Alternating current voltammetric determination of DNA damage

The conditions for alternating current (a.c.) voltammetric DNA determinations have been investigated with respect to its use with alkaline filter elution techniques at low DNA concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of DNA segments; peak II around -1.2 V caused by the native DNA (nDNA) form; peak III caused by denatured DNA (dDNA) at -1.4 V. Sonication of nDNA increases the peak current, however not with dDNA. Both dDNA and nDNA give linear peak current increments with DNA increments, their regression lines cutting the concentration axis at the origin. In filter elution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of DNA. It turns out that an unknown substance, perhaps a protein or RNA, elutes from the filters and gives rise to a current peak at about -1.3 V. This substance can interfere with the dDNA by competing for electrode surface area, since it diffuses much faster than the large molecules of the DNA. Since however, dDNA has a higher affinity for the electrode surface, after enough time, usually few minutes, the dDNA increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication, so that it will no longer interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". When EA is present at short accumulation times it prevents dDNA adsorption. By adding dDNA, the EA can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the DNA present. The conditions for voltammetric DNA determination have been investigated obeying the recognized interactions. Avoiding organic bases and using inorganic ones would simplify the determination procedure. The reproducibility of the procedure in the range of 50-60 ng DNA/ml has been found to be +/- 6%.     

Antibodies to DNA in the Blood of Patients with Tick-Borne Encephalitis

The relative levels of autoantibodies to native and denatured DNA (nDNA and dDNA, respectively) in the blood were compared in 55 patients with tick-borne encephalitis (TBE). Compared to healthy donors, the titers of antibodies (Ab) to nDNA and dDNA were significantly higher in 31% and 40% of TBE patients, respectively. The proportion of patients with an increased concentration of anti-nDNA Ab in the case of TBE (32%) was higher than among patients with multiple sclerosis (18%) and some other autoimmune diseases (6–18%) but comparable with that among patients with systemic lupus erythematosus (38%) and polymyositis (42%). In contrast to the patients with systemic lupus erythematosus and multiple sclerosis, the level of antibodies to nDNA in TBE patients was higher than that of anti-dDNA Ab. The coefficients of correlation between the levels of Ab to nDNA and dDNA were estimated for the entire group of TBE patients and for subgroups with different forms of the disease (temperature reactions, febrile form, and meningeal form). Analysis of correlation between the anti-DNA antibody titers and three standard biochemical markers of TBE (aspartate aminotransferase and alanine aminotransferase activities and total bilirubin) was carried out for 22 patients. A statistically significant correlation was revealed only between the level of Ab to nDNA and the marker enzyme activities, with the respective correlation coefficients being +0.44 and +0.48, respectively.     

Interaction of carminomycin with DNA according to data of laser polarized fluorescence

Anti-tumour antibiotic carminomycin interaction with chicken erythrocyte DNA is studied in aqueous-salt solutions by the laser polarized fluorescence method. Fluorescence quenches almost equally effectively during the antibiotic absorption on native (nDNA) and denatured (dDNA) DNAs, but the polarization degree of residual fluorescence differs about two times. Carminomycin binding to dDNA is characterized by one interaction type with a large density of occupancy sites - one antibiotic molecule per base pair. Carminomycin forms two types of complexes with nDNA, differing significantly with binding constants. Strong binding, intercalation, is saturated at one carminomycin molecule per 3 base pairs independently on the solution ionic strength. The weaker, external, interaction is characterized by the binding constant being by two orders of magnitude lower than that for intercalation, and the external interaction contribution is negligible.     

Antibodies to DNA in the blood of patients with tick-borne encephalitis

A comparison of relative levels of autoantibodies (Abs) to both native (n) and denatured (d) DNA in the blood of 55 patients with tick-borne encephalitis (TBE) was carried out. 31% of patients with TBE was shown to have an increased level Abs to nDNA and 40% of patients demonstrate increased level of Abs to dDNA. The percent of TBE patients with increased concentration of anti-nDNA Abs higher then that in patients with multiple sclerosis (18%) and some other autoimmune diseases (6-18%), but comparable with that for patients with systemic lupus erythematosus (38%) and polymyositis (42%). In contrast to patients with systemic lupus erythematosus and multiple sclerosis, the level of Abs to nDNA in patients with TBE is higher than the level of Abs to dDNA. Correlation coefficients of Ab levels to both n- and dDNA were estimated for group of patients in whole and for separate subgroups with different type of disease (temperature reaction, feverishness and meningitis). Analysis of correlation between titres of anti-DNA Abs and three standard biochemical markers of TBE (activity of aspartate- and alanine-aminotransferases, and concentration of whole bilirubin) for 22 patients with TBE was carried out. Statistically significant correlation was revealed only between the level of Abs to nDNA and activities of aspartate- and alanine-aminotransferases, correlation coefficients are equal to +0.44 and +0.48, respectively.     

