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Sample records for dna fragment size

  1. 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.

  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.

    1993-01-01

    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.

  3. 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.

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

    PubMed Central

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

    2003-01-01

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

  5. 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.

  6. Size distributions of misrejoining DNA fragments in irradiated cells.

    PubMed

    Radivoyevitch, T; Hoel, D G; Hahnfeldt, P; Sachs, R K

    1998-05-01

    When ionizing radiation strikes a cell it induces DNA double strand breaks (DSBs). Subsequently, some of the DSBs misrejoin and thus cause alterations in the size distribution of the DNA fragments. We derive a system of non-linear integro-differential equations describing the misrejoining interactions of five classes of DNA fragments, including rings and various types of linear fragments. The fragment classes are represented by density functions; the shape of a density function determines the probability that a fragment has a particular size and the amplitude (integral) equals the expected number of such fragments per cell. The equations are solved: analytically for exponentially distributed initial fragment sizes (corresponding to high doses) and numerically for arbitrary initial conditions. Computed final fragment size distributions are applied to situations representative of flow karyotypes and pulsed-field gel assays. For human flow karyotypes, the model can be used to obtain misrejoining estimates at doses too high for conventional methods of data analysis. For pulsed-field gel assays in which human chromosomes are digested with restriction endonucleases to form 'cut-somes' (restriction fragments), the model provides a means of misrejoining estimation when the cut-some sizes are non-random. The model suggests that if the cut-some size distribution for unirradiated cells is completely random, misrejoining of radiation-induced DSBs will not be detectable in the final size distribution. PMID:9621680

  7. 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

  8. 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

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

    PubMed Central

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

    1993-01-01

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

  11. Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software.

    PubMed

    McEvoy, C R; Seshadri, R; Firgaira, F A

    1998-09-01

    We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed. PMID:9762444

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

    PubMed Central

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

    1994-01-01

    We 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, we 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, we 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. Images Figure 1 Figure 2 PMID:7913584

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. In vivo cleavage of cytosine-containing bacteriophage T4 DNA to genetically distinct, discretely sized fragments

    SciTech Connect

    Carlson, K.; Wiberg, J.S.

    1983-10-01

    Mutants of bacteriophage T4D that are defective in genes 42 (dCMP hydroxymethylase), 46 (DNA exonuclease), and 56 (dCTPase) produce limited amounts of phage DNA in Escherichia coli B. In this DNA, glucosylated 5-hydroxymethylcytosine is completely replaced by cytosine. It is found that this DNA rapidly becomes fragmented in vivo to at least 16 discrete bands as visualized on agarose gels subjected to electrophoresis. The sizes of the fragments ranged from more than 20 to less than 2 kilobase pairs. When DNAs from two of these bands were radioactively labeled in vitro by nick translation and hybridized to XbaI restiction fragments of cytosine-containing T4 DNA, evidence was obtained that the two bands are genetically distinct, i.e., they contain DNA from different parts of the T4 genome. Mutational inactivation of T4 endonuclease II (gene denA) prevented the fragmentation. Three different mutations in T4 endonuclease IV (gene denB) caused the same minor changes in the pattern of fragments. It is concluded that T4 endonuclease II is required, and endonuclease IV is involved to a minor extent, in the in vivo production of these cytosine-containing T4 DNA fragments. These DNA fragments are viewed as ''restriction fragments'' since they represent degradation products of DNA ''foreign'' to T4, they are of discrete size, and they are genetically distinct.

  18. 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

  19. FRAGMENT SIZE ANALYSIS OF FREE FETAL DNA IN MATERNAL PLASMA USING Y-STR LOCI AND SRY GENE AMPLIFICATION

    PubMed Central

    KIMURA, MACHIKO; HARA, MASAAKI; ITAKURA, ATSUO; SATO, CHIAKI; IKEBUCHI, KENJI; ISHIHARA, OSAMU

    2011-01-01

    ABSTRACT Free fetal DNA (ffDNA) in maternal plasma has now become a valuable source for noninvasive prenatal diagnosis. Being able to accurately identify the size of ffDNA in maternal plasma is essential for a noninvasive prenatal diagnosis. Furthermore, it is important to investigate the molecular characteristics related to apoptosis which gives rise to ffDNA. We investigated the fragment size of ffDNA in each sample more precisely, using both Y-STR and SRY primers, in 20 maternal plasma samples from the 17th to 39th weeks of gestation. PCR was conducted with Y-STR and SRY primers which can be used to amplify 100–524 bp fragments. In samples from 10 pregnant women carrying male fetuses, the maximum fragment size detected by Y-STR and SRY primers ranged from 219 to 313 bp. As a result, the mean average maximum fragment size of free fetal DNA detected by Y-STR and SRY primers was 286±28 bp. The Y-STR alleles detected in each maternal plasma DNA sample were all in agreement with the results of their cord blood samples. We concluded that the fragment size of ffDNA comprises 2 nucleosomal complexes or less, but not exceeding 3. PMID:21928694

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  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. 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.

  11. 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.

  12. High Fragmentation Characterizes Tumour-Derived Circulating DNA

    PubMed Central

    Mouliere, Florent; Robert, Bruno; Arnau Peyrotte, Erika; Del Rio, Maguy; Ychou, Marc; Molina, Franck; Gongora, Celine; Thierry, Alain R.

    2011-01-01

    Background Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments. Methodology In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients. Conclusion/Significance Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60–100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16). PMID:21909401

  13. A stochastic model of DNA fragments rejoining.

    PubMed

    Li, Yongfeng; Qian, Hong; Wang, Ya; Cucinotta, Francis A

    2012-01-01

    When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie's stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation. PMID:23028515

  14. A Stochastic Model of DNA Fragments Rejoining

    PubMed Central

    Li, Yongfeng; Qian, Hong; Wang, Ya; Cucinotta, Francis A.

    2012-01-01

    When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie's stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation. PMID:23028515

  15. Agarose Gel Electrophoresis for the Separation of DNA Fragments

    PubMed Central

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-01-01

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments   PMID:22546956

  16. 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

  17. Molecular cloning of Renibacterium salmoninarum DNA fragments.

    PubMed

    Etchegaray, J P; Martínez, M A; Krauskopf, M; León, G

    1991-03-15

    A Renibacterium salmoninarum enriched recombinant DNA library was constructed to isolate DNA fragments which could be used as probes to detect gene sequences specific for the causative agent of bacterial kidney disease in salmonid fish. One fragment of 149 base pairs was isolated and its specificity and sequence determined. This probe may prove useful in the design of diagnostic tests for the disease in asymptomatic fish and ova. PMID:2044941

  18. 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

  19. 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

  20. Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. )

    1989-03-07

    Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

  1. Simulation of DNA fragment distributions after irradiation with photons.

    PubMed

    Friedland, W; Jacob, P; Paretzke, H G; Merzagora, M; Ottolenghi, A

    1999-05-01

    The Monte Carlo track structure code PARTRAC has been further improved by implementing electron scattering cross-sections for liquid water and by explicitly modelling the interaction of water radicals with DNA. The model of the genome inside a human cell nucleus in its interphase is based on the atomic coordinates of the DNA double helix with an additional volume for the water shell. The DNA helix is wound around histone complexes, and these nucleosomes are folded into chromatin fibres and further to fibre loops, which are interconnected to build chromosomes with a territorial organisation. Simulations have been performed for the irradiation of human fibroblast cells with carbon K and aluminium K ultrasoft x-rays, 220 kVp x-rays and 60Co gamma-rays. The ratio single-strand breaks to double-strand breaks (ssb/dsb) for both types of ultrasoft x-rays is lower than for gamma-rays by a factor of 2. The contributions of direct and indirect effects to strand break induction are almost independent of photon energy. Strand break patterns from indirect effects reflect differences in the susceptibility of the DNA helix to OH* attack inside the chromatin fibre. Distributions of small DNA fragments (<3 kbp) are determined by the chromatin fibre structure irrespective of whether direct or indirect effects are causing the breaks. In the calculated fragment size distributions for larger DNA fragments (>30 kbp), a substantial deviation from random breakage is found only for carbon K irradiation, and is attributed to its inhomogeneous dose distribution inside the cell nucleus. For the other radiation qualities, the results for larger fragments can be approximated by random breakage distributions calculated for a yield of dsb which is about 10% lower than the average for the whole genome. The excess of DNA fragments detected experimentally in the 8-300 kbp region after x-ray irradiation is not seen in our simulation results. PMID:10384954

  2. 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.

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

    NASA Astrophysics Data System (ADS)

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

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

  4. 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.

  5. Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

    PubMed

    Wenz, H M

    1994-09-25

    Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures. PMID:7937124

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

    NASA Astrophysics Data System (ADS)

    Ouchterlony, Finn

    2016-01-01

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

  7. Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA.

    PubMed Central

    Pohjanpelto, P; Hölttä, E

    1996-01-01

    In mammalian cells DNA synthesis is more complicated than in prokaryotes and less well understood. Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human fibroblasts) with [32P]orthophosphate and found that, besides high molecular weight DNA, a species of low molecular weight DNA, approximately 450 bp in size, became efficiently labeled. The short DNA was labeled first, and in pulse-chase experiments the labeling was transient. The isolated small DNA fragments (RNase A-treated) were phosphorylated by T4 polynucleotide kinase specific for polynucleotides with 5'-OH ends. A polynucleotide kinase phosphorylating these DNA pieces was also detected in nuclear extracts of the cells. Treatment with alkaline phosphatase removed most of the 32P label incorporated into the small DNA in vivo. Labeling with deoxyribonucleosides did not reveal these fragments. We hypothesize that the low molecular weight DNA represents Okazaki fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki fragments. This would imply a novel step in DNA synthesis. We also show that depriving cells of polyamines reversibly blocks synthesis of high molecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments. Images PMID:8605890

  8. [Influence of sample dilution on separation and detection of DNA fragments by capillary electrophoresis].

    PubMed

    Song, L; Chen, H; Zhang, L; Cheng, J

    1999-07-01

    Capillary electrophoresis has become an important and useful method to separate and determine DNA fragments. In molecular biochemistry, the volume of DNA sample is very small (microL level) and DNA sample is liable to be contaminated and degraded. According to theoretical inference and experiments, we propose that dilution of DNA sample solution can increase separation efficiency and resolution without evidently reducing height of peaks. By this method, the usage efficiency of DNA sample can be improved. It is also demonstrated the separation and detection of DNA fragments by capillary electrophoresis with hydroxyethyl cellulose non-gel sieving matrix and with laser-induced fluorescence charge-coupled device as detector. By using lower concentration non-gel matrix (0.4%), all 8 larger size fragments of lambda DNA/Hind III (125 bp-23 130 bp) can be completely separated. Twenty smaller size fragments of pBR322-Hae III DNA (18 bp-587 bp) can be separated by higher concentration (1.6%) non-gel matrix. As ratio of sample dilution is 10, two adjacent fragment (123 bp and 124 bp) of pBR322-Hae III DNA can be separated. PMID:12552856

  9. A mechanism of gene amplification driven by small DNA fragments.

    PubMed

    Mukherjee, Kuntal; Storici, Francesca

    2012-01-01

    DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s). Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA) occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB) external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation in nature. PMID:23271978

  10. 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.

  11. 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.

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

    PubMed Central

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

    2014-01-01

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

  13. 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.

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

    PubMed

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

    2014-10-15

    Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Because the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but they are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax) based on the sperm chromatin dispersion test and developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. The first aim of the present study was to verify the suitability of Sperm-Halomax assay, specifically developed for canine semen, for the evaluation of DNA fragmentation of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes (DFIs) obtained with Sperm-Halomax and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) were compared. The second aim was to investigate whether a correlation between DNA status, sperm head morphology, and morphometry assessed by computer-assisted semen analysis exists in cat epididymal spermatozoa. No differences were observed in DFIs obtained with Sperm-Halomax and TUNEL. This result indicates that Sperm-Halomax assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of fresh or frozen semen used in assisted reproductive technologies. PMID:25129872

  15. Bacterial natural transformation by highly fragmented and damaged DNA

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-12-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

  18. Microfluidic DNA fragmentation for on-chip genomic analysis.

    PubMed

    Shui, Lingling; Bomer, Johan G; Jin, Mingliang; Carlen, Edwin T; van den Berg, Albert

    2011-12-01

    We report a high-throughput clog-free microfluidic deoxyribonucleic acid (DNA) fragmentation chip that is based on hydrodynamic shearing. Salmon sperm DNA has been reproducibly fragmented down to ∼ 5k bp fragment lengths by applying low hydraulic pressures (≤1 bar) across micromachined constrictions positioned in larger microfluidic channels that create point-sink flow with large velocity gradients near the constriction entrance. Long constrictions (100 µm) produce shorter fragment lengths compared to shorter constrictions (10 µm), while increasing the hydrodynamic pressure requirement. Sample recirculation (10 ×) in short constrictions reduces the mean fragment length and fragment length variation, and improves yield compared to single-pass experiments without increasing the hydrodynamic pressure. PMID:22101733

  19. Electronic transport in methylated fragments of DNA

    SciTech Connect

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

    2015-11-16

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

  20. 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.

  1. 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.

  2. Dependence on radiation quality of DNA fragmentation spectra

    NASA Astrophysics Data System (ADS)

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

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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Rapid hierarchical assembly of medium-size DNA cassettes

    PubMed Central

    Schmid-Burgk, Jonathan Leo; Xie, Zhen; Frank, Stefan; Virreira Winter, Sebastian; Mitschka, Sibylle; Kolanus, Waldemar; Murray, Andrew; Benenson, Yaakov

    2012-01-01

    Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components. PMID:22422837

  12. Fragment size distributions and caloric curve in collision induced cluster fragmentation

    NASA Astrophysics Data System (ADS)

    Farizon, M.; Farizon, B.; Ouaskit, S.; Märk, T. D.

    2008-01-01

    We report on a cluster fragmentation study involving collisions of high-energy (60 keV/amu) H3+(H2)(m⩽14) hydrogen cluster ions (m = 1,14) with atomic helium. The experimental characterisation of the cluster fragmentation not only by the average fragment size distribution but also by a statistical analysis of the fragmentation events has become possible owing to a developed multi-coincidence technique in which all the fragments of all collisions occurring in the experiment are mass analysed on an event-by-event basis. Measurements and analysis are carried out on a large number of cluster ions prepared at the same total energy, as opposed to observing the evolution of a single system over time (time averaged ergodic hypothesis). By selecting specific decay reactions we can start after the energising collision with a micro-canonical cluster ion ensemble of fixed excitation energy. From the respective fragment distributions for these selected decay reactions we derive corresponding temperatures of the decaying cluster ions. The relation between this temperature and the excitation energy (caloric curve) exhibits the typical prerequisites of a first order phase transition in a finite system, in the present case signalling the transition from a bound cluster type situation to the free gas phase

  13. 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

  14. Source size scaling of fragment production in projectile breakup

    SciTech Connect

    Beaulieu, L.; Bowman, D.R.; Fox, D.; Das Gupta, S.; Pan, J.; Ball, G.C.; Djerroud, B.; Dore, D.; Galindo-Uribarri, A.; Guinet, D.; Hagberg, E.; Horn, D.; Laforest, R.; Larochelle, Y.; Lautesse, P.; Samri, M.; Roy, R.; St-Pierre, C.

    1996-09-01

    Fragment production has been studied as a function of the source mass and excitation energy in peripheral collisions of {sup 35}Cl+{sup 197}Au at 43 MeV/nucleon and {sup 70}Ge+{sup nat}Ti at 35 MeV/nucleon. The results are compared to the Au+Au data at 600 MeV/nucleon obtained by the ALADIN Collaboration. A mass scaling, by {ital A}{sub source}{approximately} 35 to 190, strongly correlated to excitation energy per nucleon, is presented, suggesting a thermal fragment production mechanism. Comparisons to a standard sequential decay model and the lattice-gas model are made. Fragment emission from a hot, rotating source is unable to reproduce the experimental source size scaling. {copyright} {ital 1996 The American Physical Society.}

  15. Phylogenetic classification of short environmental DNA fragments

    PubMed Central

    Krause, Lutz; Diaz, Naryttza N.; Goesmann, Alexander; Kelley, Scott; Nattkemper, Tim W.; Rohwer, Forest; Edwards, Robert A.; Stoye, Jens

    2008-01-01

    Metagenomics is providing striking insights into the ecology of microbial communities. The recently developed massively parallel 454 pyrosequencing technique gives the opportunity to rapidly obtain metagenomic sequences at a low cost and without cloning bias. However, the phylogenetic analysis of the short reads produced represents a significant computational challenge. The phylogenetic algorithm CARMA for predicting the source organisms of environmental 454 reads is described. The algorithm searches for conserved Pfam domain and protein families in the unassembled reads of a sample. These gene fragments (environmental gene tags, EGTs), are classified into a higher-order taxonomy based on the reconstruction of a phylogenetic tree of each matching Pfam family. The method exhibits high accuracy for a wide range of taxonomic groups, and EGTs as short as 27 amino acids can be phylogenetically classified up to the rank of genus. The algorithm was applied in a comparative study of three aquatic microbial samples obtained by 454 pyrosequencing. Profound differences in the taxonomic composition of these samples could be clearly revealed. PMID:18285365

  16. 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

  17. Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I.

    PubMed Central

    Carver, T E; Hochstrasser, R A; Millar, D P

    1994-01-01

    Fluorescence depolarization decays were measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer.27-mer oligonucleotides bound to Klenow fragment of DNA polymerase I. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with two footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3- to 4-fold increase in the equilibrium partitioning of DNA into the exonuclease site; the largest effects were observed for purine-purine mismatches. Two or more consecutive G.G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250-fold greater than that of the corresponding matched DNA sequence. Internal single mismatches produced larger effects than the same mismatch at the primer terminus, with a delta delta G relative to the matched sequence of -1.1 to -1.3 kcal/mol for mismatches located 2, 3, or 4 bases from the primer terminus. Although part of the observed effects may be attributed to the increased melting capacity of the DNA, it appears that the polymerase site also promotes movement of DNA into the exonuclease site by rejecting aberrant primer termini. These effects suggest that the polymerase and exonuclease sites act together to recognize specific errors that distort the primer terminus, such as frameshifts, in addition to proofreading misincorporated bases. Images PMID:7938011

  18. DNA fragmentation in mouse organs during endotoxic shock.

    PubMed Central

    Bohlinger, I.; Leist, M.; Gantner, F.; Angermüller, 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

  19. 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

  20. Fragmentation

    NASA Astrophysics Data System (ADS)

    Herrmann, Hans J.; Wittel, Falk K.; Kun, Ferenc

    2006-11-01

    Brittle materials fragment when exploded or under impact. The study of fragmentation is of practical importance in many areas, ranging from archaeology to milling. In the last 10 years much progress has been achieved in the understanding of the fragment size and velocity distributions as function of the total energy, the geometry and the material strength. Scaling laws, analogous to those of critical phenomena, have been formulated. Recent experiments of exploding egg shells and Christmas balls have given insight also into the fragmentation of containers. For the case of shells, new critical exponents are obtained. These results are confirmed by numerical simulations. These laws are important to understand space debris.

