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Sample records for dna fragment size

  1. DNA fragment sizing and sorting by laser-induced fluorescence

    DOEpatents

    Hammond, Mark L.; Jett, James H.; Keller, Richard A.; Marrone, Babetta L.; Martin, John C.

    1996-01-01

    A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

  2. Sizing of single fluorescently stained DNA fragments by scanning microscopy

    PubMed Central

    Laib, Stephan; Rankl, Michael; Ruckstuhl, Thomas; Seeger, Stefan

    2003-01-01

    We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates. PMID:14602931

  3. DNA fragment sizing and sorting by laser-induced fluorescence

    SciTech Connect

    Jett, J.H.; Hammond, M.L.; Keller, R.A.; Marrone, B.L.; Martin, J.C.

    1992-12-31

    A method is provided for obtaining DNA fingerprints using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a selected sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is directly proportional to the fragment length. Additional dyes can be bound to the DNA piece and DNA fragments to provide information additional to length information. Oligonucleotide specific dyes and/or hybridization probes can be bound to the DNA fragments to provide information on oligonucleotide distribution or probe hybridization to DNA fragments of different sizes.

  4. Single molecule detection: Applications to sizing of DNA fragments

    SciTech Connect

    Petty, J.T.; Johnson, M.E.; Affleck, R.L.

    1994-12-31

    Using, ultrasensitive fluorescence detection and flow cytometry, size determination of ds-DNA fragments is performed using the fluorescence intensity from samples stained with a thiazole orange homodimer TOTO-1. The stained fragments pass through a low-power (30 mW) continuous-wave laser beam. Using transit times of 1-5 ms, data were acquired in times ranging from 1 to 15 mins at a rate of 40 fragments/second. As little as 50 fg of DNA was needed for the analysis. The authors have demonstrated sizing of DNA fragments in the size range from 1.5 to 150 kbp. Future applications of this approach to DNA sizing require that the factors contributing to size resolution be understood, and the authors present simulations to address this issue. To aid in the modeling, the authors have measured the saturation intensity and the relative fluorescence quantum yield of the TOTO-1/DNA complex. Applications to physical mapping of the human genome are being investigated.

  5. Size-selective separation of DNA fragments by using lysine-functionalized silica particles

    NASA Astrophysics Data System (ADS)

    Liu, Lingling; Guo, Zilong; Huang, Zhenzhen; Zhuang, Jiaqi; Yang, Wensheng

    2016-02-01

    In this work, a facile and efficient approach has been demonstrated for size-selective separation of DNA fragments by using lysine-functionalized silica particles. At a given pH, the environmental ionic strength can be utilized to alter the electrostatic interactions of lysine-functionalized silica particles with DNA fragments and in turn the DNA fragments on the silica particle surfaces, which exhibits a clear dependence on the DNA fragment sizes. By carefully adjusting the environmental pH and salt concentration, therefore, the use of the lysine-functionalized silica particles allows effective separation of binary and ternary DNA mixtures, for example, two different DNA fragments with sizes of 101 and 1073 bp, 101 and 745 bp, 101 and 408 bp, respectively, and three different DNA fragments with sizes of 101, 408 and 1073 bp.

  6. Size-selective separation of DNA fragments by using lysine-functionalized silica particles

    PubMed Central

    Liu, Lingling; Guo, Zilong; Huang, Zhenzhen; Zhuang, Jiaqi; Yang, Wensheng

    2016-01-01

    In this work, a facile and efficient approach has been demonstrated for size-selective separation of DNA fragments by using lysine-functionalized silica particles. At a given pH, the environmental ionic strength can be utilized to alter the electrostatic interactions of lysine-functionalized silica particles with DNA fragments and in turn the DNA fragments on the silica particle surfaces, which exhibits a clear dependence on the DNA fragment sizes. By carefully adjusting the environmental pH and salt concentration, therefore, the use of the lysine-functionalized silica particles allows effective separation of binary and ternary DNA mixtures, for example, two different DNA fragments with sizes of 101 and 1073 bp, 101 and 745 bp, 101 and 408 bp, respectively, and three different DNA fragments with sizes of 101, 408 and 1073 bp. PMID:26911527

  7. Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

    PubMed Central

    Goodwin, P M; Johnson, M E; Martin, J C; Ambrose, W P; Marrone, B L; Jett, J H; Keller, R A

    1993-01-01

    Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis. Images PMID:8451182

  8. Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles.

    PubMed

    Chakraborty, R; Li, Z

    1995-01-01

    At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.

  9. Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA

    SciTech Connect

    Chakraborty, R.; Zhong, Y.; Jin, L. ); Budowle, B. )

    1994-08-01

    The authors provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, they show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, they derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. 47 refs., 2 figs., 4 tabs.

  10. Monte Carlo predictions of DNA fragment-size distributions for large sizes after HZE particle irradiation

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.

    2001-01-01

    DSBs (double-strand breaks) produced by densely ionizing space radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. DSB clustering at large scales, from >100 Mbp down to approximately 2 kbp, is modeled using a Monte-Carlo algorithm. A random-walk model of chromatin is combined with a track model, that predicts the radial distribution of energy from an ion, and the RLC (randomly-located-clusters) formalism, in software called DNAbreak. This model generalizes the random-breakage model, whose broken-stick fragment-size distribution is applicable to low-LET radiation. DSB induction due to track interaction with the DNA volume depends on the radiation quality parameter Q. This dose-independent parameter depends only weakly on LET. Multi-track, high-dose effects depend on the cluster intensity parameter lambda, proportional to fluence as defined by the RLC formalism. After lambda is determined by a numerical experiment, the model reduces to one adjustable parameter Q. The best numerical fits to the experimental data, determining Q, are obtained. The knowledge of lambda and Q allows us to give biophysically based extrapolations of high-dose DNA fragment-size data to low doses or to high LETs.

  11. Characterization of DNA size determination of small fragments by flow cytometry

    SciTech Connect

    Petty, J.T.; Johnson, M.E.; Goodwin, P.M.; Martin, J.C.; Jett, J.H.; Keller, R.A.

    1995-05-15

    DNA fragment lengths were determined using the intensity of fluorescent bursts from single fragments stained with a thiazole orange derivative. The individual stained fragments were introduced into a sheath flow cuvette and passed through a low-power (30 mW), continuous-wave laser beam with transit times in the range 3-5 ms. As little as 50 fg of DNA was analyzed at a rate of 40 fragments/s for times ranging from 1 to 15 min. A detectable lower size limit of 1.5 kilobase pairs (kbp) was demonstrated, and a linear relationship between fluorescence intensity and fragment length was observed. Issues relating to size resolution in the 2-50 kbp range are discussed. 25 refs., 7 figs., 1 tab.

  12. Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

    PubMed Central

    Ferris, Matthew M.; Yan, Xiaomei; Habbersett, Robbert C.; Shou, Yulin; Lemanski, Cheryl L.; Jett, James H.; Yoshida, Thomas M.; Marrone, Babetta L.

    2004-01-01

    The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ± 2%) and FCM (4% ± 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSDPFGE ] = 3% ± 2% and RSDFCM = 1.2% ± 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods. PMID:15131156

  13. A polymer, random walk model for the size-distribution of large DNA fragments after high linear energy transfer radiation

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Brenner, D.; Hlatky, L. R.; Sachs, R. K.

    2000-01-01

    DNA double-strand breaks (DSBs) produced by densely ionizing radiation are not located randomly in the genome: recent data indicate DSB clustering along chromosomes. Stochastic DSB clustering at large scales, from > 100 Mbp down to < 0.01 Mbp, is modeled using computer simulations and analytic equations. A random-walk, coarse-grained polymer model for chromatin is combined with a simple track structure model in Monte Carlo software called DNAbreak and is applied to data on alpha-particle irradiation of V-79 cells. The chromatin model neglects molecular details but systematically incorporates an increase in average spatial separation between two DNA loci as the number of base-pairs between the loci increases. Fragment-size distributions obtained using DNAbreak match data on large fragments about as well as distributions previously obtained with a less mechanistic approach. Dose-response relations, linear at small doses of high linear energy transfer (LET) radiation, are obtained. They are found to be non-linear when the dose becomes so large that there is a significant probability of overlapping or close juxtaposition, along one chromosome, for different DSB clusters from different tracks. The non-linearity is more evident for large fragments than for small. The DNAbreak results furnish an example of the RLC (randomly located clusters) analytic formalism, which generalizes the broken-stick fragment-size distribution of the random-breakage model that is often applied to low-LET data.

  14. Extrapolation of the dna fragment-size distribution after high-dose irradiation to predict effects at low doses

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Cucinotta, F. A.; Sachs, R. K.; Brenner, D. J.; Peterson, L. E.

    2001-01-01

    The patterns of DSBs induced in the genome are different for sparsely and densely ionizing radiations: In the former case, the patterns are well described by a random-breakage model; in the latter, a more sophisticated tool is needed. We used a Monte Carlo algorithm with a random-walk geometry of chromatin, and a track structure defined by the radial distribution of energy deposition from an incident ion, to fit the PFGE data for fragment-size distribution after high-dose irradiation. These fits determined the unknown parameters of the model, enabling the extrapolation of data for high-dose irradiation to the low doses that are relevant for NASA space radiation research. The randomly-located-clusters formalism was used to speed the simulations. It was shown that only one adjustable parameter, Q, the track efficiency parameter, was necessary to predict DNA fragment sizes for wide ranges of doses. This parameter was determined for a variety of radiations and LETs and was used to predict the DSB patterns at the HPRT locus of the human X chromosome after low-dose irradiation. It was found that high-LET radiation would be more likely than low-LET radiation to induce additional DSBs within the HPRT gene if this gene already contained one DSB.

  15. Caspase-dependent and serine protease-dependent DNA fragmentation of myocytes in the ischemia-reperfused rabbit heart: these inhibitors do not reduce infarct size.

    PubMed

    Minatoguchi, S; Kariya, T; Uno, Y; Arai, M; Nishida, Y; Hashimoto, K; Wang, N; Aoyama, T; Takemura, G; Fujiwara, T; Fujiwara, H

    2001-10-01

    Some infarcted myocytes undergo caspase-dependent DNA fragmentation, but serine protease-dependent DNA fragmentation may also be involved. There is controversy regarding whether caspase inhibitors can reduce infarct size, so the present study investigated whether serine protease inhibitor can reduce the DNA fragmentation of infarcted myocytes and whether serine protease or caspase inhibitors attenuates myocardial infarct size in Japanese white rabbits without collateral circulation. Rabbits were subjected to 30-min coronary occlusion followed by 48-h reperfusion. A vehicle (dimethylsulfoxide, control group, n=8) or Z-Val-Ala-Asp(Ome)-CH2F (ZVAD-fmk, a caspase inhibitor, ZVAD group, 0.8 mg/kg iv at 20 min before coronary occlusion and 0.8 mg/kg at 90 min after reperfusion, n=8) or 3,4-dichloroisocoumarin (DCI, a serine protease inhibitor, 2 mg/kg iv at 20 min before coronary occlusion, DCI group, n=8) was administered. Animals were killed at 48h after reperfusion for the detection of myocardial infarct size and at 4h after reperfusion for the detection of dUTP nick end-labeling (TUNEL)-positive myocytes, the electrophoretic pattern of DNA fragmentation and ultrastructural analysis. The left ventricle (LV) was excised and sliced. The myocardial infarct size as a percentage of the area at risk was assessed by triphenyltetrazolium chloride staining. DNA fragmentation was assessed by in situ TUNEL at the light microscopic level. ZVAD and DCI significantly reduced the mean blood pressure during reperfusion without affecting heart rate. There was no significant difference in the % area at risk (AAR) of LV among the 3 groups (control: 26.3+/-3.0%; ZVAD: 25.6+/-2.6%; DCI: 25.6+/-2.0%). The % infarct size as a percentage of the AAR in the ZVAD group (41.3+/-4.5%) and the DCI group (50.4+/-3.8%) was not significantly different from the control group (43.5+/-4.5%). However, the percent DNA fragmentation in the infarcted area in the ZVAD (3.5+/-0.8%) and DCI groups (4

  16. Fragment Length of Circulating Tumor DNA

    PubMed Central

    Underhill, Hunter R.; Kitzman, Jacob O.; Hellwig, Sabine; Welker, Noah C.; Daza, Riza; Gligorich, Keith M.; Rostomily, Robert C.; Shendure, Jay

    2016-01-01

    Malignant tumors shed DNA into the circulation. The transient half-life of circulating tumor DNA (ctDNA) may afford the opportunity to diagnose, monitor recurrence, and evaluate response to therapy solely through a non-invasive blood draw. However, detecting ctDNA against the normally occurring background of cell-free DNA derived from healthy cells has proven challenging, particularly in non-metastatic solid tumors. In this study, distinct differences in fragment length size between ctDNAs and normal cell-free DNA are defined. Human ctDNA in rat plasma derived from human glioblastoma multiforme stem-like cells in the rat brain and human hepatocellular carcinoma in the rat flank were found to have a shorter principal fragment length than the background rat cell-free DNA (134–144 bp vs. 167 bp, respectively). Subsequently, a similar shift in the fragment length of ctDNA in humans with melanoma and lung cancer was identified compared to healthy controls. Comparison of fragment lengths from cell-free DNA between a melanoma patient and healthy controls found that the BRAF V600E mutant allele occurred more commonly at a shorter fragment length than the fragment length of the wild-type allele (132–145 bp vs. 165 bp, respectively). Moreover, size-selecting for shorter cell-free DNA fragment lengths substantially increased the EGFR T790M mutant allele frequency in human lung cancer. These findings provide compelling evidence that experimental or bioinformatic isolation of a specific subset of fragment lengths from cell-free DNA may improve detection of ctDNA. PMID:27428049

  17. DNA fragmentation by charged particle tracks

    NASA Astrophysics Data System (ADS)

    Stenerlöw, B.; Höglund, E.; Carlsson, J.

    High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons ( 60Co) or 125 keV/μm nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.

  18. Detection of single lambda DNA fragments by flow cytometry

    SciTech Connect

    Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Keller, R.A. )

    1993-01-01

    The authors have demonstrated flow cytometric detection and sizing of single pieces of fluorescently stained lambda DNA (48.5 kb) and individual Kpn I restriction fragments of lambda DNA at 17.05 kb and 29.95 kb. DNA fragments were stained stoichiometrically with an intercalating dye such that the fluorescence from each fragment was directly proportional to fragment length. Laser powers range from 10 to 100 mW and transit times through the focused laser beam were several milliseconds. Measurements were made using time-resolved single photon counting of the detected fluorescence emission from individual stained DNA fragments. Samples were analyzed at rates of about 50 fragments per second. The measured fluorescence intensities are linearly correlated with DNA fragment length over the range measured. Detection sensitivity and resolution needed for analysis of small pieces of DNA are discussed and a comparison of single photon counting measurements of DNA fragments to measurements using more conventional flow cytometers is made. Applications of this methodology to DNA sizing and DNA fingerprinting are discussed.

  19. DNA studies using atomic force microscopy: capabilities for measurement of short DNA fragments.

    PubMed

    Pang, Dalong; Thierry, Alain R; Dritschilo, Anatoly

    2015-01-01

    Short DNA fragments, resulting from ionizing radiation induced DNA double strand breaks (DSBs), or released from cells as a result of physiological processes and circulating in the blood stream, may play important roles in cellular function and potentially in disease diagnosis and early intervention. The size distribution of DNA fragments contribute to knowledge of underlining biological processes. Traditional techniques used in radiation biology for DNA fragment size measurements lack the resolution to quantify short DNA fragments. For the measurement of cell-free circulating DNA (ccfDNA), real time quantitative Polymerase Chain Reaction (q-PCR) provides quantification of DNA fragment sizes, concentration and specific gene mutation. A complementary approach, the imaging-based technique using Atomic Force Microscopy (AFM) provides direct visualization and measurement of individual DNA fragments. In this review, we summarize and discuss the application of AFM-based measurements of DNA fragment sizes. Imaging of broken plasmid DNA, as a result of exposure to ionizing radiation, as well as ccfDNA in clinical specimens offer an innovative approach for studies of short DNA fragments and their biological functions. PMID:25988169

  20. DNA studies using atomic force microscopy: capabilities for measurement of short DNA fragments

    PubMed Central

    Pang, Dalong; Thierry, Alain R.; Dritschilo, Anatoly

    2015-01-01

    Short DNA fragments, resulting from ionizing radiation induced DNA double strand breaks (DSBs), or released from cells as a result of physiological processes and circulating in the blood stream, may play important roles in cellular function and potentially in disease diagnosis and early intervention. The size distribution of DNA fragments contribute to knowledge of underlining biological processes. Traditional techniques used in radiation biology for DNA fragment size measurements lack the resolution to quantify short DNA fragments. For the measurement of cell-free circulating DNA (ccfDNA), real time quantitative Polymerase Chain Reaction (q-PCR) provides quantification of DNA fragment sizes, concentration and specific gene mutation. A complementary approach, the imaging-based technique using Atomic Force Microscopy (AFM) provides direct visualization and measurement of individual DNA fragments. In this review, we summarize and discuss the application of AFM-based measurements of DNA fragment sizes. Imaging of broken plasmid DNA, as a result of exposure to ionizing radiation, as well as ccfDNA in clinical specimens offer an innovative approach for studies of short DNA fragments and their biological functions. PMID:25988169

  1. DNA sequence from Cretaceous period bone fragments.

    PubMed

    Woodward, S R; Weyand, N J; Bunnell, M

    1994-11-18

    DNA was extracted from 80-million-year-old bone fragments found in strata of the Upper Cretaceous Blackhawk Formation in the roof of an underground coal mine in eastern Utah. This DNA was used as the template in a polymerase chain reaction that amplified and sequenced a portion of the gene encoding mitochondrial cytochrome b. These sequences differ from all other cytochrome b sequences investigated, including those in the GenBank and European Molecular Biology Laboratory databases. DNA isolated from these bone fragments and the resulting gene sequences demonstrate that small fragments of DNA may survive in bone for millions of years.

  2. Microfluidic chip for stacking, separation and extraction of multiple DNA fragments.

    PubMed

    Wu, Ruige; Seah, Y P; Wang, Zhiping

    2016-03-11

    A disposable integrated microfluidic device was developed for rapid sample stacking, separation and extraction of multiple DNA fragments from a relatively large amount of sample. Isotachophoresis hyphenated gel electrophoresis (ITP-GE) was used to pre-concentrate and separate DNA fragments, followed by extraction of pure DNA fragments with electroelution on-chip. DNA fragments of 200bp, 500bp and 1kbp were successfully separated and collected in the extraction chamber within 25min. The extraction efficiency obtained from the chip was 49.9%, 52.1% and 53.7% for 200bp, 500bp and 1kbp DNA fragments, respectively. The extracted DNA fragments exhibited compatibility with downstream enzymatic reactions, for example PCR. The chip was also used to extract DNA fragments with specific size range from sheared genomic DNA and demonstrated similar performance to that using traditional gel cutting method. The whole assay can finish in 32min, 6 times faster than traditional method.

  3. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Neumann, Elizabeth K.; Solouki, Touradj

    2015-08-01

    Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5' phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5' phosphate group in w type ions and 5' phosphorylated DNA to generate sequence permuted [phosphopurine]- fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine]- and complementary y-B type product ions.

  4. DNA Fragmentation in mammalian cells exposed to various light ions

    NASA Astrophysics Data System (ADS)

    Belli, M.; Cherubini, R.; Dalla Vecchia, M.; Dini, V.; Esposito, G.; Moschini, G.; Sapora, O.; Signoretti, C.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/μm protons, 123 keV/μm helium-4 ions and γ-rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respct to that induced by comparable doses of γ-rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for γ-rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage reparability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by γ-rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

  5. Optical selection and collection of DNA fragments

    DOEpatents

    Roslaniec, Mary C.; Martin, John C.; Jett, James H.; Cram, L. Scott

    1998-01-01

    Optical selection and collection of DNA fragments. The present invention includes the optical selection and collection of large (>.mu.g) quantities of clonable, chromosome-specific DNA from a sample of chromosomes. Chromosome selection is based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Although more general procedures may be envisioned, the invention is demonstrated by processing chromosomes in a conventional flow cytometry apparatus, but where no droplets are generated. All chromosomes in the sample are first stained with at least one fluorescent analytic dye and bonded to a photochemically active species which can render chromosomal DNA unclonable if activated. After passing through analyzing light beam(s), unwanted chromosomes are irradiated using light which is absorbed by the photochemically active species, thereby causing photoinactivation. As desired chromosomes pass this photoinactivation point, the inactivating light source is deflected by an optical modulator; hence, desired chromosomes are not photoinactivated and remain clonable. The selection and photoinactivation processes take place on a microsecond timescale. By eliminating droplet formation, chromosome selection rates 50 times greater than those possible with conventional chromosome sorters may be obtained. Thus, usable quantities of clonable DNA from any source thereof may be collected.

  6. The Case for the Median Fragment Size as a Better Fragment Size Descriptor than the Mean

    NASA Astrophysics Data System (ADS)

    Ouchterlony, Finn

    2016-01-01

    Cunningham's use of x 50, the median fragment size, instead of the mean < x > in the main prediction equation of the Kuz-Ram model has several times been pointed out as a mistake. This paper analyses if this mistake is important using dimensional analysis and by reanalyzing the historical Soviet data behind Kuznetsov's original equation for the mean. The main findings in this paper are that: (1) Cunningham's mistake has no proven effect in practice and would only be relevant as long as he used Kuznetsov's equation for the rock factor A, i.e. till 1987. (2) Kuznetsov's equation has its roots in the characteristic size of the Rosin-Rammler (RR) functions fit to the sieving data as a way to determine the mean, not only in the mean itself. (3) The key data set behind Kuznetsov's equation just as easily provides a prediction equation for x 50 with the same goodness of fit as the equation for the mean. (4) Use of x 50 instead of the mean < x > in a dimensional analysis of fragmentation leads to considerable mathematical simplifications because the normalized mass passing at x 50 is a constant number. Non-dimensional ratios like x 50/ x max based on two percentile sizes also lead to such simplifications. The median x 50 as a fragment size descriptor thus has a sounder theoretical background than the mean < x >. It is normally less prone to measurement errors and it is not rejected by the original Soviet data. Thus, Cunningham's mistake has led the rock fragmentation community in the right direction.

  7. Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

    PubMed Central

    Papalian, M; Lafer, E; Wong, R; Stollar, B D

    1980-01-01

    Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity. Images PMID:6153184

  8. Biological and environmental conditionings for a sperm DNA fragmentation.

    PubMed

    Bojar, Iwona; Witczak, Mariusz; Wdowiak, Artur

    2013-01-01

    The objective of the presented study was determination of the effect of selected agents on sperm DNA fragmentation--superoxide dismutase in seminal plasma, the patients' age, and burdening with the tobacco smoking habit. An attempt was also undertaken to evaluate the effect of DNA fragmentation on the effectiveness of infertility treatment. The study covered 186 men who received treatment due to infertility. The database and statistical analyses were performed using computer software STATISTICA 7.1. A relationship was observed between sperm DNA fragmentation and superoxide dismutase activity, the higher the SOD activity, the lower the percentage of sperm fragmentation (rs=-0.324; P=0.000; r = -0.2110). A statistical relationship was found between sperm DNA fragmentation and the percentage of pregnancies obtained during the first year of treatment--patients with the lower DFI more frequently became fathers during the first year of trying, compared to the remainder (t=2.51; P=0.013). A statistically significant relationship was confirmed (rs=-0.370; P=0.000) consisting in an increase in the DFI with respondents' age. No significant differences were noted between the DFI and the tobacco smoking habit (Chi2=0.29; P=0.926). The percentage of sperm DNA fragmentation was inversely proportional to superoxide dismutase activity in seminal plasma. DNA fragmentation becomes intensified with patients' age. Cigarette smoking has no effect on sperm DNA fragmentation. DNA fragmentation exerts an effect on the effectiveness of infertility treatment.

  9. DNA fragmentation induced by ionizing radiation - Atomic Force Microscopy study .

    NASA Astrophysics Data System (ADS)

    Gudowska-Nowak, E.; Psonka, K.; Elsaesser, Th.; Brons, S.; Taucher-Scholz, G.

    DNA as a carrier of genetic information is considered to be the critical target for radiation induced damage Especially severe are DNA double-strand breaks DSBs formed when breaks occur in both strands of the molecule The DSBs production is determined by the spatial distribution of ionization events dependent on the physical properties of the energy deposition and the chemical environment of the DNA According to theoretical predictions high LET charged particle radiation induces lesions in close proximity forming so called clustered damage in the DNA Atomic Force Microscopy AFM was newly established as a technique allowing the direct visualization of DNA fragments resulting from DSBs induced in small DNA molecules plasmids by ionizing radiation We have used AFM to visualize the DNA fragmentation induced by heavy ions high LET radiation and to compare it to the fragmentation pattern obtained after X-rays low LET radiation Plasmid supercoiled DNA was irradiated in vitro with X-rays and 3 9 MeV u Ni ions within a dose range 0 -- 3000 Gy Afterwards the samples were analyzed using AFM which allowed the detection and length measurement of individual fragments with a nanometer resolution Recording of the length of the induced fragments allowed to distinguish between molecules broken by a single DSB or by multiple DSBs The fragment length distributions were derived for different doses and different radiation qualities The first results of the measurement of radiation-induced DNA fragmentation show an influence of radiation quality on

  10. Non-random DNA fragmentation in next-generation sequencing

    NASA Astrophysics Data System (ADS)

    Poptsova, Maria S.; Il'Icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-03-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed ``reads'' are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.

  11. Non-random DNA fragmentation in next-generation sequencing

    PubMed Central

    Poptsova, Maria S.; Il'icheva, Irina A.; Nechipurenko, Dmitry Yu.; Panchenko, Larisa A.; Khodikov, Mingian V.; Oparina, Nina Y.; Polozov, Robert V.; Nechipurenko, Yury D.; Grokhovsky, Sergei L.

    2014-01-01

    Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions. PMID:24681819

  12. Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions

    SciTech Connect

    Braun, B.; Blanch, W.; Prausnitz, J.M.

    1997-02-01

    Because the mechanism of DNA separation in capillary electrophoresis is not well understood, selection of polymers is a {open_quotes}trial-and-error{close_quotes} procedure. We investigated dilute-solution DNA separations by capillary electrophoresis using solutions of four polymers that differ in size, shape and stiffness. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp and star-(polyethylene oxide), like linear poly (ethylene oxide), provides separation of fragments up to 1353 bp.

  13. DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.

    PubMed

    Vernocchi, Valentina; Morselli, Maria Giorgia; Lange Consiglio, Anna; Faustini, Massimo; Luvoni, Gaia Cecilia

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies.

  14. Bacterial natural transformation by highly fragmented and damaged DNA.

    PubMed

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A A; Mayar, J Victor Moreno; Rasmussen, Simon; Dahl, Tais W; Rosing, Minik T; Poole, Anthony M; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-12-01

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥ 20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old.

  15. Bacterial natural transformation by highly fragmented and damaged DNA.

    PubMed

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A A; Mayar, J Victor Moreno; Rasmussen, Simon; Dahl, Tais W; Rosing, Minik T; Poole, Anthony M; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-12-01

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥ 20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old. PMID:24248361

  16. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic A. A.; Mayar, J. Victor Moreno; Rasmussen, Simon; Dahl, Tais W.; Rosing, Minik T.; Poole, Anthony M.; Sicheritz-Ponten, Thomas; Brunak, Søren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G.; Nielsen, Rasmus; Johnsen, Pål Jarle; Nielsen, Kaare Magne; Willerslev, Eske

    2013-01-01

    DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often <100 bp) and may persist in the environment for more than half a million years. Fragmented DNA is recognized as nutrient source for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escape mismatch repair. Moreover, double-nucleotide polymorphisms appear more common among genomes of transformable than nontransformable bacteria. Our findings reveal that short and damaged, including truly ancient, DNA molecules, which are present in large quantities in the environment, can be acquired by bacteria through natural transformation. Our findings open for the possibility that natural genetic exchange can occur with DNA up to several hundreds of thousands years old. PMID:24248361

  17. Clusters of DNA damage induced by ionizing radiation: formation of short DNA fragments. II. Experimental detection

    NASA Technical Reports Server (NTRS)

    Rydberg, B.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    The basic 30-nm chromatin fiber in the mammalian cell consists of an unknown (possibly helical) arrangement of nucleosomes, with about 1.2 kb of DNA per 10-nm length of fiber. Track-structure considerations suggest that interactions of single delta rays or high-LET particles with the chromatin fiber might result in the formation of multiple lesions spread over a few kilobases of DNA (see the accompanying paper: W.R. Holley and A. Chatterjee, Radiat. Res. 145, 188-199, 1996). In particular, multiple DNA double-strand breaks and single-strand breaks may form. To test this experimentally, primary human fibroblasts were labeled with [3H]thymidine and exposed at 0 degrees C to X rays or accelerated nitrogen or iron ions in the LET range of 97-440 keV/microns. DNA was isolated inside agarose plugs and subjected to agarose gel electrophoresis under conditions that allowed good separation of 0.1-2 kb size DNA. The bulk of DNA remained in the well or migrated only a small distance into the gel. It was found that DNA fragments in the expected size range were formed linearly with dose with an efficiency that increased with LET. A comparison of the yield of such fragments with the yield of total DNA double-strand breaks suggests that for the high-LET ions a substantial proportion (20-90%) of DNA double-strand breaks are accompanied within 0.1-2 kb by at least one additional DNA double-strand break. It is shown that these results are in good agreement with theoretical calculations based on treating the 30-nm chromatin fiber as the target for ionizing particles. Theoretical considerations also predict that the clusters will contain numerous single-strand breaks and base damages. It is proposed that such clusters be designated "regionally multiply damaged sites." Postirradiation incubation at 37 degrees C resulted in a decline in the number of short DNA fragments, suggesting a repair activity. The biological significance of regionally multiply damaged sites is presently unknown.

  18. Enzymatic assembly of overlapping DNA fragments.

    PubMed

    Gibson, Daniel G

    2011-01-01

    Three methods for assembling multiple, overlapping DNA molecules are described. Each method shares the same basic approach: (i) an exonuclease removes nucleotides from the ends of double-stranded (ds) DNA molecules, exposing complementary single-stranded (ss) DNA overhangs that are specifically annealed; (ii) the ssDNA gaps of the joined molecules are filled in by DNA polymerase, and the nicks are covalently sealed by DNA ligase. The first method employs the 3'-exonuclease activity of T4 DNA polymerase (T4 pol), Taq DNA polymerase (Taq pol), and Taq DNA ligase (Taq lig) in a two-step thermocycled reaction. The second method uses 3'-exonuclease III (ExoIII), antibody-bound Taq pol, and Taq lig in a one-step thermocycled reaction. The third method employs 5'-T5 exonuclease, Phusion® DNA polymerase, and Taq lig in a one-step isothermal reaction and can be used to assemble both ssDNA and dsDNA. These assembly methods can be used to seamlessly construct synthetic and natural genes, genetic pathways, and entire genomes and could be very useful for molecular engineering tools. PMID:21601685

  19. Cryopreservation increases DNA fragmentation in spermatozoa of smokers.

    PubMed

    Aydin, Mehmet Serif; Senturk, Gozde Erkanli; Ercan, Feriha

    2013-05-01

    Smoking causes subfertility due to deterioration of spermatozoa including decreased concentration and abnormal morphology. Although evidence on the deleterious effects of smoking on spermatozoa parameters is well known, its interference with cryopreservation is not clear. This study aimed to investigate the effects of cryopreservation on sperm parameters and DNA fragmentation in non-smokers and smokers. Semen samples were obtained from 40 normospermic male volunteers of whom 20 were non-smokers and 20 smokers. Samples were analyzed in terms of motility, concentration, morphology, and DNA fragmentation before freezing and 1 and 3 months after freezing and thawing. Ultrastructural alterations were investigated by transmission electron microscopy. Sperm morphology seemed to be more affected after cryopreservation in samples obtained from smokers. Ultrastructural examination showed alterations in the integrity of the membranes and increased subacrosomal swelling. Before freezing, the increase in DNA fragmentation rate in smokers was not statistically significant compared to that of non-smokers. However, after thawing, the DNA fragmentation rates were significantly high in both non-smokers and smokers compared to their respective rates before freezing. The extent of the increase in DNA fragmentation rate was significantly higher in smokers after thawing compared to that of non-smokers. In conclusion, cryopreservation causes alterations in membrane integrity and increases DNA fragmentation, thus triggering relatively negative effects on the sperm samples of smokers compared to that of non-smokers.

  20. Electronic transport in methylated fragments of DNA

    SciTech Connect

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L. Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  1. Electronic transport in methylated fragments of DNA

    NASA Astrophysics Data System (ADS)

    de Almeida, M. L.; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; de Moura, F. A. B. F.; Lyra, M. L.

    2015-11-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  2. Evolution of Particle Size Distributions in Fragmentation Over Time

    NASA Astrophysics Data System (ADS)

    Charalambous, C. A.; Pike, W. T.

    2013-12-01

    We present a new model of fragmentation based on a probabilistic calculation of the repeated fracture of a particle population. The resulting continuous solution, which is in closed form, gives the evolution of fragmentation products from an initial block, through a scale-invariant power-law relationship to a final comminuted powder. Models for the fragmentation of particles have been developed separately in mainly two different disciplines: the continuous integro-differential equations of batch mineral grinding (Reid, 1965) and the fractal analysis of geophysics (Turcotte, 1986) based on a discrete model with a single probability of fracture. The first gives a time-dependent development of the particle-size distribution, but has resisted a closed-form solution, while the latter leads to the scale-invariant power laws, but with no time dependence. Bird (2009) recently introduced a bridge between these two approaches with a step-wise iterative calculation of the fragmentation products. The development of the particle-size distribution occurs with discrete steps: during each fragmentation event, the particles will repeatedly fracture probabilistically, cascading down the length scales to a final size distribution reached after all particles have failed to further fragment. We have identified this process as the equivalent to a sequence of trials for each particle with a fixed probability of fragmentation. Although the resulting distribution is discrete, it can be reformulated as a continuous distribution in maturity over time and particle size. In our model, Turcotte's power-law distribution emerges at a unique maturation index that defines a regime boundary. Up to this index, the fragmentation is in an erosional regime with the initial particle size setting the scaling. Fragmentation beyond this index is in a regime of comminution with rebreakage of the particles down to the size limit of fracture. The maturation index can increment continuously, for example under

  3. Determination of genome size of Pseudomonas aeruginosa by PFGE: analysis of restriction fragments.

    PubMed Central

    Hector, J S; Johnson, A R

    1990-01-01

    Genomic DNA size was measured in three strains of Pseudomonas aeruginosa, ATCC 29260 (exotoxin A), ATCC 33467 (type I smooth) and ATCC 33468 (type 2 mucoid) by transverse alternating field electrophoresis of restriction fragments. Because of the high (67%) G + C content of Pseudomonas aeruginosa, restriction enzymes that recognize sequences with at least 4 AT base pairs were expected to be rare cutters. Eight enzymes produced fragments greater than 200 kb in size: Dral (TTT/AAA), Asnl (ATT/AAT), Hpal (GTT/AAC), AfIII (C/TTAAG), Xbal (T/CTAGA), Spel (A/CTAGT), Sspl (AAT/ATT) and Ndel (CA/TATG). All eight enzymes recognized one of three rare tetranucleotide sequences, TTAA, CTAG or ATAT. Pseudomonas aeruginosa strain 29260 has a genomic DNA size of 5573 kb. Strains 33467 and 33468 have identical restriction patterns and a possible deletion with a genomic size of 5407 kb. Images PMID:1972559

  4. Dependence on radiation quality of DNA fragmentation spectra

    NASA Astrophysics Data System (ADS)

    Campa, Alessandro; Ottolenghi, Andrea; Alloni, Daniele; Ballarini, Francesca; Belli, Mauro; Esposito, Giuseppe; Facoetti, Angelica; Friedland, Werner; Liotta, Marco; Paretzke, Herwig

    Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of cell damage. Here we are concerned with the damage induced by heavy charged particles; this issue is very important in the field of radioprotection of astronauts participating in long term space missions, besides being relevant also in other fields, like hadrontherapy. Galactic Cosmic Rays contain a large component of high-LET particles (HZE), e.g. helium and carbon ions, as well as highcharge particles such as iron ions. These particles are characterized by complex track structures with energy depositions not only along the path of the primary particle, but also at relatively large distance form the path, due to the presence of high energy secondary electrons. In this work we have simulated the irradiation of human fibroblasts with γ-rays, protons, helium, carbon and iron ions at a fixed dose with the biophysical Monte Carlo code PARTRAC,and calculated the induction of DSB. The PARTRAC code includes accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation. The results of a first validation of the code have been reported in A. Campa et al. (2005) and D. Alloni et al. (2007a, 2007b). DNA fragment spectra were calculated based on the DSB induction patterns and compared in particular for particles of the same specific energy and for particles of the same LET. Special emphasis has been directed to the calculation of very small fragments (< 1 kbp) that are not detectable by the most common experimental techniques and that can significantly influence the RBE

  5. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

    PubMed Central

    Schiestl, R H; Petes, T D

    1991-01-01

    DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends of BamHI fragments, this results indicates that four base pairs of homology are sufficient for some types of mitotic recombination. Images PMID:1881899

  6. Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation

    SciTech Connect

    Balch, J.; Davidson, J.; Brewer, L.; Gingrich, J.; Koo, J.; Mariella, R.; Carrano, A.

    1995-01-25

    Our efforts to develop novel technology for a rapid DNA sequencer and large fragment analysis system based upon gel electrophoresis are described. We are using microfabrication technology to build dense arrays of high speed micro electrophoresis lanes that will ultimately increase the sequencing rate of DNA by at least 100 times the rate of current sequencers. We have demonstrated high resolution DNA fragment separation needed for sequencing in polyacrylamide microgels formed in glass microchannels. We have built prototype arrays of microchannels having up to 48 channels. Significant progress has also been made in developing a sensitive fluorescence detection system based upon a confocal microscope design that will enable the diagnostics and detection of DNA fragments in ultrathin microchannel gels. Development of a rapid DNA sequencer and fragment analysis system will have a major impact on future DNA instrumentation used in clinical, molecular and forensic analysis of DNA fragments.

  7. Sperm DNA fragmentation and base oxidation.

    PubMed

    Lewis, Sheena E M

    2014-01-01

    Sperm DNA damage has been shown to be a valuable diagnostic and prognostic biomarker for male infertility and assisted reproductive treatment (ART) outcome. It is linked to every fertility checkpoint from reduced fertilization rates, lower embryo quality and pregnancy rates to higher rates of spontaneous miscarriage and childhood diseases. It is more robust than conventional semen parameters.The aim of this chapter is to provide an overview of current laboratory tests and relationships between sperm DNA damage and clinical outcomes. The conclusion is that sperm DNA damage is an important indicator of semen quality, and its routine use in the fertility clinic would improve ART success rates. PMID:23955675

  8. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    PubMed

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

  9. The Evolution of Grain Size Distribution in Explosive Rock Fragmentation - Sequential Fragmentation Theory Revisited

    NASA Astrophysics Data System (ADS)

    Scheu, B.; Fowler, A. C.

    2015-12-01

    Fragmentation is a ubiquitous phenomenon in many natural and engineering systems. It is the process by which an initially competent medium, solid or liquid, is broken up into a population of constituents. Examples occur in collisions and impacts of asteroids/meteorites, explosion driven fragmentation of munitions on a battlefield, as well as of magma in a volcanic conduit causing explosive volcanic eruptions and break-up of liquid drops. Besides the mechanism of fragmentation the resulting frequency-size distribution of the generated constituents is of central interest. Initially their distributions were fitted empirically using lognormal, Rosin-Rammler and Weibull distributions (e.g. Brown & Wohletz 1995). The sequential fragmentation theory (Brown 1989, Wohletz at al. 1989, Wohletz & Brown 1995) and the application of fractal theory to fragmentation products (Turcotte 1986, Perfect 1997, Perugini & Kueppers 2012) attempt to overcome this shortcoming by providing a more physical basis for the applied distribution. Both rely on an at least partially scale-invariant and thus self-similar random fragmentation process. Here we provide a stochastic model for the evolution of grain size distribution during the explosion process. Our model is based on laboratory experiments in which volcanic rock samples explode naturally when rapidly depressurized from initial pressures of several MPa to ambient conditions. The physics governing this fragmentation process has been successfully modelled and the observed fragmentation pattern could be numerically reproduced (Fowler et al. 2010). The fragmentation of these natural rocks leads to grain size distributions which vary depending on the experimental starting conditions. Our model provides a theoretical description of these different grain size distributions. Our model combines a sequential model of the type outlined by Turcotte (1986), but generalized to cater for the explosive process appropriate here, in particular by

  10. Direct and precise length measurement of single, stretched DNA fragments by dynamic molecular combing and STED nanoscopy.

    PubMed

    Kim, Namdoo; Kim, Hyung Jun; Kim, Younggyu; Min, Kyung Suk; Kim, Seong Keun

    2016-09-01

    A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than ∼1000 bp, the measured lengths of DNA fragments were consistently within ∼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder. PMID:27457103

  11. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    SciTech Connect

    Jackson, Christopher B.; Gallati, Sabina; Schaller, Andre

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  12. Fragment-size prediction during dynamic fragmentation of shock-melted tin: recovery experiments and modeling issues

    SciTech Connect

    Signor, L.; Roy, G.; Llorca, F.; Resseguier, T. de; Dragon, A.

    2007-12-12

    We are interested in dynamic fragmentation of shock-melted metals. The present work is devoted to laser-shock experiments in tin samples including fragments recovery and post-test evaluation of the fragment-size distribution. These results are compared with theoretical predictions from hydrocode simulations coupled with a modified formulation of a fragmentation model from the literature.

  13. Fragment-Size Prediction during Dynamic Fragmentation of Shock-Melted Tin: Recovery Experiments and Modeling Issues.

    NASA Astrophysics Data System (ADS)

    Signor, L.; de Rességuier, T.; Roy, G.; Dragon, A.; Llorca, F.

    2007-12-01

    We are interested in dynamic fragmentation of shock-melted metals. The present work is devoted to laser-shock experiments in tin samples including fragments recovery and post-test evaluation of the fragment-size distribution. These results are compared with theoretical predictions from hydrocode simulations coupled with a modified formulation of a fragmentation model from the literature.

  14. Extensive DNA fragmentation in oxyphilic cell lesions of the thyroid.

    PubMed

    Volante, M; Papotti, M; Gugliotta, P; Migheli, A; Bussolati, G

    2001-08-01

    The in situ end-labeling (ISEL) method demonstrates DNA fragmentation, commonly regarded as a marker of apoptosis. We investigated by the ISEL procedure a series of 52 thyroid lesions, including 24 lesions of mitochondrion-rich oxyphilic cells, both benign and malignant, and 28 non-oxyphilic control tumors. A high percentage of nuclear ISEL staining (approximating to 100% in most cases) was observed in the vast majority of oxyphilic cells from both adenomas and carcinomas, in the absence of morphological apoptotic changes and with no immunocytochemical evidence of caspase activation. This pattern of DNA fragmentation was not observed in non-oxyphilic lesions and was confirmed in total extracted DNA. Moreover, a peculiar cytoplasmic staining was also observed in oxyphilic cells from both benign and malignant lesions, probably related to abnormal fragmentation of mitochondrial DNA. Similar staining patterns were detected in oxyphilic cell tumors of other organs (parathyroids, salivary glands, and kidneys). These findings are consistent with an extensive DNA fragmentation peculiar to oxyphilic cells, which is not directly related to apoptosis and whose origin and biological significance are presently unknown.

  15. Development of procedures for the identification of human papilloma virus DNA fragments in laser plume

    NASA Astrophysics Data System (ADS)

    Woellmer, Wolfgang; Meder, Tom; Jappe, Uta; Gross, Gerd; Riethdorf, Sabine; Riethdorf, Lutz; Kuhler-Obbarius, Christina; Loening, Thomas

    1996-01-01

    For the investigation of laser plume for the existence of HPV DNA fragments, which possibly occur during laser treatment of virus infected tissue, human papillomas and condylomas were treated in vitro with the CO2-laser. For the sampling of the laser plume a new method for the trapping of the material was developed by use of water-soluble gelatine filters. These samples were analyzed with the polymerase chain reaction (PCR) technique, which was optimized in regard of the gelatine filters and the specific primers. Positive PCR results for HPV DNA fragments up to the size of a complete oncogene were obtained and are discussed regarding infectiousity.

  16. Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments

    SciTech Connect

    Asakawa, Jun-Ichi; Kodaira, Mieko; Satoh, Chiyoko; Kuick, R.; Hanash, S.M.; Neel, J.V.

    1994-09-13

    The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, the authors have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of {approx} 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots are the product of only one fragment, the coefficient of variation of spot intensity should be approximately {le} 0.12. At present, 482 of the spots in preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to mendelian principles. The authors have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome. 17 refs., 2 figs., 2 tabs.

  17. Size Distribution of Genesis Solar Wind Array Collector Fragments Recovered

    NASA Technical Reports Server (NTRS)

    Allton, J. H.; Stansbery, E. K.; McNamara, K. M.

    2005-01-01

    Genesis launched in 2001 with 271 whole and 30 half hexagonally-shaped collectors mounted on 5 arrays, comprised of 9 materials described in [1]. The array collectors were damaged during re-entry impact in Utah in 2004 [2], breaking into many smaller pieces and dust. A compilation of the number and approximate size of the fragments recovered was compiled from notes made during the field packaging performed in the Class 10,000 cleanroom at Utah Test and Training Range [3].

  18. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III.

    PubMed

    Arakawa, Hiroshi; Iliakis, George

    2015-01-01

    Higher eukaryotes have three types of DNA ligases: DNA ligase 1 (Lig1), DNA ligase 3 (Lig3) and DNA ligase 4 (Lig4). While Lig1 and Lig4 are present in all eukaryotes from yeast to human, Lig3 appears sporadically in evolution and is uniformly present only in vertebrates. In the classical, textbook view, Lig1 catalyzes Okazaki-fragment ligation at the DNA replication fork and the ligation steps of long-patch base-excision repair (BER), homologous recombination repair (HRR) and nucleotide excision repair (NER). Lig4 is responsible for DNA ligation at DNA double strand breaks (DSBs) by the classical, DNA-PKcs-dependent pathway of non-homologous end joining (C-NHEJ). Lig3 is implicated in a short-patch base excision repair (BER) pathway, in single strand break repair in the nucleus, and in all ligation requirements of the DNA metabolism in mitochondria. In this scenario, Lig1 and Lig4 feature as the major DNA ligases serving the most essential ligation needs of the cell, while Lig3 serves in the cell nucleus only minor repair roles. Notably, recent systematic studies in the chicken B cell line, DT40, involving constitutive and conditional knockouts of all three DNA ligases individually, as well as of combinations thereof, demonstrate that the current view must be revised. Results demonstrate that Lig1 deficient cells proliferate efficiently. Even Lig1/Lig4 double knockout cells show long-term viability and proliferate actively, demonstrating that, at least in DT40, Lig3 can perform all ligation reactions of the cellular DNA metabolism as sole DNA ligase. Indeed, in the absence of Lig1, Lig3 can efficiently support semi-conservative DNA replication via an alternative Okazaki-fragment ligation pathway. In addition, Lig3 can back up NHEJ in the absence of Lig4, and can support NER and HRR in the absence of Lig1. Supporting observations are available in less elaborate genetic models in mouse cells. Collectively, these observations raise Lig3 from a niche-ligase to a

  19. Influence of molecular size on tissue distribution of antibody fragments

    PubMed Central

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C.; Lavé, Thierry; Shah, Dhaval K.

    2016-01-01

    Biodistribution coefficients (BC) allow estimation of the tissue concentrations of proteins based on the plasma pharmacokinetics. We have previously established the BC values for monoclonal antibodies. Here, this concept is extended by development of a relationship between protein size and BC values. The relationship was built by deriving the BC values for various antibody fragments of known molecular weight from published biodistribution studies. We found that there exists a simple exponential relationship between molecular weight and BC values that allows the prediction of tissue distribution of proteins based on molecular weight alone. The relationship was validated by a priori predicting BC values of 4 antibody fragments that were not used in building the relationship. The relationship was also used to derive BC50 values for all the tissues, which is the molecular weight increase that would result in 50% reduction in tissue uptake of a protein. The BC50 values for most tissues were found to be ~35 kDa. An ability to estimate tissue distribution of antibody fragments based on the BC vs. molecular size relationship established here may allow better understanding of the biologics concentrations in tissues responsible for efficacy or toxicity. This relationship can also be applied for rational development of new biotherapeutic modalities with optimal biodistribution properties to target (or avoid) specific tissues. PMID:26496429

  20. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

    PubMed

    Hakenberg, S; Hügle, M; Meyer, P; Behrmann, O; Dame, G; Urban, G A

    2015-05-15

    With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format.

