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1

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.

1993-02-01

2

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.

1993-01-01

3

DNA fragment sizing and sorting by laser-induced fluorescence  

DOEpatents

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)

1996-01-01

4

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.

1992-12-31

5

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))

1994-08-01

6

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.

2001-01-01

7

Preparation and characterization of an anti-DNA monoclonal antibody showing size selectivity toward DNA fragments.  

PubMed

Anti-DNA monoclonal antibodies were prepared using an in vitro immunization method. Balb/c mouse splenocytes were immunized with HeLa cell nuclear extract in the presence of N-acetylmuramyl-L-alanyl-D-isoglutamine and fused with P3U1 myeloma cells using PEG 4000. After HAT selection and ELISA using fragmented HeLa genomic DNA, an anti-DNA monoclonal antibody was obtained. The monoclonal antibody D-1-1, whose isotype was IgM, interacted with a variety of double-stranded DNA. The antibody reacted only with DNA fragments longer than 0.8 kbp, and its apparent dissociation constant for a 1.0-kbp DNA fragment was 34 nM. This antibody will be a helpful tool for the detection of DNA structures. PMID:15672610

Onishi, Yoshiaki; Kato, Megumi; Hanyu, Yoshiro

2004-10-01

8

Sizing Highly Fragmented DNA in Individual Apoptotic Cells Using the Comet Assay and a DNA Crosslinking Agent  

Microsoft Academic Search

TK6 human B lymphoblast cells exposed to ionizing radiation undergo apoptosis in a time and dose-dependent manner. The resulting highly fragmented DNA is easily detected using the comet assay, a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The degree of DNA fragmentation may be indicative of different stages in the fragmentation process, responses

Peggy L. Olive; Judit P. Banáth

1995-01-01

9

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

1996-01-01

10

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

NASA Technical Reports Server (NTRS)

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

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

2000-01-01

11

Monodisperse DNA restriction fragments  

Microsoft Academic Search

We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1–11 persistence lengths. The fragments were

Karel L. Planken; Gijsberta H. Koenderink; Ramon Roozendaal; Albert P. Philipse

2005-01-01

12

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.

2001-01-01

13

SINEs and LINEs cluster in distinct DNA fragments of Giemsa band size  

Microsoft Academic Search

By in situ hybridization, short interspersed repeated DNA elements (SINEs), exemplified by Alu repeats, are located principally in Giemsa-light human metaphase chromosome bands. In contrast, the L1 family of long interspersed repeats (LINEs) preferentially cluster in Giemsa-dark bands. These SINE\\/LINE patterns also generally correspond to early and later replication band patterns. In order to provide a molecular link between structurally

Terence L. Chen; Laura Manuelidis

1989-01-01

14

Reconstructing DNA replication kinetics from small DNA fragments  

Microsoft Academic Search

In higher organisms, DNA replicates simultaneously from many origins. Recent in vitro experiments have yielded large amounts of data on the state of replication of DNA fragments. From measurements of the time dependence of the average size of replicated and nonreplicated domains, one can estimate the rate of initiation of DNA replication origins, as well as the average rate at

Haiyang Zhang; John Bechhoefer

2006-01-01

15

DNA Fragmentation in Microorganisms Assessed In Situ?  

PubMed Central

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation 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 on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions. PMID:18689511

Fernández, José Luis; Cartelle, Mónica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, María; Goyanes, Vicente; Gosálvez, Jaime; Bou, Germán

2008-01-01

16

DNA fragmentation in microorganisms assessed in situ.  

PubMed

Chromosomal DNA fragmentation may be a direct or indirect outcome of cell death. Unlike DNA fragmentation 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 on a slide, incubated in a lysis buffer to partially remove the cell walls, membranes, and proteins, and then stained with a DNA fluorochrome, SYBR Gold. Identifying cells with fragmented DNA uses peripheral diffusion of DNA fragments. Cells without DNA fragmentation show only limited spreading of DNA fiber loops. These results have been seen in several gram-negative and gram-positive bacteria, as well as in yeasts. Detection of DNA fragmentation was confirmed by fluoroquinolone treatment and by DNA breakage detection-fluorescence in situ hybridization. Proteus mirabilis with spontaneously fragmented DNA during exponential and stationary growth or Escherichia coli with DNA damaged after exposure to hydrogen peroxide or antibiotics, such as ciprofloxacin or ampicillin, was clearly detected. Similarly, fragmented DNA was detected in Saccharomyces cerevisiae after amphotericin B treatment. Our assay may be useful for the simple and rapid evaluation of DNA damage and repair as well as cell death, either spontaneous or induced by exogenous stimuli, including antimicrobial agents or environmental conditions. PMID:18689511

Fernández, José Luis; Cartelle, Mónica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, María; Goyanes, Vicente; Gosálvez, Jaime; Bou, Germán

2008-10-01

17

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))

1993-01-01

18

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

SciTech Connect

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

Clark, S.M.; Mathies, R.A. [Univ. of California, Berkeley, CA (United States)] [Univ. of California, Berkeley, CA (United States)

1997-04-01

19

A Stochastic Model of DNA Fragments Rejoining  

PubMed Central

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

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

2012-01-01

20

DNA Sequence from Cretaceous Period Bone Fragments  

Microsoft Academic Search

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

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

1994-01-01

21

DNA sequences of RAPD fragments in artiodactyls.  

