mtDNA Genotypes Research Articles

Genome Analysis of the Domestic Dog (Korean Jindo) by Massively Parallel Sequencing

Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the TCOF1 locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.

Survival and mitochondrial function in septic patients according to mitochondrial DNA haplogroup

IntroductionWe recently found that platelet cytochrome c oxidase (COX) activities and quantities in 6-month-survival septic patients are significantly higher than those of patients who died before 6 months. Other studies suggested that the mitochondrial DNA (mtDNA) genotype could play a major role in sepsis survival. Given that COX catalytic subunits are encoded by mtDNA, the objective of the present study was to explore whether mtDNA population genetic variation could affect COX activity and quantity and favors sepsis survival.MethodsA prospective, multicenter, observational study was carried out in six Spanish ICUs. We included 96 patients with severe sepsis. We determined the mtDNA haplogroup, the COX specific activity/citrate synthase specific activity (COXa/CSa) ratio and the COX quantity/citrate synthase specific activity (COXq/CSa) ratio in circulating platelets at the time of diagnosis, day 4 and day 8. We used survival at 1 and 6 months as endpoints.ResultsPatients with the JT mtDNA haplogroup (n = 15) showed higher COXq/CSa ratio at day 4 (P = 0.04) and day 8 (P = 0.02) than those with other haplogroups (n = 81). Logistic regression analysis showed that the JT mtDNA haplogroup (odds ratio = 0.18; 95% confidence interval = 0.04 to 0.94; P = 0.04) and COXq/CSa ratio (odds ratio = 0.53; 95% confidence interval = 0.30 to 0.93; P = 0.03) were associated with 1-month survival after controlling for age and lactic acid levels.ConclusionsThe novel findings of our study are that 1-month surviving septic patients showed higher COXq/CSa ratio than nonsurviving individuals, that patients from the JT mtDNA haplogroup showed a higher COXq/CSa ratio and that JT patients had a higher 1-month survival than patients from other mtDNA haplogroups.

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ 0 cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.

The effects of mitochondrial genotype on hypoxic survival and gene expression in a hybrid population of the killifish, Fundulus heteroclitus

The physiological link between oxygen availability and mitochondrial function is well established. However, whether or not fitness variation is associated with mitochondrial genotypes in the field remains a contested topic in evolutionary biology. In this study, we draw on a population of the teleost fish, Fundulus heteroclitus, where functionally distinct subspecies hybridize, likely as a result of past glacial events. We had two specific aims: (i) to determine the effect of mtDNA genotype on survivorship of male and female fish under hypoxic stress and (ii) to determine the effect of hypoxic stress, sex and mtDNA genotype on gene expression. We found an unexpected and highly significant effect of sex on survivorship under hypoxic conditions, but no significant effect of mtDNA genotype. Gene expression analyses revealed hundreds of transcripts differentially regulated by sex and hypoxia. Mitochondrial transcripts and other predicted pathways were among those influenced by hypoxic stress, and a transcript corresponding to the mtDNA control region was the most highly suppressed transcript under the conditions of hypoxia. An RT-PCR experiment on the control region was consistent with microarray results. Effects of mtDNA sequence variation on genome expression were limited; however, a potentially important epistasis between mtDNA sequence and expression of a nuclear-encoded mitochondrial translation protein was discovered. Overall, these results confirm that mitochondrial regulation is a major component of hypoxia tolerance and further suggest that purifying selection has been the predominant selective force on mitochondrial genomes in these two subspecies.