  1. Fenton fragmentation for faster electrophoretic on chip purification of amplifiable genomic DNA.

    PubMed

    Hakenberg, S; Hügle, M; Meyer, P; Behrmann, O; Dame, G; Urban, G A

    2015-05-15

    With a rapid and simple actuation protocol electrophoretic nucleic acid extraction is easy automatable, requires no moving parts, is easy to miniaturize and furthermore possesses a size dependent cut-off filter adjustable by the pore size of the hydrogel. However electrophoretic nucleic acid extraction from bacteria has so far been applied mainly for short RNA targets. One of the reasons is that electrophoretic processing of unfragmented genomic DNA strands is time-consuming, because of the length. Here DNA fragmentation would accelerate extraction and isolation. We introduce on-chip lysis and non-enzymatic DNA cleavage directly followed by a purifying step for receiving amplifiable DNA fragments from bacteria in less than 25 min. In contrast to restriction enzymes the Fenton reaction is known to cleave DNA without nucleotide specificity. The reaction mix contains iron(II) EDTA, sodium ascorbate, hydrogen peroxide and lysozyme. The degree of fragmentation can be adjusted by the concentration of reagents. The results enable electrophoretic extraction methods to unspecifically process long genomic DNA in a short time frame, e.g. for pathogen detection in a lab-on-a-chip format. PMID:24970713

  2. Coagulation-Fragmentation Model for Animal Group-Size Statistics

    NASA Astrophysics Data System (ADS)

    Degond, Pierre; Liu, Jian-Guo; Pego, Robert L.

    2016-10-01

    We study coagulation-fragmentation equations inspired by a simple model proposed in fisheries science to explain data for the size distribution of schools of pelagic fish. Although the equations lack detailed balance and admit no H-theorem, we are able to develop a rather complete description of equilibrium profiles and large-time behavior, based on recent developments in complex function theory for Bernstein and Pick functions. In the large-population continuum limit, a scaling-invariant regime is reached in which all equilibria are determined by a single scaling profile. This universal profile exhibits power-law behavior crossing over from exponent -2/3 for small size to -3/2 for large size, with an exponential cutoff.

  3. Creating Cost-Effective DNA Size Standards for Use in Teaching and Research Laboratories

    ERIC Educational Resources Information Center

    Shultz, Jeff

    2011-01-01

    I have devised a method with which a molecular size standard can be readily manufactured using Lambda DNA and PCR. This method allows the production of specific sized DNA fragments and is easily performed in a standard molecular biology laboratory. The material required to create these markers can also be used to provide a highly robust and…

  4. Detection of Irradiated Food: DNA Fragmentation in Grapefruits

    NASA Astrophysics Data System (ADS)

    Delincée, Henry

    1998-06-01

    Employing the simple microgel electrophoresis of single cells - `comet assay' - on grapefruit seeds enabled a rapid identification of irradiated fruits. Fruits were exposed to radiation doses of 0, 0.1, 0.2, 0.3, 0.4 and 0.5 kGy covering the range of potential commercial irradiation for insect disinfestation and quarantine purposes. Seeds were isolated, crushed, and the cells embedded in an agarose layer. After lysis of the cells, they were subjected to microgel electrophoresis for 2.5 minutes, and then stained. Fruits irradiated with 0.2 kGy and higher doses showed typical DNA fragmentation, the DNA fragments stretching or migrating out of the cells forming a tail towards the anode, giving the damaged cells an appearance of a comet. With increasing dose a longer extension of the DNA from the nucleus towards the anode is observed. Undamaged cells will appear as intact nuclei without tails. The DNA comet assay is thus a rapid and inexpensive screening technique to detect irradiated grapefruits. Suspected samples may subsequently be analysed by officially validated methods for detection of irradiated foods.

  5. Large-scale production of palindrome DNA fragments

    SciTech Connect

    Palmer, E.L.; Gewiess, A.; Harp, J.M.

    1995-10-10

    Our structural studies of nucleosomes necessitated the production of over 100 mg of a 146-bp perfect palindrome DNA for use in the reconstitution of perfectly symmetrical nucleosome core particles for detailed X-ray crystallographic analysis. The propagation of palindromic DNA sequences by bacterial culture is hindered by the instability of these sequences during bacterial replication and recombination. While the loss of some palindrome sequences can be elminated by the use of sbcB or sbcC mutants of Escherichia coli, not all palindrome-containing plasmids are faithfully maintained by these strains. The production of large quantities of palindrome DNA can therefore be extremely difficult. After trying several approaches, we were able to develop a reliable procedure for production of large quantities of palindrome DNA that involves production of plasmid containing multiple copies of the repeating unit of the palindrome which are isolated by restriction digestion and ligated in vitro to form the palindrome DNA. The procedure has resulted in the production of over 20 mg of a 146-bp DNA fragment in 2 weeks.

  6. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

    Telli, Onur; Sarici, Hasmet; Kabar, Mucahit; Ozgur, Berat Cem; Resorlu, Berkan; Bozkurt, Selen

    2015-01-01

    Introduction: The aims of this study were to investigate the effect of varicocelectomy on DNA fragmentation index and semen parameters in infertile patients before and after surgical repair of varicocele. Materials and Methods: In this prospective study, 72 men with at least 1-year history of infertility, varicocele and oligospermia were examined. Varicocele sperm samples were classified as normal or pathological according to the 2010 World Health Organization guidelines. The acridine orange test was used to assess the DNA fragmentation index (DFI) preoperatively and postoperatively. Results: DFI decreased significantly after varicocelectomy from 34.5% to 28.2% (P = 0.024). In addition all sperm parameters such as mean sperm count, sperm concentration, progressive motility and sperm morphology significantly increased from 19.5 × 106 to 30.7 × 106, 5.4 × 106/ml to 14.3 × 106/ml, and 19.9% to 31.2% (P < 0.001) and 2.6% to 3.1% (P = 0.017). The study was limited by the loss to follow-up of some patients and unrecorded pregnancy outcome due to short follow-up. Conclusion: Varicocele causes DNA-damage in spermatozoa. We suggest that varicocelectomy improves sperm parameters and decreases DFI. PMID:25878412

  7. Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

    PubMed

    Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

    2014-11-01

    Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk.

  8. Novel extraction method of genomic DNA suitable for long-fragment amplification from small amounts of milk.

    PubMed

    Liu, Y F; Gao, J L; Yang, Y F; Ku, T; Zan, L S

    2014-11-01

    Isolation of genomic DNA is a prerequisite for assessment of milk quality. As a source of genomic DNA, milk somatic cells from milking ruminants are practical, animal friendly, and cost-effective sources. Extracting DNA from milk can avoid the stress response caused by blood and tissue sampling of cows. In this study, we optimized a novel DNA extraction method for amplifying long (>1,000 bp) DNA fragments and used it to evaluate the isolation of DNA from small amounts of milk. The techniques used for the separation of milk somatic cell were explored and combined with a sodium dodecyl sulfate (SDS)-phenol method for optimizing DNA extraction from milk. Spectrophotometry was used to determine the concentration and purity of the extracted DNA. Gel electrophoresis and DNA amplification technologies were used for to determine DNA size and quality. The DNA of 112 cows was obtained from milk (samples of 13 ± 1 mL) and the corresponding optical density ratios at 260:280 nm were between 1.65 and 1.75. Concentrations were between 12 and 45 μg/μL and DNA size and quality were acceptable. The specific PCR amplification of 1,019- and 729-bp bovine DNA fragments was successfully carried out. This novel method can be used as a practical, fast, and economical mean for long genomic DNA extraction from a small amount of milk. PMID:25218756

  9. Acoustic stimulation promotes DNA fragmentation in the Guinea pig cochlea.

    PubMed

    Kamio, Tomonobu; Watanabe, Ken-Ichi; Okubo, Kimihiro

    2012-01-01

    Apoptosis can be described as programmed cell death. Apoptosis regulates cell turnover and is involved in various pathological conditions. The characteristic features of apoptosis are shrinkage of the cell body, chromatin condensation, and nucleic acid fragmentation. During apoptosis, double-stranded DNA is broken down into single-stranded DNA (ssDNA) by proteases. Acoustic trauma is commonly encountered in otorhinolaryngology clinics. Intense noise can cause inner ear damage, such as hearing disturbance, tinnitus, ear fullness, and decreased speech discrimination. In this study, we used immunohistochemical and electrophysiological methods to examine the fragmentation of DNA in the cochleas of guinea pigs that had been exposed to intense noise. Twenty-four guinea pigs weighing 250 to 350 g were used. The animals were divided into 4 groups: (I) a control group (n=6), (II) a group that was exposed to noise for 2 hours (n=6), (III) a group that was exposed to noise for 5 hours (n=6), and (IV) a group that was exposed to noise for 20 hours. The stimulus was a pure tone delivered at a frequency of 2 kHz. The sound pressure level was 120 dBSPL. No threshold shifts were apparent in group I. Group II showed a significant elevation of the hearing threshold (ANOVA, p<0.05(*)). The ABR threshold level was also significantly elevated immediately after the acoustic stimulation in groups III and IV (ANOVA, p<0.01(**)). In groups I, II, and IV, the lateral wall of the ear did not show immunoreactivity to ssDNA but did in group III. No immunoreactivity was apparent in the organ of Corti in group I or II. However, the supporting cells and outer hair cells in groups III and IV showed reactions for ssDNA. The fine structure of the organ of Corti had been destroyed in group IV. The lateral wall showed immunoreactivity for ssDNA only in group III, whereas the organ of Corti showed reactions for ssDNA in groups III and IV. Our study suggests that apoptotic changes occur in patients that

  10. Acoustic stimulation promotes DNA fragmentation in the Guinea pig cochlea.

    PubMed

    Kamio, Tomonobu; Watanabe, Ken-Ichi; Okubo, Kimihiro

    2012-01-01

    Apoptosis can be described as programmed cell death. Apoptosis regulates cell turnover and is involved in various pathological conditions. The characteristic features of apoptosis are shrinkage of the cell body, chromatin condensation, and nucleic acid fragmentation. During apoptosis, double-stranded DNA is broken down into single-stranded DNA (ssDNA) by proteases. Acoustic trauma is commonly encountered in otorhinolaryngology clinics. Intense noise can cause inner ear damage, such as hearing disturbance, tinnitus, ear fullness, and decreased speech discrimination. In this study, we used immunohistochemical and electrophysiological methods to examine the fragmentation of DNA in the cochleas of guinea pigs that had been exposed to intense noise. Twenty-four guinea pigs weighing 250 to 350 g were used. The animals were divided into 4 groups: (I) a control group (n=6), (II) a group that was exposed to noise for 2 hours (n=6), (III) a group that was exposed to noise for 5 hours (n=6), and (IV) a group that was exposed to noise for 20 hours. The stimulus was a pure tone delivered at a frequency of 2 kHz. The sound pressure level was 120 dBSPL. No threshold shifts were apparent in group I. Group II showed a significant elevation of the hearing threshold (ANOVA, p<0.05(*)). The ABR threshold level was also significantly elevated immediately after the acoustic stimulation in groups III and IV (ANOVA, p<0.01(**)). In groups I, II, and IV, the lateral wall of the ear did not show immunoreactivity to ssDNA but did in group III. No immunoreactivity was apparent in the organ of Corti in group I or II. However, the supporting cells and outer hair cells in groups III and IV showed reactions for ssDNA. The fine structure of the organ of Corti had been destroyed in group IV. The lateral wall showed immunoreactivity for ssDNA only in group III, whereas the organ of Corti showed reactions for ssDNA in groups III and IV. Our study suggests that apoptotic changes occur in patients that

  11. Modelization of DNA fragmentation induced in human fibroblasts by Fe-56 ions

    NASA Astrophysics Data System (ADS)

    Ballarini, F.; Belli, M.; Campa, A.; Esposito, G.; Friedland, W.; Ottolenghi, A.; Paretzke, H.

    DNA double-strand breaks DSB are widely recognized as cellular critical lesions in the pathways leading from initial energy deposition by radiation to the formation of relevant biological endpoints such as gene mutations chromosome aberrations and cell death Chromatin conformation and radiation track structure are expected to have a strong influence on the spatial modulation of DSB induction at the scale of the nucleosome i e 100 base pairs bp and of the low-level chromatin fiber organization i e 1 kbp At larger scales the DNA fragmentation pattern induced by sparsely ionizing radiation approaches a scenario resulting from a random distribution of DSB However the pattern induced by high-LET irradiation can lead to deviation from randomness also at these scales This feature can have important biological consequences since spatial correlation of DSB is thought to affect their reparability Therefore studies on fragment size distributions induced by radiations of various qualities can help to link the physical characteristics of radiation with the cellular endpoints This is an important issue for understanding the main mechanisms of cell damage induced by HZE particles In this work we have compared the pattern of DNA fragmentation in the range 1-5700 kbp induced in human fibroblasts by gamma -rays with that induced by high-energy Fe-ions which have biological significance for radiation protection issues during long term astronauts travels The study has taken into account the comparison of the experimental fragmentation spectra

  12. Phylogenomics of caspase-activated DNA fragmentation factor

    SciTech Connect

    Eckhart, Leopold . E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz; Tschachler, Erwin

    2007-04-27

    The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans.

  13. Cloning of DNA fragments: ligation reactions in agarose gel.

    PubMed

    Furtado, Agnelo

    2014-01-01

    Ligation reactions to ligate a desired DNA fragment into a vector can be challenging to beginners and especially if the amount of the insert is limiting. Although additives known as crowding agents, such as PEG 8000, added to the ligation mixes can increase the success one has with ligation reactions, in practice the amount of insert used in the ligation can determine the success or the failure of the ligation reaction. The method described here, which uses insert DNA in gel slice added directly into the ligation reaction, has two benefits: (a) using agarose as the crowding agent and (b) reducing steps of insert purification. The use of rapid ligation buffer and incubation of the ligation reaction at room temperature greatly increase the efficiency of the ligation reaction even for blunt-ended ligation. PMID:24243199

  14. Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath

    PubMed Central

    Malc, Ewa P.; Jayakody, Chatura N.; Tsuruta, James K.; Mieczkowski, Piotr A.; Janzen, William P.; Dayton, Paul A.

    2015-01-01

    A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation. PMID:26186461

  15. DNA fragmentation induced by fe ions in human cells: shielding influence on spatially correlated damage

    SciTech Connect

    Antonelli, F.; Belli, M.; Campa, A.; Chatterjee, A.; Dini, V.; Esposito, G.; Rydberg, B.; Simone, G.; Tabocchini, M.A.

    2003-11-19

    Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used small gamma, Greek-rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by small gamma, Greek-rays in the size range 123 kbp; (3) a non-random DNA DSB induction by Fe ions.

  16. DNA fragmentation induced by Fe ions in human cells: shielding influence on spatially correlated damage

    NASA Technical Reports Server (NTRS)

    Antonelli, F.; Belli, M.; Campa, A.; Chatterjee, A.; Dini, V.; Esposito, G.; Rydberg, B.; Simone, G.; Tabocchini, M. A.

    2004-01-01

    Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

  17. Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts.

    PubMed

    McArthur, C R; Clark-Walker, G D

    1983-03-01

    Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species. PMID:24173115

  18. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    NASA Astrophysics Data System (ADS)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  19. The effects of 4-MEI on cell proliferation, DNA breaking and DNA fragmentation.

    PubMed

    Tazehkand, M Norizadeh; Moridikia, A; Hajipour, O; Valipour, E; Timocin, T; Topaktas, M; Yilmaz, M B

    2016-01-01

    4-Methylimidazole (4-MEI) is a color widely found in cola drinks, roasted foods, grilled meats, coffee and other foods. This study was aimed to investigate the 4-MEI effects on the cell proliferation, purified circular DNA and DNA from cells of rats treated with the 4-MEI.In this study, mouse 3T3-L1 cell line was treated with 4-MEI at concentrations of 300, 450, 600 and 750 µg/mL for 24 hours and 48 hours periods, after that cytotoxic effect of the 4-MEI was studied by MTT test. Also, the effect of 4-MEI on purified circular DNA (pET22b) was investigated by treating of the DNA with 4-MEI concentrations of 300, 450, 600 and 750 µg/ml. DNA was extracted from liver cells of rats that have been treated with 4-MEI doses of 25 and 50 mg/kg for 10 week and it was subjected to agarose gel electrophoreses analyses.4-MEI significantly inhibited cell proliferation of 3T3-L1 cell line at highest concentration for 24 h and at all concentration for 48 h treatment time. DNA fragmentation assay showed that 4-MEI at 50 mg/kg concentration clearly produced characteristic DNA smear and no DNA laddering (200bp) was observed when mouse was exposed to 4-MEI. The results obtained from plasmid DNA damaging assay showed that 4-MEI has noeffect on the DNA, because the electrophoretic pattern of DNA treated with 4-MEI showed three bands on agarose gel electrophoresis as it was for untreated control. 4-MEI showed cytotoxic effect on 3T3-L1 cells but no effect on plasmid DNA breaking. According to DNA fragmentation assay 4-MEI has necrosis effects on mouse liver cells (Tab. 1, Fig. 4, Ref. 27). PMID:27546537

  20. Short-fragment DNA-mediated in vivo DNA electroporation delivery.

    PubMed

    Peng, Jinliang; Zhao, Yonggang; Xu, Yuhong

    2014-01-01

    Electroporation is an effective physical delivery method. A variety of factors have been shown to affect the electroporation-mediated gene delivery efficiency. Here we report the usefulness of noncoding short-fragment DNA (sf-DNA) for facilitating electroporation-mediated gene transfer. The plasmid pGL3-control encoding firefly luciferase was injected into tissue together with or without sf-DNA in different length or dose. Immediately after injection, the tissues were electroporated and the level of luciferase activity was assessed 24 h later. The results showed that plasmid DNA formulated with sf-DNA resulted in significant improvement in electroporation-mediated gene transfer efficiency. The effect is dose and length dependent, and also found in low-voltage electroporation. These results indicated that sf-DNA can be used as a helper molecule to improve the electroporation-mediated gene transfection efficiency.

  1. Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy

    PubMed Central

    Pang, Dalong; Chasovskikh, Sergey; Rodgers, James E.; Dritschilo, Anatoly

    2016-01-01

    Growing interest in proton and heavy ion therapy has reinvigorated research into the fundamental biological mechanisms underlying the therapeutic efficacy of charged-particle radiation. To improve our understanding of the greater biological effectiveness of high-LET radiations, we have investigated DNA double-strand breaks (DSBs) following exposure of plasmid DNA to low-LET Co-60 gamma photon and electron irradiation and to high-LET Beryllium and Argon ions with atomic force microscopy. The sizes of DNA fragments following radiation exposure were individually measured to construct fragment size distributions from which the DSB per DNA molecule and DSB spatial distributions were derived. We report that heavy charged particles induce a significantly larger proportion of short DNA fragments in irradiated DNA molecules, reflecting densely and clustered damage patterns of high-LET energy depositions. We attribute the enhanced short DNA fragmentation following high-LET radiations as an important determinant of the observed, enhanced biological effectiveness of high-LET irradiations. PMID:27376024

  2. Short DNA Fragments Are a Hallmark of Heavy Charged-Particle Irradiation and May Underlie Their Greater Therapeutic Efficacy.

    PubMed

    Pang, Dalong; Chasovskikh, Sergey; Rodgers, James E; Dritschilo, Anatoly

    2016-01-01

    Growing interest in proton and heavy ion therapy has reinvigorated research into the fundamental biological mechanisms underlying the therapeutic efficacy of charged-particle radiation. To improve our understanding of the greater biological effectiveness of high-LET radiations, we have investigated DNA double-strand breaks (DSBs) following exposure of plasmid DNA to low-LET Co-60 gamma photon and electron irradiation and to high-LET Beryllium and Argon ions with atomic force microscopy. The sizes of DNA fragments following radiation exposure were individually measured to construct fragment size distributions from which the DSB per DNA molecule and DSB spatial distributions were derived. We report that heavy charged particles induce a significantly larger proportion of short DNA fragments in irradiated DNA molecules, reflecting densely and clustered damage patterns of high-LET energy depositions. We attribute the enhanced short DNA fragmentation following high-LET radiations as an important determinant of the observed, enhanced biological effectiveness of high-LET irradiations. PMID:27376024

  3. Viability and DNA fragmentation in differently sorted boar spermatozoa.

    PubMed

    De Ambrogi, M; Spinaci, M; Galeati, G; Tamanini, C

    2006-11-01

    Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of genomic activation. Sex-sorting exposes spermatozoa to stress sources such as high pressure, laser beam and electrical charge. The aim of this work was to determine how sorting procedures affect viability and DNA integrity in boar spermatozoa, by using the newly developed Sperm-Sus-Halomax. Four sperm populations were considered: CONTROL (no treatment), REAL (sex-sorted semen), BULK (semen sorted without sex separation) and NO LASER (semen only exposed to the high pressure, but including also cells normally discarded from sex-sorting). A significantly (P=0.019) lower viability in NO LASER (64.71%) than in CONTROL (78.6%) and REAL (80.5%) groups was found; this was accompanied by a significantly (P=0.001) higher DNA fragmentation index (DFI) in NO LASER group (6.86%) respect to CONTROL (3.30%) and REAL (3.42%) groups. BULK group did not show any difference in viability or DFI as compared to the other groups. In conclusion, we may believe that sex-sorting procedure as a whole does not affect either viability or DFI and that shear mechanical forces are a relevant source of DNA damage for sorted semen. PMID:16814375

  4. Small Fragment Homologous Replacement (SFHR): sequence-specific modification of genomic DNA in eukaryotic cells by small DNA fragments.

    PubMed

    Luchetti, Andrea; Malgieri, Arianna; Sangiuolo, Federica

    2014-01-01

    The sequence-specific correction of a mutated gene (e.g., point mutation) by the Small Fragment Homologous Replacement (SFHR) method is a highly attractive approach for gene therapy. Small DNA fragments (SDFs) were used in SFHR to modify endogenous genomic DNA in both human and murine cells. The advantage of this gene targeting approach is to maintain the physiologic expression pattern of targeted genes without altering the regulatory sequences (e.g., promoter, enhancer), but the application of this technique requires the knowledge of the sequence to be targeted. In our recent study, an optimized SFHR protocol was used to replace the eGFP mutant sequence in SV-40-transformed mouse embryonic fibroblast (MEF-SV40), with the wild-type eGFP sequence. Nevertheless in the past, SFHR has been used to correct several mutant genes, each related to a specific genetic disease (e.g., spinal muscular atrophy, cystic fibrosis, severe combined immune deficiency). Several parameters can be modified to optimize the gene modification efficiency, as described in our recent study. In this chapter we describe the main guidelines that should be followed in SFHR application, in order to increase technique efficiency.

  5. Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA

    PubMed Central

    Sawyer, Susanna; Krause, Johannes; Guschanski, Katerina; Savolainen, Vincent; Pääbo, Svante

    2012-01-01

    DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5′-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments. PMID:22479540

  6. Flooding and fragment size interact to determine survival and regrowth after fragmentation in two stoloniferous Trifolium species.

    PubMed

    Huber, Heidrun; Visser, Eric J W; Clements, Gijs; Peters, Janny L

    2014-01-01

    Clonal plants, which reproduce by means of stolons and rhizomes, are common in frequently flooded habitats. Resilience to disturbance is an important trait enabling plants to survive in such highly disturbed habitats. Resource storage is thought to enable clonal plants to resume growth after clonal fragmentation caused by disturbance. Here we investigated if submergence prior to disturbance reduces survival and regrowth of clonal fragments and whether or not genotypes originating from highly disturbed riverine habitats are more resistant to mechanical disturbance than genotypes from less disturbed coastal dune slack habitats. We further tested if variation in survival and regrowth was affected by internode size. Clones from contrasting habitats of two closely related Trifolium species were first genotypically characterized by amplification fragment length polymorphism and then subjected to soil flooding and subsequent clonal fragmentation. These species differ with respect to their abundance in riverine and dune slack habitats, with Trifolium repens mainly occurring in riverine grasslands and Trifolium fragiferum in coastal dune slacks. Soil flooding decreased survival and regrowth by up to 80 %. Plants originating from riverine grasslands were less negatively affected by fragmentation than plants from dune slack habitats. Surprisingly, ramets did not always benefit from being attached to a larger internode, as internode size was often negatively correlated with survival after fragmentation. Regrowth, on the other hand, was generally positively correlated with internode size. These unexpected results indicate that there may be contrasting selection pressures on internode size in stoloniferous species growing in severely disturbed habitats. PMID:24887003

  7. The synthesis of new amphiphilic p-tert-butylthiacalix[4]arenes containing peptide fragments and their interaction with DNA.

    PubMed

    Padnya, Pavel L; Andreyko, Elena A; Mostovaya, Olga A; Rizvanov, Ildar Kh; Stoikov, Ivan I

    2015-06-01

    New water-soluble p-tert-butylthiacalix[4]arenes containing peptide and quaternary ammonium fragments in cone and 1,3-alternate conformations were synthesized and characterized. The interaction of the macrocycles with DNA was studied by UV-spectroscopy, DLS and TEM. It was shown that the interaction of the self-associates based on p-tert-butylthiacalix[4]arenes tetrasubstituted at the lower rim with glycine and quaternary ammonium fragments in cone and 1,3-alternate conformations with DNA led to the formation of particles of about 99-192 nm in size.

  8. In vivo assembly of DNA-fragments in the moss, Physcomitrella patens

    PubMed Central

    King, Brian Christopher; Vavitsas, Konstantinos; Ikram, Nur Kusaira Binti Khairul; Schrøder, Josephine; Scharff, Lars B.; Hamberger, Björn; Jensen, Poul Erik; Simonsen, Henrik Toft

    2016-01-01

    Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology. PMID:27126800

  9. Minifish shows high genetic variation in mtDNA size.

    PubMed

    Chen, X-W; Li, Q-L; Hu, X-J; Yuan, Y-M; Wen, M; Peng, L-Y; Liu, S-J; Hong, Y-H

    2014-01-01

    The genus Paedocypris is a newly described taxon of minifish species that are characterized by extensive chromosome evolution and one of the smallest known vertebrate nuclear genomes. Paedocypris features a tiny adult size, a short generation time, low fecundity and fragmented tropical habitats, which are factors that favor rapid speciation. Most recently, we have revealed that P. progenetica (Pp), the type species of the genus Paedocypris, has an unusual mtDNA bearing - within its D-loop - a tandem array of a 34-bp repeat sequence called the minifish repeat, which shows compromised replication efficiency in vitro. Here we report that Pp exhibits high genetic variation in mtDNA size. The efficiency of D-loop amplification was found to depend upon primers. Interestingly, Pp individuals of one and the same population differed drastically in mtDNA size resulting from varying copy numbers of the minifish repeat. We conclude that minifish has a high mutation rate and perhaps represents a rapidly evolving taxon of vertebrates.

  10. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  11. A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

    ERIC Educational Resources Information Center

    Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

    2012-01-01

    We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli" remains a fundamental…

  12. Reversible phospholipid nanogels for deoxyribonucleic acid fragment size determinations up to 1500 base pairs and integrated sample stacking.

    PubMed

    Durney, Brandon C; Bachert, Beth A; Sloane, Hillary S; Lukomski, Slawomir; Landers, James P; Holland, Lisa A

    2015-06-23

    Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix

  13. Clusters of DNA induced by ionizing radiation: formation of short DNA fragments. I. Theoretical modeling

    NASA Technical Reports Server (NTRS)

    Holley, W. R.; Chatterjee, A.

    1996-01-01

    We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber comprised of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and delta rays due to knock-on collisions involving energy transfers >100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of OH, H, eaq, etc.; (2) OH attack on sugar molecules leading to strand breaks: (3) OH attack on bases; (4) direct ionization of the sugar molecules leading to strand breaks; (5) direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 bp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. The shapes of the spectra of DNA fragment lengths depend on the symmetries or approximate symmetries of the chromatin structure. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper (B. Rydberg, Radiat, Res. 145, 200-209, 1996) after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the

  14. Clusters of DNA induced by ionizing radiation: formation of short DNA fragments. I. Theoretical modeling.

    PubMed

    Holley, W R; Chatterjee, A

    1996-02-01

    We have developed a general theoretical model for the interaction of ionizing radiation with chromatin. Chromatin is modeled as a 30-nm-diameter solenoidal fiber comprised of 20 turns of nucleosomes, 6 nucleosomes per turn. Charged-particle tracks are modeled by partitioning the energy deposition between primary track core, resulting from glancing collisions with 100 eV or less per event, and delta rays due to knock-on collisions involving energy transfers >100 eV. A Monte Carlo simulation incorporates damages due to the following molecular mechanisms: (1) ionization of water molecules leading to the formation of OH, H, eaq, etc.; (2) OH attack on sugar molecules leading to strand breaks: (3) OH attack on bases; (4) direct ionization of the sugar molecules leading to strand breaks; (5) direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 bp and over regions extending to several kilobase pairs. A characteristic feature of the regional damage predicted by our model is the production of short fragments of DNA associated with multiple nearby strand breaks. The shapes of the spectra of DNA fragment lengths depend on the symmetries or approximate symmetries of the chromatin structure. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper (B. Rydberg, Radiat, Res. 145, 200-209, 1996) after exposure to both high- and low-LET radiation. The overall measured yields agree well quantitatively with the theoretical predictions. Our theoretical results predict the existence of a strong peak at about 85 bp, which represents the revolution period about the nucleosome. Other peaks at multiples of about 1,000 bp correspond to the periodicity of the particular solenoid model of chromatin used in these calculations. Theoretical results in combination with experimental data on fragmentation spectra may help determine the consensus or average structure of the

  15. Relative stability of transgene DNA fragments from GM rapeseed in mixed ruminal cultures.

    PubMed

    Sharma, Ranjana; Alexander, Trevor W; John, S Jacob; Forster, Robert J; McAllister, Tim A

    2004-05-01

    The use of transgenic crops as feeds for ruminant animals has prompted study of the possible uptake of transgene fragments by ruminal micro-organisms and/or intestinal absorption of fragments surviving passage through the rumen. The persistence in buffered ruminal contents of seven different recombinant DNA fragments from GM rapeseed expressing the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene was tracked using PCR. Parental and transgenic (i.e. glyphosphate-tolerant; Roundup Ready, Monsanto Company, St Louis, MO, USA) rapeseed were incubated for 0, 2, 4, 8, 12, 24 and 48 h as whole seeds, cracked seeds, rapeseed meal, and as pelleted, barley-based diets containing 65 g rapeseed meal/kg. The seven transgene fragments ranged from 179 to 527 bp and spanned the entire 1363 bp EPSPS transgene. A 180 bp ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit fragment and a 466 bp 16S rDNA fragment were used as controls for endogenous rapeseed DNA and bacterial DNA respectively. The limit of detection of the PCR assay, established using negative controls spiked with known quantities of DNA, was 12.5 pg. Production of gas and NH3 was monitored throughout the incubation and confirmed active in vitro fermentation. Bacterial DNA was detected in all sample types at all time points. Persistence patterns of endogenous (Rubisco) and recombinant (EPSPS) rapeseed DNA were inversely related to substrate digestibility (amplifiable for 48, 8 and 4 h in whole or cracked seeds, meal and diets respectively), but did not differ between parental and GM rapeseed, nor among fragments. Detection of fragments was representative of persistence of the whole transgene. No EPSPS fragments were amplifiable in microbial DNA, suggesting that transformation had not occurred during the 48 h incubation. Uptake of transgenic DNA fragments by ruminal bacteria is probably precluded or time-limited by rapid degradation of plant DNA upon plant cell lysis.

  16. [Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

    PubMed

    Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

    2016-01-01

    Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

  17. Control of left ventricular mass by moxonidine involves reduced DNA synthesis and enhanced DNA fragmentation

    PubMed Central

    Paquette, P-A; Duguay, D; Ayoubi, R El-; Menaouar, A; Danalache, B; Gutkowska, J; DeBlois, D; Mukaddam-Daher, S

    2007-01-01

    Background and purpose: Left ventricular hypertrophy (LVH) is a maladaptive process associated with increased cardiovascular risk. Regression of LVH is associated with reduced complications of hypertension. Moxonidine is an antihypertensive imidazoline compound that reduces blood pressure primarily by central inhibition of sympathetic outflow and by direct actions on the heart to release atrial natriuretic peptide, a vasodilator and an antihypertrophic cardiac hormone. This study investigated the effect of moxonidine on LVH and the mechanisms involved in this effect. Experimental approach: Spontaneously hypertensive rats were treated with several doses of moxonidine (s.c.) over 4 weeks. Blood pressure and heart rate were continuously monitored by telemetry. Body weight and water and food intake were measured weekly. Measurements also included left ventricular mass, DNA content, synthesis, fragmentation, and apoptotic/anti-apoptotic pathway proteins. Key results: The decrease in mean arterial pressure stabilized at ∼ −10 mm Hg after 1 week of treatment and thereafter. Compared to vehicle-treated rats (100%), left ventricular mass was dose- and time-dependently reduced by treatment. This reduction remained significantly lower after normalizing to body weight. Moxonidine reduced left ventricular DNA content and inhibited DNA synthesis. DNA fragmentation transiently, but significantly increased at 1 week of moxonidine treatment and was paralleled by elevated active caspase-3 protein. The highest dose significantly decreased the apoptotic protein Bax and all doses stimulated anti-apoptotic Bcl-2 after 4 weeks of treatment. Conclusions and implications: These studies implicate the modulation of cardiac DNA dynamics in the control of left ventricular mass by moxonidine in a rat model of hypertension. PMID:18059325

  18. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

    PubMed

    Fernández, Jose Luis; Muriel, Lourdes; Rivero, Maria Teresa; Goyanes, Vicente; Vazquez, Rosana; Alvarez, Juan G

    2003-01-01

    Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test

  19. An innovative platform for quick and flexible joining of assorted DNA fragments

    PubMed Central

    De Paoli, Henrique Cestari; Tuskan, Gerald A.; Yang, Xiaohan

    2016-01-01

    Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. Here, we describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. This system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitry assembled from different DNA fragments. PMID:26758940

  20. Caspase-2 cleaves DNA fragmentation factor (DFF45)/inhibitor of caspase-activated DNase (ICAD).

    PubMed

    Dahal, Giri Raj; Karki, Pratap; Thapa, Arjun; Shahnawaz, Mohammad; Shin, Song Yub; Lee, Jung Sup; Cho, Byungyun; Park, Il-Seon

    2007-12-01

    To investigate the signal transduction pathway of caspase-2, cell permeable Tat-reverse-caspase-2 was constructed, characterized and utilized for biochemical and cellular studies. It could induce the cell death as early as 2h, and caspase-2-specific VDVADase activity but not other caspase activities including DEVDase and IETDase. Interestingly, nuclear DNA fragmentation occurred and consistently DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD) was cleaved inside the cell as well as in vitro, suggesting a role of caspase-2 in nuclear DNA fragmentation. PMID:17945178

  1. An innovative platform for quick and flexible joining of assorted DNA fragments.

    PubMed

    De Paoli, Henrique Cestari; Tuskan, Gerald A; Yang, Xiaohan

    2016-01-01

    Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. Here, we describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. This system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitry assembled from different DNA fragments. PMID:26758940

  2. Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

    PubMed Central

    Álvarez-Sandoval, Brenda A.; Manzanilla, Linda R.; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  3. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  4. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design.

  5. Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis.

    PubMed

    Chen, D; Stetler, R A; Cao, G; Pei, W; O'Horo, C; Yin, X M; Chen, J

    2000-12-01

    Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.

  6. Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing.

    PubMed Central

    Anderson, R A; Eliason, S L

    1986-01-01

    The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10-fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined. Images PMID:3023971

  7. Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads.

    PubMed

    Lindblad, P; Haselkorn, R; Bergman, B; Nierzwicki-Bauer, S A

    1989-01-01

    DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.

  8. Simultaneously Sizing and Quantitating Zepto-Mole DNA at High-Throughput in Free Solution

    PubMed Central

    Zhu, Zaifang; Chen, Huang; Chen, Apeng; Lu, Joann J.

    2014-01-01

    Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zepto-mole levels. We have recently developed a new technique, called Bare Narrow Capillary-Hydrodynamic Chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zepto-mole to several-molecule levels. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA having different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level. PMID:25223843

  9. Statistical methods for detecting periodic fragments in DNA sequence data

    PubMed Central

    2011-01-01

    Background Period 10 dinucleotides are structurally and functionally validated factors that influence the ability of DNA to form nucleosomes, histone core octamers. Robust identification of periodic signals in DNA sequences is therefore required to understand nucleosome organisation in genomes. While various techniques for identifying periodic components in genomic sequences have been proposed or adopted, the requirements for such techniques have not been considered in detail and confirmatory testing for a priori specified periods has not been developed. Results We compared the estimation accuracy and suitability for confirmatory testing of autocorrelation, discrete Fourier transform (DFT), integer period discrete Fourier transform (IPDFT) and a previously proposed Hybrid measure. A number of different statistical significance procedures were evaluated but a blockwise bootstrap proved superior. When applied to synthetic data whose period-10 signal had been eroded, or for which the signal was approximately period-10, the Hybrid technique exhibited superior properties during exploratory period estimation. In contrast, confirmatory testing using the blockwise bootstrap procedure identified IPDFT as having the greatest statistical power. These properties were validated on yeast sequences defined from a ChIP-chip study where the Hybrid metric confirmed the expected dominance of period-10 in nucleosome associated DNA but IPDFT identified more significant occurrences of period-10. Application to the whole genomes of yeast and mouse identified ~ 21% and ~ 19% respectively of these genomes as spanned by period-10 nucleosome positioning sequences (NPS). Conclusions For estimating the dominant period, we find the Hybrid period estimation method empirically to be the most effective for both eroded and approximate periodicity. The blockwise bootstrap was found to be effective as a significance measure, performing particularly well in the problem of period detection in the

  10. Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M J; López-Fernández, C; Johnston, S D; Blyde, D; Cooper, J; Harrison, K; de la Fuente, J; Gosálvez, J

    2015-04-01

    Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples.

  11. Effect of cryopreservation on the sperm DNA fragmentation dynamics of the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M J; López-Fernández, C; Johnston, S D; Blyde, D; Cooper, J; Harrison, K; de la Fuente, J; Gosálvez, J

    2015-04-01

    Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species-specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post-thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post-thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax(®)). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES-TRIS-fructose buffer (TTF), an egg-yolk-free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen-thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen-thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1(℗) buffer had higher levels (p < 0.05) of DNA fragmentation after 24- and 48-h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen-thawed samples. PMID:25604784

  12. A method for selective PCR-amplification of genomic DNA fragments (SAGF method)

    SciTech Connect

    Zheleznaya, L.A.; Menzenyuk, O.Y.; Matvienko, N.N.; Matvienko, N.I.

    1995-09-01

    A method is suggested for dividing into individual sets of the complex mixtures of fragments obtained by DNA cleavage with type IIS and IIN restriction endonucleases producing single-stranded termini with different sequences at the DNA fragment ends. The method is based on the ligation of short double-stranded adapters with single-stranded ends complementary to termini of the selected set of fragments followed by PCR-amplification with the primer representing one of the adapter chains. Using endonucleases BcoKI and Bli736I, recognizing sequences CTCTTC and GGTCTC and producing three- and four nucleotide 5{prime}-termini, respectively, it has been shown that amplification of a set of fragments occurs only upon attachment of the adapters to the DNA fragments with DNA-ligase. Several possible applications of the SAGF method are suggested: obtaining individual bands in DNA fingerprinting; reducing the kinetic complexity of DNA in representative difference analysis (RDA method) of complex genomes; cataloging of DNA fragments; construction of physical genome maps. 13 refs., 3 figs., 2 tabs.

  13. Clinical and legal significance of fragmentation of bullets in relation to size of wounds: retrospective analysis

    PubMed Central

    Coupland, Robin

    1999-01-01

    Objective To examine the relation between fragmentation of bullets and size of wounds clinically and in the context of the Hague Declaration of 1899. Design Retrospective analysis of prospectively collected data on hospital admissions. Setting Hospitals of the International Committee of the Red Cross. Subjects 5215 people wounded by bullets in armed conflicts (5933 wounds). Main outcome measures Grade of wound computed from the Red Cross wound classification and presence of bullet fragments on radiography. Results Of the 347 wounds with fragmentation of bullets, 251 (72%) were large wounds (grade 2 or 3)—that is, those with a clinically detectable cavity. Of the 5586 wounds without fragmentation of bullets, 2915 (52.1%) were large wounds. Only 7.9% (251/3166) of large wounds were associated with fragmentation of bullets. Conclusions Fragmentation of bullets is associated with large wounds, but most large wounds do not contain bullet fragments. In addition, bullet fragments may occur in wounds that are not defined as large. Fragmentation of bullets is neither a necessary nor sufficient cause of large wounds, and surgeons should not diagnose extensive tissue damage because of the presence of fragments on radiography. Such findings also do not necessarily represent the use of bullets which contravene the law of war. Future legislation should take into account not only the construction of bullets but also their potential to transfer energy to the human body. Key messagesThe use of certain bullets has been prohibited in warWounds from bullets are caused by transfer of kinetic energy from the bullet to the tissuesThe relation between size of wound and fragmentation of bullets can be examined using the Red Cross wound classification system Fragments of bullets seen on radiographs of wounds sustained in wars do not necessarily represent large wounds or the use of illegal bulletsExisting legislation on the construction of bullets should be supplemented by legislation on

  14. Evaluation of eruptive energy of a pyroclastic deposit applying fractal geometry to fragment size distributions

    NASA Astrophysics Data System (ADS)

    Paredes Marino, Joali; Morgavi, Daniele; Di Vito, Mauro; de Vita, Sandro; Sansivero, Fabio; Perugini, Diego

    2016-04-01

    Fractal fragmentation theory has been applied to characterize the particle size distribution of pyroclastic deposits generated by volcanic explosions. Recent works have demonstrated that fractal dimension on grain size distributions can be used as a proxy for estimating the energy associated with volcanic eruptions. In this work we seek to establish a preliminary analytical protocol that can be applied to better characterize volcanic fall deposits and derive the potential energy for fragmentation that was stored in the magma prior/during an explosive eruption. The methodology is based on two different techniques for determining the grain-size distribution of the pyroclastic samples: 1) dry manual sieving (particles larger than 297μm), and 2) automatic grain size analysis via a CamSizer-P4®device, the latter measure the distribution of projected area, obtaining a cumulative distribution based on volume fraction for particles up to 30mm. Size distribution data have been analyzed by applying the fractal fragmentation theory estimating the value of Df, i.e. the fractal dimension of fragmentation. In order to test our protocol we studied the Cretaio eruption, Ischia island, Italy. Results indicate that size distributions of pyroclastic fall deposits follow a fractal law, indicating that the fragmentation process of these deposits reflects a scale-invariant fragmentation mechanism. Matching the results from manual and automated techniques allows us to obtain a value of the "fragmentation energy" from the explosive eruptive events that generate the Cretaio deposits. We highlight the importance of these results, based on fractal statistics, as an additional volcanological tool for addressing volcanic risk based on the analyses of grain size distributions of natural pyroclastic deposits. Keywords: eruptive energy, fractal dimension of fragmentation, pyroclastic fallout.

  15. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation

    PubMed Central

    2015-01-01

    Background The assessment of cell-free circulating DNA fragments, also known as a "liquid biopsy" of the patient's plasma, is an important source for the discovery and subsequent non-invasive monitoring of cancer and other pathological conditions. Although the nucleosome-guided fragmentation patterns of cell-free DNA (cfDNA) have not yet been studied in detail, non-random representation of cfDNA sequencies may reflect chromatin features in the tissue of origin at gene-regulation level. Results In this study, we investigated the association between epigenetic landscapes of human tissues evident in the patterns of cfDNA in plasma by deep sequencing of human cfDNA samples. We have demonstrated that baseline characteristics of cfDNA fragmentation pattern are in concordance with the ones corresponding to cell lines-derived. To identify the loci differentially represented in cfDNA fragment, we mapped the transcription start sites within the sequenced cfDNA fragments and tested for association of these genomic coordinates with the relative strength and the patterns of gene expressions. Preselected sets of house-keeping and tissue specific genes were used as models for actively expressed and silenced genes. Developed measure of gene regulation was able to differentiate these two sets based on sequencing coverage near gene transcription start site. Conclusion Experimental outcomes suggest that cfDNA retains characteristics previously noted in genome-wide analysis of chromatin structure, in particular, in MNase-seq assays. Thus far the analysis of the DNA fragmentation pattern may aid further developing of cfDNA based biomarkers for a variety of human conditions. PMID:26693644

  16. Nuclear-localized plastid DNA fragments in protozoa, metazoa and fungi.

    PubMed

    Yuan, Shu; Sun, Xin; Mu, Lin-Chun; Lei, Tao; Liu, Wen-Juan; Wang, Jian-Hui; Du, Jun-Bo; Lin, Hong-Hui

    2007-01-01

    We analyzed nuclear-localized plastid-like DNA (nupDNA) fragments in protozoa, metazoa and fungi. Most eukaryotes that do not have plastids contain 40-5000 bp nupDNAs in their nuclear genomes. These nupDNA fragments are mainly derived from repeated regions of plastids and distribute through the whole genomes. A majority of nupDNA fragments is located on coding regions with very important functions. Similar to plastids, these nupDNAs most possibly originate from cyanobacteria. Analysis of them suggests that through millions of years of universal endosymbiosis and gene transfer they may have occurred in ancient protists before divergence of plants and animals/fungi, and some transferred fragments have been reserved till now even in modern mammals.

  17. Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Ng Tang, D. S.; Fermin, C. D.; Lewis, D. E.; Martin, D. S.; Garry, R. F.

    1995-01-01

    Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

  18. Size-controllable synthesis of bare gold nanoparticles by femtosecond laser fragmentation in water.

    PubMed

    Maximova, Ksenia; Aristov, Andrei; Sentis, Marc; Kabashin, Andrei V

    2015-02-13

    We report a size-controllable synthesis of stable aqueous solutions of ultrapure low-size-dispersed Au nanoparticles by methods of femtosecond laser fragmentation from preliminary formed colloids. Such approach makes possible the tuning of mean nanoparticle size between a few nm and several tens of nm under the size dispersion lower than 70% by varying the fluence of pumping radiation during the fragmentation procedure. The efficient size control is explained by 3D geometry of laser fragmentation by femtosecond laser-induced white light super-continuum and plasma-related phenomena. Despite the absence of any protective ligands, the nanoparticle solutions demonstrate exceptional stability due to electric repulsion effect associated with strong negative charging of formed nanoparticles. Stable aqueous solutions of bare gold nanoparticles present a unique object with a variety of potential applications in catalysis, surface-enhanced Raman spectroscopy, photovoltaics, biosensing and biomedicine.

  19. Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

    PubMed

    Howard, Steven; Amin, Nader; Benowitz, Andrew B; Chiarparin, Elisabetta; Cui, Haifeng; Deng, Xiaodong; Heightman, Tom D; Holmes, David J; Hopkins, Anna; Huang, Jianzhong; Jin, Qi; Kreatsoulas, Constantine; Martin, Agnes C L; Massey, Frances; McCloskey, Lynn; Mortenson, Paul N; Pathuri, Puja; Tisi, Dominic; Williams, Pamela A

    2013-12-12

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  20. Fragment-Based Discovery of 6-Azaindazoles As Inhibitors of Bacterial DNA Ligase

    PubMed Central

    2013-01-01

    Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase. PMID:24900632

  1. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

    PubMed Central

    Leclerc, Xavier; Danos, Olivier; Scherman, Daniel; Kichler, Antoine

    2009-01-01

    Background Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome. Results In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin. Conclusion Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments. PMID:19379497

  2. Simultaneous Vitality and DNA-fragmentation measurement in spermatozoa of smokers and non-smokers.

    PubMed

    De Bantel, A; Fleury-Feith, J; Poirot, C; Berthaut, I; Garcin, C; Landais, P; Ravel, C

    2014-08-30

    Background: Because cigarette smoke is a powerful ROS producer, we hypothesized that the spermatozoa of smokers would be more at risk of having increased DNA fragmentation than spermatozoa of non-smoking men. Methods: A Cross-Sectional Study was performed on consenting smokers and non-smokers, consulting in an infertility clinic for routine sperm analysis. The application of a novel TUNEL assay coupled to a vitality marker, LIVE/DEAD®, allowed both DNA fragmentation and viability measurement within spermatozoa of participants to be analyzed by flow cytometry. Results: The coupled vitality-DNA fragmentation analysis revealed that non-smokers and smokers respectively presented medians of 3.6% [0.6-36.8] and 3.3% [0.9-9.6] DNA fragmented spermatozoa among the living spermatozoa population (p>0.05). Conclusion: No deleterious effect of smoking on spermatozoa was found in our study. More studies concerning potential mutagenic capacities of cigarette smoke on spermatozoa are necessary. In addition, the coupled vitality-DNA fragmentation analysis may orient Assisted Reproductive Technologies teams when confronted with patients having a high percentage of DNA-fragmented living spermatozoa. © 2014 Clinical Cytometry Society.

