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Sample records for dna fragment size

  1. Sizing of DNA fragments by flow cytometry

    NASA Astrophysics Data System (ADS)

    Johnson, Mitchell E.; Goodwin, Peter M.; Ambrose, W. Patrick; Martin, John C.; Marrone, Babetta L.; Jett, James H.; Keller, Richard A.

    1993-06-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1, 29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis.

  2. Sizing of DNA fragments by flow cytometry

    SciTech Connect

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

    1993-02-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

  3. Sizing of DNA fragments by flow cytometry

    SciTech Connect

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

    1993-01-01

    Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1,29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis. We have demonstrated sizing of individual, stained DNA fragments with resolution approaching that of gel electrophoresis for moderately large fragments, but with significant reductions in the analysis time and the amount of sample required. Furthermore, system response is linear with DNA fragment length, in contrast to the logarithmic response in gel electrophoresis. There exists the potential to perform this sizing using relatively simple instrumentation, i.e. a continuous wave laser of low power and current mode detection.

  4. 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.

  5. 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 714%. The proposed method is straightforward and can be applied to standardized microtiter plates. PMID:14602931

  6. 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.

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

    PubMed

    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

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

    SciTech Connect

    Werner, James H.; Larson, Erica J.; Goodwin, Peter M.; Ambrose, W. Patrick; Keller, Richard A.

    2000-06-01

    We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America.

  13. GELYMAC: a Macintosh application for calculating DNA fragment size from gel electrophoresis migration data.

    PubMed

    Russell, P J; Doenias, J M; Russell, S J

    1991-04-01

    A program called GELYMAC takes data on the distances migrated by DNA fragments in a one-dimensional electrophoretic gel and, using a cubic-spline best-fit of marker fragment distance migrated versus molecular size, calculates the molecular sizes of the fragments. Written in the Rascal (Real-time Pascal) programming language, the program runs on the Macintosh family of microcomputers. Rapid entry of marker and experimental fragment migration data is afforded using a scroll bar system adjacent to a graphic representation of a gel. Output includes tabular listing of the data, graphic cartoons of the gel, and the fragment locations and molecular sizes for individual gel lanes, and the calibration curve used in data computations. PMID:2059854

  14. DNA Fragmentation Simulation Method (FSM) and Fragment Size Matching Improve aCGH Performance of FFPE Tissues

    PubMed Central

    Craig, Justin M.; Vena, Natalie; Ramkissoon, Shakti; Idbaih, Ahmed; Fouse, Shaun D.; Ozek, Memet; Sav, Aydin; Hill, D. Ashley; Margraf, Linda R.; Eberhart, Charles G.; Kieran, Mark W.; Norden, Andrew D.; Wen, Patrick Y.; Loda, Massimo; Santagata, Sandro; Ligon, Keith L.; Ligon, Azra H.

    2012-01-01

    Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice. PMID:22719973

  15. Linear induction of DNA double-strand breakage with X-ray dose, as determined from DNA fragment size distribution

    SciTech Connect

    Erixon, K.; Cedervall, B.

    1995-05-01

    Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were use to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/GY per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mb was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 60000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/GY, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy. 58 refs., 10 figs.

  16. Direct calculation of the sizes of DNA fragments separated by gel electrophoresis using programmes written for a pocket calculator.

    PubMed Central

    Gough, E J; Gough, N M

    1984-01-01

    In order to facilitate the direct computation of the sizes of DNA fragments separated by gel electrophoresis, we have written and evaluated programmes for the Hewlett-Packard 41C programmable calculator. The sizes estimated for DNA fragments of known length using some of these programmes were found to be more accurate than the estimates obtained by conventional graphical procedures. These programmes should be adaptable to other programmable calculators. Images PMID:6320110

  17. The use of biphasic linear ramped pulsed field gel electrophoresis to quantify DNA damage based on fragment size distribution

    SciTech Connect

    Lawrence, T.S.; Normolle, D.P.; Davis, M.A.; Maybaum, J.

    1993-10-20

    The development of biphasic linear pulse ramping gel electrophoresis has permitted resolution of DNA fragments from 200 Kbp to 6 Mbp in a single gel. We used this technique to measure radiation-induced DNA damage based on fragment size. Human colon cancer cells (HT29 and LS174T) and Chinese hamster ovary cells were embedded in agarose, deproteinized, irradiated with 5-80 Gy, and assessed for DNA double strand breakage using pulsed field gel electrophoresis. The frequency of DNA double strand breakage determined using a previously published method was compared to the breakage frequency calculated using the fragment size distribution. Both methods produced similar estimates for breakage frequency of approximately 5 {times} 10{sup {minus}9} breaks Gy{sup {minus}1} bp{sup {minus}1}. These findings suggest that biphasic linear pulse ramping gel electrophoresis can yield a quantitative estimate of DNA fragment distribution resulting from irradiation. The ability to quantify the distribution of DNA fragment sizes produced by irradiation should yield information concerning the mechanisms of both DNA double strand break induction and repair. 16 refs., 5 figs.

  18. Fragmentation of DNA by sonication.

    PubMed

    Sambrook, Joseph; Russell, David W

    2006-01-01

    INTRODUCTIONDNA fragmentation is often necessary prior to library construction or subcloning for DNA sequencing. This protocol describes a method for DNA fragmentation by sonication. During sonication, DNA samples are subjected to hydrodynamic shearing by exposure to brief periods of sonication. DNA that has been sonicated for excessive periods of time is extremely difficult to clone. Most sonicators will not shear DNA to a size of less than 300-500 bp, and it is tempting to continue sonication until the entire DNA population has been reduced in size. However, the yield of subclones is usually greater if sonication is stopped when the fragments of the target DNA first reach a size of ~700 bp. PMID:22485919

  19. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

    PubMed

    Devonshire, Alison S; Whale, Alexandra S; Gutteridge, Alice; Jones, Gerwyn; Cowen, Simon; Foy, Carole A; Huggett, Jim F

    2014-10-01

    Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp circulating nucleic acid (CNA) kit, NucleoSpin Plasma XS (NS) kit and FitAmp plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity. PMID:24853859

  20. 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.

  1. Effect of bromodeoxyuridine on radiation-induced DNA damage and repair based on DNA fragment size using pulsed-field gel electrophoresis

    SciTech Connect

    Lawrence, T.S.; Davis, M.A.; Normolle, D.P.

    1995-12-01

    We have used biphasic linear ramping pulsed-field gel electrophoresis (PFGE) to understand the effect of incorporation of bromodeoxyuridine (BrdUrd) on radiation-induced DNA damage and repair. This technique permits a determination of the fragment size distribution produced immediately after irradiation as well as during the repair period. We found that incorporation of BrdUrd increased the induction and decreased the repair of radiation damage. The fragment size distribution was consistent with a random breakage model. When we found that significantly more damage was detected after irradiation of deproteinized DNA compared to intact cells, we studied the effects of BrdUrd incorporation on the radiation response of cells or DNA at various phases of preparation for electrophoresis: cells adherent to the culture dish (A), trypsinized cells (B), agarose-embedded cells (C) and deproteinized DNA (D). Although there was a general tendency to detect more damage when irradiation was performed later in the preparation process, steps B and C were the only successive steps which were significantly different. These findings demonstrate that incorporation of BrdUrd randomly increases the induction of radiation damage and decreases its repair at the level of 200 kbp to 5 Mbp fragments. Furthermore, they confirm that the amount of damage detected depends upon the conditions of the cells or DNA at the time of irradiation. 34 refs., 5 figs., 2 tabs.

  2. 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.

  3. Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis

    SciTech Connect

    Cedervall, B.; Dewey, W.C.; Wong, R.

    1995-07-01

    Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes), (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/{Delta}M/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, wheras the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 {plus_minus} 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with {sub 125}IdU. 25 refs., 6 figs.

  4. Present status of radiation interaction with DNA-strand-break cross-section and fragment-size distributions

    NASA Astrophysics Data System (ADS)

    Smirnov, F. A. Castro; Rodriguez, O.; Neto, J. D. T. Arruda

    A cross-section formula for single strand brakes via radiation in the DNA molecule is reviewed and discussed, showing that it describes well-existing data for a wide range of linear energy transfer (LET). Recently published DNA fragment-size distributions (FSDs) data, obtained by irradiation of plasmids with electrons and neutrons, are presented. These results are analyzed and interpreted on the basis of a model developed by the Sao Paulo group, in which by introducing a long-range correlation among the jumps in a random walk, a power-law function is obtained for the probability distribution. The power-law exponent is a function of both the correlation potential and the degree of freedom of the system. It is shown that plasmidial DNA behaves as a nonextensive system, with FSDs described by power laws but not by exponential functions (as in extensive statistics). A pronounced crossover for electron-irradiation data and a change from 1 to 3 in the degree of freedom for neutron irradiation at a high dose were observed. A likely interpretation for these two features is presented and discussed. Perspectives for further developments in this area are presented and addressed.

  5. Fragmentation of genomic DNA using microwave irradiation.

    PubMed

    Yang, Yu; Hang, Jun

    2013-07-01

    An unconventional approach for DNA fragmentation was investigated to explore its feasibility as an alternative to the existing DNA fragmentation techniques for next-generation DNA sequencing application. Current methods are based on strong-force liquid shearing or specialized enzymatic treatments. There are shortcomings for these platforms yet to be addressed, including aerosolization of genomic materials, which may result in the cross-contamination and biohazards; the difficulty in multiplexing; and the potential sequence biases. In this proof-of-concept study, we investigated the microwave irradiation as a simple, unbiased, and easy-to-multiplex way to fragment genomic DNA randomly. In addition, heating DNA at high temperature was attempted for the same purpose and for comparison. Adaptive focused acoustic sonication was used as the control. The yield and functionality for the DNA fragments and DNA fragment libraries were analyzed to assess the feasibility and use of the proposed approach. Both microwave irradiation and thermal heating can fragment genomic DNA to the size ranges suitable for next-generation sequencing (NGS) shotgun library preparation. However, both treatments caused severe reduction in PCR amplification efficiency, which led to low production in emulsion PCR (emPCR). The result was improved by amplification prior to emPCR. Further improvements, such as DNA strand repairing, are needed for the method to be applied practically in NGS. PMID:23814501

  6. 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.

  7. Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: implications for the DNA fragmentation process during macronuclear development?

    PubMed

    Maercker, C; Lipps, H J

    1993-01-01

    The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7-9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. PMID:8293579

  8. Assembling DNA fragments by USER fusion.

    PubMed

    Annaluru, Narayana; Muller, Hlose; Ramalingam, Sivaprakash; Kandavelou, Karthikeyan; London, Viktoriya; Richardson, Sarah M; Dymond, Jessica S; Cooper, Eric M; Bader, Joel S; Boeke, Jef D; Chandrasegaran, Srinivasan

    2012-01-01

    Recent advances in DNA synthesis technology make it possible to design and synthesize DNA fragments of several kb in size. However, the process of assembling the smaller DNA fragments into a larger DNA segment is still a cumbersome process. In this chapter, we describe the use of the uracil specific excision reaction (USER)-mediated approach for rapid and efficient assembly of multiple DNA fragments both in vitro and in vivo (using Escherichia coli). For USER fusion in vitro assembly, each of the individual building blocks (BBs), 0.75 kb in size (that are to be assembled), was amplified using the appropriate forward and reverse primers containing a single uracil (U) and DNA polymerase. The overlaps between adjoining BBs were 8-13 base pairs. An equimolar of the amplified BBs were mixed together and treated by USER enzymes to generate complementary 3' single-strand overhangs between adjoining BBs, which were then ligated and amplified simultaneously to generate the larger 3-kb segments. The assembled fragments were then cloned into plasmid vectors and sequenced to confirm their identity. For USER fusion in vivo assembly in E. coli, USER treatment of the BBs was performed in the presence of a synthetic plasmid, which had 8-13 base pair overlaps at the 5'-end of the 5' BB and at the 3'-end of the 3' BB in the mixture. The USER treated product was then transformed directly into E. coli to efficiently and correctly reconstitute the recombinant plasmid containing the desired target insert. The latter approach was also used to rapidly assemble three different target genes into a vector to form a new synthetic plasmid construct. PMID:22328427

  9. 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

  10. 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.

  11. Multiplex dsDNA fragment sizing using dimeric intercalation dyes and capillary array electrophoresis: Ionic effects on the stability and electrophoretic mobility of DNA-dye complexes

    SciTech Connect

    Clark, S.M.; Mathies, R.A.

    1997-04-01

    Methods have been developed for performing accurate, high-resolution, multiplex capillary electrophoresis separations of dsDNA using dimeric intercalation dyes as noncovalent labeling reagents. The quality of these separations is highly dependent on the cation present during electrophoresis. Using buffers that contain only one cation, we show that the tetrapentylammonium (NPe{sub 4}{sup +}) ion results in high-resolution, high-sensitivity separations but that smaller ions such as sodium or the commonly used buffer ion tris produce low-resolution, low-intensity separations of DNA-dye complexes. Using an 80 mM taps-NPe{sub 4}, 1 mM H{sub 2}EDTA, pH 8.4, 0.8% HEC separation buffer, high-quality multiplex separations were performed using TOTO and buTOTIN, YOYO and TOED2, and TO and buTOTIN labeled restriction digests. In the taps-NPe{sub 4} buffer, there is no significant mobility shift when complexes are formed with DNA-dye ratios from 100 to 5 bp per dye and very little dye transfer was observed. This property permits accurate multiplex sizing of samples having a wide concentration range simply by mixing the DNA with a dye solution before electrophoresis. This capability is demonstrated by diluting unpurified PCR products 10-, 100-, and 1,000-fold before mixing with a 1 nM TOTO solution and separating these samples with a {Phi}X174 HAEIII sizing ladder complexed with buTOTIN. 62 refs., 10 figs., 1 tab.

  12. Assay for the quantification of intact/fragmented genomic DNA.

    PubMed

    Georgiou, Christos D; Papapostolou, Ioannis

    2006-11-15

    This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay. PMID:16942746

  13. Generation of Specific Repeated Fragments of Eukaryote DNA

    PubMed Central

    Mowbray, S. L.; Landy, A.

    1974-01-01

    Calf-thymus DNA, hydrolyzed with a site-specific endonuclease from Haemophilus influenzae Rd, yields 12 discrete bands on polyacrylamide-agarose gels. These range in size from 7.5 104 to 2 106 daltons, and they represent about 5% of the total DNA with individual fragments comprising 0.1-1.5%. The various DNA segments are repeated between 1500 and 220,000 times per haploid genome. Whereas the wide range of reiteration frequencies suggests different origins for some of the fragments, the bias in fragment densities in CsCl and in Ag+-Cs2SO4 toward those of known satellite DNAs suggests similar origins for some of them. Models for the possible origin of the DNA fragments can be grouped into three distinct, experimentally distinguishable, classes. Images PMID:4525302

  14. DNA repair patch size measurements with nucleosomal DNA

    SciTech Connect

    Th'ng, J.P.; Walker, I.G.

    1983-01-01

    A modified method for measuring DNA repair patch sizes by the bromodeoxyuridine buoyant density shift method is presented. This method involves the digestion of total chromatin by Staphylococcal nuclease to produce small DNA fragments with a very homogeneous size distribution. Using this method to measure the size of repair patches following treatment with u.v.-light or dimethyl sulfate, a calculated incorporation of 40 nucleotides was obtained. This value is identical to previously published patch size estimations obtained by the buoyant density shift method with sonically produced DNA fragments. It is also in close agreement with values recently obtained by the bromodeoxyuridine photolysis method. Taken together, these independent results obtained with a variety of DNA damaging agents suggest that excision repair patch size is independent of the damaging agent.

  15. 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. PMID:26879456

  16. Ultraviolet ray induces chromosomal giant DNA fragmentation followed by internucleosomal DNA fragmentation associated with apoptosis in rat glioma cells.

    PubMed

    Higuchi, Yoshiro; Mizukami, Yuji; Yoshimoto, Tanihiro

    2003-12-01

    Giant DNA fragments (1-2 Mbp) were found in C6 rat glioma cells irradiated by a lethal dose of ultraviolet-C (UV-C, 254 nm) at 50 J/m(2). After irradiation, the fragments mutated into high-molecular-weight (100-800 kbp) DNA fragments and then into ladder-formed internucleosomal DNA fragments. Poly-ADP-ribose polymerase (PARP) activity and NAD levels were reduced during DNA fragmentation. Some inhibitors of caspase and protease inhibited DNA ladder formation, but not giant DNA fragmentation, whereas antioxidants did not inhibit DNA fragmentation. These results suggest that a lethal dose of UV radiation induces giant DNA fragmentation and leads to internucleosomal DNA fragmentation associated with apoptosis through some caspases and nonreactive oxygen species in cells. PMID:15033744

  17. 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.

  18. RNA-Linked Nascent DNA Fragments in Escherichia coli*

    PubMed Central

    Sugino, Akio; Hirose, Susumu; Okazaki, Reiji

    1972-01-01

    Nucleic acid that is extracted from E. coli labeled by a brief pulse of [3H]dT and depatured by treatment with heat, formamide, or formaldehyde bands in a region with a density higher than that of single-stranded E. coli DNA in a Cs2SO4 equilibrium density gradient. If treated with alkali or RNase, it then exhibits the density of single-stranded DNA. These results suggest the presence of a short strand of RNA covalently linked to the nascent DNA. Evidence for the presence of covalently linked RNA-DNA molecules is also obtained by pulse labeling with [3H]U. Analyses of nascent nucleic acids from cells pulse labeled for various times, and of the molecules with different sizes, support the hypothesis that the short DNA fragments are formed by extension of even shorter RNA chains, which are synthesized on the parental DNA strands and are removed before ligation of the DNA fragments. The synthesis of the RNA segment of the RNA-DNA molecule is much less sensitive to rifampicin than is the synthesis of bulk RNA. Images PMID:4558661

  19. 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.

  20. 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.

  1. a Relation for Fragment Sizes of Explosive Loaded Aluminum Cylinders

    NASA Astrophysics Data System (ADS)

    Leadbetter, J.; Donahue, L.; Ripley, R. C.; Zhang, F.

    2009-12-01

    It is well established that the fragment size from explosively-loaded metal cylinders can be related to the casing expansion rate driven by high pressure explosive detonation products. During expansion, high strain rates alter material properties, and failure mechanisms influencing the fragment size distribution become dependent on dynamic phenomena such as localized shear banding. The purpose of this paper is to extend known relations for steel fragment size to aluminum cylinders. The present approach utilizes high strain rate properties of aluminum with energy-based dynamic fragmentation theory to determine the mean fragment size, and a Mott statistical distribution to account for randomness in the natural fragmentation process. To determine the strain rate, the explosive detonation and cylinder expansion are modeled using a two-way coupled CFD/FEA routine. Further analysis of a series of modeling results is shown to evaluate relationships for aluminum fragment size as a function of detonation energy and the metal-to-explosive mass ratio.

  2. GP7 induces internucleosomal DNA fragmentation independent of caspase activation and DNA fragmentation factor in NB4 cells.

    PubMed

    Qi, She-Ning; Jing, Yuan-Xue; Dong, Gen-Xi; Chen, Yan; Yoshida, Akira; Ueda, Takanori

    2007-07-01

    DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process. PMID:17549379

  3. 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.

  4. A Relation for Fragment Sizes of Explosive Loaded Aluminum Cylinders

    NASA Astrophysics Data System (ADS)

    Leadbetter, J.; Donahue, L.; Ripley, R. C.; Zhang, F.

    2009-06-01

    It is well established that the fragment size from explosively-loaded metal cylinders can be related to the expansion rate driven by high pressure explosive detonation products. During expansion, high strain rates alter material properties, and failure mechanisms influencing fragment size distribution become dependent on dynamic phenomena such as localized shear banding. The purpose of this paper is to extend known relations for steel fragment size to aluminum cylinders. The present approach utilizes high strain rate properties of aluminum with Grady and Kipp dynamic fragmentation theory to determine mean fragment size, and a statistical fragment distribution to account for randomness in the aluminum cylinder material. To determine the expansion strain rate, the explosive detonation and cylinder expansion are modeled using a two-way coupled fluid structure interaction routine. Experimental results for an aluminum-cased C4 charge are provided for model validation. Further analysis of a series of modeling results is shown to establish relationships for aluminum fragment size as a function of detonation pressure and the metal to explosive mass ratio.

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

    PubMed

    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

  6. 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.

  7. Impact and explosion crater ejecta, fragment size, and velocity

    NASA Technical Reports Server (NTRS)

    Okeefe, J. D.; Ahrens, T. J.

    1983-01-01

    A model was developed for the mass distribution of fragments that are ejected at a given velocity for impact and explosion craters. The model is semi-empirical in nature and is derived from (1) numerical calculations of cratering and the resultant mass versus ejection velocity, (2) observed ejecta blanket particle size distributions, (3) an empirical relationship between maximum ejecta fragment size and crater diameter and an assumption on the functional form for the distribution of fragements ejected at a given velocity. This model implies that for planetary impacts into competent rock, the distribution of fragments ejected at a given velocity are nearly monodisperse, e.g., 20% of the mass of the ejecta at a given velocity contain fragments having a mass less than 0.1 times a mass of the largest fragment moving at that velocity. Using this model, the largest fragment that can be ejected from asteroids, the moon, Mars, and Earth is calculated as a function of crater diameter. In addition, the internal energy of ejecta versus ejecta velocity is found. The internal energy of fragments having velocities exceeding the escape velocity of the moon will exceed the energy required for incipient melting for solid silicates and thus, constrains the maximum ejected solid fragment size.

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

    PubMed

    Overballe-Petersen, Sren; 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, Sren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G; Nielsen, Rasmus; Johnsen, Pl 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

  9. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

    Overballe-Petersen, Sren; 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, Sren; Inselmann, Sabrina; de Vries, Johann; Wackernagel, Wilfried; Pybus, Oliver G.; Nielsen, Rasmus; Johnsen, Pl 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

  10. 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.

  11. Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2013-05-01

    The generation of a uniquely (32)P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and "filling in the end" with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [?-(32)P]ATP. This generates a plasmid labeled at each end with ?-(32)P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%-8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1-2 d. PMID:23637369

  12. Polyethylene glycol derivatives of base and sequence specific DNA ligands: DNA interaction and application for base specific separation of DNA fragments by gel electrophoresis.

    PubMed Central

    Mller, W; Hattesohl, I; Schuetz, H J; Meyer, G

    1981-01-01

    Various base pair specific DNA ligands comprising a phenyl phenazinium dye, a triphenylmethan dye and Hoechst 33258 were covalently bound to polyethylene glycol (PEG) via ester or ether bonds. The DNA interactions of the PEG derivatives formed were shown to exhibit the same base pair specificity as the parent compounds. Since the PEG chains thus bound to the DNA could be expected to increase drastically the frictional coefficient of the DNA, the PEG derivatives were used for base specific DNA separations in agarose and polyacrylamide gel electrophoresis. The procedures, which do not require any special techniques, are described in detail. The resolution observed in agarose gels allows one to separate equally sized DNA fragments differing as little as 1% in base composition at mean travel distances of about 10 cm. Examples of gels showing the base compositional heterogeneity of restriction fragments obtained from lambda DNA, E. coli DNA and calf thymus DNA are given. Images PMID:6259622

  13. Aggregate Formation from Short Fragments of Plant DNA 1

    PubMed Central

    Thompson, William F.

    1976-01-01

    Large aggregates have been observed after partial reassociation of pea (Pisum sativum L.) DNA preparations sheared to mean single strand fragment lengths as short as 350 nucleotides. At high DNA concentrations and conditions of salt and temperature which require only moderate precision of base pairing, aggregates pelletable by brief centrifugation account for 30 to 40% of the total DNA from peas, while calf thymus DNA reassociated under similar conditions forms less than 10% pelletable structures. In contrast to networks formed during the reassociation of long DNA fragments containing interspersed repetitive sequences, these aggregates contain a high percentage of double-stranded DNA and are enriched in repetitive sequences. Aggregates detectable by centrifugation do not begin to appear until after extensive repetitive sequence reassociation has already occurred. The results are consistent with a model involving secondary reassociation between single-stranded regions (hanging tails) remaining after initial duplex formation. This process would lead to formation of large multimers of the original fragments, analogous to the large hyperpolymers which have been observed in extensively reassociated prokaryotic DNA. Randomly sheared fragments containing short (about 300 base pairs) repetitive sequences interspersed with single copy DNA would not be expected to hyperpolymerize significantly under these conditions. I suggest, as a working hypothesis, that much of the repetitive sequence DNA in peas is contained in regions considerably longer than 300 base pairs. PMID:16659538

  14. Sperm DNA fragmentation, recurrent implantation failure and recurrent miscarriage

    PubMed Central

    Coughlan, Carol; Clarke, Helen; Cutting, Rachel; Saxton, Jane; Waite, Sarah; Ledger, William; Li, Tinchiu; Pacey, Allan A

    2015-01-01

    Evidence is increasing that the integrity of sperm DNA may also be related to implantation failure and recurrent miscarriage (RM). To investigate this, the sperm DNA fragmentation in partners of 35 women with recurrent implantation failure (RIF) following in vitro fertilization, 16 women diagnosed with RM and seven recent fathers (control) were examined. Sperm were examined pre- and post-density centrifugation by the sperm chromatin dispersion (SCD) test and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. There were no significant differences in the age of either partner or sperm concentration, motility or morphology between three groups. Moreover, there were no obvious differences in sperm DNA fragmentation measured by either test. However, whilst on average sperm DNA fragmentation in all groups was statistically lower in prepared sperm when measured by the SCD test, this was not seen with the results from the TUNEL assay. These results do not support the hypothesis that sperm DNA fragmentation is an important cause of RIF or RM, or that sperm DNA integrity testing has value in such patients. It also highlights significant differences between test methodologies and sperm preparation methods in interpreting the data from sperm DNA fragmentation tests. PMID:25814156

  15. 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.

  16. 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.

  17. 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 ?-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 (Relative Biological Effectiveness) of high LET radiation. This work was partially supported by EU ("RISC-RAD" project, Contract no. FI6R-CT 2003- 508842, and "NOTE" project, Contract no. FI6R-036465) and ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W.Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat.Biol. 81, 841-854 (2005). D. Alloni, F. Ballarini, M. Belli, A. Campa, G. Esposito, W. Friedland, M.Liotta, A. Ottolenghi and H. G. Paretzke. Modeling of DNA fragmentation induced in human fibroblasts by 56 Fe ions. Adv. Space Res. 40, 1401-1407 (2007a). D. Alloni, F. Antonelli, F. Ballarini, M. Belli, A. Campa, V. Dini, G.Esposito, W. Friedland, M. Liotta, A. Ottolenghi, H. G. Paretzke, G. Simone, E. Sorrentino and M. A. Tabocchini. Small DNA fragments induced in human fibroblasts by 56 Fe ions: experimental data and MC simulations. Proc. "Ion Beams in biology and medicine", Heidelberg, 26-29 September 2007, edited by J. Debus, K. Henrichs, G. Kraft, p. 164 (2207b).

  18. Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation.

    PubMed

    Yan, B; Wang, H; Li, F; Li, C-Y

    2006-12-18

    Previously it was shown that horizontal DNA transfer between mammalian cells can occur through the uptake of apoptotic bodies, where genes from the apoptotic cells were transferred to neighbouring cells phagocytosing the apoptotic bodies. The regulation of this process is poorly understood. It was shown that the ability of cells as recipient of horizontally transferred DNA was enhanced by deficiency of p53 or p21. However, little is known with regard to the regulation of DNA from donor apoptotic cells. Here we report that the DNA fragmentation factor/caspase-activated DNase (DFF/CAD), which is the endonuclease responsible for DNA fragmentation during apoptosis, plays a significant role in regulation of horizontal DNA transfer. Cells with inhibited DFF/CAD function are poor donors for horizontal gene transfer (HGT) while their ability of being recipients of HGT is not affected. PMID:17146478

  19. 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 grinding conditions, or as discrete steps, such as with impact events. In both cases our model gives the energy associated with the fragmentation in terms of the developing surface area of the population. We show the agreement of our model to the evolution of particle size distributions associated with episodic and continuous fragmentation and how the evolution of some popular fractals may be represented using this approach. C. A. Charalambous and W. T. Pike (2013). Multi-Scale Particle Size Distributions of Mars, Moon and Itokawa based on a time-maturation dependent fragmentation model. Abstract Submitted to the AGU 46th Fall Meeting. Bird, N. R. A., Watts, C. W., Tarquis, A. M., & Whitmore, A. P. (2009). Modeling dynamic fragmentation of soil. Vadose Zone Journal, 8(1), 197-201. Reid, K. J. (1965). A solution to the batch grinding equation. Chemical Engineering Science, 20(11), 953-963. Turcotte, D. L. (1986). Fractals and fragmentation. Journal of Geophysical Research: Solid Earth 91(B2), 1921-1926.

  20. Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

    PubMed

    Zhang, Zijie; Liu, Juewen

    2016-03-16

    Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry. PMID:26910515

  1. 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 degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.

  2. DNA fragment editing of genomes by CRISPR/Cas9.

    PubMed

    Jinhuan, Li; Jia, Shou; Qiang, Wu

    2015-10-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system from bacteria and archaea emerged recently as a new powerful technology of genome editing in virtually any organism. Due to its simplicity and cost effectiveness, a revolutionary change of genetics has occurred. Here, we summarize the recent development of DNA fragment editing methods by CRISPR/Cas9 and describe targeted DNA fragment deletions, inversions, duplications, insertions, and translocations. The efficient method of DNA fragment editing provides a powerful tool for studying gene function, regulatory elements, tissue development, and disease progression. Finally, we discuss the prospects of CRISPR/Cas9 system and the potential applications of other types of CRISPR system. PMID:26496751

  3. 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.

  4. Novel separation and detection methods of DNA fragments in electrophoresis

    SciTech Connect

    Chan, King Cheung

    1993-01-27

    A charge-coupled device (CCD) based electrophoresis system was developed. The system allowed non-destructive, sensitive, and on-line detection of native DNA in slab-gel electrophoresis via ultraviolet absorption measurement. The detection limit of double-stranded DNA fragment was 5 ng per band. Since the amount of DNA used in this experiment was typical, the CCD-based system could be readily implemented in molecular biology. Gel-filled and non-gel sieving capillary electrophoresis was developed for rapid and efficient separation of double-stranded DNA fragments. For the gel-filled CE separation a new gel matrix, the HydroLink gel (HL), was used. The HL capillary gel was easier to cast than the polyacrylamide capillary gel. For the non-gel separation, a GC capillary was used as the separation chamber, and cellulose additive was included in the electrophoresis as the sieving medium. Indirect fluorometry was applied in non-gel and gel electrophoresis for the detection of DNA fragments. This method allowed non-destructive and on-line detection of DNA during electrophoresis. The amount of DNA used with this method was comparable to those obtained with absorption measurement.

  5. Novel separation and detection methods of DNA fragments in electrophoresis

    SciTech Connect

    Chan, K.C.

    1992-01-01

    A charge-coupled device (CCD) based electrophoresis system was developed. The system allowed non-destructive, sensitive, and on-line detection of native DNA in slab-gel electrophoresis via ultraviolet absorption measurement. The detection limit of double-stranded DNA fragment was 5 ng per band. Since the amount of DNA used in this experiment was typical, the CCD-based system could be readily implemented in molecular biology. Gel-filled and non-gel sieving capillary electrophoresis (CE) was developed for rapid and efficient separation of double-stranded DNA fragments. For the gel-filled CE separation a new gel matrix, the HydroLink gel (HL), was used. The HL capillary gel was easier to cast than the polyacrylamide capillary gel. For the non-gel separation, a GC capillary was used as the separation chamber, and cellulose additive was included in the electrophoresis as the sieving medium. Indirect fluorometry was applied in non-gel and gel electrophoresis for the detection of DNA fragments. This method allowed nondestructive and on-line detection of DNA during electrophoresis. The amount of DNA used with this method was comparable to those obtained with absorption measurement.

  6. 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.