Amplified fragment length polymorphism confirms reciprocal monophyly in Chrysomya putoria and Chrysomya chloropyga: a correction of reported shared mtDNA haplotypes

Reinvestigation of mitochondrial haplotypes previously reported to be shared between the Afrotropical blowflies Chrysomya putoria Weidemann and Chrysomya chloropyga Weidemann (Diptera: Calliphoridae) revealed an error resulting from the misidentification of specimens. Preliminary amplified fragment length polymorphism (AFLP) analysis of the original and additional individuals again failed to find reciprocal monophyly, leading to a re-examination of the specimens for diagnostic male genitalic characters that were first described following the earlier study. Four of the original study specimens were found to have been misidentified, and definitive analysis of both mtDNA and AFLP genotypes using phylogenetic analysis and genetic assignment showed that each species was indeed reciprocally monophyletic. In addition to correcting the earlier error, this study illustrates how AFLP analysis can be used for efficient and effective specimen identification through both phylogenetic analysis and genetic assignment, and suggests that the latter method has special advantages for identification when no conspecific specimens are represented in the reference database.

‘Progress’ renders detrimental an ancient mitochondrial DNA genetic variant

A human mitochondrial DNA (mtDNA) transition, m.1555A>G, in the 12S rRNA gene causes non-syndromic hearing loss. However, this pathological mutation is the wild-type allele in orangutan mtDNA. Here we rule out different genetic factors as the reason for its fixation in orangutans and show that aminoglycosides negatively affect the oxidative phosphorylation function by decreasing the synthesis of mtDNA-encoded proteins and the amount and activity of respiratory complex IV. These drugs also diminish the growth rate of orangutan cells. The m.1555G nucleotide is also the wild-type allele in other mammal species and they might be at risk of suffering a mitochondrial disorder if treated with aminoglycosides. Therefore, pharmacogenomic approaches should be used to confirm this possibility. These observations are important for human health. Due to the fact that old age and high frequency are criteria widely used in mitochondrial medicine to rule out a genetic change as being a pathological mutation, our results prevent against simplistic genetic approaches that do not consider the potential effect of environmental conditions. Hence, these results suggest that some ancient and highly frequent human population polymorphisms, such as those defining mtDNA haplogroups, in mitochondrial rRNA genes can be deleterious in association with new environmental conditions. Therefore, as the discovery of ribosomal antibiotics has allowed to fight infectious diseases and this breakthrough can be considered an important scientific advance or 'progress', our results suggest that 'progress' can also have a negative counterpart and render detrimental many of these mtDNA genotypes.

Over-expression of Tfam improves the mitochondrial disease phenotypes in a mouse model system

The phenotypes of mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) have been proposed to be strictly regulated by the proportion of wild-type and pathogenically mutated mtDNAs. More specifically, it is thought that the onset of the disease phenotype occurs when cells cannot maintain the proper mitochondrial function because of an over-abundance of pathological mtDNA. Therapies that cause a decrease in the pathogenic mtDNA population have been proposed as a treatment for mitochondrial diseases, but these therapies are difficult to apply in practice. In this report, we present a novel concept: to improve mitochondrial disease phenotypes via an increase in the absolute copy number of the wild-type mtDNA population in pathogenic cells even when the relative proportion of mtDNA genotypes remains unchanged. We have succeeded in ameliorating the typical symptoms of mitochondrial disease in a model mouse line by the over-expression of the mitochondrial transcription factor A ( Tfam) followed by an increase of the mtDNA copy number. This new concept should lead to the development of a novel therapeutic treatment for mitochondrial diseases.

Paternal Leakage and Heteroplasmy of Mitochondrial Genomes in Silene vulgaris: Evidence From Experimental Crosses

The inheritance of mitochondrial genetic (mtDNA) markers in the gynodioecious plant Silene vulgaris was studied using a series of controlled crosses between parents of known mtDNA genotype followed by quantitative PCR assays of offspring genotype. Overall, approximately 2.5% of offspring derived from crosses between individuals that were homoplasmic for different mtDNA marker genotypes showed evidence of paternal leakage. When the source population of the pollen donor was considered, however, population-specific rates of leakage varied significantly around this value, ranging from 10.3% to zero. When leakage did occur, the paternal contribution ranged from 0.5% in some offspring (i.e., biparental inheritance resulting in a low level of heteroplasmy) to 100% in others. Crosses between mothers known to be heteroplasmic for one of the markers and homoplasmic fathers showed that once heteroplasmy enters a maternal lineage it is retained by approximately 17% of offspring in the next generation, but lost from the others. The results are discussed with regard to previous studies of heteroplasmy in open-pollinated natural populations of S. vulgaris and with regard to the potential impact of mitochondrial paternal leakage and heteroplasmy on both the evolution of the mitochondrial genome and the evolution of gynodioecy.