  3. Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

    DOE PAGES

    Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth E.; Suzuki, Yo; Isalan, Mark

    2015-09-08

    Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processesmore » associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.« less

  4. DNA fragmentation occurs in skeletal muscle during tumor growth: A link with cancer cachexia?

    PubMed

    van Royen, M; Carbó, N; Busquets, S; Alvarez, B; Quinn, L S; López-Soriano, F J; Argilés, J M

    2000-04-13

    In two different experimental models of cancer cachexia, the rat Yoshida AH-130 ascites hepatoma and the mouse Lewis lung carcinoma, the implantation of the tumor caused a loss of body weight which was associated with a reduction in the weight of different skeletal muscles, as well as with their protein content. The decrease in protein content was accompanied by a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumor-bearing animals as compared with the non-tumor-bearing controls. Analysis of DNA fragmentation in skeletal muscle clearly showed enhanced laddering in the skeletal muscle of tumor-bearing animals, suggesting an apoptotic phenomenon. Interestingly, the degree of laddering (total DNA fragmented) increased with tumor burden. These results suggest that DNA fragmentation may be a primary event in cancer-associated cachexia.

  5. Dynamics of sperm DNA fragmentation in raw boar semen and fertility.

    PubMed

    Batista, C; van Lier, E; Petrocelli, H

    2016-10-01

    The aims were to evaluate sperm DNA fragmentation (SDF) in boars through the dispersion of their chromatin in raw semen samples, quantifying the extent of SDF, and to assess dynamic aspects of sperm DNA damage after incubation to obtain the rate of sperm DNA fragmentation (rSDF) under thermal conditions similar to the uterus (37°C) over a period of up to 24 hr and to correlate the reproductive outcome of the sows with the SDF of the boars at ejaculation. The study was performed on a pig-breeding farm in southern Uruguay. Sixty-one ejaculates from five of the most frequently used hybrid boars were evaluated. Semen was collected weekly from each of the boars, using the gloved-hand technique and discarding the jelly-like fraction of the ejaculate. Fresh semen was kept in a water bath at 37°C and protected from light, and was thereafter processed with Sperm-Sus-Halomax(®) to evaluate SDF. The smears for time 0 (T0) were made on farm, and thereafter smears were made at the laboratory at 4 hr of obtaining the semen (T4), then every 2 hr (T6, T8, T10, T12) and a final fixation at 24 hr (T24). Differences in SDF were observed among exposure times for all boars (p < .05), but not between T10 and T12 (p = .7751) nor T4 and T24 (p = .9113). In none of the T24 samples, sperm heads could be seen with chromatin dispersion halos. Furthermore, there were differences among boars when comparing sperm rSDF (p < .05). Farrowing rate was not affected by SDF at T0 (r = .38, p = .75), nor was litter size (r = .16, p = .70). With the present experimental conditions, we have not been able to show a relationship between sperm DNA fragmentation at ejaculation and reproductive performance. However, this could be a result of the low number of ejaculates and boars used. PMID:27546051

  6. Cluster-size dynamics: A phenomenological model for the interaction between coagulation and fragmentation processes

    NASA Astrophysics Data System (ADS)

    Rotstein, Horacio G.

    2015-06-01

    We present a novel phenomenological modeling approach to describe the growth of clusters as the result of the interaction between cluster coagulation and fragmentation. The cluster-size growth (CSG) model tracks the evolution of cluster-sizes rather than the concentrations of clusters with different sizes as in the Smoluchowski and Becker-Döring coagulation-fragmentation equations. Our modeling perspective allows for a description of cluster growth in realistic systems by using a significantly smaller number of differential equations to describe their dynamics. We used dynamical system tools (phase-plane analysis) and numerical simulations to investigate the CSG model dynamics and to understand how the model parameters describing the coagulation and fragmentation processes determine balances between these two processes that create non-zero stationary cluster size distributions.

  7. Joint effects of population size and isolation on genetic erosion in fragmented populations: finding fragmentation thresholds for management

    PubMed Central

    Méndez, María; Vögeli, Matthias; Tella, José L; Godoy, José A

    2014-01-01

    Size and isolation of local populations are main parameters of interest when assessing the genetic consequences of habitat fragmentation. However, their relative influence on the genetic erosion of local populations remains unclear. In this study, we first analysed how size and isolation of habitat patches influence the genetic variation of local populations of the Dupont's lark (Chersophilus duponti), an endangered songbird. An information-theoretic approach to model selection allowed us to address the importance of interactions between habitat variables, an aspect seldom considered in fragmentation studies, but which explained up to 65% of the variance in genetic parameters. Genetic diversity and inbreeding were influenced by the size of local populations depending on their degree of isolation, and genetic differentiation was positively related to isolation. We then identified a minimum local population of 19 male territories and a maximum distance of 30 km to the nearest population as thresholds from which genetic erosion becomes apparent. Our results alert on possibly misleading conclusions and suboptimal management recommendations when only additive effects are taken into account and encourage the use of most explanatory but easy-to-measure variables for the evaluation of genetic risks in conservation programmes. PMID:24822084

  8. The grain-size distribution of pyroclasts: Primary fragmentation, conduit sorting or abrasion?

    NASA Astrophysics Data System (ADS)

    Kueppers, U.; Schauroth, J.; Taddeucci, J.

    2013-12-01

    Explosive volcanic eruptions expel a mixture of pyroclasts and lithics. Pyroclasts, fragments of the juvenile magma, record the state of the magma at fragmentation in terms of porosity and crystallinity. The grain size distribution of pyroclasts is generally considered to be a direct consequence of the conditions at magma fragmentation that is mainly driven by gas overpressure in bubbles, high shear rates, contact with external water or a combination of these factors. Stress exerted by any of these processes will lead to brittle fragmentation by overcoming the magma's relaxation timescale. As a consequence, most pyroclasts exhibit angular shapes. Upon magma fragmentation, the gas pyroclast mixture is accelerated upwards and eventually ejected from the vent. The total grain size distribution deposited is a function of fragmentation conditions and transport related sorting. Porous pyroclasts are very susceptible to abrasion by particle-particle or particle-conduit wall interaction. Accordingly, pyroclastic fall deposits with angular clasts should proof a low particle abrasion upon contact to other surfaces. In an attempt to constrain the degree of particle interaction during conduit flow, monomodal batches of washed pyroclasts have been accelerated upwards by rapid decompression and subsequently investigated for their grain size distribution. In our set-up, we used a vertical cylindrical tube without surface roughness as conduit. We varied grain size (0.125-0.25; 0.5-1; 1-2 mm), porosity (0; 10; 30 %), gas-particle ratio (10 and 40%), conduit length (10 and 28 cm) and conduit diameter (2.5 and 6 cm). All ejected particles were collected after settling at the base of a 3.3 m high tank and sieved at one sieve size below starting size (half-Φ). Grain size reduction showed a positive correlation with starting grain size, porosity and overpressure at the vent. Although milling in a volcanic conduit may take place, porous pyroclasts are very likely to be a primary product

  9. Patch Size, Functional Isolation, Visibility and Matrix Permeability Influences Neotropical Primate Occurrence within Highly Fragmented Landscapes

    PubMed Central

    da Silva, Lucas Goulart; Ribeiro, Milton Cezar; Hasui, Érica; da Costa, Carla Aparecida; da Cunha, Rogério Grassetto Teixeira

    2015-01-01

    Forest fragmentation and habitat loss are among the major current extinction causes. Remaining fragments are mostly small, isolated and showing poor quality. Being primarily arboreal, Neotropical primates are generally sensitive to fragmentation effects. Furthermore, primates are involved in complex ecological process. Thus, landscape changes that negatively interfere with primate population dynamic affect the structure, composition, and ultimately the viability of the whole community. We evaluated if fragment size, isolation and visibility and matrix permeability are important for explaining the occurrence of three Neotropical primate species. Employing playback, we verified the presence of Callicebus nigrifrons, Callithrix aurita and Sapajus nigritus at 45 forest fragments around the municipality of Alfenas, Brazil. We classified the landscape and evaluated the metrics through predictive models of occurrence. We selected the best models through Akaike Selection Criterion. Aiming at validating our results, we applied the plausible models to another region (20 fragments at the neighboring municipality of Poço Fundo, Brazil). Twelve models were plausible, and three were validated, two for Sapajus nigritus (Area and Area+Visibility) and one for Callicebus nigrifrons (Area+Matrix). Our results reinforce the contribution of fragment size to maintain biodiversity within highly degraded habitats. At the same time, they stress the importance of including novel, biologically relevant metrics in landscape studies, such as visibility and matrix permeability, which can provide invaluable help for similar studies in the future and on conservation practices in the long run. PMID:25658108

  10. Anomalous Separation of Small Y-Chromosomal DNA Fragments on Microchip Electrophoresis

    PubMed Central

    Jabasini, Mohammad; Ewis, Ashraf; Sato, Youichi; Nakahori, Yutaka; Baba, Yoshinobu

    2016-01-01

    We investigated an anomalous DNA separation where two DNA fragments from the human Y-chromosome sY638 (64 bp) and sY592 (65 bp), with only one base pair difference, were separated. This result is abnormal since in a previous study, we found that 5 bp was the minimum difference between two DNA fragments that the microchip electrophoresis system can separate. The formation of a mini-loop in the structure of the DNA fragment of sY638 (64 bp) was strongly expected to be the reason. To investigate this, we synthesized three modified DNA fragments for sY638 (64 bp), and the modifications were in two expected locations for possible mini-loop formation. Later, the separation between sY592 (65 bp) and the three modified fragments of sY638 (64 bp) was not possible. Thus, we conclude that the formation of a mini-loop in the structure of the DNA is the reason behind this anomalous separation.

  11. Patch occupancy by stone martens Martes foina in fragmented landscapes of central Spain: the role of fragment size, isolation and habitat structure

    NASA Astrophysics Data System (ADS)

    Virgós, Emilio; García, Francisco J.

    2002-08-01

    We studied the response to forest fragmentation of a generalist carnivore, the stone marten Martes foina, in highly fragmented landscapes of central Spain. Five different areas ( n = 178 fragments) in central Spain were surveyed. This paper analyses the relationship between fragment use by martens (measured through scat presence) and a series of variables related to the size, isolation and vegetation structure of each fragment by means of stepwise logistic regression. Size and isolation have an important effect on stone marten presence in fragments. Our results were similar to those found for other marten species in landscapes with coarse-grain fragmentation, but they contrast with other studies conducted in landscapes with fine-grain fragmentation. These data suggested that in highly fragmented landscapes, size and isolation factors resulting from forest fragmentation were responsible for determining marten responses, irrespective of their habitat generalism. Management policies for the stone marten in highly fragmented scenarios require the maintenance of large forests near continuous forest tracts in mountains or riparian woodlands.

  12. Isolation and characterization of matrix associated region DNA fragments in rice (Oryza sativa L.).

    PubMed

    Nomura, K; Saito, W; Ono, K; Moriyama, H; Takahashi, S; Inoue, M; Masuda, K

    1997-09-01

    To investigate the interactions between chromosomal DNA and nuclear matrices in higher plants, matrix associated regions (MARs) of rice (Oryza sativa L.) DNAs were cloned. First, we prepared nuclear matrices from isolated nuclei by digesting them with EcoRI and then extracting with 2 M NaCl. About 6% of the total DNA remained in the nuclear matrices after this digestion and extraction. The residual DNA fragments in the nuclear matrices were cloned. Some of the cloned DNA fragments showed binding to certain nuclear proteins. One of the MAR fragments contained sequences related to known consensus motifs and a hairpin loop structure. A method is presented for isolation of matrix associated region (MAR) DNAs from plant cells.

  13. Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

    PubMed

    Wang, X; Tolstonog, G; Shoeman, R L; Traub, P

    1996-03-01

    Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global

  14. A mercury-thiol affinity system for rapid generation of overlapping labeled DNA fragments for DNA sequencing.

    PubMed Central

    Hartley, J L; Chen, K K; Donelson, J E

    1982-01-01

    We describe an in vitro protocol for quickly generating overlapping terminal-labeled restriction fragments for DNA sequence analysis via the Maxam-Gilbert technique. The protocol involves introducing mercurated nucleotides into one end of a region to be sequenced, partial digestion with several restriction enzymes and terminal-labeling, separation of the mercurated restriction enzymes and terminal-labeling, separation of the mercurated restriction fragments from non-mercurated ones on a thiol column and resolution of the different mercurated fragments on one preparative agarose gel. The protocol was used to determine the nucleotide sequence of a 980 base pair cDNA that contains the coding region for a variable surface glycoprotein of Trypanosoma brucei. It could just as quickly and easily be used to obtain many terminal-labeled overlapping restriction fragments covering a region of several kilobases. Images PMID:7050914

  15. X-ray tomography to measure size of fragments from penetration of high-velocity tungsten rods

    NASA Astrophysics Data System (ADS)

    Stone, Zach; Bless, Stephan; Tolman, John; McDonald, Jason; Levinson, Scott; Hanna, R.

    2012-03-01

    Behind-armor debris that results from tungsten rods penetrating armor steel at 2 km/s was studied by analysis of recovered fragments. Fragment recovery was by means of particle board. Individual fragments were analyzed by x-ray tomography, which provides information for fragment identification, mass, shape, and penetration down to masses of a few milligrams. The experiments were complemented by AUTODYN and EPIC calculations. Fragments were steel or tungsten generated from the channel or from the breakout through the target rear surface. Channel fragment motions were well described by Tate theory. Breakout fragments had velocities from the projectile remnant to the channel velocity, apparently depending on where in the projectile a fragment originated. The fragment size distribution was extremely broad and did not correlate well with simple uniform-fragment-size models.

  16. X-Ray Tomography to Measure Size of Fragments from Penetration of High-Velocity Tungsten Rods

    NASA Astrophysics Data System (ADS)

    Stone, Zach; Hanna, Romy; Bless, Stephan; Levinson, Scott; InstituteAdvanced Technology Collaboration; Department of Geological Sciences-UT Austin Collaboration

    2011-06-01

    Behind-armor debris that results from tungsten rods penetrating armor steel at 2 km/s was studied by analysis of recovered fragments. Fragment recovery was by means of particleboard. Individual fragments were analyzed by x-ray tomography, which provides information for fragment identification, mass, shape, and penetration down to masses of a few milligrams. The experiments were complemented by AUTODYN SPH calculations to provide the exit velocity and the strain rate at the time of particle formation. There were four types of fragments: steel or tungsten, and generated from the channel or from the breakout through the target rear surface. Channel fragment motions were well described by Tate theory. Breakout fragments had velocities from the projectile remnant to the channel velocity, apparently depending on where in the projectile a fragment originated. The fragment size distribution was extremely broad and did not correlate well with simple uniform-fragment-size models, e.g., Grady Kipp.

  17. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  18. Site-Specific Fragmentation of Polystyrene Molecule Using Size-Selected Ar Gas Cluster Ion Beam

    NASA Astrophysics Data System (ADS)

    Moritani, Kousuke; Mukai, Gen; Hashinokuchi, Michihiro; Mochiji, Kozo

    2009-04-01

    The secondary ion mass spectrum (SIMS) of a polystyrene thin film was investigated using a size-selected Ar gas cluster ion beam (GCIB). The fragmentation in the SIM spectrum varied by kinetic energy per atom (Eatom); the Eatom dependence of the secondary ion intensity of the fragment species of polystyrene can be essentially classified into three types based on the relationship between Eatom and the dissociation energy of a specific bonding site in the molecule. These results indicate that adjusting Eatom of size-selected GCIB may realize site-specific bond breaking within a molecule.

  19. [THE OPTIMAL CONDITIONS OF STORAGE OF SPERMATOZOA FOR ANALYSIS OF DNA FRAGMENTATION].

    PubMed

    Tataru, D A; Markova, E V; Osadchuk, L V; Sheina, E V; Svetlakov, A V

    2015-04-01

    The analysis of fragmentation of DNA of spermatozoons using technique of flow cytometry to evaluate male fertility more and more often begins to be applied in clinical diagnostic. However, development of optimal protocol of storage and preparation of spermatozoons for analysis still is at the stage of experimental elaboration. The studv was carried out to analyse effect of different conditions of preparation of ejaculate for adequate evaluation of index of fragmentation of DNA of spermatozoons using sperm chromatin structure assay technique. The sampling consisted of 20 patients of the Krasnoyarsk center of reproductive medicine. The sperm chromatin structure assay technique was applied to evaluate index of fragmentation of DNA of spermatozoons in fresh native ejaculate and after storage of spermatozoons under different temperature (37, 25 and 4 degrees C) and duration (1-2 and 1-3 days) and conditions of storage (-20 or -70 degrees C) of frozen spermatozoons (as native ejaculate or in TNE-buffer). It is demonstrated that index of fragmentation of DNA of spermatozoons has no significant alterations in ejaculate stored under 4 degrees C during 48 hours. In case of storage of ejaculate under 25 or 37 degrees C index of fragmentation of DNA of spermatozoons significantly increases already after first day of storage. The incubation of ejaculate under 37 degrees C results in increasing of index of fragmentation of DNA of spermatozoons already after first hour. The individual differences are established related to degree of increasing of index of fragmentation of DNA of spermatozoons because of impact of studied temperatures of ejaculate incubation. The storage of spermatozoons under temperature of - 20 and -70 degrees C in native ejaculate or in TNE-buffer has no effect of index of fragmentation of DNA of spermatozoons with measurement during 1-2 hours. Therefore, storage and transportation of native ejaculate under 4 degrees C during 1-2 days or in frozen condition

  20. Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.

    PubMed

    Wright, S C; Wei, Q S; Zhong, J; Zheng, H; Kinder, D H; Larrick, J W

    1994-12-01

    We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1-tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala-borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24-kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis. PMID:7964487

  1. Relating the microscopic rules in coalescence-fragmentation models to the cluster-size distribution

    NASA Astrophysics Data System (ADS)

    Ruszczycki, B.; Burnett, B.; Zhao, Z.; Johnson, N. F.

    2009-11-01

    Coalescence-fragmentation problems are now of great interest across the physical, biological, and social sciences. They are typically studied from the perspective of rate equations, at the heart of which are the rules used for coalescence and fragmentation. Here we discuss how changes in these microscopic rules affect the macroscopic cluster-size distribution which emerges from the solution to the rate equation. Our analysis elucidates the crucial role that the fragmentation rule can play in such dynamical grouping models. We focus our discussion on two well-known models whose fragmentation rules lie at opposite extremes. In particular, we provide a range of generalizations and new analytic results for the well-known model of social group formation developed by Eguíluz and Zimmermann, [Phys. Rev. Lett. 85, 5659 (2000)]. We develop analytic perturbation treatments of this original model, and extend the analytic analysis to the treatment of growing and declining populations.

  2. Clinical value of DNA fragmentation evaluation tests under ART treatments

    PubMed Central

    Tavukçuoğlu, İlkay Şafak; Al-Azawi, Tahani; Khaki, Amir Afshin; Khaki, Arash; Khalil, Ahmed; Al-Hasani, Safaa

    2012-01-01

    Male reproductive health has been under scrutiny recently. Many studies in the literature have concluded that semen quality is declining and that the incidence of testicular cancers is increasing. The reason for this change has been attributed to damage in sperm chromatin. During in vivo reproduction, the natural selection process ensures that only a spermatozoon with normal genomic material can fertilize an oocyte. However, the assisted reproduction technique (ART) is our selection process, leading to the possibility that abnormal spermatozoa could be used to fertilize an oocyte. We could avoid this by quantifying the amount and type of genomic damage in sperm using well-accepted laboratory methods. The sperm deoxyribonucleic acid (DNA) integrity is important for success of natural or assisted fertilization as well as normal development of the embryo, fetus and child. Intra cytoplasmic sperm injection (ICSI) is bypassing natural sperm selection mechanisms, which increases the risk of transmitting damaged DNA. The significance of required investigations and multiple techniques is that they could evaluate DNA defects in human spermatozoa. The ability of these techniques to accurately estimate sperm DNA damage depends on many technical and biological aspects. The aim of this review is to evaluate the most commonly used methods. PMID:24592055

  3. Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

    PubMed

    Regnery, R L; Fu, Z Y; Spruill, C L

    1986-01-01

    The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts. PMID:3009528

  4. Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

    PubMed Central

    Regnery, R L; Fu, Z Y; Spruill, C L

    1986-01-01

    The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts. Images PMID:3009528

  5. Accurate phylogenetic classification of DNA fragments based onsequence composition

    SciTech Connect

    McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis; Hugenholtz, Philip; Rigoutsos, Isidore

    2006-05-01

    Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequence characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.

  6. Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

    PubMed

    Klobutcher, L A; Gygax, S E; Podoloff, J D; Vermeesch, J R; Price, C M; Tebeau, C M; Jahn, C L

    1998-09-15

    During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.

  7. DNA fragmentation in developing lung fibroblasts exposed to Stachybotrys chartarum (atra) toxins.

    PubMed

    McCrae, K C; Rand, T G; Shaw, R A; Mantsch, H H; Sowa, M G; Thliveris, J A; Scott, J E

    2007-07-01

    Stachybotrys chartarum (atra) is a toxic mold that grows on water-damaged cellulose-based materials. Research has revealed also that inhalation of S. chartarum spores caused marked changes in respiratory epithelium, especially to developing lungs. We analyzed the epigenetic potential of S. chartarum spore toxins on developing rat lung fibroblasts using single cell gel electrophoresis (comet assay). Isolated fetal lung fibroblasts were exposed to S. chartarum spore toxins for 15 min, 3, 14, or 24 hr and control cells were exposed to saline under the same conditions. Cells were embedded in agarose, electrophoresed under alkaline conditions and silver stained. DNA damage was assessed in terms of fragmentation as measured by comet tail length (DNA migration) and intensity (% DNA contained within head and tail). Upon visual inspection, control fibroblasts showed no DNA fragmentation whereas S. chartarum-treated cells had definable comets of various degrees depending upon the time-course. Analyses of the comets revealed that exposure to S. chartarum spore toxins for at least 15 min to 14 hr, induced increased DNA fragmentation in a time-dependent manner. The fact that exposure to toxins for 24 hr showed less damage suggested that developing lung fibroblasts may have the capability of repairing DNA fragmentation. PMID:17534970

  8. An innovative platform for quick and flexible joining of assorted DNA fragments

    DOE PAGES

    De Paoli, Henrique Cestari; Tuskan, Gerald A.; Yang, Xiaohan

    2016-01-13

    Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. We describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. Moreover, we find that this system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitrymore » assembled from different DNA fragments.« less

  9. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  10. Particle size distributions and the sequential fragmentation/transport theory applied to volcanic ash

    SciTech Connect

    Wohletz, K.H. ); Sheridan, M.F. ); Brown, W.K. )

    1989-11-10

    The assumption that distributions of mass versus size interval for fragmented materials fit the log normal distribution is empirically based and has historical roots in the late 19th century. Other often used distributions (e.g., Rosin-Rammler, Weibull) are also empirical and have the general form for mass per size interval: {ital n}({ital l})={ital kl}{sup {alpha}} exp(-{ital l}{beta}), where {ital n}({ital l}) represents the number of particles of diameter {ital l}, {ital l} is the normalized particle diameter, and {ital k}, {alpha}, and {beta} are constants. We describe and extend the sequential fragmentation distribution to include transport effects upon observed volcanic ash size distributions. The sequential fragmentation/transport (SFT) distribution is also of the above mathematical form, but it has a physical basis rather than empirical. The SFT model applies to a particle-mass distribution formed by a sequence of fragmentation (comminution) and transport (size sorting) events acting upon an initial mass {ital m}{prime}: {ital n}({ital x}, {ital m})={ital C} {integral}{integral} {ital n}({ital x}{prime}, {ital m}{prime}){ital p}({xi}) {ital dx}{prime} {ital dm}{prime}, where {ital x}{prime} denotes spatial location along a linear axis, {ital C} is a constant, and integration is performed over distance from an origin to the sample location and mass limits from 0 to {ital m}.

  11. 2D Size Distribution of Chondrules and Chondritic Fragments of an Ordinary Chondrite from Lut Desert (Iran)

    NASA Astrophysics Data System (ADS)

    Pourkhorsandi, H.; Mirnejad, H.

    2014-09-01

    2D size measurement of chondrules and chondiritic fragments of a meteorite from Lut desert of Iran is conducted. Chondrules exhibit a size range of 55-1800 µm (average 437 µm). Chondiritic fragments show a size range of 46-1220 µm (average 261 µm).

  12. [Affinity capture of specific DNA fragments with the use of short synthetic sequences].

    PubMed

    Mikhaĭlov, V S; Potapov, V K; Amirkhanov, R N; Amirkhanov, N V; Bulanenkova, S S; Akopov, S B; Zarytova, V F; Nikolaev, L G; Sverdlov, E D

    2013-01-01

    The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine and 5-propynyl-2'-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments from mixtures. PMID:23844509

  13. Differentiation of mixed biological traces in sexual assaults using DNA fragment analysis

    PubMed Central

    Apostolov, Аleksandar

    2014-01-01

    During the investigation of sexual abuse, it is not rare that mixed genetic material from two or more persons is detected. In such cases, successful profiling can be achieved using DNA fragment analysis, resulting in individual genetic profiles of offenders and their victims. This has led to an increase in the percentage of identified perpetrators of sexual offenses. The classic and modified genetic models used, allowed us to refine and implement appropriate extraction, polymerase chain reaction and electrophoretic procedures with individual assessment and approach to conducting research. Testing mixed biological traces using DNA fragment analysis appears to be the only opportunity for identifying perpetrators in gang rapes. PMID:26019514

  14. A theoretical explanation of grain size distributions in explosive rock fragmentation

    NASA Astrophysics Data System (ADS)

    Fowler, A. C.; Scheu, Bettina

    2016-06-01

    We have measured grain size distributions of the results of laboratory decompression explosions of volcanic rock. The resulting distributions can be approximately represented by gamma distributions of weight per cent as a function of ϕ =-log2⁡d , where d is the grain size in millimetres measured by sieving, with a superimposed long tail associated with the production of fines. We provide a description of the observations based on sequential fragmentation theory, which we develop for the particular case of `self-similar' fragmentation kernels, and we show that the corresponding evolution equation for the distribution can be explicitly solved, yielding the long-time lognormal distribution associated with Kolmogorov's fragmentation theory. Particular features of the experimental data, notably time evolution, advection, truncation and fines production, are described and predicted within the constraints of a generalized, `reductive' fragmentation model, and it is shown that the gamma distribution of coarse particles is a natural consequence of an assumed uniform fragmentation kernel. We further show that an explicit model for fines production during fracturing can lead to a second gamma distribution, and that the sum of the two provides a good fit to the observed data.

  15. A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis.

    PubMed

    Miller Coyle, Heather; Shutler, Gary; Abrams, Sharon; Hanniman, Janet; Neylon, Suzanne; Ladd, Carll; Palmbach, Timothy; Lee, Henry C

    2003-03-01

    As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA.

  16. Patch size and isolation predict plant species density in a naturally fragmented forest.

    PubMed

    Munguía-Rosas, Miguel A; Montiel, Salvador

    2014-01-01

    Studies of the effects of patch size and isolation on plant species density have yielded contrasting results. However, much of the available evidence comes from relatively recent anthropogenic forest fragments which have not reached equilibrium between extinction and immigration. This is a critical issue because the theory clearly states that only when equilibrium has been reached can the number of species be accurately predicted by habitat size and isolation. Therefore, species density could be better predicted by patch size and isolation in an ecosystem that has been fragmented for a very long time. We tested whether patch area, isolation and other spatial variables explain variation among forest patches in plant species density in an ecosystem where the forest has been naturally fragmented for long periods of time on a geological scale. Our main predictions were that plant species density will be positively correlated with patch size, and negatively correlated with isolation (distance to the nearest patch, connectivity, and distance to the continuous forest). We surveyed the vascular flora (except lianas and epiphytes) of 19 forest patches using five belt transects (50×4 m each) per patch (area sampled per patch = 0.1 ha). As predicted, plant species density was positively associated (logarithmically) with patch size and negatively associated (linearly) with patch isolation (distance to the nearest patch). Other spatial variables such as patch elevation and perimeter, did not explain among-patch variability in plant species density. The power of patch area and isolation as predictors of plant species density was moderate (together they explain 43% of the variation), however, a larger sample size may improve the explanatory power of these variables. Patch size and isolation may be suitable predictors of long-term plant species density in terrestrial ecosystems that are naturally and anthropogenically fragmented.

  17. Patch Size and Isolation Predict Plant Species Density in a Naturally Fragmented Forest

    PubMed Central

    Munguía-Rosas, Miguel A.; Montiel, Salvador

    2014-01-01

    Studies of the effects of patch size and isolation on plant species density have yielded contrasting results. However, much of the available evidence comes from relatively recent anthropogenic forest fragments which have not reached equilibrium between extinction and immigration. This is a critical issue because the theory clearly states that only when equilibrium has been reached can the number of species be accurately predicted by habitat size and isolation. Therefore, species density could be better predicted by patch size and isolation in an ecosystem that has been fragmented for a very long time. We tested whether patch area, isolation and other spatial variables explain variation among forest patches in plant species density in an ecosystem where the forest has been naturally fragmented for long periods of time on a geological scale. Our main predictions were that plant species density will be positively correlated with patch size, and negatively correlated with isolation (distance to the nearest patch, connectivity, and distance to the continuous forest). We surveyed the vascular flora (except lianas and epiphytes) of 19 forest patches using five belt transects (50×4 m each) per patch (area sampled per patch = 0.1 ha). As predicted, plant species density was positively associated (logarithmically) with patch size and negatively associated (linearly) with patch isolation (distance to the nearest patch). Other spatial variables such as patch elevation and perimeter, did not explain among-patch variability in plant species density. The power of patch area and isolation as predictors of plant species density was moderate (together they explain 43% of the variation), however, a larger sample size may improve the explanatory power of these variables. Patch size and isolation may be suitable predictors of long-term plant species density in terrestrial ecosystems that are naturally and anthropogenically fragmented. PMID:25347818

  18. In vitro incubation of human spermatozoa promotes reactive oxygen species generation and DNA fragmentation.

    PubMed

    Cicaré, J; Caille, A; Zumoffen, C; Ghersevich, S; Bahamondes, L; Munuce, M J

    2015-10-01

    The aim of this study was to investigate the oxidative process associated with sperm capacitation and its impact on DNA fragmentation and sperm function. Redox activity and lipid peroxidation were analysed in human spermatozoa after 3, 6 and 22 h of incubation in Ham's F10 medium plus bovine albumin at 37° and 5% CO2 for capacitation. DNA status, tyrosine phosphorylation pattern and induced acrosome reaction were evaluated after capacitating conditions. At 22 h of incubation, there was a significant (P < 0.05) increase in oxygen-free radicals and lipid peroxidation, with no effect on sperm viability. There also was a significant (P < 0.001) increase in fragmented DNA in capacitated spermatozoa compared to semen values with higher rates being found after the occurrence of the induced acrosome reaction. Protein tyrosine phosphorylation pattern confirms that capacitation took place in parallel with the occurrence of DNA fragmentation. These results indicate that when spermatozoa are incubated for several hours (22 h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmented DNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity.

  19. Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

    PubMed Central

    Manning, F C; Blankenship, L J; Wise, J P; Xu, J; Bridgewater, L C; Patierno, S R

    1994-01-01

    Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2. Figure 3. Figure 7. PMID:7843091

  20. Charge reduced electrospray size spectrometry of mega- and gigadalton complexes: whole viruses and virus fragments.

    PubMed

    Hogan, Christopher J; Kettleson, Eric M; Ramaswami, Bala; Chen, Da-Ren; Biswas, Pratim

    2006-02-01

    The ability to analyze and identify large macromolecular complexes whose molecular weight is beyond the analyzable range of mass spectrometry is of great interest. The size of such complexes makes them suitable for analysis via mobility size spectrometry. In this work, charge reduced electrospray size spectrometry was used for the analysis of bacteriophage viruses with total molecular masses ranging from 3.6 MDa up to the gigadalton range. The electrospray source used was operated in "cone jet" mode with a mean droplet diameter of 170.56 nm. Bacteriophage MS2 was found to have a mobility diameter of 24.13 +/- 0.06 nm and remain highly viable after the electrospray process. Larger bacteriophages T2 and T4 have lengths greater than the diameter of the electrospray jet and droplets; thus, they could not be completely enclosed and were found to fragment at the virus capsid head-tail noncovalent interface during either the jet formation or jet breakup process. No viable T2 or T4 virions were detectable after being electrosprayed. While the exact mechanism of fragmentation could not be determined, it is proposed here that macromolecular fragmentation at noncovalent interfaces occurs due to mechanically and electrically induced stresses during jet formation and jet breakup. Bacteriophage T4 capsid heads were found to be statistically significantly larger than bacteriophage T2 capsid heads, with a mean peak diameter of 88.32 +/- 1.02 nm for T4 and 87.03 +/- 0.18 nm for T2. While capsid head fragments were detectable, tail and tail-fiber fragments could not be detected by size spectrometric analysis. This is attributed to the fact that the contractile tails of bacteriophage T2 and T4 virions mechanically deform to a varying degree while confined within the smaller jet and droplets. Further evidence of contractile tail deformation during the electrospray process was found by measuring the size spectrum of bacteriophage lambda, which has a noncontractile tail. Bacteriophage

  1. A Monte Carlo study of the radiation quality dependence of DNA fragmentation spectra.

    PubMed

    Alloni, D; Campa, A; Belli, M; Esposito, G; Facoetti, A; Friedland, W; Liotta, M; Mariotti, L; Paretzke, H G; Ottolenghi, A

    2010-03-01

    We simulated the irradiation of human fibroblasts with gamma rays, protons and helium, carbon and iron ions at a fixed dose of 5 Gy. The simulations were performed with the biophysical Monte Carlo code PARTRAC. From the output of the code, containing in particular the genomic positions of the radiation-induced DNA double-strand breaks (DSBs), we obtained the DNA fragmentation spectra. Very small fragments, in particular those related to "complex lesions" (few tens of base pairs), are probably very important for the late cellular consequences, but their detection is not possible with the common experimental techniques. We paid special attention to the differences among the various ions in the production of these very small fragments; in particular, we compared the fragmentation spectra for ions of the same specific energy and for ions of the same LET (linear energy transfer). As found previously for iron ions, we found that the RBE (relative biological effectiveness) for DSB production was considerably higher than 1 for all high-LET radiations considered. This is at variance with the results obtainable from experimental data, and it is due to the ability to count the contribution of small fragments. It should be noted that for a given LET this RBE decreases with increasing ion charge, due mainly to the increasing mean energy of secondary electrons. A precise quantification of the DNA initial damage can be of great importance for both radiation protection, particularly in open-space long-term manned missions, and hadrontherapy.

  2. Intraclass and interclass correlations of allele sizes within and between loci in DNA typing data

    SciTech Connect

    Chakraborty, R.; Srinivasan, M.R.; Andrade, M. de )

    1993-02-01

    Nonparametric measures of correlations of DNA fragment lengths within and between variable number of tandem repeat (VNTR) loci are proposed to test the hypothesis of random association of allele sizes at VNTR loci. Transformations of these nonparametric correlation measures are suggested to detect deviations of their null expectations caused by population subdivision and errors of measurement of VNTR fragment lengths. Analytic and permutation-based computer simulation studies are performed to show that under the hypothesis of independence of allele sizes the transformed correlation measures are normally distributed, irrespective of the VNTR fragment size distribution in the population even when the number of individuals samples is as low as 100. Power calculations are performed to establish that the current population data on six VNTR loci in the US Hispanic sample are in accordance with the hypothesis of random association of allele sizes within and between loci. Implications of these results in the context of forensic use of DNA typing are also discussed. 29 refs., 1 fig., 4 tabs.

  3. Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae.

    PubMed

    McLaughlin, G L; Brandt, F H; Visvesvara, G S

    1988-09-01

    Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

  4. Study of aneuploidy rate and sperm DNA fragmentation in large-headed, multiple-tailed spermatozoa.

    PubMed

    Brahem, S; Mehdi, M; Elghezal, H; Saad, A

    2012-04-01

    The aim of this study was to analyse the meiotic segregation and DNA fragmentation rates in ejaculated spermatozoa of Tunisian men who presented the macrocephalic sperm head syndrome and to compare the results with those from 20 fertile men with normal semen profiles. Sperm DNA fragmentation was evaluated by the terminal desoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling assay. Fluorescence in situ hybridisation for chromosomes X, Y and 18 was performed for the study of meiotic segregation. Despite a normal blood karyotype, patients with large-headed spermatozoa showed a significantly higher incidence of sperm chromosomal abnormalities compared with the control group. For all the patients, tetraploidy, triploidy and diploidy were the most observed abnormalities. A very high level of DNA fragmentation was shown for these patients. In conclusion, our results demonstrated that patients with large-headed, multiple-tailed spermatozoa had significantly higher incidence of sperm chromosomal abnormalities and very high level of DNA fragmentation. So intracytoplasmic sperm injection should not be recommended to these patients, not only because of its low chances of success rate but also because of its high genetic risk.

  5. The influence of cigarette smoking on human sperm quality and DNA fragmentation.

    PubMed

    Sepaniak, Sandrine; Forges, Thierry; Gerard, Hubert; Foliguet, Bernard; Bene, Marie-Christine; Monnier-Barbarino, Patricia

    2006-06-01

    The aim of the present study was to evaluate consequences of cigarette smoking on male gametes. In this prospective study, sperm parameters such as sperm density, motility, viability and normal morphology were measured according to the WHO criteria. In addition to these standard parameters, we analysed the degree of DNA fragmentation in spermatozoa using the TUNEL-assay with flow cytometry detection in 57 non-smokers and 51 smokers seeking for infertility counselling. The smoking intoxication was assessed by questionnaire and measured with the CO-Tester. We show that smokers' spermatozoa have a significantly higher DNA fragmentation than non-smokers (32% versus 25.9%, p<0.01). In contrast there is no significant difference in conventional parameters between smokers and non-smokers. The degree of sperm DNA fragmentation is not significantly correlated with any of the conventional parameters. These findings suggest that cigarette smoking may have deleterious effects on sperm nuclear quality and that sperm DNA fragmentation can therefore be considered as an independent parameter with diagnostic, prognostic, and strategic value in the treatment of infertility.

  6. Adenosine stimulates DNA fragmentation in human thymocytes by Ca(2+)-mediated mechanisms.

    PubMed

    Szondy, Z

    1994-12-15

    Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine

  7. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products. PMID:26556214

  8. Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds.

    PubMed

    Governini, L; Guerranti, C; De Leo, V; Boschi, L; Luddi, A; Gori, M; Orvieto, R; Piomboni, P

    2015-11-01

    This study investigated chromosomal aneuploidies and DNA damage in spermatozoa from male patients contaminated by perfluorinated compounds (PFCs) in whole blood and seminal plasma. Sperm aneuploidy and diploidy rate for chromosomes 18, X and Y were evaluated by FISH; sperm DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling technique coupled to flow cytometry. Our results indicated that PFC contamination was present in 58% of subjects included in the study. A significant increase in alterations of sperm parameters was observed in PFC-positive subjects compared to PFC-negative subjects. As regards the sperm aneuploidy, both disomy and diploidy rates resulted significantly increased in subjects positive for PFC contamination compared to PFC-negative samples. In addition, sperm DNA fragmentation index resulted significantly increased in PFC-contaminated subjects compared to PFC-non-contaminated subjects, with a significant increased level of dimmer DNA fragmentation index. Our results clearly indicate that PFC contamination may detrimentally affect spermatogenesis, disturbing both meiotic segregation and DNA integrity. We could therefore suggest cautions to reduce or eliminate any contact with these compounds because the long-term effects of PFC accumulation in the body are not predictable.

  9. Mitochondrial DNA Fragmentation to Monitor Processing Parameters in High Acid, Plant-Derived Foods.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Harris, Keith; Hassan, Hosni M; Simunovic, Josip; Sandeep, K P

    2015-12-01

    Mitochondrial DNA (mtDNA) fragmentation was assessed in acidified foods. Using quantitative polymerase chain reaction, Ct values measured from fresh, fermented, pasteurized, and stored cucumber mtDNA were determined to be significantly different (P > 0.05) based on processing and shelf-life. This indicated that the combination of lower temperature thermal processes (hot-fill at 75 °C for 15 min) and acidified conditions (pH = 3.8) was sufficient to cause mtDNA fragmentation. In studies modeling high acid juices, pasteurization (96 °C, 0 to 24 min) of tomato serum produced Ct values which had high correlation to time-temperature treatment. Primers producing longer amplicons (approximately 1 kb) targeting the same mitochondrial gene gave greater sensitivity in correlating time-temperature treatments to Ct values. Lab-scale pasteurization studies using Ct values derived from the longer amplicon differentiated between heat treatments of tomato serum (95 °C for <2 min). MtDNA fragmentation was shown to be a potential new tool to characterize low temperature (<100 °C) high acid processes (pH < 4.6), nonthermal processes such as vegetable fermentation and holding times of acidified, plant-derived products.

  10. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments

    PubMed Central

    Dabney, Jesse; Knapp, Michael; Glocke, Isabelle; Gansauge, Marie-Theres; Weihmann, Antje; Nickel, Birgit; Valdiosera, Cristina; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

    2013-01-01

    Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp. PMID:24019490

  11. In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

    PubMed

    Michikawa, Yuichi; Sugahara, Keisuke; Suga, Tomo; Ohtsuka, Yoshimi; Ishikawa, Kenichi; Ishikawa, Atsuko; Shiomi, Naoko; Shiomi, Tadahiro; Iwakawa, Mayumi; Imai, Takashi

    2008-12-15

    The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

  12. Types, Causes, Detection and Repair of DNA Fragmentation in Animal and Human Sperm Cells

    PubMed Central

    González-Marín, Clara; Gosálvez, Jaime; Roy, Rosa

    2012-01-01

    Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. PMID:23203048

  13. A linear relationship between crystal size and fragment binding time observed crystallographically: implications for fragment library screening using acoustic droplet ejection.

    PubMed

    Cole, Krystal; Roessler, Christian G; Mulé, Elizabeth A; Benson-Xu, Emma J; Mullen, Jeffrey D; Le, Benjamin A; Tieman, Alanna M; Birone, Claire; Brown, Maria; Hernandez, Jesus; Neff, Sherry; Williams, Daniel; Allaire, Marc; Orville, Allen M; Sweet, Robert M; Soares, Alexei S

    2014-01-01

    High throughput screening technologies such as acoustic droplet ejection (ADE) greatly increase the rate at which X-ray diffraction data can be acquired from crystals. One promising high throughput screening application of ADE is to rapidly combine protein crystals with fragment libraries. In this approach, each fragment soaks into a protein crystal either directly on data collection media or on a moving conveyor belt which then delivers the crystals to the X-ray beam. By simultaneously handling multiple crystals combined with fragment specimens, these techniques relax the automounter duty-cycle bottleneck that currently prevents optimal exploitation of third generation synchrotrons. Two factors limit the speed and scope of projects that are suitable for fragment screening using techniques such as ADE. Firstly, in applications where the high throughput screening apparatus is located inside the X-ray station (such as the conveyor belt system described above), the speed of data acquisition is limited by the time required for each fragment to soak into its protein crystal. Secondly, in applications where crystals are combined with fragments directly on data acquisition media (including both of the ADE methods described above), the maximum time that fragments have to soak into crystals is limited by evaporative dehydration of the protein crystals during the fragment soak. Here we demonstrate that both of these problems can be minimized by using small crystals, because the soak time required for a fragment hit to attain high occupancy depends approximately linearly on crystal size. PMID:24988328

  14. Comet Shoemaker-Levy 9 fragment size estimates: How big was the parent body?

    SciTech Connect

    Crawford, D.A.

    1995-12-31

    The impact of Comet Shoemaker-Levy 9 on Jupiter in July, 1994, was the largest, most energetic impact event on a planet ever witnessed. Because it broke up during a close encounter with Jupiter in 1992, it was bright enough to be discovered more than a year prior to impact, allowing the scientific community an unprecedented opportunity to assess the effects such an event would have. Many excellent observations were made from Earth-based telescopes, the Hubble Space Telescope (HST) and the Galileo spacecraft en route to Jupiter. In this paper, these observations are used in conjunction with computational simulations performed with the CTH shock-physics hydrocode to determine the sizes of the fifteen fragments that made discernible impact features on the planet. To do this, CTH was equipped with a radiative ablation model and a post-processing radiative ray-trace capability that enabled light-flux predictions (often called the impact flash) for the viewing geometries of Galileo and ground-based observers. The five events recorded by Galileo were calibrated to give fragment size estimates. Compared against ground-based and HST observations, these estimates were extended using a least-squares analysis to assess the impacts of the remaining ten fragments. Some of the largest impacts (L, G and K) were greater that 1 km in diameter but the density of the fragments was low, about 0.25 g/cm{sup 3}. The volume of the combined fifteen fragments would make a sphere 1.8 km in diameter. Assuming a pre-breakup density of 0.5 g/cm{sup 3}, the parent body of Shoemaker-Levy 9 had a probable diameter of 1.4 km. The total kinetic energy of all the impacts was equivalent to the explosive yield of 300 Gigatons of TNT.

  15. Comet Shoemaker-Levy 9 Fragment Size Estimates: How Big was the Parent Body?

    NASA Technical Reports Server (NTRS)

    Crawford, David A.

    1997-01-01

    The impact of Comet Shoemaker-Levy 9 on Jupiter in July, 1994 was the largest, most energetic impact event on a planet ever witnessed. Because it broke up during a close encounter with Jupiter in 1992, it was bright enough to be discovered more than a year prior to impact, allowing the scientific community an unprecedented opportunity to assess the effects such an event would have. Many excellent observations were made from Earth-based telescopes, the Hubble Space Telescope (HST), and the Galileo spacecraft en route to Jupiter. In this paper, these observations are used in conjunction with computational simulations performed with the CTH shock-physics hydrocode to determine the sizes of the fifteen fragments that made discernible impact features on the planet. To do this, CTH was equipped with a radiative ablation model and a postprocessing radiative ray-trace capability that enabled light-flux predictions (often called the impact flash) for the viewing geometries of Galileo and ground-based observers. The five events recorded by Galileo were calibrated to give fragment size estimates. Compared against ground-based and HST observations, these estimates were extended using a least-squares analysis to assess the impacts of the remaining ten fragments. Some of the largest impacts (L, G, and K) were greater that 1 km in diameter, but the density of the fragments was low, about 0.25 g/cm(exp 3). The volume of the combined fifteen fragments would make a sphere 1.8 km in diameter. Assuming a prebreakup density of 0.5 g/cm(exp 3), the parent body of Shoemaker-Levy 9 had a probable diameter of 1.4 km. The total kinetic energy of all the impacts was equivalent to the explosive yield of 300 Gigatons of TNT.

  16. [PCR-based evaluation of sequence specificity of DNA fragmentation by ultrasound].

    PubMed

    Garafutdinov, R R; Galimova, A A; Sakhabutdinova, A R; Chemeris, A V

    2016-01-01

    Ultrasonic fragmentation, which is a simple and convenient method for the mechanical degradation of DNA, is widely used in modern genome studies as one of the sample preparation steps. It has been recently found that the DNA breaks occur more often in the regions containing 5'-CG-3' dinucleotides. We studied the influence of the 5'-CG-3' dinucleotides on the efficiency of the 28S rRNA gene amplification during PCR with sonicated DNA of Mantis religiosa. It was shown that the amplification rate depends on the template length and the number of 5'-CG-3' dinucleotides. Amplification of the DNA regions with a higher 5'-CG-3' density is less efficient because of their higher sensitivity to ultrasound. The amount of the amplified DNA templates is inversely proportional to the 5'-CG-3'number. PMID:27239847

  17. Plant genome size variation: bloating and purging DNA.

    PubMed

    Michael, Todd P

    2014-07-01

    Plant genome size variation is a dynamic process of bloating and purging DNA. While it was thought plants were on a path to obesity through continual DNA bloating, recent research supports that most plants activity purge DNA. Plant genome size research has greatly benefited from the cataloguing of genome size estimates at the Kew Plant DNA C-values Database, and the recent availability of over 50 fully sequenced and published plant genomes. The emerging trend is that plant genomes bloat due to the copy-and-paste proliferation of a few long terminal repeat retrotransposons (LTRs) and aggressively purge these proliferating LTRs through several mechanisms including illegitimate and incomplete recombination, and double-strand break repair through non-homologous end joining. However, ultra-small genomes such as Utricularia gibba (Bladderwort), which is 82 megabases (Mb), purge excess DNA through genome fractionation and neofunctionalization during multiple rounds of whole genome duplication (WGD). In contrast, the largest published genome, Picea abies (Norway Spruce) at 19 800 Mb, has no detectable WGD but has bloated with diverse and diverged LTRs that either have evaded purging mechanisms or these purging mechanism are absent in gymnosperms. Finally, advances in DNA methylation studies suggest that smaller genomes have a more aggressive epigenomic surveillance system to purge young LTR retrotransposons, which is less active or missing in larger genomes like the bloated gymnosperms. While genome size may not reflect genome complexity, evidence is mounting that genome size may reflect evolutionary status.

