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Sizing of DNA fragments by flow cytometry  

SciTech Connect

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.

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



Sizing of DNA fragments by flow cytometry  

SciTech Connect

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.

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



DNA fragment sizing and sorting by laser-induced fluorescence  


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.

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)



Sizing of single fluorescently stained DNA fragments by scanning microscopy  

PubMed Central

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

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



DNA fragment sizing and sorting by laser-induced fluorescence  

SciTech Connect

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

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



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

Microsoft Academic Search

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size

Ranajit Chakraborty; Zhaojue Li



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

SciTech Connect

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

Chakraborty, R.; Zhong, Y.; Jin, L. (Univ. of Texas Health Science Center, Houston, TX (United States)); Budowle, B. (FBI Academy, Quantico, VA (United States))



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

NASA Technical Reports Server (NTRS)

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.

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



Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.  

PubMed Central

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation. PMID:8932373

Huang, Z; Petty, J T; O'Quinn, B; Longmire, J L; Brown, N C; Jett, J H; Keller, R A




E-print Network

cycles of MagNA and DNA, a 4- 5min incubation period for DNA precipitation on the beads is performed1 SUPPLEMENTARY NOTES DNA Fragmentation Human solution hybrid selection For the 189 human DNA samples for the hybrid selection (application 1), between 200 and 4,800 ng genomic DNA (final volume 200ul

Reich, David


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


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

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



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

SciTech Connect

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

Lawrence, T.S.; Davis, M.A.; Normolle, D.P. [Univ. of Michigan, Ann Arbor, MI (United States)



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

NASA Technical Reports Server (NTRS)

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.

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



DNA double-strand breaks in mammalian cells exposed to gamma-rays and very heavy ions. Fragment-size distributions determined by pulsed-field gel electrophoresis.  


The spatial distribution of DNA double-strand breaks (DSB) was assessed after treatment of mammalian cells (V79) with densely ionizing radiation. Cells were exposed to beams of heavy charged particles (calcium ions: 6.9 MeV/u, 2.1.10(3) keV/microm; uranium ions: 9.0 MeV/u, 1.4.10(4) keV/microm) at the linear accelerator UNILAC of GSI, Darmstadt. DNA was isolated in agarose plugs and subjected to pulsed-field gel electrophoresis under conditions that separated DNA fragments of size 50 kbp to 5 Mbp. The measured fragment distributions were compared to those obtained after gamma-irradiation and were analyzed by means of a convolution and a deconvolution technique. In contrast to the finding for gamma-radiation, the distributions produced by heavy ions do not correspond to the random breakage model. Their marked overdispersion and the observed excess of short fragments reflect spatial clustering of DSB that extends over large regions of the DNA, up to several mega base pairs (Mbp). At fluences of 0.75 and 1.5/microm2, calcium ions produce nearly the same shape of fragment spectrum, merely with a difference in the amount of DNA entering the gel; this suggests that the DNA is fragmented by individual calcium ions. At a fluence of 0.8/microm2 uranium ions produce a profile that is shifted to smaller fragment sizes in comparison to the profile obtained at a fluence of 0.4/microm2; this suggests cumulative action of two separate ions in the formation of fragments. These observations are not consistent with the expectation that the uranium ions, with their much larger LET, should be more likely to produce single particle action than the calcium ions. However, a consideration of the greater lateral extension of the tracks of the faster uranium ions explains the observed differences; it suggests that the DNA is closely coiled so that even DNA locations several Mbp apart are usually not separated by less than 0. 1 or 0.2 microm. PMID:9728743

Kraxenberger, F; Weber, K J; Friedl, A A; Eckardt-Schupp, F; Flentje, M; Quicken, P; Kellerer, A M



DNA Fragmentation in Microorganisms Assessed In Situ  

Microsoft Academic Search

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmen- tation in higher eukaryotic cells, DNA fragmentation in microorganisms is rarely studied. We report an adaptation of a diffusion-based assay, developed as a kit, which allows for simple and rapid discrimination of bacteria with fragmented DNA. Intact cells were embedded in an agarose microgel

JoseLuis Fernandez; M. Cartelle; L. Muriel; R. Santiso; M. Tamayo; V. Goyanes; J. Gosalvez; G. Bou



Detection of single lambda DNA fragments by flow cytometry  

SciTech Connect

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

Johnson, M.E.; Goodwin, P.M.; Ambrose, W.P.; Martin, J.C.; Marrone, B.L.; Keller, R.A. (Los Alamos National Lab., NM (United States))



Assembling DNA fragments with parallel algorithms  

Microsoft Academic Search

As more research centers embark on sequencing new genomes, the problem of DNA fragment assembly for shotgun sequencing is growing in importance and complexity. Accurate and fast assembly is a crucial part of any sequencing project and since the DNA fragment assembly problem is NP-hard, exact solutions are very difficult to obtain. Various heuristics, including genetic algorithms, were designed for

Enrique Alba; Gabriel Luque; Sami Khuri



A new thermostable DNA polymerase mixture for efficient amplification of long DNA fragments  

Microsoft Academic Search

The thermostable DNA polymerases have been used for amplification of DNA fragments since the invention of PCR. The constraint\\u000a on the maximum size of the amplified fragments can be solved to certain level by the use of unbalanced mixtures of non-proofreading\\u000a and proofreading thermostable DNA polymerases. In this study, we tested the use of a mixtures of N-terminal deletional variant

K. G. Davalieva; G. D. Efremov



Bacterial natural transformation by highly fragmented and damaged DNA  

E-print Network

environments (1). DNA degradation is initi- ated at cell death by coreleased cellular nucleases and continued-strand overhangs of the DNA fragments (2). Consequently, most free DNA fragmentsBacterial natural transformation by highly fragmented and damaged DNA Søren Overballe-Petersena,1

Nielsen, Rasmus


DNA-anti-DNA immune complexes. Antibody protection of a discrete DNA fragment from DNase digestion in vitro.  

PubMed Central

We examined the ability of DNase I to digest DNA that was contained with DNA-anti-DNA immune complexes. IgG isolated from the sera of 20 patients with systemic lupus erythematosus (SLE) and containing antibodies to DNA was incubated with double-stranded DNA to form immune complexes. Excess DNase was added, and digestion of DNA was monitored by the conversion of DNA to TCA soluble products. IgG from 8 of the 20 SLE patients protected DNA from degradation by DNase in direct proportion to the amount of DNA bound to IgG as measured in the Farr binding assay. Using IgG from these sera, we showed that the DNA protected from degradation remained bound to IgG during digestion and was 35-45 base pairs in size. The size of this fragment is the same as that which has been proposed to be the minimal size necessary for monogamous bivalent binding of IgG to DNA. We therefore compared the ability of F(ab')2 and Fab' to protect DNA from DNase digestion and demonstrated that the bivalent F(ab')2 fragments were protective, but that the univalent Fab' fragments were not. These results suggest that some antibodies to DNA that bind to DNA via monogamous bivalent binding can protect a 35-45-base pair DNA fragment from DNase digestion. The implications of this finding are discussed with regard to the in vivo behavior and potential pathogenicity of small DNA-anti-DNA immune complexes. Images PMID:6234327

Emlen, W; Ansari, R; Burdick, G



A new thermostable DNA polymerase mixture for efficient amplification of long DNA fragments.  


The thermostable DNA polymerases have been used for amplification of DNA fragments since the invention of PCR. The constraint on the maximum size of the amplified fragments can be solved to certain level by the use of unbalanced mixtures of non-proofreading and proofreading thermostable DNA polymerases. In this study, we tested the use of a mixtures of N-terminal deletional variant of Taq polymerase - Klentaq278 and Tne polymerase from Thermotoga neapolitana. Klentaq278 and Tne polymerase genes were cloned and expressed in different expression vectors under tac promoter. The most efficient ratio of Klentaq278/Tne polymerase for amplification was 10 : 1. The polymerase mixture of Klentaq278 and Tne polymerase is very effective in amplification of DNA fragments for up to 8 kb and is useful addition to a DNA polymerases used in long-range PCR. PMID:20391772

Davalieva, K G; Efremov, G D



Optical selection and collection of DNA fragments  


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

Roslaniec, Mary C. (Los Alamos, NM); Martin, John C. (Los Alamos, NM); Jett, James H. (Los Alamos, NM); Cram, L. Scott (Los Alamos, NM)



Isolation of RNA and DNA fragments using diatomaceous earth  

Microsoft Academic Search

A series of simple and phenol-free silica-based protocols for isolating and cleaning RNA or DNA fragments from different sources were developed. Cytoplasmic RNA isolated from hybridoma cells by this method was used in RT-PCR. DNA fragments obtained using this protocol were suitable for further subcloning, gene transformation and DNA sequencing. © Rapid Science Ltd. 1998

Kai Koo; Peggy M. Foegeding; Harold E. Swaisgood



Simultaneous splicing of multiple DNA fragments in one PCR reaction  

PubMed Central

Background Rapid and simultaneous splicing of multiple DNA fragments is frequently required in many recombinant DNA projects. However, former overlap extension PCRs, the most common methods for splicing DNA fragments, are not really simultaneous fusing of multiple DNA fragments. Results We performed an optimized method which allowed simultaneous splicing of multiple DNA fragments in one PCR reaction. Shorter outermost primers were prior mixed with other PCR components at the same time. A sequential thermo cycling program was adopted for overlap extension reaction and amplification of spliced DNA. Annealing temperature was relatively higher in the overlap extension reaction stage than in the fused DNA amplification. Finally we successfully harvested target PCR products deriving from fusion of two to seven DNA fragments after 5–10 cycles for overlap extension reaction and then 30 cycles for fused DNA amplification. Conclusions Our method provides more rapid, economical and handy approach to accurately splice multiple DNA fragments. We believe that our simultaneous splicing overlap extension PCR can be used to fuse more than seven DNA fragments as long as the DNA polymerase can match. PMID:24015676



The fragmented Atlantic rain forest of Brazil: size, shape and distribution of forest fragments  

Microsoft Academic Search

The geographical characteristics of a total of 1839 forest fragments surrounded by sugar cane fields were studied in the Brazilian Atlantic rain forest region of the northeastern state of Pernambuco. The size and shape of the fragments as well as inter-fragment distances and the effects of varying edge width were examined using a geographical information system. The analyses show that




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

E-print Network

We report a study of DNA (150 bp fragments) conformations in very low added salt $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 $D_p$.

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



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

NASA Technical Reports Server (NTRS)

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

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



Bacterial natural transformation by highly fragmented and damaged DNA  

PubMed Central

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

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



DNA fragmentation and sperm head morphometry in cat epididymal spermatozoa.  


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

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



Impact and explosion crater ejecta, fragment size, and velocity  

NASA Technical Reports Server (NTRS)

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.

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



Purification and cloning of DNA fragments fractionated on agarose gels  

Microsoft Academic Search

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory.\\u000a This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified\\u000a DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature\\u000a agarose

Hugh G. Griffin; Michael J. Gasson



Application of automated DNA sizing technology for genotyping microsatellite loci  

Microsoft Academic Search

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

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



Sperm DNA fragmentation in couples with unexplained recurrent spontaneous abortions.  


The aim of the present study was to evaluate the degree of sperm DNA fragmentation in couples with idiopathic recurrent spontaneous abortion (RSA) and in those with no history of infertility or abortion. In this cohort study, 30 couples with RSA and 30 fertile couples as control group completed the demographic data questionnaires, and their semen samples were analysed according to World Health Organization (WHO) standards (September 2009-March 2010) for evaluation of sperm DNA fragmentation, using sperm chromatin dispersion (SCD) technique. The percentage of morphologically normal sperm was significantly lower in RSA patients compared with control group (51.50 ± 11.60 versus 58.00 ± 9.05, P = 0.019), but not in other parameters. Additionally, the level of abnormal DNA fragmentation in the RSA group was significantly higher than in the control group (43.3% versus 16.7%, P = 0.024). Our results indicated a negative correlation between the number of sperm with progressive motility and DNA fragmentation (r = -0.613; P < 0.001). The sperm from men with a history of RSA had a higher incidence of DNA fragmentation and poor motility than those of the control group, indicating a possible relationship between idiopathic RSA and DNA fragmentation. PMID:23278374

Khadem, N; Poorhoseyni, A; Jalali, M; Akbary, A; Heydari, S T



Dependence on radiation quality of DNA fragmentation spectra  

NASA Astrophysics Data System (ADS)

Energy deposition by radiation initially gives rise to cellular critical lesions such as DNA doublestrand breaks (DSB), that later lead to the formation of relevant biological endpoints. Studies on fragment size distributions induced by radiations of various qualities can be of great help in linking the characteristics of radiation to cellular endpoints, providing information for understanding the main mechanisms of cell damage. Here we are concerned with the damage induced by heavy charged particles; this issue is very important in the field of radioprotection of astronauts participating in long term space missions, besides being relevant also in other fields, like hadrontherapy. Galactic Cosmic Rays contain a large component of high-LET particles (HZE), e.g. helium and carbon ions, as well as highcharge particles such as iron ions. These particles are characterized by complex track structures with energy depositions not only along the path of the primary particle, but also at relatively large distance form the path, due to the presence of high energy secondary electrons. In this work we have simulated the irradiation of human fibroblasts with ?-rays, protons, helium, carbon and iron ions at a fixed dose with the biophysical Monte Carlo code PARTRAC,and calculated the induction of DSB. The PARTRAC code includes accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation. The results of a first validation of the code have been reported in A. Campa et al. (2005) and D. Alloni et al. (2007a, 2007b). DNA fragment spectra were calculated based on the DSB induction patterns and compared in particular for particles of the same specific energy and for particles of the same LET. Special emphasis has been directed to the calculation of very small fragments (< 1 kbp) that are not detectable by the most common experimental techniques and that can significantly influence the RBE (Relative Biological Effectiveness) of high LET radiation. This work was partially supported by EU ("RISC-RAD" project, Contract no. FI6R-CT 2003- 508842, and "NOTE" project, Contract no. FI6R-036465) and ASI (Italian Space Agency, "Mo-Ma/COUNT" project). References A. Campa, F. Ballarini, M. Belli, R. Cherubini, V. Dini, G. Esposito, W.Friedland, S. Gerardi, S. Molinelli, A. Ottolenghi, H. G. Paretzke, G. Simone and M. A. Tabocchini. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat.Biol. 81, 841-854 (2005). D. Alloni, F. Ballarini, M. Belli, A. Campa, G. Esposito, W. Friedland, M.Liotta, A. Ottolenghi and H. G. Paretzke. Modeling of DNA fragmentation induced in human fibroblasts by 56 Fe ions. Adv. Space Res. 40, 1401-1407 (2007a). D. Alloni, F. Antonelli, F. Ballarini, M. Belli, A. Campa, V. Dini, G.Esposito, W. Friedland, M. Liotta, A. Ottolenghi, H. G. Paretzke, G. Simone, E. Sorrentino and M. A. Tabocchini. Small DNA fragments induced in human fibroblasts by 56 Fe ions: experimental data and MC simulations. Proc. "Ion Beams in biology and medicine", Heidelberg, 26-29 September 2007, edited by J. Debus, K. Henrichs, G. Kraft, p. 164 (2207b).

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


Cell nucleus and DNA fragmentation are not required for apoptosis  

Microsoft Academic Search

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytologi- cal alterations including membrane blebbing and nu- clear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into inter- nucleosomal DNA fragments is considered to be the hallmark of

Klaus Schulze-Osthoff; Herming Walczak; Peter H. Krammer



Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication*S  

E-print Network

evidence of Dna2 participating in this process is lacking. During OF processing, flap endonuclease Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication, and the **Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 633110 Background: Dna2

Monnat, Ray


Evolution of Particle Size Distributions in Fragmentation Over Time  

NASA Astrophysics Data System (ADS)

We present a new model of fragmentation based on a probabilistic calculation of the repeated fracture of a particle population. The resulting continuous solution, which is in closed form, gives the evolution of fragmentation products from an initial block, through a scale-invariant power-law relationship to a final comminuted powder. Models for the fragmentation of particles have been developed separately in mainly two different disciplines: the continuous integro-differential equations of batch mineral grinding (Reid, 1965) and the fractal analysis of geophysics (Turcotte, 1986) based on a discrete model with a single probability of fracture. The first gives a time-dependent development of the particle-size distribution, but has resisted a closed-form solution, while the latter leads to the scale-invariant power laws, but with no time dependence. Bird (2009) recently introduced a bridge between these two approaches with a step-wise iterative calculation of the fragmentation products. The development of the particle-size distribution occurs with discrete steps: during each fragmentation event, the particles will repeatedly fracture probabilistically, cascading down the length scales to a final size distribution reached after all particles have failed to further fragment. We have identified this process as the equivalent to a sequence of trials for each particle with a fixed probability of fragmentation. Although the resulting distribution is discrete, it can be reformulated as a continuous distribution in maturity over time and particle size. In our model, Turcotte's power-law distribution emerges at a unique maturation index that defines a regime boundary. Up to this index, the fragmentation is in an erosional regime with the initial particle size setting the scaling. Fragmentation beyond this index is in a regime of comminution with rebreakage of the particles down to the size limit of fracture. The maturation index can increment continuously, for example under grinding conditions, or as discrete steps, such as with impact events. In both cases our model gives the energy associated with the fragmentation in terms of the developing surface area of the population. We show the agreement of our model to the evolution of particle size distributions associated with episodic and continuous fragmentation and how the evolution of some popular fractals may be represented using this approach. C. A. Charalambous and W. T. Pike (2013). Multi-Scale Particle Size Distributions of Mars, Moon and Itokawa based on a time-maturation dependent fragmentation model. Abstract Submitted to the AGU 46th Fall Meeting. Bird, N. R. A., Watts, C. W., Tarquis, A. M., & Whitmore, A. P. (2009). Modeling dynamic fragmentation of soil. Vadose Zone Journal, 8(1), 197-201. Reid, K. J. (1965). A solution to the batch grinding equation. Chemical Engineering Science, 20(11), 953-963. Turcotte, D. L. (1986). Fractals and fragmentation. Journal of Geophysical Research: Solid Earth 91(B2), 1921-1926.

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



Fragmentation dynamics of DNA sequence duplications  

E-print Network

Motivated by empirical observations of algebraic duplicated sequence length distributions in a broad range of natural genomes, we analytically formulate and solve a class of simple discrete duplication/substitution models that generate steady-states sharing this property. Continuum equations are derived for arbitrary time-independent duplication length source distribution, a limit that we show can be mapped directly onto certain fragmentation models that have been intensively studied by physicists in recent years. Quantitative agreement with simulation is demonstrated. These models account for the algebraic form and exponent of naturally occuring duplication length distributions without the need for fine-tuning of parameters.

M. V. Koroteev; J. Miller



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

NASA Astrophysics Data System (ADS)

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.

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




Microsoft Academic Search

Particulate size distributions offer the scientist impor- tant clues about the mechanism(s) responsible for their formation. These distributions are complex, often being the combination of several subpopulations that bear the signa- ture of specific fragmentation and particulate transport processes. Historically, such distributions have been char- acterized by some empirical distribution law such as the lognormal or Weibull distributions, but such

Wilbur Brown


The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis  

Microsoft Academic Search

We report here the reconstitution of a path- way that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the

Xuesong Liu; Peng Li; Piotr Widlak; Hua Zou; Xu Luo; WILLIAM T. GARRARD; Xiaodong Wang



Reconstructing DNA replication kinetics from small DNA fragments Haiyang Zhang and John Bechhoefer*  

E-print Network

in cell-free embryos of the often-used frog Xenopus laevis have yielded enough data to begin to explore coalescence DNA replicates only once per cell cycle; two replicated domains that meet thus coalesceReconstructing DNA replication kinetics from small DNA fragments Haiyang Zhang and John Bechhoefer

Bechhoefer, John


Calcium-activated DNA fragmentation kills immature thymocytes  

Microsoft Academic Search

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



Transformation of Escherichia coli by a specific DNA restriction fragment  

Microsoft Academic Search

Specific transformation of a rifampicin sensitive strain of Escherichia coli to rifampicin resistance has been performed by a single, defined DNA restriction fragment carrying the genetic information for the ß subunit of E. coli RNA polymerase. In this transformation the transforming genetic character has been substituted for the corresponding recipient gene locus by recombination. The value of the described transformation

Silvia M. Schweitzer; Hans Matzura



Detection and size analysis of proteins with switchable DNA layers.  


We introduce a chip-compatible scheme for the label-free detection of proteins in real-time that is based on the electrically driven conformation switching of DNA oligonucleotides on metal surfaces. The switching behavior is a sensitive indicator for the specific recognition of IgG antibodies and antibody fragments, which can be detected in quantities of less than 10(-18) mol on the sensor surface. Moreover, we show how the dynamics of the induced molecular motion can be monitored by measuring the high-frequency switching response. When proteins bind to the layer, the increase in hydrodynamic drag slows the switching dynamics, which allows us to determine the size of the captured proteins. We demonstrate the identification of different antibody fragments by means of their kinetic fingerprint. The switchDNA method represents a generic approach to simultaneously detect and size target molecules using a single analytical platform. PMID:19245235

Rant, Ulrich; Pringsheim, Erika; Kaiser, Wolfgang; Arinaga, Kenji; Knezevic, Jelena; Tornow, Marc; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard



Development of mass spectrometry for rapid detection of DNA fragments  

SciTech Connect

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

Buchanan, M.V.; Hurst, G.B. [Oak Ridge National Lab., TN (United States)



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

ERIC Educational Resources Information Center

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

Shultz, Jeff



The immunomodulating agent gliotoxin causes genomic DNA fragmentation.  


Gliotoxin, a member of the class of secondary fungal metabolites characterized by the presence of an epipolythiodioxopiperazine ring, caused fragmentation of spleen cell DNA as observed by flow cytometry and gel electrophoresis. Gliotoxin was found to cause substantial double-stranded DNA breakage in spleen cells which was dose- and time-dependent. The ability of gliotoxin to cause DNA breakage was also found to be specific to cell type. DNA breakage occurred in all cell types in which gliotoxin inhibited proliferation and so provides a general explanation as to how gliotoxin prevents cell proliferation. Other results showed that gliotoxin bound to a similar extent to both sensitive and resistant cells, indicating that differential uptake is not a likely mechanism to explain cell type selectivity. The results are discussed in terms of a mechanism for gliotoxin action involving genomic DNA as the central target. PMID:2441247

Braithwaite, A W; Eichner, R D; Waring, P; Müllbacher, A




NASA Astrophysics Data System (ADS)

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

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



Body size variation of mammals in a fragmented, temperate rainforest.  


Body size is perhaps the most important trait of an organism, affecting all of its physiological and ecological processes and, therefore, fundamentally influencing its ability to survive and reproduce in different environments, including those that have been modified by human activities. We tested the hypothesis that anthropogenic transformation of old-growth forest landscapes can result in significant intraspecific changes in body size of resident biotas. We collected data on five species of nonvolant mammals (common deer mouse[Peromyscus maniculatus], northwestern deer mouse[P. keeni], southern red-backed vole[Clethrionomys gapperi], montane shrew[Sorex monticolus], and Trowbridge's shrew[S. trowbridgii]) to test whether body size (mass and length) of these species varied across types of land cover (macrohabitats) and along elevational gradients of the fragmented, temperate rainforest of Olympic National Forest (Washington, U.S.A.). We measured 2168 and 1134 individuals for body mass and body length, respectively. Three species (P. keeni, S. monticolus, and S. trowbridgii) exhibited significantly different body size among macrohabitats: individuals from fragments were smaller than those in old-growth corridors and those in more extensive stands of old-growth forest. Body size of P. keeni was significantly correlated with elevation along corridors, peaking near the medial reaches of the corridors. The effects of anthropogenic transformations of this landscape of old-growth, temperate rainforest, although not universal among the five species, were significant and rapid-developing in just a few decades following tree harvests. Thus, anthropogenic fragmentation may influence not only the diversity, species composition, and densities of local biotas, but also one of the most fundamental and defining characteristics of native species-their body size. PMID:17650255

Lomolino, Mark V; Perault, David R



Anisotropic Brownian motion in ordered phases of DNA fragments.  


Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase. PMID:22270455

Dobrindt, J; Rodrigo Teixeira da Silva, E; Alves, C; Oliveira, C L P; Nallet, F; Andreoli de Oliveira, E; Navailles, L



Cloning human telomeric DNA fragments into Saccharomyces cerevisiae using a yeast-artificial-chromosome vector  

SciTech Connect

Telomeric fragments of human DNA ranging in size from 50 to 250 kilobases were cloned into Saccharomyces cerevisiae using a yeast-artificial-chromosome (YAC) vector. Six human-telomeric YAC (HTY) strains were selected by virtue of the specific hybridization of their DNA with the human telomeric terminal-repeat sequence (TTAGGG){sub n}, and the telomeric localization of this sequence within each YAC was demonstrated by its sensitivity to nuclease BAL-31. In situ hybridization of DNA from three of these HTY strains with human metaphase chromosomes yielded discrete patterns of hybridization signals at the telomeres of a limited number of human chromosomes, different for each clone. DNA from selected cosmid subclones of one of the HTY strains was used to localize the origin of the cloned telomeric DNA by in situ hybridization to the tip of the long arm of chromosome 7.

Riethman, H.C.; Burke, D.T.; Olson, M.V. (Washington Univ. School of Medicine, St. Louis, MO (USA)); Moyzis, R.K.; Meyne, J. (Univ. of California, Los Alamos, NM (USA))



A Prototype Ultrasound Instrument To Size Stone Fragments During Ureteroscopy  

NASA Astrophysics Data System (ADS)

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

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



Large-scale production of palindrome DNA fragments  

SciTech Connect

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.

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



Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts.  


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

McArthur, C R; Clark-Walker, G D



Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men  

Microsoft Academic Search

Objective: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters.Design: Prospective, observational study.Setting: University infertility clinic.Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy.Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF.Main

Armand Zini; Ryszard Bielecki; Donna Phang; Maria Teresa Zenzes



Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication*  

PubMed Central

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G2 phase-specific DNA damage as evidenced by increased ?-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation. PMID:22570476

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



Sieving duration and sieve loading impacts on dry soil fragment size distributions  

E-print Network

based on the dry or wet separation of soil fragments, after the partial breakdown of hierarchical unitsSieving duration and sieve loading impacts on dry soil fragment size distributions M. Di; accepted 18 June 2006 Abstract Measurement of the soil fragment size distribution has been found

Perfect, Ed


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

E-print Network

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

Willems, Wolfgang


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

Microsoft Academic Search

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



A simple and effective separation and purification procedure for DNA fragments using Dodecyltrimethylammonium bromide  

Microsoft Academic Search

In this work we describe a simple two step separation procedure for the separation and purification of short DNA fragments. The first step involves precipitating the DNA using the cationic surfactant dodecyltrimethylammonium bromide. Dodecyltrimethylammonium bromide, unlike cetyltrimethylammonium bromide will not precipitate DNA before complexation is complete thus providing a high purity DNA. The second step involves dissolution of the DNA-dodecyltrimethylammonium

Daragh M. McLoughlin; Julie O'Brien; Jennifer J. McManus; Alexander V. Gorelov; Kenneth A. Dawson



Electric linear dichroism transients of bent DNA fragments.  