  7. Chromatin supraorganization, DNA fragmentation, and cell death in snake erythrocytes.

    PubMed

    Miyamoto, Maristela; Vidal, Benedicto C; Mello, Maria Luiza S

    2005-02-01

    In nucleate erythrocytes of several vertebrate groups, the frequency and intensity of DNA fragmentation associated with programmed cell death vary considerably. Although hemoglobin efficiency may be related to erythrocyte life span, and hemoglobin types and erythrocyte life spans are assumed to vary in reptiles, no data on DNA fragmentation and chromatin organization as related to cell death exist for snakes. In the present study, chromatin supraorganization, DNA fragmentation, and cell death were investigated in four snake species (Crotalus durissus terrificus, Bothrops jararaca, Bothrops alternatus, and Bothrops neuwiedii), which differ in their geographical distribution and habitats, by using image analysis of Feulgen hydrolysis kinetics, the TUNEL assay, single-cell gel electrophoresis, and transmission electron microscopy. Relatively few circulating erythrocytes were found to be simultaneously committed to cell death, although there was some variation among the snake species. Conspicuous nuclear and cytoplasmic organelles suggestive of metabolic activity were seen ultrastructurally in most snake erythrocytes. The DNA of the snake erythrocyte chromatin was much more resistant to Feulgen acid hydrolysis (DNA depurination and breakdown) than that of young adult bullfrog erythrocytes, which had a high frequency and intensity of DNA fragmentation. Of the species studied, B. neuwiedii and C. d. terrificus showed the greatest resistance to Feulgen acid hydrolysis and to the DNA fragmentation, revealed by the TUNEL assay. Although B. neuwiedii also showed the lowest frequency of cells with more damaged DNA in the single-cell gel electrophoresis assay, C. d. terrificus had the highest frequency of damaged cells, possibly because of the abundance of alkaline-sensitive DNA sites. The results for DNA fragmentation and cell death in erythrocytes of B. jararaca and B. alternatus generally differed from those for C. d. terrificus and B. neuwiedii and may reflect differences in the biology of these species selected under different geographical habitats. The differences in erythrocyte cell biology reported here may be related to hemoglobin variants selected in the mentioned snake species and that would lead the cells to different resistances to unfavorable environmental conditions. PMID:15746963

  8. Effects of pretreatment on the denaturation and fragmentation of genomic DNA for DNA hybridization.

    PubMed

    Wang, Xiaofang; Son, Ahjeong

    2013-12-01

    DNA hybridization is an important step for a number of bioassays such as fluorescence in situ hybridization, microarrays, as well as the NanoGene assay. Denaturation and fragmentation of genomic DNA are two critical pretreatments for DNA hybridization. However, no thorough and systematic characterization on denaturation and fragmentation has been carried out for the NanoGene assay so far. In this study, we investigated the denaturation and fragmentation of the bacterial gDNA with physical treatments (i.e., heating and sonication) and chemical treatments (i.e., dimethyl sulfoxide). First of all, a simple approach for indicating the denaturation fraction was developed based on the absorbance difference (i.e., hyperchromic effect) between the double-stranded DNA and single-stranded DNA fragments. Then the denaturation capabilities of the treatments to the gDNA were elucidated, followed by the examination of the possible renaturation over time. The fragmentation of the gDNA by each treatment was also investigated. Based on denaturation efficiency, minimum renaturation tendency, and fragmentation, the sonication method was found to be the best among the six methods. We further demonstrated that the sonication method produced the best result among the treatments examined for the DNA hybridization in the NanoGene assay. PMID:24162665

  9. Fragmentation of DNA in a sub-microliter microfluidic sonication device.

    PubMed

    Tseng, Qingzong; Lomonosov, Alexey M; Furlong, Eileen E M; Merten, Christoph A

    2012-11-21

    Fragmentation of DNA is an essential step for many biological applications including the preparation of next-generation sequencing (NGS) libraries. As sequencing technologies push the limits towards single cell and single molecule resolution, it is of great interest to reduce the scale of this upstream fragmentation step. Here we describe a miniaturized DNA shearing device capable of processing sub-microliter samples based on acoustic shearing within a microfluidic chip. A strong acoustic field was generated by a Langevin-type piezo transducer and coupled into the microfluidic channel via the flexural lamb wave mode. Purified genomic DNA, as well as covalently cross-linked chromatin were sheared into various fragment sizes ranging from ∼180 bp to 4 kb. With the use of standard PDMS soft lithography, our approach should facilitate the integration of additional microfluidic modules and ultimately allow miniaturized NGS workflows. PMID:23014736

  10. Fiber optic system for rapid analysis of amplified DNA fragments

    NASA Astrophysics Data System (ADS)

    Mauro, J. Matthew; Cao, Lynn K.; Golden, Joel P.

    1996-04-01

    We have developed a fiber optic sensor for rapid and direct analysis of PCR-amplified DNA fragments with minimal sample processing and real-time data readout. To accomplish this, a novel DNA-recognition system was built onto the surface of fused silica fibers. DNA fragments, labeled with a fluorophore during amplification, are bound to and detected at the fiber surface by means of evanescent wave excitation/emission. Excess unincorporated fluorescent single-stranded oligonucleotide PCR primers make only a small contribution to the signal, as the modified fiber surface only efficiently binds double-stranded DNA with the proper PCR-incorporated terminal nucleotide sequence (5'-ATGACTCAT-3'). The surface- bound double-stranded DNA recognition element utilizes a genetically engineered dimeric sequence-specific DNA binding protein. Self-assembly into the proper conformation for binding DNA occurs by means of specific interactions of the active dimer with the Fc domains of a layer of IgG molecules (antibodies) covalently attached directly to the fiber surface. The modified fiber surface is regenerated between samples by stripping away bound DNA with high salt concentrations.

  11. Efficient detection of single DNA fragments in flowing sample streams by two-photon fluorescence excitation.

    PubMed

    Van Orden, A; Cai, H; Goodwin, P M; Keller, R A

    1999-06-01

    This paper reports the demonstration of efficient single molecule detection in flow cytometry by two-photon fluorescence excitation. We have used two-photon excitation (TPE) to detect single DNA fragments as small as 383 base pairs (bp) labeled with the intercalating dye, POPO-1, at a dye:nucleotide ratio of 1:5. TPE of the dye-DNA complexes was accomplished using a mode-locked, 120 fs pulse width Ti:sapphire laser operating at 810 nm. POPO-1 labeled DNA fragments of 1.1 kilobase pairs (kbp) and larger were sequentially detected in our flow cytometry system with a detection efficiency of nearly 100%. The detection efficiency for the 383 bp DNA fragments was approximately 75%. We also demonstrate the ability to distinguish between different sized DNA fragments in a mixture by their individual fluorescence burst sizes by TPE. These studies indicate that using TPE for single molecule flow cytometry experiments lowers the intensity of the background radiation by approximately an order of magnitude compared to one-photon excitation, due to the large separation between the excitation and emission wavelengths in TPE. PMID:21662745

  12. DNA fragmentation in mouse organs during endotoxic shock.

    PubMed Central

    Bohlinger, I.; Leist, M.; Gantner, F.; Angermller, S.; Tiegs, G.; Wendel, A.

    1996-01-01

    The systemic inflammatory response syndrome has still an unpredictable outcome, and patients often die of multiple organ failure despite circulatory stabilization therapy. The still incompletely understood pathophysiological mechanisms include organ damage due to direct toxic actions of cytokines elicited by overactivation of the host response. To study this process of organ failure in experimental septic shock, we injected mice with a lethal dose of endotoxin and examined apoptotic and necrotic tissue damage biochemically, histologically, and ultrastructurally. Endotoxin administration caused oligonucleosomal as well as random DNA fragmentation in liver, lung, kidney, and intestine. In the liver, DNA fragmentation was not restricted to hepatocytes but also occurred in nonparenchymal cells. The DNA fragmentation was mediated by tumor necrosis factor and attenuated by endogenous nitric oxide release. Unlike the situation in D-galactosamine-sensitized mice, in which injection or release of tumor necrosis factor causes massive hepatocyte apoptosis, liver failure due to high doses of endotoxin was characterized by single-cell necrosis, a low incidence of apoptosis, and simultaneous damage to nonparenchymal cells. We conclude that, even though endotoxin causes cytokine-mediated DNA fragmentation in several organs including the liver, hepatocyte apoptosis itself seems to be a minor phenomenon in high-dose endotoxic shock in mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8863685

  13. 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 universal DNA ligase, which can potentially substitute or backup the repair and replication functions of all other DNA ligases in the cell nucleus. Thus, the old model of functionally dedicated DNA ligases is now replaced by one in which only Lig4 remains dedicated to C-NHEJ, with Lig1 and Lig3 showing an astounding functional flexibility and interchangeability for practically all nuclear ligation functions. The underlying mechanisms of Lig3 versus Lig1 utilization in DNA repair and replication are expected to be partly different and remain to be elucidated. PMID:26110316

  14. The orientation of DNA fragments in the agarose gels.

    PubMed

    Borejdo, J; DeFea, K

    1988-11-01

    A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic-acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10(13)g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of phi X174 DNA RF/HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA. PMID:3239744

  15. Sizing femtogram amounts of dsDNA by single-molecule counting

    PubMed Central

    Torchinsky, Dmitry; Ebenstein, Yuval

    2016-01-01

    Modern molecular-biology applications raise renewed interest in sizing minute-amounts of DNA. In this work we utilize single-molecule imaging with in situ size calibration to accurately analyze the size and mass distribution of DNA samples. We exploit the correlation between DNA length and its fluorescence intensity after staining in order to assess the length of individual DNA fragments by fluorescence microscopy. Synthetic reference DNA standards are added to the investigated sample before staining and serve as internal size calibrators, supporting a robust assay for accurate DNA sizing. Our results demonstrate the ability to reconstruct the exact length distribution in a complex DNA sample by sizing a subset containing only femtogram amounts of DNA, thus, outperforming microfluidic gel electrophoresis which is the currently accepted gold standard. This assay may find useful applications for genetic analysis where the exact size distribution of DNA molecules is critical and the availability of genetic material is limited. PMID:26365235

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

    PubMed

    Hakenberg, S; Hgle, 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

  17. CGE-laser induced fluorescence of double-stranded DNA fragments using GelGreen dye.

    PubMed

    Valds, Alberto; Garca-Caas, Virginia; Cifuentes, Alejandro

    2013-06-01

    Nowadays, new solutions focused on the replacement of reagents hazardous to human health are highly demanded in laboratories and Green Chemistry. In the present work, GelGreen, a new nonhazardous DNA staining reagent, has been assayed for the first time to analyze double-stranded DNA by CGE with LIF detection. The effect of GelGreen concentration on S/N ratio and migration time of a wide concentration range of standard DNA mixtures was evaluated. Under optimum GelGreen concentration in the sieving buffer efficient and sensitive separations of DNA fragments with sizes from 100-500 base pairs (bp) were obtained. A comparison in terms of resolution, time of analysis, LOD, LOQ, reproducibility, sizing performance, and cost of analysis was established between two optimized CGE-LIF protocols for DNA analysis, one based on the dye YOPRO-1 (typically used for CGE-LIF of DNA fragments) and another one using the new GelGreen. Analyses using YOPRO-1 were faster than those using GelGreen (ca. 31 min versus 34 min for the analysis of 100-500 bp DNA fragments). On the other side, sensitivity using GelGreen was twofold higher than that using YOPRO-1. The cost of analysis was significantly cheaper (ninefold) using GelGreen than with YOPRO-1. The resolution values and sizing performance were not significantly different between the two dyes (e.g. both dyes allowed the separation of fragments differing in only 2 bp in the 100-200 bp range). The usefulness of the separation method using GelGreen is demonstrated by the characterization of different amplicons obtained by PCR. PMID:23417332

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

    PubMed

    Li, Zhe; Krippendorff, Ben-Fillippo; Sharma, Sharad; Walz, Antje C; Lav, 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 ~35kDa. 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

  19. Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.

    PubMed

    Tsuge, Kenji; Sato, Yukari; Kobayashi, Yuka; Gondo, Maiko; Hasebe, Masako; Togashi, Takashi; Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn't require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible. PMID:25990947

  20. Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments

    PubMed Central

    Tsuge, Kenji; Sato, Yukari; Kobayashi, Yuka; Gondo, Maiko; Hasebe, Masako; Togashi, Takashi; Tomita, Masaru; Itaya, Mitsuhiro

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesnt require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible. PMID:25990947

  1. Fragment-size prediction during dynamic fragmentation of melted tin. Experimental investigation and modelling issues

    NASA Astrophysics Data System (ADS)

    Roy, Gilles; Signor, Loic; de Resseguier, Thibaut; Dragon, Andre; Llorca, Fabrice

    2007-06-01

    A triangular shock-wave of sufficient intensity propagating in a metal sample may induce melting. When it reaches the free surface, tensile stresses are generated in the liquid state and lead to the creation of an expanding continuum of liquid debris. This phenomenon called micro-spalling consists of a dynamic fragmentation process in the melted material. Relevant data are still few but important for developing robust and physics-based models. Recently, we have reported a qualitative investigation of micro-spall in tin samples submitted to laser shocks [J. Appl. Phys. 101, 013506, 2007]. The present paper contains new experimental results including fragment recovery using a low density PVC-foam and post-test evaluation of the fragment-size distribution using X-ray microtomography. These results are compared to theoretical predictions from hydrocode simulations coupled with a modified formulation of the well-known energy fragmentation model of D.E. Grady [J. Mech. Phys. Sol., 36(3), pp.353-384, 1988].

  2. Efficient double fragmentation ChIP-seq provides nucleotide resolution protein-DNA binding profiles.

    PubMed

    Mokry, Michal; Hatzis, Pantelis; de Bruijn, Ewart; Koster, Jan; Versteeg, Rogier; Schuijers, Jurian; van de Wetering, Marc; Guryev, Victor; Clevers, Hans; Cuppen, Edwin

    2010-01-01

    Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2/TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB/SOLiD platform, the same approach may be applied to other platforms. PMID:21152096

  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. Controlling DNA fragmentation in MALDI-MS by chemical modification

    SciTech Connect

    Tang, W.; Zhu, L.; Smith, L.M.

    1997-02-01

    Fragmentation has proven to be a major factor limiting accessible mass range, sensitivity, and mass resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous work has shown that this DNA fragmentation is strongly dependent on both the MALDI matrix and the nucleic acid sequence employed. Fragmentation is initiated by nucleobase protonation, leading to cleavage of the N-glycosidic bond with base loss, followed by cleavage of the phosphodiester backbone. In this study, asymmetric oligonucleotides incorporating cytidine and cytidine analogs such as 5-methyl-2{prime}-deoxycytidine, 5-bromo-2{prime}-deoxycytidine, aracytidine, and 2{prime}-fluorodeoxycytidine nucleosides were used to systematically investigate the influence of the structural changes on the stability of the N-glycosidic bond. Modifications of the deoxyribose sugar ring by replacing the 2{prime}-hydrogen with more electron-withdrawing groups such as the hydroxyl or fluoro group stabilize the N-glycosidic bond to a greater extent than the C5 nucleobase modifications. 2{prime}-Hydroxyl and 2{prime}-fluoro groups respectively are shown to partially or completely block fragmentation at the modified nucleosides. Mixtures of oligonucleotides incorporating such modifications demonstrate remarkably extended accessible mass range, as well as increased sensitivity and mass resolution. 39 refs., 11 figs.

  5. Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation.

    PubMed

    Feitosa, W B; Mendes, C M; Milazzotto, M P; Rocha, A M; Martins, L F; Simes, R; Paula-Lopes, F F; Visintin, J A; Assumpo, M E O A

    2010-09-01

    Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 x 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. PMID:20537697

  6. Analysis of different DNA fragments of Corynebacterium glutamicum complementing dapE of Escherichia coli.

    PubMed

    Wehrmann, A; Eggeling, L; Sahm, H

    1994-12-01

    In Corynebacterium glutamicum L-lysine is synthesized simultaneously via the succinylase and dehydrogenase variant of the diaminopimelate pathway. Starting from a strain with a disrupted dehydrogenase gene, three different-sized DNA fragments were isolated which complemented defective Escherichia coli mutants in the succinylase pathway. Enzyme studies revealed that in one case the dehydrogenase gene had apparently been reconstituted in the heterologous host. The two other fragments resulted in desuccinylase activity; one of them additionally in succinylase activity. However, the physical analysis showed that structural changes had taken place in all fragments. Using a probe derived from one of the fragments we isolated a 3.4 kb BamHI DNA fragment without selective pressure (by colony hybridization). This was structurally intact and proved functionally to result in tenfold desuccinylase overexpression. The nucleotide sequence of a 1966 bp fragment revealed the presence of one truncated open reading frame of unknown function and that of dapE encoding N-succinyl diaminopimelate desuccinylase (EC 3.5.1.18). The deduced amino acid sequence of the dapE gene product shares 23% identical residues with that from E. coli. The C. glutamicum gene now available is the first gene from the succinylase branch of lysine synthesis of this biotechnologically important organism. PMID:7881553

  7. FragMatch--a program for the analysis of DNA fragment data.

    PubMed

    Saari, T A; Saari, S K; Campbell, C D; Alexander, I J; Anderson, I C

    2007-03-01

    FragMatch is a user-friendly Java-supported program that automates the identification of taxa present in mixed samples by comparing community DNA fragment data against a database of reference patterns for known species. The program has a user-friendly Windows interface and was primarily designed for the analysis of fragment data derived from terminal restriction fragment length polymorphism analysis of ectomycorrhizal fungal communities, but may be adapted for other applications such as microsatellite analyses. The program uses a simple algorithm to check for the presence of reference fragments within sample files that can be directly imported, and the results appear in a clear summary table that also details the parameters that were used for the analysis. This program is significantly more flexible than earlier programs designed for matching RFLP patterns as it allows default or user-defined parameters to be used in the analysis and has an unlimited database size in terms of both the number of reference species/individuals and the number of diagnostic fragments per database entry. Although the program has been developed with mycorrhizal fungi in mind, it can be used to analyse any DNA fragment data regardless of biological origin. FragMatch, along with a full description and users guide, is freely available to download from the Aberdeen Mycorrhiza Group web page (http://www.aberdeenmycorrhizas.com). PMID:17226045

  8. Detection of Irradiated Food: DNA Fragmentation in Grapefruits

    NASA Astrophysics Data System (ADS)

    Delince, 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.

  9. Fragment size does not matter when you are well connected: effects of fragmentation on fitness of coexisting gypsophiles.

    PubMed

    Matesanz, S; Gómez-Fernández, A; Alcocer, I; Escudero, A

    2015-09-01

    Most habitat fragmentation studies have focused on the effects of population size on reproductive success of single species, but studies assessing the effects of both fragment size and connectivity, and their interaction, on several coexisting species are rare. In this study, we selected 20 fragments along two continuous gradients of size and degree of isolation in a gypsum landscape in central Spain. In each fragment, we selected 15 individuals of each of three dominant gypsophiles (Centaurea hyssopifolia, Lepidium subulatum and Helianthemum squamatum, 300 plants per species, 900 plants in total) and measured several reproductive traits: inflorescence number, fruit set, seed set and seed mass. We hypothesised that plant fitness would be lower on small and isolated fragments due to an interaction between fragment size and connectivity, and that response patterns would be species-specific. Overall, fragment size had very little effect on reproductive traits compared to that of connectivity. We observed a positive effect of fragment connectivity on C. hyssopifolia fitness, mediated by the increased seed predation in plants from isolated fragments, resulting in fewer viable seeds per capitulum and lower seed set. Furthermore, seed mass was lower in plants from isolated fragments for both C. hyssopifolia and L. subulatum. In contrast, few reproductive traits of H. squamatum were affected by habitat fragmentation. We discuss the implications of species-specific responses to habitat fragmentation for the dynamics and conservation of gypsum plant communities. Our results highlight the complex interplay among plants and their mutualistic and antagonistic visitors, and reinforce the often-neglected role of habitat connectivity as a key component of the fragmentation process. PMID:25765458

  10. 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.

  11. A wheat genomic DNA fragment reduces pollen transmission of maize transgenes by reducing pollen viability.

    PubMed

    Scott, M Paul; Peterson, Joan M; Moran, Daniel L; Sangtong, Varaporn; Smith, LaTrice

    2007-10-01

    A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal to the 1Dx5 coding sequence was replaced with the maize 27 kDa gamma-zein promoter. Like the wheat promoter-driven Glu-1Dx5 transgene, this zein promoter-driven transgene functioned to produce 1Dx5 in maize endosperm. However, with the zein promoter-driven transgene, pollen transmission of the transgene loci was normal in most self- and cross-pollinations. We concluded that the wheat genomic DNA proximal to the wheat 1Dx5 coding sequence was required for reduced pollen transmission of the transgene in maize. In two of four transformation events of the wheat promoter-driven construct examined, pollen exhibited two morphological classes. In one class, pollen was normal in morphology and displayed average viability, and in the second, pollen was reduced in size and did not germinate on artificial media. DNA from the transgene was detectable in mature pollen from plants with reduced pollen transmission of transgene loci. To explain these observations, we hypothesize that elements within the transgene construct interfere with pollen development. We demonstrated that the wheat genomic DNA fragment can be used to control pollen transmission of an herbicide resistance transgene genetically linked to it. The wheat genomic DNA fragment may contain elements that are useful for controlling pollen transmission of transgene loci in commercial maize grain and seed production. PMID:17216545

  12. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-10-17

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by ?-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120?kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery. PMID:25454071

  13. Ion induced fragmentation cross-sections of DNA constituents

    NASA Astrophysics Data System (ADS)

    Rudek, Benedikt; Arndt, Alexander; Bennett, Daniel; Wang, Mingjie; Rabus, Hans

    2015-10-01

    Proton collision with chemical analogs for the base, the sugar and the phosphor residue of the DNA, namely pyrimidine, tetrahydrofuran and trimethyl phosphate, respectively, has been investigated. The impact energies ranged from 300 keV up to 16 MeV. For the first time, relative fragmentation cross-sections for proton impact are reported for tetrahydrofuran and trimethyl phosphate; previously reported cross sections for pyrimidine are extended for energies beyond 2500 keV. Ionization of tetrahydrofuran leads to a ring break in about 80% of all events, trimethyl phosphate predominantly fragments by bond cleavage to one of the three methyl-groups and for pyrimidine the parent ion has the highest abundance. Such comparison supports earlier findings that the sugar is the weak spot for strand breaks.

  14. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    PubMed Central

    Hof, Peter; Ortmeier, Claudia; Pape, Kirstin; Reitmaier, Birgit; Regenbogen, Johannes; Goppelt, Andreas; Halle, Joern-Peter

    2002-01-01

    Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach). The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone). Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with a limited number of experimentally determined fingerprints and was able to detect differences in gene expression of transcripts representing 0.05% of the total mRNA population (e.g. medium abundant gene transcripts). PMID:11882253

  15. Fluorescence detection and size measurement of single DNA molecules

    SciTech Connect

    Castro, A.; Fairfield, F.R.; Shera, E.B. )

    1993-04-01

    We have developed a technique for the detection and size discrimination of single DNA molecules in a hydrodynamically focused flowing solution. Double-standard [lambda] DNA molecules at 3 X 10[sup [minus]15] were stained with the flourescent dye TOTO-1 and were individually detected. The technique makes use of a frequency-doubled mode-locked Nd:YAG laser to repetitively excite the molecules as they traversee the tightly focused laser beam. The flowing sample solution was hydrodynamically focused down to a 20-[mu]m-diameter stream by a rapidly flowing water sheath. The sheath flow technique is well suited for laser-induced fluorescence detection of small-volume, low-concentration samples. The emitted fluorescence photon burst originating from a single DNA molecule was detected with a microchannel plate photomultiplier based single-photon counter, which used time-gated electronics for Raman and Rayleigh scattering rejection. In addition, a mixture of [lambda] DNA and smaller single-cut fragements has been simultaneously detected and indentified by size. The advantages over other techniques for the detection and size determination of DNA fragments are discussed. 14 refs., 5 figs.

  16. 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

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

    PubMed

    Kasoji, Sandeep K; Pattenden, Samantha G; 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

  18. 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

  19. 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

  20. 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.

  1. Phylogenomics of caspase-activated DNA fragmentation factor.

    PubMed

    Eckhart, Leopold; 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. PMID:17343828

  2. 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.

  3. 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.

  4. Autonomous DNA replication in human cells is affected by the size and the source of the DNA.

    PubMed Central

    Heinzel, S S; Krysan, P J; Tran, C T; Calos, M P

    1991-01-01

    We previously developed short-term and long-term assays for autonomous replication of DNA in human cells. This study addresses the requirements for replication in these assays. Sixty-two random human genomic fragments ranging in size from 1 to 21 kb were cloned in a prokaryotic vector and tested for their replication ability in the short-term assay. We found a positive correlation between replication strength and fragment length, indicating that large size is favored for efficient autonomous replication in human cells. All large fragments replicated efficiently, suggesting that signals which can direct the initiation of DNA replication in human cells are either very abundant or have a low degree of sequence specificity. Similar results were obtained in the long-term assay. We also used the same assays to test in human cells a random series of fragments derived from Escherichia coli chromosomal DNA. The bacterial fragments supported replication less efficiently than the human fragments in the short-term and long-term assays. This result suggests that while the sequence signals involved in replication in human cells are found frequently in human DNA, they are uncommon in bacterial DNA. Images PMID:1900922

  5. 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.

  6. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  7. Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment.

    PubMed

    Gao, Hong-Wei; Liang, Cheng-Zhu; Zhang, Yi-Bing; Zhu, Lai-Hua

    2004-01-01

    Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials. PMID:15493678

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

    PubMed Central

    Sawyer, Susanna; Krause, Johannes; Guschanski, Katerina; Savolainen, Vincent; Pbo, 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

  9. Electric linear dichroism transients of bent DNA fragments

    NASA Astrophysics Data System (ADS)

    Umazano, Juan P.; Bertolotto, Jorge A.

    2013-03-01

    We study the effect of translational-rotational hydrodynamic coupling on the transient electric linear dichroism of DNA fragments in aqueous solution. As opposed to previous theoretical works, where analytic solutions valid in the limit of low electric field were reported, we present here a numerical approach which allows to obtain numerical results valid independently from the applied electric field strength. Numerical procedures here used are an extension to the transient-state of those developed in a previous work for the study of the problem in the steady-state. The molecular orientational processes induced by an electric field is characterized with statistical arguments solving the Fokker-Planck equation by means of the finite difference method to know the orientational distribution function of molecules.

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

    PubMed Central

    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

  11. Sperm DNA fragmentation is related to sperm morphological staining patterns.

    PubMed

    Sá, Rosália; Cunha, Mariana; Rocha, Eduardo; Barros, Alberto; Sousa, Mário

    2015-10-01

    In this prospective comparative study, sperm DNA fragmentation (sDNAfrag) was compared at each step of a sequential semen preparation, with semen parameters according to their degree of severity. At each step (fractions) of the sequential procedure, sDNAfrag was determined: fresh (Raw), after gradient centrifugation, washing, and swim-up (SU) for 70 infertile men enrolled in intracytoplasmic sperm injection cycles. sDNAfrag significantly (P = 0.04; P < 0.0001) decreased throughout the steps of semen preparation, with centrifugation and washing not increasing it. A negative correlation to sperm motility was observed in Raw and SU fractions, and a higher sDNAfrag was observed in samples with lower semen quality. Our results confirm that the steps of the sequential procedure do not compromise sperm DNA integrity and progressively decreased sDNAfrag regardless of the sperm abnormality and that semen parameters with lower quality present higher sDNAfrag. Four distinct patterns were observed, of which the entire sperm head staining was the pattern most expressed in all studied fractions. Additionally, the sperm head gene-rich region staining pattern was reduced by the procedure. This suggests that pattern quantification might be a useful adjunct when performing sDNAfrag testing for male infertility. PMID:26278809

  12. Dynamics of DNA polymerase I (Klenow fragment) under external force.

    PubMed

    Xie, Ping

    2013-03-01

    During DNA synthesis, high-fidelity DNA polymerase (DNAP) translocates processively along the template by utilizing the chemical energy from nucleotide incorporation. Thus, understanding the chemomechanical coupling mechanism and the effect of external mechanical force on replication velocity are the most fundamental issues for high-fidelity DNAP. Here, based on our proposed model, we take Klenow fragment as an example to study theoretically the dynamics of high-fidelity DNAPs such as the replication velocity versus different types of external force, i.e., a stretching force on the template, a backward force on the enzyme and a forward force on the enzyme. Replication velocity as a function of the template tension with only one adjustable parameter is in good agreement with the available experimental data. The replication velocity is nearly independent of the forward force, even at very low dNTP concentration. By contrast, the backward force has a large effect on the replication velocity, especially at high dNTP concentration. A small backward force can increase the replication velocity and an optimal backward force exists at which the replication velocity has maximum value; with any further increase in the backward force the velocity decreases rapidly. These results can be tested easily by future experiments and are aid our understanding of the chemomechanical coupling mechanism and polymerization dynamics of high-fidelity DNAP. PMID:23197324

  13. Impact and explosion crater ejecta, fragment size, and velocity

    NASA Technical Reports Server (NTRS)

    Okeefe, J. D.; Ahrens, T. J.

    1985-01-01

    The present investigation had the objective to develop models for the distribution of fragments which are ejected at a given velocity for both impact and explosion cratering. It is pointed out that the results have application to the physics of planetary accretion and the origin of meteorites. The impact ejection of fine dust into the earth's atmosphere has been proposed as a mechanism for extinctions which occurred at the end of the Cretaceous. A technique is developed for determining the distribution of fragments which are ejected at a given velocity. The experimental data base for the distribution fragments in the ejecta blankets of impact, explosion, and nuclear craters, are discussed. Attention is also given to impact flow field calculations, fragmentation theory, and the applications of the derived relations.

  14. 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.

  15. Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice

    PubMed Central

    Rahimipour, Marzieh; Talebi, Ali Reza; Anvari, Morteza; Abbasi Sarcheshmeh, Abolghasem; Omidi, Marjan

    2014-01-01

    Background: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans. Objective: The objective of this study was to investigate the effect of saccharin consumption on sperm parameters and apoptosis in adult mice. Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Hams F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by SCD (sperm chromatin dispersion) and terminal deoxynucleotidyl transferase (TUNEL) assay. Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.701.11 in controls vs. 12.802.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.006.40 in control animals into 63.856.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively). Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice. PMID:25031574

  16. 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…

  17. 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

  18. Nanopore-based assay for detection of methylation in double-stranded DNA fragments.

    PubMed

    Shim, Jiwook; Kim, Younghoon; Humphreys, Gwendolyn I; Nardulli, Ann M; Kosari, Farhad; Vasmatzis, George; Taylor, William R; Ahlquist, David A; Myong, Sua; Bashir, Rashid

    2015-01-27

    DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments. PMID:25569824

  19. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication

    PubMed Central

    Levikova, Maryna; Cejka, Petr

    2015-01-01

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5? DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen(PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway. PMID:26175049

  20. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication.

    PubMed

    Levikova, Maryna; Cejka, Petr

    2015-09-18

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen(PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway. PMID:26175049

  1. 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 chromatin fibers in mammalian DNA.

  2. 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. PMID:15137918

  3. Isolation and characterization of a species-specific DNA fragment for detection of Candida albicans by polymerase chain reaction.

    PubMed Central

    Miyakawa, Y; Mabuchi, T; Kagaya, K; Fukazawa, Y

    1992-01-01

    A 2-kbp DNA fragment, EO3, that was present in multiple copies in the Candida albicans genome was isolated for use in developing a detection method for C. albicans by polymerase chain reaction (PCR). Dot blot hybridization revealed that EO3 was specific for the 40 isolates of C. albicans serotypes A and B used. Using a set of primers (20-mer each) derived from the nucleotide sequence of EO3, we performed specific amplification of a 1.8-kbp DNA fragment within EO3 by PCR. All 40 isolates belonging to C. albicans serotypes A and B contained amplifiable 1.8-bkp fragments, although the DNA of the amplified products exhibited small variations in size, yielding three different fragment groups. Southern blot hybridization probed with EO3 showed that these 1.8-kbp fragments were derived from the EO3 region. Conversely, the 1.8-kbp fragment was not amplified from 38 isolates belonging to seven other medically important Candida species or from isolates of Cryptococcus neoformans, Saccharomyces cerevisiae, various bacteria, and a human cell line. The detection limit of the PCR assay for C. albicans with the EO3 fragment was shown to be approximately 2 to 10 cells and 100 cells in saline and human urine, respectively, by ethidium bromide staining and 2 and 10 cells, respectively, by Southern blot analysis. In addition, EO3 was assumed to originate from mitochondrial DNA on the basis of the results of its characterizations. These results indicate that the PCR system using the 1.8-kbp fragment as a target is a reliable method for identifying C. albicans isolates, thereby suggesting its potentials for specific and sensitive detection of C. albicans in samples from patients with candidiasis. Images PMID:1572976

  4. Development of a fast and efficient ultrasonic-based strategy for DNA fragmentation.

    PubMed

    Larguinho, Miguel; Santos, Hugo M; Doria, Gonçalo; Scholz, H; Baptista, Pedro V; Capelo, José L

    2010-05-15

    Several ultrasound-based platforms for DNA sample preparation were evaluated in terms of effective fragmentation of DNA (plasmid and genomic DNA)-ultrasonic probe, sonoreactor, ultrasonic bath and the newest Vialtweeter device. The sonoreactor showed the best efficiency of DNA fragmentation while simultaneously assuring no cross-contamination of samples, and was considered the best ultrasonic tool to achieve effective fragmentation of DNA at high-throughput and avoid sample overheating. Several operation variables were studied-ultrasonication time and amplitude, DNA concentration, sample volume and sample pre-treatment-that allowed optimisation of a sonoreactor-based strategy for effective DNA fragmentation. Optimal operating conditions to achieve DNA fragmentation were set to 100% ultrasonic amplitude, 100microL sample volume, 8min ultrasonic treatment (2min/sample) for a DNA concentration of 100microgmL(-1). The proposed ultrasonication strategy can be easily implemented in any laboratory setup, providing fast, simple and reliable means for effective DNA sample preparation when fragmentation is critical for downstream molecular detection and diagnostics protocols. PMID:20298868

  5. Effect of ultrasound on the separation of DNA fragments in agarose gel electrophoresis

    SciTech Connect

    Ma, Yinfa; Yeung, E.S. )

    1990-06-01

    Since its first use in 1966 interest in and the applications of electrophoresis of DNA fragments in agarose gel have grown rapidly. Nowadays, agarose gel electrophoresis has become a standard technique with high resolving power for the analysis of DNA structure, for example for the determination of the length of DNA fragments obtained by the action of restriction enzymes. The electrophoretic mobility ({mu}) of DNA fragments is influenced by various parameters-molecular weight, gel concentration, temperature, electric field, and DNA-agarose affinity. A comprehensive study of the influence of these main parameters has been reported. In this paper, the authors investigate a new effect on the electrophoretic mobility of DNA fragments in agarose gels, viz. the influence of ultrasound.