  1. 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

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

    NASA Technical Reports Server (NTRS)

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

    2005-01-01

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

  3. Development of mass spectrometry for rapid detection of DNA fragments

    SciTech Connect

    Buchanan, M.V.; Hurst, G.B.

    1997-12-31

    Identifying the presence of a specific DNA fragment is becoming increasingly critical in medicine, law enforcement, consumer safety, and other applications. Regions in DNA that are diagnostic for a targeted genetic disease, individual, or microorganism are amplified using the Polymerase Chain Reaction (PCR) or other reactions. These products, which contain a specific number of nucleotide units, are currently analyzed by electrophoresis or hybridization. Mass spectrometry has the potential of characterizing the PCR products faster and more confidently than these methods. We have been investigating matrix assisted laser desorption/ionization (MALDI) mass spectrometry for the detection of DNA fragments, with the goal of developing an analytical method that can be used to screen many samples quickly and reliably. We have demonstrated the efficacy of this approach by detecting PCR products isolated from both human and microbial samples. We are currently investigating approaches for improving sample preparation, enhancing ionization, extending mass range, and increasing mass resolution.

  4. 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

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

    PubMed

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

    2016-01-01

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

  6. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    PubMed Central

    Kaer, Kristel; Mätlik, Kert; Metsis, Madis; Speek, Mart

    2008-01-01

    Background In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA (<10 kb) of known sequence. Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb) fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc), separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear) by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside with the full "spectrum" of initial restriction fragments of known size. Here the strategy is used for the identification of protein-binding regions in the 5' region of the rat p75 neurotrophin receptor (p75NTR) gene. Conclusion The developed strategy is based on a combination of traditional EMSA and denaturing PAGE for the identification of protein binding regions in long fragments of genomic DNA. The identification is straightforward and can be applied to shifted bands corresponding to stable DNA-protein complexes as well as unstable complexes, which undergo dissociation during electrophoresis. PMID:18533036

  7. Process for producing shortened target DNA fragments usable in sequencing large DNA segments

    SciTech Connect

    Henikoff, S.; Gelinas, R.E.

    1989-06-27

    This patent describes a process for producing cloned, circular DNA molecules containing shortened target DNA fragments, the fragments derived from a long target DNA segment. The cloned, circular DNA molecules suitable for use in determining the nucleotide sequence of the long target DNA segment. The process consists the steps of: producing, by molecular cloning, double-stranded circular recombinant DNA molecules, each molecule containing vector DNA, a sequencing primer binding site, and a DNA region comprising a long target DNA segment, a first restriction site adjacent to the long target DNA segment adapted to be cut by a first restriction endonuclease in a manner that creates a first terminus on the DNA molecules adjacent the long target DNA segment that is susceptible to digestion by an exonuclease, and a second restriction site located between the first restriction site and the sequencing primer binding site adapted to be cut by a second restriction endonuclease in a manner that creates, without additional terminus blocking or digestion, a second terminus on the DNA molecules that is not susceptible to digestion by an exonuclease; cutting the double-stranded circular recombinant DNA molecules at the first restriction site using a first restriction endonuclease and at the second restriction site using a second restriction endonuclease to form double-stranded linear recombinant DNA molecules having a first terminus that is susceptible to digestion by an exonuclease and a second terminus that is not susceptible to digestion by an exonuclease.

  8. DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system.

    PubMed

    Liu, Ming S; Amirkhanian, Varouj D

    2003-01-01

    We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis. PMID:12652577

  9. 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].

  10. Insights into Okazaki fragment synthesis by the T4 replisome: the fate of lagging-strand holoenzyme components and their influence on Okazaki fragment size.

    PubMed

    Chen, Danqi; Yue, Hongjun; Spiering, Michelle M; Benkovic, Stephen J

    2013-07-19

    In this study, we employed a circular replication substrate with a low priming site frequency (1 site/1.1 kb) to quantitatively examine the size distribution and formation pattern of Okazaki fragments. Replication reactions by the T4 replisome on this substrate yielded a patterned series of Okazaki fragments whose size distribution shifted through collision and signaling mechanisms as the gp44/62 clamp loader levels changed but was insensitive to changes in the gp43 polymerase concentration, as expected for a processive, recycled lagging-strand polymerase. In addition, we showed that only one gp45 clamp is continuously associated with the replisome and that no additional clamps accumulate on the DNA, providing further evidence that the clamp departs, whereas the polymerase is recycled upon completion of an Okazaki fragment synthesis cycle. We found no support for the participation of a third polymerase in Okazaki fragment synthesis. PMID:23729670

  11. 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…

  12. A Prototype Ultrasound Instrument To Size Stone Fragments During Ureteroscopy

    NASA Astrophysics Data System (ADS)

    Sorensen, Mathew D.; Teichman, Joel M. H.; Bailey, Michael R.

    2008-09-01

    An intraoperative tool to measure the size of kidney stones or stone fragments during ureteroscopy would help urologists assess if a fragment is small enough to be removed through the ureter or ureteral access sheath. The goal of this study was to determine the accuracy and precision of a prototype ultrasound device used to measure in vitro stone fragments compared to caliper measurements. A 10-MHz, 10-french ultrasound transducer probe was used to send an ultrasound pulse and receive ultrasound reflections from the stone using two methods. In Method 1 the instrument was aligned over the stone and the ultrasound pulse traveled through the stone. The time between reflections from the proximal and the distal surface of the stone were used along with the sound speed to calculate the stone size. Although the sound speed varied between stones, it was unlikely to be known during surgery and thus was estimated at 3000 m/s for calculations. In Method 2 the instrument was aligned partially over the stone and the ultrasound pulse traveled through water with a sound speed of 1481 m/s. Time was determined between the reflection from the proximal stone surface and the reflection from the tissue phantom on which the stone rested. Methods 1 and 2 were compared by linear regression to caliper measurements of the size of 19 human stones of 3 different stone types. Accuracy was measured by the difference of the mean ultrasound and mean caliper measurement and precision was measured as the standard deviation in the ultrasound measurements. For Method 1, the correlation between caliper-determined stone size and ultrasound-determined stone size was r2 = 0.71 (p<0.0001). In all but two stones accuracy and precision were less than 1 mm. For Method 2, the correlation was r2 = 0.99 (p<0.0001) and measurements were accurate and precise to within 0.25 mm. We conclude that the prototype device and either method measure stone size with good accuracy.

  13. 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

  14. Detection of Irradiated Food: DNA Fragmentation in Grapefruits

    NASA Astrophysics Data System (ADS)

    Delincée, Henry

    1998-06-01

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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

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

    NASA Astrophysics Data System (ADS)

    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 γ-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 γ-rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions.

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

    PubMed

    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. PMID:15880923

  9. 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.

  10. Okazaki fragment processing-independent role for human Dna2 enzyme during DNA replication.

    PubMed

    Duxin, Julien P; Moore, Hayley R; Sidorova, Julia; Karanja, Kenneth; Honaker, Yuchi; Dao, Benjamin; Piwnica-Worms, Helen; Campbell, Judith L; Monnat, Raymond J; Stewart, Sheila A

    2012-06-22

    Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G(2) phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation. PMID:22570476

  11. Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

    PubMed Central

    Chen, F; Suttle, C A

    1995-01-01

    Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7747950

  12. Amplification of DNA polymerase gene fragments from viruses infecting microalgae.

    PubMed

    Chen, F; Suttle, C A

    1995-04-01

    Nested PCR with three highly degenerate primers was used for amplification and identification of DNA polymerase (pol) genes from viruses which infect three genera of microalgae. Group-specific primers (AVS1 and AVS2) were designed on the basis of inferred amino acid sequences unique to the DNA pol genes of viruses (PBCV-1 and NY-2A) that infect an endosymbiotic Chlorella-like alga (Chlorophyceae) and a virus (MpV-SP1) which infects the photosynthetic flagellate Micromonas pusilla (Prasinophyceae). In addition, a nested primer (POL) was designed on the basis of the highly conserved amino acid sequence YGDTDS found in most B-family (alpha-like) DNA pol genes. These primers were used to amplify DNA from the three viruses, PBCV-1, NY-2A, and MpV-SP1, for which the primers were designed, as well as eight clonal isolates of genetically distinct viruses which infect M. pusilla and others which infect Chrysochromulina spp. (Prymnesiophyceae), suggesting that these are a group of related viruses. In contrast, no product resulted from using DNA from viruses which infect the marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae), suggesting that these viruses may not be closely related to those that infect microalgae. These primers were also used to amplify DNA from natural virus communities. Our results indicate that nested PCR, even under low-stringency conditions, can be used as a rapid method to verify the presence in seawater of a group of related viruses which infect microalgae. Sequence analysis of these fragments should provide information on the genetic diversity and potentially the phyletic relationships among these viruses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7747950

  13. Yeast-based recombineering of DNA fragments into plant transformation vectors by one-step transformation.

    PubMed

    Nagano, Yukio; Takao, Syoko; Kudo, Takahiro; Iizasa, Ei'ichi; Anai, Toyoaki

    2007-12-01

    T-DNA binary vectors are often used in plant transformation experiments. Because they are usually very large and have few restriction sites suitable for DNA ligation reactions, cloning DNA fragments into these vectors is difficult. We provide herein an alternative to cloning DNA fragments into very large vectors. Our yeast-based recombineering method enables DNA fragments to be cloned into certain types of T-DNA binary vectors by one-step transformation without the requirement of specific recombination sites or precisely positioned restriction ends, thus making the cloning process more flexible. Moreover, this method is inexpensive and is applicable to multifragment cloning. PMID:17680244

  14. Differentiation of Species and Populations of Aphelenchoides and of Ditylenchus angustus Using a Fragment of Ribosomal DNA.

    PubMed

    Ibrahim, S K; Perry, R N; Burrows, P R; Hooper, D J

    1994-12-01

    The polymerase chain reaction (PCR) was used to amplify a fragment of the ribosomal DNA (rDNA) from species and undescribed populations of Aphelenchoides and Ditylenchus angustus. The PCR primers used were based on conserved sequences in the 18S and 26S ribosomal RNA genes of Caenorhabditis elegans. In C. elegans, these primers amplify a 1,292 base pair (bp) fragment, which consists of the two internal transcribed spacers and the entire 5.8S gene. Amplification products from crude DNA preparations of 12 species and populations of Aphelenchoides and from D. angustus ranged in size from approximately 860-1,100bp. Southern blots probed with a cloned ribosomal repeat from C. elegans confirmed the identity of these amplified bands as ribosomal fragments. In addition to the differing sizes of the amplified rDNA fragments, the relative intensity of hybridization with the C. elegans probe indicated varying degrees of sequence divergence between species and populations. In some cases, amplified rDNA from the fungal host was evident. Storage of A. composticola at - 45 C for 2 years did not affect the ability to obtain appropriate amplified products from crude DNA preparations. Amplified rDNA fragments were cut with six restriction enzymes, and the restriction fragments produced revealed useful diagnostic differences between species and some undescribed populations. These results were consistent with previous studies based on morphology and isoenzymes. Three undescribed populations of Aphelenchoides were found to be different from all the species examined and from each other. PMID:19279910

  15. Minifish shows high genetic variation in mtDNA size.

    PubMed

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

    2014-01-01

    The genus Paedocypris is a newly described taxon of minifish species that are characterized by extensive chromosome evolution and one of the smallest known vertebrate nuclear genomes. Paedocypris features a tiny adult size, a short generation time, low fecundity and fragmented tropical habitats, which are factors that favor rapid speciation. Most recently, we have revealed that P. progenetica (Pp), the type species of the genus Paedocypris, has an unusual mtDNA bearing - within its D-loop - a tandem array of a 34-bp repeat sequence called the minifish repeat, which shows compromised replication efficiency in vitro. Here we report that Pp exhibits high genetic variation in mtDNA size. The efficiency of D-loop amplification was found to depend upon primers. Interestingly, Pp individuals of one and the same population differed drastically in mtDNA size resulting from varying copy numbers of the minifish repeat. We conclude that minifish has a high mutation rate and perhaps represents a rapidly evolving taxon of vertebrates. PMID:25470287

  16. 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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  19. 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.

  20. 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.

  1. Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders

    PubMed Central

    Mears, J. Gregory; Ramirez, Francesco; Leibowitz, David; Nakamura, Frank; Bloom, Arthur; Konotey-Ahulu, Felix; Bank, Arthur

    1978-01-01

    Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [32P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained β-like gene sequences assayed with β globin cDNA probe. One β-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three β gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing β-like genes were absent from δβ thalassemic DNA and one new fragment containing β-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of β-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with δβ thalassemia. Images PMID:274714

  2. 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 Ham’s 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 (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 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

  3. Analysis of ionic fragment size distribution in collision of nanosecond laser with the C60 molecule

    NASA Astrophysics Data System (ADS)

    Qian, D. B.; Ma, X.; Zhang, D. C.; Li, B.; Zhu, X. L.; Wen, W. Q.; Liu, H. P.

    2014-04-01

    At the critical point of fragmentation phase transition occurring in C60 system, we found that the yield distribution of ionic fragments Cn+ (n=3-16) originating from the multi-fragmentation pattern can be approximated fairly well by a power-law attenuation form of n-λ. Under the framework of Fisher model, the experimental result implies that the ionization-degree distribution of fragments obeys a power law enhancement form as the size n.

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

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Chatterjee, A.; Esposito, G.; Rydberg, B.; Simone, G.

    Outside the magnetic field of the Earth, high energy heavy ions (HZE particles) constitute a relevant part of the biologically significant dose to astronauts during the very long travels through the space. For heavy ions the primary ionization sites occur in a correlated manner along the track of the particles and their typical pattern of energy deposition on the microscopic scale is expected to have an important role in their effects on living cells. It has been pointed out that multiple Double Strand Breaks (DSB) can be produced in local proximity by the same particle track, creating a small region of clustered damage. We have investigated the influence of the shielding on the biological effects of heavy ions, studying the initial production of very small DNA fragments in human fibroblasts irradiated with iron ions. Theoretical studies have shown that materials rich in hydrogen, such as polymethylmethacrylate (PMMA), could be more suitable in reducing the radiation risk. This is due mainly to a lower production of secondary neutrons and target fragments in hydrogen-rich materials compared to aluminium, which is the current shield used to protect astronauts. We have measured the size distribution of DNA fragments induced by high-energy Fe ions over a range from 1 kbp to 23 kpb that are produced by DSB occured over distances comparable with the chromatin fiber dimensions. 1 GeV/u Fe ion beams were obtained from the Alternating Gradient Synchrotron at the Brookhaven National Laboratory and irradiations were performed without and with a 190 mm thick PMMA shielding. Plateau phase AG1522 cells were irradiated in agarose plugs with doses up to 600 Gy and DSB induction was determined by DNA fragmentation analysis after Pulsed/Constant Field Gel Electrophoresis. The results until now obtained show that the number of DSB increases linearly either when plotted versus fluence either versus dose. The fragment distribution indicates the occurrence of a spatially correlated damage. When a PMMA shield is used, the dose-rate of the Fe ions beam decreases by a factor around 0.5 and a concomitant marked decrease in the DNA fragments production per unit dose is found.

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

    PubMed

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

    2016-03-21

    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. PMID:26932823

  6. High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

    NASA Astrophysics Data System (ADS)

    Miller, Cynthia K.; Temin, Howard M.

    1983-05-01

    DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

  7. 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…

  8. 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

  9. DNA fragmentation pattern in human fibroblasts after irradiation with iron ions

    NASA Astrophysics Data System (ADS)

    Campa, Alessandro

    In this work we studied the fragmentation pattern produced by the double stand breaks (DSB) induced in AG1522 primary human fibroblasts by two different iron beams, one of energy 414 MeV/u, and the other of energy 115 MeV/u (with dose-average LET in water equal to 202 keV/µm and 442 keV/µm, respectively). Irradiation with several doses up to 200 Gy was performed at the HIMAC facility of the National Institute of Radiological Sciences, Chiba, Japan. Experimental data, first obtained for fragments belonging to the size ranges 23-1000 kbp and 1000-5700 kbp (Belli et al., 2006), have successively been obtained also for fragments belonging to the size ranges 1-9 kbp and 9-23 kbp; the experimental analysis was performed with pulsed and constant field electrophoresis. The RBE for DSB production was evaluated in two different fragment size ranges (i.e., 23-5700 kbp and 1-5700 kbp), and it was found larger for the wider size range, especially for the beam with the higher LET. The experimental results have been compared to those computed on the basis of the Monte Carlo PARTRAC simulation code, following the line of research started in Campa et al. (2005), and exploiting the recent update of the PARTRAC code to ions heavier than helium (Friedland et al., 2006). Because the agreement has been found satisfactory for both radiation qualities, the spectra outside the experimentally observable fragment size range were also computed in order to evaluate the overall fragmentation pattern. The marked increases of the RBEs for DSB production, obtained when also the very small fragments (< 1 kbp) are included, makes them closer to the RBE values observed for the late cellular effects. This finding is a further indication for the biological significance of the spatial correlation of DSB at short distances. This work was partially supported by ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References M. Belli, A. Campa, V. Dini, G. Esposito, Y. Furusawa, G. Simone, E. Sorrentino and M. A. Tabocchini. DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of differing energies. Radiat. Res. 165, 713-720 (2006). 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). W. Friedland, P. Jacob, H. G. Paretzke, A. Ottolenghi, F. Ballarini and M. Liotta. Simulation of light ion induced DNA damge patterns. Radiat. Prot. Dosim. 122, 116-120 (2006).

  10. 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

  11. 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.

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

    PubMed

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

    2015-06-23

    Phospholipid additives are a cost-effective medium to separate deoxyribonucleic acid (DNA) fragments and possess a thermally-responsive viscosity. This provides a mechanism to easily create and replace a highly viscous nanogel in a narrow bore capillary with only a 10°C change in temperature. Preparations composed of dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble, forming structures such as nanodisks and wormlike micelles. Factors that influence the morphology of a particular DMPC-DHPC preparation include the concentration of lipid in solution, the temperature, and the ratio of DMPC and DHPC. It has previously been established that an aqueous solution containing 10% phospholipid with a ratio of [DMPC]/[DHPC]=2.5 separates DNA fragments with nearly single base resolution for DNA fragments up to 500 base pairs in length, but beyond this size the resolution decreases dramatically. A new DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1500 base pairs. This increase in the upper size limit is accomplished using commercially available phospholipids at an even lower material cost than is achieved with the 10% preparation. The separation additive is used to evaluate size markers ranging between 200 and 1500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these self-assembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency. PMID:26092346

  13. 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

  14. 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

  15. 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.