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

Umazano, Juan P; Bertolotto, Jorge A



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

PubMed Central

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

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



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


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.

Wong, Kwong-Kwok (Richland, WA)



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

SciTech Connect

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.

Wong, K.K.



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


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

Xie, Ping



Saccharin consumption increases sperm DNA fragmentation and apoptosis in mice  

PubMed Central

Background: Saccharin is an artificial non-caloric sweetener that used to sweeten products such as drinks, candies, medicines, and toothpaste, but our bodies cannot metabolize it. Sodium saccharin is considered as an important factor in tumor promotion in male rats but not in humans. Objective: The objective of this study was to investigate the effect of saccharin consumption on sperm parameters and apoptosis in adult mice. Materials and Methods: Totally 14 adult male mice were divided into 2 groups. Group 1 served as control fed on basal diet and group 2 or experimental animals received distilled water containing saccharin (0.2% w/v) for 35 days. After that, the left cauda epididymis of each mouse was cut and placed in Ham’s F10. Swimmed-out spermatozoa were used to analyze count, motility, morphology (Pap-staining) and viability (eosin-Y staining). Sperm DNA integrity, as an indicator of apoptosis, was assessed by SCD (sperm chromatin dispersion) and terminal deoxynucleotidyl transferase (TUNEL) assay. Results: Following saccharin consumption, we had a reduction in sperm motility with respect to control animals (p=0.000). In addition, the sperm count diminished (17.70±1.11 in controls vs. 12.80±2.79 in case group, p=0.003) and the rate of sperm normal morphology decreased from 77.00±6.40 in control animals into 63.85±6.81 in saccharin-treated mice (p=0.001). Also, we saw a statistically significant increase in rates of sperm DNA damage and apoptosis in experimental group when compared to control one (p=0.001, p=0.002 respectively). Conclusion: Saccharin consumption may have negative effects on sperm parameters, and increases the rate of sperm DNA fragmentation and apoptosis in mice. PMID:25031574

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



Hydrodynamics-based transfer of PCR-amplified DNA fragments into rat liver  

Microsoft Academic Search

A high level of plasmid DNA expression in rat liver can be achieved by the rapid injection of a large volume of a naked DNA solution into the tail vein, called the ‘hydrodynamics-based procedure.’ The preparation of PCR-amplified DNA fragments is easier than that of naked DNA. In this paper we evaluated the effects of expressing the erythropoietin (Epo) gene

S Kameda; H Maruyama; N Higuchi; G Nakamura; N Iino; Y Nishikawa; J Miyazaki; F Gejyo



Detection of specific sequences among DNA fragments separated by gel electrophoresis  

Microsoft Academic Search

This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.

E. M. Southern



DNA fragmentation pattern in human fibroblasts after irradiation with iron ions  

NASA Astrophysics Data System (ADS)

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).

Campa, Alessandro


Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ  

Microsoft Academic Search

Loss of nerve cells is a hallmark of the pathology of Alzheimer's disease (AD), yet the patterns of cell death are unknown. By analyzing DNA fragmentation in situ we found evidence for cell death not only of nerve cells but also of oligodendrocytes and microglia in AD brains. In average, 30 times more brain cells showed DNA fragmentation in AD

Hans Lassmann; Christian Bancher; Helene Breitschopf; Jerzy Wegiel; Maciej Bobinski; Kurt Jellinger; Henryk M. Wisniewski



DNA size separation using artificially nanostructured matrix  

NASA Astrophysics Data System (ADS)

We have demonstrated two types of size separation of biomolecules using a nanostructured matrix artificially fabricated using electron-beam lithography: sieve-type separation using a regular pillar array structure and size exclusion chromatography (SEC) type separation using a structure with narrow and wide gaps. With these devices, samples of double-stranded DNA molecules (2, 5, and 10 k base pairs) were clearly separated into bands; smaller molecules eluted earlier in the sieve type while they eluted later in the SEC type. The nanostructured matrix enables various types of molecular separation by changing the design of the nanostructure. Moreover, it should be easy to integrate the matrix with other biomolecular fluidic devices because it does not require a filling medium.

Baba, M.; Sano, T.; Iguchi, N.; Iida, K.; Sakamoto, T.; Kawaura, H.



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

NASA Technical Reports Server (NTRS)

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

Holley, W. R.; Chatterjee, A.



Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain  

PubMed Central

Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30–90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in situ using digoxigenin-dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8-hydroxy-2?-deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-positive cells in the cortex from the sham-operated control group was 3 ± 3 (mean ± SD per mm2). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS-positive cells were significantly increased by 200-fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8-hydroxy-guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 ?g/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham-operated control group. We detected few FPGSS of oh8dG-positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked-end labeling (TUNEL)-positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL. PMID:10783150




DFF, a Heterodimeric Protein That Functions Downstream of Caspase3 to Trigger DNA Fragmentation during Apoptosis  

Microsoft Academic Search

We have identified and purified from HeLa cytosol a protein that induces DNA fragmentation in coincubated nuclei after it is activated by caspase-3. This protein, designated DNA Fragmentation Factor (DFF), is a heterodimer of 40 kDa and 45 kDa subunits. The amino acid sequence of the 45 kDa subunit, determined from its cDNA sequence, reveals it to be a novel

Xuesong Liu; Hua Zou; Clive Slaughter; Xiaodong Wang



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

Microsoft Academic Search

A genomic DNA fragment from wheat carrying the Glu-1Dx5 gene has been shown to exhibit reduced pollen transmission in transgenic maize. To localize the region of the DNA fragment\\u000a responsible for this reduced pollen transmission, we produced transgenic maize plants in which the wheat genomic DNA proximal\\u000a to the 1Dx5 coding sequence was replaced with the maize 27 kDa ?-zein promoter.

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



Pulsed field gel electrophoresis and physical mapping of large DNA fragments in the Tm2a region of chromosome 9 in tomato  

Microsoft Academic Search

A method has been developed which allows the isolation of very high molecular weight DNA (>2 million bp) from leaf protoplasts of tomato (Lycopersicon esculentum). The DNA isolated in this manner was digested in agarose with rare-cutting restriction enzymes and separated by pulsed field gel electrophoresis. The size range of the reslting fragments was determined by hybridization to a number

Martin W. Ganal; Nevin D. Young; Steven D. Tanksley



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


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

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



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


Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory. PMID:12514084

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



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


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

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



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


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

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



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

PubMed Central

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

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



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


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

Anderson, R A; Eliason, S L



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


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

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



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


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

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



Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.  


Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides. PMID:6833179

Salyers, A A; Lynn, S P; Gardner, J F



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

SciTech Connect

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

Zheleznaya, L.A.; Menzenyuk, O.Y. [Institute of Theoretical and Experimental Biophysics, Pushchino (Russian Federation); Matvienko, N.N. [Branch of Shemyakin and Ovchinnikov Institute of Bioorganic, Pushchino (Russian Federation); Matvienko, N.I. [Institute of Protein Research, Pushchino (Russian Federation)




Microsoft Academic Search

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




Fracture of Brittle Solids. II. Distribution Function for Fragment Size in Single Fracture (Experimental)  

Microsoft Academic Search

The theoretical results of Gilvarry for the size distribution of the fragments in single fracture have been verified experimentally by fracturing spherical glass specimens under compression. The fragments were contained by a gelatin matrix to inhibit secondary fracture and thus make conditions conform as closely as possible to single fracture. Experimental values of the probability of fracture as obtained by

J. J. Gilvarry; B. H. Bergstrom



Hypervariable Bkm DNA Loci in a Moth, Ephestia kuehniella : Does Transposition Cause Restriction Fragment Length Polymorphism?  

PubMed Central

Bkm sequences, originally isolated from snake satellite DNA, are a component of eukaryote genomes with a preferential location on sex chromosomes. In the Ephestia genome, owing to the presence of only a few Bkm-positive BamHI restriction fragments and to extensive restriction fragment length polymorphisms between and within inbred strains, a genetic crossbreeding analysis was feasible. No sex linkage of Bkm was detected. Instead—depending on the strain—two or three autosomal Bkm DNA loci were identified. All three loci were located on different chromosomes. Fragment length and transmission of fragments was stable in some crosses. In others, changes in fragment length or loss of the Bkm component were observed, probably depending on the source strain of the fragment. The anomalous genetic behaviour is best accounted for by the assumption that Bkm sequences are included in mobile genetic elements. PMID:17246372

Traut, W.



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

PubMed Central

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

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



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

NASA Technical Reports Server (NTRS)

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

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



Cloning of Herpes Simplex Type 1 DNA Fragments in a Bacteriophage Lambda Vector  

Microsoft Academic Search

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambda gtWES\\\\cdot lambda B. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda -HSV1 hybrids were purified and amplified, and the DNA was

L. W. Enquist; M. J. Madden; P. Schiop-Stansly; G. F. Vande Woude



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


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

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



Oxygen Tension Influences DNA Fragmentation and Cell Death in Glucocorticoid-Treated Thymocytes  

Microsoft Academic Search

Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2\\/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the

C. Stefanelli; I. Stanic; F. Bonavita; C. Muscari; C. Pignatti; C. Rossoni; C. M. Caldarera



Statistical Exploration of Fragmentation Phase Space Source Sizes in Nuclear Multifragmentation  

E-print Network

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.

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



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

NASA Astrophysics Data System (ADS)

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

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


Body Size Variation of Mammals in a Fragmented, Temperate Rainforest  

Microsoft Academic Search

Body size is perhaps the most important trait of an organism, affecting all of its physiological and ecological processes and, therefore, fundamentally influencing its ability to survive and reproduce in different environments, including those that have been modified by human activities. We tested the hypothesis that anthropogenic transformation of old-growth forest landscapes can result in significant intraspecific changes in body




Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase  

Microsoft Academic Search

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as sub- strates for DNA polymerase a (pol a) and Klenow fragment (exo? ) of DNA polymerase I (Escherichia coli ). One set of analogues was designed to test the importance of the electronic nature of the base.

Kristi Kincaid; Jeff Beckman; Aleksandra Zivkovic; Randall L. Halcomb; Joachim W. Engels; Robert D. Kuchta



Repeated blast exposures cause brain DNA fragmentation in mice.  


The pathophysiology of blast-induced traumatic brain injury (TBI) and subsequent behavioral deficits are not well understood. Unraveling the mechanisms of injury is critical to derive effective countermeasures against this form of neurotrauma. Preservation of the integrity of cellular DNA is crucial for the function and survival of cells. We evaluated the effect of repeated blast exposures on the integrity of brain DNA and tested the utility of cell-free DNA (CFD) in plasma as a biomarker for the diagnosis and prognosis of blast-induced polytrauma. The results revealed time-dependent breakdown in cellular DNA in different brain regions, with the maximum damage at 24 h post-blast exposures. CFD levels in plasma showed a significant transient increase, which was largely independent of the timing and severity of brain DNA damage; maximum levels were recorded at 2 h after repeated blast exposure and returned to baseline at 24 h. A positive correlation was observed between the righting reflex time and CFD level in plasma at 2 h after blast exposure. Brain DNA damage subsequent to repeated blast was associated with decreased mitochondrial membrane potential, increased release of cytochrome C, and up-regulation of caspase-3, all of which are indicative of cellular apoptosis. Shock-wave-induced DNA damage and initiation of mitochondrial-driven cellular apoptosis in the brain after repeated blast exposures indicate that therapeutic strategies directed toward inhibition of DNA damage or instigation of DNA repair may be effective countermeasures. PMID:24074345

Wang, Ying; Arun, Peethambaran; Wei, Yanling; Oguntayo, Samuel; Gharavi, Robert; Valiyaveettil, Manojkumar; Nambiar, Madhusoodana P; Long, Joseph B



Damage to Model DNA Fragments from Very Low-Energy (Electrons  

E-print Network

Damage to Model DNA Fragments from Very Low-Energy (Electrons Joanna Berdys,, Iwona-mail: Abstract: Although electrons having enough energy to ionize or electronically suggested that even lower- energy electrons (most recently 1 eV and below) can also damage DNA. The findings

Simons, Jack


Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP).  


The sources of variance and errors in transcriptomics based on the electrophoretic separation of amplified cDNA fragments were investigated using cDNA-amplified fragment length polymorphism (AFLP). Transcriptome profiles of the plant-pathogenic fungus Verticillium longisporum were generated by a standard cDNA-AFLP protocol followed by electrophoretic separation of amplified DNA fragments in flatbed polyacrylamide gels with fluorescence detection as well as by capillary electrophoresis (DNA sequencer). The total variance was partitioned into contributions of cDNA synthesis, adapter ligation, preamplification, amplification, and electrophoresis. Parameters of computer-aided peak recognition and matching were investigated and strategies improving matching success based on double passage with different signal intensity thresholds were developed. The overall quality of data was similar for cDNA-AFLP and microarray hybridization. Variance of cDNA-AFLP was independent of signal intensity, whereas microarray data showed higher variance for low-intensity signals. Capillary electrophoresis significantly reduced the number of wrongly matched and unmatched signals as compared with flatbed gels. These results are also likely to apply to related electrophoresis-based transcriptome analysis techniques such as mRNA differential display. PMID:19588459

Weiberg, Arne; Karlovsky, Petr



Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP)  

PubMed Central

The sources of variance and errors in transcriptomics based on the electrophoretic separation of amplified cDNA fragments were investigated using cDNA-amplified fragment length polymorphism (AFLP). Transcriptome profiles of the plant-pathogenic fungus Verticillium longisporum were generated by a standard cDNA-AFLP protocol followed by electrophoretic separation of amplified DNA fragments in flatbed polyacrylamide gels with fluorescence detection as well as by capillary electrophoresis (DNA sequencer). The total variance was partitioned into contributions of cDNA synthesis, adapter ligation, preamplification, amplification, and electrophoresis. Parameters of computer-aided peak recognition and matching were investigated and strategies improving matching success based on double passage with different signal intensity thresholds were developed. The overall quality of data was similar for cDNA-AFLP and microarray hybridization. Variance of cDNA-AFLP was independent of signal intensity, whereas microarray data showed higher variance for low-intensity signals. Capillary electrophoresis significantly reduced the number of wrongly matched and unmatched signals as compared with flatbed gels. These results are also likely to apply to related electrophoresis-based transcriptome analysis techniques such as mRNA differential display. PMID:19588459

Weiberg, Arne; Karlovsky, Petr



Preparative concentration and size fractionation of DNA by porous media using a combination of flow and low electric field strength.  


The retention of DNA by porous media during chromatography with an applied axial electric field was investigated. DNA was retained by negative electric fields in columns packed with Sephadex G-75 and G-25. A negative field was defined as the electric field orientation in which the direction of electrophoresis was opposing the direction of buffer flow (positive electrode at the column inlet). A positive field was not effective at retaining the DNA. The electric field strength required to retain the DNA was dependent upon the buffer flow rate. The retention of DNA using Sephadex G-25, a gel filtration medium with a higher degree of cross-linking, required higher electric fields than the more porous Sephadex G-75. A dilute DNA solution was concentrated at the inlet of the chromatography bed by an electric field. Mixtures of DNA restriction fragments were used to determine size-dependent retention. DNA was size-fractionated by varying the electric field strength and flow rate. At a given electric field strength and flow rate, the lower molecular weight DNA fragments were not as strongly retained as the higher molecular weight fragments. Decreasing the flow rate or increasing the electric field strength resulted in increased retention of the lower molecular weight DNA fragments. In this manner, by selecting a specific set of conditions (packing material, flow rate, and electric field strength), the molecular weight of DNA fragments retained by the column can be adjusted. Efficient separation of high molecular weight DNA from bovine serum albumin, a protein with high electrophoretic mobility, was demonstrated using a field of 2 V/cm in a column packed with Sephadex G-75. PMID:9190079

Cole, K D



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


To investigate the interactions between chromosomal DNA and nuclear matrices in higher plants, matrix associated regions (MARs) of rice (Oryza sativa L.) DNAs were cloned. First, we prepared nuclear matrices from isolated nuclei by digesting them with EcoRI and then extracting with 2 M NaCl. About 6% of the total DNA remained in the nuclear matrices after this digestion and extraction. The residual DNA fragments in the nuclear matrices were cloned. Some of the cloned DNA fragments showed binding to certain nuclear proteins. One of the MAR fragments contained sequences related to known consensus motifs and a hairpin loop structure. A method is presented for isolation of matrix associated region (MAR) DNAs from plant cells. PMID:9360323

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



Cloning and sequence analysis of an XbaI fragment of rainbow trout mitochondrial DNA  

Microsoft Academic Search

A 2.4 kbp Xbal fragment of rainbow trout mitochondrial DNA was cloned into pTZ18R. DNA sequence analysis reveals that this segment of the genome encodes URF3, tRNAArg, URF4L and URF4 in the same orientation as other vertebrate mitochondrial genomes. Comparison of these segments of the rainbow trout mitochondrial genome with the corresponding sequences in human mitochondrial DNA shows that approximately

William S. Davidson; Sylvia E. Bartlett; Tim P. Birt; John M. Green



Linear contour length dependence of electrical polarizability of nucleosomal DNA fragments: implications for the flexibility of DNA.  


The transient electric birefringence of monodisperse oligonucleosomal DNA ranging from 145 to 990 base pairs has been studied. The orientation of fragments can be described in terms of an induced dipole moment with a small contribution of a permanent dipole. The electrical polarizability delta alpha was found to increase linearly with the DNA contour length. This unexpected dependence might result from a bent structure of DNA already considerable for very short segments. The observed delta alpha values agree with a segmental orientation of rigid subunits of length 13-18 nm as estimated in the elastic model of DNA with a kink angle of about 41 degrees. PMID:2306234

Hacques, M F; Marion, C



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

PubMed Central

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

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



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


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

Pedroso, M C; Durzan, D J



Influence of dsDNA fragment length on particle binding in an evanescent field biosensing system.  


Particle labels are widely used in affinity-based biosensing due to the high detection signal per label, the high stability, and the convenient biofunctionalization of particles. In this paper we address the question how the time-course of particle binding and the resulting signals depend on the length of captured target molecules. As a model system we used fragments of dsDNA with lengths of 105 bp (36 nm), 290 bp (99 nm) and 590 bp (201 nm), detected in an evanescent-field optomagnetic biosensing system. On both ends the fragments were provided with small-molecule tags to allow binding of the fragments to protein-coated particles and to the capture molecules at the sensor surface. For isolated single particles bound to the surface, we observe that the optical scattering signal per particle depends only weakly on the fragment length, which we attribute to the pivoting motion that allows the particles to get closer to the surface. Our data show a strong influence of the fragment length on the particle binding: the binding rate of particles to the sensor surface is an order of magnitude higher for the longest dsDNA fragments compared to the smallest fragment studied in this paper. We attribute the enhanced binding rate to the length and motional freedom of the fragments. These results generate a new dimension for the design of assays and systems in particle-based biosensing. PMID:24534803

Koets, Marjo; van Ommering, Kim; Wang, Liqin; Testori, Emilie; Evers, Toon H; Prins, Menno W J



Effective fragment potential study of the interaction of DNA bases.  


Hydrogen-bonded and stacked structures of adenine-thymine and guanine-cytosine nucleotide base pairs, along with their methylated analogues, are examined with the ab inito based general effective fragment potential (EFP2) method. A comparison of coupled cluster with single, double, and perturbative triple (CCSD(T)) energies is presented, along with an EFP2 energy decomposition to illustrate the components of the interaction energy. PMID:21877717

Smith, Quentin A; Gordon, Mark S; Slipchenko, Lyudmila V



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

NASA Astrophysics Data System (ADS)

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

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



DNA-Fragments Are Transcytosed across CaCo-2 Cells by Adsorptive Endocytosis and Vesicular Mediated Transport  

PubMed Central

Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >103 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system. PMID:23409196

Johannessen, Lene E.; Spilsberg, Bj?rn; Wiik-Nielsen, Christer R.; Kristoffersen, Anja B.; Holst-Jensen, Arne; Berdal, Knut G.



Seasonal changes in sperm DNA fragmentation of Murciano-Granadina goats: The compelling case for dynamic assessment  

Microsoft Academic Search

Evidence is provided of seasonal changes in the rate of DNA fragmentation dynamics of sperm collected from goats Murciano-Granadina breed), in a Spanish Breeding Centre at 41°N latitude. Results show that the initial assessment of sperm DNA fragmentation (T0) was not sufficient to differentiate sperm DNA fragmentation of bucks collected at different times of the year (Kruskal–Wallis 2.399; P=0.493). However,

C. López-Fernández; S. D. Johnston; A. Gosálbez; J. Gosálvez



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


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

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



Clinical value of DNA fragmentation evaluation tests under ART treatments  

PubMed Central

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

Tavukçuo?lu, ?lkay ?afak; Al-Azawi, Tahani; Khaki, Amir Afshin; Khaki, Arash; Khalil, Ahmed; Al-Hasani, Safaa



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

PubMed Central

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.

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



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

SciTech Connect

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.

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



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


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

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



Accurate phylogenetic classification of DNA fragments based onsequence composition  

SciTech Connect

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.

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



Identification of restriction fragment length polymorphisms in DNA from Mycobacterium paratuberculosis.  


DNAs from 34 mycobactin-dependent isolates of Mycobacterium paratuberculosis and 1 isolate of M. paratuberculosis 18 were digested with four restriction endonucleases. Southern hybridization experiments were performed with a 32P-labeled oligonucleotide DNA probe derived from the sequence of IS900, an insertion sequence present in 15 to 20 copies per M. paratuberculosis chromosome. The probe hybridized with DNA from each of the mycobactin-dependent isolates, and restriction fragment length polymorphisms were detected among the isolates with each restriction endonuclease used. Restriction fragment length polymorphism analysis may permit identification of various strains of M. paratuberculosis, which has not been possible with other techniques. PMID:1979332

Whipple, D; Kapke, P; Vary, C



Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments.  


A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization. PMID:15690452

Xu, Yan; Qin, Weidong; Li, Sam F Y



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

NASA Astrophysics Data System (ADS)

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

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



Highly Selective Separation of DNA Fragments Using Optically Directed Transport  

SciTech Connect

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.

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



Sizing bands on autoradiograms: a study of precision for scoring DNA fingerprints.  


We replicated DNA fingerprints of snapping turtles (Chelydra serpentina) and hypervariable restriction fragments of red-winged blackbirds (Agelaius phoeniceus) to estimate the between-blot and between-lane components of variance in molecular weights of restriction fragments. Molecular weight standards were included in every lane, and bands were sized using a sonic digitizer. In both studies, a strong positive correlation was found between band size and coefficient of variation (CV; mean = 0.7%). In the DNA fingerprint study, 26% of the variance in estimates of band size was due to differences between blots, 10% due to differences between lanes on the same blot, and 64% due to error in the digitizing process. In the restriction fragment length polymorphism (RFLP) study, 16% of the variance was due to difference between lanes, and 84% to digitizing. Statistical models were developed to measure the effect of sizing error on identifying identical fragments in different lanes or on different blots, in categorizing distinct alleles, and in determining the size of bins in operational allele definitions. We suggest that the distance between bands be at least 2.8 standard deviations (SD) before they are declared different at alpha = 0.05, and 3.7 SD for alpha = 0.01. A variation in CVs strongly indicates that empirical relationships between SD and band size must be used to decide if two bands represent the same allele. Alleles must be at least 3.9 SD apart before the chance of assigning new observations in error falls below 0.05. We suggest that a minimum bin width of 16 SD is necessary before the chances of assigning a band to the wrong bin falls below 0.05. PMID:1674911

Galbraith, D A; Boag, P T; Gibbs, H L; White, B N



Facile control of DNA-templated inorganic nanoshell size.  


We elaborated a facile method to control the size of CdS nanoshells obtained by DNA assisted "double templating" approach. By changing the concentration of NaCl in solution to vary the extent of DNA electrostatic deposition on cationic silica beads, we succeeded to control the density of DNA adsorbed on the beads, and further the density of CdS material grown on DNA. Further dissolution of the silica core triggers shrinking of CdS shell to a different extent depending on the CdS shell density and results in formation of CdS nanoshells of different sizes from ca. 100 nm to ca. 400 nm. Therefore, the main advantage of the proposed method is that it can be used to synthesize hollow nanoshells of various sizes, from ca. 25% to ca. 75% size of the primary template (silica bead), by using only one single primary template. PMID:22524032

Pu, Shengyan; Zinchenko, Anatoly; Murata, Shizuaki



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

PubMed Central

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

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



DNA size and conformations analysis using a synthetic nanopore  

NASA Astrophysics Data System (ADS)

Our work reveals the ability of a synthetic nanopore made in a silicon nitride membrane to discriminate between different conformations and lengths of DNA molecules and presents a comparative analysis with the electrophoretic behavior of the same DNA. Double stranded linear, supercoiled and relaxed form of the same DNA, linear restriction fragments, as well as single stranded DNA, are passed through a synthetic nanopore filled with a buffered ionic solution, and a subsequent analysis in terms of current blockage, translocation time and integrated events area shows the analytical ability of such a device. Also, we prove that an intercalating agent increases the temporal resolution by increasing the translocation time up to a factor of two.

Fologea, Daniel; Uplinger, James; Thomas, Brian; Ledden, Bradley; Brandin, Eric; Branton, Daniel; Li, Jiali



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

NASA Astrophysics Data System (ADS)

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).

Pourkhorsandi, H.; Mirnejad, H.