  6. In situ end labeling of fragmented DNA in induced ovarian atresia.

    PubMed

    D'Herde, K; De Pestel, G; Roels, F

    1994-01-01

    Apoptosis is studied in a model of induced follicular atresia in the ovary of Japanese quail (Coturnix coturnix japonica) by in situ end labeling of DNA fragments in granulosa cells using two different techniques (incorporation of labeled nucleotides by DNA polymerase I or terminal deoxynucleotidyl transferase). The most remarkable observation related to apoptosis in this model is the predominant cytoplasmic localization of labeled DNA fragments, while DNA fragmentation appears to be absent from compacted chromatin masses of apoptotic nuclei and apoptotic nuclear fragments. Unstained apoptotic bodies are present adjacent to stained ones, so that their detection rate on hematoxylin + eosin stained sections is better than on the in situ end-labeled sections. This suggests that DNA fragmentation is a late even or not obligatory in apoptotic granulosa cell death. In contrast to similar studies on atretic granulosa in mammalian models, the process of apoptosis is asynchronous in the granulosal epithelium, with a majority of nuclei with normal chromatin configuration remaining negative for DNA fragmentation. Finally it is shown that the techniques used are not specific for apoptosis, as DNA fragmentation in necrotic granulosa cells is detected as well. PMID:7654330

  7. Impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes.

    PubMed

    Esbert, Marga; Pacheco, Alberto; Vidal, Francesca; Florensa, Mireia; Riqueros, Marissa; Ballesteros, Agustn; Garrido, Nicols; Caldern, Gloria

    2011-12-01

    A prospective study was performed to assess the impact of sperm DNA fragmentation on the outcome of IVF with own or donated oocytes. The study population included 178 couples (62 cycles of IVF, 116 of intracytoplasmic sperm injection (ICSI)) with own (n=77) and donor (n=101) oocytes. DNA fragmentation was evaluated by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Correlation between DNA damage to oocyte fertilization, embryo quality and clinical pregnancy, implantation and miscarriage rates was evaluated. DNA fragmentation was not related to fertilization rates in either IVF (r=0.08) or ICSI (r=-0.04) cycles. DNA fragmentation was similar in patients with <50% embryo utilization rate compared with ?50%, in cancelled and in embryo transfer cycles and in miscarriages and in successful deliveries. Moreover, DNA fragmentation was similar in pregnant and non-pregnant women as well as in IVF with own or donor oocytes. In the multivariable analysis, the odds ratio of DNA after controlling by age was 1.0. Using a 36% sperm fragmentation threshold, results did not vary. It is concluded that DNA damage was not related to outcomes of IVF or ICSI with own or donor oocytes. PMID:22019617

  8. A unique role of the DNA fragmentation factor in maintaining genomic stability.

    PubMed

    Yan, Bin; Wang, Huili; Peng, Yuanlin; Hu, Ye; Wang, He; Zhang, Xiuwu; Chen, Qi; Bedford, Joel S; Dewhirst, Mark W; Li, Chuan-Yuan

    2006-01-31

    DNA fragmentation is a hallmark of apoptosis (programmed cell death). However, the biological function of apoptotic DNA fragmentation remains unclear. Here, we show that DNA fragmentation factor plays an important role for maintaining genomic stability. Inhibition or loss of the DNA fragmentation factor (DFF)/caspase-activated DNase (CAD), whose nuclease activity is responsible for digesting genomic DNA during apoptosis, led to significant increases in spontaneous or induced gene mutations, gene amplifications, and chromosomal instability in primary mouse cells and transformed human cell lines. The mechanism underlying genetic instability in DFF/CAD-deficient cells, at least in part, involves a small but significant elevation in the survival of cells exposed to ionizing radiation, suggesting that apoptotic DNA fragmentation factor contributes to genomic stability by ensuring the removal of cells that have suffered DNA damage. In support of this hypothesis are the observations of increased cellular transformation of mouse embryonic cells from the DFF/CAD-null mice and significantly enhanced susceptibility to radiation-induced carcinogenesis in these mice. These data, in combination with published reports on the existence of tumor-specific gene mutations/deletions in the DFF/CAD genes in human cancer samples, suggest that apoptotic DNA fragmentation factor is required for the maintenance of genetic stability and may play a role in tumor suppression. PMID:16432220

  9. 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

  10. 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

  11. Population size, habitat fragmentation, and the nature of adaptive variation in a stream fish.

    PubMed

    Fraser, Dylan J; Debes, Paul V; Bernatchez, Louis; Hutchings, Jeffrey A

    2014-09-01

    Whether and how habitat fragmentation and population size jointly affect adaptive genetic variation and adaptive population differentiation are largely unexplored. Owing to pronounced genetic drift, small, fragmented populations are thought to exhibit reduced adaptive genetic variation relative to large populations. Yet fragmentation is known to increase variability within and among habitats as population size decreases. Such variability might instead favour the maintenance of adaptive polymorphisms and/or generate more variability in adaptive differentiation at smaller population size. We investigated these alternative hypotheses by analysing coding-gene, single-nucleotide polymorphisms associated with different biological functions in fragmented brook trout populations of variable sizes. Putative adaptive differentiation was greater between small and large populations or among small populations than among large populations. These trends were stronger for genetic population size measures than demographic ones and were present despite pronounced drift in small populations. Our results suggest that fragmentation affects natural selection and that the changes elicited in the adaptive genetic composition and differentiation of fragmented populations vary with population size. By generating more variable evolutionary responses, the alteration of selective pressures during habitat fragmentation may affect future population persistence independently of, and perhaps long before, the effects of demographic and genetic stochasticity are manifest. PMID:25056619

  12. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease

    PubMed Central

    Nicholls, Thomas J.; Zsurka, Gábor; Peeva, Viktoriya; Schöler, Susanne; Szczesny, Roman J.; Cysewski, Dominik; Reyes, Aurelio; Kornblum, Cornelia; Sciacco, Monica; Moggio, Maurizio; Dziembowski, Andrzej; Kunz, Wolfram S.; Minczuk, Michal

    2014-01-01

    MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5′ ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA. PMID:24986917

  13. High-resolution liquid chromatography of DNA fragments on non-porous poly(styrene-divinylbenzene) particles.

    PubMed Central

    Huber, C G; Oefner, P J; Preuss, E; Bonn, G K

    1993-01-01

    DNA restriction fragments and PCR products were separated by means of ion-pair reversed-phase high-performance liquid chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles with a mean diameter of 2.1 microns. Optimum resolution was obtained by using an acetonitrile gradient in 100 mM of triethylammonium acetate and a column temperature of 50 degrees C. This allowed the separation of DNA fragments differing in chain length by 1-5% up to a size of 500 base pairs. PCR products could be analyzed directly in less than two minutes with a concentration sensitivity of at least 300 ng/ml. Compared with anion-exchange chromatography or gel electrophoresis no desaltation of the purified DNA molecules is required because the volatile buffer system can be readily evaporated. Subsequently, the method was used for the semiquantitative evaluation of the expression of multidrug resistance genes in mononuclear white blood cells. Images PMID:8503958

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

    PubMed

    lvarez-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

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

    PubMed Central

    lvarez-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

  16. 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. PMID:2569858

  17. 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

  18. 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.

  19. Apoptotic DNA fragmentation factor maintains chromosome stability in a P53-independent manner.

    PubMed

    Yan, B; Wang, H; Wang, H; Zhuo, D; Li, F; Kon, T; Dewhirst, M; Li, C-Y

    2006-08-31

    DNA fragmentation factor (DFF)/caspase-activated DNase (CAD) is responsible for DNA fragmentation, a hallmark event during apoptosis. Although DNA fragmentation is an evolutionarily conserved process across species, its biological function is not clearly understood. In this study, we constructed cell lines expressing a mutant ICAD (inhibitor of CAD) protein that is resistant to caspase cleavage and therefore constantly binds to DFF/CAD and inhibits DNA fragmentation. We found that irradiation of these cells led to increased chromosome aberrations and aneuploidy when compared with their parental controls. The increased chromosome instability is observed irrespective of cellular P53 status, suggesting that the effect of DFF/CAD is independent of P53. Inhibition of apoptotic DNA fragmentation resulted in increased clonogenic survival of irradiated cells and a delay in removal of cells with DNA damages induced by radiation, an effect similar to that in cells with p53 mutations. Consistent with DFF/CAD's effect on clonogenic survival, tumors established from cells deficient in DNA fragmentation showed enhanced growth in nude mice. Therefore, our results suggest that DFF/CAD plays an important and P53-independent role in maintaining chromosome stability and suppressing tumor development. PMID:16619042

  20. 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 presence of eroded periodicity. The autocorrelation method was identified as poorly suited for use with the blockwise bootstrap. Application of our methods to the genomes of two model organisms revealed a striking proportion of the yeast and mouse genomes are spanned by NPS. Despite their markedly different sizes, roughly equivalent proportions (19-21%) of the genomes lie within period-10 spans of the NPS dinucleotides {AA, TT, TA}. The biological significance of these regions remains to be demonstrated. To facilitate this, the genomic coordinates are available as Additional files 1, 2, and 3 in a format suitable for visualisation as tracks on popular genome browsers. Reviewers This article was reviewed by Prof Tomas Radivoyevitch, Dr Vsevolod Makeev (nominated by Dr Mikhail Gelfand), and Dr Rob D Knight. PMID:21527008

  1. 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

  2. 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.

  3. Unit cloning and amplification as novel and universal strategies for complex vector construction and small DNA fragment preparation.

    PubMed

    Ye, Chunjiang; Gu, Jingsong; Chen, Sunxiao; Deng, Anmei; Li, Yue Zhong; Li, Dianxiang

    2010-09-01

    With a novel and universal strategy for the cloning of multiple DNA fragments, a complex synthetic vector (pVEC100), harboring the target DNA fragments in conventional 100-bp DNA ladder, was constructed for efficient and large-scale production of 100-bp DNA marker through bacteria fermentation, plasmid extraction and restrictive digestion. Since the restrictive digestion of complex vectors yields insufficient small DNA fragments, an innovative PCR model was developed as an alternative. The PCR model comprised a specially designed template vector and a unit amplification model for producing groups of small DNA fragments. The unit amplification model improved the efficiency of the PCR protocol and made it more economical and easier for small DNA fragment amplification. The approach presented in this paper--a unit cloning model for constructing complex synthetic vectors combined with the modular design of unit amplification by PCR--is a powerful method for preparing small DNA fragments of DNA molecular weight standards. PMID:20690148

  4. Oligonucleosomal DNA fragmentation in MCF-7 cells undergoing palmitate-induced apoptosis.

    PubMed

    Semenov, D V; Aronov, P A; Kuligina, E V; Potapenko, M O; Richter, V A

    2003-12-01

    Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells. PMID:14756630

  5. 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 how much energy is transferred to tissues PMID:10445917

  6. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

    PubMed Central

    Yu, Stephanie C. Y.; Chan, K. C. Allen; Zheng, Yama W. L.; Jiang, Peiyong; Liao, Gary J. W.; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y.; Go, Attie T. J. I.; van Vugt, John M. G.; Minekawa, Ryoko; Oudejans, Cees B. M.; Nicolaides, Kypros H.; Chiu, Rossa W. K.; Lo, Y. M. Dennis

    2014-01-01

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring. PMID:24843150

  7. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

    PubMed

    Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

    2014-06-10

    Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring. PMID:24843150

  8. Cloning Should Be Simple: Escherichia coli DH5?-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.

    PubMed

    Kostylev, Maxim; Otwell, Anne E; Richardson, Ruth E; Suzuki, Yo

    2015-01-01

    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 DH5?, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5? retains sufficient recombinase activity to assemble up to six double-stranded 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. 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. PMID:26348330

  9. Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

    PubMed Central

    Richardson, Ruth E.; Suzuki, Yo

    2015-01-01

    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 double-stranded 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. 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. PMID:26348330

  10. Separation of random fragments of DNA according to properties of their sequences.

    PubMed

    Fischer, S G; Lerman, L S

    1980-08-01

    The separation of DNA fragments by electrophoresis at high temperature in a denaturing gradient is independent of the length of the fragments. We have suggested that the basis of fragment separation is that each DNA molecule undergoes partial melting as it encounters a concentration of denaturants sufficient to melt its least stable sequence, while other sequences remain double stranded; in the partially melted configuration, DNA can continue migration only slowly. This model is consistent with the observation that fragments of lambda phage DNA cleaved by different restriction endonucleases reach the same final depth in the gel if they contain the same least-stable sequence. A unique set of bands is produced from the electrophoresis of randomly fragmented DNA; this would be expected if there were a limited number of melting centers occupying discrete genetic loci. An intact DNA molecule penetrates about as deeply into the gel as the uppermost band after fragmentation; this would be expected only if the least-stable sequence controls the final depth of the whole molecule. PMID:6254023

  11. Sperm nuclear DNA fragmentation and its association with semen quality in Greek men.

    PubMed

    Evgeni, E; Lymberopoulos, G; Touloupidis, S; Asimakopoulos, B

    2015-12-01

    Due to the limitations of conventional semen analysis in predicting a man's fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. This prospective study was undertaken to investigate the associations between conventional seminal parameters and DNA fragmentation in Greek men. A total of 669 subject data were evaluated in two groups, normozoospermic (n=184) and non-normozoospermic (n=485), according to the WHO 2010 (WHO Laboratory Manual for the Examination and Processing of Human Semen, 5th edn. World Health Organization), reference limits. For all the subjects, semen volume, sperm concentration, total count, rapid and total progressive motility and morphology were recorded following the WHO 2010 methods and DNA fragmentation was assessed by the sperm chromatin dispersion assay. An inverse correlation was established between DNA fragmentation and all conventional seminal parameters except semen volume in men with seminal profiles below the reference limits, with statistical significance for rapid and total progressive motility. Normozoospermic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always below 30%. DNA fragmentation testing and traditional semen analysis should therefore be considered as complementary diagnostic tools in a comprehensive evaluation of male infertility. PMID:25586471

  12. Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis.

    PubMed

    Valenzuela, M S; Aguinaga, M D; Inman, R B

    1981-01-01

    By the use of a restriction enzyme digestion of gently lysed E. coli or B. subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient. Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed. Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques. The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates. In B. subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker. PMID:6268941

  13. Investigations of Bacterial Inactivation and DNA Fragmentation Induced by Flowing Humid Argon Post-discharge

    NASA Astrophysics Data System (ADS)

    Odic, Emmanuel; Limam, S.; Kirkpatrick, M. J.; Dodet, B.; Salamitou, S.; DuBow, M. S.

    Bio-contaminated surfaces were exposed to an atmospheric pressure flowing post-discharge, i.e. without direct contact of the plasma with the surface. The non-thermal plasma source was a dielectric barrier discharge. Using humid argon as a feed gas, a reduction of six orders of magnitude of survivors could be obtained for Escherichia coli. An investigation of bacterial inactivation mechanisms during the plasma induced treatment was conducted. For this purpose, DNA (plasmid and genomic DNA in aqueous solution) degradation by the plasma process was studied, assuming that the bacterial inactivation is obtained when the bacterial DNA is fragmented. According to the operating conditions (feed gas, reactor geometry and discharge input power), DNA fragmentation was evaluated in correlation with aqueous phase hydrogen peroxide concentration measurements. It appears that hydrogen peroxide is not the only factor responsible for DNA fragmentation and that short-lived species produced by water dissociation are major contributors.

  14. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil.

    PubMed

    Lee, Sin Hang

    2014-03-01

    Gardasil is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results. PMID:24083601

  15. A Microfabricated Device for Sizing and Sorting DNA Molecules

    NASA Astrophysics Data System (ADS)

    Chou, Hou-Pu; Spence, Charles; Scherer, Axel; Quake, Stephen

    1999-01-01

    We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.

  16. Novel PTP1B inhibitors identified by DNA display of fragment pairs.

    PubMed

    Barluenga, Sofia; Zambaldo, Claudio; Ioannidou, Heraklidia A; Ciobanu, Mihai; Morieux, Pierre; Daguer, Jean-Pierre; Winssinger, Nicolas

    2016-02-01

    DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay. PMID:26691757

  17. The size distributions of fragments ejected at a given velocity from impact craters

    NASA Technical Reports Server (NTRS)

    Okeefe, J. D.; Ahrens, T. J.

    1986-01-01

    The mass distribution of fragments that are ejected at a given velocity for impact craters is modeled to allow extrapolation of laboratory, field, and numerical results to large scale planetary events. The model is semi-empirical in nature and is derived from: (1) numerical calculations of cratering and the resultant mass versus ejection velocity, (2) observed ejecta blanket particle size distributions, (3) an empirical relationship between maximum ejecta fragment size and crater diameter, (4) measurements and theory of maximum ejecta size versus ejecta velocity, and (5) an assumption on the functional form for the distribution of fragments ejected at a given velocity. This model implies that or planetary impacts into competent rock, the distribution of fragments ejected at a given velocity is broad, e.g., 68% of the mass of the ejecta at a given velocity contains fragments having a mass less than 0.1 times a mass of the largest fragment moving at that velocity. The broad distribution suggests that in impact processes, additional comminution of ejecta occurs after the upward initial shock has passed in the process of the ejecta velocity vector rotating from an initially downward orientation. This additional comminution produces the broader size distribution in impact ejecta as compared to that obtained in simple brittle failure experiments.

  18. CONSTRUCTION OF CONTIGS OF AEGILOPS TAUSCHII GENOMIC DNA FRAGMENTS CLONED IN BAC AND BIBAC VECTORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput, fully automated, multi-color fluorescent fingerprinting technique for large-insert genomic DNA clones was developed. The technique was used to fingerprint 200,000 genomic DNA fragments of Aegilops tauschii line genetically closely related to the D genome of Chinese Spring wheat. T...

  19. 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-Dring 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.

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

    PubMed

    Rotstein, Horacio G

    2015-06-14

    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-Dring 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. PMID:26071695

  1. Differences in Electrostatic Potential Around DNA Fragments Containing Guanine and 8-oxo-Guanine

    SciTech Connect

    Haranczyk, Maciej; Gutowski, Maciej S.

    2007-02-01

    hanges of electrostatic potential (EP) around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through large scale density functional theory computations. Quantum mechanical structural optimizations of DNA fragments with three pairs of nucleotoides and accompanying counteractions were performed with a B3LYP exchange-correlation functional and 6-31G** basis sets. The intact DNA fragment contained guanine in the middle layer, while the damaged fragment had the guanine replaced with 8-oxo-guanine. The electrostatic potential around these DNA fragments was projected on a surface around the double helix. The 2D maps of EP of intact and damaged DNA fragments were analyzed to identify these modifications of EP that result from the occurrence of 8-oxo-guanine. It was found that distortions of the phosphate groups and displacements of the accompanying countercations are clearly reflected in the EP maps.

  2. 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

  3. Shrinking the apparatus size for DNA analysis

    NASA Astrophysics Data System (ADS)

    Zimmer, Klaus-Peter; Braun, Alexander; Kostrzewa, M.

    2001-03-01

    Miniaturization of chemical and/or biological analytical systems requires an innovative design and new manufacturing methods. This includes the fabrication of components or structures, the assembly of these parts, and a testing strategy. The separation of an entire device into a disposable microfluidic system and a multi-use supply unit and housing allows an easy fabrication as well as low cost of operation. A simple, replicated, micro-sized, and disposable unit guarantees the same initial conditions for every analytic cycle, whereas, on the other hand all microfluidic actuators and other key elements can remain outside of the microsystem. In order to drive the implemented passive elements of the microfluidic system by external forces of the base unit, elasticity is a crucial material property. Thus silicone was used as material for the microsystem. A microfluidic system intended for use in DNA analysis employing the principles of the polymerase chain reaction (PCR) is presented. All functional units have been integrated into a complex module using a CAD-program. The 3D-drawing was converted into several machining layers for a direct laser writing CNC-code. A focussed excimer laser beam was used in order to micromachine the negative channel and reservoir system in a polycarbonate slab employing ablative photo-decomposition. Excimer laser micromachining proofed to be an ideal prototyping technique for this purpose with sufficient lateral and depth control. Its rather low throughput was bypassed with an additional hot embossed intermediate positive polyethylene master which, in turn, replicated produces the negative fluidic system in the target material PDMS (polydimethylsiloxane) as an elastomeric material. The components of the fluidic systems have been sealed with flat slabs or other microsystem parts of either PDMS or glass. In either case both parts were exposed to a plasma discharge for some seconds in order to clean, oxidize and activate the surface. This enabled an irreversible seal when two oxidized

  4. 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-?). 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 of magma fragmentation at or close to the fragmentation level. Given the high abrasiveness of pumice, hemispherical clasts should be observed if clast break-up followed efficient clast abrasion. As a consequence, finer grained pyroclastic fall deposits do not necessarily proof efficient secondary fragmentation in the conduit but may rather reveal the influence of conduit length on 'What size of pyroclasts can be erupted'?

  5. Patch size, functional isolation, visibility and matrix permeability influences neotropical primate occurrence within highly fragmented landscapes.

    PubMed

    da Silva, Lucas Goulart; Ribeiro, Milton Cezar; Hasui, rica; da Costa, Carla Aparecida; da Cunha, Rogrio 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 Poo 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

  6. 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, rica; da Costa, Carla Aparecida; da Cunha, Rogrio 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 Poo 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

  7. 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

  8. Menadione-induced DNA fragmentation without 8-oxo-2'-deoxyguanosine formation in isolated rat hepatocytes.

    PubMed

    Fischer-Nielsen, A; Corcoran, G B; Poulsen, H E; Kamendulis, L M; Loft, S

    1995-05-17

    Menadione (2-methyl-1,4-naphthoquinone) induces oxidative stress in cells causing perturbations in the cytoplasm as well as nicking of DNA. The mechanisms by which DNA damage occurs are still unclear, but a widely discussed issue is whether menadione-generated reactive oxygen species (ROS) directly damage DNA. In the present study, we measured the effect of menadione on formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG), an index of oxidative DNA base modifications, and on DNA fragmentation. Isolated hepatocytes from phenobarbital-pretreated rats were exposed to menadione, 25-400 microM, for 15, 90 or 180 min with or without prior depletion of reduced glutathione (GSH) by diethyl maleate. Menadione caused profound GSH depletion and internucleosomal DNA fragmentation, which was demonstrated by a prominent fragmentation ladder on agarose gel electrophoresis. We found no oxidative modification of DNA in terms of increased 8-oxodG formation. In contrast, the positive control of sunlamp light increased 8-oxodG 5-fold in rat hepatocytes. We conclude that oxidative modification of DNA bases is unlikely to be important in menadione-induced DNA damage. PMID:7763290

  9. 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)

    Virgs, Emilio; Garca, 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.

  10. Identification of novel DNA fragments and partial sequence of a genomic island specific of Brucella pinnipedialis.

    PubMed

    Maquart, Marianne; Fardini, Yann; Zygmunt, Michel S; Cloeckaert, Axel

    2008-11-25

    Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammals and were recently recognized as two different species, i.e. Brucella pinnipedialis for pinniped isolates and Brucella ceti for cetacean isolates. The aim of this study was to identify specific DNA fragments of marine mammal Brucella strains using a previously described infrequent restriction site-PCR (IRS-PCR) method but with three new couples of restriction enzymes applied on a larger panel of marine mammal Brucella isolates (n=74) and one human isolate from New Zealand likely from marine mammal origin. This study revealed five DNA fragments specific of Brucella strains isolated from marine mammals. Among them two new DNA fragments were specific of B. pinnipedialis but were not detected in hooded seal isolates. DNA fragment I identified in the previous IRS-PCR study and fragment VI of this study were located on a cloned and sequenced 6kb SacI fragment. Its nucleotide sequence revealed that it is likely part of a putative genomic island. Sequence analysis showed that it carries four ORFs coding for putative metabolic functions. Although hooded seal isolates are classified within B. pinnipedialis it was shown in this study that they do not carry this genomic island and this raises the question about their evolutionary history within B. pinnipedialis. PMID:18514443

  11. [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 under temperature of -20 degrees C or -70 degrees C can be recommended for adequate evaluation of level of fragmentation of DNA of spermatozoons. PMID:26189292

  12. Oligomerization state of the DNA fragmentation factor in normal and apoptotic cells.

    PubMed

    Lechardeur, Delphine; Dougaparsad, Sam; Nemes, Csilla; Lukacs, Gergely L

    2005-12-01

    The caspase-activated DNase (CAD) is the primary nuclease responsible for oligonucleosomal DNA fragmentation during apoptosis. The DNA fragmentation factor (DFF) is composed of the 40-kDa CAD (DFF40) in complex with its cognate 45-kDa inhibitor (inhibitor of CAD: ICAD or DFF45). The association of ICAD with CAD not only inhibits the DNase activity but is also essential for the co-translational folding of CAD. Activation of CAD requires caspase-3-dependent proteolysis of ICAD. The tertiary structures of neither the inactive nor the activated DFF have been conclusively established. Whereas the inactive DFF is thought to consist of the CAD/ICAD heterodimer, activated CAD has been isolated as a large (>MDa) multimer, as well as a monomer. To establish the subunit stoichiometry of DFF and some of its structural determinants in normal and apoptotic cells, we utilized size-exclusion chromatography in combination with co-immunoprecipitation and mutagenesis techniques. Both endogenous and heterologously expressed DFF have an apparent molecular mass of 160-190 kDa and contain 2 CAD and 2 ICAD molecules (CAD/ICAD)2 in HeLa cells. Although the N-terminal (CIDE-N) domain of CAD is not required for ICAD binding, it is necessary but not sufficient for ICAD homodimerization in the DFF. In contrast, the CIDE-N domain of ICAD is required for CAD/ICAD association. Using bioluminescence resonance energy transfer (BRET), dimerization of ICAD in DFF was confirmed in live cells. In apoptotic cells, endogenous and exogenous CAD forms limited oligomers, representing the active nuclease. A model is proposed for the rearrangement of the DFF subunit stoichiometry in cells undergoing programmed cell death. PMID:16204257

  13. Relationship of spermatozoal DNA fragmentation with semen quality in varicocele-positive men.

    PubMed

    Moazzam, A; Sharma, R; Agarwal, A

    2015-10-01

    The aim of the study was to assess the semen quality and levels of spermatozoal nuclear DNA fragmentation in subfertile subjects clinically diagnosed with varicocele, subfertile subjects without varicocele and healthy fertile controls. Semen samples were obtained from 302 subjects. Of them, 115 were healthy fertile controls having normal semen characteristics, 121 subfertile men diagnosed with varicocele, both, clinically and on ultrasonography, while 66 subjects were subfertile with no varicocele. Spermatozoal concentration, percentage motility, morphology and DNA fragmentation were measured. In the study population, deterioration in semen quality-decreased spermatozoal concentration, percentage motility and normal morphology was seen in subfertile subjects, especially with varicocele. Highest spermatozoal DNA fragmentation was observed in varicocele-positive subjects as compared with varicocele-negative subjects and healthy fertile controls. Significant negative correlation was seen between spermatozoal DNA fragmentation and concentration (r=-0.310), motility (r=-0.328) normal morphology, WHO method (r=-0.221) and Tygerberg strict criteria (r=-0.180) in the varicocele-positive subfertile subjects. In conclusion, this study suggests existence of a negative relationship between spermatozoal DNA fragmentation and semen quality in varicocele-positive subfertile subjects. PMID:25346327

  14. Junction between Z and B conformations in a DNA restriction fragment: evaluation by Raman spectroscopy.

    PubMed Central

    Wartell, R M; Klysik, J; Hillen, W; Wells, R D

    1982-01-01

    Raman vibrational spectra were obtained from two DNA restriction fragments and the DNA polymer (dG-dC)n . (dG-dC)n in 0.01 and 4.5 M NaCl. One fragment contained 95 base pairs (bp) of the Escherichia coli lactose operator-promoter region (95-bp fragment). The other fragment consisted of the 95-bp region flanked by 26 and 32 bp of dC-dG sequences and BamHI ends (157-bp fragment). In 0.01 M NaCl all three DNAs have Raman spectra characteristic of a right-handed B conformation. The high salt spectrum of the 95-bp fragment is also characteristic of a B conformation. However, the spectrum of the 157-bp fragment in 4.5 M NaCl shows major intensity changes from the 0.01 M NaCl spectrum. These changes are also observed in the high salt spectra of (dG-dC)n . (dG-dC)n and are correlated with the presence of a left-handed Z conformation. Comparisons between the high salt Raman spectra of the 157-bp fragment and spectra calculated from (dG-dC)n . (dG-dC)n and the 95-bp fragment indicated that essentially all of the dC-dG regions in the 157-bp fragment are in the Z conformation and a large part (approximately 80%) of the 95-bp region no longer has a B-type backbone vibration. However, this non-B-DNA-like character of the central region is not indicated by base vibrations. PMID:7045865

  15. 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

  16. Polymerization behavior of Klenow fragment and Taq DNA polymerase in short primer extension reactions.

    PubMed

    Zhao, Guojie; Guan, Yifu

    2010-10-01

    DNA polymerases amplify DNA fragments through primer extension reactions. However, polymerization behavior of short primers in the primer extension process has not been systematically explored. In this study, we examined the minimal primer length required for primer extension, and the effect of primer length, mismatches and other conditions on DNA polymerization using a non-radioactive method. Under the condition we conducted, the shortest primers polymerized by Klenow fragment (KF) and Taq DNA polymerase in our experiments were respectively heptamer and octamer. The extension efficiency was also affected by the up-stream overhanging structure of the primer-template complex. We hypothesized a simple model to interpret these observations based on the polymerase structures. Furthermore, it was found that the longer the primer, the more efficient is the primer extension. These polymerization behavior of short primers lay foundation about DNA polymerization mechanism and development of novel nucleic acid detection assays. PMID:20829187

  17. Detection of herpes simplex virus type-2 DNA restriction fragments in human cervical carcinoma tissue.

    PubMed Central

    Park, M; Kitchener, H C; Macnab, J C

    1983-01-01

    DNA extracted from eight human cervical carcinomas, one lymph node metastasis and related control tissue was examined for the presence of herpes simplex virus (HSV) DNA sequences. Southern blot transfers of tumour and control DNA were hybridised with radioactively labelled cloned probes representing 70% of the HSV-2 genome. Specific hybridisation to HSV DNA sequences was observed in one of eight carcinoma tissues analysed. Hybridisation of HSV-2 DNA probes to BamHI and XhoI restriction enzyme fragments of tumour cell DNA which co-migrated with authentic HSV-2 viral fragments identified co-linear HSV-2 DNA sequences comprising 3% of the HSV-2 genome, between map coordinates 0.582 and 0.612. The remaining eight tumour and all control tissues analysed, showed no specific hybridisation to any of the probes used at levels of sensitivity which would detect 0.5 copies/cell of HSV-2 DNA restriction fragments of 2 kb or greater. Images Fig. 2. Fig. 3. Fig. 5. PMID:6313349

  18. Concentration and size separation of DNA samples at liquid-liquid interfaces.

    PubMed

    Hahn, Thomas; Hardt, Steffen

    2011-07-15

    This report introduces a new analytical concept utilizing the mass transfer resistance of a liquid-liquid interface to concentrate and separate DNA samples. DNA molecules can be electrophoretically accumulated at a liquid-liquid interface of an aqueous two-phase system (ATPS) of poly(ethylene glycol) (PEG) and dextran, two polymers that form two immiscible phases in aqueous electrolyte solutions. The detachment of DNA from the interface into the other phase can be triggered by increasing the applied electric field. We experimentally study the size dependence of the detachment process for a broad spectrum of DNA fragments. In a regime where the coiling of the chains does not play a significant role, the process shows a linear dependence on the diffusion coefficient, with shorter DNA chains detaching at lower electric field strengths than larger ones. The concept may enable novel separation protocols for preparative and analytical purposes. PMID:21682284

  19. DNA fragmentation is not associated with apoptosis in zerumbone-induced HepG2 cells.

    PubMed

    Kamalidehghan, Behnam; Ahmadipour, Fatemeh; Noordin, Mohamed Ibrahim

    2012-01-01

    Zerumbone is a cytotoxic compound isolated from the herbal plant, Zingiber zerumbet Smith, which exhibits antitumor activity [1-2], anti-inflammatory effects and possesses anti-proliferative potentials in a variety of cell lines [3-4]. DNA fragmentation indicates an early event of apoptosis leading to cell death due to the absence of new cellular proteins synthesizing for cell survival. Previous studies indicated that the cleavage of double-stranded DNA in apoptotic DNA degradation occurs via the activation of endogenous Ca2+/Mg2+-dependent endonuclease that specifically cleaves between nucleosomes to produce DNA fragments that are multiples of ~180 base pairs [5]. In order to investigate DNA fragmentation, we treated HepG2 cells with zerumbone (IC50: 3.45 ± 0.026 µg/mL) in both dose-dependent (2, 4, 6 and 8 µg/mL) and time-dependent manner (4, 8, 12, 16, 24, 48 and 72 h). The assay was performed using the Suicide Track™ DNA Ladder Isolation Kit (Calbio-chem, CA, USA), according to the manufacturer's instructions. DNA was analyzed using 1.5% agarose gel electrophoresis, observed under UV illumination and visualized using a gel documentation system (UVP Biospectrum HR410, USA). To furthur confirm the induction of apoptosis, the protein of zerumbone-induced HepG2 cells using Western-blotting indicated a low and high expression of Bcl2 and Bax proteins, respectively. In conclusion, these results indicate that no DNA fragmentation in the human hepatocellular liver carcinoma (HepG2) cells was observed even in the presence of caspase-3 during apoptosis. Therefore, we hypothesize that not all compounds necessairly indicate fragmentation of condensed chromatin into several discrete mass in cell lines as in vitro condition. PMID:23183623

  20. 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.

  1. Performance of heuristic methods driven by chaotic dynamics for ATSP and applications to DNA fragment assembly

    NASA Astrophysics Data System (ADS)

    Kato, Tomohiro; Hasegawa, Mikio

    Chaotic dynamics has been shown to be effective in improving the performance of combinatorial optimization algorithms. In this paper, the performance of chaotic dynamics in the asymmetric traveling salesman problem (ATSP) is investigated by introducing three types of heuristic solution update methods. Numerical simulation has been carried out to compare its performance with simulated annealing and tabu search; thus, the effectiveness of the approach using chaotic dynamics for driving heuristic methods has been shown. The chaotic method is also evaluated in the case of a combinatorial optimization problem in the real world, which can be solved by the same heuristic operation as that for the ATSP. We apply the chaotic method to the DNA fragment assembly problem, which involves building a DNA sequence from several hundred fragments obtained by the genome sequencer. Our simulation results show that the proposed algorithm using chaotic dynamics in a block shift operation exhibits the best performance for the DNA fragment assembly problem.

  2. 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.

  3. Specific sperm defects are differentially correlated with DNA fragmentation in both normozoospermic and teratozoospermic subjects.

