Literatura académica sobre el tema "MtDNA segregation"

Abstract To investigate the interactions between mtDNA and nuclear genomes, we produced heteroplasmic maternal lineages by transferring the cytoplasts between the embryos of two mouse strains, C57BL/6 (B6) and RR. A total of 43 different nucleotides exist in the displacement-loop (D-loop) region of mtDNA between B6 and RR. Heteroplasmic embryos were reconstructed by electrofusion using a blastomere from a two-cell stage embryo of one strain and an enucleated blastomere from a two-cell stage embryo of the other strain. Equivalent volumes of both types of mtDNAs were detected in blastocyst stage embryos. However, the mtDNA from the RR strain became biased in the progeny, regardless of the source of the nuclear genome. The RR mtDNA population was very high in most of the tissues examined but was relatively low in the brain and the heart. An age-related increase of RR mtDNA was also observed in the blood. The RR mtDNAs in the reconstructed embryos and in the embryos collected from heteroplasmic mice showed a different segregation pattern during early embryonic development. These results suggest that the RR mtDNA has a replicative advantage over B6 mtDNA during embryonic development and differentiation, regardless of the type of nuclear genome.

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type–specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues.

Abstract Mitochondrial DNA (mtDNA) encodes cellular machinery vital for cell and organism survival. Mutations, genetic manipulation, and gene therapies may produce cells where different types of mtDNA coexist in admixed populations. In these admixtures, one mtDNA type is often observed to proliferate over another, with different types dominating in different tissues. This ‘segregation bias’ is a long-standing biological mystery that may pose challenges to modern mtDNA disease therapies, leading to substantial recent attention in biological and medical circles. Here, we show how an mtDNA sequence’s balance between replication and transcription, corresponding to molecular ‘selfishness’, in conjunction with cellular selection, can potentially modulate segregation bias. We combine a new replication-transcription-selection (RTS) model with a meta-analysis of existing data to show that this simple theory predicts complex tissue-specific patterns of segregation in mouse experiments, and reversion in human stem cells. We propose the stability of G-quadruplexes in the mtDNA control region, influencing the balance between transcription and replication primer formation, as a potential molecular mechanism governing this balance. Linking mtDNA sequence features, through this molecular mechanism, to cellular population dynamics, we use sequence data to obtain and verify the sequence-specific predictions from this hypothesis on segregation behaviour in mouse and human mtDNA.

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for ~60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from ~20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.Key words: mtDNA deletion, Caenorhabditis elegans, heteroplasmy, inheritance, mtDNA copy number.

Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)–induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H2O2), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation correlated with mtDNA-concatemer production and enabled transmission of multiple identical mt-genome copies as a single unit. Our results support a mechanism by which mt-allele segregation toward mt-homoplasmy is mediated by concatemers.

Abstract Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (ρ+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous Δabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf 2p is known to be required for the maintenance of mtDNA in ρ+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in ρ− cells grown on the same medium. The content of both ρ+ and ρ− mtDNAs is increased in cells by 50–150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf 2p by ≥10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of ρ+ mtDNA and a quantitative conversion of ρ+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in ρ− cells also leads to a loss of mtDNA, but at a slower rate than was observed for ρ+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf 2p because a mutant Abf 2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements Δabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf 2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.

Green fluorescent protein (GFP) was used to tag proteins of the mitochondrial matrix, inner, and outer membranes to examine their sorting patterns relative to mtDNA in zygotes of synchronously mated yeast cells in ρ+ × ρ0 crosses. When transiently expressed in one of the haploid parents, each of the marker proteins distributes throughout the fused mitochondrial reticulum of the zygote before equilibration of mtDNA, although the membrane markers equilibrate slower than the matrix marker. A GFP-tagged form of Abf2p, a mtDNA binding protein required for faithful transmission of ρ+ mtDNA in vegetatively growing cells, colocalizes with mtDNA in situ. In zygotes of a ρ+ × ρ+ cross, in which there is little mixing of parental mtDNAs, Abf2p–GFP prelabeled in one parent rapidly equilibrates to most or all of the mtDNA, showing that the mtDNA compartment is accessible to exchange of proteins. In ρ+ × ρ0 crosses, mtDNA is preferentially transmitted to the medial diploid bud, whereas mitochondrial GFP marker proteins distribute throughout the zygote and the bud. In zygotes lacking Abf2p, mtDNA sorting is delayed and preferential sorting is reduced. These findings argue for the existence of a segregation apparatus that directs mtDNA to the emerging bud.