Apparent lack of restriction fragment length polymorphisms in chromosome specific arrays of gamma satellite DNA  

SciTech Connect

The centromeric regions of human chromosomes are known to be composed primarily of repeated satellite DNA sequences which are tandemly organized into large arrays. A new class of centromeric satellite DNA, designated as human gamma satellite DNA, was originally identified in the centromeric region of human chromosome 8 (gamma 8) and found to be characterized by monomeric units of 220 bp. More recently, a cosmid clone was localized by fluorescence in situ hybridization to the centromeric region of the human X chromosome and was found to lack alpha satellite DNA. A 1205 bp EcoRI subclone (2D12/E2) of this X-centromere specific cosmid clone revealed approximately 5 monomers of 220 bp which had approximately 62% DNA sequence similarly to the gamma 8 monomers. Genomic DNA from ten unrelated individuals (6 males & 4 females) were digested in agarose plugs with Sstl, separated by pulsed field gel electrophoresis and probed with {sup 32}P-labeled 2D12/E2. A high molecular weight fragment of {approximately}34 kb was detected in all individuals. A similar experiment using Hpal revealed high molecular weight fragments of {approximately} 54 kb and {approximately}70 kb in all individuals. When the Southern blots were stripped and reprobed with the 704 bp, gamma-8 satellite DNA probe (50E1), Sstl fragments of {approximately} 34 kb were seen in all individuals tested. This apparent lack of polymorphisms in chromosome specific arrays of gamma satellite DNA among unrelated individuals is contrasting to the high frequency of polymorphisms seen in arrays of alpha satellite DNA. This may indicate a physical or functional constraint against sequence variation in gamma satellite DNA arrays.

Lin, C.C.; Lee, C. [Univ. of Alberta, Edmonton (Canada)]|[Univ. of Alberta Hospitals, Edmonton (Canada)



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

SciTech Connect

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}.

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))



Size-dependent enrichment of waste slag aggregate fragments abraded from asphalt concrete  

Microsoft Academic Search

Authors consider the environmental prospects of using melted waste slag as the aggregate for asphalt pavement. In particular, the enrichment of slag-derived fragments in fine abrasion dust particles originated from slag asphalt concrete and its size dependency were concerned. A series of surface abrasion tests for asphalt concrete specimens, containing only natural aggregates as reference or 30wt% of substituted slag

Fumitake Takahashi; Takayuki Shimaoka; Kevin Gardner; Akiko Kida



46 astronomy /// may 05 MOON-SIZE FRAGMENTS FLY as bodies in the  

E-print Network

46 astronomy /// may 05 MOONS MOON-SIZE FRAGMENTS FLY as bodies in the Kuiper Belt collide. Some;Around each gas-giant planet orbit dozens of small moons born elsewhere. These far-ranging bodies HAMILTON elusive quarry were new uran- ian moons, small objects orbit- ing far from their giant blue

Hamilton, Douglas P.


Fracture of Brittle Solids. I. Distribution Function for Fragment Size in Single Fracture (Theoretical)  

Microsoft Academic Search

For single fracture of a brittle solid, the distribution function for fragment size is obtained on the basis of Griffith's theory of brittle strength (which postulates crack propagation when pre-existent flaws are activated by stress). Three assumptions are made: (a) Fracture proceeds by activation of flaws in the volume of the specimen, in fracture surfaces through the specimen, and in

J. J. Gilvarry



A ring fragmentation approach to medium-sized cyclic 2-alkynones  

PubMed Central

Bicyclic ?-silyloxy-?-hydroxy-?-diazoketones in which the C?-C? bond is the ring fusion bond productively fragment when treated with tin(IV) chloride to provide medium-sized cyclic 2-alkynones. This method provides good to excellent yields of 10-membered, 11-membered and 12-membered alkynone products. PMID:22132946

Tsvetkov, Nikolay P.; Bayir, Ali; Schneider, Samuel; Brewer, Matthias



Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.  


The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy. PMID:3912509

Warwicker, J; Ollis, D; Richards, F M; Steitz, T A



Topological defects and the optimum size of DNA condensates.  

PubMed Central

Under a wide variety of conditions, the addition of condensing agents to dilute solutions of random-coil DNA gives rise to highly compact particles that are toroidal in shape. The size of these condensates is remarkably constant and is largely independent of DNA molecular weight and basepair sequence, and of the nature of condensing agent (e.g., multivalent cation, polymers, or added cosolvent). We show how this optimum size is determined by the interactions between topological defects, which unavoidably strain the circumferentially wound DNA strands in the torus. PMID:9675173

Park, S Y; Harries, D; Gelbart, W M



On-chip fraction collection for multiple selected ssDNA fragments using isolated extraction channels.  


High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay. PMID:21227429

Li, Zheyu; Sun, Kai; Sunayama, Misato; Matsuo, Yasutaka; Mizeikis, Vygantas; Araki, Ryoko; Ueno, Kosei; Abe, Masumi; Misawa, Hiroaki



Oak chloroplast-DNA polymorphisms detected by restriction fragment length polymorphism (RFLP)  

E-print Network

de fragment de restriction (RFLP) INTRODUCTION Climatic variations as well as human activ- ities (eg pressures. Therefore, we need better insight into the genetics and ecology of populations in or- der sequences of plants. cpDNA polymorphisms could provide infor- mation on evolutionary distances among oak

Paris-Sud XI, Université de


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

Microsoft Academic Search

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

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



Species relationships in the Hordeum murinum aggregate viewed from chloroplast DNA restriction fragment patterns  

Microsoft Academic Search

Three annual widespread species of Hordeum were investigated by the fragment pattern method on their chloroplast (cp) DNA. The species were H. glaucum, H. leporinum and H. murinum; H. vulgare was surveyed for comparison. Twelve restriction enzymes were used, nine recognizing 6 bp, one 5 bp and two 4 bp, thus, randomly surveyed, a total of 2,113 bp or 1.6%

B. R. Baum; L. G. Bailey



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

PubMed Central

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

Munguia-Rosas, Miguel A.; Montiel, Salvador



Non-monotonic density dependence of the diffusion of DNA fragments in low-salt suspensions  

E-print Network

The high linear charge density of 20-base-pair oligomers of DNA is shown to lead to a striking non-monotonic dependence of the long-time self-diffusion on the concentration of the DNA in low-salt conditions. This generic non-monotonic behavior results from both the strong coupling between the electrostatic and solvent-mediated hydrodynamic interactions, and from the renormalization of these electrostatic interactions at large separations, and specifically from the dominance of the far-field hydrodynamic interactions caused by the strong repulsion between the DNA fragments.

M. G. McPhie; G. Naegele



G Protein-Mediated Neuronal DNA Fragmentation Induced by Familial Alzheimer's Disease-Associated Mutants of APP  

Microsoft Academic Search

Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation

Tomoki Yamatsuji; Takashi Matsui; Takashi Okamoto; Katsumi Komatsuzaki; Shizu Takeda; Hiroaki Fukumoto; Takeshi Iwatsubo; Nobuhiro Suzuki; Asano Asami-Odaka; Scott Ireland; T. Bernard Kinane; Ugo Giambarella; Ikuo Nishimoto



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

PubMed Central

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

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



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


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

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



DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat  

Microsoft Academic Search

Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment

N. P. Harberd; D. Bartels; R. D. Thompson



Response of DNA fragments to potentiometric sensors studied using HPLC.  


Potentiometric sensors are studied as viable candidates for the construction of high throughput DNA arrays. For preliminary investigations, such sensors were used in an HPLC setup in the present work. This avoided errors due to ionic contaminants or additives in the commercial samples. The oligonucleotides dT(10), dT(20) and dT(30) were used as test substances. The potentiometric sensors were of the coated wire type, containing PVC, DOP, MTDDACl and a synthetic podand urea receptor. The HPLC system consisted of a reversed phase column eluted with a phosphate buffer, triethylammoniumacetate (TEAA), and an acetonitrile gradient. Molar responses and sensitivities increased with increasing chain length of oligonucleotides, yielding detection limits as low as 10(-6)M (dT(30), injected concentration). The slopes of the calibration graphs were at least 23 mV/decade (dT(10)), which was much higher than expected. The results are discussed in view of the potential use of this sensor type in high throughput microarrays. PMID:17979638

Nagels, Luc J; Everaert, Joseph; Bohets, Hugo; Del Favero, Jurgen; Goossens, Dirk; Robbens, Johan; Pietraszkiewicz, Marek; Pietraszkiewicz, Oksana



Genome size and endonuclear DNA replication in spiders.  


Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development. PMID:15971267

Rasch, Ellen M; Connelly, Barbara A



Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA.  


An optimized DNA extraction protocol for animal tissues coupled with sensitive PCR methods was used to determine whether trace levels of feed-derived DNA fragments, plant and/or transgenic, are detectable in animal tissue samples including dairy milk and samples of muscle (meat) from chickens, swine, and beef steers. Assays were developed to detect DNA fragments of both the high copy number chloroplast-encoded maize rubisco gene (rbcL) and single copy nuclear-encoded transgenic elements (p35S and a MON 810-specific gene fragment). The specificities of the two rbcL PCR assays and two transgenic DNA PCR assays were established by testing against a range of conventional plant species and genetically modified maize crops. The sensitivities of the two rbcL PCR assays (resulting in 173 and 500 bp amplicons) were similar, detecting as little as 0.08 and 0.02 genomic equivalents, respectively. The sensitivities of the p35S and MON 810 PCR assays were approximately 5 and 10 genomic equivalents for 123 bp and 149 bp amplicons, respectively, which were considerably less than the sensitivity of the rbcL assays in terms of plant cell equivalents, but approximately similar when the higher numbers of copies of the chloroplast genome per cell are taken into account. The 173 bp rbcL assay detected the target plant chloroplast DNA fragment in 5%, 15%, and 53% of the muscle samples from beef steers, broiler chickens, and swine, respectively, and in 86% of the milk samples from dairy cows. Reanalysis of new aliquots of 31 of the pork samples that were positive in the 173 bp rbcL PCR showed that 58% of these samples were reproducibly positive in this same PCR assay. The 500 bp rbcL assay detected DNA fragments in 43% of the swine muscle samples and 79% of the milk samples. By comparison, no statistically significant detections of transgenic DNA fragments by the p35S PCR assay occurred with any of these animal tissue samples. PMID:15453677

Nemeth, Anne; Wurz, Andreas; Artim, Lori; Charlton, Stacy; Dana, Greg; Glenn, Kevin; Hunst, Penny; Jennings, James; Shilito, Ray; Song, Ping



Computational analysis of DNA replicases in double-stranded DNA viruses: relationship with the genome size  

PubMed Central

Genome duplication in free-living cellular organisms is performed by DNA replicases that always include a DNA polymerase, a DNA sliding clamp and a clamp loader. What are the evolutionary solutions for DNA replicases associated with smaller genomes? Are there some general principles? To address these questions we analyzed DNA replicases of double-stranded (ds) DNA viruses. In the process we discovered highly divergent B-family DNA polymerases in phiKZ-like phages and remote sliding clamp homologs in Ascoviridae family and Ma-LMM01 phage. The analysis revealed a clear dependency between DNA replicase components and the viral genome size. As the genome size increases, viruses universally encode their own DNA polymerases and frequently have homologs of DNA sliding clamps, which sometimes are accompanied by clamp loader subunits. This pattern is highly non-random. The absence of sliding clamps in large viral genomes usually coincides with the presence of atypical polymerases. Meanwhile, sliding clamp homologs, not accompanied by clamp loaders, have an elevated positive electrostatic potential, characteristic of non-ring viral processivity factors that bind the DNA directly. Unexpectedly, we found that similar electrostatic properties are shared by the eukaryotic 9-1-1 clamp subunits, Hus1 and, to a lesser extent, Rad9, also suggesting the possibility of direct DNA binding. PMID:21742758

Kazlauskas, Darius; Venclovas, Ceslovas



DNA vaccination with VP2 gene fragment confers protection against Infectious Bursal Disease Virus in chickens.  


Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens by destruction of antibody producing B cells in the Bursa of Fabricius and poses a potential threat to the poultry industry. We have examined the protective efficacy of a subunit DNA vaccine against IBDV infection in chickens in this study. An immunodominant VP2 gene fragment (VP252-417) was cloned into CMV promoter based DNA vaccine vector pVAX1 and in vitro expression of the DNA encoded antigens was confirmed by transfection of CHO cells with vaccine constructs followed by RT-PCR and western blot analysis using IBDV-antiserum. Two weeks old chickens were immunized intramuscularly with pVAXVP252-417 and the in vivo transcription of the plasmid DNA was confirmed by RT-PCR analysis of DNA injected muscle tissue at different intervals of post immunization. Tissue distribution analysis revealed that the plasmid DNA was extensively distributed in muscle, spleen, kidney, liver, and bursa tissues. Chickens immunized with pVAXVP252-417 developed high titer (1:12,000) of anti-VP252-417 antibodies. Further, chicken splenocytes from pVAXVP252-417 immunized group showed a significantly high proliferation to the whole viral and recombinant antigen (P<0.01) compared to control groups, which implies that pVAXVP252-417 codes for immunogenic fragment which has epitopes capable of eliciting both B and T cell responses. This is evident by the fact that, pVAXVP252-417 immunized chicken conferred 75% protection against virulent IBDV (vIBDV) challenge compared to the control group. Thus, the present study confirms that the immunodominant VP2 fragment can be used as a potential DNA vaccine against IBDV infection in chickens. PMID:24745626

Pradhan, Satya Narayan; Prince, Prabhu Rajaiah; Madhumathi, Jayaprakasam; Arunkumar, Chakkaravarthy; Roy, Parimal; Narayanan, Rangarajan Badri; Antony, Usha



Growth and fragmentation of centimetre-sized dust aggregates: the dependence on aggregate size and porosity  

E-print Network

We carry out three-dimensional Smoothed Particle Hydrodynamics simulations of spherical homogeneous SiO2 dust aggregates to investigate how the mass and the porosity of the aggregates affects their ability to survive an impact at various different collision velocities (between 1 - 27.5m/s). We explore how the threshold velocities for fragmentation vary with these parameters. Crucially, we find that the porosity plays a part of utmost importance in determining the outcome of collisions. In particular, we find that aggregates with filling factors >37% are significantly weakened and that the velocity regime in which the aggregates grow is reduced or even non-existent (instead, the aggregates either rebound off each other or break apart). At filling factors less than ~37% we find that more porous objects are weaker but not as weak as highly compact objects with filling factors >37%. In addition we find that (for a given aggregate density) collisions between very different mass objects have higher threshold veloci...

Meru, Farzana; Schaefer, Christoph; Speith, Roland; Kley, Wilhelm



Human Sperm DNA Fragmentation and its Correlation with Conventional Semen Parameters  

PubMed Central

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

Evgeni, Evangelini; Charalabopoulos, Konstantinos; Asimakopoulos, Byron



Correlation between human clusterin in seminal plasma with sperm protamine deficiency and DNA fragmentation.  


Seminal proteins can be considered as factors that control fertilization. Clusterin is one such protein that has been implicated in many activities, including apoptosis inhibition, cell cycle control, DNA repair, and sperm maturation. In this study, the relationship between human secretory clusterin (sCLU) in seminal plasma with sperm parameters, protamine deficiency, and DNA fragmentation was investigated. Semen samples were collected from 63 Iranian men, and semen analysis was performed according to World Health Organization criteria and computer aided semen analysis (CASA). The concentration of sCLU in seminal plasma was measured by enzyme-linked immunosorbant assay (ELISA), protamine deficiency was determined by chromomycin A3 staining (CMA3 ), and sperm DNA fragmentation was checked by sperm chromatin dispersion (SCD) assay. The level of sCLU in seminal fluid of fertile patients was 48.3 ± 38.6 ng/ml and in infertile patients was 15.5 ± 9.7 ng/ml; this difference was significant (P < 0.001). sCLU correlated negatively with protamine deficiency, sperm DNA fragmentation, and abnormal morphology. In conclusion, seminal clusterin can be considered as a marker for the quick assessment of semen quality in male infertility studies. PMID:23740886

Salehi, Mohammad; Akbari, Hakimeh; Heidari, Mohammad Hassan; Molouki, Aidin; Murulitharan, Kavitha; Moeini, Hassan; Novin, Marefat Ghaffari; Aabed, Farhang; Taheri, Hossein; Fadaei, Fateme; Mohsenzadeh, Mehdi; Jafari, Mohammad; Pirouzi, Aliyar; Heidari, Reihane



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


This study investigated meiotic segregation in spermatozoa to determine if severe teratozoospermia should prevent the use of intracytoplasmic sperm injection (ICSI) because of the high production of gametes with chromosomal aneuploidies and analysed DNA fragmentation in gametes from the same semen to determine if DNA integrity was worse in patients with severe teratozoospermia. Sperm samples from 12 infertile patients were studied by fluorescence in-situ hybridization for chromosomes X, Y, 13, 18 and 21 and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling. Four patients with a majority of macrocephalic forms with multiple flagella had more than 99% spermatozoa with abnormal chromosomal content. The other patients (globozoospermia or other abnormalities concerning sperm heads) had no increased aneuploidy or a slightly significant increase (P<0.05). The rate of DNA fragmentation was significantly higher in infertile patients than in the controls (P<0.001; 14.3% versus 1.20%, respectively) but presented important variability. Therefore, ICSI should not be attempted if men have macrocephalic gametes with multiple flagella but morphology is not always a good predictor of chromosomal content, depending upon the kind of teratozoospermia. Evaluation of the rate of aneuploidy and DNA fragmentation in gametes of patients with severe teratozoospermia is recommended. PMID:21233018

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



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

PubMed Central

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

Birone, Claire; Brown, Maria; Hernandez, Jesus; Neff, Sherry; Williams, Daniel; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.



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

SciTech Connect

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

Crawford, D.A.



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

SciTech Connect

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.

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



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


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

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



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

SciTech Connect

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

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



Isolation of specific DNA fragments of Mycobacterium avium and their possible use in diagnosis.  

PubMed Central

We cloned and sequenced two DNA fragments (DT1 and DT6) from Mycobacterium avium serotype 2 for use in the identification of members of the M. avium-M. intracellulare complex (MAC). Reference strains of MAC belonging to serovars 1 to 28 were examined by using these DNA fragments as probes. The study revealed that the DT6 probe hybridized with DNAs from M. avium strains (serovars 1 to 6, 8 to 11, and 21), while the DT1 probe hybridized with DNAs from serovars 2, 3, 7, 12 to 20, and 23 to 25. DT1- and DT6-derived oligonucleotides were selected for use as primers in a polymerase chain reaction test. Amplification of the DT1 and DT6 sequences may provide the basis for a rapid and reliable assay for the detection of mycobacteria belonging to MAC. Images PMID:8501206

Thierry, D; Vincent, V; Clement, F; Guesdon, J L



Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes  

Microsoft Academic Search

Bacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.

Tom Mattson; Griet Van Houwe; Antoinette Bolle; Gerald Selzer; Richard Epstein



Species relationships in the Hordeum murinum aggregate viewed from chloroplast DNA restriction fragment patterns.  


Three annual widespread species of Hordeum were investigated by the fragment pattern method on their chloroplast (cp) DNA. The species were H. glaucum, H. leporinum and H. murinum; H. vulgare was surveyed for comparison. Twelve restriction enzymes were used, nine recognizing 6 bp, one 5 bp and two 4 bp, thus, randomly surveyed, a total of 2,113 bp or 1.6% of the cp genome. Differences in patterns were found in three enzymes, HindIII, CfoI and MspI. CfoI characterizes H. glaucum from the other two species. HindIII and MspI revealed polymorphisms within species. These results confirm previous numerical taxonomic relationships among these three closely related species. Furthermore, cpDNA polymorphism in Hordeum is discussed in view of earlier reports on cpDNA polymorphism in H. vulgare. The taxonomic implications of cpDNA polymorphism are discussed after reviewing several articles using the fragment pattern method on cpDNA. The importance of using material from several populations representative of a species is stressed. PMID:24227234

Baum, B R; Bailey, L G



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

PubMed Central

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

Lima, Fernanda B.; Bethea, Cynthia L.



The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress.  


The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair. PMID:24107631

Kim, Ekaterina R; Selyutina, Anastasia A; Buldakov, Ilya A; Evdokimova, Valentina; Ovchinnikov, Lev P; Sorokin, Alexey V



The proteolytic YB-1 fragment interacts with DNA repair machinery and enhances survival during DNA damaging stress  

PubMed Central

The Y-box binding protein 1 (YB-1) is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA and RNA-dependent events is determined by its localization in the cell. We have shown previously that YB-1 is cleaved by 20S proteasome between E219 and G220, and the truncated N-terminal YB-1 fragment accumulates in the nuclei of cells treated with DNA damaging drugs. We proposed that appearance of truncated YB-1 in the nucleus may predict multiple drug resistance. Here, we compared functional activities of the full-length and truncated YB-1 proteins and showed that the truncated form was more efficient in protecting cells against doxorubicin treatment. Both forms of YB-1 induced changes in expression of various genes without affecting those responsible for drug resistance. Interestingly, although YB-1 cleavage did not significantly affect its DNA binding properties, truncated YB-1 was detected in complexes with Mre11 and Rad50 under genotoxic stress conditions. We conclude that both full-length and truncated YB-1 are capable of protecting cells against DNA damaging agents, and the truncated form may have an additional function in DNA repair. PMID:24107631

Kim, Ekaterina R; Selyutina, Anastasia A; Buldakov, Ilya A; Evdokimova, Valentina; Ovchinnikov, Lev P; Sorokin, Alexey V



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

SciTech Connect

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.

Tee, Thiam-Tsui, E-mail: [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)



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


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

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



Long-fragment DNA as a potential marker for stool-based detection of colorectal cancer  

PubMed Central

Neoplastic cells that are exfoliated from the colorectal epithelium exhibit dysfunctional apoptotic mechanisms, and thus it is possible to identify high-molecular-weight DNA fragments (long DNA) in feces. In the present study, the sensitivity and specificity of fecal-based long DNA assays were evaluated for the detection of colorectal cancer (CRC). Feces were collected from 54 healthy volunteers and 130 patients with CRC prior to surgical treatment. The presence of long DNA of the adenomatosis polyposis coli, Kirsten rat sarcoma viral oncogene homolog (KRAS), B-raf proto-oncogene, serine/threonine kinase and p53 genes was assessed by polymerase chain reaction followed by electrophoresis. The identification of long DNA in feces was found to exhibit a sensitivity of 56.2% and specificity of 96.3% for CRC detection. In addition, long DNA was identified in the feces of 58/90 (64.4%) patients with distal CRC and 15/40 (37.5%) patients with proximal CRC. This study indicates the potential of the fecal long DNA assay as a non-invasive and easily performed method for the detection of individuals with CRC.




Characterization of DNA-Hv1 histone interactions; discrimination of DNA size and shape.  


We have studied the formation of histone Hv1-DNA complexes using an acoustic biosensor and AFM imaging. Our results show that DNA and histone molecules aggregate into amorphous accumulations which form a compact rigid layer on the sensor's surface. By measuring changes in the acoustic wave amplitude, it was possible to titrate surface bound DNA with Hv1 and discriminate between DNA molecules of different size and shape. From the kinetic analysis of real time data, Keq was found equal to 3x10(5) M(-1). PMID:20085758

Papadakis, George; Tsortos, Achilleas; Mitsakakis, Konstantinos; Gizeli, Electra



Tracing a phase transition with fluctuations of the largest fragment size: Statistical multifragmentation models and the ALADIN S254 data  

Microsoft Academic Search

A phase transition signature associated with cumulants of the largest fragment size distribution has been identified in statistical multifragmentation models and examined in analysis of the ALADIN S254 data on fragmentation of neutron-poor and neutron-rich projectiles. Characteristics of the transition point indicated by this signature are weakly dependent on the A\\/Z ratio of the fragmenting spectator source. In particular, chemical

T. Pietrzak; P. Adrich; T. Aumann; C. O. Bacri; T. Barczyk; R. Bassini; S. Bianchin; C. Boiano; A. S. Botvina; A. Boudard; J. Brzychczyk; A. Chbihi; J. Cibor; B. Czech; M. De Napoli; J.-E. Ducret; H. Emling; J. D. Frankland; M. Hellstrom; D. Henzlova; G. Imme; I. Iori; H. Johansson; K. Kezzar; A. Lafriakh; A. Le Fevre; E. Le Gentil; Y. Leifels; J. Luhning; J. Lukasik; W. G. Lynch; U. Lynen; Z. Majka; M. Mocko; W. F. J. Muller; A. Mykulyak; H. Orth; A. N. Otte; R. Palit; P. Pawlowski; A. Pullia; G. Raciti; E. Rapisarda; H. Sann; C. Schwarz; C. Sfienti; H. Simon; K. Summerer; W. Trautmann; M. B. Tsang; G. Verde; C. Volant; M. Wallace; H. Weick; J. Wiechula; A. Wieloch; B. Zwieglinski



DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid.  


Five chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the same Comet assay, were obtained after a 20-h exposure to the following subtoxic concentrations of the five test compounds: methimazole from 2.5 to 10mM; nitrobenzene, potassium bromate, N,N'-diethylthiourea and ethylenethiourea from 1.25 to 5mM. Under the same experimental conditions, DNA repair synthesis, as evaluated by quantitative autoradiography, was present in potassium bromate-exposed thyroid cells from all the three donors and in those from two of three donors with either nitrobenzene or ethylenethiourea, but did not match the criteria for a positive response in thyroid cells from any of the donors with methimazole and N,N'-diethylthiourea. Consistently with their ability to induce thyroid tumors, all the five test compounds, administered p.o. in rats in a single dose corresponding to 1/2 LD50, induced a statistically significant degree of DNA fragmentation in the thyroid. These findings suggest that the five test compounds might be carcinogenic to thyroid in humans. PMID:16942904

Mattioli, Francesca; Martelli, Antonietta; Gosmar, Marzia; Garbero, Claudia; Manfredi, Valeria; Varaldo, Emanuela; Torre, Gian Carlo; Brambilla, Giovanni



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

SciTech Connect

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.

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:; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)



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


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

Deaville, Eddie R; Maddison, Ben C



Pyrrolamide DNA gyrase inhibitors: fragment-based nuclear magnetic resonance screening to identify antibacterial agents.  


DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 ?M. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications. PMID:22183167

Eakin, Ann E; Green, Oluyinka; Hales, Neil; Walkup, Grant K; Bist, Shanta; Singh, Alok; Mullen, George; Bryant, Joanna; Embrey, Kevin; Gao, Ning; Breeze, Alex; Timms, Dave; Andrews, Beth; Uria-Nickelsen, Maria; Demeritt, Julie; Loch, James T; Hull, Ken; Blodgett, April; Illingworth, Ruth N; Prince, Bryan; Boriack-Sjodin, P Ann; Hauck, Sheila; MacPherson, Lawrence J; Ni, Haihong; Sherer, Brian



Prediction of fragment size and ejection distance of masonry wall under blast load using homogenized masonry material properties  

Microsoft Academic Search

The fragment hazard resulting from a nearby explosion is a major concern in the design of structures which may be subjected to blast loads. This paper presents a predictive method based on the theories of continuum damage mechanics and mechanics of micro-crack development, and numerical simulation to determine the probabilistic fragment size distribution and the launch distances. Theoretical derivations are

Ming Wang; Hong Hao; Yang Ding; Zhong-Xian Li



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

SciTech Connect

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

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso (NWU)



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

Microsoft Academic Search

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-

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



Rapid purification of truncated Taq DNA polymerase Stoffel fragment by boiling lysis of bacterial expression cultures.  


