintain metabolic function and regions that evolve uniquely on lineages. Evolutionary rate varies across intergenic regions of the mtDNA. Rapidly evolving intergenic regions harbor the majority of mitochondrial indel divergence. In contrast, patterns of nearly perfect conservation within intergenic regions reveal a refined set of nucleotides underlying the binding of transcription termination factors. Sequencing of 5' cDNA ends indicates that cytochrome C oxidase I (CoI) has a novel (T/C)CG start codon and that perfectly conserved regions upstream of two NADH dehydrogenase (ND) genes are transcribed and likely extend these protein sequences. Substitutions at synonymous sites in the Drosophila mitochondrial proteomes reflect a mutation process that is biased toward A and T nucleotides and differs between mtDNA strands. Differences in codon usage bias across genes reveal that weak selection at silent sites may offset the mutation bias. The mutation-selection balance at synonymous sites has also diverged between the Drosophila and Sophophora lineages. Rates of evolution are highly heterogeneous across the mitochondrial proteome, with ND accumulating many more amino acid substitutions than CO. These oxidative phosphorylation complex-specific rates of evolution vary across lineages and may reflect physiological and ecological change across the Drosophila phylogeny.

Evolution of the Mitochondrial Genome in Mammals Living at High Altitude: New Insights from a Study of the Tribe Caprini (Bovidae, Antilopinae)

Organisms living at high altitude are exposed to severe environmental stress associated with decreased oxygen pressure, cold temperatures, increased levels of UV radiation, steep slopes, and scarce food supplies, which may have imposed important selective constraints on the evolution of the mitochondrial genome. Within mammals, the tribe Caprini is of particular interest for studying the evolutionary effects of life at high altitude, as most species live in mountain regions, where they developed morphological and physiological adaptations for climbing. In this report, we analyzed the complete mitochondrial genome of 24 ruminants, including 20 species of Caprini. The phylogenetic analyses based on 16,117 nucleotides suggested the existence of a new large clade, here named subtribe Caprina, containing all genera, but Pantholops (Pantholopina), Capricornis, Naemorhedus, and Ovibos (Ovibovina). The alignment of the control region showed that all Caprini have between two and four tandem repeats of ~75 bp in the RS2 region, and that several of these copies emerged from recent and independent duplication events. We proposed therefore that the maintenance of at least two RS2 repeats in the control region of Caprini is positively selected, probably for producing a higher number of D-loop strands 3'-ending at different locations. The analyses of base composition at third-codon positions of protein-coding genes revealed that Caprini have the highest percentage of A nucleotide and the lowest percentage of G nucleotide, a pattern which suggests increased rates of cytosine deamination (C-->T transitions) on the H strand of mtDNA. Two nonexclusive hypotheses related to high-altitude life can explain such a mutational pattern: more severe oxidative stress (ROS) and higher metabolic rates. By comparing the relative rates of nonsynonymous and synonymous substitutions in protein-coding genes, we identified that Caprini have higher levels of adaptive variation in the ATPase complex. In addition, we detected several changes in mitochondrial genes that should be tested for their potential role in mountain adaptation.

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.