  16. Incorporation of polyamidoamine sweeping and electrokinetic supercharging for in-line DNA fragment preconcentration.

    PubMed

    Tian, Jing; Qiao, Jinping; Gao, Jingjie; Qin, Weidong

    2013-03-01

    We report an approach for in-line preconcentration of DNA fragments using dendritic polyamidoamine generation 2.0 (PAMAM G 2.0) as sweeping agent. During the experiment, a plug of PAMAM G 2.0 was hydrodynamically injected first, followed by field-amplified sample injection (FASI) of DNA fragments, which were concurrently swept by PAMAMs via DNA-PAMAM complexation. Then, a segment of releasing agent, sodium dodecyl sulfate (SDS), was hydrodynamically introduced and subsequently electrophoretically driven to interact with the DNA-PAMAM complexes, forming more stable supramolecular SDS-PAMAM complexes and releasing the initially bound DNA fragments. The excess dodecyl sulfate anion also acted as terminating electrolyte in the separation, thereby the DNA fragments were enriched by the joint effects of FASI, sweeping and transient isotachophoresis. We term the approach PAMAM sweeping-electrokinetic supercharging (EKS). Because the mobility of the DNA-PAMAM complex was very low, the proposed method allowed long-time sample injection without notable loss in separation efficiency. Under the optimum conditions, the PAMAM sweeping-EKS strategy improved the detection sensitivity of DNA fragments by more than 30 folds relative to the conventional FASI. Moreover, due to the sweeping process incorporated, the approach can be applied to enrichment of DNA fragments in highly saline matrix. PMID:23374369

  17. Impact of forest fragment size on between-group encounters in lion-tailed macaques.

    PubMed

    Kumara, Honnavalli Nagaraj; Singh, Mewa; Sharma, Anantha Krishna; Santhosh, Kumar; Pal, Arijit

    2014-10-01

    Between-group encounters are an obvious outcome of intergroup competition. Between-group encounters in primates range from avoidance to fatally aggressive. The prevailing hypotheses explain such encounters as mate defense strategy by males and resource defense strategy by females. However, the rate and nature of between-group encounters may also be influenced by habitat and demographic characteristics. We studied the effect of forest fragment size on group encounters in lion-tailed macaques in the Western Ghats of southern India. The encounter rate decreased as the fragment size increased. Group density and home range overlap correlated positively with the encounter rate. The aggressive encounters were more in the relatively medium-sized fragment where the observed frequency of between-group encounters was higher than the expected frequency than in the small fragment and the large forest complex. Together, these results indicate a complex pattern of effects of fragment size on between-group encounters in primates. PMID:25028052

  18. Two distinct human DNA diesterases that hydrolyze 3'-blocking deoxyribose fragments from oxidized DNA.

    PubMed Central

    Chen, D S; Herman, T; Demple, B

    1991-01-01

    Mammalian cells were investigated for enzymes that help correct oxidative damages in DNA. We focused on 3'-repair diesterases, which process DNA ends at oxidative strand breaks by removing 3'-blocking fragments of deoxyribose that prevent DNA repair synthesis. Two enzymes were found in a variety of mouse, bovine and human tissues and cultured cells. The two activities were purified to differing degrees from HeLa cells. One enzyme had the properties of the known HeLa AP endonuclease (Mr approximately 38,000, with identical substrate specificity and reaction requirements, and cross-reactivity with anti-HeLa AP endonuclease antiserum) and is presumed identical to that protein. The second activity did not interact with anti-HeLa AP endonuclease antibodies and had relatively less AP endonuclease activity. This second enzyme may have been detected in other studies but never characterized. In addition to the 3'-repair diesterase and AP endonuclease, this partially purified preparation also harbored DNA 3'-phosphatase and 3'-deoxyribose diesterase activities. It is unknown whether all activities detected in the second preparation are due to a single protein, although activity against undamaged DNA was not detected. The in vivo roles of these two widely distributed 3'-repair diesterase/AP endonucleases have not been determined, but with the characterizations presented here such questions may now be focused. Images PMID:1719484

  19. Simultaneously sizing and quantitating zeptomole-level DNA at high throughput in free solution.

    PubMed

    Zhu, Zaifang; Chen, Huang; Chen, Apeng; Lu, Joann J; Liu, Shaorong; Zhao, Meiping

    2014-10-20

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

  20. 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

  1. 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

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

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

    2014-01-01

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

  4. 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

  5. Fibril fragmentation in amyloid assembly and cytotoxicity: when size matters.

    PubMed

    Xue, Wei-Feng; Hellewell, Andrew L; Hewitt, Eric W; Radford, Sheena E

    2010-01-01

    Amyloid assemblies are associated with several debilitating human disorders. Understanding the intra- and extracellular assembly of normally soluble proteins and peptides into amyloid aggregates and how they disrupt normal cellular functions is therefore of paramount importance. In a recent report, we demonstrated a striking relationship between reduced fibril length caused by fibril fragmentation and enhanced ability of fibril samples to disrupt membranes and to reduce cell viability. These findings have important implications for our understanding of amyloid disease in that changes in the physical dimensions of fibrils, without parallel changes in their composition or molecular architecture, could be sufficient to alter the biological responses to their presence. These conclusions provide a new hypothesis that the physical dimensions and surface interactions of fibrils play key roles in amyloid disease. Controlling fibril length and stability toward fracturing, and thereby the biological availability of fibril material, may provide a new target for future therapeutic strategies towards combating amyloid disease. PMID:20305394

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  17. 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.

  18. Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size.

    PubMed

    Sun, Ying; Makarava, Natallia; Lee, Cheng-I; Laksanalamai, Pongpan; Robb, Frank T; Baskakov, Ilia V

    2008-02-29

    Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-beta-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions--perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including alpha B-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease. PMID:18206163

  19. Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns.

    PubMed Central

    Beverley, S M; Ismach, R B; Pratt, D M

    1987-01-01

    Restriction endonuclease DNA fragment patterns have been used to examine the relationships among 28 isolates of Leishmania as well as Crithidia, Endotrypanum, and Trypanosoma cruzi. Fragments of nuclear DNA were generated with six restriction enzymes, and blots were hybridized with probes from three loci. Among the major lineages the fragment patterns are essentially completely different, while within the major lineages various degrees of divergence are found. Molecular evolutionary trees were constructed using the method of Nei and Li to estimate the percent nucleotide sequence divergence among strains from the fraction of fragments shared. Defined groups, such as species or subspecies within the major lineages, are also grouped by nuclear DNA comparisons. Within the donovani complex, we find Leishmania donovani chagasi and Leishmania donovani infantum to be as similar as strains within Leishmania donovani donovani, consistent with the proposal by other workers that New World visceral leishmaniasis originated quite recently. Images PMID:3025876

  20. 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...

  1. Interaction of fragmented double-stranded DNA with carbon nanotubes in aqueous solution

    NASA Astrophysics Data System (ADS)

    Gladchenko, G. O.; Karachevtsev, M. V.; Leontiev, V. S.; Valeev, V. A.; Glamazda, A. Yu.; Plokhotnichenko, A. M.; Stepanian, S. G.

    Aqueous suspensions of ultrasonically fragmented double-stranded (fds-) DNA and single-walled carbon nanotubes (SWNTs) have been investigated by UV- and IR-absorption, NIR-emission and Raman spectroscopy. According to gel-electrophoresis, the lengths of the polymer fragments were 100-500 base pairs. Analysis of IR and UV data indicates the presence of both double-stranded (ds) and single-stranded (ss)-regions in the fragments. SWNT complex with DNA was revealed by NIR-emission and Raman spectroscopy. It turned out that fds-DNA is less efficient in holding nanotubes in the aqueous solution than ss-DNA. From the UV-data, the character of the helix-coil transition is seen to be like that for fds-DNA off and on nanotube, however, DNA thermostability increased in this latter case. The effective charge density on the DNA sugar-phosphate backbone of the fds-DNA:SWNT hybrid was less than that of DNA alone. Spectroscopic data can be explained by a model in which the formation of hybrids starts due to the interaction between untwisted ss-regions of DNA and the nanotube: the strands wrap on the tube and thus create an 'anchor' for the whole polymer. The ds-part of the polymer is located close to the nanotube.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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'?

  8. 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

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

    PubMed Central

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

    2015-01-01

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

  10. The trans-autostimulatory activity of Rad27 suppresses dna2 defects in Okazaki fragment processing.

    PubMed

    Munashingha, Palinda Ruvan; Lee, Chul-Hwan; Kang, Young-Hoon; Shin, Yong-Keol; Nguyen, Tuan Anh; Seo, Yeon-Soo

    2012-03-16

    Dna2 and Rad27 (yeast Fen1) are the two endonucleases critical for Okazaki fragment processing during lagging strand DNA synthesis that have been shown to interact genetically and physically. In this study, we addressed the functional consequences of these interactions by examining whether purified Rad27 of Saccharomyces cerevisiae affects the enzymatic activity of Dna2 and vice versa. For this purpose, we constructed Rad27DA (catalytically defective enzyme with an Asp to Ala substitution at amino acid 179) and found that it significantly stimulated the endonuclease activity of wild type Dna2, but failed to do so with Dna2Δ405N that lacks the N-terminal 405 amino acids. This was an unexpected finding because dna2Δ405N cells were still partially suppressed by overexpression of rad27DA in vivo. Further analyses revealed that Rad27 is a trans-autostimulatory enzyme, providing an explanation why overexpression of Rad27, regardless of its catalytic activity, suppressed dna2 mutants as long as an endogenous wild type Rad27 is available. We found that the C-terminal 16-amino acid fragment of Rad27, a highly polybasic region due to the presence of multiple positively charged lysine and arginine residues, was sufficient and necessary for the stimulation of both Rad27 and Dna2. Our findings provide further insight into how Dna2 and Rad27 jointly affect the processing of Okazaki fragments in eukaryotes. PMID:22235122

  11. 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

  12. 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

  13. [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

  14. Effect of different gravity environments on DNA fragmentation and cell death in Kalanchoe leaves.

    PubMed

    Pedroso, M C; Durzan, D J

    2000-11-01

    Different gravity environments have been shown to significantly affect leaf-plantlet formation and asexual reproduction in Kalanchoë daigremontiana Ham. and Perr. In the present work, we investigated the effect of gravity at tissue and cell levels. Leaves and leaf-plantlets were cultured for different periods of time (min to 15 d) in different levels of gravity stimulation: simulated hypogravity (1 rpm clinostats; 2 x 10(-4) g), 1 g (control) and hypergravity (centrifugation; 20 and 150 g). Both simulated hypogravity and hypergravity affected cell death (apoptosis) in this species, and variations in the number of cells showing DNA fragmentation directly correlated with nitric oxide (NO) formation. Apoptosis in leaves was more common as gravity increased. Apoptotic cells were localized in the epidermis, mainly guard cells, in leaf parenchyma, and in tracheary elements undergoing terminal differentiation. Exposures to acute hypergravity (up to 60 min) showed that chloroplast DNA fragmentation occurred prior to nuclear DNA fragmentation, marginalization of chromatin, nuclear condensation, and nuclear blebbing. Addition of sodium nitroprusside (NO donor) mimicked centrifugation. NO and DNA fragmentation decreased with N(G)-monomethyl-L-arginine (NO-synthase inhibitor). The variations in NO levels, nucleoid DNA fragmentation, and cell death show how chloroplasts, cells and leaves may respond (and adapt) to gravity changes. PMID:11762440

  15. 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

  16. Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells.

    PubMed Central

    Oldenburg, DJ; Bendich, AJ

    1996-01-01

    The BY-2 tobacco cell line was used to study the size and structure of replicating mitochondrial DNA (mtDNA). Approximately 70 to 90% of the newly synthesized mtDNA did not migrate during pulsed-field gel electrophoresis. Moving pictures of the fluorescently labeled molecules showed that most of the immobile well-bound DNA was in structures larger than the size of the BY-2 mitochondrial genome of ~270 kb. Most of the structures appeared as complex forms with multiple DNA fibers. The sizes of the circular molecules that were also observed ranged continuously from ~20 to 560 kb without prominent size classes. Pulse-chase and mung bean nuclease experiments showed that the well-bound DNA contained single-stranded regions and was converted to linear molecules of between 50 and 150 kb. MtDNA replication in plants may be initiated by recombination events that create branched structures of multigenomic concatemers that are then processed to 50- to 150-kb subgenomic fragments. PMID:12239390

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  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. 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

  20. TNF-α is involved in activating DNA fragmentation in skeletal muscle

    PubMed Central

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

    2002-01-01

    Intraperitoneal administration of 100 μg kg−1 (body weight) of tumour necrosis factor-α to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-α-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-α-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-α receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-α partly mediates DNA fragmentation during experimental cancer-associated cachexia. British Journal of Cancer (2002) 86, 1012–1016. DOI: 10.1038/sj/bjc/6600167 www.bjcancer.com © 2002 Cancer Research UK PMID:11953838

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. [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

  6. Highly Selective Separation of DNA Fragments Using Optically Directed Transport

    SciTech Connect

    Braiman, Avital; Rudakov, Fedor M; Thundat, Thomas George

    2010-01-01

    We present a design that allows selective separation of biomolecules of a particular size without performing complete separation of the sample by size. By focusing a laser beam onto a photoelectrode in contact with an electrolyte medium, a highly localized and optically controlled photoelectrophoretic trap is created. Moving the light beam along the photoelectrode consequently moves the trap. We demonstrate that by manipulating the speed of the photoelectrophoretic trap biomolecules of a particular size can be selectively separated from the mixture. We achieve a qualitative agreement between our experimental results and numerical simulations.

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

    PubMed

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

    2003-03-01

    As a first step in developing a molecular method for the individualization of marijuana samples, we evaluated a plant DNA extraction kit. The QIAGEN plant DNeasy method uses a spin column format for recovery of DNA and is effective for obtaining high molecular weight DNA from leaf, flower (bud), and seed samples of marijuana. The average DNA yield was 125-500 ng per 100 milligrams of fresh plant tissue. The recovered DNA was of polymerase chain reaction (PCR) quality as measured by the ability to generate reproducible amplified fragment length polymorphism (AFLP) profiles. AFLP is a technique used to create a DNA profile for plant varieties and is being applied to marijuana samples by the authors to link growers and distributors of clonal material. The QIAGEN plant DNeasy method was simple, efficient, and reproducible for processing small quantities of marijuana into DNA. PMID:12664992

  8. 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

  9. Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

    PubMed Central

    Millard, J T; Weidner, M F; Kirchner, J J; Ribeiro, S; Hopkins, P B

    1991-01-01

    A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency. Images PMID:1903204

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

    SciTech Connect

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

    1989-11-10

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

  11. [Sperm DNA fragmentation index and the success rate of IVF/ICSI].

    PubMed

    Xi, Di; Chen, Yun; Dai, Yu-tian

    2016-01-01

    Sperm DNA fragmentation index (DFI) refers to the percentage of DNA strand breaks in the total sperm. Many studies suggest that elevated DFI can lead to male infertility and early spontaneous abortion. High-DFI patients are more likely to fail in assisted reproduction and preliminary treatment or prevention methods have been developed for this population. This review focuses on the impact of DFI on clinical pregnancy outcomes and progress in the studies of its treatment. PMID:26931032

  12. 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).

  13. Early stage intercalation of doxorubicin to DNA fragments observed in molecular dynamics binding simulations.

    PubMed

    Lei, Hongxing; Wang, Xiaofeng; Wu, Chun

    2012-09-01

    The intercalation mode between doxorubicin (an anticancer drug) and two 6-base-pair DNA model fragments (d(CGATCG)₂ and d(CGTACG)₂) has been well studied by X-ray crystallography and NMR experimental methods. Yet, the detailed intercalation pathway at molecular level remains elusive. In this study, we conducted molecular dynamics binding simulations of these two systems using AMBER DNA (parmbsc0) and drug (GAFF) force fields starting from the unbound state. We observed outside binding (minor groove binding or end-binding) in all six independent binding simulations (three for each DNA fragment), followed by the complete intercalation of a drug molecule in two simulations (one for each DNA fragment). First, our data directly supported that the minor groove binding is the dominant pre-intercalation step. Second, we observed that the opening and flipping of a local base pair (A3-T10 for d(CGATCG)₂ and C1-G12 for d(CGTACG)₂) in the two intercalation trajectories. This locally cooperative flipping-intercalation mechanism was different from the previously proposed rise-insertion mechanism by which the distance between two neighboring intact base pairs increases to create a space for the drug insertion. Third, our simulations provided the first set of data to support the applicability of the AMBER DNA and drug force fields in drug-DNA atomistic binding simulations. Implications on the kinetics pathway and drug action are also discussed. PMID:23079648

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

    PubMed

    Munguía-Rosas, Miguel A; Montiel, Salvador

    2014-01-01

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

  16. 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...

  17. Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from gram-negative soil bacteria indistinguishable by DNA-DNA hybridization.

    PubMed Central

    Osborn, A M; Bruce, K D; Strike, P; Ritchie, D A

    1993-01-01

    Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high levels of mercury (sites T2, SE, and SO) and one uncontaminated site (SB). The frequencies of Hgr bacteria in the total cultivable populations were 0.05% (SB), 0.69% (SO), 4.8% (SE), and 25% (T2). Between 35 and 100% of the isolates from the four sites contained DNA sequences homologous to a DNA probe from the mercury resistance (mer) operon of the Tn501 Hgr determinant. The mer sequences of 10 Tn501-homologous Hgr determinants from each site were amplified by the polymerase chain reaction, with primers designed to consensus sequences of the mer determinants of Tn501, Tn21, and pMJ100, and were classified on the basis of the size of the amplified product and the restriction fragment length polymorphism pattern. Two main groups of amplification product were identified. The first, represented by the T2 and SB isolates and one SE isolate, gave an amplification product indistinguishable in size from that amplified from Tn501 (approximately 1,010 bp). The second group, represented by the SO isolates and the majority of the SE isolates, produced larger amplification products of 1,040 or 1,060 bp. Restriction fragment length polymorphism analysis revealed that each amplification product size group could be further subdivided into five subgroups. Images PMID:7904439

  18. Fragment size information from evolution of Shoemaker-Levy 9 impact fireballs

    SciTech Connect

    Boslough, M.B.; Crawford, D.A.; Robinson, A.C.