A simplified and low-cost method, boiling lysis, was developed and used to purify the truncated DNA polymerase Stoffel fragment after overproduction of the enzyme in Escherichia coli. The method is based on the thermostable properties of the Stoffel fragment. Conditions such as the boiling time and number of cycles employed were identified as determinants with which to achieve a high yield and activity of this truncated Taq enzyme. Under established conditions, a period of 8 min consisting of two cycles of boiling and centrifuging was ideal for the purification of the Taq protein, followed by dibasic phosphate and ethanol extraction in a single type of storage buffer to remove contaminating nucleic acid. The whole purification process could be achieved within 1 or 2 h. A total yield of 80 mg of truncated Taq enzyme with an activity of 1.6x10(7) units was purified from l litre of bacterial culture. The purified Taq enzyme had a molecular mass of 61 kDa, a value consistent with the truncated DNA polymerase Stoffel fragment reported previously. PMID:18184110

Yang, Zhengan; Ding, Yumei; Zhang, Yinghua; Liu, Feihu



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

NASA Astrophysics Data System (ADS)

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

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



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

PubMed Central

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

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



DNA fragment encoding human IL-1beta 163-171 peptide enhances the immune responses elicited in mice by DNA vaccine against foot-and-mouth disease.  


DNA vaccine has been tested for protection against foot-and-mouth disease. However, the relatively low efficacy of DNA vaccine in inducing immune responses in large animals has restricted its practical use. Interleukin-1 plays an essential role in amplifying both the cellular and humoral immune responses to foreign antigens, and may therefore represent a good candidate as an adjuvant of DNA vaccines. Since the inflammatory activity of IL-I may restrict its application in DNA vaccine treatment, we explored the possibilities of augmenting immune responses without unwanted inflammatory effects using the IL-1beta fragment (amino acids (aa) 163-171), which is essential for IL-1 receptor-1 binding. The DNA fragment encoding the human IL-1beta fragment (aa 163-171) was fused to foot-and-mouth disease virus (FMDV) DNA vaccine, and injected into mice to analyse its immune response. Compared with control mice receiving FMDV DNA vaccine alone, significant increases in the FMDV-specific antibody response and also in T cell proliferation were observed in mice receiving IL-1beta (163-171)-FMDV. These results suggested that DNA fragment encoding IL-1beta 163-171 peptide might represent a good candidate for an adjuvant of FMDV DNA vaccine. PMID:15727290

Shao, H J; Chen, L; Su, Y B



Mitochondrial DNA size diversity in the Dekkera\\/Brettanomyces yeasts  

Microsoft Academic Search

Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera\\/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis\\/B. lambicus; B. abstinens\\/B. custersii and B. anomalus\\/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp

C. R. McArthur; G. D. Clark-Walker



Evaluation of DNA fragmentation in llama (Lama glama) sperm using the sperm chromatin dispersion test.  


The integrity of sperm chromatin is now viewed as an important factor in male fertility and in early embryonic development. The objectives of this study were: (1) adapt the simple and inexpensive sperm chromatin dispersion (SCD) test to evaluate DNA fragmentation in llama sperm and establish the halo patterns observed in this species, (2) determine an effective and reliable positive control for this technique and (3) evaluate correlation between the SCD test and the toluidine blue (TB) stain. To adapt the SCD test, three different mercaptoethanol (ME) concentrations were assayed (2.5%, 5% and 10% ME). To determine an effective positive control, three treatments (incubation at 100 °C for 30 min, incubation with 0.3 M NaOH for 30 min at room temperature and exposure to UV light for 2h) were assayed. The concentration selected to use in the SCD test was 5% ME, because it produced the largest halo while still conserving the structure of the core. Four DNA dispersion patterns were clearly observed: (I) nuclei with large DNA dispersion halos; (II) nuclei with medium halos; (III) nuclei with very small halos and (IV) nuclei with no halo. All treatments used as positive controls were effective in producing DNA fragmentation. A high correlation (r=0.84, P=0.03) was observed between spermatozoa without halos and TB positive cells. To conclude, SCD patterns in llama sperm have been established as well as a repeatable positive control for the assay. The SCD test and TB stain are simple and inexpensive techniques that can be used to evaluate DNA damage in llama sperm. PMID:22437148

Carretero, M I; Lombardo, D; Arraztoa, C C; Giuliano, S M; Gambarotta, M C; Neild, D M



Size-related auto-fragment production and carbohydrate storage in auto-fragment of Myriophyllum spicatum L. in response to sediment nutrient and plant density  

Microsoft Academic Search

Size-related asexual reproduction of submersed macrophytes is still poorly understood. Here, we investigate how size-related\\u000a auto-fragmentation in Myriophyllum spicatum L. responds to sediment nutrients and plant density. An experiment was carried out with sediments containing two different\\u000a nutrient levels and with two levels of plant density. The results show that sediment nutrients and plant density brought about\\u000a a strong dependency

Dong Xie; Dan Yu



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

PubMed Central

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

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



Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)  

PubMed Central

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

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



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

NASA Astrophysics Data System (ADS)

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.

Kiviniemi, Katariina



Detection of a mouse OGG1 fragment during caspase-dependent apoptosis: oxidative DNA damage and apoptosis.  


We investigated the expression of mouse 8-oxoguanine DNA glycosylase 1 (mOGG1) in mouse non-parenchymal hepatocytes (NCTC) during etoposide- or mitomycin C (MMC)-induced apoptosis. We observed mOGG1 fragmentation in apoptotic cells. The apoptosis accompanying the fragmentation of mOGG1 was caspase-dependent. The mOGG1 fragment existed in both the cytoplasm and nucleus of the etoposide-treated NCTC, indicating that the mOGG1 fragment could be transferred into the nucleus. In addition, 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) accumulated in the DNA of NCTC treated with etoposide, suggesting that the mOGG1 fragment might not function as a normal repair enzyme in etoposide-treated NCTC. Although we have not clarified in detail the mechanism and the significance of the mOGG1 fragmentation, further study of the fragmentation of DNA repair enzymes might provide insights into the relationship between oxidative DNA damage and apoptosis. PMID:15298724

Hirano, Takeshi; Kawai, Kazuaki; Ootsuyama, Yuko; Orimo, Hiroshi; Kasai, Hiroshi



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

SciTech Connect

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.

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



Exploring the Limits of DNA Size: Naphtho-homologated DNA Bases and Pairs  

PubMed Central

A new design for DNA bases and base pairs is described in which the pyrimidine bases are widened by naphtho-homologation. Two naphtho-homologated deoxyribosides, dyyT (1) and dyyC (2) were synthesized and could be incorporated into oligonucleotides as suitably protected phosphoramidite derivatives. The deoxyribosides were found to be fluorescent, with emission maxima at 446 and 433 nm, respectively. Studies with single substitutions of 1 and 2 in the natural DNA context revealed exceptionally strong base stacking propensity for both. Sequences containing multiple substitutions of 1 and 2 paired opposite adenine and guanine were subsequently mixed and studied by several analytical methods. Data from UV mixing experiments, FRET measurements, fluorescence quenching experiments, and hybridizations on beads suggest that complementary “doublewide DNA” (yyDNA) strands may self-assemble into helical complexes with 1:1 stoichiometry. Data from thermal denaturation plots and CD spectra were less conclusive. Control experiments in one sequence context gave evidence that yyDNA helices, if formed, are preferentially antiparallel and are sequence selective. Hypothesized base pairing schemes are analogous to Watson-Crick pairing, but with glycosidic C1?-C1? distances widened by over 45%, to ca. 15.2 Å. The possible self-assembly of the double-wide DNA helix establishes a new limit for the size of information-encoding, DNA-like molecules, and the fluorescence of yyDNA bases suggests uses as reporters in monomeric and oligomeric forms. PMID:16834396

Lee, Alex H. F.; Kool, Eric T.



Characterization of Drug Resistance-Associated Mutations in the Human Cytomegalovirus DNA Polymerase Gene by Using Recombinant Mutant Viruses Generated from Overlapping DNA Fragments  

Microsoft Academic Search

A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts,




Species-specific variation in efficacy of yeast genomic DNA isolation techniques assessed using amplified fragment length polymorphism  

Microsoft Academic Search

Methods for isolating genomic DNA from yeasts are optimized for strains of Saccharomyces cerevisiae. The DNeasy tissue kit proved to be effective with 65 additional yeast species, providing 0.1 to 4.7?g DNA\\/ml culture with sufficient purity to give reproducible amplified fragment length polymorphism (AFLP) profiles, but was unsuccessful with 13 other species. Two alternative yeast DNA purification kits, MasterPure and

Linda J. Fuller; Donald A. MacKenzie; Ian N. Roberts



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

E-print Network

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.

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



Restriction fragment primed phi X174 single-stranded DNA as template for DNA polymerase alpha and beta. Detection and partial purification of a polymerase alpha stimulating factor.  


Template-primers constructed of phiX174 single-stranded viral DNA hybridized to a restriction fragment of phiX174 RF DNA can be used for extensive polymerization by DNA polymerase alpha. Polymerization is dependent upon a restriction fragment containing a 3'OH. The products of the reaction have been identified by agarose gel electrophoresis. Polymerization of 150--400 nucleotides can be obtained in 1h depending upon the restriction fragment used as primer. Synthesis may be limited by barriers in the primary or secondary structure of the template. A factor which stimulates the rate of alpha polymerase activity on these templates was partially purified. This factor does not stimulate alpha polymerase on activated DNA. The stimulating factor sediments at 5.5 S in glycerol gradients containing 0.4M potassium phosphate and has an apparent molecular weight of 70 000 on Sephadex G-100. PMID:6250616

Burke, J F; Plummer, J; Huberman, A J; Evans, M J



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


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

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



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

PubMed Central

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

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



Bulky DNA adducts in human sperm associated with semen parameters and sperm DNA fragmentation in infertile men: a cross-sectional study  

PubMed Central

Background DNA adducts are widely used marker of DNA damage induced by environmental pollutants. The present study was designed to explore whether sperm polycyclic aromatic hydrocarbon-DNA adducts were associated with sperm DNA integrity and semen quality. Methods A total of 433 Han Chinese men were recruited from an infertility clinic. Immunofluorescence was applied to analyze sperm PAH-DNA adducts. Sperm DNA fragmentation was detected by terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay. Results After adjustment for potential confounders using linear regression, sperm PAH-DNA adducts were negatively associated with sperm concentration, total sperm count, sperm motility, and curvilinear velocity (VCL). In addition, a positive relationship between sperm PAH-DNA adducts and sperm DNA fragmentation was found. Conclusions Our findings suggested an inverse association between sperm PAH-DNA adducts and semen quality, and provided the first epidemiologic evidence of an adverse effect of PAH-DNA adducts on sperm DNA integrity. PMID:24073787



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


This study aimed to assess the influence of smoking duration and intensity on sperm vitality, sperm DNA fragmentation, reactive oxygen species (ROS) and zinc (Zn) levels in oligoasthenoteratozoospermic (OAT) men with varicocele (Vx). A total of 246 men were investigated who were divided into OAT nonsmokers, OAT smokers, OAT nonsmokers and OAT smokers with Vx. They were subjected to history taking, clinical examination and semen analysis. In their semen, sperm hypo-osmotic swelling (HOS) test, sperm DNA fragmentation test, seminal ROS and seminal Zn were assessed. The results demonstrated significantly decreased HOS test, seminal Zn level and significantly increased sperm DNA fragmentation, seminal ROS levels in OAT smokers with Vx more than OAT smokers compared with OAT nonsmokers. Smoking intensity, smoking duration and Vx grade demonstrated significant negative correlations with sperm motility, HOS test percentage and significant positive correlations with sperm DNA fragmentation, seminal ROS level. It is concluded that smoking has a negative impact on sperm progressive motility, HOS test, seminal Zn and positive impact on sperm DNA fragmentation, semen ROS level that are exaggerated if Vx is associated being correlated with smoking intensity, smoking duration and Vx grade. PMID:23866014

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



Fracture of Brittle Solids. IV. Two-Dimensional Distribution Function for Fragment Size in Single Fracture (Theoretical)  

Microsoft Academic Search

Prior work in paper I on the size distribution of the fragments in single fracture of a three-dimensional solid is extended to the two-dimensional case (that of a thin plate). The underlying physical assumptions are the two-dimensional analogs of those made in I. These assumptions yield directly the probability d? (c,a) of formation of a fragment with perimeter and area

J. J. Gilvarry



Tracing a phase transition with fluctuations of the largest fragment size: Statistical multifragmentation models and the ALADIN S254 data  

E-print Network

A phase transition signature associated with cumulants of the largest fragment size distribution has been identified in statistical multifragmentation models and examined in analysis of the ALADIN S254 data on fragmentation of neutron-poor and neutron-rich projectiles. Characteristics of the transition point indicated by this signature are weakly dependent on the A/Z ratio of the fragmenting spectator source. In particular, chemical freeze-out temperatures are estimated within the range 5.9 to 6.5 MeV. The experimental results are well reproduced by the SMM model.

T. Pietrzak; P. Adrich; T. Aumann; C. O. Bacri; T. Barczyk; R. Bassini; S. Bianchin; C. Boiano; A. S. Botvina; A. Boudard; J. Brzychczyk; A. Chbihi; J. Cibor; B. Czech; M. De Napoli; J. -E. Ducret; H. Emling; J. D. Frankland; M. Hellstrom; D. Henzlova; G. Imme; I. Iori; H. Johansson; K. Kezzar; A. Lafriakh; A. Le Fevre; E. Le Gentil; Y. Leifels; J. Luhning; J. Lukasik; W. G. Lynch; U. Lynen; Z. Majka; M. Mocko; W. F. J. Muller; A. Mykulyak; H. Orth; A. N. Otte; R. Palit; P. Pawlowski; A. Pullia; G. Raciti; E. Rapisarda; H. Sann; C. Schwarz; C. Sfienti; H. Simon; K. Summerer; W. Trautmann; M. B. Tsang; G. Verde; C. Volant; M. Wallace; H. Weick; J. Wiechula; A. Wieloch; B. Zwieglinski



A multiple-staining procedure for the detection of different DNA fragments on a single blot.  


We have developed a method that implies the use of a particular type of substrate which can be used in combination with alkaline phosphatase in detecting nucleic acid on filters. The method allows the detection of several different nucleic acid sequences on a single filter. In consecutive steps, the target DNA molecules are hybridized with different digoxigenin-labeled DNA probes. After each hybridization step, digoxigenin is detected with an antibody-alkaline phosphatase conjugate. This enzyme is subsequently visualized by a color reaction using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS) phosphates as substrates in combination with varying diazonium salts. The multiple-staining procedure is based on the fact that the probe DNA-antibody complex can be removed while the color precipitate remains stably bound at its place on the filter. This allows several repeated hybridizations with other digoxigenin-labeled probes followed by antibody detection and color reaction with other naphthol AS phosphate-diazonium salt combinations. Aside from the ability to simultaneously visualize different target DNAs on a single filter, this new method provides several important features that are more powerful than the conventional 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium (BCIP-NBT) color reaction for alkaline phosphatase. The colors are more stable and brilliant than BCIP-NBT; their development is faster, the resolution of closely spaced bands is greater, and the background is much lower. The detection limit for alkaline phosphates is as good as with BCIP-NBT (0.1 pg of DNA). One major advantage is the simplicity of removing the colors by ethanol incubation. In this paper, the method is described using the example of Southern blotted DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1705397

West, S; Schröder, J; Kunz, W



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

USGS Publications Warehouse

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.

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



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

SciTech Connect

We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 {mu}g range) spread out over a large surface area (42 cm{sup 2}) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar{sup +} ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair.

Sellami, L.; Lacombe, S.; Hunting, D.; Wagner, R. J.; Huels, M. A. [Ion Reaction Laboratory, Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec J1H 5N4 (Canada)



Species-specific variation in efficacy of yeast genomic DNA isolation techniques assessed using amplified fragment length polymorphism.  


Methods for isolating genomic DNA from yeasts are optimized for strains of Saccharomyces cerevisiae. The DNeasy tissue kit proved to be effective with 65 additional yeast species, providing 0.1 to 4.7 microg DNA/ml culture with sufficient purity to give reproducible amplified fragment length polymorphism (AFLP) profiles, but was unsuccessful with 13 other species. Two alternative yeast DNA purification kits, MasterPure and Y-DER, were effective with 6 of these and 2 additional species, leaving only 9 species that remained recalcitrant to yielding sufficient amounts of DNA with the required purity. PMID:18601896

Fuller, Linda J; MacKenzie, Donald A; Roberts, Ian N



Bioactive bead-mediated transformation of plants with large DNA fragments.  


The efficient transformation of plants with large DNA molecules containing a set of useful genes would provide vast possibilities for the genetic improvement of agricultural as well as nonagricultural plants. The development of the bioactive beads (BABs) transformation method has proven useful for introduction of large DNA molecules into plant cells. In this chapter, the BABs transformation method used for the transformation of a 100-kb BAC DNA construct containing wheat genes into rice will be presented. Furthermore, the improved production method for BABs will be described. With the conventional method for producing BABs, the bead size varies, and the larger beads tend to carry fewer DNA molecules than the smaller beads. Thus, in order to facilitate the preparation of BABs with more uniform sizes, a simple set-up -composed of a sine wave sound generator and microsyringe pump was fabricated. Using this bead-maker set-up, uniform and smaller beads could be produced which enhance the transformation efficiency. PMID:22351002

Wada, Naoki; Cartagena, Joyce A; Khemkladngoen, Naruemon; Fukui, Kiichi



Sequence-based analysis of enzymatically amplified DNA fragments by mutation detection techniques.  


The accurate analysis of molecular variation is important in a range of disciplines of parasitology. Although conventional DNA techniques have overcome some of the limitations of traditional approaches, some can be relatively expensive and/or cumbersome to use when large sample sizes require analysis, and some cannot accurately resolve or define nucleotide variation. Using selected examples of applications to parasites, Robin Gasser and Xingquan Zhu discuss some PCR-based mutation detection techniques and their advantages over conventional analytical methods. PMID:10511690

Gasser, R B; Zhu, X Q



Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus  

Microsoft Academic Search

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments

Susan M. Elsevier; Hans Joachim Lipps; Günther Steinbrück



Characterization of HIFU ablation using DNA fragmentation labeling as apoptosis stain  

NASA Astrophysics Data System (ADS)

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.

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



Storing DNA, 2D animationSite: DNA Interactive (  

NSDL National Science Digital Library

To store DNA, you need unusual storage containers. Organisms such as bacteria and viruses were the Human Genome Project's unconventional libraries and duplicating systems. "DNA libraries" give researchers a way to store and access genes of interest. These libraries consist of large numbers of DNA vectors each containing a different DNA fragment. Different vectors are used for DNA fragments of different sizes.



Cloned single- and double-stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co-inoculated subgenomic DNA fragments are infectious.  

PubMed Central

A set of monomeric and oligomeric potato spindle tuber viroid (PSTV) specific DNA forms representing complete DNA copies of the circular PSTV RNA genome were constructed and cloned in plasmid pBR322 and bacteriophage M13. Both single- and double-stranded PSTV DNAs are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the RNA-RNA pathway without DNA being involved. All dimeric and higher multimeric forms were infectious irrespective of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA. The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised. Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious. These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses. Images Fig. 2. Fig. 3. PMID:6549294

Tabler, M; Sänger, H L



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

PubMed Central

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



Cloning and expression of small cDNA fragment encoding strong antiviral peptide from Celosia cristata in Escherichia coli.  


A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714. PMID:16266271

Gholizadeh, A; Kohnehrouz, B Baghban; Santha, I M; Lodha, M L; Kapoor, H C



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


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

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



Microfabricated Devices for Sizing DNA and Sorting Cells Hou-Pu Choua  

E-print Network

to its speed and low material requirement. One possible application is DNA fingerprinting. A portableMicrofabricated Devices for Sizing DNA and Sorting Cells Hou-Pu Choua , Charles Spenceb , Axel and sort microscopic biological objects, ranging from cells to single molecules of DNA. Sizing

Quake, Stephen R.


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

Microsoft Academic Search

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

Katariina Kiviniemi



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

PubMed Central

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

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



Isolation and Characterization of a Species-Specific DNA Fragment for Identification of Candida (Torulopsis) glabrata by PCR  

PubMed Central

A PCR specific for Candida glabrata that amplifies a mitochondrial rRNA gene fragment was developed by analysis of C. glabrata-specific agarose gel bands, which were generated by arbitrarily primed PCR. The expected PCR product was successfully amplified with genomic DNA from 95 C. glabrata isolates but not from a number of other fungal isolates. PMID:11526177

Becker, Karsten; Badehorn, Dorte; Keller, Birgit; Schulte, Martina; Bohm, Karl Heinz; Peters, Georg; Fegeler, Wolfgang



Radiation-induced double strand breaks and subsequent apoptotic DNA fragmentation in human peripheral blood mononuclear cells.  


In case of accidental radiation exposure or a nuclear incident, physical dosimetry is not always complete. Therefore, it is important to develop tools that allow dose estimates and determination that are based on biological markers of radiation exposure. Exposure to ionizing radiation triggers a large-scale activation of specific DNA signaling and repair mechanisms. This includes the phosphorylation of ?H2AX in the vicinity of a double-strand break (DSB). A DNA DSB is a cytotoxic form of DNA damage, and if not correctly repaired can initiate genomic instability, chromosome aberrations, mutations or apoptosis. Measurements of DNA DSBs and their subsequent repair after in vitro irradiation has been suggested to be of potential use to monitor cellular responses. The bone marrow and the blood are known to be the most radiosensitive tissues of the human body and can therefore be of particular importance to find radiation-induced biological markers. In the present study, changes in H2AX phosphorylation and apoptosis of irradiated human peripheral blood mononuclear cells (PBMCs) were analyzed. Freshly isolated PBMCs from healthy donors were irradiated with X-rays (0.1, 0.25, 0.5, 1, 2 and 4 Gy). The phosphorylation of ?H2AX was measured at different time points (0, 0.25, 1, 2, 4, 6 and 24 h) after irradiation. We detected a linear dose-dependency of ?H2AX phosphorylation measured by ?H2AX foci scoring using immunofluorescence microscopy as well as by ?H2AX fluorescence detection using flow cytometry. Apoptosis was detected by measuring DNA fragmentation at different time points (0, 24, 48, 72, 96 h) after X-irradiation using DNA ladder gel electrophoresis. The apoptotic DNA fragmentation increased in a dose-dependent manner. In conclusion, DNA DSBs and subsequent apoptotic DNA fragmentation monitoring have potential as biomarkers for assessing human exposure in radiation biodosimetry. PMID:22322361

Ghardi, Myriam; Moreels, Marjan; Chatelain, Bernard; Chatelain, Christian; Baatout, Sarah



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

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

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



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

NASA Astrophysics Data System (ADS)

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

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



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


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

Muriel, Sandra B; Kattan, Gustavo H



Selection of normal spermatozoa with a vacuole-free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates.  


Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole-free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n = 8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole-free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1 ± 1.1%, n = 191) than all other types of spermatozoa: non-selected spermatozoa (n = 8000; 26.1 ± 1.5% versus 4.1 ± 1.1%; P < 0.005), motile spermatozoa (n = 444; 20.8 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and motile, normal spermatozoa selected at 200× magnification (n = 370; 18.7 ± 2.7% versus 4.1 ± 1.1%; P < 0.001) and then motile, morphometrically normal spermatozoa with anterior vacuoles (n = 368; 15.9 ± 2.9% versus 4.1 ± 1.1%; P < 0.05) or posterior vacuoles (n = 402; 22.5 ± 3.6% versus 4.1 ± 1.1%; P < 0.001) selected at 6300× magnification. For patients with high sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole-free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non-fragmented DNA. PMID:22731614

Hammoud, I; Boitrelle, F; Ferfouri, F; Vialard, F; Bergere, M; Wainer, B; Bailly, M; Albert, M; Selva, J



Enhancement of immune responses to the hepatitis B virus core protein through DNA vaccines with a DNA fragment encoding human IL-1beta 163-171 peptide.  


DNA vaccines have been widely used as effective means of eradicating a variety of viruses, parasites, bacteria as well as means of alleviating allergic and autoimmune diseases and tumors. As interleukin 1 (IL-1) plays an essential role in augmenting both cellular and humoral immune responses to foreign antigens, it may represent a good candidate for an adjuvant to DNA vaccines. Since the inflammatory activity of IL-1 may have a restricted application to DNA vaccines, we explored the possibility of augmenting immune response without unwanted inflammatory effect using IL-1beta 163-171 peptide, which is essential for IL-1 receptor 1 binding. A DNA fragment encoding the human IL-1beta 163-171 peptide of concern was fused to the Hepatitis B virus (HBV) core DNA vaccine, and injected into mice to analyze its immune responses. Compared with the control mice which received hepatitis B virus core antigen (HBcAg) alone, significant increase in not only the HBcAg-specific antibody response but also in T cell proliferation was observed in mice which received IL-1beta 163-171-HBcAg. These results suggest that the DNA fragment encoding the IL-1beta polypeptide of aa 163-171 might represent a good candidate for an adjuvant of DNA vaccines. PMID:15068376

Shao, H J; Chen, L; Shen, M S; Yu, G F



Applications of mass spectrometry to DNA fingerprinting and DNA sequencing.  

National Technical Information Service (NTIS)

DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and ...

