Showing papers on "Nuclear DNA published in 2012"

The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation.

Abstract: Acetaminophen (APAP) overdose is the predominant cause of acute liver failure in the United States. Toxicity begins with a reactive metabolite that binds to proteins. In rodents, this leads to mitochondrial dysfunction and nuclear DNA fragmentation, resulting in necrotic cell death. While APAP metabolism is similar in humans, the later events resulting in toxicity have not been investigated in patients. In this study, levels of biomarkers of mitochondrial damage (glutamate dehydrogenase [GDH] and mitochondrial DNA [mtDNA]) and nuclear DNA fragments were measured in plasma from APAP-overdose patients. Overdose patients with no or minimal hepatic injury who had normal liver function tests (LTs) (referred to herein as the normal LT group) and healthy volunteers served as controls. Peak GDH activity and mtDNA concentration were increased in plasma from patients with abnormal LT. Peak nuclear DNA fragmentation in the abnormal LT cohort was also increased over that of controls. Parallel studies in mice revealed that these plasma biomarkers correlated well with tissue injury. Caspase-3 activity and cleaved caspase-3 were not detectable in plasma from overdose patients or mice, but were elevated after TNF-induced apoptosis, indicating that APAP overdose does not cause apoptosis. Thus, our results suggest that mitochondrial damage and nuclear DNA fragmentation are likely to be critical events in APAP hepatotoxicity in humans, resulting in necrotic cell death.

The half-life of DNA in bone: measuring decay kinetics in 158 dated fossils

Abstract: Claims of extreme survival of DNA have emphasized the need for reliable models of DNA degradation through time. By analysing mitochondrial DNA (mtDNA) from 158 radiocarbon-dated bones of the extinct New Zealand moa, we confirm empirically a long-hypothesized exponential decay relationship. The average DNA half-life within this geographically constrained fossil assemblage was estimated to be 521 years for a 242 bp mtDNA sequence, corresponding to a per nucleotide fragmentation rate (k) of 5.50 × 10(-6) per year. With an effective burial temperature of 13.1°C, the rate is almost 400 times slower than predicted from published kinetic data of in vitro DNA depurination at pH 5. Although best described by an exponential model (R(2) = 0.39), considerable sample-to-sample variance in DNA preservation could not be accounted for by geologic age. This variation likely derives from differences in taphonomy and bone diagenesis, which have confounded previous, less spatially constrained attempts to study DNA decay kinetics. Lastly, by calculating DNA fragmentation rates on Illumina HiSeq data, we show that nuclear DNA has degraded at least twice as fast as mtDNA. These results provide a baseline for predicting long-term DNA survival in bone.

Mesenchymal Stem Cells Transfer Mitochondria to the Cells with Virtually No Mitochondrial Function but Not with Pathogenic mtDNA Mutations

Abstract: It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine− and bromodeoxyuridine [BrdU]+) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ0 cells under uridine-free condition, induced increased chemotaxis in MSC, which was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction.

The oxidative DNA lesions 8,5′‐cyclopurines accumulate with aging in a tissue‐specific manner

Abstract: Accumulation of DNA damage is implicated in aging. This is supported by the fact that inherited defects in DNA repair can cause accelerated aging of tissues. However, clear-cut evidence for DNA damage accumulation in old age is lacking. Numerous studies report measurement of DNA damage in nuclear and mitochondrial DNA from tissues of young and old organisms, with variable outcomes. Variability results from genetic differences between specimens or the instability of some DNA lesions. To control these variables and test the hypothesis that elderly organisms have more oxidative DNA damage than young organisms, we measured 8,5'-cyclopurine-2'-deoxynucleosides (cPu), which are relatively stable, in tissues of young and old wild-type and congenic progeroid mice. We found that cPu accumulate spontaneously in the nuclear DNA of wild-type mice with age and to a greater extent in DNA repair-deficient progeroid mice, with a similar tissue-specific pattern (liver > kidney > brain). These data, generated under conditions where genetic and environmental variables are controlled, provide strong evidence that DNA repair mechanisms are inadequate to clear endogenous lesions over the lifespan of mammals. The similar, although exaggerated, results obtained from progeroid, DNA repair-deficient mice and old normal mice support the conclusion that DNA damage accumulates with, and likely contributes to, aging.