    1995-04-01

    The authors have used the CTH Eulerian shock physics code to perform 2-D and 3-D computational simulations of fragment penetration into Jupiter`s atmosphere and of the resulting fireball growth and evolution. These simulations have shown that information about the fragment diameters at the time of impact can be extracted from the trajectories of the fireballs, the maximum plume heights, and the size of the ejecta blanked. The fireball/plume evolution is dominated by the impact energy that is deposited above 50 km beneath the 1 bar level and is not sensitive to penetration depth. Fragments with diameters of 2-3 km at the time of impact produce fireballs similar to those that were observed, regardless of whether they were a single solid object or a low-density rubble pile. Uniform plume heights imply consistent diameters, but not necessarily equal masses.

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

    PubMed

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

    2015-12-01

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

  20. 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

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

    PubMed Central

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

    2013-01-01

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

  2. Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

    PubMed Central

    van Bergen, B G; van der Ley, P A; van Driel, W; van Mansfeld, A D; van der Vliet, P C

    1983-01-01

    Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication. Images PMID:6300787

  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. Cloning a selected fragment from a human DNA 'fingerprint': isolation of an extremely polymorphic minisatellite.

    PubMed Central

    Wong, Z; Wilson, V; Jeffreys, A J; Thein, S L

    1986-01-01

    A large hypervariable DNA fragment from a human DNA fingerprint was purified by preparative gel electrophoresis and molecular cloning. The cloned fragment contained a 6.3 kb long minisatellite consisting of multiple copies of a 37 bp repeat unit. Each repeat contained an 11 bp copy of the "core" sequences, a putative recombination signal in human DNA. The cloned minisatellite hybridized to a single locus in the human genome. This locus is extremely polymorphic, with at least 77 different alleles containing 14 to 525 repeat units per allele being resolved in a sample of 79 individuals. All alleles except the shortest are rare and the resulting heterozygosity is very high (approximately 97%). Cloned minisatellites should therefore provide a panel of extremely informative locus-specific probes ideal for linkage analysis in man. Images PMID:2423969

  5. 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

  6. Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity.

    PubMed

    Robbiano, Luigi; Baroni, Debora; Novello, Luca; Brambilla, Giovanni

    2006-06-16

    Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5-10 mM), hydrazine (HZ; 0.5-4 mM), cadmium sulfate (CD; 31.2 and 62.5 μM), 4,4'-methylene bis (2-chloroaniline) (MOCA; 31.2-125 μM), isobutyl nitrite (IBN; 7.8-31.2 μM) and tetranitromethane (TNM; 1.9-15.6 μM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats. PMID:16690349

  7. Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

    PubMed Central

    Moyer, R C; Moyer, M P; Gerodetti, M H

    1978-01-01

    Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent. PMID:207888

  8. Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

    PubMed Central

    Van Dyke, M W; Dervan, P B

    1983-01-01

    DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA. Images PMID:6225070

  9. 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

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

    PubMed

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

    2014-01-01

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

  11. 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

  12. 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

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. Human Sperm DNA Fragmentation and its Correlation with Conventional Semen Parameters

    PubMed Central

    Evgeni, Evangelini; Charalabopoulos, Konstantinos; Asimakopoulos, Byron

    2014-01-01

    Background The initial step in the diagnostic investigation of male infertility has been traditionally based on the conventional seminal profile. However, there are significant limitations regarding its ability to determine the underlying mechanisms that cause the disorder. Sperm DNA fragmentation has emerged as a potential causative factor of reproductive failure and its assessment has been suggested as a useful adjunct to the laboratory methodology of male infertility evaluation, especially before the application of assisted reproduction technology (ART). Methods A review of recent bibliography was carried out in PubMed by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation. Results A comprehensive diagnostic approach of male infertility should be based on a combination of diagnostic attributes, derived from the conventional semen analysis, as well as the investigation of genomic integrity testing. Conclusion Due to its strong correlation with several aspects of ART procedures and further consequences for the offspring, sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies focusing on specific subgroups of infertile men who may benefit from an efficient therapeutic management based on the optimization of sperm DNA integrity are needed. PMID:24696791

  16. 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.

  17. 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.

  18. 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.

  19. Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

    PubMed

    Bounartzi, Theofania; Dafopoulos, Konstantinos; Anifandis, George; Messini, Christina I; Koutsonikou, Chrysoula; Kouris, Spyros; Satra, Maria; Sotiriou, Sotirios; Vamvakopoulos, Nicholas; Messinis, Ioannis E

    2016-04-01

    The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p < 0.05) than f-spDNA (AUC = 0.6, p > 0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success. PMID:27006263

  20. 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.

  1. 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

  2. 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.

  3. Reverse-phase HPLC of DNA restriction fragments and ribooligonucleotides on uncoated Kel-F powder.

    PubMed Central

    Usher, D A

    1979-01-01

    Uncoated Kel-F powder offers some unique features as a support for reverse-phase HPLC of oligonucleotides and DNA restriction fragments. Compounds are eluted from the column by a gradient of acetonitrile (0 tto 18% v/v) in 0.1 M aqueous triethylammonium acetate. In contrast to RPC-5 chromatography, oligonucleotides are not eluted by aqueous salt solutions alone, and the separation of restriction fragments depends only on the chainlength. The packing material is cheap, easy to pack, chemically inert, and does not bleed, so that separations are highly reproducible. The DNA loading capacity for Kel-F is presently inferior to RPC-5, but recovery of microgram amounts of material is typically better than 50%. Images PMID:461189

  4. Evidence that the DNA mismatch repair system removes 1-nucleotide Okazaki fragment flaps.

    PubMed

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

    2015-10-01

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

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. GOGOT: a method for the identification of differentially expressed fragments from cDNA-AFLP data

    PubMed Central

    2007-01-01

    Background One-dimensional (1-D) electrophoretic data obtained using the cDNA-AFLP method have attracted great interest for the identification of differentially expressed transcript-derived fragments (TDFs). However, high-throughput analysis of the cDNA-AFLP data is currently limited by the need for labor-intensive visual evaluation of multiple electropherograms. We would like to have high-throughput ways of identifying such TDFs. Results We describe a method, GOGOT, which automatically detects the differentially expressed TDFs in a set of time-course electropherograms. Analysis by GOGOT is conducted as follows: correction of fragment lengths of TDFs, alignment of identical TDFs across different electropherograms, normalization of peak heights, and identification of differentially expressed TDFs using a special statistic. The output of the analysis is a highly reduced list of differentially expressed TDFs. Visual evaluation confirmed that the peak alignment was performed perfectly for the TDFs by virtue of the correction of peak fragment lengths before alignment in step 1. The validity of the automated ranking of TDFs by the special statistic was confirmed by the visual evaluation of a third party. Conclusion GOGOT is useful for the automated detection of differentially expressed TDFs from cDNA-AFLP temporal electrophoretic data. The current algorithm may be applied to other electrophoretic data and temporal microarray data. PMID:17535446

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Characterization of a cDNA clone corresponding to a transcript from the Epstein-Barr virus BamHI M fragment: evidence for overlapping mRNAs.

    PubMed Central

    Pfitzner, A J; Strominger, J L; Speck, S H

    1987-01-01

    A 1.95-kilobase cDNA clone was isolated by screening a size-selected lambda gt10 cDNA library prepared from an Epstein-Barr virus-transformed B-cell line, IB4, with the Epstein-Barr virus BamHI M fragment. Sequence analysis revealed that this clone contains about 75% of the BMRF1 and the complete BMRF2 open reading frames. The transcript is not spliced, and the polyadenylation signal at base pair 2641 of the BamHI M fragment is used. Northern blots (RNA blots) indicate that this polyadenylation signal is used for three overlapping mRNAs. The sizes of these transcripts are 3.5, 2.6, and 1.5 kilobases. Images PMID:2441081

  16. Fragment size-ejection speed correlation in impactor-ejecta processes

    NASA Astrophysics Data System (ADS)

    Sachse, M.

    2014-04-01

    Ejecta created in high velocity impacts (v > 10 km s ) of micro-meteoroids on atmosphereless cosmic bodies is an efficient source for interplanetary dust. The impact erodes the target surface and releases material into space. The ejecta are typically micron-sized and populate a dust cloud whose number density decreases with increasing distance from the target. Unbound particles escape and add to the planetary dust environment. However, even mesoscopic particles (R > 100 μm) can severely damage manmade space hardware as they have high kinetic energies when they encounter spacecraft with high relative velocities. Here we investigate the influence of a correlation between the fragment size R and the ejection speed u in the form stating that larger fragments are (in average) launched with slower speeds as suggested by theoretical considerations and impact experiments (Melosh, 1984; Miljkovíc et al., 2012). We found that such a correlation constitutes a dynamical filter which removes large ejecta from high altitudes. For large moons they are always bound and restricted to regions close to the target surface. The effect is stronger for bigger ejecta and for more massive target bodies. Our results show that the risk to encounter dangerous particles during close flybys around large moons is lower than expected from the uncorrelated model of Krivov et al. (2003). Further changes due to strong planetary magnetic fields at the other end of the size range are discussed.

  17. 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

  18. 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

  19. Relatedness of Strains of Xanthomonas fragariae by Restriction Fragment Length Polymorphism, DNA-DNA Reassociation, and Fatty Acid Analyses†

    PubMed Central

    Roberts, P. D.; Hodge, N. C.; Bouzar, H.; Jones, J. B.; Stall, R. E.; Berger, R. D.; Chase, A. R.

    1998-01-01

    The levels of relatedness of strains of Xanthomonas fragariae collected over several years from locations in Canada and the United States were compared by determining fatty acid methyl ester profiles, restriction fragment length polymorphisms (RFLP) based on pulsed-field gel electrophoresis (PFGE) analysis, and DNA-DNA reassociation values. Based on qualitative and quantitative differences in fatty acid profiles, the strains were divided into nine groups and four groups by the MIDI “10% rule” and unweighted pair analysis, respectively. Restriction analysis of genomic DNA by PFGE with two endonucleases (XbaI and SpeI) revealed four distinct profiles. When a third endonuclease (VspI) was used, one group was divided into three subgroups. The profile of the American Type Culture Collection type strain differed from the profile of every other strain of X. fragariae. Considerable diversity was observed within X. fragariae, although the majority of the strains represented a clonal population. The four groups based on fatty acid profiles were similar to the four groups based on RFLP, but neither method related groups to the geographic origins of the strains. The DNA-DNA reassociation values were high for representative strains, providing evidence that all of the strains belong to the same species. PMID:9758826

  20. DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice.

    PubMed

    Shyu, R H; Shaio, M F; Tang, S S; Shyu, H F; Lee, C F; Tsai, M H; Smith, J E; Huang, H H; Wey, J J; Huang, J L; Chang, H H

    2000-01-01

    Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins. PMID:10644889

  1. 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

  2. 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, Jürgen

    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

  3. 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.

  4. 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].

  5. 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

  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. 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

  8. Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: A Design of Experiments approach

    PubMed Central

    Sun, Mingyun; Lin, Jennifer S.

    2012-01-01

    Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses (HECs) with average molar masses of ~27 kDa and ~1 MDa were blended with a second class of polymer, high-molar mass (~7 MDa) linear polyacrylamide (LPA). Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1 Kb DNA extension ladder (200 bp to 40,000 bp) was completed in 2 minutes. An orthogonal Design of Experiments (DOE) was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 kbp and 10 kbp, and large dsDNA fragments above 10 kbp. PMID:22009451

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  11. 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.

  12. Catastrophic Disruption of comet ISON: Determination of Size and Drift Velocity of ISON Fragments

    NASA Astrophysics Data System (ADS)

    Keane, Jacqueline V.; Milam, Stefanie; Coulson, Iain; Steckloff, Jordan; Knight, Matthew

    2015-08-01

    We report submillimeter dust continuum observations for comet C/2012 S1(ISON) obtained during the time period immediately before perihelion on 2013 November 28 (r = 0.0125AU). Prior to perihelion passage on 28 November 2013, the observed right ascension (RA) and declination (Dec) coordinates of comet C/2012 S1 (ISON) significantly lagged the predicted JPL (# 53) ephemeris. We show that this “braking effect” is due to a dynamic pressure exerted by sublimating gases on the sunward side of the nucleus. When comet ISON was first detected at 850 μm, the 1-mm-sized dust particles were tightly bound to the comet nucleus until at least November 23. Three days later, the dust was less tightly bound, elongated and diffuse, spread out over as much as 120 arc seconds (80,000 km) in the anti-solar direction, suggesting a fragmentation event. We calculate the average braking velocity of the nucleus of comet ISON by comparing the central RA position with the predicted JPL ephemeris. The difference in the observed nucleus distance from the predicted ephemeris in the elapsed time between two observations yields an average drift velocity for the comet. We apply a sublimation mass-loss model to determine the size and fragmentation of the comet ISON's nucleus over time.

  13. Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum.

    PubMed

    Schillberg, S; Zimmermann, S; Voss, A; Fischer, R

    1999-08-01

    We compared the expression of a functional recombinant TMV-specific full-size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full-size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full-size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full-size rAb29 expression were high in the apoplast (up to 8.5 micrograms per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody of scFv29, which was expressed in the periplasmic space of E. coli and showed the same binding specificity as full-size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV-coat protein monomers as rAb29. PMID:10621973

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

    PubMed Central

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

    2014-01-01

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

  15. 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

    2015-01-01

    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

  16. Nitric oxide-mediated proteasome-dependent oligonucleosomal DNA fragmentation in Leishmania amazonensis amastigotes.

    PubMed

    Holzmuller, Philippe; Sereno, Denis; Cavaleyra, Mireille; Mangot, Isabelle; Daulouede, Sylvie; Vincendeau, Philippe; Lemesre, Jean-Loup

    2002-07-01

    Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through the L-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoan Leishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO(2) or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets. PMID:12065515

  17. Nitric Oxide-Mediated Proteasome-Dependent Oligonucleosomal DNA Fragmentation in Leishmania amazonensis Amastigotes

    PubMed Central

    Holzmuller, Philippe; Sereno, Denis; Cavaleyra, Mireille; Mangot, Isabelle; Daulouede, Sylvie; Vincendeau, Philippe; Lemesre, Jean-Loup

    2002-01-01

    Resistance to leishmanial infections depends on intracellular parasite killing by activated host macrophages through the l-arginine-nitric oxide (NO) metabolic pathway. Here we investigate the cell death process induced by NO for the intracellular protozoan Leishmania amazonensis. Exposure of amastigotes to moderate concentrations of NO-donating compounds (acidified sodium nitrite NaNO2 or nitrosylated albumin) or to endogenous NO produced by lipopolysaccharide or gamma interferon treatment of infected macrophages resulted in a dramatic time-dependent cell death. The combined use of several standard DNA status analysis techniques (including electrophoresis ladder banding patterns, YOPRO-1 staining in flow cytofluorometry, and in situ recognition of DNA strand breaks by TUNEL [terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling] assay) revealed a rapid and extensive fragmentation of nuclear DNA in both axenic and intracellular NO-treated amastigotes of L. amazonensis. Despite some similarities to apoptosis, the nuclease activation responsible for characteristic DNA degradation was not under the control of caspase activity as indicated by the lack of involvement of cell-permeable inhibitors of caspases and cysteine proteases. In contrast, exposure of NO-treated amastigotes with specific proteasome inhibitors, such as lactacystin or calpain inhibitor I, markedly reduced the induction of the NO-mediated apoptosis-like process. These data strongly suggest that NO-induced oligonucleosomal DNA fragmentation in Leishmania amastigotes is, at least in part, regulated by noncaspase proteases of the proteasome. The determination of biochemical pathways leading up to cell death might ultimately allow the identification of new therapeutic targets. PMID:12065515

  18. 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.

  19. 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)

  20. 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

  1. Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

    PubMed Central

    Friedl, Anna A.; Kiechle, Markus; Maxeiner, Horst G.; Eckardt-Schupp, Friederike

    2010-01-01

    The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence. PMID:20677012

  2. 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

  3. 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

  4. 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

  5. Sequence context effects on 8-methoxypsoralen photobinding to defined DNA fragments

    SciTech Connect

    Sage, E.; Moustacchi, E.

    1987-06-16

    The photoreaction of 8-methoxypsoralen (8-MOP) with DNA fragments of defined sequence was studied. The authors took advantage of the blockage by bulky adducts of the 3'-5'-exonuclease activity associated with the T4 DNA polymerase. The action of the exonuclease is stopped by biadducts as well as by monoadducts. The termination products were analyzed on sequencing gels. A strong sequence specificity was observed in the DNA photobinding of 8-MOP. The exonuclease terminates its digestion near thymine residues, mainly at potentially cross-linkable sites. There is an increasing reactivity of thymine residues in the order T < TT << TTT in a GC environment. For thymine residues in cross-linkable sites, the reactivity follows the order AT << TA approx. TAT << ATA < ATAT < ATATAA. Repeated A-T sequences are hot spots for the photochemical reaction of 8-MOP with DNA. Both monoadducts and interstrand cross-links are formed preferentially in 5'-TpA sites. The results highlight the role of the sequence and consequently of the conformation around a potential site in the photobinding of 8-MOP to DNA.

  6. Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ.

    PubMed

    Lin, Szu Hua Sharon; Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y C; Lee, Marietta Y W T

    2013-11-01

    Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5'-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1-10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells. PMID:24035200

  7. 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.

  8. The roles of family B and D DNA polymerases in Thermococcus species 9°N 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 9°N 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

  9. [Inhibitory Properties of Nitrogen-Containing Adamantane Derivatives with Monoterpenoid Fragments Against Tyrosyl-DNA Phosphodiesterase I].

    PubMed

    Zakharenko, A L; Ponomarev, K U; Suslov, E V; Korchagina, D V; Volcho, K P; Vasil'eva, I A; Salakhutdinov, N F; Lavrik, O I

    2015-01-01

    It was found that compounds combining diazaadamantane and monoterpenoid fragments are potent inhibitors of new structural type of human recombinant DNA repair enzyme Tyrosyl-DNA phosphodiesterase I (Tdp1). It was demonstrated that the inhibition efficiency depended on the length and flexibility of the aliphatic chain of the substituent. PMID:27125028

  10. Simultaneous monitoring of DNA fragments separated by electrophoresis in a multiplexed array of 100 capillaries

    SciTech Connect

    Ueno, Kyoji; Yeung, E.S. )

    1994-05-01

    Various excitation schemes for distributing a laser beam to a large number of capillaries in an array are evaluated. This led to the construction of a multiplexed system for monitoring the electrophoresis of DNA fragments in 100 capillaries. The laser-excited fluorescence signals from each capillary are simultaneously recorded at the rate of 0.6 frame/s by a CCD camera. The reconstructed electropherograms show excellent reproducibility and minimal cross-talk. The system provides for two simultaneous excitation wavelengths so that it can be adapted for two-color, two-intensity DNA sequencing based on the commercial four-dye chemistry. Only 20 mW per laser line was employed. Further development of this system to accommodate up to 4096 independent sequencing channels at a time is discussed. 26 refs., 6 figs., 2 tabs.