K. B. Jacobson, M. V. Buchanan, C. H. Chen, M. J. Doktycz, S. A. McLuckey



DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line  

PubMed Central

Objectives: Apoptosis, an important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus (Saffron) in different cancer types. However, limited effort has been made to correlate these effects to the active ingredients of saffron. The present study was designed to elucidate cytotoxic and apoptosis induction by safranal, the major coloring compound in saffron, in a human prostate cancer cell line (PC-3). Materials and Methods: PC-3 and human fetal lung fibroblast (MRC-5) cells were cultured and exposed to safranal (5, 10, 15, and 20 ?g/ml). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cytotoxicity. DNA fragmentation was assessed by gel electrophoresis. Cells were incubated with different concentrations of safranal, and cell morphologic changes and apoptosis were determined by the normal inverted microscope, Annexin V, and propidium iodide, followed by flow cytometric analysis, respectively. Results: MTT assay revealed a remarkable and concentration-dependent cytotoxic effect of safranal on PC-3 cells in comparison with non-malignant cell line. The morphologic alterations of the cells confirmed the MTT results. The IC50 values against PC-3 cells were found to be 13.0 ? 0.07 and 6.4 ? 0.09 ?g/ml at 48 and 72 h, respectively. Safranal induced an early and late apoptosis in the flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. DNA analysis revealed typical ladders as early as 48 and 72 h after treatment, indicative of apoptosis. Conclusions: Our preclinical study demonstrated a prostate cancer cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not clearly understood, it appears to have potential as a therapeutic agent. PMID:24082436

Samarghandian, Saeed; Shabestari, Mahmoud M



A versatile bacterial expression vector designed for single-step cloning of multiple DNA fragments using homologous recombination.  


Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, we have developed an efficient cloning strategy based on yeast homologous recombination cloning (YHRC) into the new pET-based bacterial expression vector pSUMO-YHRC. The vector supports cloning for untagged expression as well as fusions to His6-SUMO or His6 tags. We demonstrate that YHRC from single PCR products of 6 independent genes into the vector results in virtually no background. Importantly, in a quantitative assay for functional expression we find that single-step YHRC of 7 DNA fragments can be performed with very high cloning efficiencies. The method and reagents described in this paper significantly simplifies the construction of expression plasmids from multiple DNA fragments, including complex gene fusions, chimeric genes and polycistronic constructs. PMID:24631626

Holmberg, Mats A; Gowda, Naveen Kumar Chandappa; Andréasson, Claes



Analysis of Sequence Variations in Several Human Genes Using Phosphoramidite Bond DNA Fragmentation and Chip-Based MALDI-TOF  

PubMed Central

The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5? phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3?UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples. PMID:14707175

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



Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion.  


Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype-phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction-dependent destabilization of the underlying double helix, and a self-structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self-structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size-dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1-12, 2014. PMID:23494673

Völker, Jens; Plum, G Eric; Gindikin, Vera; Klump, Horst H; Breslauer, Kenneth J



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

PubMed Central

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

Lopez, Gemma; Lafuente, Rafael; Checa, Miguel A; Carreras, Ramon; Brassesco, Mario



Efficiency of genomic DNA extraction dependent on the size of magnetic nanoclusters  

NASA Astrophysics Data System (ADS)

We report the efficiency of genomic DNA extraction as a function of particle size and quantity. For DNA extraction, we synthesized magnetic nanoclusters of various sizes and coated the surface of these magnetic nanoclusters with meso-2,3-dimercaptosuccinic acid. We showed that the nanoclusters had a tight particle size distribution and high crystallinity. Furthermore, we observed that the three types of magnetic nanoclusters studied exhibited ferrimagnetic behavior and that larger nanoclusters showed larger saturation magnetization values. The resultant efficiency of DNA extraction is inversely proportional to particle size in the range of nanoclusters tested, due to the fact that the surface-to-volume ratio decreases as particle size increases.

Cho, Hyun Ah; Hyun Min, Ji; Hua Wu, Jun; Woo Jang, Jin; Lim, Chae-Seung; Keun Kim, Young



Chromosome size and DNA content of species of anemone L. and related genera (Ranunculaceae)  

Microsoft Academic Search

Relative amounts of DNA were determined by Feulgen cytophotometry in 22 diploid species of Ranunculaceae (n=7, 8, 9) representing six genera, and exhibiting large differences in chromosome size, but no marked differences in karyotype pattern. Chemical determination of absolute amounts of DNA for six of these species, allowed conversion of all the photometric data into absolute units of DNA. The

Klaus Rothfels; Elizabeth Sexsmith; Margaret Heimburger; Margarida O. Krause



Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments.  

PubMed Central

Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions. Images PMID:1531871

Holzmayer, T A; Pestov, D G; Roninson, I B



Potential utility of DNA sequence analysis of long-term-stored plant leaf fragments for forensic discrimination and identification.  


This study examined the potential utility of DNA sequence analysis to discriminate and identify plant material in forensic investigations. DNA was extracted from plant leaf fragments of 11 species stored for 5 to 22 years after collection. The trnH-psbA intergenic spacer and 316 bp of the rbcL gene were successfully amplified and sequenced for all fragments except for the trnH-psbA spacer of one sample. All of the plant samples were discriminated in pairwise comparisons of the sequences. Using a combination of local and global genetic databases is likely to provide greater reliability in search results to identify forensic samples from sequence data. PMID:20702948

Kikkawa, Hitomi S; Sugita, Ritsuko; Matsuki, Rikyu; Suzuki, Shinichi



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

SciTech Connect

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.

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:



Structure functions of rod-like DNA fragment and polystyrenesulfonate solutions in the modified Poisson-Boltzmann theory  

Microsoft Academic Search

The partial structure functions of aqueous solutions of rod-like DNA fragments and polystyrenesulfonic acid are calculated in the modified Poisson-Boltzmann theory. The cylindrical cell model appropriate for linear polyelectrolyte solutions with monovalent counterions and without any added salt is utilized. The predicted results are compared with the corresponding results from the classical Poisson-Boltzmann theory, and experimental small angle neutron scattering

L. B. Bhuiyan; C. W. Outhwaite; J. R. C. van der Maarel



Ebselen Reduces Cytochrome c Release From Mitochondria and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice  

Microsoft Academic Search

Background and Purpose—The seleno-organic compound ebselen has both antioxidant and anti-inflammatory properties. Although ebselen has been shown to protect the brain against stroke, it is unclear how ebselen provides neuroprotection. In the present study the authors examined whether ebselen inhibits neuronal apoptosis resulting from transient focal cerebral ischemia in mice. The cytochrome c release and DNA fragmentation, both of which

Hiroyuki Masayasu; Haruhiko Kikuchi


Amplified fragment length polymorphisms as a tool for DNA fingerprinting sunflower germplasm: genetic diversity among oilseed inbred lines  

Microsoft Academic Search

Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints. The\\u000a AFLP diversity of sunflower has not been described, and much of the public germ plasm of sunflower has not yet been fingerprinted.\\u000a Our objectives were to: (1) estimate genetic similarities, polymorphism rates, and polymorphic information contents (PICs)\\u000a for AFLP markers among elite public

Vipa Hongtrakul; Gordon M. Huestis; Steven J. Knapp



Fracture of Brittle Solids. III. Experimental Results on the Distribution of Fragment Size in Single Fracture  

Microsoft Academic Search

In papers I and II of this series, it was concluded on the basis of theory and a limited number of experimental samples that the over-all differential probability p? of single fracture, as a function of the mean fragment dimension x, should show the presence of three peaks. These correspond to the effect of internal flaws of facial and volume

J. J. Gilvarry; B. H. Bergstrom



Is thioredoxin reductase involved in the defense against DNA fragmentation in varicocele?  

PubMed Central

We aimed to investigate the role of thioredoxin reductase (TR) and inducible heat shock protein 70 (iHsp70) and their relationship with sperm quality in varicocele (VAR) patients. Semen samples were obtained from 16 subfertile men diagnosed as VAR and 10 fertile men who applied to the Andrology Laboratory of Istanbul Medical Faculty of Istanbul University. The sperm TR and iHsp 70 expression levels were determined using Western blot analysis. The TR activity of the sperm was assayed spectrophometrically. The sperm quality was evaluated both by conventional sperm analysis and by a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) technique that assayed DNA-fragmented spermatozoa in semen samples. The percentage of TUNEL-positive spermatozoa in the VAR group (16.3%±5.6%) was higher than that in the fertile group (5.5%±1.9%). Significant inverse correlations were detected between the percentage of TUNEL-positive cells and both the concentration (r=?0.609; P=0.001) and motility (r=?0.550; P=0.004) of spermatozoa. Both the TR expression and activity were increased significantly in the VAR group (U=22.0; P=0.001 and U=33.5; P=0.012, respectively) as analyzed using the Mann–Whitney U Wilcoxon rank sum W test. Furthermore, significant positive correlations were found between TR expression and activity (r=0.406; P=0.040) and between TR expression and the percentage of TUNEL-positive cells (r=0.665; P=0.001). Sperm iHsp70 expression did not differ between the VAR and fertile groups. In conclusion, increased sperm TR expression might be a defense mechanism against apoptosis in the spermatozoa of men with VAR. PMID:23603921

Ozdemirler Erata, Gul; Kucukgergin, Canan; Aktan, Gulsan; Kadioglu, Ates; Uysal, Mujdat; Kocak-Toker, Necla



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


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

Cao, Huachuan; Wang, Yinsheng



DNA Detectives  

NSDL National Science Digital Library

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.

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



[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].  


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

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



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


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

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



Influence of Heteroanion and Ammonium Cation Size on the Composition and Gas-Phase Fragmentation of Polyoxovanadates  

SciTech Connect

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.

Johnson, Grant E.; Al Hasan, Naila M.; Laskin, Julia



Size and shape effects in point load tests of irregular rock fragments  

Microsoft Academic Search

Summary Point-load tests were performed on three hard rocks of the Lake Superior district, ironformation, metadiabase, and ophitic basalt. More than 500 irregular, mine-run fragments ranging in diameter up to about 250 mm were tested in the field, using a specially designed, semi-portable test rig. Results were analyzed by multiple regression techniques, seeking a “best” expression for the point-load strength

L. A. Panek; T. A. Fannon



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

E-print Network

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...

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



Molecular-Sized DNA or RNA Sequencing Machine

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.


Secondary Craters and the Size-Velocity Distribution of Ejected Fragments around Lunar Craters Measured Using LROC Images  

NASA Astrophysics Data System (ADS)

Title: Secondary Craters and the Size-Velocity Distribution of Ejected Fragments around Lunar Craters Measured Using LROC Images Authors: Kelsi N. Singer1, Bradley L. Jolliff1, and William B. McKinnon1 Affiliations: 1. Earth and Planetary Sciences, Washington University in St Louis, St. Louis, MO, United States. We report results from analyzing the size-velocity distribution (SVD) of secondary crater forming fragments from the 93 km diameter Copernicus impact. We measured the diameters of secondary craters and their distances from Copernicus using LROC Wide Angle Camera (WAC) and Narrow Angle Camera (NAC) image data. We then estimated the velocity and size of the ejecta fragment that formed each secondary crater from the range equation for a ballistic trajectory on a sphere and Schmidt-Holsapple scaling relations. Size scaling was carried out in the gravity regime for both non-porous and porous target material properties. We focus on the largest ejecta fragments (dfmax) at a given ejection velocity (?ej) and fit the upper envelope of the SVD using quantile regression to an equation of the form dfmax = A*?ej ^- ?. The velocity exponent, ?, describes how quickly fragment sizes fall off with increasing ejection velocity during crater excavation. For Copernicus, we measured 5800 secondary craters, at distances of up to 700 km (15 crater radii), corresponding to an ejecta fragment velocity of approximately 950 m/s. This mapping only includes secondary craters that are part of a radial chain or cluster. The two largest craters in chains near Copernicus that are likely to be secondaries are 6.4 and 5.2 km in diameter. We obtained a velocity exponent, ?, of 2.2 × 0.1 for a non-porous surface. This result is similar to Vickery's [1987, GRL 14] determination of ? = 1.9 × 0.2 for Copernicus using Lunar Orbiter IV data. The availability of WAC 100 m/pix global mosaics with illumination geometry optimized for morphology allows us to update and extend the work of Vickery [1986, Icarus 67, and 1987], who compared secondary crater SVDs for craters on the Moon, Mercury, and Mars. Additionally, meter-scale NAC images enable characterization of secondary crater morphologies and fields around much smaller primary craters than were previously investigated. Combined results from all previous studies of ejecta fragment SVDs from secondary crater fields show that ? ranges between approximately 1 and 3. First-order spallation theory predicts a ? of 1 [Melosh 1989, Impact Cratering, Oxford Univ. Press]. Results in Vickery [1987] for the Moon exhibit a generally decreasing ? with increasing primary crater size (5 secondary fields mapped). In the same paper, however, this trend is flat for Mercury (3 fields mapped) and opposite for Mars (4 fields mapped). SVDs for craters on large icy satellites (Ganymede and Europa), with gravities not too dissimilar to lunar gravity, show generally low velocity exponents (? between 1 and 1.5), except for the very largest impactor measured: the 585-km-diameter Gilgamesh basin on Ganymede (? = 2.6 × 0.4) [Singer et al., 2013, Icarus 226]. The present work, focusing initially on lunar craters using LROC data, will attempt to confirm or clarify these trends, and expand the number of examples under a variety of impact conditions and surface materials to evaluate possible causes of variations.

Singer, K. N.; Jolliff, B. L.; McKinnon, W. B.



[Role of ryanodine receptors in hydrogen peroxide-induced DNA fragmentation and thymidine incorporation in cultured rat astrocytes].  


In the CNS, reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemia-reperfusion injury, Alzheimer disease, Parkinson disease and aging. However, the exact mechanism is unknown, and there is little information on possible roles of ROS in cell injury and the process on recovery of astrocytes, the most abundant glial cells in the brain. We examined hydrogen peroxide (H2O2)-induced DNA fragmentation and thymidine incorporation into cultured astrocytes as an indicator of the process of recovery from astrocytic DNA injury. Astrocytes were isolated from cerebral cortices of 0-day-old rats and treated with 1 mM dibutyryl cyclic AMP for 4 days. H2O2 of 100 microM stimulated thymidine incorporation into astrocytes. Caffeine, ryanodine, cyclic ADP-ribose (endogenous ryanodine receptor agonist) and beta-NAD+ (precursor of cyclic ADP-ribose) suppressed partially the stimulatory effect of H2O2. Ruthenium red (ryanodine receptor antagonist) facilitated further the stimulatory effect of H2O2. The facilitated effect of ruthenium red on H2O2-induced thymidine incorporation was suppressed by caffeine, ryanodine, cyclic ADP-ribose and beta-NAD+. H2O2-induced DNA fragmentation and astrocytic death were suppressed by ruthenium red. These findings suggest that the process of recovery from astrocytic DNA injury by H2O2 may be regulated by Ca2+ efflux from ryanodine-sensitive intracellular Ca2+ stores. PMID:10190145

Tanaka, K; Ohnishi, Y; Sato, T; Nishikawa, T



Ring-opening metathesis polymerization-derived monolithic anion exchangers for the fast separation of double-stranded DNA fragments.  


Ring-opening metathesis polymerization- (ROMP) derived monoliths were prepared from 5-norborn-2-enemethyl bromide (NBE-CH(2)Br) and tris(5-norborn-2-enemethoxy)methylsilane ((NBE-CH(2)O)(3)SiCH(3)) within the confines of surface-silanized borosilicate columns (100×3 mm I.D.), applying Grubbs' first generation benzylidene-type catalyst [RuCl(2)(PCy(3))(2)(CHPh)]. Monoliths were converted into weak anion exchangers via reaction with diethyl amine. The resulting monolithic anion exchangers demonstrated a very good potential for the anion-exchange separation of nucleic acids applying a phosphate buffer (0.05 mol/L, pH 7) and NaCl (1.0 mol/L) as a gradient former. Fast and efficient separations, indicated by sharp and highly symmetric analyte peaks, were established. Except for the 267 and 298 base pair fragments, the eleven fragments of a ds-pUC18 DNA Hae III digest were baseline separated within ?8 min. Nineteen fragments of a ds-pBR322 Hae III digest were separated within ?12 min. There, only the 192 and 213 base pair fragments and the 458, 504 and 540 base pair fragments coeluted. A ds-pUC18 DNA Hae III digest was used as a control analyte in evaluating the influence of organic additives on the mobile phase such as methanol and acetonitrile on nucleic acid separation. Methanol, and even better, acetonitrile improved the separation efficiency and shortened the analysis time. PMID:20850125

Lubbad, Said H; Buchmeiser, Michael R



DNA fragmentation and DNA repair synthesis induced in rat and human thyroid cells by chemicals carcinogenic to the rat thyroid  

Microsoft Academic Search

Five chemicals that are known to induce in rats thyroid follicular-cell adenomas and carcinomas were assayed for their ability to induce DNA damage and DNA repair synthesis in primary cultures of human thyroid cells. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the same Comet assay, were obtained after a 20-h exposure

Francesca Mattioli; Antonietta Martelli; Marzia Gosmar; Claudia Garbero; Valeria Manfredi; Emanuela Varaldo; Gian Carlo Torre; Giovanni Brambilla



Critical behavior of megabase-size DNA toward the transition into a compact state  

NASA Astrophysics Data System (ADS)

We studied the changes in the higher-order structure of a megabase-size DNA (S120-1 DNA) under different spermidine (SPD) concentrations through single-molecule observations using fluorescence microscopy (FM) and atomic force microscopy (AFM). We examined the difference between the folding transitions in S120-1 DNA and sub-megabase-size DNA, T4 DNA (166 kbp). From FM observations, it is found that S120-1 DNA exhibits intra-chain segregation as the intermediate state of transition, in contrast to the all-or-none nature of the transition on T4 DNA. Large S120-1 DNA exhibits a folding transition at lower concentrations of SPD than T4 DNA. AFM observations showed that DNA segments become aligned in parallel on a two-dimensional surface as the SPD concentration increases and that highly intense parallel alignment is achieved just before the compaction. S120-1 DNA requires one-tenth the SPD concentration as that required by T4 DNA to achieve the same degree of parallel ordering. We theoretically discuss the cause of the parallel ordering near the transition into a fully compact state on a two-dimensional surface, and argue that such parallel ordering disappears in bulk solution.

Yoshikawa, Yuko; Suzuki, Yuki; Yamada, Kozo; Fukuda, Wakao; Yoshikawa, Kenichi; Takeyasu, Kunio; Imanaka, Tadayuki



Evidence for Variation in the Effective Population Size of Animal Mitochondrial DNA  

E-print Network

Evidence for Variation in the Effective Population Size of Animal Mitochondrial DNA Gwenael across animals of diverse census population sizes and ecologies, which has led to the suggestion, Eyre-Walker A (2009) Evidence for Variation in the Effective Population Size of Animal Mitochondrial

Eyre-Walker, Adam


The Size and Shape of Caldesmon and Its Fragments in Solution Studied by Dynamic Light Scattering and Hydrodynamic Model Calculations  

PubMed Central

The size and the shape of caldesmon as well as its 50-kDa central and 19-kDa C-terminal fragments were investigated by photon correlation spectroscopy. The hydrodynamic radii, which have been calculated from the experimentally obtained translational diffusion coefficients, are 9.8 nm, 6.0 nm, and 2.9 nm, respectively. Moreover, the experimental values for the translational diffusion coefficients are compared with results obtained from hydrodynamic model calculations. Detailed models for the structure of caldesmon in solution are derived. The contour length is about 64 nm for all of the models used for caldesmon. ImagesFIGURE 3FIGURE 4 PMID:9017208

Czurylo, Edward A.; Hellweg, Thomas; Eimer, Wolfgang; Dabrowska, Renata



Paternal inheritance of plastids in interspecific hybrids of the genus Actinidia revealed by PCR-amplification of chloroplast DNA fragments.  


RFLPs (restriction fragment length polymorphisms) of PCR (polymerase chain reaction) -amplified fragments were used to trace the pattern of plastid DNA inheritance in the genus Actinidia. A total of 51 progeny originating from interspecific crosses between three A. arguta cultivars and A. deliciosa, the kiwifruit, and 12 progeny originating from the cross between A. kolomikta and A. chinensis were analysed together with their parents. No reciprocal crosses could be tested since they all failed to set viable seeds. Attempts to rescue immature embryos failed in all cases as well. The A. argutaXA. deliciosa crosses were checked for the RFLP patterns of a sequence encoding part of the Rubisco large subunit (rbcL), using either AluI or MseI, and for a sequence encoding part of the photosystem II D1 protein (psbA), using HinfI. The A. kolomiktaXA. chinensis cross was checked for the RFLP patterns of sequences encoding the spacers between trnT and the 5'-trnL exon (a-b spacer DNA) and the trnL 3' exon and trnF (e-f spacer DNA), respectively. The first spacer revealed a natural polymorphism between the two parent species due to a large deletion occurring in A. kolomikta detectable without further restriction enzyme treatment. The e-f spacer DNA was digested with HinfI. The comparison of the RFLP patterns in the parents and their progeny showed a strictly paternal inheritance of chloroplast DNA in Actinidia, with no exception found in any of the crosses examined. As the reciprocal crosses were not available, we do not know whether paternal inheritance of plastids is restricted to the crosses we analysed or if this is the general rule for plastid inheritance in the genus Actinidia. Actinidia is dioecious and is the first purely outbreeding species for which a paternal plastid inheritance has so far been documented. PMID:7616960

Cipriani, G; Testolin, R; Morgante, M



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

PubMed Central

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

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



Electric dichroism and bending amplitudes of DNA fragments according to a simple orientation function for weakly bent rods.  


The linear dichroism is calculated for DNA fragments in their thermal bending equilibrium. These calculations are given for relatively short fragments, where bent molecules can be described by an arc model. Using the measured value of 350 A for the persistence length, the limit dichroism (corresponding to complete alignment) decreases due to thermal bending, e.g., for a fragment with 100 base pairs to 80% of the value expected for straight molecules. Thermal bending should lead to a strong continuous decrease of the dichroism with increasing chain length, which is not observed, however, in electric dichroism experiments due to electric stretching. The influence of the electric field on the bending equilibrium is described by a contribution to the bending energy, which is calculated from the movement of charge equivalents against the potential gradient upon bending. The charge equivalents, which are assigned to the helix ends, are derived from the dipole moments causing the stationary degree of orientation. By this procedure the energy term inducing DNA stretching is given for induced, permanent, and saturating induced dipole models without introduction of any additional parameter. The stationary dichroism at a given electric field strength is then calculated according to an arc model by integration over all angles of orientation of helix axes or chords with respect to the field vector, and at each of these angles the contribution to the dichroism is calculated by integration over all helices with different degrees of bending. Orientation functions obtained by this procedure are fitted to dichroism data measured for various restriction fragments. Optimal fits are found for an induced dipole model with saturation of the polarizability. The difference between orientation functions with and without electric stretching is used to evaluate dichroism bending amplitudes. Both chain length and field strength dependence of bending amplitudes are consistent with experimental amplitudes derived from the dichroism decay in low salt buffers containing multivalent ions like Mg2+, spermine, or [CoNH3)6]3+. Bending amplitudes can be used to evaluate the persistence length from electrooptical data obtained for a single DNA restriction fragment. Bending and stretching effects are considerable already at relatively low chain length, and thus should not be neglected in any quantitative evaluation of experimental data. PMID:2752096

Porschke, D



DNA evidence for historic population size and past ecosystem impacts of gray whales  

E-print Network

. Eastern Pacific gray whales play a key ecological role in their Arctic feeding grounds and are widelyDNA evidence for historic population size and past ecosystem impacts of gray whales S. Elizabeth abundance of gray whales and report DNA variability at 10 loci that is typical of a population of 76

Palumbi, Stephen


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


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

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



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

PubMed Central

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



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

Microsoft Academic Search

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

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



Interaction of short-fragmented DNA with dipalmitoylphosphatidylcholine bilayers in presence of zinc.  


The structure and temperature behaviour of the DNA+dipalmitoylphosphatidylcholine (DPPC) bilayer as a function of ZnCl2 concentration were examined using differential scanning calorimetry (DSC), small-angle neutron scattering (SANS) and small-angle X-ray diffraction (SAXD). Experiments revealed the coexistence of two lamellar phases in the mixture: the L(PC) phase, formed due to Zn(2+) binding to the DPPC bilayers, and the condensed lamellar phase L(DNA+PC) with DNA strands packed between the DPPC bilayers. With increasing concentration of zinc, the temperature of the gel - liquid-crystal phase transition of DPPC increases in both phases, and the volume fraction of L(DNA+PC) phase decreases. In the gel state (at 20 degrees C), the repeat distance of L(DNA+PC) phase is constant, d(DNA+PC) approximately 8.3 nm, up to 20 mmol/l of ZnCl2, and increases for higher concentrations of the salt. The periodicity of the L(PC) lamellar phase decreases substantially with the increasing concentration of the salt in the mixture. In the liquid-crystalline state, concentrations above 20 mmol/l ZnCl2 promote the dissolution of the L(DNA+PC) phase into DPPC + Zn(2+) unilamellar vesicles and DNA is neutralized by Zn(2+) ions. The screening of Zn(2+) charge and the formation of a diffuse double layer due to increasing ionic strength of solution are responsible for the observed changes. PMID:19592711

Uhríková, Daniela; Pullmannová, Petra; Bastos, Margarida; Funari, Sérgio S; Teixeira, José



Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis.  


A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative T06 as they passed through the approximately 2 micrometer diameter focused laser beam. Amplified photoelectron pulses from the photomultiplier are grouped into bins of 360-450 micros in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. Statistical analyses showed that (i) the number of M13 molecules counted versus concentration was linear with slope = 1, (ii) the average burst duration was consistent with the expected transit time of a single molecule through the laser beam, and (iii) the number of detected molecules was consistent with single molecule detection. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The autocorrelation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of approximately 100 DNA molecules/band or better. PMID:11407410

Haab, B B; Mathies, R A



Crystal structures of open and closed forms of binary and ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I: structural basis for nucleotide incorporation  

Microsoft Academic Search

The crystal structures of two ternary complexes of the large fragment of Thermus aquaticus DNA polymerase I (Klentaq1) with a primer\\/template DNA and dideoxy- cytidine triphosphate, and that of a binary complex of the same enzyme with a primer\\/template DNA, were determined to a resolution of 2.3, 2.3 and 2.5 Å, respectively. One ternary complex structure differs markedly from the

Ying Li; Sergey Korolev; Gabriel Waksman



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


Ejaculates from nine Asian and two African elephants were analysed to gain a further understanding of mechanisms underlying variable semen quality after transrectal massage. Semen analysis was performed after collection (0 h; subjective motility parameters only) and after 24 h of chilled storage at 10 °C (24 h; all ejaculate and sperm characteristics). Ejaculates with ?50% total motility (TM) at 24 h, which represented >90% of collection attempts, contained a sperm population with a high degree of DNA damage (64.2 ± 19.2% fragmented DNA) and an elevated incidence of detached heads (43.3 ± 22.5%). In contrast, good quality ejaculates designated as those with >50% TM at 24 h displayed higher (p < 0.05) values of sperm kinetic parameters, DNA integrity and normal morphology. Fertility potential was high for good quality ejaculates from two males (one Asian and one African bull) based on in vitro characteristics after chilled storage for up to 48 h post-collection. Urine contamination of semen, as assessed quantitatively by creatinine concentration, was confirmed as a significant factor in reduced elephant ejaculate quality. However, the identification of considerable DNA damage and morphological degeneration in the majority of ejaculates after only 24 h of chilled storage indicates that sperm ageing could be a primary contributor to inconsistent semen quality in the elephant. PMID:23536498

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



A localized transition in the size variation of circular DNA in nanofluidic slitlike confinement  

NASA Astrophysics Data System (ADS)

We report strong evidence for a localized transition in the size variation of circular DNA between strong and moderate regimes of slitlike confinement. A novel and rigorous statistical analysis was applied to our recent experimental measurements of DNA size for linear and circular topologies in nanofluidic slits with depths around ? 2p, where p is the persistence length. This empirical approach revealed a localized transition between confinement regimes for circular DNA at a slit depth of ? 3p but neither detected nor ruled out the possibility for such a transition for linear DNA. These unexpected results provide the first indication of the localized influence of polymer topology on size variation in slitlike confinement. Improved understanding of differences in polymer behavior related to topology in this controversial and relevant system is of fundamental importance in polymer science and will inform nanofluidic methods for biopolymer analysis.