Quantitative aspects of lupus anti-DNA autoantibody specificity

In this study we have attempted to define the cross-reactive potential of SLE anti-DNA antibodies (in 19 representative sera and plasmas) in both the solution phase and the solid phase. We used the Farr and RBC-CF solution phase assays to measure quantitatively the ability of a variety of negatively charged structurally unrelated molecules to inhibit antibody binding to both native DNA (nDNA) and denatured DNA (dDNA). The inhibitors used were of two types: 1) phospholipids (cardiolipin, phosphatidyl glycerol, and phosphatidic acid) and 2) repeating negatively charged molecules (poly-glutamic acid, heparin sulfate, and chondroitin sulfate). We found in both assays that the phospholipids could inhibit antibody binding to nDNA and dDNA, but a large excess (about 1500-fold) of these molecules was needed relative to DNA to achieve equivalent levels of inhibition. The repeating negatively charged molecules did not inhibit DNA binding at equivalent molar levels as the phospholipids; generally, at least a 10,000-fold excess was needed relative to the nucleic acids to achieve any appreciable inhibition. Results of a dDNA binding-inhibition solid-phase ELISA for cross-reactivity of the anti-DNA antibodies gave quite similar results. Finally, we found that eight of the SLE samples did have anti-cardiolipin antibodies, as demonstrated in a cardiolipin-based ELISA. These results suggest that previous reports describing an apparent cross-reactivity of anti-DNA antibodies may not represent physiologically relevant interactions between anti-DNA antibodies and non-nucleic acid antigens.     

Diversity and metabolism of marine bacteria cultivated on dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Diversity and Metabolism of Marine Bacteria Cultivated on Dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Hybridoma anti-DNA autoantibodies from patients with rheumatoid arthritis and systemic lupus erythematosus demonstrate similar nucleic acid binding characteristics

Hybridoma anti-DNA antibodies have been generated from the fusion of the GM 4672 lymphoblastoid line with peripheral blood lymphocytes from four normal subjects, nine patients with rheumatoid arthritis (RA), and 13 patients with systemic lupus erythematosus (SLE). A total of 441 hybridoma clones were obtained, of which 37 secreted anti-DNA autoantibodies. The nucleic acid binding characteristics of the anti-DNA antibodies produced by two hybridomas from normal subjects, nine hybridomas from RA patients, and 18 hybridomas from SLE patients are reported. The hybridoma anti-DNA antibodies from all three groups showed similar antigen-binding characteristics for denatured DNA (dDNA), native DNA (nDNA), poly(I), poly(dT), and cardiolipin, by both direct binding and competitive binding analyses. One difference noted between normal-derived anti-DNA antibodies and autoimmune-derived antibodies was the inability of the former to react with z-DNA. However, this requires further substantiation with larger numbers of normal-derived clones. The broad overlap of reactivity to nucleic acid antigens among individual anti-DNA autoantibodies found in two clinically different autoimmune diseases, namely RA and SLE, suggests that the pathogenicity of anti-DNA autoantibodies may bear no relationship to their nucleic acid antigen-binding characteristics.     

Physical map of the origin of defective DNA in herpes simplex virus type 1 DNA.

The origin of defective DNA (dDNA) of the Patton strain of herpes simplex virus type 1 (HSV-1) was physically mapped with BamHI in the parental DNA. The dDNA obtained from virus passaged at high multiplicities of infection was resistant to cleavage with HindIII, whereas digestion with EcoRI yielded a cluster of fragments 5.4 to 5.7 megadaltons (Mdal) in size. Cleavage with BamHI gave a cluster of fragments 2.6 to 3.2 Mdal in size, plus two homogeneous, comigrating 1-Mdal fragments. One of the latter fragments contained the single EcoRI site approximately 65 base pairs from one end. Hybridization of in vitro labeled dDNA probe to EcoRI, HindIII, BamHI, and Hpa I digests of nondefective HSV-1 DNA demonstrated that, in addition to the S-region terminal repeat, only one end of the S region was involved in the generation of this class of dDNA. Thus, the dDNA probe did not hybridize to either the S region 3.0-Mdal HindIIIN fragment or a 3.0-Mdal BamHI fragment of the adjacent 8.7-Mdal HindIIIG fragment, but did hybridize to four BamHI fragments of HindIII G (approximately 5.7 Mdal). The cluster of 2.6- to 3.2-Mdal fragments obtained with BamHI digestion of dDNA appears to represent a novel junction between the termination of dDNA adjacent to the 3.0-Mdal BamHI fragment in HindIII G and the 2.0- to 2.3-Mdal BamHI fragment terminal in HSV-1 DNA.     

