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. DNA fragment sizing and sorting by laser-induced fluorescence

    DOEpatents

    Hammond, Mark L. (Angier, NC); Jett, James H. (Los Alamos, NM); Keller, Richard A. (Los Alamos, NM); Marrone, Babetta L. (Los Alamos, NM); Martin, John C. (Los Alamos, NM)

    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.

  3. Size fractionation of DNA fragments by liquid-liquid chromatography

    PubMed Central

    Müller, Werner; Schuetz, Hans-Jürgen; Guerrier-Takada, Cecilia; Cole, Patricia E.; Potts, Russell

    1979-01-01

    A methods for the fractionation of double-stranded DNA fragments from 150 to 22000 b.p. in size by liquid-liquid chromatography is described. The procedure makes use of the fact that the partitioning of DNA in a polyethylene glycol-dextran system is size dependent and can be altered by alkali metal cations. Cellulose or celite are used as supports for the stationary, dextran-rich phase. Examples show the fractionation of digests of T7 DNA produced by Dpn II and Hind II restriction endonucleases as well as ? DNA digests produced by Hind III and Eco RI restriction endonucleases. Images PMID:160546

  4. Estimation of DNA fragment size and generation of DNA restriction endonuclease maps using linear models.

    PubMed

    Holford, T R; Brown, S E; Knudson, D L

    1985-02-01

    A method for the estimation of DNA fragment size and for the generation of DNA restriction endonuclease maps using linear models is discussed, and a computer program which utilizes the SAS (SAS Institute Inc., 1982) statistical package is presented. PMID:2984227

  5. 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

  6. Size-selected genomic libraries: the distribution and size-fractionation of restricted genomic DNA fragments by gel electrophoresis.

    PubMed

    Gondo, Y

    1995-02-01

    By using one-dimensional genome scanning, it is possible to directly identify the restricted genomic DNA fragment that reflects the site of genetic change. The subsequent strategies to obtain the molecular clones of the corresponding restriction fragment are usually as follows: (i) the restriction of a mass quantity of an appropriate genomic DNA, (ii) the size-fractionation of the restricted DNA on a preparative electrophoresis gel in order to enrich the corresponding restriction fragment, (iii) the construction of the size-selected libraries from the fractionated genomic DNA, and (iv) the screening of the library to obtain an objective clone which is identified on the analytical genome scanning gel. A knowledge of the size distribution pattern of restriction fragments of the genomic DNA makes it possible to calculate the heterogeneity or complexity of the restriction fragment in each size-fraction. This manuscript first describes the distribution of the restriction fragments with respect to their length. Some examples of the practical application of this theory to genome scanning is then discussed using presumptive genome scanning gels. The way to calculate such DNA complexities in the prepared size-fractionated samples is also demonstrated. Such information should greatly facilitate the design of experimental strategies for the cloning of a certain size of genomic DNA after digestion with restriction enzyme(s) as is the case with genome scanning. PMID:7774556

  7. 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.

  8. 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.

  9. Performance assessment of DNA fragment sizing by high-sensitivity flow cytometry and pulsed-field gel electrophoresis.

    PubMed

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

    2004-05-01

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

  10. Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

    PubMed Central

    Lin, Edward A.; Zhang, Xue-Song; Levine, Steven M.; Gill, Steven R.; Falush, Daniel; Blaser, Martin J.

    2009-01-01

    Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950–1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus. PMID:19282979

  11. Natural transformation of helicobacter pylori involves the integration of short DNA fragments interrupted by gaps of variable size.

    PubMed

    Lin, Edward A; Zhang, Xue-Song; Levine, Steven M; Gill, Steven R; Falush, Daniel; Blaser, Martin J

    2009-03-01

    Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950-1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus. PMID:19282979

  12. 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.

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

    Microsoft Academic Search

    Artem L. Ponomarev; David Brenner; Lynn R. Hlatky; Rainer K. Sachs

    2000-01-01

    DNA double-strand breaks (DSBs) produced by densely ionizing radiation are not located randomly in the genome: recent data\\u000a indicate DSB clustering along chromosomes. Stochastic DSB clustering at large scales, from >100 Mbp down to <0.01 Mbp, is\\u000a modeled using computer simulations and analytic equations. A random-walk, coarse-grained polymer model for chromatin is combined\\u000a with a simple track structure model in

  14. Agarose gel electrophoresis for the separation of DNA fragments.

    PubMed

    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 kb(1). Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits(2). 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 weight(3). 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 along(4). 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 conformation(5); 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: Understand the mechanism by which DNA fragments are separated within a gel matrix Understand how conformation of the DNA molecule will determine its mobility through a gel matrix Identify an agarose solution of appropriate concentration for their needs Prepare an agarose gel for electrophoresis of DNA samples Set up the gel electrophoresis apparatus and power supply Select an appropriate voltage for the separation of DNA fragments Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands Determine the sizes of separated DNA fragments. PMID:22546956

  15. Capillary electrophoresis of DNA restriction fragments: effect of polymer properties.

    PubMed

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

    1997-10-01

    The mechanism of DNA separation by dilute polymer solutions in capillary electrophoresis is not well understood. To provide information on the effect of polymer properties on DNA separations, four polymers that differ in size, shape and stiffness were examined. Hydroxyethylcellulose of high molecular weight provides excellent separation of large DNA fragments (2027 bp - 23,130 bp). Polyvinylpyrrolidone separates DNA from 72 bp to 23 kbp; star-poly(ethylene oxide) and linear poly(ethylene oxide) provide separation of fragments to 1353 bp. PMID:9420158

  16. Glucocorticosteroids induce DNA fragmentation in human lymphoid leukemia cells.

    PubMed

    Distelhorst, C W

    1988-10-01

    The present study was undertaken to investigate the potential role of glucocorticoid-induced DNA damage in the lysis of human lymphoid leukemia cells by glucocorticoids. Lymphoblasts were isolated from patients with acute lymphoblastic leukemia (ALL) or chronic myelogenous leukemia (CML) in blast crisis and cultured in vitro with or without dexamethasone. DNA was then purified from the cells and analyzed by agarose gel electrophoresis. Only high molecular weight (mol wt) DNA was present in cells cultured without dexamethasone, but a ladder of DNA fragments ranging in size from 180 to 200 base pairs (bp) to greater than 1,500 bp was present in cells cultured with dexamethasone. The DNA fragments were multiples of 180 to 200 bp, suggesting an internucleosomal site of DNA cleavage. The same pattern of DNA fragmentation was detected in normal thymocytes isolated from adrenalectomized rats following in vivo treatment with dexamethasone and in S49 mouse thymoma cells after in vitro incubation with dexamethasone. Dexamethasone-induced DNA fragmentation preceded overt loss of viability in glucocorticoid-sensitive cells but was not detected in two variants of the S49 cell line that are glucocorticoid resistant owing to glucocorticoid receptor defects. The results suggest that glucocorticoids kill human lymphoblastic leukemia cells and both normal and malignant murine thymocytes by a common mechanism that involves glucocorticoid induction of an endonucleolytic activity with cleavage of genomic DNA. PMID:3262387

  17. DNA Sequence from Cretaceous Period Bone Fragments

    Microsoft Academic Search

    Scott R. Woodward; Nathan J. Weyand; Mark Bunnell

    1994-01-01

    DNA was extracted from 80-million-year-old bone fragments found in strata of the Upper Cretaceous Blackhawk Formation in the roof of an underground coal mine in eastern Utah. This DNA was used as the template in a polymerase chain reaction that amplified and sequenced a portion of the gene encoding mitochondrial cytochrome b. These sequences differ from all other cytochrome b

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

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. (Univ. of North Carolina, Chapel Hill (USA))

    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.

  19. Bacterial Species Determination from DNA-DNA Hybridization by Using Genome Fragments and DNA Microarrays

    Microsoft Academic Search

    JAE-CHANG CHO; JAMES M. TIEDJE

    2001-01-01

    Whole genomic DNA-DNA hybridization has been a cornerstone of bacterial species determination but is not widely used because it is not easily implemented. We have developed a method based on random genome fragments and DNA microarray technology that overcomes the disadvantages of whole-genome DNA-DNA hybridization. Reference genomes of four fluorescent Pseudomonas species were fragmented, and 60 to 96 genome fragments

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

    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.

  1. 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/?m protons, 123 keV/?m helium-4 ions and ?-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 ?-rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for ?-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 ?-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.

  2. Coagulation, Fragmentation and Growth Processes in a Size Structured Population.

    E-print Network

    Mottram, Nigel

    Coagulation, Fragmentation and Growth Processes in a Size Structured Population. Jacek Banasiak the effects of cell division and aggregration are incorporated by coupling the coagulation- fragmentation in the literature. Key words. semigroups of operators, semilinear Cauchy problem, coagulation, fragmentation, algal

  3. Bacterial natural transformation by highly fragmented and damaged DNA

    E-print Network

    Nielsen, Rasmus

    Bacterial natural transformation by highly fragmented and damaged DNA Søren Overballe-Petersena,1 for review August 14, 2013) DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often

  4. DNA Extraction Strategies for Amplified Fragment Length Polymorphism Analysis

    Microsoft Academic Search

    Catherine Theisen Comey

    ABSTRACT: A polymerase chain reaction-based DNA typing method, amplified fragment length polymorphism (AMP-FLP) analysis, has shown promise as a means of analyzing forensic biological evidence. A variety of DNA extraction methods,were evaluated,for their suitability for AMP-FLP analysis. Factors that were considered in the evaluation included DNA yield, ability of DNA to be amplified, the presence of DNA fragments other than

  5. Internucleosomal DNA fragmentation during deprived and non-deprived olfactory development

    Microsoft Academic Search

    Joseph Najbauer; Xiao-Xin Yan; Michael Leon

    2002-01-01

    DNA fragmentation is a key marker of neuronal death during development, yet little is known about the size, pattern or quantities of fragments generated during normal and sensory-deprived development. Since there are few neurons dying at any particular time, it has not been possible to obtain sufficient quantities of material to make such a determination. By using a highly sensitive

  6. Internucleosomal DNA fragmentation during deprived and non-deprived olfactory development.

    PubMed

    Najbauer, Joseph; Yan, Xiao-Xin; Leon, Michael

    2002-02-01

    DNA fragmentation is a key marker of neuronal death during development, yet little is known about the size, pattern or quantities of fragments generated during normal and sensory-deprived development. Since there are few neurons dying at any particular time, it has not been possible to obtain sufficient quantities of material to make such a determination. By using a highly sensitive Taq polymerase-based technique, we revealed DNA fragments of 180 base pairs and multiples thereof both in bulbs and cortex of young rats (P4-P31). The bulbs subjected to olfactory deprivation at P1 had higher levels of internucleosomal DNA fragmentation at P16 than the contra-lateral, non-deprived bulbs. Interestingly, the DNA fragmentation induced by olfactory deprivation displayed a characteristic internucleosomal fragmentation pattern, suggesting that the cells induced to die may do so by apoptosis. A significant inverse correlation between DNA fragmentation and the natural variation in normal bulb size was found, suggesting that bulb size may be related to cell death. PMID:11814413

  7. DNA fragment assembly using an ant colony system algorithm

    Microsoft Academic Search

    Prakit Meksangsouy; N. Chaiyaratana

    2003-01-01

    This work presents the use of an ant colony system algorithm in a DNA (deoxyribonucleic acid) fragment assembly. The assembly problem is a combinatorial optimisation problem where the aim of the search is to find the right order and orientation of each fragment in the fragment ordering sequence that leads to the formation of a consensus sequence. In this paper,

  8. 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.

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

    Microsoft Academic Search

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

    1996-01-01

    We have developed a fiber optic sensor for rapid and direct analysis of PCR-amplified DNA fragments with minimal sample processing and real-time data readout. To accomplish this, a novel DNA-recognition system was built onto the surface of fused silica fibers. DNA fragments, labeled with a fluorophore during amplification, are bound to and detected at the fiber surface by means of

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

    PubMed Central

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

    1981-01-01

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

  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. Intermediate DNA at low added salt: DNA bubbles slow the diffusion of short DNA fragments

    E-print Network

    Tomislav Vuletic; Sanja Dolanski Babic; Ticijana Ban; Joachim Raedler; Francoise Livolant; Silvia Tomic

    2011-01-05

    We report a study of DNA (150 bp fragments) conformations in very low added salt $DNA concentration range $0.0015\\leq c \\leq 8$~mM (bp). We found an intermediate DNA conformation in the region $0.05 DNA has the diffusion coefficient, $D_p$ reduced below the values for both ssDNA coils and native dsDNA helices of similar polymerization degree $N$. Thus, this DNA population can not be a simple mix of dsDNA and of ssDNA which results from DNA melting. Here, melting occurs due to a reduction in screening concomitant with DNA concentration being reduced, in already very low salt conditions. The intermediate DNA is rationalized through the well known concept of fluctuational openings (DNA bubbles) which we postulate to form in AT-rich portions of the sequence, without the strands coming apart. Within the bubbles, DNA is locally stretched, while the whole molecule remains rod-like due to very low salt environment. Therefore, such intermediate DNA is elongated, in comparison to dsDNA, which accounts for its reduced $D_p$.

  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. Application of automated DNA sizing technology for genotyping microsatellite loci

    Microsoft Academic Search

    J. S. Ziegle; K. P. Corcoran; P. E. Mayrand; L. B. Hoff; L. J. McBride; M. N. Kronick; Ying Su; S. R. Diehl

    1992-01-01

    Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, the authors report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. They capitalize on the availability of three distinct

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

    SciTech Connect

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

    1995-01-25

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

  17. SEARCHING FOR AGROBACTERIAL T-DNA FRAGMENTS IN PLANT GENOMES

    Microsoft Academic Search

    SUMMARY Motivation: The aim of this work was to search for the nucleotide sequences in plant genome data banks similar to agrobacterial T-DNA fragments, in order to evaluate the role of naturally associated soilborne agrobacteria in plant evolution. Results: Depending on the variant and length of the T-DNA right-border fragment, we found from 2 to 115 nucleotide sequences within different

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

    PubMed

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

    2015-01-01

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

  19. Neuronal nuclear DNA fragmentation in the aged canine brain: apoptosis or nuclear DNA fragility?

    PubMed

    Borràs, D; Pumarola, M; Ferrer, I

    2000-04-01

    Neuronal DNA fragmentation, as revealed with the method of in situ end-labeling of nuclear DNA fragmentation (TUNEL), has been reported in both the canine and human brains in normal ageing, and in some human age-related neurodegenerative diseases. These results have suggested that apoptosis plays an important role in age-related neuronal loss. It is not clear, however, whether the TUNEL method is highly specific for apoptosis, as DNA fragmentation also occurs in the late stages o necrosis. In this study we have examined 27 dogs aged from 8 to 18 years, to investigate the occurrence of nuclear DNA fragmentation. An autolysis index based on current histological criteria was assigned to each animal to evaluate the effects of autolysis on nuclear DNA integrity. Our results have shown that neuronal nuclear DNA fragmentation is frequent in aged dogs, although it is not accompanied by apoptotic morphology. Yet, a positive relation between TUNEL labelling and the degree of tissue autolysis was observed. In contrast, no TUNEL labelling was detected in young control dogs despite autolysis indices being similar to those in aged dogs. Taken together, these results suggest that neuronal nuclear DNA fragmentation is an age-related phenomenon, not due to apoptosis, whenever other factors render neuronal DNA more susceptible to autolytic fragmentation. We confirm the effect of autolysis in a subpopulation of neurons in the aged canine brain, inducing nuclear DNA fragmentation. PMID:10787039

  20. 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.

  1. 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.

  2. Size and physical organization of chloroplast DNA from mustard ( Sinapis alba L.)

    Microsoft Academic Search

    Gerhard Link; Susan E. Chambers; John A. Thompson; Heinz Falk

    1981-01-01

    Intact chloroplast (cp)DNA from mustard cotyledons (Sinapis alba L.) was found by electron microscopy to be a uniform population of circular molecules with a contour length corresponding to 158 kilobase pairs. This size was confirmed by restriction endonuclease analysis. Nucleases SalI and XhoI each generate a small number of cpDNA fragments. The sizes of all fragments generated by each enzyme

  3. Multiple displacement amplification for complex mixtures of DNA fragments

    PubMed Central

    Shoaib, Muhammad; Baconnais, Sonia; Mechold, Undine; Le Cam, Eric; Lipinski, Marc; Ogryzko, Vasily

    2008-01-01

    Background A fundamental requirement for genomic studies is the availability of genetic material of good quality and quantity. The desired quantity and quality are often hard to obtain when target DNA is composed of complex mixtures of relatively short DNA fragments. Here, we sought to develop a method to representatively amplify such complex mixtures by converting them to long linear and circular concatamers, from minute amounts of starting material, followed by phi29-based multiple displacement amplification. Results We report here proportional amplification of DNA fragments that were first converted into concatamers starting from DNA amounts as low as 1 pg. Religations at low concentration (< 1 ng/?L) preferentially lead to fragment self-circularization, which are then amplified independently, and result in non-uniform amplification. To circumvent this problem, an additional (stuffer) DNA was added during religation (religation concentration > 10 ng/?L), which helped in the formation of long concatamers and hence resulted in uniform amplification. To confirm its usefulness in research, DP1 bound chromatin was isolated through ChIP and presence of DHFR promoter was detected using q-PCR and compared with an irrelevant GAPDH promoter. The results clearly indicated that when ChIP material was religated in presence of stuffer DNA (improved MDA), it allowed to recover the original pattern, while standard MDA and MDA without stuffer DNA failed to do so. Conclusion We believe that this method allows for generation of abundant amounts of good quality genetic material from a complex mixture of short DNA fragments, which can be further used in high throughput genetic analysis. PMID:18793430

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

    PubMed Central

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

    2015-01-01

    In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method 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. Calcium-activated DNA fragmentation kills immature thymocytes

    Microsoft Academic Search

    DAVID J. MCCONKEY; PIA HARTZELL; PIERLUIGI NICOTERA; STEN ORRENIUS

    Glucocorticoid hormones kill immature thymocytes by activating a self-destructive process that involves exten- sive DNA fragmentation. It has been demonstrated that thymocyte suicide is dependent on an early, sustained increase in cytosolic Ca2 concentration, and new protein synthesis, but the biochemical lesion that leads to cell death has not been established. To determine whether endonuclease activation or activation of another

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

    SciTech Connect

    Signor, L.; Roy, G.; Llorca, F. [Commissariat a l'Energie Atomique-Centre de Valduc-21120 Is-sur-Tille (France); Resseguier, T. de [LCD - UPR 9028, ENSMA, 1 ave. Clement Ader, 86961 Futuroscope Cedex (France); Dragon, A. [LMPM - UMR 6617, ENSMA, 1 ave. Clement Ader, 86961 Futuroscope Cedex (France)

    2007-12-12

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

  7. Alternative Okazaki Fragment Ligation Pathway by DNA Ligase III

    PubMed Central

    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

  8. 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

  9. Influence of char fragmentation on ash particle size distributions

    Microsoft Academic Search

    J. J. Helble; A. F. Sarofim

    1989-01-01

    The influence of char fragmentation on the size distribution of combustion-generated ash has been investigated in a detailed laboratory study utilizing both coals and synthetic chars. Combustion of three different coal types resulted in production of ash particles in the 1-10-..mu..m size range at all conditions considered (1500 and 1750 K, 8% Oâ and above). The amount of ash in

  10. Plasmid vectors capable of transferring large DNA fragments to yeast.

    PubMed

    Morris, D W; Noti, J D; Osborne, F A; Szalay, A A

    1981-01-01

    We have constructed several cloning vectors which can be used in vitro packaging and yeast transformation. These plasmids have been designed for the convenient cloning of large segments of DNA and their transfer to yeast. They contain bacterial plasmid DNA sequences for replication and selection in Escherichia coli, yeast 2-microns plasmid DNA sequences or chromosomal replicators and yeast markers necessary for replication and selection in yeast, and the cohesive ends of bacteriophage lambda which allow packaging of recombinant molecules into lambda phage heads. Large fragments (22-38 kb) of Klebsiella pneumoniae and Zea mays DNA were ligated into plasmid vector pBTI-1 to make complete genome libraries. One clone from the K. pneumoniae library was amplified in E. coli and the purified DNA used to transform yeast cells. Transformation of yeast by large DNA fragments occurred at high frequencies. The recombinant plasmid was stably maintained in yeast, provided selective pressure for Leu+ transformants was maintained. The structurally complete recombinant plasmid can be recovered from yeast by transforming E. coli to ampicillin resistance. Fewer than 5% of the recovered plasmids had undergone recombination with endogenous yeast 2-microns plasmid. PMID:6299664

  11. 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. [Laboratoire de Physique Nucleaire, Departement de Physique, Universite Laval, Sainte-Foy, Quebec, G1K 7P4 (CANADA)] [Laboratoire de Physique Nucleaire, Departement de Physique, Universite Laval, Sainte-Foy, Quebec, G1K 7P4 (CANADA); [AECL, Chalk River Laboratories, Chalk River, Ontario, K0J 1J0 (CANADA); [Institut de Physique Nucleaire de Lyon, 46 Bd du 11 Novembre 1918, F-69622, Villeurbanne Cedex (France); [Department of Physics, McGill University, 3600 University St., Montreal, Quebec, H3A 2T8 (CANADA)

    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.}

  12. Source size scaling of fragment production in projectile breakup

    E-print Network

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

    1996-07-15

    Fragment production has been studied as a function of the source mass and excitation energy in peripheral collisions of $^{35}$Cl+$^{197}$Au at 43 MeV/nucleon and $^{70}$Ge+$^{nat}$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 $A_{source} \\sim$ 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.

  13. DNA fragment conformations in adducts with Kiteplatin.

    PubMed

    Margiotta, Nicola; Petruzzella, Emanuele; Platts, James A; Mutter, Shaun T; Deeth, Robert J; Ranaldo, Rosa; Papadia, Paride; Marzilli, Patricia A; Marzilli, Luigi G; Hoeschele, James D; Natile, Giovanni

    2015-02-28

    The anticancer activity of cisplatin is triggered by its formation of intrastrand adducts involving adjacent G residues of DNA. To obtain information on the different conformers that can be formed, carrier ligands such as 2,2'-bipiperidine, which provide large steric bulk near the platinum coordination plane and decrease the dynamic motion about the Pt-N7 bonds, were introduced ("retro-modelling" approach). In the present study we investigate the effect of cis-1,4-diaminocyclohexane (cis-1,4-DACH) on the formation, stability, and stereochemistry of (cis-1,4-DACH)Pt(ss-oligo) adducts (ss-oligo = d(GpG) with 3'- and/or 5'-substituents). Interesting features of this ligand, absent in previous retro-modelling studies, include the large bite angle (expected to impede the ease of interconversion between possible conformers), the presence of two protons on each nitrogen (a characteristic associated with antitumor activity), and the absence of chiral centres. The use of cis-1,4-DACH has made it possible to detect different conformers in a system containing a primary diamine carrier ligand associated with anticancer activity and to confirm the previous hypothesis that the coexistence of different conformers established in studies of retro models having relatively bulky ligands is not an artefact resulting from carrier-ligand bulk. Moreover, the data for the (cis-1,4-DACH)Pt(d(GpG)) and (cis-1,4-DACH)Pt(d(GGTTT)) adducts indicate that at a temperature close to the physiological one (40 °C) HH1 and ?HT1 conformers are present in comparable amounts. In contrast, at low temperature (close to 0 °C) the equilibrium shifts dramatically toward the more stable HH1 conformer (for the (cis-1,4-DACH)Pt(d(TGGT)) adduct the HH1 conformer is always dominant, even at high temperature). Notably, (cis-1,4-DACH)PtCl2 (Kiteplatin) has been recently reinvestigated and found to be particularly active against colorectal cancer (including oxaliplatin-resistant phenotypes). PMID:25144401

  14. 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].

  15. Controlling DNA fragmentation in MALDI-MS by chemical modification

    SciTech Connect

    Tang, W.; Zhu, L.; Smith, L.M. [Univ. of Wisconsin, Madison, WI (United States)] [Univ. of Wisconsin, Madison, WI (United States)

    1997-02-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  17. Large-scale production of palindrome DNA fragments

    SciTech Connect

    Palmer, E.L.; Gewiess, A.; Harp, J.M. [Oak Ridge National Lab., TN (United States)] [and others] [Oak Ridge National Lab., TN (United States); and others

    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.

  18. Application of automated DNA sizing technology for genotyping microsatellite loci

    SciTech Connect

    Ziegle, J.S.; Corcoran, K.P.; Mayrand, P.E.; Hoff, L.B.; McBride, L.J.; Kronick, M.N. (Applied Biosystems, Foster City, CA (United States)); Su, Ying; Diehl, S.R. (Virginia Commonwealth Univ., Richmond, VA (United States))

    1992-12-01

    Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, the authors report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. They capitalize on the availability of three distinct fluorescent dyes to uniquely label loci that overlap in size. This innovation increases by threefold the number of loci that can be analyzed simultaneously. They label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy. 29 refs., 3 figs.

  19. 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.

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

  1. 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

  2. Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

    PubMed Central

    Vlassov, V V; Zarytova, V F; Kutiavin, I V; Mamaev, S V; Podyminogin, M A

    1986-01-01

    A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment. Images PMID:3714471

  3. Does varicocelectomy affect DNA fragmentation in infertile patients?

    PubMed Central

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

    2015-01-01

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

  4. Ca2 antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen

    Microsoft Academic Search

    SIDHARTHA D. RAY; LISA M KAMENDULIS; MARK W. GURULE; ROBERT D. YORKIN; GEORGE B. CORCORAN

    1993-01-01

    Ca2 accumulates in the nucleus and DNA undergoes enzymatic cleavage into internucleosome- length fragments before acetaminophen and dimethyl- nitrosamine produce hepatic necrosis in vivo and toxic cell death in vitro. However, Ca2-endonudease fragmen- tation of DNA is characteristic of apoptosis, a type of cell death considered biochemically and functionally distinct from toxic cell death. The present studies investigate DNA fragmentation

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

    Microsoft Academic Search

    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

  6. DNA fragment assembly: an application of graph theory in molecular biology

    E-print Network

    Willems, Wolfgang

    DNA fragment assembly: an application of graph theory in molecular biology Martin Mascher Leibniz Technology Since the central importance of the DNA in storing biological informa- tion had been recognised limitations permit scientists only to obtain contigu- ous DNA fragments whose lengths range from a few dozen

  7. Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

    Microsoft Academic Search

    Zhi-Hong Xian; Wen-Ming Cong; Shu-Hui Zhang; Meng-Chao Wu

    2005-01-01

    Abstract Abstract Abstract Abstract Abstract AIM:T o study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS:DNA isolated from tumors and corresponding

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

    DOEpatents

    Wong, Kwong-Kwok (Richland, WA)

    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.

  9. Differentiation ofCampylobacter jejuniandCampylobacter coli Strains by Using Restriction Endonuclease DNA Profiles and DNA Fragment Polymorphisms

    Microsoft Academic Search

    VICTORIA KOROLIK; LISA MOORTHY; ANDPETER J. COLOE

    The chromosomal DNA fragment patterns from a total of 169 Campylobacter jejuni and Campylobacter coli isolatesfrompoultryandhumanswereanalyzedbyusingDNArestrictionendonucleasesClaIandEcoRV.The DNA restriction patterns produced byClaI andEcoRV consisted of unique DNA fragments of 9 to 9.5 kb and 3.5 kb generated with ClaI and a single unique fragment of 3.0 kb produced by EcoRV. These patterns were obtained with all strains ofC. jejunitested. The DNA

  10. Electric linear dichroism transients of bent DNA fragments.

    PubMed

    Umazano, Juan P; Bertolotto, Jorge A

    2013-03-01

    We study the effect of translational-rotational hydrodynamic coupling on the transient electric linear dichroism of DNA fragments in aqueous solution. As opposed to previous theoretical works, where analytic solutions valid in the limit of low electric field were reported, we present here a numerical approach which allows to obtain numerical results valid independently from the applied electric field strength. Numerical procedures here used are an extension to the transient-state of those developed in a previous work for the study of the problem in the steady-state. The molecular orientational processes induced by an electric field is characterized with statistical arguments solving the Fokker-Planck equation by means of the finite difference method to know the orientational distribution function of molecules. PMID:23485329

  11. Inhibition of Apoptosis-Associated DNA Fragmentation Activity in Nonapoptotic Cells: The Role of DNA Fragmentation Factor45 (DFF45\\/ICAD)

    Microsoft Academic Search

    Steven L. Sabol; Renee Li; Tamara Y. Lee; Rolla Abdul-Khalek

    1998-01-01

    We have investigated the mechanism whereby nuclear DNA fragmentation activity emerging during early apoptosis is inhibited during normal cell life. In a cell-free system, cytosol fractions from diverse nonapoptotic human cell lines (Jurkat T-cell leukemia, HeLa carcinoma, SK-N-MC neuroblastoma, and WI-38 embryonic lung fibroblast) potently neutralized the nuclear DNA fragmentation activity of cytosol from apoptotic anti-Fas treated Jurkat cells. Recombinant

  12. 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.

  13. Late, but not early, paternal effect on human embryo development is related to sperm DNA fragmentation

    Microsoft Academic Search

    Jan Tesarik; Ermanno Greco; Carmen Mendoza

    BACKGROUND: It is known that repeated failure of assisted reproduction treatment (ART) can be due to a paternal effect. This study was undertaken to analyse the possible relationship between ART failure and sperm DNA fragmentation. METHODS: Zygote morphology and the percentage of spermatozoa with fragmented DNA (assessed by TUNEL) were compared in two groups using donor oocytes for ICSI attempts.

  14. 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).

  15. Photochemistry of DNA fragments via semiclassical nonadiabatic dynamics.

    PubMed

    Alexandrova, Anastassia N; Tully, John C; Granucci, Giovanni

    2010-09-23

    Forming upon absorption of a UV photon, excited states of DNA are subject to nonadiabatic evolution, via either internal conversion (IC) back to the ground state or mutagenesis. Nonadiabatic processes following the formation of the first singlet excited states, S1, in 10 different small DNA fragments--4 single 4'H-nucleosides, 2 Watson-Crick base pairs, and 4 nucleotide quartets--have been investigated. Simulations were done via the nonadiabatic direct trajectory surface hopping semiclassical dynamics. The electronic wave function was obtained with configuration interaction, based on the semiempirical AM1 and PM3 Hamiltonians with fractional orbital occupation numbers. The evolution of the electronic wave function was governed by the time-dependent Schrödinger equation with a locally diabatic representation, intrinsically stable near surface crossings. The nuclei evolved on adiabatic potential energy surfaces, as prescribed by classical Newtonian dynamics, with sudden hops between potential energy surfaces to account for nonadiabatic transitions. The "fewest switches" surface hopping algorithm coupled the quantum and classical parts of the system. The dynamics simulations revealed several routes of nonadiabatic relaxation in these systems, which were not reported previously, and also recovered known routes of IC. PMID:20795696

  16. 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

  17. Effects of population size on genetic diversity, fitness and pollinator community composition in fragmented populations of Anthericum liliago L

    Microsoft Academic Search

    Angela Peterson; Igor V. Bartish; Jens Peterson

    2008-01-01

    The genetic and fitness consequences of habitat fragmentation on the dry grassland species Anthericum liliago L. (Anthericaceae) were examined. We used random amplified polymorphic DNA (RAPD) markers to determine the distribution of\\u000a genetic diversity within and among 10 German A. liliago populations, ranging in size from 116 to over 2 million ramets. The genetic diversity of an A. liliago population was highly

  18. cDNA selection: efficient PCR approach for the selection of cDNAs encoded in large chromosomal DNA fragments.

    PubMed Central

    Parimoo, S; Patanjali, S R; Shukla, H; Chaplin, D D; Weissman, S M

    1991-01-01

    Identification of coding segments in large fragments of genomic DNA is a recurrent problem in genome mapping and positional cloning studies. We have developed a rapid and efficient protocol to achieve this goal, based on hybridization of cDNA fragments to immobilized DNA and recovery of the selected cDNAs by the PCR. The procedure permits rapid cloning of cDNA fragments encoded by large genomic DNA fragments, groups of yeast artificial chromosomes, or cosmids and has the potential to directly enrich cDNAs encoded in chromosome segments. By this approach we have been able to identify several non-major histocompatibility complex class I clones from a yeast artificial chromosome that includes the HLA-A locus. Images PMID:1946377

  19. Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids.

    PubMed

    Huang, Ming-Feng; Huang, Chih-Ching; Chang, Huan-Tsung

    2003-09-01

    The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems. PMID:12973792

  20. Computer Simulation of Packing of Particles with Size Distributions Produced by Fragmentation Processes

    NASA Astrophysics Data System (ADS)

    Martín, Miguel Angel; Muñoz, Francisco J.; Reyes, Miguel; Taguas, F. Javier

    2015-01-01

    Fragmentation schemes inspired by theoretical results and conjectures of Kolmogorov are applied to produce particle size distributions of different natures, depending on fragmentation parameters. A two-dimensional computer simulation method of packing is applied to the resulting distributions and the void fraction is evaluated. The relationship between the void fraction and characteristic parameters of the fragmentation process is studied.

  1. DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

    Microsoft Academic Search

    Rui Yan; Mark Ottenbreit; Bharati Hukku; Michael Mally; Sharong Chou; Joseph Kaplan

    1996-01-01

    Summary  Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme\\u000a phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used\\u000a restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method\\u000a of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms.

  2. RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.

    PubMed

    Lin, Qiuhui; Jia, Bin; Mitchell, Leslie A; Luo, Jingchuan; Yang, Kun; Zeller, Karen I; Zhang, Wenqian; Xu, Zhuwei; Stracquadanio, Giovanni; Bader, Joel S; Boeke, Jef D; Yuan, Ying-Jin

    2015-03-20

    We describe rapid assembly of DNA overlapping multifragments (RADOM), an improved assembly method via homologous recombination in Saccharomyces cerevisiae, which combines assembly in yeasto with blue/white screening in Escherichia coli. We show that RADOM can successfully assemble ?3 and ?10 kb DNA fragments that are highly similar to the yeast genome rapidly and accurately. This method was tested in the Build-A-Genome course by undergraduate students, where 125 ?3 kb "minichunks" from the synthetic yeast genome project Sc2.0 were assembled. Here, 122 out of 125 minichunks achieved insertions with correct sizes, and 102 minichunks were sequenced verified. As this method reduces the time-consuming and labor-intensive efforts of yeast assembly by improving the screening efficiency for correct assemblies, it may find routine applications in the construction of DNA fragments, especially in hierarchical assembly projects. PMID:24895839

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    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

    2014-12-01

    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

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

    NASA Astrophysics Data System (ADS)

    O'Keefe, J. D.; Ahrens, T. J.

    1985-05-01

    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.

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

    PubMed Central

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

    2009-01-01

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

  7. A mini-library of sequenced human DNA fragments: linking bench experiments with informatics

    Microsoft Academic Search

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

    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 skill commonly taught to undergraduate biology students. The individual procedures

  8. Characterization of pearl millet mitochondrial DNA fragments rearranged by reversion from cytoplasmic male sterility to fertility

    Microsoft Academic Search

    R. L. Smith; M. K. U. Chowdhury

    1991-01-01

    Cloned pearl millet [Pennisetum glaucum (L.) R. Br.] mitochondrial (mt) DNA fragments rearranged by spontaneous reversion from cytoplasmic male sterility (cms) to fertility were characterized by restriction mapping, hybridization with maize mt genes, and transcription analyses. The clones characterized were a 4.7-kb fragment found only in the male-sterile cytoplasm and lost upon reversion to fertility, a 10.9-kb fragment found in

  9. NEST SURVIVAL RELATIVE TO PATCH SIZE IN A HIGHLY FRAGMENTED SHORTGRASS PRAIRIE LANDSCAPE

    Microsoft Academic Search

    SUSAN K. SKAGEN; AMY A. YACKEL ADAMS; ROD D. ADAMS

    2005-01-01

    Understanding the influences of habitat fragmentation on vertebrate populations is essential for the protection and ecological restoration of strategic sites for native species. We examined the effects of prairie fragmentation on avian reproductive success using artificial and natural nests on 26 randomly selected, privately owned patches of shortgrass prairie ranging in size from 7 to 454 ha within a cropland

  10. DNA Fragmentation and DSB correlation Induced in Human Fibroblasts by Accelerated 56Fe Ions of Differing Energies

    NASA Astrophysics Data System (ADS)

    Antonelli, F.; Belli, M.; Campa, A.; Dini, V.; Esposito, G.; Furusawa, Y.; Simone, G.; Sorrentino, E.; Tabocchini, M. A.

    HZE particles from space radiation raise an important protection concern during long-term astronauts travels Although these particles are less abundant than protons they are more effective in damaging biological systems It is thought that this is due to the frequent production of spatially correlated DNA damaged sites particularly double strand breaks DSB since this correlation can strongly affect the repair capability of the cells In this work we have studied the DNA fragmentation induced in human fibroblasts by accelerated 56 Fe ions of four different energies i e 115 MeV u 414 MeV u 1 GeV u and 5 GeV u and by gamma-rays used as reference radiation DNA fragmentation was studied in various size ranges varying from 1 to 5700 kbp using Pulsed or Constant Field Gel Electrophoresis The DSB yields have been derived from fragmentation in the overall range as well as in the two ranges 1-23 and 23-5700 kbp The overall DSB yield slightly increased with the ion energy maily due to the contribution of the 23-5700 kbp fragments while that of small fragments 1-23 kbp was almost constant Accordingly the relative biological effectiveness RBE for DSB induction increased with energy from about 1 3 at 115 MeV u to about 1 8 at about 5 GeV u i e less than the RBE for chromosome aberration and cell inactivation The degree of spatial correlation of DSB was evaluated through the departure from the randomness of the fragment distribution with a simple theoretical tool that we have recently introduced To this aim a parameter R was used

  11. 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...