Strychalski, Elizabeth A.; Stavis, Samuel M.; Geist, Jon



Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.  


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

Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc



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

PubMed Central

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

Sivikova, Katarina; Dianovsky, Jan; Holeckova, Beata



Aromaticity-induced changes in electronic properties of size-expanded DNA bases: Case of xC  

Microsoft Academic Search

Size-expanded DNA bases are analogues of natural bases that can be described as a synthesis between benzene and a natural base. Size-expanded bases have been combined with natural bases to form xDNA and yDNA, a new class of synthetic nucleic acids. We are interested in xDNA and yDNA because they might function as molecular wires. Recently, we also became intrigued

Miguel A Fuentes-Cabrera; Pawel Lipkowski; Oscar Huertas; Bobby G. Sumpter; Modesto Orozco; F. Javier Luque; Jack C. Wells; Jerzy Leszczynski



Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats  

PubMed Central

Background In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene. Methodology/Principal Findings We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I. Conclusions/Significance “Super-long” CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD. PMID:21347256

Duzdevich, Daniel; Li, Jinliang; Whang, Jhoon; Takahashi, Hirohide; Takeyasu, Kunio; Dryden, David T. F.; Morton, A. Jennifer; Edwardson, J. Michael



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.  


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

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



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

E-print Network

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 ...

Sen, Yi-Heng



New insight into power-law behavior of fragment size distributions in the C60 multifragmentation regime  

NASA Astrophysics Data System (ADS)

Previous experimental work has shown that a phase transition in C60 multifragmentation induced by nanosecond laser occurs at almost constant temperature covering a wide range of laser fluency. Here the relative yields of ionic fragments (IFs) Cn+ (n = 1-20) resulting from the multifragmentation are measured within the phase transition region. By excluding two small IFs and magic IFs due to their abnormal behavior, the data for residual IFs are used to estimate the size distributions of primary intermediate-mass IFs in the multifragmentation regime. The distributions are found to obey power laws n-?. Furthermore, the exponent ? values have sensitive dependence on lower laser fluency and converge to a constant of about 2.4 ± 0.2 for larger fluencies. These observations are in good agreement with an explanation based on the Fisher droplet model, offering the tantalizing possibility of a liquid-to-gas phase transition in C60 systems.

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



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


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

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



Large-restriction-fragment analysis of Mycobacterium kansasii genomic DNA and its application in molecular typing.  

PubMed Central

Large-restriction-fragment (LRF) polymorphisms in Mycobacterium kansasii isolates from 84 patients with bronchopulmonary infections in Japan between the 1960s and 1995 were studied by pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with VspI were most suitable for PFGE separation of 16 to 21 fragments of between 40 and 550 kbp. All 84 isolates and the type strain M. kansasii ATCC 12478 were successfully typed by LRF analysis with VspI digestion. Twenty-one distinctive LRF types were identified, and the LRF patterns tested over time were reproducible and stable. A computer-assisted dendrogram of the percent similarity demonstrated that isolates of 18 LRF types had relatively close genetic relatedness, while isolates of the remaining 3 types showed divergence. Sequence analysis of the 16S rRNA gene in the isolates showing divergent genetic relatedness revealed a sequence identical to that of a previously reported subspecies of M. kansasii. In the Chugoku district of Japan, 11 cases of M. kansasii infection which occurred in workers in a coastal industrial zone between 1982 and 1993 were caused by one particular strain tentatively named LRF type M. When both detailed demographic data for the patients and ecologic data for the M. kansasii isolates are obtained, LRF typing may be of potential use for investigating the source and transmission of M. kansasii infection. PMID:9041396

Iinuma, Y; Ichiyama, S; Hasegawa, Y; Shimokata, K; Kawahara, S; Matsushima, T



Exclusion of cytoplasmic fragments in flow cytometric analysis of lymph node samples from dogs with lymphoma using membrane-permeable violet laser-excitable DNA-binding fluorescent dye (DyeCycle Violet)  

PubMed Central

Background Cytoplasmic fragments derived from fragile neoplastic lymphocytes are common in samples of lymph nodes collected from dogs with lymphoma. These cytoplasmic fragments interfere with accurate gating of target cells and quantification protocols used for flow cytometry because of their variable size and expression of lymphoid cell surface antigens on their membranes. Objective The aim of this study was to develop a method to efficiently exclude cytoplasmic fragments from flow cytometric analysis of canine lymph nodes in which lymphoma was present. Methods Single-cell suspensions of neoplastic cells were prepared from biopsy samples and fine-needle aspirates of lymph nodes from 23 dogs with lymphoma. Suspensions were stained using a violet laser-excitable (405 nm) membrane-permeable DNA-binding fluorescent dye (DyeCycle Violet, DCV), incubated with antibodies against CD3, CD5, CD21, CD22, and CD45, and then stained with 7-amino-actinomycin D, an argon-excitable (488 nm) membrane-impermeable DNA-binding fluorescent dye. Multi-parameter flow cytometry was used for analysis based on selective uptake and laser-activated fluorescence of these dyes. Results Cytoplasmic fragments, which were DCV-negative and CD45-positive, and dead cells, which were positive for 7-amino-actinomycin D, were efficiently separated from neoplastic cells. Conclusion Staining with DyeCycle Violet is a useful method to improve flow cytometric gating methods and quantitative analyses of lymph node samples from dogs with lymphoma. PMID:21198735

Ito, Daisuke; O'Brien, Timothy D.; Modiano, Jaime F.



Quantitative determination of size and shape of surface-bound DNA using an acoustic wave sensor.  


DNA bending plays a significant role in many biological processes, such as gene regulation, DNA replication, and chromosomal packing. Understanding how such processes take place and how they can, in turn, be regulated by artificial agents for individual oriented therapies is of importance to both biology and medicine. In this work, we describe the application of an acoustic wave device for characterizing the conformation of DNA molecules tethered to the device surface via a biotin-neutravidin interaction. The acoustic energy dissipation per unit mass observed upon DNA binding is directly related to DNA intrinsic viscosity, providing quantitative information on the size and shape of the tethered molecules. The validity of the above approach was verified by showing that the predesigned geometries of model double-stranded and triple-helix DNA molecules could be quantitatively distinguished: the resolution of the acoustic measurements is sufficient to allow discrimination between same size DNA carrying a bent at different positions along the chain. Furthermore, the significance of this analysis to the study of biologically relevant systems is shown during the evaluation of DNA conformational change upon protein (histone) binding. PMID:18178642

Tsortos, Achilleas; Papadakis, George; Mitsakakis, Konstantinos; Melzak, Kathryn A; Gizeli, Electra



Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes  

PubMed Central

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates. PMID:14532186

Mochizuki, Takashi; Ishizaki, Hiroshi; Barton, Richard C.; Moore, Mary K.; Jackson, Colin J.; Kelly, Steven L.; Evans, E. Glyn V.



A Rapid Chromosome-Mapping Method for Cloned Fragments of Yeast DNA  

PubMed Central

A rapid and generally applicable method is described for mapping a cloned yeast DNA segment to the chromosome(s) from which it originated. The method is based upon the recent finding that the integration into a yeast chromosome of a segment of the 2µ plasmid DNA results, in heterozygous diploids, in the specific loss of genetic information from the chromosome into which the 2µ DNA was integrated (Falco et al. 1982). After verification of the accuracy of the method using several genes whose position was known in advance, the method was used to locate the yeast actin gene, which lies on the left arm of chromosome VI, about 50 cM distal to CDC4. PMID:6357946

Falco, S. Carl; Botstein, David



Identification of Freshwater Phycodnaviridae and Their Potential Phytoplankton Hosts, Using DNA pol Sequence Fragments and a Genetic-Distance Analysis? †  

PubMed Central

Viruses that infect phytoplankton are an important component of aquatic ecosystems, yet in lakes they remain largely unstudied. In order to investigate viruses (Phycodnaviridae) infecting eukaryotic phytoplankton in lakes and to estimate the number of potential host species, samples were collected from four lakes at the Experimental Lakes Area in Ontario, Canada, during the ice-free period (mid-May to mid-October) of 2004. From each lake, Phycodnaviridae DNA polymerase (pol) gene fragments were amplified using algal-virus-specific primers and separated by denaturing gradient gel electrophoresis; 20 bands were extracted from the gels and sequenced. Phylogenetic analysis indicated that freshwater environmental phycodnavirus sequences belong to distinct phylogenetic groups. An analysis of the genetic distances “within” and “between” monophyletic groups of phycodnavirus isolates indicated that DNA pol sequences that differed by more than 7% at the inferred amino acid level were from viruses that infect different host species. Application of this threshold to phylogenies of environmental sequences indicated that the DNA pol sequences from these lakes came from viruses that infect at least nine different phytoplankton species. A multivariate statistical analysis suggested that potential freshwater hosts included Mallomonas sp., Monoraphidium sp., and Cyclotella sp. This approach should help to unravel the relationships between viruses in the environment and the phytoplankton hosts they infect. PMID:19088313

Clasen, Jessica L.; Suttle, Curtis A.



Characterization of Escherichia hermannii by ribosomal DNA restriction fragment length polymorphism.  


Ribosomal DNA polymorphism was used to characterize strains of Escherichia hermannii and to differentiate them from E. coli. DNA from 11 E. hermannii strains previously separated into three zymotypes by enzyme electrophoretic polymorphism was digested with HindIII and EcoRI restriction enzymes and analyzed by Southern blotting. The 10 ribotypes obtained with EcoRI fell into 3 groups which correlated with the corresponding zymotypes, and the 5 ribotypes obtained with HindIII were clearly distinct from those of E. coli strains. PMID:7910695

Picard-Pasquier, N; Picard, B; Krishnamoorthy, R; Goullet, P



Early gene expression associated with the commitment and differentiation of a plant tracheary element is revealed by cDNA-amplified fragment length polymorphism analysis.  


Isolated mesophyll cells from Zinnia elegans are induced by auxin and cytokinin to form tracheary elements (TEs) in vitro with high synchrony. To reveal the changing patterns of gene expression during the 48 h of transdifferentiation from mesophyll to TE cell fate, we used a cDNA-amplified fragment length polymorphism approach to generate expression profiles of >30,000 cDNA fragments. Transcriptional changes of 652 cDNA fragments were observed, of which 304 have no previously described function or sequence identity. Sixty-eight genes were upregulated within 30 min of induction and represent key candidates for the processes that underlie the early stages of commitment and differentiation to a TE cell fate. PMID:12417703

Milioni, Dimitra; Sado, Pierre-Etienne; Stacey, Nicola J; Roberts, Keith; McCann, Maureen C



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

PubMed Central

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

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



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

PubMed Central

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

Vesely, Pavel; Bures, Petr; Smarda, Petr; Pavlicek, Tomas



Cloning of an M. tuberculosis DNA Fragment Associated with Entry and Survival Inside Cells  

Microsoft Academic Search

Mycobacterium tuberculosis infects one-third of the world's human population. This widespread infection depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. DNA sequences of M. tuberculosis have been cloned; these confer on a nonpathogenic Escherichia coli strain an ability to invade HeLa cells, augment macrophage phagocytosis, and survive for at least 24

Sergio Arruda; Gloria Bomfim; Ronald Knights; Tellervo Huima-Byron; Lee W. Riley



Identification of Neural Programmed Cell Death through the Detection DNA Fragmentation In Situ and by PCR  

PubMed Central

Programmed cell death is a fundamental process for the development and somatic maintenance of organisms. This unit describes methods for visualizing both dying cells in situ and for detection of nucleosomal ladders. A description of various current detection strategies is provided, as well as support protocols for preparing positive and negative controls and for preparing genomic DNA. PMID:18428472

Yung, Yun C.; Kennedy, Grace; Chun, Jerold



Methods for rapid cloning and detection for sequencing of cloned inverse PCR-generated DNA fragments adjacent to known sequences in bacterial chromosomes.  


Since the invention of PCR, many adaptation techniques have been developed for sequencing DNA fragments flanking known sequences. Of them, inverse PCR is a matter of interest because of the simplicity of its principle. However, the protocols for inverse PCR introduced so far consist of some time-consuming procedures, and with them, we cannot "walk" chromosomes too far since the number of suitable restriction enzymes is limited. Our experiments led to confirming simpler technical approaches applicable to the case of bacterial chromosomes, that is, designing two end-specific "contextual" sequences with which we can quickly detect the desired clones of targeted DNA fragments by simply analyzing PCR products, employing "the minimum value of the desired fragments" as a "discriminating minimum" value to decrease contaminant DNA fragments, and creating a new tandem of two cleaved end fragments of a known sequence ("reordering") for PCR amplification in combination with cloning of the inverse PCR-generated DNA. With the improvements, we could both simplify the procedures and broaden the capacity of the inverse PCR in "walking" chromosomes. PMID:10553675

Pham, H S; Kiuchi, A; Tabuchi, K



Fracture of Brittle Solids. V. Two-Dimensional Distribution Function for Fragment Size in Single Fracture (Experimental)  

Microsoft Academic Search

Experimental data corresponding to the exoclastic fragments from spherical glass specimens fractured in gelatin in the course of the work on paper II are used to test the theoretical predictions of paper IV; the fragments in question are those engendered by surficial flaws of the specimen. The data on the fragments consist of measurements of the spherical surface corresponding to

J. J. Gilvarry



The nucleolar size is associated to the methylation status of ribosomal DNA in breast carcinomas  

PubMed Central

Background There is a body of evidence that shows a link between tumorigenesis and ribosome biogenesis. The precursor of mature 18S, 28S and 5.8S ribosomal RNAs is transcribed from the ribosomal DNA gene (rDNA), which exists as 300–400 copies in the human diploid genome. Approximately one half of these copies are epigenetically silenced, but the exact role of epigenetic regulation on ribosome biogenesis is not completely understood. In this study we analyzed the methylation profiles of the rDNA promoter and of the 5’ regions of 18S and 28S in breast cancer. Methods We analyzed rDNA methylation in 68 breast cancer tissues of which the normal counterpart was partially available (45/68 samples) using the MassARRAY EpiTYPER assay, a sensitive and quantitative method with single base resolution. Results We found that rDNA locus tended to be hypermethylated in tumor compared to matched normal breast tissues and that the DNA methylation level of several CpG units within the rDNA locus was associated to nuclear grade and to nucleolar size of tumor tissues. In addition we identified a subgroup of samples in which large nucleoli were associated with very limited or absent rDNA hypermethylation in tumor respect to matched normal tissue. Conclusions In conclusion, we suggest that rDNA is an important target of epigenetic regulation in breast tumors and that rDNA methylation level is associated to nucleolar size. PMID:24884608



Nitric oxide co-operates with hydrogen peroxide in inducing DNA fragmentation and cell lysis in murine lymphoma cells.  

PubMed Central

We examined whether NO and H2O2 could interact in inducing DNA fragmentation and cell death. H2O2 and the NO-releasing compounds sodium nitroprusside (SNP) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) by themselves elicited lysis of YAC-1 murine lymphoma cells in a concentration-dependent manner. Exposure of the cells to a combination of sublytic concentrations of SNP (0.78 mM) plus H2O2 (7.8 microM) or SNAP (0.18 mM) plus H2O2 (7.8 microM) resulted in cell death which is mediated, in part, through apoptosis. Evidence for this direction is provided by fluorescence microscopic evaluation of the cells, which revealed the presence of changes in nuclear morphology characteristic of apoptosis in 30-40% of lymphoma cells and by the specific pattern of internucleosomal DNA fragmentation detected by gel electrophoresis. The cytotoxic effect of SNP plus H2O2 could be effectively inhibited by either oxyhaemoglobin, which binds NO, or catalase, which eliminates H2O2. Partial protection from SNP-plus-H2O2-induced cell lysis was observed with the poly(ADP-ribose) polymerase inhibitors, nicotinamide and 3-aminobenzamide, parallelling their ability to reverse depletion of cellular NAD+ pools. These results indicate an interaction between NO and H2O2 which leads to a markedly enhanced cytotoxic activity, in part, via induction of apoptosis and suggest that poly(ADP-ribosylation) and subsequent NAD+ depletion mediate, at least in part, this cytotoxic activity. PMID:9032481

Filep, J G; Lapierre, C; Lachance, S; Chan, J S



Anti-genotoxic effect of the Sargassum dentifolium extracts: prevention of chromosomal aberrations, micronuclei, and DNA fragmentation.  


The alga Sargassum dentifolium (Turner) C. Agardh, belongs to Sargassaceae, is a brown seaweed in red sea shores in Egypt. This work aimed to extract different water-soluble polysaccharide extracts (E1, E2, and E3) from S. dentifolium and to investigate their protective effect against cyclophosphamide (CP)-induced genotoxicity. Mice bone marrow cells (BMCs) were collected and analyzed for the chromosomal aberration, micronucleated BMCs (MN-BMCs), the mitotic index, DNA fragmentation by comet assay, and histone deacetylases (HDACs), and radical scavenging capacity of extracts was evaluated by the oxygen radical absorbance capacity assay. The results indicated that E2 and E3 significantly inhibited CP-induced multiple chromosomal aberrations, where E1 and E3 significantly suppressed the number of CP-induced formation of tetraploidy. The extracts prohibited the cytotoxic effect of CP and recovered the mitotic activity, whereas E1 possessed the highest recovery and mitosis. In absence of MN, CP induced formation of bi- and poly-nucleated BMCs. E1 prohibited CP-induced formation of bi-nucleated BMCs, while E2 and E3 prohibited CP-induced formation of poly-nucleated BMCs. CP-induced MN-BMCs were accompanied with mono-, bi- and poly-nucleated cells. E1 and E3 remarkably suppressed mono-nucleated MN-BMCs, while E2 inhibited bi-nucleated MN-BMCs. All the extracts significantly inhibited the CP-induced formation of poly-nucleated MN-BMCs. CP-induced DNA fragmentation was inhibited by all extracts, where E1 was the strongest inhibitor as concluded from the comet tail moment. All the extracts were strong OH scavengers, while only E3 was ROO scavenger. The results revealed a drastic decline in HDACs activity by E1 and E3. In conclusion, S. dentifolium polysaccharide extracts E1 and E3 possessed a potential anti-genotoxic and a promising anti-mutagenic activity. PMID:21652192

Gamal-Eldeen, Amira M; Abo-Zeid, Mona A M; Ahmed, Eman F



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

NASA Astrophysics Data System (ADS)

Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials". The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; Akyürek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Pérez Urquiza, M.; Bautista, J. L.; Garibay, A.; Fuller, B.; Baoutina, A.; Partis, L.; Emslie, K.; Holden, M.; Chum, W. Y.; Kim, H.-H.; Phunbua, N.; Milavec, M.; Zel, J.; Vonsky, M.; Konopelko, L. A.; Lau, T. L. T.; Yang, B.; Hui, M. H. K.; Yu, A. C. H.; Viroonudomphol, D.; Prawettongsopon, C.; Wiangnon, K.; Takabatake, R.; Kitta, K.; Kawaharasaki, M.; Parkes, H.



Selection against spermatozoa with fragmented DNA after postovulatory mating depends on the type of damage  

Microsoft Academic Search

BACKGROUND: Before ovulation, sperm-oviduct interaction mechanisms may act as checkpoint for the selection of fertilizing spermatozoa in mammals. Postovulatory mating does not allow the sperm to attach to the oviduct, and spermatozoa may only undergo some selection processes during the transport through the female reproductive tract and\\/or during the zona pellucida (ZP) binding\\/penetration. METHODS: We have induced DNA damage in

Juan D Hourcade; Miriam Pérez-Crespo; Raúl Fernández-González; Belén Pintado; Alfonso Gutiérrez-Adán




Microsoft Academic Search

Purpose: The antitumor agent paclitaxel (Taxolt) has been shown to arrest cells in mitosis through microtubule stabilization and to induce apoptosis. The tumor suppressor gene p53 is implicated in the regulation of cell cycle checkpoints and can mediate apoptotic cell death. Although initial studies demonstrated that various DNA-damaging agents can induce p53, more recent studies have also shown p53 induction




Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats  

Microsoft Academic Search

BackgroundIn the R6\\/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.Methodology\\/Principal FindingsWe analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats

Daniel Duzdevich; Jinliang Li; Jhoon Whang; Hirohide Takahashi; Kunio Takeyasu; David T. F. Dryden; A. Jennifer Morton; J. Michael Edwardson



Cation-size-dependent DNA adsorption kinetics and packing density on gold nanoparticles: an opposite trend.  


The property of DNA is strongly influenced by counterions. Packing a dense layer of DNA onto a gold nanoparticle (AuNP) generates an interesting colloidal system with many novel physical properties such as a sharp melting transition, protection of DNA against nucleases, and enhanced complementary DNA binding affinity. In this work, the effect of monovalent cation size is studied. First, for free AuNPs without DNA, larger group 1A cations are more efficient in inducing their aggregation. The same trend is observed with group 2A metals using AuNPs capped by various self-assembled monolayers. After establishing the salt range to maintain AuNP stability, the DNA adsorption kinetics is also found to be faster with the larger Cs(+) compared to the smaller Li(+). This is attributed to the easier dehydration of Cs(+), and dehydrated Cs(+) might condense on the AuNP surface to reduce the electrostatic repulsion effectively. However, after a long incubation time with a high salt concentration, Li(+) allows ?30% more DNA packing compared to Cs(+). Therefore, Li(+) is more effective in reducing the charge repulsion among DNA, and Cs(+) is more effective in screening the AuNP surface charge. This work suggests that physicochemical information at the bio/nanointerface can be obtained by using counterions as probes. PMID:25329233

Liu, Biwu; Kelly, Erin Y; Liu, Juewen



Size and DNA distributions of electrophoretically separated cultured human kidney cells  

NASA Technical Reports Server (NTRS)

Electrophoretic purification of purifying cultured cells according to function presumes that the size of cycle phase of a cell is not an overriding determinant of its electrophoretic velocity in an electrophoretic separator. The size distributions and DNA distributions of fractions of cells purified by density gradient electrophoresis were determined. No systematic dependence of electrophoretic migration upward in a density gradient column upon either size or DNA content were found. It was found that human leukemia cell populations, which are more uniform function and found in all phases of the cell cycle during exponential growth, separated on a vertical sensity gradient electrophoresis column according to their size, which is shown to be strictly cell cycle dependent.

Kunze, M. E.; Plank, L. D.; Todd, P. W.



DNA fragment of Penaeus monodon baculovirus PmNOBII gives positive in situ hybridization with white-spot viral infections in six penaeid shrimp species  

Microsoft Academic Search

PmNOBII was first described from experimentally infected shrimp, but contemporary reports showed that white-spot virus infections in several penaeid shrimp species exhibited similar gross signs and histopathology. Using laboratory infected specimens of Penaeus monodon, DNA of the non-occluded baculovirus PmNOBII was extracted and digested with BamHI and EcoRI. Resulting DNA fragments were ligated with Bluescribe vector using T4 ligase and

Chainarong Wongteerasupaya; Sriwan Wongwisansri; Vichai Boonsaeng; Sakol Panyim; Pusit Pratanpipat; Gary L. Nash; Boonsirm Withyachumnarnkul; T. W. Flegel



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

Microsoft Academic Search

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

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



Heterogeneity of rDNA distribution and genome size in Silene spp.  


Genus Silene L. (Caryophyllaceae) contains about 700 species divided into 44 sections. According to recent taxonomic classification this genus also includes taxa previously classified in genera Lychnis and Melandrium. In this work, four Silene species belonging to different sections were studied: S. latifolia (syn. Melandrium album, Section Elisanthe), S. vulgaris (Inflatae), S. pendula (Erectorefractae), and S. chalcedonica (syn. Lychnis chalcedonica, Lychnidiformes). Flow cytometric analysis revealed a genome size of 2.25 and 2.35 pg/2C for S. vulgaris and S. pendula and of 5.73 and 6.59 pg/2C for S. latifolia and S. chalcedonica. All four species have the same chromosome number including the pair of sex chromosomes of the dioecious S. latifolia (2n = 2x = 24). Double target fluorescence in-situ hybridization revealed the chromosomal locations of 25S rDNA and 5S rDNA. A marked variation in number and localization of rDNA loci but no correlation between the numbers of rDNA clusters and genome size was found. FISH and genome size data indicate that nuclear genomes of Silene species are highly diversified as a result of numerous DNA amplifications and translocations. PMID:11448040

Siroký, J; Lysák, M A; Dolezel, J; Kejnovský, E; Vyskot, B



Towards the onset of fruit tree growing north of the Alps: ancient DNA from waterlogged apple (Malus sp.) seed fragments.  


Wild apples (Malus sp.) have been a major food source in the northern Alpine region since prehistory and their use is well understood. The onset of deliberate fruit tree growing in the area is, however, less clear. It is generally assumed that horticulture was practised in Roman times, but it might be even earlier. In the archaeological record seed testa and pericarp remains are particularly frequent at sites with waterlogged preservation such as lakeshore settlements or wells, pits and ditches, but the distinction between wild and domestic plants is not morphologically possible. With waterlogged remains being one main source of information about past fruit cultivation, we have tested the feasibility of analysing ancient DNA from waterlogged preserved bulk samples of testa fragments. We studied apple seeds from three Neolithic and three Roman sites with waterlogged preservation in the Alpine foreland. Chloroplast markers failed in all samples, but nuclear ITS1 (internal transcribed spacer region 1) of the ribosomal DNA was successfully typed in two Roman samples from the site Oedenburg/Biesheim-Kunheim (Haut-Rhin, F). The retrieved ITS1 sequences are identical to each other and are shared with wild Malus sylvestris and Malus sieversii, and with domestic apple cultivars, supporting the potential of using waterlogged remains for identifying the genetic status of apple diachronically. PMID:21501956

Schlumbaum, Angela; van Glabeke, Sabine; Roldan-Ruiz, Isabel



Identification of Thiobacillus ferrooxidans strains based on restriction fragment length polymorphism analysis of 16S rDNA.  