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

    PubMed

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

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines. PMID:27033773

  12. 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

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

    PubMed

    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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

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

    SciTech Connect

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

    1994-07-01

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

  1. DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

    PubMed Central

    Wang, F. J.; Ripley, L. S.

    1994-01-01

    Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis. PMID:8005428

  2. 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.

  3. 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

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

  5. 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.

  6. 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

  7. A Virtual Screen Discovers Novel, Fragment-Sized Inhibitors of Mycobacterium tuberculosis InhA

    PubMed Central

    Perryman, Alexander L.; Yu, Weixuan; Wang, Xin; Ekins, Sean; Forli, Stefano; Li, Shao-Gang; Freundlich, Joel S.; Tonge, Peter J.; Olson, Arthur J.

    2015-01-01

    Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections, and is used in combination therapy to treat active tuberculosis disease (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria project (GO FAM) were designed to discover new scaffolds that display base stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the sixteen soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 μM, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and, thus, help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat. PMID:25636146

  8. 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

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

    PubMed Central

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

    1998-01-01

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

  10. Structure of monoubiquitinated PCNA: implications for DNA polymerase switching and Okazaki fragment maturation.

    PubMed

    Zhang, Zhongtao; Zhang, Sufang; Lin, Szu Hua Sharon; Wang, Xiaoxiao; Wu, Licheng; Lee, Ernest Y C; Lee, Marietta Y W T

    2012-06-01

    Ubiquitination of proliferating cell nuclear antigen (PCNA) to ub-PCNA is essential for DNA replication across bulky template lesions caused by UV radiation and alkylating agents, as ub-PCNA orchestrates the recruitment and switching of translesion synthesis (TLS) polymerases with replication polymerases. This allows replication to proceed, leaving the DNA to be repaired subsequently. Defects in a TLS polymerase, Pol η, lead to a form of Xeroderma pigmentosum, a disease characterized by severe skin sensitivity to sunlight damage and an increased incidence of skin cancer. Structurally, however, information on how ub-PCNA orchestrates the switching of these two classes of polymerases is lacking. We have solved the structure of ub-PCNA and demonstrate that the ubiquitin molecules in ub-PCNA are radially extended away from the PCNA without structural contact aside from the isopeptide bond linkage. This unique orientation provides an open platform for the recruitment of TLS polymerases through ubiquitin-interacting domains. However, the ubiquitin moieties, to the side of the equatorial PCNA plane, can place spatial constraints on the conformational flexibility of proteins bound to ub-PCNA. We show that ub-PCNA is impaired in its ability to support the coordinated actions of Fen1 and Pol δ in assays mimicking Okazaki fragment processing. This provides evidence for the novel concept that ub-PCNA may modulate additional DNA transactions other than TLS polymerase recruitment and switching. PMID:22592530

  11. 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.

  12. DNA Sequence Fragment Containing C to A Mutation as a Convenient Mutation Standard for DHPLC Analysis

    PubMed Central

    Dastsooz, Hassan; Vahedi, Nazanin; Fardaei, Majid

    2013-01-01

    Objective(s): Denaturing high performance liquid chromatography (DHPLC) is a high throughput approach for screening DNA sequence variations. To assess oven calibration, cartridge performance, buffer composition and stability, the WAVE Low and High Range Mutation Standards are employed to ensure reproducibility and accuracy of the chromatographic analysis. The purpose of this study was to provide a cost-effective homemade mutation standard for DHPLC analysis. Materials and Methods: DHPLC was performed to evaluate different elution temperatures of a 374 bp DNA fragment with C>A mutation at position of 59 to achieve a peak profile similar to the Low Mutation Standard. In order to verify the reproducibility of the homemade mutation standard using DHPLC, 15 different experiments were performed to compare the homemade mutation standard, the WAVE Low Range Mutation Standard with a positive DNA control sample. Results: We identified a comparable elution temperature and a peak profile with the WAVE Low Range Mutation Standard. Conclusion: This study confirmed the reproducibility of the peak profile of our homemade mutation standard compared to the Low Mutation Standard using DHPLC analysis. PMID:24106601

  13. 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.

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

    PubMed

    Wanek, Johann; Rühli, Frank Jakobus

    2016-05-01

    Computed tomography represents the gold standard in forensic and palaeopathological diagnosis. However, the X-rays used may affect the DNA quality through fragmentation and loss of genetic information. Previous work showed that the effects of ionizing radiation on dry DNA are non-significant with P < 10(-8), which cannot be detected by means of polymerase chain reaction methods. In the present paper, complete analytical model that characterizes radiation effects on fragmented DNA in dry, wet, and frozen states is described. Simulation of radiation tracks in water phantom cells was performed using the Geant4-DNA toolkit. Cell hits by electrons with energies between 5 and 20 keV were simulated, and the formation of radiolytic products was assessed at a temperature of 298 K. The diffusion coefficient and the mean square displacement of reactive species were calculated by Stokes-Einstein-Smoluchowski relations at 273 K. Finally, DNA fragment damage was estimated using the density distribution of fragments calculated from atomic force microscopy images. The lowest probability of radiation-induced DNA damage was observed for dry state, with a range from 2.5 × 10(-9) to 7.8 × 10(-12) at 298 K, followed by that for frozen state, with a range from 0.9 to 4 × 10(-7) at 273 K. The highest probability of radiation-induced DNA damage was demonstrated for fragmented DNA in wet state with a range from 2 to 9 × 10(-7) at 298 K. These results significantly improve the interpretation of CT imaging in future studies in forensic and palaeopathological science. PMID:26883247

  15. 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

  16. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

    PubMed

    Inaba, Yasushi; Miyashita, Satoshi; Somfai, Tamás; Geshi, Masaya; Matoba, Satoko; Dochi, Osamu; Nagai, Takashi

    2016-04-01

    This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells. PMID:26996887

  17. Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes.

    PubMed

    Ouspenski, I I; Brinkley, B R

    1993-06-01

    Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster cells. One of the clones thus obtained was characterized in detail. It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes. PMID:8408270

  18. 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

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

    PubMed Central

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

    2013-01-01

    In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species. PMID:23527060

  20. 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.

  1. 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, ɛ', and imaginary, ɛ'', 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.

  2. Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida)

    PubMed Central

    2014-01-01

    Background The 5’ region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks. Methods In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods. Results Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise). Conclusions As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks. PMID:24589289

  3. 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

  4. Effect of in vitro exposure to lead chloride on semen quality and sperm DNA fragmentation.

    PubMed

    Gomes, M; Gonçalves, A; Rocha, E; Sá, R; Alves, A; Silva, J; Barros, A; Pereira, M L; Sousa, M

    2015-06-01

    Exposure to lead may cause changes in the male reproductive system. We evaluated the effect of lead chloride (PbCl2) in vitro on semen quality from 31 individuals. Samples were incubated at room temperature for two exposure times (4 h and 8 h) and with two concentrations of PbCl2 (15 μg/ml or 30 μg/ml). Results showed that PbCl2 significantly inhibited rapid progressive motility and caused an increase in the percentage of tail anomalies in both times and concentrations assessed, as well as a decrease in vitality in the group exposed to 30 μg/ml PbCl2. A significant increase in immotile sperm was also observed between the group control and the groups submitted to lead. Total motility and DNA fragmentation also showed a significant decrease and increase, respectively, after 4 h of incubation in the group exposed to 30 μg/ml and in both groups after 8 h of incubation. In conclusion, PbCl2 affected sperm parameters and DNA integrity, which are essential for male fertility. PMID:24521979

  5. On the size distribution of collision fragments of NLC dust particles and their relevance to meteoric smoke particles

    NASA Astrophysics Data System (ADS)

    Havnes, O.; Gumbel, J.; Antonsen, T.; Hedin, J.; La Hoz, C.

    2014-10-01

    We present the results from a new dust probe MUDD on the PHOCUS payload which was launched in July 2011. In the interior of MUDD all the incoming NLC/PMSE icy dust particles will collide, at an impact angle ~70° to the surface normal, with a grid constructed such that no dust particles can directly hit the bottom plate of the probe. Only collision fragments will continue down towards the bottom plate. We determine an energy distribution of the charged fragments by applying a variable electric field between the impact grid and the bottom plate of MUDD. We find that ~30% of the charged fragments have kinetic energies less than 10 eV, ~20% have energies between 10 and 20 eV while ~50% have energies above 20 eV. The transformation of limits in kinetic energy for ice or meteoric smoke particles (MSP) to radius is dependent on many assumptions, the most crucial being fragment velocity. We find, however, that the sizes of the charged fragments most probably are in the range of 1 to 2 nm if meteoric smoke particles (MSP), and slightly higher if ice particles. The observed high charging fraction and the dominance of fragment sizes below a few nm makes it very unlikely that the fragments can consist mainly of ice but that they must be predominantly MSP as predicted by Havnes and Næsheim (2007) and recently observed by Hervig et al. (2012). The MUDD results indicate that MSP are embedded in NLC/PMSE ice particles with a minimum volume filling factor of ~.05% in the unlikely case that all embedded MSP are released and charged. A few % volume filling factor (Hervig et al., 2012) can easily be reached if ~10% of the MSP are released and that their charging probability is ~0.1.

  6. Effects of patch size and type of coffee matrix on ithomiine butterfly diversity and dispersal in cloud-forest fragments.

    PubMed

    Muriel, Sandra B; Kattan, Gustavo H

    2009-08-01

    Determining the permeability of different types of landscape matrices to animal movement is essential for conserving populations in fragmented landscapes. We evaluated the effects of habitat patch size and matrix type on diversity, isolation, and dispersal of ithomiine butterflies in forest fragments surrounded by coffee agroecosystems in the Colombian Andes. Because ithomiines prefer a shaded understory, we expected the highest diversity and abundance in large fragments surrounded by shade coffee and the lowest in small fragments surrounded by sun coffee. We also thought shade coffee would favor butterfly dispersal and immigration into forest patches. We marked 9675 butterflies of 39 species in 12 forest patches over a year. Microclimate conditions were more similar to the forest interior in the shade-coffee matrix than in the sun-coffee matrix, but patch size and matrix type did not affect species richness and abundance in forest fragments. Furthermore, age structure and temporal recruitment patterns of the butterfly community were similar in all fragments, independent of patch size or matrix type. There were no differences in the numbers of butterflies flying in the matrices at two distances from the forest patch, but their behavior differed. Flight in the sun-coffee matrix was rapid and directional, whereas butterflies in shade-coffee matrix flew slowly. Seven out of 130 recaptured butterflies immigrated into patches in the shade-coffee matrix, and one immigrated into a patch surrounded by sun coffee. Although the shade-coffee matrix facilitated movement in the landscape, sun-coffee matrix was not impermeable to butterflies. Ithomiines exhibited behavioral plasticity in habitat use and high mobility. These traits favor their persistence in heterogeneous landscapes, opening opportunities for their conservation. Understanding the dynamics and resource requirements of different organisms in rural landscapes is critical for identifying management options that address both animals' and farmers' needs. PMID:19627322

  7. 1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions.

    PubMed Central

    Burkitt, M J; Milne, L; Nicotera, P; Orrenius, S

    1996-01-01

    Isolated rat-liver nuclei were incubated with a series of membrane-permeable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OP) and neocuproine (NC) were found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleosomal cleavage, which is widely considered to be a hallmark for the enzymic DNA digestion that occurs during apoptosis. Ascorbate, particularly in the presence of hydrogen peroxide, increased the levels of fragmentation induced by OP. As well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the hydroxyl radical. Complementary experiments in vitro involving ESR determinations of hydroxyl radical formation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP or NC (but not the other complexing agents tested) that stimulates hydroxyl radical formation and DNA damage in the presence of hydrogen peroxide and ascorbate. It is therefore proposed that OP in the nuclei incubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demonstrate that the internucleosomal DNA 'laddering' that is often used as an indicator of apoptosis may also result from DNA fragmentation by non-enzymic processes. PMID:8546678

  8. 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.

  9. 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.

  10. 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 τclosed. Under Vmax conditions, the average time of τclosed 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 τopen, which includes molecular recognition of the incoming dNTP. All α-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

  11. Crystal- and fragment- size distributions of quartz and zircon in pumice: growth and fragmentation conditions in large and small-volume magma chambers

    NASA Astrophysics Data System (ADS)

    Bindeman, I.

    2003-12-01

    I describe an acid (HF and HBF4) technique to extract phenocrysts from individual vesiculated pumice clasts, coupled with camera- and computer-assisted measurements of phenocryst length, width, 3D shape, and vol abundance. CSDs of quartz and zircon are presented for several well-known voluminous ash-flow tuffs and small-volume lavas: Bishop, Lava Creek, Lower Bandelier, Toba, Katmai, and Timber Mt. Measured CSDs of quartz and zircon from these clasts provide a quenched "snapshot" view of growth conditions in preclimactic magma chambers. A common feature of CSDs of unfragmented phenocrysts is a concave-down, lognormal shape in contrast to the reported linear CSDs in more mafic systems.In addition, there are no crystals smaller than a threshold size. These features in silicic magmas are interpreted to be a general result of surface-controlled crystal growth (with growth rate dispersion) by layer nucleation. CSD slopes on log-linear frequency- size graphs in large volume tuffs, and smaller volume intracaldera lavas are similar, and do not simply correlate to the eruptive volume, or SHRIMP-determined zircon ages. CSDs of quartz in clasts with known stratigraphic positions document single evolving reservoir, fingerprint different magma batches (L Bandelier and Lava Creek), and overgrowth and gravitational redistribution (Bishop). Fragment size distributions (FSDs) in the same clasts document fragmentation due to 1) decrepitation of melt inclusions decompression- and heating-induced), and 2)syneruptive breakage. FSDs are treated with lognormal, Weibull, and fractal distributions. Among studied clasts, asymptotic and fractal FSDs are found to be more common. However, the genesis mechanisms (e.g. fractal, scale-invariant vs. size-dependent lognormal) inferred from CSD or FSD should be treated with caution. Decrepitation results in a smaller number of fragments (2-6) than crushing and in shapes that can be distinguished on perimeter/area vs. length diagrams. CSD and FSD have potential implications as fingerprinting tools to identify/correlate different magma batches in ash-flow tuffs. FSD serves as a novel tool for trace elemental and isotopic exchange in magma chambers.

  12. 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

  13. 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-Ш (SGШ) 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

  14. 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 28–32 cycles). The first round (the first three cycles) is named the “One by One Annealing Round”. The second round (28–32 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

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

    PubMed Central

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

    2014-01-01

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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. Diagnostic value of sperm DNA fragmentation and sperm high-magnification for predicting outcome of assisted reproduction treatment

    PubMed Central

    López, Gemma; Lafuente, Rafael; Checa, Miguel A; Carreras, Ramón; Brassesco, Mario

    2013-01-01

    Over the last years, major improvements in the field of male infertility diagnosis have been achieved. The aim of this study was to determine the diagnostic usefulness of sperm DNA integrity and sperm vacuolisation for predicting outcome in infertile couples undergoing in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) treatments. A cohort study from 152 infertile couples undergoing sperm DNA fragmentation and high-magnification tests prior to an assisted reproduction treatment was designed. We found that the most predictive cutoff for pregnancy was 25.5% of DNA fragmentation with a negative predictive value of 72.7% (P=0.02). For the degree of vacuolisation, the best predictor of pregnancy was 73.5% of vacuolated sperm grades III+IV with a negative predictive value of 39.4% (P=0.09), which was not statistically significant. In conclusion, sperm DNA fragmentation greater than 25.5% could be associated with higher probability of failure IVF treatment. Regarding the results of the sperm analysis at high magnification, they do not allow us to predict whether or not patients will become pregnant. PMID:23912311

  2. Helical DNA origami tubular structures with various sizes and arrangements.

    PubMed

    Endo, Masayuki; Yamamoto, Seigi; Emura, Tomoko; Hidaka, Kumi; Morone, Nobuhiro; Heuser, John E; Sugiyama, Hiroshi

    2014-07-14

    We developed a novel method to design various helical tubular structures using the DNA origami method. The size-controlled tubular structures which have 192, 256, and 320 base pairs for one turn of the tube were designed and prepared. We observed the formation of the expected short tubes and unexpected long ones. Detailed analyses of the surface patterns of the tubes showed that the short tubes had mainly a left-handed helical structure. The long tubes mainly formed a right-handed helical structure and extended to the directions of the double helical axes as structural isomers of the short tubes. The folding pathways of the tubes were estimated by analyzing the proportions of short and long tubes obtained at different annealing conditions. Depending on the number of base pairs involved in one turn of the tube, the population of left-/right-handed and short/long tubes changed. The bending stress caused by the stiffness of the bundled double helices and the non-natural helical pitch determine the structural variety of the tubes. PMID:24888699

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm chromatin dispersion test.

    PubMed

    Urbano, M; Dorado, J; Ortiz, I; Morrell, J M; Demyda-Peyrás, S; Gálvez, M J; Alcaraz, L; Ramírez, L; Hidalgo, M

    2013-12-01

    The aims of this study were: 1) to assess the effect of freezing and thawing on dog sperm DNA fragmentation index (sDFI) using the sperm chromatin dispersion test (SCDt); and 2) to determine whether or not the sperm selection by single layer centrifugation (SLC) using Androcoll-C improves sperm DNA longevity in SLC-selected frozen-thawed dog semen samples. Semen samples were collected from 4 dogs using digital manipulation. After collection, ejaculates were pooled and cryopreserved following a standard protocol. Sperm motility and morphology were assessed before freezing and after thawing as a control for the cryopreservation method used. In experiment 1, sDFI was analyzed immediately before freezing and after thawing (baseline values), showing no significant differences between fresh and frozen-thawed semen samples. In experiment 2, frozen-thawed semen samples were processed or not by SLC using Androcoll-C and longevity of DNA were assessed in terms of sDFI after 24h of in vitro incubation at physiological temperature (38°C). The results showed low values of sDFI in SLC-selected semen in comparison to unselected samples. In conclusion, no effect of cryopreservation was observed on baseline values of dog sperm DNA fragmentation. Additionally, SLC-selection using Androcoll-C improved longevity of frozen-thawed sperm DNA assessed by the SCDt. PMID:24210910

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67. 7-kDa carboxyl-terminal fragment

    SciTech Connect

    D'Arpa, P.; Machlin, P.S.; Ratrie, H. III; Rothfield, N.F.; Cleveland, D.W.; Earnshaw, W.C.

