Showing papers on "Nuclear DNA published in 2014"
TL;DR: In this article, the authors analyzed mice deficient for the lysosomal nuclease Dnase2a and observed elevated levels of undegraded DNA in both phagocytic and nonphagocytic cells.
171 citations
TL;DR: This work reports metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract, which projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.
Abstract: We report metrics from complete genome capture of nuclear DNA from extinct mammoths using biotinylated RNAs transcribed from an Asian elephant DNA extract. Enrichment of the nuclear genome ranged from 1.06- to 18.65-fold, to an apparent maximum threshold of ∼80% on-target. This projects an order of magnitude less costly complete genome sequencing from long-dead organisms, even when a reference genome is unavailable for bait design.
120 citations
TL;DR: It is shown that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens.
115 citations
TL;DR: The role of DNA repair in mitochondrial function and a possible cross-talk with other pathways that may potentially participate in mitochondrial genomic stability are addressed, such as mitochondrial dynamics and nuclear-mitochondrial signaling.
81 citations
TL;DR: Evidence of both negative and adaptive (positive) selection acting on the mtDNA and nDNA-encoded genes and the effect of both types of selection on mito-nuclear interacting factors are discussed.
Abstract: Most cell functions are carried out by interacting factors, thus underlying the functional importance of genetic interactions between genes, termed epistasis. Epistasis could be under strong selective pressures especially in conditions where the mutation rate of one of the interacting partners notably differs from the other. Accordingly, the order of magnitude higher mitochondrial DNA (mtDNA) mutation rate as compared to the nuclear DNA (nDNA) of all tested animals, should influence systems involving mitochondrial-nuclear (mito-nuclear) interactions. Such is the case of the energy producing oxidative phosphorylation (OXPHOS) and mitochondrial translational machineries which are comprised of factors encoded by both the mtDNA and the nDNA. Additionally, the mitochondrial RNA transcription and mtDNA replication systems are operated by nDNA-encoded proteins that bind mtDNA regulatory elements. As these systems are central to cell life there is strong selection towards mito-nuclear co-evolution to maintain their function. However, it is unclear whether (A) mito-nuclear co-evolution befalls only to retain mitochondrial functions during evolution or, also, (B) serves as an adaptive tool to adjust for the evolving energetic demands as species’ complexity increases. As the first step to answer these questions we discuss evidence of both negative and adaptive (positive) selection acting on the mtDNA and nDNA-encoded genes and the effect of both types of selection on mito-nuclear interacting factors. Emphasis is given to the crucial role of recurrent ancient (nodal) mutations in such selective events. We apply this point-of-view to the three available types of mito-nuclear co-evolution: protein-protein (within the OXPHOS system), protein-RNA (mainly within the mitochondrial ribosome) and protein DNA (at the mitochondrial replication and transcription machineries).
78 citations
TL;DR: Based on a large data set of nuclear and plastid DNA sequences, a reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods, showing better resolution at both inter- and intra-sectional level than previous studies.
Abstract: Populus (Salicaceae) is one of the most economically and ecologically important genera of forest trees. The complex reticulate evolution and lack of highly variable orthologous single-copy DNA markers have posed difficulties in resolving the phylogeny of this genus. Based on a large data set of nuclear and plastid DNA sequences, we reconstructed robust phylogeny of Populus using parsimony, maximum likelihood and Bayesian inference methods. The resulting phylogenetic trees showed better resolution at both inter- and intra-sectional level than previous studies. The results revealed that (1) the plastid-based phylogenetic tree resulted in two main clades, suggesting an early divergence of the maternal progenitors of Populus; (2) three advanced sections (Populus, Aigeiros and Tacamahaca) are of hybrid origin; (3) species of the section Tacamahaca could be divided into two major groups based on plastid and nuclear DNA data, suggesting a polyphyletic nature of the section; and (4) many species proved to be of hybrid origin based on the incongruence between plastid and nuclear DNA trees. Reticulate evolution may have played a significant role in the evolution history of Populus by facilitating rapid adaptive radiations into different environments.