The 16S rDNA sequences from ten strains of Thiobacillus ferrooxidans were amplified by PCR. The products were compared by performing restriction fragment length polymorphism (RFLP) analysis with restriction endonucleases Alu I, Hap II, Hha I, and Hae III. The RFLP patterns revealed that T. ferrooxidans could be distinguished from other iron- or sulphur-oxidizing bacteria such as T. thiooxidans NB1-3, T. caldus GO-1, Leptospirillum ferrooxidans and the marine iron-oxidizing bacterium strain KU2-11. The RFLP patterns obtained with Alu I, Hap II, and Hae III were the same for nine strains of T. ferrooxidans except for strain ATCC 13661. The RFLP patterns for strains NASF-1 and ATCC 13661 with Hha I were distinct from those for other T. ferrooxidans strains. The 16S rDNA sequence of T. ferrooxidans NASF-1 possessed an additional restriction site for Hha I. These results show that iron-oxidizing bacteria isolated from natural environments were rapidly identified as T. ferrooxidans by the method combining RFLP analysis with physiological analysis. PMID:11414499

Kamimura, K; Wakai, S; Sugio, T



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

PubMed Central

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



Carnosol delays chemotherapy-induced DNA fragmentation and morphological changes associated with apoptosis in leukemic cells.  


Carnosol, from the herb rosemary, has been shown to induce apoptotic cell death in high-risk pre-B acute lymphoblastic leukemia (ALL). In the present study, carnosol was tested for its ability to sensitize leukemia cells to chemotherapeutic agents. Carnosol reduced the percentage of cell death in the pre-B ALL lines SEM, RS4;11, and REH when combined with cytarabine, methotrexate, or vincristine compared to the chemotherapeutic agents alone. Analysis of DNA strand breaks by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling showed that carnosol delayed DNA cleavage in the cells when combined with chemotherapeutic drugs. Co-treatment of the cells with carnosol and chemotherapeutic drugs did not reduce mitochondrial membrane depolarization compared to the drug treatment alone. Time course analysis of caspase-3 activation by flow cytometry showed co-treatment with carnosol and drugs increased the activation of caspase-3 above that observed for the chemotherapeutic drugs alone. A lower percentage of caspase-3 positive cells progressed to an apoptotic phenotype when co-treated with carnosol and the chemotherapeutic drugs compared to drugs alone. These data show that carnosol blocks the terminal apoptotic events induced by chemotherapeutic drugs and suggest that increased dietary intake of carnosol may potentially decrease the effectiveness of some standard chemotherapy treatments used for leukemia. PMID:19116879

Zunino, Susan J; Storms, David H



The Poly(ADP-ribose) polymerase-1-regulated endonuclease DNAS1L3 is required for etoposide-induced internucleosomal DNA fragmentation and increases etoposide cytotoxicity in transfected osteosarcoma cells.  


The cytotoxic effect of the chemotherapeutic drug etoposide (VP-16) is thought to result from its ability to induce DNA damage and thereby to trigger apoptosis. Internucleosomal DNA fragmentation occurs late during apoptosis in many cell types. However, whereas human osteosarcoma cells undergo internucleosomal DNA fragmentation during staurosporine-induced apoptosis, they fail to do so in response to VP-16. Recently, we showed that these cells also do not express the poly(ADP-ribosyl)ation-regulated Ca(2+)- and Mg(2+)-dependent endonuclease DNAS1L3. The possibility that this deficiency underlies the failure of these cells to undergo internucleosomal DNA fragmentation in response to VP-16 was investigated. The proteolytic processing and consequent activation of procaspase-3, cleavage of the inhibitory subunit of DNA fragmentation factor, and the degradation of DNA into 50-kb fragments occurred similarly in osteosarcoma cells exposed to either staurosporine or VP-16. However, the additional processing of the 50-kb DNA fragments to oligonucleosomal fragments was not apparent in the VP-16-treated cells. Ectopic expression of DNAS1L3 conferred on osteosarcoma cells the ability to undergo VP-16-induced internucleosomal DNA fragmentation. Furthermore, expression of DNAS1L3 markedly potentiated the cytotoxic effect of VP-16 in these cells. Both DNAS1L3-mediated and staurosporine-induced internucleosomal DNA fragmentation were Ca(2+) dependent, but only the DNAS1L3-mediated DNA cleavage was blocked by expression of a caspase-3-resistant mutant of poly(ADP-ribose) polymerase-1. The present work results suggest a direct relation between the activity of a chemotherapeutic drug (VP-16) and a specific endonuclease (DNAS1L3). They also indicate that internucleosomal DNA fragmentation plays an active role in apoptosis and that the failure of cancer cells to undergo such DNA degradation may contribute to the development of resistance to chemotherapeutic drugs. PMID:12154052

Boulares, A Hamid; Zoltoski, Anna J; Sherif, Zaki A; Yakovlev, Alexander G; Smulson, Mark E



Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella fastidiosa  

PubMed Central

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

Kung, Stephanie H.; Retchless, Adam C.; Kwan, Jessica Y.



Transposable elements (TEs) are fragments of DNA that can insert into new chromosomal locations and  

E-print Network

A triploid nutritive tissue in flowering plants. EPIGENETIC Any heritable change in gene expression starchy ENDOSPERM and that is the site of pigment biosynthesis. TE-induced alleles of several genes in the anthocyanin (pigment) biosynthetic path- way give rise to distinct patterns with respect to the size

Feschotte, Cedric


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

NASA Technical Reports Server (NTRS)

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

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



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

E-print Network

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.

Nicolas Douarche; Simona Cocco



Reconstructing the history of a fragmented and heavily exploited red deer population using ancient and contemporary DNA  

PubMed Central

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



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

PubMed Central

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

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



A Laboratory Introduction to DNA Restriction Analysis  

NSDL National Science Digital Library

This workshop will serve as an introduction to laboratory exercises in molecular biology. DNA from lambda virus will be digested with various restriction enzymes, and the resulting fragments separated using agarose gel electrophoresis. The separation patterns will be visualized, photographed and used to illustrate the relationship between DNA fragment size and electrophoretic mobility.

David A. Micklos (DNA Learning Center of Cold Spring Harbor Laboratory;); Greg A. Freyer (Columbia University College of Physicians and Surgeons;)



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

SciTech Connect

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.

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



Manganese Superoxide Dismutase Mediates the Early Release of Mitochondrial Cytochrome C and Subsequent DNA Fragmentation after Permanent Focal Cerebral Ischemia in Mice  

Microsoft Academic Search

Recent studies have shown that release of mitochondrial cyto- chrome c is a critical step in the apoptosis process. We have reported that cytosolic redistribution of cytochrome c in vivo occurred after transient focal cerebral ischemia (FCI) in rats and preceded the peak of DNA fragmentation. Although the involve- ment of reactive oxygen species in the cytosolic redistribution of cytochrome

Miki Fujimura; Yuiko Morita-Fujimura; Makoto Kawase; Jean-Christophe Copin; Bernard Calagui; Charles J. Epstein; Pak H. Chan



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


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

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



Interleukin-1 beta fragment (163-171) modulates bovine granulosa cell proliferation in vitro: dependence on size of follicle.  


The biological effects of IL-1 on ovarian function have been considered as an inflammatory-like reaction. The peptide fragment of IL-1 (163-171 aa), which belongs to the active site of the whole protein, has been shown to exert an immunostimulatory activity without inducing inflammatory effects. The aim of this study was to investigate whether IL 163-171, alone or associated with bFSH, modulates granulosa cell proliferation; in addition, we wanted to assess if IL 163-171 interferes with FSH binding to receptors. Bovine follicles were divided according to their size. Cell proliferation, assessed by [3H]-thymidine uptake and [125I]-FSH binding, were studied in cells treated with IL 163-171(0, 0.5, 5 and 20 ng/ml) with or without bFSH (100 ng/ml) for 24, 48 or 72 h. Basal incorporation of [3H]-thymidine into granulosa cells from small follicles was always 3-fold higher (P < 0.01) than that by cells from large follicles. IL 163-171 did not show any effect in granulosa cells from large follicles but stimulated (P < 0.01) [3H]-thymidine uptake into granulosa cells from small follicles; furthermore IL 163-171 interacted positively with bFSH (P < 0.01) after 48 and 72 h. IL 163-171 significantly reduced (P < 0.05) FSH binding in cells from small follicles after 24 h, but not after 48 and 72 h. This data demonstrates that: (1) Follicular size and cell proliferation are inversely related; (2) IL 163-171 modulates granulosa cell proliferation only in cells from small follicles; and (3) the interaction between IL 163-171 and FSH does not mainly occur at receptor level. PMID:9571568

Basini, G; Baratta, M; Bussolati, S; Tamanini, C



Secuencia parcial de un fragmento de ADN de patos silvestres homólogo al complejo mayor de histocompatibilidad de Gallus gallus Partial sequence of a DNA fragment from wild ducks homologous to the Gallus gallus major histocompatibility complex  

Microsoft Academic Search

In this study, a genomic DNA fragment from domestic duck (Anas domesticus) was amplified by PCR. Such fragment shared 93 % identity to the chicken (Gallus gallus) MHC class II, which correspond to DAB1- (Disabled 1)-like sequence. The DAB1 sequence was also amplified from nine wild duck species of the Anas genus, which after performing a restriction fragment length polymorphism

Sofía González-Guzmán; Elizabeth Loza-Rubio; Virginia León-Régagnonc; Gary García-Espinosa


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

PubMed Central

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

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



Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events.  


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

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



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

PubMed Central

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

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



Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing  

E-print Network

Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing H: Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging be- cause only a small of the small quantity of cell-free DNA. METHODS: We used high-throughput paired-end se- quencing to directly

Quake, Stephen R.


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

SciTech Connect

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.

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))



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

SciTech Connect

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.

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)



Early DNA sequencing, 2D animationSite: DNA Interactive (  

NSDL National Science Digital Library

Two sequencing techniques were developed independently in the 1970s. The method developed by Fred Sanger used chemically altered "dideoxy" bases to terminate newly synthesized DNA fragments at specific bases (either A, C, T, or G). These fragments are then size-separated, and the DNA sequence can be read.



Thromboxane A2 receptor is highly expressed in mouse immature thymocytes and mediates DNA fragmentation and apoptosis  

PubMed Central

We have recently revealed that the thymus is the organ showing the highest expression of thromboxane (TX) A2 receptor in mice. In this study, thymic cell populations expressing the receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 receptor. The receptor density in these cells was comparable to that seen in blood platelets. This receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration- dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 receptor on their cell surface and suggest a role for the TXA2/TXA2 receptor system in the thymic micro-environment. PMID:8228829



On the importance of size of plastic fragments and pellets on the strandline: a snapshot of a Brazilian beach  

Microsoft Academic Search

Virgin plastic pellets and plastic fragments are reported as ubiquitous beach contaminants in the peer-reviewed literature.\\u000a A surface density of 0.3 virgin plastic pellets and plastic fragments per square centimeter of the strandline area was registered\\u000a on an urban beach of the northeast of Brazil. This beach is presently not affected by petrochemical facilities or pellet processing\\u000a plants. The main

Monica F. Costa; Juliana A. Ivar do Sul; Jacqueline S. Silva-Cavalcanti; Maria Christina B. Araújo; Ângela Spengler; Paula S. Tourinho



Size-expanded DNA bases: An ab-initio study of their structural and electronic properties  

SciTech Connect

The size-expanded DNA bases, xA, xC, xG, and xT, are benzo-homologue forms of the natural DNA bases; i.e., their structure can be seen as the fusion of a natural base and a benzene ring. Recently, a variety of DNAs, known as xDNAs, have been synthesized in which size-expanded and natural bases are paired. In this paper we use second-order Moeller-Plesset perturbation theory and density functional theory to investigate the structural and electronic properties of xA, xC, xG, and xT and their natural counterparts. We find that whereas natural and size-expanded bases have both nonplanar amino groups the latter have also nonplanar aromatic rings. When density functional theory is used to investigate the electronic properties of size-expanded and natural bases, it is found that the HOMO-LUMO gap of the size-expanded bases is smaller than that of the natural bases. Also, xG should be easier to oxidize than G.

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



Power law and exponential ejecta size distributions from the dynamic fragmentation of shock-loaded Cu and Sn metals under melt conditions  

SciTech Connect

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.

Durand, O.; Soulard, L. [CEA, DAM, DIF, F-91297 Arpajon (France)



Size-Expanded yDNA bases: An Ab Initio Study  

SciTech Connect

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.

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



Distribution of telomeric DNA sequences on the X-radiation-induced chromosome fragments observed in the genome of androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814).  


Cytogenetic screening of the androgenetic brook trout (Salvelinus fontinalis, Mitchill 1814) offspring hatched from eggs exposed to 420 Gy of X-radiation before insemination exhibited residues of the irradiated maternal nuclear genome in the form of small chromosome fragments. Remnants of the irradiated chromosomes had different sizes, and their number varied intraindividually from 1 to 15. To efficiently pass through the series of the cell divisions, such chromosome fragments must have had functional kinetochores. Distribution patterns of the telomeric hybridization signals on the chromosome fragments enabled us to distinguish their 3 groups: (i) telomere-less ring chromosomes with fused broken chromosome arms, (ii) rings formed in the course of fusion of the radiation-broken chromosome arm with the opposite telomeric region and exhibiting interstitial telomeric signals at the fusion point, and (iii) chromosome fragments with fused unprotected sister chromatids of 1 broken arm and intact telomeres from the other arm. Disturbances during segregation of such fragments, mainly breakages during anaphase, may partially explain intraindividual variation in the number and size of the chromosome fragments observed in the androgenetic brook trout. PMID:22777065

Ocalewicz, K; Dobosz, S; Kuzminski, H



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

SciTech Connect

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.

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)



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

NASA Technical Reports Server (NTRS)

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

Sekanina, Z.



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

PubMed Central

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

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



Effect of peptide fragment size on the propensity of cyclization in collision-induced dissociation: oligoglycine b(2)-b(8).  


The chemistry of peptide fragmentation by collision-induced dissociation (CID) is currently being reviewed, as a result of observations that the amino acid sequence of peptide fragments can change upon activation. This rearrangement mechanism is thought to be due to a head-to-tail cyclization reaction, where the N-terminal and C-terminal part of the fragment are fused into a macrocycle (= cyclic peptide) structure, thus "losing" the memory of the original sequence. We present a comprehensive study for a series of b fragment ions, from b(2) to b(8), based on the simplest amino acid residue glycine, to investigate the effect of peptide chain length on the appearance of macrocycle fragment structures. The CID product ions are structurally characterized with a range of gas-phase techniques, including isotope labeling, infrared photodissociation spectroscopy, gas-phase hydrogen/deuterium exchange (using CH(3)OD), and computational structure approaches. The combined insights from these results yield compelling evidence that smaller b(n) fragments (n = 2, 3) exclusively adopt oxazolone-type structures, whereas a mixture of oxazolone and macrocycle b fragment structures are formed for midsized b(n) fragments, where n = 4-7. As each of these chemical structures exchanges at different rates, it is possible to approximate the relative abundances using kinetic fits to the H/D exchange data. Under the conditions used here, the "slow"-exchanging macrocycle structure represents approximately 30% of the b ion population for b(6)-b(7), while the "fast"-exchanging oxazolone structure represents the remainder (70%). Intriguingly, for b(8) only the macrocycle structure is identified, which is also consistent with the "slow" kinetic rate in the HDX results. In a control experiment, a protonated cyclic peptide with 6 amino acid residues, cyclo(Gln-Trp-Phe-Gly-Leu-Met), is confirmed not to adopt an oxazolone structure, even upon collisional activation. These results demonstrate that in some cases larger macrocycle structures are surprisingly stable. While more studies are required to establish the general propensity for cyclization in b fragments, the implications from this study are troubling in terms of faulty sequence identification. PMID:19947633

Chen, Xian; Yu, Long; Steill, Jeffrey D; Oomens, Jos; Polfer, Nick C



DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma  

Microsoft Academic Search

Vaccination with idiotypic protein protects against B-cell lymphoma, mainly through anti-idiotypic antibody. For use in patients, DNA vaccines containing single-chain Fv derived from tumor provide a convenient alternative vaccine delivery system. However, single-chain Fv sequence alone induces low anti-idiotypic response and poor protection against lymphoma. Fusion of the gene encoding fragment C of tetanus toxin to single-chain Fv substantially promotes

Catherine A. King; Myfanwy B. Spellerberg; Delin Zhu; Jason Rice; Surinder S. Sahota; Andrew R. Thompsett; Terry J. Hamblin; Jiri Radl; Freda K. Stevenson



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

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

Vizcaino, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernandez-Lago, Luis



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

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

Vizcaino, Nieves; Cloeckaert, Axel; Zygmunt, Michel S.; Fernandez-Lago, Luis



Trapping of megabase-sized DNA molecules during agarose gel electrophoresis  

PubMed Central

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated ?-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed. PMID:9892654

Gurrieri, Sergio; Smith, Steven B.; Bustamante, Carlos



Molecular assessment of post-BMT chimerism using various biologic specimens and automated DNA sizing technology.  


Highly polymorphic microsatellite markers provide useful genetic markers for detection of complete or mixed chimerism in patients after allogeneic BMT (allo-BMT). We report application of automated DNA sizing technology for detection of post-BMT chimerism using fresh peripheral blood, BM, or archival blood smears and various DNA isolation techniques. Donors' and recipients' DNA was amplified with fluorescent PCR primers specific for short tandem repeat (STR) marker loci: FGA, VWA, TH01, F13A1, D21S11. Chimerism was assessed in 14 recipients after allo-BMT. A complete chimerism was detected in 10 patients, in 3 patients we observed fluctuations of chimerism status, and mixed chimerism was assessed in 1 patient. We show that DNA from different types of biologic specimens (whole peripheral blood, BM suspension, archival blood smears), prepared according to the various isolation techniques (salting-out method, phenol chloroform extraction, Chelex procedure) and amplified with fluorescent PCR primers for microsatellite markers, enable identification of chimerism status following allo-BMT in children. PMID:10813540

Jó?kowska, J; Wachowiak, J; Lange, A; Kwissa, M; Witt, M



A novel transchromosomic system: stable maintenance of an engineered Mb-sized human genomic fragment translocated to a mouse chromosome terminal region.  


Transchromosomic (Tc) technology using human chromosome fragments (hCFs), or human artificial chromosomes (HACs), has been used for generating mice containing Mb-sized segments of the human genome. The most significant problem with freely segregating chromosomes with human centromeres has been mosaicism, possibly due to the instability of hCFs or HACs in mice. We report a system for the stable maintenance of Mb-sized human chromosomal fragments following translocation to mouse chromosome 10 (mChr.10). The approach utilizes microcell-mediated chromosome transfer and a combination of site-specific loxP insertion, telomere-directed chromosome truncation, and precise reciprocal translocation for the generation of Tc mice. Human chromosome 21 (hChr.21) was modified with a loxP site and truncated in homologous recombination-proficient chicken DT40 cells. Following transfer to mouse embryonic stem cells harboring a loxP site at the distal region of mChr.10, a ~4 Mb segment of hChr.21 was translocated to the distal region of mChr.10 by transient expression of Cre recombinase. The residual hChr.21/mChr.10ter fragment was reduced by antibiotic negative selection. Tc mice harboring the translocated ~4 Mb fragment were generated by chimera formation and germ line transmission. The hChr.21-derived Mb fragment was maintained stably in tissues in vivo and expression profiles of genes on hChr.21 were consistent with those seen in humans. Thus, Tc technology that enables translocation of human chromosomal regions onto host mouse chromosomes will be useful for studying in vivo functions of the human genome, and generating humanized model mice. PMID:24488595

Takehara, Shoko; Schulz, Thomas C; Abe, Satoshi; Takiguchi, Masato; Kazuki, Kanako; Kishigami, Satoshi; Wakayama, Teruhiko; Tomizuka, Kazuma; Oshimura, Mitsuo; Kazuki, Yasuhiro



Universal elements of fragmentation  

SciTech Connect

A fragmentation theory is proposed that explains the universal asymptotic behavior of the fragment-size distribution in the large-size range, based on simple physical principles. The basic principles of the theory are the total mass conservation in a fragmentation process and a balance condition for the energy expended in increasing the surface of fragments during their breakup. A flux-based approach is used that makes it possible to supplement the basic principles and develop a minimal theory of fragmentation. Such a supplementary principle is that of decreasing fragment-volume flux with increasing energy expended in fragmentation. It is shown that the behavior of the decreasing flux is directly related to the form of a power-law fragment-size distribution. The minimal theory is used to find universal asymptotic fragment-size distributions and to develop a natural physical classification of fragmentation models. A more general, nonlinear theory of strong fragmentation is also developed. It is demonstrated that solutions to a nonlinear kinetic equation consistent with both basic principles approach a universal asymptotic size distribution. Agreement between the predicted asymptotic fragment-size distributions and experimental observations is discussed.

Yanovsky, V. V., E-mail: yanovsky@isc.kharkov.u [National Academy of Sciences of Ukraine, Institute for Single Crystals (Ukraine); Tur, A. V. [CNRC-UPS, Center d'Etude Spatiale Des Rayonnements (France); Kuklina, O. V. [National Academy of Sciences of Ukraine, Institute for Single Crystals (Ukraine)



Movements and Habitat Use of Eastern Foxsnakes (Pantherophis gloydi) in Two Areas Varying in Size and Fragmentation  

E-print Network

that vary in their degree of fragmentation. As predators, snakes are an important component of ecosystems scale. Within the smaller habitat patch, however, these preferences were accentuated with snakes and landscapes. Snakes are significant predators of birds, mammals, am- phibians, and reptiles (Schwaner

Blouin-Demers, Gabriel


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

Microsoft Academic Search

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

Thomas M. Gehring; Robert K. Swihart



The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities.  


Saporin-6 produced by the plant Saponaria officinalis belongs to the family of single chain ribosome-inactivating proteins. It potently inhibits protein synthesis in eukaryotic cells, by cleaving the N-glycosidic bond of a specific adenine in 28 S rRNA, which results in the cell death. Saporin-6 has also been shown to be active on DNA and induces apoptosis. In the current study, we have investigated the roles of rRNA depurination and the activity of saporin-6 on genomic DNA in its cytotoxic activity. The role of putative active site residues, Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208), and two invariant residues, Tyr(16) and Arg(24), proposed to be important for structural stability of saporin-6, has been investigated in its catalytic and cytotoxic activity. These residues were mutated to alanine to generate seven mutants, Y16A, R24A, Y72A, Y120A, E176A, R179A, and W208A. We show that for the RNA N-glycosidase activity of saporin-6, residues Tyr(16), Tyr(72), and Arg(179) are absolutely critical; Tyr(120) and Glu(176) can be partially dispensed with, whereas Trp(208) and Arg(24) do not appear to be involved in this activity. The residues Tyr(72), Tyr(120), Glu(176), Arg(179), and Trp(208) were found to be essential for the genomic DNA fragmentation activity, whereas residues Tyr(16) and Arg(24) do not appear to be required for the DNA fragmentation. The study shows that saporin-6 possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both activities are required. PMID:12466280

Bagga, Shveta; Seth, Divya; Batra, Janendra K



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

Microsoft Academic Search

IntroductionRecent 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.MethodsTotal 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

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



Intracellular forms of adenovirus DNA: integrated form of adenovirus DNA appears early in productive infection.  

PubMed Central

In KB cells productively infected with adenovirus type 2, alkali-stable greater than 100S and 40-100S viral DNAs are synthesized starting 2-4 hr postinfection, i.e., before unit length (34 S) viral DNA is made. The amount of greater than 100S and 40-100S viral DNA increases when 34S viral DNA synthesis begins, and at 16-18 hr postinfection, the 40-100S viral DNA represents 5-20% of the total intracellular viral DNA. The 40-100S viral DNA is synthesized throughout infection. Part of the 40-100S DNA synthesized 5-8 hr postinfection has a density in alkaline CsCl gradients intermediate between those of viral and cellular DNAs. This finding indicates that newly synthesized viral DNA is covalently linked to cellular DNA. Viral sequences can be excised from the cellular DNA of infected cells with the EcoRI restriction endonuclease. Fragments of viral DNA are detected in polyacrylamide-agarose gels by DNA-DNA hybridization, and these fragments correspond in size to most of the known EcoRI fragments of adenovirus 2 DNA. Viral DNA sequences in size-classes between the EcoRI-A and -C fragments are also found and probably represent viral DNA linked to cellular sequences. PMID:1063388

Schick, J; Baczko, K; Fanning, E; Groneberg, J; Burger, H; Doerfler, W



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

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

Hilde Nybom; Steven H. Rogstad



Applications of mass spectrometry to DNA fingerprinting and DNA sequencing  

SciTech Connect

DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. [Oak Ridge National Lab., TN (United States); Arlinghaus, H.F. [Atom Sciences, Inc., Oak Ridge, TN (United States)



Applications of mass spectrometry to DNA fingerprinting and DNA sequencing  

SciTech Connect

DNA fingerprinting and sequencing rely on polyacrylamide gel electrophoresis to determine the sizes of the DNA fragments. Innovative altematives to polyacrylamide gel electrophoresis are under investigation for characterization of such fingerprinting and sequencing. One method uses stable isotopes of tin and other elements to label the DNAwhereas other procedures do not require labels. The detectors in each case are mass spectrometers that detect either the stable isotopes or the DNA fragments themselves. If successful, these methods will speed up the rate of DNA analysis by one or two orders of magnitude.

Jacobson, K.B.; Buchanan, M.V.; Chen, C.H.; Doktycz, M.J.; McLuckey, S.A. (Oak Ridge National Lab., TN (United States)); Arlinghaus, H.F. (Atom Sciences, Inc., Oak Ridge, TN (United States))



Controlled Fragmentation  

NASA Astrophysics Data System (ADS)

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.

Arnold, Werner



Controlled Fragmentation  

NASA Astrophysics Data System (ADS)

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.

Arnold, Werner



Pyrazinamide potential effects on male rats DNA fragmentation, bone type I collagen amino acid composition, reproductive capability and posterity antenatal and postnatal development.  