    1988-04-01

    cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambdagt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. Molecular characterization of a DNA fragment harboring the replicon of pBMB165 from Bacillus thuringiensis subsp. tenebrionis

    PubMed Central

    Huang, Junyan; Guo, Suxia; Mahillon, Jacques; Van der Auwera, Géraldine A; Wang, Li; Han, Dongmei; Yu, Ziniu; Sun, Ming

    2006-01-01

    Background Bacillus thuringiensis belongs to the Bacillus cereus sensu lato group of Gram-positive and spore-forming bacteria. Most isolates of B. thuringiensis can bear many endogenous plasmids, and the number and size of these plasmids can vary widely among strains or subspecies. As far as we know, the replicon of the plasmid pBMB165 is the first instance of a plasmid replicon being isolated from subsp. tenebrionis and characterized. Results A 20 kb DNA fragment containing a plasmid replicon was isolated from B. thuringiensis subsp. tenebrionis YBT-1765 and characterized. By Southern blot analysis, this replicon region was determined to be located on pBMB165, the largest detected plasmid (about 82 kb) of strain YBT-1765. Deletion analysis revealed that a replication initiation protein (Rep165), an origin of replication (ori165) and an iteron region were required for replication. In addition, two overlapping ORFs (orf6 and orf10) were found to be involved in stability control of plasmid. Sequence comparison showed that the replicon of pBMB165 was homologous to the pAMβ1 family replicons, indicating that the pBMB165 replicon belongs to this family. The presence of five transposable elements or remnants thereof in close proximity to and within the replicon control region led us to speculate that genetic exchange and recombination are potentially responsible for the divergence among the replicons of this plasmid family. Conclusion The replication and stability features of the pBMB165 from B. thuringiensis subsp. tenebrionis YBT-1765 were identified. Of particular interest is the homology and divergence shared between the pBMB165 replicon and other pAMβ1 family replicons. PMID:17059605

  1. 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.

  2. 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.

  3. Fragmentation, merging, and internal dynamics for PIC simulation with finite size particles

    NASA Astrophysics Data System (ADS)

    Hewett, Dennis W.

    2003-08-01

    Components of a new type of "smart PIC" algorithm, intended to bridge the gap between Eulerian fluid regimes and kinetic regimes, are developed. Enlarging the scope of PIC, the CPK method (Complex Particle Kinetic) concept uses an ensemble of small, fluid-like macro-particles to represent particle distributions in phase space. These macro-particles are Gaussian-shaped in both position and velocity compared to the phase-space delta functions used in standard PIC and the spatial "sugar cube" particles used in an early version of this model [1]. Time evolution is modeled by a combination of the Lagrangian motion and internal evolution within each individual macro-particle. An analytic term is added to each particle's shape that represents internal evolution consistent with the collisionless, free-streaming of each macro-particle. Collision-dominated, γ-law gas internal evolution is also developed to define the opposite limit of collisionality. Similar to our initial effort, macro-particles are aggressively fragmented in phase space to probe for emerging kinetic features and aggressively merged, for economy, if interesting features fail to materialize. With CPK, fragmentation in both position and velocity space can be accomplished without loss of significant phase space information. Fragmentation preserves the kinetic capabilities of PIC; merging dramatically shrinks the number of particles in non-kinetic or collisional regions. In collision-dominated regimes, merging naturally produces a few Lagrangian particles that act much as nodes in Free-Lagrangian hydrodynamics. The only interaction between neutral particles is through merging; no mesh-dependent pressure gradients are needed. Finally, a linked-list data structure significantly reduces time spent "sorting" nearest neighbors for potential merging—and should lead to straightforward MPP operation.

  4. Fragmentation, Merging, and Internal Dynamics for Collisional PIC Simulation with Finite Size Particles

    NASA Astrophysics Data System (ADS)

    Hewett, Dennis; Larson, Dennis

    2002-11-01

    Components of a new type of "smart PIC" algorithm, intended to bridge the gap between Eulerian fluid regimes and kinetic regimes, are developed. Enlarging the scope of PIC, the KEYDRO (Kinetically-Extended hYDROdynamics) concept uses an ensemble of small, fluid-like macro-particles to represent particle distributions in phase space. These macro-particles are Gaussian-shaped in both position and velocity compared to the phase-space delta functions used in standard PIC and the spatial "sugar cube" particles used in an early version of this model [1]. Time evolution is modeled by a combination of the Lagrangian motion of each of these macro-particles and internal evolution within each individual macro-particle. An analytic term is now added to each particle's shape that represents internal evolution consistent with the collision-less, free-streaming of each macro-particle. Collision-dominated, -law gas internal evolution is also developed to define the opposite limit of collisionality. Recent developments now indicate that a consistent, partially collisional algorithm, encompassing the collision-dominated limit, can be incorporated as an integral part of the merging procedure. KEYDRO macro-particles are aggressively fragmented in phase space to probe for emerging kinetic features and aggressively merged, for economy, if interesting features fail to materialize. Fragmentation in both position and velocity space can be accomplished without loss of significant phase space information. Fragmentation preserves the kinetic capabilities of PIC; merging dramatically shrinks the number of particles in non-kinetic or collisional regions, and, in collision-dominated regimes, merging naturally produces a few Lagrangian particles that act much as nodes in Free-Lagrangian hydrodynamics. The only interaction between neutral particles is through merging; no mesh-dependent pressure gradients are needed. Finally, a double-linked-list data structure significantly reduces time spent "sorting" nearest neighbors for potential merging--and should lead to straightforward MPP operation. 1. Hewett, Submitted JCP 2002

  5. 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

  6. 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

  7. 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

  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. Short prokaryotic DNA fragment binning using a hierarchical classifier based on linear discriminant analysis and principal component analysis.

    PubMed

    Zheng, Hao; Wu, Hongwei

    2010-12-01

    Metagenomics is an emerging field in which the power of genomic analysis is applied to an entire microbial community, bypassing the need to isolate and culture individual microbial species. Assembling of metagenomic DNA fragments is very much like the overlap-layout-consensus procedure for assembling isolated genomes, but is augmented by an additional binning step to differentiate scaffolds, contigs and unassembled reads into various taxonomic groups. In this paper, we employed n-mer oligonucleotide frequencies as the features and developed a hierarchical classifier (PCAHIER) for binning short (≤ 1,000 bps) metagenomic fragments. The principal component analysis was used to reduce the high dimensionality of the feature space. The hierarchical classifier consists of four layers of local classifiers that are implemented based on the linear discriminant analysis. These local classifiers are responsible for binning prokaryotic DNA fragments into superkingdoms, of the same superkingdom into phyla, of the same phylum into genera, and of the same genus into species, respectively. We evaluated the performance of the PCAHIER by using our own simulated data sets as well as the widely used simHC synthetic metagenome data set from the IMG/M system. The effectiveness of the PCAHIER was demonstrated through comparisons against a non-hierarchical classifier, and two existing binning algorithms (TETRA and Phylopythia). PMID:21121023

  10. Assessment of sperm DNA fragmentation in stallion (Equus caballus) and donkey (Equus asinus) using the sperm chromatin dispersion test.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Serres-Dalmau, C; Gutiérrez de las Rozas, A L; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2009-10-01

    Sperm DNA fragmentation (sDF) is an important parameter to assessing sperm quality. Information about sperm quality is not available for donkeys, especially in some breeds at risk of extinction. The objectives of this research were to test the four commercial variants of sperm chromatin dispersion test (SCD; sperm Halomax test), originally developed to assess sDF in boars, bulls, rams and stallions, in order to scrutinize their applicability in the study of sDF in a donkey breed at risk of extinction (Zamorano-Leonesa), for which there is no specific test available to analyze sperm at present. Only the SCD test, originally developed for stallions, produced stable and consistent results, and was deemed suitable to assess DNA fragmentation in sperm samples from donkeys. Image analysis was used to compare differences between the SCD methodology applied to stallion and donkey semen samples processed under the same experimental conditions. The extent of SCD in the SCD test was approximately 20% lower in donkey sperm than in stallion sperm. Yet, the ratio of chromatin sperm dispersion achieved in fragmented and unfragmented nuclei did not differ significantly between species. These data suggest that a similar protein depletion treatment can cause differences in protein removal in equivalent cells from different species and that sperm chromatin may be organized differently in stallions and donkeys. PMID:19019071

  11. Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1

    PubMed Central

    Michaillat, Lydie; Baars, Tonie Luise; Mayer, Andreas

    2012-01-01

    Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles—the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment. PMID:22238359

  12. The Sperm Chromatin Structure Assay (SCSA(®)) and other sperm DNA fragmentation tests for evaluation of sperm nuclear DNA integrity as related to fertility.

    PubMed

    Evenson, Donald P

    2016-06-01

    Thirty-five years ago the pioneering paper in Science (240:1131) on the relationship between sperm DNA integrity and pregnancy outcome was featured as the cover issue showing a fluorescence photomicrograph of red and green stained sperm. The flow cytometry data showed a very significant difference in sperm DNA integrity between fertile and subfertile bulls and men. This study utilized heat (100°C, 5min) to denature DNA at sites of DNA strand breaks followed by staining with acridine orange (AO) and measurements of 5000 individual sperm of green double strand (ds) DNA and red single strand (ss) DNA fluorescence. Later, the heat protocol was changed to a low pH protocol to denature the DNA at sites of strand breaks; the heat and acid procedures produced the same results. SCSA data are very advantageously dual parameter with 1024 channels (degrees) of both red and green fluorescence. Hundreds of publications on the use of the SCSA test in animals and humans have validated the SCSA as a highly useful test for determining male breeding soundness. The SCSA test is a rapid, non-biased flow cytometer machine measurement providing robust statistical data with exceptional precision and repeatability. Many genotoxic experiments showed excellent dose response data with very low coefficient of variation that further validated the SCSA as being a highly powerful assay for sperm DNA integrity. Twelve years following the introduction of the SCSA test, the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labelling (TUNEL) test (1993) for sperm was introduced as the only other flow cytometric assay for sperm DNA fragmentation. However, the TUNEL test can also be done by light microscopy with much less statistical robustness. The COMET (1998) and Sperm Chromatin Dispersion (SCD; HALO) (2003) tests were introduced as light microscope tests that don't require a flow cytometer. Since these tests measure only 50-200 sperm per sample, they suffer from the lack of the statistical robustness of flow cytometric measurements. Only the SCSA test has an exact standardization of a fixed protocol. The many variations of the other tests make it very difficult to compare data and thresholds for risk of male factor infertility. Data from these four sperm DNA fragmentation tests plus the light microscope acridine orange test (AOT) are correlated to various degrees. PMID:26919909

  13. Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments.

    PubMed Central

    Ryerson, DE; Heath, MC

    1996-01-01

    It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants. PMID:12239388

  14. 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

  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. 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.

  17. 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 Sidrón 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

  18. [Polymorphism of DNA fragments flanked by microsatellite loci (ISSR-PCR) in cattle reproduced under low-dose irradiation conditions].

    PubMed

    Triapitsyna, N V; Glazko, V I

    2005-01-01

    Polymorphism of DNA fragments flanked by microsatellite loci (ISSR-PCR) in cattle reproduced under low-dose irradiation conditions. In this study the family analysis was performed of amplicon heredity (ISSR-PCR markers) in ancestral F0 generation and daughter's F1 and F2 generations of Holstein cattle reproduced in alienation zone of Chernobyl accident for investigation of genetic structure changes and polymorphism peculiarities under influence of low-dose irradiation. Increasing of the heterozygosity calculated (Hc) in F2 generation has been found that may be considered as response to ecological stress factor. No new amplicons have been revealed which could be evaluated as mutation events. PMID:16398145

  19. Investigation of hospital-acquired infections due to Alcaligenes denitrificans subsp. xylosoxydans by DNA restriction fragment length polymorphism.

    PubMed Central

    Cheron, M; Abachin, E; Guerot, E; el-Bez, M; Simonet, M

    1994-01-01

    We demonstrate that DNA restriction fragment length polymorphism determined by pulsed-field gel electrophoresis is very useful in the investigation of the epidemiology of hospital-acquired infections caused by Alcaligenes denitrificans subsp. xylosoxydans. This approach showed that hospital-acquired infections caused by this opportunistic pathogen over a 6-month period in 10 patients hospitalized in an intensive care unit and a surgical unit were not a true outbreak. In addition, this molecular typing method established that the respiratory therapy equipment was the source of the contamination of two patients. Images PMID:7913093

  20. 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

  1. Highly efficient synthesis of DNA-binding polyamides using a convergent fragment-based approach.

    PubMed

    Fallows, Andrew J; Singh, Ishwar; Dondi, Ruggero; Cullis, Paul M; Burley, Glenn A

    2014-09-01

    Two advances in the synthesis of hairpin pyrrole-imidazole polyamides (PAs) are described. First, the application of a convergent synthetic strategy is shown, involving the Boc-based solid phase synthesis of a C-terminal fragment and the solution phase synthesis of the N-terminal fragment. Second a new hybrid resin is developed that allows for the preparation of hairpin PAs lacking a C-terminal β-alanine tail. Both methods are compatible with a range of coupling reagents and provide a facile, modular route to prepare PA libraries in high yield and crude purity. PMID:25162625

  2. Effect of vesicle size on tissue localization and immunogenicity of liposomal DNA vaccines.

    PubMed

    Carstens, Myrra G; Camps, Marcel G M; Henriksen-Lacey, Malou; Franken, Kees; Ottenhoff, Tom H M; Perrie, Yvonne; Bouwstra, Joke A; Ossendorp, Ferry; Jiskoot, Wim

    2011-06-24

    The formulation of plasmid DNA (pDNA) in cationic liposomes is a promising strategy to improve the potency of DNA vaccines. In this respect, physicochemical parameters such as liposome size may be important for their efficacy. The aim of the current study was to investigate the effect of vesicle size on the in vivo performance of liposomal pDNA vaccines after subcutaneous vaccination in mice. The tissue distribution of cationic liposomes of two sizes, 500 nm (PDI 0.6) and 140 nm (PDI 0.15), composed of egg PC, DOPE and DOTAP, with encapsulated OVA-encoding pDNA, was studied by using dual radiolabeled pDNA-liposomes. Their potency to elicit cellular and humoral immune responses was investigated upon application in a homologous and heterologous vaccination schedule with 3 week intervals. It was shown that encapsulation of pDNA into cationic lipsomes resulted in deposition at the site of injection, and strongest retention was observed at large vesicle size. The vaccination studies demonstrated a more robust induction of OVA-specific, functional CD8+ T-cells and higher antibody levels upon vaccination with small monodisperse pDNA-liposomes, as compared to large heterodisperse liposomes or naked pDNA. The introduction of a PEG-coating on the small cationic liposomes resulted in enhanced lymphatic drainage, but immune responses were not improved when compared to non-PEGylated liposomes. In conclusion, it was shown that the physicochemical properties of the liposomes are of crucial importance for their performance as pDNA vaccine carrier, and cationic charge and small size are favorable properties for subcutaneous DNA vaccination. PMID:21565240

  3. Antibody fragments

    PubMed Central

    2010-01-01

    The antibody molecule is modular and separate domains can be extracted through biochemical or genetic means. It is clear from review of the literature that a wave of novel, antigen-specific molecular forms may soon enter clinical evaluation. This report examines the developmental histories of therapeutics derived from antigen-specific fragments of antibodies produced by recombinant processes. Three general types of fragments were observed, antigen-binding fragments (Fab), single chain variable fragments (scFv) and “third generation” (3G), each representing a successive wave of antibody fragment technology. In parallel, drug developers have explored multi-specificity and conjugation with exogenous functional moieties in all three fragment types. Despite high hopes and an active pipeline, enthusiasm for differentiating performance of fragments should, perhaps, be tempered as there are yet few data that suggest these molecules have distinct clinical properties due only to their size. PMID:20093855

  4. 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.

  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. 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

  8. Screening relevant genes of tolerance to low phosphorus in maize using cDNA-amplified fragment length polymorphism.

    PubMed

    Jiang, H Y; Li, Z; Zhao, J; Ma, Q; Cheng, B J; Zhu, S W

    2015-01-01

    Soil contains a large amount of phosphorus, but plants cannot absorb most of this phosphorus effectively. Low inorganic phosphorus has been singled out as a major constraint that leads to a perpetually low Zea mays (maize) grain yield. The fundamental approach to solving this problem is to screen new genes of low phosphorous (LP) tolerance. Consequently, the exploration and utilization of LP-tolerant genes are of great significance in plants. The maize inbred line 178 is an inbred LP-tolerant line. In the current study, the expression of this inbred line was induced under the stress of LP conditions. We applied cDNA-amplified fragment length polymorphism to screen LP-tolerant genes and obtained and sequenced 78 differentially expressed gene fragments. Their functions were predicted via bioinformatic analysis. There were no function annotations for 8 differentially expressed fragments. Nine genes exhibited high homology to Arabidopsis thaliana and Oryza sativa genes involved in phosphorus metabolism. This study lays a good foundation for further cloning and verification of the genes involved in phosphorus metabolism in maize. PMID:26125772

  9. 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

  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. Increase in the astaxanthin synthase gene (crtS) dose by in vivo DNA fragment assembly in Xanthophyllomyces dendrorhous

    PubMed Central

    2013-01-01

    Background Xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous. Results In this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced. Conclusions This methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods. PMID:24103677

  12. 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

  13. 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

  14. Successful carnivore identification with faecal DNA across a fragmented Amazonian landscape.

    PubMed

    Michalski, Fernanda; Valdez, Fernanda Pedone; Norris, Darren; Zieminski, Chris; Kashivakura, Cyntia Kayo; Trinca, Cristine S; Smith, Heath B; Vynne, Carly; Wasser, Samuel K; Metzger, Jean Paul; Eizirik, Eduardo

    2011-09-01

    The use of scat surveys to obtain DNA has been well documented in temperate areas, where DNA preservation may be more effective than in tropical forests. Samples obtained in the tropics are often exposed to high humidity, warm temperatures, frequent rain and intense sunlight, all of which can rapidly degrade DNA. Despite these potential problems, we demonstrate successful mtDNA amplification and sequencing for faeces of carnivores collected in tropical conditions and quantify how sample condition and environmental variables influence the success of PCR amplification and species identification. Additionally, the feasibility of genotyping nuclear microsatellites from jaguar (Panthera onca) faeces was investigated. From October 2007 to December 2008, 93 faecal samples were collected in the southern Brazilian Amazon. A total of eight carnivore species was successfully identified from 71% of all samples obtained. Information theoretic analysis revealed that the number of PCR attempts before a successful sequence was an important negative predictor across all three responses (success of species identification, success of species identification from the first sequence and PCR amplification success), whereas the relative importance of the other three predictors (sample condition, season and distance from forest edge) varied between the three responses. Nuclear microsatellite amplification from jaguar faeces had lower success rates (15-44%) compared with those of the mtDNA marker. Our results show that DNA obtained from faecal samples works efficiently for carnivore species identification in the Amazon forest and also shows potential for nuclear DNA analysis, thus providing a valuable tool for genetic, ecological and conservation studies. PMID:21676206

  15. 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

  16. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

    PubMed Central

    Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

    2016-01-01

    Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

  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. Cell-free DNA in maternal plasma: is it all a question of size?