  12. DNA fragmentation and BCL2 expression in infantile spinal muscular atrophy

    Microsoft Academic Search

    D. S. Tews; H. H. Goebel

    1996-01-01

    Chromatin cleavage, a hallmark of apoptosis, was identified by in situ labeling in 55 ± 7% of the muscle fibers in infantile spinal muscular atrophy (ISMA) and, to a lesser extent, in peripheral neuropathy indicating that DNA fragmentation is not specific to ISMA but a common feature in defect innervation. However, as DNA breaks are also known as a temporary

  13. Statistical Exploration of Fragmentation Phase Space Source Sizes in Nuclear Multifragmentation

    E-print Network

    L. G. Moretto; L. Beaulieu; L. Phair; G. J. Wozniak

    2000-02-15

    The multiplicity distributions for individual fragment Z values in nuclear multifragmentation are binomial. The extracted maximum value of the multiplicity is found to depend on Z according to m=Z_0/Z, where Z_0 is the source size. This is shown to be a strong indication of statistical coverage of fragmentation phase space. The inferred source sizes coincide with those extracted from the analysis of fixed multiplicity charge distributions.

  14. Absence of cinoxacin-induced DNA fragmentation and mutations in the rat granuloma pouch.

    PubMed

    Pino, A; Maura, A; Grillo, P

    1989-01-01

    The mutagenic and DNA-damaging activities of cinoxacin were evaluated in the rat granuloma pouch assay (GPA) in order to assess its genotoxic potential in vivo. High doses of this antimicrobial quinolone, either directly injected into the pouch or administered by gavage, did not induce mutation at the hgprt locus or DNA fragmentation in granuloma cells. Moreover, DNA damage was absent in the liver and kidney of rats given cinoxacin by the oral route. PMID:2917551

  15. Restriction fragment polymorphisms in the rDNA region among seven species of Alnus and Betula papyrifera

    Microsoft Academic Search

    Jean Bousquet; Eric Girouard; Curtis Strobeck; Bruce P. Dancik; Maurice Lalonde

    1989-01-01

    A study of restriction fragment polymorphisms of ribosomal DNA among seven actinorhizal species (Alnus spp.) and a non-actinorhizal species (Betula papyrifera Marsh.) of the Betulaceae was conducted, using a simple method for the extraction of high molecular weight restrictable nuclear DNA from leaf tissues of perennial angiosperms and nine restriction endonucleases. rDNA restriction fragments were variable within and among the

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

    PubMed Central

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

    1986-01-01

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

  17. Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA.

    PubMed

    Ito, M; Yamamoto, T; Watanabe, M; Ihara, T; Kamiya, H; Sakurai, M

    1996-09-01

    Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway. PMID:8880136

  18. BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium

    Microsoft Academic Search

    J. Song; J. M. Bradeen; S. K. Naess; J. P. Helgeson; J. Jiang

    2003-01-01

    Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes. The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation. Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium.

  19. Sperm DNA fragmentation and its role in wildlife conservation.

    PubMed

    Gosálvez, Jaime; Holt, William V; Johnston, Stephen D

    2014-01-01

    Until about 20 years ago, sperm assessment in the laboratory was focused on motility, morphology and acrosomal integrity. Then came the gradual realisation that, because the main objective of a spermatozoon is to deliver an intact genetic payload of DNA to the egg, being able to check DNA quality of spermatozoa would be equally important, if not more so. Research over the last two decades has therefore led to the development of several techniques for reliably detecting DNA strand breaks, and the more recent focus has been directed towards understanding the fertility implications of DNA damage. It is now clear that evolutionary history has played an important role in determining the stability of sperm DNA under stressful conditions, and that the nature of the DNA-protein interactions also influence the extent to which fertility is affected by both technical procedures involved in sperm preservation and the basic biology of the species concerned. Here we present an overview of the principles involved in DNA assessment and also provide some cases studies that illustrate the influences of species diversity. PMID:25091917

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

    SciTech Connect

    Chakraborty, R.; Srinivasan, M.R.; Andrade, M. de (Univ. of Texas Graduate School of Biomedical Sciences, Houston (United States))

    1993-02-01

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

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

    PubMed Central

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

    2014-01-01

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

  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. Phylogenetic relationships of turfgrasses as revealed by restriction fragment analysis of chloroplast DNA.

    PubMed

    Yaneshita, M; Ohmura, T; Sasakuma, T; Ogihara, Y

    1993-10-01

    Chloroplast DNAs (cpDNAs) were analyzed in order to clarify the phylogenetic relationships among turfgrasses. Physical maps of cpDNAs from Agrostis stolonifera and Zoysia japonica, which are representative species of cool (C3 type) and warm (C4 type) season turfgrasses, respectively, were constructed with four restriction enzymes, i.e., PstI, SalI, SacI, and XhoI. The genome structures of these cpDNAs were found to be similar to each other in terms of genome size and gene orders, showing thereby a similarity to other grass cpDNAs. CpDNAs of 5 species of cool season turfgrasses and 6 species of warm season turfgrasses as well as four species of cereals, distributed among 14 genera of Gramineae, were digested with PstI, XhoI, and BamHI, and their restriction fragment patterns were compared. Their genome sizes were estimated to be 135-140 kbp. Each species showed characteristic RFLP patterns. On the basis of the frequency of commonly shared fragments, a dendrogram showing the phylogenetic relationships among their cpDNAs was constructed. This dendrogram shows that turfgrasses can be divided into three major groups; these correspond to the subfamilies. Cool and warm season turfgrasses are clearly distinguishable from each other, and the latter can be further classified into two subgroups that correspond to Eragrostoideae and Panicoideae. Our classification of turfgrasses and cereals by RFLP analysis of cpDNA agreed in principal with their conventional taxonomy, except for the location of Festuca and Lolium. PMID:24190204

  4. Estimating Dataset Size Requirements for Classifying DNA Microarray Data

    E-print Network

    Poggio, Tomaso

    Estimating Dataset Size Requirements for Classifying DNA Microarray Data S. Mukherjee*+#1 , P methodology for estimating dataset size requirements for classifying microarray data using learning curves is introduced. The goal is to use existing classification results to estimate dataset size requirements

  5. Electronic and molecular structure of M-DNA fragments

    Microsoft Academic Search

    Yury V. Rubin; Leonid F. Belous; ?natolij ?. Yakuba

    2011-01-01

    To study M-DNA molecular structure (such DNA with transition metal ions placed between the nucleic bases is able to conduct\\u000a the electric current) and its conductivity mechanisms, we carried out ab initio quantum-mechanical calculations of electronic and spatial structures, thermodynamic characteristics of adenine-thymine (??)\\u000a and guanine-cytosine (GC) base pair complexes with Zn2+ and Ni2+. To take into account the influence

  6. 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-Döring coagulation-fragmentation equations. Our modeling perspective allows for a description of cluster growth in realistic systems by using a significantly smaller number of differential equations to describe their dynamics. We used dynamical system tools (phase-plane analysis) and numerical simulations to investigate the CSG model dynamics and to understand how the model parameters describing the coagulation and fragmentation processes determine balances between these two processes that create non-zero stationary cluster size distributions. PMID:26071695

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

    PubMed

    da Silva, Lucas Goulart; Ribeiro, Milton Cezar; Hasui, 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. Monte Carlo evaluation of DNA fragmentation spectra induced by different radiation qualities.

    PubMed

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

    2011-02-01

    The PARTRAC code has been developed constantly in the last several years. It is a Monte Carlo code based on an event-by-event description of the interactions taking place between the ionising radiation and liquid water, and in the present version simulates the transport of photons, electrons, protons, helium and heavier ions. This is combined with an atom-by-atom representation of the biological target, i.e. the DNA target model of a diploid human fibroblast in its interphase (genome of 6 Gigabase pairs). DNA damage is produced by the events of energy depositions, either directly, if they occur in the volume occupied by the sugar-phosphate backbone, or indirectly, if this volume is reached by radiation-induced radicals. This requires the determination of the probabilities of occurrence of DNA damage. Experimental data are essential for this determination. However, after the adjustment of the relevant parameters through the comparison of the simulation data with the DNA fragmentation induced by photon irradiation, the code has been used without further parameter adjustments, and the comparison with the fragmentation induced by charged particle beams has validated the code. In this paper, the results obtained for the DNA fragmentation induced by gamma rays and by charged particle beams of various LET are shown, with a particular attention to the production of very small fragments that are not detected in experiments. PMID:21084331

  11. Size heterogeneity of Salmonella typhimurium lipopolysaccharides in outer membranes and culture supernatant membrane fragments.

    PubMed Central

    Munford, R S; Hall, C L; Rick, P D

    1980-01-01

    Enterobacteriaceae cells growing in liquid media shed fragments of their outer membranes. These fragments, which may constitute a biologically important form of gram-negative bacterial endotoxin, have been reported to contain proteins, phospholipids, and lipopolysaccharides (LPS). In this study we compared the sizes of LPS molecules in shed membrane fragments and outer membranes from cells growing in broth cultures. Using conditional mutants of Salmonella typhimurium which incorporate specific sugars into LPS, we analyzed radiolabeled LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique revealed that S. typhimurium LPS are more heterogeneous than previously known; molecules possessing from 0 to more than 30 O-chain repeat units were identified in outer membranes, supernatant fragments, and purified LPS. The size distributions of LPS molecules in outer membranes and supernatant fragments were similar; supernatant fragments appeared to be slightly enriched in molecules with long O-polysaccharide chains. Our results indicate the LPS molecules of many sizes are synthesized, translocated to outer membranes, and released into culture supernatants. Since the hydrophilic O-polysaccharides extend from bacterial surfaces into the aqueous environment, our findings suggest that the cell surface topography of this bacterium may be very irregular. We also speculate that heterogeneity in the degree of polymerization of O-antigenic side chains may influence the interactions of the toxic moiety of LPS (lipid A) with host constituents. Images PMID:7000751

  12. Sorting Short Fragments of Single-Stranded DNA with an Evolving Electric Double Layer

    PubMed Central

    Wu, Jiamin; Zhao, Shuang-Liang; Gao, Lizeng; Wu, Jianzhong; Gao, Di

    2013-01-01

    We demonstrate a new procedure for separation of single-stranded DNA (ssDNA) fragments that are anchored to the surface of a gold electrode by end hybridization. The new separation procedure takes the advantage of the strong yet evolving non-uniform electric field near the gold surface in contact with a buffer solution gradually being diluted with deionized water. Separation of short ssDNA fragments is demonstrated by monitoring the DNA at the gold surface with in situ fluorescence measurement. The experimental results can be rationalized with a simple theoretical model of electric double layer that relates the strength of the surface pulling force to the ionic concentration of the changing buffer solution. PMID:23356906

  13. Acetaldehyde and ethanol are responsible for mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) in flor yeasts.

    PubMed

    Castrejón, Francisco; Codón, Antonio C; Cubero, Beatriz; Benítez, Tahía

    2002-10-01

    Flor yeasts grow and survive in fino sherry wine where the frequency of respiratory-deficient (petite) mutants is very low. Mitochondria from flor yeasts are highly acetaldehyde- and ethanol-tolerant, and resistant to oxidative stress. However, restriction fragment length polymorphism (RFLP) of mtDNA from flor yeast populations is very high and reflects variability induced by the high concentrations of acetaldehyde and ethanol of sherry wine on mtDNA. mtDNA RFLP increases as the concentration of these compounds also increases, but is followed by a total loss of mtDNA in petite cells. Yeasts with functional mitochondria (grande) are target of continuous variability, so that flor yeast mtDNA can evolve extremely rapidly and may serve as a reservoir of genetic diversity, whereas petite mutants are eventually eliminated because metabolism in sherry wine is oxidative. PMID:12421085

  14. Analysis of DNA size, content and cell cycle in leaves of Napier grass (Pennisetum purpureum Schum.).

    PubMed

    Taylor, M G; Vasil, I K

    1987-10-01

    Mesophyll cell nuclei isolated from leaves of Pennisetum purpureum were analysed by flow cytometry to determine the nuclear DNA content and the percentage of cells in different phases of the cell cycle. Samples taken from base, middle and tip regions of leaves 2 to 8 (leaf 1, which was adjacent to the meristem, was too small to sample) showed no significant differences in the amount of DNA per G1 nucleus due to either age or position. The average amount of DNA per G1 nucleus was 5.78 pg. Although the majority of cells for each sample were in G1, samples taken from older leaves had higher percentages of cells in G2 and S phases. More specifically, base and middle regions of older leaves had a higher percentage of cells in G2 than all three positions in younger leaves. Electrophoretic analysis of nuclear DNA from leaves 2 to 7 showed no evidence of degradation or difference in fragment size for any sample or position. This study was compared to previous work on the relationship between leaf age and embryogenic competence in Pennisetum purpureum. The results suggest that changes in the cell cycle, and/or a loss or fragmentation of the nuclear DNA, are not responsible for loss of embryogenic competence in mature leaf tissue. PMID:24240324

  15. 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.

  16. Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3?-OH Single-strand DNA Breaks*

    PubMed Central

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

    2013-01-01

    Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD?/? cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3?-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3?-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

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

    SciTech Connect

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

    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.

  18. Temporal Profile of In Situ DNA Fragmentation After Transient Middle Cerebral Artery Occlusion in the Rat

    Microsoft Academic Search

    Yi Li; Michael Chopp; Ning Jiang; Fayi Yao; Cecylia Zaloga

    1995-01-01

    Summary: We measured the temporal profile and anatomic distribution of cells exhibiting DNA fragmentation at various durations of reperfusion after middle cerebral artery (MCA) occlusion in the rat. Focal cerebral ischemia was induced in male Wistar rats (n = 62) using an intraluminal monofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different

  19. DNA restriction fragment length polymorphisms and heterozygosity in the human genome

    Microsoft Academic Search

    David N. Cooper; Jörg Schmidtke

    1984-01-01

    A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented

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

    Microsoft Academic Search

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

    2006-01-01

    The aim of the present study was to evaluate consequences of cigarette smoking on male gametes. In this prospective study, sperm parameters such as sperm density, motility, viability and normal morphology were measured according to the WHO criteria. In addition to these standard parameters, we analysed the degree of DNA fragmentation in spermatozoa using the TUNEL-assay with flow cytometry detection

  1. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously

    Microsoft Academic Search

    Nikolai A. Shevchuk; Anton V. Bryksin; Yevgeniya A. Nusinovich; Felipe C. Cabello; Margaret Sutherland; Stephan Ladisch

    2004-01-01

    A procedure for precise assembly of linear DNA constructs as long as 20 kb is proposed. The method, which we call long multiple fusion, has been used to assemble up to four fragments simul- taneously (for a 10.8 kb final product), offering an additional improvement on the combination of long PCR and overlap extension PCR. The method is based on

  2. 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...

  3. Are the Fragments of Comet Shoemaker-Levy 9 Swarms of Meter-Sized Planetesimals?

    NASA Astrophysics Data System (ADS)

    Rettig, T. W.; Mumma, M. J.; Tegler, S. C.; Hahn, J.

    1994-05-01

    The tidally fragmented comet S-L 9 presently exhibits 21 major concentrations of material (sub-nuclei) as well as extended dust wings. Comet S-L 9 is the first inactive comet observed to fragment under the influence of tidal forces alone. In contrast to the analysis of Weaver et al. (1994) where each sub-nucleus is interpreted to contain a compact body 3-4 km in diameter, we investigate the possibility that each sub-nucleus is composed of tidally fragmented `swarms' of ~ 50-m sized planetesimals. Our swarm model for the subnuclei suggests the upcoming impact of comet S-L 9 with Jupiter will result in vaporization of these bolides high in the Jovian stratosphere. Recent models of planetesimal accumulation in the solar nebula favor development of a bi-modal size distribution before the onset of gravitational instability triggers formation of larger bodies (Weidenschilling 1993). The dominant modal sizes at Uranus-Neptune are ~ 50-m and < 10-cm. The former agrees well with the size estimates of outbursting volumes in cometary nuclei (Whitney 1955, Rettig et al. 1992, Mumma et al. 1993). We thus might expect 50-m sized cometesimals and cm-sized particles to have accreted into km-sized bodies, several of which then combined to form a larger cometary nucleus. It is likely that tidal disruption of S-L 9 occurred episodically, with the weaker bound units disrupting first. The fragment cloud would have stretched out in a tube along the tidal direction. After perijove, we show the decreasing tidal force permits self-gravity to re-focus material forming ( ~ 20) fragments. We also demonstrate that after apjove there will be a critical distance where tidal dispersal begins again. This critical distance can be used to determine the individual swarm masses. Our tidally disrupted swarm model explains the dust wings, the 21 subnuclei, and predicts only slight modification to the Jovian atmosphere during the July 1994 impact.

  4. 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).

  5. 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

  6. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    PubMed

    Vtiurina, N N; Grokhovski?, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed. PMID:21786693

  7. The Effect of Change in Population Size on DNA Polymorphism

    Microsoft Academic Search

    Fumio Tajima

    The expected number of segregating sites and the expectation of the average number of nucleotide differences among DNA sequences randomly sampled from a population, which is not in equilibrium, have been developed. The results obtained indicate that, in the case where the population size has changed drastically, the number of segregating sites is influenced by the size of the current

  8. Microscopic Varicocelectomy Significantly Decreases the Sperm DNA Fragmentation Index in Patients with Infertility

    PubMed Central

    Kadioglu, Teoman Cem; Aliyev, Emin; Celtik, Murad

    2014-01-01

    Background. Varicocele is associated with high levels of DNA damage in spermatozoa due to oxidative stress and elevated levels of sperm DNA fragmentation, which has been currently proposed to be an essential additional diagnostic test to be recommended for patients with clinical varicocele. The aim of this study was to evaluate the parameters of semen and the DNA fragmentation index (DFI) in patients with varicocele before and after varicocelectomy. Methods. The details of 92 consecutive patients were retrospectively analyzed from January 2010 to December 2012. The sperm samples were evaluated according to the World Health Organization Guidelines. Sperm DNA damage, characterized as DFI, was evaluated by sperm chromatin structure assay using flow cytometry. Results. There was a statistically significant improvement in the semen concentration, the total motile count, the total normal sperm count, and the sperm DNA fragmentation index (DFI; the percentage of sperm with denatured DNA) after varicocelectomy. There was a large decrease in DFI from a preoperative mean of 42.6% to a postoperative mean of 20.5% (P < 0.001). A higher preoperative DFI was associated with a larger decrease in postoperative DFI, and significant negative correlations were observed between the DFI and sperm motility (r = ?0.42, P < 0.01). Conclusion. Our data suggest that varicocelectomy can improve multiple semen parameters and sperm DNA damage in infertile men with varicocele. The patients with preoperative defects in those parameters showed greater improvement postoperatively. Further research in this area is needed to understand the exact mechanisms of DNA damage in infertile men with varicocele. PMID:24712000

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

    SciTech Connect

    Wohletz, K.H. (Earth and Space Science Division Los Alamos National Laboratory, New Mexico (USA)); Sheridan, M.F. (Department of Geology, Arizona State University, Tempe (USA)); Brown, W.K. (Math/Science Division, Lassen College, Susanville, California (USA))

    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}.

  10. 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

  11. 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.

  12. [Level of DNA fragmentation in human sperm cells in varicocele and prostatitis].

    PubMed

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

    2014-01-01

    Varicocele and prostatitis are the most common andrological diseases, which may be accompanied by a decrease in the production of sperm cells, the deterioration of their quality and increased risk of infertility. This work was aimed to the evaluation of sperm DNA fragmentation index (DFI) and main indices of sperm fertility (concentration, motility and morphology), and the relationship between these parameters in the men of active reproductive age suffering from prostatitis or varicocele. Assessment of sperm DNA fragmentation was performed by SCSA (sperm chromatin structure assay) using flow cytometry; sperm parameters were evaluated according to WHO recommendations. It was shown that men with prostatitis (n = 9) and varicocele (n = 22) had significantly higher DFI compared with men in the control group (n = 22). Negative influence of these diseases on the concentration and the percentage of motile sperm cells in the ejaculate was revealed. These data suggest that the deterioration in the quality of semen in varicocele and prostatitis may be caused not only by pathospermia, but also, at least partially, by violation of the integrity of the sperm DNA. Evaluation of sperm DNA fragmentation can be recommended for use in laboratory diagnostics for prediction of fertility in infertile men. PMID:25211925

  13. Nest survival relative to patch size in a highly fragmented shortgrass prairie landscape

    USGS Publications Warehouse

    Skagen, S.K.; Yackel Adams, A.A.; Adams, R.D.

    2005-01-01

    Understanding the influences of habitat fragmentation on vertebrate populations is essential for the protection and ecological restoration of strategic sites for native species. We examined the effects of prairie fragmentation on avian reproductive success using artificial and natural nests on 26 randomly selected, privately owned patches of shortgrass prairie ranging in size from 7 to 454 ha within a cropland matrix in Washington County, Colorado, summer 2000. Survival trends of artificial and natural nests differed. Daily survival of artificial nests increased with patch size up to about 65 ha and differed little at larger patch sizes, whereas daily survival of Lark Bunting (Calamospiza melanocorys) and Horned Lark (Eremophila alpestris) nests decreased with increasing size of the grassland patch. We hypothesize that our unexpected findings of lower survival of natural nests with increasing patch sizes and different trends between artificial and natural nests are due to the particular structure of predator communities in our study area and the ways in which individual predators respond to artificial and natural nests. We recommend that the value of small habitat patches in highly fragmented landscapes not be overlooked.

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

    PubMed

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

    2014-04-01

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

  15. Abundance of Virus-Sized Non-DNase-Digestible DNA (Coated DNA) in Eutrophic Seawater

    PubMed Central

    Maruyama, A.; Oda, M.; Higashihara, T.

    1993-01-01

    Total DNA concentration in 0.2-?m-pore-size Nuclepore filter filtrates (<0.2-?m fraction) of Tokyo Bay water was estimated to be 9 to 19 ng/ml by an immunochemical quantification method. Almost 90% of the DNA in the <0.2-?m fraction was found in the size fractions larger than 3.0 × 105 Da and 0.03 ?m, and most was not susceptible to DNase digestion, that is, consisted of non-DNase-digestible DNA (coated DNA). A significant amount of DNA was obtained from the <0.2-?m fraction of the seawater by three different methods: polyethylene glycol precipitation, direct ethanol precipitation, and ultrafilter concentration. Gel electrophoresis analysis of the isolated DNAs showed that they consisted mainly of coated DNAs with a similar molecular sizes (20 to 30 kb [1.3 × 107 to 2.0 × 107 Da). The abundance of the ultramicron virus-sized coated DNA in natural seawater suggests that these DNA-rich particles can be attributed to marine DNA virus assemblages and that they may be a significant phosphorus reservoir in the environment. Images PMID:16348887

  16. DNA Electrophoresis on patterned Surfaces The separation of DNA molecules based on their size and mass has many applications in

    E-print Network

    Singh, Jayant K.

    DNA Electrophoresis on patterned Surfaces The separation of DNA molecules based on their size and mass has many applications in biotechnology. Traditionally gel electrophoresis and capillary electrophoresis techniques are used to separate the DNA molecules based on their molecular weight

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

    PubMed

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

    2010-01-01

    Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage. PMID:19896957

  18. 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.

  19. Size dependent fragmentation of argon clusters in the soft x-ray ionization regime

    SciTech Connect

    Gisselbrecht, Mathieu; Lindgren, Andreas; Burmeister, Florian; Tchaplyguine, Maxim; Oehrwall, Gunnar; Lundin, Magnus; Naves de Brito, Arnaldo; Svensson, Svante; Bjoerneholm, Olle; Sorensen, Stacey L. [MAX-lab, Lund University, P.O. Box 118, S-221 00 Lund, Sweden and LIXAM, UMR8624, Universite Paris-Sud, Bat 350, 91405 Orsay (France); Department of Synchrotron Radiation Research, Institute of Physics, P.O. Box 118, S-221 00 Lund (Sweden); Division for Electricity, Uppsala University, P.O. Box 534, S-75121 Uppsala (Sweden); MAX-lab, Lund University, P.O. Box 118, S-221 00 Lund (Sweden); Department of Physics, Uppsala University, P.O. Box 534, S-75121 Uppsala (Sweden); MAX-lab, Lund University, Box 118, S-221 00 Lund (Sweden); Laboratorio Nacional de Luz Sincrotron (LNLS), P.O. Box 6192, Campinas, Sao Paulo, 13084-971 (Brazil); Department of Physics, Uppsala University, P.O. Box 534, S-75121 Uppsala (Sweden); Department of Synchrotron Radiation Research, Institute of Physics, P.O. Box 118, S-221 00 Lund (Sweden)

    2008-01-28

    Photofragmentation of argon clusters of average size ranging from 10 up to 1000 atoms is studied using soft x-ray radiation below the 2p threshold and multicoincidence mass spectroscopy technique. For small clusters (=10), ionization induces fast fragmentation with neutral emission imparting a large amount of energy. While the primary dissociation takes place on a picosecond time scale, the fragments undergo slow degradation in the spectrometer on a microsecond time scale. For larger clusters ({>=}100) we believe that we observe the fragmentation pattern of multiply charged species on a time-scale which lasts a few hundred nanoseconds. The reason for these slower processes is the large number of neutral atoms which act as an efficient cooling bath where the excess energy ('heat') dissipates among all degrees of freedom. Further degradation of the photoionic cluster in spectrometer then takes place on the microsecond time scale, similar to small clusters.

  20. Specific amino acid substitutions in bacterioopsin: replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons

    SciTech Connect

    Lo, K.M.; Jones, S.S.; Hackett, N.R.; Khorana, H.G.

    1984-04-01

    To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed. 32 references, 6 figures.

  1. 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

  2. Ejaculate oxidative stress is related with sperm DNA fragmentation and round cells.

    PubMed

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

    2015-01-01

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

  3. 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. Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

    PubMed Central

    Freund, J; di Mayorca, G; Subramanian, K N

    1979-01-01

    A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level. Images PMID:221680

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

    Microsoft Academic Search

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

    2000-01-01

    Programmed cell death or apoptosis leads to the activation of the caspase-activated DNase (CAD), which degrades chromosomal DNA into nucleosomal fragments. Biochemical studies revealed that CAD forms an inactive heterodimer with the inhibitor of caspase-activated DNase (ICAD), or its alternatively spliced variant, ICAD-S, in the cytoplasm. It was ini- tially proposed that proteolytic cleavage of ICAD by activated caspases causes

  6. A comparative analysis of DNA barcode microarray feature size

    Microsoft Academic Search

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

    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

  7. Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas.

    PubMed

    Jordan, N D; Franklin, F C; Franklin-Tong, V E

    2000-08-01

    Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen. PMID:10972873

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

    SciTech Connect

    Tee, Thiam-Tsui, E-mail: thiamtsu@yahoo.com [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)] [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Cheah, Yew-Hoong [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia) [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia); Bioassay Unit, Herbal Medicine Research Center, Institute for Medical Research, Jalan Pahang, Kuala Lumpur (Malaysia); Meenakshii, Nallappan [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia)] [Biology Department, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Mohd Sharom, Mohd Yusof; Azimahtol Hawariah, Lope Pihie [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)] [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor (Malaysia)

    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.

  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. Cell-free DNA Fragmentation Patterns in Amniotic Fluid Identify Genetic Abnormalities and Changes due to Storage

    PubMed Central

    Peter, Inga; Tighiouart, Hocine; Lapaire, Olav; Johnson, Kirby L.; Bianchi, Diana W.; Terrin, Norma

    2015-01-01

    Circulating cell-free DNA (cfDNA) has become a promising biomarker in prenatal diagnosis. However, despite extensive studies in different body fluids, cfDNA predictive value is uncertain owing to the confounding factors that can affect its levels, such as gestational age, maternal weight, smoking status, and medications. Residual fresh and archived amniotic fluid (AF) supernatants were obtained from gravid women (mean gestational age 17 wk) carrying euploid (N = 36) and aneuploid (N = 29) fetuses, to characterize cfDNA-fragmentation patterns with regard to aneuploidy and storage time (?80°C). AF cfDNA was characterized by the real-time quantitative polymerase chain reaction amplification of glyceraldehyde-3-phosphate dehydrogenase, gel electrophoresis, and pattern recognition of the DNA fragmentation. The distributions of cfDNA fragment lengths were compared using 6 measures that defined the locations and slopes for the first and last peaks, after elimination of the confounding variables. This method allowed for the unique classification of euploid and aneuploid cfDNA samples in AF, which had been matched for storage time. In addition, we showed that archived euploid AF samples gradually lose long cfDNA fragments: this loss accurately distinguishes them from the fresh samples. We present preliminary data using cfDNA-fragmentation patterns, to uniquely distinguish between AF samples of pregnant women with regard to aneuploidy and storage time, independent of gestational age and initial DNA amount. In addition to potential applications in prenatal diagnosis, these data suggest that archived AF samples consist of large amounts of short cfDNA fragments, which are undetectable using standard real-time polymerase chain reaction amplification. PMID:18382362

  11. Optimized Quantification of Fragmented, Free Circulating DNA in Human Blood Plasma Using a Calibrated Duplex Real-Time PCR

    Microsoft Academic Search

    Martin Horlitz; Annabelle Lucas; Markus Sprenger-Haussels; Jörg Hoheisel

    2009-01-01

    BackgroundDuplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers

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

    SciTech Connect

    Evenson, Donald P. [HCLD, Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD 57007 (United States) and SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)]. E-mail: scsa@brookings.net; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)

    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.

  13. 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.

  14. Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic cDNA fragments

    Microsoft Academic Search

    Loic Lepiniec; Lynn Dalgarno; V. T. Q. Huong; T. P. Monath; J.-P. Digoutte; V. Deubel

    1994-01-01

    We have compared the nucleotide sequence of an envelope protein gene fragment encoding amino acids 291 to 406 of 22 yellow fever (YF) virus strains of diverse geographic and host origins isolated over a 63 year time span. The nucleotide fragment of viral RNA was examined by direct sequencing ofa PCR product derived from complementary DNA. Alignment with the proto-

  15. Fragmentation of extracellular DNA by long-term exposure to radiation from uranium in aquatic environments.

    PubMed

    Arruda-Neto, J D T; Nieto, L; Righi, H; Cotta, M A; Carrer, H; Rodrigues, T E; Genofre, G C

    2012-08-01

    Persistent harmful scenarios associated with disposal of radioactive waste, high-background radiation areas and severe nuclear accidents are of great concern regarding consequences to both human health and the environment. Of particular concern is the extracellular DNA in aquatic environments contaminated by radiological substances. Strand breaks induced by radiation promote decrease in the transformation efficiency for extracellular DNA. The focus of this study is the quantification of DNA damage following long-term exposure (over one year) to low doses of natural uranium (an alpha particle emitter) to simulate natural conditions, since nothing is known about alpha radiation induced damage to extracellular DNA. A high-resolution Atomic Force Microscope was used to evaluate DNA fragments. Double-stranded plasmid pBS as a model for extracellular DNA was exposed to different amounts of natural uranium. It was demonstrated that low concentrations of U in water (50 to 150 ppm) produce appreciable numbers of double strand breaks, scaling with the square of the average doses. The importance of these findings for environment monitoring of radiological pollution is addressed. PMID:22760469

  16. Multiple determinations of sperm DNA fragmentation show that varicocelectomy is not indicated for infertile patients with subclinical varicocele.

    PubMed

    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

  17. 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

  18. 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

  19. Dust size distributions in coagulation/fragmentation equilibrium: numerical solutions and analytical fits

    NASA Astrophysics Data System (ADS)

    Birnstiel, T.; Ormel, C. W.; Dullemond, C. P.

    2011-01-01

    Context. Grains in circumstellar disks are believed to grow by mutual collisions and subsequent sticking due to surface forces. Results of many fields of research involving circumstellar disks, such as radiative transfer calculations, disk chemistry, magneto-hydrodynamic simulations largely depend on the unknown grain size distribution. Aims: As detailed calculations of grain growth and fragmentation are both numerically challenging and computationally expensive, we aim to find simple recipes and analytical solutions for the grain size distribution in circumstellar disks for a scenario in which grain growth is limited by fragmentation and radial drift can be neglected. Methods: We generalize previous analytical work on self-similar steady-state grain distributions. Numerical simulations are carried out to identify under which conditions the grain size distributions can be understood in terms of a combination of power-law distributions. A physically motivated fitting formula for grain size distributions is derived using our analytical predictions and numerical simulations. Results: We find good agreement between analytical results and numerical solutions of the Smoluchowski equation for simple shapes of the kernel function. The results for more complicated and realistic cases can be fitted with a physically motivated “black box” recipe presented in this paper. Our results show that the shape of the dust distribution is mostly dominated by the gas surface density (not the dust-to-gas ratio), the turbulence strength and the temperature and does not obey an MRN type distribution.

  20. 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. [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy)] [and others] [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy); and others

    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.

  1. 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

  2. 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.

  3. Presence of DNA Fragmentation and Lack of Neuroprotective Effect in DFF45 Knockout Mice Subjected to Traumatic Brain Injury

    Microsoft Academic Search

    Alexander G. Yakovlev; Xiao Di; Vilen Movsesyan; Paul G. M. Mullins; Geping Wang; Hamid Boulares; Jianhua Zhang; Ming Xu; Alan I. Faden

    2001-01-01

    Background: Apoptosis plays an important pathophysio- logic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA frag- mentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after ex- perimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. Materials and Methods: We used mice

  4. 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.

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

    PubMed Central

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

    2015-01-01

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 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

  6. Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields.

    PubMed

    Heiger, D N; Cohen, A S; Karger, B L

    1990-09-01

    This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation. PMID:1962784

  7. Manning free counterions fraction for a rod-like polyion - short DNA fragments in very low salt

    E-print Network

    Tomislav Vuletic; Sanja Dolanski Babic; Danijel Grgicin; Damir Aumiler; Joachim Raedler; Francoise Livolant; Silvia Tomic

    2011-01-05

    We quantified the Manning free (uncondensed) counterions fraction $\\theta$ for dilute solutions of rod-like polyions - 150bp DNA fragments, in very low salt $salt environment, with the decrease in DNA concentration itself. The extremes of the experimental $\\theta(c)$ range occur towards the highest, above 1 mM and the lowest, below 0.05 mM, DNA concentrations, and correspond to the theoretical $\\theta$ values for dsDNA and ssDNA, respectively. Therefore, we confirmed Manning condensation and conductivity models to be valuable in description of dilute solutions of rod-like polyions.

  8. Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

    PubMed

    Rill, R L; Nelson, D A; Oosterhof, D K; Hozier, J C

    1977-04-01

    DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed. PMID:866190

  9. Assessment of Subtractive Hybridization to Select Species and Subspecies Specific DNA Fragments for the Identification of Xylophilus ampelinus by Polymerase Chain Reaction (PCR)

    Microsoft Academic Search

    Charles Manceau; Marie-Germaine Coutaud; Richard Guyon

    2000-01-01

    Eighteen Bsp143I digested DNA fragments specific to Xylophilus ampelinus were cloned from a library enriched for X. ampelinus obtained after a subtractive hybridization step. It was also possible to clone specific DNA sequences directly after DNA digestion with Bsp143I probably because X. ampelinus is a unique bacterium. Nucleotidic sequences of four cloned specific fragments were determined. They did not share

  10. True ternary fission of 252Cf(sf), the collinear decay into fragments of similar size

    NASA Astrophysics Data System (ADS)

    von Oertzen, W.; Nasirov, A. K.

    2014-12-01

    The ternary decay in 252Cf(sf, fff), with three cluster fragments of different masses (e.g.132Sn,52-48Ca,68-72Ni), has been observed by the FOBOS group in JINR. This work has established a new decay mode of heavy nuclei, the collinear cluster tripartition, (CCT). This "true ternary fission" of heavy nuclei has been predicted many times in theoretical works during the last decades. In the present report we discuss true ternary fission (FFF) into three nuclei of almost equal size (e.g. Z=98 ? Zi = 32, 34, 32) and other fission modes in the same system. The possible fission channels for 252 Cf(sf) are predicted from potential-energy (PES) calculations. These PES's show pronounced minima for several ternary fragmentation decays, suggesting a variety of collinear ternary fission modes. The FFF-decays have very similar dynamical features as the previously observed collinear CCT-decays, the central fragment has very small kinetic energy. The data of the cited experiment allow the extraction of the yield for some FFF-decays, by using specific gates on the measured parameters.

  11. 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

  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. Effects of fragment size and isolation on the occurrence of four short-lived plants in semi-natural grasslands

    NASA Astrophysics Data System (ADS)

    Kiviniemi, Katariina

    2008-01-01

    Habitat fragmentation is predicted to lead to an area-related reduction in population size and a decreasing colonisation rate due to isolation. A reduction in grassland size may promote a "run-away-decline process" leading to reduced individual fitness and viability of the populations originally inhabiting the grassland. To circumvent the problems of time-lags associated with the slow response of long-lived plants to semi-natural grassland fragmentation, four short-lived grassland species were studied. During three years, data on population sizes were gathered for Carum carvi, Rhinanthus minor, Trifolium arvense and Viola tricolor in Swedish semi-natural grasslands varying in size and degree of isolation. A seed-sowing experiment was conducted to assess dispersal and seed limitation at a local and regional scale, respectively. Overall, the presence/absence of species was not related to fragment size and isolation (connectivity). However, for the fragments where the species were present, positive relationships between grassland size and population size were detected for three species. No significant relationships between isolation and population size were detected for any species. This study thus demonstrates that short-lived plant species, confined to semi-natural grasslands, respond to decreases in fragment size by forming smaller populations. Seed sowing indicated that the species are both dispersal and seed limited in the study area, and that disturbances are important for establishment. In order to maintain characteristic grassland species in fragmented (isolated) semi-natural grasslands, it may therefore be of interest to preserve large intact fragments instead of several small ones.