Current therapeutic regimens with first-line antitubercular agents are associated with a high rate of adverse effects which can lead to therapeutic failure. Understanding the nature and the severity of these effects is important for treatment optimization. The aim of present study was to investigate pyrazinamide potential effects on male rats DNA fragmentation, amino acid composition of bone type I collagen, reproductive capability and their posterity antenatal and postnatal development. Wistar albino male rats (160-200 g b.w.) were divided into three groups: I--received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, II--at a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; III--control. After 60 days of the experiment, rats of the experimental (groups I and II) and control groups were mated with intact virgin females. The amino acids contents of male rat bone type I collagens were determined using amino acid analyzer, epididymis and testis DNA fragmentation--electrophoretically; posterity antenatal development indices and postnatal development--by standard procedures. The study of pyrazinamide effects (administered in different doses) on males bone type I collagen amino acid contents and testis DNA fragmentation demonstrated the presence of dose-dependent pyrazinamide-mediated quantitative and qualitative changes in male rat reproductive organs DNA and extracellular matrix proteins in comparison with control. Changes in nucleic acids and proteins structure were accompanied by alterations in processes of fertilization (with intact females), embryogenesis and by lowering of posterity survival. PMID:23061279

Bondarenko, Larysa B; Shayakhmetova, Ganna M; Byshovets, Taisiya F; Kovalenko, Valentina M



The fate of transgenic sequences present in genetically modified plant products in fish feed, investigating the survival of GM soybean DNA fragments during feeding trials in Atlantic salmon, Salmo salar L  

Microsoft Academic Search

Vegetable protein sources like soybeans, canola and maize gluten are good alternatives to fish meal. However, a large proportion of such products available on the international market may possess genetically modified (GM) components. This report concerns a study to investigate the fate and survival of ingested GM soy DNA fragments (120 and 195 bp) and a 180-bp fragment of the

Monica Sanden; Ian J Bruce; M. Azizur Rahman; Gro-Ingunn Hemre



Aneuploidogenic effects and DNA oxidation induced in vitro by differently sized gold nanoparticles  

PubMed Central

Gold nanoparticles (Au NPs) are used in many fields, including biomedical applications; however, no conclusive information on their potential cytotoxicity and genotoxicity mechanisms is available. For this reason, experiments in human primary lymphocytes and murine macrophages (Raw264.7) were performed exposing cells to spherical citrate-capped Au NPs with two different nominal diameters (5 nm and 15 nm). The proliferative activity, mitotic, apoptotic, and necrotic markers, as well as chromosomal damage were assessed by the cytokinesis-block micronucleus cytome assay. Fluorescence in situ hybridization with human and murine pancentromeric probes was applied to distinguish between clastogenic and aneuploidogenic effects. Our results indicate that 5 nm and 15 nm Au NPs are able to inhibit cell proliferation by apoptosis and to induce chromosomal damage, in particular chromosome mis-segregation. DNA strand breaks were detected by comet assay, and the modified protocol using endonuclease-III and formamidopyrimidine-DNA glycosylase restriction enzymes showed that pyrimidines and purines were oxidatively damaged by Au NPs. Moreover, we show a size-independent correlation between the cytotoxicity of Au NPs and their tested mass concentration or absolute number, and genotoxic effects which were more severe for Au NP 15 nm compared to Au NP 5 nm. Results indicate that apoptosis, aneuploidy, and DNA oxidation play a pivotal role in the cytotoxicity and genotoxicity exerted by Au NPs in our cell models. PMID:24855356

Di Bucchianico, Sebastiano; Fabbrizi, Maria Rita; Cirillo, Silvia; Uboldi, Chiara; Gilliland, Douglas; Valsami-Jones, Eugenia; Migliore, Lucia



Quantifying Double-Strand Breaks and Clustered Damages in DNA by Single-Molecule Laser Fluorescence Sizing  

PubMed Central

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels. PMID:12547808

Filippova, Elena M.; Monteleone, Denise C.; Trunk, John G.; Sutherland, Betsy M.; Quake, Stephen R.; Sutherland, John C.



The use of nano-sized acicular material, sliding friction, and antisense DNA oligonucleotides to silence bacterial genes  

PubMed Central

Viable bacterial cells impaled with a single particle of a nano-sized acicular material formed when a mixture containing the cells and the material was exposed to a sliding friction field between polystyrene and agar gel; hereafter, we refer to these impaled cells as penetrons. We have used nano-sized acicular material to establish a novel method for bacterial transformation. Here, we generated penetrons that carried antisense DNA adsorbed on nano-sized acicular material (?-sepiolite) by providing sliding friction onto the surface of agar gel; we then investigated whether penetron formation was applicable to gene silencing techniques. Antisense DNA was artificially synthesized as 15 or 90mer DNA oligonucleotides based on the sequences around the translation start codon of target mRNAs. Mixtures of bacterial cells with antisense DNA adsorbed on ?-sepiolite were stimulated by sliding friction on the surface of agar gel for 60 s. Upon formation of Escherichia coli penetrons, ?-lactamase and ?-galactosidase expression was evaluated by counting the numbers of colonies formed on LB agar containing ampicillin and by measuring ?-galactosidase activity respectively. The numbers of ampicillin resistant colonies and the ?-galactosidase activity derived from penetrons bearing antisense DNA (90mer) was repressed to 15% and 25%, respectively, of that of control penetrons which lacked antisense DNA. Biphenyl metabolite, ring cleavage yellow compound produced by Pseudomonas pseudoalcaligenes penetron treated with antisense oligonucleotide DNA targeted to bphD increased higher than that lacking antisense DNA. This result indicated that expression of bphD in P. pseudoalcaligenes penetrons was repressed by antisense DNA that targeted bphD mRNA. Sporulation rates of Bacillus subtilis penetrons treated with antisense DNA (15mer) targeted to spo0A decreased to 24.4% relative to penetrons lacking antisense DNA. This novel method of gene silencing has substantial promise for elucidation of gene function in bacterial species that have been refractory to experimental introduction of exogenous DNA.



Large size and complex structure of mitochondrial DNA in two nonflowering land plants.  


We report the first estimates of genome size and complexity for mitochondrial DNAs (mtDNAs) from nonflowering land plants. The mtDNA of Onoclea sensibilis (sensitive fern) is approximately 300 kb in size, while that of Equisetum arvense (common horsetail) is at least 200 kb. Sufficient mtDNA of Onoclea was available to permit an estimation of the copy number and a linkage analysis of nine mitochondrial genes. Six of these genes appear to be present in only one or two copies in the Onoclea genome, whereas three other genes are present in multiple copies. Five of the approximately ten genes encoding 26S rRNA are located on a large, greater than 10 kb, dispersed repeat that also contains closely linked genes for 18S rRNA and the alpha subunit of ATPase (atpA). The other 26S genes belong to a second dispersed repeat family of greater than 8 kb whose elements do not contain any other identified genes. Because flowering plant mtDNAs are also large and contain dispersed, gene-containing, repeats, it appears that these features arose early in the evolution of land plants, or perhaps even in their green algal ancestors. PMID:1568256

Palmer, J D; Soltis, D; Soltis, P



The detection of HPV DNA, the size of tampon specimens and the menstrual cycle.  

PubMed Central

OBJECTIVE--To determine if HPV detection or the size of a tampon specimen is affected by the menstrual cycle. MATERIALS--Two hundred and eighty women between 18-35 years of age attending a gynaecology clinic at The Royal Women's Hospital were enrolled. Each woman completed a questionnaire on the risk factors of HPV infection and provided a tampon specimen. Specimens were analysed for the presence of HPV DNA (polymerase chain reaction with the L1 consensus primers) after the pellet volume and number of cells was assessed. RESULTS--The mean age of the 298 women enrolled in this study was 27.0 years (SD 4.5, range 18-35). Ninety two (30.9%) of the tampon specimens were positive for HPV using the L1 consensus primer. The detection of HPV DNA was not associated with the quartiles of the menstrual cycle (p = 0.32). Both the pellet volume and the number of cells from a tampon specimen were greater during the mid cycle, although this was significant for the pellet volume only (p = 0.002 and 0.1 respectively). The pellet volume was not significantly associated with other variables assessed by the questionnaire. The number of cells from a tampon specimen increased with the numbers of life time sexual partners (p = 0.02) and was higher for a single marital status (p = 0.0008). CONCLUSION--The timing of the menstrual cycle effects the size of tampon specimens but not the probability of detecting HPV DNA. PMID:8039780

Fairley, C K; Robinson, P M; Chen, S; Tabrizi, S N; Garland, S M



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


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

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



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

PubMed Central

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

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



Conformational analysis of a 139 base-pair DNA fragment containing a single-stranded break and its interaction with human poly(ADP-ribose) polymerase.  


The conformational changes induced by the introduction of a central and unique single-stranded break in a 139 base-pair DNA duplex have been analysed by means of polyacrylamide gel electrophoresis, HPLC and dark-field electron microscopy. Compared to the control DNA, the disruption of the covalent sugar-phosphate backbone induces a retardation detected both by gel electrophoresis and anion exchange based HPLC. Electron microscopic visualization of the DNA molecules reveals that most of them present a central fracture at the position of the nick. Measures of the angle at the apex were very well fitted by a simple model of isotropic flexible junction assuming spatial Hooke's law and simple basic Boltzmann statistics. This amounts to using a folded Gaussian distribution. The fit yields an angle equilibrium value phi 0 = 122 degrees for the nicked fragment. The angle distribution could also result from an equilibrium between two forms of the molecule with isotropic flexibility at the nicked site: a stacked and a very flexible unstacked form. The majority of bound poly(ADP-ribose) polymerase, a zinc-finger enzyme involved in DNA break detection, was localized at the apex of the V-shaped DNA duplex, with an accentuation of its general V-shaped conformation (phi 0 = 102 degrees). PMID:8289308

Le Cam, E; Fack, F; Ménissier-de Murcia, J; Cognet, J A; Barbin, A; Sarantoglou, V; Révet, B; Delain, E; de Murcia, G



Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor.  

PubMed Central

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone. Images PMID:8380453

Ban, J; Portetelle, D; Altaner, C; Horion, B; Milan, D; Krchnak, V; Burny, A; Kettmann, R




E-print Network


Hagiya, Masami


Observation of fast collinear partitioning of the {sup 197}Au + {sup 197}Au system into three and four fragments of comparable size  

SciTech Connect

Collisions of a very heavy nonfusing nuclear system {sup 197}Au+{sup 197}Au were studied at an energy of 15 MeV/nucleon. An interesting process of violent reseparation of this heavy system into three or four fragments of comparable size was observed. In the case of ternary partitioning, either the projectile-like fragment (PLF) or target-like fragment (TLF) breaks up almost collinearly with the PLF-TLF separation axis. In the case of quaternary reactions, both PLF and TLF were observed breaking up along this direction. By comparison with a dynamical model of deep inelastic collisions it was concluded that the ternary and quaternary reactions occur in semiperipheral collisions, in a range of angular momenta corresponding to about 0.5-0.7 of the maximum L value for grazing collisions. The time elapsing from the scission of the binary PLF + TLF system to the secondary scission of PLF or TLF was estimated to be of about 70-80 fm/c for the ternary reactions and 80-100 fm/c for the quaternary reactions.

Wilczynski, J.; Swiderski, L. [Andrzej Soltan Institute for Nuclear Studies, Swierk/Warsaw (Poland); Skwira-Chalot, I.; Siwek-Wilczynska, K. [Institute of Experimental Physics, University of Warsaw, Warsaw (Poland); Pagano, A.; Cardella, G.; De Filippo, E.; Guidara, E. La; Papa, M.; Pirrone, S. [INFN, Sezione di Catania, Catania (Italy); Amorini, F.; Anzalone, A.; Cavallaro, S.; Colonna, M.; Toro, M. Di; Maiolino, C.; Porto, F.; Rizzo, F.; Russotto, P. [INFN, Laboratori Nazionali del Sud, Catania (Italy); Dipartimento di Fisica e Astronomia, Universita di Catania, Catania (Italy); Auditore, L. [INFN, Gruppo Collegato di Messina and Dipartimento di Fisica, Universita di Messina, Messina (Italy)



Type V collagen-induced upregulation of capn2 (large subunit of m-calpain) gene expression and DNA fragmentation in 8701-BC breast cancer cells.  


Type V collagen is known to be over-deposited in the stroma of ductal infiltrating carcinomas of the breast. When used as a substrate, type V collagen restrains growth and invasion, and affects gene expression of 8701-BC ductal infiltrating carcinomas cells. Here we supplement existing data by demonstrating type V collagen dependent upregulation of capn2 gene expression in 8701-BC cells through differential display-PCR and Western blot assays. Furthermore, we suggest that our data obtained by centrifugal sedimentation and electrophoresis strongly suggest a correlation between calpain overproduction and DNA fragmentation, since the incubation with calpain inhibitor partly reverts the latter. PMID:21585285

Luparello, Claudio; Sirchia, Rosalia



Effects of Caffeine and Chlorogenic Acid on Propidium Iodide Accessibility to DNA: Consequences on Genome Size Evaluation in Coffee Tree  

PubMed Central

Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo?intraspecific variation in genome size. Two important compounds present in coffee trees—caffeine and chlorogenic acid—modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes. PMID:12876189




Toward metrological traceability for DNA fragment ratios in GM quantification. 3. Suitability of DNA calibrants studied with a MON 810 corn model.  


The quantification of GMOs by real-time PCR relies on an external calibrant. In this paper the suitability of two DNA calibrants, genomic DNA from plant leaves and plasmidic DNA, was investigated. The PCR efficiencies, the correlation coefficients of the calibration curves, and the ratios between PCR efficiencies of transgenic and endogenous sequences were compared for both calibrants using 59 data sets produced by 43 laboratories. There were no significant differences between plasmidic and genomic DNA except for the PCR efficiencies of the calibration curves for the transgene of the construct-specific real-time PCR method. In the GM system investigated, PCR efficiencies of plasmidic calibrants were slightly closer to the PCR efficiencies observed for the unknowns than those of the genomic DNA calibrant. Therefore, plasmidic DNA was the more suitable calibrant for the PCR measurements on genomic DNA extracted from MON 810 seeds. It is shown that plasmidic DNA is an appropriate choice for the calibration of measurements of MON 810 corn with respect to the DNA copy number ratio. PMID:17407307

Charels, Diana; Broeders, Sylvia; Corbisier, Philippe; Trapmann, Stefanie; Schimmel, Heinz; Emons, Hendrik



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.



RNA transcripts, miRNA-sized fragments and proteins produced from D4Z4 units: new candidates for the pathophysiology of facioscapulohumeral dystrophy  

PubMed Central

Deletion of a subset of the D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q causes facioscapulohumeral muscular dystrophy (FSHD) when occurring on a specific haplotype of 4qter (4qA161). Several genes have been examined as candidates for causing FSHD, including the DUX4 homeobox gene in the D4Z4 repeat, but none have been definitively shown to cause the disease, nor has the full extent of transcripts from the D4Z4 region been carefully characterized. Using strand-specific RT–PCR, we have identified several sense and antisense transcripts originating from the 4q D4Z4 units in wild-type and FSHD muscle cells. Consistent with prior reports, we find that the DUX4 transcript from the last (most telomeric) D4Z4 unit is polyadenylated and has two introns in its 3-prime untranslated region. In addition, we show that this transcript generates (i) small si/miRNA-sized fragments, (ii) uncapped, polyadenylated 3-prime fragments that encode the conserved C-terminal portion of DUX4 and (iii) capped and polyadenylated mRNAs that contain the double-homeobox domain of DUX4 but splice-out the C-terminal portion. Transfection studies demonstrate that translation initiation at an internal methionine can produce the C-terminal polypeptide and developmental studies show that this peptide inhibits myogenesis at a step between MyoD transcription and the activation of MyoD target genes. Together, we have identified new sense and anti-sense RNA transcripts, novel mRNAs and mi/siRNA-sized RNA fragments generated from the D4Z4 units that are new candidates for the pathophysiology of FSHD. PMID:19359275

Snider, Lauren; Asawachaicharn, Amy; Tyler, Ashlee E.; Geng, Linda N.; Petek, Lisa M.; Maves, Lisa; Miller, Daniel G.; Lemmers, Richard J.L.F.; Winokur, Sara T.; Tawil, Rabi; van der Maarel, Silvere M.; Filippova, Galina N.; Tapscott, Stephen J.



Codon Optimization of Gene Fragments Encoding Plasmodium falciparum Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice  

Microsoft Academic Search

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (80%) is much greater than that of Homo sapiens (59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA




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

PubMed Central

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

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



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

PubMed Central

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

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



Fragments of Cell-Free DNA (cfDNA) Enhance Transcription Activity in Human Mesenchymal Stem Cells (hMSCs) and Inhibit Their In Vitro Differentiation  

Microsoft Academic Search

\\u000a CpG-enriched rDNA accumulating in human cfDNA significantly stimulate gene transcription in mesenchymal stem cells by activating\\u000a TLR9 and MyD88-dependent signaling pathways and inhibiting differentiation of mesenchymal stem cells into adipocytes. These\\u000a data are important for stem cell therapy.

Elena M. Malinovskaya; Svetlana V. Kostyuk; Aleksey V. Ermakov; Marina S. Konkova; Tatjana D. Smirnova; Larisa V. Kameneva; Liudmila V. Efremova; Anna Yu. Alekseeva; Liudmila N. Lyubchenko; Natalya N. Veiko


Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene  

Microsoft Academic Search

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase- positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of




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

Microsoft Academic Search

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,

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



Size Dependent Free Solution DNA Electrophoresis in Structured Micro Fluidic Systems  

E-print Network

. The electrophoretic migration of single DNA molecules stained with the intercalator YOYO was investigated by real intercalators) labelled DNA molecules. This has been used by several groups to enlighten DNA migration of the migration of individual DNA molecules stained with the fluorescent intercalator YOYO. 4 #12;Materials


Cloning, Molecular Characterization, and Application of Rice Epiphytic Bacillus pumilus Promoter Fragments  

Microsoft Academic Search

To establish a constitutive, high-efficiency expression system for Bacillus pumilus (B.P), we cloned random chromosomal DNA into promoter probe shuttle vector ECE7 and selected for strong promoter activity\\u000a by chloramphenicol resistance of transformed B. pumilus cells. The nucleotide sequences of nine chromosomal fragments were determined. These DNA fragments range from 300 to 2200\\u000a bp in size. The transcription strength of

Qingyu Cao; Zhicai Qu; Youzhong Wan; Hongwei Zhang; Daleng Shen



Validation of a New Test for Schistosoma haematobium Based on Detection of Dra1 DNA Fragments in Urine: Evaluation through Latent Class Analysis  

PubMed Central

Background Diagnosis of urogenital schistosomiasis in chronically infected adults is challenging but important, especially because long term infection of the bladder and urinary tract can have dire consequences. We evaluated three tests for viable infection: detection of parasite specific DNA Dra1 fragments, haematuria and presence of parasite eggs for sensitivity (Se) and specificity (Sp). Methods Over 400 urine specimens collected from adult volunteers in an endemic area in Western Nigeria were assessed for haematuria then filtered in the field, the filter papers dried and later examined for eggs and DNA. The results were stratified according to sex and age and subjected to Latent Class analysis. Conclusions Presence of Dra1 in males (Se?=?100%; Sp?=?100%) exceeded haematuria (Se?=?87.6%: Sp?=?34.7%) and detection of eggs (Se?=?70.1%; Sp?=?100%). In females presence of Dra1 was Se?=?100%: Sp?=?100%, exceeding haematuria (Se?=?86.7%: Sp?=?77.0%) and eggs (Se?=?70.1%; Sp?=?100%). Dra1 became undetectable 2 weeks after praziquantel treatment. We conclude detection of Dra1 fragment is a definitive test for the presence of Schistosoma haematobium infection. PMID:22235360

Asaolu, Samuel; Moustaki, Irini; Shiff, Clive



The crystal structure of EcoRV endonuclease and of its complexes with cognate and non-cognate DNA fragments.  

PubMed Central

The crystal structure of EcoRV endonuclease has been determined at 2.5 A resolution and that of its complexes with the cognate DNA decamer GGGATATCCC (recognition sequence underlined) and the non-cognate DNA octamer CGAGCTCG at 3.0 A resolution. Two octamer duplexes of the non-cognate DNA, stacked end-to-end, are bound to the dimeric enzyme in B-DNA-like conformations. The protein--DNA interactions of this complex are prototypic for non-specific DNA binding. In contrast, only one cognate decamer duplex is bound and deviates considerably from canonical B-form DNA. Most notably, a kink of approximately 50 degrees is observed at the central TA step with a concomitant compression of the major groove. Base-specific hydrogen bonds between the enzyme and the recognition base pairs occur exclusively in the major groove. These interactions appear highly co-operative as they are all made through one short surface loop comprising residues 182-186. Numerous contacts with the sugar phosphate backbone extending beyond the recognition sequence are observed in both types of complex. However, the total surface area buried on complex formation is > 1800 A2 larger in the case of cognate DNA binding. Two acidic side chains, Asp74 and Asp90, are close to the reactive phosphodiester group in the cognate complex and most probably provide oxygen ligands for binding the essential cofactor Mg2+. An important role is also indicated for Lys92, which together with the two acidic functions appears to be conserved in the otherwise unrelated structure of EcoRI endonuclease. The structural results give new insight into the physical basis of the remarkable sequence specificity of this enzyme. Images PMID:8491171

Winkler, F K; Banner, D W; Oefner, C; Tsernoglou, D; Brown, R S; Heathman, S P; Bryan, R K; Martin, P D; Petratos, K; Wilson, K S



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

Microsoft Academic Search

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

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



DNA driven self-assembly of micron-sized rods using DNA-grafted bacteriophage fd virions  

E-print Network

We have functionalized the sides of fd bacteriophage virions with oligonucleotides to induce DNA hybridization driven self-assembly of high aspect ratio filamentous particles. Potential impacts of this new structure range from an entirely new building block in DNA origami structures, inclusion of virions in DNA nanostructures and nanomachines, to a new means of adding thermotropic control to lyotropic liquid crystal systems. A protocol for producing the virions in bulk is reviewed. Thiolated oligonucleotides are attached to the viral capsid using a heterobifunctional chemical linker. A commonly used system is utilized, where a sticky, single-stranded DNA strand is connected to an inert double-stranded spacer to increase inter-particle connectivity. Solutions of fd virions carrying complementary strands are mixed, annealed, and their aggregation is studied using dynamic light scattering (DLS), fluorescence microscopy, and atomic force microscopy (AFM). Aggregation is clearly observed on cooling, with some degr...

Unwin, R R; Yanagishima, T; Blower, T R; Takahashi, H; Salmond, G P C; Edwardson, J M; Fraden, S; Eiser, E



Behavioral and physiological responses to subgroup size and number of people in howler monkeys inhabiting a forest fragment used for nature-based tourism.  


Animals' responses to potentially threatening factors can provide important information for their conservation. Group size and human presence are potentially threatening factors to primates inhabiting small reserves used for recreation. We tested these hypotheses by evaluating behavioral and physiological responses in two groups of mantled howler monkeys (Alouatta palliata mexicana) at the "Centro Ecológico y Recreativo El Zapotal", a recreational forest reserve and zoo located in the Mexican state of Chiapas. Both groups presented fission-fusion dynamics, splitting into foraging subgroups which varied in size among, but not within days. Neither subgroup size nor number of people had an effect on fecal cortisol. Out of 16 behavioral response variables tested, the studied factors had effects on six: four were affected by subgroup size and two were affected by number of people. With increasing subgroup size, monkeys increased daily path lengths, rested less, increased foraging effort, and used more plant individuals for feeding. As the number of people increased, monkeys spent more time in lower-quality habitat, and less time engaged in social interactions. Although fecal cortisol levels were not affected by the factors studied, one of the monkey groups had almost twice the level of cortisol compared to the other group. The group with higher cortisol levels also spent significantly more time in the lower-quality habitat, compared to the other group. Our results suggest that particular behavioral adjustments might allow howler monkeys at El Zapotal to avoid physiological stress due to subgroup size and number of people. However, the fact that one of the monkey groups is showing increased cortisol levels may be interpreted as a warning sign, indicating that an adjustment threshold is being reached, at least for part of the howler monkey population in this forest fragment. PMID:23801542

Aguilar-Melo, Adriana R; Andresen, Ellen; Cristóbal-Azkarate, Jurgi; Arroyo-Rodríguez, Victor; Chavira, Roberto; Schondube, Jorge; Serio-Silva, Juan Carlos; Cuarón, Alfredo D



Aromaticity-induced changes in the electronic properties of size-expanded DNA bases: Case of xC.  

SciTech Connect

Size-expanded DNA bases are analogues of natural bases that can be described as a synthesis between benzene and a natural base. Size-expanded bases have been combined with natural bases to form xDNA and yDNA, a new class of synthetic nucleic acids. We are interested in xDNA and yDNA because they might function as molecular wires. Recently, we also became intrigued by the possibility of altering the electronic conductivity of xDNA and yDNA by means of structural changes in the constituent bases. This possibility appeared after we noticed that the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) gap of the base yG can be increased dramatically, {approx}0.73 eV, by changing the aromaticity of its benzene ring. Therefore, if one is able to alter the HOMO-LUMO gap of size-expanded bases, it should be possible to change the electronic conductivity of xDNAs and yDNAs as well. In the present work, we extend our study on aromaticity-induced changes on the electronic properties of size-expanded bases by investigating the HOMO-LUMO gap of all possible tautomers of xC. We have found that, as for yG, the HOMO-LUMO gap of xC can be modified by {approx} 0.74 eV, and that this can be accomplished by changing the aromaticity of its benzene ring.

Fuentes-Cabrera, Miguel A [ORNL; Lipkowski, Pawel [Wroclaw University of Technology, Poland; Huertas, Oscar [Universitat de Barcelona; Sumpter, Bobby G [ORNL; Orozco, Modesto [Institut de Recerca Biomedica, Parc Cientific de Barcelona, Barcelona, Spain; Luque, Javier [Universitat de Barcelona; Wells, Jack C [ORNL; Leszczynski, Jerzy [Computational Center for Molecular Structure and Interactions, Jackson, MS



Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species  

PubMed Central

Summary Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious. PMID:15998412

Baack, Eric J.; Whitney, Kenneth D.; Rieseberg, Loren H.



Effects of interleukin-1 beta fragment (163-171) on progesterone and estradiol-17 beta release by bovine granulosa cells from different size follicles.  


Recent studies indicate that interleukin-1 beta may play a role in ovarian function. To explore this possibility, we examined the effects of a