  18. Heterogeneity of DNA fragments associated with the sickle-globin gene.

    PubMed

    Feldenzer, J; Mears, J G; Burns, A L; Natta, C; Bank, A

    1979-09-01

    We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.

  19. Description of the entire mRNA population by a 3' end cDNA fragment generated by class IIS restriction enzymes.

    PubMed Central

    Kato, K

    1995-01-01

    A novel means of recording the expression status of the total gene population is described. Digestion of cDNA by class IIS restriction enzymes produces a fragment with a poly (A) stretch and a 5' overhang with an unknown sequence. This fragment contains information such as the class IIS enzyme that cuts cDNA nearest to the poly (A) stretch, the sequence of the 5' overhang, and the size of the fragment. Expressed genes can be discriminated and displayed by the fragment as follows: (i) cut the cDNA with one class IIS restriction enzyme; (ii) ligate the digested cDNA to that from a pool of 64 biotinylated adaptors cohesive to all possible overhangs; (iii) digest by other two class IIS enzymes; (iv) recover the ligated molecules with streptavidin-coated paramagnetic beads; (v) perform PCR with the adaptor-primer and an anchored oligo-dT primer; (vi) separate the amplified fragments by denaturing polyacrylamide gel electrophoresis. Repeat the experiment with 64 adaptors, three enzymes and three anchored oligo-dT primers displays most of the expressed genes. Because redundancy is minimized, this technique is also ideal for generating tags for expressed genes, with which to construct a transcript map of the genome. Images PMID:7478997

  20. Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea hongkongensis.

    PubMed

    Xiang, Zhiming; Qu, Fufa; Qi, Lin; Ying, Tong; Li, Jun; Shu, Xiao; Yu, Ziniu

    2014-05-01

    Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis.

  1. Relationship between phospholipase C zeta immunoreactivity and DNA fragmentation and oxidation in human sperm

    PubMed Central

    Park, Ju Hee; Kim, Seul Ki; Kim, Jayeon; Kim, Ji Hee; Chang, Jae Hoon; Kim, Seok Hyun

    2015-01-01

    Objective The study aimed to evaluate the feasibility and reproducibility of measuring phospholipase C zeta (PLCζ) using immunostaining in human sperm and to investigate the relationship between PLCζ immunoreactivity and DNA fragmentation and oxidation in human sperm. Methods Semen samples were obtained from participants (n=44) and processed by the conventional swim-up method. Sperm concentration, motility, normal form by strict morphology, DNA fragmentation index assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling method and immunofluorescent expression for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and PLCζ were assessed. Results When duplicate PLCζ tests were performed on two sperm samples from each of the 44 participants, similar results were obtained (74.1±9.4% vs. 75.4±9.7%). Two measurements of PLCζ were found to be highly correlated with each other (r=0.759, P<0.001). Immunoreactivity of PLCζ was not associated with donor's age, sperm concentration, motility, and the percentage of normal form as well as DNA fragmentation index. However, immunoreactivity of PLCζ showed a significant negative relationship with 8-OHdG immunoreactivity (r=-0.404, P=0.009). Conclusion Measurement of PLCζ by immunostaining is feasible and reproducible. Lower expression of PLCζ in human sperm may be associated with higher sperm DNA oxidation status. PMID:26023673

  2. Study of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia.

    PubMed

    Perrin, A; Louanjli, N; Ziane, Y; Louanjli, T; Le Roy, C; Gueganic, N; Amice, V; De Braekeleer, M; Morel, F

    2011-02-01

    This study investigated meiotic segregation in spermatozoa to determine if severe teratozoospermia should prevent the use of intracytoplasmic sperm injection (ICSI) because of the high production of gametes with chromosomal aneuploidies and analysed DNA fragmentation in gametes from the same semen to determine if DNA integrity was worse in patients with severe teratozoospermia. Sperm samples from 12 infertile patients were studied by fluorescence in-situ hybridization for chromosomes X, Y, 13, 18 and 21 and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling. Four patients with a majority of macrocephalic forms with multiple flagella had more than 99% spermatozoa with abnormal chromosomal content. The other patients (globozoospermia or other abnormalities concerning sperm heads) had no increased aneuploidy or a slightly significant increase (P<0.05). The rate of DNA fragmentation was significantly higher in infertile patients than in the controls (P<0.001; 14.3% versus 1.20%, respectively) but presented important variability. Therefore, ICSI should not be attempted if men have macrocephalic gametes with multiple flagella but morphology is not always a good predictor of chromosomal content, depending upon the kind of teratozoospermia. Evaluation of the rate of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia is recommended.

  3. A cost for high levels of sperm competition in rodents: increased sperm DNA fragmentation.

    PubMed

    delBarco-Trillo, Javier; García-Álvarez, Olga; Soler, Ana Josefa; Tourmente, Maximiliano; Garde, José Julián; Roldan, Eduardo R S

    2016-03-16

    Sperm competition, a prevalent evolutionary process in which the spermatozoa of two or more males compete for the fertilization of the same ovum, leads to morphological and physiological adaptations, including increases in energetic metabolism that may serve to propel sperm faster but that may have negative effects on DNA integrity. Sperm DNA damage is associated with reduced rates of fertilization, embryo and fetal loss, offspring mortality, and mutations leading to genetic disease. We tested whether high levels of sperm competition affect sperm DNA integrity. We evaluated sperm DNA integrity in 18 species of rodents that differ in their levels of sperm competition using the sperm chromatin structure assay. DNA integrity was assessed upon sperm collection, in response to incubation under capacitating or non-capacitating conditions, and after exposure to physical and chemical stressors. Sperm DNA was very resistant to physical and chemical stressors, whereas incubation in non-capacitating and capacitating conditions resulted in only a small increase in sperm DNA damage. Importantly, levels of sperm competition were positively associated with sperm DNA fragmentation across rodent species. This is the first evidence showing that high levels of sperm competition lead to an important cost in the form of increased sperm DNA damage.

  4. Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions.

    PubMed

    Yokota, Yuichiro; Yamada, Shinya; Hase, Yoshihiro; Shikazono, Naoya; Narumi, Issay; Tanaka, Atsushi; Inoue, Masayoshi

    2007-01-01

    The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.

  5. Discussion of "The Case for the Median Fragment Size as a Better Fragment Size Descriptor than the Mean" by Finn Ouchterlony, Rock Mech Rock Eng, 2015. doi:10.1007/s00603-015-0722-1

    NASA Astrophysics Data System (ADS)

    Spathis, A. T.

    2016-01-01

    Ouchterlony (The Case for the Median Fragment Size as a Better Fragment Size Descriptor than the Mean, Rock Mech Rock Eng, 2015. doi: 10.1007/s00603-015-0722-1) argues that the median is preferred over the mean as a measure of central tendency of the rock fragmentation size distribution produced by blasting. This discussion suggests that neither is favoured over the other. Indeed, for distributions where both exist, they may be found in terms of each other, either by an analytical expression or by numerical calculation.

  6. Patch size effects on plant species decline in an experimentally fragmented landscape.

    PubMed

    Collins, Cathy D; Holt, Robert D; Foster, Bryan L

    2009-09-01

    Understanding local and global extinction is a fundamental objective of both basic and applied ecology. Island biogeography theory (IBT) and succession theory provide frameworks for understanding extinction in changing landscapes. We explore the relative contribution of fragment size vs. succession on species' declines by examining distributions of abundances for 18 plant species declining over time in an experimentally fragmented landscape in northeast Kansas, U.S.A. If patch size effects dominate, early-successional species should persist longer on large patches, but if successional processes dominate, the reverse should hold, because in our system woody plant colonization is accelerated on large patches. To compare the patterns in abundance among patch sizes, we characterize joint shifts in local abundance and occupancy with a new metric: rank occupancy-abundance profiles (ROAPs). As succession progressed, statistically significant patch size effects emerged for 11 of 18 species. More early-successional species persisted longer on large patches, despite the fact that woody encroachment (succession) progressed faster in these patches. Clonal perennial species persisted longer on large patches compared to small patches. All species that persisted longer on small patches were annuals that recruit from the seed bank each year. The degree to which species declined in occupancy vs. abundance varied dramatically among species: some species declined first in occupancy, others remained widespread or even expanded their distribution, even as they declined in local abundance. Consequently, species exhibited various types of rarity as succession progressed. Understanding the effect of fragmentation on extinction trajectories requires a species-by-species approach encompassing both occupancy and local abundance. We propose that ROAPs provide a useful tool for comparing the distribution of local abundances among landscape types, years, and species.

  7. Patch size effects on plant species decline in an experimentally fragmented landscape.

    PubMed

    Collins, Cathy D; Holt, Robert D; Foster, Bryan L

    2009-09-01

    Understanding local and global extinction is a fundamental objective of both basic and applied ecology. Island biogeography theory (IBT) and succession theory provide frameworks for understanding extinction in changing landscapes. We explore the relative contribution of fragment size vs. succession on species' declines by examining distributions of abundances for 18 plant species declining over time in an experimentally fragmented landscape in northeast Kansas, U.S.A. If patch size effects dominate, early-successional species should persist longer on large patches, but if successional processes dominate, the reverse should hold, because in our system woody plant colonization is accelerated on large patches. To compare the patterns in abundance among patch sizes, we characterize joint shifts in local abundance and occupancy with a new metric: rank occupancy-abundance profiles (ROAPs). As succession progressed, statistically significant patch size effects emerged for 11 of 18 species. More early-successional species persisted longer on large patches, despite the fact that woody encroachment (succession) progressed faster in these patches. Clonal perennial species persisted longer on large patches compared to small patches. All species that persisted longer on small patches were annuals that recruit from the seed bank each year. The degree to which species declined in occupancy vs. abundance varied dramatically among species: some species declined first in occupancy, others remained widespread or even expanded their distribution, even as they declined in local abundance. Consequently, species exhibited various types of rarity as succession progressed. Understanding the effect of fragmentation on extinction trajectories requires a species-by-species approach encompassing both occupancy and local abundance. We propose that ROAPs provide a useful tool for comparing the distribution of local abundances among landscape types, years, and species. PMID:19769135

  8. Observed changes in the block size ditribution as consequence of the rockfall fragmentation

    NASA Astrophysics Data System (ADS)

    Ruiz-Carulla, Roger; Corominas, Jordi; Mavrouli, Olga

    2015-04-01

    The fragmentation of the rock mass during a rockfall is a complex phenomenon which is poorly understood. A fragmental rockfall is defined by the separation of a mass into several smaller pieces upon the first impact(s) with the ground surface, leading to individual trajectories of the resultant blocks, affecting the redistribution of the initial mass and energy. This should be considered in the quantitative assessment of the rockfall hazard. A rock mass detached from the slope face at a rockfall event is composed of intact rock (blocks) and discontinuities and its volume can be characterized by an In situ Block Size Distribution (IBSD). After the first impact(s), both the disaggregation of the rock mass along preexisting discontinuities and the block breakage modify the original distribution of the block volumes resulting in a new one, the Rockfall Block Size Distribution (RBSD). The scope of this work is the study of the fragmentation process by comparing the changes between the IBSD and the RBSD, with the ultimate goal of obtaining the latter from the former based on a fractal fragmentation model. We have analyzed the RBSD generated in a large fragmental rockfall in the Cadí Sierra (Eastern Pyrenees) over 10000 m3 of rock mass and compared it to the ISBD derived from the scar.The RBSD was obtained by measuring more than 1500 blocks in the field and the biggest ones were also georeferenced. To obtain the IBSD, a digital surface model (DSM) of the cliff has been generated by means of digital photogrammetry. The main joint sets have been identified from the DSM, which has been also used to reconstruct the detached rockfall volume. The difference in volumes is less than 20%. The detached volume has been cut by all the observed joint sets, preserving their spatial location and assuming infinite persistence. Thus, the volume distribution of the in-situ rock blocks has been generated. The IBSD and the RBSD can be well fitted with an exponential and power law

  9. Three-dimensional imaging of DNA fragments during electrophoresis using a confocal detector

    NASA Astrophysics Data System (ADS)

    Brewer, Laurence R.; Davidson, Courtney; Balch, Joseph W.; Carrano, Anthony

    1995-04-01

    We have measured the 3D distribution of DNA fragments within an electrophoretic band. The measurements were made using a confocal microscope and a photon counting photomultiplier detector. A DNA sequencing standard was loaded into glass microchannel plates containing polyacrylamide gel. The measurements were made by scanning the plates in three dimensions using a mechanical stage under computer control, while electrophoresis was taking place. We found that the distribution of DNA was the same for all the bands measured in the sequencing ladder with an approximate Gaussian distribution along all three axis. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also as an aid in the design of high throughput DNA sequencers.

  10. Evidence That the DNA Mismatch Repair System Removes 1-Nucleotide Okazaki Fragment Flaps*♦

    PubMed Central

    Kadyrova, Lyudmila Y.; Dahal, Basanta K.; Kadyrov, Farid A.

    2015-01-01

    The DNA mismatch repair (MMR) system plays a major role in promoting genome stability and suppressing carcinogenesis. In this work, we investigated whether the MMR system is involved in Okazaki fragment maturation. We found that in the yeast Saccharomyces cerevisiae, the MMR system and the flap endonuclease Rad27 act in overlapping pathways that protect the nuclear genome from 1-bp insertions. In addition, we determined that purified yeast and human MutSα proteins recognize 1-nucleotide DNA and RNA flaps. In reconstituted human systems, MutSα, proliferating cell nuclear antigen, and replication factor C activate MutLα endonuclease to remove the flaps. ATPase and endonuclease mutants of MutLα are defective in the flap removal. These results suggest that the MMR system contributes to the removal of 1-nucleotide Okazaki fragment flaps. PMID:26224637

  11. DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure.

    PubMed

    Focke, Frauke; Schuermann, David; Kuster, Niels; Schär, Primo

    2010-01-01

    Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage.

  12. DNA fragmentation in morphologically normal spermatozoa: how much should we be concerned in the ICSI era?

    PubMed

    Avendaño, Conrado; Oehninger, Sergio

    2011-01-01

    Intracytoplasmic sperm injection (ICSI) has revolutionized the treatment of male infertility. However, there are still unanswered questions about the safety of this technique. During ICSI, only morphologically normal and motile spermatozoa are typically used to fertilize an oocyte. We recently reported that in infertile men, spermatozoa with apparently normal morphology may have DNA fragmentation. This finding consequently raised the possibility that spermatozoa with normal-shaped appearance but with DNA fragmentation could be mistakenly selected to fertilize oocytes during ICSI. This concern became more clinically significant following the subsequent finding that the presence of an increased proportion of normal spermatozoa with damaged DNA was negatively associated with embryo quality and pregnancy outcome after ICSI. Herein, we propose and discuss the hypothesis that the examination of DNA integrity in the subpopulation of highly motile (hence viable) and morphologically normal cells (and not in the total sperm population) may provide optimized information in prediction of ICSI success. More importantly, this new way of evaluation may provide reassurance about genomic normalcy and minimal risk of transmission of genetic disease and guide the development of improved methods of selection of spermatozoa with intact DNA to be used in assisted reproduction.

  13. DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

    PubMed Central

    2012-01-01

    Background Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. Results Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. Conclusions We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells. PMID:22480385

  14. Cloning and Analysis of a DNA Fragment Stimulating Avermectin Production in Various Streptomyces avermitilis Strains

    PubMed Central

    Hwang, Yong-Soon; Kim, Eung-Soo; Biró, Sándor; Choi, Cha-Yong

    2003-01-01

    To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis. PMID:12571055

  15. Cloning and analysis of a DNA fragment stimulating avermectin production in various Streptomyces avermitilis strains.

    PubMed

    Hwang, Yong-Soon; Kim, Eung-Soo; Biró, Sándor; Choi, Cha-Yong

    2003-02-01

    To isolate a gene for stimulating avermectin production, a genomic library of Streptomyces avermitilis ATCC 31267 was constructed in Streptomyces lividans TK21 as the host strain. An 8.0-kb DNA fragment that significantly stimulated actinorhodin and undecylprodigiosin production was isolated. When wild-type S. avermitilis was transformed with the cloned fragment, avermectin production increased approximately 3.5-fold. The introduction of this fragment into high-producer (ATCC 31780) and semi-industrial (L-9) strains also resulted in an increase of avermectin production by more than 2.0- and 1.4-fold, respectively. Subclones were studied to locate the minimal region involved in stimulation of pigmented-antibiotic and avermectin production. An analysis of the nucleotide sequence of the entire DNA fragment identified eight complete and one incomplete open reading frame. All but one of the deduced proteins exhibited strong homology (68 to 84% identity) to the hypothetical proteins of Streptomyces coelicolor A3(2). The orfX gene product showed no significant similarity to any other protein in the databases, and an analysis of its sequence suggested that it was a putative membrane protein. Although the nature of the stimulatory effect is still unclear, the disruption of orfX revealed that this gene was intrinsically involved in the stimulation of avermectin production in S. avermitilis. PMID:12571055

  16. Xanthorrhizol induced DNA fragmentation in HepG2 cells involving Bcl-2 family proteins

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Meenakshii, Nallappan; Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We isolated xanthorrhizol, a sesquiterpenoid compound from Curcuma xanthorrhiza. Black-Right-Pointing-Pointer Xanthorrhizol induced apoptosis in HepG2 cells as observed using SEM. Black-Right-Pointing-Pointer Apoptosis in xanthorrhizol-treated HepG2 cells involved Bcl-2 family proteins. Black-Right-Pointing-Pointer DNA fragmentation was observed in xanthorrhizol-treated HepG2 cells. Black-Right-Pointing-Pointer DNA fragmentation maybe due to cleavage of PARP and DFF45/ICAD proteins. -- Abstract: Xanthorrhizol is a plant-derived pharmacologically active sesquiterpenoid compound isolated from Curcuma xanthorrhiza. Previously, we have reported that xanthorrhizol inhibited the proliferation of HepG2 human hepatoma cells by inducing apoptotic cell death via caspase activation. Here, we attempt to further elucidate the mode of action of xanthorrhizol. Apoptosis in xanthorrhizol-treated HepG2 cells as observed by scanning electron microscopy was accompanied by truncation of BID; reduction of both anti-apoptotic Bcl-2 and Bcl-X{sub L} expression; cleavage of PARP and DFF45/ICAD proteins and DNA fragmentation. Taken together, these results suggest xanthorrhizol as a potent antiproliferative agent on HepG2 cells by inducing apoptosis via Bcl-2 family members. Hence we proposed that xanthorrhizol could be used as an anti-liver cancer drug for future studies.

  17. [An efficient genetic knockout system based on linear DNA fragment homologous recombination for halophilic archaea].

    PubMed

    Xiaoli, Wang; Chuang, Jiang; Jianhua, Liu; Xipeng, Liu

    2015-04-01

    With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea.

  18. Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends

    NASA Technical Reports Server (NTRS)

    Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    1995-01-01

    An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

  19. Size distribution of particles in Saturn’s rings from aggregation and fragmentation

    PubMed Central

    Brilliantov, Nikolai; Krapivsky, P. L.; Bodrova, Anna; Spahn, Frank; Hayakawa, Hisao; Stadnichuk, Vladimir; Schmidt, Jürgen

    2015-01-01

    Saturn’s rings consist of a huge number of water ice particles, with a tiny addition of rocky material. They form a flat disk, as the result of an interplay of angular momentum conservation and the steady loss of energy in dissipative interparticle collisions. For particles in the size range from a few centimeters to a few meters, a power-law distribution of radii, ∼r−q with q≈3, has been inferred; for larger sizes, the distribution has a steep cutoff. It has been suggested that this size distribution may arise from a balance between aggregation and fragmentation of ring particles, yet neither the power-law dependence nor the upper size cutoff have been established on theoretical grounds. Here we propose a model for the particle size distribution that quantitatively explains the observations. In accordance with data, our model predicts the exponent q to be constrained to the interval 2.75≤q≤3.5. Also an exponential cutoff for larger particle sizes establishes naturally with the cutoff radius being set by the relative frequency of aggregating and disruptive collisions. This cutoff is much smaller than the typical scale of microstructures seen in Saturn’s rings. PMID:26183228

  20. Assessing landscape structure and pattern fragmentation in semiarid ecosystems using patch-size distributions.

    PubMed

    Moreno-de Las Heras, Mariano; Saco, Patricia M; Willgoose, Garry R; Tongway, David J

    2011-10-01

    Spatial vegetation patterns are recognized as sources of valuable information that can be used to infer the state and functionality of semiarid ecosystems, particularly in the context of both climate and land use change. Recent studies have suggested that the patch-size distribution of vegetation in drylands can be described using power-law metrics, and that these scale-free distributions deviate from power-law linearity with characteristic scale lengths under the effects of increasing aridity or human disturbance, providing an early sign of desertification. These findings have been questioned by several modeling approaches, which have identified the presence of characteristic scale lengths on the patch-size distribution of semiarid periodic landscapes. We analyze the relationship between fragmentation of vegetation patterns and their patch-size distributions in semiarid landscapes showing different degree of periodicity (i.e., banding). Our assessment is based on the study of vegetation patterns derived from remote sensing in a series of semiarid Australian Mulga shrublands subjected to different disturbance levels. We use the patch-size probability density and cumulative probability distribution functions from both nondirectional and downslope analyses of the vegetation patterns. Our results indicate that the shape of the patch-size distribution of vegetation changes with the methodology of analysis applied and specific landscape traits, breaking the universal applicability of the power-law metrics. Characteristic scale lengths are detected in (quasi) periodic banded ecosystems when the methodology of analysis accounts for critical landscape anisotropies, using downslope transects in the direction of flow paths. In addition, a common signal of fragmentation is observed: the largest vegetation patches become increasingly less abundant under the effects of disturbance. This effect also explains deviations from power-law behavior in disturbed vegetation which

  1. Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism

    SciTech Connect

    Shields, P.G.; Bowman, E.D.; Weston, A.; Harris, C.C.; Sugimura, H.; Caporaso, N.E.; Petruzzelli, S.F. ); Trump, B.F. )

    1992-11-01

    Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the [sup 32]P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a US lung cancer case-control study, no association with lung cancer was found. 17 refs., 3 figs.

  2. Size-selective DNA separation: recovery spectra help determine the sodium chloride (NaCl) and polyethylene glycol (PEG) concentrations required.

    PubMed

    He, Zhangyong; Xu, Hong; Xiong, Min; Gu, Hongchen

    2014-10-01

    In the presence of sodium chloride (NaCl), DNA fragments can be size-selectively separated by varying the final concentration of polyethylene glycol (PEG). This separation strategy in combination with the use of paramagnetic particles provides a valuable platform for achieving the desired DNA size interval, which is important in automated library preparation for high-throughput DNA sequencing. Here, we report the establishment of recovery spectra of DNA fragments that enable the determination of suitable NaCl and PEG concentrations for size-selective separation. Firstly, at a given NaCl concentration, the recovery equation was obtained by fitting the DNA recovery ratios versus the PEG concentrations using the logistic function to determine the required parameters. Secondly, the slope function of the recovery equation was achieved by deducing its first derivative. Therefore, the recovery spectrum can be generated using the slope function based on those parameters. According to the recovery spectra of different length DNA fragments, suitable NaCl and PEG concentrations can be determined, respectively, by calculating their resolution values and recovery ratios. The strategy was effectively applied to the size-selective separation of 532-, 400-, and 307-bp fragments at the selected reagent concentrations with recoveries of 96.9, 64.7, and 85.9%, respectively. Our method enables good predictions of NaCl and PEG concentrations for size-selective DNA separation.

  3. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

    PubMed

    Hamilton, Thais Rose dos Santos; de Castro, Letícia Signori; Delgado, Juliana de Carvalho; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Mendes, Camilla Mota; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; D'Ávila Assumpção, Mayra Elena Ortiz

    2016-04-01

    Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm. PMID:26811546

  4. Antiserum to Nitrogenase Generated from an Amplified DNA Fragment from Natural Populations of Trichodesmium spp

    PubMed Central

    Zehr, Jonathan P.; Limberger, Ronald J.; Ohki, Kaori; Fujita, Yoshihiko

    1990-01-01

    A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA. Images PMID:16348356

  5. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

    PubMed

    Hamilton, Thais Rose dos Santos; de Castro, Letícia Signori; Delgado, Juliana de Carvalho; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Mendes, Camilla Mota; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; D'Ávila Assumpção, Mayra Elena Ortiz

    2016-04-01

    Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

  6. Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

    PubMed

    Oscorbin, Igor P; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2015-10-01

    A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.

  7. Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

    PubMed

    Oscorbin, Igor P; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2015-10-01

    A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA. PMID:26289299

  8. Characterization of DNA fragment from Chlamydia psittaci avian strain which shows high homology with hypB gene of Chlamydia.

    PubMed

    Sato, C; Katumata, A; Takashima, I; Hashimoto, N

    1991-12-01

    A study was performed to characterize DNA fragment No. 17 of C. psittaci strain P-1041 which encoded 42 KD beta-galactosidase fusion protein with type-specific antigenicity. Sequence determination identified a partial open reading frame that spanned about 1,200b. p. nucleotides. Screening the literatures for the nucleotide and deduced amino acid sequences revealed extensive similarity between the DNA fragment of P-1041 and two chlamydial hypB genes. This DNA showed 91.5% homology with C. psittaci GPIC hypB gene in nucleotide sequence and 96.4% homology in deduced amino acid sequence. The hypB gene of C. trachomatis serovar A and the P-1041 DNA fragment showed 81.2% and 91.3% homology in nucleotide and amino acid sequences, respectively. Dot enzyme-linked immunosorbent assay, for the products of deleted DNA fragments defined the coding region for type-specific antigenic polypeptide. In addition, the P-1041 DNA fragment carried a sequence highly homologous (greater than 49%) with other bacterial and plant genes called chaperonin which responds to various stress in cells. From these results, the P-1041 DNA fragment was found to be a part of hypB gene and to encode the region critical for type-specific antigenicity.

  9. Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco.

    PubMed Central

    Breyne, P; van Montagu, M; Depicker, N; Gheysen, G

    1992-01-01

    Using a low-salt extraction procedure, we isolated nuclear scaffolds from tobacco that bind specific plant DNA fragments in vitro. One of these fragments was characterized in more detail; this characterization showed that it contains sequences with structural properties analogous to animal scaffold attachment regions (SARs). We showed that scaffold attachment is evolutionarily conserved between plants and animals, although different SARs have different binding affinities. Furthermore, we demonstrated that flanking a chimeric transgene with the characterized SAR-containing fragment reduces significantly the variation in expression in series of transformants with an active insertion, whereas a SAR fragment from the human beta-globin locus does not. Moreover, the frequency distribution patterns of transgene activities showed that most of the transformants containing the plant SAR fragment had expression levels clustered around the mean. These data suggest that the particular plant DNA fragment can insulate the reporter gene from expression-influencing effects exerted from the host chromatin. PMID:1498604

  10. Fragmentation of DNA components by hyperthermal heavy ion (Ar+ and Xe+) impact in the condensed phase

    NASA Astrophysics Data System (ADS)

    Sarabipour, Sarvenaz; Sarvenaz Sarabipour, Ms; Michaud, Marc; Deng, Zongwu; Huels, Michael A.

    The overriding environmental factor that presently limits human endeavors in space is exposure to heavy ion radiation. While knowledge of its damage to living tissue is essential for radiation protection and risk estimates for astronauts, very little data exists at the molecular level regarding the nascent DNA damage by the primary particle track, or by secondary species during subsequent reaction cascades. This persistent lack of a basic understanding of nascent damage induced by such low dose, high LET radiation, introduces unacceptable errors in radiation risk estimates (based mainly on extrapolation from high dose, low LET radiation), particularly for long term exposure. Mutagenic effects induced by heavy ion radiation to cells are largely due to DNA damage by secondary transient species, i.e. secondary ballistic ions, electrons and radicals generated along the ion tracks; the secondary ions have hyperthermal energies up to several 100 eV, which they will deposit within a few nm in the surrounding medium; thus their LET is very high, and yields lethal clustered DNA lesions. We present measurements of molecular damage induced in films of DNA components by ions with precisely such low energies (1-100 eV) and compare results to conventional electron impact measurements. Experiments are conducted in UHV using a mass selected low energy ion source, and a high-resolution quadrupole MS to monitor ion yields desorbing from molecular films. Among the major fragments, NH4 + is identified in the desorption mass spectra of irradiated films of Adenine, Guanine, Cytosine, indicating efficient deamination; in cells this results in pre-mutagenic lesions. Experiments with 5-amino-Uracil, and comparison to previous results on uracil and thymine show that deamination is a key step in the NH4 + fragment formation. For Adenine, we also observe formation of amine aducts in the films, viz. amination of Adenine, and global fragmentation in all ion impact mass spectra, attributed

  11. Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding.

    PubMed

    Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

    2010-07-14

    Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA. PMID:20637419

  12. Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

    PubMed

    Künne, Tim; Kieper, Sebastian N; Bannenberg, Jasper W; Vogel, Anne I M; Miellet, Willem R; Klein, Misha; Depken, Martin; Suarez-Diez, Maria; Brouns, Stan J J

    2016-09-01

    Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. PMID:27546790

  13. Lack of in vivo transcription of Acetabularia mediterranea 1175 bp ctDNA fragment homologous to the Drosophila per locus.

    PubMed

    Yang, X P; Mittelsten Scheid, O

    1992-01-01

    The period (per) locus of Drosophila melanogaster has a fundamental role in the expression of biological rhythms. A DNA sequence homologous to a short region of the Drosophila per locus was detected in the chloroplast of Acetabularia mediterranea. A 1175 bp DNA fragment containing the sequence was used as a probe in 'Northern' hybridization experiments. It was found that this DNA was not transcribed or only marginally transcribed in A. mediterranea, at least at the developmental stage just prior to cap formation. It seems that the 1175 bp ctDNA fragment is not involved in the Acetabularia biological rhythm mechanism.

  14. Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA[reg])

    SciTech Connect

    Evenson, Donald P. . E-mail: scsa@brookings.net; Wixon, Regina

    2005-09-01

    Studies over the past two decades have clearly shown that reproductive toxicants cause sperm DNA fragmentation. This DNA fragmentation can usually be detected prior to observing alterations of metaphase chromosomes in embryos. Thus, Sperm Chromatin Structure Assay (SCSA)-detected DNA damage is viewed as the molecular precursor to later gross chromosome damage observed under the light microscope. SCSA measurements of animal or human sperm consist of first obtaining a fresh or flash frozen neat semen sample in LN2 or dry ice. Samples are then sent to a SCSA diagnostic laboratory where the samples are thawed, diluted to {approx}1-2 x 106 sperm/ml, treated for 30 s with a pH 1.2 detergent buffer and then stained with acridine orange (AO). The low pH partially denatures DNA at the sites of DNA strand breaks and the AO-ssDNA fluoresces red while the AO-dsDNA fluoresces green. Flow cytometry measurements of 5000 sperm/sample provide statistically robust data on the ratio of red to green sperm, the extent of the DNA fragmentation and the standard deviations of measures. Numerous experiments on rodents treated with reproductive toxicants clearly showed that SCSA measures are highly dose responsive and have a very low CV. Different agents that act on germ cells at various stages of development usually showed sperm DNA fragmentation when that germ cell fraction arrived in the epididymis or ejaculate. Some of these treated samples were capable of successful in vitro fertilization but with frequent embryo failure. A 2-year longitudinal study of men living a valley town with a reported abnormal level of infertility and spontaneous miscarriages and also a seasonal atmospheric smog pollution, showed, for the first time, that SCSA measurements of human sperm DNA fragmentation were detectable and correlated with dosage of air pollution while the classical semen measures were not correlated. Also, young men spraying pesticides without protective gear are at an increased risk for

  15. DNA sequence analysis of the Hind III M fragment from Chinese vaccine strain of vaccinia virus.

    PubMed

    Liu, V J; Jin, Q; Jin, D Y; Hou, Y D

    1989-01-01

    The complete DNA sequence of the Hind III M fragment of vaccinia virus (VV) Tian Tan strain genome was determined by the dideoxynucleotide chain termination method. Three open reading frames (ORFs) were identified in the complementary strand of the sequence, comprised of 2218bp. Among them, ORF K1 initiates its transcription at -45 of the Hind III K fragment. The deduced peptide encoded by K1 contains 284 amino acids with a calculated molecular weight of 32.48 KDa. Its sequence is homologous to the host range protein of VV Copenhagen strain; the variation is only 2.46% at the amino acid level. ORF M2 could encode a peptide of 21.94 KDa with 196 amino acids. This gene was shown to be homologous to that of the 23 KDa peptide of herpes simplex virus type I. A non-coding region of 204bp located between K1 and M2 is rich in palindromic structures. ORF M1 extends its 3' terminus into the Hind III N fragment. Within the M fragment, M1 can only encode 212 amino acids. The major part of ORF M1 is very similar to the M portion of a possible alpha-amanitin resistance gene isolated from VV-WR strain. This work provides a molecular foundation in the construction of a new insertion vector for the preparation of a recombinant vaccinia virus to be used as a polyvalent live vaccine.

  16. Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C.

    PubMed

    McConkey, D J; Hartzell, P; Jondal, M; Orrenius, S

    1989-08-15

    Glucocorticoid hormones and Ca2+ ionophores stimulate a suicide process in immature thymocytes, known as apoptosis or programmed cell death, that involves extensive DNA fragmentation. We have recently shown that a sustained increase in cytosolic Ca2+ concentration stimulates DNA fragmentation and cell killing in glucocorticoid- or ionophore-treated thymocytes. However, a sustained increase in the cytosolic Ca2+ level also mediates lymphocyte proliferation, suggesting that apoptosis is blocked in proliferating thymocytes. In this study we report that phorbol esters, which selectively stimulate protein kinase C (PKC), blocked DNA fragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone. The T cell mitogen, concanavalin A, which stimulates thymocytes by a mechanism that involves PKC activation, caused concentration-dependent increases in the cytosolic Ca2+ level that did not result in DNA fragmentation, but incubation with concanavalin A and the PKC inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) resulted in both DNA fragmentation and cell death. Phorbol ester directly inhibited Ca2+-dependent DNA fragmentation in isolated thymocyte nuclei. Our results strongly suggest that PKC activation blocks thymocyte apoptosis by preventing Ca2+-stimulated endonuclease activation. PMID:2503500

  17. Cloning should be simple: Escherichia coli DH5α-mediated assembly of multiple DNA fragments with short end homologies

    SciTech Connect

    Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth E.; Suzuki, Yo; Isalan, Mark

    2015-09-08

    Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six doublestranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. In addition, we demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work.

  18. Deoxyribonuclease I is Essential for DNA Fragmentation Induced by Gamma Radiation in Mice

    PubMed Central

    Apostolov, Eugene O.; Soultanova, Izoumroud; Savenka, Alena; Bagandov, Osman O.; Yin, Xiaoyan; Stewart, Anna G.; Walker, Richard B.; Basnakian, Alexei G.

    2009-01-01

    Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates γ-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in γ-radiation-induced cell death in radiosensitive organs. PMID:19772469

  19. Amyloid-β peptide structure in aqueous solution varies with fragment size

    NASA Astrophysics Data System (ADS)

    Wise-Scira, Olivia; Xu, Liang; Kitahara, Taizo; Perry, George; Coskuner, Orkid

    2011-11-01

    Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends - to some extent - with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.

  20. DNA fragmentation and nuclear phenotype in tendons exposed to low-intensity infrared laser

    NASA Astrophysics Data System (ADS)

    de Paoli, Flavia; Ramos Cerqueira, Larissa; Martins Ramos, Mayara; Campos, Vera M.; Ferreira-Machado, Samara C.; Geller, Mauro; de Souza da Fonseca, Adenilson

    2015-03-01

    Clinical protocols are recommended in device guidelines outlined for treating many diseases on empirical basis. However, effects of low-intensity infrared lasers at fluences used in clinical protocols on DNA are controversial. Excitation of endogenous chromophores in tissues and free radicals generation could be described as a consequence of laser used. DNA lesions induced by free radicals cause changes in DNA structure, chromatin organization, ploidy degrees and cell death. In this work, we investigated whether low-intensity infrared laser therapy could alter the fibroblasts nuclei characteristics and induce DNA fragmentation. Tendons of Wistar rats were exposed to low-intensity infrared laser (830 nm), at different fluences (1, 5 and 10 J/cm2), in continuous wave (power output of 10mW, power density of 79.6 mW/cm2). Different frequencies were analyzed for the higher fluence (10 J/cm2), at pulsed emission mode (2.5, 250 and 2500 Hz), with the laser source at surface of skin. Geometric, densitometric and textural parameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed on the nuclear phenotype of tendons after exposure to laser, as well as, high cell death percentages was observed for all fluences and frequencies analyzed here, exception 1 J/cm2 fluence. Our results indicate that low-intensity infrared laser can alter geometric, densitometric and textural parameters in tendon fibroblasts nuclei. Laser can also induce DNA fragmentation, chromatin lost and consequently cell death, using fluences, frequencies and emission modes took out from clinical protocols.

  1. Multiple Determinations of Sperm DNA Fragmentation Show That Varicocelectomy Is Not Indicated for Infertile Patients with Subclinical Varicocele

    PubMed Central

    García-Peiró, Agustín; Ribas-Maynou, Jordi; Oliver-Bonet, María; Navarro, Joaquima; Checa, Miguel A.; Nikolaou, Alexandros; Amengual, María J.; Abad, Carlos; Benet, Jordi

    2014-01-01

    Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient's fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA) were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment). TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele. PMID:24967335

  2. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

    PubMed

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-01-01

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process. PMID:27104527

  3. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size

    PubMed Central

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-01-01

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of −0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process. PMID:27104527

  4. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

    PubMed

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-04-20

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.

  5. Correlation between sperm DNA fragmentation index and CMA3 positive spermatozoa in globozoospermic patients.

    PubMed

    Hosseinifar, H; Yazdanikhah, S; Modarresi, T; Totonchi, M; Sadighi Gilani, M A; Sabbaghian, M

    2015-05-01

    The absence of the acrosome causes the situation which is called globozoospermia. There are a few studies, mostly as case reports, about correlation between levels of sperm DNA damage in patients with total round-headed spermatozoa. We investigated this correlation as well as CMA3 positive spermatozoa in 20 globozoospermic men (with more than 90% round-headed spermatozoa) attending to Royan Institute. Semen samples divided into three parts to semen analysis, to measure DNA fragmentation index (DFI) using sperm chromatin structure assay (SCSA) and to detect CMA3(+) sperm cells by chromomycin A3 staining and fluorescent microscopy. Our results showed that there were significant differences in sperm concentration, total sperm motility, and normal morphology between patients and controls group (p < 0.001). Moreover, the average of DFI and CMA3 positive spermatozoa in patients group significantly increases compared with control group (p < 0.001). A significant correlation between DFI and CMA3(+) in total population was also detected in patients group (r = 0.45, p = 0.046). To our knowledge, this is the largest study about correlation between DNA damage levels and CMA3 positive spermatozoa with round head sperm cells in total globozoospermic men. It seems that the increase in DNA damage may be because of defective sperm DNA compaction, as we detected CMA3 positive sperm cells in these patients.

  6. A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

    PubMed Central

    Hooker, David J.; Mobarok, Masqura; Anderson, Jenny L.; Rajasuriar, Reena; Gray, Lachlan R.; Ellett, Anne M.; Lewin, Sharon R.; Gorry, Paul R.; Cherry, Catherine L.

    2012-01-01

    Several critical events of apoptosis occur in the cell nucleus, including inter-nucleosomal DNA fragmentation (apoptotic DNA) and eventual chromatin condensation. The generation of apoptotic DNA has become a biochemical hallmark of apoptosis because it is a late ‘point of no return’ step in both the extrinsic (cell-death receptor) and intrinsic (mitochondrial) apoptotic pathways. Despite investigators observing apoptotic DNA and understanding its decisive role as a marker of apoptosis for over 20 years, measuring it has proved elusive. We have integrated ligation-mediated PCR and qPCR to design a new way of measuring apoptosis, termed ApoqPCR, which generates an absolute value for the amount (picogram) of apoptotic DNA per cell population. ApoqPCR’s advances over current methods include a 1000-fold linear dynamic range yet sensitivity to distinguish subtle low-level changes, measurement with a 3- to 4-log improvement in sample economy, and capacity for archival or longitudinal studies combined with high-throughput capability. We demonstrate ApoqPCR’s utility in both in vitro and in vivo contexts. Considering the fundamental role apoptosis has in vertebrate and invertebrate health, growth and disease, the reliable measurement of apoptotic nucleic acid by ApoqPCR will be of value in cell biology studies in basic and applied science. PMID:22544708

  7. Effects of zinc and cadmium on apoptotic DNA fragmentation in isolated bovine liver nuclei.

    PubMed Central

    Lohmann, R D; Beyersmann, D

    1994-01-01

    Isolated nuclei from mammalian cells contain a calcium-dependent endonuclease. The produced DNA fragmentation is a necessary step in the sequence of events resulting in apoptosis (programmed cell death). We report here that zinc and cadmium inhibit the calcium-dependent endonuclease. The essential metal ion zinc may counterbalance the calcium-mediated apoptosis. In contrast to zinc, cadmium alone stimulates the endonuclease by replacing calcium. Thus cadmium exerts a dual effect: micromolar concentrations inhibit the apoptotic endonuclease in the presence but activate the enzyme in the absence of calcium. Images Figure 2. PMID:7843111

  8. Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress

    PubMed Central

    Muratori, Monica; Tamburrino, Lara; Marchiani, Sara; Cambi, Marta; Olivito, Biagio; Azzari, Chiara; Forti, Gianni; Baldi, Elisabetta

    2015-01-01

    Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies. PMID:25786204

  9. Structural and Thermodynamic Properties of Amyloid-β Peptides: Impact of Fragment Size

    NASA Astrophysics Data System (ADS)

    Kitahara, T.; Wise-Scira, O.; Coskuner, O.

    2010-10-01

    Alzheimer's disease is a progressive neurodegenerative disease whose physiological characteristics include the accumulation of amyloid-containing deposits in the brain and consequent synapse and neuron loss. Unfortunately, most widely used drugs for the treatment can palliate the outer symptoms but cannot cure the disease itself. Hence, developing a new drug that can cure it. Most recently, the ``early aggregation and monomer'' hypothesis has become popular and a few drugs have been developed based on this hypothesis. Detailed understanding of the amyloid-β peptide structure can better help us to determine more effective treatment strategies; indeed, the structure of Amyloid has been studied extensively employing experimental and theoretical tools. Nevertheless, those studies have employed different fragment sizes of Amyloid and characterized its conformational nature in different media. Thus, the structural properties might be different from each other and provide a reason for the existing debates in the literature. Here, we performed all-atom MD simulations and present the structural and thermodynamic properties of Aβ1-16, Aβ1-28, and Aβ1-42 in the gas phase and in aqueous solution. Our studies show that the overall structures, secondary structures, and the calculated thermodynamic properties change with increasing peptide size. In addition, we find that the structural properties of those peptides are different from each other in the gas phase and in aqueous solution.

  10. DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA-liposome complexes in IVF bovine zygotes.

    PubMed

    Vichera, G; Moro, L N; Buemo, C; Salamone, D

    2014-05-01

    Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

  11. Universal real-time PCR assay for quantitation and size evaluation of residual cell DNA in human viral vaccines.

    PubMed

    André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines.

  12. Behavioral response of the coachwhip (Masticophis flagellum) to habitat fragment size and isolation in an urban landscape

    USGS Publications Warehouse

    Mitrovich, Milan J.; Diffendorfer, Jay E.; Fisher, Robert N.

    2009-01-01

    Habitat fragmentation is a significant threat to biodiversity worldwide. Habitat loss and the isolation of habitat fragments disrupt biological communities, accelerate the extinction of populations, and often lead to the alteration of behavioral patterns typical of individuals in large, contiguous natural areas. We used radio-telemetry to study the space-use behavior of the Coachwhip, a larger-bodied, wide-ranging snake species threatened by habitat fragmentation, in fragmented and contiguous areas of coastal southern California. We tracked 24 individuals at three sites over two years. Movement patterns of Coachwhips changed in habitat fragments. As area available to the snakes was reduced, individuals faced increased crowding, had smaller home-range sizes, tolerated greater home-range overlap, and showed more concentrated movement activity and convoluted movement pathways. The behavioral response shown by Coachwhips suggests, on a regional level, area-effects alone cannot explain observed extinctions on habitat fragments but, instead, suggests changes in habitat configuration are more likely to explain the decline of this species. Ultimately, if "edge-exposure" is a common cause of decline, then isolated fragments, appropriately buffered to reduce emigration and edge effects, may support viable populations of fragmentation-sensitive species.

  13. The AT-Hook motif as a versatile minor groove anchor for promoting DNA binding of transcription factor fragments

    PubMed Central

    Rodríguez, Jéssica; Mosquera, Jesús; Couceiro, Jose R.; Vázquez, M. Eugenio; Mascareñas, José L.

    2015-01-01

    We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the AT-Hook motif also favors the cell internalization of the conjugates. PMID:26290687

  14. Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa.

    PubMed

    Dietrich, G J; Szpyrka, A; Wojtczak, M; Dobosz, S; Goryczko, K; Zakowski, L; Ciereszko, A

    2005-11-01

    Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.

  15. Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M-J; López-Fernández, C; Martínez-Nevado, E; Pérez-Gutiérrez, J F; de la Fuente, J; Johnston, S D; Blyde, D; Harrison, K; Gosálvez, J

    2014-10-01

    Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.

  16. Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

    PubMed

    Sánchez-Calabuig, M-J; López-Fernández, C; Martínez-Nevado, E; Pérez-Gutiérrez, J F; de la Fuente, J; Johnston, S D; Blyde, D; Harrison, K; Gosálvez, J

    2014-10-01

    Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field. PMID:25130370

  17. High-sensitivity capillary electrophoresis of double-stranded DNA fragments using monomeric and dimeric fluorescent intercalating dyes

    SciTech Connect

    Zhu, H.; Clark, S.M.; Benson, S.C.; Rye, H.S.; Glazer, A.N.; Mathies, R.A. )

    1994-07-01

    Fluorescence-detected capillary electrophoresis separations of [phi]X174/HaeIII DNA restriction fragments have been performed using monomeric and dimeric intercalating dyes. Replaceable hydroxyethyl cellulose solutions were used as the separation medium. Confocal fluorescence detection was performed following 488-nm laser excitation. The limits of DNA detection for on-column staining with monomeric dyes (ethidium bromide, two propidium dye derivatives, oxazole yellow, thiazole orange, and a polycationic thiazole orange derivative) were determined. The thiazole orange dyes provide the most sensitive detection with limiting sensitivities of 2-4 amol of DNA base pairs per band, and detection of the 603-bp fragment was successful, injecting from [phi]X174/HaeIII samples containing only 1-2 fg of this fragment per microliter. Separations of preformed DNA-dimeric dye complexes were also performed. The breadth of the bands observed in separations of preformed DNA-dimeric dye complexes is due to the presence of DNA fragments with different numbers of bound dye molecules that can be resolved as closely spaced subbands in many of our separations. The quality of these DNA-dye complex separations can be dramatically improved by performing the electrophoresis with 9-aminoacridine (9AA) in the column and running buffers. 43 refs., 10 figs., 1 tab.

  18. Phenotypic changes in Cyprinus carpiovar var. Jian introduced by sperm-mediated transgenesis of rearranged homologous DNA fragments.

    PubMed

    Cao, Zheming; Ding, Weidong; Ren, Hongtao

    2013-09-01

    Common carp, specifically the Jian variety (Cyprinus carpiovar var. Jian), is an important Chinese and global aquatic stock for commercial foodstuff. Homologous recombination of carp gene sequences has been widely used in population genetics to broadly screen for beneficial phenotypical variations, thus optimizing artificially engineered carp stocks with Jian variety and native stock varieties. Random rearrangement of homologous DNA fragments from parent specimens of C. carpiovar var. Jian were attained by digestion of genomic DNA with MspI followed by religation and redigestion with EcoR I to specifically rearrange homologous DNA fragments of myostatin and microsatellite genes. Based on known characteristics of myostatin gene function, growth pattern changes in resultant carp mutant varieties was expected. DNA fragments were introduced into metaphase-II oocytes, resulting in one to several dozen insertions of homologous fragments into the host genome by sperm-mediated transgenesis. Introduction of rearranged homologous DNA fragments often resulted in phenotypic changes in C. carpiovar var. Jian, including significant phenotypic changes linked to growth rate at 4 months.

