Immobilization of DNA on microporous membranes using UV-irradiation]

Various techniques of DNA immobilization onto nitrocellulose and nylon microporous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10(-4). UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc = (22.3 +/- 4.8) c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8-1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.     

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.     

Potential repair of Escherichia coli DNA following exposure to UV radiation from both medium- and low-pressure UV sources used in drinking water treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm(2) from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm(2) from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37 degrees C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm(2). This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.     

Photo-regulation of DNA/RNA duplex formation by azobenzene-tethered DNA towards antisense strategy.

Modified DNA carrying an azobenzene was successfully applied to the photo-regulation of DNA/RNA hybridization. When the azobenzene was isomerized from trans- to cis-form on UV-irradiation, the melting temperature of the duplex was significantly lowered. This process was totally reversible so that the Tm increased by cis-->trans isomerization induced by visible light irradiation.     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

Potential Repair of Escherichia coli DNA following Exposure to UV Radiation from Both Medium- and Low-Pressure UV Sources Used in Drinking Water Treatment

The increased use of UV radiation as a drinking water treatment technology has instigated studies of the repair potential of microorganisms following treatment. This study challenged the repair potential of an optimally grown nonpathogenic laboratory strain of Escherichia coli after UV radiation from low- and medium-pressure lamps. Samples were irradiated with doses of 5, 8, and 10 mJ/cm² from a low-pressure lamp and 3, 5, 8, and 10 mJ/cm² from a medium-pressure UV lamp housed in a bench-scale collimated beam apparatus. Following irradiation, samples were incubated at 37°C under photoreactivating light or in the dark. Sample aliquots were analyzed for up to 4 h following incubation using a standard plate count. Results of this study showed that E. coli underwent photorepair following exposure to the low-pressure UV source, but no repair was detectable following exposure to the medium-pressure UV source at the initial doses examined. Minimal repair was eventually observed upon medium-pressure UV lamp exposure when doses were lowered to 3 mJ/cm². This study clearly indicates differences in repair potential under laboratory conditions between irradiation from low-pressure and medium-pressure UV sources of the type used in water treatment.     

Survival and DNA Repair of Somatic Cell Hybrids after Ultraviolet Irradiation

THE technique of somatic cell hybridization has opened up studies on genetic regulation¹ and human genetic analysis^2–5. Hybrid cells are isolated in conditions that select against parental cells while allowing hybrids to survive by genomic complementation. In xeroderma pigmentosum (XP), a human disease with an autosomal recessive defect in an early stage of DNA repair⁶, the skin is extremely sensitive to sunlight in vivo⁷ and skin fibroblasts show sharply reduced survival following ultraviolet irradiation in vitro^8,9. This communication concerns the use of ultraviolet irradiation in combination with a chemical method to produce hybrids between fibroblasts from XP and a hamster line, followed by analysis of these cells for their capacity to survive and repair DNA after exposure to ultraviolet. Methods for initiation and propagation of skin fibroblasts from two subjects, male and female siblings with XP, have been described⁸. Details on the origin of the TG2 line of golden hamster fibroblasts, which has a non-reverting mutation in the gene for hypoxanthine-guanine phosphoribosyltransferase (HGPRT), the general hybridization procedure¹⁰ and methods for cell survival and DNA repair by unscheduled synthesis⁸ were also described previously. Hybrids were produced by fusion with Sendai virus and selected by ultraviolet irradiation followed by culture on HAT medium (Fig. 1).     

DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae

Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 gamma irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following gamma irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by gamma irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after gamma irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.     

Large scale preparation of DNA for vast DNA excess hybridization

A method for the preparation of relatively large quantities of sonicated DNA for use in the vast DNA excess hybridization technique is reported. The procedure, applied here to rat liver DNA, involves centrifugation of DNA from sonicated nuclei to equilibrium in a CsCl gradient, RNase and pronase digestion, and phenol extraction. Compared with DNA purified by the Marmur procedure, the product has a lower contamination with protein and RNA, takes about one third the time to prepare, and has better hybridization characteristics.     