  14. Vaccination of mice with DNA encoding a large fragment of botulinum neurotoxin serotype A.

    PubMed

    Clayton, J; Middlebrook, J L

    2000-03-01

    The potential utility of using DNA vaccination to protect mice from the microbial neurotoxin, botulinum toxin type A, was evaluated. A synthetically derived gene encoding a carboxyl-terminal 50 kDa fragment of the toxin was placed in two sites in the DNA inoculation vehicle pCMVint-BL (Vical), one predicted to lead to MHC I processing (pJT-1 construct) and the other to direct MHC II processing (pJT-2 construct). Mice were then inoculated at 3 week intervals with these two constructs and with the vehicle alone and evaluated for protection from botulinum toxin by i.p. challenges with various toxin doses. Protection was observed at about week 10-11 from toxin doses of 25-100 LD(50). Only animals inoculated with pJT-2 exhibited protection. In dose-response experiments, 50 micrograms of DNA was the minimal dose required to elicit a protective response against serotype A, while protection against serotypes B or E was not obtained. With standard ELISA testing, a relationship was observed between the level of protection and the level of ELISA reactive antibody. Our results support the concept that DNA vaccination is a viable methodology to use in cases where protection from toxins is the goal. PMID:10699334

  15. Patterns of Genomic Integration of Nuclear Chloroplast DNA Fragments in Plant Species

    PubMed Central

    Yoshida, Takanori; Furihata, Hazuka Y.; Kawabe, Akira

    2014-01-01

    The transfer of organelle DNA fragments to the nuclear genome is frequently observed in eukaryotes. These transfers are thought to play an important role in gene and genome evolution of eukaryotes. In plants, such transfers occur from plastid to nuclear [nuclear plastid DNAs (NUPTs)] and mitochondrial to nuclear (nuclear mitochondrial DNAs) genomes. The amount and genomic organization of organelle DNA fragments have been studied in model plant species, such as Arabidopsis thaliana and rice. At present, publicly available genomic data can be used to conduct such studies in non-model plants. In this study, we analysed the amount and genomic organization of NUPTs in 17 plant species for which genome sequences are available. The amount and distribution of NUPTs varied among the species. We also estimated the distribution of NUPTs according to the time of integration (relative age) by conducting sequence similarity analysis between NUPTs and the plastid genome. The age distributions suggested that the present genomic constitutions of NUPTs could be explained by the combination of the rapidly eliminated deleterious parts and few but constantly existing less deleterious parts. PMID:24170805

  16. Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

    Microsoft Academic Search

    E. B. Brosalina; V. V. Vlasov; I. V. Kutyavin; S. V. Mamaev; A. G. Pletnev; M. A. Podyminogin

    1986-01-01

    The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-(N-methyl-N-(2-chloroethyl)amino)benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are

  17. Complementarily addressed modification and cleavage of a single-stranded fragment of DNA with the aid of alkylating derivatives of oligonucleotides

    SciTech Connect

    Brosalina, E.B.; Vlasov, V.V.; Kutyavin, I.V.; Mamaev, S.V.; Pletnev, A.G.; Podyminogin, M.A.

    1986-12-01

    The chemical modification of a 303-nucleotide single-stranded fragment of DNA by alkylating oligonucleotide derivatives bearing 4-(N-methyl-N-(2-chloroethyl)amino)benzyl groups in the 5'-terminal phosphate of the 3'-terminal ribose residue has been investigated. It has been shown that under the conditions of the formation of a complex with the DNA fragment both types of derivatives specifically alkylate nucleotides of the DNA fragments that are located directly adjacent to the sections complementary to the oligonucleotides bearing the reactive groups. Alkylation takes place with a high efficiency, and the DNA fragment can be cleaved specifically at the position of the alkylated nucleotides.

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

    PubMed

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

    2014-01-01

    Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17-30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50 °C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90-95%. PMID:25551825

  19. 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 ?m features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 ?m feature size is of comparable quality to the 30 ?m 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

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

    SciTech Connect

    Okano, Kazuhiro [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States); Vanarsdall, Adam L. [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States); Rohrmann, George F. [Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804 (United States)]. E-mail: rohrmanng@orst.edu

    2007-03-01

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

  1. Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

    Microsoft Academic Search

    Lijuan Li; Linda B. McGown

    2001-01-01

    Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly\\u000a fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with\\u000a dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The\\u000a sieving

  2. Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

    Microsoft Academic Search

    Christina Hnida; Elisabete Engenheiro; Søren Ziebe

    BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients

  3. 'Size leap' algorithm: an efficient extraction of the longest common motifs from a molecular sequence set. Application to the DNA sequence reconstruction.

    PubMed

    Danckaert, A; Chappey, C; Hazout, S

    1991-10-01

    We propose a new method, called 'size leap' algorithm, of search for motifs of maximum size and common to two fragments at least. It allows the creation of a reduced database of motifs from a set of sequences whose size obeys the series of Fibonacci numbers. The convenience lies in the efficiency of the motif extraction. It can be applied in the establishment of overlap regions for DNA sequence reconstruction and multiple alignment of biological sequences. The method of complete DNA sequence reconstruction by extraction of the longest motifs ('anchor motifs') is presented as an application of the size leap algorithm. The details of a reconstruction from three sequenced fragments are given as an example. PMID:1747784

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

    PubMed

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

    2007-08-01

    We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(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(+) 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. PMID:17764359

  5. High transmission of paternal plastid DNA in alfalfa plants demonstrated by restriction fragment polymorphic analysis.

    PubMed

    Masoud, S A; Johnson, L B; Sorensen, E L

    1990-01-01

    A high frequency of paternal plastid transmission occurred in progeny from crosses among normal green alfalfa plants. Plastid transmission was analyzed by hybridization of radiolabeled alfalfa plastid DNA (cpDNA) probes to Southern blots of restriction digests of the progeny DNA. Each probe revealed a specific polymorphism differentiating the parental plastid genomes. Of 212 progeny, 34 were heteroplastidic, with their cpDNAs ranging from predominantly paternal to predominantly maternal. Regrowth of shoots from heteroplasmic plants following removal of top growth revealed the persistence of mixed plastids in a given plant. However, different shoots within a green heteroplasmic plant exhibited paternal, maternal, or mixed cpDNAs. Evidence of maternal nuclear genomic influence on the frequency of paternal plastid transmission was observed in some reciprocal crosses. A few tetraploid F1 progeny were obtained from tetraploid (2n=4x=32) Medicago sativa ssp. sativa x diploid (2n=2x=16) M. sativa ssp. falcata crosses, and resulted from unreduced gametes. Here more than the maternal genome alone apparently functioned in controlling plastid transmission. Considering all crosses, only 5 of 212 progeny cpDNAs lacked evidence of a definitive paternal plastid fragment. PMID:24226119

  6. Initial decay of woody fragments in soil is influenced by size, vertical position, nitrogen availability and soil origin

    Microsoft Academic Search

    Annemieke van der Wal; Wietse de Boer; Wiecher Smant; Johannes A. van Veen

    2007-01-01

    Fast-growing bacteria and fungi are expected to cause the initial stage of decomposition of woody fragments in and on soils,\\u000a i.e. the respiration of sugars, organic acids, pectin and easily accessible cellulose and hemi-cellulose. However, little\\u000a is known about the factors regulating initial wood decomposition. We examined the effect of wood fragment size, vertical position,\\u000a nitrogen addition and soil origin

  7. Highly Selective Isolation of Unknown Mutations in Diverse DNA Fragments: Toward New Multiplex Screening in Cancer1

    Microsoft Academic Search

    Subrata Chakrabarti; Brendan D. Price; Sotirios Tetradis; Edward A. Fox; Yuzhi Zhang; Gautam Maulik; Mike Makrigiorgos

    2000-01-01

    Cancer research would greatly benefit from technologies that allow simul- taneous screening of several unknown gene mutations. Lack of such methods currently hampers the large-scale detection of genetic alterations in complex DNA samples. We present a novel mismatch-capture methodology for the highly efficient isolation and amplification of mutation-containing DNA from diverse nucleic acid fragments of unknown sequence. To demonstrate the

  8. Electrochemical Sensors Based on Stationary Electrodes and Immobilized DNA or Its Fragments and the Assessment of Their Analytical Potentials

    Microsoft Academic Search

    S. S. Babkina; E. Palecek; F. Jelen; M. Fojta

    2005-01-01

    Amperometric biosensors were developed on the basis of stationary mercury-film and glassy-carbon electrodes and DNA or its fragments, oligodeoxynucleotides (ODNs), immobilized in a nitrocellulose matrix. Taking into account the high affinity of Cu(II) and Fe(III) ions to denatured DNA ((19.1 ± 0.1) ×105 and (1.4 ± 0.3) × 105 L\\/mol, respectively), a procedure was proposed for the voltammetric determination of

  9. 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.

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

    PubMed

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

    2012-04-20

    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(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. PMID:22465013

  11. 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.

  12. 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

  13. Fatal Outcome in Bacteremia is Characterized by High Plasma Cell Free DNA Concentration and Apoptotic DNA Fragmentation: A Prospective Cohort Study

    PubMed Central

    Huttunen, Reetta; Kuparinen, Taru; Jylhävä, Juulia; Aittoniemi, Janne; Vuento, Risto; Huhtala, Heini; Laine, Janne; Syrjänen, Jaana; Hurme, Mikko

    2011-01-01

    Introduction Recent studies have shown that apoptosis plays a critical role in the pathogenesis of sepsis. High plasma cell free DNA (cf-DNA) concentrations have been shown to be associated with sepsis outcome. The origin of cf-DNA is unclear. Methods Total plasma cf-DNA was quantified directly in plasma and the amplifiable cf-DNA assessed using quantitative PCR in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-hemolytic streptococcae or Escherichia coli. The quality of cf-DNA was analyzed with a DNA Chip assay performed on 8 survivors and 8 nonsurvivors. Values were measured on days 1–4 after positive blood culture, on day 5–17 and on recovery. Results The maximum cf-DNA values on days 1–4 (n?=?132) were markedly higher in nonsurvivors compared to survivors (2.03 vs 1.26 ug/ml, p<0.001) and the AUCROC in the prediction of case fatality was 0.81 (95% CI 0.69–0.94). cf-DNA at a cut-off level of 1.52 ug/ml showed 83% sensitivity and 79% specificity for fatal disease. High cf-DNA (>1.52 ug/ml) remained an independent risk factor for case fatality in a logistic regression model. Qualitative analysis of cf-DNA showed that cf-DNA displayed a predominating low-molecular-weight cf-DNA band (150–200 bp) in nonsurvivors, corresponding to the size of the apoptotic nucleosomal DNA. cf-DNA concentration showed a significant positive correlation with visually graded apoptotic band intensity (R?=?0.822, p<0.001). Conclusions Plasma cf-DNA concentration proved to be a specific independent prognostic biomarker in bacteremia. cf-DNA displayed a predominating low-molecular-weight cf-DNA band in nonsurvivors corresponding to the size of apoptotic nucleosomal DNA. PMID:21747948

  14. A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

    Microsoft Academic Search

    Ranulfo González; Richard Wilkerson; Marco Fidel Suárez; Felipe García; Gerardo Gallego; Heiber Cárdenas; Carmen Elisa Posso; Myriam Cristina Duque

    2007-01-01

    The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymor- phism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer com- binations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence

  15. Study of mechanisms of some caffeine biological effects via computer simulation of its interactions with DNA fragments

    Microsoft Academic Search

    A. S. Deriabina; T. I. Grokhlina; N. A. Polteva; E. González; V. I. Poltev

    2006-01-01

    To approach atomic level mechanisms of caffeine biological activity, molecular mechanics computations of interactions between caffeine molecule and DNA duplex fragments, containing four nucleotide pairs, have been performed. The calculations reveal a set of energy minima referring to rather compact caffeine arrangement in both major and minor grooves of the helix. The set includes the minima correspondent to the H-bond

  16. Analysis on DNA sequence of GPR54 gene and its association with litter size in goats.

    PubMed

    Cao, G L; Chu, M X; Fang, L; Feng, T; Di, R; Li, N

    2011-08-01

    The kisspeptin/GPR54 pathway is crucial in the process of puberty onset. Six pairs of primers were designed to clone goat GPR54 and scan polymorphisms and one pair of primers to detect polymorphisms of GPR54 in sexual precocious and sexual late-maturing goat breeds. A DNA fragment of 4258 bp of goat GPR54 was obtained, which contains an open reading frame (ORF) of 1137 bp and encodes 378 amino acids, having a high homology with other mammals. The protein was predicted to have seven transmembrane regions. There were no base pair variation in exons 1-4 and three base changes (G4014A, G4136A and C4152T) in exon 5 by sequencing and the three mutations may have some correlation with sexual precocity in goats. For the 4152 locus, the Jining Grey goat does with genotype TT and CT had 1.02 and 0.84 (P<0.01) kids more than those with genotype CC, respectively. No significant difference (P>0.05) was found in litter size between TT and CT genotypes in Jining Grey goat. For the other two loci, no significant difference (P>0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele T of the 4152 locus in GPR54 and high litter size in Jining Grey goats. PMID:21110113

  17. Structural and Thermodynamic Properties of Amyloid-? 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-? 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 A?1-16, A?1-28, and A?1-42 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.

  18. Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments.

    PubMed

    Li, L; McGown, L B

    2001-02-01

    Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide. PMID:11293703

  19. Denaturing high-performance liquid chromatography using the WAVE DNA fragment analysis system.

    PubMed

    Donohoe, Elizabeth

    2005-01-01

    Denaturing high-performance liquid chromatography (DHPLC) is a chromatographic mutation analysis technique that is based on temperature-dependent separation of DNA containing mismatched base pairs from polymerase chain reaction (PCR)-amplified DNA fragments. The WAVE system, developed for DHPLC analysis, allows for unattended analysis of 96 samples directly from a PCR plate under a number of different conditions. It utilizes a Peltier cooling platform to maintain sample integrity. Sample detection is achieved via UV absorbance at 260 nm, thereby avoiding the cost, safety, variability, or waste disposal issues associated with radioisotopic, enzyme-linked, or fluorescence detection systems. There are four key aspects to successfully detecting mutations on the WAVE: (1) PCR primer design, (2) PCR protocol, (3) separation gradient, and (4) separation temperature. Provided these procedures are carried out correctly, almost 100% detection of single-nucleotide polymorphisms (SNPs) and small deletion/insertion mutations can be achieved. For this reason, DHPLC is a powerful tool for identifying mutations in candidate genes for hypertension. PMID:16028684

  20. 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. [Lawrence Berkeley Lab., CA (United States)

    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.

  1. Elevated dietary intake of Zn-methionate is associated with increased sperm DNA fragmentation in the boar.

    PubMed

    García-Contreras, Adelfa; De Loera, Yasmin; García-Artiga, Carlos; Palomo, Antonio; Guevara, Jesús A; Herrera-Haro, José; López-Fernández, Carmen; Johnston, Steve; Gosálvez, Jaime

    2011-05-01

    Boars fed on ration of 200 ppm Zn methionate showed a significant increase (P < 0.001) in sperm DNA fragmentation when compared to animals fed on non-supplemented and rations containing 150 ppm Zn methionate. There was a positive correlation (R2 = 0.207; P = 0.002) between % sperm DNA fragmentation (SDF) and the concentration of Zn in spermatozoa. Increased Zn in the diet also resulted in a non-proportional increase in Zn concentration in the testis and spermatozoa but not in the epididymis; Zn in sperm accumulated at levels up to 50 times higher than that found in the seminal plasma and 10-13 times that found in the epididymis and testis, respectively. These results show that supplementation of dietary Zn at a concentration of 200 ppm had an adverse effect on boar sperm DNA quality and may be related to the ability of spermatozoa to accumulate Zn during spermiogenesis. PMID:21182932

  2. DNA Detectives

    NSDL National Science Digital Library

    BEGIN:VCARD VERSION:2.1 FN:Suzanne Black N:Black; Suzanne ORG:Inglemoor High School REV:2005-04-09 END:VCARD

    1995-06-30

    Many of the revolutionary changes that have occurred in biology since 1970 can be attributed directly to the ability to manipulate DNA in defined ways. The principal tools for this recombinant DNA technology are enzymes that can "cut and "paste" DNA. Restriction enzymes are the "chemical scissors" of the molecular biologist; these enzymes cut DNA at specific nucleotide sequences. A sample of someone's DNA, incubated with restriction enzymes, is reduced to millions of DNA fragments of varying sizes. A DNA sample from a different person would have a different nucleotide sequence and would thus be enzymatically "chopped up" into a very different collection of fragments. We have been asked to apply DNA fingerprinting to determine which suspect should be charged with a crime perpetrated in our city.

  3. Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site.

    PubMed

    Livak, K J; Whitehorn, E A

    1986-01-01

    Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence. PMID:3006543

  4. Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900.

    PubMed Central

    Moss, M T; Green, E P; Tizard, M L; Malik, Z P; Hermon-Taylor, J

    1991-01-01

    This paper describes the evaluation of a newly developed DNA probe for Mycobacterium paratuberculosis. DNA probe PCR278 is a 278 bp fragment obtained by polymerase chain reaction (PCR) amplification of the 5'-region of IS900, an insertion element contained in the genome of M paratuberculosis. This DNA probe can specifically distinguish M paratuberculosis from a wide range of other organisms, including members of the M avium-M intracellulare complex. When used in conjunction with the PCR amplification technique DNA probe PCR278 could detect as little as 10 fg (equivalent to two genomes) starting material of M paratuberculosis genomic DNA. Use of PCR amplification assays based on IS900, for the detection of M paratuberculosis, and homologous IS elements found in disease isolates of M avium should greatly help our understanding of the role of these organisms in Crohn's disease and other chronic inflammatory disorders. Images Figure 1 Figure 2 Figure 3 PMID:1851124

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

    PubMed Central

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

    2015-01-01

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

  6. Challenges in the Chemical Synthesis of Average Sized Proteins: Sequential vs. Convergent Ligation of Multiple Peptide Fragments

    E-print Network

    Bang, Duhee

    ? There are some issues in the routine chemical synthesis of protein molecules. These include Fmoc-based peptide chemical synthesis of large proteins involves the liga- tion of multiple peptide fragments; consequentlyChallenges in the Chemical Synthesis of Average Sized Proteins: Sequential vs. Convergent Ligation

  7. Effects of fragment size and isolation on the occurrence of four short-lived plants in semi-natural grasslands

    Microsoft Academic Search

    Katariina Kiviniemi

    2008-01-01

    Habitat fragmentation is predicted to lead to an area-related reduction in population size and a decreasing colonisation rate due to isolation. A reduction in grassland size may promote a “run-away-decline process” leading to reduced individual fitness and viability of the populations originally inhabiting the grassland. To circumvent the problems of time-lags associated with the slow response of long-lived plants to

  8. 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

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

    PubMed

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

    2013-11-01

    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

  10. 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.

  11. 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

  12. 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

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

    PubMed Central

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

    2011-01-01

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

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

    SciTech Connect

    Beverley, S.M. (Harvard Medical School, Boston, MA (USA))

    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.

  15. Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

    SciTech Connect

    Al-Gubory, Kais H. [Unite de Biologie du Developpement et de la Reproduction, Departement de Physiologie Animale, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex (France)]. 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.

  16. The effect of indwelling endoprosthesis on stone size or fragmentation after long-term treatment with biliary stenting for large stones

    Microsoft Academic Search

    P. Katsinelos; I. Galanis; I. Pilpilidis; G. Paroutoglou; P. Tsolkas; B. Papaziogas; S. Dimiropoulos; E. Kamperis; D. Katsiba; M. Kalomenopoulou; A. Papagiannis

    2003-01-01

    Background: Endoscopic biliary stenting is often used for large or difficult common bile duct (CBD) stones, but the effect of indwelling endoprosthesis on size or fragmentation of stones after long-term treatment with biliary stenting has not been formally established. We compared the stone size or fragmentation of common bile duct stones after a long period of biliary stenting. Methods: Endoscopic

  17. Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

    PubMed Central

    Kuijpers, W H; Huskens, J; Koole, L H; van Boeckel, C A

    1990-01-01

    A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included. PMID:2402444

  18. Cloning and expression in Escherichia coli of a DNA fragment from Bacillus sphaericus coding for biocidal activity against mosquito larvae

    Microsoft Academic Search

    Sathyam Ganesan; Haresch Kamdar; Kunthala Jayaraman; Jekisiel Szulmajster

    1983-01-01

    A 3.7 kb DNA fragment from Bacillus sphaericus 1593 was cloned and expressed in E. coli using the plasmid pHV33. The level of larvicidal activity of the hybrid plasmid, pGsp03, against two species of mosquito larvae was comparable to that found with sporulating cell suspensions of B. sphaericus. A limited restriction cleavage map of the cloned insert is given.

  19. Cell-specific DNA fragmentation may be attenuated by a survivin-dependent mechanism after traumatic brain injury in rats

    Microsoft Academic Search

    Erik A. Johnson; Stanislav I. Svetlov; Kevin K. W. Wang; Ronald L. Hayes; Jose A. Pineda

    2005-01-01

    Survivin attenuates apoptosis by inhibiting cleavage of some cell proteins by activated caspase-3. We recently discovered\\u000a strong up-regulation of survivin, primarily in astrocytes and a sub-set of neurons, after traumatic brain injury (TBI) in\\u000a rats. In this study we characterized co-expression of survivin with activated caspase-3 and downstream DNA fragmentation (TUNEL)\\u000a in astrocytes and neurons after TBI. Western blot analysis

  20. Multi-Niche Crowding in Genetic Algorithms and its Application to the Assembly of DNA Restriction-Fragments

    Microsoft Academic Search

    Walter Cedeño; V. Rao Vemuri; Tom Slezak

    1994-01-01

    The determination of the sequence of all nucleotide base-pairs in a DNA molecule, from restriction-fragment data, is a complex t ask and can be posed as the problem of finding the optima of a multi-modal function. A genetic algorithm that uses multi-niche crowding permits us to do t his. Performance of this algorithm is first t ested using a standard

  1. Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation

    Microsoft Academic Search

    Chisato Mori; Noriko Nakamura; Yo Okamoto; Makoto Osawa; Kohei Shiota

    1994-01-01

    The process of palate fusion was examined in 13- and 14-day-old mouse fetuses by using in situ staining for nuclear DNA fragmentation (TUNEL method) and immunofluorescent staining for keratin, with special reference to the disruption of the midline epithelial seam. TUNEL-positive cells were found in the disappearing midline seam and the oral and nasal epithelial triangles at some late stages

  2. Nuclear DNA content variation in Helianthus annus and the detection of repetitive DNA sequences 

    E-print Network

    Michaelson, Martin Joseph

    1986-01-01

    restriction fragments of five H. annuus library clones and the EMBL 3 bacteriophage . . ~ 36 Interpretive drawing of Figure 2, giving sizes of restriction fragments Results of hybridization of biotinylated, whole Ha89 DHA to clone restriction fragments... transferred to a nitrocellulose filter 38 Interpretive drawinp identifying restriction fragments that hybridized the probe 39 Possible restriction maps of clone 7 based on fragment sizes from Figures 3 and 5 INTRODUCTION DNA content varies over 8 orders...

  3. [Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs].

    PubMed

    Nevinski?, G A; Potapova, I A; Tarusova, N B; Khalabuda, O V; Khomov, V V

    1990-01-01

    AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization. PMID:2161489

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  5. Diversity of fragment sizes in multifragmentation of gold nuclei induced by relativistic 3He ions

    Microsoft Academic Search

    J. Brzychczyk; E. C. Pollacco; C. Volant; D. Lacroix; R. Legrain; K K Kwiatkowski; D. S. Bracken; K. B. Morley; E. Renshaw Foxford; V. E. Viola; N. R. Yoder; J. Cugnon; R. G. Korteling; H. Breuer

    1998-01-01

    The charge-moment technique has been used to study the fragment charge distribution for the 3He(4.8 GeV)+197Au reaction. A large variety of fragment charges characterized by a relative variance ~2.3, is observed for excitation energies around 5.5 MeV\\/nucleon. Similar signals related to a phase transition are predicted by the percolation model and the statistical multifragmentation model. Effects of detector acceptance and

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

    PubMed Central

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

    1985-01-01

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

  7. Nuclear DNA Content Varies with Cell Size across Human Cell Types.

    PubMed

    Gillooly, James F; Hein, Andrew; Damiani, Rachel

    2015-01-01

    Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity. PMID:26134319

  8. Fragmentation of isomeric intrastrand crosslink lesions of DNA in an ion-trap mass spectrometer.

    PubMed

    Cao, Huachuan; Wang, Yinsheng

    2009-04-01

    The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand crosslink-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. A Dimroth-like rearrangement, which was reported for protonated 2'-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5-8]G, C[5-2]A, and C[5-8]A, but not C[5-N(2)]G or C[5-N(6)]A. In these two sets of crosslinks, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N(2)]G, C[5-N(6)]A, and unmodified cytosine, but not C[5-8]G, C[5-2]A, or C[5-8]A. In addition, ammonia could be lost more readily from C[5-N(2)]G and C[5-N(6)]A than from C[5-8]G, C[5-2]A, and C[5-8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand crosslink lesions. PMID:19103496

  9. The effect of sperm DNA fragmentation on the dynamics of the embryonic development in intracytoplasmatic sperm injection.

    PubMed

    Wdowiak, Artur; Bakalczuk, Szymon; Bakalczuk, Grzegorz

    2015-06-01

    The genetic integrity of sperm DNA can contribute to the infertility problems experienced by couples. Sperm DNA fragmentation (SDF) is the most common DNA abnormality in male gametes, and yet its effect on embryo kinetics has not been widely studied. Embryo morphokinetic parameters during the first days of embryo culture after intracytoplasmatic sperm injection (ICSI) are weakly predictive of both embryo development and pregnancy outcome. This study investigated the effect of SDF on embryo morphokinetic parameters following ICSI. The DNA fragmentation index (DFI) in spermatozoa from all males in the study (n=165) was determined before ICSI and the morphokinetic parameters of the embryos (n=165) were monitored via time-lapse recording. We found that a low DFI index in spermatozoa corresponded with embryos that reached the blastocyst stage at a faster rate after ICSI. Overall, lower SDF levels were also found in the group of patients that achieved pregnancy. Thus, higher SDF levels can slow down embryo morphokinetic parameters, and may be predictive of pregnancy outcomes after ICSI. PMID:26051457

  10. Cloning and Identification of lilyturf (Ophiopogon japonicus) RbcL cDNA fragment

    Microsoft Academic Search

    Tianqi Guo; Cong Li; Yingbo Wang; Liebao Han; Wenpan Zhang

    2011-01-01

    This study was aimed to isolate ribulose-1,5- bisphosphate carboxylase\\/oxygenase large subunit (rbcL) from lilyturf (Ophiopogon japonicus). A pair of primers used in amplifying chloroplast fragment from lilyturf was designed according to the corresponding sequences in tobacco, spinach chloroplast genomes. The fragment containing rbcL gene was cloned from lilyturf chloroplast genome. Sequencing analysis indicated that this fragment was 800 base pairs

  11. 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...

  12. Small variations in crucial steps of TUNEL assay coupled to flow cytometry greatly affect measures of sperm DNA fragmentation.

    PubMed

    Muratori, Monica; Tamburrino, Lara; Tocci, Valentina; Costantino, Antonietta; Marchiani, Sara; Giachini, Claudia; Laface, Ilaria; Krausz, Csilla; Meriggiola, Maria Cristina; Forti, Gianni; Baldi, Elisabetta

    2010-01-01

    Techniques for assessing sperm DNA damage are numerous and various. There are 2 main types of assay: direct and indirect. The former directly detects the amount of sperm DNA damage, whereas the latter reveals the effects of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal or quantify sperm DNA damage, or both, are used. Finally, these techniques, except for sperm chromatin structure assay (SCSA), lack standardized protocols to which all users can adhere to minimize interlaboratory variations. In this study, we investigated the effects of some of the many ways the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay is performed when measuring sperm DNA fragmentation by flow cytometry. In addition, by using an established procedure, we determined the precision of the technique by calculating intra-assay coefficients of variation (CVs). We found that concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks, and the method used to analyze flow cytometric data all greatly affect the measures of sperm DNA fragmentation. In particular, we found that treatment with paraformaldehyde produced additional damage in most samples, suggesting that TUNEL also can be considered an indirect assay when performed in semen samples treated with such a fixative reagent. We also showed that 2 different methods used to analyze data yielded results that, albeit correlating, were different and associated differently to semen quality. On the contrary, the TUNEL assay, as measured here, showed low intraassay CVs, resulting in a quite precise technique when performed in established conditions. PMID:19959824

  13. 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

  14. Chromosome size and DNA values in sundews ( Droseraceae )

    Microsoft Academic Search

    K. ROTIIFELS; M. Heimburger

    1968-01-01

    Relative DNA content has been determined Feulgen cytophotometrically and autoradiographically for roottip nuclei of Drosophyllum lusitanicum L., (2n=12), Drosera rotundifolia L. (2x=20), D. intermediaHayne (2x=20), D. linearisGoldie (2x= 20), D. binataLabill. (3x=32), D. capensis L. (4x= 40), D. spathulataLabill. (8x=80), all Droseraceae. Relative DNA values per diploid genome for Drosophyllum and diploid, triploid, and higher polyploid Drosera were approximately as

  15. Molecular-Sized DNA or RNA Sequencing Machine

    Cancer.gov

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

  16. Diversity of fragment sizes in multifragmentation of gold nuclei induced by relativistic {sup 3}He ions

    SciTech Connect

    Brzychczyk, J.; Pollacco, E.C.; Volant, C.; Legrain, R. [DAPNIA/SPhN CEA/Saclay, F-91191 Gif-sur-Yvette Cedex (France)] [DAPNIA/SPhN CEA/Saclay, F-91191 Gif-sur-Yvette Cedex (France); Lacroix, D. [GANIL, BP5027 F-14021 Caen Cedex (France)] [GANIL, BP5027 F-14021 Caen Cedex (France); Kwiatkowski, K.; Bracken, D.S.; Morley, K.B.; Renshaw Foxford, E.; Viola, V.E.; Yoder, N.R. [Departments of Chemistry and Physics and Indiana University Cyclotron Facility, Indiana University, Bloomington, Indiana 47405 (United States)] [Departments of Chemistry and Physics and Indiana University Cyclotron Facility, Indiana University, Bloomington, Indiana 47405 (United States); Cugnon, J. [Universite de Liege, Institut de Physique, B-4000 Liege 1 (Belgium)] [Universite de Liege, Institut de Physique, B-4000 Liege 1 (Belgium); Korteling, R.G. [Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, (Canada) V5A S16] [Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, (Canada) V5A S16; Breuer, H. [Department of Physics, University of Maryland, College Park, Maryland 20742 (United States)] [Department of Physics, University of Maryland, College Park, Maryland 20742 (United States)

    1998-09-01

    The charge-moment technique has been used to study the fragment charge distribution for the {sup 3}He(4.8thinspGeV)+{sup 197}Au reaction. A large variety of fragment charges characterized by a relative variance {approximately}2.3, is observed for excitation energies around 5.5 MeV/nucleon. Similar signals related to a phase transition are predicted by the percolation model and the statistical multifragmentation model. Effects of detector acceptance and contribution from fission are discussed. {copyright} {ital 1998} {ital The American Physical Society}

  17. Isolate and sequence ribosome-protected mRNA fragments using size-exclusion chromatography

    E-print Network

    Cai, Long

    is to isolate and sequence the nuclease-resistant ribosome-protected fragments or footprints (RPFs), which and significantly reduces the time and labor to move from cell lysis to a library ready for Illumina sequencing into an Illumina-compatible sequencing library.To generate the RPFs, cells are lysed with the included polysome

  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. fied fragments of cloned alleles were used for size determina-tion at the respective loci.

    E-print Network

    Provan, Jim

    , Chourrout D (1996) Rapid one tube DNA extraction for reliable PCR detection of fish poly- morphic markers) Inter-nest distance and sneaking in the three-spined stickleback. Animal Behaviour, 44, 793Ð795. Raymond

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

    E-print Network

    T. Hamada; R. Fujimoto; S. F. Shimobayashi; M. Ichikawa; M. Takagi

    2015-04-13

    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.

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

    E-print Network

    Hamada, T; Shimobayashi, S F; Ichikawa, M; Takagi, M

    2015-01-01

    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 molec...

  2. 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.

  3. DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction.

    PubMed

    Cortés-Gutiérrez, E I; Crespo, F; Gosálvez, A; Dávila-Rodríguez, M I; López-Fernández, C; Gósalvez, J

    2008-05-01

    The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen-thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 degrees C, 25 degrees C, and 4 degrees C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20+/-14.77% and did not differ significantly from the results of a neutral comet assay (22.0+/-19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 degrees C than when incubated at 25 degrees C or 4 degrees C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing. PMID:18367243

  4. Cloning and Stable Maintenance of 300-Kilobase-Pair Fragments of Human DNA in Escherichia coli Using an F-Factor-Based Vector

    Microsoft Academic Search

    Hiroaki Shizuya; Bruce Birren; Ung-Jin Kim; Valeria Mancino; Tatiana Slepak; Yoshiaki Tachiiri; Melvin Simon

    1992-01-01

    A bacterial cloning system for mapping and analysis of complex genomes has been developed. The BAC system (for bacterial artificial chromosome) is based on Escherichia coli and its single-copy plasmid F factor. It is capable of maintaining human genomic DNA fragments of >300 kilobase pairs. Individual clones of human DNA appear to be maintained with a high degree of structural

  5. CD34+ Cells in the Peripheral Blood Transport Herpes Simplex Virus DNA Fragments to the Skin of Patients with Erythema Multiforme (HAEM)

    Microsoft Academic Search

    Fumitake Ono; Bhuvnesh K. Sharma; Cynthia C. Smith; Joseph W. Burnett; Laure Aurelian

    2005-01-01

    Herpes simplex virus (HSV)-associated erythema multiforme (HAEM) is a recurrent disease characterized by the presence and expression of HSV DNA fragments in lesional skin. Our studies examined the mechanism of viral DNA transport to the skin of HAEM patients. CD34+ cells were isolated from the blood of normal subjects and HSV and HAEM patients during acute lesions and at quiescence.

  6. A computer simulation analysis of the accuracy of partial genome sequencing and restriction fragment analysis in estimating genetic relationships: an application to papillomavirus DNA sequences

    Microsoft Academic Search

    Baozhen Qiao; Ronald M. Weigel

    2004-01-01

    Background: Determination of genetic relatedness among microorganisms provides information necessary for making inferences regarding phylogeny. However, there is little information available on how well the genetic relationships inferred from different genotyping methods agree with true genetic relationships. In this report, two genotyping methods - restriction fragment analysis (RFA) and partial genome DNA sequencing - were each compared to complete DNA

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

    PubMed

    Abad, C; Amengual, M J; Gosálvez, J; Coward, K; Hannaoui, N; Benet, J; García-Peiró, A; Prats, J

    2013-06-01

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

  8. Constructing a DNA ladder Range for Lambda Phage by multiplex PCR

    PubMed Central

    Gopalakrishnan, R; Joseph, S; Sellappa, S

    2010-01-01

    Background and Objectives DNA ladder contains DNA fragments of different length but with known size, used to determine the size of unknown DNA molecules. Different DNA ladders are available for expected DNA length. Conserved sequences were selected for design of primers to generate DNA fragments of known specific size. Materials and Methods In this study, we describe a method by which DNA ladder was prepared based on multiplex PCR technique. Different lengths of DNA fragments were amplified using the primers designed according to the 1216-2136 sequence extent of lambda phage DNA. Target DNA fragments were amplified using multiplex PCR and extracted. Results The results showed an amplified lambda phage DNA at particular target sites by using 1 forward and 6 different reverse primers (for 100, 200, 400, 600, 800, 1000bp) for the successful amplification. Conclusion This method would be more cost effective than commercial DNA molecular weight markers. PMID:22347574

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

    PubMed

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

    2013-12-01

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

  10. 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

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

    E-print Network

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

    2009-09-01

    cannabi- September 2009 2581FRAGMENTATION AND PLANT SPECIES DECLINES num, Solidago canadensis, Melilotus spp., Helianthus annuus, Aster praealtus) increased between the initial survey (1985) and 1995, but then showed substantial declines in 2000. For three... range of the Iberian Lynx. Ecography 25:314–328. Schoener, T. W. In press. The MacArthuer-Wilson equilibrium model: a chronicle of theoretical modification and real-world evaluation. In J. Losos and R. Ricklefs, editors. Island bio- geography at 40...

  12. Estimating Dataset Size Requirements for Classifying DNA Microarray Data

    Microsoft Academic Search

    Sayan Mukherjee; Pablo Tamayo; Simon Rogers; Ryan M. Rifkin; Anna Engle; Colin Campbell; Todd R. Golub; Jill P. Mesirov

    2003-01-01

    A statistical methodology for estimating dataset size requirements for classifying microarray data using learning curves is introduced. The goal is to use existing classié cation results to estimate dataset size requirements for future classié cation experiments and to evaluate the gain in accuracy and signié cance of classié ers built with additional data. The method is based on é tting

  13. 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.

  14. Detection of Genomic DNA Fragmentation during Apoptosis (DNA Ladder) and the Simultaneous Isolation of RNA from Low Cell Numbers

    Microsoft Academic Search

    Peter T. Daniel; Isrid Sturm; Silke Ritschel; Katrin Friedrich; Bernd Dörken; Peter Bendzko; Timo Hillebrand

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the

  15. DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine.

    PubMed Central

    Kwok, J. B.; Tattersall, M. H.