PubMed

A bovine RAPD profile, generated by a 10-mer primer, was analysed by sequencing the major fragments. Three of four different fragments showed homologies to previously characterized mammalian sequences. One was 61-66% identical to LINE sequences and another was 78.5% identical to a human chromosome 2 sequence tagged site. The third fragment was 93.1% identical to the human type 2 inositol 1,4,5-trisphosphate receptor gene. This fragment had counterparts in white-tailed deer and reindeer; fragments of slightly different size in these species showed high sequence similarity and the size differences were due to varying numbers of dinucleotide microsatellite repeats inside the fragment. PMID:8984008

Kostia, S; Palo, J; Varvio, S L

1996-04-01

22

Dependence on radiation quality of DNA fragmentation spectra  

Microsoft Academic Search

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

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

2008-01-01

23

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

1984-01-01

24

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

Microsoft Academic Search

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

JAE-CHANG CHO; JAMES M. TIEDJE

2001-01-01

25

Separation of DNA restriction fragments using capillary electrophoresis  

SciTech Connect

Gel-filled and non-gel' capillary electrophoresis (CE) have been applied to the separation of various DNA restriction fragments. 30% HydroLink gel, polymerized inside a 75[mu]m i.d. fused-silica capillary, was used in the gel-filled CE. Primary results show that the HL capillary gel was simple to cast, and its stability was reasonably good under the running conditions. In the non-gel CE experiment, a buffer containing the sieving additive hydroxypropylmethyl cellulose was used to affect the size-dependent separation. The use of GC capillaries eliminates the inconvenience of separately coating the capillary walls for efficient non-gel separation. Finally, the authors demonstrate that it is feasible to detect native DNA fragments using indirect fluorometry in non-gel capillary electrophoresis.

Chan, K.C.; Whang, Chenwen; Yeung, E.S. (Iowa State Univ., Ames (United States))

1993-01-01

26

Optical selection and collection of DNA fragments  

DOEpatents

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)

1998-01-01

27

a Relation for Fragment Sizes of Explosive Loaded Aluminum Cylinders  

NASA Astrophysics Data System (ADS)

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

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

2009-12-01

28

Bacterial natural transformation by highly fragmented and damaged DNA  

E-print Network

replication. We show that the adjacent nucleotide variations generated by uptake of short DNA fragments escapeBacterial natural transformation by highly fragmented and damaged DNA Søren Overballe-Petersena,1 for review August 14, 2013) DNA molecules are continuously released through decomposition of organic matter

Nielsen, Rasmus

29

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

2013-01-01

30

A Prototype Ultrasound Instrument To Size Stone Fragments During Ureteroscopy  

Microsoft Academic Search

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

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

2008-01-01

31

Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions  

SciTech Connect

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

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

1997-02-01

32

Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA  

Microsoft Academic Search

Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the \\

Lorena S. Beese; Victoria Derbyshire; Thomas A. Steitz

1993-01-01

33

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

E-print Network

Reconstructing DNA replication kinetics from small DNA fragments Haiyang Zhang and John Bechhoefer 2006; published 5 May 2006 In higher organisms, DNA replicates simultaneously from many origins. Recent in vitro experiments have yielded large amounts of data on the state of replication of DNA fragments. From

Bechhoefer, John

34

Hyperglycemia Enhances DNA Fragmentation After Transient Cerebral Ischemia  

Microsoft Academic Search

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular

Ping-An Li; Ingrid Rasquinha; Qing Ping He; Katalin Csiszár; Charles D. Boyd; John P. MacManus

2001-01-01

35

A Mechanism of Gene Amplification Driven by Small DNA Fragments  

PubMed Central

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

Mukherjee, Kuntal; Storici, Francesca

2012-01-01

36

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning  

Microsoft Academic Search

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes

Elena R. Lozovskaya; Dmitri A. Petrov; Daniel L. Hartl

1993-01-01

37

Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes  

SciTech Connect

An early event in death of interphase lymphocytes exposed in vivo or in vitro to low doses of gamma-irradiation is the degradation of DNA into nucleosome-sized fragments. Induction of fragmentation required RNA and protein synthesis because actinomycin D and cycloheximide, respectively, are able to inhibit DNA fragmentation in irradiated lymphocytes. Studies adding cycloheximide and actinomycin D at various times postirradiation suggest that once the metabolic process is initiated within an individual cell it proceeds to completion. The reversible RNA synthesis inhibitor, 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole inhibits DNA fragmentation in irradiated thymocytes. When this drug is removed after 6 hr, irradiated thymocytes proceed to fragment their DNA; this suggests that an inducing signal that is not simply mRNA persists within the irradiated cell for at least 6 hr after irradiation. In contrast to mitogen-activated T and B lymphoblasts, resting T and B cells show significant DNA fragmentation after exposure to 100 to 500 rad. At 2000 rad, all of the splenic subpopulations die rapidly via a different mechanism. By studying the mechanism of DNA fragmentation induced during the interphase death of lymphocytes, we hope to understand better the extreme sensitivity of resting lymphocytes to radiation and what may be the common final pathway of programmed cell death.

Sellins, K.S.; Cohen, J.J.

1987-11-15

38

Non-random DNA fragmentation in next-generation sequencing  

PubMed Central

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

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

2014-01-01

39

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)

1996-01-01

40

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

2013-01-01

41

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

1992-01-01

42

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

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

2011-01-05

43

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.

1983-01-01

44

SEARCHING FOR AGROBACTERIAL T-DNA FRAGMENTS IN PLANT GENOMES  

Microsoft Academic Search

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

45

Advanced microinstrumentation for rapid DNA sequencing and large DNA fragment separation  

SciTech Connect

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

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

1995-01-25

46

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

47

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

1994-01-01

48

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

SciTech Connect

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

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)] [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

2012-07-06

49

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.

2013-12-01

50

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

1996-01-01

51

Novel separation and detection methods of DNA fragments in electrophoresis  

SciTech Connect

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

Chan, King Cheung.

1993-01-27

52

Novel separation and detection methods of DNA fragments in electrophoresis  

SciTech Connect

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

Chan, K.C.