High-sensitivity hybridization assay for quantitation of residual E. coli DNA

Impurity assays for recombinant protein therapeutics are essential to ensure batch-to-batch consistency and to meet the FDA's criteria for a well-characterized biopharmaceutical. For determination of residual host cell DNA, membrane hybridization assays utilizing radiolabeled DNA probes prepared from the host cell's genomic DNA have traditionally been used for products derivedfrom bacterial expression systems to obtain the required low picogram sensitivity. Nonradioactive methods, while desirable to eliminate radioactive waste disposal and safety issues, typically suffer from poor sensitivity and high backgrounds. We report the development of a suitably sensitive, nonradioactive assay to quantitate residual E. coli DNA levels in purified protein drugs by means of a slot-blot hybridization method. The assay utilizes digoxigenin-labeled E. coli DNA probes and SuperSignal chemiluminescent substrate. The optimized chemiluminescent hybridization assay has both low background and high sensitivity, allowing routine detection of 2.5 pg E. coli DNA. The method can be tailored for detection/quantitation of DNA contamination in recombinant protein products expressed in E. coli or other bacterial expression systems.     

The recovery of normal DNA replication kinetics in ultraviolet-irradiated Chinese hamster cells

The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m², the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation.     

Effects of homogeneous and inhomogeneous static magnetic fields combined with gamma radiation on DNA and DNA repair

The aim of this study was to reveal whether static magnetic fields (SMFs) influence the repair of radiation‐damaged DNA on leukocytes or has any effect on DNA. After 4 Gy of ⁶⁰Co‐γ irradiation, some of the samples were exposed to inhomogeneous SMFs with a lateral magnetic flux density gradient of 47.7, 1.2, or 0.3 T/m by 10 mm lateral periodicity, while other samples were exposed to homogeneous SMF of 159.2 ± 13.4 mT magnetic flux density for a time period of 0.5 min, 1, 2, 4, 6, 18, 20, or 24 h. Another set of samples was exposed to the aforementioned SMFs before gamma irradiation. The following three groups were examined: (i) exposed to SMF only, (ii) exposed to SMF following irradiation by ⁶⁰Co‐γ, and (iii) exposed to SMF before ⁶⁰Co‐γ irradiation. The analysis of the DNA damage was made by single‐cell gel electrophoresis technique (comet assay). Statistically significant differences were found at 1 h (iSMF), 4 h (hSMF), and 18 h (hSMF) if samples were exposed to only SMF, compared to control. When the SMF exposure followed the ⁶⁰Co‐γ irradiation, statistically significant differences were found at 1 h (iSMF) and 4 h (hSMF). If exposure to SMF preceded ⁶⁰Co‐γ irradiation, no statistically significant difference was found compared to 4 Gy gamma‐irradiated group. Bioelectromagnetics 31:488–494, 2010. © 2010 Wiley‐Liss, Inc.     

Impact of enrichment conditions on cross‐species capture of fresh and degraded DNA

By combining high‐throughput sequencing with target enrichment (‘hybridization capture’), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination‐rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter – hybridization temperature – on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect^? arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on‐target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments.     

DNA repair in cultured human fibroblasts does not decline with donor age

Human fibroblasts from young (3 days to 3 years) and old (84–94 years) donors were tested for their ability to repair DNA damage by measuring survival of colony formation following irradiation with ultraviolet (UV) light. Repair was also measured by the ability to reactivate herpes simplex virus following treatment of the virus with UV light, methyl methane sulfonate or 4,5′,8-trimethylpsoralen plus light. This virus was used as a probe of cellular repair capacity because survival of damaged virus is lower in repair-deficient cell lines [1]. Cell lines from both age groups exhibited comparable survivals following UV irradiation and failed to show increased sensitivity to irradiation in the presence of caffeine. Cells from both groups repaired damaged virus to equal extents. Proficient viral repair was observed under conditions in which cells were infected by either single or multiple viral genomes. These results suggest that DNA repair mechanisms which act on a variety of lesions (e.g. pyrimidine dimers, apurinic sites, alkylated bases, cross-links, etc.) do not decline with age. A model for biological aging resulting from the accumulation with age of unrepaired DNA damage is discussed.     