    PubMed

    Mangiarini, A; Paffoni, A; Restelli, L; Ferrari, S; Guarneri, C; Ragni, G; Somigliana, E

    2013-11-01

    A positive effect of selecting spermatozoa under high magnification during intracytoplasmic sperm injection (ICSI) has been described, but a clear explanation has not been given yet. Previous works have shown that high magnification selected spermatozoa have significantly better chromatin status than unselected cells; on the other hand, it has been reported that spermatozoa with no morphological defects can also be negatively associated with embryo quality and pregnancy outcome attributable to DNA fragmentation. The aim of this study was to investigate whether sperm morphology is correlated with DNA fragmentation, both in normozoospermic and teratozoospermic patients. A prospective cohort study involving 32 subjects was recruited over a 3-month period. Spermatozoa were fixed on a slide for TUNEL assay and evaluated using an epifluorescent light microscope equipped with a video monitor. Single TUNEL-positive or -negative cells were evaluated for morphology at 4400 magnification. Each spermatozoon was then classified according to morphological normalcy or specific defects. The median percentage of typical forms was 11 and 0%, in the normozoospermic and teratozoospermic groups respectively (p=0.001). In normozoospermic samples, the percentage of TUNEL-positive morphologically normal spermatozoa was 4%. By comparison, spermatozoa showing a vacuolated head or a small non-oval head had a significantly higher incidence of DNA fragmentation in both groups (12 and 13%, 19 and 13% respectively; p<0.05). In contrast, spermatozoa showing a pyriform head had a DNA fragmentation rate similar to typical forms (3 and 5%, in normozoospermic and teratozoospermic respectively). This study shows that specific defects evaluated in fixed spermatozoa under high-power magnification are more likely to be associated with DNA fragmentation. High-magnification evaluation of spermatozoa can therefore reduce the probability of selecting cells carrying fragmented DNA during ICSI. PMID:24115574

  4. [Amplification of mitochondrial DNA fragments from ancient human teeth and bones].

    PubMed

    Hänni, C; Laudet, V; Sakka, M; Bègue, A; Stéhelin, D

    1990-01-01

    We extracted and visualized DNA from ancient human teeth and bones of 150 to 5,500 years B.P. from three deposits from the south of France. The DNA extracted was used as template for PCR with specific primers corresponding to a portion of the human mitochondrial genome. In our samples, we have amplified a specific DNA fragment of 121 bp which, in the case of one bone of 150 years B.P. has been cloned and sequenced. We show that this sequence is identical to the homologous region of human mitochondrial DNA. The striking implications of this new method for archaeological and paleontological studies are exposed. PMID:2113826

  5. Molecular Cloning and Sequence Analysis of Novel Cytochrome P450 cDNA Fragments from Dastarcus helophoroides

    PubMed Central

    Wang, Hai-Dong; Li, Fei-Fei; He, Cai; Cui, Jun; Song, Wang; Li, Meng-Lou

    2014-01-01

    The predatory beetle Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is a natural enemy of many longhorned beetles and is mainly distributed in both China and Japan. To date, no research on D. helophoroides P450 enzymes has been reported. In our study, for the better understanding of P450 enzymes in D. helophoroides, 100 novel cDNA fragments encoding cytochrome P450 were amplified from the total RNA of adult D. helophoroides abdomens using five pairs of degenerate primers designed according to the conserved amino acid sequences of the CYP6 family genes in insects through RT-PCR. The obtained nucleotide sequences were 250 bp, 270 bp, and 420 bp in length depending on different primers. Ninety-six fragments were determined to represent CYP6 genes, mainly from CYP6BK, CYP6BQ, and CYP6BR subfamilies, and four fragments were determined to represent CYP9 genes. Twenty-two fragments, submitted to GenBank, were selected for further homologous analysis, which revealed that some fragments of different sizes might be parts of the same P450 gene. PMID:25373175

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

    PubMed Central

    Apostolov, ?leksandar

    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

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

    PubMed

    Cicar, 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 22h 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 22h 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 (22h), a common practice in assisted reproductive techniques, an increase in oxidative sperm metabolism and in the proportion of fragmentedDNA should be expected. However, there was no effect on any of the other functional parameters associated with sperm fertilising capacity. PMID:25233794

  8. 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}.

  9. 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).

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

    PubMed

    Cheng, Xin; Ivessa, Andreas S

    2010-10-01

    Migration of fragmented mitochondrial DNA (mtDNA) to the nucleus has been shown to occur in multiple species including yeast, plants, and mammals. Several human diseases, including Pallister-Hall syndrome and mucolipidosis, can be initiated by mtDNA insertion mutagenesis of nuclear DNA. In yeast, we demonstrated that the rate of mtDNA fragments translocating to the nucleus increases during chronological aging. The yeast chronological lifespan (CLS) is determined by the survival of nondividing cell populations. Whereas yeast strains with elevated migration rates of mtDNA fragments to the nucleus showed accelerated chronological aging, strains with decreased mtDNA transfer rates to the nucleus exhibited an extended CLS. Although one of the most popular theories of aging is the free radical theory, migration of mtDNA fragments to the nucleus may also contribute to the chronological aging process by possibly increasing nuclear genomic instability in cells with advanced age. PMID:20626726

  11. Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments.

    PubMed

    Palecek, E; Brzdov, M; Cernock, H; Vlk, D; Brzda, V; Vojtesek, B

    1999-06-17

    Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these hands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 microM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5-20 microM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 microM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo. PMID:10380883

  12. 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.

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

    PubMed

    Mungua-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 (504 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

  14. 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

  15. Plant DNA Flow Cytometry and Estimation of Nuclear Genome Size

    PubMed Central

    DOLEEL, JAROSLAV; BARTO, JAN

    2005-01-01

    Background DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. Scope The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. Conclusions Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal. PMID:15596459

  16. Genetic analysis of an aphid endosymbiont DNA fragment homologous to the rnpA-rpmH-dnaA-dnaN-gyrB region of eubacteria.

    PubMed

    Lai, C Y; Baumann, P

    1992-04-15

    Buchnera aphidicola is a Gram- eubacterium with a DNA G+C content of 28-30 mol%. This organism is an obligate intracellular symbiont of aphids. To determine its similarity to or difference from other eubacteria, a 4.9-kb DNA fragment from B. aphidicola containing the gene homologous to Escherichia coli dnaA (a gene involved in the initiation of chromosome replication) was cloned into E. coli and sequenced. The order of genes on this fragment, 60K-10K-rnpA-rpmH-dnaA-dnaN-gyrB, was similar to that found in other eubacteria. The sole difference was the absence of recF between dnaN and gyrB. The deduced amino acid sequence of these proteins resembled those of E. coli by a 41 to 83% identity. Except for E. coli, in all the eubacteria so far examined, dnaA is preceded by multiple 9-nucleotide repeats known as a DnaA boxes. No DnaA boxes were detected in the endosymbiont DNA. The possibility that this observation is a consequence of the low G+C content of this DNA fragment (14 mol% G+C) is unlikely since in Mycoplasma capricolum this fragment (19 mol% G+C) has eight DnaA boxes (Fujita et al., 1992). The presence of the sequence, GATC, recognized by the Dam methyl-transferase system, only within six regions coding for proteins suggests that methylation is not a factor in the regulation of the initiation of endosymbiont chromosome replication. PMID:1572539

  17. Correlation between aneuploidy, apoptotic markers and DNA fragmentation in spermatozoa from normozoospermic patients.

    PubMed

    Vendrell, Xavier; Ferrer, Minerva; Garca-Mengual, Elena; Muoz, Patricia; Trivio, Juan Carlos; Calatayud, Carmen; Rawe, Vanesa Y; Ruiz-Jorro, Miguel

    2014-04-01

    Genetic and biochemical sperm integrity is essential to ensure the reproductive competence. However, spermatogenesis involves physiological changes that could endanger sperm integrity. DNA protamination and apoptosis have been studied extensively. Furthermore, elevated rates of aneuploidy and DNA injury correlate with reproductive failures. Consequently, this study applied the conventional spermiogram method in combination with molecular tests to assess genetic integrity in ejaculate from normozoospermic patients with implantation failure by retrospectively analysing aneuploidy (chromosomes 18, X, Y), DNA fragmentation, externalization of phosphatidylserine and mitochondrial membrane potential status before and after magnetic activated cell sorting (MACS). Aneuploid, apoptotic and DNA-injured spermatozoa decreased significantly after MACS. A positive correlation was detected between reduction of aneuploidy and decreased DNA damage, but no correlation was determined with apoptotic markers. The interactions between apoptotic markers, DNA integrity and aneuploidy, and the effect of MACS on these parameters, remain unknown. In conclusion, use of MACS reduced aneuploidy, DNA fragmentation and apoptosis. A postulated mechanism relating aneuploidy and DNA injury is discussed; on the contrary, cell death markers could not be related. An 'apoptotic-like' route could explain this situation. PMID:24581602

  18. Transient massive DNA fragmentation in nervous system during the early course of a murine neurodegenerative disease.

    PubMed

    Blondet, B; At-Ikhlef, A; Murawsky, M; Rieger, F

    2001-06-15

    In neurodegenerative diseases, such as Alzheimer's disease or HIV encephalitis, neuronal DNA fragmentation has been observed at unexpected high frequencies, without definitive evidence for activation of an irreversible apoptotic pathway. The wobbler mouse is a suggested genetic model of neurodegenerative disease. The mutant mouse develops normally until the fourth week of age when atrophy and weakness of forelimb muscles become apparent. There is a slow progression of the disease and wobbler mice may survive for several months. Spinal cord examination reveals the presence of several motoneurons with perikaryal vacuolar degeneration. In this study, we observed, using terminal dUTP nick-end-labelling staining in mutant spinal cord sections, a massive although very transient DNA fragmentation in different cell types, including glial cells and motoneurons, before the apparition of any clinical symptoms. In older wobbler mice, this DNA fragmentation had completely disappeared and the majority of motoneurons survived. To our knowledge, this is the first example of a massive and transient DNA fragmentation in the central nervous system during the early course of a neurodegenerative disease. PMID:11403940

  19. Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

    PubMed

    Strege, M; Lagu, A

    1991-07-01

    The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity. PMID:1654750

  20. A WHEAT DNA FRAGMENT EXHIBITS REDUCED POLLEN TRANSMISSION IN TRANSGENIC MAIZE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An 8.2 kb fragment of wheat genomic DNA containing the Glu1-Dx5 gene has been transferred to maize using biolistic transformation. The Glu1-Dx5 gene encodes the 1Dx5 high molecular weight glutenin subunit, a seed storage protein associated with good bread making properties. The transgenic maize plan...

  1. Fragment-based discovery of DNA gyrase inhibitors targeting the ATPase subunit of GyrB.

    PubMed

    Mesleh, Michael F; Cross, Jason B; Zhang, Jing; Kahmann, Jan; Andersen, Ole A; Barker, John; Cheng, Robert K; Felicetti, Brunella; Wood, Michael; Hadfield, Andrea T; Scheich, Christoph; Moy, Terence I; Yang, Qingyi; Shotwell, Joseph; Nguyen, Kien; Lippa, Blaise; Dolle, Roland; Ryan, M Dominic

    2016-02-15

    Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265?M inhibitor is described herein. PMID:26786695

  2. A strategy for seamless cloning of large DNA fragments from Streptomyces.

    PubMed

    Huang, Jun; Yu, Zhen; Li, Mei-Hong; Li, Na; Zhou, Jun; Zheng, Yu-Guo

    2015-10-01

    We report a novel method for the seamless cloning of large DNA fragments (SCLF) of up to 44 kb or larger from Streptomyces chromosomal DNA. SCLF is based on homologous recombination in Streptomyces and is easy to perform. The strategy of SCLF is to flank the target sequence in the chromosomal DNA with two identical restriction sites by the insertion of plasmids containing that site at either end of the fragment, which is then isolated by plasmid rescue through the self-ligation of restriction digested genomic DNA. The method involves three steps: (i) placing a certain restriction site (CRS) at the 3'-end of the target sequence by insertion through homologous recombination of a plasmid containing the CRS; (ii) inserting through homologous recombination at the 5'-end of the target sequence a linearized self-suicide vector with the identical CRS; (iii) digesting the genomic DNA with the certain restriction enzyme followed by self-ligation in order to plasmid rescue the target fragment. SCLF can be applied to other Actinomycetales, and further optimizations may reduce the amount of time required to perform this technique. PMID:26458547

  3. 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

  4. Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment.

    PubMed Central

    Nicholls, R D; Hill, A V; Clegg, J B; Higgs, D R

    1985-01-01

    We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta-globin alleles from an individual with beta-thalassaemia. Images PMID:2999697

  5. 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. PMID:24651721

  6. Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization.

    PubMed

    Wang, Shanquan; He, Jianzhong

    2011-09-01

    Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (1) bp and 514 (1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities. PMID:21824243

  7. A Linear Relationship between Crystal Size and Fragment Binding Time Observed Crystallographically: Implications for Fragment Library Screening Using Acoustic Droplet Ejection

    PubMed Central

    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

  8. 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; Mul, 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

  9. On the accuracy of fragment size measurement by image analysis in combination with some distribution functions

    NASA Astrophysics Data System (ADS)

    Sanchidrin, J. A.; Segarra, P.; Ouchterlony, F.; Lpez, L. M.

    2009-02-01

    Size distributions of fragments of crushed rock in conveyor belts and of blasted rock in a muckpile obtained by sieving are compared with the size distributions obtained by digital image analysis of photographs of the same materials taken on-site. Several calculation methods are tested, based on the raw distribution of fragment areas and on the volume-transformed ones. The influence of the calibration of the system on the results and the performance of the system in a non-calibrated mode are evaluated. The capacity of some distributions (Rosin-Rammler, Swebrec and lognormal) to fit the data in the coarse region (where particles can be delineated, i.e. discriminated individually) and to extrapolate to the non-delineated fines (where particles cannot be outlined and their contour delineated) is assessed. The error between the sizes measured and the sizes of the reference distributions (determined by sieving) increases from the coarse to the fines region. The maximum error at a given size depends primarily on its value relative to the fines cut-off (FCO) of the image analysis. In general, at sizes greater than the FCO, where the system is able to delineate fragments reliably, both volume and surface-based, calibrated, calculations can determine the sizes with maximum error expectancy of about 30%. Below the FCO, only the calibrated, volume calculation maintains a maximum error of 30%, down to sizes of about one fourth the FCO, rapidly increasing for smaller sizes. Where the calibration is done based on data above the FCO, errors can be large below this point, in excess of 80% at sizes half the FCO. In the fines range (sizes smaller than 0.2 times the FCO) the maximum errors can be close to or greater than 100% for most of the calculations and function fittings. Of the distributions tested, all of them are acceptable at sizes above the FCO; below that, the Swebrec function seems to adapt better towards the fines than the Rosin-Rammler and lognormal.

  10. 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. PMID:26936246

  11. 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.

  12. 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.

  13. 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. PMID:24642253

  14. Microchip capillary electrophoresis protocol to evaluate quality and quantity of mtDNA amplified fragments for DNA sequencing in forensic genetics.

    PubMed

    Fernández, Coro; Alonso, Antonio

    2012-01-01

    Here, we describe a microcapillary electrophoresis technique with application to the quantitative analysis of mtDNA hypervariable regions HVR1, HVR2, and HVR3 PCR amplicons previous to sequence analysis, which yields several important advantages compared to traditional separation and detection methods. Based on laser-induced fluorescence (LIF) detection, and performed in a microchip, this analysis system enables the handling of very small volumes via microchannels etched in the chip. Moreover it is faster than traditional methods; chip priming and sample loading are the only manual interventions, as the rest of the process is fully automated by software control: injection, electrophoretic separation, detection of the fluorescent signal, and calculation of both quantity and size. MtDNA amplicons are separated in microchannels with an effective length of 15 mm and detected by means of the fluorescence displayed by an intercalated dye. A software records the fluorescence and entails the data into size and concentration through the use of two internal standards and an external ladder of 11 fragments. The effectiveness of this procedure has been illustrated with a validation experiment carried out in our laboratory, in order to assess the detection limit of mtDNA sequencing by determining the minimal amount of PCR amplicon needed to edit a reproducible and high quality mtDNA sequence from complementary sequence data obtained using forward and reverse primers. PMID:22139673

  15. Pulsed-field electrophoresis of megabase-sized DNA

    SciTech Connect

    Gunderson, K.; Chu, G. )

    1991-06-01

    Success in constructing a physical map of the human genome will depend on two capabilities: rapid resolution of very large DNA and identification of migration anomalies. To address these issues, a systematic exploration of pulsed-field electrophoresis conditions for separating multimegabase-sized DNA was undertaken. Conditions were found for first liberating and then separating DNA up to 6 megabases at higher field strengths and more rapidly than previously reported. In addition, some conditions for transversely pulsed fields produced mobility inversion, in which increased size was accompanied by faster rather than slower migration. Importantly, anomalous migration could be identified by the presence of lateral band spreading, in which the DNA band remained sharply defined but spread laterally while moving down the gel. These results have implications for both practical applications and theoretical models of pulsed-field electrophoresis.

  16. Mutations in the C-terminal fragment of DnaK affecting peptide binding.

    PubMed Central

    Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

    1996-01-01

    Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

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

    SciTech Connect

    Brewer, L.R.; Davidson, C.; Balch, J.; Carrano, A.

    1995-01-30

    We have measured the three dimensional 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 axes. These measurements are important to understand what physical forces shape electrophoretic bands confined by a channel and also to aid in the design of high throughput DNA sequencers.

  18. An efficient algorithm for DNA fragment assembly in MapReduce.

    PubMed

    Xu, Baomin; Gao, Jin; Li, Chunyan

    2012-09-28

    Fragment assembly is one of the most important problems of sequence assembly. Algorithms for DNA fragment assembly using de Bruijn graph have been widely used. These algorithms require a large amount of memory and running time to build the de Bruijn graph. Another drawback of the conventional de Bruijn approach is the loss of information. To overcome these shortcomings, this paper proposes a parallel strategy to construct de Bruijin graph. Its main characteristic is to avoid the division of de Bruijin graph. A novel fragment assembly algorithm based on our parallel strategy is implemented in the MapReduce framework. The experimental results show that the parallel strategy can effectively improve the computational efficiency and remove the memory limitations of the assembly algorithm based on Euler superpath. This paper provides a useful attempt to the assembly of large-scale genome sequence using Cloud Computing. PMID:22960169

  19. Characterization of breaker efficiency based upon size distribution of polymeric fragments

    SciTech Connect

    Brannon, H.D.; Tjon Joe Pin, R.M.

    1995-12-31

    Damage to proppant packs is known to negatively impact well productivity. The damage is often the result of inadequate degradation of the polymers used to viscosity fracturing fluids. Gel breakers are used to degrade the polymer by cleaving the macro-molecule into smaller fragments, ideally to a size which can easily be produced during load recovery. Fluid viscosity reduction is commonly used to gauge polymer degradation. However, although viscosity reduction indicates polymer degradation, it is misleading to conclude that this reduced viscosity equates to improved fracture conductivity. Polymer fragments which are desolublized from the gelled fluid no longer contribute to fluid viscosity but do, unfortunately contribute significantly to proppant pack damage. Several new breaker technologies have been introduced in efforts to improve polymer degradation, and hence, improve fracture conductivity and ultimately well productivity. Many production case histories have been offered as evidence of the utility of the new technologies to improve well productivity. However, the facility to quantitatively determine the polymer degrading efficiency of the breakers has heretofore been lacking. Laboratory procedures, both wet chemical and instrumental, have recently been developed to address characterization of the relative degrading efficiency of the various breakers and breaking mechanisms. The analysis of the combined data provide both a qualitative size distribution, as well as a quantitative profile of the polymer fragments. Extensive studies were conducted employing the new procedures to compare the degrading efficiency of various oxidative and enzymatic breakers. Detailed analysis of the results are provided.

  20. Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

    SciTech Connect

    Elkin, Christopher; Kapur, Hitesh; Smith, Troy; Humphries, David; Pollard, Martin; Hammon, Nancy; Hawkins, Trevor

    2001-09-15

    We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620 phred20 bases as part of Joint Genome Institutes Production Process.

  1. Chromosome loss caused by DNA fragmentation induced in main nuclei and micronuclei of human lymphoblastoid cells treated with colcemid.

    PubMed

    Yamamoto, Mika; Wakata, Akihiro; Aoki, Yoshinobu; Miyamae, Yoichi; Kodama, Seiji

    2014-04-01

    Aneuploidy, a change in the number of chromosomes, plays an essential role in tumorigenesis. Our previous study demonstrated that a loss of a whole chromosome is induced in human lymphocytes by colcemid, a well-known aneugen. Here, to clarify the mechanism for colcemid-induced chromosome loss, we investigated the relationship between chromosome loss and DNA fragmentation in human lymphoblastoid cells treated with colcemid (an aneugen) compared with methyl methanesulfonate (MMS; a clastogen). We analyzed the number of fluorescence in situ hybridization (FISH) signals targeted for a whole chromosome 2 in cytokinesis-blocked binucleated TK6 cells and WTK-1 cells treated with colcemid and MMS, and concurrently detected DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Results revealed that DNA fragmentation occurred in 60% of all binucleated TK6 cells harboring colcemid-induced chromosome loss (30% of micronuclei and 30% of main nuclei). DNA fragmentation was observed in colcemid-induced micronuclei containing a whole chromosome but not in MMS-induced micronuclei containing chromosome fragments. In contrast, colcemid-induced nondisjunction had no effect on induction of DNA fragmentation, suggesting that DNA fragmentation was triggered by micronuclei containing a whole chromosome but not by micronuclei containing chromosome fragments or nondisjunction. In addition, the frequency of binucleated cells harboring chromosome loss with DNA fragmentation in micronuclei or main nuclei was higher in wild-type p53 TK6 cells than in mutated-p53 WTK-1 cells treated with colcemid. Taken together, these present and previous results suggest that colcemid-induced chromosome loss is caused by DNA fragmentation, which is triggered by a micronucleus with a whole chromosome and controlled by the p53-dependent pathway. PMID:24582839

  2. Ejaculate Oxidative Stress Is Related with Sperm DNA Fragmentation and Round Cells

    PubMed Central

    Iommiello, Valeria Maria; Albani, Elena; Di Rosa, Alessandra; Marras, Alessandra; Menduni, Francesca; Morreale, Giovanna; Levi, Shanti Lia; Pisano, Benedetta; Levi-Setti, Paolo Emanuele

    2015-01-01

    Oxidative stress (OS) plays an essential role in male infertility aetiology by affecting sperm quality, function, and also the integrity of sperm DNA. The assessment of oxidative stress in semen may be an important tool to improve the evaluation of sperm reproductive capacity. The purpose of this study was the evaluation of any possible relation between the unbalance of oxidative stress caused by superoxide anion in the ejaculate with the presence of sperm DNA fragmentation and high concentration of round cells. 56 semen samples from males from couples suffering from infertility were evaluated according to World Health Organisation (WHO) 2010 guidelines. Oxidative stress levels from N1 (low) to N4 (high) were assessed in ejaculates using oxiSperm; DFI (sperm DNA fragmentation index) as assessed by the SCSA (Sperm Chromatin Structure Assay) was used for evaluation of sperm chromatin integrity. Our data show that high oxidative stress (N3-N4 levels) correlated positively with a DFI ≥ 30% (P = 0.0379) and round cells ≥1.500.000/mL (P = 0.0084). In conclusion, OS increases sperm DNA damage. Thus evaluation of semen OS extent of sperm DNA damage in infertile man could be useful to develop new therapeutic strategies and improve success of assisted reproduction techniques (ART). PMID:25802519

  3. In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation.

    PubMed

    Fraczek, Monika; Szumala-Kakol, Anna; Dworacki, Grzegorz; Sanocka, Dorota; Kurpisz, Maciej

    2013-11-01

    The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6 + IL-8 and IL-12 + IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy. PMID:24344359

  4. One-step DNA fragment assembly and circularization for gene cloning.

    PubMed

    Zuo, Peijun; Rabie, Bakr M

    2010-01-01

    This article describes a one-step procedure based on Taq polymerase for the precise assembly of DNA fragments into circular constructs as long as 6 kb. The only prior step needed was the amplification of the gene to be cloned and the linear vector backbone, and the whole process up to assembly and circularization lasted only 2 days, compared with the conventional method's 2 weeks. Furthermore, the final DNA construct was used to transform Escherichia coli directly without any further treatment. By circumventing the need for DNA ligase, our "Quick Assemble" method offers an improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-directed mutagenesis and whole-DNA library gene shuffling, as well as the construction of new plasmids with any promoter, resistance gene marker, restriction site, or any DNA tag. PMID:19494420

  5. Identification of novel putative regulators of the major apoptotic nuclease DNA Fragmentation Factor.

    PubMed

    Hanus, Jakub; Kalinowska-Herok, Magdalena; Widlak, Piotr

    2010-01-01

    Yeast two- and three-hybrid systems were used to screen cDNA libraries from HeLa cells and human brain tissue to identify novel protein partners of DNA Fragmentation Factor, the major apoptotic nuclease. The two-hybrid system revealed the DFF45 inhibitory subunit of the nuclease as the only identified partner of the DFF40 catalytic subunit. Similar analysis revealed several protein candidates that potentially interact with the DFF45 subunit: FBXO28, FOSL1, PGK1, PCNT, FHL1 and GFAP. Recombinant GFAP protected DFF45 against cleavage with caspase-3 and prevented activation of the DFF nuclease in vitro. In addition, three-hybrid system results revealed a putative novel protein partner of the DFF40-DFF45 heterodimer. The candidate cDNA contained two open reading frames that mapped to an intron of the GBF1 gene. Products of the candidate cDNA derived from a cell-free transcription/translation system inhibited DNA cleavage by recombinant caspase-activated DFF. This putative partner of DFF may have functional importance in regulating the apoptotic response because its RNAi silencing facilitated cleavage of the DFF45 inhibitor subunit and affected chromatin fragmentation in HeLa cells undergoing apoptosis. This hypothetical protein, named DRIG based on an acronym specifying its genomic location, could be a novel factor involved in regulation of DFF40 apoptotic nuclease. PMID:21152448

  6. 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.

  7. Sperm DNA Fragmentation and Standard Semen Parameters in Algerian Infertile Male Partners

    PubMed Central

    Boushaba, Sarah

    2015-01-01

    Purpose To date, standard semen parameters have been the only parameters investigated in sperm samples of infertile men in Algeria. We investigated, for the first time, semen parameters according to sperm DNA fragmentation (SDF) in these subjects. Materials and Methods SDF was determined by a validated sperm chromatin dispersion test in 26 infertile men. Patients were split into two groups according to the SDF level estimated by the DNA fragmentation index (DFI): the low fragmentation group (LFG; LFG with DFI ?18%) and high fragmentation group (HFG; HFG with DFI >18%). The standard semen parameters were measured in both groups. Results We found that semen concentration and motility were negatively correlated with DFI (r=-0.65, r=-0.45, respectively; p<0.05), while morphology and semen volume were not correlated with it (r=0.24, r=-0.18, respectively; p>0.05). Comparison of the sperm concentration revealed that it was significantly higher in LFG than in HFG (37.57%13.16% vs. 7.32%3.59%, respectively; p<0.05), whereas no significant difference was observed regarding sperm motility and morphology. Conclusions Our findings suggest that SDF correlates well with both sperm motility and concentration but not with morphology. Thus, we conclude that SDF evaluation provides additional information regarding sperm quality, and should be used as a complementary test for assessing semen characteristics in infertile males. PMID:25927056

  8. 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

  9. Ovarian Steroids Decrease DNA Fragmentation In Serotonin Neurons of Non-injured Rhesus Macaques

    PubMed Central

    Lima, Fernanda B.; Bethea, Cynthia L.

    2009-01-01

    We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys received Silastic implants that were empty (placebo) or containing estradiol (E), progesterone (P) or estradiol plus progesterone (E+P) for one month. Eight levels of the dorsal raphe nucleus in a rostral to caudal direction were immunostained with TUNEL (terminal deoxynucleotidyl transferase nick end labeling). Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area. A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2 and TUNEL positive cells were counted. In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL positive cells (Mann Whitney test, both treatments p=0.04) and E+P treatment reduced the number of TUNEL positive cells/cubic millimeter (Mann Whitney test, p=0.04). Double immunocytochemistry for TUNEL and TPH indicated that DNA fragmentation was prominent in serotonin neurons. These data suggest that in the absence of ovarian steroids, a cascade of gene and protein expression leads to an increase in DNA fragmentation in serotonin neurons. Conversely, ovarian steroids have a neuroprotective role in the non-injured brain and prevent DNA fragmentation and cell death in serotonin neurons of nonhuman primates. PMID:19823180

  10. 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.

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

    SciTech Connect

    Tee, Thiam-Tsui; Cheah, Yew-Hoong; Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur ; 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.

  12. [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. PMID:25881705

  13. Terminal strand-switching of E. coli RNA polymerase transcribing a truncated DNA fragment.

    PubMed

    Oostra, B A; Arnberg, A C; Ab, G; Gruber, M

    1981-10-27

    When transcribing a restriction fragment containing the promoters and the first part of the rrnE operon of Escherichia coli, RNA polymerase holoenzyme starts exclusively on the promoters. Besides run-off transcripts, molecules longer than template-size are formed by terminal strand switch. PMID:6269631

  14. 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, respectively. By comparing them in terms of cumulative number of blocks it is observed a significant reduction of blocks bigger than one cubic meter, and a sharp increase of blocks with volumes smaller than a cubic meter. The difference between the area defined by the IBSD and the RBSD is typically attributed to the fragmentation energy in blastability studies. Even though rock fall blocks can be generated by disaggregation of the originally detached rock mass, in the Cadí case we interpret that block breakage is the predominant mechanism.

  15. 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. PMID:25044673

  16. Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

    PubMed Central

    Roychoudhury, R; Jay, E; Wu, R

    1976-01-01

    Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA. Images PMID:765970

  17. 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.

  18. 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 592aa protein with a predicted molecular mass of 69.8kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10nmol of dNTP into acid insoluble material in 30min at 65C). 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

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

    PubMed

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

    2015-08-01

    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. 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

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

    PubMed

    Hamilton, Thais Rose Dos Santos; Castro, Letícia Signori de; 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

  2. Restriction fragment length polymorphism detected by cDNA and genomic DNA clones in Stylosanthes.

    PubMed

    Liu, C J; Musial, J M

    1995-12-01

    A DNA isolation method suitable for genomic library construction and RFLP analyses of the forage legume Stylosanthes was developed. Probes isolated using this method were used to investigate the feasibility of constructing RFLP-based genetic maps in this genus. Two hundred and seventy-one PstI genomic DNA and 134 cDNA clones were analysed against four Stylosanthes accessions, including two tetraploids and two diploids, with the use of two restriction enzymes, DraI and HindIII. The proportion of clones which detected single-copy sequences from the PstI genomic library was higher than that from the cDNA library, but the percentage of clones which detected low-copy sequences was doubled in the latter. There was no significant difference in the level of RFLPs detected by gDNA and cDNA probes, although the level of polymorphism was lower in the diploids. A large proportion of RFLPs seemed to have resulted from mutation/base substitution events, and this was especially the case in diploids. PMID:24170048

  3. Detection of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers.

    PubMed

    Deaville, Eddie R; Maddison, Ben C

    2005-12-28

    The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal. PMID:16366726

  4. Structures of Minimal Catalytic Fragments of Topoisomerase V Reveals Conformational Changes Relevant for DNA Binding

    SciTech Connect

    Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

    2010-12-03

    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.

  5. 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 mainly to kinetic & potential ion scattering.[Funded by NSERC and the Canadian Space Agency].

  6. 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 elevated sperm DNA fragmentation. Extensive DNA fragmentation probably cannot be repaired by the egg and the spontaneous abortion rate is {approx}2x higher if a man has more than 30% of sperm showing DNA fragmentation. DNA fragmentation is an excellent marker for exposure to potential reproductive toxicants and a diagnostic/prognostic tool for potential male infertility.

  7. Reconstructing Pre-Fragmentation Bubble Size Distributions from Volcanic Ash using Stereo SEM Analysis

    NASA Astrophysics Data System (ADS)

    Sahagian, D. L.; Proussevitch, A. A.; Mulukutla, G. K.; Genareau, K.

    2010-12-01

    We have conducted an analysis of bubble (BSD) and ash particle (PSD) size distributions for ashes from two contrasting eruptions. The first is the May, 1980 eruption of Mt. St. Helens (MSH), a dacitic plinian eruption that spread ash over a large area of the Western U.S. The second is the basaltic sub-plinian 1974 eruption of Fuego (Guatemala), which was confined to local deposition with less variation of ash PSDs. Four successive small explosive eruptions of Fuego produced less than 0.02 km3 of dense rock equivalent (DRE) in a dispersal area of 80 km from the volcano. In contrast, the May 1980 plinian eruption of Mount St. Helens resulted in a distal fallout leading to a large subaerial ash deposit as far away as 325 km from the volcano. Pyroclastic flows added extensive fine material to the eruption column resulting in extensive ash dispersal. MSH samples were collected from a range of distances away from the vent, while collection of samples from Fuego was limited to nearer regions due to the lesser dispersal of the ash. Technique- Stereo SEM analysis of BSD of eruptions products (ash) to determine the pre-fragmentation properties of ash-producing magma bodies. This information is normally considered lost due to fragmentation of bubbles in late stages of eruptions. However, using SSEM, we have devised a technique to determine the pre-fragmentation BSDs that reflect the conduit processes of bubble nucleation and growth, and magma rise history. Using standard off-the-shelf software (Alicona MeX) to create Digital Elevation Models (DEMs) of individual ash particles, we built a database of ash surface characteristics. These surfaces include imprints of bubbles that exploded during fragmentation. We use the curvature of these imprints to reconstruct the complete bubbles, using newly developed software we call Bubblemaker that extrapolates the measured DEMs using best-fit ellipsoids of revolution (not necessarily spherical). We have now reconstructed the bubble volumes. These data are used in turn to characterize the statistical parameters of the bubble population, including size distribution, distribution function type (log-normal), its moments, and bubble number density. Our results show that the silicic energetic MSH eruption ashes contain smaller bubbles and higher number densities than do the ashes collected from the more basaltic Fuego eruption. From these results, it is possible to speculate regarding eruption processes. It appears that within a single eruption, there is relatively little variability of bubble sizes as a function of depositional distance from the vent, although other ash characteristics such as PSD vary more strongly with distance.