1992-01-01

53

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

PubMed

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

Wang, Xiaofang; Son, Ahjeong

2013-12-01

54

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

2013-10-29

55

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

2011-01-01

56

Modeling the dust size distribution in comets with dust fragmentation  

NASA Technical Reports Server (NTRS)

A hydrodynamic model was developed of a spherically symmetric dusty gas flow in a cometary atmosphere assuming a single fluid, inviscid, perfect gas. The hydrodynamics for gas and dust, which involves the gas drag force (momentum transfer), heat exchange between gas and dust, photodissociation energy for H2O gas, and radiative heating and cooling terms for dust particles, are solved using the Gear method for stiff, coupled differential equations. Calculations were done with a dust size distribution for radii alpha = 0.01 micron to 10 cm with densities variable with the size. A nucleus size of 4.0 km radius with a density of 0.5 g/cu cm and a total dust-to-gas mass ratio chi = 1 was adopted. There are indications from in situ observations that dust particle fragment into smaller ones. Fragmentation of dust particles was incorporated into the model. This is done by adding source and sink terms in the continuity equations for the dust. Lifetimes for the decay of dust particles were assumed as a function of particle size. It is also assumed that dust particles always fragment only into the next smaller size.

Konno, Ichishiro; Huebner, Walter F.

1990-01-01

57

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

PubMed

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

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

2015-05-15

58

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

PubMed

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

2007-08-01

59

Cell nucleus and DNA fragmentation are not required for apoptosis  

PubMed Central

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 cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death. PMID:7523418

1994-01-01

60

Rearrangement of DNA fragments: a branch-and-cut algorithm  

Microsoft Academic Search

We consider a problem called minimum k-contig problem (MkCP), whose specialization to an alphabet with four symbols can be seen as a problem that arises in the process of arranging DNA fragments to reconstruct a molecule. We present a graph theoretical formulation of MkCP and mention some extensions. We show this problem to be NP-hard for every k?1 (for an

C. E. Ferreira; C. Carvalho De Souza; Yoshiko Wakabayashi

2002-01-01

61

Diffusional spinning as a probe of DNA fragments conformation  

NASA Astrophysics Data System (ADS)

The dependence of the spinning diffusion coefficient of a wormlike chain upon contour length L, persistence length P, and radius R is shown here to follow a ``Lorentzian'' law of width ? vs ??L/R, where ?2?=l0/P is the variance of the bending angles distribution of Monte Carlo simulated chains with bond length l0. This description is equivalent to that of a spinning cylinder of length L and effective radius Reff=R(L,P), with Reff?R. When considering experimental data it is found that fluorescence polarization anisotropy (FPA), a technique very sensitive to spinning, also yields apparent DNA radii depending upon fragment length. In order to derive DNA parameters which are independent of fragment length, we introduce a procedure for fitting FPA data which takes into account thermal distortions and employs the parametric expressions for rigid body rotations, spinning and tumbling, depending only upon L, P, and the actual DNA radius, R. Then the apparent persistence length P can be estimated once a value of R is assumed together with the value of the dynamic persistence length, the latter affecting the internal bending motions of the fragments. Fitting the FPA data is easily accomplished with the value of R=10 Å as suggested by a number of recent measurements.

Collini, Maddalena; Chirico, Giuseppe; Baldini, Giancarlo

1996-04-01

62

Small DNA Pieces in C. elegans Are Intermediates of DNA Fragmentation during Apoptosis  

PubMed Central

While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, ?10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment. While the small DNA pieces are also present in a number of strains with mutations in predicted nucleases, they are undetectable in strains containing mutations in nuc-1, which encodes a DNase II endonuclease. We find that the small DNA pieces can be labeled with terminal deoxynucleotidyltransferase only after phosphatase treatment, as expected if they are products of DNase II cleavage, which generates a 3? phosphate. Our studies reveal a previously unknown intermediate in the process of apoptotic DNA fragmentation and thus bring us closer to defining this important pathway. PMID:20585459

Aruscavage, P. Joseph; Hellwig, Sabine; Bass, Brenda L.

2010-01-01

63

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

SciTech Connect

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

Heinzel, S.S.; Krysan, P.J.; Tran, C.T.; Calos, M.P. (Stanford University School of Medicine, CA (United States))

1991-04-01

64

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.

2008-09-01

65

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

PubMed

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

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

2014-10-17

66

Detection of Irradiated Food: DNA Fragmentation in Grapefruits  

NASA Astrophysics Data System (ADS)

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

Delincée, Henry

1998-06-01

67

Structure of DNA polymerase I Klenow fragment bound to duplex DNA.  

PubMed

Klenow fragment of Escherichia coli DNA polymerase I, which was cocrystallized with duplex DNA, positioned 11 base pairs of DNA in a groove that lies at right angles to the cleft that contains the polymerase active site and is adjacent to the 3' to 5' exonuclease domain. When the fragment bound DNA, a region previously referred to as the "disordered domain" became more ordered and moved along with two helices toward the 3' to 5' exonuclease domain to form the binding groove. A single-stranded, 3' extension of three nucleotides bound to the 3' to 5' exonuclease active site. Although this cocrystal structure appears to be an editing complex, it suggests that the primer strand approaches the catalytic site of the polymerase from the direction of the 3' to 5' exonuclease domain and that the duplex DNA product may bend to enter the cleft that contains the polymerase catalytic site. PMID:8469987

Beese, L S; Derbyshire, V; Steitz, T A

1993-04-16

68

DNA Flexibility Studied by Covalent Closure of Short Fragments into Circles  

NASA Astrophysics Data System (ADS)

The ring closure probability, or j factor, has been measured for DNA restriction fragments of defined sequence bearing EcoRI cohesive ends and ranging in size from 126 to 4361 base pairs (bp). The j factor is defined as the ratio of the equilibrium constants for cyclization and for bimolecular association via the cohesive ends. The end-joining reactions are fast compared to covalent closure of the cohesive ends by T4 DNA ligase. The rate of ligase closure is shown to be proportional to the equilibrium fraction of DNA molecules with joined cohesive ends, both in cyclization and in bimolecular association reactions. The j factor changes by less than 10-fold between 242 and 4361 bp, whereas it decreases by more than 100-fold between 242 and 126 bp as the DNA reaches the size range of the persistence length (150 bp). As regards ring closure, short DNA fragments are surprisingly flexible. These data are in good agreement with predictions by others for the ring closure probability of a wormlike chain.

