Showing papers on "Nuclear DNA published in 2010"

Molecular Poltergeists: Mitochondrial DNA Copies (numts) in Sequenced Nuclear Genomes

Abstract: The natural transfer of DNA from mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts) and is an ongoing evolutionary process, as genome sequences attest. In humans, five different numts cause genetic disease and a dozen human loci are polymorphic for the presence of numts, underscoring the rapid rate at which mitochondrial sequences reach the nucleus over evolutionary time. In the laboratory and in nature, numts enter the nuclear DNA via non-homolgous end joining (NHEJ) at double-strand breaks (DSBs). The frequency of numt insertions among 85 sequenced eukaryotic genomes reveal that numt content is strongly correlated with genome size, suggesting that the numt insertion rate might be limited by DSB frequency. Polymorphic numts in humans link maternally inherited mitochondrial genotypes to nuclear DNA haplotypes during the past, offering new opportunities to associate nuclear markers with mitochondrial markers back in time.

Mitochondrial DNA is released by shock and activates neutrophils via p38 map kinase.

Abstract: Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.

G-Quadruplex DNA Sequences Are Evolutionarily Conserved and Associated with Distinct Genomic Features in Saccharomyces cerevisiae

Abstract: G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

A rapid column-based ancient DNA extraction method for increased sample throughput

Abstract: Genetic analyses using museum specimens and ancient DNA from fossil samples are becoming increasingly important in phylogenetic and especially population genetic studies. Recent progress in ancient DNA sequencing technologies has substantially increased DNA sequence yields and, in combination with barcoding methods, has enabled large-scale studies using any type of DNA. Moreover, more and more studies now use nuclear DNA sequences in addition to mitochondrial ones. Unfortunately, nuclear DNA is, due to its much lower copy number in living cells compared to mitochondrial DNA, much more difficult to obtain from low-quality samples. Therefore, a DNA extraction method that optimizes DNA yields from low-quality samples and at the same time allows processing many samples within a short time frame is immediately required. In fact, the major bottleneck in the analysis process using samples containing low amounts of degraded DNA now lies in the extraction of samples, as column-based methods using commercial kits are fast but have proven to give very low yields, while more efficient methods are generally very time-consuming. Here, we present a method that combines the high DNA yield of batch-based silica extraction with the time-efficiency of column-based methods. Our results on Pleistocene cave bear samples show that DNA yields are quantitatively comparable, and in fact even slightly better than with silica batch extraction, while at the same time the number of samples that can conveniently be processed in parallel increases and both bench time and costs decrease using this method. Thus, this method is suited for harvesting the power of high-throughput sequencing using the DNA preserved in the millions of paleontological and museums specimens.

Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells

Abstract: Plant mitochondrial genomes are split into sub-genomes, i.e. genes are distributed across various sub-genomic molecules. To investigate whether copy numbers vary between individual mitochondrial genes, we used quantitative real-time PCR in combination with flow cytometric determination of nuclear DNA quantities to determine absolute per-cell-copy numbers of four mitochondrial genes in various Arabidopsis organs and the leaves of tobacco (Nicotiana tabacum) and barley (Hordeum vulgare). The copy numbers of the investigated mitochondrial genes (atp1, rps4, nad6 and cox1) not only differed from each other, but also varied between organs and changed during the development of cotyledons and leaves in Arabidopsis. We found no correlation between altered gene copy numbers, transcript levels and O(2) consumption. However, per cell, both the number of mitochondria and the number of gene copies increased with growing cell size. Gene copy numbers varied from approximately 40 (cox1 in young leaves) to approximately 280 (atp1 in mature leaves), and the mean number of mitochondria was approximately 300 in young leaves and 450 in mature leaves. Thus, cells are polyploid with respect to their mitochondrial genomes, but individual mitochondria may contain only part of the genome or even no DNA at all. Our data supports structural models of the mitochondrial genome in non-dividing cells of angiosperms that predict localization of the genes on sub-genomic molecules rather than master chromosomes. The data indicate control of the number of individual genes according to the genotype and developmental program(s) via amplification and/or degradation of sub-genomic molecules.

Recruitment of phosphorylated NPM1 to sites of DNA damage through RNF8-dependent ubiquitin conjugates.