Mitochondrial genotypes have been shown to segregate both rapidly and slowly when transmitted to consecutive generations in mammals. Our objective was to develop an animal model to analyze the patterns of mammalian mitochondrial DNA (mtDNA) segregation and transmission in an intraspecific heteroplasmic maternal lineage to investigate the mechanisms controlling these phenomena. Heteroplasmic progeny were obtained from reconstructed blastocysts derived by transplantation of pronuclear-stage karyoplasts to enucleated zygotes with different mtDNA. Although the reconstructed zygotes contained on average 19% mtDNA of karyoplast origin, most progeny contained fewer mtDNA of karyoplast origin and produced exclusively homoplasmic first generation progeny. However, one founder heteroplasmic adult female had elevated tissue heteroplasmy levels, varying from 6% (lung) to 69% (heart), indicating that stringent replicative segregation had occurred during mitotic divisions. First generation progeny from the above female were all heteroplasmic, indicating that, despite a meiotic segregation, they were derived from heteroplasmic founder oocytes. Some second and third generation progeny contained exclusively New Zealand Black/BINJ mtDNA, suggesting, but not confirming, an origin from an homoplasmic oocyte. Moreover, several third to fifth generation individuals maintained mtDNA from both mouse strains, indicating a slow or persistent segregation pattern characterized by diminished tissue and litter variability beyond second generation progeny. Therefore, although some initial lineages appear to segregate rapidly to homoplasmy, within two generations other lineages transmit stable amounts of both mtDNA molecules, supporting a mechanism where mitochondria of different origin may fuse, leading to persistent intraorganellar heteroplasmy.

Abstract Despite the widespread use of mitochondrial DNA by evolutionary geneticists, relatively little effort has been spent assessing the magnitude of forces maintaining mtDNA sequence diversity. In this study the influence of cytoplasmic variation on viability in Drosophila was examined by analysis of second chromosome segregation. A factorial experiment with balancer chromosomes permitted the effects of cytoplasma and reciprocal crosses to be individually distinguished. The first test used six lines of diverse geographic origin, testing the segregation of all six second chromosomes in all six cytoplasms. The second and third tests were also factorial designs, but used flies from one population in central Pennsylvania. The fourth test was a large chain cross, using 28 lines from the same Pennsylvania population. Only the first test detected a significant nuclear-cytoplasmic effect. Restriction site variation in the mtDNA of all of these lines was assayed by Southern blotting, and statistical tests were performed in an effort to detect an influence of mtDNA type on fitness components. Posterior linear contrasts revealed an effect of mtDNA on segregation only among lines of diverse geographic origin. Within a population, no such influence was detected, even though the experiment was sufficiently large to have revealed statistical significance of a 0.5% segregation difference with a 57% probability.

Our fascination for mitochondria relates to their origin as symbiotic, semi-independent organisms on which we, as eukaryotic beings, rely nearly exclusively to produce energy for every cell function. Therefore, it is not surprising that these organelles play an essential role in many events during early development and in artificial reproductive technologies (ARTs) applied to humans and domestic animals. However, much needs to be learned about the interactions between the nucleus and the mitochondrial genome (mtDNA), particularly with respect to the control of transcription, replication and segregation during preimplantation. Nuclear-encoded factors that control transcription and replication are expressed during preimplantation development in mice and are followed by mtDNA transcription, but these result in no change in mtDNA copy number. However, in cattle, mtDNA copy number increases during blastocyst expansion and hatching. Nuclear genes influence the mtDNA segregation patterns in heteroplasmic animals. Because many ARTs markedly modify the mtDNA content in embryos, it is essential that their application is preceded by careful experimental scrutiny, using suitable animal models.