Shore, David; Langowski, Jorg; Baldwin, Robert L.

1981-08-01

69

Ribosomal RNA genes in Euglena gracilis mitochondrial DNA: fragmented genes in a seemingly fragmented genome  

Microsoft Academic Search

Because relatively little information is available about mtDNA in the euglenid protozoa, distant relatives of the kinetoplastid\\u000a protozoa, we investigated mitochondrial genome structure and expression in Euglena gracilis. We found that isolated E. gracilis mtDNA comprises a heterodisperse collection of short molecules (modal size ~4 kbp) and that the mitochondrial large subunit\\u000a (LSU) and small subunit (SSU) rRNAs are each split

David F. Spencer; Michael W. Gray

2011-01-01

70

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

NASA Technical Reports Server (NTRS)

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

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

2004-01-01

71

Does varicocelectomy affect DNA fragmentation in infertile patients?  

PubMed Central

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

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

2015-01-01

72

Replicon size of yeast ribosomal DNA  

Microsoft Academic Search

The ribosomal RNAs of the yeast Saccharomyces cerevisiae are transcribed from a 9Kbp stretch of DNA which is reiterated about 120-fold in a continuous array, about 360 µm long, on chromosome XII. Although ARS activity has been detected in the repeat unit, the size and disposition of replicons along this array of identical genes has not hitherto been determined. We

Richard M. Walmsley; Leland H. Johnston; Donald H. Williamson; Stephen G. Oliver

1984-01-01

73

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

2001-01-01

74

Phylogenomics of caspase-activated DNA fragmentation factor.  

PubMed

The degradation of nuclear DNA by DNA fragmentation factor (DFF) is a key step in apoptosis of mammalian cells. Using comparative genomics, we have here determined the evolutionary history of the genes encoding the two DFF subunits, DFFA (also known as ICAD) and DFFB (CAD). Orthologs of DFFA and DFFB were identified in Nematostella vectensis, a representative of the primitive metazoan clade cnidarians, and in various vertebrates and insects, but not in representatives of urochordates, echinoderms, and nematodes. The domains mediating the interaction of DFFA and DFFB, a caspase cleavage site in DFFA, and the amino acid residues critical for endonuclease activity of DFFB were conserved in Nematostella. These findings suggest that DFF has been a part of the primordial apoptosis system of the eumetazoan common ancestor and that the ancient cell death machinery has degenerated in several evolutionary lineages, including the one leading to the prototypical apoptosis model, Caenorhabditis elegans. PMID:17343828

Eckhart, Leopold; Fischer, Heinz; Tschachler, Erwin

2007-04-27

75

Phylogenomics of caspase-activated DNA fragmentation factor  

SciTech Connect

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

Eckhart, Leopold [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)]. E-mail: leopold.eckhart@meduniwien.ac.at; Fischer, Heinz [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria); Tschachler, Erwin [Department of Dermatology, Medical University of Vienna, A-1090 Vienna (Austria)

2007-04-27

76

A Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome  

E-print Network

for isolation and lab cultivation of individual species [4]. The whole genome(DNA) or metagenome population canA Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome Sara Nasser, Adrienne classifier is used to group shotgun sequence fragments as small as 500 base pairs according to their DNA

Nicolescu, Monica

77

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

78

Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA  

PubMed Central

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

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

2012-01-01

79

RegionSpecific Interrelations between Apoptotic Proteins Expression and DNA Fragmentation in the Neonatal Rat Brain  

Microsoft Academic Search

DNA fragmentation, mRNA and protein levels of Bcl-XL, Bax and caspase-3 were determined to characterize interrelations between expression of these apoptotic markers in the neonatal brain regions. High DNA fragmentation intensity in the cortex was in consonance with the lowest Bcl-XL\\/Bax expression ratio, the highest procaspase-3 and active caspase-3 levels. Low and intermediate DNA fragmentation levels in the cerebellum and

Petr N. Menshanov; Anita V. Bannova; Nikolay N. Dygalo

2006-01-01

80

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

PubMed

The sequence-specific correction of a mutated gene (e.g., point mutation) by the Small Fragment Homologous Replacement (SFHR) method is a highly attractive approach for gene therapy. Small DNA fragments (SDFs) were used in SFHR to modify endogenous genomic DNA in both human and murine cells. The advantage of this gene targeting approach is to maintain the physiologic expression pattern of targeted genes without altering the regulatory sequences (e.g., promoter, enhancer), but the application of this technique requires the knowledge of the sequence to be targeted. In our recent study, an optimized SFHR protocol was used to replace the eGFP mutant sequence in SV-40-transformed mouse embryonic fibroblast (MEF-SV40), with the wild-type eGFP sequence. Nevertheless in the past, SFHR has been used to correct several mutant genes, each related to a specific genetic disease (e.g., spinal muscular atrophy, cystic fibrosis, severe combined immune deficiency). Several parameters can be modified to optimize the gene modification efficiency, as described in our recent study. In this chapter we describe the main guidelines that should be followed in SFHR application, in order to increase technique efficiency. PMID:24557898

Luchetti, Andrea; Malgieri, Arianna; Sangiuolo, Federica

2014-01-01

81

Single-cell pulsed-field gel electrophoresis to detect the early stage of DNA fragmentation in human sperm nuclei.  