The Spectrum of Mitochondrial Mutation Differs across Species

Mutations are ubiquitous, and many arise during the very process of replicating and transmitting genomes. This process is the source of the genetic variation that provides the raw material for both evolutionary novelty and human disease. Mutation rates are known to vary among nucleotides, across genomic regions, and between taxa. It is conventional wisdom that animal mitochondrial DNA (mtDNA) is one genomic region that has a particularly high mutation rate. Until recently, this high rate of mutation has been predominantly inferred from high levels of mitochondrial sequence divergence between species. However, the apparently simple process of mutation and sequence divergence is intriguingly complex in mitochondria, due to the unique biology of these extrachromosomal genomes. Unlike nuclear DNA, where a new mutation arises on one of four possible DNA strands that can be passed to a diploid offspring, a new mtDNA mutation exists on one of many thousands of mtDNA strands that might (or might not) get incorporated into an egg. With a complex cellular pedigree of mtDNA molecules per mitochondrion, mitochondria per egg cell, egg cells per female, and an even more complex pedigree of females per population, it is a complicated path from mtDNA mutation to fixed mtDNA difference between species [1]. The basic biology of this problem was sketched out more than 30 years ago in a pioneering study of mtDNA sequence variation in sheep and goats by Upholt and Dawid [2]. They recognized the clonal nature of mtDNA inheritance, the random drift process acting on mutations within cytoplasms, and the likelihood that mutations may contribute to variation within species but not become fixed substitutions between species. In short order, mtDNA became a powerful tool of population and evolutionary biologists when it was realized that the rapid rate of mitochondrial mutation and evolution was useful for evolutionary inference [3,4]. In the mid-1980s, mtDNA mutations became candidates for human disease as several papers attributed a variety of disorders to specific point mutations and deletions in the mitochondrial genome [5–7]. In the ensuing years, mutation in the mitochondrial genome has been studied intensively by two different camps: evolutionary biologists, who assumed that mtDNA mutations had no significant functional effects and would serve as reliable neutral markers, and molecular and cell biologists, who saw mtDNA mutations as an underappreciated source of human pathologies. However, it is becoming increasingly popular to apply evolutionary models to problems in mitochondrial disease [8,9] and to examine molecular mechanisms of mutation among strains of model organisms that have been allowed to mutate and evolve in the lab. What we are learning after three decades of extensive study is that the spectrum of mitochondrial mutations varies widely across taxa, with important consequences for the mutation-selection balance maintaining nucleotide composition. However, a new flurry of papers quantifying mitochondrial mutation rates in mutation accumulation studies across model organisms is showing us just how much we still have to learn about mtDNA mutation, variation, and evolution.

Mutations at position 7445 in the precursor of mitochondrial tRNA Ser(UCN) gene in three maternal Chinese pedigrees with sensorineural hearing loss

We report here the clinical, genetic and molecular characterization of three Chinese pedigrees with nonsyndromic bilateral hearing loss. Clinical and genetic evaluations revealed the variable severity and age-of-onset in hearing impairment in these families. Strikingly, there were extremely low penetrances of hearing impairment in these Chinese families. Sequence analysis of the complete mitochondrial DNA (mtDNA) showed the known A7445C mutation in two pedigrees and the novel A7445T mutation in another pedigree, in addition to distinct sets of mtDNA polymorphisms belong to Asian haplogroups D4j and F4. Indeed, the A7445C or A7445T mutation in the CO1 and the precursor of tRNA Ser(UCN) genes was present in homoplasmy only in the maternal lineage of those pedigrees but not other members of these families and 164 Chinese controls. In fact, the A7445C or A7445T mutation results in a read-through of the stop condon AGA of the CO1 message on the H strand of mtDNA, thereby adding three amino acids (Ser-Gln-Lys) to the C-terminal of the polypeptide. However, the mutated polypeptide may retain a partial function. Alternatively, the A7445C or A7445T mutation is adjacent to the site of 3′ end endonucleolytic processing of L-strand RNA precursor, spanning tRNA Ser(UCN) and ND6 mRNA. Thus, the A7445C or A7445T mutation may also cause a defect in the processing of the L-strand RNA precursor, thus causing mitochondrial dysfunctions. Furthermore, the occurrence of the mutations at position 7445 in these genetically unrelated subjects affected by hearing impairment strongly indicates that mutations at the position 7445 are involved in the pathogenesis of hearing impairment.

MTERF3 Is a Negative Regulator of Mammalian mtDNA Transcription

Regulation of mammalian mtDNA gene expression is critical for altering oxidative phosphorylation capacity in response to physiological demands and disease processes. The basal machinery for initiation of mtDNA transcription has been molecularly defined, but the mechanisms regulating its activity are poorly understood. In this study, we show that MTERF3 is a negative regulator of mtDNA transcription initiation. The MTERF3 gene is essential because homozygous knockout mouse embryos die in midgestation. Tissue-specific inactivation of MTERF3 in the heart causes aberrant mtDNA transcription and severe respiratory chain deficiency. MTERF3 binds the mtDNA promoter region and depletion of MTERF3 increases transcription initiation on both mtDNA strands. This increased transcription initiation leads to decreased expression of critical promoter-distal tRNA genes, which is possibly explained by transcriptional collision on the circular mtDNA molecule. To our knowledge, MTERF3 is the first example of a mitochondrial protein that acts as a specific repressor of mammalian mtDNA transcription initiation in vivo.