Abstract: Protein accumulation at DNA double-strand breaks (DSB) is essential for genome stability; however, the mechanisms governing these events are not fully understood. Here, we report a new role for the nucleophosmin protein NPM1 in these mechanisms. Thr199-phosphorylated NPM1 (pT199-NPM1) is recruited to nuclear DNA damage foci induced by ionizing radiation (IR). Foci formation is impaired by depletion of the E3 ubiquitin ligases RNF8 and RNF168 or the E2 Ubc13, and pT199-NPM1 binds to Lys63-linked ubiquitin polymers in vitro. Thus, phosphorylated NPM1 may interact with RNF8-dependent ubiquitin conjugates at sites of DNA damage. The interaction was found to rely on T199 phosphorylation, an acidic tract, and an adjacent ubiquitin-interacting motif-like domain. Depletion of the breast cancer suppressor BRCA1 or its partner, RAP80, enhanced IR-induced NPM1 foci and prolonged persistence of the foci, possibly implicating BRCA1 in pT199-NPM1 action and dynamics. Replacement of endogenous NPM1 with its nonphosphorylable T199A mutant prolonged persistence of IR-induced RAD51 foci accompanied by unrepaired DNA damage. Collectively, our findings suggest that phosphorylated NPM1 is a novel component in DSB repair that is recruited by ubiquitin conjugates downstream of RNF8 and RNF168.

Anhydrobiosis-Associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage with Radioresistance

Abstract: Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions (4He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3–4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.

Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells

Abstract: Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC(50) values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.

Nuclear mitochondrial pseudogenes

Abstract: Transfer of genetic material from mitochondria to the nucleus and their integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) in the nuclear genome are incorporated as non-encoded sequences, which are called nuclear mitochondrial pseudogenes (NUMT-pseudogenes). At present, the formation NUMT-pseudogenes in the nuclear genome is shown in many eukaryotes. They are distributed on different chromosomes, form a "library" of mtDNA fragments, migrated into the nuclear genome and provide important information on the history of the evolution of genomes. Escape of mtDNA from the mitochondria most is associated with damage and mitophagy these organelles. The integration of mtDNA fragments into the nuclear genome may occur during repair of double strand breaks of nuclear DNA (nDNA) arising under the action of endogenous and exogenous agents. Reparation of nDNA double strand breaks with "capture" fragments of mtDNA, occurs by non-homologous end joining and a similar mechanism, but with the involvement microhomology, located on the terminal sequences. Analysis of data allows us to suppose that the rate of formation NUMT-pseudogenes will depend on the rate of double strand breaks in nDNA, activity systems, their repair, as well--the number of mtDNA fragments that have emerged from the organelles, with their further migration into the nucleus. Such situations can be expected, most often after exposure to the damaging agents, in the first place--ionizing radiation. The emergence of new NUMT-pseudogenes, obviously, is changing not only the structure of the genome in the areas of their implementation, but may have a significant impact on the realization of genetic information. Integration NUMT-pseudogenes in the nuclear genome de novo may play a role in the development of various pathologies and aging. NUMT-pseudogenes can make serious errors in analyzing free mtDNA of total cellular DNA (using PCR), as a result of their co-amplification.

The migration of mitochondrial DNA fragments to the nucleus affects the chronological aging process of Saccharomyces cerevisiae.

Abstract: Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister-Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological life span (CLS) is determined by the survival of non-dividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age.

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Abstract: Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (≤1 mm) and leaves of different developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10−2 pg DNA for plastids of 1→8 μm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.

Genome Size in Diploids, Allopolyploids, and Autopolyploids of Mediterranean Triticeae

Abstract: Nuclear DNA amount, determined by the flow cytometry method, in diploids, natural and synthetic allopolyploids, and natural and synthetic autopolyploids of the tribe Triticeae (Poaceae) is reviewed here and discussed. In contrast to the very small and nonsignificant variation in nuclear DNA amount that was found at the intraspecific level, the variation at the interspecific level is very large. Evidently changes in genome size are either the cause or the result of speciation. Typical autopolyploids had the expected additive DNA amount of their diploid parents, whereas natural and synthetic cytologically diploidized autopolyploids and natural and synthetic allopolyploids had significantly less DNA than the sum of their parents. Thus, genome downsizing, occurring during or immediately after the formation of these polyploids, provides the physical basis for their cytological diploidization, that is, diploid-like meiotic behavior. Possible mechanisms that are involved in genome downsizing and the biological significance of this phenomenon are discussed.

Nuclear copies of mitochondrial genes: another problem for ancient DNA.