    1992-01-01

    Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death. Images Figure 3 PMID:1562458

  16. 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

  17. Characterization of DNA in cell culture supernatant by fluorescence-detection size-exclusion chromatography.

    PubMed

    Tan, Lihan; Yeo, Veronica; Yang, Yuansheng; Gagnon, Pete

    2015-05-01

    A fluorescence-detection size-exclusion chromatography (FSEC) method was developed to characterize DNA in cell culture supernatant. Samples stained with Picogreen were fractionated by size-exclusion chromatography (SEC) and monitored simultaneously by UV absorbance and fluorescence. SEC provided a size-characterization capability absent from bulk fluorescent assays, and was also free from interference from other fluorescent and UV-absorbing small-molecule cell culture components. FSEC revealed that DNA in mammalian cell culture supernatant exists mostly in the form of nucleosomal arrays. FSEC combined with agarose electrophoresis revealed spontaneous degradation of DNA in mammalian cell culture supernatant over a 30 day period at 4 °C: from arrays containing up to ~40 nucleosomes, down to arrays containing three or fewer nucleosomes. It also detected nucleosomal DNA in wheat, soy, and yeast hydrolysates commonly used to enhance cell culture productivity. PMID:25821116

  18. Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

    PubMed Central

    Woods, D E; Edge, M D; Colten, H R

    1984-01-01

    Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases. Images PMID:6086718

  19. Distribution and size of capercaillie leks in relation to old forest fragmentation

    Microsoft Academic Search

    J. Rolstad; P. Wegge

    1987-01-01

    Distribution and size of 38 capercaillie Tetrao urogallus leks were related to amount and configuration of old forest patches in two south-east Norwegian coniferous forests. The smallest occupied patch was 48 ha containing a solitary displaying cock. All patches larger than 1 km2 contained leks. Number of cocks per lek increased with increasing patch size. Number of leks per patch

  20. 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

  1. Relationship of seminal reactive nitrogen and oxygen species and total antioxidant capacity with sperm DNA fragmentation in infertile couples with normal and abnormal sperm parameters.

    PubMed

    Khosravi, F; Valojerdi, M R; Amanlou, M; Karimian, L; Abolhassani, F

    2012-11-01

    The objective of this study was to investigate the association between the amount of superoxide anion, peroxynitrite as oxidative stress (OS) markers and total antioxidant capacity (TAC) with sperm DNA fragmentation in infertile men with abnormal semen parameters. Semen samples were obtained from 102 infertile couples and divided into groups with normal and abnormal semen parameters according to the World Health Organization (WHO). Peroxynitrite and superoxide anions were detected using spectrofluorometric assays combined with 2,7 dicholorofluorescein (DCF)-DA and 4-chloro-7-nitrobenzo-2-oxa -1, 3-diazole (NBD-CL). Colorimetric assay was used for evaluation of TAC, while DNA fragmentation was studied by using sperm chromatin dispersion test. Superoxide anion, peroxynitrite and DNA fragmentation were significantly higher in infertile couples with abnormal semen parameters as compared to infertile couples with normal semen (P < 0.01). TAC was significantly lower in infertile men with abnormal semen parameters (P < 0.01). There was also a significant positive correlation between OS markers with sperm DNA fragmentation (r = 0.59, P < 0.01 and r = 0.67, P < 0.01, respectively). We have found that imbalance between superoxide anion and peroxynitrite with antioxidant capacity in infertile men with abnormal sperm parameters is associated with higher sperm DNA fragmentation. PMID:23126684

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

    PubMed

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

    2008-05-01

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

  3. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation.

    PubMed Central

    Waxman, A B; Einarsson, O; Seres, T; Knickelbein, R G; Warshaw, J B; Johnston, R; Homer, R J; Elias, J A

    1998-01-01

    Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O2 on transgenic mice that overexpress IL-11 in the lung and transgene (-) controls. IL-11 markedly enhanced survival in 100% O2 with 100% of transgene (-) animals dying within 72-96 h and > 90% of transgene (+) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene (+) and (-) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene (+) as versus (-) mice after 100% O2 exposure, and IL-11 diminished hyperoxia-induced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation. PMID:9576762

  4. 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

  5. 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 ?-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

  6. Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

    PubMed

    Daniel, P T; Sturm, I; Ritschel, S; Friedrich, K; Dörken, B; Bendzko, P; Hillebrand, T

    1999-01-01

    In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor. PMID:9887219

  7. Strongly structured DNA sequences as targets for genosensing: sensing phase design and coupling to PCR amplification for a highly specific 33-mer gliadin DNA fragment.

    PubMed

    Martín-Fernández, Begoña; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús; Frutos-Cabanillas, Gloria; de-los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz

    2014-10-15

    Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour. PMID:24813914

  8. DNA Diffusion in Mucus: Effect of Size, Topology of DNAs, and Transfection Reagents

    PubMed Central

    Shen, Hong; Hu, Yueyue; Saltzman, W. M.

    2006-01-01

    DNA represents a promising therapeutic and prophylactic macromolecule in treating genetic diseases, infectious diseases and cancers. The therapeutic potential of DNA is directly related to how DNA transports within the targeted tissue. In this study, fluorescence photobleaching recovery was used to examine the diffusion of plasmid DNAs with various size (2.7 ? 8.3 kb), topology, and in the presence of transfection reagents in mucus. We observed that DNAs diffused slower when size of DNAs increased; supercoiled DNAs diffused faster than linear ones; mucus did not reduce the diffusion of linear DNAs but retarded the diffusion of supercoiled DNAs. Diffusion data were fitted to models of a polymer chain diffusing in gel systems. Diffusion of linear DNAs in mucus were better described by the Zimm model with a scaling factor of ?0.8, and supercoiled DNAs showed a reptational behavior with a scaling factor of ?1.3. Based on the Zimm model, the pore size of bovine mucus was estimated and agreed well with previous experimental data. In the presence of transfection reagents, e.g., liposomes, the diffusion of DNAs increased by a factor of 2 in mucus. By using bovine mucus as a model system, this work suggests that DNA size, topology, and the presence of transfection reagents may affect the diffusion of DNA in tissues, and thus the therapeutic effects of DNA. PMID:16632500

  9. Summary. We present a method of extracting DNA from medium-sized paper wasps without sacrificing the animal.

    E-print Network

    Starks, Philip

    the animal. From the distal half of a single leg, enough DNA is extracted for 125 PCR reactions. This DNA animals appeared to behave normally. This DNA extraction method is well suited for studies that combineSummary. We present a method of extracting DNA from medium-sized paper wasps without sacrificing

  10. Interspecific rDNA restriction fragment length polymorphism in Globodera species, parasites of Solanaceous plants

    Microsoft Academic Search

    Murielle THIÉRY; Didier MUGNIÉRY

    1996-01-01

    Swnmary - The status of the Globodera species that are parasites of Solanaceous is still unclear due ta low morphological polymorphism. Two 21-mers oligonucleotide primers were used ta amplify the internai Transcribed Spacer (ITS). The amplified product was aproximately 1.2 Kb in the 26 Globodera populations examined. Thirty-one restriction enzymes were tested, of which twelve produced some fragment length polymorphism.

  11. DNA ruler : enhancing nanopore sizing resolution by multiple measurements on the same DNA molecule

    E-print Network

    Sen, Yi-Heng

    2012-01-01

    Nanopores are versatile sensors for label-free detection of single molecules and particles that have attracted attention for applications such as DNA sequencing and nanoparticle analysis. Detection of single molecules or ...

  12. Gene organization in a central DNA fragment of Oenococcus oeni bacteriophage fOg44 encoding lytic, integrative and non-essential functions

    Microsoft Academic Search

    Ricardo Parreira; Carlos São-José; Anabela Isidro; Susana Domingues; Graça Vieira; Mário A. Santos

    1999-01-01

    The nucleotide sequence of a DNA fragment previously shown to contain the attachment site (attP) of Oenococcus oeni phage fOg44 (Santos et al., 1998. Arch. Virol. 143, 523–536) has been determined. Sequence analysis indicated that this 6226bp EcoRI fragment harbours an integrase gene, in the vicinity of a direct repeat rich region defining attP, as well as genes encoding a

  13. 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. (Los Alamos National Lab., NM (United States)); Haces, A.; Shih, P.J.; Harding, J.D. (Life Technologies, Inc., Gaithersburg, MD (United States))

    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.

  14. 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. [Los Alamos National Lab., NM (United States); Haces, A.; Shih, P.J.; Harding, J.D. [Life Technologies, Inc., Gaithersburg, MD (United States)

    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.

  15. Polymer Size Effect on Shape and Position in DNA Trap by Electric and Hydrodynamic Force Fields

    NASA Astrophysics Data System (ADS)

    Ueno, Kunimitsu; Nagasaka, Wako; Tomizawa, Yuichi; Nakamori, Yoshiteru; Tamiya, Eiichi; Takamura, Yuzuru

    2007-08-01

    In recent years, a phenomenon has been discovered wherein charged particles are trapped near the constricted position of a microfluidic tapering channel when hydropressure and electric field are applied in opposite directions. By this phenomenon, the existence of various types of trapping patterns has already been demonstrated in a T4 phage DNA trapping experiment. In this study, we aim to explain the mechanism of this phenomenon. The relationship between DNA size and trapping pattern was examined using five different sizes of DNA samples. As a result, the trapping pattern and trapping condition extremely different with size when the most stable and clearest trapping image is observed. That is, the main trapping patterns change markedly in shapes and trapped positions, and large DNA traps occur under the conditions of low pressure and low voltage, whereas small size traps occur under the conditions of high pressure and high voltage. Another important result is that the ratio of the pressure and the voltage is independent of size when the main trap is observed. These findings suggest that this trap has size selectivity and molecular selectivity for extracting target molecules from a mixed solution.

  16. Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments.

    PubMed Central

    Renders, N; Römling, Y; Verbrugh, H; van Belkum, A

    1996-01-01

    Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms. PMID:8940470

  17. Insights on genome size evolution from a miniature inverted repeat transposon driving a satellite DNA.

    PubMed

    Scalvenzi, Thibault; Pollet, Nicolas

    2014-12-01

    The genome size in eukaryotes does not correlate well with the number of genes they contain. We can observe this so-called C-value paradox in amphibian species. By analyzing an amphibian genome we asked how repetitive DNA can impact genome size and architecture. We describe here our discovery of a Tc1/mariner miniature inverted-repeat transposon family present in Xenopus frogs. These transposons named miDNA4 are unique since they contain a satellite DNA motif. We found that miDNA4 measured 331 bp, contained 25 bp long inverted terminal repeat sequences and a sequence motif of 119 bp present as a unique copy or as an array of 2-47 copies. We characterized the structure, dynamics, impact and evolution of the miDNA4 family and its satellite DNA in Xenopus frog genomes. This led us to propose a model for the evolution of these two repeated sequences and how they can synergize to increase genome size. PMID:25193611

  18. An Interface for a Fragment Assembly Kernel

    Microsoft Academic Search

    Susan Larson; Mudita Jain; Eric Anson; Gene Myers

    ABSTRACT This document,describes the C programming,language interface to our Fragment Assembly Kernel library. Inputs to the Fragment Assembly Kernel are (1) DNA fragment sequences from potentially inaccurate sequencing experiments, and (2) optional constraints on fragment assembly such as known fragment overlaps or relative fragment orientation. Fragment sequence version control is supported. The Fragment Assembly Kernel produces the most probable reconstructions

  19. dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model.

    PubMed

    Paris, Roberta; Dondini, Luca; Zannini, Graziano; Bastia, Daniela; Marasco, Elena; Gualdi, Valentina; Rizzi, Valeria; Piffanelli, Pietro; Mantovani, Vilma; Tartarini, Stefano

    2012-05-01

    cDNA-AFLP analysis for transcript profiling has been successfully applied to study many plant biological systems, particularly plant-microbe interactions. However, the separation of cDNA-AFLP fragments by gel electrophoresis is reported to be labor-intensive with only limited potential for automation, and the collection of differential bands for gene identification is even more cumbersome. In this work, we present the use of dHPLC (denaturing high performance liquid chromatography) and automated DNA fragment collection using the WAVE(®) System to analyze and recover cDNA-AFLP fragments. The method is successfully applied to the Malus-Venturia inaequalis interaction, making it possible to collect 66 different transcript-derived fragments for apple genes putatively involved in the defense response activated by the HcrVf2 resistance gene. The results, validated by real time quantitative RT-PCR, were consistent with the plant-pathogen interaction under investigation and this further supports the suitability of dHPLC for cDNA-AFLP transcript profiling. Features and advantages of this new approach are discussed, evincing that it is an almost fully automatable and cost-effective solution for processing large numbers of samples and collecting differential genes involved in other biological processes and non-model plants. PMID:22270558

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

    PubMed Central

    Akita, Hidetaka; Kurihara, Dai; Schmeer, Marco; Schleef, Martin; Harashima, Hideyoshi

    2015-01-01

    The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer. PMID:26066769

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

    PubMed

    Akita, Hidetaka; Kurihara, Dai; Schmeer, Marco; Schleef, Martin; Harashima, Hideyoshi

    2015-01-01

    The nuclear transfer process is one of the critical rate-limiting processes in transgene expression. In the present study, we report on the effect of compaction and the size of the DNA molecule on nuclear transfer efficiency by microinjection. A DNA/protamine complex- or variously-sized naked DNA molecules were injected into the cytoplasm or nucleus of synchronized HeLa cells. To evaluate the nuclear transfer process, a nuclear transfer score (NT score), calculated based on transgene expression after cytoplasmic microinjection divided by that after nuclear microinjection, was employed. The compaction of DNA with protamine decreased the NT score in comparison with the injection of naked DNA when the N/P ratio was increased to >2.0. Moreover, when naked DNA was microinjected, gene expression increased in parallel with the size of the DNA in the following order: minicircle DNA (MC07.CMV-EGFP; 2257 bp) > middle-sized plasmid DNA (pBS-EGFP; 3992 bp) > conventional plasmid DNA (pcDNA3.1-EGFP; 6172 bp), while the level of gene expression was quite comparable among them when the DNAs were injected into the nucleus. The above findings suggest that the intrinsic size of the DNA molecule is a major determinant for nuclear entry and that minicircle DNA has a great advantage in nuclear transfer. PMID:26066769

  2. Rapid parallel mutation scanning of gene fragments using a microelectronic protein–DNA chip format

    PubMed Central

    Behrensdorf, Heike A.; Pignot, Marc; Windhab, Norbert; Kappel, Andreas

    2002-01-01

    We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research. PMID:12136112

  3. A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

    PubMed Central

    Emond, E; Fliss, I; Pandian, S

    1993-01-01

    cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate. Images PMID:8368854

  4. Restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from mapped loci of rice ( Oryza sativa L.) genomic DNA

    Microsoft Academic Search

    M. N. V. Williams; N. Pande; S. Nair; M. Mohan; J. Bennett

    1991-01-01

    Thirty mapped Indica rice genomic (RG) clones were partially sequenced from each end. From such sequence data, pairs of oligonucleotides were synthesized to act as primers for polymerase chain reaction (PCR) amplification of the corresponding loci in crude total DNA preparations. The PCR products from DNA of Indica varieties were of the sizes expected from the sizes of the corresponding

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

    Microsoft Academic Search

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

    1991-01-01

    The extent and type of chloroplast DNA restriction fragment length polymorphism was determined among individual tree samples of coast redwood, Douglas fir, incense-cedar, and loblolly pine. A total of 107 trees was surveyed for three restriction enzymes (BamHI, EcoRI, HindIII) and six chloroplast DNA probes from petunia (P3, P4, P6, P8, P10, S8). The probes comprise 64% of the petunia

  6. Damage to model DNA fragments from very low-energy (<1 eV) electrons.

    PubMed

    Berdys, Joanna; Anusiewicz, Iwona; Skurski, Piotr; Simons, Jack

    2004-05-26

    Although electrons having enough energy to ionize or electronically excite DNA have long been known to cause strand breaks (i.e., bond cleavages), only recently has it been suggested that even lower-energy electrons (most recently 1 eV and below) can also damage DNA. The findings of the present work suggest that, while DNA bases can attach electrons having kinetic energies in the 1 eV range and subsequently undergo phosphate-sugar O-C sigma bond cleavage, it is highly unlikely (in contrast to recent suggestions) that electrons having kinetic energies near 0 eV can attach to the phosphate unit's P=O bonds. Electron kinetic energies in the 2-3 eV range are required to attach directly to DNA's phosphate group's P=O pi orbital and induce phosphate-sugar O-C sigma bond cleavages if the phosphate groups are rendered neutral (e.g., by nearby counterions). Moreover, significant activation barriers to C-O bond breakage render the rates of both such damage mechanisms (i.e., P=O-attached and base-attached) slow as compared to electron autodetachment and to other damage processes. PMID:15149241

  7. A Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome

    E-print Network

    Nicolescu, Monica

    is influenced by their habitat. Most microorganisms genomes are known from pure cul- tures of organisms isolated natural habitat as part of a community. Research has broadened from studying single species for isolation and lab cultivation of individual species [4]. The whole genome(DNA) or metagenome population can

  8. Multiplex analysis of DNA

    DOEpatents

    Church, George M. (Boston, MA); Kieffer-Higgins, Stephen (Dorchester, MA)

    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.

  9. Analysis of conflicting experimental studies of DNA size in nanofluidic slits

    NASA Astrophysics Data System (ADS)

    Stavis, Samuel M.; Strychalski, Elizabeth A.; Nablo, Brian J.; Geist, Jon

    2013-03-01

    Recent experimental studies have reported conflicting accounts of the size variation of DNA in nanofluidic slitlike confinement; [Bonthuis et al., Physical Review Letters 101, 10, 108303 (2008)], [Tang et al., Macromolecules 43, 17, 7368 (2010)], [Strychalski et al., Macromolecules 45, 3, 1602 (2012)], [Lin et al., Macromolecules 45, 6, 2920 (2012)], [Dai et al., Soft Matter 8, 10, 2972 (2012)]. In an effort to resolve this controversy, these studies are analyzed by a reductive as opposed to predictive approach. Minimum references for DNA size (baselines) are simulated by a Monte Carlo methodology and quantitatively compared to measured and inferred DNA sizes. The measurements of Tang et al., Strychalski et al., and Lin et al. are consistent with the related baselines and in semi-quantitative agreement with each other. The inferences of Tang et al. and Dai et al. are consistent with the related baseline and in qualitative agreement with the measurements of Tang et al., Strychalski et al., and Lin et al. The measurements of Bonthuis et al. are inconsistently larger than the related baseline and the other experimental measurements and inferences of DNA size around the transition from moderate to weak slitlike confinement. A variety of physical and chemical differences between the experimental systems are examined in detail to elucidate this inconsistency. Detailed analyses of the baseline distribution and variation clarify several core physical attributes of the system related to excluded volume effects and chain dimensionality.

  10. Fragmentation of condensed-phase DNA components by hyperthermal He{sup +} impact

    Microsoft Academic Search

    Deng Zongwu; Marjorie Imhoff; Ilko Bald; Eugen Illenberger; Michael A. Huels

    2006-01-01

    We have observed severe damage to films of DNA components (thymine, D-ribose, 2-deoxy-D-ribose, and thymidine) induced by 10 to 100 eV He{sup +} ions (2.5-25 eV\\/amu). The damage is attributed to the kinetic and potential energies, as well as the chemical reactivity of the He{sup +} projectiles. Hyperthermal He{sup +} ion impact on these films results in the complete destruction

  11. Protein-Mediated DNA Loops: Effects of Protein Bridge Size and Kinks

    E-print Network

    Nicolas Douarche; Simona Cocco

    2005-11-16

    This paper focuses on the probability that a portion of DNA closes on itself through thermal fluctuations. We investigate the dependence of this probability upon the size r of a protein bridge and/or the presence of a kink at half DNA length. The DNA is modeled by the Worm-Like Chain model, and the probability of loop formation is calculated in two ways: exact numerical evaluation of the constrained path integral and the extension of the Shimada and Yamakawa saddle point approximation. For example, we find that the looping free energy of a 100 base pairs DNA decreases from 24 kT to 13 kT when the loop is closed by a protein of r = 10 nm length. It further decreases to 5 kT when the loop has a kink of 120 degrees at half-length.

  12. 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

  13. Structural, Dynamical and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Sumpter, Bobby G [ORNL; Fuentes-Cabrera, Miguel A [ORNL

    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. Multiphoton Dissociation of Electrosprayed MegaDalton-Sized DNA Ions in a Charge-Detection Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Doussineau, Tristan; Paletto, Pierre; Dugourd, Philippe; Antoine, Rodolphe

    2015-01-01

    Charge detection mass spectrometry in combination with a linear electrostatic ion trap coupled to a continuous wavelength infrared CO2 laser has been used to study the multiphoton dissociation of DNA macromolecular ions. Samples, with masses ranging from 2.23 to 31.5 MDa, include single strand circular M13mp18, double strand circular M13mp18, and double strand linear LambdaPhage DNA fragments. Their activation energies for unimolecular dissociation were determined. Activation energy values slightly increase as a function of the molecular weight. The most important result is the difference between the fragmentations observed for hybridized double-strands and dimers of single strands.

  15. 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

  16. Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients

    PubMed Central

    Frenkel, Niza; Roizman, Bernard

    1972-01-01

    Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 × 106 daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 × 106 to 39 × 106 daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with 3H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments. PMID:4343538

  17. Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis.

    PubMed

    Shen, Xuan; Chen, Xiaomu; Tabor, David E; Liu, Yi; Albarghouthi, Methal; Zhang, Yi-Fan; Galinski, Mark S

    2013-05-01

    Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples. PMID:23313102

  18. Sorbent Inventory and Particle Size Distribution in Air-Blown Circulating Fluidized Bed Combustors: The Influence of Particle Attrition and Fragmentation

    NASA Astrophysics Data System (ADS)

    Montagnarn, Fabio; Salatino, Piero; Scala, Fabrizio; Urcluokr, Massimo

    Attrition and fragmentation of limestone during FB combustion of sulphur-bearing fuels have a profound influence on sorbent inventory and particle size distribution establishing at steady state in the bed. A population balance model is presented aiming at the prediction of the inventory and of the particle size distribution of sorbent particles establishing at steady state in the bed of an air-blown CFBC. The core of the model is represented by a population balance equation on sorbent particles which embodies terms expressing the extent/rate of each attrition/fragmentation process. The effect of the progress of sulphation on attrition/fragmentation is also taken into account. Constitutive equations needed to quantify attrition/fragmentation are developed on the basis of published data. Model results are presented and discussed with the aim of clarifying the influence of particle attrition/fragmentation on sorbent inventory and particle size distribution in a CFBC and on the closely related variables. Research needs and priorities within the specific field of investigation are also discussed.

  19. High content, size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.)

    Microsoft Academic Search

    Steffen Backert; Rudi Lurz; Omar A. Oyarzabal; Thomas Börner

    1997-01-01

    Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknownfeature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be

  20. 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

  1. DNA “fingerprints” detect genetic variation in Acer negundo ( Aceraceae )

    Microsoft Academic Search

    Hilde Nybom; Steven H. Rogstad

    1990-01-01

    Genomic DNA samples from 21 box elder plants collected in Missouri (U.S.A.) were digested with restriction enzyme and southern blot hybridized with the M13 minisatellite probe. Each plant was found to have a unique DNA fragment pattern. Moreover, levels of genetic variation estimated from a similarity index appear to be related to sampling distances. However, size of the fragments utilized

  2. How realistic is the pore size distribution calculated from adsorption isotherms if activated carbon is composed of fullerene-like fragments?w

    E-print Network

    Harris, Peter J F

    of adsorption on fullerene-like carbon models. 1. Introduction and the aim of the study Activated carbonsHow realistic is the pore size distribution calculated from adsorption isotherms if activated carbon is composed of fullerene-like fragments?w Artur P. Terzyk,*a Sylwester Furmaniak,a Peter J. F

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

    PubMed Central

    Rotman, Ella; Kuzminov, Andrei

    2007-01-01

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

  4. Probability of double-strand breaks in genome-sized DNA by {gamma}-ray decreases markedly as the DNA concentration increases

    SciTech Connect

    Shimobayashi, Shunsuke F. [Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan); Iwaki, Takafumi [Fukui Institute for Fundamental Chemistry, Kyoto University, Kyoto 606-8103 (Japan); Mori, Toshiaki [Radiation Research Center, Osaka Prefecture University, Sakai 599-8570 (Japan); Yoshikawa, Kenichi [Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502 (Japan); Faculty of Life and Medical Sciences, Doshisha University, Kyoto 610-0394 (Japan)

    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.

  5. An amphioxus RAG1-like DNA fragment encodes a functional central domain of vertebrate core RAG1

    PubMed Central

    Zhang, Yanni; Xu, Ke; Deng, Anqi; Fu, Xing; Xu, Anlong; Liu, Xiaolong

    2014-01-01

    The highly diversified repertoire of antigen receptors in the vertebrate immune system is generated via proteins encoded by the recombination activating genes (RAGs) RAG1 and RAG2 by a process known as variable, diversity, and joining [V(D)J] gene recombination. Based on the study of vertebrate RAG proteins, many hypotheses have been proposed regarding the origin and evolution of RAG. This issue remains unresolved, leaving a significant gap in our understanding of the evolution of adaptive immunity. Here, we show that the amphioxus genome contains an ancient RAG1-like DNA fragment (bfRAG1L) that encodes a virus-related protein that is much shorter than vertebrate RAG1 and harbors a region homologous to the central domain of core RAG1 (cRAG1). bfRAG1L also contains an unexpected retroviral type II nuclease active site motif, DXN(D/E)XK, and is capable of degrading both DNA and RNA. Moreover, bfRAG1L shares important functional properties with the central domain of cRAG1, including interaction with RAG2 and localization to the nucleus. Remarkably, the reconstitution of bfRAG1L into a cRAG1-like protein yielded an enzyme capable of recognizing recombination signal sequences and performing V(D)J recombination in the presence of mouse RAG2. Moreover, this reconstituted cRAG1-like protein could mediate the assembly of antigen receptor genes in RAG1-deficient mice. Together, our results demonstrate that amphioxus bfRAG1L encodes a protein that is functionally equivalent to the central domain of cRAG1 and is well prepared for further evolution to mediate V(D)J recombination. Thus, our findings provide unique insights into the evolutionary origin of RAG1. PMID:24368847

  6. The Opisthorchis felineus paramyosin: cDNA sequence and characterization of its recombinant fragment.

    PubMed

    Shustov, A V; Kotelkin, A T; Sorokin, A V; Ternovoi, V A; Loktev, V B

    2002-08-01

    The cDNA sequence of Opisthorchis felineus paramyosin (PM) was determined and shown to have 66-70% homology with two schistosomes and two cestodes. Phylogenetic analysis revealed an almost equal distance between O. felineus and these distinct clades. Because of its relatively low conservation, the PM gene may be a convenient genetic marker for studying phylogenetic relationships among platyhelminthes.A 25-kDa recombinant polypeptide corresponding to the central part of the full-length PM was produced. In Western blot analysis, murine hyperimmune serum against recombinant PM (recPM) detected 100-kDa polypeptides in the O. felineusegg and somatic antigens. Interactions of recPM with polyclonal anti-parasite antibodies and anti-recPM sera in ELISA with native antigens demonstrated that recPM carries a B cell epitope identical to the O. felineusnative antigen. Our sequence and immunologic data may be helpful in developing new diagnostic tools and candidate vaccines for O. felineus infection. PMID:12122429

  7. DNA variation in the wild plant Arabidopsis thaliana revealed by amplified fragment length polymorphism analysis.

    PubMed Central

    Miyashita, N T; Kawabe, A; Innan, H

    1999-01-01

    To investigate the level and pattern of DNA variation of Arabidopsis thaliana at the entire genome level, AFLP analysis was conducted for 38 ecotypes distributed throughout the world. Ten pairs of selective primers were used to detect a total of 472 bands, of which 374 (79. 2%) were polymorphic. The frequency distribution of polymorphic bands was skewed toward an excess of singleton variation. On the basis of AFLP variation, nucleotide diversity for the entire genome was estimated to be 0.0106, which was within the range reported previously for specific nuclear genes. The frequency distribution of pairwise distance was bimodal because of an ecotype (Fl-3) with a large number of unique bands. Linkage disequilibrium between polymorphic AFLPs was tested. The proportion of significant linkage disequilibria was close to random expectation after neglecting the ecotype Fl-3. This result indicates that the effect of recombination could not be ignored in this selfing species. A neighbor-joining tree was constructed on the basis of the AFLP variation. This tree has a star-like topology and shows no clear association between ecotype and geographic origin, suggesting a recent spread of this plant species and limited migration between its habitats. PMID:10430596

  8. EGassembler: online bioinformatics service for large-scale processing, clustering and assembling ESTs and genomic DNA fragments.

    PubMed

    Masoudi-Nejad, Ali; Tonomura, Koichiro; Kawashima, Shuichi; Moriya, Yuki; Suzuki, Masanori; Itoh, Masumi; Kanehisa, Minoru; Endo, Takashi; Goto, Susumu

    2006-07-01

    Expressed sequence tag (EST) sequencing has proven to be an economically feasible alternative for gene discovery in species lacking a draft genome sequence. Ongoing large-scale EST sequencing projects feel the need for bioinformatics tools to facilitate uniform EST handling. This brings about a renewed importance for a universal tool for processing and functional annotation of large sets of ESTs. EGassembler (http://egassembler.hgc.jp/) is a web server, which provides an automated as well as a user-customized analysis tool for cleaning, repeat masking, vector trimming, organelle masking, clustering and assembling of ESTs and genomic fragments. The web server is publicly available and provides the community a unique all-in-one online application web service for large-scale ESTs and genomic DNA clustering and assembling. Running on a Sun Fire 15K supercomputer, a significantly large volume of data can be processed in a short period of time. The results can be used to functionally annotate genes, to facilitate splice alignment analysis, to link the transcripts to genetic and physical maps, design microarray chips, to perform transcriptome analysis and to map to KEGG metabolic pathways. The service provides an excellent bioinformatics tool to research groups in wet-lab as well as an all-in-one-tool for sequence handling to bioinformatics researchers. PMID:16845049

  9. 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. (Thomas Jefferson Univ., Philadelphia, PA (United States)); Lia, K.; Silverman, G.A. (Harvard Medical School, Boston, MA (United States))

    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.

  10. Size-Expanded yDNA bases: An Ab Initio Study

    SciTech Connect

    Fuentes-Cabrera, Miguel A [ORNL; Sumpter, Bobby G [ORNL; Lipkowski, Pawel [Wroclaw University of Technology, Poland; Wells, Jack C [ORNL

    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.

  11. Impact of plasmid size on the purification of model plasmid DNA vaccines by phenyl membrane adsorbers.

    PubMed

    Raiado-Pereira, Luís; Prazeres, D Miguel F; Mateus, Marília

    2013-11-01

    Plasmid DNA (pDNA) offers a versatile platform for the development of new pharmaceuticals. This versatility also adds in variability among plasmid products most of the times sharing only the same basic molecular structure. Membrane chromatography experiments performed with a Sartorius(®) Phenyl 3 mL spiral cartridge and differently sized plasmids (3.70 kbp, 6.05 kbp and 10.4 kbp) show that the strength of interaction of pDNA isoforms with HIC membrane adsorbers depends on size. These differences in relative binding strength were explored using a stepwise elution strategy of decreasing buffer conductivities in order to increase the purity of supercoiled (SC) pDNA isoforms. The open circular (OC) isoforms of all plasmids eluted earlier at a similar conductivity of 190 mS/cm, independently of the hydrodynamic diameter (Dh). A drop in conductivity of 16.0 mS/cm, 23 mS/cm and 19 mS/cm had to be imposed to elute the supercoiled (SC) counterparts of the 3.70 kbp, 6.05 kbp and 10.4 kbp, respectively. This corresponds to relative binding strengths of the SC over OC isoforms of 1.09, 1.14 and 1.11. Unlike the OC isoforms, the behavior of SC isoforms was dependent of the Dh. The purified and pooled plasmid fractions were assayed and demonstrated high degree of purity, compliant with regulatory agencies criteria: over 99% RNA removal, endotoxin levels below 0.001 EU/?g pDNA and undetectable protein content by BCA assay. PMID:24103809

  12. 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

  13. Molecular Characterization of a Brucella Species Large DNA Fragment Deleted in Brucella abortus Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide

    PubMed Central

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

    1999-01-01

    A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably 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 described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the sequenced B. melitensis DNA fragment. PMID:10338472

  14. An RAPD (Random Amplified Polymorphic DNA) Analysis of Genetic Population Structure of Balea biplicata (Gastropoda: Clausiliidae) in Fragmented Floodplain Forests of the Elster\\/Saale Riparian System

    Microsoft Academic Search

    A. Hille; K. Liebal; B. Mosch; H. Pellmann; M. Schlegel

    2003-01-01

    Eight German populations of the land snail Balea biplicata(Mollusca: Clausiliidae) were studied using the randomly amplified polymorphic DNA-polymerase chain reaction and morphometrics (principal component and discriminant analysis) to examine population structure and gene flow patterns in a fragmented landscape mosaic along the Elster\\/Saale riparian system, Germany. A variety of population genetic analyses targeting either more on the geographic scale of

  15. Protective Effects of Grape Seed Proanthocyanidins and Selected Antioxidants against TPA-Induced Hepatic and Brain Lipid Peroxidation and DNA Fragmentation, and Peritoneal Macrophage Activation in Mice

    Microsoft Academic Search

    D Bagchi; A Garg; R. L Krohn; M Bagchi; D. J Bagchi; J Balmoori; S. J Stohs

    1998-01-01

    1.The comparative protective abilities of a grape seed proanthocyanidin extract (GSPE) (25–100 mg\\/kg), vitamin C (100 mg\\/kg), vitamin E succinate (VES) (100 mg\\/kg) and ?-carotene (50 mg\\/kg) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation and DNA fragmentation in the hepatic and brain tissues, as well as production of reactive oxygen species by peritoneal macrophages, were assessed.2.Treatment of mice with GSPE (100 mg\\/kg),

  16. DNA sequence polymorphisms in the genus saccharomyces III. Restriction endonuclease fragment patterns of chromosomal regions in brewing and other yeast strains

    Microsoft Academic Search

    Mogens Bohl Pedersen

    1986-01-01

    30 bottom fermenting strains, 13 hybrid lager strains, 11 top fermenting strains. In addition, 24 other yeast species and\\u000a strains have been analysed in this study. Four DNA regions have been monitored by restriction endonuclease fragment pattern\\u000a polymorphisms. The regions analysed are:RDN1 (encoding cytosolic ribosomal RNA molecules) located on chromosome XII in S. cerevisiae,HIS4 (histidine 4) andLEU2 (leucine 2) both

  17. 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. [CEA, DAM, DIF, F-91297 Arpajon (France)

    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.

  18. Clear Genetic Structure of Pinus kwangtungensis (Pinaceae) Revealed by a Plastid DNA Fragment with a Novel Minisatellite

    PubMed Central

    Tian, Shuang; Luo, Lai-Chun; Ge, Song; Zhang, Zhi-Yong

    2008-01-01

    Background and Aims Pinus kwangtungensis is a five-needled pine, inhabiting isolated mountain tops, cliffs or slopes in the montane areas of southern China and northern Vietnam. Global warming and long-term deforestation in southern China threaten its existence and genetic integrity, and this species is listed as vulnerable in the China Species Red List. However, the level and distribution of genetic diversity in this vulnerable species are completely unknown. In this paper, the genetic diversity and structure are examined using paternally inherited plastid markers to shed light on its evolutionary history and to provide a genetic perspective for its conservation. Methods By means of direct sequencing, a new polymorphic fragment containing a minisatellite site was identified within the plastid genome of P. kwangtungensis. Using the minisatellite site along with five SNPs (one indel and four substitutions) within the same fragment, the population genetic structure and pollen flow were analysed in 17 populations of P. kwangtungensis in southern China. Key Results Analysis of 227 individuals from 17 populations revealed ten haplotypes at the minisatellite site. The haplotype diversity at species level was relatively high (0·629). Genetic diversity of each population ranged from 0 to 0·779, and the western populations harboured more genetic variation than the eastern and Hainan populations, although the former appeared to have experienced a bottleneck in recent history. Population subdivision based on this site was high (FST = 0·540 under IAM; RST = 0·677 under SMM). Three major clusters (eastern, western and Hainan) were identified based on a neighbor-joining dendrogram generated from genetic distances among the populations. The genetic structures inferred from all the polymorphic sites and the SNPs were in concordance with that from the minisatellite site. Conclusions The results suggest that there are at least three refugia for P. kwangtungensis and that populations in these refugia should be treated as separate evolutionarily significant units or conservation units. The high diversities in the western populations suggest that these were much larger in the past (e.g. glacial stages) and that the shrinking population size might have been caused by recent events (e.g. deforestation, global warming, etc.). The western populations should be given priority for conservation due to their higher genetic diversity and limited population sizes. It is concluded that the newly found minisatellite may serve as a novel and applicable molecular marker for unravelling evolutionary processes in P. kwangtungensis. PMID:18463112

  19. DNA DNA DNA (d)DNA DNA DNA

    E-print Network

    Hagiya, Masami

    DNA DNA DNA DNA DNA DNA DNA DNA [ 2008] (d)DNA DNA DNA DNA 2 3 DNA DNA DNA DNA DNA DNA DNA (a) (c) (b) (d) #12;DNA DNA DNA DNA DNA DNA DNA DNA (b) DNA [Tanaka et al.2008] DNA DNA DNA DNA DNA DNA DNA #12;iGEM MIT MIT

  20. Phylogenetic Analysis of DNA C-values provides Evidence for a Small Ancestral Genome Size in Flowering Plants

    Microsoft Academic Search

    ILIA J. LEITCH; MARK W. CHASE; MICHAEL D. BENNETT

    1998-01-01

    DNA C-value is a highly variable aspect of plant biodiversity whose origin and significance has often attracted general interest. Evaluation of the phylogenetic component of genome size variation is essential for a full explanation of its evolutionary significance but was previously prevented by insufficient data and lack of phylogenetic consensus. However, the recent development for the angiosperms of a DNA

  1. Fragmentation of Isomeric Intrastrand Cross-link Lesions of DNA in an Ion-trap Mass Spectrometer

    PubMed Central

    Cao, Huachuan; Wang, Yinsheng

    2009-01-01

    The collision-induced dissociation pathways of isomeric cytosine-guanine and cytosine-adenine intrastrand cross-link-containing dinucleoside monophosphates were investigated with the stable isotope-labeled compounds to gain insights into the effects of chemical structure on the fragmentation pathways of these DNA modifications. Dimroth-like rearrangement, which was reported for protonated 2?-deoxycytidine and involved the switching of the exocyclic N4 with the ring N3 nitrogen atom, was also observed for the cytosine component in the protonated ions of C[5–8]G, C[5-2]A and C[5 – 8]A, but not C[5-N2]G or C[5-N6]A. In these two sets of cross-links, the C5 of cytosine is covalently bonded with its neighboring purine base via a carbon atom on the aromatic ring and an exocyclic nitrogen atom, respectively. On the contrary, the rearrangement could occur for the deprotonated ions of C[5-N2]G, C[5-N6]A and unmodified cytosine, but not C[5 – 8]G, C[5-2]A or C[5 – 8]A. In addition, ammonia could be lost more readily from C[5-N2]G and C[5-N6]A than from C[5 – 8]G, C[5-2]A and C[5 – 8]A. The results from the present study afforded important guidance for the application of mass spectrometry for the structure elucidation of other intrastrand/interstrand cross-link lesions. PMID:19103496

  2. 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

  3. DNA Restriction

    NSDL National Science Digital Library

    The discovery of enzymes that could cut and paste DNA made genetic engineering possible. Restriction enzymes, found naturally in bacteria, can be used to cut DNA fragment at specific sequences, while another enzyme, DNA ligase, can attach or rejoin DNA fragments with complementary ends. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents DNA restriction through a series of illustrations of processes involved.