Generation of mtDNA Homoplasmic Cloned Lambs

Generally in mammals, individual animals contain only maternally inherited mitochondrial DNA (mtDNA), as paternal (sperm)-derived mitochondria are usually eliminated during early development. Somatic cell nuclear transfer (SCNT) bypasses the normal routes of mtDNA inheritance and introduces not only a different nuclear genome into the recipient cytoplast (in general an enucleated oocyte) but also somatic mitochondria. Differences in mtDNA genotype between recipient oocytes and potential mtDNA heteroplasmy due to persistence and replication of somatic mtDNA means that offspring generated by SCNT are not true clones. However, more importantly, the consequences of the presence of somatic mtDNA, mtDNA heteroplasmy, or possible incompatibility between nuclear and mtDNA genotypes on subsequent development and function of the embryo, fetus and offspring are unknown. Following sexual reproduction, mitochondrial function requires the biparental control of maternally inherited mtDNA, whereas following SCNT incompatibility between the recipient cell mitochondrial and transplanted nuclear genomes, or mtDNA heteroplasmy, may result in energy imbalance and initiate the onset of mtDNA-type disease, or disruption of normal developmental events. To remove the potentially adverse effects of somatic mtDNA following SCNT we have previously produced embryos using donor cells depleted to residual levels of mtDNA (mtDNA). We now report that these cells support development to term and produced live lambs in which no donor somatic mtDNA was detected, the lambs being homoplasmic for recipient oocyte DNA.

Erratum: Drawing the history of the Hutterite population on a genetic landscape: inference from Y-chromosome and mtDNA genotypes

Erratum: Drawing the history of the Hutterite population on a genetic landscape: inference from Y-chromosome and mtDNA genotypes

ECOMPAGT integrates mtDNA: import, validation and export of mitochondrial DNA profiles for population genetics, tumour dynamics and genotype-phenotype association studies

BackgroundMitochondrial DNA (mtDNA) is widely being used for population genetics, forensic DNA fingerprinting and clinical disease association studies. The recent past has uncovered severe problems with mtDNA genotyping, not only due to the genotyping method itself, but mainly to the post-lab transcription, storage and report of mtDNA genotypes.DescriptioneCOMPAGT, a system to store, administer and connect phenotype data to all kinds of genotype data is now enhanced by the possibility of storing mtDNA profiles and allowing their validation, linking to phenotypes and export as numerous formats. mtDNA profiles can be imported from different sequence evaluation programs, compared between evaluations and their haplogroup affiliations stored. Furthermore, eCOMPAGT has been improved in its sophisticated transparency (support of MySQL and Oracle), security aspects (by using database technology) and the option to import, manage and store genotypes derived from various genotyping methods (SNPlex, TaqMan, and STRs). It is a software solution designed for project management, laboratory work and the evaluation process all-in-one.ConclusionsThe extended mtDNA version of eCOMPAGT was designed to enable error-free post-laboratory data handling of human mtDNA profiles. This software is suited for small to medium-sized human genetic, forensic and clinical genetic laboratories. The direct support of MySQL and the improved database security options render eCOMPAGT a powerful tool to build an automated workflow architecture for several genotyping methods. eCOMPAGT is freely available at http://dbis-informatik.uibk.ac.at/ecompagt.