Abstract: The application of ancient DNA techniques is subject to many problems caused primarily by low quality and by low quantity of DNA. For these reasons most studies employing ancient DNA rely on the characterization of mitochondrial DNA, which is present in many more copies per cell than nuclear DNA and hence more copies are likely to survive. We used universal and taxon specific mitochondrial primers to amplify DNA from museum specimens, and found many instances where the amplification of nuclear copies of the mitochondrial gene (numts) instead of the targeted mitochondrial fragment had occurred. Furthermore, the likelihood of amplifying numts increased dramatically when universal primers were utilized. Here we suggest that ancient DNA practitioners must consider the possibility that numts can be amplified at higher rates than previously thought. This is another complication for ancient DNA studies, but it also suggests that more extensive inclusion of nuclear markers in ancient DNA studies should be feasible.

DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions

Abstract: Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed. We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclar mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain. A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.

Characterisation of insect and plant origins using DNA extracted from small volumes of bee honey

Abstract: A DNA-based tool was validated that potentially enables the characterisation of both plant and insect of origin of small (approximately 1 ml) samples of bee honey. Using this method, mitochondrial, nuclear and chloroplast DNA (mtDNA, nuDNA, cpDNA) markers were successfully extracted, PCR amplified, and sequenced from a range of honeys, and the relative amount of plant nuDNA and cpDNA, and bee mtDNA in the samples was quantified using quantitative real-time PCR. Short, but taxonomically informative lengths of insect and plant organelle DNA could be routinely recovered from all honey samples tested, and longer organelle, and nuclear DNA sequences can be recovered from many. The data also enabled preliminary characterisation of the quality of these different DNA sources in honey. Although the absolute quantity of the different genetic markers varied considerably between sample, a general trend was observed of insect mtDNA dominating over plant organelle DNA, and with plant nuclear DNA at the lowest levels. Furthermore there was a clear correlation between the plant DNA content and the success of the PCR assays. To maximise successful characterisation of samples, future studies are recommended to focus on the use of organelle markers, and limit the size of PCR amplicons targeted, although with appropriate sample selection and assay optimisation, other approaches may be possible.

Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans

Abstract: A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism Phytophthora infestans is the pathogen that causes potato and tomato late blight The genetic flexibility of P infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P infestans The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates Nuclear DNA content varies from 175x to 075x that of nuclei in a standard strain from central Mexico Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen

Genome-wide mapping of nuclear mitochondrial DNA sequences links DNA replication origins to chromosomal double-strand break formation in Schizosaccharomyces pombe

Abstract: Chromosomal double-strand breaks (DSBs) threaten genome integrity and repair of these lesions is often mutagenic. How and where DSBs are formed is a major question conveniently addressed in simple model organisms like yeast. NUMTs, nuclear DNA sequences of mitochondrial origin, are present in most eukaryotic genomes and probably result from the capture of mitochondrial DNA (mtDNA) fragments into chromosomal breaks. NUMT formation is ongoing and was reported to cause de novo human genetic diseases. Study of NUMTs is likely to contribute to the understanding of naturally occurring chromosomal breaks. We show that Schizosaccharomyces pombe NUMTs are exclusively located in noncoding regions with no preference for gene promoters and, when located into promoters, do not affect gene transcription level. Strikingly, most noncoding regions comprising NUMTs are also associated with a DNA replication origin (ORI). Chromatin immunoprecipitation experiments revealed that chromosomal NUMTs are probably not acting as ORI on their own but that mtDNA insertions occurred directly next to ORIs, suggesting that these loci may be prone to DSB formation. Accordingly, induction of excessive DNA replication origin firing, a phenomenon often associated with human tumor formation, resulted in frequent nucleotide deletion events within ORI3001 subtelomeric chromosomal locus, illustrating a novel aspect of DNA replication-driven genomic instability. How mtDNA is fragmented is another important issue that we addressed by sequencing experimentally induced NUMTs. This highlighted regions of S. pombe mtDNA prone to breaking. Together with an analysis of human NUMTs, we propose that these fragile sites in mtDNA may correspond to replication pause sites.

DNA damage and impairment of DNA repair in Alzheimer's disease.

Abstract: Deoxyribonucleic acid (DNA) damage has been implicated in ageing and neurodegenerative disorders including Alzheimer's disease (AD) for a few decades. Although it is an established finding, yet there are limited studies on DNA damage. In both nucleus and mitochondria, DNA damage is primarily free radical mediated. It has been proven that mitochondrial DNA is more vulnerable to damage compared to the nuclear DNA. A few studies summarized in this review throw light on the mechanisms of free radical mediated DNA damage and impairment of DNA repair mechanisms in AD. There is a growing need to initiate studies on DNA damage and repair and unravel the molecular underpinnings entailed in the etiopathogenesis of the disease. The outcome of such studies substantiates the corner stone streamlined to employ therapeutic strategies.