72 citations
TL;DR: The mitochondrial unfolded protein response is a relatively recently discovered PQC pathway, which senses the proteostatic disturbances specifically in the mitochondria and resolves the stress by retrograde signaling to the nucleus and consequent transcriptional activation of protective genes.
Abstract: Mitochondria, the main site of cellular energy harvesting, are derived from proteobacteria that evolved within our cells in endosymbiosis. Mitochondria retained vestiges of their proteobacterial genome, the circular mitochondrial DNA, which encodes 13 subunits of the oxidative phosphorylation multiprotein complexes in the electron transport chain (ETC), while the remaining ~80 ETC components are encoded in the nuclear DNA (nDNA). A further ~1,400 proteins, which are essential for mitochondrial function are also encoded in nDNA. Thus, a majority of mitochondrial proteins are translated in the cytoplasm, then imported, processed, and assembled in the mitochondria. An intricate protein quality control (PQC) network, constituted of chaperones and proteases that refold or degrade defective proteins, maintains mitochondrial proteostasis and ensures the cell and organism health. The mitochondrial unfolded protein response is a relatively recently discovered PQC pathway, which senses the proteostatic disturbances specifically in the mitochondria and resolves the stress by retrograde signaling to the nucleus and consequent transcriptional activation of protective genes. This PQC system does not only transiently resolve the local stress but also can have long-lasting effects on whole body metabolism, fitness, and longevity. A delicate tuning of its activation levels might constitute a treatment of various diseases, such as metabolic diseases, cancer, and neurodegenerative disorders.
67 citations
TL;DR: The available data for enzymes related to organellar genome replication in green plants and red algae are summarized and the transitional history of replication enzymes in the organelles of plants is discussed.
Abstract: Plastids and mitochondria possess their own genomes. Although the replication mechanisms of these organellar genomes remain unclear in photosynthetic eukaryotes, several organelle-localized enzymes related to genome replication, including DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, primer removal enzyme, and several DNA recombination-related enzymes, have been identified. In the reference Eudicot plant Arabidopsis thaliana, the replication-related enzymes of plastids and mitochondria are similar because many of them are dual targeted to both organelles, whereas in the red alga Cyanidioschyzon merolae, plastids and mitochondria contain different replication machinery components. The enzymes involved in organellar genome replication in green plants and red algae were derived from different origins, including proteobacterial, cyanobacterial, and eukaryotic lineages. In the present review, we summarize the available data for enzymes related to organellar genome replication in green plants and red algae. In addition, based on the type and distribution of replication enzymes in photosynthetic eukaryotes, we discuss the transitional history of replication enzymes in the organelles of plants.
49 citations
TL;DR: The results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.
Abstract: DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.
46 citations
TL;DR: The relative pitfalls associated with using FUdR and germline deficient mutant strains as tools for the experimental elimination of progeny are discussed and the half-life of mtDNA in somatic cells of adult C. elegans is determined to be between 8 and 13 days; this long half- life very likely explains the small or undetectable impact of F UdR on mitochondrial endpoints in the authors' experiments.
43 citations
TL;DR: Results indicate that neuropathy is highly predictive of a nuclear DNA defect and that it is rarely associated with single mitochondrial DNA deletions.