Mitochondrial inheritance in haploid × non-haploid crosses in Cryptococcus neoformans

In the basidiomycetous yeast Cryptococcus neoformans, fusants and meiotic progeny from haploid-haploid (HH) crosses between strains of mating type a (MAT a) and mating type alpha (MATalpha) typically inherit mitochondrial DNA (mtDNA) from the MAT a parent. In this study, we investigated the mtDNA inheritance pattern in haploid x non-haploid crosses. A total of 420 meiotic progeny and 173 fusants were obtained from five crosses and analyzed for two polymorphic mitochondrial markers. The percentage of meiotic progeny and fusants inheriting mtDNA from MATalpha or MATalpha/alpha parents ranged from 8 to 50%. The leakage was significantly greater than those observed in HH crosses, indicating that mtDNA inheritance is not uniparental in haploid x non-haploid crosses in C. neoformans. In addition, mtDNA leakage in the fusants, but not the meiotic progeny, of the MATalpha/alpha x MAT a cross was significantly higher than that in the MAT a/a x MATalpha cross, suggesting that the diploid parents with different mating types contribute differently in determining fusant mtDNA genotype in these crosses. Flow cytometry analysis revealed that meiotic progeny population of each cross was of mixed ploidy while the ploidy level of the selected fusants ranged from diploid to triploid.

56 EFFECTS OF PHYLOGENIC GENERA OF RECIPIENT CYTOPLASTS ON DEVELOPMENT AND VIABILITY OF CANADA LYNX (LYNX CANADENSIS) CLONED EMBRYOS

Canada lynx (Lynx canadensis; CL) once occupied 16 states in the Unites States of America, but small populations remain in only 3 states. Interspecies-somatic cell nuclear transfer (Is-SCNT) offers the possibility of preventing their extinction; however, developmental constraints on Is-SCNT embryos are proportional to the phylogenetic distance between the donor cell and the recipient oocyte. Mitochondrial DNA (mtDNA) heteroplasmy may be involved in nuclear-cytoplasmic incompatibilities, thus inhibiting development of cloned embryos at the time of genomic activation. Minimizing the phylogenetic distance between the donor cell and recipient oocyte may enhance development of clone embryos. Caracal (Caracal caracal) may be suitable as an oocyte donor for SCNT and a recipient of CL cloned embryos because caracals hybridize with other felid species and share physical characteristics with the lynx family, marked by being previously classified in the lynx genera and having similar gestational length. To ensure compatibilities between the donor nuclei of the CL and the mitochondria of recipient oocytes, we (1) compared in vitro development of CL cloned embryos reconstructed with domestic cat (Felis catus; DSH) or caracal cytoplasts, (2) examined the mtDNA genotypes in CL cloned embryos, and (3) evaluated in vivo developmental competence of CL cloned embryos after transfer into caracal recipients. A total of 160 and 217 preovulatory oocytes were collected by laparoscopy from gonadotropin-treated caracals (n = 8) and DSH (n = 10) and used as recipient cytoplasts for reconstructing CL embryos. Results indicated that the phylogenetic genera of recipient cytoplasts did not affect embryo cleavage at Day 2 (caracal 50/55, 91% v. DSH 63/65, 97%), but development of CL cloned embryos to the blastocyst stage was higher when caracal oocytes were used as recipient cytoplasts (15/50; 30%) than with DSH cytoplasts (9/63, 14%; P < 0.05). The extent of mtDNA homoplasmy or heteroplasmy in CL cloned embryos was calculated by the number of single nucleotide polymorphisms (SNP) derived from the DSH or caracal oocyte donors and from the somatic cell donor CL. DNA was isolated from 25 and 35 CL cloned embryos reconstructed with caracal or DSH cytoplasts, respectively. All amplified products after PCR were sequenced and SNP analyzed. All CL embryos reconstructed with DSH cytoplasts were homoplasmic, carrying mtDNA only from the DSH oocyte donor (n = 35; SNP DSH = 2-6). Embryos reconstructed with caracal cytoplasts were homoplasmic for CL mtDNA (n = 9; SNPCL = 10-12) or heteroplasmic (caracal × CL, n = 17; SNPCL = 7-9; SNP caracal = 2-3). A total of 69 (mean = 34.5 ± 4.9 per caracal) and 70 (mean = 35.0 ± 9.8 per caracal) CL cloned embryos reconstructed with caracal and DSH cytoplasts, respectively, were transferred into 4 caracal recipients; however, no pregnancies were established. In summary, Is-SCNT between 2 phylogenetically closer species favors retention of the donor’s mitochondria, which might lead to a better nucleo-cytoplasmic interaction for reprogramming of donor nucleus.