Environmental dissolved DNA harbours meaningful biological information on microbial community structure

Extracellular DNA (eDNA) comprises all the DNA molecules outside cells. This component of microbial ecosystems may serve as a source of nutrients and genetic information. Hypersaline environments harbour one of the highest concentrations of eDNA reported for natural systems, which has been attributed to the physicochemical preservative effect of salts and to high viral abundance. Here, we compared centrifugation and filtration protocols for the extraction of dissolved DNA (dDNA, as opposed to eDNA that also includes DNA from free viral particles) from a solar saltern crystallizer pond (CR30) water sample. The crystallizer dDNA fraction has been characterized, for the first time, and compared with cellular and viral metagenomes from the same location. High-speed centrifugation affected CR30 dDNA concentration and composition due to cell lysis, highlighting that protocol optimization should be the first step in dDNA studies. Crystallizer dDNA, which accounted for lower concentrations than those previously reported for hypersaline anoxic sediments, had a mixed viral and cellular origin, was enriched in archaeal DNA and had a distinctive taxonomic composition compared to that from the cellular assemblage of the same sample. Bioinformatic analyses indicated that nanohaloarchaeal viruses could be a cause for these differences.     

Synchronous synthesis of DNA in coleoptiles and the initial leaf of developing etiolated wheat shoots: the nature and correlation of nuclear and mitochondrial DNA syntheses

In etiolated coleoptiles and initial leaf of developing wheat shoots the DNA synthesis is periodical and synchronous. In the initial leaf each step of DNA synthesis results in a stepwise increase of DNA content and is doubled at the first three steps. During the leaf plane formation the synthesis of nuclear DNA (nDNA) is decreased, while that of mitochondrial DNA (mitDNA) continues in synchronous cycles. This is the cause of relative stabilization of DNA content per unit of leaf plane length. The DNA increase in this organ occurs due to synchronous synthesis of nDNA and mitDNA in intercalary meristem cells. In coleoptiles a marked replication of nDNA is observed at the first three steps of the synthesis; in each cycle nDNA synthesis precedes that of mitDNA. With completion of coleoptile formation the nDNA synthesis in it practically ceases, whereas that of mitDNA continues in synchronous cycles. MitDNA is non-methylated and its composition (56 mol.% GC) differs significantly from that of the newly synthesized nDNA (44 mol.% GC; 100 X m5C/(C + m5C) = 16-17%). It may be concluded that in various organs of wheat shoots the composition and methylation of newly synthesized DNA depend on the age of the shoot and on the ratio of nDNA/mitDNA syntheses.     

Mechanism of action of DNA-hydrolyzing antibodies to DNA from blood of patients with systemic lupus erythematosus

Four fractions of IgG antibodies to native DNA (nDNA) were obtained from blood of patients with systemic lupus erythematosus (SLE). These antibodies displayed a thermostable DNA-hydrolyzing activity and were different in affinity for DNA-cellulose and sorption on DEAE-cellulose. DNA-hydrolyzing antibodies to nDNA are metal-dependent endonucleases, cause mainly single-strand breaks in DNA, and are active over a wide range of pH. By atomic-force microscopy, three-dimensional images of DNA complexes with DNA-hydrolyzing antibodies to nDNA were obtained with nanometer resolution, and a nonprocessive action mechanism was shown for the DNase activity of antibodies to nDNA.     

Disturbed population genetics: suspected introgressive hybridization between two Mnais damselfly species (Odonata)

Mnais costalis and M. pruinosa are damselflies (Odonata: Calopterygidae) with low dispersal abilities, both during their aquatic stream-living immature stage and their flying adult stage. A previous nuclear DNA (nDNA) sequencing and morphology study showed that these two species are very closely related, and cohabit widely in western Japan. The two species, however, segregate microhabitats along a stream: M. costalis lives in the lower reaches, and M. pruinosa in the upper reaches. In this study, our analyses were based on mitochondrial DNA (mtDNA), which usually mutates faster and is more variable among individuals than nDNA, and which is inherited maternally. We found that most COI haplotypes were shared between the two species, and that for most study sites interspecific riverine genetic structures were not clarified by mtDNA analysis. Incongruent population genetic structures based on nDNA and mtDNA suggested hybridization and introgression of mtDNA between the two species.     