Abstract: Progressive external ophthalmoplegia is a common clinical feature in mitochondrial disease caused by nuclear DNA defects and single, large-scale mitochondrial DNA deletions and is less frequently associated with point mutations of mitochondrial DNA. Peripheral neuropathy is also a frequent manifestation of mitochondrial disease, although its prevalence and characteristics varies considerably among the different syndromes and genetic aetiologies. Based on clinical observations, we systematically investigated whether the presence of peripheral neuropathy could predict the underlying genetic defect in patients with progressive external ophthalmoplegia. We analysed detailed demographic, clinical and neurophysiological data from 116 patients with genetically-defined mitochondrial disease and progressive external ophthalmoplegia. Seventy-eight patients (67%) had a single mitochondrial DNA deletion, 12 (10%) had a point mutation of mitochondrial DNA and 26 (22%) had mutations in either POLG, C10orf2 or RRM2B, or had multiple mitochondrial DNA deletions in muscle without an identified nuclear gene defect. Seventy-seven patients had neurophysiological studies; of these, 16 patients (21%) had a large-fibre peripheral neuropathy. The prevalence of peripheral neuropathy was significantly lower in patients with a single mitochondrial DNA deletion (2%) as compared to those with a point mutation of mitochondrial DNA or with a nuclear DNA defect (44% and 52%, respectively; P<0.001). Univariate analyses revealed significant differences in the distribution of other clinical features between genotypes, including age at disease onset, gender, family history, progressive external ophthalmoplegia at clinical presentation, hearing loss, pigmentary retinopathy and extrapyramidal features. However, binomial logistic regression analysis identified peripheral neuropathy as the only independent predictor associated with a nuclear DNA defect (P=0.002; odds ratio 8.43, 95% confidence interval 2.24-31.76). Multinomial logistic regression analysis identified peripheral neuropathy, family history and hearing loss as significant predictors of the genotype, and the same three variables showed the highest performance in genotype classification in a decision tree analysis. Of these variables, peripheral neuropathy had the highest specificity (91%), negative predictive value (83%) and positive likelihood ratio (5.87) for the diagnosis of a nuclear DNA defect. These results indicate that peripheral neuropathy is a rare finding in patients with single mitochondrial DNA deletions but that it is highly predictive of an underlying nuclear DNA defect. This observation may facilitate the development of diagnostic algorithms. We suggest that nuclear gene testing may enable a more rapid diagnosis and avoid muscle biopsy in patients with progressive external ophthalmoplegia and peripheral neuropathy.
TL;DR: The most recent line of evidence indicates the presence of 5-methylcytosine and 5-hydroxymethylcyTosine in mitochondrial DNA (mtDNA); thus, the level of gene expression can be regulated by direct epigenetic modifications.
TL;DR: A promising new system that can be used to visualize DNA replication in isolated maize root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU).
Abstract: The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chro-matin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA rep-lication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ . Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize ( Zea mays L.) root tip nuclei after in planta pulse labelling with the thymi-dine analogue, 5-ethynyl-2 ′ -deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation.Key words:
TL;DR: This RECQ4 mutant, which is no longer regulated by p32 and is enriched in the mitochondria, interacts with the mitochondrial replication helicase PEO1 and induces abnormally high levels of mtDNA synthesis.
TL;DR: A3A-related enzymes from the rhesus and tamarin monkey, horse, sheep, dog, and panda proved to be orthologous to the human enzyme in all these activities revealing strong conservation more than 148 My, suggesting their singular role in DNA catabolism is a well-established mechanism probably outweighing any deleterious or pathological roles.
Abstract: The human APOBEC3 gene cluster locus encodes polynucleotide cytidine deaminases. Although many act as viral restriction factors through mutation of single-stranded DNA, recent reports have shown that human APOBEC3A was capable of efficiently hypermutating nuclear DNA and inducing DNA breaks in genomic DNA. In addition, the enzyme was unique in efficiently deaminating 5-methylcytidine in single-stranded DNA. To appreciate the evolutionary relevance of these activities, we analyzed A3A-related enzymes from the rhesus and tamarin monkey, horse, sheep, dog, and panda. All proved to be orthologous to the human enzyme in all these activities revealing strong conservation more than 148 My. Hence, their singular role in DNA catabolism is a well-established mechanism probably outweighing any deleterious or pathological roles such as genomic instability and cancer formation.
TL;DR: The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in the study reveal the complex topology of the Phasianidae, and combines multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids.