    PubMed

    Li, Ying; Holzgreve, Wolfgang; DI Naro, Edoardo; Vitucci, Angeloantonio; Hahn, Sinuhe

    2006-09-01

    Fetal cell-free DNA (cf-DNA) represents only a small fraction of the total cf-DNA in maternal plasma. This feature has rendered it difficult to reliably distinguish fetal alleles which are not very disparate from maternal ones, such as those involving point mutations, by conventional polymerase chain reaction (PCR)-based approaches. It has recently been shown that cell-free fetal DNA molecules have a smaller size than comparable cf-DNA molecules of maternal origin, and that this feature can be exploited for the selective enrichment of fetal DNA sequences, thereby permitting the detection of otherwise masked fetal genetic traits. By the use of this approach, we have shown that it is possible to detect fetal genetic loci for microsatellite markers, as well as point mutations involved in disorders such as achondroplasia and beta-thalassemia. PMID:17108195

  19. Nucleotide sequence analysis of a cloned DNA fragment from human cells reveals homology to retrotransposons.

    PubMed Central

    Flügel, R M; Maurer, B; Bannert, H; Rethwilm, A; Schnitzler, P; Darai, G

    1987-01-01

    During molecular cloning of proviral DNA of human spumaretrovirus, various recombinant clones were established and analyzed. Blot hybridization revealed that one of the recombinant plasmids had the characteristic features of a member of the long interspersed repetitive sequences family. The DNA element was analyzed by restriction mapping and nucleotide sequencing. It showed a high degree of amino acid sequence homology of 54.3% when compared with the 5'-terminal part of the pol gene product of the murine retrotransposon LIMd. The 3' region of the cloned DNA element encodes proteins with an even higher degree of homology of 67.4% in comparison to the corresponding parts of a member of the primate KpnI sequence family. Images PMID:3031462

  20. Identification of a Renibacterium salmoninarum DNA fragment associated with bacterial internalization into CHSE-cultured cells.

    PubMed

    Maulén, N P; Morales, P J; Aruti, D; Figueroa, J E; Concha, M I; Krauskopf, M; León, G

    1996-01-01

    We report here the isolation of a Renibacterium salmoninarum DNA sequence capable of transforming a non-invasive Escherichia coli strain into a microorganism able to enter the fish cell line, CHSE-214. Immunofluorescence and electron microscopy techniques were used to assess the acquired invasive phenotype by HB101 E. coli cells, upon transformation with pPMV-189. This plasmid carries a 2282-bp R. salmoninarum DNA segment. The invasive phenotype is conserved upon deletion of approximately 1000 bp at the 3' end of the insert. The remaining segment contains an ORF region encoding a putative protein of about 30 kDa. PMID:8598275

  1. Grand-canonical simulation of DNA condensation with two salts, effect of divalent counterion size

    NASA Astrophysics Data System (ADS)

    Nguyen, Toan T.

    2016-02-01

    The problem of DNA- DNA interaction mediated by divalent counterions is studied using a generalized grand-canonical Monte-Carlo simulation for a system of two salts. The effect of the divalent counterion size on the condensation behavior of the DNA bundle is investigated. Experimentally, it is known that multivalent counterions have strong effect on the DNA condensation phenomenon. While tri- and tetra-valent counterions are shown to easily condense free DNA molecules in solution into toroidal bundles, the situation with divalent counterions is not as clear cut. Some divalent counterions like Mg+2 are not able to condense free DNA molecules in solution, while some like Mn+2 can condense them into disorder bundles. In restricted environment such as in two dimensional system or inside viral capsid, Mg+2 can have strong effect and able to condense them, but the condensation varies qualitatively with different system, different coions. It has been suggested that divalent counterions can induce attraction between DNA molecules but the strength of the attraction is not strong enough to condense free DNA in solution. However, if the configuration entropy of DNA is restricted, these attractions are enough to cause appreciable effects. The variations among different divalent salts might be due to the hydration effect of the divalent counterions. In this paper, we try to understand this variation using a very simple parameter, the size of the divalent counterions. We investigate how divalent counterions with different sizes can lead to varying qualitative behavior of DNA condensation in restricted environments. Additionally, a grand canonical Monte-Carlo method for simulation of systems with two different salts is presented in detail.

  2. 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

  3. Short-Fragment DNA Residue from Vaccine Purification Processes Promotes Immune Response to the New Inactivated EV71 Vaccine by Upregulating TLR9 mRNA

    PubMed Central

    Shao, Jie; Gao, Fan; Lin, Hui-Juan; Mao, Qun-Ying; Chen, Pan; Wu, Xing; Yao, Xin; Kong, Wei; Liang, Zheng-Lun

    2016-01-01

    To reduce potential oncogenic long genomic DNA in vaccines, nuclease treatment has been applied in the purification processes. However, this action increased the residue of short-fragment DNA and its effect on vaccine potency was still elusive. In this study, we found residual sf-DNA in an inactivated EV71 vaccine could enhance humoral immune response in mice. Ag stimulation in vitro and vaccine injection in vivo revealed that TLR9 transcription level was elevated, indicating that sf-DNA could activate TLR9. These new findings will help us to understand the molecular mechanism induced by vero-cell culture-derived vaccines. PMID:27082865

  4. Genetic relationships among the members of the family rickettsiaceae as shown by DNA restriction fragment polymorphism analysis.

    PubMed

    Ralph, D; Pretzman, C; Daugherty, N; Poetter, K

    1990-01-01

    The genetic diversity of members of the genus Rickettsia was examined using restriction site polymorphisms found within a series of DNA fragments scattered throughout the genome. Rickettsia belli, R. akari, and R. australis were the most divergent species when compared to the other species examined. These three species were also not closely related to each other. The other examined species were more tightly clustered. This survey also examined the genetic diversity within several species. The unexpected finding of this survey is that several species of rickettsia are as closely related to the surveyed strains of R. rickettsii as these strains are to each other. These results indicate that R. sibirica, R. parkeri, R. rickettsii, and an unnamed isolate from Africa are likely to be strains of a single rickettsial species of worldwide distribution. R. conorii was very closely related to this R. rickettsii-containing group but is likely to remain in a genetically distinct category as the data base expands. PMID:1974127

  5. Absence of SV40 antibodies or DNA fragments in prediagnostic mesothelioma serum samples.

    PubMed

    Kjaerheim, Kristina; Røe, Oluf Dimitri; Waterboer, Tim; Sehr, Peter; Rizk, Raeda; Dai, Hong Yan; Sandeck, Helmut; Larsson, Erik; Andersen, Aage; Boffetta, Paolo; Pawlita, Michael

    2007-06-01

    The rhesus monkey virus Simian Virus 40 (SV40) is a member of the polyomavirus family. It was introduced inadvertently to human populations through contaminated polio vaccine during the years 1956-1963, can induce experimental tumors in animals and transform human cells in culture. SV40 DNA has been identified in mesothelioma and other human tumors in some but not all studies. We tested prediagnostic sera from 49 mesothelioma cases and 147 matched controls for antibodies against the viral capsid protein VP1 and the large T antigen of SV40 and of the closely related human polyomaviruses BK and JC, and for SV40 DNA. Cases and controls were identified among donors to the Janus Serum Bank, which was linked to the Cancer Registry of Norway. Antibodies were analyzed by recently developed multiplex serology based on recombinantly expressed fusions of glutathione-S transferase with viral proteins as antigens combined with fluorescent bead technology. BKV and JCV specific antibodies cross- reactive with SV40 were preabsorbed with the respective VP1 proteins. Sera showing SV40 reactivity after preabsorption with BKV and JCV VP1 were further analyzed in SV40 neutralization assays. SV40 DNA was analyzed by SV40 specific polymerase chain reactions. The odds ratio for being a case when tested positive for SV40 VP1 in the antibody capture assay was 1.5 (95% CI 0.6-3.7) and 2.0 (95% CI 0.6-7.0) when only strongly reactive sera where counted as positive. Although some sera could neutralize SV40, preabsorption with BKV and JCV VP1 showed for all such sera that this neutralizing activity was due to cross-reacting antibodies and did not represent truly SV40-specific antibodies. No viral DNA was found in the sera. No significant association between SV40 antibody response in prediagnostic sera and risk of mesothelioma was seen. PMID:17315193

  6. Identification of a 118-kb DNA fragment containing the locus of blast resistance gene Pi-2(t) in rice.

    PubMed

    Jiang, J; Wang, S

    2002-10-01

    Rice blast disease, caused by the fungal pathogen Pyricularia grisea Sacc., is one of the most devastating crop diseases worldwide. Previous studies have shown that the dominant blast resistance gene Pi-2(t) confers resistance to a broad spectrum of pathogenic strains. Using a population of 292 recombinant inbred lines combined with bioinformatic analysis, we mapped Pi-2(t) between the SSR (simple-sequence repeat) marker SSR140 and the RFLP (restriction fragment length polymorphism) marker JSH12, 0.9 cM from both SSR140 and JSH12. A physical map consisting of six overlapping BAC (bacterial artificial chromosome) clones was anchored to the region containing the Pi-2(t) locus. By analyzing recombination events in this region, the Pi-2(t) locus was localized to a DNA fragment of 118 kb in length. The detailed genetic and physical maps of the Pi-2(t) locus will facilitate both molecular isolation of the gene and marker-assisted transfer of the gene in breeding programs. PMID:12395199

  7. 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

  8. CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

    NASA Astrophysics Data System (ADS)

    Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; 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).

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

    PubMed Central

    Veselý, Pavel; Bureš, Petr; Šmarda, Petr; Pavlíček, Tomáš

    2012-01-01

    Background and Aims Genome size is known to affect various plant traits such as stomatal size, seed mass, and flower or shoot phenology. However, these associations are not well understood for species with very large genomes, which are laregly represented by geophytic plants. No detailed associations are known between DNA base composition and genome size or species ecology. Methods Genome sizes and GC contents were measured in 219 geophytes together with tentative morpho-anatomical and ecological traits. Key Results Increased genome size was associated with earliness of flowering and tendency to grow in humid conditions, and there was a positive correlation between an increase in stomatal size in species with extremely large genomes. Seed mass of geophytes was closely related to their ecology, but not to genomic parameters. Genomic DNA GC content showed a unimodal relationship with genome size but no relationship with species ecology. Conclusions Evolution of genome size in geophytes is closely related to their ecology and phenology and is also associated with remarkable changes in DNA base composition. Although geophytism together with producing larger cells appears to be an advantageous strategy for fast development of an organism in seasonal habitats, the drought sensitivity of large stomata may restrict the occurrence of geophytes with very large genomes to regions not subject to water stress. PMID:22021815

  10. [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

  11. AB112. Detection of human sperm DNA fragmentation by alkaline comet and neutral comet improved by research center for genetics and reproductive health (CGRH)

    PubMed Central

    Mai, Ma Pham Que; An, Nguyen Thi Thuy; Ha, Nguyen Truong Thai; Tram, Nguyen Bao; Bao, Nguyen Hong Quoc; Tuong, Ho Manh

    2015-01-01

    Objective To optimize the specificity and sensitivity of protocols to detect single-strand and double-strand DNA fragmentation of human sperm by using Neutral Comet and Alkaline Comet assay respectively. In both assays, the same conditions of lysis solution and gel electrophoresis were applied. Materials and methods Thirty samples of semen were collected to assess DNA fragmentation. The inclusion criteria were 18-40-year-old men and the exclusion criteria was azoospermia. Results The sample was prepared on lame at the concentration of 1×106 sperm/mL. The duration of cell lysis was successfully decrease to 30 minutes using the optimized solutions of 1.5 M NaCl and 1 mM DTT. After lysing and removing saline solutions, the gel electrophoresis was run at 20 V/70 mA in 10 minutes. The positive control sample was well-prepared by using 2% H2O2 to detect specificity and sensitivity of lysing and electrophoresis. In assay of Alkaline Comet, the sample was covered in alkaline solution (pH>13), 4 °C in 5 minutes after lysing, then moved into gel electrophoresis. The sample was dyed with SYBR and observed the sperm DNA fragmentation under fluorescent microscope. Four hundred of sperms were randomly counted in every sample. The images were well-captured in terms of detection of fragmented and nonfragmented sperms. Conclusions The optimized protocol allowed to detect the single- and double-strand DNA fragmentation in human sperms by only using the same conditions of lysis solutions and gel electrophoresis; moreover, to reduce the duration of lab performance and the cost. The protocols could be easily applied in andrology labs to provide for useful information together with semen analysis (WHO, 2010). This is the first result in Vietnam to detect DNA fragmentation by using Comet assays.

  12. 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

  13. 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

  14. 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

  15. Development of capillary size exclusion chromatography for the analysis of monoclonal antibody fragments extracted from human vitreous humor.

    PubMed

    Rea, Jennifer C; Lou, Yun; Cuzzi, Joel; Hu, Yuhua; de Jong, Isabella; Wang, Yajun Jennifer; Farnan, Dell

    2012-12-28

    Recombinant antigen-binding fragments (Fabs) are currently on the market and in development for the treatment of ophthalmologic indications. Recently, Quality by Design (QbD) initiatives have been implemented that emphasize understanding the relationship between quality attributes of the product and their impact on safety and efficacy. In particular, changes in product quality once the protein is administered to the patient are of particular interest. Knowledge of protein aggregation in vivo is of importance due to the possibility of antibody aggregates eliciting an immunogenic response in the patient. Presently, there are few analytical methods with adequate sensitivity to analyze Fab aggregates in human vitreous humor (HVH) because the Fab amount available for analysis is often quite low. Here, we report the development of a highly sensitive capillary size exclusion chromatography (SEC) methodology for Fab aggregate analysis in HVH. We demonstrate a process to perform capillary SEC to analyze Fabs with picogram sensitivity and an RSD of less than 8% for the relative peak area of high molecular weight species (HMWS). In addition, we have developed a Protein G affinity chromatography method to capture Fabs from HVH for capillary SEC analysis. Recovery efficiencies ranging from 86 to 99% were achieved using this recovery method with 300 μL HVH samples containing Fab1. Finally, we demonstrate the applicability of the methodology by quantifying Fab aggregates in HVH, which can potentially be used for aggregate analysis of clinically relevant samples. PMID:23177154

  16. 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

  17. Historic cycles of fragmentation and expansion in Parnassius smintheus (papilionidae) inferred using mitochondrial DNA.

    PubMed

    DeChaine, Eric G; Martini, Andrew P

    2004-01-01

    Climate oscillations of the Quaternary drove the repeated expansion and contraction of ecosystems. Alpine organisms were probably isolated in sky island refugia during warm interglacials, such as now, and expanded their range by migrating down-slope during glacial periods. We used population genetic and phylogenetic approaches to infer how paleoclimatic events influenced the distribution of genetic variation in the predominantly alpine butterfly Parnassius smintheus. We sequenced a 789 bp region of cytochrome oxidase I for 385 individuals from 20 locations throughout the Rocky Mountains, ranging from southern Colorado to northern Montana. Analyses revealed at lease two centers of diversity in the northern and southern Rocky Mountains and strong population structure. Nested clade analysis suggested that the species experienced repeated cycles of population expansion and fragmentation. The estimated ages of these events, assuming a molecular clock, corresponded with paleoclimatic data on habitat expansion and contraction over the past 400,000 years. We propose that alpine butterflies persisted in an archipelago of isolated sky islands during interglacials and that populations expanded and became more connected during cold glacial periods. An archipelago model implies that the effects of genetic drift and selection varied among populations, depending on their latitude, area, and local environment. Alpine organisms are sensitive indicators of climate change and their history can be used to predict how high-elevation ecosystems might respond to further climate warming. PMID:15058724

  18. DNA Based Micelles: Synthesis, Micellar Properties and Size-dependent Cell Permeability

    PubMed Central

    Liu, Haipeng; Zhu, Zhi; Kang, Huaizhi; Wu, Yanrong; Sefan, Kwame

    2012-01-01

    Functional nanomaterials based on molecular self-assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self-assemble into monodispersed, three dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while remain their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size-dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity, are all excellent features that make our DNA-micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems. PMID:20162643

  19. 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.

  20. 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

  1. 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

  2. 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

  3. Molecular Typing of a Suspected Cluster of Nocardia farcinica Infections by Use of Randomly Amplified Polymorphic DNA, Pulsed-Field Gel Electrophoresis, and Amplified Fragment Length Polymorphism Analyses▿

    PubMed Central

    Kalpoe, J. S.; Templeton, K. E.; Horrevorts, A. M.; Endtz, H. P.; Kuijper, E. J.; Bernards, A. T.; Klaassen, C. H. W.

    2007-01-01

    Randomly amplified polymorphic DNA (RAPD), pulsed-field gelelectrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analyses were used to investigate a possible outbreak of Nocardia farcinica. RAPD and PFGE analyses yielded irreproducible and unsatisfactory results, respectively. AFLP analysis seem to be a promising and welcome addition for molecular analysis of Nocardia isolates. PMID:17913932

  4. 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

  5. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo−, but not by DNA polymerase ζ

    PubMed 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) α, β, ζ, η, ι, κ and REV1. We found that KF exo− and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3′ Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3′ or 5′ Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ. PMID:26491064

  6. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    PubMed

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

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ. PMID:26491064

  7. [Characteristics of DNA adsorption on different sizes red soil colloidal particles].

    PubMed

    Liao, Min; Xie, Xiao-Mei; Fang, Shu; Qiu, Xiao-Bai; Chen, Na; Xu, Ya-Qian; Jiang, Chun-Yan; Chen, Xue-fang

    2013-03-01

    By using balance reaction method, this paper studied the adsorption characteristics and thermodynamic properties of DNA on four kinds of red soil colloids (organic matter-contained coarse clay, organic matter-removed coarse clay, organic matter-contained fine clay, and organic matter-removed fine clay). The DNA adsorption on the four red soil colloids was a process of fast reaction, and the adsorption isotherms were conformed to the Langmuir equation, with the corresponding correlation coefficient (r2) being 0.974, 0. 991, 0. 958, and 0. 975, respectively. The maximum adsorption amount of DNA on the colloidal particles followed the order of organic matter-contained fine clay > organic matter-removed fine clay > organic matter-contained coarse clay > organic matter-removed coarse clay, implying that the size and organic matter content of colloidal particles played an important role in DNA adsorption. Electrolyte concentration and type and adsorption system pH were the main factors affecting the DNA adsorption on the four soil colloids. Within a definite electrolyte concentration range (NaCl < 60 mmol . L-1 and CaCl2 <10 mmol L-1) , the adsorption amount of DNA on the red soil colloids increased significantly with the increase of electrolyte concentration. As compared with sodium ion, calcium ion had a greater promotion effect on the DNA adsorption, but the effect decreased significantly with the increase of adsorption system pH. The DNA adsorption on the organic matter-contained red soil colloids was an endothermic reaction, while the DNA adsorption on the organic matter-removed red soil colloids was an exothermic reaction. The DNA adsorption on the red soil colloids was a process of entropy increase. PMID:23755493

  8. 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

  9. Finite-size effects on long-range correlations: Implications for analyzing DNA sequences

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Simons, M.; Stanley, H. E.