Intraspecific genome size variation and morphological differentiation of Ranunculus parnassifolius (Ranunculaceae), an Alpine–Pyrenean–Cantabrian polyploid group

Abstract: The aim of this study was to assess genome size variation and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation within the Ranunculus parnassifolius group in the Pyrenees and the Alps. Although divergences in nuclear DNA content among different species within a genus are widely acknowledged, intraspecific variation is still a somewhat controversial issue. Holoploid and monoploid genome sizes (C- and Cx-values) were determined using propidium iodide flow cytometry in 125 plants of R. parnassifolius s.l. distributed across four European countries. Three different DNA ploidy levels were revealed in the study area: diploid (2n ∼ 2x, 57.14%), triploid (2n ∼ 3x, 1.19%), and tetraploid (2n ∼ 4x, 41.67%). The mean population 2C-values ranged from 8.15 pg in diploids to 14.80 pg in tetraploids, representing a ratio of 1 : 1.8. Marked intraspecific/interpopulation differences in nuclear DNA content were found. Diploid populations prevail in the Pyrenees, although tetraploid cytotypes were reported throughout the distribution area. In general, mixed-cytotype populations were not found. The Spearman correlation coefficient did not reveal significant correlations between genome size and altitude, longitude, or latitude. Morphometric analyses and cluster analyses based on genome size variation revealed the presence of three major groups, which exhibited a particular biogeographical pattern. A new cytotype, DNA triploid, was found for the first time. Tetraploid populations showed constant nuclear DNA levels, whereas diploid populations from the Pyrenees, in which introgressive hybridization is suggested as a presumable trigger for genome size variation, did not. Scenarios for the evolution of geographical parthenogenesis in R. parnassifolius s.l. are discussed. Finally, the different levels of effectiveness between plant and animal reference standards are analysed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101, 251–271.

Expression changes in DNA repair enzymes and mitochondrial DNA damage in aging rat lens.

Abstract: Purpose To determine if there is increased mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage with age in the lenses of rats. We also explored the immunolocalization of 8-oxoguanine DNA glycosylase 1 (OGG1) and AP endonuclease 1 (APE1) in the lens and studied three of the predominant base excision repair (BER) enzymes: OGG1, APE1, and DNA polymerase γ (Polγ).

The value of serial plasma nuclear and mitochondrial DNA levels in adult community-acquired bacterial meningitis

Abstract: Background: Increased levels of plasma nuclear and mitochondrial DNA have been reported in critically ill patients. We tested the hypothesis that plasma nuclear and mitochondrial DNA are substantially increased in acute bacterial meningitis and decrease after antimicrobial therapy, and that plasma nuclear and mitochondrial DNA levels can predict treatment outcomes. Methods: We examined serial plasma nuclear and mitochondrial DNA levels in 22 adult community-acquired bacterial meningitis (ACABM) patients. The plasma nuclear and mitochondrial DNA levels were also evaluated in 11 aseptic meningitis patients and 22 volunteer subjects during the study period. Results: All of the both bacterial and aseptic meningitis groups had a higher plasma DNA levels on admission as compared with those of volunteer groups. Levels of plasma nuclear and mitochondrial DNA in ACABM cases were significantly increased initially and substantially decreased thereafter. Both plasma nuclear DNA and plasma mitochondrial DNA levels at presentation are significantly negative correlate with modified Barthel Index (average) ( r = −0.639, P = 0.004 and r = −0.551, P = 0.018) at 3 months after discharge (average), respectively, in this study. Both higher plasma nuclear (cutoff value of >169 ng/ml) and mitochondrial DNA levels (cutoff value of >58.9 ng/ml) at presentation were associated with poor outcome in ACABM patients. Conclusions: Based on our results, the higher plasma DNA levels were associated with a poorer outcome. Therefore, we look forward to more prospective multicenter investigations specifically to confirm the predictive value of plasma DNA levels in outcome prediction.