Abstract: The phylogenetic relationships of species in the Phasianidae, Order Galliformes, are the object of intensive study. However, convergent morphological evolution and rapid species radiation result in much ambiguity in the group. Further, matrilineal (mtDNA) genealogies conflict with trees based on nuclear DNA retrotransposable elements. Herein, we analyze 39 nearly complete mitochondrial genomes (three new) and up to seven nuclear DNA segments. We combine these multiple unlinked, more informative genetic markers to infer historical relationships of the major groups of phasianids. The nuclear DNA tree is largely congruent with the tree derived from mt genomes. However, branching orders of mt/nuclear trees largely conflict with those based on retrotransposons. For example, Gallus/Bambusicola/Francolinus forms the sister-group of Coturnix/Alectoris in the nuclear/mtDNA trees, yet the tree based on retrotransposable elements roots the former at the base of the tree and not with the latter. Further, while peafowls cluster with Gallus/Coturnix in the mt tree, they root at the base of the phasianids following Gallus in the tree based on retrotransposable elements. The conflicting branch orders in nuclear/mtDNA and retrotransposons-based trees in our study reveal the complex topology of the Phasianidae.
TL;DR: A model on the succession of OREs in eukaryotes is proposed on the basis of the results of localization and phylogenetic analyses, and it is proposed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteob bacterial, cyanobacterial, and eUKaryotic, in both red algae and green plants.
Abstract: Plants and algae possess plastids and mitochondria harboring their own genomes, which are replicated by the apparatus consisting of DNA polymerase, DNA primase, DNA helicase, DNA topoisomerase, single-stranded DNA maintenance protein, DNA ligase, and primer removal enzyme. In the higher plant Arabidopsis thaliana, organellar replication-related enzymes (OREs) are similar in plastids and mitochondria because many of them are dually targeted to plastids and mitochondria. In the red algae, there is a report about a DNA replicase, plant/protist organellar DNA polymerase, which is localized to both plastids and mitochondria. However, other OREs remain unclear in algae. Here, we identified OREs possibly localized to organelles in the unicellular rhodophyte Cyanidioschyzon merolae. We then examined intracellular localization of green fluorescent protein-fusion proteins of these enzymes in C. merolae, whose cell has a single plastid and a single mitochondrion and is suitable for localization analysis, demonstrating that the plastid and the mitochondrion contain markedly different components of replication machinery. Phylogenetic analyses revealed that the organelle replication apparatus was composed of enzymes of various different origins, such as proteobacterial, cyanobacterial, and eukaryotic, in both red algae and green plants. Especially in the red alga, many enzymes of cyanobacterial origin remained. Finally, on the basis of the results of localization and phylogenetic analyses, we propose a model on the succession of OREs in eukaryotes.
TL;DR: It is shown that a chloroplast promoter, 16S rrn, drives nuclear transcription, suggesting that a transferred organellar gene may become active without obtaining a nuclear promoter.
Abstract: Endosymbiotic gene transfer from cytoplasmic organelles (chloroplasts and mitochondria) to the nucleus is an ongoing process in land plants. Although the frequency of organelle DNA migration is high, functional gene transfer is rare because a nuclear promoter is thought necessary for activity in the nucleus. Here we show that a chloroplast promoter, 16S rrn, drives nuclear transcription, suggesting that a transferred organellar gene may become active without obtaining a nuclear promoter. Examining the chromatin status of a known de novo chloroplast integrant indicates that plastid DNA inserts into open chromatin and that this relaxed condition is maintained after integration. Transcription of nuclear organelle DNA integrants was explored at the whole genome level by analyzing RNA-seq data of Oryza sativa subsp. japonica, and utilizing sequence polymorphisms to unequivocally discriminate nuclear organelle DNA transcripts from those of bona fide cytoplasmic organelle DNA. Nuclear copies of organelle DNA that are transcribed show a spectrum of transcriptional activity but at comparatively low levels compared with the majority of other nuclear genes.