  19. Smoking influence on sperm vitality, DNA fragmentation, reactive oxygen species and zinc in oligoasthenoteratozoospermic men with varicocele.

    PubMed

    Taha, E A; Ezz-Aldin, A M; Sayed, S K; Ghandour, N M; Mostafa, T

    2014-08-01

    This study aimed to assess the influence of smoking duration and intensity on sperm vitality, sperm DNA fragmentation, reactive oxygen species (ROS) and zinc (Zn) levels in oligoasthenoteratozoospermic (OAT) men with varicocele (Vx). A total of 246 men were investigated who were divided into OAT nonsmokers, OAT smokers, OAT nonsmokers and OAT smokers with Vx. They were subjected to history taking, clinical examination and semen analysis. In their semen, sperm hypo-osmotic swelling (HOS) test, sperm DNA fragmentation test, seminal ROS and seminal Zn were assessed. The results demonstrated significantly decreased HOS test, seminal Zn level and significantly increased sperm DNA fragmentation, seminal ROS levels in OAT smokers with Vx more than OAT smokers compared with OAT nonsmokers. Smoking intensity, smoking duration and Vx grade demonstrated significant negative correlations with sperm motility, HOS test percentage and significant positive correlations with sperm DNA fragmentation, seminal ROS level. It is concluded that smoking has a negative impact on sperm progressive motility, HOS test, seminal Zn and positive impact on sperm DNA fragmentation, semen ROS level that are exaggerated if Vx is associated being correlated with smoking intensity, smoking duration and Vx grade.

  20. A baculovirus alkaline nuclease knockout construct produces fragmented DNA and aberrant capsids

    SciTech Connect

    Okano, Kazuhiro; Vanarsdall, Adam L.; Rohrmann, George F. . E-mail: rohrmanng@orst.edu

    2007-03-01

    DNA replication of bacmid-derived constructs of the Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) was analyzed by field inversion gel electrophoresis (FIGE) in combination with digestion at a unique Eco81I restriction enzyme site. Three constructs were characterized: a parental bacmid, a bacmid deleted for the alkaline nuclease gene, and a bacmid from which the gp64 gene had been deleted. The latter was employed as a control for comparison with the alkaline nuclease knockout because neither yields infectious virus and their replication is limited to the initially transfected cells. The major difference between DNA replicated by the different constructs was the presence in the alkaline nuclease knockout of high concentrations of relatively small, subgenome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more subgenome size DNA than the other constructs. Electron microscopic examination of cells transfected with the alkaline nuclease knockout indicated that, in addition to a limited number of normal-appearing electron-dense nucleocapsids, numerous aberrant capsid-like structures were observed indicating a defect in nucleocapsid maturation or in a DNA processing step that is necessary for encapsidation. Because of the documented role of the baculovirus alkaline nuclease and its homologs from other viruses in homologous recombination, these data suggest that DNA recombination may play a major role in the production of baculovirus genomes.

  1. DNA fingerprinting of Mycobacterium leprae strains using variable number tandem repeat (VNTR) - fragment length analysis (FLA).

    PubMed

    Jensen, Ronald W; Rivest, Jason; Li, Wei; Vissa, Varalakshmi

    2011-07-15

    presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.

  2. DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

    PubMed Central

    Jensen, Ronald W.; Rivest, Jason; Li, Wei; Vissa, Varalakshmi

    2011-01-01

    gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types. PMID:21775969

  3. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  4. Characterization of highly and moderately repetitive 500 bp Eco RI fragments from Xenopus laevis DNA.

    PubMed Central

    Hummel, S; Meyerhof, W; Korge, E; Knöchel, W

    1984-01-01

    Three different types of repetitive Eco RI fragments, which comigrate within a visible band of approximately 500 bp at gel electrophoresis of Xenopus laevis DNA Eco RI digests have been cloned and sequenced. These sequences are designated as Repetitive Eco RI Monomers: REM 1, REM 2 and REM 3. The sequences contain direct repeats, inverted repeats and palindromic elements. Genomic organization of the most abundant sequence (REM 1; 0.4% of total DNA) is that of an interspersed sequence. REM 2 (0.08%) is partly organized as an interspersed element and partly found in tandem arrangement, whereas REM 3 (0.02%) represents the tandemly repeated monomeric unit of a satellite DNA. In situ hybridization has shown that REM 1 and REM 2 sequences are found on most chromosomes, REM 1 being preferentially located on specific chromosomal loci. REM 3 is located near the centromere region of only one chromosome pair (presumably number 1). Hybridization of Northern blots from RNAs of different developmental stages revealed that REM 1, REM 2 and REM 3 sequences are transcribed and that transcription is under developmental control. Images PMID:6330690

  5. Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

    PubMed Central

    Li, Y.; Kong, Y.; Korolev, S.; Waksman, G.

    1998-01-01

    The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant. PMID:9605316

  6. Novel apparatus to measure hyperthermal heavy ion damage to DNA: Strand breaks, base loss, and fragmentation

    NASA Astrophysics Data System (ADS)

    Sellami, L.; Lacombe, S.; Hunting, D.; Wagner, R. J.; Huels, M. A.

    2007-08-01

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100μg range) spread out over a large surface area (42cm2) with low energy (kT-100eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar+ ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair.

  7. Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.

    PubMed

    Fu, Changlin; Donovan, William P; Shikapwashya-Hasser, Olga; Ye, Xudong; Cole, Robert H

    2014-01-01

    Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%.

  8. Flexible bent rod model with a saturating induced dipole moment to study the electric linear dichroism of DNA fragments

    NASA Astrophysics Data System (ADS)

    Bertolotto, Jorge A.; Umazano, Juan P.

    2016-06-01

    In the present work we make a theoretical study of the steady state electric linear dichroism of DNA fragments in aqueous solution. The here developed theoretical approach considers a flexible bent rod model with a saturating induced dipole moment. The electric polarizability tensor of bent DNA fragments is calculated considering a phenomenological model which theoretical and experimental backgroung is presented here. The model has into account the electric polarizability longitudinal and transversal to the macroion. Molecular flexibility is described using an elastic potential. We consider DNA fragments originally bent with bending fluctuations around an average bending angle. The induced dipole moment is supposed constant once the electric field strength grows up at critical value. To calculate the reduced electric linear dichroism we determine the optical factor considering the basis of the bent DNA perpendicular to the molecular axis. The orientational distribution function has into account the anisotropic electric properties and the molecule flexibility. We applied the present theoretical background to fit electric dichroism experimental data of DNA fragments reported in the bibliography in a wide range of molecular weight and electric field. From these fits, values of DNA physical properties are estimated. We compare and discuss the results here obtained with the theoretical and experimental data presented by other authors. The original contributions of this work are: the inclusion of the transversal electric polarizability saturating with the electric field, the description of the electric properties with an electric polarizability tensor dependant on the bending angle and the use of an arc model originally bent.

  9. A comparative analysis of DNA barcode microarray feature size

    PubMed Central

    Ammar, Ron; Smith, Andrew M; Heisler, Lawrence E; Giaever, Guri; Nislow, Corey

    2009-01-01

    Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 μm features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 μm feature size is of comparable quality to the 30 μm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density. PMID:19825181

  10. Risk to fragmented DNA in dry, wet, and frozen states from computed tomography: a comparative theoretical study.

    PubMed

    Wanek, Johann; Rühli, Frank Jakobus

    2016-05-01

    Computed tomography represents the gold standard in forensic and palaeopathological diagnosis. However, the X-rays used may affect the DNA quality through fragmentation and loss of genetic information. Previous work showed that the effects of ionizing radiation on dry DNA are non-significant with P < 10(-8), which cannot be detected by means of polymerase chain reaction methods. In the present paper, complete analytical model that characterizes radiation effects on fragmented DNA in dry, wet, and frozen states is described. Simulation of radiation tracks in water phantom cells was performed using the Geant4-DNA toolkit. Cell hits by electrons with energies between 5 and 20 keV were simulated, and the formation of radiolytic products was assessed at a temperature of 298 K. The diffusion coefficient and the mean square displacement of reactive species were calculated by Stokes-Einstein-Smoluchowski relations at 273 K. Finally, DNA fragment damage was estimated using the density distribution of fragments calculated from atomic force microscopy images. The lowest probability of radiation-induced DNA damage was observed for dry state, with a range from 2.5 × 10(-9) to 7.8 × 10(-12) at 298 K, followed by that for frozen state, with a range from 0.9 to 4 × 10(-7) at 273 K. The highest probability of radiation-induced DNA damage was demonstrated for fragmented DNA in wet state with a range from 2 to 9 × 10(-7) at 298 K. These results significantly improve the interpretation of CT imaging in future studies in forensic and palaeopathological science.

  11. Exhaustive de novo design of low-molecular-weight fragments against the ATP-binding site of DNA-gyrase.

    PubMed

    Firth-Clark, Stuart; Todorov, Nikolay P; Alberts, Ian L; Williams, Anthony; James, Timothy; Dean, Philip M

    2006-01-01

    We present a de novo design approach to generating small fragments in the DNA-gyrase ATP-binding site using the computational drug design platform SkelGen. We have generated an exhaustive number of structural possibilities, which were subsequently filtered for site complementarity and synthetic tractability. A number of known active fragments are found, but most of the species created are potentially novel and could be valuable for further elaboration and development into lead-like structures.

  12. Effects of prey quality and predator body size on prey DNA detection success in a centipede predator.

    PubMed

    Eitzinger, B; Unger, E M; Traugott, M; Scheu, S

    2014-08-01

    Predator body size and prey quality are important factors driving prey choice and consumption rates. Both factors might affect prey detection success in PCR-based gut content analysis, potentially resulting in over- or underestimation of feeding rates. Experimental evidence, however, is scarce. We examined how body size and prey quality affect prey DNA detection success in centipede predators. Due to metabolic rates increasing with body size, we hypothesized that prey DNA detection intervals will be shorter in large predators than in smaller ones. Moreover, we hypothesized that prey detection intervals of high-quality prey, defined by low carbon-to-nitrogen ratio will be shorter than in low-quality prey due to faster assimilation. Small, medium and large individuals of centipedes Lithobius spp. (Lithobiidae, Chilopoda) were fed Collembola and allowed to digest prey for up to 168 h post-feeding. To test our second hypothesis, medium-sized lithobiids were fed with either Diptera or Lumbricidae. No significant differences in 50% prey DNA detection success time intervals for a 272-bp prey DNA fragment were found between the predator size groups, indicating that body size does not affect prey DNA detection success. Post-feeding detection intervals were significantly shorter in Lumbricidae and Diptera compared to Collembola prey, apparently supporting the second hypothesis. However, sensitivity of diagnostic PCR differed between prey types, and quantitative PCR revealed that concentration of targeted DNA varied significantly between prey types. This suggests that both DNA concentration and assay sensitivity need to be considered when assessing prey quality effects on prey DNA detection success.

  13. Effects of prey quality and predator body size on prey DNA detection success in a centipede predator.

    PubMed

    Eitzinger, B; Unger, E M; Traugott, M; Scheu, S

    2014-08-01

    Predator body size and prey quality are important factors driving prey choice and consumption rates. Both factors might affect prey detection success in PCR-based gut content analysis, potentially resulting in over- or underestimation of feeding rates. Experimental evidence, however, is scarce. We examined how body size and prey quality affect prey DNA detection success in centipede predators. Due to metabolic rates increasing with body size, we hypothesized that prey DNA detection intervals will be shorter in large predators than in smaller ones. Moreover, we hypothesized that prey detection intervals of high-quality prey, defined by low carbon-to-nitrogen ratio will be shorter than in low-quality prey due to faster assimilation. Small, medium and large individuals of centipedes Lithobius spp. (Lithobiidae, Chilopoda) were fed Collembola and allowed to digest prey for up to 168 h post-feeding. To test our second hypothesis, medium-sized lithobiids were fed with either Diptera or Lumbricidae. No significant differences in 50% prey DNA detection success time intervals for a 272-bp prey DNA fragment were found between the predator size groups, indicating that body size does not affect prey DNA detection success. Post-feeding detection intervals were significantly shorter in Lumbricidae and Diptera compared to Collembola prey, apparently supporting the second hypothesis. However, sensitivity of diagnostic PCR differed between prey types, and quantitative PCR revealed that concentration of targeted DNA varied significantly between prey types. This suggests that both DNA concentration and assay sensitivity need to be considered when assessing prey quality effects on prey DNA detection success. PMID:24383982

  14. Reduction of the DNA base excision repair protein, XRCC1, may contribute to DNA fragmentation after cold injury-induced brain trauma in mice.

    PubMed

    Fujimura, M; Morita-Fujimura, Y; Noshita, N; Yoshimoto, T; Chan, P H

    2000-06-30

    The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.

  15. Total reactive antioxidant potential and DNA fragmentation index as fertility sperm parameters.

    PubMed

    Miciński, Piotr; Pawlicki, Krzysztof; Wielgus, Ewa; Bochenek, Michał; Gogol, Piotr; Ficek, Beata

    2011-07-01

    There is a growing evidence that oxidative stress play a major role in the etiology of defective sperm function including impaired morphology, motility, metabolism and fertility. The aim of the present study was to examine: 1/ total reactive antioxidant potential (TRAP) in seminal plasma; 2/ sperm DNA fragmentation index (DFI), 3/ sperm morphology and motility and 4/ cellular membrane integrity (hypoosmotic swelling test: HOS test) in patients attending in vitro fertilization/intracytoplasmatic sperm injection ( IVF/ICSI) program. According to the DFI value, the men were divided into: group 1 with DFI ≤15% (n=38) and group 2 with DFI ≥15% (n=37). Significant differences between the two groups were found in TRAP, sperm motility, morphology and concentration as well as HOS test scores. In group 1, DFI was negatively correlated with sperm motility and HOS test scores (p<0.05). The sperm morphology was positively correlated with sperm motility and HOS test scores in both groups. There was no correlation between TRAP and sperm chromatin fragmentation. Our results suggest that seminal plasma TRAP level may be a DFI independent parameter of sperm fertility. PMID:21804634

  16. Characterization of HIFU ablation using DNA fragmentation labeling as apoptosis stain

    NASA Astrophysics Data System (ADS)

    Anquez, Jeremie; Corréas, Jean-Michel; Pau, Bernard; Lacoste, François; Yon, Sylvain

    2012-11-01

    The goal of this work was to compare modalities to precisely quantify the extent of thermally induced lesions: gross pathology vs. histopathology vs. devascularization. Liver areas of 14 rabbits were targeted with HIFU and RF ablations in an acute study. Contrast enhanced computorized tomography (CE-CT) scan images were acquired two hours after HIFU and RF treatment to obtain the devascularized volumes of the livers. The animals were then euthanized and deep frozen. The livers were sliced and each slice was photographed and stacked yielding a volume of gross pathology. The volume VGP of the HIFU lesions were derived. The area AGP of the lesions were computed on a particular slice. The lesions were segmented as hypo intense (devascularized) regions on CE-CT images and their volumes VC were computed. The ratios VC/VGP were computed for all the HIFU lesions on all the 14 subjects with a mean value of 1.2. Histology was performed on the livers using Hematoxyline Eosine Staining (HES) and DNA Fragmentation labeling (TUNEL® technology) which characterizes apoptosis. Apoptotic regions of area AT were segmented on the images stained by TUNEL®. No necrosis was identified on the HES data. While TUNEL® did not mark the cores of the RF lesions as apoptotic, the periphery of HIFU and RF lesions was always recognized with TUNEL® as apoptotic. The ratio AGP/AT was computed. The mean value was 0.95 and 0.25 for HIFU and RF lesions respectively. These findings show that the devascularized territory seen on CE-CT scan coincide with the coagulated territories seen with gross pathology. Those actually correspond to cells in apoptosis. It is confirmed that HES stain does not show necrosis 2 hours after thermal ablation. TUNEL® technology for DNA fragmentation labeling appears as a useful marker for thermally induced acute lesions in the liver.

  17. A DNA Metabarcoding Study of a Primate Dietary Diversity and Plasticity across Its Entire Fragmented Range

    PubMed Central

    Quéméré, Erwan; Hibert, Fabrice; Miquel, Christian; Lhuillier, Emeline; Rasolondraibe, Emmanuel; Champeau, Julie; Rabarivola, Clément; Nusbaumer, Louis; Chatelain, Cyrille; Gautier, Laurent; Ranirison, Patrick; Crouau-Roy, Brigitte; Taberlet, Pierre; Chikhi, Lounès

    2013-01-01

    In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the

  18. N400 solution effect of Chinese character fragments: an orthographic neighborhood size effect.

    PubMed

    Wang, Quanhong; Zhang, Jiewei

    2011-10-10

    In this study, event-related brain potentials (ERPs) elicited by fragments of Chinese single-character words were simultaneously recorded while participants completed a delayed character-matching task as fast as possible, wherein probe characters were matched with their prior fragments. The number of solutions for such fragments was manipulated. Results indicate that fragments completed with several characters elicited greater N400 than did fragments with a single solution. Behavioral results demonstrated that "multiple-solution" responses were slower and had lower accuracy rates than the "one-solution" responses. In this article, both behavioral and N400 solution-effects are interpreted to provide evidence that supports the interactive activation model (IAM) but counters the efficacy of serial search models.

  19. Characterization of a fragment containing a putative TLP cDNA sequence.

    PubMed

    Tarro, G

    2002-01-01

    With the aim of isolating the Tumor Liberated Protein (TLP) gene, reverse transcription-polymerase chain reaction (RT-PCR) was used to isolate a approximately equal to 500 bp fragment containing a putative TLP cDNA sequence. Total RNAs were extracted from several cell lines with RNAzol B reagent (Tel-Test, Inc) and reverse transcribed using the Reverse Transcription System (Promega). PCR was carried out for 35 cycles (1 minute at 95 degrees C, 2 minutes at 40 degrees C and 1 minute at 72 degrees C) using an upstream degenerate oligonucleotide, ACN AAY AAR GAR GCN TCN ATG TG, which corresponds to the amino acid sequence T N K E A S I, and random hexamers as the downstream primer. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide. The PCR products were cloned in the pGEM-T easy vector (PROMEGA) and the resulting plasmid clones were sequenced with the chain termination method using the Applied Biosystems model 373A DNA sequencer. A putative open reading frame was deducted. The results obtained can be considered as preliminary data that will require more investigation in order to confirm them. We propose to continue the studies to verify that TLP could be a diagnostic marker in human cancer.

  20. Random rapid amplification of cDNA ends (RRACE) allows for cloning of multiple novel human cDNA fragments containing (CAG)n repeats.

    PubMed

    Carney, J P; McKnight, C; VanEpps, S; Kelley, M R

    1995-04-01

    We describe a new technique for isolating cDNA fragments in which (i) either a partial sequence of the cDNA is known or (ii) a repeat sequence is utilized. We have used this technique, termed random rapid amplification of cDNA ends (random RACE), to isolate a number of trinucleotide repeat (CAG)n-containing genes. Using the random RACE (RRACE) technique, we have isolated over a hundred (CAG)n-containing genes. The results of our initial analysis of ten clones indicate that three are identical to previously cloned (CAG)n-containing genes. Three of our clones matched with expressed sequence tags, one of which contained a CA repeat. The remaining four clones did not match with any sequence in GenBank. These results indicate that this approach provides a rapid and efficient method for isolating trinucleotide repeat-containing cDNA fragments. Finally, this technique may be used for purposes other than cloning repeat-containing cDNA fragments. If only a partial sequence of a gene is known, our system, described here, provides a rapid and efficient method for isolating a fragment of the gene of interest. PMID:7536696

  1. Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis.

    PubMed

    Wang, Rong; Xie, Hua; Xu, Yue-Bing; Jia, Zheng-Ping; Meng, Xian-Dong; Zhang, Juan-Hong; Ma, Jun; Wang, Juan; Wang, Xian-Hua

    2012-03-01

    The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis.

  2. Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

    PubMed

    Suhardiman, Maman; Kramyu, Jarin; Narkpuk, Jaraspim; Jongkaewwattana, Anan; Wanasen, Nanchaya

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines.

  3. A model to predict the conditions for liquid drop breakup and the resultant mean fragment size

    NASA Technical Reports Server (NTRS)

    Wert, K. L.; Jacobs, H. R.

    1994-01-01

    The potential significance of drop fragmentation in sprays and other propulsion-related multiphase flows has been noted in the literature. This has motivated recent experimental and theoretical works to: better understand the fundamentals of physics of drop breakup processes, and develop models of drop fragmentation suitable for use in multiphase flow codes. The works summarized below aim to contribute to both sides of this two-pronged attack.

  4. Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens).

    PubMed

    Quinn, T W; White, B N

    1987-03-01

    A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment-length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist. PMID:2895887

  5. Restriction fragment length polymorphism DNA analysis by the FBI Laboratory protocol using a simple, convenient hardware system.

    PubMed

    Lewis, M E; Kouri, R E; Latorra, D; Berka, K M; Lee, H C; Gaensslen, R E

    1990-09-01

    Restriction fragment length polymorphism analysis of human deoxyribonucleic acid (DNA) using two probes, pYNH24 and CMM101, was performed on the BIOS Timeframe system following the Federal Bureau of Investigation (FBI) Laboratory protocol and some variations of it. Comparable results were obtained by the different methods used.

  6. Effect of Sperm DNA Fragmentation on Clinical Outcome of Frozen-Thawed Embryo Transfer and on Blastocyst Formation

    PubMed Central

    Ni, Wuhua; Xiao, Shiquan; Qiu, Xiufang; Jin, Jianyuan; Pan, Chengshuang; Li, Yan; Fei, Qianjin; Yang, Xu; Zhang, Liya; Huang, Xuefeng

    2014-01-01

    During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles. PMID:24733108

  7. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture.

  8. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture. PMID:25052520

  9. The nucleotide sequence of a DNA fragment, 71 base pairs in length, near the origin of DNA replication of bacteriophage 0X174.

    PubMed Central

    Mansfeld, A D; Vereijken, J M; Jansz, H S

    1976-01-01

    Part of the nucleotide sequence of a restriction fragment covering the origin of phiX174 DNA replication 1 has been determined. The fragment A7c was obtained by digestion of phiX174 RF DNA by the restriction enzyme from Arthrobacter luteus, Alu 1. It was further cleaved into two fragments, one large and one small, by the action of the restriction enzyme from Haemophilus aegyptius, Hae 111. The nucleotide sequence of the small fragment has been determined by analysis of the transcription products obtained by the action of Escherichia coli DNA-dependent RNA polymerase on denaturated template under conditions of low salt. Transcripts longer than the template were found. The whole sequence of 71 nucleotide pairs could be derived from complementary oligonucleotides, obtained after digestion of the transcripts with T1 or pancreatic RNAase. The sequence suggests that at least 4 of the 5 amber mutants 2 that have been mapped on this fragment are identical. On account of this and other evidence a reading frame is proposed. Images PMID:995652

  10. Ambivalent incorporation of the fluorescent cytosine analogues tC and tCo by human DNA polymerase alpha and Klenow fragment.

    PubMed

    Stengel, Gudrun; Purse, Byron W; Wilhelmsson, L Marcus; Urban, Milan; Kuchta, Robert D

    2009-08-11

    We studied the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase alpha and Klenow fragment of DNA polymerase I (Escherichia coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly expanded in size toward the major groove. Despite the size alteration, both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only approximately 4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs and can incorporate at least four consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. Klenow fragment inserts dGTP with a 4-9-fold higher probability than dATP, while polymerase alpha favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as a templating base and as an incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o). PMID:19580325

  11. Effects of logging, hunting, and forest fragment size on physiological stress levels of two sympatric ateline primates in Colombia.

    PubMed

    Rimbach, Rebecca; Link, Andrés; Heistermann, Michael; Gómez-Posada, Carolina; Galvis, Nelson; Heymann, Eckhard W

    2013-01-01

    Habitat fragmentation and anthropogenic disturbances are of major concern to the conservation of endangered species because of their potentially negative impact on animal populations. Both processes can impose physiological stress (i.e. increased glucocorticoid output) on animals, and chronically elevated stress levels can have detrimental effects on the long-term viability of animal populations. Here, we investigated the effect of fragment size and human impact (logging and hunting pressure) on glucocorticoid levels of two sympatric Neotropical primates, the red howler monkey (Alouatta seniculus) and the critically endangered brown spider monkey (Ateles hybridus). These two species have been reported to contrast strongly in their ability to cope with anthropogenic disturbances. We collected faecal samples from eight spider monkey groups and 31 howler monkey groups, living in seven and 10 different forest fragments in Colombia, respectively. We measured faecal glucocorticoid metabolite (FGCM) levels in both species using previously validated methods. Surprisingly, fragment size did not influence FGCM levels in either species. Spider monkeys showed elevated FGCMs in fragments with the highest level of human impact, whereas we did not find this effect in howler monkeys. This suggests that the two species differ in their physiological responsiveness to anthropogenic changes, further emphasizing why brown spider monkeys are at higher extinction risk than red howler monkeys. If these anthropogenic disturbances persist in the long term, elevated FGCM levels can potentially lead to a state of chronic stress, which might limit the future viability of populations. We propose that FGCM measurements should be used as a tool to monitor populations living in disturbed areas and to assess the success of conservation strategies, such as corridors connecting forest fragments.

  12. Effects of logging, hunting, and forest fragment size on physiological stress levels of two sympatric ateline primates in Colombia

    PubMed Central

    Rimbach, Rebecca; Link, Andrés; Heistermann, Michael; Gómez-Posada, Carolina; Galvis, Nelson; Heymann, Eckhard W.

    2013-01-01

    Habitat fragmentation and anthropogenic disturbances are of major concern to the conservation of endangered species because of their potentially negative impact on animal populations. Both processes can impose physiological stress (i.e. increased glucocorticoid output) on animals, and chronically elevated stress levels can have detrimental effects on the long-term viability of animal populations. Here, we investigated the effect of fragment size and human impact (logging and hunting pressure) on glucocorticoid levels of two sympatric Neotropical primates, the red howler monkey (Alouatta seniculus) and the critically endangered brown spider monkey (Ateles hybridus). These two species have been reported to contrast strongly in their ability to cope with anthropogenic disturbances. We collected faecal samples from eight spider monkey groups and 31 howler monkey groups, living in seven and 10 different forest fragments in Colombia, respectively. We measured faecal glucocorticoid metabolite (FGCM) levels in both species using previously validated methods. Surprisingly, fragment size did not influence FGCM levels in either species. Spider monkeys showed elevated FGCMs in fragments with the highest level of human impact, whereas we did not find this effect in howler monkeys. This suggests that the two species differ in their physiological responsiveness to anthropogenic changes, further emphasizing why brown spider monkeys are at higher extinction risk than red howler monkeys. If these anthropogenic disturbances persist in the long term, elevated FGCM levels can potentially lead to a state of chronic stress, which might limit the future viability of populations. We propose that FGCM measurements should be used as a tool to monitor populations living in disturbed areas and to assess the success of conservation strategies, such as corridors connecting forest fragments. PMID:27293615

  13. Distinguishing between the bone fragments of medium-sized mammals and children. A histological identification method for archaeology.

    PubMed

    Cuijpers, Saddha A G F M

    2009-06-01

    In archaeology, it is not always possible to identify bone fragments. A novel approach was chosen to assess the potential of histology as an identification tool. Instead of studying a few bones of different categories from many species, this study concentrated on the diaphyses of long bones in four species of comparable size which are relevant to archaeology; young humans, pigs, sheep and goats, to broaden the insight into variations in diaphyseal bone structure within and between these species. A general difference in the primary bone structure was found between children older than one year and the three medium-sized mammals, namely lamellar vs. fibro-lamellar primary bone. Although, the diaphyseal bone structure of children below the age of one year also showed (developing) fibro-lamellar bone, its composition was distinctive from the medium-sized mammals. A difference in the secondary bone structure was also observed. Connecting (Volkmann's) canals, giving the secondary bone a reticular aspect, were seen in the medium-sized mammals but not in the young human long bones. To confirm the validity and applicability of these observed histological differences, a blind test was conducted on 14 diaphyseal fragments of identified long bones from archaeological sites. The results were very promising. All the bone fragments were correctly attributed using the difference in primary bone structure, even when the bone was severely degraded.

  14. Design of chimeric proteins by combination of subdomain-sized fragments.

    PubMed

    Rico, José Arcadio Farías; Höcker, Birte

    2013-01-01

    Hybrid proteins or chimeras are generated by recombination of protein fragments. In the course of evolution, this mechanism has led to major diversification of protein folds and their functionalities. Similarly, protein engineers have taken advantage of this attractive strategy to build new proteins. Methods that use homologous recombination have been developed to (semi) randomly create chimeras from which the best can be selected. We wanted to recombine very divergent or even unrelated fragments, which is not possible with these methods. Consequently, based on the observation that nature evolves new proteins also through illegitimate recombination, we developed a strategy to design chimeras using protein fragments from different folds. For this approach, we employ detailed structure comparisons, and based on structural similarities, we choose the fragments used for recombination. Model building and minimization can be used to assess the design, and further optimization can be performed using established computational design methodologies. Here, we outline a general approach to rational protein chimera design based on our experience, and provide considerations for the selection of the fragments, the evaluation, and possible redesign of the constructs. PMID:23422440

  15. Design of chimeric proteins by combination of subdomain-sized fragments.

    PubMed

    Rico, José Arcadio Farías; Höcker, Birte

    2013-01-01

    Hybrid proteins or chimeras are generated by recombination of protein fragments. In the course of evolution, this mechanism has led to major diversification of protein folds and their functionalities. Similarly, protein engineers have taken advantage of this attractive strategy to build new proteins. Methods that use homologous recombination have been developed to (semi) randomly create chimeras from which the best can be selected. We wanted to recombine very divergent or even unrelated fragments, which is not possible with these methods. Consequently, based on the observation that nature evolves new proteins also through illegitimate recombination, we developed a strategy to design chimeras using protein fragments from different folds. For this approach, we employ detailed structure comparisons, and based on structural similarities, we choose the fragments used for recombination. Model building and minimization can be used to assess the design, and further optimization can be performed using established computational design methodologies. Here, we outline a general approach to rational protein chimera design based on our experience, and provide considerations for the selection of the fragments, the evaluation, and possible redesign of the constructs.

  16. Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.

    PubMed

    Kitazumi, Ikuko; Maseki, Yoko; Nomura, Yoshiko; Shimanuki, Akiko; Sugita, Yumi; Tsukahara, Masayoshi

    2010-01-01

    DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

  17. A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.

    PubMed

    Tomimoto, Kazuya; Fujita, Kosuke; Ishibashi, Jun; Imanishi, Shigeo; Yamakawa, Minoru; Tanaka, Hiromitsu

    2012-01-01

    Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library. PMID:21516519

  18. A novel method to convert a DNA fragment inserted into a plasmid to an inverted repeat structure.

    PubMed

    Tomimoto, Kazuya; Fujita, Kosuke; Ishibashi, Jun; Imanishi, Shigeo; Yamakawa, Minoru; Tanaka, Hiromitsu

    2012-01-01

    Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

  19. Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.

    PubMed

    Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz

    2012-03-01

    Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot.

  20. Clinical Factors Associated with Sperm DNA Fragmentation in Male Patients with Infertility

    PubMed Central

    Komiya, Akira; Kato, Tomonori; Kawauchi, Yoko; Watanabe, Akihiko; Fuse, Hideki

    2014-01-01

    Objective. The clinical factors associated with sperm DNA fragmentation (SDF) were investigated in male patients with infertility. Materials and Methods. Fifty-four ejaculates from infertile Japanese males were used. Thirty-three and twenty-one were from the patients with varicoceles and idiopathic causes of infertility, respectively. We performed blood tests, including the serum sex hormone levels, and conventional and computer-assisted semen analyses. The sperm nuclear vacuolization (SNV) was evaluated using a high-magnification microscope. The SDF was evaluated using the sperm chromatin dispersion test (SCDt) to determine the SDF index (SDFI). The SDFI was compared with semen parameters and other clinical variables, including lifestyle factors. Results. The SDFI was 41.3 ± 22.2% (mean ± standard deviation) and did not depend on the cause of infertility. Chronic alcohol use increased the SDFI to 49.6 ± 23.3% compared with 33.9 ± 18.0% in nondrinkers. The SDFI was related to adverse conventional semen parameters and sperm motion characteristics and correlated with the serum FSH level. The SNV showed a tendency to increase with the SDFI. The multivariate analysis revealed that the sperm progressive motility and chronic alcohol use were significant predictors of the SDF. Conclusion. The SCDt should be offered to chronic alcohol users and those with decreased sperm progressive motility. PMID:25165747

  1. Quantitative study of Helicobacter pylori in gastric mucus by competitive PCR using synthetic DNA fragments.

    PubMed

    Furuta, T; Kaneko, E; Suzuki, M; Arai, H; Futami, H

    1996-10-01

    Helicobacter pylori is closely related to upper gastrointestinal diseases, and the precise evaluation of H. pylori infection is necessary for the treatment of these diseases. The aim of the present study was to establish a method for the quantitative detection of H. pylori. We applied a competitive PCR method using various amounts of synthetic DNA fragments containing the same primer-binding and a subset of the same template sequences as the target competing for primer binding and amplification in order to quantify H. pylori in gastric mucus. The results obtained by this method were compared with the results of histological examination, the rapid urease test, bacterial culture, the [13C]urea breath test, and urea and ammonia measurements in gastric juice. As the quantity of H. pylori in gastric mucus increased, the rates of positivity of histological examination, the rapid urease test, and bacterial culture increased. The quantity of H. pylori in gastric mucus was also significantly correlated with the results of the [13C]urea breath test and was negatively correlated with the urea/ammonia ratio in gastric juice. The competitive PCR method provides an objective measure of the quantity of H. pylori and makes it possible to distinguish true negatives from false negatives due to incomplete PCR and true positives from false positives due to contamination. This method is very useful for the precise evaluation of gastric H. pylori infection. PMID:8880492

  2. Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF.

    PubMed

    Smylie, Kevin J; Cantor, Charles R; Denissenko, Mikhail F

    2004-01-01

    The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.

  3. Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

    PubMed

    Sheikh, Inayat Hussain; Kaushal, Deep C; Chandra, Deepak; Kaushal, Nuzhat A

    2016-10-01

    Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses. PMID:27311385

  4. [The discovery of a specific DNA fragment associated with maize cytoplasmic male sterility and its differential display].

    PubMed

    Cao, Mo-Ju; Rong, Ting-Zhao; Zhu, Ying-Guo

    2005-09-01

    Three pairs of PCR primers were designed according to the mitochondrial DNA sequence. PCR amplification was applied to 3 sets of isonuclear alloplasm materials and 3 sets of isoplasm allonuclear materials. Multiplex PCR and general PCR protocol were adopted with total genomic DNA. As for the primers having detected polymorphsim between male sterility and its maintainers, differential display was conducted with mRNA from different development stage of microspore. The results showed as follows: with total genomic DNA template, primer P1-P2 has amplified a specific fragment only in all the male sterile materials, primer P5-P6 has amplified a specific fragment only in maintainer Huangzaosi, primer P3-P4 has no amplification in all the experiment materials. So primer P1-P2 can be used to distinguish male sterile cytoplasm and normal cytoplasm. RT-PCR was conducted with primer P1-P2 in inbred line huangzaosi and 48-2 with male sterile cytoplasm and normal cytoplasm, mRNA was separately isolated from tetrad stage, uninucleate stage and binucleate stage of microspore development, cDNA was obtained with random hexanucleotide primers. With the cDNA template, specific amplified fragments were also detected by primer P1-P2 in the male sterile materials at different development stage of microspore, but there was no amplification by primer P1-P2 in the 2 maintainer lines. This result indicated that primer P1-P2 can be transcripted at 3 development stages of microspore in all male sterile materials, and same transcript was produced by primer P1-P2 among all male sterile materials include 3 sets of isonuclear alloplasm and 3 sets of isoplasm allonuclear. It was suggested from this experiment that the specific DNA sequence detected by primer P1-P2 in all male sterile material total genomic DNA might be related to the cytoplasmic male sterile character.

  5. Biophysical and chemical handles to control the size of DNA nanoparticles produced by rolling circle amplification.

    PubMed

    Lee, So Yeon; Kim, Kyoung-Ran; Bang, Duhee; Bae, Se Won; Kim, Hak Joong; Ahn, Dae-Ro

    2016-08-16

    Although rolling circle amplification (RCA) is an efficient method to produce DNA materials for biomedical applications, it does not yield nano-sized products suitable for intracellular delivery. We here provide the ways to control the size of RCA products and show a potential application of the size-controlled DNA nanoparticles. PMID:27464359

  6. Biophysical and chemical handles to control the size of DNA nanoparticles produced by rolling circle amplification.

    PubMed

    Lee, So Yeon; Kim, Kyoung-Ran; Bang, Duhee; Bae, Se Won; Kim, Hak Joong; Ahn, Dae-Ro

    2016-08-16

    Although rolling circle amplification (RCA) is an efficient method to produce DNA materials for biomedical applications, it does not yield nano-sized products suitable for intracellular delivery. We here provide the ways to control the size of RCA products and show a potential application of the size-controlled DNA nanoparticles.

  7. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction.

    PubMed

    Birla, Bhagyashree S; Chou, Hui-Hsien

    2015-01-01

    Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.

  8. Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    PubMed Central

    Birla, Bhagyashree S.; Chou, Hui-Hsien

    2015-01-01

    Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly. PMID:26716828

  9. Cloning and characterization of a highly reiterated 5.8-kilobase pair nucleolar EcoRI DNA fragment found in Novikoff hepatoma ascites cells.

    PubMed

    Parker, D L; Busch, H; Rothblum, L I

    1981-02-17

    The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control.

  10. Pikeperch Sander lucioperca egg quality cannot be predicted by total antioxidant capacity and mtDNA fragmentation.

    PubMed

    Schaefer, Fabian J; Overton, Julia L; Wuertz, Sven

    2016-04-01

    In farmed pikeperch, there is a high variability in egg quality restraining the propagation of this species in aquaculture. The identification of reliable biomarkers for predicting successful embryo development already at an early stage (unfertilized oocyte) could help improve production efficiency. Total antioxidant capacity (TAC) and the quantification of mitochondrial DNA (mtDNA) fragmentation have been established as biomarkers for oxidative stress and damage of macromolecules, potentially influencing embryo development. Therefore, we evaluated these biomarkers in eggs of commercially farmed pikeperch (44 females). We measured egg TAC, as well as lesion rates per 10 kb of 12S and cytochrome b (cytb) as target regions within the mitochondrial genome by qPCR. It was tested whether these markers correlate with embryo development (fertilization rate, embryo survival, hatching rate). There was no significant relation of mtDNA lesion rates or TAC with these egg quality parameters. We detected average lesion rates (± SD) of 1.50 (± 1.57) and 1.89 (± 2.14) in 12S and cytb mtDNA respectively. Lesion rates in 12S and cytb were highly correlated within samples (P < 0.0001) and were independent of the observed TAC. The results suggest that TAC does not prevent mtDNA fragmentation and that embryos rather seem to be able to cope with the observed fragmentation of mtDNA. However, in post-ovulatory aged eggs of three females with little to no fertilization success, lesion rates of cytb were significantly elevated, whereas TAC was significantly lower compared to other females, suggesting a possible role of oxidative stress during post-ovulatory ageing. PMID:26922635

  11. Effects of habitat fragmentation, population size and demographic history on genetic diversity: the Cross River gorilla in a comparative context.

    PubMed

    Bergl, Richard A; Bradley, Brenda J; Nsubuga, Anthony; Vigilant, Linda

    2008-09-01

    In small and fragmented populations, genetic diversity may be reduced owing to increased levels of drift and inbreeding. This reduced diversity is often associated with decreased fitness and a higher threat of extinction. However, it is difficult to determine when a population has low diversity except in a comparative context. We assessed genetic variability in the critically endangered Cross River gorilla (Gorilla gorilla diehli), a small and fragmented population, using 11 autosomal microsatellite loci. We show that levels of diversity in the Cross River population are not evenly distributed across the three genetically identified subpopulations, and that one centrally located subpopulation has higher levels of variability than the others. All measures of genetic variability in the Cross River population were comparable to those of the similarly small mountain gorilla (G. beringei beringei) populations (Bwindi and Virunga). However, for some measures both the Cross River and mountain gorilla populations show lower levels of diversity than a sample from a large, continuous western gorilla population (Mondika, G. gorilla gorilla). Finally, we tested for the genetic signature of a bottleneck in each of the four populations. Only Cross River showed strong evidence of a reduction in population size, suggesting that the reduction in size of this population was more recent or abrupt than in the two mountain gorilla populations. These results emphasize the need for maintaining connectivity in fragmented populations and highlight the importance of allowing small populations to expand. PMID:18521886

  12. Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques

    PubMed Central

    Muratori, Monica; Tarozzi, Nicoletta; Cambi, Marta; Boni, Luca; Iorio, Anna Lisa; Passaro, Claudia; Luppino, Benedetta; Nadalini, Marco; Marchiani, Sara; Tamburrino, Lara; Forti, Gianni; Maggi, Mario; Baldi, Elisabetta; Borini, Andrea

    2016-01-01

    Abstract Predicting the outcome of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) is one main goal of the present research on assisted reproduction. To understand whether density gradient centrifugation (DGC), used to select sperm, can affect sperm DNA integrity and impact pregnancy rate (PR), we prospectively evaluated sperm DNA fragmentation (sDF) by TUNEL/PI, before and after DGC. sDF was studied in a cohort of 90 infertile couples the same day of IVF/ICSI treatment. After DGC, sDF increased in 41 samples (Group A, median sDF value: 29.25% [interquartile range, IQR: 16.01–41.63] in pre- and 60.40% [IQR: 32.92–93.53] in post-DGC) and decreased in 49 (Group B, median sDF value: 18.84% [IQR: 13.70–35.47] in pre- and 8.98% [IQR: 6.24–15.58] in post-DGC). PR was 17.1% and 34.4% in Group A and B, respectively (odds ratio [OR]: 2.58, 95% confidence interval [CI]: 0.95–7.04, P = 0.056). After adjustment for female factor, female and male age and female BMI, the estimated OR increased to 3.12 (95% CI: 1.05–9.27, P = 0.041). According to the subgroup analysis for presence/absence of female factor, heterogeneity in the association between the Group A and B and PR emerged (OR: 4.22, 95% CI: 1.16–15.30 and OR: 1.53, 95% CI: 0.23–10.40, respectively, for couples without, n = 59, and with, n = 31, female factor). This study provides the first evidence that the DGC procedure produces an increase in sDF in about half of the subjects undergoing IVF/ICSI, who then show a much lower probability of pregnancy, raising concerns about the safety of this selection procedure. Evaluation of sDF before and after DGC configures as a possible new prognostic parameter of pregnancy outcome in IVF/ICSI. Alternative sperm selection strategies are recommended for those subjects who undergo the damage after DGC. PMID:27196465

  13. Generation of porcine reproductive and respiratory syndrome virus by in vitro assembly of viral genomic cDNA fragments.

    PubMed

    Suhardiman, Maman; Kramyu, Jarin; Narkpuk, Jaraspim; Jongkaewwattana, Anan; Wanasen, Nanchaya

    2015-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines. PMID:25300804

  14. Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation

    PubMed Central

    Cho, Chak-Lam; Esteves, Sandro C; Agarwal, Ashok

    2016-01-01

    Varicocele has been associated with reduced male reproductive potential. With the advances in biomolecular techniques, it has been possible to better understand the mechanisms involved in testicular damage provoked by varicocele. Current evidence suggests the central role of reactive oxygen species (ROS) and the resultant oxidative stress (OS) in the pathogenesis of varicocele-associated male subfertility although the mechanisms have not yet been fully described and it is likely to be multifactorial. Excessive ROS is associated with sperm DNA fragmentation, which may mediate the clinical manifestation of poor sperm function and fertilization outcome related to varicocele. Testing of ROS/OS and DNA fragmentation has the potential to provide additional diagnostic and prognostic information compared to conventional semen analysis and may guide therapeutic management strategies in individual patient. PMID:26732105

  15. Influence of Heteroanion and Ammonium Cation Size on the Composition and Gas-Phase Fragmentation of Polyoxovanadates

    SciTech Connect

    Johnson, Grant E.; Al Hasan, Naila M.; Laskin, Julia

    2013-11-15

    This paper describes the results of a systematic experimental investigation of the influence of different size cationic ammonium ligands and heteroanions on the composition, ionic charge state and gas-phase fragmentation pathways of anionic polyoxovanadates synthesized in solution. Four separate solutions of olyoxometalates (POMs) were prepared using all possible combinations of the tetraethylammonium [(C2H5)4N+] ligand, chloride (Cl-) heteroanion, tetrabutylammonium [(C4H9)4N+] ligand and acetate (CH3CO2-) heteroanion. Employing electrospray ionization combined with high-resolution mass spectrometry (ESI-MS) we demonstrate that POM solutions synthesized using the small [(C2H5)4N+] ligand and Cl-heteroanion are composed predominately of large doubly and triply charged chlorine containing clusters with a size distribution centered at fourteen vanadium atoms. POM solutions prepared using the Cl- anion and [(C4H9)4N+] ligand are shown to contain slightly larger clusters with fifteen and sixteen vanadium atoms, thereby indicating that the size of the cationic ammonium ligand exerts only a weak influence on the polymerization of polyoxovanadates. POM solutions prepared using (C2H5)4NCl and (C4H9)4NCl also produced peaks consistent with the attachment of one and two ammonium cations to the larger clusters. Solutions prepared using the large CH3CO2 - heteroanion, in contrast, are demonstrated to contain much smaller singly and doubly *Manuscript Click here to view linked References 2 charged clusters with a size distribution centered at six vanadium atoms. In addition, while incorporation of one and two ammonium ligands into the smaller clusters was observed, no POMs containing the CH3CO2 - heteroanion were identified. The gas-phase fragmentation pathways of representative POMs containing one and two ammonium ligands were examined using collision induced dissociation (CID) and mass spectrometry. Similar primary fragmentation pathways involving partial loss of a ligand

  16. Secondary Craters and the Size-Velocity Distribution of Ejected Fragments around Lunar Craters Measured Using LROC Images

    NASA Astrophysics Data System (ADS)

    Singer, K. N.; Jolliff, B. L.; McKinnon, W. B.

    2013-12-01

    Title: Secondary Craters and the Size-Velocity Distribution of Ejected Fragments around Lunar Craters Measured Using LROC Images Authors: Kelsi N. Singer1, Bradley L. Jolliff1, and William B. McKinnon1 Affiliations: 1. Earth and Planetary Sciences, Washington University in St Louis, St. Louis, MO, United States. We report results from analyzing the size-velocity distribution (SVD) of secondary crater forming fragments from the 93 km diameter Copernicus impact. We measured the diameters of secondary craters and their distances from Copernicus using LROC Wide Angle Camera (WAC) and Narrow Angle Camera (NAC) image data. We then estimated the velocity and size of the ejecta fragment that formed each secondary crater from the range equation for a ballistic trajectory on a sphere and Schmidt-Holsapple scaling relations. Size scaling was carried out in the gravity regime for both non-porous and porous target material properties. We focus on the largest ejecta fragments (dfmax) at a given ejection velocity (υej) and fit the upper envelope of the SVD using quantile regression to an equation of the form dfmax = A*υej ^- β. The velocity exponent, β, describes how quickly fragment sizes fall off with increasing ejection velocity during crater excavation. For Copernicus, we measured 5800 secondary craters, at distances of up to 700 km (15 crater radii), corresponding to an ejecta fragment velocity of approximately 950 m/s. This mapping only includes secondary craters that are part of a radial chain or cluster. The two largest craters in chains near Copernicus that are likely to be secondaries are 6.4 and 5.2 km in diameter. We obtained a velocity exponent, β, of 2.2 × 0.1 for a non-porous surface. This result is similar to Vickery's [1987, GRL 14] determination of β = 1.9 × 0.2 for Copernicus using Lunar Orbiter IV data. The availability of WAC 100 m/pix global mosaics with illumination geometry optimized for morphology allows us to update and extend the work of Vickery

  17. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. . E-mail: kais.algubory@jouy.inra.fr

    2005-11-01

    The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals.