PubMed

Single-cell pulsed-field gel electrophoresis (SCPFGE) with dual electrode pairs was developed to detect the early stage of DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugation technique and subsequent swim-up. The sperm were embedded in a thin film of agarose containing bovine trypsin (20 µg/mL) and were then lysed. Prior to SCPFGE, proteolysis of DNA-binding components, such as protamine and the nuclear matrix was essential to separate the long chain fibers from the fibrous and granular fragments derived from a single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragments were observed at the beginning of the process, however, as the fragmentation advanced, the long chain fibers decreased and shortened, and, conversely, the granular fragments increased until finally almost all the DNA was shredded. Although the ejaculate contained sperm with heterogeneous stages, the purified motile sperm exhibited several dozens of uniformly elongated fibers arising from the tangled DNA at the origin, whereas a part of these fibers gave rise to fibrous fragments beyond the tip of the elongated fibers, and their numbers and sizes varied among the sperm. Conventional intra-cytoplasmic sperm injection (ICSI) usually depends on intra-operative light microscopic observation to select a sperm for injection. The present results revealed that sperm motility could not give full assurance of DNA integrity. SCPFGE is likely to serve an important role in the preoperative differential diagnosis to determine the competence of the sperm population provided for injection. PMID:22848752

Kaneko, Satoru; Yoshida, Joji; Ishikawa, Hiromichi; Takamatsu, Kiyoshi

2012-01-01

82

Cocrystal Structure of an Editing Complex of Klenow Fragment with DNA  

Microsoft Academic Search

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydophobic interactions between Phe-473, Leu-361, and

P. S. Freemont; J. M. Friedman; L. S. Beese; M. R. Sanderson; T. A. Steitz

1988-01-01

83

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

DOEpatents

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)

2000-01-01

84

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

PubMed Central

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

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

1978-01-01

85

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

PubMed

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

2014-01-01

86

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

87

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

1975-01-01

88

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

ERIC Educational Resources Information Center

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

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

2012-01-01

89

Structural analyses of DNA fragments integrated by illegitimate recombination in Schizosaccharomyces pombe  

Microsoft Academic Search

In order to elucidate the mechanisms of illegitimate recombination in eukaryotes, we have studied the structure of DNA fragments integrated by illegitimate recombination into the genome of fission yeast. Nonhomologous recombination was rarely identified when a long region of homology with the chromosomal leu1+ gene was present in the introduced leu1::ura4+ DNA fragment; but a decrease in length of homology

Kazuo Tatebayashi; Jun-ichi Kato; Hideo Ikeda

1994-01-01

90

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

2014-01-01

91

Key morphological features of apoptosis may occur in the absence of internucleosomal DNA fragmentation.  

PubMed Central

Apoptosis, a major form of cell death, is characterized by chromatin condensation, a reduction in cell volume and endonuclease cleavage of DNA into oligonucleosomal length fragments. The detection of these fragments by gel electrophoresis, as a DNA ladder, is currently used as the major biochemical index of apoptosis. Here we report that key morphological changes of apoptosis can be dissociated experimentally from the DNA fragmentation produced by endonuclease activity. Internucleosomal cleavage of DNA is thus likely to be a later event in the apoptotic process. Images Fig. 2. Fig. 3. PMID:1530564

Cohen, G M; Sun, X M; Snowden, R T; Dinsdale, D; Skilleter, D N

1992-01-01

92

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.

1996-01-01

93

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.  

PubMed Central

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment. PMID:2453027

de Vroom, E; Roelen, H C; Saris, C P; Budding, T N; van der Marel, G A; van Boom, J H

1988-01-01

94

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

1997-01-01

95

RADOM, an Efficient In Vivo Method for Assembling Designed DNA Fragments up to 10 kb Long in Saccharomyces cerevisiae.  

PubMed

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

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

2015-03-20

96

Okazaki Fragment Maturation in Yeast I. DISTRIBUTION OF FUNCTIONS BETWEEN FEN1 AND DNA2*  

E-print Network

/s). The Dna2 nuclease/helicase alone did not efficiently promote nick translation, nor did it affect nick translation with FEN1. Maturation in the presence of DNA ligase was studied with various downstream primersOkazaki Fragment Maturation in Yeast I. DISTRIBUTION OF FUNCTIONS BETWEEN FEN1 AND DNA2* Received

Burgers, Peter M.

97

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

2014-01-01

98

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I.  

PubMed

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA. PMID:9485315

Carver, T E; Millar, D P

1998-02-17

99

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

PubMed Central

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

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

2014-01-01

100

Effect of Cryopreservation on the Sperm DNA Fragmentation Dynamics of the Bottlenose Dolphin (Tursiops truncatus).  

PubMed

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

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

2015-04-01

101

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks  

PubMed Central

Resolution of DNA fragments separated by electrophoresis in polymer solutions (“matrices”) is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (> 150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependences of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms. PMID:22648809

Fredlake, Christopher P.; Hert, Daniel G.; Niedringhaus, Thomas P.; Lin, Jennifer S.; Barron, Annelise E.

2015-01-01

102

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)

1995-09-01

103

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

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

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

2005-01-01

104

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.

1995-01-01

105

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

1995-01-01

106

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

PubMed Central

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

2013-01-01

107

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

2000-02-15

108

Two-dimensional conformation-dependent electrophoresis (2D-CDE) to separate DNA fragments containing unmatched bulge from complex DNA samples  

PubMed Central

DNA fragments containing mispaired and modified bases, bulges, lesions and specific sequences have altered conformation. Methods for separating complex samples of DNA fragments based on conformation but independent of length have many applications, including (i) separation of mismatched or unmatched DNA fragments from those perfectly matched; (ii) simultaneous, diagnostic, mismatch scanning of multiple fragments; (iii) isolation of damaged DNA fragments from undamaged fragments; and (iv) estimation of reannealing efficiency of complex DNA samples. We developed a two-dimensional conformation-dependent electrophoresis (2D-CDE) method for separating DNA fragments based on length and conformation in the first dimension and only on length in the second dimension. Differences in migration velocity due to conformation were minimized during second dimension electrophoresis by introducing an intercalator. To test the method, we constructed 298 bp DNA fragments containing cytosine bulges ranging from 1 to 5 nt. Bulge-containing DNA fragments had reduced migration velocity in the first dimension due to altered conformation. After 2D-CDE, bulge-containing DNA fragments had migrated in front of an arc comprising heterogeneous fragments with regular conformation. This simple and robust method could be used in both analytical and preparative applications involving complex DNA samples. PMID:14762200

Gunnarsson, Gudmundur H.; Thormar, Hans G.; Gudmundsson, Bjarki; Akesson, Lina; Jonsson, Jon J.