  4. Mitochondrial DNA polymorphism in Japanese

    Microsoft Academic Search

    Satoshi Horai; Ei Matsunaga

    1986-01-01

    Mitochondrial DNA (mtDNA) from 116 Japanese was analyzed with nine restriction enzymes that recognize a four or five base pair sequence. The sizes of the mtDNA fragments produced by digestion by each enzyme were compared after gel electrophoresis. Double digestion experiments indicated that, in the coding region from URF2 (unidentified reading frame) to tRNAAsn (bp 5274–5691), there is an insertion

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

    PubMed

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

    1991-01-01

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

  6. Panax ginseng extract modulates oxidative stress, DNA fragmentation and up-regulate gene expression in rats sub chronically treated with aflatoxin B1 and fumonisin B 1.

    PubMed

    Hassan, Aziza M; Abdel-Aziem, Sekena H; El-Nekeety, Aziza A; Abdel-Wahhab, Mosaad A

    2014-04-19

    Aflatoxins and fumonisins are important food-borne mycotoxins implicated in human health and have cytotoxic effects. The aims of the current study were to evaluate the protective role of Panax ginseng extract (PGE) against the synergistic effect of subchronic administration of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) on DNA and gene expression in rat. Female Sprague-Dawley rats were divided into eight groups (ten rats/group) and treated for 12 weeks including the control group, the group having received AFB1 (80 µg/kg bw), the group having received FB1 (100 µg/kg bw), the group having received AFB1 plus FB1 and the groups having received PGE (20 mg/kg bw) alone or with AFB1 and/or FB1. At the end of experiment, liver and kidney were collected for the determination of DNA fragmentation, lipid peroxidation (LP), glutathione (GSH) contents and alterations in gene expression. The results indicated that these mycotoxins increased DNA fragmentation, LP and decreased GSH content in liver and kidney and down-regulated gene expression of antioxidants enzymes. The combined treatments with AFB1 and/or FB1 plus PGE suppressed DNA fragmentation only in the liver, normalized LP and increased GSH in the liver and kidney as well as up-regulated the expression of GPx, SOD1 and CAT mRNA. It could be concluded that AFB1 and FB1 have synergistic genotoxic effects. PGE induced protective effects against their oxidative stress and genotoxicity through its antioxidant properties. PMID:24748134

  7. Relationships between sperm DNA fragmentation, sperm apoptotic markers and serum levels of CB-153 and p,p'-DDE in European and Inuit populations.

    PubMed

    Stronati, A; Manicardi, G C; Cecati, M; Bordicchia, M; Ferrante, L; Spanò, M; Toft, G; Bonde, J P; Jönsson, B A G; Rignell-Hydbom, A; Rylander, L; Giwercman, A; Pedersen, H S; Bonefeld-Jørgensen, E C; Ludwicki, J K; Lesovoy, V; Sakkas, D; Bizzaro, D

    2006-12-01

    Persistent organochlorine pollutants (POPs) are suspected to interfere with hormone activity and the normal homeostasis of spermatogenesis. We investigated the relationships between sperm DNA fragmentation, apoptotic markers identified on ejaculated spermatozoa and POP levels in the blood of 652 adult males (200 Inuits from Greenland, 166 Swedish, 134 Polish and 152 Ukrainian). Serum levels of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl (CB-153), as a proxy of the total POP burden, and of 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE), as a proxy of the total DDT exposure were determined. Sperm DNA fragmentation was measured by using the TUNEL assay, whereas immunofluorescence methods were utilized for detecting pro-apoptotic (Fas) and anti-apoptotic (Bcl-xL) markers. Both TUNEL assay and apoptotic markers were statistically differed across the four populations. No correlation between neither sperm DNA fragmentation nor apoptotic sperm parameters and the large variations in POPs exposure was observed for the separate study groups. However, considering the European populations taken together, we showed that both %TUNEL positivity and Bcl-xL were related to CB-153 serum levels, whereas our study failed to demonstrate any relations between DDE and %TUNEL positivity and apoptotic sperm biomarkers (Fas and Bcl-xL) in any region or overall regions. These results suggest that CB-153 and related chemicals might alter sperm DNA integrity and Bcl-xL levels in European adult males, but not in the highly exposed Inuit men. Additional issues (genetic background, lifestyle habits and characterization of total xeno-hormonal activities) need to be investigated in order to fully assess the population variations observed. PMID:17127755

  8. [Line class retroposon is the component of the DNA polymorphic fragments pattern of trematode Himasthla elongata parthenitae].

    PubMed

    Solov'eva, A I; Galaktionov, N K; Podgornaia, O I

    2013-01-01

    We have determined that S-SAP method (Sequence specific amplification polymorphism) reveals clonal variability in the genomes of larvae of flatworm Himasthla elongata (Trematoda, Echinostomatidae). Being parthenogenetic the larvae were previously considered to be genetically homogeneous. Cloning and sequencing of a -500 bp conservative fragment (B1) from the fragments' pattern has been performed. Sequence analysis of B1 has shown that this fragment has maximum homology with LINE elements from CR1 family of Hydra and sparrow. In situ hybridization (FISH) has detected dispersed distribution of B1. Several other fragments cloned from the same lane of agarose electrophoresis correspond to conservative domain of reverse transcriptase (RT) from CR1 family. Thus, we have shown that 1) cercariae of trematode H. elongata have clonal variability; 2) the S-SAP method allows to obtaining patterns of fragment distribution characteristic of individual cercariae; 3) conservative domain of RT of CR1 family participates in the pattern of polymorphic fragments generation. Identification of the CR1 transcripts in cercariae of H. elongata transcriptome is the aim of the future work. Cloning of the variable fragments from the fragments' pattern is in progress. PMID:25509118

  9. Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques

    Microsoft Academic Search

    C. Cattaneo; S. DiMartino; S. Scali; O. E. Craig; M. Grandi; R. J. Sokol

    1999-01-01

    In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative

  10. Preparative and Analytical Purification of DNA from Agarose

    Microsoft Academic Search

    Bert Vogelstein; David Gillespie

    1979-01-01

    Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and

  11. TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments

    Microsoft Academic Search

    Beiyu Liu; Jianyang Wang; Gokben Yildirir; Paul T. Englund

    2009-01-01

    Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its

  12. Estimating Size and Trend of the North Interlake Woodland Caribou Population Using Fecal-DNA and Capture–Recapture Models

    PubMed Central

    Hettinga, Peter N; Arnason, Arni Neil; Manseau, Micheline; Cross, Dale; Whaley, Kent; Wilson, Paul J

    2012-01-01

    A critical step in recovery efforts for endangered and threatened species is the monitoring of population demographic parameters. As part of these efforts, we evaluated the use of fecal-DNA based capture–recapture methods to estimate population sizes and population rate of change for the North Interlake woodland caribou herd (Rangifer tarandus caribou), Manitoba, Canada. This herd is part of the boreal population of woodland caribou, listed as threatened under the federal Species at Risk Act (2003) and the provincial Manitoba Endangered Species Act (2006). Between 2004 and 2009 (9 surveys), we collected 1,080 fecal samples and identified 180 unique genotypes (102 females and 78 males). We used a robust design survey plan with 2 surveys in most years and analysed the data with Program MARK to estimate encounter rates (p), apparent survival rates (?), rates of population change (?), and population sizes (N). We estimated these demographic parameters for males and females and for 2 genetic clusters within the North Interlake. The population size estimates were larger for the Lower than the Upper North Interlake area and the proportion of males was lower in the Lower (33%) than the Upper North Interlake (49%). Population rate of change for the entire North Interlake area (2005–2009) using the robust design Pradel model was significantly <1.0 (? = 0.90, 95% CI: 0.82–0.99) and varied between sex and area with the highest being for males in Lower North Interlake (? = 0.98, 95% CI: 0.83–1.13) and the lowest being for females in Upper North Interlake (? = 0.83, 95% CI: 0.69–0.97). The additivity of ? between sex and area is supported on the log scale and translates into males having a ? that is 0.09 greater than females and independent of sex, Lower North Interlake having a ? that is 0.06 greater than Upper North Interlake. Population estimates paralleled these declining trends, which correspond to trends observed in other fragmented populations of woodland caribou along the southern part of their range. The results of this study clearly demonstrate the applicability and success of non-invasive genetic sampling in monitoring populations of woodland caribou. © 2012 The Wildlife Society. PMID:22973066

  13. Estimating Size and Trend of the North Interlake Woodland Caribou Population Using Fecal-DNA and Capture-Recapture Models.

    PubMed

    Hettinga, Peter N; Arnason, Arni Neil; Manseau, Micheline; Cross, Dale; Whaley, Kent; Wilson, Paul J

    2012-08-01

    A critical step in recovery efforts for endangered and threatened species is the monitoring of population demographic parameters. As part of these efforts, we evaluated the use of fecal-DNA based capture-recapture methods to estimate population sizes and population rate of change for the North Interlake woodland caribou herd (Rangifer tarandus caribou), Manitoba, Canada. This herd is part of the boreal population of woodland caribou, listed as threatened under the federal Species at Risk Act (2003) and the provincial Manitoba Endangered Species Act (2006). Between 2004 and 2009 (9 surveys), we collected 1,080 fecal samples and identified 180 unique genotypes (102 females and 78 males). We used a robust design survey plan with 2 surveys in most years and analysed the data with Program MARK to estimate encounter rates (p), apparent survival rates (?), rates of population change (?), and population sizes (N). We estimated these demographic parameters for males and females and for 2 genetic clusters within the North Interlake. The population size estimates were larger for the Lower than the Upper North Interlake area and the proportion of males was lower in the Lower (33%) than the Upper North Interlake (49%). Population rate of change for the entire North Interlake area (2005-2009) using the robust design Pradel model was significantly <1.0 (? = 0.90, 95% CI: 0.82-0.99) and varied between sex and area with the highest being for males in Lower North Interlake (? = 0.98, 95% CI: 0.83-1.13) and the lowest being for females in Upper North Interlake (? = 0.83, 95% CI: 0.69-0.97). The additivity of ? between sex and area is supported on the log scale and translates into males having a ? that is 0.09 greater than females and independent of sex, Lower North Interlake having a ? that is 0.06 greater than Upper North Interlake. Population estimates paralleled these declining trends, which correspond to trends observed in other fragmented populations of woodland caribou along the southern part of their range. The results of this study clearly demonstrate the applicability and success of non-invasive genetic sampling in monitoring populations of woodland caribou. © 2012 The Wildlife Society. PMID:22973066

  14. [Cloning of PCR-copies of Streptomyces coelicolor A3(2) chromosome fragment].

    PubMed

    Luk'ianchuk, V V

    2009-01-01

    Copies of chromosome Streptomyces coelicolor A3(2) fragment (gene coding polyketide syntase of III type) were obtained to use polymerase cycle reaction. Amplified DNA was cloned in shuttle vector pWHM4 on sites for HindIII and EcoRI. The restriction analysis of plasmid DNAs of transformants has demonstrated that molecular size of cloned DNA fragment was 1134 bp. PMID:20458937

  15. Body size, niche breadth, and ecologically scaled responses to habitat fragmentation: mammalian predators in an agricultural landscape

    Microsoft Academic Search

    Thomas M. Gehring; Robert K. Swihart

    2003-01-01

    The ability to make a priori assessments of a species' response to fragmentation, based on its distribution in the landscape, would serve as a valuable conservation and management tool. During 1997–1999, we monitored 717 scent stations to examine seasonal use of forest patches, corridors, and crop fields by coyotes (Canis latrans), domestic cats (Felis catus), foxes (Vulpes vulpes and Urocyon

  16. Controlled Fragmentation

    NASA Astrophysics Data System (ADS)

    Arnold, Werner

    2001-06-01

    Contrary to natural fragmentation, controlled fragmentation offers the possibility to adapt fragment parameters like size and mass to the performance requirements in a very flexible way. Known mechanisms like notches inside the casing, weaken the structure. This is, however, excluded for applications with high accelerations during launch or piercing requirements for example on a semi armor piercing penetrator. Another method to achieve controlled fragmentation with an additional grid layer is presented with which the required notches are produced "just in time" inside the casing during detonation of the high explosive. The process of generating the notches aided by the grid layer was studied using the hydrocode HULL with respect to varying grid designs and material combinations. Subsequent to this, a large range of these theoretically investigated combinations was contemplated in substantial experimental tests. With an optimised grid design and a suitable material selection the controlled fragment admits a very flexible adaptation to the set requirements. Additional advantages like the increase of perforation performance or incendiary amplification can be realized with the grid layer.

  17. Controlled Fragmentation

    NASA Astrophysics Data System (ADS)

    Arnold, Werner

    2002-07-01

    Contrary to natural fragmentation, controlled fragmentation offers the possibility to adapt fragment parameters like size and mass to the performance requirements in a very flexible way. Known mechanisms like grooves inside the casing, weaken the structure. This is, however, excluded for applications with high accelerations during launch or piercing requirements for example on a semi armor piercing penetrator. Another method to achieve controlled fragmentation with an additional grid layer is presented with which the required grooves are produced "just in time" inside the casing during detonation of the high explosive. The process of generating the grooves aided by the grid layer was studied using the hydrocode HULL with respect to varying grid designs and material combinations. Subsequent to this, a large range of these theoretically investigated combinations was contemplated in substantial experimental tests. With an optimised grid design and a suitable material selection, the controlled fragment admits a very flexible adaptation to the set requirements. Additional advantages like the increase of perforation performance or incendiary amplification can be realized with the grid layer.

  18. Electrochemical detection of synthetic DNA and native 16S rRNA fragments on a microarray using a biotinylated intercalator as coupling site for an enzyme label.

    PubMed

    Zimdars, Andreas; Gebala, Magdalena; Hartwich, Gerhard; Neugebauer, Sebastian; Schuhmann, Wolfgang

    2015-10-01

    The direct electrochemical detection of synthetic DNA and native 16S rRNA fragments isolated from Escherichia coli is described. Oligonucleotides are detected via selective post-labeling of double stranded DNA and DNA-RNA duplexes with a biotinylated intercalator that enables high-specific binding of a streptavidin/alkaline phosphatase conjugate. The alkaline phosphatase catalyzes formation of p-aminophenol that is subsequently oxidized at the underlying gold electrode and hence enables the detection of complementary hybridization of the DNA capture strands due to the enzymatic signal amplification. The hybridization assay was performed on microarrays consisting of 32 individually addressable gold microelectrodes. Synthetic DNA strands with sequences representing six different pathogens which are important for the diagnosis of urinary tract infections could be detected at concentrations of 60nM. Native 16S rRNA isolated from the different pathogens could be detected at a concentration of 30fM. Optimization of the sensing surface is described and influences on the assay performance are discussed. PMID:26078123

  19. Sperm DNA fragmentation measured by Halosperm does not impact on embryo quality and ongoing pregnancy rates in IVF/ICSI treatments.

    PubMed

    Anifandis, G; Bounartzi, T; Messini, C I; Dafopoulos, K; Markandona, R; Sotiriou, S; Tzavella, A; Messinis, I E

    2015-04-01

    Sperm DNA fragmentation (SDF) has been proposed to be one of the main markers regarding male infertility. A prospective study was performed to assess primarily whether sperm DNA damage has any impact on embryological data and secondarily on pregnancy rates. This prospective study evaluated the sperm DNA damage in fresh ejaculated sperm samples from couples undergoing IVF/ICSI treatments, using the improved SCD method, known as Halosperm(®) . The results were evaluated by performing statistical analysis with the statistical package of SPSS v17. A total of 156 fresh semen samples derived from 156 couples undergoing 156 IVF/ICSI cycles. From the 156 couples, 139 finally reached the embryo transfer (ET) procedure. Overall, SDF did not correlate with embryological data, while ongoing pregnancy rate/ET was 21.6%. SDF only correlated with sperm characteristics. After the categorisation of SDF (?35% and >35%), according to the specific references of the method used, embryological data were comparable as also ongoing pregnancy rates. Using the SCD method, sperm DNA damage is associated neither with embryological data nor to pregnancy rates. However, we should not rule out the fact that extremely high DNA damages are associated with total pregnancy failure. PMID:24621442

  20. Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

    PubMed

    Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

    2015-05-01

    DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. PMID:25746798

  1. Restriction-based Multiple-fragment Assembly Strategy to Avoid Random Mutation during Long cDNA Cloning

    PubMed Central

    Wang, Shang; Chen, Wen; Zhang, Kai; Jiao, Peng; Mo, Lihua; Yang, Xiaoxu; Hu, Xiang; Zhang, Jian; Wei, Chenxi; Xiang, Shuanglin

    2015-01-01

    Long fragment cloning is a challenge for its difficulty in accurate amplifying and tendency to get unwanted mutation. Here we discuss Restriction-based Multiple-fragment Assembly Strategy's advantages and limitations. In this strategy, rather than PCR amplifying the entire coding sequence (CDS) at one time, we amplified and sequenced smaller fragments which are shorter than 1.5kb spanning the CDS. After that, the sequence-proved fragments were assembled by digestion-ligation cloning to the target vector. We test its universality in our script programmed in Python. Our data shows that, among the entire human and mouse CDS, at least 70% of long CDS cloning will benefit from this strategy.

  2. The impact of partial manganese superoxide dismutase (SOD2)-deficiency on mitochondrial oxidant stress, DNA fragmentation and liver injury during acetaminophen hepatotoxicity

    SciTech Connect

    Ramachandran, Anup; Lebofsky, Margitta [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS 66160 (United States); Weinman, Steven A. [Department of Medicine and Microbiology and Immunology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, MS 1018, Kansas City, KS 66160 (United States)

    2011-03-15

    Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure in many countries. The mechanism of cell death is initiated by formation of a reactive metabolite that binds to mitochondrial proteins and promotes mitochondrial dysfunction and oxidant stress. Manganese superoxide dismutase (SOD2) is a critical defense enzyme located in the mitochondrial matrix. The objective of this investigation was to evaluate the functional consequences of partial SOD2-deficiency (SOD2+/-) on intracellular signaling mechanisms of necrotic cell death after APAP overdose. Treatment of C57Bl/6J wild type animals with 200 mg/kg APAP resulted in liver injury as indicated by elevated plasma alanine aminotransferase activities (2870 {+-} 180 U/L) and centrilobular necrosis at 6 h. In addition, increased tissue glutathione disulfide (GSSG) levels and GSSG-to-GSH ratios, delayed mitochondrial GSH recovery, and increased mitochondrial protein carbonyls and nitrotyrosine protein adducts indicated mitochondrial oxidant stress. In addition, nuclear DNA fragmentation (TUNEL assay) correlated with translocation of Bax to the mitochondria and release of apoptosis-inducing factor (AIF). Furthermore, activation of c-jun-N-terminal kinase (JNK) was documented by the mitochondrial translocation of phospho-JNK. SOD2+/- mice showed 4-fold higher ALT activities and necrosis, an enhancement of all parameters of the mitochondrial oxidant stress, more AIF release and more extensive DNA fragmentation and more prolonged JNK activation. Conclusions: the impaired defense against mitochondrial superoxide formation in SOD2+/- mice prolongs JNK activation after APAP overdose and consequently further enhances the mitochondrial oxidant stress leading to exaggerated mitochondrial dysfunction, release of intermembrane proteins with nuclear DNA fragmentation and more necrosis.

  3. THE MINERALOGY AND SIZE OF AIRBORNE CHRYSOTILE AND ROCK FRAGMENTS: RAMIFICATIONS OF USING THE NIOSH 7400 METHOD

    Microsoft Academic Search

    Ann G. Wylie; Kelly F. Bailey

    1992-01-01

    The length and width of chrysotile and rock fragments that were collected on nine air-monitoring filters in the mine and plant of the Lowell asbestos mine in Vermont have been measured by transmission electron microscopy (TEM). Selective area electron diffraction (SAED) and energy dispersive x-ray analysis (EDS) were used to identify particles longer than 5?µim with a length-to-width aspect ratio

  4. Molecular cloning and characterization of CIDE-3, a novel member of the cell-death-inducing DNA-fragmentation-factor (DFF45)-like effector family.

    PubMed Central

    Liang, Liang; Zhao, Mujun; Xu, Zhenhua; Yokoyama, Kazunari K; Li, Tsaiping

    2003-01-01

    DNA fragmentation is one of the critical steps in apoptosis, which is induced by DNA fragmentation factor (DFF). DFF is composed of two subunits, a 40 kDa caspase-activated nuclease (DFF40) and a 45 kDa inhibitor (DFF45). Recently a novel family of cell-death-inducing DFF45-like effectors (CIDEs) has been identified. Among CIDEs, two from human (CIDE-A and CIDE-B) and three from mouse (CIDE-A, CIDE-B and FSP27) have been reported. In this study human CIDE-3, a novel member of CIDEs, was identified upon sequence analysis of a previously unidentified cDNA that encoded a protein of 238 amino acids. It was shown to be a human homologue of mouse FSP27, and shared homology with the CIDE-N and CIDE-C domains of CIDEs. Apoptosis-inducing activity was clearly shown by DNA-fragmentation assay of the nuclear DNA of CIDE-3 transfected 293T cells. The expression pattern of CIDE-3 was different from that of CIDE-B. As shown by Northern-blot analysis, CIDE-3 was expressed mainly in human small intestine, heart, colon and stomach, while CIDE-B showed strong expression in liver and small intestine and at a lower level in colon, kidney and spleen. Green-fluorescent-protein-tagged CIDE-3 was revealed in some cytosolic corpuscles. Alternative splicing of the CIDE-3 gene was also identified by reverse transcription PCR, revealing that two transcripts, CIDE-3 and CIDE-3alpha, were present in HepG2 and A375 cells. CIDE-3 comprised a full-length open reading frame with 238 amino acids; in CIDE-3alpha exon 3 was deleted and it encoded a protein of 164 amino acids. Interestingly the CIDE-3alpha isoform still kept the apoptosis-inducing activity and showed the same pattern of subcellular localization as CIDE-3. Consistent with its chromosome localization at 3p25, a region associated with high frequency loss of heterozygosity in many tumours, CIDE-3 may play an important role in prevention of tumorigenesis. PMID:12429024

  5. Prokaryotic Genome Size and SSU rDNA Copy Number: Estimation of Microbial Relative Abundance from a Mixed Population

    Microsoft Academic Search

    G. B. Fogel; C. R. Collins; J. Li; C. F. Brunk

    1999-01-01

    Determination of the relative abundance of a specific prokaryote in an environmental sample is of major interest in applied\\u000a and environmental microbiology. Relative abundance can be calculated using knowledge of SSU rDNA copy number, amount of SSU\\u000a rDNA in the sample, and a weighted average estimate of the genome sizes for organisms in the original sample. By surveying\\u000a the literature,

  6. Differential Gene Expression in Response to Papaya ringspot virus Infection in Cucumis metuliferus Using cDNA- Amplified Fragment Length Polymorphism Analysis

    PubMed Central

    Lin, Chia-Wei; Chung, Chien-Hung; Chen, Jo-Chu; Yeh, Shy-Dong; Ku, Hsin-Mei

    2013-01-01

    A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future. PMID:23874746

  7. Differential gene expression in response to Papaya ringspot virus infection in Cucumis metuliferus using cDNA-amplified fragment length polymorphism analysis.

    PubMed

    Lin, Yu-Tsung; Jan, Fuh-Jyh; Lin, Chia-Wei; Chung, Chien-Hung; Chen, Jo-Chu; Yeh, Shy-Dong; Ku, Hsin-Mei

    2013-01-01

    A better understanding of virus resistance mechanisms can offer more effective strategies to control virus diseases. Papaya ringspot virus (PRSV), Potyviridae, causes severe economical losses in papaya and cucurbit production worldwide. However, no resistance gene against PRSV has been identified to date. This study aimed to identify candidate PRSV resistance genes using cDNA-AFLP analysis and offered an open architecture and transcriptomic method to study those transcripts differentially expressed after virus inoculation. The whole genome expression profile of Cucumis metuliferus inoculated with PRSV was generated using cDNA-amplified fragment length polymorphism (cDNA-AFLP) method. Transcript derived fragments (TDFs) identified from the resistant line PI 292190 may represent genes involved in the mechanism of PRSV resistance. C. metuliferus susceptible Acc. 2459 and resistant PI 292190 lines were inoculated with PRSV and subsequently total RNA was isolated for cDNA-AFLP analysis. More than 400 TDFs were expressed specifically in resistant line PI 292190. A total of 116 TDFs were cloned and their expression patterns and putative functions in the PRSV-resistance mechanism were further characterized. Subsequently, 28 out of 116 candidates which showed two-fold higher expression levels in resistant PI 292190 than those in susceptible Acc. 2459 after virus inoculation were selected from the reverse northern blot and bioinformatic analysis. Furthermore, the time point expression profiles of these candidates by northern blot analysis suggested that they might play roles in resistance against PRSV and could potentially provide valuable information for controlling PRSV disease in the future. PMID:23874746

  8. Widespread distribution of extensive chromosomal fragmentation in ciliates.

    PubMed

    Riley, J L; Katz, L A

    2001-07-01

    Ciliates are a diverse group of eukaryotes characterized by their division of nuclear function into a "germ line" micronucleus and a "somatic" macronucleus. After conjugation, chromosomes in the transcriptionally active macronucleus develop by fragmentation, elimination, and amplification of germ line chromosomes. Extensive chromosomal processing that generates a macronucleus with gene-sized fragments has thus far been well documented in members of only one class of ciliates, the Spirotrichea. Here we establish the broad distribution of extensive fragmentation among members of the class Phyllopharyngea and the genera Metopus (order Armophorida) and Nyctotherus (order Clevelandellida). Moreover, analyses of small-subunit rDNA genealogies indicate that gene-sized chromosomes occur in members of the three separate clades: (1) the class Spirotrichea, (2) the class Phyllopharyngea, and (3) the two orders Clevelandellida and Armophorida. Together, these data indicate that the generation of gene-sized chromosomes is widespread and demonstrate multiple origins of extensive fragmentation within ciliates. PMID:11420375

  9. Size-dependent trajectories of DNA macromolecules due to insulative dielectrophoresis in submicrometer-deep fluidic channels

    PubMed Central

    Parikesit, Gea O. F.; Markesteijn, Anton P.; Piciu, Oana M.; Bossche, Andre; Westerweel, Jerry; Young, Ian T.; Garini, Yuval

    2008-01-01

    In this paper, we demonstrate for the first time that insulative dielectrophoresis can induce size-dependent trajectories of DNA macromolecules. We experimentally use ? (48.5 kbp) and T4GT7 (165.6 kbp) DNA molecules flowing continuously around a sharp corner inside fluidic channels with a depth of 0.4 ?m. Numerical simulation of the electrokinetic force distribution inside the channels is in qualitative agreement with our experimentally observed trajectories. We discuss a possible physical mechanism for the DNA polarization and dielectrophoresis inside confining channels, based on the observed dielectrophoresis responses due to different DNA sizes and various electric fields applied between the inlet and the outlet. The proposed physical mechanism indicates that further extensive investigations, both theoretically and experimentally, would be very useful to better elucidate the forces involved at DNA dielectrophoresis. When applied for size-based sorting of DNA molecules, our sorting method offers two major advantages compared to earlier attempts with insulative dielectrophoresis: Its continuous operation allows for high-throughput analysis, and it only requires electric field strengths as low as ?10 V?cm. PMID:19693406

  10. Size effect on transfection and cytotoxicity of nanoscale plasmid DNA/polyethyleneimine complexes for aerosol gene delivery

    SciTech Connect

    Hoon Byeon, Jeong, E-mail: jbyeon@purdue.edu [Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 (United States); Kim, Jang-Woo, E-mail: jwkim@hoseo.edu [Department of Digital Display Engineering, Hoseo University, Asan 336-795 (Korea, Republic of)

    2014-02-03

    Nanoscale plasmid DNA (pDNA)/polyethyleneimine (PEI) complexes were fabricated in the aerosol state using a nebulization system consisting of a collison atomizer and a cool-walled diffusion dryer. The aerosol fabricated nanoscale complexes were collected and employed to determine fundamental properties of the complexes, such as size, structure, surface charge, and in vitro gene transfection efficiency and cytotoxicity. The results showed that mass ratio between pDNA and PEI should be optimized to enhance gene transfection efficiency without a significant loss of cell viability. These findings may support practical advancements in the field of nonviral gene delivery.

  11. Restriction fragment length polymorphism of 195 bp repeated satellite dna of Trypanosoma cruzi supports the existence of two phylogenetic groups

    Microsoft Academic Search

    Brigitte Bastrenta; Marie France Bosseno; Christian Barnabé; Michel Tibayrenc; Simone Frédérique Brenière

    1999-01-01

    Hinf I) and high molecular weight DNA (Hae III), while group 2 presents a ladder profile for each enzyme, which is a characteristic of tandemly repeated DNA. The two groups, respectively, clustered stocks pertaining to the two principal lineages evidenced by isoenzyme and RAPD markers. The congruence among these three independent genomic markers corroborates the existence of two real phylogenetic

  12. Layer-by-layer coated gold nanoparticles: size-dependent delivery of DNA into cells.

    PubMed

    Elbakry, Asmaa; Wurster, Eva-Christina; Zaky, Alaa; Liebl, Renate; Schindler, Edith; Bauer-Kreisel, Petra; Blunk, Torsten; Rachel, Reinhard; Goepferich, Achim; Breunig, Miriam

    2012-12-21

    Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size-dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer-by-layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC(50) value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface. PMID:22911477

  13. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

    PubMed Central

    Kuo, W S; Chak, K F

    1996-01-01

    Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains. PMID:8919799

  14. Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA.

    PubMed

    Kuo, W S; Chak, K F

    1996-04-01

    Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains. PMID:8919799

  15. REMA: A computer-based mapping tool for analysis of restriction sites in multiple DNA sequences

    Microsoft Academic Search

    Jan Szubert; Caroline Reiff; Andrew Thorburn; Brajesh K. Singh

    2007-01-01

    REMA is an interactive web-based program which predicts endonuclease cut sites in DNA sequences. It analyses multiple sequences simultaneously and predicts the number and size of fragments as well as provides restriction maps. The users can select single or paired combinations of all commercially available enzymes. Additionally, REMA permits prediction of multiple sequence terminal fragment sizes and suggests suitable restriction

  16. Blade fragment energy analysis

    NASA Technical Reports Server (NTRS)

    Oconnor, M. A., Jr.

    1977-01-01

    Two classes of fan blade fragments were considered in an analysis of blade fragment energy. The first, of relatively small size (.15 pound) and energy, tends to rebound from the fan and case when liberated in an FOD encounter. These small fragments have relatively low secondary damage potential and are less demanding in terms of protection. The larger fan blade fragments are ejected in a more direct release trajectory with higher energy and hence can represent a higher potential hazard. Simplified analytical methods were used to describe blade fragment energy transfer kinematics, establish fragment energy levels, evaluate damage potential and configure protection. The approach, methodology, and application are discussed as a possible building block for other applications. Development of effective local protection using Kevlar is also discussed. Analysis methods developed and applied to the rebound fragment problem and to the large direct release fragment problem are described.

  17. Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

    PubMed Central

    Pawlowski, Wojciech P.; Somers, David A.

    1998-01-01

    Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35–280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci. PMID:9770447

  18. Influence of particle size and antacid on release and stability of plasmid DNA from uniform PLGA microspheres.

    PubMed

    Varde, Neelesh K; Pack, Daniel W

    2007-12-20

    PLGA microspheres are attractive DNA delivery vehicles due to their controlled release capabilities. One major problem with PLGA microspheres is that they develop an acidic microclimate as the polymer degrades, lowering the intraparticle pH, and potentially damaging the DNA. Antacids have recently shown promise in buffering this acidic microclimate and enhancing protein stability. We manufactured uniform plasmid DNA-encapsulating PLGA microspheres of two sizes (47, 80 microm diameter) and antacid concentrations (0, 3% Mg(OH)2). Microspheres with antacid had a homogeneous surface coverage of small pores, which resulted in a significant reduction of the burst effect. The 47 microm microspheres exhibited complete release of plasmid DNA over the course of two months. Incomplete release was observed from 80 microm spheres, though microspheres with 3% Mg(OH)2 showed a higher cumulative release, suggesting that the antacid at least partially aids in increasing the stability of DNA. SEM was used to visualize the surface pore evolution and cross-sectional microsphere structure over time. Subsequent image analysis was used to quantify the increase of surface pore sizes. Cross-sectional images showed increasing internal degradation and erosion, which resulted in a hollowing-out of microspheres. Our studies show that the incorporation of antacid into the microsphere structure has potential in addressing some of the major problems associated with DNA encapsulation and release in PLGA microspheres. PMID:17928089

  19. Influence of particle size and antacid on release and stability of plasmid DNA from uniform PLGA microspheres

    PubMed Central

    Varde, Neelesh K.; Pack, Daniel W.

    2007-01-01

    PLGA microspheres are attractive DNA delivery vehicles due to their controlled release capabilities. One major problem with PLGA microspheres is that they develop an acidic microclimate as the polymer degrades, lowering the intraparticle pH, and potentially damaging the DNA. Antacids have recently shown promise in buffering this acidic microclimate and enhancing protein stability. We manufactured uniform plasmid DNA-encapsulating PLGA microspheres of two sizes (47, 80 ?m diameter) and antacid concentrations (0, 3% Mg(OH)2). Microspheres with antacid had a homogeneous surface coverage of small pores, which resulted in a significant reduction of the burst effect. The 47 ?m microspheres exhibited complete release of plasmid DNA over the course of two months. Incomplete release was observed from 80 ?m spheres, though microspheres with 3% Mg(OH)2 showed a higher cumulative release, suggesting that the antacid at least partially aids in increasing the stability of DNA. SEM was used to visualize the surface pore evolution and cross-sectional microsphere structure over time. Subsequent image analysis was used to quantify the increase of surface pore sizes. Cross-sectional images showed increasing internal degradation and erosion, which resulted in a hollowing-out of microspheres. Our studies show that the incorporation of antacid into the microsphere structure has potential in addressing some of the major problems associated with DNA encapsulation and release in PLGA microspheres. PMID:17928089

  20. Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny

    Microsoft Academic Search

    Michael W. Lassner; Peter Peterson; John I. Yoder

    1989-01-01

    We describe the simultaneous amplification of different segments of foreign DNA in transgenic plants using the polymerase\\u000a chain reaction (PCR). We used PCR to simultaneously amplify different regions of transformed T-DNA in order to assay the integrity\\u000a of transformed constructions in primary tomato transformants. We also used simultaneous PCR amplification to examine the segregation\\u000a of transformed sequences in progeny of

  1. Analysis of mitochondrial DNA and its inheritance in Populus

    Microsoft Academic Search

    R. Radetzky

    1990-01-01

    The mitochondrial genome of several poplar clones has been characterized by restriction analysis and hybridization with gene probes from Oenothera. The mitochondrial (mt) DNA of Populus has a complex restriction fragment pattern and its genome size was estimated to be about 450 kilobase pairs (kb). Restriction fragment length polymorphisms (RFLPs) could be detected only between, and not within, species of

  2. C-Terminal Domain Swapping of SSB Changes the Size of the ssDNA Binding Site

    PubMed Central

    Huang, Yen-Hua

    2014-01-01

    Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, including DNA replication, repair, and recombination, and is therefore essential for cell survival. Bacterial SSB consists of an N-terminal ssDNA-binding/oligomerization domain and a flexible C-terminal protein-protein interaction domain. We characterized the ssDNA-binding properties of Klebsiella pneumoniae SSB (KpSSB), Salmonella enterica Serovar Typhimurium LT2 SSB (StSSB), Pseudomonas aeruginosa PAO1 SSB (PaSSB), and two chimeric KpSSB proteins, namely, KpSSBnStSSBc and KpSSBnPaSSBc. The C-terminal domain of StSSB or PaSSB was exchanged with that of KpSSB through protein chimeragenesis. By using the electrophoretic mobility shift assay, we characterized the stoichiometry of KpSSB, StSSB, PaSSB, KpSSBnStSSBc, and KpSSBnPaSSBc, complexed with a series of ssDNA homopolymers. The binding site sizes were determined to be 26 ± 2, 21 ± 2, 29 ± 2, 21 ± 2, and 29 ± 2 nucleotides (nt), respectively. Comparison of the binding site sizes of KpSSB, KpSSBnStSSBc, and KpSSBnPaSSBc showed that the C-terminal domain swapping of SSB changes the size of the binding site. Our observations suggest that not only the conserved N-terminal domain but also the C-terminal domain of SSB is an important determinant for ssDNA binding. PMID:25162017

  3. Salt ions and related parameters affect PEI-DNA particle size and transfection efficiency in Chinese hamster ovary cells.