  18. Amplification of a species-specific DNA fragment of Mycobacterium tuberculosis and its possible use in diagnosis.

    PubMed Central

    Del Portillo, P; Murillo, L A; Patarroyo, M E

    1991-01-01

    In recent work, a species-specific Mycobacterium tuberculosis DNA fragment was cloned and sequenced. On the basis of its nucleotide sequence, two oligonucleotides were synthesized and used as primers for polymerase chain reaction (PCR) amplification. A 396-bp fragment was specifically amplified from the M. tuberculosis genome. No amplification was observed from any of 10 different mycobacterial strains, included those belonging to the M. tuberculosis complex. Neither was this fragment amplified from genomes of humans or different species of clinically important bacteria. The PCR product was detected by dot blot hybridization even when as little as 10 fg of purified M. tuberculosis DNA was used. This amplification method was subsequently used to detect and identify bacilli in different clinical samples, such as sputum, urine, and cerebrospinal fluid. A good correlation was observed between the results obtained with the PCR method that we describe and other diagnostic tests currently used. Thus, PCR amplification of this genomic fragment is proposed as a specific, rapid, and sensitive test for the diagnosis of infection with M. tuberculosis. Images PMID:1939567

  19. Does the marine biotoxin okadaic acid cause DNA fragmentation in the blue mussel and the pacific oyster?

    PubMed

    McCarthy, Moira; O'Halloran, John; O'Brien, Nora M; van Pelt, Frank F N A M

    2014-10-01

    Two bivalve species of global economic importance: the blue mussel, Mytilus edulis and the pacific oyster, Crassostrea gigas were exposed in vivo, to the diarrhoetic shellfish toxin okadaic acid (OA), and impacts on DNA fragmentation were measured. Shellfish were exposed using two different regimes, the first was a single (24 h) exposure of 2.5 nM OA (∼0.1 μg/shellfish) and algal feed at the beginning of the trial (T0), after which shellfish were only fed algae. The second was daily exposure of shellfish to two different concentrations of OA mixed with the algal feed over 7 days; 1.2 nM OA (∼0.05 μg OA/shellfish/day) and 50 nM OA (∼2 μg OA/shellfish/day). Haemolymph and hepatopancreas cells were extracted following 1, 3 and 7 days exposure. Cell viability was measured using the trypan blue exclusion assay and remained above 85% for both cell types. DNA fragmentation was examined using the single-cell gel electrophoresis (comet) assay. A significant increase in DNA fragmentation was observed in the two cell types from both species relative to the controls. This increase was greater in the pacific oyster at the higher toxin concentration. However, there was no difference in the proportion of damage measured between the two cell types, and a classic dose response was not observed, increasing toxin concentration did not correspond to increased DNA fragmentation. PMID:25440785

  20. Efficiency of genomic DNA extraction dependent on the size of magnetic nanoclusters

    NASA Astrophysics Data System (ADS)

    Cho, Hyun Ah; Hyun Min, Ji; Hua Wu, Jun; Woo Jang, Jin; Lim, Chae-Seung; Keun Kim, Young

    2014-05-01

    We report the efficiency of genomic DNA extraction as a function of particle size and quantity. For DNA extraction, we synthesized magnetic nanoclusters of various sizes and coated the surface of these magnetic nanoclusters with meso-2,3-dimercaptosuccinic acid. We showed that the nanoclusters had a tight particle size distribution and high crystallinity. Furthermore, we observed that the three types of magnetic nanoclusters studied exhibited ferrimagnetic behavior and that larger nanoclusters showed larger saturation magnetization values. The resultant efficiency of DNA extraction is inversely proportional to particle size in the range of nanoclusters tested, due to the fact that the surface-to-volume ratio decreases as particle size increases.

  1. Spent fuel waste form characteristics: Grain and fragment size statistical dependence for dissolution response

    SciTech Connect

    Stout, R.B.; Leider, H.; Weed, H.; Nguyen, S.; McKenzie, W.; Prussin, S.; Wilson, C.N.; Gray, W.J.

    1991-04-01

    The Yucca Mountain Project of the US Department of Energy is investigating the suitability of the unsaturated zone at Yucca Mountain, NV, for a high-level nuclear waste repository. All of the nuclear waste will be enclosed in a container package. Most of the nuclear waste will be in the form of fractured UO{sub 2} spent fuel pellets in Zircaloy-clad rods from electric power reactors. If failure of both the container and its enclosed clad rods occurs, then the fragments of the fractured UO{sub 2} spent fuel will be exposed to their surroundings. Even though the surroundings are an unsaturated zone, a possibility of water transport exists, and consequently, UO{sub 2} spent fuel dissolution may occur. A repository requirement imposes a limit on the nuclide release per year during a 10,000 year period; thus the short term dissolution response from fragmented fuel pellet surfaces in any given year must be understood. This requirement necessitates that both experimental and analytical activities be directed toward predicting the relatively short term dissolution response of UO{sub 2} spent fuel. The short term dissolution response involves gap nuclides, grain boundary nuclides, and grain volume nuclides. Analytical expressions are developed that describe the combined geometrical influences of grain boundary nuclides and grain volume nuclides on the dissolution rate of spent fuel. 7 refs., 1 fig.

  2. Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

    PubMed Central

    Zarrin, Majid; Erfaninejad, Maryam

    2016-01-01

    Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085

  3. DNA templates silver clusters with magic sizes and colors for multi-cluster fluorescent assemblies

    NASA Astrophysics Data System (ADS)

    Copp, Stacy

    2015-03-01

    The natural inclusion of information in DNA, a vital part of life's rich complexity, can also be exploited to create diverse structures with multiple scales of complexity. Now emerging in novel photonic applications, DNA-stabilized silver clusters (AgN-DNA) are compelling examples of multi-scale DNA-directed assembly: individual fluorescent clusters, each templated by specific DNA base motifs, can then be arranged together in DNA-mediated multi-cluster assemblies with nanoscale precision. We discuss how DNA imbues AgN-DNA with unique features. Our optical data on pure AgN-DNA show that DNA base-cationic silver ligands impose rod-like shapes for neutral silver clusters, whose length primarily determines fluorescence color. This shape anisotropy leads to the aspherical AgN-DNA magic number cluster sizes and ``magic color'' groupings. We exploit DNA's sequence properties to extract multi-base motifs that select certain magic cluster sizes, using machine learning algorithms applied to large data sets. With these base motifs, we design DNA scaffolds to arrange multiple atomically precise AgN together in nanoscale proximity. We demonstrate that clusters are stable when held at separations below 10 nm, both in bicolor, dual cluster DNA clamp assemblies and in one-dimensional assemblies of atomically precise clusters arrayed on DNA nanotubes. Supported by NSF-CHE-1213895 and NSF-DMR-1309410. SMC acknowledges NSF-DGE-1144085, a NSF GRFP.

  4. Submicron-sized boron carbide particles encapsulated in turbostratic graphite prepared by laser fragmentation in liquid medium.

    PubMed

    Ishikawa, Yoshie; Sasaki, Takeshi; Koshizaki, Naoto

    2010-08-01

    Submicron-sized B4C spherical particles were obtained by laser fragmentation of large B4C particles dispersed in ethyl acetate. The irradiated surface of large B4C raw particles was heated and melted by laser energy absorption. B4C droplets were then cooled down, and finally B4C spherical particles were obtained. Moreover, each B4C particle obtained was encapsulated in a graphitic layer that is useful for medical functionalization of particles. Thus, obtained B4C particles encapsulated in graphitic layer may have potential uses in boron neutron capture therapy.

  5. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate.

    PubMed Central

    Wahl, G M; Stern, M; Stark, G R

    1979-01-01

    We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly. Images PMID:291033

  6. Size-controllable DNA nanoribbons assembled from three types of reusable brick single-strand DNA tiles.

    PubMed

    Shi, Xiaolong; Chen, Congzhou; Li, Xin; Song, Tao; Chen, Zhihua; Zhang, Zheng; Wang, Yanfeng

    2015-11-21

    Precise control of nanostructure is a significant goal shared by supramolecular chemistry, nanotechnology and materials science. In DNA nanotechnology, methods of constructing desired DNA nanostructures using programmable DNA strands have been studied extensively and have become a promising branch of research, but developing universal and low-cost (in the sense of using fewer types of DNA strands) methods remains a challenge. In this work, we propose a novel approach to assemble size-controllable DNA nanoribbons with three types of reusable brick SSTs (single-stranded DNA tiles), where the control of ribbon size is achieved by regulating the concentration ratio between manipulative strands and packed single-stranded DNA tiles. In our method, three types of brick SSTs are sufficient in assembling DNA nanoribbons of different sizes, which is much less than the number of types of unique tile-programmable assembling strategy, thus achieving a universal and low-cost method. The assembled DNA nanoribbons are observed and analyzed by atomic force microscopy (AFM). Experimental observations strongly suggest the feasibility and reliability of our method. PMID:26367111

  7. Size-controllable DNA nanoribbons assembled from three types of reusable brick single-strand DNA tiles.

    PubMed

    Shi, Xiaolong; Chen, Congzhou; Li, Xin; Song, Tao; Chen, Zhihua; Zhang, Zheng; Wang, Yanfeng

    2015-11-21

    Precise control of nanostructure is a significant goal shared by supramolecular chemistry, nanotechnology and materials science. In DNA nanotechnology, methods of constructing desired DNA nanostructures using programmable DNA strands have been studied extensively and have become a promising branch of research, but developing universal and low-cost (in the sense of using fewer types of DNA strands) methods remains a challenge. In this work, we propose a novel approach to assemble size-controllable DNA nanoribbons with three types of reusable brick SSTs (single-stranded DNA tiles), where the control of ribbon size is achieved by regulating the concentration ratio between manipulative strands and packed single-stranded DNA tiles. In our method, three types of brick SSTs are sufficient in assembling DNA nanoribbons of different sizes, which is much less than the number of types of unique tile-programmable assembling strategy, thus achieving a universal and low-cost method. The assembled DNA nanoribbons are observed and analyzed by atomic force microscopy (AFM). Experimental observations strongly suggest the feasibility and reliability of our method.

  8. Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

    PubMed

    Michaillat, Lydie; Baars, Tonie Luise; Mayer, Andreas

    2012-03-01

    Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

  9. Viability and DNA fragmentation of rainbow trout embryos (Oncorhynchus mykiss) obtained from eggs stored at 4 °C.

    PubMed

    Ubilla, A; Valdebenito, I; Árias, M E; Risopatrón, J

    2016-05-01

    In vitro storage of salmonid eggs leads to aging of the cells causing a decline in quality and reducing their capacity to develop and produce embryos. The quality of salmonid embryos is assessed by morphologic analyses; however, data on the application of biomarkers to determine the cell viability and DNA integrity of embryos in these species are limited. The aim of this study was to evaluate the effect on embryo development, viability and DNA fragmentation in the embryonic cells of in vitro storage time at 4 °C of rainbow trout (Oncorhynchus mykiss) eggs. The embryos were obtained by IVF from eggs stored for 0 (control), 48, and 96 hours at 4 °C. At 72 hours after fertilization, dechorionated embryos were examined to determine percentages of developed embryos (embryos with normal cell division morphology), viability (LIVE/DEAD sperm viability kit), and DNA integrity (terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay). The percentage of developing embryos decreased (P < 0.05) with storage time of the eggs (95.10 ± 2.55; 88.14 ± 4.50; 79.99 ± 6.60 for 0, 48, and 96 hours, respectively). Similarly, cell viability decreased (P < 0.05; 96.07 ± 7.15; 80.42 ± 8.55; 77.47 ± 7.88 for 0, 48, and 96 hours, respectively), and an increase (P < 0.05) in DNA fragmentation in the embryos was observed at 96-hour storage. A positive correlation was found between cell DNA fragmentation and storage time (r = 0.8173; P < 0.0001). The results revealed that terminal deoxynucleotidyl transferase [TdT] dUTP nick-end labeling assay technique is reliable mean to assess the state of the DNA in salmonid embryos and that in vitro eggs storage for 96h reduces embryo development and cell DNA integrity. DNA integrity evaluation constitutes a biomarker of the quality of the ova and resulting embryos so as to predict their capacity to produce good-quality embryos in salmonids, particularly under culture conditions. PMID:26893166

  10. Selection of normal spermatozoa with a vacuole-free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates.

    PubMed

    Hammoud, I; Boitrelle, F; Ferfouri, F; Vialard, F; Bergere, M; Wainer, B; Bailly, M; Albert, M; Selva, J

    2013-06-01

    Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole-free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n = 8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole-free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1 ± 1.1%, n = 191) than all other types of spermatozoa: non-selected spermatozoa (n = 8000; 26.1 ± 1.5% versus 4.1 ± 1.1%; P < 0.005), motile spermatozoa (n = 444; 20.8 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and motile, normal spermatozoa selected at 200× magnification (n = 370; 18.7 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and then motile, morphometrically normal spermatozoa with anterior vacuoles (n = 368; 15.9 ± 2.9% versus 4.1 ± 1.1%; P < 0.05) or posterior vacuoles (n = 402; 22.5 ± 3.6% versus 4.1 ± 1.1%; P < 0.001) selected at 6300× magnification. For patients with high sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole-free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non-fragmented DNA.

  11. Characterization of 2-mum DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid.

    PubMed Central

    Hollenberg, C P; Degelmann, A; Kustermann-Kuhn, B; Royer, H D

    1976-01-01

    Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-mum supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2-mum yeast DNA in E. coli plasmid pCR1 directly showed that existence of two types of molecules as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-mum circles is due to an inversion of about 1.6 X 10(6) daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 10(6) daltons. Possible implications of this structure are dicussed. Images PMID:778854

  12. Entamoeba histolytica: target cells killed by trophozoites undergo DNA fragmentation which is not blocked by Bcl-2.

    PubMed

    Ragland, B D; Ashley, L S; Vaux, D L; Petri, W A

    1994-11-01

    Amebic destruction of neutrophils and macrophages is contact-dependent. Adherence is mediated by a galactose-specific surface lectin on the amebic membrane. The pathway by which contact-dependent cytolysis of the target cell occurs is unknown. We hypothesized that target cell death is due to the triggering of apoptosis (programmed cell death) by the amebae. The purpose of this study was to determine whether target cell DNA is fragmented into a ladder pattern characteristic of apoptosis and to test whether overexpression of Bcl-2, a protein that confers resistance to apoptotic death from some stimuli, blocks target cell killing. The murine myeloid cell line FDC-P1 transfected with a retrovirus construct expressing the Bcl-2 protein was shown to be resistant to the apoptotic death that the parental line undergoes upon growth factor deprivation. 51Cr-labeled FDC-P1 control or bcl-2-transfected cells were incubated with Entamoeba histolytica (4:1 cell/ameba ratio) and killing of the cells was assessed by 51Cr release. Both cell lines were susceptible to contact-dependent killing. Death induced by the amebae in the bcl-2-transfected cells resulted in a DNA ladder fragmentation pattern (using [125I]iododeoxyuridine-labeled target cell DNA) identical to that seen in the control cells undergoing apoptosis upon growth factor withdrawal. Target cell DNA fragmentation was inhibited by blocking adherence with galactose. Our data suggest that target cell killing by E. histolytica can occur via Bcl-2-independent apoptotic mechanism. PMID:7957763

  13. How do the residual fragments after SWL affect the health-related quality of life? A critical analysis in a size-based manner.

    PubMed

    Sahin, Cahit; Kafkasli, Alper; Cetinel, Cihangir A; Narter, Fehmi; Saglam, Erkin; Sarica, Kemal

    2015-04-01

    This study aimed at evaluating the possible effects of residual fragments (RF) after shockwave lithotripsy (SWL) on the health-related quality of life (QOL) of the patients on a size-related basis. Eighty six patients with RF after SWL were divided into three groups: Group 1 (n:30 with fragments ≤ 2 mm), Group 2 (n:21 2-≤ 4 mm) and Group 3 (n:35 > 4 mm). During a 3-month follow-up, spontaneous passage rates, emergency department visits, mean analgesic required, additional procedures and the QOL were all evaluated. QOL was evaluated using the Short Form-36 survey. Of the 30 patients with fragments ≤ 2 mm all cases passed the fragments spontaneously. Of the 21 cases with fragments 2-≤ 4, however, 76% were stone free. Last, of the 35 cases with fragments >4 mm, 52% passed them spontaneously in 3 months. While no patient with fragments ≤ 2 mm required emergency department visit, 19% of the cases with fragments 2-≤ 4 mm and 51.4 % with fragments >4 mm did require this visit. Mean analgesic need (mg) values were higher in cases with larger fragments. Evaluation of the QOL score data in a subgroup comparison base showed that cases with larger fragments had prominently lower scores during both 1- and 3-month evaluation. RF after SWL could pose an impact on the QOL of the cases in a size-related basis. While fragments ≤ 2 mm had nearly no impact on this aspect larger fragments could significantly affect the QOL.

  14. Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

    PubMed Central

    Jin, H; Ren, Z; Pozsgay, J M; Elkins, C; Whitby, P W; Morton, D J; Stull, T L

    1996-01-01

    Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo. PMID:8757844

  15. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21.3) by cDNA hybridization selection.

    PubMed Central

    Goei, V. L.; Parimoo, S.; Capossela, A.; Chu, T. W.; Gruen, J. R.

    1994-01-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. Images Figure 2 Figure 4 PMID:8304341

  16. Quantitative predictivity of carcinogenicity for four short-term parameters, evaluated in rat liver: alkaline DNA fragmentation, autoradiographic repair, DNA adducts, preneoplastic nodules.

    PubMed

    Parodi, S; Taningher, M; Santi, L

    1984-01-01

    The possibility of the study of a quantitative correlation between short-term tests and carcinogenicity, instead of a qualitative one, is discussed. Four tests related to the target organ, rat liver, were considered: alkaline DNA fragmentation, DNA repair, DNA adducts and the formation of preneoplastic nodules. All the four tests showed a similar level of correlation with carcinogenic potency (r approximately equal to 0.4). With this level of correlation, the dispersion of the data appeared too large to offer a meaningful degree of quantitative predictivity of carcinogenicity, in reference to a single test. It appeared however, that the use of a battery of two or three independent short-term tests, with the above level of simple correlation, could generate a multiple correlation high enough to be potentially useful for some degree of quantitative predictivity of carcinogenic potency.

  17. Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques.

    PubMed

    Degheidy, T; Abdelfattah, H; Seif, A; Albuz, F K; Gazi, S; Abbas, S

    2015-10-01

    Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P < 0.05). It can be concluded that MACs technique is a simple, noninvasive, technique that can efficiently reduce DNA fragmentation in infertile varicocele patients prior to ART.

  18. A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter.

    PubMed

    Qiu, Bin; Zhang, Ya-shan; Lin, Yi-bing; Lu, Yu-Jing; Lin, Zhen-yu; Wong, Kwok-Yin; Chen, Guo-nan

    2013-03-15

    In this paper, we reported a convenient fluorescence method for the detection of genetically modified organisms (GMOs). As it is known that the cauliflower mosaic virus (CaMV) 35S promoter is widely used in most transgenic plants (Schnurr and Guerra, 2000), we thus design a simple method based on the detection of a section target DNA (DNA-T) from the transgene CaMV 35S promoter. In this method, the full-length guanine-rich single-strand sequences were split into fragments (Probe 1 and 2) and each part of the fragment possesses two GGG repeats. In the presence of K(+) ion and berberine, if a complementary target DNA of the CaMV 35S promoter was introduced to hybridize with Probe 1 and 2, a G-quadruplex-berberine complex was thus formed and generated a strong fluorescence signal. The generation of fluorescence signal indicates the presence of CaMV 35S promoter. This method is able to identify and quantify Genetically Modified Organisms (GMOs), and it shows wide linear ranges from 5.0×10(-9) to 9.0×10(-7) mol/L with a detection limit of 2.0×10(-9) mol/L. PMID:22959013

  19. A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter.

    PubMed

    Qiu, Bin; Zhang, Ya-shan; Lin, Yi-bing; Lu, Yu-Jing; Lin, Zhen-yu; Wong, Kwok-Yin; Chen, Guo-nan

    2013-03-15

    In this paper, we reported a convenient fluorescence method for the detection of genetically modified organisms (GMOs). As it is known that the cauliflower mosaic virus (CaMV) 35S promoter is widely used in most transgenic plants (Schnurr and Guerra, 2000), we thus design a simple method based on the detection of a section target DNA (DNA-T) from the transgene CaMV 35S promoter. In this method, the full-length guanine-rich single-strand sequences were split into fragments (Probe 1 and 2) and each part of the fragment possesses two GGG repeats. In the presence of K(+) ion and berberine, if a complementary target DNA of the CaMV 35S promoter was introduced to hybridize with Probe 1 and 2, a G-quadruplex-berberine complex was thus formed and generated a strong fluorescence signal. The generation of fluorescence signal indicates the presence of CaMV 35S promoter. This method is able to identify and quantify Genetically Modified Organisms (GMOs), and it shows wide linear ranges from 5.0×10(-9) to 9.0×10(-7) mol/L with a detection limit of 2.0×10(-9) mol/L.

  20. The kinetics of force-dependent hybridization and strand-peeling of short DNA fragments

    NASA Astrophysics Data System (ADS)

    Yang, ZhouJie; Yuan, GuoHua; Zhai, WeiLi; Yan, Jie; Chen, Hu

    2016-08-01

    Deoxyribonucleic acid (DNA) carries the genetic information in all living organisms. It consists of two interwound single-stranded (ss) strands, forming a double-stranded (ds) DNA with a right-handed double-helical conformation. The two strands are held together by highly specific basepairing interactions and are further stabilized by stacking between adjacent basepairs. A transition from a dsDNA to two separated ssDNA is called melting and the reverse transition is called hybridization. Applying a tensile force to a dsDNA can result in a particular type of DNA melting, during which one ssDNA strand is peeled away from the other. In this work, we studied the kinetics of strand-peeling and hybridization of short DNA under tensile forces. Our results show that the force-dependent strand-peeling and hybridization can be described with a simple two-state model. Importantly, detailed analysis of the force-dependent transition rates revealed that the transition state consists of several basepairs dsDNA.

  1. Tandem mass spectrometry-based detection of c4'-oxidized abasic sites at specific positions in DNA fragments.

    PubMed

    Chowdhury, Goutam; Guengerich, F Peter

    2009-07-01

    Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and ionizing radiation have been shown to cause oxidative DNA sugar damage. Detection of DNA lesions is important for understanding the toxicological or therapeutic consequences associated with such agents. C4'-oxidized abasic sites (C4-AP) are produced by the antitumor drug bleomycin and ionizing radiation. The currently available methods for the detection of C4-AP cannot provide both structural and sequence information. We have developed an LC-ESI-MS-based approach for specific detection and mapping of C4-AP from a mixture of lesions. We show using Fe-bleomycin-damaged DNA that C4-AP can be detected at cytosine and thymine sites by direct MS analysis. Our results reveal that collision-induced dissociation of C4-AP-containing oligonucleotides results in preferential fragmentation at C4-AP sites with the formation of the unique a* ions (18 amu more than the a-B ions) that allow mapping of the C4-AP sites. Various chemical modification strategies (e.g., reduction with NaBH4 and NaBD4 and derivatization with methoxyamine and hydrazine, followed by LC-MS analysis) were also used for unambiguous detection of C4-AP sites. Finally, we show that the methods described here can detect the presence of C4-AP at specific sites in a complex sample such as hydroxyl radical-damaged DNA. The LC-MS approach was also used for the simultaneous detection of the other C4'-oxidation end product, 3'-phosphoglycolate, at a specific site in hydroxyl radical-damaged DNA. Thus, LC-MS provides a rapid and direct approach for the detection and mapping of oxidative DNA lesions. PMID:19496605

  2. Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis

    SciTech Connect

    Haab, B.B.; Mathies, R.A.

    1995-09-15

    A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative TO6 as they passed through the nearly 2-{mu}m diameter focused laser beam. Amplified photo-electron pulses from the photomultiplier are grouped into bins of 360-450 {mu}s in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The auto-correlation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of nearly 100 DNA molecules/band or better. 45 refs., 10 figs.

  3. Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape.

    PubMed

    Michalski, Fernanda; Valdez, Fernanda Pedone; Norris, Darren; Zieminski, Chris; Kashivakura, Cyntia Kayo; Trinca, Cristine S; Smith, Heath B; Vynne, Carly; Wasser, Samuel K; Metzger, Jean Paul; Eizirik, Eduardo

    2011-09-01

    The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies.

  4. New insight into power-law behavior of fragment size distributions in the C₆₀ multifragmentation regime.

    PubMed

    Qian, D B; Ma, X; Chen, Z; Li, B; Zhang, D C; Zhu, X L; Wen, W Q; Liu, H P

    2014-08-01

    Previous experimental work has shown that a phase transition in C60 multifragmentation induced by nanosecond laser occurs at almost constant temperature covering a wide range of laser fluency. Here the relative yields of ionic fragments (IFs) C(n)(+) (n = 1-20) resulting from the multifragmentation are measured within the phase transition region. By excluding two small IFs and magic IFs due to their abnormal behavior, the data for residual IFs are used to estimate the size distributions of primary intermediate-mass IFs in the multifragmentation regime. The distributions are found to obey power laws n(-τ). Furthermore, the exponent τ values have sensitive dependence on lower laser fluency and converge to a constant of about 2.4 ± 0.2 for larger fluencies. These observations are in good agreement with an explanation based on the Fisher droplet model, offering the tantalizing possibility of a liquid-to-gas phase transition in C60 systems. PMID:25106587

  5. Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

    SciTech Connect

    Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet

    2014-02-07

    Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

  6. A localized transition in the size variation of circular DNA in nanoslits

    NASA Astrophysics Data System (ADS)

    Strychalski, Elizabeth A.; Stavis, Samuel M.; Geist, Jon

    2013-03-01

    We observe a localized transition in the size variation of circular DNA between strong and moderate regimes of nanofluidic slitlike confinement. We applied a rigorous statistical analysis to our recent experimental measurements of DNA size for linear and circular topologies in nanoslits with depths around ~2p, where p is the DNA persistence length [E. A. Strychalski, J. Geist, M. Gaitan, L. E. Locascio, S. M. Stavis. Macromolecules, 45, 1602-1611 (2012)]. Our empirical approach revealed a localized transition between confinement regimes for circular DNA at a nanoslit depth of ~3p but detected no such transition for linear DNA with a similar contour length. These results provide the first indication of the localized influence of polymer topology on size variation across changing nanoslit depths. Improved understanding of differences in polymer behavior due to topology in this controversial system is of fundamental importance in polymer science and will inform new nanofluidic methods for biopolymer analysis.

  7. Screening relevant genes of tolerance to low phosphorus in maize using cDNA-amplified fragment length polymorphism.

    PubMed

    Jiang, H Y; Li, Z; Zhao, J; Ma, Q; Cheng, B J; Zhu, S W

    2015-01-01

    Soil contains a large amount of phosphorus, but plants cannot absorb most of this phosphorus effectively. Low inorganic phosphorus has been singled out as a major constraint that leads to a perpetually low Zea mays (maize) grain yield. The fundamental approach to solving this problem is to screen new genes of low phosphorous (LP) tolerance. Consequently, the exploration and utilization of LP-tolerant genes are of great significance in plants. The maize inbred line 178 is an inbred LP-tolerant line. In the current study, the expression of this inbred line was induced under the stress of LP conditions. We applied cDNA-amplified fragment length polymorphism to screen LP-tolerant genes and obtained and sequenced 78 differentially expressed gene fragments. Their functions were predicted via bioinformatic analysis. There were no function annotations for 8 differentially expressed fragments. Nine genes exhibited high homology to Arabidopsis thaliana and Oryza sativa genes involved in phosphorus metabolism. This study lays a good foundation for further cloning and verification of the genes involved in phosphorus metabolism in maize. PMID:26125772

  8. Screening relevant genes of tolerance to low phosphorus in maize using cDNA-amplified fragment length polymorphism.

    PubMed

    Jiang, H Y; Li, Z; Zhao, J; Ma, Q; Cheng, B J; Zhu, S W

    2015-05-29

    Soil contains a large amount of phosphorus, but plants cannot absorb most of this phosphorus effectively. Low inorganic phosphorus has been singled out as a major constraint that leads to a perpetually low Zea mays (maize) grain yield. The fundamental approach to solving this problem is to screen new genes of low phosphorous (LP) tolerance. Consequently, the exploration and utilization of LP-tolerant genes are of great significance in plants. The maize inbred line 178 is an inbred LP-tolerant line. In the current study, the expression of this inbred line was induced under the stress of LP conditions. We applied cDNA-amplified fragment length polymorphism to screen LP-tolerant genes and obtained and sequenced 78 differentially expressed gene fragments. Their functions were predicted via bioinformatic analysis. There were no function annotations for 8 differentially expressed fragments. Nine genes exhibited high homology to Arabidopsis thaliana and Oryza sativa genes involved in phosphorus metabolism. This study lays a good foundation for further cloning and verification of the genes involved in phosphorus metabolism in maize.

  9. Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines.

    PubMed

    Carstens, Myrra G; Camps, Marcel G M; Henriksen-Lacey, Malou; Franken, Kees; Ottenhoff, Tom H M; Perrie, Yvonne; Bouwstra, Joke A; Ossendorp, Ferry; Jiskoot, Wim

    2011-06-24

    The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination. PMID:21565240

  10. Crocin Effects on Human Myeloma Cells Regarding Intracellular Redox State, DNA Fragmentation, and Apoptosis or Necrosis Profile

    PubMed Central

    Rezaee, Ramin; Jamialahmadi, Khadijeh; Riahi Zanjani, Bamdad; Mahmoudi, Mahmoud; Abnous, Khalil; Zamani Taghizadeh Rabe, Shahrzad; Tabasi, Nafiseh; Zali, Marjan; Rezaee, Marjan; Amin, Bahareh; Karimi, Gholamreza

    2014-01-01

    Background: Well-documented studies reported several pharmacological properties for crocin, the active compound of Crocus sativus, such as its antitumor, radical scavenging, antidepressant, and memory-enhancing effects. Objectives: We aimed to evaluate the possible cytotoxic activity of crocin on B lymphocytes in human myeloma (U266 cell line) after 24- and 48-hour treatment. Materials and Methods: For this purpose, cell viability was determined by the colorimetric MTT assay and cell death pattern was evaluated using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit. ROS (reactive oxygen species) production and DNA fragmentation were assessed using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) kit and PI staining, respectively. Results: The highest concentration of crocin significantly decreased ROS production after 48 hours of treatment. However, crocin had no effect on the expression level of HSP (Heat shock protein). Additionally, its administration caused a mild decline in cell viability and a mild increase in the population of DNA fragmented cells as well as apoptosis. Conclusions: In our study, no prominent effect was seen; therefore, in order to have a better perspective of crocin activity against cancerous cell lines, further studies are highly recommended. PMID:25625054

  11. Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous

    PubMed Central

    2013-01-01

    Background Xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous. Results In this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced. Conclusions This methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods. PMID:24103677

  12. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells.

    PubMed

    Arora, Shagun; Tandon, Simran

    2015-01-01

    In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

  13. Lophirones B and C prevent aflatoxin B1-induced oxidative stress and DNA fragmentation in rat hepatocytes.

    PubMed

    Ajiboye, Taofeek Olakunle; Yakubu, Musa Toyin; Oladiji, Adenike Temidayo

    2016-10-01

    Context Despite the reported anticarcinogenic activity of lophirones B and C, no scientific information exists for its activity in rat hepatocytes. Objective Effect of lophirones B and C on aflatoxin B1 (AFB1)-induced oxidative stress, and DNA fragmentation in rat hepatocytes was investigated. Materials and methods Wistar rat hepatocytes were incubated with lophirones B and C (1 mg/mL) or sylimarin (1 mg/mL) in the presence or absence of AFB1. For an in vivo study, rats were orally administered with lophirones B and C, and/or AFB1 (20 μg/d) for 9 weeks. Results Lophirones B and C lowered AFB1-mediated increase in nitric oxide, superoxide anion radicals, caspase-3 and fragmented DNA. Lophirones B and C attenuated AFB1-mediated decrease in superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and reduced glutathione. Also, lophirones B and C attenuated AFB1-mediated increase in conjugated dienes, lipid hydroperoxides and malondialdehyde in rat hepatocytes. Furthermore, AFB1-mediated alterations in alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin and globulin in rat serum were significantly annulled in lophirones B and C-treated rats. Conclusion This study revealed that lophirones B and C prevented AFB1-induced oxidative damage in rat hepatocytes.

  14. Multi-Scale Particle Size Distributions of Mars, Moon and Itokawa based on a time-maturation dependent fragmentation model

    NASA Astrophysics Data System (ADS)

    Charalambous, C. A.; Pike, W. T.

    2013-12-01

    We present the development of a soil evolution framework and multiscale modelling of the surface of Mars, Moon and Itokawa thus providing an atlas of extra-terrestrial Particle Size Distributions (PSD). These PSDs are profoundly based on a tailoring method which interconnects several datasets from different sites captured by the various missions. The final integrated product is then fully justified through a soil evolution analysis model mathematically constructed via fundamental physical principles (Charalambous, 2013). The construction of the PSD takes into account the macroscale fresh primary impacts and their products, the mesoscale distributions obtained by the in-situ data of surface missions (Golombek et al., 1997, 2012) and finally the microscopic scale distributions provided by Curiosity and Phoenix Lander (Pike, 2011). The distribution naturally extends at the magnitudinal scales at which current data does not exist due to the lack of scientific instruments capturing the populations at these data absent scales. The extension is based on the model distribution (Charalambous, 2013) which takes as parameters known values of material specific probabilities of fragmentation and grinding limits. Additionally, the establishment of a closed-form statistical distribution provides a quantitative description of the soil's structure. Consequently, reverse engineering of the model distribution allows the synthesis of soil that faithfully represents the particle population at the studied sites (Charalambous, 2011). Such representation essentially delivers a virtual soil environment to work with for numerous applications. A specific application demonstrated here will be the information that can directly be extracted for the successful drilling probability as a function of distance in an effort to aid the HP3 instrument of the 2016 Insight Mission to Mars. Pike, W. T., et al. "Quantification of the dry history of the Martian soil inferred from in situ microscopy

  15. Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

    PubMed

    Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

    2004-03-01

    Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

  16. Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

    PubMed Central

    Duzdevich, Daniel; Li, Jinliang; Whang, Jhoon; Takahashi, Hirohide; Takeyasu, Kunio; Dryden, David T. F.; Morton, A. Jennifer; Edwardson, J. Michael

    2011-01-01

    Background In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene. Methodology/Principal Findings We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I. Conclusions/Significance “Super-long” CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD. PMID:21347256

  17. Molecular behavior of DNA in a cell-sized compartment coated by lipids

    NASA Astrophysics Data System (ADS)

    Hamada, Tsutomu; Fujimoto, Rie; Shimobayashi, Shunsuke F.; Ichikawa, Masatoshi; Takagi, Masahiro

    2015-06-01

    The behavior of long DNA molecules in a cell-sized confined space was investigated. We prepared water-in-oil droplets covered by phospholipids, which mimic the inner space of a cell, following the encapsulation of DNA molecules with unfolded coil and folded globule conformations. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space. Globular DNA showed a cell-size-dependent unfolding transition after adsorption on the membrane. Furthermore, when DNA interacted with a two-phase membrane surface, DNA selectively adsorbed on the membrane phase, such as an ordered or disordered phase, depending on its conformation. We discuss the mechanism of these trends by considering the free energy of DNA together with a polyamine in the solution. The free energy of our model was consistent with the present experimental data. The cooperative interaction of DNA and polyamines with a membrane surface leads to the size-dependent behavior of molecular systems in a small space. These findings may contribute to a better understanding of the physical mechanism of molecular events and reactions inside a cell.

  18. Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding.

    PubMed

    Agha-Rahimi, Azam; Khalili, Mohammad Ali; Nabi, Ali; Ashourzadeh, Sareh

    2014-03-01

    Human sperm vitrification is a new cryopreservation method. This study compared the effects of rapid freezing and vitrification on various sperm parameters, hyaluronan-binding assay and DNA fragmentation and assessed the impact of cryoprotectant agents (CPA) with vitrification. A total of 30 normo-ejaculates were prepared by swim up and the motile sperm fraction was divided into four: fresh (control), rapid freezing, and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held just above the liquid nitrogen surface, and for vitrification, 30μl suspension was dropped directly into liquid nitrogen. Sperm parameters, including motility, viability and morphology, declined after cryopreservation in both groups. DNA fragmentation was not significantly higher in the vitrification (15.7±4.4%) or rapid freezing (16.6±5.6%) groups when compared with controls (11.6±4.5%). The rates of hyaluronan binding were similar between the control and cryopreserved groups. Moreover, addition of CPA for vitrification had a neutral effect on rates of sperm recovery. In conclusion, vitrification has great potential for human sperm cryopreservation and does not require CPA, with its possible toxicity. However, it is not superior to rapid cryopreservation regarding sperm recovery rate in normozoospermia. Human sperm vitrification is a new cryopreservation method that has been introduced recently. This study compared the effects of rapid freezing with vitrification on rates of sperm parameters, hyaluronan-binding assay and DNA fragmentation after thawing/warming and assessed the impact of cryoprotectant agent (CPA) on vitrification. The study was performed on 30 ejaculates prepared using the swim-up technique. Each motile sperm suspension was divided into four: control (fresh); rapid freezing; and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held above the surface of

  19. Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape.

    PubMed

    Michalski, Fernanda; Valdez, Fernanda Pedone; Norris, Darren; Zieminski, Chris; Kashivakura, Cyntia Kayo; Trinca, Cristine S; Smith, Heath B; Vynne, Carly; Wasser, Samuel K; Metzger, Jean Paul; Eizirik, Eduardo

    2011-09-01

    The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies. PMID:21676206

  20. Size-specific concentration of DNA to a nanostructured tip using dielectrophoresis and capillary action.

    PubMed

    Yeo, Woon-Hong; Chung, Jae-Hyun; Liu, Yaling; Lee, Kyong-Hoon

    2009-08-01

    One of the critical challenges in the fields of disease diagnostics and environmental monitoring is to concentrate extracellular DNA from a sample mixture rapidly. Unlike genomic DNA in normal cells, extracellular DNA dissolved in a biological sample can potentially offer crucial information about pathogens and toxins. The current concentration methods, however, are not able to directly concentrate extracellular DNA due to aggressive sample preparation steps. This paper presents a concentration mechanism of extracellular DNA onto a nanostructured tip using dielectrophoresis (DEP) in conjunction with capillary action. DNA immersed in a solution is captured onto a nanotip by two sequential actions: (1) attraction of DNA and other bioparticles in the vicinity of a nanotip by DEP and (2) size-specific capture of DNA onto the nanotip by capillary action. To investigate the size-specific capturing mechanism, an analytical model for the capillary action on a nanotip is presented, which is compared to the experiment for capturing polystyrene nanospheres. This analysis predicts the capture of a spherical particle smaller than 0.39 times a nanotip diameter, whereas our experiment shows that polystyrene spheres smaller than 0.84 times a nanotip diameter are captured. This discrepancy can be caused by the increase of the capturing force due to attractive DEP force. In addition, the diameter of the captured spheres can be increased by other experimental conditions including the tip geometry, the multiple particle interaction, and the contact angles. When a nanotip is used for concentrating lambda-DNA, 6.7 pg/mL (210 aM) of DNA is selectively extracted from a sample mixture containing lambda-DNA and Drosophila cells in one minute. The captured DNA is investigated by fluorescence microscopy, scanning electron microscopy (SEM), and X-ray analysis. This nanotip-based DNA concentrating method is a rapid and highly sensitive technique to detect extracellular DNA from a sample mixture.

  1. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

    PubMed Central

    Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

    2016-01-01

    Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

  2. Strongly structured DNA sequences as targets for genosensing: sensing phase design and coupling to PCR amplification for a highly specific 33-mer gliadin DNA fragment.

    PubMed

    Martín-Fernández, Begoña; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús; Frutos-Cabanillas, Gloria; de-los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz

    2014-10-15

    Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour.

  3. The effect of two pre-cryopreservation single layer colloidal centrifugation protocols in combination with different freezing extenders on the fragmentation dynamics of thawed equine sperm DNA

    PubMed Central

    2012-01-01

    Background Variability among stallions in terms of semen cryopreservation quality renders it difficult to arrive at a standardized cryopreservation method. Different extenders and processing techniques (such us colloidal centrifugation) are used in order to optimize post-thaw sperm quality. Sperm chromatin integrity analysis is an effective tool for assessing such quality. The aim of the present study was to compare the effect of two single layer colloidal centrifugation protocols (prior to cryopreservation) in combination with three commercial freezing extenders on the post-thaw chromatin integrity of equine sperm samples at different post-thaw incubation (37°C) times (i.e., their DNA fragmentation dynamics). Results Post-thaw DNA fragmentation levels in semen samples subjected to either of the colloidal centrifugation protocols were significantly lower (p<0.05) immediately after thawing and after 4 h of incubation at 37°C compared to samples that underwent standard (control) centrifugation. The use of InraFreeze® extender was associated with significantly less DNA fragmentation than the use of Botu-Crio® extender at 6 h of incubation, and than the use of either Botu-Crio® or Gent® extender at 24 h of incubation (p<0.05). Conclusions These results suggest that single layer colloidal centrifugation performed with extended or raw semen prior to cryopreservation reduces DNA fragmentation during the first four hours after thawing. Further studies are needed to determine the influence of freezing extenders on equine sperm DNA fragmentation dynamics. PMID:23217215

  4. Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.

    PubMed

    Belcheva, Antoaneta; Ivanova-Kicheva, Maria; Tzvetkova, Petia; Marinov, Mihail

    2004-10-01

    Cigarette smoking is a serious health problem of our society. It is known that cigarette smoke is a cell mutagen and carcinogen, and that it may affect adversely male fertility. The possible detrimental effects on sperm cells are of great interest but the data available to support this statement are somewhat elusive. To approach this problem we examined conventional semen parameters, plasma membrane translocation of phosphatidylserine (PS) (annexin V/6-CFDA cell staining) and sperm DNA integrity (comet assay) in a group of healthy man smoking cigarettes on a regular basis. The results of the study were compared with the results of the same tests in healthy non-smoking donors. Significant difference in standard sperm parameters between the two groups was not found. Intensive expression of PS on the sperm plasma membrane surface (assayed by annexin V positive staining) was detected in the smokers group. There is a significant increase of population of apoptotic spermatozoa in ejaculates of smokers. Albeit DNA damages (high frequencies of double- and single- stranded DNA breaks) in spermatozoa of smokers are increased compared with non-smokers, but this difference is not statistically significant. Sperm DNA integrity of healthy smokers remains in the normal range, but a clear negative trend is observed, especially in respect of disturbance of plasma membrane phospholipid asymmetry.

  5. Hydrodynamic size of DNA/cationic gemini surfactant complex as a function of surfactant structure.

    PubMed

    Devínsky, Ferdinand; Pisárcik, Martin; Lacko, Ivan

    2009-06-01

    The present study deals with the determination of hydrodynamic size of DNA/cationic gemini surfactant complex in sodium bromide solution using the dynamic light scattering method. Cationic gemini surfactants with polymethylene spacer of variable length were used for the interaction with DNA. The scattering experiments were performed at constant DNA and sodium bromide concentrations and variable surfactant concentration in the premicellar and micellar regions as a function of surfactant spacer length. It was found that the DNA conformation strongly depends on the polymethylene spacer length as well as on the surfactant concentration relative to the surfactant critical micelle concentration. Gemini surfactant molecules with 4 methylene groups in the spacer were found to be the least efficient DNA compacting agent in the region above the surfactant cmc. Gemini molecules with the shortest spacer length (2 methylene groups) and the longest spacer length (8 methylene groups) investigated showed the most efficient DNA compaction ability. PMID:19592712

  6. Molecular cloning of a human histocompatibility antigen cDNA fragment.

    PubMed Central

    Ploegh, H L; Orr, H T; Strominger, J L

    1980-01-01

    A clone (pHLA-1) containing HLA-specific cDNA was constructed by reverse transcription of partially purified HLA mRNA from the human lymphoblastoid cell line LKT. The identity of pHLA-1 was established by its ability to hybridize to HLA heavy chain mRNA and by nucleotide sequence analysis. The pHLA-1 cDNA insert (approximately 525 base pairs) corresponds to the COOH-terminal 46 amino acids of an HLA-A, -B, or -C antigen (15 residues from the hydrophobic region and the remainder from the COOH-termial hydrophilic region), together with a portion of the 3' untranslated region of the mRNA. Images PMID:6934534

  7. Efficient DNA Fingerprinting of Clostridium botulinum Types A, B, E, and F by Amplified Fragment Length Polymorphism Analysis

    PubMed Central

    Keto-Timonen, Riikka; Nevas, Mari; Korkeala, Hannu

    2005-01-01

    Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification. PMID:15746312

  8. CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

    NASA Astrophysics Data System (ADS)

    Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; Akyürek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Pérez Urquiza, M.; Bautista, J. L.; Garibay, A.; Fuller, B.; Baoutina, A.; Partis, L.; Emslie, K.; Holden, M.; Chum, W. Y.; Kim, H.-H.; Phunbua, N.; Milavec, M.; Zel, J.; Vonsky, M.; Konopelko, L. A.; Lau, T. L. T.; Yang, B.; Hui, M. H. K.; Yu, A. C. H.; Viroonudomphol, D.; Prawettongsopon, C.; Wiangnon, K.; Takabatake, R.; Kitta, K.; Kawaharasaki, M.; Parkes, H.

    2012-01-01

    Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials". The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  9. Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA.

    PubMed

    Shao, Jie; Gao, Fan; Lin, Hui-Juan; Mao, Qun-Ying; Chen, Pan; Wu, Xing; Yao, Xin; Kong, Wei; Liang, Zheng-Lun

    2016-01-01

    To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines.

  10. Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA

    PubMed Central

    Shao, Jie; Gao, Fan; Lin, Hui-Juan; Mao, Qun-Ying; Chen, Pan; Wu, Xing; Yao, Xin; Kong, Wei; Liang, Zheng-Lun

    2016-01-01

    To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines. PMID:27082865

  11. Grand-canonical simulation of DNA condensation with two salts, effect of divalent counterion size.

    PubMed

    Nguyen, Toan T

    2016-02-14

    The problem of DNA- DNA interaction mediated by divalent counterions is studied using a generalized grand-canonical Monte-Carlo simulation for a system of two salts. The effect of the divalent counterion size on the condensation behavior of the DNA bundle is investigated. Experimentally, it is known that multivalent counterions have strong effect on the DNA condensation phenomenon. While tri- and tetra-valent counterions are shown to easily condense free DNA molecules in solution into toroidal bundles, the situation with divalent counterions is not as clear cut. Some divalent counterions like Mg(+2) are not able to condense free DNA molecules in solution, while some like Mn(+2) can condense them into disorder bundles. In restricted environment such as in two dimensional system or inside viral capsid, Mg(+2) can have strong effect and able to condense them, but the condensation varies qualitatively with different system, different coions. It has been suggested that divalent counterions can induce attraction between DNA molecules but the strength of the attraction is not strong enough to condense free DNA in solution. However, if the configuration entropy of DNA is restricted, these attractions are enough to cause appreciable effects. The variations among different divalent salts might be due to the hydration effect of the divalent counterions. In this paper, we try to understand this variation using a very simple parameter, the size of the divalent counterions. We investigate how divalent counterions with different sizes can lead to varying qualitative behavior of DNA condensation in restricted environments. Additionally, a grand canonical Monte-Carlo method for simulation of systems with two different salts is presented in detail.

  12. Grand-canonical simulation of DNA condensation with two salts, effect of divalent counterion size

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan T.

    2016-02-01

    The problem of DNA- DNA interaction mediated by divalent counterions is studied using a generalized grand-canonical Monte-Carlo simulation for a system of two salts. The effect of the divalent counterion size on the condensation behavior of the DNA bundle is investigated. Experimentally, it is known that multivalent counterions have strong effect on the DNA condensation phenomenon. While tri- and tetra-valent counterions are shown to easily condense free DNA molecules in solution into toroidal bundles, the situation with divalent counterions is not as clear cut. Some divalent counterions like Mg+2 are not able to condense free DNA molecules in solution, while some like Mn+2 can condense them into disorder bundles. In restricted environment such as in two dimensional system or inside viral capsid, Mg+2 can have strong effect and able to condense them, but the condensation varies qualitatively with different system, different coions. It has been suggested that divalent counterions can induce attraction between DNA molecules but the strength of the attraction is not strong enough to condense free DNA in solution. However, if the configuration entropy of DNA is restricted, these attractions are enough to cause appreciable effects. The variations among different divalent salts might be due to the hydration effect of the divalent counterions. In this paper, we try to understand this variation using a very simple parameter, the size of the divalent counterions. We investigate how divalent counterions with different sizes can lead to varying qualitative behavior of DNA condensation in restricted environments. Additionally, a grand canonical Monte-Carlo method for simulation of systems with two different salts is presented in detail.

  13. Genotyping of Madurella mycetomatis by selective amplification of restriction fragments (amplified fragment length polymorphism) and subtype correlation with geographical origin and lesion size.