TL;DR: To study the evolution of different cytogenetic characters in species of Solanum sect.
Abstract: In order to study the evolution of different cytogenetic characters in species of Solanum sect. Acanthophora in relationship to the known phylogeny for this group, the following techniques were used: CMA/DAPI chromosome banding; fluorescent in situ hybridization with probes for the 18-5.8-26S and the 5S rDNA genes in mitotic chromosomes; nuclear DNA quantification by flow cytometry. Depending on the species, 2–6 of the 12 basic chromosome pairs were identified. The heterochromatic banding patterns were shown to be species-specific. All species presented one chromosome pair bearing a 18-5.8-26S signal and one pair (rarely two) with a 5S signal, the two rDNA sites being non-syntenic. The techniques employed allowed us to establish two species groups within sect. Acanthophora: one with small, symmetric chromosomes, little heterochromatin and lower DNA content, and the other one with larger and more asymmetric chromosomes, more heterochromatin CMA+/DAPI− (associated with NOR or not) and a higher DNA content. An elevated karyotype asymmetry would be associated with a high amount of heterochromatin and a high DNA content. The trend within sect. Acanthophora would be towards a loss of heterochromatin, a reduction of chromosome size, and an increase in symmetry.
TL;DR: K-type cytoplasm affected the methylation to a much greater degree than T-type and S- type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated Sites, and polymorphism between A lines and B line for these cytopLasms.
TL;DR: A multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken, duck and turkey nuclear DNA was developed, able to robustly detect and quantify DNA from each of the three poultry species in mixed samples.
Abstract: DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.
TL;DR: A model in which NNK exposure causes damage to both C. elegans nuclear and mitochondrial genomes is suggested, and the hypothesis that the mitochondrial damage is functionally important in this model is supported.
Abstract: The metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) form DNA adducts in animal models. While there are many reports of formation of nuclear DNA adducts, one report also detected NNK-induced damage to the mitochondrial genome in rats. Using a different DNA damage detection technology, we tested whether this finding could be repeated in the nematode Caenorhabditis elegans. We treated N2 strain (wild-type) nematodes with NNK in liquid culture, and applied quantitative PCR to analyze NNK-induced nuclear and mitochondrial DNA (mtDNA) damage. Our results confirm that NNK causes both nuclear and mtDNA damage. However, we did not detect a difference in the level of nuclear versus mtDNA damage in C. elegans. To test whether the mtDNA damage was associated with mitochondrial dysfunction, we used a transgenic nematode strain that permits in vivo measurement of ATP levels and found lower levels of ATP in NNK-exposed animals when compared with the unexposed controls. To test whether the lower levels of ATP could be attributed to inhibition of respiratory chain components, we investigated oxygen consumption in whole C. elegans and found reduced oxygen consumption in exposed animals when compared with the unexposed controls. Our data suggest a model in which NNK exposure causes damage to both C. elegans nuclear and mitochondrial genomes, and support the hypothesis that the mitochondrial damage is functionally important in this model. These results also represent a first step in developing this genetically tractable organism as a model for assessing NNK toxicity.
TL;DR: Five new nuclear genetic markers will be useful for phylogeographic studies in a range of odonate species, but also for phylogenetic studies, providing a particularly useful complement to the existing mitochondrial and nuclear loci.
Abstract: While phylogeographic data provide valuable information to inform conservation plans, there are comparatively few Odonata phylogeographic studies. This lack of research is partially due to a lack of independent DNA markers with appropriate levels of polymorphism that PCR-amplify in a range of species. We followed an exon-primed, intron-crossing (EPIC) PCR strategy to develop five new, polymorphic nuclear DNA sequence loci (six distinct DNA fragments) for the southern damselfly Coenagrion mercuriale. These markers were: cell division cycle 5 protein (CDC5), arginine methyltransferase (PRMT), acetylglucosaminyl-transferase (AgT), myosin light chain (MLC) and phosphoglucose isomerase (PGI). Between three and five of these new markers could be PCR-amplified in five other species from the genus Coenagrion; one locus (PRMT) can be used in 26 other species of odonates that we examined, including three species of Anisoptera belonging to the genus Onychogomphus. These new nuclear genetic markers will be useful for...