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100-0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.     

Accumulation of nuclear DNA damage or neuron loss: molecular basis for a new approach to understanding selective neuronal vulnerability in neurodegenerative diseases

According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.     

Nuclear and mitochondrial DNA repair: similar pathways?

Mitochondrial DNA (mtDNA) alterations are implicated in a broad range of human diseases and alterations of the mitochondrial genome are assumed to be a result of its high susceptibility to oxidative damage and its limited DNA repair compared to nuclear DNA (nDNA). Characterization of DNA repair mechanisms has generally focused on these processes in nDNA but increasing interest and research effort have contributed to our knowledge of the mechanisms underlying DNA repair in mitochondria. In this review, we make comparisons between nDNA and mtDNA repair pathways and propose a model for how these pathways interact in mitochondria.     

Genetic diversity of the Andean tetraploid cultivated potato (Solanum tuberosum L. subsp. andigena Hawkes) evaluated by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are native farmer-selected important cultivars that form a primary gene pool of the common potato (Solanum tuberosum L. subsp. tuberosum). The genetic diversity of 185 Andigena accessions and 6 Chilean native potatoes (S. tuberosum subsp. tuberosum) was studied using chloroplast DNA (ctDNA) microsatellites and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Andigena potatoes had 14 ctDNA haplotypes and showed higher variability in the central Andes, particularly in Bolivia, whereas those in the northern regions of the distribution area were remarkably uniform with A1 ctDNA and Chilean subsp. tuberosum with T ctDNA. Most of 123 clearly scored RFLP bands using 30 single-copy probes were randomly distributed throughout the distribution area and proved the same gene pool shared among these widely collected accessions. Nevertheless, the geographic trend of the nDNA differentiation from north to south along the Andes and the correlated differentiation between nDNA and ctDNA (r = 0.120) could also be revealed by canonical variates analysis. These results suggest that the genetic diversity in Andigena was brought about primarily from cultivated diploid species but considerably modified through sexual polyploidization and intervarietal and (or) introgressive hybridization and long-distance dispersal of seed tubers by humans.     

Antigen binding diversity of affinity purified autoantibodies against DNA.

Naturally occurring autoantibodies against native DNA (nDNA) in SLE sera showed diverse antigen binding characteristics. The antibodies isolated by affinity chromatography using nDNA linked to Sepharose 4B exhibited specificity towards nDNA and showed strong reactivity with DNA-psoralen photoadduct by direct binding assay and competitive ELISA. The anti-DNA antibody belong to both IgG and IgM immunoglobulin classes and their ratio was 5:1. The possible significance of altered conformation of nDNA in the etiology of SLE has been discussed.     

DNA-binding proteins of mammalian mitochondria

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.     

Origin and evolution of Andigena potatoes revealed by chloroplast and nuclear DNA markers.

Andigena potatoes (Solanum tuberosum L. subsp. andigena Hawkes) (2n = 4x = 48) are important, native-farmer-selected cultivars in the Andes, which form a primary gene pool for improving a worldwide grown potato (S. tuberosum subsp. tuberosum). To elucidate the origin of Andigena, 196 Andigena accessions were compared with 301 accessions of 33 closely related cultivated and wild species using several types of chloroplast DNA (ctDNA) markers and nuclear DNA (nDNA) restriction fragment length polymorphism (RFLP) markers. Fourteen ctDNA types (haplotypes) and 115 RFLP bands were detected in Andigena, of which the main haplotypes and frequent RFLP bands were mostly shared with a cultivated diploid species, S. stenotomum Juz. et Buk. Principal component analysis of nDNA polymorphisms revealed a progressive and continuous variation from Peruvian wild species with C-type ctDNA to a group of wild species having S-type ctDNA in its variation range (S. bukasovii, S. canasense, S. candolleanum, and S. multidissectum), to cultivated diploid potatoes (S. phureja and S. stenotomum), and to cultivated tetraploid potatoes (Andigena and Chilean S. tuberosum subsp. tuberosum). These results suggest that the initial Andigena population arose with multiple origins exclusively from S. stenotomum. The overall evolutionary process toward the present-day Andigena was discussed.