    PubMed

    van de Sande, Wendy W J; Gorkink, Roy; Simons, Guus; Ott, Alewijn; Ahmed, Abdalla O A; Verbrugh, Henri; van Belkum, Alex

    2005-09-01

    One of the causative organisms of mycetoma is the fungus Madurella mycetomatis. Previously, extensive molecular typing studies identified Sudanese isolates of this fungus as clonal, but polymorphic genetic markers have not yet been identified. Here, we report on the selective amplification of restriction fragment (AFLP) analysis of 37 Sudanese clinical isolates of M. mycetomatis. Of 93 AFLP fragments generated, 25 were polymorphic, and 12 of these 25 polymorphic fragments were found in a large fraction of the strains. Comparative analysis resulted into a tree, composed of two main (clusters I and II) and one minor cluster (cluster III). Seventy-five percent of the strains found in cluster I originated from central Sudan, while the origin of the strains in cluster II was more heterogeneous. Furthermore, the strains found in cluster I were generally obtained from lesions larger than those from which the strains found in cluster II were obtained (chi-square test for trend, P = 0.03). Among the 12 more commonly found polymorphisms, 4 showed sequence homology with known genes. Marker A7 was homologous to an endo-1,4-beta-glucanase from Aspergillus oryzae, 97% identical markers A12 and B3 matched a hypothetical protein from Gibberella zeae, and marker B4 was homologous to casein kinase I from Danio rerio. The last marker seemed to be associated with strains originating from central Sudan (P = 0.001). This is the first report on a genotypic study where genetic markers which may be used to study pathogenicity in M. mycetomatis were obtained.

  14. Effects of cyanoacrylate fuming, time after recovery, and location of biological material on the recovery and analysis of DNA from post-blast pipe bomb fragments*.

    PubMed

    Bille, Todd W; Cromartie, Carter; Farr, Matthew

    2009-09-01

    This study investigated the effects of time, cyanoacrylate fuming, and location of the biological material on DNA analysis of post-blast pipe bomb fragments. Multiple aliquots of a cell suspension (prepared by soaking buccal swabs in water) were deposited on components of the devices prior to assembly. The pipe bombs were then deflagrated and the fragments recovered. Fragments from half of the devices were cyanoacrylate fumed. The cell spots on the fragments were swabbed and polymerase chain reaction/short tandem repeat analysis was performed 1 week and 3 months after deflagration. A significant decrease in the amount of DNA recovered was observed between samples collected and analyzed within 1 week compared with the samples collected and analyzed 3 months after deflagration. Cyanoacrylate fuming did not have a measurable effect on the success of the DNA analysis at either time point. Greater quantities of DNA were recovered from the pipe nipples than the end caps. Undeflagrated controls showed that the majority (>95%) of the DNA deposited on the devices was not recovered at a week or 3 months. PMID:19737244

  15. Effects of cyanoacrylate fuming, time after recovery, and location of biological material on the recovery and analysis of DNA from post-blast pipe bomb fragments*.

    PubMed

    Bille, Todd W; Cromartie, Carter; Farr, Matthew

    2009-09-01

    This study investigated the effects of time, cyanoacrylate fuming, and location of the biological material on DNA analysis of post-blast pipe bomb fragments. Multiple aliquots of a cell suspension (prepared by soaking buccal swabs in water) were deposited on components of the devices prior to assembly. The pipe bombs were then deflagrated and the fragments recovered. Fragments from half of the devices were cyanoacrylate fumed. The cell spots on the fragments were swabbed and polymerase chain reaction/short tandem repeat analysis was performed 1 week and 3 months after deflagration. A significant decrease in the amount of DNA recovered was observed between samples collected and analyzed within 1 week compared with the samples collected and analyzed 3 months after deflagration. Cyanoacrylate fuming did not have a measurable effect on the success of the DNA analysis at either time point. Greater quantities of DNA were recovered from the pipe nipples than the end caps. Undeflagrated controls showed that the majority (>95%) of the DNA deposited on the devices was not recovered at a week or 3 months.

  16. Genome size and DNA base composition of geophytes: the mirror of phenology and ecology?

    PubMed Central

    Veselý, Pavel; Bureš, Petr; Šmarda, Petr; Pavlíček, Tomáš

    2012-01-01

    Background and Aims Genome size is known to affect various plant traits such as stomatal size, seed mass, and flower or shoot phenology. However, these associations are not well understood for species with very large genomes, which are laregly represented by geophytic plants. No detailed associations are known between DNA base composition and genome size or species ecology. Methods Genome sizes and GC contents were measured in 219 geophytes together with tentative morpho-anatomical and ecological traits. Key Results Increased genome size was associated with earliness of flowering and tendency to grow in humid conditions, and there was a positive correlation between an increase in stomatal size in species with extremely large genomes. Seed mass of geophytes was closely related to their ecology, but not to genomic parameters. Genomic DNA GC content showed a unimodal relationship with genome size but no relationship with species ecology. Conclusions Evolution of genome size in geophytes is closely related to their ecology and phenology and is also associated with remarkable changes in DNA base composition. Although geophytism together with producing larger cells appears to be an advantageous strategy for fast development of an organism in seasonal habitats, the drought sensitivity of large stomata may restrict the occurrence of geophytes with very large genomes to regions not subject to water stress. PMID:22021815

  17. Historic cycles of fragmentation and expansion in Parnassius smintheus (papilionidae) inferred using mitochondrial DNA.

    PubMed

    DeChaine, Eric G; Martini, Andrew P

    2004-01-01

    Climate oscillations of the Quaternary drove the repeated expansion and contraction of ecosystems. Alpine organisms were probably isolated in sky island refugia during warm interglacials, such as now, and expanded their range by migrating down-slope during glacial periods. We used population genetic and phylogenetic approaches to infer how paleoclimatic events influenced the distribution of genetic variation in the predominantly alpine butterfly Parnassius smintheus. We sequenced a 789 bp region of cytochrome oxidase I for 385 individuals from 20 locations throughout the Rocky Mountains, ranging from southern Colorado to northern Montana. Analyses revealed at lease two centers of diversity in the northern and southern Rocky Mountains and strong population structure. Nested clade analysis suggested that the species experienced repeated cycles of population expansion and fragmentation. The estimated ages of these events, assuming a molecular clock, corresponded with paleoclimatic data on habitat expansion and contraction over the past 400,000 years. We propose that alpine butterflies persisted in an archipelago of isolated sky islands during interglacials and that populations expanded and became more connected during cold glacial periods. An archipelago model implies that the effects of genetic drift and selection varied among populations, depending on their latitude, area, and local environment. Alpine organisms are sensitive indicators of climate change and their history can be used to predict how high-elevation ecosystems might respond to further climate warming.

  18. Historic cycles of fragmentation and expansion in Parnassius smintheus (papilionidae) inferred using mitochondrial DNA.

    PubMed

    DeChaine, Eric G; Martini, Andrew P

    2004-01-01

    Climate oscillations of the Quaternary drove the repeated expansion and contraction of ecosystems. Alpine organisms were probably isolated in sky island refugia during warm interglacials, such as now, and expanded their range by migrating down-slope during glacial periods. We used population genetic and phylogenetic approaches to infer how paleoclimatic events influenced the distribution of genetic variation in the predominantly alpine butterfly Parnassius smintheus. We sequenced a 789 bp region of cytochrome oxidase I for 385 individuals from 20 locations throughout the Rocky Mountains, ranging from southern Colorado to northern Montana. Analyses revealed at lease two centers of diversity in the northern and southern Rocky Mountains and strong population structure. Nested clade analysis suggested that the species experienced repeated cycles of population expansion and fragmentation. The estimated ages of these events, assuming a molecular clock, corresponded with paleoclimatic data on habitat expansion and contraction over the past 400,000 years. We propose that alpine butterflies persisted in an archipelago of isolated sky islands during interglacials and that populations expanded and became more connected during cold glacial periods. An archipelago model implies that the effects of genetic drift and selection varied among populations, depending on their latitude, area, and local environment. Alpine organisms are sensitive indicators of climate change and their history can be used to predict how high-elevation ecosystems might respond to further climate warming. PMID:15058724

  19. Rapid identification and classification of bacteria by 16S rDNA restriction fragment melting curve analyses (RFMCA).

    PubMed

    Rudi, Knut; Kleiberg, Gro H; Heiberg, Ragnhild; Rosnes, Jan T

    2007-08-01

    The aim of this work was to evaluate restriction fragment melting curve analyses (RFMCA) as a novel approach for rapid classification of bacteria during food production. RFMCA was evaluated for bacteria isolated from sous vide food products, and raw materials used for sous vide production. We identified four major bacterial groups in the material analysed (cluster I-Streptococcus, cluster II-Carnobacterium/Bacillus, cluster III-Staphylococcus and cluster IV-Actinomycetales). The accuracy of RFMCA was evaluated by comparison with 16S rDNA sequencing. The strains satisfying the RFMCA quality filtering criteria (73%, n=57), with both 16S rDNA sequence information and RFMCA data (n=45) gave identical group assignments with the two methods. RFMCA enabled rapid and accurate classification of bacteria that is database compatible. Potential application of RFMCA in the food or pharmaceutical industry will include development of classification models for the bacteria expected in a given product, and then to build an RFMCA database as a part of the product quality control. PMID:17367680

  20. Towards the onset of fruit tree growing north of the Alps: ancient DNA from waterlogged apple (Malus sp.) seed fragments.

    PubMed

    Schlumbaum, Angela; van Glabeke, Sabine; Roldan-Ruiz, Isabel

    2012-01-20

    Wild apples (Malus sp.) have been a major food source in the northern Alpine region since prehistory and their use is well understood. The onset of deliberate fruit tree growing in the area is, however, less clear. It is generally assumed that horticulture was practised in Roman times, but it might be even earlier. In the archaeological record seed testa and pericarp remains are particularly frequent at sites with waterlogged preservation such as lakeshore settlements or wells, pits and ditches, but the distinction between wild and domestic plants is not morphologically possible. With waterlogged remains being one main source of information about past fruit cultivation, we have tested the feasibility of analysing ancient DNA from waterlogged preserved bulk samples of testa fragments. We studied apple seeds from three Neolithic and three Roman sites with waterlogged preservation in the Alpine foreland. Chloroplast markers failed in all samples, but nuclear ITS1 (internal transcribed spacer region 1) of the ribosomal DNA was successfully typed in two Roman samples from the site Oedenburg/Biesheim-Kunheim (Haut-Rhin, F). The retrieved ITS1 sequences are identical to each other and are shared with wild Malus sylvestris and Malus sieversii, and with domestic apple cultivars, supporting the potential of using waterlogged remains for identifying the genetic status of apple diachronically.

  1. Rapid radiation events in the family Ursidae indicated by likelihood phylogenetic estimation from multiple fragments of mtDNA.

    PubMed

    Waits, L P; Sullivan, J; O'Brien, S J; Ward, R H

    1999-10-01

    The bear family (Ursidae) presents a number of phylogenetic ambiguities as the evolutionary relationships of the six youngest members (ursine bears) are largely unresolved. Recent mitochondrial DNA analyses have produced conflicting results with respect to the phylogeny of ursine bears. In an attempt to resolve these issues, we obtained 1916 nucleotides of mitochondrial DNA sequence data from six gene segments for all eight bear species and conducted maximum likelihood and maximum parsimony analyses on all fragments separately and combined. All six single-region gene trees gave different phylogenetic estimates; however, only for control region data was this significantly incongruent with the results from the combined data. The optimal phylogeny for the combined data set suggests that the giant panda is most basal followed by the spectacled bear. The sloth bear is the basal ursine bear, and there is weak support for a sister taxon relationship of the American and Asiatic black bears. The sun bear is sister taxon to the youngest clade containing brown bears and polar bears. Statistical analyses of alternate hypotheses revealed a lack of strong support for many of the relationships. We suggest that the difficulties surrounding the resolution of the evolutionary relationships of the Ursidae are linked to the existence of sequential rapid radiation events in bear evolution. Thus, unresolved branching orders during these time periods may represent an accurate representation of the evolutionary history of bear species. PMID:10508542

  2. Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLink CBH351.

    PubMed

    Chowdhury, E H; Mikami, O; Nakajima, Y; Hino, A; Kuribara, H; Suga, K; Hanazumi, M; Yomemochi, C

    2003-03-01

    We tried to detect DNA fragments derived from maize in the intestinal contents of pigs fed genetically modified (GM) StarLink CBH351 maize (SL) or non-GM maize. Intestinal contents of 8 SL and 8 non-GM maize-fed pigs were collected at slaughter, and the genes of the recombinant cry9C and the maize intrinsic zein (Zel) were assayed by polymerase chain reaction (PCR) 3 times with a total of 4 primer pairs of different expected lengths. The cry9C gene (either 103 or 170 bp) was detected in the rectal contents (with a frequency of 25-37.5%) and in the cecal contents (25-50%) of the pigs fed SL. In a similar fashion, the zein (Zel) gene (either 242 or 329 bp) was detected in the rectal contents (with a frequency of 31.3%) and in the cecal contents (25-37.5%) of pigs fed on SL non-GM maize. These results suggested that ingested DNA was not totally degraded, but is present in a form detectable by PCR.

  3. DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction.

    PubMed

    Palittapongarnpim, P; Chomyc, S; Fanning, A; Kunimoto, D

    1993-04-01

    Strain identification of Mycobacterium tuberculosis would prove whether transmission had occurred between individuals. A method to characterize strains of M. tuberculosis has been developed utilizing polymerase chain reaction (PCR). Purified chromosomal DNA of cultured clinical samples of M. tuberculosis were subjected to PCR using short (10-12 nucleotide) oligonucleotide primers. PCR products visualized after agarose gel electrophoresis and ethidium bromide staining demonstrated that different strains of M. tuberculosis give different banding patterns. This technique was used to confirm the relationship between cases of tuberculosis in several clusters, prove the lack of relationship between 2 isolates with the same antibiotic-resistance pattern, confirm a suspected mislabeling event, and suggest the source of infection in a case of tuberculous meningitis. This method is rapid and simple and does not require radioactive probes.

  4. The Protein Kinase Cδ Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint*

    PubMed Central

    LaGory, Edward L.; Sitailo, Leonid A.; Denning, Mitchell F.

    2010-01-01

    Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G2/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G2/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. PMID:19917613

  5. Fragment charge difference method for estimating donor-acceptor electronic coupling: Application to DNA π-stacks

    NASA Astrophysics Data System (ADS)

    Voityuk, Alexander A.; Rösch, Notker

    2002-09-01

    The purpose of this communication is two-fold. We introduce the fragment charge difference (FCD) method to estimate the electron transfer matrix element HDA between a donor D and an acceptor A, and we apply this method to several aspects of hole transfer electronic couplings in π-stacks of DNA, including systems with several donor-acceptor sites. Within the two-state model, our scheme can be simplified to recover a convenient estimate of the electron transfer matrix element HDA=(1-Δq2)1/2(E2-E1)/2 based on the vertical excitation energy E2-E1 and the charge difference Δq between donor and acceptor. For systems with strong charge separation, Δq≳0.95, one should resort to the FCD method. As favorable feature, we demonstrate the stability of the FCD approach for systems which require an approach beyond the two-state model. On the basis of ab initio calculations of various DNA related systems, we compared three approaches for estimating the electronic coupling: the minimum splitting method, the generalized Mulliken-Hush (GMH) scheme, and the FCD approach. We studied the sensitivity of FCD and GMH couplings to the donor-acceptor energy gap and found both schemes to be quite robust; they are applicable also in cases where donor and acceptor states are off resonance. In the application to π-stacks of DNA, we demonstrated for the Watson-Crick pair dimer [(GC),(GC)] how structural changes considerably affect the coupling strength of electron hole transfer. For models of three Watson-Crick pairs, we showed that the two-state model significantly overestimates the hole transfer coupling whereas simultaneous treatment of several states leads to satisfactory results.

  6. Reconstructing the history of a fragmented and heavily exploited red deer population using ancient and contemporary DNA

    PubMed Central

    2012-01-01

    Background Red deer (Cervus elaphus) have been an important human resource for millennia, experiencing intensive human influence through habitat alterations, hunting and translocation of animals. In this study we investigate a time series of ancient and contemporary DNA from Norwegian red deer spanning about 7,000 years. Our main aim was to investigate how increasing agricultural land use, hunting pressure and possibly human mediated translocation of animals have affected the genetic diversity on a long-term scale. Results We obtained mtDNA (D-loop) sequences from 73 ancient specimens. These show higher genetic diversity in ancient compared to extant samples, with the highest diversity preceding the onset of agricultural intensification in the Early Iron Age. Using standard diversity indices, Bayesian skyline plot and approximate Bayesian computation, we detected a population reduction which was more prolonged than, but not as severe as, historic documents indicate. There are signs of substantial changes in haplotype frequencies primarily due to loss of haplotypes through genetic drift. There is no indication of human mediated translocations into the Norwegian population. All the Norwegian sequences show a western European origin, from which the Norwegian lineage diverged approximately 15,000 years ago. Conclusions Our results provide direct insight into the effects of increasing habitat fragmentation and human hunting pressure on genetic diversity and structure of red deer populations. They also shed light on the northward post-glacial colonisation process of red deer in Europe and suggest increased precision in inferring past demographic events when including both ancient and contemporary DNA. PMID:23009643

  7. Size and DNA distributions of electrophoretically separated cultured human kidney cells

    NASA Technical Reports Server (NTRS)

    Kunze, M. E.; Plank, L. D.; Todd, P. W.

    1985-01-01

    Electrophoretic purification of purifying cultured cells according to function presumes that the size of cycle phase of a cell is not an overriding determinant of its electrophoretic velocity in an electrophoretic separator. The size distributions and DNA distributions of fractions of cells purified by density gradient electrophoresis were determined. No systematic dependence of electrophoretic migration upward in a density gradient column upon either size or DNA content were found. It was found that human leukemia cell populations, which are more uniform function and found in all phases of the cell cycle during exponential growth, separated on a vertical sensity gradient electrophoresis column according to their size, which is shown to be strictly cell cycle dependent.

  8. The RSC chromatin remodelling ATPase translocates DNA with high force and small step size.

    PubMed

    Sirinakis, George; Clapier, Cedric R; Gao, Ying; Viswanathan, Ramya; Cairns, Bradley R; Zhang, Yongli

    2011-06-15

    ATP-dependent chromatin remodelling complexes use the energy of ATP hydrolysis to reposition and reconfigure nucleosomes. Despite their diverse functions, all remodellers share highly conserved ATPase domains, many shown to translocate DNA. Understanding remodelling requires biophysical knowledge of the DNA translocation process: how the ATPase moves DNA and generates force, and how translocation and force generation are coupled on nucleosomes. Here, we characterize the real-time activity of a minimal RSC translocase 'motor' on bare DNA, using high-resolution optical tweezers and a 'tethered' translocase system. We observe on dsDNA a processivity of ∼35 bp, a speed of ∼25 bp/s, and a step size of 2.0 (±0.4, s.e.m.) bp. Surprisingly, the motor is capable of moving against high force, up to 30 pN, making it one of the most force-resistant motors known. We also provide evidence for DNA 'buckling' at initiation. These observations reveal the ATPase as a powerful DNA translocating motor capable of disrupting DNA-histone interactions by mechanical force.

  9. Cation-size-dependent DNA adsorption kinetics and packing density on gold nanoparticles: an opposite trend.

    PubMed

    Liu, Biwu; Kelly, Erin Y; Liu, Juewen

    2014-11-11

    The property of DNA is strongly influenced by counterions. Packing a dense layer of DNA onto a gold nanoparticle (AuNP) generates an interesting colloidal system with many novel physical properties such as a sharp melting transition, protection of DNA against nucleases, and enhanced complementary DNA binding affinity. In this work, the effect of monovalent cation size is studied. First, for free AuNPs without DNA, larger group 1A cations are more efficient in inducing their aggregation. The same trend is observed with group 2A metals using AuNPs capped by various self-assembled monolayers. After establishing the salt range to maintain AuNP stability, the DNA adsorption kinetics is also found to be faster with the larger Cs(+) compared to the smaller Li(+). This is attributed to the easier dehydration of Cs(+), and dehydrated Cs(+) might condense on the AuNP surface to reduce the electrostatic repulsion effectively. However, after a long incubation time with a high salt concentration, Li(+) allows ∼30% more DNA packing compared to Cs(+). Therefore, Li(+) is more effective in reducing the charge repulsion among DNA, and Cs(+) is more effective in screening the AuNP surface charge. This work suggests that physicochemical information at the bio/nanointerface can be obtained by using counterions as probes.

  10. DNA variation in the wild plant Arabidopsis thaliana revealed by amplified fragment length polymorphism analysis.

    PubMed Central

    Miyashita, N T; Kawabe, A; Innan, H

    1999-01-01

    To investigate the level and pattern of DNA variation of Arabidopsis thaliana at the entire genome level, AFLP analysis was conducted for 38 ecotypes distributed throughout the world. Ten pairs of selective primers were used to detect a total of 472 bands, of which 374 (79. 2%) were polymorphic. The frequency distribution of polymorphic bands was skewed toward an excess of singleton variation. On the basis of AFLP variation, nucleotide diversity for the entire genome was estimated to be 0.0106, which was within the range reported previously for specific nuclear genes. The frequency distribution of pairwise distance was bimodal because of an ecotype (Fl-3) with a large number of unique bands. Linkage disequilibrium between polymorphic AFLPs was tested. The proportion of significant linkage disequilibria was close to random expectation after neglecting the ecotype Fl-3. This result indicates that the effect of recombination could not be ignored in this selfing species. A neighbor-joining tree was constructed on the basis of the AFLP variation. This tree has a star-like topology and shows no clear association between ecotype and geographic origin, suggesting a recent spread of this plant species and limited migration between its habitats. PMID:10430596

  11. Comparative study and prediction of DNA fragments associated with various elements of the nuclear matrix.

    PubMed

    Glazko, G V; Rogozin, I B; Glazkov, M V

    2001-02-16

    Scaffold/matrix-associated region (S/MAR) sequences are DNA regions that are attached to the nuclear matrix, and participate in many cellular processes. The nuclear matrix is a complex structure consisting of various elements. In this paper we compared frequencies of simple nucleotide motifs in S/MAR sequences and in sequences extracted directly from various nuclear matrix elements, such as nuclear lamina, cores of rosette-like structures, synaptonemal complex. Multivariate linear discriminant analysis revealed significant differences between these sequences. Based on this result we have developed a program, ChrClass (Win/NT version, ftp.bionet.nsc.ru/pub/biology/chrclass/chrclass.zip), for the prediction of the regions associated with various elements of the nuclear matrix in a query sequence. Subsequently, several test samples were analyzed by using two S/MAR prediction programs (a ChrClass and MAR-Finder) and a simple MRS criterion (S/MAR recognition signature) indicating the presence of S/MARs. Some overlap between the predictions of all MAR prediction tools has been found. Simultaneous use of the ChrClass, MRS criterion and MAR-Finder programs may help to obtain a more clearcut picture of S/MAR distribution in a query sequence. In general, our results suggest that the proportion of missed S/MARs is lower for ChrClass, whereas the proportion of wrong S/MARs is lower for MAR-Finder and MRS.

  12. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  13. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

  14. Fractals and fragmentation

    NASA Technical Reports Server (NTRS)

    Turcotte, D. L.

    1986-01-01

    The use of renormalization group techniques on fragmentation problems is examined. The equations which represent fractals and the size-frequency distributions of fragments are presented. Method for calculating the size distributions of asteriods and meteorites are described; the frequency-mass distribution for these interplanetary objects are due to fragmentation. The application of two renormalization group models to fragmentation is analyzed. It is observed that the models yield a fractal behavior for fragmentation; however, different values for the fractal dimension are produced . It is concluded that fragmentation is a scale invariant process and that the fractal dimension is a measure of the fragility of the fragmented material.

  15. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    PubMed

    Suzuki, Masayo; Kino, Katsuhito; Kawada, Taishu; Oyoshi, Takanori; Morikawa, Masayuki; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

  16. [Molecular cloning and primary structure of cDNA fragment for alpha-latrocrustatoxin from black widow spider venom].

    PubMed

    Volynskiĭ, K E; Volkova, T M; Galkina, T G; Krasnoperov, V G; Pluzhnikov, K A; Khvoshchev, M V; Grishin, E V

    1999-01-01

    A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.

  17. Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells

    PubMed Central

    Akita, Hidetaka; Kurihara, Dai; Schmeer, Marco; Schleef, Martin; Harashima, Hideyoshi

    2015-01-01

    The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer. PMID:26066769

  18. Intervention improves assisted conception intracytoplasmic sperm injection outcomes for patients with high levels of sperm DNA fragmentation: a retrospective analysis.

    PubMed

    Bradley, C K; McArthur, S J; Gee, A J; Weiss, K A; Schmidt, U; Toogood, L

    2016-09-01

    Sperm DNA fragmentation (SDF) is used in assisted reproductive technology (ART) programs as an indicator for sperm quality, although there is still a lack of consensus as to its clinical utility. In this retrospective study, we examined intracytoplasmic sperm injection (ICSI) outcomes of 1924 infertile patients who underwent SDF analysis using the sperm chromatin integrity test. ART patients were classified as having low [DNA fragmentation index (DFI) <29%] or high SDF (DFI ≥29%) and by whether or not an intervention [physiological intracytoplasmic sperm injection (PICSI), intracytoplasmic morphologically selected sperm injection (IMSI), testicular sperm extraction (TESE)/testicular sperm aspiration (TESA), frequent ejaculation] was performed. High SDF patients who did not have an intervention had a lower fertilization rate and poorer clinical outcomes from blastocyst transfers as compared with low SDF patients; the fertilization rate was 66.0% vs. 70.2% (p = 0.042), single embryo transfer (SET) fetal heart pregnancy rate was 28.5% vs. 45.2% (p = 0.042), and SET live birth rate was 24.9% vs. 40.6% (p = 0.060), respectively. Furthermore, high SDF patients who had an intervention had significantly improved blastocyst transfer outcomes, similar to those of low SDF patients; the SET live birth rate for high SDF intervention patients was 43.8% as compared with 24.9% for high SDF no intervention patients (p = 0.037) and 40.6% for low SDF patients (p = 0.446). Analysis of the three main intervention subgroups for high SDF patients revealed that TESE/TESA patients had the highest SET live birth rate; in comparison with 24.2% for high SDF patients who did not have an intervention, PICSI patients had 38.3% (p = 0.151), IMSI patients had 28.7% (p = 0.680), and TESE/TESA patients had 49.8% (p = 0.020). Our data suggest that SDF results indicate ICSI outcomes and that patients who have high SDF benefit from an intervention.

  19. Analysis of conflicting experimental studies of DNA size in nanofluidic slits

    NASA Astrophysics Data System (ADS)

    Stavis, Samuel M.; Strychalski, Elizabeth A.; Nablo, Brian J.; Geist, Jon

    2013-03-01

    Recent experimental studies have reported conflicting accounts of the size variation of DNA in nanofluidic slitlike confinement; [Bonthuis et al., Physical Review Letters 101, 10, 108303 (2008)], [Tang et al., Macromolecules 43, 17, 7368 (2010)], [Strychalski et al., Macromolecules 45, 3, 1602 (2012)], [Lin et al., Macromolecules 45, 6, 2920 (2012)], [Dai et al., Soft Matter 8, 10, 2972 (2012)]. In an effort to resolve this controversy, these studies are analyzed by a reductive as opposed to predictive approach. Minimum references for DNA size (baselines) are simulated by a Monte Carlo methodology and quantitatively compared to measured and inferred DNA sizes. The measurements of Tang et al., Strychalski et al., and Lin et al. are consistent with the related baselines and in semi-quantitative agreement with each other. The inferences of Tang et al. and Dai et al. are consistent with the related baseline and in qualitative agreement with the measurements of Tang et al., Strychalski et al., and Lin et al. The measurements of Bonthuis et al. are inconsistently larger than the related baseline and the other experimental measurements and inferences of DNA size around the transition from moderate to weak slitlike confinement. A variety of physical and chemical differences between the experimental systems are examined in detail to elucidate this inconsistency. Detailed analyses of the baseline distribution and variation clarify several core physical attributes of the system related to excluded volume effects and chain dimensionality.

  20. Power and sample size estimation for epigenome-wide association scans to detect differential DNA methylation

    PubMed Central

    Tsai, Pei-Chien; Bell, Jordana T

    2015-01-01

    Background: Epigenome-wide association scans (EWAS) are under way for many complex human traits, but EWAS power has not been fully assessed. We investigate power of EWAS to detect differential methylation using case-control and disease-discordant monozygotic (MZ) twin designs with genome-wide DNA methylation arrays. Methods and Results: We performed simulations to estimate power under the case-control and discordant MZ twin EWAS study designs, under a range of epigenetic risk effect sizes and conditions. For example, to detect a 10% mean methylation difference between affected and unaffected subjects at a genome-wide significance threshold of P = 1 × 10−6, 98 MZ twin pairs were required to reach 80% EWAS power, and 112 cases and 112 controls pairs were needed in the case-control design. We also estimated the minimum sample size required to reach 80% EWAS power under both study designs. Our analyses highlighted several factors that significantly influenced EWAS power, including sample size, epigenetic risk effect size, the variance of DNA methylation at the locus of interest and the correlation in DNA methylation patterns within the twin sample. Conclusions: We provide power estimates for array-based DNA methylation EWAS under case-control and disease-discordant MZ twin designs, and explore multiple factors that impact on EWAS power. Our results can help guide EWAS experimental design and interpretation for future epigenetic studies. PMID:25972603

  1. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. PMID:26733203

  2. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.

  3. Structural, Dynamical and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Sumpter, Bobby G; Fuentes-Cabrera, Miguel A

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, size-expanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. The most relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMO-LUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  4. “Cre/loxP plus BAC”: a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens

    PubMed Central

    Hu, Shengbiao; Liu, Zhengqiang; Zhang, Xu; Zhang, Guoyong; Xie, Yali; Ding, Xuezhi; Mo, Xiangtao; Stewart, A. Francis; Fu, Jun; Zhang, Youming; Xia, Liqiu

    2016-01-01

    Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for direct cloning gene clusters. A type three secretion system (T3SS) gene cluster (~32 kb) from Photorhabdus luminescens TT01 and DNA fragment (~78 kb) containing the siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58 have been successfully cloned into pBeloBAC11 with “Cre/loxP plus BAC” strategy. Based on the fact that Cre/loxP system has successfully used for genomic engineering in a wide range of organisms, we believe that this strategy could be widely used for direct cloning of large DNA fragment. PMID:27364376

  5. "Cre/loxP plus BAC": a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens.

    PubMed

    Hu, Shengbiao; Liu, Zhengqiang; Zhang, Xu; Zhang, Guoyong; Xie, Yali; Ding, Xuezhi; Mo, Xiangtao; Stewart, A Francis; Fu, Jun; Zhang, Youming; Xia, Liqiu

    2016-01-01

    Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for direct cloning gene clusters. A type three secretion system (T3SS) gene cluster (~32 kb) from Photorhabdus luminescens TT01 and DNA fragment (~78 kb) containing the siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58 have been successfully cloned into pBeloBAC11 with "Cre/loxP plus BAC" strategy. Based on the fact that Cre/loxP system has successfully used for genomic engineering in a wide range of organisms, we believe that this strategy could be widely used for direct cloning of large DNA fragment. PMID:27364376

  6. Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat.

    PubMed

    Lan, J; Henshall, D C; Simon, R P; Chen, J

    2000-01-01

    The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively. Systemic KA (11 mg/kg) significantly increased levels of 8-OHdG within the thalamus after 2 h, within the amygdala/piriform cortex after 4 h, and within the hippocampus after 8 h. Levels remained elevated up to sevenfold within these areas for 72 h. Smaller increases in 8-OHdG levels were also detected within the parietal cortex and striatum. PANT-positive cells were detected within the thalamus, amygdala/piriform cortex, and hippocampus 24-72 h following KA injection. TUNEL-positive cells appeared within the same brain regions and over a similar time course (24-72 h) but were generally lower in number. The present data suggest oxidative damage to DNA may be an early consequence of epileptic seizures and a possible initiation event in the progression of seizure-induced injury to DNA fragmentation and cell death.

  7. The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

    PubMed Central

    Pierandrei, Silvia; Luchetti, Andrea; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica; Lucarelli, Marco

    2016-01-01

    Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR. PMID:27045208

  8. An amphioxus RAG1-like DNA fragment encodes a functional central domain of vertebrate core RAG1.

    PubMed

    Zhang, Yanni; Xu, Ke; Deng, Anqi; Fu, Xing; Xu, Anlong; Liu, Xiaolong

    2014-01-01

    The highly diversified repertoire of antigen receptors in the vertebrate immune system is generated via proteins encoded by the recombination activating genes (RAGs) RAG1 and RAG2 by a process known as variable, diversity, and joining [V(D)J] gene recombination. Based on the study of vertebrate RAG proteins, many hypotheses have been proposed regarding the origin and evolution of RAG. This issue remains unresolved, leaving a significant gap in our understanding of the evolution of adaptive immunity. Here, we show that the amphioxus genome contains an ancient RAG1-like DNA fragment (bfRAG1L) that encodes a virus-related protein that is much shorter than vertebrate RAG1 and harbors a region homologous to the central domain of core RAG1 (cRAG1). bfRAG1L also contains an unexpected retroviral type II nuclease active site motif, DXN(D/E)XK, and is capable of degrading both DNA and RNA. Moreover, bfRAG1L shares important functional properties with the central domain of cRAG1, including interaction with RAG2 and localization to the nucleus. Remarkably, the reconstitution of bfRAG1L into a cRAG1-like protein yielded an enzyme capable of recognizing recombination signal sequences and performing V(D)J recombination in the presence of mouse RAG2. Moreover, this reconstituted cRAG1-like protein could mediate the assembly of antigen receptor genes in RAG1-deficient mice. Together, our results demonstrate that amphioxus bfRAG1L encodes a protein that is functionally equivalent to the central domain of cRAG1 and is well prepared for further evolution to mediate V(D)J recombination. Thus, our findings provide unique insights into the evolutionary origin of RAG1.

  9. An amphioxus RAG1-like DNA fragment encodes a functional central domain of vertebrate core RAG1.

    PubMed

    Zhang, Yanni; Xu, Ke; Deng, Anqi; Fu, Xing; Xu, Anlong; Liu, Xiaolong

    2014-01-01

    The highly diversified repertoire of antigen receptors in the vertebrate immune system is generated via proteins encoded by the recombination activating genes (RAGs) RAG1 and RAG2 by a process known as variable, diversity, and joining [V(D)J] gene recombination. Based on the study of vertebrate RAG proteins, many hypotheses have been proposed regarding the origin and evolution of RAG. This issue remains unresolved, leaving a significant gap in our understanding of the evolution of adaptive immunity. Here, we show that the amphioxus genome contains an ancient RAG1-like DNA fragment (bfRAG1L) that encodes a virus-related protein that is much shorter than vertebrate RAG1 and harbors a region homologous to the central domain of core RAG1 (cRAG1). bfRAG1L also contains an unexpected retroviral type II nuclease active site motif, DXN(D/E)XK, and is capable of degrading both DNA and RNA. Moreover, bfRAG1L shares important functional properties with the central domain of cRAG1, including interaction with RAG2 and localization to the nucleus. Remarkably, the reconstitution of bfRAG1L into a cRAG1-like protein yielded an enzyme capable of recognizing recombination signal sequences and performing V(D)J recombination in the presence of mouse RAG2. Moreover, this reconstituted cRAG1-like protein could mediate the assembly of antigen receptor genes in RAG1-deficient mice. Together, our results demonstrate that amphioxus bfRAG1L encodes a protein that is functionally equivalent to the central domain of cRAG1 and is well prepared for further evolution to mediate V(D)J recombination. Thus, our findings provide unique insights into the evolutionary origin of RAG1. PMID:24368847

  10. Power law and exponential ejecta size distributions from the dynamic fragmentation of shock-loaded Cu and Sn metals under melt conditions

    SciTech Connect

    Durand, O.; Soulard, L.

    2013-11-21

    Large scale molecular dynamics (MD) simulations are performed to study and to model the ejecta production from the dynamic fragmentation of shock-loaded metals under melt conditions. A generic 3D crystal in contact with vacuum containing about 10{sup 8} atoms and with a sinusoidal free surface roughness is shock loaded so as to undergo a solid-liquid phase change on shock. The reflection of the shock wave at the interface metal/vacuum gives rise to the ejection of 2D jets/sheets of atoms (Richtmyer-Meshkov instabilities in the continuum limit), which develop and break up, forming ejecta (fragments) of different volumes (or mass). The fragmentation process is investigated by analyzing the evolution of the resulting volume distribution of the ejecta as a function of time. Two metals are studied (Cu and Sn) and the amplitude of the roughness is varied. The simulations show that the associated distributions exhibit a generic behavior with the sum of two distinct terms of varying weight, following the expansion rate of the jets: in the small size limit, the distribution obeys a power law dependence with an exponent equal to 1.15 ± 0.08; and in the large size limit, it obeys an exponential form. These two components are interpreted, with the help of additional simple simulations, as the signature of two different basic mechanisms of fragmentation. The power law dependence results from the fragmentation of a 2D network of ligaments arranged following a fractal (scale free) geometry and generated when the sheets of liquid metal expand and tear. The exponential distribution results from a 1D Poisson fragmentation process of the largest ligaments previously generated. Unlike the power law distribution, it is governed by a characteristic length scale, which may be provided by energy balance principle.

  11. Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

    PubMed

    Loaces, Inés; Amarelle, Vanesa; Muñoz-Gutierrez, Iván; Fabiano, Elena; Martinez, Alfredo; Noya, Francisco

    2015-11-01

    With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed β-glucosidase had a positie effect on cell growth in agreement with the fact that no putative β-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

  12. Assessment of sperm function parameters and DNA fragmentation in ejaculated alpaca sperm (Lama pacos) by flow cytometry.

    PubMed

    Cheuquemán, C; Merino, O; Giojalas, L; Von Baer, A; Sánchez, R; Risopatrón, J

    2013-06-01

    Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.

  13. Interleukin-15 is able to suppress the increased DNA fragmentation associated with muscle wasting in tumour-bearing rats.

    PubMed

    Figueras, Maite; Busquets, Sílvia; Carbó, Neus; Barreiro, Esther; Almendro, Vanessa; Argilés, Josep M; López-Soriano, Francisco J

    2004-07-01

    Administration of interleukin-15 (IL-15) to rats bearing the Yoshida AH-130 ascites hepatoma (a tumour that induces an important cachectic response) resulted in a significant reduction of muscle wasting, both measured as muscle weight and as protein content of different types of skeletal muscle. In addition, the administration of the cytokine completely reversed the increased DNA fragmentation observed in skeletal muscle of tumour-bearing animals. Concerning the mechanism(s) involved in the anti-apoptotic effects of IL-15 on skeletal muscle, the administration of the cytokine resulted in a considerable decrease in both R1 (43%) and R2 (64%) TNF-alpha receptors (TNFRs), and therefore it may be suggested that IL-15 decreases apoptosis by affecting TNF-alpha signalling. Formation of NO could be the signalling event associated with the activation of apoptosis in muscle of tumour-bearing rats; indeed, administration of IL-15 decreased the inducible nitric oxide synthase protein levels by 73%, suggesting that NO formation and muscle apoptosis during tumour growth are related. In conclusion, IL-15 seems to be able to reduce/suppress protein loss and apoptosis related to muscle wasting during cancer cachexia in experimental animals.

  14. Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Orozco, Modesto; Luque, Javier; Sumpter, Bobby G; Blas, Jose; Ordejon, Pablo J; Huertas, Oscar; Tabares, Carolina

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, sizeexpanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. Themost relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMOLUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  15. Analysis of Nuclear Mitochondrial DNA Segments of Nine Plant Species: Size, Distribution, and Insertion Loci

    PubMed Central

    Ko, Young-Joon

    2016-01-01

    Nuclear mitochondrial DNA segment (Numt) insertion describes a well-known phenomenon of mitochondrial DNA transfer into a eukaryotic nuclear genome. However, it has not been well understood, especially in plants. Numt insertion patterns vary from species to species in different kingdoms. In this study, the patterns were surveyed in nine plant species, and we found some tip-offs. First, when the mitochondrial genome size is relatively large, the portion of the longer Numt is also larger than the short one. Second, the whole genome duplication event increases the ratio of the shorter Numt portion in the size distribution. Third, Numt insertions are enriched in exon regions. This analysis may be helpful for understanding plant evolution. PMID:27729838

  16. The development of reduced size STR amplicons as tools for analysis of degraded DNA.

    PubMed

    Butler, John M; Shen, Yin; McCord, Bruce R

    2003-09-01

    New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.

  17. The influence of ginger (Zingiber officinale) on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial

    PubMed Central

    Hosseini, Jalil; Mardi Mamaghani, Azar; Hosseinifar, Hani; Sadighi Gilani, Mohammad Ali; Dadkhah, Farid; Sepidarkish, Mahdi

    2016-01-01

    Background: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. Objective: This study was designed to investigate the effects of ginger (Zingiber officinale) on sperm DNA fragmentation (SDF) in infertile men. Materials and Methods: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group). Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. Results: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02) in cases and (56.75, 95%CI: 40.01-73.5) in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39) was lower compared with placebo (40.54, 95%CI: 23.94-57.13) after three month of treatment (p=0.02). In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009). There were no significant differences between two groups regarding to semen parameters. Conclusion: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men. PMID:27679829

  18. The influence of ginger (Zingiber officinale) on human sperm quality and DNA fragmentation: A double-blind randomized clinical trial

    PubMed Central

    Hosseini, Jalil; Mardi Mamaghani, Azar; Hosseinifar, Hani; Sadighi Gilani, Mohammad Ali; Dadkhah, Farid; Sepidarkish, Mahdi

    2016-01-01

    Background: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. Objective: This study was designed to investigate the effects of ginger (Zingiber officinale) on sperm DNA fragmentation (SDF) in infertile men. Materials and Methods: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group). Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. Results: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02) in cases and (56.75, 95%CI: 40.01-73.5) in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39) was lower compared with placebo (40.54, 95%CI: 23.94-57.13) after three month of treatment (p=0.02). In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009). There were no significant differences between two groups regarding to semen parameters. Conclusion: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

  19. Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

    PubMed Central

    Hajian, Mahdi; Hosseini, Seyed Morteza; Asgari, Vajiheh; Ostadhoosseini, Somayyeh; Forouzanfar, Mohsen; Nasr Esfahani, Mohammad Hossein

    2011-01-01

    Background This study investigated the effect of two in vitro embryo culture systems (co-culture system versus cell-free sequential-media) on developmental competence, cryosurvival and DNA- fragmentation of in vitro developed bovine blastocysts. Materials and Methods Bovine presumptive zygotes were cultured in Ménézo's B2 (B2) plus vero-cells or sequential synthetic oviductal fluid (SOF) for eight days. Subsequently, half of the expanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and post- warming embryos along with their corresponding non-vitrified embryos were cultured for two additional days in the same medium used before vitrification. Embryo development, cryosurvival and apoptosis were compared between the groups. Results For non-vitrified embryos, culture in SOF significantly promoted the potency of embryos to develop into blastocysts compared with the co-culture system. The difference in post vitrification survival rate of SOF blastocysts (83.3%) was insignificant compared with co-culture (84.3%). However, while total cell number of warmed blastocysts in the co-culture system was significantly higher in the co-culture versus the sequential system (215.4 vs. 170.4), the quality of survived embryos in terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF (65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05). Conclusion Although co-culture system may increase the viability of embryos following cryopreservation, the potency and dynamics of blastocyst formation significantly increased with sequential media compared to the co-culture system which can compensate for the lower efficiency of sequential media for vitrification/warming purposes. PMID:24917920

  20. Assessment of sperm function parameters and DNA fragmentation in ejaculated alpaca sperm (Lama pacos) by flow cytometry.

    PubMed

    Cheuquemán, C; Merino, O; Giojalas, L; Von Baer, A; Sánchez, R; Risopatrón, J

    2013-06-01

    Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry. PMID:23082871

  1. Insulin-sensitive glucose transporter transcript levels in calf muscles assessed with a bovine GLUT4 cDNA fragment.

    PubMed

    Hocquette, J F; Graulet, B; Castiglia-Delavaud, C; Bornes, F; Lepetit, N; Ferre, P

    1996-07-01

    Previous studies have shown that the expression of the insulin-sensitive glucose transporter (GLUT4) is lower in oxidative muscles than in glycolytic muscles in bovines and goats in contrast to observations in rats. Additional experiments in this work provide very strong arguments that the immunoreactive band detected does represent GLUT4 protein, which further validates our previous results. Therefore, to determine the level of regulation, the main objective of the present study was to measure GLUT4 mRNA amounts in various bovine muscles. A 241-bp fragment of the bovine GLUT4 cDNA was cloned by polymerase chain reaction (PCR). It shares 80-90% sequence identity with related sequences in other species. This PCR-amplified bovine GLUT4 probe was used to determine the distribution of GLUT4 mRNA in bovine tissues in comparison with that of GLUT1 mRNA. Moreover, GLUT4 mRNA amounts were quantified by Northern-blot analysis in heart and seven skeletal muscles with various oxidative and glycolytic activities from seven ruminant calves. GLUT4 mRNA was detected by Northern-blot analysis only in calf insulin-sensitive tissues. In contrast, GLUT1 mRNA was detected in all tissues studied except liver. GLUT4 mRNA amount was the highest in masseter and heart, which are oxidative muscles (1.67 +/- 0.16 and 1.53 +/- 0.19 units/g wet tissue weight, respectively) and the lowest in glycolytic or oxido-glycolytic muscles (0.31 +/- 0.04 to 1.00 +/- 0.09 units/g wet tissue weight; SEM, n = 7). These data and our previous results provide evidence for translational and/or post-translational control mechanisms of bovine GLUT4 protein expression in a muscle type-specific manner.

  2. DNA restriction fragment length polymorphism of HLA-DR2 haplotypes in normal individuals and in patients with rheumatoid arthritis.

    PubMed Central

    Singal, D P; Reid, B; Green, D; Bensen, W G; D'Souza, M

    1990-01-01

    A strong association between HLA-DR4 and rheumatoid arthritis (RA) has been found in a number of populations. In contrast, the incidence of DR2 is decreased in patients with RA, suggesting that this specificity may confer some protection against the disease. A number of subtypes of DR2 have been defined by serology, by responses in mixed lymphocyte culture reaction, and, more recently, by restriction fragment length polymorphism. These subtypes of DR2 are in linkage disequilibrium with different subspecificities of DQw1. It is thus likely that the distribution of these subtypic DR,DQ haplotypes in DR2 positive patients with RA may be important in understanding the genetic basis of susceptibility/resistance to RA. In this paper a study of the subtypes of DR2,DQw1 haplotypes in 18 patients with RA, who required sodium aurothiomalate as a disease remitting drug, and unrelated healthy individuals is reported. Three subtypes of DR2 haplotypes, DRw15 (Dw2),DQw1.2(DQw6), DRw15(Dw12),DQw1.12(DQw6), and DRw16(Dw21),DQw1, AZH (DQw5), were analysed with a cDNA probe for the DQ beta gene. The data show that DR2 positive patients with RA carried either the DRw15(Dw2),DQw6 or DRw15(Dw12),DQw6 haplotype. No patient with RA was positive for the DRw16(Dw21),DQw5 subspecificity. In contrast, six of 29 (21%) normal healthy DR2,DQw1 positive individuals carried the DRw16(Dw21),DQw5 haplotype. These data together with earlier results on the distribution of the DR4,DQw7 haplotype in patients with RA support the hypothesis that DQB1 chain polymorphism may be important in determining susceptibility to severe RA. Images PMID:1969727

  3. Size-Expanded yDNA bases: An Ab Initio Study

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Sumpter, Bobby G; Lipkowski, Pawel; Wells, Jack C

    2006-01-01

    xDNA and yDNA are new classes of synthetic nucleic acids characterized by having base-pairs with one of the bases larger than the natural congeners. Here these larger bases are called x- and y-bases. We recently investigated and reported the structural and electronic properties of the x-bases (Fuentes-Cabrera et al. J. Phys. Chem. B 2005, 109, 21135-21139). Here we extend this study by investigating the structure and electronic properties of the y-bases. These studies are framed within our interest that xDNA and yDNA could function as nanowires, for they could have smaller HOMO-LUMO gaps than natural DNA. The limited amount of experimental structural data in these synthetic duplexes makes it necessary to first understand smaller models and, subsequently, to use that information to build larger models. In this paper, we report the results on the chemical and electronic structure of the y-bases. In particular, we predict that the y-bases have smaller HOMO-LUMO gaps than their natural congeners, which is an encouraging result for it indicates that yDNA could have a smaller HOMO-LUMO gap than natural DNA. Also, we predict that the y-bases are less planar than the natural ones. Particularly interesting are our results corresponding to yG. Our studies show that yG is unstable because it is less aromatic and has a Coulombic repulsion that involves the amino group, as compared with a more stable tautomer. However, yG has a very small HOMO-LUMO gap, the smallest of all the size-expanded bases we have considered. The results of this study provide useful information that may allow the synthesis of an yG-mimic that is stable and has a small HOMO-LUMO gap.