Naturally occurring mitochondrial dna haplotypes exhibit metabolic differences: insight into functional properties of mitochondria

Abstract: Linking the mitochondrial genotype and the organismal phenotype is of paramount importance in evolution of mitochondria. In this study, we determined the differences in catalytic properties of mitochondria dictated by divergences in the siII and siIII haplogroups of Drosophila simulans using introgressions of siII mtDNA type into the siIII nuclear background. We used a novel in situ method (permeabilized fibers) that allowed us to accurately measure the consumption of oxygen by mitochondria in constructed siII-introgressed flies and in siIII-control flies. Our results showed that the catalytic capacity of the electron transport system is not impaired by introgressions, suggesting that the functional properties of mitochondria are tightly related to the mtDNA haplogroup and not to the nuclear DNA or to the mito-nuclear interactions. This is the first study, to our knowledge, that demonstrates a naturally occurring haplogroup can confer specific functional differences in aspects of mitochondrial metabolism. This study illustrates the importance of mtDNA changes on organelle evolution and highlights the potential bioenergetic and metabolic impacts that divergent mitochondrial haplogroups may have upon a wide variety of species including humans.

The mitochondrial genome of Malus domestica and the import-driven hypothesis of mitochondrial genome expansion in seed plants.

Abstract: Mitochondrial genomes of spermatophytes are the largest of all organellar genomes. Their large size has been attributed to various factors; however, the relative contribution of these factors to mitochondrial DNA (mtDNA) expansion remains undetermined. We estimated their relative contribution in Malus domestica (apple). The mitochondrial genome of apple has a size of 396 947 bp and a one to nine ratio of coding to non-coding DNA, close to the corresponding average values for angiosperms. We determined that 71.5% of the apple mtDNA sequence was highly similar to sequences of its nuclear DNA. Using nuclear gene exons, nuclear transposable elements and chloroplast DNA as markers of promiscuous DNA content in mtDNA, we estimated that approximately 20% of the apple mtDNA consisted of DNA sequences imported from other cell compartments, mostly from the nucleus. Similar marker-based estimates of promiscuous DNA content in the mitochondrial genomes of other species ranged between 21.2 and 25.3% of the total mtDNA length for grape, between 23.1 and 38.6% for rice, and between 47.1 and 78.4% for maize. All these estimates are conservative, because they underestimate the import of non-functional DNA. We propose that the import of promiscuous DNA is a core mechanism for mtDNA size expansion in seed plants. In apple, maize and grape this mechanism contributed far more to genome expansion than did homologous recombination. In rice the estimated contribution of both mechanisms was found to be similar.

Specific mitochondrial DNA mutation in mice regulates diabetes and lymphoma development

Abstract: It has been hypothesized that respiration defects caused by accumulation of pathogenic mitochondrial DNA (mtDNA) mutations and the resultant overproduction of reactive oxygen species (ROS) or lactates are responsible for aging and age-associated disorders, including diabetes and tumor development. However, there is no direct evidence to prove the involvement of mtDNA mutations in these processes, because it is difficult to exclude the possible involvement of nuclear DNA mutations. Our previous studies resolved this issue by using an mtDNA exchange technology and showed that a G13997A mtDNA mutation found in mouse tumor cells induces metastasis via ROS overproduction. Here, using transmitochondrial mice (mito-mice), which we had generated previously by introducing G13997A mtDNA from mouse tumor cells into mouse embryonic stem cells, we provide convincing evidence supporting part of the abovementioned hypothesis by showing that G13997A mtDNA regulates diabetes development, lymphoma formation, and metastasis—but not aging—in this model.

Nuclear envelope defects impede a proper response to micronuclear DNA lesions

Abstract: When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.