    PubMed

    Sang, Yunxia; Xie, Kui; Mu, Yubin; Lei, Yun; Zhang, Baohong; Xiong, Sheng; Chen, Yantian; Qi, Nianmin

    2015-01-01

    Transfection efficiency is directly associated with the expression level and quantity of recombinant protein after the transient transfection of animal cells. The transfection process can be influenced by many still-unknown factors, so it is valuable to study the precise mechanism and explore these factors in gene delivery. Polyethylenimine (PEI) is considered to have high transfection efficiency and endosome-disrupting capacity. Here we aimed to investigate optimal conditions for transfection efficiency by setting different parameters, including salt ion concentration, DNA/PEI ratio, and incubation time. We examined the PEI-DNA particle size using a Malvern particle size analyzer and assessed the transfection efficiency using flow cytometry in Chinese hamster ovary-S cells. Salt ions, higher amounts of PEI tended to improve the aggregation of PEI-DNA particles and the particle size of PEI-DNA complexes and the transfection efficiency were increased. Besides, the particle size was also found to benefit from longer incubation time. However, the transfection efficiency increased to maximum of 68.92 % at an incubation time of 10 min, but decreased significantly thereafter to 23.71 %, when incubating for 120 min (P < 0.05). Besides, PEI-DNA complexes formed in salt-free condition were unstable. Our results suggest DNA and PEI incubated in 300 mM NaCl at a ratio of 1:4 for 10 min could achieve the optimal transfection efficiency. Our results might provide guidance for the optimization of transfection efficiency and the industrial production of recombinant proteins. PMID:24166598

  4. A DNA Fragment Mapped within the Submicroscopic Deletion of Ph1, a Chromosome Pairing Regulator Gene in Polyploid Wheat

    PubMed Central

    Gill, K. S.; Gill, B. S.

    1991-01-01

    Bread wheat is an allohexaploid consisting of three genetically related (homoeologous) genomes. The homoeologous chromosomes are capable of pairing but strict homologous pairing is observed at metaphase 1. The diploid-like pairing is regulated predominantly by Ph1, a gene mapped on long arm of chromosome 5B. We report direct evidence that a mutant of the gene (ph1b) arose from a submicroscopic deletion. A probe (XksuS1-5) detects the same missing fragment in two independent mutants ph1b and ph1c and a higher intensity fragment in a duplication of the Ph1 gene. It is likely that XksuS1-5 lies adjacent to Ph1 on the same chromosome fragment that is deleted in ph1b and ph1c. XksuS1-5 can be used to tag Ph1 gene to facilitate incorporation of genetic material from homoeologous genomes of the Triticeae. It may also be a useful marker in cloning Ph1 gene by chromosome walking. PMID:1936962

  5. Anthocyanin Inhibits Propidium Iodide DNA Fluorescence in Euphorbia pulcherrima: Implications for Genome Size Variation and Flow Cytometry

    PubMed Central

    Bennett, Michael D.; Price, H. James; Johnston, J. Spencer

    2008-01-01

    Background Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics. Methods DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia). Key Results There were large differences in PI staining (35–70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2·8–6·9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1·69–1·76 pg) than green leaves (1·81 pg). Chopping pea or poinsettia tissue in buffer with 0–200 µm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin. Conclusions Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or correlated with environmental factors known to induce variation in pigmentation. PMID:18158306

  6. Characterization of primary biogenic aerosol particles in urban, rural, and high-alpine air by DNA sequence and restriction fragment analysis of ribosomal RNA genes

    NASA Astrophysics Data System (ADS)

    Després, V. R.; Nowoisky, J. F.; Klose, M.; Conrad, R.; Andreae, M. O.; Pöschl, U.

    2007-12-01

    This study explores the applicability of DNA analyses for the characterization of primary biogenic aerosol (PBA) particles in the atmosphere. Samples of fine particulate matter (PM2.5) and total suspended particulates (TSP) have been collected on different types of filter materials at urban, rural, and high-alpine locations along an altitude transect in the south of Germany (Munich, Hohenpeissenberg, Mt. Zugspitze). From filter segments loaded with about one milligram of air particulate matter, DNA could be extracted and DNA sequences could be determined for bacteria, fungi, plants and animals. Sequence analyses were used to determine the identity of biological organisms, and terminal restriction fragment length polymorphism analyses (T-RFLP) were applied to estimate diversities and relative abundances of bacteria. Investigations of blank and background samples showed that filter materials have to be decontaminated prior to use, and that the sampling and handling procedures have to be carefully controlled to avoid artifacts in the analyses. Mass fractions of DNA in PM2.5 were found to be around 0.05% in urban, rural, and high-alpine aerosols. The average concentration of DNA determined for urban air was on the order of ~7 ng m-3, indicating that human adults may inhale about one microgram of DNA per day (corresponding to ~108 haploid bacterial genomes or ~105 haploid human genomes, respectively). Most of the bacterial sequences found in PM2.5 were from Proteobacteria (42) and some from Actinobacteria (10) and Firmicutes (1). The fungal sequences were characteristic for Ascomycota (3) and Basidiomycota (1), which are known to actively discharge spores into the atmosphere. The plant sequences could be attributed to green plants (2) and moss spores (2), while animal DNA was found only for one unicellular eukaryote (protist). Over 80% of the 53 bacterial sequences could be matched to one of the 19 T-RF peaks found in the PM2.5 samples, but only 40% of the T-RF peaks did correspond to one of the detected bacterial sequences. The results demonstrate that the T-RFLP analysis covered more of the bacterial diversity than the sequence analysis. Shannon-Weaver indices calculated from both sequence and T-RFLP data indicate that the bacterial diversity in the rural samples was higher than in the urban and alpine samples. Two of the bacterial sequences (Gammaproteobacteria) and five of the T-RF peaks were found at all sampling locations.

  7. Abundance, body size and movement patterns of a tropical treefrog in continuous and fragmented forests in the Brazilian Amazon

    Microsoft Academic Search

    Selvino Neckel-Oliveira; Claude Gascon

    2006-01-01

    Phyllomedusa tarsius is a hylid frog that breeds in ponds located in a range of habitats from continuous forests to severely disturbed matrix habitats in Central Amazon. During three reproductive seasons, we followed the movement patterns, measured body size and registered abundance and residency time of this species in five habitats: pasture, Vismia regrowth, Cecropia regrowth, 1 and 10ha forest

  8. Population sizes and within-deme movement of Trimerotropis saxatilis (Acrididae), a grasshopper with a fragmented distribution

    Microsoft Academic Search

    Anne S. Gerber; Alan R. Templeton

    1996-01-01

    Capture-mark-recapture studies were initiated in 1990 on four Missouri populations of the lichen grasshopper, Trimerotropis saxatilis. This grasshopper lives only on glade habitat, predominantly in the Ozark Mountains. Genetic data suggest that no gene flow occurs among T. saxatilis populations. Lichen grasshopper population size (both present and historical), and the likelihood of movement within and between glades, are the subjects

  9. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors

    Microsoft Academic Search

    D. T. Burke; G. F. Carle; M. V. Olson

    1987-01-01

    Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with

  10. A new sensitive short pentaplex (ShoP) PCR for typing of degraded DNA

    Microsoft Academic Search

    C. Meissner; P. Bruse; E. Mueller; M. Oehmichen

    2007-01-01

    Analysis of short tandem repeat makers has become the most powerful tool for DNA typing in forensic casework analysis. Unfortunately, typing of DNA extracted from telogen shed hairs, bones buried in the soil or from paraffin-embedded, formalin-fixed tissue often reveals no results due to the degradation of DNA. The reduction in size of the target fragments by development of new

  11. Biochemical profiles, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA) for typing Staphylococcus aureus isolated from dairy products.

    PubMed

    Morandi, S; Brasca, M; Lodi, R; Brusetti, L; Andrighetto, C; Lombardi, A

    2010-06-01

    The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains. PMID:19926103

  12. A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV1 for targeted episomal and chromosomal gene repair

    Microsoft Academic Search

    Xavier Leclerc; Olivier Danos; Daniel Scherman; Antoine Kichler

    2009-01-01

    BACKGROUND: Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double

  13. Size–frequency distributions of fragments from SPH\\/ N-body simulations of asteroid impacts: Comparison with observed asteroid families

    Microsoft Academic Search

    Daniel D. Durda; William F. Bottke; David Nesvorný; Brian L. Enke; William J. Merline; Erik Asphaug; Derek C. Richardson

    2007-01-01

    We investigate the morphology of size–frequency distributions (SFDs) resulting from impacts into 100-km-diameter parent asteroids, represented by a suite of 161 SPH\\/N-body simulations conducted to study asteroid satellite formation [Durda, D.D., Bottke, W.F., Enke, B.L., Merline, W.J., Asphaug, E., Richardson, D.C., Leinhardt, Z.M., 2004. Icarus 170, 243–257]. The spherical basalt projectiles range in diameter from 10 to 46 km (in

  14. Patch occupancy in the endangered butterfly Lycaena helle in a fragmented landscape: effects of habitat quality, patch size and isolation

    Microsoft Academic Search

    Stephanie S. Bauerfeind; Anett Theisen; Klaus Fischer

    2009-01-01

    While there is agreement that both habitat quality and habitat network characteristics (such as patch size and isolation)\\u000a contribute to the occupancy of patches by any given species, the relative importance of these factors is under debate. This\\u000a issue is of fundamental ecological importance, and moreover of special concern for conservation biologists aiming at preserving\\u000a endangered species. Against this background

  15. Effects of natural habitat fragmentation on an endemic scrub lizard (Sceloporus woodi): an historical perspective based on a mitochondrial DNA gene genealogy.

    PubMed

    Clark, A M; Bowen, B W; Branch, L C

    1999-07-01

    The Florida scrub lizard, Sceloporus woodi, is endemic to scrub habitat patches along the central portion of the Florida peninsula and xeric coastal regions. Scrub ecosystems are the patchily distributed remnants of previously widespread habitats formed during the Pleiocene and early Pleistocene. Scrub lizards appear to have limited dispersal capabilities due to high habitat specificity and low mobility. To assess the population structure and phylogeography of S. woodi, 135 samples were collected from 16 patches on five major ridges in Florida, USA. Analysis of 273 bp of mitochondrial DNA (mtDNA) cytochrome b reveals a very strong geographic distribution of genetic diversity. Haplotype frequencies are significantly different in 63 of 66 comparisons between patches. With one exception, samples from the five major ridges are characterized by fixed differences in haplotype distribution and deep evolutionary separations (3-10%). Fixed genetic differences were also observed between northern and southern segments of several ridges. Analysis of molecular variance (AMOVA) shows an estimated 10.4% total genetic variation within patches, 17.5% among patches (within ridges), and 72.1% among ridges. This strong population structure among patches within ridges indicates that the distribution of S. woodi is tightly linked to sandy scrub habitat and that the discontinuous distribution of scrub habitats significantly inhibits dispersal and gene flow. Phylogeographic analyses indicate a pattern of dispersal down the Florida peninsula during the late Pliocene-early Pleistocene, followed by habitat fragmentation and variant isolation events. Therefore, the deep genetic structuring among scrub lizard populations on separate ridges is attributed to ancient isolation events induced by a shift from dry (xeric) to wet (mesic) conditions on the Florida peninsula. These findings indicate that some scrub lizard populations have persisted in isolation for time frames in excess of 1 Myr, providing a case history on the genetic consequences of habitat fragmentation. PMID:10447851

  16. Sequence of a 10.5 kbp fragment of Clostridium pasteurianum genomic DNA encompassing the hydrogenase I gene and two spore germination genes.

    PubMed

    Meyer, J

    1995-06-01

    The gene encoding hydrogenase I from Clostridium pasteurianum had previously been cloned and sequenced as part of a 2.3 kbp Sau3A genomic DNA fragment. The analysis of the regions surrounding the hydrogenase gene has been extended by cloning and sequencing two EcoRI fragments, both partially overlapping the 2.3 kbp Sau3A fragment. An uninterrupted genomic sequence of 10.5 kbp has thus been obtained, including 6.7 kbp upstream of the hydrogenase gene, and 2 kbp downstream. The hydrogenase gene is separated by 473 bp from the next open reading frame on its 5' side and by 366 bp from the next open reading frame on its 3' side. It is preceded by putative promoter regions, and followed by a strong transcription termination signal. Therefore the hydrogenase I gene from C. pasteurianum probably belongs to a monocistronic operon. This is consistent with previous biochemical evidence showing that the enzyme is monomeric. The sequence data also show that in the case of this Fe-hydrogenase, in contrast to the NiFe-hydrogenases, there are no accessory genes cotranscribed with, or located near, the structural genes. The sequenced region contains six open reading frames in addition to the hydrogenase gene. One of these probably encodes phosphatidylserine decarboxylase, and two others are homologous to two of the three genes of the gerA and gerB loci (spore germination) from Bacillus subtilis. These are the first spore germination gene sequences obtained from a bacterium of the genus Clostridium. PMID:16887524

  17. Auxin-induced rapid degradation of inhibitor of caspase-activated DNase (ICAD) induces apoptotic DNA fragmentation, caspase activation, and cell death: a cell suicide module.

    PubMed

    Samejima, Kumiko; Ogawa, Hiromi; Ageichik, Alexander V; Peterson, Kevin L; Kaufmann, Scott H; Kanemaki, Masato T; Earnshaw, William C

    2014-11-01

    Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms. PMID:25248749

  18. Auxin-induced Rapid Degradation of Inhibitor of Caspase-activated DNase (ICAD) Induces Apoptotic DNA Fragmentation, Caspase Activation, and Cell Death

    PubMed Central

    Samejima, Kumiko; Ogawa, Hiromi; Ageichik, Alexander V.; Peterson, Kevin L.; Kaufmann, Scott H.; Kanemaki, Masato T.; Earnshaw, William C.

    2014-01-01

    Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms. PMID:25248749

  19. Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

    Microsoft Academic Search

    Edward A. Lin; Xue-Song Zhang; Steven M. Levine; Steven R. Gill; Daniel Falush; Martin J. Blaser

    2009-01-01

    Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organism's ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products

  20. Dielectrophoresis of DNA: Quantification by impedance measurements

    PubMed Central

    Henning, Anja; Bier, Frank. F.; Hölzel, Ralph

    2010-01-01

    Dielectrophoretic properties of DNA have been determined by measuring capacitance changes between planar microelectrodes. DNA sizes ranged from 100 bp to 48 kbp, DNA concentrations from below 0.1 to 70 ?g?ml. Dielectrophoretic spectra exhibited maximum response around 3 kHz and 3 MHz. The strongest response was found for very long DNA (above 10 kbp) and for short 100 bp fragments, which corresponds to the persistence length of DNA. The method allows for an uncomplicated, automatic acquisition of the dielectrophoretic properties of submicroscopical objects without the need for labeling protocols or optical accessibility. PMID:20697597

  1. Optical/Near-infrared Polarization Survey of Sh 2-29: Magnetic Fields, Dense Cloud Fragmentations, and Anomalous Dust Grain Sizes

    NASA Astrophysics Data System (ADS)

    Santos, Fábio P.; Franco, Gabriel A. P.; Roman-Lopes, Alexandre; Reis, Wilson; Román-Zúñiga, Carlos G.

    2014-03-01

    Sh 2-29 is a conspicuous star-forming region marked by the presence of massive embedded stars as well as several notable interstellar structures. In this research, our goals were to determine the role of magnetic fields and to study the size distribution of interstellar dust particles within this turbulent environment. We have used a set of optical and near-infrared polarimetric data obtained at OPD/LNA (Brazil) and CTIO (Chile), correlated with extinction maps, Two Micron All Sky Survey data, and images from the Digitized Sky Survey and Spitzer. The region's most striking feature is a swept out interstellar cavity whose polarimetric maps indicate that magnetic field lines were dragged outward, piling up along its borders. This led to a higher magnetic strength value (?400 ?G) and an abrupt increase in polarization degree, probably due to an enhancement in alignment efficiency. Furthermore, dense cloud fragmentations with peak AV between 20 and 37 mag were probably triggered by its expansion. The presence of 24 ?m point-like sources indicates possible newborn stars inside this dense environment. A statistical analysis of the angular dispersion function revealed areas where field lines are aligned in a well-ordered pattern, seemingly due to compression effects from the H II region expansion. Finally, Serkowski function fits were used to study the ratio of the total-to-selective extinction, revealing a dual population of anomalous grain particle sizes. This trend suggests that both effects of coagulation and fragmentation of interstellar grains are present in the region. Based on observations collected at the National Optical Astronomy Observatory (CTIO, Chile) and Observatório do Pico dos Dias, operated by Laboratório Nacional de Astrofísica (LNA/MCT, Brazil).

  2. Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

    PubMed Central

    Yolov, A A; Gromova, E S; Kubareva, E A; Potapov, V K; Shabarova, Z A

    1985-01-01

    Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate. Images PMID:3001655

  3. In vitro nucleotide misinsertion opposite the oxidized guanosine lesions spiroiminodihydantoin and guanidinohydantoin and DNA synthesis past the lesions using Escherichia coli DNA polymerase I (Klenow fragment).

    PubMed

    Kornyushyna, Olga; Berges, Aym M; Muller, James G; Burrows, Cynthia J

    2002-12-24

    The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation. Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers. Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts. dAMP and dGMP were found to be inserted opposite these OG oxidation products. Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp). Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs. A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment. Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As. Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context. UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes. These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe. PMID:12484769

  4. A viral packaging motor varies its DNA rotation and step size to preserve subunit coordination as the capsid fills.

    PubMed

    Liu, Shixin; Chistol, Gheorghe; Hetherington, Craig L; Tafoya, Sara; Aathavan, K; Schnitzbauer, Joerg; Grimes, Shelley; Jardine, Paul J; Bustamante, Carlos

    2014-04-24

    Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per base pair increases with filling. This change accompanies a reduction in the motor's step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the downregulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated. PMID:24766813

  5. Size-frequency distributions of fragments from SPH/ N-body simulations of asteroid impacts: Comparison with observed asteroid families

    NASA Astrophysics Data System (ADS)

    Durda, Daniel D.; Bottke, William F.; Nesvorný, David; Enke, Brian L.; Merline, William J.; Asphaug, Erik; Richardson, Derek C.

    2007-02-01

    We investigate the morphology of size-frequency distributions (SFDs) resulting from impacts into 100-km-diameter parent asteroids, represented by a suite of 161 SPH/N-body simulations conducted to study asteroid satellite formation [Durda, D.D., Bottke, W.F., Enke, B.L., Merline, W.J., Asphaug, E., Richardson, D.C., Leinhardt, Z.M., 2004. Icarus 170, 243-257]. The spherical basalt projectiles range in diameter from 10 to 46 km (in equally spaced mass increments in logarithmic space, covering six discrete sizes), impact speeds range from 2.5 to 7 km/s (generally in 1 km/s increments), and impact angles range from 15° to 75° (nearly head-on to very oblique) in 15° increments. These modeled SFD morphologies match very well the observed SFDs of many known asteroid families. We use these modeled SFDs to scale to targets both larger and smaller than 100 km in order to gain insights into the circumstances of the impacts that formed these families. Some discrepancies occur for families with parent bodies smaller than a few tens of kilometers in diameter (e.g., 832 Karin), however, so due caution should be used in applying our results to such small families. We find that ˜20 observed main-belt asteroid families are produced by the catastrophic disruption of D >100 km parent bodies. Using these data as constraints, collisional modeling work [Bottke Jr., W.F., Durda, D.D., Nesvorný, D., Jedicke, R., Morbidelli, A., Vokrouhlický, D., Levison, H.F., 2005b. Icarus 179, 63-94] suggests that the threshold specific energy, QD?, needed to eject 50% of the target body's mass is very close to that predicted by Benz and Asphaug [Benz, W., Asphaug, E., 1999. Icarus 142, 5-20].

  6. Genetic Diversity among Strains of Moraxella catarrhalis: Analysis Using Multiple DNA Probes and a Single-Locus PCR-Restriction Fragment Length Polymorphism Method

    PubMed Central

    Walker, Elaine S.; Preston, Robert A.; Post, J. Christopher; Ehrlich, Garth D.; Kalbfleisch, John H.; Klingman, Karin L.

    1998-01-01

    Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce ?-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites. PMID:9650948

  7. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals

    PubMed Central

    Nabholz, Benoit; Glémin, Sylvain; Galtier, Nicolas

    2009-01-01

    Background During the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial DNA, the most popular marker of molecular biodiversity. Several important results were recently reported using mammals as model organisms, including (i) the absence of relationship between mitochondrial DNA diversity and life-history or ecological variables, (ii) the absence of prominent adaptive selection, contrary to what was found in invertebrates, and (iii) the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. We propose to challenge these results thanks to the bird/mammal comparison. Direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals (higher mass-specific metabolic rate and longevity). These properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation. Results A phylogenetic analysis of cytochrome b third-codon position confirms that the mitochondrial DNA mutation rate is quite variable in birds, passerines being the fastest evolving order. On average, mitochondrial DNA evolves slower in birds than in mammals of similar body size. This result is in agreement with the longevity hypothesis, and contradicts the hypothesis of a metabolic rate-dependent mutation rate. Birds show no footprint of adaptive selection on cytochrome b evolutionary patterns, but no link between direct estimates of population size and cytochrome b diversity. The mutation rate is the best predictor we have of within-species mitochondrial diversity in birds. It partly explains the differences in mitochondrial DNA diversity patterns observed between mammals and birds, previously interpreted as reflecting Hill-Robertson interferences with the W chromosome. Conclusion Mitochondrial DNA diversity patterns in birds are strongly influenced by the wide, unexpected variation of mutation rate across species. From a fundamental point of view, these results are strongly consistent with a relationship between species maximal longevity and mitochondrial mutation rate, in agreement with the mitochondrial theory of ageing. Form an applied point of view, this study reinforces and extends the message of caution previously expressed for mammals: mitochondrial data tell nothing about species population sizes, and strongly depart the molecular clock assumption. PMID:19284537

  8. Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples.

    PubMed

    Uyaguari-Diaz, Miguel I; Slobodan, Jared R; Nesbitt, Matthew J; Croxen, Matthew A; Isaac-Renton, Judith; Prystajecky, Natalie A; Tang, Patrick

    2015-01-01

    Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads. PMID:25938488

  9. Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

    PubMed Central

    Cohen, S; Lavi, S

    1996-01-01

    Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed. PMID:8628266

  10. Self-assembly of DNA nanohydrogels with controllable size and stimuli-responsive property for targeted gene regulation therapy.

    PubMed

    Li, Juan; Zheng, Cheng; Cansiz, Sena; Wu, Cuichen; Xu, Jiehua; Cui, Cheng; Liu, Yuan; Hou, Weijia; Wang, Yanyue; Zhang, Liqin; Teng, I-ting; Yang, Huang-Hao; Tan, Weihong

    2015-02-01

    Here, we report the synthesis and characterization of size-controllable and stimuli-responsive DNA nanohydrogels as effective targeted gene delivery vectors. DNA nanohydrogels were created through a self-assembly process using three kinds of building units, respectively termed Y-shaped monomer A with three sticky ends (YMA), Y-shaped monomer B with one sticky end (YMB), and DNA linker (LK) with two sticky ends. Hybridization at the sticky ends of monomers and LK leads to nanohydrogel formation. DNA nanohydrogels are size-controllable by varying the ratio of YMA to YMB. By incorporating different functional elements, such as aptamers, disulfide linkages, and therapeutic genes into different building units, the synthesized aptamer-based nanohydrogels (Y-gel-Apt) can be used for targeted and stimuli-responsive gene therapy. Y-gel-Apt strongly inhibited cell proliferation and migration in target A549 cells, but not in control cells. By taking advantage of facile modular design and assembly, efficient cellular uptake, and superior biocompatibility, this Y-gel-Apt holds great promise as a candidate for targeted gene or drug delivery and cancer therapy. PMID:25581100

  11. A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index.

    PubMed

    Hamidi, Jamal; Frainais, Christophe; Amar, Edouard; Bailly, Eric; Clément, Patrice; Ménézo, Yves

    2015-08-01

    The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB. PMID:24988915

  12. Next-generation sequencing of genomic DNA fragments bound to a transcription factor in vitro reveals its regulatory potential.

    PubMed

    Kurihara, Yukio; Makita, Yuko; Kawashima, Mika; Hamasaki, Hidefumi; Yamamoto, Yoshiharu Y; Matsui, Minami

    2014-01-01

    Several transcription factors (TFs) coordinate to regulate expression of specific genes at the transcriptional level. In Arabidopsis thaliana it is estimated that approximately 10% of all genes encode TFs or TF-like proteins. It is important to identify target genes that are directly regulated by TFs in order to understand the complete picture of a plant's transcriptome profile. Here, we investigate the role of the LONG HYPOCOTYL5 (HY5) transcription factor that acts as a regulator of photomorphogenesis. We used an in vitro genomic DNA binding assay coupled with immunoprecipitation and next-generation sequencing (gDB-seq) instead of the in vivo chromatin immunoprecipitation (ChIP)-based methods. The results demonstrate that the HY5-binding motif predicted here was similar to the motif reported previously and that in vitro HY5-binding loci largely overlapped with the HY5-targeted candidate genes identified in previous ChIP-chip analysis. By combining these results with microarray analysis, we identified hundreds of HY5-binding genes that were differentially expressed in hy5. We also observed delayed induction of some transcripts of HY5-binding genes in hy5 mutants in response to blue-light exposure after dark treatment. Thus, an in vitro gDNA-binding assay coupled with sequencing is a convenient and powerful method to bridge the gap between identifying TF binding potential and establishing function. PMID:25534860

  13. A method to simultaneously construct up to 12 differently sized Illumina Nextera long mate pair libraries with reduced DNA input, time, and cost.

    PubMed

    Heavens, Darren; Accinelli, Gonzalo Garcia; Clavijo, Bernardo; Clark, Matthew Derek

    2015-01-01

    Long mate pair (LMP) or "jump" libraries are invaluable for producing contiguous genome assemblies and assessing structural variation. However the consistent production of high quality (low duplication rate, accurately sized) LMP libraries has proven problematic in many genome projects. Input DNA length and quantity are key issues that can affect success. Here we demonstrate how 12 libraries covering a wide range of jump sizes can be constructed from <10 µg of DNA, thus ensuring production of the best LMP libraries from a given DNA sample. Finally, we demonstrate the accuracy of the insert sizes by mapping reads from each library back to an existing assembly. PMID:26156783

  14. Nucleotide sequence of XhoI O fragment of ectromelia virus DNA reveals significant differences from vaccinia virus.

    PubMed

    Senkevich, T G; Muravnik, G L; Pozdnyakov, S G; Chizhikov, V E; Ryazankina, O I; Shchelkunov, S N; Koonin, E V; Chernos, V I

    1993-10-01

    The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions. PMID:8266721

  15. The Innate Immunity Adaptor SARM Translocates to the Nucleus to Stabilize Lamins and Prevent DNA Fragmentation in Response to Pro-Apoptotic Signaling

    PubMed Central

    Sethman, Chad R.; Hawiger, Jacek

    2013-01-01

    Sterile alpha and armadillo-motif containing protein (SARM), a highly conserved and structurally unique member of the MyD88 family of Toll-like receptor adaptors, plays an important role in innate immunity signaling and apoptosis. Its exact mechanism of intracellular action remains unclear. Apoptosis is an ancient and ubiquitous process of programmed cell death that results in disruption of the nuclear lamina and, ultimately, dismantling of the nucleus. In addition to supporting the nuclear membrane, lamins serve important roles in chromatin organization, epigenetic regulation, transcription, nuclear transport, and mitosis. Mutations and other damage that destabilize nuclear lamins (laminopathies) underlie a number of intractable human diseases. Here, we report that SARM translocates to the nucleus of human embryonic kidney cells by using its amino-terminal Armadillo repeat region. Within the nucleus, SARM forms a previously unreported lattice akin to the nuclear lamina scaffold. Moreover, we show that SARM protects lamins from apoptotic degradation and reduces internucleosomal DNA fragmentation in response to signaling induced by the proinflammatory cytokine Tumor Necrosis Factor alpha. These findings indicate an important link between the innate immunity adaptor SARM and stabilization of nuclear lamins during inflammation-driven apoptosis in human cells. PMID:23923041

  16. DNA sequence analysis and restriction fragment length polymorphisms of the P1 gene of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    PubMed Central

    Reed, R B; Frost, J B; Kort, K; Myers, S D; Lesse, A J

    1996-01-01

    Brazilian purpuric fever (BPF) is a fulminant pediatric disease caused by specific strains of Haemophilus influenzae biogroup aegyptius. A conserved epitope on the P1 protein of strains of H. influenzae biogroup aegyptius is seen on most virulent isolates. The P1 protein from a Brazilian case-clone strain of H. influenzae biogroup aegyptius was analyzed by cloning and sequencing the gene. Three major variable regions are present within the P1 gene of the BPF clone in an architecture similar to that of the previously sequenced P1 genes from H. influenzae. The DNA sequence data of the P1 gene provided information for restriction fragment length polymorphism analyses among strains of H. influenzae biogroup aegyptius. Using PCR for amplification of the P1 gene, we found that AlwI restriction of this gene allowed for a highly accurate segregation of virulent strains of H. influenzae biogroup aegyptius associated with BPF. The strong association of virulent phenotypes with specific AlwI restriction patterns of the P1 gene provides a basis for the convenient and accurate identification of strains of H. influenzae biogroup aegyptius which cause BPF. PMID:8751915

  17. A Discrete-Time Model for Cell-Age, Size, and DNA Distributions of Proliferating Cells, and its Application to the Movement of the Labeled Cohort

    Microsoft Academic Search

    M. Kim; Khosrow Bahrami; Kwang B. Woo

    1974-01-01

    The dynamics of cell cycle and proliferating kinetics are characterized by the state equations, which are linear difference matrix equations with their state vectors consisting of variables representing cells in a particular compartment for cell-age, cell-size, and cell-DNA content. The transformation required from the cell-age distribution to the cell-size and DNA distributions in this discrete-time model under various assumptions is

  18. Analysis of DNA size, content and cell cycle in leaves of Napier grass ( Pennisetum purpureum Schum.)

    Microsoft Academic Search

    M. G. Taylor; I. K. Vasil

    1987-01-01

    Mesophyll cell nuclei isolated from leaves of Pennisetum purpureum were analysed by flow cytometry to determine the nuclear DNA content and the percentage of cells in different phases of the cell cycle. Samples taken from base, middle and tip regions of leaves 2 to 8 (leaf 1, which was adjacent to the meristem, was too small to sample) showed no

  19. Fabrication of Size-Controllable Nanofluidic Channels by Nanoimprinting and Its Application for DNA Stretching

    Microsoft Academic Search

    L. Jay Guo; Xing Cheng; Chia-Fu Chou

    2004-01-01

    We report a new approach to greatly simplify the fabrication of nanofluidic channels with well-controlled dimensions. It is achieved by imprinting a channel template into a thin polymer film cast on a glass cover slip in a single step, offering a much higher throughput than previous methods. We demonstrated effective DNA stretching in these nanochannels. This method provides a simple

  20. Fragmentation of Shells

    NASA Astrophysics Data System (ADS)

    Wittel, F.; Kun, F.; Herrmann, H. J.; Kröplin, B. H.

    2004-07-01

    We present a theoretical and experimental study of the fragmentation of closed thin shells made of a disordered brittle material. Experiments were performed on brown and white hen egg shells under two different loading conditions: impact with a hard wall and explosion by a combustible mixture. Both give rise to power law fragment size distributions. A three-dimensional discrete element model of shells is worked out. Based on simulations of the model, we give evidence that power law fragment mass distributions arise due to an underlying phase transition which proved to be abrupt for explosion and continuous for impact. We demonstrate that the fragmentation of closed shells defines a new universality class of fragmentation phenomena.

  1. Genetic (RAPD) diversity in Peromyscus maniculatus populations in a naturally fragmented landscape

    Microsoft Academic Search

    L. M. Vucetich; J. A. Vucetich; C. P. Joshi; T. A. Waite; R. O. Peterson

    2001-01-01

    We assessed the effects of long-term habitat fragmentation on genetic (random amplified polymorphic DNA) diversity in 11 Peromyscus maniculatus populations in the Lake Superior watershed. We analysed genetic structure at two spatial scales and the effect of island size and isolation on genetic diversity. At the regional scale, island populations differed from mainland populations ( F ST = 0.36), but

  2. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  3. Species richness and food web structure of soil decomposer community as affected by the size of habitat fragment and habitat corridors

    Microsoft Academic Search

    R ANT A LAINEN; J ARI H AIMI; T A I N A P E N N A N E Nw; H E I K K I S E T ALAz

    2005-01-01

    While most ecologists agree that the effects of fragmentation on diversity of organisms are predominantly negative and that the scale of fragmentation defines their severity, the role of habitat corridors in mitigating those effects still remains controversial. This ambiguousness rests largely on various difficulties in experimentation, a problem partially solved in the present paper by the use of easily manipulated

  4. Genome analysis of ground squirrels of the genus Citellus (Rodentia, Sciuridae) I. DNA reassociation kinetics and genome size of eight species

    Microsoft Academic Search

    L. K. Ginatulina; A. A. Ginatulin; E. A. Lvapunova; N. N. Vorontsov

    1982-01-01

    Kinetic of reassociation of short DNA fragments were measured in eight ground squirrel species: Citellus undulatus, C. parryi, C. relictus, C. dauricus, C. citellus, C. pygmaeus, C. fulvus and C. major. It was shown that 30–50% of their genome were represented by repeated sequences forming three kinetic fractions, i.e., very fast (Cot<10-3), fast (Cot 10-3-3×10-1) and intermediate (Cot 6×10-1-6×101). Based

  5. LOCALIZATION IN THE TOMATO GENOME OF DNA RESTRICTION FRAGMENTS CONTAINING SEQUENCES HOMOLOGOUS TO THE RRNA (45S), THE MAJOR CHLOROPHYLL A\\/B BINDING POLYPEPTIDE AND THE RIBULOSE BISPHOSPHATE CARBOXYLASE GENES

    Microsoft Academic Search

    C. E. VALLEJOS; S. D. TANKSLEY; R. BERNATZKY

    DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a\\/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm

  6. Differentiation of Spotted Fever Group Rickettsiae by Sequencing and Analysis of Restriction Fragment Length Polymorphism of PCR Amplified DNA of the Gene Encoding the Protein rOmpA

    Microsoft Academic Search

    VERONIQUE ROUX; PIERRE-EDOUARD FOURNIER; ANDDIDIER RAOULT

    1996-01-01

    Currently, the genotypic identification of the spotted fever group (SFG) rickettsiae is based on restriction fragmentlengthpolymorphismanalysisofPCR-amplifiedgenescodingfortheenzymecitratesynthaseandthe surfaceproteinsrOmpAandrOmpB.Asetofusefulrestrictionendonucleaseswasfoundfollowingcomparison ofRickettsiarickettsiiandR.prowazekiisequences.However,byusingthreePCRamplificationsandfourenzyme digestions with this set, it was impossible to differentiate between all of the known serotypes of the SFG rickettsiae. We amplified by PCR and sequenced using an automated laser fluorescent DNA sequencer a fragment of the gene encoding the protein rOmpA from

  7. Taxonomy and phylogeny of some Eimeria (Apicomplexa: Eimeriidae) species of rodents as determined by polymerase chain reaction\\/restriction-fragment-length polymorphism analysis of 18S rDNA

    Microsoft Academic Search

    J. A. Hnida; D. W. Duszynski

    1999-01-01

    The 18S rDNA genes of 10 Eimeria species from rodents (E. albigulae, E. arizonensis, E. falciformis, E. langebarteli, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis) were polymerase-chain-reaction (PCR)-amplified, digested with 12 restriction endonucleases, and electophoresed in agarose\\u000a gels. The resulting fragment patterns (riboprints) distinguished all species except E. sevilletensis from E.?falciformis, and E. arizonensis from

  8. The size of the internal loop in DNA hairpins influences their targeting with partially complementary strands.

    PubMed

    Prislan, Iztok; Lee, Hui-Ting; Lee, Cynthia; Marky, Luis A

    2015-01-01

    Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, "T5" is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression. PMID:25486129

  9. Levels and size complexity of DNA polymerase beta mRNA in rat regenerating liver and other organs.

    PubMed

    Nowak, R; Siedlecki, J A; Kaczmarek, L; Zmudzka, B Z; Wilson, S H

    1989-07-01

    A cDNA probe encoding DNA polymerase beta (beta-pol) was used to study the level and size complexity of beta-pol mRNA in regenerating rat liver and other rat tissues. An almost 2-fold increase in beta-pol mRNA was observed 18-24 h after partial hepatectomy. In most adult rat tissues (liver, heart, kidney, stomach, spleen, thymus, lung and brain) the abundance of beta-pol mRNA was low. In contrast, young brain and testes exhibited beta-pol mRNA levels 5- and 15-times higher, respectively. The observed changes in the level of beta-pol mRNA in regenerating rat liver and in developing brain are correlated with reported changes in DNA polymerase beta activity. Four different (4.0, 2.5, 2.2, 1.4 kb) transcripts hybridizing to beta-pol probe were found in all tissues examined. The 4.0 kb transcript was dominant for young and adult brain, whereas the 1.4 kb transcript was dominant for testes. The significance of these transcripts is discussed. PMID:2736248

  10. A Viral Packaging Motor Varies Its DNA Rotation and Step Size to Preserve Subunit Coordination as the Capsid Fills

    PubMed Central

    Tafoya, Sara; Aathavan, K.; Schnitzbauer, Joerg; Grimes, Shelley; Jardine, Paul J.; Bustamante, Carlos

    2014-01-01

    SUMMARY Multimeric, ring-shaped molecular motors rely on the coordinated action of their subunits to perform crucial biological functions. During these tasks, motors often change their operation in response to regulatory signals. Here, we investigate a viral packaging machine as it fills the capsid with DNA and encounters increasing internal pressure. We find that the motor rotates the DNA during packaging and that the rotation per basepair increases with filling. This change accompanies a reduction in the motor’s step size. We propose that these adjustments preserve motor coordination by allowing one subunit to make periodic, specific, and regulatory contacts with the DNA. At high filling, we also observe the down-regulation of the ATP-binding rate and the emergence of long-lived pauses, suggesting a throttling-down mechanism employed by the motor near the completion of packaging. This study illustrates how a biological motor adjusts its operation in response to changing conditions, while remaining highly coordinated. PMID:24766813

  11. Finding of widespread viral and bacterial revolution dsDNA translocation motors distinct from rotation motors by channel chirality and size

    PubMed Central

    2014-01-01

    Background Double-stranded DNA translocation is ubiquitous in living systems. Cell mitosis, bacterial binary fission, DNA replication or repair, homologous recombination, Holliday junction resolution, viral genome packaging and cell entry all involve biomotor-driven dsDNA translocation. Previously, biomotors have been primarily classified into linear and rotational motors. We recently discovered a third class of dsDNA translocation motors in Phi29 utilizing revolution mechanism without rotation. Analogically, the Earth rotates around its own axis every 24 hours, but revolves around the Sun every 365 days. Results Single-channel DNA translocation conductance assay combined with structure inspections of motor channels on bacteriophages P22, SPP1, HK97, T7, T4, Phi29, and other dsDNA translocation motors such as bacterial FtsK and eukaryotic mimiviruses or vaccinia viruses showed that revolution motor is widespread. The force generation mechanism for revolution motors is elucidated. Revolution motors can be differentiated from rotation motors by their channel size and chirality. Crystal structure inspection revealed that revolution motors commonly exhibit channel diameters larger than 3 nm, while rotation motors that rotate around one of the two separated DNA strands feature a diameter smaller than 2 nm. Phi29 revolution motor translocated double- and tetra-stranded DNA that occupied 32% and 64% of the narrowest channel cross-section, respectively, evidencing that revolution motors exhibit channel diameters significantly wider than the dsDNA. Left-handed oriented channels found in revolution motors drive the right-handed dsDNA via anti-chiral interaction, while right-handed channels observed in rotation motors drive the right-handed dsDNA via parallel threads. Tethering both the motor and the dsDNA distal-end of the revolution motor does not block DNA packaging, indicating that no rotation is required for motors of dsDNA phages, while a small-angle left-handed twist of dsDNA that is aligned with the channel could occur due to the conformational change of the phage motor channels from a left-handed configuration for DNA entry to a right-handed configuration for DNA ejection for host cell infection. Conclusions The revolution motor is widespread among biological systems, and can be distinguished from rotation motors by channel size and chirality. The revolution mechanism renders dsDNA void of coiling and torque during translocation of the lengthy helical chromosome, thus resulting in more efficient motor energy conversion. PMID:24940480

  12. Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell.