Mammalian mitochondrial DNA (mtDNA) is a maternally inherited, multi-copy, small circular genome approximately 16 kb in size that codes for 13 polypeptides of the mitochondrial respiratory chain. Typically, mtDNA is present as only one genotype in a cell, a state known as homoplasmy. In rare circumstances, two or more mtDNA sequence variants can be present in a cell, a state known as heteroplasmy, and these sequence variants can segregate during mitosis and meiosis. In this thesis, I have investigated the basis for a novel tissue-specific segregation of mtDNA in a heteroplasmic mouse model that had been previously constructed from two inbred strains (NZB/BinJ and BALB/c mtDNA). In these mice, four tissues showed directional selection for an mtDNA genotype: in the liver and kidney for the NZB mtDNA genotype; and in the spleen and blood for the BALB mtDNA genotype. I investigated the mechanism responsible for selection of NZB mtDNA, focusing on the liver which showed the strongest effect. In this tissue, selection for the NZB mtDNA genotype is constant with time, independent of allele frequency and does not appear to be mediated through an advantage of respiratory chain function or replication rate of mtDNA. To identify the genetic basis for this mtDNA selection, I set up an intersubspecific intercross and used quantitative trait loci (QTL) mapping to map three QTL in F2 mice that are strong gene effects in the liver, kidney, and spleen. These QTLs, Smdq-1 (liver), Smdq-2 (kidney), and Smdq-3 (kidney & spleen) (s&barbelow;egregation of m&barbelow;itochondrial D&barbelow;NA Q&barbelow;TL-#) map to chromosome 5, 2, and 6 respectively. Smdq-1 was a dominant QTL in the liver that mapped to a 2 LOD support interval of approximately 1 cM and accounted for 34% of the variation in the trait. To reduce the interval size of Smdq-1 and confirm the map position, BALB chromosome 5 interval-specific congenic mice lines are being generated across a 20 cM interval. This is the fir

Pendant longtemps, il n'était pas possible de manipuler le génome mitochondrial des plantes (mtDNA), jusqu'aux récentes avancées en édition des génomes utilisant des "Transcription Activator-Like Effector Nucleases" (TALEN). Dans ce travail, j'ai utilisé des TALENs spécifiquement ciblés sur les mitochondries (mitoTALENs) pour étudier la réparation et la ségrégation du mtDNA des plantes. Les constructions de mitoTALEN ont été introduites dans 10 lignées mutantes différentes d'Arabidopsis thaliana, déficientes en divers facteurs impliqués dans la réparation mitochondriale des plantes par recombinaison homologue. Les lignées résultantes ont eté analysées par séquençage Illumina et par des approches de qPCR. Chez les plantes de type sauvage, les cassures double brin (DSB) de l'ADN mitochondrial induites par les mitoTALENs ont été réparées par recombinaison homologue, entraînant le remplacement de la région contenant la DSB par une séquence distale, non-affectée, du mtDNA, flanquée par les mêmes séquences répétées. Chez les mutants déficients en facteurs de réparation, la réparation pourrait se dé placer vers des voies alternatives, telles que le "Single-Strand Annealing" (SSA) et "Microhomology-mediated recombination" (MHMR). De plus, chez certains mutants, les données n'ont révélé aucune trace de réparation des DSB, mais ont plutôt suggéré que les plantes déficientes en facteurs de réparation essentiels pourraient survivre en reconstituant un génome mitochondrial alternatif viable, à partir de sous- génomes pré existants se répliquant de manière autonome
For long, the plant mitochondrial genome (mtDNA) was not amenable to manipulation, until recent advancements in genome engineering using Transcription Activator-Like Effector Nucleases (TALEN). In this work I used TALENs specifically targeted to mitochondria (mitoTALENs) to study plant mtDNA repair and segregation. MitoTALEN constructs were transformed into the background of 10 different Arabidopsis thaliana mutant lines, deficient in various factors involved in plant mitochondrial repair by homologous recombination. The resulting lines were analysed by Illumina sequencing and qPCR approaches. In wild type plants, the mtDNA double-strand-break (DSB) induced by MitoTALENs was repaired by homologous recombination, resulting in the replacement of the region containing the DSB by a distal unaffected sequence of the mtDNA, flanked by the same set of repeated sequences. In mutants deficient in repair factors, repair could shift to alternative pathways, such as Single-Strand Annealing (SSA) and Microhomology-mediated recombination (MHMR). Furthermore, in some mutants, the data revealed no evidence of DSB repair, but rather suggested that plants deficient in key repair factors could survive by reconstituting an alternative viable mitochondrial genome, from pre-existing autonomously replicating sub-genomes