  4. Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

    PubMed

    Hummer, G; García, A E; Soumpasis, D M

    1995-05-01

    A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution.

  5. A study of aneuploidy and DNA fragmentation in spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line.

    PubMed

    Nguyen, Minh Huong; Morel, Frederic; Bujan, Louis; May-Panloup, Pascale; De Braekeleer, Marc; Perrin, Aurore

    2015-06-01

    Meiotic segregation of mosaic males with a 45,X cell line has been little examined. In this study, we evaluated the risk of aneuploid gametes using fluorescence in situ hybridization (FISH) and DNA fragmentation in ejaculated spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line. Triple- and dual-color FISH were performed. Sperm DNA fragmentation was detected using the TUNEL assay. A significantly increased frequency of XY disomic spermatozoa was observed for patients (P)1 and P2. A significant increase in diploidy and autosomal aneuploidy was found in P2 and P3, respectively. The rate of DNA fragmentation was not different from that observed in a control group. Data from the literature are scarce (only 3 cases reported), making comparison of the present data difficult, especially as the frequencies of the cell lines comprising the mosaicism differed between patients. Furthermore, the proportion of the different cell lines can differ from one tissue to another in the same patient. Whether the relative levels of the several cell lines present in the mosaicism can influence the rate of aneuploid spermatozoa remains unknown.

  6. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A. ); Haces, A.; Shih, P.J.; Harding, J.D. )

    1993-01-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  7. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A.; Haces, A.; Shih, P.J.; Harding, J.D.

    1993-02-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  8. [Line class retroposon is the component of the DNA polymorphic fragments pattern of trematode Himasthla elongata parthenitae].

    PubMed

    Solov'eva, A I; Galaktionov, N K; Podgornaia, O I

    2013-01-01

    We have determined that S-SAP method (Sequence specific amplification polymorphism) reveals clonal variability in the genomes of larvae of flatworm Himasthla elongata (Trematoda, Echinostomatidae). Being parthenogenetic the larvae were previously considered to be genetically homogeneous. Cloning and sequencing of a -500 bp conservative fragment (B1) from the fragments' pattern has been performed. Sequence analysis of B1 has shown that this fragment has maximum homology with LINE elements from CR1 family of Hydra and sparrow. In situ hybridization (FISH) has detected dispersed distribution of B1. Several other fragments cloned from the same lane of agarose electrophoresis correspond to conservative domain of reverse transcriptase (RT) from CR1 family. Thus, we have shown that 1) cercariae of trematode H. elongata have clonal variability; 2) the S-SAP method allows to obtaining patterns of fragment distribution characteristic of individual cercariae; 3) conservative domain of RT of CR1 family participates in the pattern of polymorphic fragments generation. Identification of the CR1 transcripts in cercariae of H. elongata transcriptome is the aim of the future work. Cloning of the variable fragments from the fragments' pattern is in progress. PMID:25509118

  9. Probability of double-strand breaks in genome-sized DNA by {gamma}-ray decreases markedly as the DNA concentration increases

    SciTech Connect

    Shimobayashi, Shunsuke F.; Iwaki, Takafumi; Mori, Toshiaki; Yoshikawa, Kenichi

    2013-05-07

    By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by {gamma}-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P{sub 1}, the number of double-strand breaks (DSBs) per base pair per unit Gy, is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. The inverse relationship implies that the total number of DSBs remains essentially constant. We give a theoretical interpretation of our experimental results in terms of attack of reactive species upon DNA molecules, indicating the significance of the characteristics of genome-sized giant DNA as semiflexible polymers for the efficiency of DSBs.

  10. Protective role of vanadium on the early process of rat mammary carcinogenesis by influencing expression of metallothionein, GGT-positive foci and DNA fragmentation.

    PubMed

    Sankar Ray, Rajarshi; Roy, Souvik; Samanta, Shaonly; Maitra, Dilip; Chatterjee, Malay

    2005-01-01

    Vanadium, a dietary micronutrient, is now proving to be a promising anti-tumour agent. The present study was conducted to ascertain its anti-neoplastic potential against an experimental mammary carcinogenesis. Female Sprague-Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(alpha)anthracene (DMBA; 0.5 mg per 100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 p.p.m. was supplemented in the drinking water and given ad libitum to the experimental group immediately after the carcinogen treatment and it continued until the termination of the study (24 weeks for histological, immunological and biochemical observations and 35 weeks for morphological findings). It was found that vanadium treatment brought about substantial protection against DMBA-induced mammary carcinogenesis. This was evident from histological findings that showed substantial repair of hyperplastic lesions following supplementation of vanadium alone. There was a significant reduction in incidence (P<0.05), total number, multiplicity (P<0.01), size of palpable mammary tumours and delay in mean latency period of tumour appearance (P<0.001) following vanadium supplementation compared to the DMBA control. The immunohistochemical localization of metallothionein (a prognostic marker for breast cancer) showed reduced expression with vanadium treatment. Further, DNA fragmentation in the mammary tissue of the vanadium-treated group indicated apoptosis. In this group, vanadium also caused a significant decrease in the number (P<0.002) and focal area (P<0.05) of gamma-glutaminetranspeptidase-positive hepatic foci. The results clearly show the anti-neoplastic potential of vanadium.

  11. Separation of large DNA molecules by size exclusion chromatography-based microchip with on-chip concentration structure

    NASA Astrophysics Data System (ADS)

    Azuma, Naoki; Itoh, Shintaro; Fukuzawa, Kenji; Zhang, Hedong

    2016-06-01

    The separation of DNA molecules according to their size represents a fundamental bioanalytical procedure. Here, we report the development of a chip-sized device, consisting of micrometer-sized fence structures fabricated in a microchannel, for the separation of large DNA molecules (over 10 kbp) based on the principle of size exclusion chromatography (SEC). In order to achieve separation, two approaches were utilized: first, the DNA samples were concentrated immediately prior to separation using nanoslit structures, with the aim of improving the resolution. Second, a theoretical model of SEC-based separation was established and applied in order to predict the optimal voltage range for separation. In this study, we achieved separation of λ DNA (48.5 kbp) and T4 DNA (166 kbp) using the present SEC-based microchip.

  12. Multiplex analysis of DNA

    DOEpatents

    Church, George M.; Kieffer-Higgins, Stephen

    1992-01-01

    This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

  13. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

    PubMed

    Zhou, Jianting; Wu, Ronghai; Xue, Xiaoli; Qin, Zhongjun

    2016-08-19

    Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions. PMID:27220470

  14. DNA fragmentation and oxidative stress compromise sperm motility and survival in late pregnancy exposure to omega-9 fatty acid in rats

    PubMed Central

    Oluwakemi, Oyelowo; Olufeyisipe, Adegoke

    2016-01-01

    Objective(s): The aim of this study was to evaluate the oxidative status and DNA integrity in testes of wistar rat offspring exposed to omega-9 monounsaturated (MUFA) at different times of late organogenesis. Materials and Methods: Sixty female rats were divided into six groups of 10 animals. The first group served as control and received the drug vehicle, olive oil (1 ml/kg/day). The second, third, fourth, fifth and sixth group received 1000 mg/kg of oleic acid on gestation day 15 (D15), 16 (D16), 17 (D17), 18 (D18) and 19 (D19), respectively. Male pups were allowed to attain puberty and thereafter, blood was taken for hormonal analyses. Sperm count and motility were assessed. Testes homogenate was used for the determination of biochemical variables. Testes DNA was also determined. Results: The results showed that sperm count and motility were significantly decreased in the treated groups as compared to the control. There was a marked increase in the malondialdehyde level in rat testes from all of the treated groups as compared to the control (P<0.05). DNA from the testes of rats of D19 had the highest level of fragmentation as compared to the control. Conclusion: Omega-9 MUFA exposure in utero imposes negative effects on sperm variables and increases the level of sperm DNA fragmentation and oxidative stress. PMID:27403258

  15. Paramecium putrinum (Ciliophora, Protozoa): the first insight into the variation of two DNA fragments - molecular support for the existence of cryptic species.

    PubMed

    Tarcz, Sebastian; Rautian, Maria; Potekhin, Alexey; Sawka, Natalia; Beliavskaya, Alexandra; Kiselev, Andrey; Nekrasova, Irina; Przyboś, Ewa

    2014-04-01

    Paramecium putrinum (Claparede & Lachmann 1858) is one of the smallest (80-140 μm long) species of the genus Paramecium. Although it commonly occurs in freshwater reservoirs, no molecular studies of P. putrinum have been conducted to date. Herein we present an assessment of molecular variation in 27 strains collected from widely separated populations by using two selected DNA fragments (ITS1-5.8S-ITS2-5'LSU rDNA and COI mtDNA). Both the trees and haplotype networks reconstructed for both genome fragments show that the studied strains of P. putrinum form five main haplogroups. The mean distance between the studied strains is p-distance=0.007/0.068 (rDNA/COI) and exhibits similar variability as that between P. bursaria syngens. Based on these data, one could hypothesize that the clusters revealed in the present study may correspond to previously reported syngens and that there are at least five cryptic species within P. putrinum.

  16. Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

    PubMed

    Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

    2015-06-01

    Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis

  17. Self-assembly of size-controlled liposomes on DNA nanotemplates

    PubMed Central

    Yang, Yang; Wang, Jing; Shigematsu, Hideki; Xu, Weiming; Shih, William M; Rothman, James E; Lin, Chenxiang

    2016-01-01

    Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as studying vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometer precision is challenging and approaches to achieve this can be heavily affected by lipid composition. Here we present a bio-inspired templating method to generate highly monodispersed sub-100nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method we produced homogenous liposomes with four distinct pre-defined sizes. We also show that the method can be used with a variety of lipid compositions and probed the mechanism of the templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide the lipid bilayer formation, and could be generalized to engineer complex membrane/protein structures with nanoscale precision. PMID:27102682

  18. Self-assembly of size-controlled liposomes on DNA nanotemplates

    NASA Astrophysics Data System (ADS)

    Yang, Yang; Wang, Jing; Shigematsu, Hideki; Xu, Weiming; Shih, William M.; Rothman, James E.; Lin, Chenxiang

    2016-05-01

    Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision.

  19. The antigenotoxic activities of cactus (Opuntia ficus-indica) cladodes against the mycotoxin zearalenone in Balb/c mice: prevention of micronuclei, chromosome aberrations and DNA fragmentation.

    PubMed

    Zorgui, Lazhar; Ayed-Boussema, Imen; Ayed, Yosra; Bacha, Hassen; Hassen, Wafa

    2009-03-01

    Zearalenone (ZEN) is a potent estrogenic metabolite. Evidence of its cytotoxicity and genotoxicity has recently emerged from several reports. This study was conducted to evaluate the ability of cactus (Opuntia ficus-indica) cladodes to protect Balb/c mice against ZEN induced genotoxicity. To this end, the effect of a single dose of ZEN (40 mg/kg b.w.) alone and with extract of cactus cladodes (25, 50 and 100 mg/kg b.w.) was monitored by measuring: (i) micronuclei induction in bone marrow cells, (ii) chromosome aberrations mainly breaks and gaps in bone marrow cells also and finally and (iii) DNA fragmentation in liver and kidney. Our results clearly show that ZEN is genotoxic to Balb/c mice. It induces DNA damage as indicated by DNA fragmentation, micronuclei and chromosomal aberrations in bone marrow cells. It is of note that cactus cladodes extract assayed alone at high dose (100 mg/kg b.w.) was found completely safe and did not induce any genotoxic effects. The simultaneous administration of cactus cladodes extract with ZEN resulted in an efficient prevention of micronuclei (the number of PCE MN decreased from 71.3+/-6.1 for animals treated with Zen to 32.6+/-15.5 for animals treated with cactus cladodes), chromosomal aberrations frequency (the % of chromosomal aberrations decreased from 38.3+/-3.0 to 18.6+/-1.1) in bone marrow cells and of DNA fragmentation compared to the group treated with ZEN alone. It could be concluded that cactus cladodes extract was effective in the protection against ZEN genotoxicity. This could be relevant, particularly with the emergent demand for natural products which may neutralize the genotoxic effects of the multiple food contaminants.

  20. The antigenotoxic activities of cactus (Opuntia ficus-indica) cladodes against the mycotoxin zearalenone in Balb/c mice: prevention of micronuclei, chromosome aberrations and DNA fragmentation.

    PubMed

    Zorgui, Lazhar; Ayed-Boussema, Imen; Ayed, Yosra; Bacha, Hassen; Hassen, Wafa

    2009-03-01

    Zearalenone (ZEN) is a potent estrogenic metabolite. Evidence of its cytotoxicity and genotoxicity has recently emerged from several reports. This study was conducted to evaluate the ability of cactus (Opuntia ficus-indica) cladodes to protect Balb/c mice against ZEN induced genotoxicity. To this end, the effect of a single dose of ZEN (40 mg/kg b.w.) alone and with extract of cactus cladodes (25, 50 and 100 mg/kg b.w.) was monitored by measuring: (i) micronuclei induction in bone marrow cells, (ii) chromosome aberrations mainly breaks and gaps in bone marrow cells also and finally and (iii) DNA fragmentation in liver and kidney. Our results clearly show that ZEN is genotoxic to Balb/c mice. It induces DNA damage as indicated by DNA fragmentation, micronuclei and chromosomal aberrations in bone marrow cells. It is of note that cactus cladodes extract assayed alone at high dose (100 mg/kg b.w.) was found completely safe and did not induce any genotoxic effects. The simultaneous administration of cactus cladodes extract with ZEN resulted in an efficient prevention of micronuclei (the number of PCE MN decreased from 71.3+/-6.1 for animals treated with Zen to 32.6+/-15.5 for animals treated with cactus cladodes), chromosomal aberrations frequency (the % of chromosomal aberrations decreased from 38.3+/-3.0 to 18.6+/-1.1) in bone marrow cells and of DNA fragmentation compared to the group treated with ZEN alone. It could be concluded that cactus cladodes extract was effective in the protection against ZEN genotoxicity. This could be relevant, particularly with the emergent demand for natural products which may neutralize the genotoxic effects of the multiple food contaminants. PMID:19152824

  1. Panax ginseng extract modulates oxidative stress, DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B 1.

    PubMed

    Hassan, Aziza M; Abdel-Aziem, Sekena H; El-Nekeety, Aziza A; Abdel-Wahhab, Mosaad A

    2015-10-01

    Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on DNA and gene expression in rat. Female Sprague-Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB1 (80 µg/kg bw), the group having received FB1 (100 µg/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20 mg/kg bw) alone or with AFB1 and/or FB1. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB1 and/or FB1 plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB1 and FB1 have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties. PMID:24748134

  2. Number and size of human X chromosome fragments transferred to mouse cells by chromosome-mediated gene transfer

    SciTech Connect

    Olsen, A.S.; McBride, O.W.; Moore, D.E.

    1981-05-01

    Labeled probes of unique-sequence human X chromosomal deoxyribonucleic acid, prepared by two different procedures, were used to measure the amount of human X chromosomal deoxyribonucleic acid in 12 mouse cell lines expressing human hypoxanthine phosphoribosyltransferase after chromosome-mediated gene transfer. The amount of X chromosomal deoxyribonucleic acid detected by this procedure ranged from undetectable levels in the three stable transformants and some unstable transformants examined to about 20% of the human X chromosome in two unstable transformants. Reassociation kinetics of the X chromosomal probe with deoxyribonucleic acid from the two unstable transformants containing 15 to 20% of the human X chromosome indicate that a single copy of these sequences is present. In one of these lines, the X chromosomal sequences exist as multiple fragments which were not concordantly segregated when the cells were selected for loss of hprt.

  3. Characterization of the genome of molluscum contagiosum virus type 1 between the genome coordinates 0.045 and 0.075 by DNA nucleotide sequence analysis of a 5.6-kb HindIII/MluI DNA fragment.

    PubMed

    Hadasch, R P; Bugert, J J; Janssen, W; Darai, G

    1993-01-01

    The complete DNA nucleotide sequence of a HindIII/MluI genomic DNA fragment (0.045-0.075 viral map units) from molluscum contagiosum virus type 1 (MCV-1) was determined. The HindIII/MluI DNA fragment comprises 5,646 bp with a base composition of 64.4% G + C and 35.6% A + T. The DNA sequence contains many perfect direct repeats. A cluster of three repetitive DNA elements R1, R2 and R3, with a complex structural arrangement was detected between nucleotide positions 1802 and 2107. The unit length (box) of the repetitive DNA sequences was found to be 6 bp (15 boxes) and 9 bp (24 boxes) for R1 and R2, respectively. The repetitive DNA element R3 is organized in fifteen boxes (15 bp) in which a unit length of R1 is combined with a unit length of R2. The arrangement of the repetition R3 within the DNA sequences of this particular region of the MCV-1 genome was found to be (5 x R3) + (2 x R2) + (1 x R3) + (6 x R2) + (1 x R3) + (1 x R2) + (8 x R3). Twenty-three open reading frames (ORFs) of 60-1,175 amino acid (AA) residues were detected. The largest ORF (number 17) comprises 1,175 AA with a predicted molecular weight of 126 kD. This ORF harbors a promoter signal which is located 21 nucleotides upstream from the start codon and is very similar to the early promoter signals known for vaccinia virus. This putative protein contains glutamine-enriched regions between AA residues 427 and 682 which show homologies to the corresponding glutamine-enriched regions of a variety of cellular genes like human transcriptional initiation factor (TFIID: TATA box factor).

  4. Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery.

    PubMed

    Lamichhane, Tek N; Raiker, Rahul S; Jay, Steven M

    2015-10-01

    Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles.

  5. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  6. Bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex a new apoptotic agent through Flk-1 down regulation, caspase-3 activation and oligonucleosomes DNA fragmentation.

    PubMed

    Azab, Hassan A; Hussein, Belal H M; El-Azab, Mona F; Gomaa, Mohamed; El-Falouji, Abdullah I

    2013-01-01

    New bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex was synthesized and characterized. In vivo anti-angiogenic activities of bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex against Ehrlich ascites carcinoma (EAC) cells are described. The newly synthesized complex resulted in inhibition of proliferation of EAC cells and ascites formation. The anti-tumor effect was found to be through anti-angiogenic activity as evident by the reduction of microvessel density in EAC solid tumors. The anti-angiogenic effect is mediated through down-regulation of VEGF receptor type-2 (Flk-1). The complex was also found to significantly increase the level of caspase-3 in laboratory animals compared to the acridine ligand and to the control group. This was also consistent with the DNA fragmentation detected by capillary electrophoresis that proved the apoptotic effect of the new complex. Our complex exhibited anti-angiogenic and apoptotic activity in vivo, a thing that makes it a potential effective chemotherapeutic agent. The interaction of calf thymus DNA (ct-DNA) with bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex has been investigated using fluorescence technique. A competitive experiment of the europium(III)-acridine complex with ethidium bromide (EB) to bind DNA revealed that interaction between the europium(III)-acridine and DNA was via intercalation. The interaction of the synthesized complex with tyrosine kinases was also studied using molecular docking simulation to further substantiate its mode of action.

  7. Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA

    PubMed Central

    Stabile, J.; Waldman, J. R.; Parauka, F.; Wirgin, I.

    1996-01-01

    Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids. PMID:8889537

  8. Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

    PubMed

    Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

    2015-10-01

    The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60 nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30 fM. Optimization of the sensing surface is described and influences on the assay performance are discussed.

  9. The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity

    SciTech Connect

    Ramachandran, Anup; Lebofsky, Margitta; Weinman, Steven A.; Jaeschke, Hartmut

    2011-03-15

    Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200 mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870 {+-} 180 U/L) and centrilobular necrosis at 6 h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. Conclusions: the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.

  10. Protective role of probiotic lactic acid bacteria against dietary fumonisin B1-induced toxicity and DNA-fragmentation in sprague-dawley rats.

    PubMed

    Khalil, Ashraf A; Abou-Gabal, Ashgan E; Abdellatef, Amira A; Khalid, Ahmed E

    2015-08-18

    The genus Fusarium, especially F. verticillioides and F. proliferatum, has been found in several agricultural products worldwide, especially in maize. Regardless the occurrence of symptoms, the presence of Fusarium in maize constitutes an imminent risk due to its ability to produce fumonisins, mycotoxins with proven carcinogenic effect on rats, swine, and equines and already classified as possible carcinogens to humans. The toxicity of incremental levels of fumonisin B1 (FB1), that is, 50, 100, and 200 mg FB1/kg diet, and the role of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL) and Pediococcus acidilactici NNRL B-5627 (PA) supplementation in counteracting the FB1 effects in intoxicated rats were monitored over a period of 4 weeks. Effects on the feed intake and body weight gain were noticed. A significant (p ≤ 0.05) increase in the level of liver and kidney functions markers and DNA fragmentation was also noticed in rat groups T100 and T200. The lactic acid bacteria (LAB) supplementation could bring back the normal serum biochemical parameters in rats fed on fumonisin B1-contaminated diets (T50 and T100) compared to FB1-treated groups. In rats of high-dosage dietary groups supplemented with LAB (T200-LL and T200-PA), the supplementation reduced the serum activity levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and creatinine by 11.3, 11.9, 32, and 20%, respectively. DNA fragmentations were observed in the rat group treated with 200 mg FB1 after 3 weeks, while fragmentation was noticed in treated groups with 100 and 200 mg FB1 after 4 weeks. No DNA fragmentation was apparent in FB1-treated rats co-administered the LL or PA strain. These results suggest that in male rats consuming diets containing FB1, there is a time- and dose-dependent increase in serum enzyme activities and DNA lesions. Moreover, Lb. delbrueckii subsp. lactis (LL) and P. acidilactici (PA) strains have a protective effect

  11. Estimating Size and Trend of the North Interlake Woodland Caribou Population Using Fecal-DNA and Capture–Recapture Models

    PubMed Central

    Hettinga, Peter N; Arnason, Arni Neil; Manseau, Micheline; Cross, Dale; Whaley, Kent; Wilson, Paul J

    2012-01-01

    A critical step in recovery efforts for endangered and threatened species is the monitoring of population demographic parameters. As part of these efforts, we evaluated the use of fecal-DNA based capture–recapture methods to estimate population sizes and population rate of change for the North Interlake woodland caribou herd (Rangifer tarandus caribou), Manitoba, Canada. This herd is part of the boreal population of woodland caribou, listed as threatened under the federal Species at Risk Act (2003) and the provincial Manitoba Endangered Species Act (2006). Between 2004 and 2009 (9 surveys), we collected 1,080 fecal samples and identified 180 unique genotypes (102 females and 78 males). We used a robust design survey plan with 2 surveys in most years and analysed the data with Program MARK to estimate encounter rates (p), apparent survival rates (ϕ), rates of population change (λ), and population sizes (N). We estimated these demographic parameters for males and females and for 2 genetic clusters within the North Interlake. The population size estimates were larger for the Lower than the Upper North Interlake area and the proportion of males was lower in the Lower (33%) than the Upper North Interlake (49%). Population rate of change for the entire North Interlake area (2005–2009) using the robust design Pradel model was significantly <1.0 (λ = 0.90, 95% CI: 0.82–0.99) and varied between sex and area with the highest being for males in Lower North Interlake (λ = 0.98, 95% CI: 0.83–1.13) and the lowest being for females in Upper North Interlake (λ = 0.83, 95% CI: 0.69–0.97). The additivity of λ between sex and area is supported on the log scale and translates into males having a λ that is 0.09 greater than females and independent of sex, Lower North Interlake having a λ that is 0.06 greater than Upper North Interlake. Population estimates paralleled these declining trends, which correspond to trends observed in other fragmented populations of

  12. Universality of fragment shapes

    PubMed Central

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  13. Universality of fragment shapes.

    PubMed

    Domokos, Gábor; Kun, Ferenc; Sipos, András Árpád; Szabó, Tímea

    2015-01-01

    The shape of fragments generated by the breakup of solids is central to a wide variety of problems ranging from the geomorphic evolution of boulders to the accumulation of space debris orbiting Earth. Although the statistics of the mass of fragments has been found to show a universal scaling behavior, the comprehensive characterization of fragment shapes still remained a fundamental challenge. We performed a thorough experimental study of the problem fragmenting various types of materials by slowly proceeding weathering and by rapid breakup due to explosion and hammering. We demonstrate that the shape of fragments obeys an astonishing universality having the same generic evolution with the fragment size irrespective of materials details and loading conditions. There exists a cutoff size below which fragments have an isotropic shape, however, as the size increases an exponential convergence is obtained to a unique elongated form. We show that a discrete stochastic model of fragmentation reproduces both the size and shape of fragments tuning only a single parameter which strengthens the general validity of the scaling laws. The dependence of the probability of the crack plan orientation on the linear extension of fragments proved to be essential for the shape selection mechanism. PMID:25772300

  14. Trapping of 27 bp 8 kbp DNA and immobilization of thiol-modified DNA using dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Tuukkanen, Sampo; Kuzyk, Anton; Jussi Toppari, J.; Häkkinen, Hannu; Hytönen, Vesa P.; Niskanen, Einari; Rinkiö, Marcus; Törmä, Päivi

    2007-07-01

    Dielectrophoretic trapping of six different DNA fragments, sizes varying from 27 to 8416 bp, has been studied using confocal microscopy. The effect of the DNA length and the size of the constriction between nanoscale fingertip electrodes on the trapping efficiency have been investigated. Using finite element method simulations in conjunction with the analysis of the experimental data, the polarizabilities of the different size DNA fragments have been calculated for different frequencies. Also the immobilization of trapped hexanethiol- and DTPA-modified 140 nm long DNA to the end of gold nanoelectrodes was experimentally quantified and the observations were supported by density functional theory calculations.

  15. Differential response of human thymus cells to CD2 antibodies: fragmentation of DNA of CD45RO+ and proliferation of CD45RO- subsets.

    PubMed Central

    Li, J; Campbell, D; Hayward, A R

    1992-01-01

    Human thymocytes bearing the CD45RO 'memory' cell phenotype do not proliferate in concanavalin A (Con A)-stimulated cultures and may be destined for intrathymic death. To determine whether this subset would exhibit characteristics of programmed cell death (apoptosis), we examined the integrity of the nuclear DNA by gel electrophoresis. DNA fragmentation was restricted to the CD45RO+ subset of human thymocytes following exposure to stimulating concentrations of anti-CD2 antibodies. Both CD45RO- and CD45RO+ subsets mobilized cytoplasmic Ca2+ following cell-surface CD2 ligation, but entry into the cell cycle and vigorous thymidine uptake were restricted to the CD45RO- subset. Our results provide a mechanism which may account for the failure of thymic CD45RO+ cells to respond to stimuli which elicit proliferation by the reciprocal CD45RA+ subset. Images Figure 1 Figure 2 PMID:1348052

  16. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A.; Arlinghaus, H.F.

    1993-06-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  17. Applications of mass spectrometry to DNA fingerprinting and DNA sequencing

    SciTech Connect

    Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. ); Arlinghaus, H.F. )

    1993-01-01

    DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

  18. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family.

    PubMed

    Liang, Liang; Zhao, Mujun; Xu, Zhenhua; Yokoyama, Kazunari K; Li, Tsaiping

    2003-02-15

    DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40 kDa caspase-activated nuclease (DFF40) and a 45 kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3alpha, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3alpha exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3alpha isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis.

  19. Protective effect of dietary curcumin in Anabas testudineus (Bloch) with a special note on DNA fragmentation assay on hepatocytes and micronucleus assay on erythrocytes in vivo.

    PubMed

    Manju, Maniyan; Vijayasree, Appiyathu Saraswathy; Akbarsha, Mohammad Abdulkader; Oommen, Oommen Vilaverthottathil

    2013-10-01

    The present study was conducted to evaluate the safety of long-term dietary curcumin at doses 0.5 and 1% in Anabas testudineus employing hematological and cytological techniques. The fish were fed with curcumin-supplemented feed for 6 months. Fine blood smears were prepared and subjected to three different staining techniques. The erythrocyte micronucleus frequency (MN) and the cytometric measurements of erythrocytes were determined. Blood from the control and treated fish was subjected to the assessment of several hematological parameters. Also, DNA fragmentation assay on hepatocytes was conducted. The results showed that hemoglobin content, RBC count and hematocrit increased in the curcumin-fed fish compared to control, whereas WBC count, platelet count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were unaffected. WBC/RBC ratio was lower in the case of curcumin-treated fish. The cytometric measurements revealed no change in the erythrocytes and their nuclei after curcumin treatment. DNA fragmentation assay revealed intact DNA in curcumin-fed group, ruling out the possibility of curcumin-induced DNA damage. The positive control group showed a significant increase in MN frequency compared to negative control and curcumin-fed groups. In fact, the MN frequency decreased in 1% curcumin-fed group compared to the negative control and 0.5% curcumin groups. All these indicated a state of well-being of the curcumin-treated fish. Therefore, it is concluded that curcumin could be used as a safe feed ingredient to improve the growth of finfish in aquaculture.

  20. Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Sandeep, K P; Simunovic, Josip; Harris, Keith; Osborne, Jason A; Hassan, Hosni M

    2015-08-01

    Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products. PMID:26235411

  1. Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials.

    PubMed

    Caldwell, Jane M; Pérez-Díaz, Ilenys M; Sandeep, K P; Simunovic, Josip; Harris, Keith; Osborne, Jason A; Hassan, Hosni M

    2015-08-01

    Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products.

  2. Channel Size Conversion of Phi29 DNA-Packaging Nanomotor for Discrimination of Single- and Double-Stranded Nucleic Acids

    PubMed Central

    Geng, Jia; Wang, Shaoying; Fang, Huaming; Guo, Peixuan

    2013-01-01

    Nanopores have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological pores are extremely reproducible at the atomic level with uniform channel sizes. The channel of the bacterial virus phi29 DNA packaging motor is a natural conduit for the transportation of double-stranded DNA (dsDNA), and has the largest diameter among the well-studied biological channels. The larger channel facilitates translocation of dsDNA, and offers more space for further channel modification and conjugation. Interestingly, the relatively large wild type channel, which translocates dsDNA, cannot detect single-stranded nucleic acids (ssDNA or ssRNA) under the current experimental conditions. Herein, we reengineered this motor channel by removing the internal loop segment of the channel. The modification resulted in two classes of channels. One class was the same size as the wild type channel, while the other class had a cross-sectional area about 60% of the wild type. This smaller channel was able to detect the real-time translocation of single stranded nucleic acids at single-molecule level. While the wild type connector exhibited a one-way traffic property with respect to dsDNA translocation, the loop deleted connector was able to translocate ssDNA and ssRNA with equal competencies from both termini. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA packaging motor in nanomedicine, nanobiotechnology, and high-throughput single pore DNA sequencing. PMID:23488809

  3. Behavioral and physiological responses to subgroup size and number of people in howler monkeys inhabiting a forest fragment used for nature-based tourism.

    PubMed

    Aguilar-Melo, Adriana R; Andresen, Ellen; Cristóbal-Azkarate, Jurgi; Arroyo-Rodríguez, Victor; Chavira, Roberto; Schondube, Jorge; Serio-Silva, Juan Carlos; Cuarón, Alfredo D

    2013-11-01

    Animals' responses to potentially threatening factors can provide important information for their conservation. Group size and human presence are potentially threatening factors to primates inhabiting small reserves used for recreation. We tested these hypotheses by evaluating behavioral and physiological responses in two groups of mantled howler monkeys (Alouatta palliata mexicana) at the "Centro Ecológico y Recreativo El Zapotal", a recreational forest reserve and zoo located in the Mexican state of Chiapas. Both groups presented fission-fusion dynamics, splitting into foraging subgroups which varied in size among, but not within days. Neither subgroup size nor number of people had an effect on fecal cortisol. Out of 16 behavioral response variables tested, the studied factors had effects on six: four were affected by subgroup size and two were affected by number of people. With increasing subgroup size, monkeys increased daily path lengths, rested less, increased foraging effort, and used more plant individuals for feeding. As the number of people increased, monkeys spent more time in lower-quality habitat, and less time engaged in social interactions. Although fecal cortisol levels were not affected by the factors studied, one of the monkey groups had almost twice the level of cortisol compared to the other group. The group with higher cortisol levels also spent significantly more time in the lower-quality habitat, compared to the other group. Our results suggest that particular behavioral adjustments might allow howler monkeys at El Zapotal to avoid physiological stress due to subgroup size and number of people. However, the fact that one of the monkey groups is showing increased cortisol levels may be interpreted as a warning sign, indicating that an adjustment threshold is being reached, at least for part of the howler monkey population in this forest fragment.

  4. Deceleration of single-stranded DNA passing through a nanopore using a nanometre-sized bead structure

    PubMed Central

    Goto, Yusuke; Haga, Takanobu; Yanagi, Itaru; Yokoi, Takahide; Takeda, Ken-ichi

    2015-01-01

    DNA sequencing with a solid-state nanopore requires a reduction of the translocation speeds of single-stranded DNA (ssDNA) over 10 μs/base. In this study, we report that a nanometre-sized bead structure constructed around a nanopore can reduce the moving speed of ssDNA to 270 μs/base by adjusting the diameter of the bead and its surface chemical group. This decelerating effect originates from the strong interaction between ssDNA and the chemical group on the surface of the bead. This nanostructure was simply prepared by dip coating in which a substrate with a nanopore was immersed in a silica bead solution and then dried in an oven. As compared with conventional approaches, our novel method is less laborious, simpler to perform and more effective in reducing ssDNA translocation speed. PMID:26559466

  5. Deceleration of single-stranded DNA passing through a nanopore using a nanometre-sized bead structure

    NASA Astrophysics Data System (ADS)

    Goto, Yusuke; Haga, Takanobu; Yanagi, Itaru; Yokoi, Takahide; Takeda, Ken-Ichi

    2015-11-01

    DNA sequencing with a solid-state nanopore requires a reduction of the translocation speeds of single-stranded DNA (ssDNA) over 10 μs/base. In this study, we report that a nanometre-sized bead structure constructed around a nanopore can reduce the moving speed of ssDNA to 270 μs/base by adjusting the diameter of the bead and its surface chemical group. This decelerating effect originates from the strong interaction between ssDNA and the chemical group on the surface of the bead. This nanostructure was simply prepared by dip coating in which a substrate with a nanopore was immersed in a silica bead solution and then dried in an oven. As compared with conventional approaches, our novel method is less laborious, simpler to perform and more effective in reducing ssDNA translocation speed.

  6. Size-frequency distributions of fragments from SPH/ N-body simulations of asteroid impacts: Comparison with observed asteroid families

    NASA Astrophysics Data System (ADS)

    Durda, Daniel D.; Bottke, William F.; Nesvorný, David; Enke, Brian L.; Merline, William J.; Asphaug, Erik; Richardson, Derek C.

    2007-02-01

    We investigate the morphology of size-frequency distributions (SFDs) resulting from impacts into 100-km-diameter parent asteroids, represented by a suite of 161 SPH/N-body simulations conducted to study asteroid satellite formation [Durda, D.D., Bottke, W.F., Enke, B.L., Merline, W.J., Asphaug, E., Richardson, D.C., Leinhardt, Z.M., 2004. Icarus 170, 243-257]. The spherical basalt projectiles range in diameter from 10 to 46 km (in equally spaced mass increments in logarithmic space, covering six discrete sizes), impact speeds range from 2.5 to 7 km/s (generally in 1 km/s increments), and impact angles range from 15° to 75° (nearly head-on to very oblique) in 15° increments. These modeled SFD morphologies match very well the observed SFDs of many known asteroid families. We use these modeled SFDs to scale to targets both larger and smaller than 100 km in order to gain insights into the circumstances of the impacts that formed these families. Some discrepancies occur for families with parent bodies smaller than a few tens of kilometers in diameter (e.g., 832 Karin), however, so due caution should be used in applying our results to such small families. We find that ˜20 observed main-belt asteroid families are produced by the catastrophic disruption of D >100 km parent bodies. Using these data as constraints, collisional modeling work [Bottke Jr., W.F., Durda, D.D., Nesvorný, D., Jedicke, R., Morbidelli, A., Vokrouhlický, D., Levison, H.F., 2005b. Icarus 179, 63-94] suggests that the threshold specific energy, QD∗, needed to eject 50% of the target body's mass is very close to that predicted by Benz and Asphaug [Benz, W., Asphaug, E., 1999. Icarus 142, 5-20].

  7. Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

    PubMed

    Krueger, Andrew T; Kool, Eric T

    2008-03-26

    We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis

  8. Orthogonal enzyme arrays on a DNA origami scaffold bearing size-tunable wells

    NASA Astrophysics Data System (ADS)

    Yamazaki, Takahiro; Heddle, Jonathan Gardiner; Kuzuya, Akinori; Komiyama, Makoto

    2014-07-01

    A new waffle-like DNA origami assembly (DNA waffle) with nine nanometer-scale wells in a 3 × 3 matrix pattern has been successfully constructed and used as a scaffold for selective nano-patterning of individual protein molecules. The folding pattern of the scaffold was specially designed so that the dimensions of each well could be independently tuned according to the dimensions of the guest nanoparticles. We demonstrated that two distinct proteins, streptavidin (SA) tetramer (d = 5 nm) and anti-fluorescein antibody (IgG) (inter-paratope distance ~14.0 nm), could be selectively captured in size-variable wells of dimensions 6.8 × 12 × 2.0 nm for SA and 6.8 × 12 × 2.0 nm or 10.2 × 12 × 2.0 nm for IgG, respectively, through the attachment of two biotins or two fluoresceins at the two edges of each well. This allowed the formation of a heterogeneous protein nanoarray of individual molecules. The position of SA or IgG capture can be fully controlled by placement of biotins or fluoresceins in the nanoarray well. Moreover, a hetero-nanoarray consisting of two kinds of enzyme: horseradish peroxidase-labeled streptavidin (HRP-SA) and alkaline phosphatase-labeled anti-FITC antibody (AP-IgG) was successfully constructed through selective attachment of biotin or fluorescein in any desired wells. Successful enzyme-heteroarray formation was confirmed by enzymatic activity analyses after purification of mixtures of enzymes and DNA waffles.A new waffle-like DNA origami assembly (DNA waffle) with nine nanometer-scale wells in a 3 × 3 matrix pattern has been successfully constructed and used as a scaffold for selective nano-patterning of individual protein molecules. The folding pattern of the scaffold was specially designed so that the dimensions of each well could be independently tuned according to the dimensions of the guest nanoparticles. We demonstrated that two distinct proteins, streptavidin (SA) tetramer (d = 5 nm) and anti-fluorescein antibody (IgG) (inter

  9. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.

    PubMed

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael

    2010-01-01

    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  10. Expression of genes derived from the genomic DNA fragments of the brown-winged green bug (Plautia stali) symbiont in Escherichia coli.

    PubMed

    Fujii-Muramatsu, Rika; Kobayashi, Hideaki; Noda, Hiroaki; Takeishi, Keiichi

    2013-08-01

    Many insect species harbour symbiotic microorganisms (symbionts) that are generally unculturable in media. To utilize symbionts as genome resources, we examined whether insect symbiont genes can be expressed in Escherichia coli. 144 plasmid clones were isolated from gene libraries, which were constructed from the genomic DNA of the intestinal bacterial symbiont in the brown-winged green bug, Plautia stali, using an E. coli system. Proteins prepared from a culture of each clone were analysed using SDS-PAGE. A discrete symbiont-specific band was detected in six clones. From the structural analyses of the insert in each clone, the candidate gene encoding the symbiont-specific protein was predicted and the amino acid sequence of the protein was deduced. The amino acid sequence in the N-terminal region of each protein was identical to that deduced from the genomic DNA sequence of the symbiont, but not of the host. The promoter sequences of the symbiont genes, very similar to those of the corresponding E. coli genes, were found in the insert DNA. These findings clearly indicate that genes derived from genomic DNA fragments of the P. stali symbiont can be expressed in E. coli. PMID:23613025

  11. True ternary fission, the collinear decay into fragments of similar size in the 252Cf(sf) and 235U(nth, f) reactions

    NASA Astrophysics Data System (ADS)

    von Oertzen, W.; Nasirov, A. K.

    2014-06-01

    The collinear cluster decay in 252Cf(sf, fff), with three cluster fragments of different masses (e.g. 132Sn, 52-48Ca, 68-72Ni), which has been observed by the FOBOS group in JINR, has established a new decay mode of heavy nuclei, the collinear cluster tripartition (CCT). The same type of ternary fission decay has been observed in the reaction 235U(nth, fff). This kind of “true ternary fission” of heavy nuclei has been predicted many times in theoretical works during the last decades. In the present note we discuss true ternary fission (TFFF) into three nuclei of almost equal size (e.g. Z=98→Zi=32, 34, 32) in the same systems. The possible fission channels are predicted from potential-energy (PES) calculations. These PES's show pronounced minima for several ternary fragmentation decays, e.g. for 252Cf(sf) and for 235U(nth, f). They suggest the existence of a variety of collinear ternary fission modes. The TFFF-decays chosen in this letter have very similar dynamical features as the previously observed collinear CCT-decays. The data obtained in the above mentioned experiments allow us to extract the yield for these TFFF-decays in both systems by using specific gates on the measured parameters. These yields are a few 1.0ṡ10-6/(binary fission).

  12. Protective effect of nicotine through nicotinic acetylcholine receptor alpha 7 on hypoxia-induced membrane disintegration and DNA fragmentation of cultured PC12 cells.

    PubMed

    Tohgi, H; Utsugisawa, K; Nagane, Y

    2000-05-12

    To investigate the effect of nicotine on hypoxic neuronal damage, cultured PC12 cells were exposed to hypoxia for 9 h and then reoxygenated for 72 h. The cells were stained by propidium iodide (PI), a marker of cell membrane disintegration and the TUNEL method, which indicates DNA fragmentation. In control cultures, the ratio of PI-positive cells to total cells progressively increased during and after exposure to hypoxia, constituting 39% of total cells at 72 h posthypoxia. This increase in PI-positive cells was completely inhibited by nicotine until 12 h posthypoxia, and was partially and dose-dependently inhibited thereafter. The ratio of TUNEL-positive cells to total cells started to increase at 24 h posthypoxia and reached 36% at 72 h in control cultures. This ratio was also dose-dependently inhibited by nicotine. These inhibitory effects of nicotine on the increase in PI-positive and TUNEL-positive cells were abolished by the addition to the medium of alpha-bungarotoxin, an antagonistic ligand for nicotinic acetylcholine receptor (AChR) alpha7. These findings suggest that nicotine inhibits, through AChR alpha7, hypoxia-induced cell membrane disintegration and DNA fragmentation of cultured PC12 cells exposed to hypoxia.

  13. The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.

    PubMed Central

    Winkler, F K; Banner, D W; Oefner, C; Tsernoglou, D; Brown, R S; Heathman, S P; Bryan, R K; Martin, P D; Petratos, K; Wilson, K S

    1993-01-01

    The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme. Images PMID:8491171

  14. [Construction of large fragment metagenome library of natural mangrove soil].

    PubMed

    Jiang, Yun-Xia; Zheng, Tian-Ling

    2007-11-01

    Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significant increased. The large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. All of the clones had recombinant Cosmids and each differed in their fragment profiles when Cosmid DNA was extracted from 12 randomly picked colonies and digested with BamHI. The average insert size for this library was larger than 35 kbp. This culturing-independent library at least encompassed 335 Mbp valuable genetic information of mangrove soil microbes. It allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.

  15. Optimized sequence retrieval from single bands of temperature gradient gel electrophoresis profiles of the amplified 16S rDNA fragments from an activated sludge system.

    PubMed

    Zhang, Xueli; Yan, Xing; Gao, Pingping; Wang, Linghua; Zhou, Zhihua; Zhao, Liping

    2005-01-01

    Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single bands were excised, and a clone library was constructed for each. Sequence heterogeneity in each single band was found to be significantly overestimated due to single-stranded DNA (ssDNA) contamination formed during the PCR amplification, since only 10-60% of library clones of each single TGGE band had identical migration behavior compared with the parent band. Three methods, digestion with mung bean nuclease, optimization of PCR amplification, and purification via denatured polyacrylamide gel electrophoresis (d-PAGE), were compared for their ability to minimize ssDNA contamination, with the last one being the most efficient. After using d-PAGE to minimize ssDNA to a nearly nondetectable level, 70-100% of library clones for each single TGGE band had identical migration compared with the parent band. Several sequences were found in each of six single bands, and this co-migration could be predicted with the Poland software. The predominant bacteria of the activated sludge were assessed via a combination of sequence retrieval from each single TGGE band and band intensity analysis. Only beta and alpha subclasses of the Proteobacteria were detected, 93.8% and 6.2%, respectively. Our work suggests that prior to constructing a clone library to retrieve the actual sequence diversity of a single DGE band, it is advisable to minimize ssDNA contamination to a nondetectable level.

  16. Effects of Caffeine and Chlorogenic Acid on Propidium Iodide Accessibility to DNA: Consequences on Genome Size Evaluation in Coffee Tree

    PubMed Central

    NOIROT, M.; BARRE, P.; DUPERRAY, C.; LOUARN, J.; HAMON, S.

    2003-01-01

    Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo‐intraspecific variation in genome size. Two important compounds present in coffee trees—caffeine and chlorogenic acid—modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes. PMID:12876189

  17. A DNA Fragment Mapped within the Submicroscopic Deletion of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

    PubMed Central

    Gill, K. S.; Gill, B. S.

    1991-01-01

    Bread wheat is an allohexaploid consisting of three genetically related (homoeologous) genomes. The homoeologous chromosomes are capable of pairing but strict homologous pairing is observed at metaphase 1. The diploid-like pairing is regulated predominantly by Ph1, a gene mapped on long arm of chromosome 5B. We report direct evidence that a mutant of the gene (ph1b) arose from a submicroscopic deletion. A probe (XksuS1-5) detects the same missing fragment in two independent mutants ph1b and ph1c and a higher intensity fragment in a duplication of the Ph1 gene. It is likely that XksuS1-5 lies adjacent to Ph1 on the same chromosome fragment that is deleted in ph1b and ph1c. XksuS1-5 can be used to tag Ph1 gene to facilitate incorporation of genetic material from homoeologous genomes of the Triticeae. It may also be a useful marker in cloning Ph1 gene by chromosome walking. PMID:1936962

  18. Technical Reproducibility of Single-Nucleotide and Size-Based DNA Biomarker Assessment Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Tissues

    PubMed Central

    Zhang, Shenli; Tan, Iain B.; Sapari, Nur S.; Grabsch, Heike I.; Okines, Alicia; Smyth, Elizabeth C.; Aoyama, Toru; Hewitt, Lindsay C.; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P.; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-01-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5′ untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5′ untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%–47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%–14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. PMID:25746798

  19. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    PubMed

    Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-05-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies.

  20. Optical/near-infrared polarization survey of Sh 2-29: Magnetic fields, dense cloud fragmentations, and anomalous dust grain sizes

    SciTech Connect

    Santos, Fábio P.; Franco, Gabriel A. P.; Reis, Wilson; Roman-Lopes, Alexandre; Román-Zúñiga, Carlos G. E-mail: franco@fisica.ufmg.br E-mail: roman@dfuls.cl

    2014-03-01

    Sh 2-29 is a conspicuous star-forming region marked by the presence of massive embedded stars as well as several notable interstellar structures. In this research, our goals were to determine the role of magnetic fields and to study the size distribution of interstellar dust particles within this turbulent environment. We have used a set of optical and near-infrared polarimetric data obtained at OPD/LNA (Brazil) and CTIO (Chile), correlated with extinction maps, Two Micron All Sky Survey data, and images from the Digitized Sky Survey and Spitzer. The region's most striking feature is a swept out interstellar cavity whose polarimetric maps indicate that magnetic field lines were dragged outward, piling up along its borders. This led to a higher magnetic strength value (≈400 μG) and an abrupt increase in polarization degree, probably due to an enhancement in alignment efficiency. Furthermore, dense cloud fragmentations with peak A{sub V} between 20 and 37 mag were probably triggered by its expansion. The presence of 24 μm point-like sources indicates possible newborn stars inside this dense environment. A statistical analysis of the angular dispersion function revealed areas where field lines are aligned in a well-ordered pattern, seemingly due to compression effects from the H II region expansion. Finally, Serkowski function fits were used to study the ratio of the total-to-selective extinction, revealing a dual population of anomalous grain particle sizes. This trend suggests that both effects of coagulation and fragmentation of interstellar grains are present in the region.