Species delineation using Bayesian model-based assignment tests: a case study using Chinese toad-headed agamas (genus Phrynocephalus)

Abstract: Species are fundamental units in biology, yet much debate exists surrounding how we should delineate species in nature. Species discovery now requires the use of separate, corroborating datasets to quantify independently evolving lineages and test species criteria. However, the complexity of the speciation process has ushered in a need to infuse studies with new tools capable of aiding in species delineation. We suggest that model-based assignment tests are one such tool. This method circumvents constraints with traditional population genetic analyses and provides a novel means of describing cryptic and complex diversity in natural systems. Using toad-headed agamas of the Phrynocephalus vlangalii complex as a case study, we apply model-based assignment tests to microsatellite DNA data to test whether P. putjatia, a controversial species that closely resembles P. vlangalii morphologically, represents a valid species. Mitochondrial DNA and geographic data are also included to corroborate the assignment test results. Assignment tests revealed two distinct nuclear DNA clusters with 95% (230/243) of the individuals being assigned to one of the clusters with > 90% probability. The nuclear genomes of the two clusters remained distinct in sympatry, particularly at three syntopic sites, suggesting the existence of reproductive isolation between the identified clusters. In addition, a mitochondrial ND2 gene tree revealed two deeply diverged clades, which were largely congruent with the two nuclear DNA clusters, with a few exceptions. Historical mitochondrial introgression events between the two groups might explain the disagreement between the mitochondrial and nuclear DNA data. The nuclear DNA clusters and mitochondrial clades corresponded nicely to the hypothesized distributions of P. vlangalii and P. putjatia. These results demonstrate that assignment tests based on microsatellite DNA data can be powerful tools for distinguishing closely related species and support the validity of P. putjatia. Assignment tests have the potential to play a significant role in elucidating biodiversity in the era of DNA data. Nonetheless, important limitations do exist and multiple independent datasets should be used to corroborate results from assignment programs.

Karyological and Nuclear DNA Content Variation in Some Iranian Endemic Thymus Species (Lamiaceae)

Abstract: Thymus is a medicinal plant which contains one of the world's top ten essential oils, exhibiting antibacterial, antioxidative, food preservative and mammalian age-delaying properties. This work was aimed at identifying between-species variations requiring for selecting appropriate parents for hybridization. Six Iranian endemic Thymus accessions belonging to Thymus daenensis , T. eriocalyx and T. migricus were studied. Root tips were examined for karyological studies and fresh young leaves of the standard reference (Parsley, Petroselinum crispum , 2C DNA= 4.45 pg) and the Thymus samples stained with propidium iodide (PI) for flow cytometric (FCM) measurements. Two ploidy levels (diploid and tetraploid) and 3 chromosome numbers (30, 56, 60) were recognized. The latter chromosome number is being reported for the first time on T. daenensis accession. FCM measurements showed that 2C DNA contents varied from 1.02 to 2.42 pg, verifying more than 2-fold variations and showing a genome size range of 499 to 1182 Mbp, correspondingly. The mean amount of 2C DNA/chromosome and mean of monoploid genome size were not proportional to ploidy. 2C-values were correlated with, and linearly regressed upon somatic metaphase, considering either total chromosome volume (TCV) or total chromatin length (X).

Replication of Nuclear DNA

Abstract: In common with other eukaryotes, plant DNA is organised for replication as multiple replicons. A key event is the recognition of origins by the origin-recognition complex (ORC). Despite earlier indications, it is now likely that as in mammals, plant replication origins are not defined strictly by sequence; it is thus important to ascertain what features of origins are recognised by the ORC. Activation of origins occurs by stepwise loading of the pre-replication complex, all components of which have been identified in plants. This step is important in restricting DNA replication to once per cell cycle, although in plants, this restriction is relatively easily overcome, thus permitting DNA endoreduplication. It is also important to note the flexibility of origin use in relation to aspects of plant development. Is this flexibility mediated by ORC binding or at the pre-replication step? Following origin activation, the origin is prepared for initiation, again by the stepwise loading of several proteins, including the initiating DNA polymerase, polymerase-α-primase. The last pre-initiation step is strand separation by GINS-CDC45-MCM2-7 to generate a replication bubble. Synthesis itself is initiated by DNA polymerase-α-primase (which has been extensively researched in plants). This then “hands over” to DNA polymerase-e on the leading strand and to DNA polymerase-δ on each Okazaki fragment. Polymerases -α, -δ and -e are three of the 14 DNA polymerases known at protein and/or gene level in plants. Intriguingly, polymerase-β (a repair enzyme in animals) is associated in plants with DNA endoreduplication, in which replication is repeated without an intervening mitosis. Whether synthesised during replication or endoreduplication, the newly synthesised strands are completed by the ligation of Okazaki fragments and replicon-length pieces by DNA ligase 1. What will now be especially interesting is the integration of these molecular events into higher levels of control, such as the specification of meristem identity.

Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells.

Abstract: This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real-time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF-exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty-four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis. Bioelectromagnetics 31:630–639, 2010. © 2010 Wiley-Liss, Inc.