2004-01-01

109

Estimating Dataset Size Requirements for Classifying DNA Microarray Data  

E-print Network

Estimating Dataset Size Requirements for Classifying DNA Microarray Data S. Mukherjee*+#1 , P words: Gene expression profiling, molecular pattern recognition, DNA microarrays, microarray analysis;1. Introduction Over the last few years the routine use of DNA microarrays has made possible the creation of large

Poggio, Tomaso

110

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

PubMed

We report a size-controllable synthesis of stable aqueous solutions of ultrapure low-size-dispersed Au nanoparticles by methods of femtosecond laser fragmentation from preliminary formed colloids. Such approach makes possible the tuning of mean nanoparticle size between a few nm and several tens of nm under the size dispersion lower than 70% by varying the fluence of pumping radiation during the fragmentation procedure. The efficient size control is explained by 3D geometry of laser fragmentation by femtosecond laser-induced white light super-continuum and plasma-related phenomena. Despite the absence of any protective ligands, the nanoparticle solutions demonstrate exceptional stability due to electric repulsion effect associated with strong negative charging of formed nanoparticles. Stable aqueous solutions of bare gold nanoparticles present a unique object with a variety of potential applications in catalysis, surface-enhanced Raman spectroscopy, photovoltaics, biosensing and biomedicine. PMID:25605000

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

2015-02-13

111

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

NASA Astrophysics Data System (ADS)

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

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

2015-02-01

112

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

NASA Astrophysics Data System (ADS)

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

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

113

Isolation and characterization of two repetitive DNA fragments located near the centromere of the mouse X chromosome  

Microsoft Academic Search

Two repetitive DNA fragments located on the mouse X chromosome are described. The fragments were isolated from a lambda phage library enriched in X-chromosomal sequences by flow sorting. Both fragments, which are repeated 20 to 50 times in the genome, were mapped to the mouse X chromosome by Southern blot hybridization to DNA from hybrid cells retaining the mouse X

C. M. Disteche; U. Tantravahi; S. Gandy; M. Eisenhard; D. Adler; L. M. Kunkel

1985-01-01

114

Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation  

Microsoft Academic Search

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Cur- rently, its existence is inferred mainly from gel elec- trophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the devel- opment of a

Yael Gavrieli; Yoav Sherman; Shmuel A. Ben-Sasson

1992-01-01

115

CONFORMATIONAL STABILITY OF PrP AMYLOID FIBRILS CONTOLS THEIR SMALLEST POSSIBLE FRAGMENT SIZE  

PubMed Central

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

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

2008-01-01

116

Size Matters: Origin of Binomial Scaling in Nuclear Fragmentation Experiments  

E-print Network

The relationship between measured transverse energy, total charge recovered in the detector, and size of the emitting system is investigated. Using only very simple assumptions, we are able to reproduce the observed binomial emission probabilities and their dependences on the transverse energy.

Wolfgang Bauer; Scott Pratt

1998-08-27

117

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

MARK V. LOMOLINO; DAVID R. PERAULT

2007-01-01

118

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

NASA Astrophysics Data System (ADS)

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

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

119

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

PubMed

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

2009-07-01

120

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

NASA Technical Reports Server (NTRS)

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

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

1986-01-01

121

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

1986-01-01

122

Viability and DNA fragmentation in differently sorted boar spermatozoa  

Microsoft Academic Search

Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of

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

2006-01-01

123

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

PubMed Central

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

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

2015-01-01

124

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

NASA Astrophysics Data System (ADS)

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

Virgós, Emilio; García, Francisco J.

2002-08-01

125

Mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets.  

PubMed

We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets. PMID:18253826

Chiang, Ming-Chou; Ashraf, Qazi M; Mishra, Om P; Delivoria-Papadopoulos, Maria

2008-07-01

126

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

PubMed Central

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

1994-01-01

127

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

PubMed

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

Moazzam, A; Sharma, R; Agarwal, A

2014-10-24

128

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

PubMed Central

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

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

2014-01-01

129

Effect of magnesium ions and temperature on the sequence-dependent curvature of DNA restriction fragments  

PubMed Central

Transient electric birefringence has been used to quantitate the curvature of two DNA restriction fragments, a 199-base pair fragment taken from the origin of replication of the M13 bacteriophage and a 207-base pair fragment taken from the VP1 gene in the SV40 minichromosome. Stable curvature in the SV40 and M13 restriction fragments is due a series of closely spaced A-tracts, runs of 4 to 6 contiguous adenine residues located within 40 or 60 base pair “curvature modules” near the center of each fragment. The M13 and SV40 restriction fragments exhibit bends of ~45° in solutions containing monovalent cations and ~58° in solutions containing Mg2+ ions. The curvature is not localized at a single site but is distributed over the various A-tracts in the curvature modules. Thermal denaturation studies indicate that the curvature in the M13 and SV40 restriction fragments remains constant up to 30°C in solutions containing monovalent cations, and up to 40°C in solutions containing Mg2+ ions, before beginning to decrease slowly with increasing temperature. Hence, stable curvature in these DNA restriction fragments exists at the biologically important temperature of 37°C. PMID:21406776

Stellwagen, Nancy C.; Lu, Yongjun

2011-01-01

130

The flux of Tunguska-sized fragments from the main asteroid belt  

Microsoft Academic Search

The numerical model described in Menichella et al. (Earth, Moon and Planets72, 133–149, 1996) is used to investigate the flux of Tunguska-sized asteroid fragments into chaotic resonant orbits leading them to attain an Earth-crossing status. The assumed main-belt size distribution is derived from that of known asteroids, extrapolated down to sizes ? 1 m and modified in such a way

Paolo Farinella; Mario Menichella

1998-01-01

131

DNA-fragments are transcytosed across CaCo-2 cells by adsorptive endocytosis and vesicular mediated transport.  