Drawing the history of the Hutterite population on a genetic landscape: inference from Y-chromosome and mtDNA genotypes

Although the North American Hutterites trace their origins to South Tyrol, no attempts have been made to examine the genetic migration history of the Hutterites before emigrating to the United States in the 1870s. To investigate this, we studied 9 microsatellite loci and 11 unique event polymorphism (UEP) markers on the Y-chromosome, the hypervariable region I (HVRI) of the mitochondrial DNA (mtDNA), as well as the complete mtDNA genome of Hutterite and South Tyrolean samples. Only 6 out of 14 Y-chromosome UEP+microsatellite haplotypes and 3 out of 11 mitochondrial haplotypes that were present in the Hutterites were also present in the South Tyrolean population. The phylogenetic relationships inferred from Y-chromosome and mtDNA databases show that the Hutterites have a unique genetic background related to a similar extent to central and eastern European populations. An admixture analysis indicates, however, a relatively high genetic contribution of central European populations to the Hutterite gene pool. These results are consistent with historical records on Hutterite migrations and demographic history. In addition, our data reveal similar numbers of Y and mitochondrial haplotypes in Hutterite male and female founders, respectively. The Hutterite male and female gene pools are similar with respect to genetic diversity and genetic distance measures and comparable with respect to their origins, suggesting a similar evolutionary history.

Mitochondrial DNA and gastrointestinal motor and sensory functions in health and functional gastrointestinal disorders

Nerve, muscle, and inflammatory cells involved in gastrointestinal (GI) function have high-energy requirements and are affected in mitochondrial disorders. Familial aggregation of irritable bowel syndrome (IBS) frequently involves mothers and their children. Since mitochondrial DNA (mtDNA) is maternally inherited, mtDNA single nucleotide polymorphisms (SNPs) could confer risk to the development of IBS. The mtDNA SNPs, 16519C>T and 3010G>A, are associated with migraine and childhood cyclic vomiting syndrome. Our hypothesis is that these mtDNA SNPs are associated with functional GI disorders (FGIDs) and GI functions. The mt genome was first tested for the 7028C polymorphism (defining haplogroup H) in 699 patients or controls, and those with 7028C were further genotyped at 16519 and 3010. Phenotypes were based on symptoms (validated questionnaires and criteria) and GI physiology using validated motor and sensory studies. Constipation-predominant IBS and alternating constipation and diarrhea IBS are less prevalent in individuals with the 7028C mtDNA polymorphism than in individuals with 7028T. Conversely, 7028C is associated with higher maximum tolerated volume (lower satiation) compared with 7028T. Among those with 7028C, nonspecific abdominal pain (chronic abdominal pain or dyspepsia) was significantly associated with 3010A compared with 3010G (odds ratio 3.3, P=0.02), and slower gastric emptying was statistically associated with 3010G. There were no significant associations of mtDNA genotypes tested and stomach volumes, small bowel or colonic transit, rectal compliance, and motor or sensory functions. Thus variation in mtDNA may be associated with satiation, gastric emptying, and possibly pain; further studies of mtDNA in appetite regulation and larger numbers of patients with FGIDs are warranted.

Characterization of mitochondrial genotypes in the foundation herd of the Canchim beef cattle breed