  8. DNA methylation Landscape of body size variation in sheep

    PubMed Central

    Cao, Jiaxue; Wei, Caihong; Liu, Dongming; Wang, Huihua; Wu, Mingming; Xie, Zhiyuan; Capellini, Terence D.; Zhang, Li; Zhao, Fuping; Li, Li; Zhong, Tao; Wang, Linjie; Lu, Jian; Liu, Ruizao; Zhang, Shifang; Du, Yongfei; Zhang, Hongping; Du, Lixin

    2015-01-01

    Sub-populations of Chinese Mongolian sheep exhibit significant variance in body mass. In the present study, we sequenced the whole genome DNA methylation in these breeds to detect whether DNA methylation plays a role in determining the body mass of sheep by Methylated DNA immunoprecipitation – sequencing method. A high quality methylation map of Chinese Mongolian sheep was obtained in this study. We identified 399 different methylated regions located in 93 human orthologs, which were previously reported as body size related genes in human genome-wide association studies. We tested three regions in LTBP1, and DNA methylation of two CpG sites showed significant correlation with its RNA expression. Additionally, a particular set of differentially methylated windows enriched in the “development process” (GO: 0032502) was identified as potential candidates for association with body mass variation. Next, we validated small part of these windows in 5 genes; DNA methylation of SMAD1, TSC1 and AKT1 showed significant difference across breeds, and six CpG were significantly correlated with RNA expression. Interestingly, two CpG sites showed significant correlation with TSC1 protein expression. This study provides a thorough understanding of body size variation in sheep from an epigenetic perspective. PMID:26472088

  9. DNA methylation Landscape of body size variation in sheep.

    PubMed

    Cao, Jiaxue; Wei, Caihong; Liu, Dongming; Wang, Huihua; Wu, Mingming; Xie, Zhiyuan; Capellini, Terence D; Zhang, Li; Zhao, Fuping; Li, Li; Zhong, Tao; Wang, Linjie; Lu, Jian; Liu, Ruizao; Zhang, Shifang; Du, Yongfei; Zhang, Hongping; Du, Lixin

    2015-01-01

    Sub-populations of Chinese Mongolian sheep exhibit significant variance in body mass. In the present study, we sequenced the whole genome DNA methylation in these breeds to detect whether DNA methylation plays a role in determining the body mass of sheep by Methylated DNA immunoprecipitation - sequencing method. A high quality methylation map of Chinese Mongolian sheep was obtained in this study. We identified 399 different methylated regions located in 93 human orthologs, which were previously reported as body size related genes in human genome-wide association studies. We tested three regions in LTBP1, and DNA methylation of two CpG sites showed significant correlation with its RNA expression. Additionally, a particular set of differentially methylated windows enriched in the "development process" (GO: 0032502) was identified as potential candidates for association with body mass variation. Next, we validated small part of these windows in 5 genes; DNA methylation of SMAD1, TSC1 and AKT1 showed significant difference across breeds, and six CpG were significantly correlated with RNA expression. Interestingly, two CpG sites showed significant correlation with TSC1 protein expression. This study provides a thorough understanding of body size variation in sheep from an epigenetic perspective. PMID:26472088

  10. Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

    PubMed Central

    Gray, A J; Beecher, D E; Olson, M V

    1984-01-01

    A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

  11. A simulation study of sample size for DNA barcoding.

    PubMed

    Luo, Arong; Lan, Haiqiang; Ling, Cheng; Zhang, Aibing; Shi, Lei; Ho, Simon Y W; Zhu, Chaodong

    2015-12-01

    For some groups of organisms, DNA barcoding can provide a useful tool in taxonomy, evolutionary biology, and biodiversity assessment. However, the efficacy of DNA barcoding depends on the degree of sampling per species, because a large enough sample size is needed to provide a reliable estimate of genetic polymorphism and for delimiting species. We used a simulation approach to examine the effects of sample size on four estimators of genetic polymorphism related to DNA barcoding: mismatch distribution, nucleotide diversity, the number of haplotypes, and maximum pairwise distance. Our results showed that mismatch distributions derived from subsamples of ≥20 individuals usually bore a close resemblance to that of the full dataset. Estimates of nucleotide diversity from subsamples of ≥20 individuals tended to be bell-shaped around that of the full dataset, whereas estimates from smaller subsamples were not. As expected, greater sampling generally led to an increase in the number of haplotypes. We also found that subsamples of ≥20 individuals allowed a good estimate of the maximum pairwise distance of the full dataset, while smaller ones were associated with a high probability of underestimation. Overall, our study confirms the expectation that larger samples are beneficial for the efficacy of DNA barcoding and suggests that a minimum sample size of 20 individuals is needed in practice for each population. PMID:26811761

  12. Ejecta from experimental impact craters: Particle size distribution and fragmentation energy

    NASA Astrophysics Data System (ADS)

    Buhl, Elmar; Sommer, Frank; Poelchau, Michael H.; Dresen, Georg; Kenkmann, Thomas

    2014-07-01

    The particle size distribution (PSD) of impact crater ejecta is an important parameter that is useful for understanding the formation of natural craters, the distribution of space debris, the influence of impact events on climate and energy partitioning in impact events. 11 impact experiments into dry and water-saturated sandstone were performed and analyzed. The experiments span a range of impact velocities from 2.5 to 5.3 km s-1 using projectile sizes from 2.5 to 12 mm. Kinetic impact energies between 874 and 80,338 J were achieved. Ejecta of these experiments was collected and the PSD was measured and quantified with power law fits. The resulting power law exponents lie between 2.54 and 2.74. Our results do not show an influence of impact energy or impact velocity on the PSD of impact ejecta. A significant increase in the PSD values was found from dry to water-saturated sandstone targets. We suggest that water saturation of the target has multiple effects on ejecta fragmentation. A comparison of our experimental data with data from the literature shows no correlation between the target material lithology and the ejecta PSD. Interestingly, literature data for disruption experiments revealed a strong influence imparted energy density on the D-values. PSD values were used to calculate the energy spent for target fragmentation and show that the fraction of impact energy used for comminution is in the lower single-digit percentage.

  13. Protein-DNA interactions in vitro with 5'-flanking DNA fragments from the chicken vitellogenin gene.

    PubMed

    Bakker, O; Das, A T; Ab, G

    1988-01-01

    The expression of the vitellogenin gene in the liver of oviparous animals is under strict control of estrogen. We have studied the interaction of proteins extracted from nuclei of different estrogen responsive tissues with two fragments (-728 to -470 and -625 to -470) of the upstream region of the chicken vitellogenin gene, using the gel-retardation technique. We found a complex pattern of retarded bands using nuclear extracts from laying hen liver, rooster liver and MCF-7 cells. The patterns observed display differences in the position and intensities of some of the bands, depending on the source of the extract used. The possible significance of these findings will be discussed. PMID:2838691

  14. Do Pilea Microphylla Improve Sperm DNA Fragmentation and Sperm Parameters in Varicocelized Rats?

    PubMed

    Heidari, Reza; Alizadeh, Rafieh; Abbasi, Niloufar; Pasbakhsh, Parichehr; Hedayatpour, Azim; Farajpour, Mostafa; Khaleghi, Mohammad Reza; Abbasi, Mehdi; Dehpour, Ahmad Reza

    2015-09-01

    Varicocele is one of the most common causes of primary male infertility. Pilea microphylla (PM) is being used as folk medicine. This study was aimed to investigate the effects of PM in a rat model of varicocele. A total of 30 male Wistar rats were divided into control, sham, varicocele, accessory varicocele and PM-treated groups. After 10 weeks of varicocele induction, sperm parameters and chromatin (Aniline blue, acridine orange and toluidine blue) were evaluated, except for the treated and accessory groups that received 50 mg/kg PM orally daily for 10 weeks and then were sacrificed. Sperm parameters significantly decreased in varicocele groups (P < 0.01). Moreover, there was a negative correlation between the DNA fragmentation and sperm parameters in varicocelized rats. Administration of PM led to significantly increased sperm parameters and AO staining (P < 0.05). These findings suggest that PM improves sperm parameters and DNA fragmentation in varicocelized rats. PM can reduce the damage to sperm DNA but not chromatin condensation. PMID:26553082

  15. 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.

  16. Low energy electron induced fragmentation and reactions of DNA and its molecular components

    NASA Astrophysics Data System (ADS)

    Bass, Andrew

    2005-05-01

    Much research has been stimulated by the recognition that ionizing radiation can, in condensed matter, generate large numbers of secondary electrons with energies less than 20 eV [1] and by the experimental demonstration that such electrons may induce both single and double strand breaks in plasmid DNA [2]. Identifying the underlying mechanisms involves several research methodologies, from further experiments with DNA to studies of the electron interaction with the component `sub-units' of DNA in both the gas and condensed phases [3]. In particular, understanding electron-induced strand break damage, the type of damage most difficult for organisms to repair, necessitates study of the sub-units of DNA back-bone, and here Tetrahyrofuran (THF) and its derivatives, provide a useful model for the furyl ring at the centre of the deoxyribose sugar. In this contribution, we review with particular reference to DNA and related molecules, the use of electron spectroscopy and mass spectrometry to study electron-induced fragmentation and reactions in thin molecular solids. We describe a newly completed instrument that combines laser post-ionization with a time-of-flight mass analyzer for highly sensitive ion and neutral detection. Use of the instrument is illustrated with results for THF and derivatives. Anion desorption measurements reveal the role of transient negative ions (TNI) and Dissociative Electron Attachment in significant molecular fragmentation and permit effective cross sections for this electron-induced damage to be obtained. The neutral yield functions also illustrate the importance of TNI, mirroring features seen in recently measured cross sections for electron induced aldehyde production in THF [4]. 1. J. A. Laverne and S. M. Pimblott, Radiat. Res. 141, 208 (1995) 2. B. Boudaiffa, et al, Science 287, 1658 (2000) 3. L. Sanche. Physica Scripta. 68, C108, (2003) 4. S.-P. Breton, et al.,J. Chem. Phys. 121, 11240 (2004)

  17. Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209.

    PubMed

    Ramachander, T V N; Rohini, D; Belhekar, A; Rawal, S K

    2002-12-20

    An approximately 5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and introduced into Escherichia coli. The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions. The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85x10(5). Purified PHB granules were spherical with an average size of 1.1 microm and of stable configuration. DSC thermogram suggested high crystalline nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 degrees C. Recombinant E. coli cells preferentially utilized glycerol as the carbon source and accumulated 25-28 times more PHB than the native S. aureofaciens. PMID:12559428

  18. Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment.

    PubMed

    Kira, Nozomu; Ohnishi, Kohei; Miyagawa-Yamaguchi, Arisa; Kadono, Takashi; Adachi, Masao

    2016-02-01

    We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a "gene cassette"). Then the various lengths of linear vectors (+0-+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500bp at both ends of the gene cassette (+0-+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧50bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5-300fmol/shot), we observed that transformation efficiencies using 37.5fmol (52.2ng)/shot of the linear vector were not significantly different from those obtained using 300fmol of the linear vector. The 300fmol quantity was set considering the quantity of the circular plasmid (1μg=approx. 300fmol) and the 37.5fmol quantity was set for quick and easy preparation of approximately 500ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50μl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications. PMID:26711090

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

    PubMed

    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

    2012-08-01

    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

  20. 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

  1. Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment).

    PubMed

    Olsen, Tivoli J; Choi, Yongki; Sims, Patrick C; Gul, O Tolga; Corso, Brad L; Dong, Chengjun; Brown, William A; Collins, Philip G; Weiss, Gregory A

    2013-05-29

    Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10,000 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme's open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme's closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme's open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I. PMID:23631761

  2. The Roles of Family B and D DNA Polymerases in Thermococcus Species 9N Okazaki Fragment Maturation*

    PubMed Central

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

    2015-01-01

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

  3. The roles of family B and D DNA polymerases in Thermococcus species 9N Okazaki fragment maturation.

    PubMed

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F

    2015-05-15

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

  4. The effects of chromium(III) on DNA replication across O{sup 6}. Methylguanine by DNA polymerase {beta} and E. coli DNA polymerase I-Klenow fragment

    SciTech Connect

    Singh, J.; Su, L.; Snow, E.T.

    1995-11-01

    We are investigating the molecular mechanisms of how metal ions affect the fidelity of DNA replication. In our DNA replication system primed templates site-specifically modified with a model mutagenic lesion. O{sup 6}-methyldeoxyguanosine (O{sup 6}mG), are replicated in vitro by various purified DNA polymerases. O{sup 6}mG blocks DNA replication by human DNA polymerase {beta} but is less inhibitory to E. coli DNA Polymerase I-Klenow Fragment (KF) and its 3`-5` exonuclease deficient counterpart [KF (exo{sup {minus}})]. All three DNA polymerases exhibit a strong prelesion block and decreased rates of nucleotide extension. Polymerase {beta} exhibits discrimination against the incorporation of the right (dC) versus the wrong (dT) base. dT is incorporated in preference to dC opposite O{sup 6}mG-dT. KF (exo{sup {minus}}), on the other hand, extends the O{sup 6}mG-dT base pair more efficiently than O{sup 6}mG-dC. Thus individual polymerases may have opposing preferences for incorporation versus extension. Our previous studies have shown that chromium (III) [Cr(III)] increases DNA polymerase processivity and lowers the fidelity of DNA replication. At low final concentrations (about 0.1 {mu}M) Cr(III) stimulates the rate of nucleotide incorporation opposite O{sup 6}mG by KF(exo{sup {minus}}) and, to a lesser extent, by polymerase {beta}. Cr(III) does not affect incorporation of dT opposite dA, but decreases by 10-fold the K{sub M} for incorporation of dT opposite O{sup 6}mG. This constitutes an important mutagenic effect. Further experiments are underway to determine how Cr(III) affects the DNA binding and kinetic parameters of these exonuclease deficient DNA repair polymerases.

  5. Roles of the major apoptotic nuclease-DNA fragmentation factor-in biology and disease.

    PubMed

    Widlak, P; Garrard, W T

    2009-01-01

    It has now been more than ten years since the discovery of the major apoptotic nuclease, DNA fragmentation factor (DFF), also known as caspase-activated DNase (CAD). Here we review the recent literature that has uncovered new insight into DFF's regulation, and both its positive and negative roles in human disease. Cells from mice deficient in DFF still undergo apoptotic death without significant cell-autonomous DNA degradation. Their corpses' genomes are subsequently degraded by lysosomal DNase II after phagocytosis. However,DFF-deficient mice are more susceptible to cancer. Indeed, several different cancers in humans are associated with defects in DFF expression and it has been proposed that DFF is a p53-independent tumor suppressor. Negative aspects of DFF expression include contributing to susceptibility to acquire systemic lupus erythematosus, to chromosomal translocations that result in mixed lineage leukemias, and in the possible spreading of oncogenes and HIV due to horizontal gene transfer. PMID:18810317

  6. 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

  7. Impact of forest fragment size on the population structure of three palm species (Arecaceae) in the Brazilian Atlantic rainforest.

    PubMed

    Portela, Rita de Cssia Quitete; dos Santos, Flavio Antonio Maes

    2014-06-01

    The main threats to natural populations in terrestrial ecosystems have been widly recognized to be the habitat fragmentation and the exploitation of forest products. In this study, we compared the density of the populations and the structure of three tropical palm species, Astrocaryum aculeatissimum, Euterpe edulis and Geonoma schottiana. For this, we selected five forest fragments of different sizes (3 500ha, 2 400ha, 57ha, 21ha and 19ha) where palms were censused in nine 30 x 30m plots. We tracked the palms survival from 2005 to 2007, and recorded all new individuals encountered. Each individual was assigned in one of the five ontogenetic stages: seedling, infant, juvenile, immature and reproductive. The demographic structure of each palm species was analyzed and compared by a generalized linear model (GLM). The analysis was performed per palm species. The forest fragment area and the year of observation were explanatory variables, and the proportion of individuals in each ontogenetic class and palm density were response variables. The total number of individuals (from seedlings to reproductives, of all species) monitored was 6 450 in 2005, 7 268 in 2006, and 8 664 in 2007. The densities of two palm species were not influenced by the size of the fragment, but the population density of A. aculeatissimum was dependent on the size of the fragment: there were more individuals in the bigger than in the smaller forest fragments. The population structure of A. aculeatissimum, E. edulis, and G. schottiana was not altered in the smaller fragments, except the infants of G. schottiana. The main point to be drawn from the results found in this study is that the responses of density and population structure seem not to be dependent on fragment size, except for one species that resulted more abundant in bigger fragments. PMID:25102629

  8. Induction of apoptosis by dietary polyunsaturated fatty acids in human leukemic cells is not associated with DNA fragmentation.

    PubMed

    Chiu, L C; Wan, J M; Ooi, V E

    2000-10-01

    Apoptosis is important in anticancer strategy. In this study, bivariate annexin V/PI flow cytometry showed that dietary polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), induced apoptosis in human leukemic HL-60 but not K-562 cells. Results from DNA-PI flow cytometry and TUNEL flow cytometry illustrated that neither AA nor EPA induced DNA fragmentation in the leukemic cells. These findings suggested that the AA- and EPA-induced apoptosis might not associate with endonucleases activation, and DNA fragmentation could not be used as a sole criterion to identify apoptotic cells. PMID:10995893

  9. Collisional energy transfer and fragmentation of size selected CO2 clusters

    NASA Astrophysics Data System (ADS)

    Buck, U.; Lauenstein, Ch.; Sroka, R.; Tolle, M.

    1988-06-01

    Carbon dioxide clusters are generated in a supersonic molecular beam and size selected by scattering from a He beam. By analyzing the measured time-of-flight spectra as a function of the deflection angle, differential energy loss spectra for (CO2)2 — He are obtained which show a rotational rainbow structure with a maximal energy transfer of Δ E/E=0.4. This result is compatible with the slipped parallel structure of dimer but not with the T-shaped geometry. The scattering analysis is also used to derive information about the pressure dependence of cluster formation and the fragmentation by electron impact ionisation. The latter process leads preferably to the monomer product ion CO{2/+} with a small but finite probability for other ionic channels.

  10. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    SciTech Connect

    Murru, S.; Casula, L.; Moi, P.

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  11. Enhanced recovery and restriction mapping of DNA fragments cloned in a new lambda vector.

    PubMed Central

    Whittaker, P A; Campbell, A J; Southern, E M; Murray, N E

    1988-01-01

    In this paper we describe a modification to the lambda vector EMBL3 which greatly expedites the construction of restriction maps of cloned DNA sequences. In the modified vector, EMBL3cos, all the phage coding sequences are placed to the right of the cloning sites so that the left cohesive end is separated by only 200bp, rather than 20kb (as in conventional lambda vectors), from the inserted DNA fragment. We show that reliable restriction maps can be rapidly constructed from partial digests of clones made in this vector by labelling the left cohesive end with a complementary 32P-labelled oligonucleotide. In addition, we quantify the restriction of clones containing human DNA by the McrA and McrB systems of E. coli and show that the use of Mcr- plating strains can increase the yield of recombinant phage up to tenfold, to give cloning efficiencies of greater than or equal to 10(7) pfu/microgram of human DNA. Images PMID:2841642

  12. ERp57/PDIA3 binds specific DNA fragments in a melanoma cell line.

    PubMed

    Aureli, Cristina; Gaucci, Elisa; Arcangeli, Valentina; Grillo, Caterina; Eufemi, Margherita; Chichiarelli, Silvia

    2013-07-25

    ERp57/PDIA3 is a ubiquitously expressed disulfide isomerase protein, which acts in concert with calreticulin and calnexin in the folding of glycoproteins destined to the plasma membrane or to be secreted. Its canonical compartment is the endoplasmic reticulum, where it acts as a chaperone and redox catalyst, but non canonical locations have been described as well, and ERp57 has been found associated with DNA and nuclear proteins. In previous work performed in HeLa cells, ERp57 has been demonstrated to bind specific DNA sequences involved in the stress response. The direct interaction with the DNA sequences identified as ERp57-targeted regions in HeLa cells has now been confirmed in a melanoma cell line. Furthermore, the ERp57 silencing, achieved by RNA interference, has produced a significant down-regulation of the expression of target genes. The possible involvement of other proteins in complex with ERp57 has been studied by an in vitro biotin-streptavidin based binding assay and the interacting protein APE/Ref-1 has been also assessed for its direct association with the ERp57 target regions. In conclusion, nuclear ERp57 interacts in vivo with DNA fragments in melanoma cells and is potentially involved in the transcriptional regulation of its target genes. PMID:23587917

  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. Klenow Fragment Discriminates against the Incorporation of the Hyperoxidized dGTP Lesion Spiroiminodihydantoin into DNA.

    PubMed

    Huang, Ji; Yennie, Craig J; Delaney, Sarah

    2015-12-21

    Defining the biological consequences of oxidative DNA damage remains an important and ongoing area of investigation. At the foundation of understanding the repercussions of such damage is a molecular-level description of the action of DNA-processing enzymes, such as polymerases. In this work, we focus on a secondary, or hyperoxidized, oxidative lesion of dG that is formed by oxidation of the primary oxidative lesion, 2'-deoxy-8-oxo-7,8-dihydroguanosine (8-oxodG). In particular, we examine incorporation into DNA of the diastereomers of the hyperoxidized guanosine triphosphate lesion spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP). Using kinetic parameters, we describe the ability of the Klenow fragment of Escherichia coli DNA polymerase I lacking 3' → 5' exonuclease activity (KF(-)) to utilize (S)-dSpTP and (R)-dSpTP as building blocks during replication. We find that both diastereomers act as covert lesions, similar to a Trojan horse: KF(-) incorporates the lesion dNTP opposite dC, which is a nonmutagenic event; however, during the subsequent replication, it is known that dSp is nearly 100% mutagenic. Nevertheless, using kpol/Kd to define the nucleotide incorporation specificity, we find that the extent of oxidation of the dGTP-derived lesion correlates with its ability to be incorporated into DNA. KF(-) has the highest specificity for incorporation of dGTP opposite dC. The selection factors for incorporating 8-oxodGTP, (S)-dSpTP, and (R)-dSpTP are 1700-, 64000-, and 850000-fold lower, respectively. Thus, KF(-) is rigorous in its discrimination against incorporation of the hyperoxidized lesion, and these results suggest that the specificity of cellular polymerases provides an effective mechanism to avoid incorporating dSpTP lesions into DNA from the nucleotide pool. PMID:26572218

  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. PMID:25130370

  16. 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. PMID:23824532

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    PubMed Central

    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%. PMID:25551825

  2. 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 (1-100?g 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.

  3. The measurement of molecular fragments from DNA components using synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Fujii, K.; Akamatsu, K.; Yokoya, A.

    2003-03-01

    Photon-stimulated desorption of positive ions from thin film DNA components, 2-deoxy- D-ribose, thymine and guanine, were investigated in the oxygen K-edge excitation region. H +, CH 2+, C 2H 2+, CHO +, C 3H 3+ and C 2HO + were desorbed mainly from the 2-deoxy- D-ribose thin film following oxygen K-edge excitation. The ion yields were obtained as a function of the photon energy. Each spectrum showed a prominent peak structure coinciding with the O 1 s? ??(C-O) excitation energy. These results indicate that the observed ions are produced not only by direct photodecomposition but also by the impact of secondary electrons that the core excitation generates. On the other hand, H + has been observed by irradiation of thymine and guanine thin films, while only insignificant amounts of the other ions were observed. It is shown that the core excitation more drastically degraded the 2-deoxy- D-ribose molecule into small fragments than is the case with the nucleobases. The sugar moiety in DNA is likely to be one of the nor fragile molecular sites, conducive to a single-strand DNA break.

  4. Sequence elements in both subunits of the DNA fragmentation factor are essential for its nuclear transport.

    PubMed

    Neimanis, Sonja; Albig, Werner; Doenecke, Detlef; Kahle, Joerg

    2007-12-01

    DNA cleavage is a biochemical hallmark of apoptosis. In humans, apoptotic DNA cleavage is executed by DNA fragmentation factor (DFF) 40. In proliferating cells DFF40 is expressed in the presence of its chaperone and inhibitor DFF45, which results in the formation of the DFF complex. Here, we present a systematic analysis of the nuclear import of the DFF complex. Our in vitro experiments demonstrate that the importin alpha/beta-heterodimer mediates the translocation of the DFF complex from the cytoplasm to the nucleus. Both DFF subunits interact directly with the importin alpha/beta-heterodimer. However, importin alpha/beta binds more tightly to the DFF complex compared with the individual subunits. Additionally, the isolated C-terminal regions of both DFF subunits together bind importin alpha/beta more strongly than the individual C termini. Our results from in vivo studies reveal that the C-terminal regions of both DFF subunits harbor nuclear localization signals. Furthermore, nuclear import of the DFF complex requires the C-terminal regions of both subunits. In more detail, one basic cluster in the C-terminal region of each subunit, DFF40 (RLKRK) and DFF45 (KRAR), is essential for nuclear accumulation of the DFF complex. Based on these findings two alternative models for the interaction of importin alpha/beta with the DFF complex are presented. PMID:17938174

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

    SciTech Connect

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

    2007-08-15

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 {mu}g range) spread out over a large surface area (42 cm{sup 2}) with low energy (kT-100 eV) 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., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar{sup +} 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.

  6. 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. PMID:16711736

  7. Oxidative DNA damage in mouse sperm chromosomes: Size matters.

    PubMed

    Kocer, Ayhan; Henry-Berger, Joelle; Noblanc, Anais; Champroux, Alexandre; Pogorelcnik, Romain; Guiton, Rachel; Janny, Laurent; Pons-Rejraji, Hanae; Saez, Fabrice; Johnson, Graham D; Krawetz, Stephen A; Alvarez, Juan G; Aitken, R John; Drevet, Jol R

    2015-12-01

    Normal embryo and foetal development as well as the health of the progeny are mostly dependent on gamete nuclear integrity. In the present study, in order to characterize more precisely oxidative DNA damage in mouse sperm we used two mouse models that display high levels of sperm oxidative DNA damage, a common alteration encountered both in in vivo and in vitro reproduction. Immunoprecipitation of oxidized sperm DNA coupled to deep sequencing showed that mouse chromosomes may be largely affected by oxidative alterations. We show that the vulnerability of chromosomes to oxidative attack inversely correlated with their size and was not linked to their GC richness. It was neither correlated with the chromosome content in persisting nucleosomes nor associated with methylated sequences. A strong correlation was found between oxidized sequences and sequences rich in short interspersed repeat elements (SINEs). Chromosome position in the sperm nucleus as revealed by fluorescent in situ hybridization appears to be a confounder. These data map for the first time fragile mouse sperm chromosomal regions when facing oxidative damage that may challenge the repair mechanisms of the oocyte post-fertilization. PMID:26510519

  8. DNA Methylation Patterns in Cord Blood DNA and Body Size in Childhood

    PubMed Central

    Relton, Caroline L.; Groom, Alexandra; St. Pourcain, Beate; Sayers, Adrian E.; Swan, Daniel C.; Embleton, Nicholas D.; Pearce, Mark S.; Ring, Susan M.; Northstone, Kate; Tobias, Jon H.; Trakalo, Joseph; Ness, Andy R.; Shaheen, Seif O.; Davey Smith, George

    2012-01-01

    Background Epigenetic markings acquired in early life may have phenotypic consequences later in development through their role in transcriptional regulation with relevance to the developmental origins of diseases including obesity. The goal of this study was to investigate whether DNA methylation levels at birth are associated with body size later in childhood. Principal Findings A study design involving two birth cohorts was used to conduct transcription profiling followed by DNA methylation analysis in peripheral blood. Gene expression analysis was undertaken in 24 individuals whose biological samples and clinical data were collected at a mean ± standard deviation (SD) age of 12.35 (0.95) years, the upper and lower tertiles of body mass index (BMI) were compared with a mean (SD) BMI difference of 9.86 (2.37) kg/m2. This generated a panel of differentially expressed genes for DNA methylation analysis which was then undertaken in cord blood DNA in 178 individuals with body composition data prospectively collected at a mean (SD) age of 9.83 (0.23) years. Twenty-nine differentially expressed genes (>1.2-fold and p<10−4) were analysed to determine DNA methylation levels at 1–3 sites per gene. Five genes were unmethylated and DNA methylation in the remaining 24 genes was analysed using linear regression with bootstrapping. Methylation in 9 of the 24 (37.5%) genes studied was associated with at least one index of body composition (BMI, fat mass, lean mass, height) at age 9 years, although only one of these associations remained after correction for multiple testing (ALPL with height, pCorrected = 0.017). Conclusions DNA methylation patterns in cord blood show some association with altered gene expression, body size and composition in childhood. The observed relationship is correlative and despite suggestion of a mechanistic epigenetic link between in utero life and later phenotype, further investigation is required to establish causality. PMID:22431966

  9. Septic sera induces apoptosis and DNA fragmentation factor 40 activation in fibroblasts.

    PubMed

    Brabant, Danielle; Michael, Paul; Bleiblo, Farag; Saleh, Mazen; Narain, Ravin; Tai, T C; Ramana, Chilakamarti V; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

    2011-08-26

    Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction. PMID:21820410

  10. A DNA fragment from the cyanobacterium Synechocystis sp. PCC 6803 mediates gene expression inducible by osmotic stress in E. coli.

    PubMed

    Milkowski, C; Quinones, A; Hagemann, M

    1998-08-01

    Fragments of Synechocystis-DNA driving salt-induced gene expression in E. coli were isolated with translational fusions to a 'lacZ gene. One fragment (fragment 19) showed a NaCl-dependent activation of betaGal expression with the maximum of a ninefold increase in enzyme activity. A similar induction was triggered by the nonionic osmolyte sucrose, indicating an osmotically dependent activation. On the contrary, transcriptional activity of the DNA fragment 19 was only slightly enhanced under salt stress conditions, suggesting a posttranscriptional mechanism of induction. Primer extension assay was performed to identify the transcription initiation site. Upstream regions share weak homology to the "-10" hexamer consensus of E. coli sigma70 promoters. The most thermodynamically stable secondary structure for the nontranslated part of the mRNA indicated that potential translation initiation sites might be blocked, leading to a low basal translation, whereas osmotic stress-induced changes of mRNA structure could be involved to increase translation. In order to analyze the function of fragment 19 in Synechocystis, promoter-probe plasmids were constructed allowing the stable integration of transcriptional and translational reporter gene fusions into the cyanobacterial chromosome. Quantitative assessment of reporter gene expression revealed a weak constitutive promoter activity of fragment 19 in Synechocystis. Sequence analysis showed that fragment 19 comprises 223 bp of the ORF sll0747 of the Synechocystis genome. PMID:9662610

  11. End-joining of DNA fragments in adenovirus transfection of human cells.

    PubMed

    Munz, P L; Young, C S

    1991-07-01

    Overlapping terminal fragments of adenovirus DNA transfected into human cells either recombine to form standard unit-length genomes, or can join end-to-end to produce internally redundant, viable, genomes. The end-joining reaction in human HeLa and A549 cells is almost as efficient as the recombination reaction, and is relatively insensitive to the nature of the ends, as pairs of fragments terminating in several different single strands or in blunt ends can join. In contrast to the results from transfection with SV40, the ends are usually modified, for example by the loss of 3' single strands or the repair of 5' single strands. The ability to recover viable redundant molecules is not confined to any one area of the adenovirus genome, but can occur in the E1 and L2 regions as well as in the E2b region. The redundant genomes contain extra splice signals and may have the capacity to encode fusion proteins. PMID:1647067

  12. Identification of a premature termination of DNA polymerization in vitro by Klenow fragment mutants.

    PubMed

    Zhao, Guojie; Wei, Hua; Guan, Yifu

    2013-06-01

    DNA polymerization products by Klenow fragment (KF) are blunt-ended. In the present study, we found that the Klenow fragment mutants with partial deletions of thumb subdomain were unable to extend primers to the 5' terminal of templates, thus creating 5' overhanging sticky ends 2 nt long. We termed this phenomenon as PmTP (premature termination of polymerization). The KF mutants produced homogenous sticky-ended products only under mild reaction conditions, whereas under vigorous reaction conditions, the sticky ends were prone to be blunt-ended. It was also identified that deletions of more than four residues of KF thumb subdomain could induce PmTP, and tworesidue deletion of KF thumb subdomain only induced PmTP in a lower-concentration situation. Structure modelling analysis suggested that shortening or destruction of alpha helix H1 at the tip of the thumb subdomain was crucial to PmTP, while the conserved residues in front of alpha helix was less important. PmTP might be caused by the reduced DNAbinding affinity of the mutants. The sticky ends made by PmTP have potential applications in gene splicing and molecular cloning techniques. PMID:23660662

  13. A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range.

    PubMed

    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 dispersal ability of this species. PMID:23527060

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

    PubMed Central

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

    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 dispersal ability of this species. PMID:23527060

  15. 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.

  16. Effect of size of radiolabeled antibody and fragments on tumor uptake and distribution in nephrectomized mice

    SciTech Connect

    Halpern, S.E.; Buchegger, F.; Schreyer, M.; Mach, J.P.

    1984-01-01

    The importance of molecular size in tumor (T) uptake of intact monoclonal antibody (MAb) of MAb fragments (frag.) is difficult to assess because frag. are excreted by the kidney. To obviate this problem nephrectomized nude mice (M) bearing carcinoembryonic (CEA) secreting human colon (T) were used in the following experiments. Following nephrectomy 3 groups of M were injected intravenously with intact In-111 anti-CEA MAb and simultaneously with I-125 intact, F(ab')2 or Fab anti-CEA-MAb. Iodination was by chloramine T and In-111 labeling by bifunctional chelation. All M were killed 8 h after injection, T and normal tissues (NT) dissected, weighed, and counted against a standard of the injectate. The distribution of intact I-125 and In-111-MAb were nearly identical allowing In-111-MAb to be used for comparison with the I-125 frag. T concentration (conc.) of I-125-F(ab')2 were 25% greater and L conc. were 63% lower than for intact In-111-MAb. T conc. of I-125-Fab were 86% greater and L conc. 64% lower than for intact In-111-MAb. Large T, which produce higher serum CEA levels, increased the L conc. of intact MAb but not I-125 frag. The authors conclude that when renal excretion is prevented there is (a) an inverse relationship between size of a MAb moiety and its T conc. indicating that the improved T/NT ratios observed with frag. are not due only to renal excretion, and (b) the Fc portion of the MAb appears to be critical for L uptake of MAb or immune complexes.