Nuclear and mitochondrial DNA oxidation in Alzheimer's disease

Abstract: The study of Alzheimer's disease neuropathology has been intimately associated with the field of oxidative stress for nearly 20 years. Indeed, increased markers of oxidative stress have been associated with this neurodegenerative condition, resulting from oxidation of lipids, proteins and nucleic acids. Increased nuclear and mitochondrial DNA oxidation are observed in Alzheimer's disease, stemming from increased reactive oxygen species attack to DNA bases and from the impairment of DNA repair mechanisms. Moreover, mitochondrial DNA is found to be more extensively oxidized than nuclear DNA. This review is intended to summarizes the most important cellular reactive oxygen species producers and how mitochondrial dysfunction, redox-active metals dyshomeostasis and NADPH oxidases contribute to increased oxidative stress in Alzheimer's disease. A summary of the antioxidant system malfunction will also be provided. Moreover, we will highlight the mechanisms of DNA oxidation and repair. Importantly, we will discuss evidence relating the DNA repair machinery and accumulated DNA oxidation with Alzheimer's disease.

Mitochondrial DNA: A Blind Spot in Neuroepigenetics.

Abstract: Neuroepigenetics, which includes nuclear DNA modifications such as 5-methylcytosine and 5-hydoxymethylcytosine and modifications of nuclear proteins such as histones, is emerging as the leading field in molecular neuroscience. Historically, a functional role for epigenetic mechanisms, including in neuroepigenetics, has been sought in the area of the regulation of nuclear transcription. However, one important compartment of mammalian cell DNA, different from nuclear but equally important for physiological and pathological processes (including in the brain), mitochondrial DNA has for the most part not had a systematic epigenetic characterization. The importance of mitochondria and mitochondrial DNA (particularly its mutations) in central nervous system physiology and pathology has long been recognized. Only recently have mechanisms of mitochondrial DNA methylation and hydroxymethylation, including the discovery of mitochondrial DNA-methyltransferases and the presence and the functionality of 5-methylcytosine and 5-hydroxymethylcytosine in mitochondrial DNA (e.g., in modifying the transcription of mitochondrial genome), been unequivocally recognized as a part of mammalian mitochondrial physiology. Here we summarize for the first time evidence supporting the existence of these mechanisms and we propose the term "mitochondrial epigenetics" to be used when referring to them. Currently, neuroepigenetics does not include mitochondrial epigenetics - a gap that we expect to close in the near future.

An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila

Abstract: Programmed DNA rearrangements are important processes present in many organisms. In the ciliated protozoan Tetrahymena thermophila, DNA rearrangements occur during the sexual conjugation process and lead to the deletion of thousands of specific DNA segments and fragmentation of the chromosomes. In this study, we found that the Ku80 homologue, a conserved component of the nonhomologous end-joining process of DNA repair, was essential for these two processes. During conjugation, TKU80 was highly expressed and localized to the new macronucleus, where DNA rearrangements occur. Homokaryon TKU80-knockout mutants are unable to complete conjugation and produce progeny and are arrested at the two-micronuclei/two-macronuclei stage. Analysis of their DNA revealed failure to complete DNA deletion. However, the DNA-cutting step appeared to have occurred, as evidenced by the presence of circularized excised DNA. Moreover, chromosome breakage or de novo telomere addition was affected. The mutant appears to accumulate free DNA ends detectable by terminal deoxynucleotidyl transferase dUTP nick end labeling assays that led to the degradation of most DNA in the developing macronucleus. These findings suggest that Tku80p may serve an end-protective role after DNA cleavage has occurred. Unexpectedly, the large heterochromatin structures that normally associate with DNA rearrangements failed to form without TKU80. Together the results suggest multiple roles for Tku80p and indicate that a Ku-dependent DNA-repair pathway is involved in programmed DNA rearrangements in Tetrahymena.

DNA methylation and genome rearrangement characteristics of phase change in cultured shoots of Sequoia sempervirens.