MtDNA mutations in mammalian cells are implicated in cellular ageing and encephalomyopathies, although mechanisms involved are not completely understood. The mitochondrial genetic bottleneck has puzzled biologists for a long time. Approximate models of genetic bottleneck proposed in the literature do not accurately model underlying biology. Recent studies indicate mitochondrial morphology changes during cellular aging in culture. In particular, the rates of mitochondrial fission and fusion are shown to be in tight balance, though this rate decreases with age. Some proteins involved in mitochondrial morphology maintenance are implicated in apoptosis. Hence, mitochondrial genetic and morphologic dynamics are critical to the life and death of cells. By working closely with experimental collaborators and by utilizing data derived from literature, we have developed stochastic simulation models of mitochondrial genetic and morphologic dynamics. Hypotheses from the mitochondrial genetic dynamics model include: (1) the decay of mtDNA heteroplasmy in blood is exponential and not linear as reported in literature. (2) Blood heteroplasmy measurements are a good proxy for the blood stem cell heteroplasmy. (3) By analyzing our simulation results in tandem with published longitudinal clinical data, we propose for the first time, a way to correct for the patient's age in the analysis of heteroplasmy data. (4) We develop a direct model of the genetic bottleneck process during mouse embryogenesis. (5) Partitioning of mtDNA into daughter cells during blastocyst formation and relaxed replication of mtDNA during the exponential growth phase of primordial germ cells leads to the variation in heteroplasmy inherited by offspring from the same mother. (6) We develop a “simulation control” for experimental studies on mtDNA heteroplasmy variation in cell cultures. Hypothesis from the mitochondrial morphologic dynamics model: (7) A cell adjusts the mitochondrial fusion rate to compensate for the fluctuations in the fission rate, but not vice versa. A deterministic model for this control is proposed. Contributions: extensible simulation models of mitochondrial genetic and morphologic dynamics to aide in the powerful analysis of published and new experimental data. Our results have direct relevance to cell biology and clinical diagnosis. The work also illustrates scientific success by tight integration of theory with practice.
Ph. D.