  17. Calorimetric and Low-Frequency Dielectric Studies of Mesoscopic Ordering in Solutions of Engineered DNA Hairpin Fragments

    NASA Astrophysics Data System (ADS)

    Kashuri, K.; Kashuri, H.; Iannacchione, G. S.

    2012-02-01

    Calorimetry (both AC and MDSC) from 20 to 100 ^oC, as well as low-frequency (0.1 to 100 kHz) isothermal dielectric measurements have been performed on solutions of DNA fragments as a function of concentration. Custom hairpin DNA fragments were obtained with 13-base unit length and samples made in solution at various concentration. Results show a reproducible heat capacity Cp signature on heating and cooling scans. This thermal behavior of a diluted oligonucleotide chain is very different from that seen for mesoscopic ordering of liquid crystals. The AC Cp peak vanishes and new features are revealed as the temperature scan rate is lowered to 0.017 K min-1. The observed real, ?', and imaginary, ?'', permittivity of the suspended DNA show features indicating low-frequency dynamics that in turn suggests large-scale ordering or agglomeration of the DNA hairpin loops.

  18. DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments.

    PubMed

    Wu, Feinan; Olson, Brennan G; Yao, Jie

    2016-01-01

    The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq. PMID:26862720

  19. The metabolic enhancer piracetam attenuates mitochondrion-specific endonuclease G translocation and oxidative DNA fragmentation.

    PubMed

    Gupta, Sonam; Verma, Dinesh Kumar; Biswas, Joyshree; Rama Raju, K Siva; Joshi, Neeraj; Wahajuddin; Singh, Sarika

    2014-08-01

    This study was performed to investigate the involvement of mitochondrion-specific endonuclease G in piracetam (P)-induced protective mechanisms. Studies have shown the antiapoptotic effects of piracetam but the mechanism of action of piracetam is still an enigma. To assess the involvement of endonuclease G in piracetam-induced protective effects, astrocyte glial cells were treated with lipopolysaccharide (LPS) and piracetam. LPS treatment caused significantly decreased viability, mitochondrial activity, oxidative stress, chromatin condensation, and DNA fragmentation, which were attenuated by piracetam cotreatment. Cotreatment of astrocytes with piracetam showed its significantly time-dependent absorption as observed with high-performance liquid chromatography. Astrocytes treated with piracetam alone showed enhanced mitochondrial membrane potential (MMP) in comparison to control astrocytes. However, in LPS-treated cells no significant alteration in MMP was observed in comparison to control cells. Protein and mRNA levels of the terminal executor of the caspase-mediated pathway, caspase-3, were not altered significantly in LPS or LPS + piracetam-treated astrocytes, whereas endonuclease G was significantly translocated to the nucleus in LPS-treated astrocytes. Piracetam cotreatment attenuated the LPS-induced endonuclease G translocation. In conclusion this study indicates that LPS treatment of astrocytes caused decreased viability, oxidative stress, mitochondrial dysfunction, chromatin condensation, DNA damage, and translocation of endonuclease G to the nucleus, which was inhibited by piracetam cotreatment, confirming that the mitochondrion-specific endonuclease G is one of the factors involved in piracetam-induced protective mechanisms. PMID:24882422

  20. Separation of fragments up to 570 bases in length by use of 6% T non-cross-linked polyacrylamide for DNA sequencing in capillary electrophoresis

    SciTech Connect

    Best, N.; Arriaga, E.; Chen, D.Y.; Dovichi, N.J. )

    1994-11-15

    Non-cross-linked polyacrylamide is a very convenient medium for the separation of DNA sequencing fragments in capillary electrophoresis. We demonstrate DNA sequencing with this matrix at an electric field of 200 V/cm and at room temperature. Resolution is observed to decrease exponentially with fragment length. Fragments 570 bases in length generate a resolution of 0.5, which is adequate for sequence identification. 49 refs., 5 figs.

  1. DNA double-strand breaks induced by high-energy neon and iron ions in human fibroblasts. II. Probing individual NotI fragments by hybridization

    SciTech Connect

    Loebrich, M.; Rydberg, B.; Cooper, P.K.

    1994-08-01

    The initial yields of DNA double-strand breaks induced by energetic heavy ions (425 MeV/u neon and 250, 400 and 600 MeV/u iron) in comparison to X rays were measured in normal human diploid fibroblast cells within three small areas of the genome, defined by NotI fragments of 3.2, 2.0 and 1.2 Mbp. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated cells, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with probes recognizing single-copy sequences within the three NotI fragments. The gradual disappearance of a smear of broken DNA molecules are quantified. Assuming Poisson statistics for the number of double-strand breaks induced per NotI fragment of known size, absolute yields of DNA double-strand breaks were calculated and determined to be linear with dose in all cases, with the neon ion (LET 32 keV/{mu}m) producing 4.4 x 10{sup {minus}3} breaks/Mbp/Gy and all three iron-ion beams (LETs from 190 to 350 keV/{mu}m) producing 2.8 x 10{sup {minus}3} breaks/Mbp/Gy, giving RBE values for production of double-strand breaks of 0.76 for neon and 0.48 for iron in comparison to our previously determined X-ray induction rate of 5.8 x 10{sup {minus}3} breaks/Mbp/Gy. These RBE values are in good agreement with results of measurements over the whole genome as reported in the accompanying paper. The distribution of broken DNA molecules was similar for the various radiations, supporting a random distribution of double-strand breaks induced by the heavy ions over Mbp distances; however, correlated breaks (clusters) over much smaller distances are not ruled out. Reconstitution of the 3.2 Mbp NotI fragment was studied during postirradiation incubation of the cells as a measure of rejoining of correct DNA ends. The proportion of breaks repaired decreased with increasing LET. 41 refs., 6 figs., 1 tab.

  2. Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.

    PubMed

    Pugliese, Kaitlin M; Gul, O Tolga; Choi, Yongki; Olsen, Tivoli J; Sims, Patrick C; Collins, Philip G; Weiss, Gregory A

    2015-08-01

    DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by ?closed. Under Vmax conditions, the average time of ?closed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by ?open, which includes molecular recognition of the incoming dNTP. All ?-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair. PMID:26147714

  3. Investigation of fragment sizes in laser-driven shock-loaded tin with improved watershed segmentation method.

    PubMed

    He, Weihua; Xin, Jianting; Chu, Genbai; Li, Jing; Shao, Jianli; Lu, Feng; Shui, Min; Qian, Feng; Cao, Leifeng; Wang, Pei; Gu, Yuqiu

    2014-08-11

    Studying dynamic fragmentation in shock-loaded metals and evaluating the geometrical and kinematical properties of the resulting fragments are of significant importance in shock physics, material science as well as microstructural modeling. In this paper, we performed the laser-driven shock-loaded experiment on the Shenguang-? (SG?) prototype laser facility, and employed X-ray micro-tomography technique to give a whole insight into the actual fragmentation process. To investigate the size distribution of the soft recovered fragments from Poly 4-methyl-1-pentene (PMP) foam sample, we further developed an automatic analysis approach based on the improved watershed segmentation. Comparison results of segmenting fragments in slices with different methods demonstrated that our proposed segmentation method can overcome the drawbacks of under-segmentation and over-segmentation, and has the best performance in both segmentation accuracy and robustness. With the proposed automatic analysis approach, other parameters such as the position distribution and penetration depth are also obtained, which are very helpful for understanding the dynamic failure mechanisms. PMID:25320978

  4. Thymoma exhibiting spontaneous regression in size, pleural effusion and serum cytokeratin fragment level: A case report

    PubMed Central

    FURUYA, KENTA; ISOBE, KAZUTOSHI; SANO, GO; KABURAKI, KYOHEI; GOCHO, KYOKO; ISHIDA, FUMIAKI; KIKUCHI, NAOSHI; SUGINO, KEISHI; SAKAMOTO, SUSUMU; TAKAI, YUJIRO; OTSUKA, HAJIME; HATA, YOSHINOBU; IYODA, AKIRA; WAKAYAMA, MEGUMI; SHIBUYA, KAZUTOSHI; HOMMA, SAKAE

    2015-01-01

    A 30-year-old man was admitted to Toho University Omori Medical Center for assessment of right chest pain and fever. Chest computed tomography (CT) revealed an anterior mediastinal tumor sized 11.0×6.0×5.0 cm, with right pleural effusion. The laboratory analysis revealed elevated white blood cell count (11,000/µl), C-reactive protein (4.1 mg/dl) and cytokeratin fragment (CYFRA; 12.7 ng/ml; normal, <2 ng/ml). The level of CYFRA in the pleural effusion was also markedly elevated (143 ng/ml). On the first day after admission (6 days after the initial CT), there was a mild regression on CT (10.0×5.5×4.4 cm; reduction rate, 26.7%), with decrease of the pleural effusion volume. A CT-guided needle biopsy was performed, but the findings were not conclusive, as most of the tissue was necrotic. Seven days later (13 days after the initial CT), a CT revealed further regression (9.5×5.4×4.2 cm; reduction rate, 34.7%) with disappearance of the pleural effusion. The patient was followed up on an outpatient basis. At 35 days after the initial CT, the tumor continued to shrink without treatment (8.0×3.6×3.0 cm; reduction rate, 73.8%) and the serum CYFRA level had decreased to 0.8 ng/ml, although it had not returned to normal levels. At 62 days after the initial CT, the patient underwent surgical resection. The resected specimen was diagnosed as thymoma (World Health Organization type B2; Masaoka classification, stage II), with prominent degeneration and necrosis. One possible cause of the spontaneous regression may be increased internal pressure, probably associated with rapid tumor growth, leading to massive necrosis with resulting chest pain, inflammatory reaction with pleural effusion and subsequent tumor regression. The serum CYFRA level may be a useful marker for the evaluation of the clinical course of thymoma with extensive necrosis. PMID:26623050

  5. Circulating Bacterial-Derived DNA Fragment Level Is a Strong Predictor of Cardiovascular Disease in Peritoneal Dialysis Patients

    PubMed Central

    Szeto, Cheuk-Chun; Kwan, Bonnie Ching-Ha; Chow, Kai-Ming; Kwok, Jeffrey Sung-Shing; Lai, Ka-Bik; Cheng, Phyllis Mei-Shan; Pang, Wing-Fai; Ng, Jack Kit-Chung; Chan, Michael Ho-Ming; Lit, Lydia Choi-Wan; Leung, Chi-Bon; Li, Philip Kam-Tao

    2015-01-01

    Background Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD) patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients. Methods We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival. Results The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001). After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 – 42.5%) excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001) and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001). Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV), but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005). Conclusions Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients. PMID:26010741

  6. Report on the effects of fragment size, indexing, and read length on HLA sequencing on the Illumina MiSeq.

    PubMed

    Profaizer, T; Coonrod, E M; Delgado, J C; Kumnovics, A

    2015-12-01

    Single-molecule sequencing should allow for unambiguous, accurate, and high-throughput HLA typing. In this proof of principle study, we investigated the effects of fragment size for library preparation, indexing strategy, and read length on HLA typing. Whole gene amplicons of HLA-A, B, C, DRB1, and DQB1 were obtained by long-range PCR. For library preparation, two fragment sizes were evaluated: 100-300bp and 300-600bp. For sample multiplexing, two indexing strategies were compared: indexing-by-amplicon, where each individual amplicon is barcoded, and indexing-by-patient, where each patient's five loci are equimolarly pooled after PCR and indexed with the same barcode. Sequencing was performed on an Illumina MiSeq instrument using paired-end 150bp and 250bp read lengths. Our results revealed that the 300-600bp fragments in the 2250 MiSeq group gave the most accurate sequencing results. There was no difference in HLA typing results between the two indexing strategies, suggesting that indexing-by-patient, which is much simpler, is a viable option. In conclusion, enzymatic fragmentation of pooled whole gene amplicons is a suitable strategy for HLA typing by next-generation sequencing on the Illumina MiSeq. PMID:26303189

  7. Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

    PubMed Central

    2012-01-01

    Background Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. Results A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed Two Rounds Mode (three and 2832 cycles). The first round (the first three cycles) is named the One by One Annealing Round. The second round (2832 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. Conclusions UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species. PMID:22894545

  8. Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (Icad/Cad)

    PubMed Central

    Lechardeur, Delphine; Drzymala, Luke; Sharma, Manu; Zylka, Danuta; Kinach, Robert; Pacia, Joanna; Hicks, Christopher; Usmani, Nawaid; Rommens, Johanna M.; Lukacs, Gergely L.

    2000-01-01

    Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was initially proposed that proteolytic cleavage of ICAD by activated caspases causes the dissociation of the ICAD/CAD heterodimer and the translocation of active CAD into the nucleus in apoptotic cells. Here, we show that endogenous and heterologously expressed ICAD and CAD reside predominantly in the nucleus in nonapoptotic cells. Deletional mutagenesis and GFP fusion proteins identified a bipartite nuclear localization signal (NLS) in ICAD and verified the function of the NLS in CAD. The two NLSs have an additive effect on the nuclear targeting of the CADICAD complex, whereas ICAD-S, lacking its NLS, appears to have a modulatory role in the nuclear localization of CAD. Staurosporine-induced apoptosis evoked the proteolysis and disappearance of endogenous and exogenous ICAD from the nuclei of HeLa cells, as monitored by immunoblotting and immunofluorescence microscopy. Similar phenomenon was observed in the caspase-3deficient MCF7 cells upon expressing procaspase-3 transiently. We conclude that a complex mechanism, involving the recognition of the NLSs of both ICAD and CAD, accounts for the constitutive accumulation of CAD/ICAD in the nucleus, where caspase-3dependent regulation of CAD activity takes place. PMID:10908575

  9. FLOW CYTOMETRIC DETECTION OF SUBHAPLOID NUCLEI IN HUMAN SPERM AS A MEASURE OF DNA FRAGMENTATION AND APOPTOSIS.

    PubMed

    Grbner, S; Franz, M; Hoberg, U; Wetzka, B; Schweizer, T

    2015-01-01

    The use of assisted reproductive technologies (ARTs) is increasing worldwide. In order to predict the rate of pregnancy after ART the DNA fragmentation index (DFI) of ejaculated spermatocytes may be a better marker than conventional semen quality parameters. Spermatocytes with fragmented DNA are associated with apoptotic stages and are characterized by a low DNA content. The subhaploid nuclei of DNA-damaged spermatocytes can be easily detected by flow cytometry. We here analyzed the percentage of subhaploid nuclei of semen samples from 163 patients aged 26 to 74 years who consulted one of the ten centres for reproductive medicine which routinely send sperm samples to our laboratory in order to determine special sperm parameters. The percentage of subhaploid nuclei indicating the DFI of spermatocytes did not correlate with age and sperm volume, but inversely correlated with sperm concentration and the percentage of motile spermatocytes. This is in concordance with previous studies which demonstrated that DNA damage of spermatozoa correlates with conventional semen quality parameters. Since DNA-damaged spermatocytes are associated with an impaired outcome of assisted conception technologies, this method could help to monitor sperm quality of subfertile men after measures to increase sperm quality and to improve selection criteria of cryopreserved sperm samples in assisted reproduction medicine. PMID:26122219

  10. DNA large restriction fragment patterns of sporadic and epidemic nosocomial strains of Mycobacterium chelonae and Mycobacterium abscessus.

    PubMed Central

    Wallace, R J; Zhang, Y; Brown, B A; Fraser, V; Mazurek, G H; Maloney, S

    1993-01-01

    Large restriction fragment (LRF) pattern analysis of genomic DNA using pulsed-field gel electrophoresis was performed on three reference strains, 32 sporadic isolates, and 92 nosocomial isolates from 12 epidemics of Mycobacterium chelonae and Mycobacterium abscessus. Only 17 of 30 (57%) unrelated strains of M. abscessus, compared with 10 of 11 (91%) of M. chelonae strains, gave satisfactory DNA extractions, with the remainder resulting in highly fragmented DNA. DraI, AsnI, XbaI, and SpeI gave satisfactory LRF patterns. Sporadic isolates of the two species had highly variable LRF patterns, except for one reference strain and one sporadic isolate of M. chelonae that differed by only two to five bands. Evaluation of repeat isolates from five patients monitored for 8 months to 13 years (mean, 5.8 years) revealed LRF patterns to be stable, with changes of not more than two bands. LRF analysis of the seven nosocomial outbreaks with evaluable DNA revealed identical patterns in most or all of the patient isolates and in three outbreaks revealed identity with environmental isolates. These outbreaks included endoscope contamination, postinjection abscesses, and surgical wound infections. LRF analysis of genomic DNA is a useful technique for epidemiologic studies of M. abscessus and M. chelonae, although improved technology is needed for the approximately 50% of strains of M. abscessus with unsatisfactory DNA extractions. Images PMID:8253968

  11. Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers.

    PubMed

    Ahuja, M R; Devey, M E; Groover, A T; Jermstad, K D; Neale, D B

    1994-06-01

    A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers. PMID:24186006

  12. 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

  13. Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells

    PubMed Central

    Liu, Wei; Lu, Xiangyi; He, Guangyang; Gao, Xiang; Xu, Maonian; Zhang, Jingkai; Li, Meiling; Wang, Lifeng; Li, Zhenjing; Wang, Likui; Luo, Cheng

    2013-01-01

    Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed. PMID:24177565

  14. Protective roles of Gadd45 and MDM2 in blueberry anthocyanins mediated DNA repair of fragmented and non-fragmented DNA damage in UV-irradiated HepG2 cells.

    PubMed

    Liu, Wei; Lu, Xiangyi; He, Guangyang; Gao, Xiang; Xu, Maonian; Zhang, Jingkai; Li, Meiling; Wang, Lifeng; Li, Zhenjing; Wang, Likui; Luo, Cheng

    2013-01-01

    Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed. PMID:24177565

  15. Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase.

    PubMed Central

    Baillon, J G; Nashed, N T; Sayer, J M; Jerina, D M

    1991-01-01

    Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy. In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20. The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods. Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay. The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment. Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive. PMID:1704125

  16. 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 10kb 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

  17. Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

    PubMed Central

    2011-01-01

    The hepatitis B virus (HBV) has been classified into eight genotypes (A-H) based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR) on the eight (A-H) standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI) and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%), 25 cases (50%), and 14 cases (28%) respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype. PMID:21693069

  18. Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

    PubMed

    Watanabe, K; Kodama, Y; Harayama, S

    2001-04-01

    Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers. PMID:11240048

  19. Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

    TOXLINE Toxicology Bibliographic Information

    Watanabe K; Kodama Y; Harayama S

    2001-04-01

    Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.

  20. Novel insights into the pathophysiology of varicocele and its association with reactive oxygen species and sperm DNA fragmentation.

    PubMed

    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

  1. 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

  2. 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.

  3. A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited.

    PubMed

    Zeltz, P; Kadowaki, K; Kubo, N; Maier, R M; Hirai, A; Kssel, H

    1996-06-01

    The RNA editing processes in chloroplasts and mitochondira of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. An rpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrial rpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potential rpoB editing sites previously detected in maize were found to be edited in the rice chloroplast rpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events. PMID:8790296

  4. Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

    PubMed

    Haghpanah, Tahereh; Salehi, Mohammad; Ghaffari Novin, Marefat; Masteri Farahani, Reza; Fadaei-Fathabadi, Fatemeh; Dehghani-Mohammadabadi, Maryam; Azimi, Hadi

    2016-02-01

    Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ?30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p??0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p?>?0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte. PMID:26678043

  5. Herpes simplex virus cloned DNA fragments induce coumermycin A1 resistance in Escherichia coli.

    PubMed Central

    Pearson, R E; Conley, A J

    1984-01-01

    We have taken a new approach to identify and fine map previously undescribed herpes simplex virus (HSV) functions. In experiments described in this report the antibiotic coumermycin A1 was used to select two HSV type 1 BamHI fragments cloned in pBR322 that confer partial resistance to drug-susceptible Escherichia coli. The genes encoding these HSV functions have been designated cour-1 and cour-2 and have been fine mapped to the HSV sequences. HSV-cour1 is located at the left end of BamHI-F near HSV type 1 genomic map coordinate 0.645. cour-2 maps to BamHI-M', which is a 159-base-pair internal component of the alpha ICP4-coding sequence located in the reiterated sequences of the S component. In pBR322 both inserts apparently rely on the tet promoter for expression. Additionally, cour-2 functions when present as a BamHI insert in pUC7. The analysis of cour-2 "maxi" cell proteins reveals the presence of proteins produced by the fusion of HSV-1 BamHI-M' sequences and the sequences of the vector genes, i.e., the major tet product for pBR322 and the modified beta-galactosidase for pUC7. These data suggest that the development of bacterial assays for fusion products of eucaryotic DNA open reading frames in plasmid vectors may be a useful technique for initiating gene function studies. Images PMID:6092666

  6. 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

  7. In Vitro Effect of Cell Phone Radiation on Motility, DNA Fragmentation and Clusterin Gene Expression in Human Sperm

    PubMed Central

    Zalata, Adel; El-Samanoudy, Ayman Z; Shaalan, Dalia; El-Baiomy, Youssef; Mostafa, Taymour

    2015-01-01

    Background Use of cellular phones emitting radiofrequency electromagnetic field (RF-EMF) has been increased exponentially and become a part of everyday life. This study aimed to investigate the effects of in vitro RF-EMF exposure emitted from cellular phones on sperm motility index, sperm DNA fragmentation and seminal clusterin (CLU) gene expression. Materials and Methods In this prospective study, a total of 124 semen samples were grouped into the following main categories: i. normozoospermia (N, n=26), ii. asthenozoospermia (A, n=32), iii. asthenoteratozoospermia (AT, n=31) and iv. oligoasthenoteratozoospermia (OAT, n=35). The same semen samples were then divided into two portions non-exposed and exposed samples to cell phone radiation for 1 hour. Before and immediately after exposure, both aliquots were subjected to different assessments for sperm motility, acrosin activity, sperm DNA fragmentation and CLU gene expression. Statistical differences were analyzed using paired t student test for comparisons between two sub-groups where p<0.05 was set as significant. Results There was a significant decrease in sperm motility, sperm linear velocity, sperm linearity index, and sperm acrosin activity, whereas there was a significant increase in sperm DNA fragmentation percent, CLU gene expression and CLU protein levels in the exposed semen samples to RF-EMF compared with non-exposed samples in OAT>AT>A>N groups, respectively (p<0.05). Conclusion Cell phone emissions have a negative impact on exposed sperm motility index, sperm acrosin activity, sperm DNA fragmentation and seminal CLU gene expression, especially in OAT cases. PMID:25918601

  8. 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

  9. 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.

  10. Improved separation of chromosome-sized DNA from Trypanosoma brucei, stock 427-60.

    PubMed Central

    Van der Ploeg, L H; Smith, C L; Polvere, R I; Gottesdiener, K M

    1989-01-01

    Separation of chromosome-sized DNA from the parasitic protozoan Trypanosoma brucei had previously resulted in the fractionation of DNA molecules that ranged in size from 50 kb up to roughly 1.5 Mb. The number of larger chromosomes and their size, accounting for 80% of the DNA of T. brucei remained unclear. We have now size separated these larger DNA molecules by pulsed field gel electrophoresis (PFG) and resolve a total of 20 bands, accounting for roughly 120 chromosomes, ranging in size from 50 kb up to the size of the largest, 5.7 Mb chromosome of Schizosaccharomyces pombe. Three different VSG gene expression sites were located to chromosomes of 430 kb, 1.5 Mb and 3 Mb, respectively. We have not been able to identify additional, previously cryptic DNA rearrangements, that could explain the activation or inactivation of the expression sites. Images PMID:2726456

  11. Probability of double-strand breaks in genome-sized DNA by ?-ray decreases markedly as the DNA concentration increases.

    PubMed

    Shimobayashi, Shunsuke F; Iwaki, Takafumi; Mori, Toshiaki; Yoshikawa, Kenichi

    2013-05-01

    By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by ?-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P1, 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. PMID:23656159

  12. Determination of the coding capacity of the BamHI DNA fragment B of apathogenic Herpes simplex virus type 1 strain HFEM by DNA nucleotide sequence analysis.

    PubMed

    Rsen-Wolff, A; Frank, S; Raab, K; Moyal, M; Becker, Y; Darai, G

    1992-09-15

    Herpes simplex virus type 1 (HSV-1) strain HFEM acquired an apathogenic phenotype due to a deletion within the DNA sequences of the BamHI DNA fragment B of the viral genome. In order to investigate the coding strategy of this particular region of the genome of HSV-1 strain HFEM the DNA nucleotide sequence of the BamHI DNA fragment B was determined. This analysis revealed that the BamHI DNA fragment B of HSV-1 strain HFEM comprises 6593 bp, corresponding to the nucleotide positions (np) 113322 to 117088 and np 120643 to 123465 of the genome of HSV-1 strain 17. According to these data the deletion of the genome of HSV-1 strain HFEM occurred between the np 117089 and 120642. The promoter region of the UL56 gene of HSV-1 strain HFEM is a part of the deleted DNA sequences. Therefore, this gene of HSV-1 strain HFEM is affected and cannot be expressed. The first 35 amino acid (AA) residues of the deduced amino acid sequence of the UL56 open reading frame (ORF) were found to be identical to the amino acid sequence of the UL56 genes of HSV-1 strains 17 and F. However, due to a deletion at np 3494 of the BamHI DNA fragment B of HSV-1 strain HFEM the amino acid composition of the predicted UL56 gene of HSV-1 strain HFEM is different from HSV-1 strain 17 between amino acid positions 36 and 233. In addition the deduced amino acid sequence of the IRL (inverted repeat of the long segment) copy of the IE110 gene of HSV-1 strain HFEM was found to be about 342 amino acids shorter than the amino acid sequence of IE110 gene of HSV-1 strain 17 (775 AA). This was based on a point mutation which was detected within the DNA sequences of Exon 3 of this copy of IE110 gene of HSV-1 strain HFEM. PMID:1332274

  13. 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.

  14. 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 -[(CxHy)3N+ x = 2,4; y = 5,9] were observed for clusters containing both one and two ligands largely independent of the size, composition and charge state of the precursor ion. The [(C4H9)4N+] ligand was found to exhibit stronger interactions with the core of the POMs resulting in higher abundances of fragment ions containing (C4H9) units compared to (C2H5) units from [(C2H5)4N+]. These results provide fundamental insight into the interactions between anionic metal oxide clusters, heteroanions and cationic ammonium ligands that are responsible for the size and composition controlled synthesis of POMs in solution.

  15. 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 [1986, Icarus 67, and 1987], who compared secondary crater SVDs for craters on the Moon, Mercury, and Mars. Additionally, meter-scale NAC images enable characterization of secondary crater morphologies and fields around much smaller primary craters than were previously investigated. Combined results from all previous studies of ejecta fragment SVDs from secondary crater fields show that β ranges between approximately 1 and 3. First-order spallation theory predicts a β of 1 [Melosh 1989, Impact Cratering, Oxford Univ. Press]. Results in Vickery [1987] for the Moon exhibit a generally decreasing β with increasing primary crater size (5 secondary fields mapped). In the same paper, however, this trend is flat for Mercury (3 fields mapped) and opposite for Mars (4 fields mapped). SVDs for craters on large icy satellites (Ganymede and Europa), with gravities not too dissimilar to lunar gravity, show generally low velocity exponents (β between 1 and 1.5), except for the very largest impactor measured: the 585-km-diameter Gilgamesh basin on Ganymede (β = 2.6 × 0.4) [Singer et al., 2013, Icarus 226]. The present work, focusing initially on lunar craters using LROC data, will attempt to confirm or clarify these trends, and expand the number of examples under a variety of impact conditions and surface materials to evaluate possible causes of variations.

  16. The hidden component of size in two-dimensional fragment descriptors: side effects on sampling in bioactive libraries.

    PubMed

    Dixon, S L; Koehler, R T

    1999-07-29

    We have carried out a number of sampling experiments in libraries of bioactive compounds to illustrate how size biases introduced by two-dimensional (2D) fragment distance functions may provide misleading information about the diversity of compound subsets. The number of different biological targets covered by a given subset is used as a measure of bioactive diversity, and it is considered to be the relevant property with which 2D diversity should correlate. Since the nature of the size biases depends on the way in which 2D distance is computed, we investigated three different methods of calculating distance. Use of 1-Tanimoto as a dissimilarity measure leads to the spurious conclusion that collections of structurally small compounds are inherently more diverse than other collections which may cover a broader range of sizes and more biological targets. XOR or squared Euclidean distance, by contrast, shows a preference for subsets of structurally larger compounds, but this does not appear to have as many adverse consequences in terms of target coverage. A simple product of 1-Tanimoto and XOR tends to equalize the opposing size effects of the two component distance functions and leads to a relatively unbiased means of comparing structures. Results here suggest that careful consideration should be given to the way in which chemical structures are compared whenever 2D fragment descriptors are used. PMID:10425098

  17. A systematic study of HLA class II-beta DNA restriction fragments in insulin-dependent diabetes mellitus.

    PubMed Central

    Cohen-Haguenauer, O; Robbins, E; Massart, C; Busson, M; Deschamps, I; Hors, J; Lalouel, J M; Dausset, J; Cohen, D

    1985-01-01

    DNA restriction fragments of the genes encoding HLA class II-beta antigens were compared in 34 patients with insulin-dependent diabetes mellitus and 34 HLA-DR-matched healthy individuals. Ninety-three fragments, determined by six restriction enzymes (EcoRI, EcoRV, HindIII, BamHI, Pvu II, and Taq I), were analyzed: (i) A DR Taq I 12.7-kilobase-pair fragment might be a marker for the extended haplotype HLA-B8, DR3. (ii) In controls, DR4 haplotypes are associated with two distinct clusters of DQ restriction fragments (DQR4 and DQR5). Almost all (94%) DR4 patients belong to the DQR4 and not to the DQR5 cluster. This suggests that, among HLA-DR4 haplotypes, only DQR4 haplotypes are involved in susceptibility to insulin-dependent diabetes mellitus. (iii) A DR Taq I 14.5-kilobase-pair fragment was found to be strongly associated with DQR4, mainly in DR3/DR4 heterozygous patients (P = 5 X 10(-4). However, these results must be interpreted with caution, taking into account the high number of statistical tests performed. Images PMID:2987920

  18. 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

  19. Distinct complexes of DNA polymerase I (Klenow fragment) for base and sugar discrimination during nucleotide substrate selection.

    PubMed

    Garalde, Daniel R; Simon, Christopher A; Dahl, Joseph M; Wang, Hongyun; Akeson, Mark; Lieberman, Kate R

    2011-04-22

    During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the ?-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions. PMID:21362617

  20. High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 1. DNA size standards and the effect of agarose and temperature

    SciTech Connect

    Mathew, M.K.; Smith, C.L.; Cantor, C.R. )

    1988-12-27

    Pulsed-field gel electrophoresis (PGF) subjects DNA alternately to two electrical fields to resolve DNA ranging from 10,000 base pairs (10 kb) to 10,000 kb in size. The separations are quite sensitive to a variety of experimental variables. This makes it critical to have a wide range of reliable size standards. A technique is described for preparing mixtures of bacteriophage DNA oligomers that span a size range from monomer to more than 30-mer. The relationship between size and mobility of oligomers of different bacteriophage DNA monomers is generally self-consistent. Thus, these samples can serve as primary length standards for DNAs ranging from 10 kb to more than 1,500 kb. They have been used to estimate the size of the chromosomal DNAs from various Saccharomyces cerevisiae strains and to test the effect of gel concentration and temperature on PFG. DNA resolution during PFG is slightly improved in agarose gels with small pore sizes, in contrast to continuous electrophoresis where the opposite is observed. PFG mobility is surprisingly sensitive to changes in the running temperature.

  1. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

    SciTech Connect

    Beverley, S.M. )

    1989-02-15

    A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis.

  2. 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.

  3. 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

  4. Molecular-Sized DNA or RNA Sequencing Machine

    Cancer.gov

    Current high-throughput DNA sequencing methods suffer from several limitations. Many methods require multiple fluid handling steps, fixing of molecules on beads or a 2D surface, and provide very short read-lengths. Researchers at the National Cancer Institute's Gene Regulation and Chromosome Biology Laboratory offer a potential DNA or RNA sequencing device that drastically simplifies the process by combining all elements for sequence detection in a single molecule.

  5. Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3.