    PubMed

    Kononowicz, H; Janick, J

    1988-01-01

    There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm. PMID:3220146

  13. New Scalings in Nuclear Fragmentation

    SciTech Connect

    Bonnet, E. [GANIL (DSM-CEA/CNRS/IN2P3), F-14076 Caen cedex (France); Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); Borderie, B.; Rivet, M. F. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); Le Neindre, N. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); LPC, CNRS/IN2P3, Ensicaen, Universite de Caen, F-14050 Caen cedex (France); Raduta, Ad. R. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); National Institute for Physics and Nuclear Engineering, RO-76900 Bucharest-Magurele (Romania); Bougault, R. [LPC, CNRS/IN2P3, Ensicaen, Universite de Caen, F-14050 Caen cedex (France); Chbihi, A.; Frankland, J. D.; Wieleczko, J. P. [GANIL (DSM-CEA/CNRS/IN2P3), F-14076 Caen cedex (France); Galichet, E. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); Conservatoire National des Arts et Metiers, F-75141 Paris cedex 03 (France); Gagnon-Moisan, F. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); Laboratoire de Physique Nucleaire, Departement de Physique, de Genie Physique et d'Optique, Universite Laval, Quebec, G1K 7P4 (Canada); Guinet, D.; Lautesse, P. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Claude Bernard Lyon 1, F-69622 Villeurbanne cedex (France); Lukasik, J. [Institute of Nuclear Physics IFJ-PAN, PL-31342 Krakow (Poland); Marini, P. [Institut de Physique Nucleaire, CNRS/IN2P3, Universite Paris-Sud 11, F-91406 Orsay cedex (France); GANIL (DSM-CEA/CNRS/IN2P3), F-14076 Caen cedex (France); Parlog, M. [LPC, CNRS/IN2P3, Ensicaen, Universite de Caen, F-14050 Caen cedex (France); National Institute for Physics and Nuclear Engineering, RO-76900 Bucharest-Magurele (Romania)

    2010-10-01

    Fragment partitions of fragmenting hot nuclei produced in central and semiperipheral collisions have been compared in the excitation energy region 4-10 MeV per nucleon where radial collective expansion takes place. It is shown that, for a given total excitation energy per nucleon, the amount of radial collective energy fixes the mean fragment multiplicity. It is also shown that, at a given total excitation energy per nucleon, the different properties of fragment partitions are completely determined by the reduced fragment multiplicity (i.e., normalized to the source size). Freeze-out volumes seem to play a role in the scalings observed.

  14. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery.

    PubMed

    Dauty, Emmanuel; Verkman, A S

    2005-03-01

    The cytosol of mammalian cells is a crowded environment containing soluble proteins and a network of cytoskeletal filaments. Gene delivery by synthetic vectors involves the endocytosis of DNA-polycation complexes, escape from endosomes, and diffusion of non-complexed DNA through the cytosol to reach the nucleus. We found previously that the translational diffusion of large DNAs (>250 bp) in cytoplasm was greatly slowed compared with that of smaller DNAs (Lukacs, G. L., Haggie, P., Seksek, O., Lechardeur, D., Freedman, N., and Verkman, A. S. (2000) J. Biol. Chem. 275, 1625-1629). To determine the mechanisms responsible for size-dependent DNA diffusion, we used fluorescence correlation spectroscopy to measure the diffusion of single fluorophore-labeled DNAs in crowded solutions, cytosol extracts, actin network, and living cells. DNA diffusion (D) in solutions made crowded with Ficoll-70 (up to 40 weight percentage) or soluble cytosol extracts (up to 100 mg/ml) relative to diffusion of the same sized DNAs in saline (D/D(o)) was approximately independent of DNA size (20-4500 bp), quite different from the strong reduction in D/D(o) in the cytoplasm of living cells. However, the reduced D/D(o) with increasing DNA size was closely reproduced in solutions containing cross-linked actin filaments assembled with gelsolin, whereas soluble macromolecules of the same size and concentration did not reduce D/D(o). In intact cells microinjected with fluorescent DNAs and studied by fluorescence correlation spectroscopy or photobleaching methods, D/D(o) was reduced by 5-150-fold (20-6000 bp); however, the size-dependent reduction in D/D(o) was abolished after actin cytoskeleton disruption. Our results identify the actin cytoskeleton as a major barrier restricting cytoplasmic transport of non-complexed DNA in non-viral gene transfer. PMID:15632160

  15. [Analysis of DNA polymorphism in populations of the Volga-Ural region using genome fingerprinting with phage M13 DNA].

    PubMed

    Khusnutdinova, E K; Khidiiatova, I M; Viktorova, T V; Fatkhlislamova, R I; Limborskaia, S A

    1999-04-01

    The hyperpolymorphism of minisatellite DNA hybridizing with DNA of bacteriophage M13 was analyzed in seven Turkic and Finno-Ugric populations from the Volga-Urals region. In total, hybridization revealed 80 BspRI genomic DNA fragments ranging in size from 1.7 to 10 kb; the average frequency of an individual fragment was 0.299 +/- 0.020. The average number of hybridization fragments per pattern (varying from 14 to 20 in different populations) and frequencies of individual fragments showed significant interpopulation differences. Parameters of this polymorphic system were assumed to reflect phenotypic diversity of populations. Genome fingerprinting with the use of phage M13 can be employed in the studies of population genetic structure and differentiation and in forensic medicine, for more accurate personal identification. PMID:10420275

  16. Quantification of HIV1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR

    Microsoft Academic Search

    Ubaldo Visco Comandini; Anders Sönnerborg; Anders Vahlne; Zhibing Yun

    1997-01-01

    A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard

  17. Auroral fragmentation into patches

    NASA Astrophysics Data System (ADS)

    Shiokawa, Kazuo; Hashimoto, Ayumi; Hori, Tomoaki; Sakaguchi, Kaori; Ogawa, Yasunobu; Donovan, Eric; Spanswick, Emma; Connors, Martin; Otsuka, Yuichi; Oyama, Shin-Ichiro; Nozawa, Satonori; McWilliams, Kathryn

    2014-10-01

    Auroral patches in diffuse auroras are very common features in the postmidnight local time. However, the processes that produce auroral patches are not yet well understood. In this paper we present two examples of auroral fragmentation which is the process by which uniform aurora is broken into several fragments to form auroral patches. These examples were observed at Athabasca, Canada (geomagnetic latitude: 61.7°N), and Tromsø, Norway (67.1°N). Captured in sequences of images, the auroral fragmentation occurs as finger-like structures developing latitudinally with horizontal-scale sizes of 40-100 km at ionospheric altitudes. The structures tend to develop in a north-south direction with speeds of 150-420 m/s without any shearing motion, suggesting that pressure-driven instability in the balance between the earthward magnetic-tension force and the tailward pressure gradient force in the magnetosphere is the main driving force of the auroral fragmentation. Therefore, these observations indicate that auroral fragmentation associated with pressure-driven instability is a process that creates auroral patches. The observed slow eastward drift of aurora during the auroral fragmentation suggests that fragmentation occurs in low-energy ambient plasma.

  18. DNA fingerprinting intron-sizing method to accomplish a specific, rapid, and sensitive identification of carotenogenic Dunaliella species.

    PubMed

    Olmos-Soto, Jorge; Paniagua-Michel, J; Contreras, Rosalía; Ochoa, Leonel

    2012-01-01

    Dunaliella salina has become the most important microorganism for the production of ?-carotene around the world. Natural carotenoids are a source of active metabolites utilized in different areas of food nutrition and pharmaceuticals, both in humans and also in animals. Identification of Dunaliella species from natural environments or certified culture collections is not precise and it is time consuming. However, accurate identification is extremely important because a slight difference in Dunaliella species generates great differences in carotenoids production. Here, we describe an intron-sizing method to make a rapid and precise identification for each of the most important carotenogenic species, showing that each hyperproducer species has an exclusive 18S rDNA fingerprint profile. PMID:22623309

  19. Thermodynamics of fragment binding.

    PubMed

    Ferenczy, György G; Keser?, György M

    2012-04-23

    The ligand binding pockets of proteins have preponderance of hydrophobic amino acids and are typically within the apolar interior of the protein; nevertheless, they are able to bind low complexity, polar, water-soluble fragments. In order to understand this phenomenon, we analyzed high resolution X-ray data of protein-ligand complexes from the Protein Data Bank and found that fragments bind to proteins with two near optimal geometry H-bonds on average. The linear extent of the fragment binding site was found not to be larger than 10 Å, and the H-bonding region was found to be restricted to about 5 Å on average. The number of conserved H-bonds in proteins cocrystallized with multiple different fragments is also near to 2. These fragment binding sites that are able to form limited number of strong H-bonds in a hydrophobic environment are identified as hot spots. An estimate of the free-energy gain of H-bond formation versus apolar desolvation supports that fragment sized compounds need H-bonds to achieve detectable binding. This suggests that fragment binding is mostly enthalpic that is in line with their observed binding thermodynamics documented in Isothermal Titration Calorimetry (ITC) data sets and gives a thermodynamic rationale for fragment based approaches. The binding of larger compounds tends to more rely on apolar desolvation with a corresponding increase of the entropy content of their binding free-energy. These findings explain the reported size-dependence of maximal available affinity and ligand efficiency both behaving differently in the small molecule region featured by strong H-bond formation and in the larger molecule region featured by apolar desolvation. PMID:22458364

  20. Food Fragmentation by Human Mastication

    Microsoft Academic Search

    Naoki Kobayashi; Kaoru Kohyama; Chiharu Kobori; Yo Sasaki; Mitsugu Matsushita

    2006-01-01

    Fragment-size distribution has been studied experimentally by masticating raw carrot and fish gel. Lognormal distribution shows partly-good fit with various data. We have found that the fit of two-lognormals distribution is extremely good up to the entire region for masticated food fragments. The excellent data fitting by two-lognormals distribution implies that two main functions of mastication, a sequential fragmentation with

  1. Micron-sized pillars for ion-pair reversed-phase DNA separations.

    PubMed

    De Malsche, Wim; Zhang, Lei; Op De Beeck, Jeff; Vangelooven, Joris; Majeed, Bivragh; Desmet, Gert

    2010-12-01

    In the present paper, the feasibility to construct micron-sized silicon pillar channels to be used in HPLC is studied. For this, a channel with flow-through pores of 1??m and with critical sidewall dimensions below 1??m was constructed using advanced deep-UV lithographic equipment. Integrating a 3-nL injection system on the chip directly in front of the separation channel and using elongated distribution structures, a very controlled and high aspect ratio sample definition across the relatively wide separation channel was accomplished. The system was evaluated in isocratic ion-pair RP mode, allowing the separation of a mixture of two components with, respectively, 300 and 400 base pairs in 5?s only. PMID:21031463

  2. Enhanced discrimination of DNA molecules in nanofluidic channels through multiple measurements

    PubMed Central

    Sen, Yi-Heng; Jain, Tarun; Aguilar, Carlos A.; Karnik, Rohit

    2012-01-01

    Nanofluidic sensing elements have been the focus of recent experiments for numerous applications from nucleic acid fragment sizing to single-molecule DNA sequencing. These applications critically rely on high measurement fidelity, and methods to increase resolution are required. Herein, we describe fabrication and testing of a nanochannel device that enhances measurement resolution by performing multiple measurements (>100) on single DNA molecules. The enhanced measurement resolution enabled length discrimination between a mixture of ?-DNA (48.5 kbp) and T7 DNA (39.9 kbp) molecules, which were detected as transient current changes during translocation of the molecules through the nanochannel. As long DNA molecules are difficult to resolve quickly and with high fidelity with conventional electrophoresis, this approach may yield potentially portable, direct electrical sizing of DNA fragments with high sensitivity and resolution. PMID:22298224

  3. DNA fingerprinting for forensic identification: potential effects on data interpretation of subpopulation heterogeneity and band number variability.

    PubMed Central

    Cohen, J E

    1990-01-01

    Some methods of statistical analysis of data on DNA fingerprinting suffer serious weaknesses. Unlinked Mendelizing loci that are at linkage equilibrium in subpopulations may be statistically associated, not statistically independent, in the population as a whole if there is heterogeneity in gene frequencies between subpopulations. In the populations where DNA fingerprinting is used for forensic applications, the assumption that DNA fragments occur statistically independently for different probes, different loci, or different fragment size classes lacks supporting data so far; there is some contrary evidence. Statistical association of alleles may cause estimates based on the assumption of statistical independence to understate the true matching probabilities by many orders of magnitude. The assumptions that DNA fragments occur independently and with constant frequency within a size class appear to be contradicted by the available data on the mean and variance of the number of fragments per person. The mistaken use of the geometric mean instead of the arithmetic mean to compute the probability that every DNA fragment of a randomly chosen person is present among the DNA fragments of a specimen may substantially understate the probability of a match between blots, even if other assumptions involved in the calculations are taken as correct. The conclusion is that some astronomically small probabilities of matching by chance, which have been claimed in forensic applications of DNA fingerprinting, presently lack substantial empirical and theoretical support. PMID:2301401

  4. Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542

    Microsoft Academic Search

    Toshihiko Komari

    1990-01-01

    A 15.2-kb KpnI fragment from the virulence region of pTiBo542, the Ti plasmid harbored by Agrobacterium tumefaciens strain A281, was introduced into binary vectors. The fragment contained the virB, virC and virG genes, and it is known to have the ability to increase the virulence of strains of A. tumefaciens. The strains of A. tumefaciens that carried the resulting plasmids

  5. A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus

    Microsoft Academic Search

    M. Kool; R. W. Goldbach; J. M. Vlak

    1994-01-01

    In addition to the seven known homologous regions (hrs) of Autographa ealiforniea multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindlII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this or~ contains several segments essential for its activity and other 'auxiliary' sequences that enhance the or~ activity. Sequence analysis

  6. Comparison of CpG s-ODNs, chromatin immune complexes, and dsDNA fragment immune complexes in the TLR9-dependent activation of rheumatoid factor B cells

    Microsoft Academic Search

    Ann Marshak-Rothstein; Liliana Busconi; Christina M. Lau; Abigail S. Tabor; Elizabeth A. Leadbetter; Shizuo Akira; Arthur M. Krieg; Grayson B. Lipford; Gregory A. Viglianti; Ian R. Rifkin

    2004-01-01

    Synthetic single-stranded oligodeoxynucleotides (15—30 bp) containing CpG motifs and phosphorothioate backbones (CpG s-ODN), immune complexes consisting of anti-nucleosome mAbs and mammalian chromatin (chromatin IC), and immune complexes consisting of anti-hapten mAbs and haptenated-double stranded DNA fragments (~600 bp) can all effectively stimulate transgenic B cells expressing a rheumatoid factor receptor by a TLR9-dependent process. However, differential sensitivity to both s-ODN

  7. DNA Extraction from Museum Specimens of Parasitic Hymenoptera

    PubMed Central

    Andersen, Jeremy C.; Mills, Nicholas J.

    2012-01-01

    At the same time that molecular researchers are improving techniques to extract DNA from museum specimens, this increased demand for access to museum specimens has created tension between the need to preserve specimens for maintaining collections and morphological research and the desire to conduct molecular analyses. To address these concerns, we examined the suitability of non-invasive DNA extraction techniques on three species of parasitic Hymenoptera (Braconidae), and test the effects of body size (parasitoid species), age (time since collection), and DNA concentration from each extract on the probability of amplifying meaningful fragments of two commonly used genetic loci. We found that age was a significant factor for determining the probability of success for sequencing both 28S and COI fragments. While the size of the braconid parasitoids significantly affected the total amount of extracted DNA, neither size nor DNA concentration were significant factors for the amplification of either gene region. We also tested several primer combinations of various lengths, but were unable to amplify fragments longer than ?150 base pairs. These short fragments of 28S and COI were however sufficient for species identification, and for the discovery of within species genetic variation. PMID:23077493

  8. The shape distribution of fragments in dynamic fragmentation: Geometric approach

    NASA Astrophysics Data System (ADS)

    Michikami, Tatsuhiro; Sirono, Sin-Iti

    Dynamic fragmentation is a complex physical phenomenon in many natural systems. The shape of fragments is one of the most interesting aspects of dynamic fragmentation. That is, in laboratory impact experiments, the shape of ? fragments over a broad size range is distributed around the mean value of the axial ratio 2: 2: 1, which is independent of a wide range of experimental conditions. The shape distributions of the boulders on asteroid Eros and the small-and fast-rotating asteroids (diameter <200 m and rotation period <1h) are similar to those of laboratory fragments (Michikami et al., Icarus, in press). However, there are few studies to explain the shape distribution of fragments. No simple fragmentation model can reproduce the experimental shape distribution satisfactory. Many geometric approaches have focused on the size distribution of fragments. In this study, a geometric approach by numerical simulation has been performed, but we consider the effect of faults and the growth of cracks on the shape of fragments. Our model can reproduce the experimental shape distribution. The results show that the shape distribution is independent of target's shape, the growth of cracks and the number of inherent faults in the target.

  9. Programmed size-selected permeation of ssDNA into ZnS mesoporous hollow Dara Van Gough,* Juliet L. Defino and Paul V. Braun

    E-print Network

    Braun, Paul

    Programmed size-selected permeation of ssDNA into ZnS mesoporous hollow spheres Dara Van Goughsm00053a The permeability of liquid crystal templated ZnS mesoporous hollow spheres is programmed work, we combined colloid and LLC templating to obtain ZnS mesoporous hollow spheres through a process

  10. The Presence of the DNA Repair Genes mutM, mutY, mutL, and mutS is Related to Proteome Size in Bacterial Genomes

    PubMed Central

    Garcia-Gonzalez, Aurian; Rivera-Rivera, Ruben J.; Massey, Steven E.

    2012-01-01

    DNA repair is expected to be a modulator of underlying mutation rates, however the major factors affecting the distribution of DNA repair pathways have not been determined. The Proteomic Constraint theory proposes that mutation rates are inversely proportional to the amount of heredity information contained in a genome, which is effectively the proteome. Thus, organisms with larger proteomes are expected to possess more efficient DNA repair. We show that an important factor influencing the presence or absence of four DNA repair genes mutM, mutY, mutL, and mutS is indeed the size of the bacterial proteome. This is true both of intracellular and other bacteria. In addition, the relationship of DNA repair to genome GC content was examined. In principle, if a DNA repair pathway is biased in the types of mutations it corrects, this may alter the genome GC content. The presence of the mismatch repair genes mutL and mutS was not correlated with genome GC content, consistent with their involvement in an unbiased DNA repair pathway. In contrast, the presence of the base excision repair genes mutM and mutY, whose products both correct GC???AT mutations, was positively correlated with genome GC content, consistent with their biased repair mechanism. Phylogenetic analysis however indicates that the relationship between the presence of mutM and mutY genes and genome GC content is not a simple one. PMID:22403581

  11. Use of restriction fragment length polymorphisms resolved by pulsed-field gel electrophoresis for subspecies identification of mycobacteria in the Mycobacterium avium complex and for isolation of DNA probes.

    PubMed Central

    Coffin, J W; Condon, C; Compston, C A; Potter, K N; Lamontagne, L R; Shafiq, J; Kunimoto, D Y

    1992-01-01

    Mycobacterial strains from the Mycobacterium avium complex were compared with each other and with Mycobacterium phlei isolates by restriction endonuclease digestion of chromosomal DNA with SspI and analysis by pulsed-field gel electrophoresis. Characteristic profiles were observed for known typed strains, and five groups were identified. Primary bovine isolates identified as Mycobacterium paratuberculosis by classical methods were shown to fall into both the M. paratuberculosis- and M. avium-like groups. M. paratuberculosis 18 was in the latter category. Two Mycobacterium intracellulare strains of different Schaefer serotypes had different digestion profiles. In addition, this system was exploited for the preparation of DNA probes by the isolation, digestion, and subcloning of DNA fragments separated by pulsed-field gel electrophoresis. Probe JC12 hybridized only to M. avium complex strains, but not to M. phlei, showing characteristic hybridization profiles for each of the groups previously identified by pulsed-field gel electrophoresis. The approach taken in the study lends itself to the comparative analysis of members of the M. avium complex and to the isolation and characterization of DNA probes with specificity for these mycobacteria. Images PMID:1352787

  12. Magma Fragmentation

    NASA Astrophysics Data System (ADS)

    Gonnermann, Helge M.

    2015-05-01

    Magma fragmentation is the breakup of a continuous volume of molten rock into discrete pieces, called pyroclasts. Because magma contains bubbles of compressible magmatic volatiles, decompression of low-viscosity magma leads to rapid expansion. The magma is torn into fragments, as it is stretched into hydrodynamically unstable sheets and filaments. If the magma is highly viscous, resistance to bubble growth will instead lead to excess gas pressure and the magma will deform viscoelastically by fracturing like a glassy solid, resulting in the formation of a violently expanding gas-pyroclast mixture. In either case, fragmentation represents the conversion of potential energy into the surface energy of the newly created fragments and the kinetic energy of the expanding gas-pyroclast mixture. If magma comes into contact with external water, the conversion of thermal energy will vaporize water and quench magma at the melt-water interface, thus creating dynamic stresses that cause fragmentation and the release of kinetic energy. Lastly, shear deformation of highly viscous magma may cause brittle fractures and release seismic energy.

  13. Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.

    PubMed

    Nachamkin, I; Panaro, N J; Li, M; Ung, H; Yuen, P K; Kricka, L J; Wilding, P

    2001-02-01

    The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed. PMID:11158144

  14. Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars.

    PubMed Central

    Laguerre, G; Mavingui, P; Allard, M R; Charnay, M P; Louvrier, P; Mazurier, S I; Rigottier-Gois, L; Amarger, N

    1996-01-01

    Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient. PMID:8787401

  15. Quantitative and Rapid DNA Detection by Laser Transmission Spectroscopy

    PubMed Central

    Li, Frank; Mahon, Andrew R.; Barnes, Matthew A.; Feder, Jeffery; Lodge, David M.; Hwang, Ching-Ting; Schafer, Robert; Ruggiero, Steven T.; Tanner, Carol E.

    2011-01-01

    Laser transmission spectroscopy (LTS) is a quantitative and rapid in vitro technique for measuring the size, shape, and number of nanoparticles in suspension. Here we report on the application of LTS as a novel detection method for species-specific DNA where the presence of one invasive species was differentiated from a closely related invasive sister species. The method employs carboxylated polystyrene nanoparticles functionalized with short DNA fragments that are complimentary to a specific target DNA sequence. In solution, the DNA strands containing targets bind to the tags resulting in a sizable increase in the nanoparticle diameter, which is rapidly and quantitatively measured using LTS. DNA strands that do not contain the target sequence do not bind and produce no size change of the carboxylated beads. The results show that LTS has the potential to become a quantitative and rapid DNA detection method suitable for many real-world applications. PMID:22195026

  16. Quantitative and rapid DNA detection by laser transmission spectroscopy.

    PubMed

    Li, Frank; Mahon, Andrew R; Barnes, Matthew A; Feder, Jeffery; Lodge, David M; Hwang, Ching-Ting; Schafer, Robert; Ruggiero, Steven T; Tanner, Carol E

    2011-01-01

    Laser transmission spectroscopy (LTS) is a quantitative and rapid in vitro technique for measuring the size, shape, and number of nanoparticles in suspension. Here we report on the application of LTS as a novel detection method for species-specific DNA where the presence of one invasive species was differentiated from a closely related invasive sister species. The method employs carboxylated polystyrene nanoparticles functionalized with short DNA fragments that are complimentary to a specific target DNA sequence. In solution, the DNA strands containing targets bind to the tags resulting in a sizable increase in the nanoparticle diameter, which is rapidly and quantitatively measured using LTS. DNA strands that do not contain the target sequence do not bind and produce no size change of the carboxylated beads. The results show that LTS has the potential to become a quantitative and rapid DNA detection method suitable for many real-world applications. PMID:22195026

  17. Can Finite Size Effects in the Poland-Scheraga Model Explain Simulations of a Simple Model for DNA Denaturation ?

    E-print Network

    Lothar Schäfer

    2005-02-28

    We compare results of previous simulations of a simple model of DNA denaturation to the predictions of the Poland-Scheraga model. Concentrating on the critical region of the latter model we calculate both thermodynamic quantities and the distribution functions measured in the simulations. We find that the Poland-Scheraga model yields an excellent fit to the data, provided (i) we include a (singular) factor weighting the open ends of the doubly stranded chain, and (ii) we keep the leading corrections to the finite size scaling limit. The exponent c_1, which governs the end-weighting factor, is fairly well determined: 0.1 <~ c_1 <~ 0.15. The exponent c, which governs the length distribution of large loops, is determined only poorly. The data are compatible with values of c in at least the range 1.9 <~ c <~ 2.2. From the data it therefore cannot be decided whether the denaturation transition asymptotically is of first or of second order. We suggest that simulations of doubly stranded chains closed at both ends might allow for a more precise determination of c.

  18. On the Fragmentation Front in Conduit

    Microsoft Academic Search

    S. Kudomi; K. Kurita

    2001-01-01

    Vesiculation and fragmentation of magma in the conduit are the most fundamental processes to characterize explosive eruption. The interval from the vesiculation front to the fragmentation front critically controls the size distribution of pyroclasts and hence the style of eruption. The vesiculation process has been extensively investigated through experimental and model-simulational approaches. As for the fragmentation process, on the other

  19. Application of DNA barcoding for identification of freshwater carnivorous fish diets: Is number of prey items dependent on size class for Micropterus salmoides?

    PubMed Central

    Jo, Hyunbin; Gim, Jeong-An; Jeong, Kwang-Seuk; Kim, Heui-Soo; Joo, Gea-Jae

    2014-01-01

    Understanding predator–prey interactions is a major challenge in ecological studies. In particular, the accurate identification of prey is a fundamental requirement in elucidating food-web structure. This study took a molecular approach in determining the species identity of consumed prey items of a freshwater carnivorous fish (largemouth bass, Micropterus salmoides), according to their size class. Thirty randomly selected gut samples were categorized into three size classes, based on the total length of the bass. Using the universal primer for the mtDNA cytochrome oxidase I (COI) region, polymerase chain reaction (PCR) amplification was performed on unidentified gut contents and then sequenced after cloning. Two gut samples were completely empty, and DNA materials from 27 of 28 gut samples were successfully amplified by PCR (success rate: 96.4%). Sequence database navigation yielded a total of 308 clones, containing DNA from 26 prey items. They comprised four phyla, including seven classes, 12 orders, and 12 families based on BLAST and BOLD database searches. The results indicate that largemouth bass show selective preferences in prey item consumption as they mature. These results corroborate a hypothesis, presence of ontogenetic diet shift, derived through other methodological approaches. Despite the practical limitations inherent in DNA barcoding analysis, high-resolution (i.e., species level) identification was possible, and the predation patterns of predators of different sizes were identifiable. The utilization of this method is strongly recommended for determining specific predator–prey relationships in complex freshwater ecosystems. PMID:24558577

  20. Colonizing populations of Candida albicans are clonal in origin but undergo microevolution through C1 fragment reorganization as demonstrated by DNA fingerprinting and C1 sequencing.

    PubMed Central

    Lockhart, S R; Fritch, J J; Meier, A S; Schröppel, K; Srikantha, T; Galask, R; Soll, D R

    1995-01-01

    The genetic homogeneity of nine commensal and infecting populations of Candida albicans has been assessed by fingerprinting multiple isolates from each population by Southern blot hybridization first with the Ca3 probe and then with the 0.98-kb C1 fragment of the Ca3 probe. The isolates from each population were highly related, demonstrating the clonal origin of each population, but each population contained minor variants, demonstrating microevolution. Variation in each case was limited to bands of the Ca3 fingerprint pattern which hybridized with the 0.98-kb C1 fragment. The C1 fragment was therefore sequenced and demonstrated to contain an RPS repetitive element. The C1 fragment also contained part or all of a true end of the RPS element. These results, therefore, demonstrate that most colonizing C. albicans populations in nonimmuno-suppressed patients are clonal, that microevolution can be detected in every colonizing population by C1 hybridization, and that C1 contains the repeat RPS element. PMID:7650175

  1. Identification and Typing of Malassezia Species by Amplified Fragment Length Polymorphism and Sequence Analyses of the Internal Transcribed Spacer and Large-Subunit Regions of Ribosomal DNA

    Microsoft Academic Search

    Aditya K. Gupta; Teun Boekhout; Bart Theelen; Richard Summerbell; Roma Batra

    2004-01-01

    Malassezia yeasts are associated with several dermatological disorders. The conventional identification of Malassezia species by phenotypic methods is complicated and time-consuming, and the results based on culture methods are difficult to interpret. A comparative molecular approach based on the use of three molecular tech- niques, namely, amplified fragment length polymorphism (AFLP) analysis, sequencing of the internal tran- scribed spacer, and

  2. Computer models of dynamic fracture and fragmentation

    Microsoft Academic Search

    D. R. Curran

    1994-01-01

    In the defense community, fracture and fragmentation theory and associated computational models are used to produce descriptions of material damage as well as fragment sizes, velocities, and trajectories for fragmenting warheads and back-of-the-armor debris. Whereas previously such models depended almost exclusively on empirical correlations, emerging physics models of the fracture and fragmentation process that are based on laboratory-measured properties of

  3. Live Cell Characterization of DNA Aggregation Delivered through Lipofection

    PubMed Central

    Mieruszynski, Stephen; Briggs, Candida; Digman, Michelle A.; Gratton, Enrico; Jones, Mark R

    2015-01-01

    DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation. PMID:26013547

  4. HindIII and Sst I restriction sites mapped on rabbit poxvirus and vaccinia virus DNA.

    PubMed Central

    Wittek, R; Menna, A; Schümperli, D; Stoffel, S; Müller, H K; Wyler, R

    1977-01-01

    The DNAs of two closely related orthopoxviruses, rabbit poxvirus (RPV) and vaccinia virus (VV), were mapped by overlapping-fragment analysis using restriction endonucleases HindIII and Sst I. The exact arrangement of these fragments was accomplished by total digestion of isolated partial restriction products and by end-fragment determination. RPV and VV DNAs showed identical restriction patterns in an internal region comprising approximately 60% of the genome. The size, by electrophoretical analysis of the RPV DNA, was 118 X 10(6) daltons, some 6 X 10(6) daltons less than VV DNA. The two opposite terminal restriction fragments of RPV DNA cross-hybridized to each other. Images PMID:197263

  5. Computerized Analysis of Restriction Fragment Length Polymorphism Patterns: Comparative Evaluation of Two Commercial Software Packages

    PubMed Central

    Gerner-Smidt, P.; Graves, L. M.; Hunter, Susan; Swaminathan, B.

    1998-01-01

    Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared. The two systems use different approaches to compare patterns from different gels. In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern. In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel. The molecular size estimates obtained with the two systems for 12 restriction fragments of phage ? were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments. At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of HhaI-restricted DNA of Listeria monocytogenes. Both systems require the user to make critical decisions in the analysis. It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing. It is also important to verify results obtained in the pattern matching or clustering portions of the analysis. PMID:9574697

  6. Disposable polyester-toner electrophoresis microchips for DNA analysis.

    PubMed

    Duarte, Gabriela R M; Coltro, Wendell K T; Borba, Juliane C; Price, Carol W; Landers, James P; Carrilho, Emanuel

    2012-06-01

    Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a direct-printing process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm(-1). Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215,000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device. PMID:22545263

  7. Nucleic acid fragmentation on the millisecond timescale using a conventional X-ray rotating anode source: application to protein-DNA footprinting

    Microsoft Academic Search

    Arnon Henn; Jacob Halfon; Itai Kela; Itzhak Orion; Irit Sagi

    2001-01-01

    Nucleic acid fragmentation (footprinting) by ·OH radicals is used often as a tool to probe nucleic acid structure and nucleic acid-protein interactions. This method has proven valuable because it provides structural information with single base pair reso- lution. Recent developments in the field introduced the 'synchrotron X-ray footprinting' method, which uses a high-flux X-ray source to produce single base pair

  8. Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling

    Microsoft Academic Search

    G. B. Rogers; M. P. Carroll; D. J. Serisier; P. M. Hockey; G. Jones; K. D. Bruce

    2004-01-01

    Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data

  9. Protein-mediated DNA loops: Effects of protein bridge size and kinks Nicolas Douarche and Simona Cocco

    E-print Network

    Cocco, Simona

    steps translation, transcription, etc and do involve interactions between several biological mol- ecules gene transcription by interacting with each other. By bringing those proteins closer, DNA looping canR transcriptional repres- sors 7 . Two units of such proteins bind at two specific positions along the same DNA

  10. The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals

    Microsoft Academic Search

    Benoit Nabholz; Sylvain Glémin; Nicolas Galtier

    2009-01-01

    BACKGROUND: During the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial DNA, the most popular marker of molecular biodiversity. Several important results were recently reported using mammals as model organisms, including (i) the absence of relationship between mitochondrial DNA diversity and life-history or ecological variables, (ii) the absence of prominent adaptive selection,

  11. Velocity of finer fragments from impact

    Microsoft Academic Search

    Akiko M. Nakamura; Akira Fujiwara; Toshihiko Kadono

    1994-01-01

    We describe the method and the result of a new experiment to obtain velocity distribution of fine ejecta fragments, from a few to a hundred microns in size, produced from basalt targets by impacts of nylon projectiles at a velocity of 3.7 km\\/s. The size distribution of holes perforated by the ejecta fragments on thin films and foils placed around

  12. Capillary electrophoresis of small ssDNA molecules

    NASA Astrophysics Data System (ADS)

    Kopecka, Katerina; Slater, Gary W.; Drouin, Guy

    2004-03-01

    Recently, the electrophoretic separation of small ssDNA fragments (bellow 250 bases) has attracted a lot of attention because of applications related to Single Nucleotide Polymorphisms. In order to optimize these systems, we require a better understanding of DNA migration behavior in this size range. While the reptation model provides an excellent understanding of the dynamics of long DNA fragments in gel electrophoresis, the properties of small DNA fragments has not been studied extensively yet. At least three theoretical formulas have been proposed to explain the mobility of short ssDNA molecules in this regime. Specifically, the Ogston regime was introduced for small molecules having radii-of-gyration comparable to or smaller than the pore size of the sieving matrix. We introduce these three different formulas and discuss how their free parameters are related to actual physical parameters. We then test these formulas with new data obtained by capillary electrophoresis in our laboratory using poly(dimethylacrylamide) sieving matrices. Our results show that all three formulas provide decent fits, and that their fitting parameters are consistent with one another. This is the first step towards the development of a systematic approach to optimizing sequencing systems for this size range.

  13. Chloroplast DNA from the fern Osmunda cinnamomea : physical organization, gene localization and comparison to angiosperm

    Microsoft Academic Search

    Jeffrey D. Palmer; Diana B. Stein

    1982-01-01

    Chloroplast DNA from the fern Osmunda einnamomea was isolated by a sucrose gradient procedure utilizing PEG to stabilize chloroplasts. Analysis with the restriction endonucleases PvuII, Sacl and BstEII indicates a chloroplast genome size of 144 kb. A physical map of the fragments produced by these three enzymes was constructed by filter hybridizations using purified PvuII fragments as hybridization probes. The

  14. Grain size and flow volume effects on granular flow mobility in numerical simulations: 3-D discrete element modeling of flows of angular rock fragments

    NASA Astrophysics Data System (ADS)

    Cagnoli, B.; Piersanti, A.