La nature a développé diverses stratégies afin d’assurer le commencement de la vie dans des conditions d’homoplasmie, c’est-à-dire des conditions telles que les cellules sont dotées du même ADN mitochondrial. Toutefois, des nouveaux haplotypes de l’acide désoxyribonucléique mitochondrial (ADNmt) peuvent apparaitre et croître de plusieurs façons tout au long de la durée d’une vie menant à l’hétéroplasmie. Par exemple, l’hétéroplasmie de l’ADNmt peut être créée artificiellement par des technologies reproductives assistées, ainsi que naturellement par le processus de vieillissement. De ce fait, la thèse de ce doctorat fut divisée en deux principaux objectifs. Le premier étant celui d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt produit par le transfert nucléaire des cellules somatiques (SCNT) lors du développement de l’embryon jusqu’au fœtus et aux tissus adultes de bovins clonés. En ce qui concerne le second objectif, il s’agit d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt causés par le vieillissement dans une cellule somatique adulte et dans des tissus germinaux durant l’ovogénèse, ainsi qu’au début de l’embryogenèse et dans la procédure de culture in vitro sur des souris. Dans la première série d’expériences sur des bovins, des fibroblastes fœtaux transportant une mutation d’ADNmt (insertion de 66 pb) furent fusionnés avec des ovocytes receveurs transportant l’ADNmt du type sauvage. La présence d’ADNmt venant de la cellule donneuse a été analysée à différents stades de développement, soit sur des embryons âgés de 17 jours (n=17), des fœtus âgés de 40 jours (n=3), des fœtus âgés de 60 jours (n=3), un fœtus âgé de 240 jours et 3 clones post-nataux âgés de 18 à 24 mois. Chaque individu s’est avéré être hétéroplasmique et 99 % (103/104) des échantillons de tissus analysés étaient également hétéroplasmiques. Cependant, l’ovaire venant du fœtus de 240 jours fut le seul à être homoplasmique pour l’ADNmt de l’ovocyte receveur. Dans la plupart des échantillons analysés (95,2 %, soit 99/104) la moyenne d’hétéroplasmie était de 1,46 %. Par contre, un fœtus âgé de 40 jours a présenté un niveau élevé d’hétéroplasmie (20,9 %), indiquant ainsi que des évènements rares d’augmentation de l’ADNmt des cellules donneuses peuvent survenir. Étant donné que la majorité des clones SCNT montrait de l’hétéroplasmie de l’ADNmt à des proportions comparables à celles des cellules donneuses au moment de la reconstruction de l’embryon, on a pu conclure que l’hétéroplasmie produite par des techniques de transfert nucléaire utilisant des cellules somatiques est due à une ségrégation neutre de l’ADNmt. Dans la seconde série d’expériences sur des souris, des femelles de différents âges, c.à.d. jeunes (0 – 8 mois), moyennes (8 – 16 mois) et vieilles (16 – 24 mois), ont été synchronisées (gonadotrophines) et sacrifiées dans le but d’obtenir des ovocytes au stade de vésicule germinal, et des ovocytes au stade métaphase-II produits in vivo et in vitro. De plus, des embryons in vivo et in vitro au stade de deux-cellules et des embryons au stade de blastocystes ont été obtenus de femelles jeunes. Différents tissus somatiques, venant de femelles des trois stades d’âge ont été obtenus : cerveau, foie, muscle et du cumulus ovocytaire. De plus, l’effet du vieillissement a été mesuré selon la fertilité de la femelle. En effet, les effets sur l’hétéroplasmie du vieillissement, du stade de développement et de la culture in vitro ont été mesurés dans des ovocytes et dans des embryons. Les effets du vieillissement sur les mitochondries ont été mesurés par rapport au nombre total de copies de l’ADNmt, au pourcentage des délétions communes et sur l’expression de trois gènes : Ndufs4, Mt-nd2 and Mt-nd4. Il a été possible d’observer que la fertilité des femelles dans la colonie de souris diminuait avec l’âge. En fait, le vieillissement affectait l’ADNmt dans les tissus somatiques, cependant il n’avait pas d’effet sur le cumulus, les ovocytes et les embryons. Le nombre de délétions de l’ADNmt augmentait pendant la reprise de la méiose et celui-ci diminuait au début du développement embryonnaire. La culture in vitro n’affectait pas la quantité d’ADNmt dans la plupart des tissus germinaux. Puisque nous n’avons pas trouvé d’effet de l’âge dans la majorité des paramètres mitochondriaux analysés dans les ovocytes et les embryons, il est suggéré que la délétion commune de l’ADNmt dans les tissus germinaux est davantage reliée au statut cellulaire de la production d’énergie qu’au processus de vieillissement. Deux sources différentes de mutations de l’ADNmt produites dans les ovocytes normaux ou reconstitués ont produit différents résultats d’hétéroplasmie au début de l’embryogénèse. Chez les bovins, l’hétéroplasmie artificielle impliquant une petite insertion (66 pb) dans la région non codante (D-loop) de l’ADNmt a été vraisemblablement non nocive pour l’embryon, tolérant la persistance de l’ADNmt étranger pendant les différents stades du développement des clones. Chez les souris, l’hétéroplasmie naturelle produite par une grande délétion (4974 pb délétion commune) dans la région codante de l’ADNmt a été vraisemblablement nocive pour l’embryon et par conséquent éliminée pour assurer l’homoplasmie au début du développement embryonnaire.
Nature has developed strategies to ensure the beginning of life in conditions of homoplasmy, i.e. cells harboring the same mitochondrial DNA (mtDNA). However, novel mtDNA haplotypes can arise by many means during life, leading to heteroplasmy. For instance, mtDNA heteroplasmy can originate artificially through assisted reproductive technologies and naturally by the process of aging. Therefore, this doctoral thesis was divided into two general objectives: Firstly, to analyze the changes in mtDNA heteroplasmy produced by somatic cell nuclear transfer (SCNT) during development from embryos, to fetuses and adult tissues, in cattle. Secondly, to analyze the changes in mtDNA heteroplasmy caused by aging in adult germinal and somatic tissues, during oogenesis and early embryogenesis, and in in vitro culture procedures in mice. In the first series of experiments in cattle, fetal fibroblasts carrying an mtDNA mutation (insertion of 66 bp) were fused to host oocytes carrying wild type mtDNA. The presence of mtDNA from the donor cell was analyzed in 30 SCNT clones at different stages of development: 17-day-old embryos (n=17); 40-day-old fetuses (n=3); 60-day-old fetuses (n=3); one 240 day-old fetus; and 3 post-natal clones (18-24 months). Every individual clone proved to be heteroplasmic and 99% (103/104) of the analyzed tissue samples were heteroplasmic as well. Only the ovary coming from a 240 day old fetus was homoplasmic for the mtDNA of the recipient oocyte. In most (95.2%) of the analyzed tissue samples (99/104) the mean of heteroplasmy was 1.46%. In contrast, one 40-day-old fetus presented high levels of heteroplasmy (20.9%) indicating rare events of donor mtDNA increases. Since most SCNT clones showed heteroplasmy at proportions comparable to the donor mtDNA at the moment of embryo reconstruction, we concluded that heteroplasmy produced by nuclear transfer techniques using somatic cells is due to the neutral segregation of the mtDNA. In the second series of experiments, performed in mice, females of different ages, i.e. young (0-8 months), middle (8-16 months) and old (16-24 months), were synchronized (gonadotropins) and sacrificed to obtain germinal vesicle oocytes, metaphase-II oocytes in vivo and in vitro. Also, 2-cell and blastocyst stage embryos were obtained from young females in vivo and in vitro. Somatic tissues from females of the three age periods were obtained: brain, granulosa, liver and muscle and the effect of aging was measured on fertility. The effects of aging, stage of development and in vitro culture on the heteroplasmy were measured in oocytes and embryos. Also, the effects of aging were measured in somatic and germinal tissues on total copies of mtDNA, percentage of mtDNA common deletion and the expression of three genes: Ndufs4, Mt-nd2 and Mt-nd4. We observed that female fertility in the mouse colony decreases with age. Aging affected mtDNA in somatic tissues but no effect was observed in granulosa, oocytes and embryos. MtDNA deletions increased during the resumption of meiosis and decreased during early embryo development; and culture in vitro did not affect the mtDNA in most germinal tissues. Because we did not find effects of age in most mitochondrial parameters analyzed in oocytes and embryos, we suggest that mtDNA common deletion in germinal tissues is more related with the cellular status of energy production than with the process of aging. Two different sources of mutations in the mtDNA generated in normal or reconstructed oocytes produced different heteroplasmy outcomes at the beginning of embryogenesis. In cattle, artificial heteroplasmy involving a small insertion (66 bp) in the non coding region (D-loop) of the mitochondrial DNA was apparently not harmful to the embryo, allowing persistence of the foreign mtDNA during the different stages of clonal development. In mice, the natural heteroplasmy of a large deletion (4974 bp, common deletion) in the coding region of the mtDNA was apparently harmful to the embryo and, therefore, may have been eliminated to ensure homoplasmy at the beginning of embryonic development.