    PubMed

    Park, Ok Kyeung; Park, Hyun Ho

    2012-09-21

    DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes. PMID:22850116

  6. 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. PMID:21125920

  7. DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

    PubMed Central

    Makarova, Olga; Contaldo, Nicoletta; Paltrinieri, Samanta; Kawube, Geofrey; Bertaccini, Assunta; Nicolaisen, Mogens

    2012-01-01

    Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification. PMID:23272216

  8. A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting f...

  9. Delivery of Plasmid DNA Encoding Bone Morphogenetic Protein-2 with a Biodegradable Branched Polycationic Polymer in a Critical-Size Rat Cranial Defect Model

    PubMed Central

    Chew, Sue Anne; Kretlow, James D.; Spicer, Patrick P.; Edwards, Austin W.; Baggett, L. Scott; Tabata, Yasuhiko; Kasper, F. Kurtis

    2011-01-01

    This study investigated the delivery of plasmid DNA (pDNA) encoding bone morphogenetic protein-2 in the form of polyplexes with a biodegradable branched triacrylate/amine polycationic polymer (TAPP) that were complexed with gelatin microparticles (GMPs) loaded within a porous tissue engineering scaffold. More specifically, the study investigated the interplay between TAPP degradation, gelatin degradation, pDNA release, and bone formation in a critical-size rat cranial defect model. The pDNA release kinetics in vitro were not affected by the crosslinking density of the GMPs but depended, rather, on the degradation rates of the TAPPs. Besides the initial release of polyplexes not bound to the GMPs and the minimal release of polyplexes through diffusion or dissociation from the GMPs, the pDNA was likely released as naked pDNA or as part of an incomplete polyplex, after the degradation of fragments of the polycationic polymer. After 30 days, significantly higher amounts of pDNA were released (93%98%) from composite scaffolds containing naked pDNA or pDNA complexed with P-AEPZ (synthesized with 1-[2-aminoethyl]piperazine, a faster degrading TAPP) compared with those containing pDNA complexed with P-DED (synthesized with N,N-dimethylethylenediamine, a slower degrading TAPP) (74%82%). Composite scaffolds containing GMPs complexed with TAPP/pDNA polyplexes did not result in enhanced bone formation, as analyzed by microcomputed tomography and histology, in a critical-size rat cranial defect at 12 weeks postimplantation compared with those loaded with naked pDNA. The results demonstrate that polycationic polymers with a slow degradation rate can prolong the release of pDNA from the composite scaffolds and suggest that a gene delivery system comprising biodegradable polycationic polymers should be designed to release the pDNA in an intact polyplex form. PMID:20964581

  10. Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA.

    PubMed

    Boyd, Victoria L; Moody, Kristina I; Karger, Achim E; Livak, Kenneth J; Zon, Gerald; Burns, John W

    2006-07-15

    Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus. PMID:16725101

  11. 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 (80.646.97%) compared with control samples (80.977.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.615.62%) compared with pre-MACs controls (12.436.29%) (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. PMID:25209213

  12. 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

  13. Blood levels of histone-complexed DNA fragments are associated with coagulopathy, inflammation and endothelial damage early after trauma

    PubMed Central

    Johansson, Pr I; Windelv, Nis A; Rasmussen, Lars S; Srensen, Anne Marie; Ostrowski, Sisse R

    2013-01-01

    Background: Tissue injury increases blood levels of extracellular histones and nucleic acids, and these may influence hemostasis, promote inflammation and damage the endothelium. Trauma-induced coagulopathy (TIC) may result from an endogenous response to the injury that involves the neurohumoral, inflammatory and hemostatic systems. Aims: To study the contribution of extracellular nucleic constituents to TIC, inflammation and endothelial damage. Setting and Design: Prospective observational study. Materials and Methods: We investigated histone-complexed DNA fragments (hcDNA) along with biomarkers of coagulopathy, inflammation and endothelial damage in plasma from 80 trauma patients admitted directly to the Trauma Centre from the scene of the accident. Blood was sampled a median of 68 min (IQR 48-88) post injury. Trauma patients with hcDNA levels >median or ?median were compared. Results: Trauma patients with high plasma hcDNA had higher Injury Severity Score (ISS) and level of sympathoadrenal activation (higher adrenaline and noradrenaline) and a higher proportion of prolonged activated partial thromboplastin time (APTT) and higher D-dimer, tissue-type plasminogen activator (tPA), Annexin V and soluble CD40 ligand (sCD40L) concurrent with lower plasminogen activator inhibitor (PAI)-1) and prothrombin fragment (PF) 1 + 2 (all P < 0.05), all indicative of impaired thrombin generation, hyperfibrinolysis and platelet activation. Furthermore, patients with high hcDNA had enhanced inflammation and endothelial damage evidenced by higher plasma levels of terminal complement complex (sC5b-9), IL-6, syndecan-1, thrombomodulin and tissue factor pathway inhibitor (all P < 0.05). Conclusions: Excessive release of extracellular histones and nucleic acids seems to contribute to the hypocoagulability, inflammation and endothelial damage observed early after trauma. PMID:23960372

  14. Effective source size, radial, angular and energy spread of therapeutic 11C positron emitter beams produced by 12C fragmentation

    NASA Astrophysics Data System (ADS)

    Lazzeroni, Marta; Brahme, Anders

    2014-02-01

    The use of positron emitter light ion beams in combination with PET (Positron Emission Tomography) and PET-CT (Computed Tomography) imaging could significantly improve treatment verification and dose delivery imaging during radiation therapy. The present study is dedicated to the analysis of the beam quality in terms of the effective source size, as well as radial, angular and energy spread of the 11C ion beam produced by projectile fragmentation of a primary point monodirectional and monoenergetic 12C ion beam in a dedicated range shifter of different materials. This study was performed combining analytical methods describing the transport of particles in matter and the Monte Carlo code SHIELD-HIT+. A high brilliance and production yield of 11C fragments with a small effective source size and emittance is best achieved with a decelerator made of two media: a first liquid hydrogen section of about 20 cm followed by a hydrogen rich section of variable length. The calculated intensity of the produced 11C ion beam ranges from about 5% to 8% of the primary 12C beam intensity depending on the exit energy and the acceptance of the beam transport system. The angular spread is lower than 1 degree for all the materials studied, but the brilliance of the beam is the highest with the proposed mixed decelerator.

  15. Effect of alkali on the size dispersity of mammalian DNA measured by filter elution.

    PubMed Central

    van Ankeren, S C; Wheeler, K T

    1984-01-01

    DNA from unirradiated and irradiated cultured 9L rat brain tumor cells was held for varying times in low ionic strength solutions at pH 11.0, 12.3, or 12.9. The effect of this exposure to alkali on the DNA size distribution was determined by comparing the DNA filter elution profiles obtained experimentally with those theoretically predicted for monodispersed and random distributions. At pH 12.3 or 12.9, DNA from cells irradiated with 300 rad eluted with first-order kinetics corresponding to a random DNA size distribution. The median size of the distribution decreased if the irradiated DNA was exposed to pH 12.3 for 24 h. At pH 12.3 or 12.9, DNA from unirradiated cells eluted initially with complex kinetics that later became linear (18-21 h for pH 12.3 or 13-15 h for pH 12.9), characteristic of a monodispersed DNA size distribution. Holding either unirradiated or irradiated DNA at pH 11.0, below the critical unwinding pH, produced no effect on the elution profiles. Analysis of these filter elution data indicated that after sufficient exposure to pH 12.3 or 12.9, undamaged DNA molecules from mammalian cells elute as a single-stranded monodispersed size distribution of approximately 1 X 10(10) daltons. While the possibility cannot be completely eliminated that this monodispersed size represents an upper limit determined by physical forces, these results, in conjunction with those obtained using other techniques, lend credence to the existence of a nonrandom higher-order structure in mammalian chromosomal DNA. PMID:6696969

  16. Fragmentation of Contaminant and Endogenous DNA in Ancient Samples Determined by Shotgun Sequencing; Prospects for Human Palaeogenomics

    PubMed Central

    Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

    2011-01-01

    Background Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. Methodology/Principals Findings To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrn site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. Conclusions We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5? and 3? ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone. PMID:21904610

  17. Actions of the Klenow fragment of DNA polymerase I and some DNA glycosylases on chemically stable analogues of N7-methyl-2'-deoxyguanosine.

    PubMed

    Rana, Jagruti; Huang, Haidong

    2013-11-15

    N7-methyl-9-deaza-dG was synthesized and incorporated into oligonucleotides. Thermal melting studies showed that replacement of dG by N7-methyl-9-deaza-dG only slightly decreased DNA duplex stability. Replication of DNA templates containing N7-methyl-9-deaza-dG and the related 7-methyl-7-deaza-dG and 7-deaza-dG by the Klenow fragment of Escherichia coli DNA polymerase I was examined. The dNTP misinsertion frequencies on all three templates were comparably low, although the 7-methyl group significantly slowed down the turnover rates of the polymerase when dCTP was incorporated. The stabilities of N7-methyl-9-deaza-dG and 7-methyl-7-deaza-dG against the actions of formamidopyrimidine DNA glycosylase (Fpg) and human alkyladenine DNA glycosylase (hAAG) were also examined. N7-methyl-9-deaza-dG was stable in the presence of both enzymes. In contrast, 7-methyl-7-deaza-dG was cleaved by Fpg, and possibly by hAAG but at an extremely slow rate. This study suggests that N7-alkyl-9-deaza-dG is a better analogue than 7-alkyl-7-deaza-dG for cellular studies. PMID:24100157

  18. Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA.

    PubMed

    Abad, C; Amengual, M J; Goslvez, J; Coward, K; Hannaoui, N; Benet, J; Garca-Peir, A; Prats, J

    2013-06-01

    The primary aim of this study was to determine the effect of oral antioxidant treatment (1500mg of l-Carnitine; 60mg of vitamin C; 20mg of coenzyme Q10; 10mg of vitamin E; 10mg of zinc; 200?g of vitamin B9; 50?g of selenium; 1?g of vitamin B12) during a time period of 3months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0h, 2h, 6h, 8h and 24h) at 37C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P<0.01). The proportion of DDS was also significantly reduced (P<0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques. PMID:22943406

  19. PCR-restriction fragment length polymorphism analysis of a diagnostic 452-base-pair DNA fragment discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum isolates of human and animal origin.

    PubMed

    Guyot, K; Follet-Dumoulin, A; Recourt, C; Lelièvre, E; Cailliez, J C; Dei-Cas, E

    2002-04-01

    Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes. PMID:11916736

  20. 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.

  1. Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E. coli

    PubMed Central

    Zhu, Fen-Lu; Lu, Hao-Ying; Li, Zhuo; Qi, Zhong-Tian

    1998-01-01

    AIM: To obtain greater antigenicity of HCV NS3 protein. METHODS: The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy-mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli. RESULTS: Sequence analysis indicated that the HCV isolate of this study belongs to HCV-II; SDS-PAGE demonstrated an Mr 23800 and an Mr 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION: Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents. PMID:11819265

  2. A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4.

    PubMed

    Park, Ok Kyung; Park, Hyun Ho

    2013-04-01

    DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4. PMID:23417746

  3. 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.

  4. Molecular behavior of DNA in a cell-sized compartment coated by lipids.

    PubMed

    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. PMID:26172746

  5. Genome size expansion and the relationship between nuclear DNA content and spore size in the Asplenium monanthes fern complex (Aspleniaceae)

    PubMed Central

    2013-01-01

    Background Homosporous ferns are distinctive amongst the land plant lineages for their high chromosome numbers and enigmatic genomes. Genome size measurements are an under exploited tool in homosporous ferns and show great potential to provide an overview of the mechanisms that define genome evolution in these ferns. The aim of this study is to investigate the evolution of genome size and the relationship between genome size and spore size within the apomictic Asplenium monanthes fern complex and related lineages. Results Comparative analyses to test for a relationship between spore size and genome size show that they are not correlated. The data do however provide evidence for marked genome size variation between species in this group. These results indicate that Asplenium monanthes has undergone a two-fold expansion in genome size. Conclusions Our findings challenge the widely held assumption that spore size can be used to infer ploidy levels within apomictic fern complexes. We argue that the observed genome size variation is likely to have arisen via increases in both chromosome number due to polyploidy and chromosome size due to amplification of repetitive DNA (e.g. transposable elements, especially retrotransposons). However, to date the latter has not been considered to be an important process of genome evolution within homosporous ferns. We infer that genome evolution, at least in some homosporous fern lineages, is a more dynamic process than existing studies would suggest. PMID:24354467

  6. 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.

  7. Sperm DNA fragmentation and morphological degeneration in chilled elephant (Elephas maximus and Loxodonta Africana) semen collected by transrectal massage.

    PubMed

    O'Brien, J K; Steinman, K J; Montano, G A; Love, C C; Robeck, T R

    2013-05-01

    Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0h; subjective motility parameters only) and after 24h of chilled storage at 10C (24h; all ejaculate and sperm characteristics). Ejaculates with ?50% total motility (TM) at 24h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.219.2% fragmented DNA) and an elevated incidence of detached heads (43.322.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24h displayed higher (p<0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant. PMID:23536498

  8. Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.

    PubMed

    Tuomela, Johanna M; Sandholm, Jouko A; Kaakinen, Mika; Hayden, Katherine L; Haapasaari, Kirsi-Maria; Jukkola-Vuorinen, Arja; Kauppila, Joonas H; Lehenkari, Petri P; Harris, Kevin W; Graves, David E; Selander, Katri S

    2016-01-01

    Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying. PMID:26780557

  9. Human and yeast DNA damage recognition complexes bind with high affinity DNA structures mimicking in size transcription bubble.

    PubMed

    Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Anarbaev, Rashid O; Pestryakov, Pavel E; Sugasawa, Kaoru; Min, Jung-Hyun; Lavrik, Olga I

    2013-12-01

    The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. In this study, two types of DNA binding assays were used for the detailed analysis of interaction of these proteins with damaged DNA. An electrophoretic mobility shift assay revealed that human and yeast orthologs behave similarly in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed using fluorescent depolarization measurements. The XPC-RAD23B and the Rad4-Rad23 proteins bind to the damaged 15?nt bubble-DNA structure mimicking in size the "transcription bubble" DNA intermediate with the highest affinity (KD values ~10(-10) ?M or less) that is reduced in the following order: damaged bubble?>?undamaged bubble?>?damaged duplex?>?undamaged duplex. The affinity of XPC/Rad4 for various DNAs was shown to correlate with DNA bending angle. The results obtained show clearly that more deviation from regular DNA structure leads to higher XPC/Rad4 affinity. PMID:24277610

  10. 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. PMID:14689155

  11. Induction of SCEs and DNA fragmentation in bovine peripheral lymphocytes by in vitro exposure to tolylfluanid-based fungicide

    PubMed Central

    Sivikov, Katarna; Dianovsky, Jn; Holeckov, Beta

    2011-01-01

    The potential for genotoxic and cytotoxic effects of tolylfluanid-based fungicide (50% active agent) was evaluated using sister chromatid exchange (SCE) and proliferation indices (PI) in cultured bovine peripheral lymphocytes. For the detection of possible genetic damage, DNA fragmentation assay was also applied. Bovine lymphocytes cultured for 72 h were treated with the fungicide at the final concentrations of 1.75, 3.5, 8.75, and 17.5 ?g/mL for the last 24 and 48 h of culture without S9 metabolic activation, and during the last 2 h of culture with S9 metabolic activation. In the SCE assays no evidence for genotoxic activity of the fungicide was found in treatments of 24 h without and 2 h with S9. After the 24 h exposure to tolylfluanid, a weak decrease in the PI was observed. With the prolonged exposure time (48 h), dose dependence in the increase of SCE frequencies was observed. Moreover, after 48 h exposure slight fragmentation of DNA at the concentrations of 3.5 and 8.75 ?g/mL was demonstrated. SCE quantification is the most widely used approach for the assessment of genotoxic/cytogenetic effects of chemical compounds. Positive results in the assay at 48 h exposure indicated a potential of the fungicide to increase frequency of chromosomal damage (replication injuries) that is the confirmation of early effect of exposure. PMID:21637552

  12. Study of a novel sample injection method (floating electrokinetic supercharging) for high-performance microchip electrophoresis of DNA fragments.

    PubMed

    Hirokawa, Takeshi; Takayama, Yoichi; Arai, Akihiro; Xu, Zhongqi

    2008-05-01

    Aiming to achieve high-performance analysis of DNA fragments using microchip electrophoresis, we developed a novel sample injection method, which was given the name of floating electrokinetic supercharging (FEKS). In the method, electrokinetic injection (EKI) and ITP preconcentration of samples was performed in a separation channel, connecting two reservoir ports (P3 and P4) on a cross-geometry microchip. At these two stages, side channels, crossing the separation channel, and their ports (P1 and P2) were electrically floated. After the ITP-stacked zones passed the cross-part, they were eluted for detection by using leading ions from P1 and P2 that enabled electrophoresis mode changing rapidly from ITP to zone electrophoresis (ZE). Possible sample leakage at the cross-part toward P1 and P2 was studied in detail on the basis of computer simulation using a CFD-ACE+ software and real experiments, through which it was validated that the analyte recovery to the separation channel was almost complete. The FEKS method successfully contributed to higher resolution and shorter analysis time of DNA fragments on the cross-microchip owing to more rapid switching from ITP status to ZE separation in comparison with our previous EKS procedure realized on a single-channel microchip. Without any degradation of resolution, the achieved LODs were on average ten times better than using conventional pinched injection. PMID:18393341

  13. 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

  14. 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

  15. Transient transgene transmission to piglets by intrauterine insemination of spermatozoa incubated with DNA fragments.

    PubMed

    Wu, Zhenfang; Li, Zicong; Yang, Jinzeng

    2008-01-01

    An efficient and low-cost production of transgenic pigs has significant applications to the pig industry and biomedical science. Generation of transgenic pig by sperm-mediated gene transfer (SMGT) was inexpensive and convenient, and reported with high efficiency. To test the method of SMGT in pigs, we employed deep post-cervical intrauterine insemination of incubated spermatozoa in this study. A test of sperm motility of semen from nine Landrace boars after incubation with radioactively labeled DNA construct indicated that DNA uptake of the sperm was highly correlated with sperm motility at the time of collection. DNA concentration of 50 and 300 microg per one billion sperm was incubated with washed high-motility sperm at 17 degrees C for 2 hr. Twenty one hybrid gilts and sows of Meishan crossed with Large White were inseminated with transgene-incubated sperm and produced 156 piglets. Transgene DNA sequences were identified in 31 piglets by PCR amplification of genomic DNA isolated from piglet ears at the age of 3 days. The deep intrauterine insemination had a higher rate of positive transgenic piglets than regular insemination (29.6% of 98 piglets vs. 3.4% of 58 piglets). However, the exogenous transgene DNA was not detected in any piglets at the age of 70-100 days. Therefore, the results further demonstrated that transgene through incubation with spermatozoa was mostly transiently transmitted to the offspring at early growing stage and lost in adulthood, which may result from episomal DNA replications during cell divisions only at the early stage of development. PMID:17546633

  16. 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

    Martn-Fernndez, Begoa; Miranda-Ordieres, Arturo J; Lobo-Castan, Mara Jess; Frutos-Cabanillas, Gloria; de-los-Santos-lvarez, Noem; Lpez-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. PMID:24813914

  17. 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." Geophysical Research Letters 38.24 (2011). C. A. Charalambous and W. T. Pike (2013). 'Evolution of Particle Size Distributions in Fragmentation Over Time' Abstract Submitted to the AGU 46th Fall Meeting. Charalambous, C., Pike, W. T., Goetz, W., Hecht, M. H., & Staufer, U. (2011, December). 'A Digital Martian Soil based on In-Situ Data.' In AGU Fall Meeting Abstracts (Vol. 1, p. 1669). Golombek, M., & Rapp, D. (1997). 'Size-frequency distributions of rocks on Mars and Earth analog sites: Implications for future landed missions.' Journal of Geophysical Research, 102(E2), 4117-4129. Golombek, M., Huertas, A., Kipp, D., & Calef, F. (2012). 'Detection and characterization of rocks and rock size-frequency distributions at the final four Mars Science Laboratory landing sites.' Mars, 7, 1-22.

  18. 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 (37C) 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 37C 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

  19. Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

    NASA Astrophysics Data System (ADS)

    Brancolini, Giorgia; Di Felice, Rosa

    2011-05-01

    Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

  20. Winding single-molecule double-stranded DNA on a nanometer-sized reel

    PubMed Central

    You, Huijuan; Iino, Ryota; Watanabe, Rikiya; Noji, Hiroyuki

    2012-01-01

    A molecular system of a nanometer-sized reel was developed from F1ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.96.0 pN) and the diameter of the wound loop (21.48.5?nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54??9?nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands. PMID:22772992

  1. Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria

    PubMed Central

    Grzymski, Joseph J.; Carter, Brandon J.; DeLong, Edward F.; Feldman, Robert A.; Ghadiri, Amir; Murray, Alison E.

    2006-01-01

    Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (? and ?), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters. PMID:16461708

  2. 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. PMID:26874503

  3. Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA.

    PubMed

    Breskvar, K; Cresnar, B; Plaper, A; Hudnik-Plevnik, T

    1991-08-15

    The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family. PMID:1872831

  4. Heat-Shocked Monocytes Are Resistant to Staphylococcus aureus-Induced Apoptotic DNA Fragmentation due to Expression of HSP72

    PubMed Central

    Guzik, Krzysztof; Bzowska, Ma?gorzata; Dobrucki, Jerzy; Pryjma, Juliusz

    1999-01-01

    Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis. PMID:10417194

  5. Quantitative Fluorescence Determination of Long-Fragment DNA in Stool as a Marker for the Early Detection of Colorectal Cancer

    PubMed Central

    Calistri, Daniele; Rengucci, Claudia; Molinari, Chiara; Ricci, Enrico; Cavargini, Elena; Scarpi, Emanuela; Milandri, Gian Luigi; Fabbri, Carla; Ravaioli, Alberto; Russo, Antonio; Amadori, Dino; Silvestrini, Rosella

    2009-01-01

    Background: A variety of molecular markers have been evaluated for the development of a non-invasive approach to the diagnosis of colorectal cancer. We aimed to validate the diagnostic accuracy, using the same threshold as in the previous pilot study, of fluorescent long DNA test as a relatively simple and inexpensive tool for colorectal cancer detection. Methods: A case-control study was conducted on 100 healthy subjects and 100 patients at first diagnosis of colorectal cancer. Human long-fragment DNA in stool was quantified by fluorescence primers and a standard curve and expressed in DNA nanograms. Results: We validated the 25-ng value, which emerged as the most accurate cut-off in the pilot study, obtaining 79% (95% CI, 7187%) sensitivity and 89% (95% CI, 8395%) specificity. Specificity was very high for all cut-off values (1540 ng) analyzed, ranging from 78 to 96%. Sensitivity was only slightly lower, reaching 84% at the lowest cut-off and maintaining a good level at the higher values. Diagnostic potential was independent of gender, age and tumor site. Conclusion: Fecal DNA analysis is a non-invasive and fairly simple test showing high diagnostic potential. These characteristics, together with the small amount of stool required, make it potentially suitable to be used alongside or as an alternative to current non-invasive screening approaches. Our next step will be to validate these results in a large-scale cohort study of a screening population, which is needed prior to implementation into clinical practice. PMID:19096146

  6. Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif.

    PubMed

    Leroy, J L; Guéron, M; Mergny, J L; Hélène, C

    1994-05-11

    In the recently discovered i-motif, four stretches of cytosine form two parallel-stranded duplexes whose C.C+ base pairs are fully intercalated. The i-motif may be recognized by characteristic Overhauser cross-peaks of the proton NMR spectrum, reflecting short H1'-H1' distances across the minor groove, and short internucleotide amino-proton-H2'/H2" across the major groove. We report the observation of such cross-peaks in the spectra of a fragment of the C-rich telomeric strand of vertebrates, d[CCCTAA]3CCC. The spectra also demonstrate that the cytosines are base-paired and that proton exchange is very slow, as reported previously for the i-motif. From UV absorbance and gel chromatography measurements, we assign these properties to an i-motif which includes all or nearly all the cytosines, and which is formed by intramolecular folding at slightly acid or neutral pH. A fragment of telomeric DNA of Tetrahymena, d[CCCCAA]3CCCC, has the same properties. Hence four consecutive C stretches of a C-rich telomeric strand can fold into an i-motif. Hypothetically, this could occur in vivo. PMID:8202359

  7. 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.; Akyrek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Prez 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 Comit Consultatif pour la Quantit de Matire (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), TBITAK 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).

  8. Identification of heterotrophic nanoflagellates by restriction fragment length polymorphism analysis of small subunit ribosomal DNA.

    PubMed

    Lim, E L; Dennett, M R; Caron, D A

    2001-01-01

    Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated. PMID:11411833

  9. [Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

    PubMed

    Geng, Li-xia; Zheng, Rui; Ren, Jie; Niu, Zhi-tao; Sun, Yu-long; Xue, Qing-yun; Liu, Wei; Ding, Xiao-yu

    2015-08-01

    In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous. PMID:26669009

  10. Sulindac activates nuclear translocation of AIF, DFF40 and endonuclease G but not induces oligonucleosomal DNA fragmentation in HT-29 cells.

    PubMed

    Park, Young Chul; Jeong, Jin Hee; Park, Ki Jae; Choi, Hong Jo; Park, Yeong Min; Jeong, Byung Kap; Higuchi, Yoshihiro; Yoo, Young Hyun

    2005-09-01

    Sulindac is one of the most widely studied nonsteroidal anti-inflammatory drugs in the prevention of colon cancer. Thus, from the viewpoint of colon cancer chemotherapy it is important to reveal the mechanism of sulindac-induced cell death. This study was undertaken to dissect the molecular mechanism underlying sulindac-induced apoptosis in human colon cancer cell line HT-29 (mutant p53), focusing on nuclear translocation of AIF, DFF and endonuclease G. On induction of apoptosis by sulindac, it was associated with decreased mitochondrial membrane potential, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of mitochondrial proteins to the nucleus, and morphological evidence of nuclear condensation. However, sulindac led to only disintegration of nuclear DNA into high molecular weight DNA fragments of about 100-300 kbp as determined by a pulse-field gel electrophoresis, suggesting a predominantly AIF-mediated cell death process. In summary, our findings indicate that sulindac induces large-scale DNA fragmentation without oligonucleosomal DNA fragmentation. This result suggests that nuclear translocation of DFF and endonuclease G are not sufficient for the induction of oligonucleosomal DNA fragmentation in HT-29 cells. PMID:15946692

  11. Genome size and DNA base composition of geophytes: the mirror of phenology and ecology?

    PubMed Central

    Vesel, Pavel; Bure, Petr; marda, Petr; Pavl?ek, Tom

    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

  12. Mechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells ?

    PubMed Central

    Kim, Jung-Sik; Xu, Xuehua; Li, Huifang; Solomon, David; Lane, William S.; Jin, Tian; Waldman, Todd

    2011-01-01

    Following DNA damage, human cells undergo arrests in the G1 and G2 phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN+/+ cells recapitulated the lack of size checkpoint control seen in PTEN?/? cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size. PMID:21536651

  13. Cytoplasmic RNA sequences complementary to cloned chick delta-crystallin cDNA show size heterogeneity.

    PubMed Central

    Bower, D J; Errington, L H; Wainwright, N R; Sime, C; Morris, S; Clayton, R M

    1982-01-01

    Double-stranded complementary DNA (cDNA) sequences were prepared from day-old chick lens total polysomal RNA and inserted into the unique PstI restriction site of the plasmid pBR322. Colonies containing sequences complementary to abundant lens poly(A)-containing RNA sequences were identified by using lens 32P-labelled cDNA. Some of these clones have been characterized as containing delta-crystallin mRNA coding sequences by genomic DNA blot hybridization and RNA blot hybridizations. Hybridization of labelled DNA from such clones to RNA blots detected four size classes of delta-crystallin RNA sequences, although Southern blots indicated that there are probably only two delta-crystallin genes. Images Fig. 1. Fig. 2. Fig. 4. PMID:6282266

  14. Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci.

    PubMed Central

    Arnheim, N; Strange, C; Erlich, H

    1985-01-01

    A rapid method has been developed and used to search for restriction fragment length polymorphisms (RFLPs) that are in linkage disequilibrium with disease-associated loci. By using genomic blot-hybridization analysis with DQ beta-chain and DR beta-chain cDNA probes, we examined DNA polymorphisms within the HLA class II loci associated with susceptibility to insulin-dependent mellitus (IDDM). To facilitate the search for informative RFLPs, we compared pooled DNA samples from IDDM patients with pooled DNA samples from randomly selected control individuals, instead of using the conventional approach of examining DNA samples from individuals in two groups. (The conditions under which this approach is useful are treated theoretically in the Appendix.) Several specific polymorphic restriction fragments associated with IDDM were revealed by using this economical and rapid approach. The restriction enzymes and probes identified as informative in this screening were then used to analyze HLA-DR-typed IDDM families, homozygous typing cells, and unrelated individuals to determine the association of the specific restriction fragments with HLA-DR serological type and the frequency in control and IDDM populations. Some individual polymorphic fragments for which the IDDM population was enriched correlated strongly with HLA-DR3, whereas others correlated strongly with HLA-DR4. Some fragments (e.g., a 10-kilobase Taq I fragment detected with the DR beta probe) that were more prevalent in the IDDM population subdivided the serologically defined HLA-DR type and may be informative markers for IDDM susceptibility. Images PMID:2995996

  15. A Model of Post-Traumatic Epilepsy After Penetrating Brain Injuries: Effect of Lesion Size and Metal Fragments

    PubMed Central

    Kendirli, M. Tansel; Rose, Dominique T.; Bertram, Edward H.

    2014-01-01

    Objective Penetrating brain injury (PBI) has the highest risk for inducing post-traumatic epilepsy and retained foreign materials such as bullet fragments carry the greatest risk. This study examines the potential contribution of copper, a major component of bullets, to the development of epilepsy following PBI. Methods Anesthetized adult male rats received a penetrating injury from the dorsal cortex to the ventral hippocampus from a high speed small bit drill. In one group of animals, copper wire was inserted into the lesion. Control animals had only the lesion or the lesion plus stainless steel wire (biologically inert foreign body). From 6 to up to 11 months following the injury the rats were monitored intermittently for the development of epilepsy with video-EEG. A separate set of animals was examined for possible acute seizures in the week following the injury. Results 22 of the 23 animals with copper wire developed chronic epilepsy compared to 3 of the 20 control rats (lesion and lesion with stainless steel). Copper was associated with more extensive injury. The control rats with epilepsy had larger lesions. In the acute injury group, there was no difference in the incidence of seizures (83% lesion plus stainless steel, 70% lesion plus copper). Conclusions Copper increases the risk for epilepsy and may increase damage over time, but there were no differences between the groups in the incidence of acute post-injury seizures. Lesion size may contribute to epilepsy development in lesion only animals. Copper maybe an independent risk factor for the development of epilepsy and possible secondary injury, but lesion size also contributes to the development of epilepsy. The consequences of prolonged exposure of the brain to copper observed in these animals may have clinical implications that require further evaluation. PMID:25470332

  16. Investigation of interaction between magnetic silica particles and lambda phage DNA fragment.

    PubMed

    Smerkova, Kristyna; Dostalova, Simona; Vaculovicova, Marketa; Kynicky, Jindrich; Trnkova, Libuse; Kralik, Miroslav; Adam, Vojtech; Hubalek, Jaromir; Provaznik, Ivo; Kizek, Rene

    2013-12-01

    Nucleic acids belong to the most important molecules and therefore the understanding of their properties, function and behavior is crucial. Even though a range of analytical and biochemical methods have been developed for this purpose, one common step is essential for all of them - isolation of the nucleic acid from the from complex sample matrix. The use of magnetic particles for the separation of nucleic acids has many advantages over other isolation methods. In this study, an isolation procedure for extraction of DNA was optimized. Each step of the isolation process including washing, immobilization and elution was optimized and therefore the efficiency was increased from 1.7% to 28.7% and the total time was shortened from 75 to 30min comparing to the previously described method. Quantification of the particular parameter influence was performed by square-wave voltammetry using hanging drop mercury electrode. Further, we compared the optimized method with standard chloroform extraction and applied on isolation of DNA from Staphylococcus aureus and Escherichia coli. PMID:23994762

  17. 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

  18. 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. PMID:21501956

  19. The Relationship between Seminal Melatonin with Sperm Parameters, DNA Fragmentation and Nuclear Maturity in Intra-Cytoplasmic Sperm Injection Candidates

    PubMed Central

    Sharbatoghli, Mina; Rezazadeh Valojerdi, Mojtaba; Bahadori, Mohammad Hadi; Salman Yazdi, Reza; Ghaleno, Leila Rashki

    2015-01-01

    Objective Melatonin, the chief secretory product of the pineal gland, regulates dynamic physiological adaptations that occur in seasonally breeding mammals as a response to changes in daylight hours. Because of the presence of melatonin in semen and the mem- brane melatonin receptor in spermatozoa, the impact of melatonin on the regulation of male infertility is still questionable. The aim of this study was to determine the effects of endogenous melatonin on human semen parameters (sperm concentration, motility and normal morphology), DNA fragmentation (DF) and nuclear maturity. Materials and Methods In this clinical prospective study, semen samples from 75 infer- tile men were routinely analyzed and assessed for melatonin and total antioxidant capac- ity (TAC) levels using the enzyme-linked immunosorbent assay (ELISA) and colorimetric assay kits, respectively. DF was examined by the sperm chromatin dispersion (SCD) test. Acidic aniline blue staining was used to detect chromatin defects in the sperm nuclei. Results There was no significant correlation between seminal plasma melatonin and TAC with sperm parameters and nuclear maturity. However, we observed a positive significant correlation between DF and melatonin level (r=0.273, P<0.05). Conclusion Melatonin in seminal plasma is positively correlated with damaged sperm DNA of infertile patients. The mechanism of this phenomenon needs further study. PMID:26464827

  20. Identification of Clostridium Species and DNA Fingerprinting of Clostridium perfringens by Amplified Fragment Length Polymorphism Analysis?