    2015-04-01

    The results of three-dimensional discrete element modeling presented in this paper confirm the grain size and flow volume effects on granular flow mobility that were observed in laboratory experiments where batches of granular material traveled down a curved chute. Our numerical simulations are able to predict the correct relative mobility of the granular flows because they take into account particle interactions and, thus, the energy dissipated by the flows. The results illustrated here are obtained without prior fine-tuning of the parameter values to get the desired output. The grain size and flow volume effects can be expressed by a linear relationship between scaling parameters where the finer the grain size or the smaller the flow volume, the more mobile the center of mass of the granular flows. The numerical simulations reveal also the effect of the initial compaction of the granular masses before release. The larger the initial compaction, the more mobile the center of mass of the granular flows. Both grain size effect and compaction effect are explained by different particle agitations per unit of flow mass that cause different energy dissipations per unit of travel distance. The volume effect is explained by the backward accretion of the deposits that occurs wherever there is a change of slope (either gradual or abrupt). Our results are relevant for the understanding of the travel and deposition mechanisms of geophysical flows such as rock avalanches and pyroclastic flows.

  15. Sex in Escherichia coli does not disrupt the clonal structure of the population: evidence from random amplified polymorphic DNA and restriction-fragment-length polymorphism

    Microsoft Academic Search

    Patricia Desjardins; Bertrand Picard; Bernhard Kaltenbiick; Jacques Elion; Erick Denamurl

    1995-01-01

    Analysis of the Escherichia coli population by multilocus enzyme electrophoresis (MLEE) has established its clonal organization, but there is increasing evidence that horizontal DNA transfer occurs in E. coli. We have assessed the genetic structure of the species E. coli and determined the extent to which recombination can affect the clonal structure of bacteria. A panel of 72 E. coli

  16. Development of a real-time PCR method for the detection of fossil 16S rDNA fragments of phototrophic sulfur bacteria

    E-print Network

    Gilli, Adrian

    of phototrophic sulfur bacteria in the sediments of Lake Cadagno D. F. RAVASI,1 S. PEDUZZI,1 , 2 V. GUIDI,2 , 3 R sulfur bacteria in the chemocline has been monitored since 1994 with molecular methods such as 16S r sulfur bacteria populations from sediment samples. We detected fossil 16S rDNA of nine populations

  17. Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence

    Microsoft Academic Search

    Alain Bonnin; Marie Noelle Fourmaux; Jean François Dubremetz; Richard Gordon Nelson; Philippe Gobet; Géraldine Harly; Marielle Buisson; Dominique Puygauthier-Toubas; Florence Gabriel-Pospisil; Muriel Naciri; Patrick Camerlynck

    1996-01-01

    In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed

  18. Statistical Laws for Food Fragmentation by Human Mastication

    Microsoft Academic Search

    Naoki Kobayashi; Kaoru Kohyama; Yo Sasaki; Mitsugu Matsushita

    2006-01-01

    Fragment-size distribution has been studied experimentally by masticating raw carrot. For a few chews a single lognormal distribution well fits the entire region for masticated food fragments. Furthermore, the fragment-size distribution changes from a single lognormal structure to a double-size-group structure of lognormal with a power-law tail as the number of chews increases. The most of the fragments belong to

  19. Apoptotic DNA Fragmentation May Be a Cooperative Activity between Caspase-activated Deoxyribonuclease and the Poly(ADP-ribose) Polymerase-regulated DNAS1L3, an Endoplasmic Reticulum-localized Endonuclease That Translocates to the Nucleus during Apoptosis*

    PubMed Central

    Errami, Youssef; Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Suzuki, Yasuhiro; El-Bahrawy, Ali H.; Ghonim, Mohamed A.; Hemeida, Ramadan A.; Mansy, Moselhy S.; Zhang, Jianhua; Xu, Ming; Smulson, Mark E.; Brim, Hassan; Boulares, A. Hamid

    2013-01-01

    Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca2+-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca2+ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca2+ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca2+ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor ?-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca2+-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis. PMID:23229555

  20. Long-term effects of fragmentation and fragment properties on bird species richness in Hawaiian forests

    Microsoft Academic Search

    David J. Flaspohler; Christian P. Giardina; Gregory P. Asner; Patrick Hart; Jonathan Price; Cassie Ka’apu Lyons; Xeronimo Castaneda

    2010-01-01

    Forest fragmentation is a common disturbance affecting biological diversity, yet the impacts of fragmentation on many forest processes remain poorly understood. Forest restoration is likely to be more successful when it proceeds with an understanding of how native and exotic vertebrates utilize forest patches of different size. We used a system of forest fragments isolated by volcanic activity 153years ago

  1. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

    PubMed Central

    La, Jennifer; Latham, Catherine F.; Tinetti, Ricky N.; Johnson, Adam; Tyssen, David; Huber, Kelly D.; Sluis-Cremer, Nicolas; Simpson, Jamie S.; Headey, Stephen J.; Chalmers, David K.; Tachedjian, Gilda

    2015-01-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  2. Gold and gold–silver core-shell nanoparticle constructs with defined size based on DNA hybridization

    Microsoft Academic Search

    Andrea Steinbrück; Andrea Csaki; Kathrin Ritter; Martin Leich; J. Michael Köhler; Wolfgang Fritzsche

    2009-01-01

    Nanoparticles represent versatile building blocks in material science and nanotechnology. Thereby, the defined assembly of\\u000a nanostructures (13 and 56 nm in diameter, respectively) is of significant importance. Short DNA sequences can be bound to\\u000a the nanoparticle surface thus enabling highly specific DNA hybridization-driven events that direct the formation of nanoparticle\\u000a constructs.\\u000a \\u000a In this paper, examples for the defined formation of gold

  3. Diel Patterns of UVBR-Induced DNA Damage in Picoplankton Size Fractions from the Gulf of Aqaba, Red Sea

    Microsoft Academic Search

    P. Boelen; A. F. Post; M. J. W. Veldhuis; A. G. J. Buma

    2002-01-01

      This study focuses on the impact of natural levels of UVBR (ultraviolet-B radiation: 280 to 315 nm) on bacterio- and phytoplankton\\u000a (<10 mm) from the Gulf of Aqaba, Red Sea. Incident biologically effective doses (BEDs) and attenuation of biologically effective\\u000a radiation in the water column were measured using a DNA biodosimeter. UVBR-induced DNA damage was measured as cyclobutane\\u000a pyrimidine dimers

  4. Galactose-PEI–DNA complexes for targeted gene delivery: degree of substitution affects complex size and transfection efficiency

    Microsoft Academic Search

    Klaus Kunath; Anke von Harpe; Dagmar Fischer; Thomas Kissel

    2003-01-01

    Complexes of galactosylated polyethylenimines (gal-PEI) with DNA have been proposed for gene delivery to hepatocytes. We synthesized gal-PEI with a broad range of degrees of substitution (DS) ranging from 3.5 to 31% of all PEI amino groups by reductive amination to determine physico–chemical and biological properties with respect to the DS. Gel retardation assay for herring testes DNA–polymer polyplexes showed

  5. Introducing foreign DNA into tiger shrimp (Penaeus monodon) by electroporation.

    PubMed

    Tseng, F S; Tsai, H J; Liao, I C; Song, Y L

    2000-12-01

    Electroporation was used to introduce pFLAG-CMV-1-BAP, a DNA fragment that includes a bacterial alkaline phosphatase gene driven by a human cytomegalovirus (CMV) promoter, into Penaeus monodon zygotes. The transgenic tiger shrimp was achievedby using 10kV, 28 pulses, 120 g sec pulse time, 10 cycles, and a DNA concentration of 37.5 microg/mL. The hatching rate of electroporated zygotes (46%) was significantly lower than that of zygotes in the untreated group (89%). The survival rate of postlarvae in the electroporated group using a DNA concentration of 37.5 microg/mL decreased from 0.6% for postlarva 45 to 0.4% for postlarva 120. Based on dot blot analysis, the rate of gene transfer was 37% in mysis-stage, 23% postlarva 15(PL15), 19% postlarva 45(PL45), and 21% 4-month-old (about PL120). Genomic Southern blotting demonstrated that DNA from transgenic tiger shrimp contained fragments of exogenous DNA that were smaller, larger and of the same molecular size as pFLAG-CMV-1-BAP. Transferred DNA fragments were integrated into the genomes of 31% of the transgenic tiger shrimp. The exogenous DNA was mosaically distributed in a wide variety of tissues. Immunohistochemical staining revealed that the FLAG-BAP fused-protein encoded by pFLAG-CMV-1-BAP was present in the ovaries of some transgenic tiger shrimp. PMID:11191866

  6. Large Gap Size Paired-end Library Construction for Second Generation Sequencing

    SciTech Connect

    Peng, Ze; Hamilton, Matthew; Froula, Jeff; Ewing, Aren; Foster, Brian; Cheng, Jan-Fang

    2010-05-28

    Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 8 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 PE Library protocol. With the increasing fragment sizes, we found a general trend of decreasing library quality in several areas. First, redundant reads and reads containing multiple loxP linkers increase when the average fragment size increases. Second, the contamination of short distance pairs (<10Kb) increases as the fragment size increases. Third, chimeric rate increases with the increasing fragment sizes. We have modified several steps to improve the quality of the long span PE libraries. The modification includes (1) the use of special PFGE program to reduce small fragment contamination; (2) the increase of DNA samples in the circularization step and prior to the PCR to reduce redundant reads; and (3) the decrease of fragment size in the double SPRI size selection to get a higher frequency of LoxP linker containing reads. With these modifications we have generated large gap size PE libraries with a much better quality.

  7. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments

    Microsoft Academic Search

    P. Garbeva; L. S. van Overbeek; J. W. L. van Vuurde; J. D. van Elsas

    2001-01-01

    The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desirée) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization\\u000a of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities\\u000a detected in potato stem bases as well as in roots were in most cases

  8. Coagulation and fragmentation dynamics of inertial particles

    E-print Network

    Jens C. Zahnow; Rafael D. Vilela; Ulrike Feudel; Tamás Tél

    2009-08-20

    Inertial particles suspended in many natural and industrial flows undergo coagulation upon collisions and fragmentation if their size becomes too large or if they experience large shear. Here we study this coagulation-fragmentation process in time-periodic incompressible flows. We find that this process approaches an asymptotic, dynamical steady state where the average number of particles of each size is roughly constant. We compare the steady-state size distributions corresponding to two fragmentation mechanisms and for different flows and find that the steady state is mostly independent of the coagulation process. While collision rates determine the transient behavior, fragmentation determines the steady state. For example, for fragmentation due to shear, flows that have very different local particle concentrations can result in similar particle size distributions if the temporal or spatial variation of shear forces is similar.

  9. DNA Amplification from Pin Mounted Bumble Bees (Bombus) in a Museum Collection: Effects of Fragment Size and Specimen Age on Successful PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Historic data in the form of pinned specimens in entomological collections offer the potential to determine trends in genetic diversity of bumble bees (Bombus). We screened eight microsatellite loci for amplification success in pinned bumble bee specimens from the U.S. National Pollinating Insects C...

  10. Sequential fragmentation/transport theory, pyroclast size-density relationships, and the emplacement dynamics of pyroclastic density currents — A case study on the Mt. St. Helens (USA) 1980 eruption

    NASA Astrophysics Data System (ADS)

    Mackaman-Lofland, Chelsea; Brand, Brittany D.; Taddeucci, Jacopo; Wohletz, Kenneth

    2014-04-01

    Pyroclastic density currents (PDCs) are the most dangerous hazard associated with explosive volcanic eruptions. Despite recent advancements in the general understanding of PDC dynamics, limited direct observation and/or outcrop scarcity often hinder the interpretation of specific transport and depositional processes at many volcanoes. This study explores the potential of sequential fragmentation/transport theory (SFT; cf. Wohletz et al., 1989), a modeling method capable of predicting particle mass distributions based on the physical principles of fragmentation and transport, to retrieve the transport and depositional dynamics of well-characterized PDCs from the size and density distributions of individual components within the deposits. The extensive vertical and lateral exposures through the May 18th, 1980 PDC deposits at Mt. St. Helens (MSH) provide constraints on PDC regimes and flow boundary conditions at specific locations across the depositional area. Application to MSH deposits suggests that SFT parameter distributions can be effectively used to characterize flow boundary conditions and emplacement processes for a variety of PDC lithofacies and deposit locations. Results demonstrate that (1) the SFT approach reflects particle fragmentation and transport mechanisms regardless of variations in initial component distributions, consistent with results from previous studies; (2) SFT analysis reveals changes in particle characteristics that are not directly observable in grain size and fabric data; and (3) SFT parameters are more sensitive to regional transport conditions than local (outcrop-scale) depositional processes. The particle processing trends produced using SFT analysis are consistent with the degree of particle processing inferred from lithofacies architectures: for all lithofacies examined in this study, suspension sedimentation products exhibit much better processing than concentrated current deposits. Integrated field observations and SFT results provide evidence for increasing density segregation within the depositional region of the currents away from source, as well as for comparable density-segregation processes acting on lithic concentrations and pumice lenses within the current. These findings further define and reinforce the capability of SFT analysis to complement more conventional PDC study methods, significantly expanding the information gained regarding flow dynamics. Finally, this case study demonstrates that the SFT methodology has the potential to constrain regional flow conditions at volcanoes where outcrop exposures are limited.

  11. Contemporary effective population and metapopulation size (Ne and meta-Ne): comparison among three salmonids inhabiting a fragmented system and differing in gene flow and its asymmetries

    PubMed Central

    Gomez-Uchida, Daniel; Palstra, Friso P; Knight, Thomas W; Ruzzante, Daniel E

    2013-01-01

    We estimated local and metapopulation effective sizes ( and meta-) for three coexisting salmonid species (Salmo salar, Salvelinus fontinalis, Salvelinus alpinus) inhabiting a freshwater system comprising seven interconnected lakes. First, we hypothesized that might be inversely related to within-species population divergence as reported in an earlier study (i.e., FST: S. salar> S. fontinalis> S. alpinus). Using the approximate Bayesian computation method implemented in ONeSAMP, we found significant differences in () between species, consistent with a hierarchy of adult population sizes (). Using another method based on a measure of linkage disequilibrium (LDNE: ), we found more finite values for S. salar than for the other two salmonids, in line with the results above that indicate that S. salar exhibits the lowest among the three species. Considering subpopulations as open to migration (i.e., removing putative immigrants) led to only marginal and non-significant changes in , suggesting that migration may be at equilibrium between genetically similar sources. Second, we hypothesized that meta- might be significantly smaller than the sum of local s (null model) if gene flow is asymmetric, varies among subpopulations, and is driven by common landscape features such as waterfalls. One ‘bottom-up’ or numerical approach that explicitly incorporates variable and asymmetric migration rates showed this very pattern, while a number of analytical models provided meta- estimates that were not significantly different from the null model or from each other. Our study of three species inhabiting a shared environment highlights the importance and utility of differentiating species-specific and landscape effects, not only on dispersal but also in the demography of wild populations as assessed through local s and meta-s and their relevance in ecology, evolution and conservation. PMID:23532448

  12. Stable overexpression of DNA fragmentation factor in T-47D cells: sensitization of breast cancer cells to apoptosis in response to acetazolamide and sulfabenzamide.

    PubMed

    Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

    2014-11-01

    Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers. PMID:25086620

  13. Size Wheel

    NSDL National Science Digital Library

    2012-06-26

    In this fun sticker activity, learners will create a size wheel with images of objects of different size, from macroscopic scale (like an ant) to nanoscale (like DNA). Learners will be able to understand the difference in sizes and also learn about how small objects look when examined with special imaging technology such as a Scanning Electron Microscope. The activity includes images of: ant, dust mite, hair, virus, chromosome, spider web, penny, red blood cell, DNA, optic fiber, pollen grain, microchip, flagellum, plant cell, and silk threads.

  14. Time of flight mass spectrometry of DNA laser-ablated from frozen aqueous solutions: applications to the Human Genome Project

    NASA Astrophysics Data System (ADS)

    Williams, Peter

    1994-02-01

    Time of flight mass spectrometry offers an extremely rapid and accurate alternative to gel electrophoresis for sizing DNA fragments in the Sanger sequencing process, if large single-stranded DNA molecules can be volatilized and ionized without fragmentation. A process based on pulsed laser ablation of thin frozen films of DNA solutions has been shown to ablate intact DNA molecules up to [approximate]400 kDa in mass, and also has been shown to yield molecular ions of single-stranded DNA up to [approximate]18 500 Da. The theoretical basis and the progress to date in this approach are described and the potential impact of mass spectrometry on large-scale DNA sequencing is discussed.

  15. Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity

    Microsoft Academic Search

    Miloslava Fojtova ´; Jana Fulneckova ´

    2002-01-01

    It has been shown previously that apoptosis of tobacco cells induced by cadmium ions shows a rela- tively long lag period between exposure and cell death. This lag phase lasts for 3 d in TBY-2 cell cultures and is characterized by the maintenance of full cell viability despite extensive fragmentation of DNA into pieces of chromatin loop size. Experiments reported

  16. Microsatellite DNA markers for Australian geckos M. Hoehn1,2,

    E-print Network

    Canberra, University of

    Microsatellite DNA markers for Australian geckos M. Hoehn1,2, * & S. D. Sarre1 1 Applied Ecology September 2005; accepted 17 October 2005 Key words: dispersal, effective population size, gecko, Gehyra. Different gecko species exhibit varying rates of extinction in fragmented landscapes and the genetic markers

  17. C-banding and DNA content in three species of Belostoma (Heteroptera) with large differences in chromosome size and number

    Microsoft Academic Search

    A. G. Papeschi

    1988-01-01

    C-banding was carried out on Belostoma elegans (2n=26+X1X2Y) (?), B. micantulum (2n=14+XY) (?) and B. oxyurum (2n=6+XY) (?) (Belostomatidae, Heteroptera). The C-bands always have a telomeric localization and no interstitial bands were detected. An inverse relationship between chromosome size and chromosome number exists, and besides, an inverse relationship between chromosome size and the size of the C-bands was observed. The

  18. Assisted large fragment insertion by Red/ET-recombination (ALFIRE)—an alternative and enhanced method for large fragment recombineering

    PubMed Central

    Rivero-Müller, Adolfo; Laji?, Svetlana; Huhtaniemi, Ilpo

    2007-01-01

    Functional genomics require manipulation and modification of large fragments of the genome. Such manipulation has only recently become more efficient due to the discovery of different techniques based on homologous recombination. However, certain limitations of these strategies still exist since insertion of homology arms (HAs) is often based on amplification of DNA sequences with PCR. Large quantities of PCR products longer than 4–5?kb can be difficult to obtain and the risk of mutations or mismatches increases with the size of the template that is being amplified. This can be overcome by adding HAs by conventional cloning techniques, but with large fragments such as entire genes the procedure becomes time-consuming and tedious. Second, homologous recombination techniques often require addition of antibiotic selection genes, which may not be desired in the final construct. Here, we report a method to overcome the size and selection marker limitations by a two- or three-step procedure. The method can insert any fragment into small or large episomes, without the need of an antibiotic selection gene. We have humanized the mouse luteinizing hormone receptor gene (Lhcgr) by inserting a ?55?kb fragment from a BAC clone containing the human Lhcgr gene into a 170?kb BAC clone comprising the entire mouse orthologue. The methodology is based on the rationale to introduce a counter-selection cassette flanked by unique restriction sites and HAs for the insert, into the vector that is modified. Upon enzymatic digestion, in vitro or in Escherichia coli, double-strand breaks are generated leading to recombination between the vector and the insert. The procedure described here is thus an additional powerful tool for manipulating large and complex genomic fragments. PMID:17517785

  19. Physical mappings of chloroplast DNA from liverwort Marchantia polymorpha L. cell suspension cultures

    Microsoft Academic Search

    Kanji Ohyama; Yoshiaki Yamano; Hideya Fukuzawa; Tohru Komano; Hideo Yamagishi; Sinji Fujimoto; Masahiro Sugiura

    1983-01-01

    Physical maps of liverwort Marchantia polymorpha chloroplast DNA were constructed with restriction endonucleases, BamHI, SmaI, KpnI, and XhoI. The molecular size, calculated from the sum of the restriction fragments, is 121.0±1.0 kilobase (kb) pairs. This value is in good agreement with that of 118.7±2.0 kb pairs obtained from contour length measurements of the chloroplast DNA electron micrographs. The physical map

  20. Random Amplified Polymorphic Dna-PCR Typing of Vibrio parahaemolyticus Isolated from Local Cockles (Anadara granosa)

    Microsoft Academic Search

    Lesley Maurice Bilung; Son Radu; Abdul Rani Bahaman; Raha Abdul Rahim; Suhaimi Napis; Cheah Yoke Kqueen; Chandrika Murugaiah; Yousr Abdul Hadi; Tunung Robin; Mitsuaki Nishibuchi

    2005-01-01

    Randomly amplified polymorphic DNA (RAPD) was used in this study to examine the genetic relatedness among the Vibrio parahaemolyticus strains. In the analysis by RAPD-PCR, the size for RAPD fragments ranged from 0.25 to 10.0 kb with average number of ten bands. The RAPD profiles revealed a high level of DNA sequence diversity within the Vibrio parahaemolyticus strains tested. Hence,

  1. Lasing the DNA fragments through ?-diketimine framed Knoevenagel condensed Cu(II) and Zn(II) complexes - An in vitro and in vivo approach

    NASA Astrophysics Data System (ADS)

    Raman, Natarajan; Pravin, Narayanaperumal

    2014-01-01

    The syntheses, structures and spectroscopic properties of Cu(II) and Zn(II) complexes having Knoevenagel condensate ?-diketimine Schiff base ligands have been investigated in this paper. Characterization of these complexes was carried out using FTIR, NMR, UV-Vis, elemental analysis, mass and EPR techniques. Absorption titration, electrochemical analyses and viscosity measurements have also been carried out to determine the mode of binding. The shift in ?Ep, E1/2 and Ipc values explores the interaction of CT DNA with the above metal complexes. Interaction of ligands and their complexes with DNA revealed an intercalative mode of binding between them. Antimicrobial studies showed an effective antimicrobial activity of the metal ions after coordination with the ligands. The antioxidant properties of the Schiff base ligands and their complexes were evaluated in a series of in vitro tests by using 1,1-diphenyl-2-picrylhydrazyl (DPPHrad ) and H2O2 free radical scavengers. In vivo and in vitro antitumor functions of the complexes against Ehrlich ascites carcinoma tumor model have also been investigated. All the results support that ?-diketone derived Knoevenagel condensate Schiff base complexes may act as novel antitumor drugs and suggest that their potent cell life inhibition may contribute to their anti-cancer efficacy.

  2. Effect of divalent metal ions on DNA studied by capillary electrophoresis.

    PubMed

    Hartzell, Brittany; McCord, Bruce

    2005-03-01

    Divalent metal ions, such as Zn(2+), Co(2+), and Ni(2+), are capable of incorporating into DNA under certain conditions to form complexes termed M-DNA. To better understand the effects of these cations on DNA we used capillary electrophoresis (CE). The presence of these metal ions in a typical genotyping buffer led to broad peaks with low fluorescence intensities. In addition, some of the metal-complexed DNA molecules had different electrophoretic mobilities than their normal DNA counterparts. It is likely that the mobility shifts observed in the electropherograms of these affected fragments are due to the divalent cations causing structural changes in the single-stranded DNA. However, as can be seen from the resulting peak shapes, the structure, charge, and/or mass changes due to metal binding are not conserved among all of the DNA fragments. The extent of both peak-broadening and mobility shifts were found to be dependent on the metal cation and its concentration, the length of time that the DNA sample existed in formamide prior to injection into the capillary, and also the fragment size and sequence. These results suggest that the presence of metal ions might be responsible for the poor CE performance that occurs when genotyping certain kinds of DNA samples. PMID:15712361

  3. Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis.

    PubMed

    Buraczynska, M J; Van Keuren, M L; Buraczynska, K M; Chang, Y S; Crombez, E; Kurnit, D M

    1995-01-01

    We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was > or = 650 base pairs and the average initial complexity of the given libraries was 10(6). We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development. PMID:7656596

  4. Genetic structure in a fragmented Northern Hemisphere rainforest: large effective sizes and high connectivity among populations of the epiphytic lichen Lobaria pulmonaria.

    PubMed

    Hilmo, Olga; Lundemo, Sverre; Holien, Håkon; Stengrundet, Kirsti; Stenøien, Hans K

    2012-07-01

    An extraordinary diversity of epiphytic lichens is found in the boreal rainforest of central Norway, the highest-latitude rainforest in the world. These rainforest relicts are located in ravine systems, and clear cutting has increased the distance between remaining patches. We hypothesized that the relatively small lichen populations in the remaining forest stands have suffered a depletion of genetic diversity through bottlenecks and founder events. To test this hypothesis, we assessed genetic diversity and structure in the populations of the tripartite lichen Lobaria pulmonaria using eight SSR loci. We sampled thalli growing on Picea abies branches and propagules deposited in snow at three localities. Contrary to expectations, we found high genetic diversity in lichen and snow samples, and high effective sizes of the studied populations. Also, limited genetic differentiation between populations, high historical migration rates, and a high proportion of first generation immigrants were estimated, implying high connectivity across distances <30km. Almost all genetic variation was attributed to variation within sites; spatial genetic structures within populations were absent or appeared on small scales (5-10m). The high genetic diversity in the remaining old boreal rainforests shows that even relict forest patches might be suitable for conservation of genetic diversity. PMID:22571538

  5. Reproductive success and genetic diversity of Psychotria hastisepala (Rubiaceae), in fragmented Atlantic forest, Southeastearn Brazil.

    PubMed

    Silva, Celice A; Vieira, Milene F; Carvalho-Okano, Rita M de; Oliveira, Luiz O de

    2014-03-01

    The impacts of forest fragmentation on both reproductive biology and genetic diversity of native plant species is hardly understood, despite some studies have analyzed this current worldwide problem. Since this constitutes one of the main threats to seasonal semi-deciduous forests in Southeastern Brazil, we investigated the reproductive success and the genetic diversity of a distylous, understory shrub (Psychotria hastisepala) within this context of forest fragmentation. For this study, a set of seven forest fragments of sizes ranging from 4.1 to 168.7 hectares were chosen. The intervenient matrix comprised pastures (25-50%), monocultures (33-50%) and rural roads and buildings (14-28.5%). Overall, 91 plants (54 for the short-styled morph and 37 for the long-styled morph; mean of 6.5 plants per fragment) were investigated. To evaluate reproductive success, we quantified fruit and seed production under natural pollination; to evaluate genetic diversity and population structure, we employed ISSR markers on genomic DNA. Plants with the short-styled morph exhibited a significantly higher reproductive success than those with the long-styled morph; there was no association between seed production and size of the forest fragment. Levels of genetic diversity were positively associated with the number of plants per fragment; but they were not related to flower morph. AMOVA showed that about 65% of the overall genetic variation was attributed to the differences between plants within fragments. The results suggested that populations of P. hastisepala were susceptible to decline owing to forest fragmentation. PMID:24912360

  6. The isolation and characterization of plastid DNA from rice (Oryza sativa) 

    E-print Network

    Scheuring, Chantel Fougeron

    1987-01-01

    ; and SstII. 7. Several bands were found as doublets indicating the presence of the inverted repeats found in plastid genomes of other cereals. Three rice plastid Pstl fragments of sizes 2. 1, 5. 0, and 7. 6 kb were cloned into the plasmid pUC8.... The cloned fragments were individually mapped with Sall, PvuII, Sstl, and SstII. The cloned fragments were also nick-translated with biotinylated dUTP and used as probes to Southern blots of total plastid DNA digested with Pstl and Sall and to Southern...

  7. Isolation and characterization of 25 unique DNA markers for human chromosome 22

    SciTech Connect

    Van Biezen, N.A.; Deprez, R.H.L.; Thijs, A.; Zwarthoff, E.C. (Erasmus Univ., Rotterdam (Netherlands)); Heutink, P.; Oostra, B.A.; Kessel, A.H.M.G. van (Univ. of Nijmegen (Netherlands))

    1993-01-01

    Twenty-five single-copy anonymous DNA markers for human chromosome 22 were isolated. These markers were assigned to four different regions on the chromosome. Six markers recognize restriction fragment length polymorphisms. The relative positions of five of these polymorphic markers on the framework map of chromosome 22 were determined by linkage analysis. The sizes of the NotI fragments recognized by 22 markers were determined by pulsed-field gel analysis. The total length of the NotI fragments identified is at least 12 Mb, which represents about 20% of the entire chromosome. 15 refs., 1 fig.

  8. Selectable fragmentation warhead

    Microsoft Academic Search

    C. S. Bryan; D. L. Paisley; N. I. Montoya; D. B. Stahl

    1993-01-01

    A selectable fragmentation warhead is described comprising: a case having proximal and distal ends; a fragmenting plate mounted in said distal end of said casing; first explosive means cast adjacent to said fragmenting plate for creating a predetermined number of fragments from said fragmenting plate; three or more first laser-driven slapper detonators located adjacent to said first explosive means for

  9. Fragment Flow and the Multifragmentation Phase Space

    Microsoft Academic Search

    G. J. Kunde; W. C. Hsi; W. D. Kunze; A. Schuettauf; A. Woerner; M. Begemann-Blaich; Th. Blaich; D. R. Bowman; R. J. Charity; A. Cosmo; A. Ferrero; C. K. Gelbke; J. Hubele; G. Immé; I. Iori; P. Kreutz; V. Lindenstruth; M. A. Lisa; W. G. Lynch; U. Lynen; M. Mang; T. Moehlenkamp; A. Moroni; W. F. J. Mueller; M. Neumann; B. Ocker; C. A. Ogilvie; G. F. Peaslee; J. Pochodzalla; G. Raciti; T. Rubehn; H. Sann; W. Seidel; V. Serfling; L. G. Sobotka; J. Stroth; L. Stuttge; S. Tomasevic; W. Trautmann; M. B. Tsang; A. Tucholski; G. Verde; C. W. Williams; E. Zude; B. Zwieglinski

    1995-01-01

    Fragment distributions have been measured for Au+Au collisions at E\\/A = 100 and 1000 MeV. A high detection efficiency for fragments was obtained by combining the ALADIN spectrometer and the MSU-Miniball\\/WU-Miniwall array. At both energies the maximum multiplicity of intermediate mass fragments (IMF) normalized to the size of the decaying system is about one IMF per 30 nucleons but the

  10. Chapter 7: Habitat Fragmentation Fragmentation & Heterogeneity

    E-print Network

    Gottgens, Hans

    into smaller & isolated parcels. Consequences (Lindenmayer & Franklin 2002) · Habitat loss · Subdivision of habitat · Patch isolation · Edge effects FRAMENTATION #12;A conceptual illustration of habitat loss, isolation, and fragmentation #12;NE China Miombo Africa Bayfield (WI) Maraba Africa Fragmentation: Habitat

  11. Variable sizes of introns in the SSU rDNA in three species of Roccella (Arthoniales, Euascomycetes)

    Microsoft Academic Search

    Leena Myllys; Mari Källersjö; Anders Tehler

    1999-01-01

    Three species of the lichenized fungus Roccella (Arthoniales, Euascomycetes), R. canariensis, R. tuberculata and R. montagnei, were found to possess insertions in the first half of the nuclear SSU rDNA. Both the number and the type of these insertions\\u000a varied between and within species. At position 516 the insertions belonged to group-IC1 introns with a characteristic secondary\\u000a structure and conserved

  12. Fragmentation in the branching coral Acropora palmata (Lamarck): growth, survivorship, and reproduction of colonies and fragments.

    PubMed

    Lirman

    2000-08-23

    Acropora palmata, a branching coral abundant on shallow reef environments throughout the Caribbean, is susceptible to physical disturbance caused by storms. Accordingly, the survivorship and propagation of this species are tied to its capability to recover after fragmentation. Fragments of A. palmata comprised 40% of ramets within populations that had experienced recent storms. While the survivorship of A. palmata fragments was not directly related to the size of fragments, removal of fragments from areas where they settled was influenced by size. Survivorship of fragments was also affected by type of substratum; the greatest mortality (58% loss within the first month) was observed on sand, whereas fragments placed on top of live colonies of A. palmata fused to the underlying tissue and did not experience any losses. Fragments created by Hurricane Andrew on a Florida reef in August 1992 began developing new growth (proto-branches) 7 months after the storm. The number of proto-branches on fragments was dependent on size, but growth was not affected by the size of fragments. Growth-rates of proto-branches increased exponentially with time (1.7 cm year(-1) for 1993-1994, 2.7 cm year(-1) for 1994-1995, 4.2 cm year(-1) for 1995-1996, and 6.5 cm year(-1) for 1996-1997), taking over 4 years for proto-branches to achieve rates comparable to those of adult colonies on the same reef (6.9 cm year(-1)). In addition to the initial mortality and reduced growth-rates, fragmentation resulted in a loss of reproductive potential. Neither colonies that experienced severe fragmentation nor fragments contained gametes until 4 years after the initial damage. Although A. palmata may survive periodic fragmentation, the long-term effects of this process will depend ultimately on the balance between the benefits and costs of this process. PMID:10958900

  13. Chloroplast DNA inheritance in Populus

    Microsoft Academic Search

    O. P. Rajora; B. P. Dancik

    1992-01-01

    The inheritance of chloroplast (cp) DNA was examined in F1 hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen

  14. Modification of the GS LT Paired-end Library Protocol for Constructing Longer Insert Size Libraries

    SciTech Connect

    Peng, Ze; Peng, Ze; Hamilton, Matthew; Ting, Sara; Tu, Hank; Goltsman, Eugene; Lapidus, Alla; Lucas, Susan; Cheng, Jan-Fang

    2008-05-22

    Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

  15. FRAGMENTATION OF CONTINENTAL UNITES STATES FORESTS

    EPA Science Inventory

    We report a multiple-scale analysis of forest fragmentation based on 30-m land-cover maps for the conterminous United States. Each 0.09-ha unit of forest was classified according to fragmentation indices measured within the surrounding landscape, for five landscape sizes from 2....

  16. Detection of transgenic and endogenous plant DNA fragments and proteins in the digesta, blood, tissues, and eggs of laying hens fed with phytase transgenic corn.

    PubMed

    Ma, Qiugang; Gao, Chunqi; Zhang, Jianyun; Zhao, Lihong; Hao, Wenbo; Ji, Cheng

    2013-01-01

    The trials were conducted to assess the effects of long-term feeding with phytase transgenic corn (PTC) to hens on laying performance and egg quality, and investigate the fate of transgenic DNA and protein in digesta, blood, tissues, and eggs. Fifty-week old laying hens (n?=?144) were fed with a diet containing 62.4% PTC or non-transgenic isogenic control corn (CC) for 16 weeks. We observed that feeding PTC to laying hens had no adverse effect on laying performance or egg quality (P>0.05) except on yolk color (P<0.05). Transgenic phyA2 gene and protein were rapidly degraded in the digestive tract and were not detected in blood, heart, liver, spleen, kidney, breast muscle, and eggs of laying hens fed with diet containing PTC. It was concluded that performance of hens fed diets containing PTC, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with CC. There was no evidence of phyA2 gene or protein translocation to the blood, tissues, and eggs of laying hens. PMID:23577200

  17. Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

    PubMed Central

    Sandin, Peter; Stengel, Gudrun; Ljungdahl, Thomas; Börjesson, Karl; Macao, Bertil; Wilhelmsson, L. Marcus

    2009-01-01

    Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5?-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication. PMID:19401439

  18. Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits.

    PubMed

    Di Cagno, R; Minervini, G; Sgarbi, E; Lazzi, C; Bernini, V; Neviani, E; Gobbetti, M

    2010-10-15

    The diversity of 72 isolates of Lactobacillus plantarum, previously identified from different raw vegetables and fruits, was studied based on phenotypic (Biolog System) and genotypic (randomly amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) approaches. A marked phenotypic and genotypic variability was found. Eight clusters were formed at the similarity level of 92% based on Biolog System analysis. The most numerous clusters grouped isolates apart from the original habitat. Almost all isolates fermented maltose, D,L-lactic acid, N-acetyl-D-mannosamine and dextrin, and other typical carbon sources which are prevalent in raw vegetables and fruits. None of the isolates fermented lactose and free amino acids. At high values of linkage distance, two main clusters were obtained from both UPGMA (unweighted pair group with arithmetic average) dendrograms of RAPD-PCR and AFLP analyses. The two clusters mainly separated isolates from tomatoes and carrots from those isolated from pineapples. At 2.5 linkage distance, a high polymorphism was found and several sub-clusters were formed with both analyses. In particular, AFLP allowed the differentiation of 55 of the 72 isolates of L. plantarum. The discriminatory power of each technique used was calculated through the Simpson's index of diversity (D). The values of the D index were 0.65, 0.92 and 0.99 for Biolog System, RAPD-PCR and AFLP analyses, respectively. PMID:20850884

  19. The Neandertal genome and ancient DNA authenticity

    PubMed Central

    Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

    2009-01-01

    Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

  20. DNA terminal mismatch-induced stabilization of polymer micelles from RAFT-generated poly(N-isopropylacrylamide)-DNA block copolymers.

    PubMed

    Isoda, Kyosuke; Kanayama, Naoki; Fujita, Masahiro; Takarada, Tohru; Maeda, Mizuo

    2013-12-01

    Temperature-responsive diblock copolymers made of poly(N-isopropylacrylamide) (PNIPAAm) generated by reversible addition-fragmentation chain transfer (RAFT) polymerization and a single-stranded DNA (ssDNA) self-assembled into polymer micelles. The micelles consisted of the PNIPAAm core surrounded by the ssDNA corona with a hydrodynamic diameter up to 300 nm in an aqueous medium above the lower critical solution temperature. In a medium of high ionic strength, the formation of the fully matched duplex with the complementary ssDNA on the surface of the polymer micelles induced rapid and spontaneous aggregation. By contrast, the micelles remained dispersed under the identical conditions when single-base-substituted ssDNA was added to form the corresponding terminal-mismatched duplex on the micellar surface. This highly sequence-selective process took place irrespective of the size of the PNIPAAm core. PMID:24006358