Abstract A key aim of mitochondrial genetics is to understand the rules that govern mtDNA segregation. To this end, cells carrying more than one mtDNA genotype have been generated and the partitioning of mtDNA to daughter cells monitored. Typically, these experiments investigated the segregation of mtDNAs carrying a pathological mutation versus apparently normal mtDNA. Another approach was to create animals with two mtDNA genotypes that were non-pathological and analyse the segregation pattern of the two types of mtDNA in different tissues at various ages. Collectively, these studies demonstrate that mtDNA segregation can be non-random, yet the molecular mechanisms underlying the complex patterns of mtDNA segregation remain to be elucidated.

Abstract Both mitochondrial division and mtDNA replication are unrelated to the cell cycle or to the timing of nuclear DNA replication. Thus, a dividing cell has the potential to donate a variable number of organelles and genomes to its daughter cells, a phenomenon termed mitotic segregation. This process becomes important clinically if a patient is heteroplasmic, that is, he or she harbours two populations of mtDNA—normal mtDNAs and mutated mtDNAs causing a mitochondrial disease. Because of mitotic segregation, the phenotypic expression of a pathogenic mtDNA mutation may vary both in time (during development or over the course of a life-span) and in space (among tissues or cells).

Abstract Faithful copying and maintenance of the mitochondrial genome is fundamental to aerobic metabolism and therefore life. Many of the factors involved in mitochondrial DNA metabolism are unknown, or poorly characterized and even the basic mechanism of mitochondrial DNA replication is not well understood. Nevertheless, considerable progress has been made in recent years and this is undoubtedly a field that will benefit enormously from the characterization of the human genome sequence. Current knowledge of the machinery of mtDNA replication is discussed together with a review of the often-contentious areas of repair and recombination. Other topics covered here include the organization and segregation of mtDNA, and auxiliary requirements for mtDNA maintenance, notably nucleotide metabolism.