    1993-05-01

    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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Role of increased male age in IVF and egg donation: is sperm DNA fragmentation responsible?

    PubMed

    Humm, Kathryn C; Sakkas, Denny

    2013-01-01

    The well documented increase in age that women conceive their first child has detracted from a similar change observed in males. As both males and females decide to conceive later, the question of whether this may impact their fertility individually and as a couple becomes even more crucial. A paternal age of over 40 years at the time of conception is a frequently quoted male age threshold, however, currently there is no clearly accepted definition of advanced paternal age or even a consensus on the implications of advancing male age. In this paper, we review some of the potential risks to the offspring of advancing male age and examine. The data available regarding pregnancy outcomes based on paternal age in both the fertile and infertile populations. Within the infertile population specifically, we examine the association between male age and outcomes based on treatment modality, including intrauterine insemination (IUI), in vitro fertilization (IVF), and donor oocyte IVF. Finally, we discuss the various mechanisms by which male age may impact sperm and fertility potential, including sperm DNA damage. PMID:23273987

  15. DNA Analysis of Insect Iridescent Virus 6: Evidence for Circular Permutation and Terminal Redundancy

    PubMed Central

    Delius, H.; Darai, G.; Flügel, R. M.

    1984-01-01

    DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 × 106 daltons), duplex DNA circles of a smaller size (140 × 106 daltons) with protruding tails were formed. Images PMID:16789247

  16. Chloroplast DNA phylogeography of a distylous shrub (Palicourea padifolia, Rubiaceae) reveals past fragmentation and demographic expansion in Mexican cloud forests.

    PubMed

    Gutiérrez-Rodríguez, Carla; Ornelas, Juan Francisco; Rodríguez-Gómez, Flor

    2011-12-01

    Several phylogeographic studies in northern Mesoamerica have examined the influence of Pleistocene glaciations on the genetic structure of temperate tree species with their southern limit by the contact zone between species otherwise characteristic of North or South America, but few have featured plant species that presumably colonized northern Mesoamerica from South America. A phylogeographical study of Palicourea padifolia, a fleshy-fruited, bird dispersed distylous shrub, was conducted to investigate genetic variation at two chloroplast regions (trnS-trnG and rpl32-trnL) across cloud forest areas to determine if such patterns are consistent with the presence of Pleistocene refugia and/or with the historical fragmentation of the Mexican cloud forests. We conducted population and spatial genetic analyses as well as phylogenetic and isolation with migration analyses on 122 individuals from 22 populations comprising the distribution of P. padifolia in Mexico to gain insight of the evolutionary history of these populations. Twenty-six haplotypes were identified after sequencing 1389 bp of chloroplast DNA. These haplotypes showed phylogeographic structure (N(ST) = 0.508, G(ST) = 0.337, N(ST) > G(ST), P < 0.05), including a phylogeographic break at the Isthmus of Tehuantepec, with private haplotypes at either side of the isthmus, and a divergence time of the split in the absence of gene flow dating back c. 309,000-103,000 years ago. The patterns of geographic structure found in this study are consistent with past fragmentation and demographic range expansion, supporting the role of the Isthmus of Tehuantepec as a biogeographical barrier in the dispersal of P. padifolia. Our data suggest that P. padifolia populations were isolated throughout glacial cycles by the Isthmus of Tehuantepec, accumulating genetic differences due to the lack of migration across the isthmus in either direction, but the results of our study are not consistent with the existence of the previously proposed Pleistocene refugia for rain forest plant species in the region. PMID:21930221

  17. Nanoscale structure of protamine/DNA complexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Motta, Simona; Brocca, Paola; Del Favero, Elena; Rondelli, Valeria; Cantù, Laura; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2013-02-01

    Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

  18. 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

  19. 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

  20. The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes.

    PubMed

    Pierandrei, Silvia; Luchetti, Andrea; Sanchez, Massimo; Novelli, Giuseppe; Sangiuolo, Federica; Lucarelli, Marco

    2016-01-01

    Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR. PMID:27045208

  1. Evaluation of the ALF DNA sequencer for high-speed sizing of short tandem repeat alleles.

    PubMed

    Decorte, R; Cassiman, J J

    1996-10-01

    DNA typing of short tandem repeat (STR) loci with automated real-time analysis of the fluorescent polymerase chain reaction (PCR) products has given forensic DNA analysis a new dimension. In the present work, the ALF DNA sequencer was evaluated for automated size determination of tetra-nucleotide STRs at high speed. Short gel plates, with a well-to-laser distance of 10 cm, allowed for the analysis of four STR loci (HUMvWF, HUMTH01, D21S11 and HPRT) in one gel lane in less than 75 min. Allele size determination was done with two external allelic ladders for each locus. Lane-to-lane variations were overcome by the inclusion in each lane of two fluorescent PCR products of constant size (123 and 375 bp) that migrated below and above the multiplex of the four STR loci. The accuracy of sizing and allele detection within and between different gels was high (99.89%) for all four STR systems investigated and the gels could be reloaded without a decrease in accuracy of the allele size estimation. This way, the throughput of the system was increased, which is of interest for linkage studies, gene mapping, and population diversity studies. PMID:8957177

  2. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. PMID:26733203

  3. 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.

  4. Aloe-emodin-induced DNA fragmentation in the mouse in vivo comet assay.

    PubMed

    Nesslany, Fabrice; Simar-Meintières, Sophie; Ficheux, Hervé; Marzin, Daniel

    2009-08-01

    Aloe-emodin (AE) and derivatives may be present as undesired components co-extracted during extraction of plants containing anthraquinonic derivatives for preparation of diacetylrhein. AE is a well-known in vitro mutagen, but up to now it failed to induce any clear in vivo genotoxic activity in the chromosome aberration assay in rat bone marrow or the in vivo/in vitro UDS test in liver. However, the two target organs noted during rodent carcinogenicity studies with danthron and emodin, two other well-known anthraquinone derivatives, are the colon and the kidney. Therefore, the choice of the organs for testing the genotoxicity of AE, i.e. bone marrow and liver, may be considered inadequate to demonstrate a possible in vivo genotoxic activity. In this context, the in vivo mouse comet assay was performed on both isolated kidney and colon cells in order to demonstrate a possible organospecific genotoxicity after oral administration of AE. Concurrently, the Ames test and the in vitro micronucleus assay with TK6 human lymphoblastoid cells were performed in their microscale version both with S9 from Aroclor 1254-induced liver or kidney, and without S9. AE induced primary DNA damage in the liver and in the kidney as observed between 3 and 6h after two oral administrations at 500, 1000 and 2000mg/kg bw, underlining an in vivo genotoxic mechanism of action. Furthermore, AE induced a clear genotoxic activity both in the Salmonella typhimurium strains TA1537 and TA98 and in the in vitro micronucleus assay in the absence as well as in the presence of metabolic activation. As no significant variation in the genotoxic activity of AE was noted when using either liver or kidney S9-mix, it seems that no quantitatively and/or qualitatively specific renal metabolism occurs. The kidney may be a target organ of AE as it is the major route of excretion. Under such conditions the separation of AE components should take place and the residual content of undesired AE derivatives should be made as low as reasonably achievable. AE present in plant extracts should be considered as an in vivo genotoxin and this property should be taken into account in the risk assessment for human exposure. PMID:19559101

  5. In situ measurement of the particle size distribution of the fragmentation product of laser-shock-melted aluminum using in-line picosecond holography

    NASA Astrophysics Data System (ADS)

    Li, Ying-Hua; Zhao, Yu; Li, Xue-Mei; Zhang, Zu-Gen; Ye, Xiang-Ping; Zhong, Jie; Cai, Ling-Cang; Zhang, Lin

    2016-02-01

    The dynamic fragmentation of shock-melted metal is a topic of increasing interest in shock physics. However, high-quality experimental studies of the phenomenon are limited, and data that are essential for developing predictive models of the phenomenon, such as the mass and particle sizes distributions, are quite sparse. In-line holography is an effective non-contact technique for measuring particle size distribution, but critical technical requirements, in particular, particle density limits, complicate its application to the subject phenomenon. These challenges have been reasonably overcome in the present study, allowing for successful in situ measurements of the size distribution of the fragmentation product from laser-shock-melted aluminum. In this letter, we report on our experiments and present the measured data.

  6. 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

  7. 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.

  8. 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

  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. 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

  11. 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.

  12. 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.

  13. Evidence on sizes and fragmentation of the nuclei of comet Shoemaker-Levy 9 from Hubble Space Telescope images.

    NASA Astrophysics Data System (ADS)

    Sekanina, Z.

    1995-12-01

    Digital maps of the central regions of 13 nuclear condensations of Comet Shoemaker-Levy 9, obtained with the Wide Field Planetary Camera-2 of the Hubble Space Telescope on January 24-25, March 28-30, and July 4, 1994, were 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, an extensive analysis of the errors involved is presented, and the results are summarized. They include the finding that sizable fragments did 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 regarding the geometric albedo and the phase coefficient, the largest fragments are found to have had effective diameters of ~4km as late as March and even early July 1994. In most condensations, several sizable companions (~1km or more across) have been detected within ~1000km 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, most probably due to effects of solar radiation pressure.

  14. Defined-size DNA triple crossover construct for molecular electronics: modification, positioning and conductance properties

    NASA Astrophysics Data System (ADS)

    Linko, Veikko; Leppiniemi, Jenni; Paasonen, Seppo-Tapio; Hytönen, Vesa P.; Jussi Toppari, J.

    2011-07-01

    We present a novel, defined-size, small and rigid DNA template, a so-called B-A-B complex, based on DNA triple crossover motifs (TX tiles), which can be utilized in molecular scale patterning for nanoelectronics, plasmonics and sensing applications. The feasibility of the designed construct is demonstrated by functionalizing the TX tiles with one biotin-triethylene glycol (TEG) and efficiently decorating them with streptavidin, and furthermore by positioning and anchoring single thiol-modified B-A-B complexes to certain locations on a chip via dielectrophoretic trapping. Finally, we characterize the conductance properties of the non-functionalized construct, first by measuring DC conductivity and second by utilizing AC impedance spectroscopy in order to describe the conductivity mechanism of a single B-A-B complex using a detailed equivalent circuit model. This analysis also reveals further information about the conductivity of DNA structures in general.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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.

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

    PubMed

    Cheuquemán, C; Merino, O; Giojalas, L; Von Baer, A; Sánchez, R; Risopatrón, J

    2013-06-01

    Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR-14⁄PI staining; acrosomal membrane integrity using FITC-conjugated Pisum Sativum Agglutinin⁄PI labelling; mitochondrial membrane potential (Δψm) by staining with JC-1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = -0.41) and with plasma membrane integrity (p = 0.01; r = -0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry. PMID:23082871

  5. Characterization of a Brucella Species 25-Kilobase DNA Fragment Deleted from Brucella abortus Reveals a Large Gene Cluster Related to the Synthesis of a Polysaccharide

    PubMed Central

    Vizcaíno, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernández-Lago, Luis

    2001-01-01

    In the present study we completed the nucleotide sequence of a Brucella melitensis 16M DNA fragment deleted from B. abortus that accounts for 25,064 bp and show that the other Brucella spp. contain the entire 25-kb DNA fragment. Two short direct repeats of four nucleotides, detected in the B. melitensis 16M DNA flanking both sides of the fragment deleted from B. abortus, might have been involved in the deletion formation by a strand slippage mechanism during replication. In addition to omp31, coding for an immunogenic protein located in the Brucella outer membrane, 22 hypothetical genes were identified. Most of the proteins that would be encoded by these genes show significant homology with proteins involved in the biosynthesis of polysacharides from other bacteria, suggesting that they might be involved in the synthesis of a Brucella polysaccharide that would be a heteropolymer synthesized by a Wzy-dependent pathway. This polysaccharide would not be synthesized in B. abortus and would be a polysaccharide not identified until present in the genus Brucella, since all of the known polysaccharides are synthesized in all smooth Brucella species. Discovery of a novel polysaccharide not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes detected in the DNA fragment deleted in B. abortus no longer lead to the synthesis of a polysaccharide must not be excluded. They might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, with some of its members synthesizing extracellular polysaccharides and, as Brucella spp., living in association with eukaryotic cells. PMID:11598046

  6. Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation

    PubMed Central

    2013-01-01

    Background Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation. Methods Semen samples, obtained from forty-four patients (23–30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation. Results Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation. Conclusions Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture. PMID:23958080

  7. 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.

  8. Hydration of nucleic acid fragments: comparison of theory and experiment for high-resolution crystal structures of RNA, DNA, and DNA-drug complexes.

    PubMed Central

    Hummer, G; García, A E; Soumpasis, D M

    1995-01-01

    A computationally efficient method to describe the organization of water around solvated biomolecules is presented. It is based on a statistical mechanical expression for the water-density distribution in terms of particle correlation functions. The method is applied to analyze the hydration of small nucleic acid molecules in the crystal environment, for which high-resolution x-ray crystal structures have been reported. Results for RNA [r(ApU).r(ApU)] and DNA [d(CpG).d(CpG) in Z form and with parallel strand orientation] and for DNA-drug complexes [d(CpG).d(CpG) with the drug proflavine intercalated] are described. A detailed comparison of theoretical and experimental data shows positional agreement for the experimentally observed water sites. The presented method can be used for refinement of the water structure in x-ray crystallography, hydration analysis of nuclear magnetic resonance structures, and theoretical modeling of biological macromolecules such as molecular docking studies. The speed of the computations allows hydration analyses of molecules of almost arbitrary size (tRNA, protein-nucleic acid complexes, etc.) in the crystal environment and in aqueous solution. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 9 FIGURE 12 FIGURE 13 PMID:7542034

  9. Separation of large DNA molecules by size exclusion chromatography-based microchip with on-chip concentration structure

    NASA Astrophysics Data System (ADS)

    Azuma, Naoki; Itoh, Shintaro; Fukuzawa, Kenji; Zhang, Hedong

    2016-06-01

    The separation of DNA molecules according to their size represents a fundamental bioanalytical procedure. Here, we report the development of a chip-sized device, consisting of micrometer-sized fence structures fabricated in a microchannel, for the separation of large DNA molecules (over 10 kbp) based on the principle of size exclusion chromatography (SEC). In order to achieve separation, two approaches were utilized: first, the DNA samples were concentrated immediately prior to separation using nanoslit structures, with the aim of improving the resolution. Second, a theoretical model of SEC-based separation was established and applied in order to predict the optimal voltage range for separation. In this study, we achieved separation of λ DNA (48.5 kbp) and T4 DNA (166 kbp) using the present SEC-based microchip.

  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. Bayes estimation of species divergence times and ancestral population sizes using DNA sequences from multiple loci.

    PubMed Central

    Rannala, Bruce; Yang, Ziheng

    2003-01-01

    The effective population sizes of ancestral as well as modern species are important parameters in models of population genetics and human evolution. The commonly used method for estimating ancestral population sizes, based on counting mismatches between the species tree and the inferred gene trees, is highly biased as it ignores uncertainties in gene tree reconstruction. In this article, we develop a Bayes method for simultaneous estimation of the species divergence times and current and ancestral population sizes. The method uses DNA sequence data from multiple loci and extracts information about conflicts among gene tree topologies and coalescent times to estimate ancestral population sizes. The topology of the species tree is assumed known. A Markov chain Monte Carlo algorithm is implemented to integrate over uncertain gene trees and branch lengths (or coalescence times) at each locus as well as species divergence times. The method can handle any species tree and allows different numbers of sequences at different loci. We apply the method to published noncoding DNA sequences from the human and the great apes. There are strong correlations between posterior estimates of speciation times and ancestral population sizes. With the use of an informative prior for the human-chimpanzee divergence date, the population size of the common ancestor of the two species is estimated to be approximately 20,000, with a 95% credibility interval (8000, 40,000). Our estimates, however, are affected by model assumptions as well as data quality. We suggest that reliable estimates have yet to await more data and more realistic models. PMID:12930768

  12. 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

  13. Intercomparison of DNA sizing ladders in electrophoretic separation matrices and their potential for accurate typing of the D1S8O locus.

    PubMed

    Kline, M C; Redman, J W; Reeder, D J; Duewer, D L

    1996-01-01

    The 100-1,000 basepair size typical of PCR-amplified DNA fragments demands high resolution electrophoretic gels for the adequate characterization of small differences among samples. We have studied the behavior of a number of commercial sizing ladders in three classes of separation systems: polyacrylamides with discontinuous buffer, proprietary acrylamides with continuous buffer, and agarose-like materials with continuous buffer. None of the ladders examined perform adequately in any of these systems using vendor-supplied nominal ladder component basepair sizes. All ladders successfully typed D1S8O alleles after calibration with the allelic ladder (replacing the nominal size values with the least squares estimate of allele/matrix-specific apparent sizes). Some ladders and matrices are qualitatively better than others. No one ladder proved consistently better than others; a polyacrylamide gel with ribose modifier provided the most precise results in this study. Appropriately calibrated electrophoretic apparent sizes must be used for results to be validly exchanged among laboratories. Appropriate allelic ladders or a well defined subset of known alleles can serve as the calibration system. PMID:9072079

  14. Self-assembly of size-controlled liposomes on DNA nanotemplates

    NASA Astrophysics Data System (ADS)

    Yang, Yang; Wang, Jing; Shigematsu, Hideki; Xu, Weiming; Shih, William M.; Rothman, James E.; Lin, Chenxiang

    2016-05-01

    Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision.

  15. Self-assembly of size-controlled liposomes on DNA nanotemplates.

    PubMed

    Yang, Yang; Wang, Jing; Shigematsu, Hideki; Xu, Weiming; Shih, William M; Rothman, James E; Lin, Chenxiang

    2016-05-01

    Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision. PMID:27102682

  16. Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

    PubMed

    Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

    2015-06-01

    Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis 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

  17. 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 restrictio