Abstract: Epigenetic machinery regulates the expression of individual genes and plays a crucial role in globally shaping and maintaining developmental patterning. We studied the extent of DNA methylation in the nucleus, mitochondrion and chloroplast in cultured Sequoia sempervirens (coast redwood) adult, juvenile and rejuvenated shoots by measuring the ratio of methylcytosine to total cytosine using high-performance liquid chromatography (HPLC). We also analyzed nuclear DNA (nuDNA) polymorphisms of different shoot types by methylation-sensitive amplified fragment length polymorphism (MSAP) and Southern blot analysis. The extent of nuDNA methylation was greater in the adult vegetative than juvenile and rejuvenated shoots (8% vs 6.5-7.5%). In contrast, the proportion of methylcytosine was higher in mitochondrial DNA (mDNA) of juvenile and rejuvenated shoots than adult shoots (6.6% vs 7.8-8.2%). MSAP and Southern blot analyses identified three MSAP fragments which could be applied as phase-specific molecular markers. We also found nuclear genome and mtDNA rearrangement may be as important as DNA methylation status during the phase change. Our findings strongly suggest that DNA methylation and genome rearrangement may affect the dynamic tissue- and cell type-specific changes that determine the developmental phase of S. sempervirens shoots.

Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

Abstract: In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan ® quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor ( MC1R ) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human–dog–cat origin with as little as 0.4pg of human and cat nuclear DNA, respectively, and 4.0pg of dog nuclear DNA.

Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

Abstract: Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process.

TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells.

Abstract: Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations.

In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair.

Abstract: Hair fibers are formed by keratinocytes of the hair follicle in a process that involves the breakdown of the nucleus including DNA. Accordingly, DNA can be isolated with high yield from the hair bulb which contains living keratinocytes, whereas it is difficult to prepare from the distal portions of hair fibers and from shed hair. Nevertheless, forensic investigations are successful in a fraction of shed hair samples found at crime scenes. Here, we report that interindividual differences in the completeness of DNA removal from hair corneocytes are major determinants of DNA content and success rates of forensic investigations of hair. Distal hair samples were permeabilized with ammonia and incubated with the DNA-specific dye Hoechst 33258 to label DNA in situ. Residual nuclear DNA was visualized under the fluorescence microscope. Hair from some donors did not contain any stainable nuclei, whereas hair of other donors contained a variable number of DNA-positive nuclear remnants. The number of DNA-containing nuclear remnants per millimeter of hair correlated with the amount of DNA that could be extracted and amplified by quantitative PCR. When individual hairs were investigated, only hairs in which DNA could be labeled in situ gave positive results in short tandem repeat typing. This study reveals that the completeness of DNA degradation during cornification of the hair is a polymorphic trait. Furthermore, our results suggest that in situ labeling of DNA in hair may be useful for predicting the probability of success of forensic analysis of nuclear DNA in shed hair.

Involvement of autophagy and mitochondrial dynamics in determining the fate and effects of irreparable mitochondrial DNA damage.

Abstract: Mitochondrial DNA (mtDNA) is different in many ways from nuclear DNA. A key difference is that certain types of DNA damage are not repaired in the mitochondrial genome. What, then, is the fate of such damage? What are the effects? Both questions are important from a health perspective because irreparable mtDNA damage is caused by many common environmental stressors including ultraviolet C radiation (UVC). We found that UVC-induced mtDNA damage is removed slowly in the nematode Caenorhabditis elegans via a mechanism dependent on mitochondrial fusion, fission, and autophagy. However, knockdown or knockout of genes involved in these processes—many of which have homologs involved in human mitochondrial diseases—had very different effects on the organismal response to UVC. Reduced mitochondrial fission and autophagy caused no or small effects, while reduced mitochondrial fusion had dramatic effects.