    PubMed Central

    Keto-Timonen, Riikka; Heikinheimo, Annamari; Eerola, Erkki; Korkeala, Hannu

    2006-01-01

    An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species. PMID:16971642

  1. Species mtDNA genetic diversity explained by infrapopulation size in a host-symbiont system.

    PubMed

    Doa, Jorge; Moreno-Garca, Marina; Criscione, Charles D; Serrano, David; Jovani, Roger

    2015-12-01

    Understanding what shapes variation in genetic diversity among species remains a major challenge in evolutionary ecology, and it has been seldom studied in parasites and other host-symbiont systems. Here, we studied mtDNA variation in a host-symbiont non-model system: 418 individual feather mites from 17 feather mite species living on 17 different passerine bird species. We explored how a surrogate of census size, the median infrapopulation size (i.e., the median number of individual parasites per infected host individual), explains mtDNA genetic diversity. Feather mite species genetic diversity was positively correlated with mean infrapopulation size, explaining 34% of the variation. As expected from the biology of feather mites, we found bottleneck signatures for most of the species studied but, in particular, three species presented extremely low mtDNA diversity values given their infrapopulation size. Their star-like haplotype networks (in contrast with more reticulated networks for the other species) suggested that their low genetic diversity was the consequence of severe bottlenecks or selective sweeps. Our study shows for the first time that mtDNA diversity can be explained by infrapopulation sizes, and suggests that departures from this relationship could be informative of underlying ecological and evolutionary processes. PMID:26811755

  2. 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. PMID:25329233

  3. 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

  4. The Protein Kinase C? 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 C? (PKC?) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKC? is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKC?-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKC?-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKC?-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 PKC?-cat-induced G2/M arrest, suggesting that PKC?-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKC? and PKC?-cat in the cell cycle response to DNA damage, we exposed wild-type and PKC? 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 PKC? null MEFs were resistant to these effects. Expression of PKC?-green fluorescent protein, but not caspase-resistant or kinase-inactive PKC?, was able to restore G2/M checkpoint integrity in PKC? null MEFs. The function of PKC? in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. PMID:19917613

  5. Molecular cloning of a mouse submaxillary gland renin cDNA fragment.

    PubMed Central

    Rougeon, F; Chambraud, B; Foote, S; Panthier, J J; Nageotte, R; Corvol, P

    1981-01-01

    The mRNA encoding mouse renin has been partially purified from total poly(A)-containing RNA of submaxillary glands of male Swiss mice. Corresponding cDNAs were cloned in the Pst I site of pBR322. Recombinants have been characterized by differential screening and hybrid-arrested translation. The DNA of clone pRn3-5 has been used to study the expression of renin mRNA in the submaxillary gland and in the kidney of different mouse strains. The renin mRNA from submaxillary gland and kidney have the same length (1600 nucleotides) and appear to be the products of the same gene. In vitro translation of mRNAs and RNA blotting experiments have shown that renin mRNA sequences are accumulated in the submaxillary gland of males of AKR and Swiss strains but not in the gland of male BALB/c. Images PMID:6171818

  6. 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.

  7. 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

  8. Effect of the Compaction and the Size of DNA on the Nuclear Transfer Efficiency after Microinjection in Synchronized Cells.

    PubMed

    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

  9. Primary structure of a gene-sized DNA encoding calmodulin from the hypotrichous ciliate Stylonychia lemnae.

    PubMed

    Gaunitz, C; Witte, H; Gaunitz, F

    1992-10-01

    We have isolated and characterized a gene-sized DNA encoding calmodulin (Clm) from macronuclear (MA) DNA of the hypotrichous ciliate, Stylonychia lemnae. The gene has 3500 copies per macronucleus. The length of the gene was deduced by agarose-gel electrophoresis of MA DNA and Southern blot analysis using a Clm cDNA probe from chicken. We then isolated the gene from a MA library. The overall length of the gene is 821 bp with a 450-bp intronless coding region. The deduced amino acid (aa) sequence of ciliate Clm has 149 aa and an M(r) of 16,819. Both ends of the cloned gene have the hypotrichous telomeric C4A4 repeat. The coding region is flanked by a 158-bp 5'-leader sequence and a 3'-trailer sequence of 213 bp. S1 analysis was used to locate the transcription start point (tsp) 49 bp upstream from the start codon. No common eukaryotic transcription signals were found upstream from the tsp. A second gene-sized DNA, detected by its cross-hybridization with the Clm DNA, predicts the existence of a second Ca(2+)-binding protein with only one Ca(2+)-binding site. It's function and biological significance is yet unknown. PMID:1398099

  10. Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA

    PubMed Central

    2014-01-01

    Background Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. Results We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. Conclusions A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities. PMID:25183040

  11. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases ?, ?, ?, ?, ?, REV1 and Klenow Fragment exo?, but not by DNA polymerase ?

    PubMed Central

    Suzuki, Masayo; Kino, Katsuhito; Kawada, Taishu; Oyoshi, Takanori; Morikawa, Masayuki; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2016-01-01

    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) ?, ?, ?, ?, ?, ? and REV1. We found that KF exo? and Pols ?, ?, ? and REV1 inserted one nucleotide opposite the 3? Oz of OzOz and stalled at the subsequent extension, and that Pol ? incorporated no nucleotide. Pol ? 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 ? 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 ? is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ?. PMID:26491064

  12. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases ?, ?, ?, ?, ?, REV1 and Klenow Fragment exo-, but not by DNA polymerase ?.

    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) ?, ?, ?, ?, ?, ? and REV1. We found that KF exo(-) and Pols ?, ?, ? and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol ? incorporated no nucleotide. Pol ? 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 ? 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 ? is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ?. PMID:26491064

  13. Effects of DNA size on transformation and recombination efficiencies in Xylella fastidiosa.

    PubMed

    Kung, Stephanie H; Retchless, Adam C; Kwan, Jessica Y; Almeida, Rodrigo P P

    2013-03-01

    Horizontally transferred DNA acquired through transformation and recombination has the potential to contribute to the diversity and evolution of naturally competent bacteria. However, many different factors affect the efficiency with which DNA can be transformed and recombined. In this study, we determined how the size of both homologous and nonhomologous regions affects transformation and recombination efficiencies in Xylella fastidiosa, a naturally competent generalist pathogen responsible for many emerging plant diseases. Our experimental data indicate that 96 bp of flanking homology is sufficient to initiate recombination, with recombination efficiencies increasing exponentially with the size of the homologous flanking region up to 1 kb. Recombination efficiencies also decreased with the size of the nonhomologous insert, with no recombination detected when 6 kb of nonhomologous DNA was flanked on either side by 1 kb of homologous sequences. Upon analyzing sequenced X. fastidiosa subsp. fastidiosa genomes for evidence of allele conversion, we estimated the mean size of recombination events to be 1,906 bp, with each event modifying, on average, 1.79% of the nucleotides in the recombined region. There is increasing evidence that horizontally acquired genes significantly affect the genetic diversity of X. fastidiosa, and DNA acquired through natural transformation could be a prominent mode of this horizontal transfer. PMID:23315739

  14. 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.

  15. Finite-size effects on long-range correlations: implications for analyzing DNA sequences

    NASA Technical Reports Server (NTRS)

    Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Simons, M.; Stanley, H. E.

    1993-01-01

    We analyze the fluctuations in the correlation exponents obtained for noncoding DNA sequences. We find prominent sample-to-sample variations as well as variations within a single sample in the scaling exponent. To determine if these fluctuations may result from finite system size, we generate correlated random sequences of comparable length and study the fluctuations in this control system. We find that the DNA exponent fluctuations are consistent with those obtained from the control sequences having long-range power-law correlations. Finally, we compare our exponents for the DNA sequences with the exponents obtained from power-spectrum analysis and correlation-function techniques, and demonstrate that the original "DNA-walk" method is intrinsically more accurate due to reduced noise.

  16. Charge density and particle size effects on oligonucleotide and plasmid DNA binding to nanosized hydrotalcite.

    PubMed

    Sanderson, Brian A; Sowersby, Drew S; Crosby, Sergio; Goss, Marcus; Lewis, L Kevin; Beall, Gary W

    2013-12-01

    Hydrotalcite (HT) and other layered double metal hydroxides are of great interest as gene delivery and timed release drug delivery systems and as enteric vehicles for biologically active molecules that are sensitive to gastric fluids. HT is a naturally occurring double metal hydroxide that can be synthesized as a nanomaterial consisting of a brucite structure with isomorphous substitution of aluminum ions. These positively charged nanoparticles exhibit plate-like morphology with very high aspect ratios. Biomolecules such as nucleic acids and proteins form strong associations with HT because they can associate with the positively charged layers. The binding of nucleic acids with HT and other nanomaterials is currently being investigated for potential use in gene therapy; however, the binding of specific nucleic acid forms, such as single- and double-stranded DNA, has been little explored. In addition, the effects of charge density and particle size on DNA adsorption has not been studied. In this paper, the binding of different forms of DNA to a series of HTs prepared at different temperatures and with different anion exchange capacities has been investigated. Experiments demonstrated that HTs synthesized at higher temperatures associate with both single- and double-stranded oligomers and circular plasmid DNA more tightly than HTs synthesized at room temperature, likely due to the hydrothermal conditions promoting larger particle sizes. HT with an anion exchange capacity of 300 meq/100 g demonstrated the highest binding of DNA, likely due to the closer match of charge densities between the HT and DNA. The details of the interaction of various forms of DNA with HT as a function of charge density, particle size, and concentration are discussed. PMID:24706120

  17. Structural, dynamical, and electronic transport properties of modified DNA duplexes containing size-expanded nucleobases.

    PubMed

    Blas, Jos Ramn; Huertas, Oscar; Tabares, Carolina; Sumpter, Bobby G; Fuentes-Cabrera, Miguel; Orozco, Modesto; Ordejn, Pablo; Luque, F Javier

    2011-10-20

    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. PMID:21888322

  18. 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.

  19. The RSC chromatin remodelling ATPase translocates DNA with high force and small step size

    PubMed Central

    Sirinakis, George; Clapier, Cedric R; Gao, Ying; Viswanathan, Ramya; Cairns, Bradley R; Zhang, Yongli

    2011-01-01

    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. PMID:21552204

  20. The Tertiary DNA Structure in the Single-Stranded hTERT Promoter Fragment Unfolds and Refolds by Parallel Pathways via Cooperative or Sequential Events

    PubMed Central

    Yu, Zhongbo; Gaerig, Vanessa; Cui, Yunxi; Kang, HyunJin; Gokhale, Vijay; Zhao, Yuan; Hurley, Laurence H.; Mao, Hanbin

    2012-01-01

    The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells. PMID:22372563

  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. Micrometer-sized network structure of novel DNA-lipid conjugates induced by heat stimulation.

    PubMed

    Takahashi, K; Matsuo, M; Banno, T; Toyota, T

    2015-09-21

    We have developed a novel lipid-bearing DNA that forms hairpin modules, including a single RNA monomer; this can be used to create micrometer-sized structures from nanometer-sized building blocks during breakage at the RNA site. In the presence of divalent metal ions and heat stimulation, we observed transition of the self-assembly, which results in the formation of a three-dimensional network structure. To our knowledge, this is also the first report of heat-induced micrometer-sized molecular self-assembly of molecules that carry biological information. PMID:26249035

  3. 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.

  4. 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.

  5. 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

  6. First principles effective electronic couplings for hole transfer in natural and size-expanded DNA

    PubMed Central

    Migliore, Agostino; Corni, Stefano; Varsano, Daniele; Klein, Michael L.; Di Felice, Rosa

    2009-01-01

    Hole transfer processes between base pairs in natural DNA and size-expanded DNA (xDNA) are studied and compared, by means of an accurate first principles evaluation of the effective electronic couplings (also known as transfer integrals), in order to assess the effect of the base augmentation on the efficiency of charge transport through double-stranded DNA. According to our results, the size expansion increases the average electronic coupling, and thus the CT rate, with potential implications in molecular biology and in the implementation of molecular nanoelectronics. Our analysis shows that the effect of the nucleobase expansion on the charge-transfer (CT) rate is sensitive to the sequence of base pairs. Furthermore, we find that conformational variability is an important factor for the modulation of the CT rate. From a theoretical point of view, this work offers a contribution to the CT chemistry in ?-stacked arrays. Indeed, we compare our methodology against other standard computational frameworks that have been adopted to tackle the problem of CT in DNA, and unravel basic principles that should be accounted for in selecting an appropriate theoretical level. PMID:19537767

  7. Antigenic analysis of group I house dust mite allergens using random fragments of Der p I expressed by recombinant DNA libraries.

    PubMed

    Greene, W K; Chua, K Y; Stewart, G A; Thomas, W R

    1990-01-01

    Antigenic regions of a major house dust mite allergen, Der p I, were identified by a recombinant DNA strategy employing the technique of random fragmentation. Fragments of cDNA coding for Der p I were produced by sonication and used to construct lambda gt 11 expression libraries. Analyses of recombinant fragments reactive with a rabbit anti-Der p I antiserum showed that the B cell determinants expressed in Escherichia coli were limited, with the majority (86%) of antigenic clones isolated mapping to the region comprising amino acid sequence position 60-80. To define antigenic regions of Der p I more precisely, selected overlapping fragments were subcloned into the expression vector pGEX-1. Dot blot immunoassay and immunoabsorption studies using individual fusion proteins revealed five regions - 34-47, 60-72, 82-99, 112-140, and 166-194 - to contain B cell determinants responsible for the antigenicity of recombinant Der p I. Absorption of the antiserum with Dermatophagoides pteronyssinus extract removed reactivity to all fragments, whereas absorption with an extract from the related mite Dermatophagoides farinae removed reactivity to peptides containing residues 34-47, 60-72, and 166-194, but not 82-99 and 112-140. Similarly, rabbit anti-D.farinae reacted strongly with peptides containing residues 34-47, 60-72, and 166-194, but not residues 82-99 and 112-140 which again showed antigenic differences in these residues between the group I allergens. PMID:2246073

  8. Fabrication and Characterization of Quartz Nanopillars for DNA Separation by Size

    NASA Astrophysics Data System (ADS)

    Ogawa, Ryo; Kaji, Noritada; Hashioka, Shingi; Baba, Yoshinobu; Horiike, Yasuhiro

    2007-04-01

    We fabricated two different distributions of nanopillarstilted and square distributions. There are difficulties in fabricating a chip containing these nanopillars, particularly in Ni plating and quartz bonding. We improved the Ni plating by applying stable electric current and the reliability of the quartz bonding. In nanopillars with a square distribution, DNA showed conformational changes, and did not show any reptile motion, but moved in a straight line in contrast with its behavior in nanopillars with a tilted distribution. Nanopillars with a tilted distribution acted effectively as sieves for DNA separation by size.

  9. Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

    PubMed

    Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2014-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. PMID:24393541

  10. Genotypic analyses of Mycoplasma gallisepticum isolates from songbirds by random amplification of polymorphic DNA and amplified-fragment length polymorphism.

    PubMed

    Cherry, John J; Ley, David H; Altizer, Sonia

    2006-04-01

    Mycoplasma gallisepticum (MG) conjunctivitis emerged in 1994 as a disease of free-ranging house finches (Carpodacus mexicanus) in North America and has also been isolated from other songbirds with conjunctivitis. Random amplification of polymorphic DNA (RAPD) of house finch and other songbird isolates has suggested that a single 'strain' initiated this outbreak. To explore the possibility of genomic variability among house finch isolates of MG and to evaluate the utility of a second technique for MG genotyping, we selected samples from our archive of reference strains and wild songbird isolates to analyze using both RAPD and amplified-fragment length polymorphism (AFLP); this is a newer technique that has been successfully used to explore the genomic variability of several Mycoplasma species. Both RAPD and AFLP results confirmed previous observations that during the initial stages of the MG epidemic in songbirds, isolates from different geographic locations and songbird species had genotypes that appeared to be highly similar, further supporting a single point source of origin. One 2001 isolate from New York was clearly different from the other songbird samples and clustered together with the vaccine and reference strains, indicating that substantial molecular evolution or a separate introduction has occurred. PMID:16870869

  11. STS map of genes and anonymous DNA fragments on human chromosome 18 using a panel of somatic cell hybrids

    SciTech Connect

    Overhauser, J.; Mewar, R.; Rojas, K.; Kline, A.D. ); Lia, K.; Silverman, G.A. )

    1993-02-01

    Somatic cell hybrids containing different deleted regions of chromosome 18 derived form patients with balanced translocations or terminal deletions were used to create a deletion mapping panel. Twenty-four sequence-tagged sites (STSs) for 17 genes and 7 anonymous polymorphic DNA fragments were identified. These STSs were used to map the 24 loci to 18 defined regions of chromosome 18. Both ERV1, previously mapped to 18q22-q23, and YES1, previously mapped to 18q21.3, were found to map to 18p11.21-pter. Several genes previously mapped to 18q21 were found to be in the order cen-SSAV1-DCC-FECH-GRP-BCL2-PLANH2-tel. The precise mapping of genes to chromosome 18 should help in determining whether these genes may be involved in the etiology of specific chromosomal syndromes associated with chromosome 18. The mapping of the poloymorphic loci will assist in the integration of the physical map with the recombination map of chromosome 18. 43 refs., 2 figs., 1 tab.

  12. 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. PMID:26175105

  13. Assessment of sperm function parameters and DNA fragmentation in ejaculated alpaca sperm (Lama pacos) by flow cytometry.

    PubMed

    Cheuquemn, C; Merino, O; Giojalas, L; Von Baer, A; Snchez, R; Risopatrn, 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 (??m) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 578 %. Spermatozoa with intact acrosome membrane was 87.95%, and viable sperm with intact acrosomal membrane was 46.89%, high mitochondrial membrane potential (??m) was detected in 66.329.51% of spermatozoa and mean DFI value was 0.910.9%. The DFI was inversely correlated with high ??m (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

  14. 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.

  15. Evidence on Sizes and Fragmentation of the Nuclei of Comet Shoemaker-Levy 9 from Hubble Space Telescope Images

    NASA Technical Reports Server (NTRS)

    Sekanina, Z.

    1995-01-01

    Central regions on the digital maps of 13 nuclear condensations of Comet Shoemaker-Levy 9, obtained with the Planetary Camera of the Hubble Space Telescope on January 24-25, March 28-30, and July 4, 1994, have been analyzed with the aim to identify the presence of distinct, major fragments in each condensation, to deconvolve their contributions to the signal that also includes the contribution from a surrounding cloud of dust (modeled as an extended source, using two different laws), to estimate the dimensions of the fragments and to study their temporal variations, and to determine the spatial distributions of the fragments as projected onto the plane of the sky. The deconvolution method applied is described and the results of the analysis are summarized, including the finding that sizable fragments die survive until the time of atmospheric entry. This result does not contradict evidence of the comet's continuing, apparently spontaneous fragmentation, which still went on long after the extremely close approach to Jupiter in July 1992 and which, because of the jovian tidal effects, may even have intensified in the final days before the crash on Jupiter. On plausible assumptions, the largest fragments are found to have had effect diameters of 4 km as late as March and event early July 1994. In most condensations, several sizable companions ( 1 km or more across) have been detected within 1000 km of the projected location of the brightest fragment and the surrounding dust cloud has been found to be centered on a point that is shifted in the general direction of the tail, probably due to effects of solar radiation pressure. Since the developed approach is based on certain premises and involves approximations, the results should be viewed as preliminary and the problem should be a subject of further investigation.

  16. The mutT Defect Does Not Elevate Chromosomal Fragmentation in Escherichia coli Because of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications?

    PubMed Central

    Rotman, Ella; Kuzminov, Andrei

    2007-01-01

    Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT?CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we found mutT recBC double mutants viable with no signs of chromosomal fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated with MutM in vitro, shows no increased relaxation, indicating no additional 8-oxo-G modifications. Our ?mutT allele elevates the AT?CG transversion rate 27,000-fold, consistent with published reports. However, the rate of AT?CG transversions in our mutT+ progenitor strain is some two orders of magnitude lower than in previous studies, which lowers the absolute rate of mutagenesis in ?mutT derivatives, translating into less than four 8-oxo-G modifications per genome equivalent, which is too low to cause the expected effects. Introduction of various additional mutations in the ?mutT strain or treatment with oxidative agents failed to increase the mutagenesis even twofold. We conclude that, in contrast to the previous studies, there is not enough 8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would trigger chromosomal fragmentation. PMID:17616589

  17. DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E.

    PubMed

    Abid-Essefi, Salwa; Baudrimont, Isabelle; Hassen, Wafa; Ouanes, Zouhour; Mobio, Thophile A; Anane, Rachid; Creppy, Edmond E; Bacha, Hassen

    2003-11-01

    Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by several Fusarium species growing on cereals. ZEN and its metabolites bind to human oestrogen receptors and hence display oestrogenic and anabolic properties. Several lines of investigation suggest that ZEN may be genotoxic in vivo. ZEN damages DNA in Bacillus subtilis recombination tests, and it induces sister chromatid exchange and chromosomal aberration in CHO cells. ZEN also induces DNA-adduct formation in mouse tissues and SOS repair process in lysogenic bacteria. In the present study, ZEN genotoxicity has been confirmed in three cell-lines, Vero, Caco-2 and DOK at concentrations of 10, 20 and 40 microM. Under these conditions, ZEN induces concentration-dependent DNA fragmentation resulting in DNA laddering patterns on agarose gel electrophoresis. This observation is consistent with apoptosis, which was confirmed by observations of formation of apoptotic bodies. Moreover, ZEN induces cell cycle arrest in the three cell-lines characterised by an increase of the number of cells in the G2/M phase of the cell cycle. Vitamin E (25 microM) added simultaneously with ZEN partially reduces DNA fragmentation and apoptotic body formation after 24h incubation. Vitamin E may act by maintaining prolonged cell cycle arrest during which time DNA repair takes place. PMID:14580790

  18. Drug-induced death of leukaemic cells after G2/M arrest: higher order DNA fragmentation as an indicator of mechanism.

    PubMed Central

    Sleiman, R. J.; Catchpoole, D. R.; Stewart, B. W.

    1998-01-01

    Many reports have documented apoptotic death in different cell types within hours of exposure to cytotoxic drugs; lower drug concentrations may cause cell cycle arrest at G2/M and subsequent death, which has been distinguished from 'classic' apoptosis. We have analysed etoposide-induced cell death in two lymphoblastoid T-cell lines, CCRF-CEM and MOLT-4, specifically in relation to DNA cleavage as indicated by pulse-field gel and conventional electrophoresis. High (5 microM) concentration etoposide causes 50-kb cleavage of DNA that occurs at the same time as apoptotic morphology and internucleosomal cleavage. At lower concentrations (0.5-0.05 microM), sequential change may be discerned with altered gene expression being similar to that at high dose, but preceding cell cycle arrest and 50-kb cleavage. These last changes, in turn, clearly precede internucleosomal fragmentation of DNA, vital dye staining and morphological evidence cell death. The pattern of higher order fragmentation constitutes a sensitive indicator of commitment to cell death in these cells. Morphological evidence of cell death is associated with internucleosomal fragmentation in one of the lines, but the pattern of 50-kb DNA cleavage provides the clearest evidence of commonality in death processes occurring at low and high drug concentration. Images Figure 2 Figure 3 Figure 5 Figure 6 PMID:9459144

  19. Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory.

    PubMed Central

    Schaeffer, F; Kolb, A; Buc, H

    1982-01-01

    To understand the denaturation process of short DNA segments we have chosen a 203-base pair (bp) restriction fragment containing the lactose control region. A steady decrease in GC content exists between its i proximal and z proximal ends. We confirm that this fragment melts at low salt in two subtransitions. A GC to AT mutation in the AT-rich region (mutation UV5) increases the number of denatured base pairs in the first subtransition and decreases the cooperativity of the melting process. A GC to AT mutation in the GC-rich region (mutation L8) decreases the number of denatured base pairs in the first subtransition and increases the cooperativity. These mutations induce the same shift in the temperature of half denaturation. The effects of both mutations are additive. A short deletion at the z end of the fragment affects only the first subtransition. When four GC pairs are added to both end, the fragment melts in one transition. Comparison with the results obtained with a larger 789-bp lac fragment reveals strong end effects on base pair stability and suggests that denaturation of the 203-bp fragment proceeds unidirectionally from the z end. Good agreement is shown with the predictions made with the "z ipper model" of Crothers et al. (1965). PMID:7188180

  20. 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.

  1. 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.

  2. DNA sequencing by single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    NASA Astrophysics Data System (ADS)

    Goodwin, Peter M.; Schecker, Jay A.; Wilkerson, Charles W., Jr.; Hammond, Mark L.; Ambrose, W. Patrick; Jett, James H.; Martin, John C.; Marrone, Babetta L.; Keller, Richard A.; Haces, Alberto; Shih, Po-Jen; Harding, John D.

    1993-06-01

    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.

  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. C1q tumor necrosis factor ?-related protein isoform 5 attenuates palmitate-induced DNA fragmentation in myocytes through an AMPK-dependent mechanism

    PubMed Central

    Yang, Won-Mo; Min, Kyung-Ho; Lee, Wan

    2015-01-01

    This article reports the data for the effects of C1q tumor necrosis factor ?-related protein isoform 5 (CTRP5) on the palmitate-induced apoptosis in myocytes. The data obtained from in vitro cultured myocytes shows that the cellular treatment with the globular domain of CTRP5 (gCTRP5) significantly inhibits the palmitate-induced MTT reduction, caspase-3 activation, and DNA fragmentation in a time-dependent manner. The data presented in this article also shows that AraA, an inhibitor of AMPK, almost completely abolished the protective effect of gCTRP5 on the DNA fragmentation induced by palmitate in myocytes. Interpretation of our data and further extensive insights into the protective role of CTRP5 in palmitate-induced apoptosis in myocytes can be found in Yang and Lee (2014) [1]. PMID:26693510

  5. Backbone (1)H, (15)N, (13)C NMR assignment of the 518-627 fragment of the androgen receptor encompassing N-terminal and DNA binding domains.

    PubMed

    Meyer, Sandra; Wang, Ying-Hui; Pérez-Escrivà, Pau; Kieffer, Bruno

    2016-04-01

    Androgen receptor (AR) belongs to the nuclear receptor superfamily that are ligand dependent transcription factors. This protein binds to steroid hormones such as dihydrotestosterone, to specific DNA sequences as well as to a number of co-regulatory factors. A number of these interactions involve the N-terminal domain (NTD), that is predicted to be intrinsically disordered. In order to provide functional information about possible cross-talk mechanisms between the AR NTD and its DNA binding domain (DBD), we have undertaken the NMR study of a fragment of human AR encompassing the last 37 residues of the NTD and the DBD (NTD-DBD518-627). The backbone (1)H, (15)N, (13)C NMR resonance assignments of this fragment indicate the presence of residual helical secondary structure within the AR NTD. PMID:26732902

  6. Optimization of PCR conditions to amplify Cyt b, COI and 12S rRNA gene fragments of Malayan gaur (Bos gaurus hubbacki) mtDNA.

    PubMed

    Rosli, M K A; Zamzuriada, A S; Syed-Shabthar, S M F; Mahani, M C; Abas-Mazni, O; Md-Zain, B M

    2011-01-01

    PCR has been extensively used for amplification of DNA sequences. We conducted a study to obtain the best amplification conditions for cytochrome b (Cyt b), cytochrome c oxidase I (COI) and 12S rRNA (12S) gene fragments of Malayan gaur mtDNA. DNA from seven Malayan gaur samples were extracted for PCR amplification. Various trials and combinations were tested to determine the best conditions of PCR mixture and profile to obtain the best PCR products for sequencing purposes. Four selected target factors for enhancing PCR, annealing temperature, concentration of primer pairs, amount of Taq polymerase, and PCR cycle duration, were optimized by keeping the amount of DNA template (50 ng/?L) and concentration of PCR buffer (1X), MgCl(2) (2.5 mM) and dNTP mixture (200 ?M each) constant. All genes were successfully amplified, giving the correct fragment lengths, as assigned for both forward and reverse primers. The optimal conditions were determined to be: 0.1 ?M primers for Cyt b and COI, 0.3 ?M primers for 12S, 1 U Taq polymerase for all genes, 30 s of both denaturation and annealing cycles for Cyt b, 1 min of both stages for 12S and COI and annealing temperature of 58.4 C for Cyt b, 56.1 C for 12S and 51.3 C for COI. PCR products obtained under these conditions produced excellent DNA sequences. PMID:22033937

  7. 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.

  8. 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.

  9. Differences in Electrostatic Potential Around DNA Fragments Containing Adenine and 8-oxo-Adenine. An Analysis Based on Regular Cylindrical Projection

    SciTech Connect

    Haranczyk, Maciej; Miller, John H; Gutowski, Maciej S

    2007-07-01

    Changes of electrostatic potential (EP) around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through large scale density functional theory computations. Quantum mechanical structural optimizations of DNA fragments with three pairs of nucleotides and accompanying counteractions were performed with a B3LYP exchange-correlation functional and 6-31G** basis sets. The “intact” DNA fragment contained adenine in the middle layer, while the “damaged” fragment had the adenine replaced with 8-oxo-adenine. The electrostatic potential around these DNA fragments was projected on a cylindrical surface around the double helix. The two-dimensional maps of EP of the intact and damaged DNA fragments were analyzed to identify these modifications of EP that result from the occurrence of 8-oxo-adenine (8oA). It was found that distortions of a phosphate group neighboring 8oA and displacements of the accompanying countercation are clearly reflected in the EP maps. Helpful discussions Michel Dupuis are gratefully acknowledged. Authors wish to thank Marcel Swart for directing us to a compilation of van der Waals radii. This work was supported by the: (i) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G. and M.H.), (ii) the Office of Science (BER), U. S. Department of Energy, Grant No. DE-FG03-02ER63470 (JHM), (iii) Polish State Committee for Scientific Research (KBN) Grant DS/8221-4-0140-6 (MG), (iv) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic Computer Center in Gdansk (TASK) and at the Molecular Science Computing Facility (MSCF) in the William R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory, which is operated by Battelle for the US Department of Energy. The MSCF resources were available through a pilot project.

  10. 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. PMID:25545806

  11. Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome.

    PubMed

    Kaufman, R M; Pham, C T; Ley, T J

    1999-11-01

    To date, the normal transcriptional regulation of the human beta-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human beta-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human beta-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F(1) mice showed that 2 lines express human beta globin at levels approximately equivalent to the endogenous mouse beta-major genes. One line expresses no human beta globin, while the remaining 6 lines show intermediate expression levels. Complete gamma-->beta-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic-->adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size. PMID:10556205

  12. 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 in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. PMID:25828663

  13. Chloroplast DNA restriction fragment length polymorphism in Sequoia sempervirens D. Don Endl., Pseudotsuga menziesii (Mirb.) Franco, Calocedrus decurrens (Torr.), and Pinus taeda L.

    PubMed

    Ali, I F; Neale, D B; Marshall, K A

    1991-01-01

    The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia chloroplast genome. Polymorphisms were detected in all species but loblolly pine. Coast redwood and incense-cedar had a small number of rare variants, whereas Douglas fir had one highly polymorphic region of insertions/deletions in sequences revealed by the P6 probe from petunia. The mutation hotspot is currently being studied by DNA sequence analysis. PMID:24221163

  14. Ultrasonic degradation of DNA.

    PubMed

    Elsner, H I; Lindblad, E B

    1989-12-01

    Different results are obtained when DNA in aqueous solution and DNA in biological tissue are exposed to ultrasound. At intensities of ultrasound comparable to those applied clinically, ultrasonication is able to degrade purified DNA in aqueous solution, making ultrasonication a useful tool for preparing DNA fragments in vitro. Ultrasonic degradation of DNA in solution occurs by breaking hydrogen bonds and by single-strand and double-strand ruptures of the DNA helix. Two mechanisms are mainly responsible: cavitation and a thermal or mechanical effect. Stable cavitation is seen at low intensities of ultrasound. Increasing the intensity of the ultrasound above 2 W/cm2 is followed by increases in single-strand ruptures due to the creation of free radicals by transient cavitation. Following sonication, the distribution of the resulting DNA fragments approaches a lower size limit of 100-500 bp. Breaks in the DNA helix occur mainly between oxygen and carbon atoms, resulting in DNA fragments with a phosphorylated 5' end and a free alcohol at the 3' end. The relative lack of specificity in degrading the DNA helix makes ultrasonication a complementary alternative to the highly specific fragmentation obtained by restriction endonucleases. PMID:2693020

  15. Genotyping bovine milk proteins using allele discrimination by primer length and automated DNA sizing technology.

    PubMed

    Lindersson, M; Lundn, A; Andersson, L

    1995-04-01

    A method for genotyping kappa-casein (A, B, E), beta-casein (A1, A2, A3, A5, B) and beta-lactoglobulin (A, B) simultaneously by the use of allele discrimination by primer length combined with automated detection of fragments with a sequencing instrument is described. Seven different mutations within the milk protein genes were analysed in order to distinguish between the alleles examined. The samples were amplified in two separate multiplex polymerase chain reactions (PCRs), which were then pooled and separated according to size in a single lane on the gel. By using stringent PCR conditions, we have been able to achieve allele-specific amplifications and minimize amplification of mis-matched primer for all seven mutations. PMID:7733509

  16. Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

    PubMed Central

    Clerc, Agathe; Manceau, Charles; Nesme, Xavier

    1998-01-01

    Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique. PMID:16349533

  17. Curcumin induces caspase-3-dependent apoptotic pathway but inhibits DNA fragmentation factor 40/caspase-activated DNase endonuclease in human Jurkat cells.

    PubMed

    Sikora, Ewa; Bielak-Zmijewska, Anna; Magalska, Adriana; Piwocka, Katarzyna; Mosieniak, Grazyna; Kalinowska, Magdalena; Widlak, Piotr; Cymerman, Iwona A; Bujnicki, Janusz M

    2006-04-01

    Curcumin is a natural pigment that has been shown to induce cell death in many cancer cells; however, the death mode depends on the cell type and curcumin concentration. Here we show that, in Jurkat cells, 50 micromol/L curcumin severely lowers cell survival and induces initial stage of chromatin condensation. It also induces caspase-3, which is sufficient to cleave DNA fragmentation factor 45 [DFF45/inhibitor of caspase-activated DNase (ICAD)], the inhibitor of DFF40/CAD endonuclease. However, the release of DFF40/CAD from its inhibitor does not lead to oligonucleosomal DNA degradation in curcumin-treated cells. Moreover, curcumin treatment protects cells from UVC-induced oligonucleosomal DNA degradation. In biochemical experiments using recombinant DFF activated with caspase-3, we show that curcumin inhibits plasmid DNA and chromatin degradation although it does not prevent activation of DFF40/CAD endonuclease after its release from the inhibitor. Using DNA-binding assay, we show that curcumin does not disrupt the DNA-DFF40/CAD interaction. Instead, molecular modeling indicates that the inhibitory effect of curcumin on DFF40/CAD activity results from curcumin binding to the active center of DFF40/CAD endonuclease. PMID:16648563

  18. 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 12weeks including the control group, the group having received AFB1 (80g/kg bw), the group having received FB1 (100g/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20mg/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