PubMed

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 >10(3) 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, Bjørn; Wiik-Nielsen, Christer R; Kristoffersen, Anja B; Holst-Jensen, Arne; Berdal, Knut G

2013-01-01

132

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

PubMed Central

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

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

1983-01-01

133

Sperm chromatin dispersion test in the assessment of DNA fragmentation and aneuploidy in human spermatozoa.  

PubMed

Sperm DNA damage is thought to be increased in men with male factor infertility. Previous studies suggest a correlation between sperm DNA fragmentation and aneuploidy. The sperm chromatin dispersion (SCD) test was modified to produce the Halosperm Kit. The SCD-fluorescent in-situ hybridization (FISH) test allows the simultaneous detection of DNA fragmentation and aneuploidy on the same sperm cell. The objectives of this study were to validate the SCD, SCD-FISH and Halosperm tests for the analysis of sperm DNA fragmentation and compare them to the sperm chromatin structure assay (SCSA). Semen samples from 20 males undergoing IVF/intracytoplasmic sperm injection were processed using FISH, SCD-FISH, SCD and Halosperm, and compared with SCSA results. There was a significant difference between FISH and SCD-FISH results in the detection of aneuploidy (P=0.000) and the level of sperm DNA fragmentation in the samples subjected to SCSA and SCD (P=0.001) or SCSA and SCD-FISH (P=0.001). There was no significant correlation between DNA fragmentation and aneuploidy. If sperm aneuploidy is to be determined, more reliable results will be obtained if FISH is performed rather than SCD-FISH. A lack of validation and unknown clinical significance question the value of DNA fragmentation assays. DNA damage in the male germ line may result in adverse clinical outcomes and the pathophysiology and clinical consequences of sperm DNA damage are being actively researched. Many DNA fragmentation assays such as the Halosperm Kit have been developed recently and are now available at a commercial level. Unfortunately, aimed at vulnerable couples with difficulty conceiving, many of these tests have not been clinically validated. Despite its plausible appeal and fervour of its supporters, the benefits of widespread DNA testing that only achieves the distressing of couples with the knowledge that effectual therapeutic strategies are absent are questionable. Commercially, however, it is no doubt lucrative. Analysis of gametes prior to the initiation of an IVF cycle may improve the quality of embryos transferred. The clinical and scientific community considers it a matter of urgency to translate the basic science behind how a cell prepares for fertilization into routine clinical practice. However, it is equally important, if not more, to allow the science behind such applications to draw level with its practice before its widespread implementation. PMID:21397561

Balasuriya, A; Speyer, B; Serhal, P; Doshi, A; Harper, J C

2011-05-01

134

DNA-Tract Curvature Profile Reconstruction: A Fragment Flipping Algorithm  

Microsoft Academic Search

At nanometric level of resolution DNA molecules can be idealized with one dimensional curved line. The curvature value along\\u000a this line is composed by static and dynamic contributions. The first ones constitute the intrinsic curvature, vectorial function\\u000a of the sequence of DNA nucletides, while the second ones, caused by thermal energy, constitute the flexibility. The analysis\\u000a of intrinsic curvature are

Daniele Masotti; Viale Risorgimento

2002-01-01

135

Efficient combination of large DNA in vitro: in gel site specific recombination (IGSSR) of PAC fragments containing alpha satellite DNA and the human HPRT gene locus.  

PubMed Central

In an attempt to combine a cloned genomic copy of a selectable gene with different cloned centromeric sequences to develop mammalian artificial chromosomes (MAC) we used site specific recombination mediated by purified Cre recombinase acting on the loxP sequence in PAC vector DNA. A new method was required to purify highly concentrated, virtually 100% intact PAC DNA which could be stored for a long period. Here we show the efficient linking of linearized PACs containing alpha satellite DNA from chromosomes X and 17 with sizes of 125 and 140 kb, respectively, to a 95 kb restriction fragment derived from a 175 kb PAC containing the intact human HPRT gene locus. PMID:9153331

Schindelhauer, D; Cooke, H J

1997-01-01

136

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. PMID:25373175

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

2014-01-01

137

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

PubMed

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

1991-01-01

138

Comprehensive Study On The Metastable Negative Ion Fragmentation Of Individual Dna Components And Larger Oligonucleotides  

NASA Astrophysics Data System (ADS)

Here we present a systematic study on the unimolecular decay pathways of the deprotonated building blocks of DNA and RNA to address the following questions: 1. Are the negative ion fragmentation patterns observed in the metastable decay of individual DNA components still evident when these are combined to larger oligonucleotides? 2. What is the significance of the charge location in determining the fragmentation pathways in the metastable decay process? 3. Are those metastable decay channels relevant in dissociative electron attachment to DNA components? To address these questions we have studied the fragmentation patterns of the deprotonated ribose and ribose 5'-monophosphate, the fragmentation patterns of the individual bases, all nucleosides and all 2'-deoxynucleosides as well as the individual nucleotides and several combinations of hexameric oligonucleotides. Furthermore, to understand the significance of the charge location in determining the fragmentation path in the metastable decay process of these deprotonated ions we have also studied modified uridine and guanosine. These have been modified to block different deprotonation sites and thus to control the initial step in the in the fragmentation process i.e. the site of deprotonation. In addition to our experimental approach we have also simulated the metastable fragmentation of the deprotonated uridine and 2'-deoxyguanosine to clarify the mechanisms and fragmentation patterns observed. Where data is available, the results are compared to dissociative electron attachment to DNA components and discussed in context to the underlying mechanism. Experiments on modified nucleosides where selected deprotonation sites have been blocked are used to verify the predicted reaction paths and imulations on uridine and 2'-deoxyguanosine are compared to the experimental results and used to shed light on the mechanisms involved.

Ingolfsson, O.; Flosadottir, H. D.; Omarsson, B.; Ilko, B.