Mitochondrial Base Excision Repair Assays

Abstract: Mitochondrial DNA (mtDNA) is constantly exposed to oxidative injury Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage The accumulation of DNA damage is thought to be particularly deleterious in post-mitotic cells, including neurons, and to play a critical role in the aging process and in a variety of diseases Thus, efficient mtDNA repair is important for the maintenance of genomic integrity and a healthy life The base excision repair (BER) mechanism was the first to be described in mitochondria, and consequently it is the best known This chapter outlines protocols for isolating mitochondria from mammalian cells in culture and from rodent tissues including liver and brain It also covers the isolation of synaptic mitochondria BER takes place in four distinct steps, and protocols describing in vitro assays for measuring these enzymatic steps in lysates of isolated mitochondria are included

Evolution of the primate APOBEC3A cytidine deaminase gene and identification of related coding regions.

Abstract: The APOBEC3 gene cluster encodes six cytidine deaminases (A3A-C, A3DE, A3F-H) with single stranded DNA (ssDNA) substrate specificity. For the moment A3A is the only enzyme that can initiate catabolism of both mitochondrial and nuclear DNA. Human A3A expression is initiated from two different methionine codons M1 or M13, both of which are in adequate but sub-optimal Kozak environments. In the present study, we have analyzed the genetic diversity among A3A genes across a wide range of 12 primates including New World monkeys, Old World monkeys and Hominids. Sequence variation was observed in exons 1-4 in all primates with up to 31% overall amino acid variation. Importantly for 3 hominids codon M1 was mutated to a threonine codon or valine codon, while for 5/12 primates strong Kozak M1 or M13 codons were found. Positive selection was apparent along a few branches which differed compared to positive selection in the carboxy-terminal of A3G that clusters with A3A among human cytidine deaminases. In the course of analyses, two novel non-functional A3A-related fragments were identified on chromosome 4 and 8 kb upstream of the A3 locus. This qualitative and quantitative variation among primate A3A genes suggest that subtle differences in function might ensue as more light is shed on this increasingly important enzyme.

Genome sizes of all 19 Araucaria species are correlated with their geographical distribution

Abstract: Nuclear genome size was determined to investigate the relationships between all 19 species of Araucaria de Jussieu. Species from the two other genera of Araucariaceae, Wollemia and Agathis, were also studied. The genome size of 17 out of the 19 species of Araucaria are reported here for the first time. All Araucariaceae have the same chromosome number 2n = 26. However, the nuclear DNA contents (2C value) for Araucaria range from 31.3 to 45.4 pg. There is a good correlation between genome size and division in sections, and geographical distribution. The two species from South America have 44.7 and 45.4 pg, the two species from Australia have 35.7 and 44.4 pg and the two species from New Guinea 34.7 and 40.4 pg. All 13 species of New Caledonia and the one from Norfolk Island have a similar, if not identical, amount of nuclear DNA of, on average, 31.9 pg. This corroborates the identical DNA rbcL sequences found for the New Caledonian araucarias. It suggests that the species from New Caledonia diversified more recently and it questions their status as separate species. Compared with this 31.9 pg a strong increase seems to have occurred in the genome size of the “mainland” araucarias. Genome sizes are evaluated and compared with available taxonomic treatments and extant geographic spreading. The nuclear DNA contents found within the sections are close, making it possible to assign an unknown plant to a section. A difference of 1 pg, which amounts to a difference of 978 Mbp, far exceeds a single character. Nuclear DNA content, as measured by flow cytometry, may conveniently be used to produce systematic data. It is applicable even with young plants or seeds for monitoring the trade in endangered species.

Transmitochondrial Cybrids: Tools for Functional Studies of Mutant Mitochondria

Abstract: Mitochondrial functions are controlled by both mitochondrial DNA (mtDNA) and nuclear DNA. Hence, it is difficult to identify whether mitochondrial or nuclear genome is responsible for a particular mitochondrial defect. Cybrid is a useful tool to overcome this difficulty, where we can compare mitochondria from different sources in a defined nuclear background. Cybrids are constructed by fusing enucleated cells harboring wild type or altered mtDNA of interest with ρ(0) cells (cells lacking mtDNA) in which the endogenous mtDNA has been depleted. Therefore, cybrids are very useful in studying consequences of mtDNA alterations or other mitochondrial defects at the cellular level by excluding the influence of nuclear DNA mutations.