Identification of bacterial DNA markers for the detection of human fecal pollution in water

We used genome fragment enrichment and bioinformatics to identify several microbial DNA sequences with high potential for use as markers in PCR assays for detection of human fecal contamination in water. Following competitive solution-phase hybridization of total DNA from human and pig fecal samples, 351 plasmid clones were sequenced and were determined to define 289 different genomic DNA regions. These putative human-specific fecal bacterial DNA sequences were then analyzed by dot blot hybridization, which confirmed that 98% were present in the source human fecal microbial community and absent from the original pig fecal DNA extract. Comparative sequence analyses of these sequences suggested that a large number (43.5%) were predicted to encode bacterial secreted or surface-associated proteins. Deoxyoligonucleotide primers capable of annealing to a subset of 26 of the candidate sequences predicted to encode factors involved in interactions with host cells were then used in the PCR and did not amplify markers in DNA from any additional pig fecal specimens. These 26 PCR assays exhibited a range of specificity in tests with 11 other animal sources, with more than half amplifying markers only in specimens from dogs or cats. Four assays were more specific, detecting markers only in specimens from humans, including those from 18 different human populations examined. We then demonstrated the potential utility of these assays by using them to detect human fecal contamination in several impacted watersheds.     

Variation in the ribosomal DNA intergenic spacer of a maize population mass-selected for high grain yield

Summary Variation in the intergenic spacer of ribosomal DNA (rDNA) was detected among individual plants of the open-pollinated maize variety Hays Golden and populations derived from this variety. rDNA intergenic spacer-length variants were detected at approximately 200 bp intervals, consistent with the number of 200 bp subrepeats as the basis for this variation. Inheritance data revealed that more than one spacer-length class may be present on an individual chromosome. Fourteen different predominant rDNA intergenic spacer hybridization fragment patterns were detected. C-29, a population developed by 29 cycles of mass-selecting Hay Golden for high grain yield, exhibited a significant change in rDNA intergenic spacer hybridization fragment pattern composition in comparison to Hays Golden. This change included a reduction in frequency of the shortest predominant space-length variant (3.4 kb) and an increase in a 5.2 -kb hybridization fragment. I-31, a population developed through thermal neutron irradiation of Hays Golden and 31 generations of mass selection for high grain yield, did not exhibit a significant change in overall rDNA intergenic spacer composition. I-31 did exhibit an increase in frequency of the 5.2-kb hybridization fragment and a significant change in two specific hybridization fragment patterns that had also changed in C-29. These data, particularly for the C-29 population, suggest that rDNA intergenic spacer-length variants and/or associated loci were influenced by selection.Paper No. 8701, Journal Series, Nebraska Agricultural Research Division     

Mutant copies of mitochondrial DNA in tissues and plasma of mice subjected to X-ray irradiation

Impairments of mitochondrial genome are associated with a wide spectrum of degenerative diseases, development of tumors, aging, and cell death. We studied the content of mitochondrial DNA (mtDNA) with mutations and the total content of mutations in the brain and the spleen of mice subjected to X-ray irradiation at a dose of 1–5 Gy at 8–28 days after treatment. In these mice, we studied the number of mutant copies of extracellular mtDNA (ec-mtDNA) and its total content in blood plasma. We estimated mutations in control and irradiated mice using cleavage of heteroduplexes prepared by hybridization of PCR amplicons of mtDNA (D-loop region) mediated by CEL-I endonuclease, an enzyme that specifically cleaves unpaired bases. Changes in the total number of mtDNA copies relative to nuclear DNA were assessed by real time PCR using the ND-4 and GAPDH genes, respectively. We found that the number of mutant mtDNA copies was significantly increased in the brain and the spleen of irradiated mice and reached the maximum level at the eighth day after treatment; it then decreased by the 28th day after treatment. In nuclear genes similar to mutagenesis, mutagenesis of mtDNA in the brain and spleen tissues linearly depended on irradiation dose. In contrast to mutant nuclear genes, most mutant mtDNA copies were eliminated in the brain and spleen tissues, whereas the total content of mtDNA did not change within 28 days after irradiation. Our data show that, during this period, a high level of ec-mtDNA with mutations was observed in DNA circulating in blood plasma with the maximum level found at the 14th day. We suppose that mutant mtDNA copies are eliminated from cells of animals subjected to irradiation during the posttreatment period. Higher content of ec-mtDNA in blood plasma can be considered as a potential marker of radiation damage to the body.