The Canchim (5/8 Charolais + 3/8 Zebu) beef cattle breed was developed at Southeast-Embrapa Cattle to take advantage of hybrid vigor and to combine the higher growth rate and beef quality of Charolais with tropical adaptations of Zebu. The development of three lineages (old, new, and crossbred) has increased its genetic basis. The genotypic origin (Bos taurus or Bos indicus) of the mitochondrial DNA (mtDNA) of the Canchim breed was unknown. We characterized the mtDNA genotype of this founder herd by allele-specific polymerase chain reaction. The 173 founder Zebu females (62 Indubrasil, 3 Guzerat, and 108 Nellore) and their 6749 offspring were identified. The frequency of B. indicus mtDNA ranged from 1.15 to 2.05% among the descendants (n= 6404) of each maternal line with available DNA, and among animals that were alive (n= 689) in December 2007 among the three lineages. Though mtDNA characterization can be used to direct animal selection, the low frequency of B. indicus mtDNA impairs the evaluation of its effects on production traits in these animals. The high prevalence of B. taurus mtDNA in Canchim proves that the founder Zebu females from the Indubrasil, Guzerat and Nellore breeds were obtained from crosses of Zebu sires with local B. taurus dams.

Clonal expansion of different mtDNA variants without selective advantage in solid tumors

In search of tumor-specific mitochondrial DNA (mtDNA) mutations in head and neck squamous cell cancer, we found heteroplasmy in the blood of two individuals, i.e., these individuals carried two alleles of mtDNA. In both cases, the tumor was found to be homoplasmic, i.e., it contained only one of the two mtDNA alleles present in blood. More interestingly, in one case the tumor had acquired the wild-type allele, while in the other case it contained the mutant allele only. Sequencing of the whole 16.5 kb mtDNA showed that the observed heteroplasmic positions in the D-loop region, nucleotides 152 and 16187, respectively, were the only differences between tumor and blood mtDNA genotypes in these individuals. Our findings thus strongly support the hypothesis that accumulation of mtDNA mutations in solid tumors occurs by clonal and random expansion of pre-existing alleles and is not necessary for the metabolic changes generally associated with tumor formation, the Warburg effect.

Genotype-dependent variation of mitochondrial transcriptional profiles in interpopulation hybrids

Hybridization between populations can disrupt gene expression, frequently resulting in deleterious hybrid phenotypes. Reduced fitness in interpopulation hybrids of the marine copepod Tigriopus californicus has been traced to interactions between the nuclear and mitochondrial genomes. Here, we determine transcript levels of four to six genes involved in the mitochondrial oxidative phosphorylation pathway for a series of parental and inbred hybrid lines using RT-qPCR. Both nuclear and mitochondrial-encoded genes are included in the analysis. Although all genes studied are up-regulated under salinity stress, only expression of genes located on the mtDNA differed among lines. Because mitochondrial genes are transcribed by a dedicated RNA polymerase encoded in the nuclear genome, we compare transcript levels among hybrid lines with different combinations of mitochondrial RNA polymerase and mtDNA genotypes. Lines bearing certain mtDNA-mitochondrial RNA polymerase genotypic combinations show a diminished capacity to up-regulate mitochondrial genes in response to hypoosmotic stress. Effects on the transcriptional profile depend on the specific interpopulation cross and are correlated with viability effects. We hypothesize that disruption of the mitochondrial transcriptional system in F(2) hybrids may play a central role in hybrid breakdown.

A Thirty Million Year-Old Inherited Heteroplasmy

Due to essentially maternal inheritance and a bottleneck effect during early oogenesis, newly arising mitochondrial DNA (mtDNA) mutations segregate rapidly in metazoan female germlines. Consequently, heteroplasmy (i.e. the mixture of mtDNA genotypes within an organism) is generally resolved to homoplasmy within a few generations. Here, we report an exceptional transpecific heteroplasmy (predicting an alanine/valine alloacceptor tRNA change) that has been stably inherited in oniscid crustaceans for at least thirty million years. Our results suggest that this heteroplasmy is stably transmitted across generations because it occurs within mitochondria and therefore escapes the mtDNA bottleneck that usually erases heteroplasmy. Consistently, at least two oniscid species possess an atypical trimeric mitochondrial genome, which provides an adequate substrate for the emergence of a constitutive intra-mitochondrial heteroplasmy. Persistence of a mitochondrial polymorphism on such a deep evolutionary timescale suggests that balancing selection may be shaping mitochondrial sequence evolution in oniscid crustaceans.

The Layered Structure of Human Mitochondrial DNA Nucleoids

Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.