TL;DR: Impaired BCKD activity as in MSUD, results in accumulation of DNA damage and corresponding mitochondrial dysfunction, suggesting that the genotoxic effect is ascribed to accumulating metabolites.
Abstract: Objective The mitochondrial branched-chain ketoacid dehydrogenase (BCKD) catalyzes the degradation of branched-chain amino acids (BCAA), which have been shown to induce oxidative stress. Maple Syrup Urine Disease (MSUD) is caused by impaired activity of BCKD, suggesting that oxidative stress and resulting DNA damage could contribute to pathology. We evaluated the potential effect of BCKD deficiency on genome integrity and mitochondrial function as a downstream target. Methods Primary fibroblasts from MSUD patients and controls were either cultivated under normal conditions or exposed to metabolic or oxidative stress. DNA was analyzed for damage and mitochondrial function was evaluated by gene expression analyses, functional assays and immunofluorescent methods. Results Patient fibroblasts accumulated damage in mitochondrial DNA (mtDNA) and nuclear DNA, with a corresponding reduction in mitochondrial transcription, mtDNA copy number and pyruvate dehydrogenase. We found no evidence of increased level of reactive oxygen species (ROS) in patient fibroblasts under normal conditions, suggesting that the genotoxic effect is ascribed to accumulating metabolites. Conclusions Impaired BCKD activity as in MSUD, results in accumulation of DNA damage and corresponding mitochondrial dysfunction.
TL;DR: The results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level, and found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events.
Abstract: Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events.
TL;DR: The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.
Abstract: Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus—as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.
TL;DR: Results provided direct evidence supporting that JBP proteins play an important role in oxidizing thymidine to form 5-HmdU, which facilitated the generation of dJ, the first report about the application of LC-MS/MS for the quantification of base J.
TL;DR: It is found that under mtDNA guidance in one of the two study cases niche divergence is overestimated, whereas in the other it is underestimated, and the role of estimated niche divergence in species delineation is discussed.
Abstract: If potential morphologically cryptic species, identified based on differentiated mitochondrial DNA, express ecological divergence, this increases support for their treatment as distinct species However, mitochondrial DNA introgression hampers the correct estimation of ecological divergence We test the hypothesis that estimated niche divergence differs when considering nuclear DNA composition or mitochondrial DNA type as representing the true species range We use empirical data of two crested newt species (Amphibia: Triturus) which possess introgressed mitochondrial DNA from a third species in part of their ranges We analyze the data in environmental space by determining Fisher distances in a principal component analysis and in geographical space by determining geographical overlap of species distribution models We find that under mtDNA guidance in one of the two study cases niche divergence is overestimated, whereas in the other it is underestimated In the light of our results we discuss the role of estimated niche divergence in species delineation
TL;DR: Here it is shown that the 3DPCR technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5′GpCpR context, and cannot be used to identify fixed C ->T transitions in cancer genomes.
Abstract: The revolution in cancer genomics shows that the dominant mutations are CG->TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C->T mutations in the signature 5′TpC context. These can be copied as G->A transitions in the 5′GpA context. DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA. Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C->T and G->A mutations in a 5′GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR. Differential DNA denaturation PCR cannot be used to identify fixed C->T transitions in cancer genomes. Presently, the overall mutation frequency is ∼104–105 base substitutions per cancer genome, or 0.003–0.03 kb−1. By contrast, the 3DPCR error rate is of the order of 4–20 kb−1 owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C->T and G->A mutations associated with the 5′GpC represent artefacts.
TL;DR: This study addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias and found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation betweenThe results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.
Abstract: It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4–30.7 pg in M. truncata and 32.0–64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.