2010-07-01

139

A Y-chromosomal DNA Fragment Is Conserved in Human and Chimpanzee1  

Microsoft Academic Search

A human male-specific Y-chromosomal DNA fragment (hYH2D6) has been iso- lated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq 11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fmgments were detected

B. K. A. Rasheed; E. C. Whisenant; R. Fernandez; H. Ostrer

140

Size exclusion chromatography of plasmid DNA isoforms  

Microsoft Academic Search

There is considerable interest in using size exclusion chromatography (SEC) to analyze and purify specific plasmid isoforms, but there is currently no fundamental understanding of the effects of plasmid size and morphology on plasmid behavior in SEC. Experiments were performed for plasmids from 3.0 to 17.0kbp in size. The linear and open-circular isoforms were generated from the supercoiled plasmid by

David R. Latulippe; Andrew L. Zydney

2009-01-01

141

Cell Size and the Initiation of DNA Replication in Bacteria  

PubMed Central

In eukaryotes, DNA replication is coupled to the cell cycle through the actions of cyclin-dependent kinases and associated factors. In bacteria, the prevailing view, based primarily from work in Escherichia coli, is that growth-dependent accumulation of the highly conserved initiator, DnaA, triggers initiation. However, the timing of initiation is unchanged in Bacillus subtilis mutants that are ?30% smaller than wild-type cells, indicating that achievement of a particular cell size is not obligatory for initiation. Prompted by this finding, we re-examined the link between cell size and initiation in both E. coli and B. subtilis. Although changes in DNA replication have been shown to alter both E. coli and B. subtilis cell size, the converse (the effect of cell size on DNA replication) has not been explored. Here, we report that the mechanisms responsible for coordinating DNA replication with cell size vary between these two model organisms. In contrast to B. subtilis, small E. coli mutants delayed replication initiation until they achieved the size at which wild-type cells initiate. Modest increases in DnaA alleviated the delay, supporting the view that growth-dependent accumulation of DnaA is the trigger for replication initiation in E. coli. Significantly, although small E. coli and B. subtilis cells both maintained wild-type concentration of DnaA, only the E. coli mutants failed to initiate on time. Thus, rather than the concentration, the total amount of DnaA appears to be more important for initiation timing in E. coli. The difference in behavior of the two bacteria appears to lie in the mechanisms that control the activity of DnaA. PMID:22396664

Hill, Norbert S.; Kadoya, Ryosuke; Chattoraj, Dhruba K.; Levin, Petra Anne

2012-01-01

142

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.

2009-11-01

143

Clinical value of DNA fragmentation evaluation tests under ART treatments.  

PubMed

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

2012-01-01

144

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

PubMed Central

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

Van Dyke, M W; Dervan, P B

1983-01-01

145

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

2006-05-01

146

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

1998-01-01

147

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

Microsoft Academic Search

The importance of molecular size in tumor (T) uptake of intact monoclonal antibody (MAb) of MAb fragments (frag.) is difficult to assess because frag. are excreted by the kidney. To obviate this problem nephrectomized nude mice (M) bearing carcinoembryonic (CEA) secreting human colon (T) were used in the following experiments. Following nephrectomy 3 groups of M were injected intravenously with

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

1984-01-01

148

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))

1989-11-10

149

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.

150

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

PubMed

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

1985-12-01

151

Rapid Large-segment DNA Sizing by Fluorescent Flow Cytometry  

NASA Astrophysics Data System (ADS)

Rapid determination of the size of DNA segments is an important technique in biology and is usually accomplished with gel electrophoresis. We are developing microfabricated alternatives to gel electrophoresis that allow fast DNA sizing with no inherent length limitation. By using micromachined T-channels made of silicon oxide, silicone rubber (RTV) or polyimide material and a sensitive avalanche photodiode (APD) detection system, we are able to image single molecules of DNA stained with the fluorescent dye YoYo. The microfluidic system allows the use of high numerical aperture optics for the efficient detection of weak signals. We have demonstrated rapid sizing of single lambda DNA segments (50kbps) with 10% coefficient of variance at a rate of up to 5 DNA per second throughput. Large segments of HindIII-digest lambda DNAs can also be identified under our system setup. The typical size of the T-shaped microchannels is 5 ? m wide and 3 ? m deep and they are sealed with either Si-SiO2 anodic bonding or the natural adhesion of RTV to SiO2 or plastic glass. Simutaneous sizing and sorting of single DNA can be achieved with an integrated detection and switching T-channel system.

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

1998-03-01

152

[Optimization of non-gel sieving capillary electrophoretic separation of DNA fragments of hundreds of base pairs].  

PubMed

Non-gel sieving capillary electrophoresis has been employed in the biological sciences for the size-based separation of macromolecules such as nucleic acids. In this paper, four factors i.e. electric field strength, capillary length, capillary diameter and hydroxy-propylmethylcellulose (HPMC) concentration were integratively evaluated to select the optimal condition of separating DNA fragments of a few hundreds of base pairs through orthogonal analysis. Conclusion was made through comprehensive analysis: better separation could be achieved in longer capillary, smaller inside diameter of capillary and less field strength. In practical application, effective separation in short time is important. We preferred to employ 8 g/L HPMC in coated capillary (37 cm x 75 microns i.d.) and electric field strength of 324 V/cm in separating DNA fragments of hundreds of base pairs. From sampling to getting results only a few more than ten minutes were needed in capillary electrophoresis. It needs less amount of sample (a few nanoliters) and shows higher sensitivity (0.1 pmol in UV detection) than polyacrylamide gel electrophoresis (PAGE). pBR322/Msp I fragments were completely resolved in both capillary electrophoresis and PAGE, and four-bases resolution was obtained. When the concentration of the PCR product is too low (< 20 mg/L), PCR reaction system is used as a negative control to make sure of the peak generated by the unpurified PCR products, avoiding interference made by PCR buffer and polymerase. PMID:11938911

Ding, X; Liao, J; Liu, X; Wang, Q; Ma, L

1998-11-01

153

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

PubMed

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

Taylor, M G; Vasil, I K

1987-10-01

154

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

Munguía-Rosas, Miguel A.; Montiel, Salvador

2014-01-01

155

Tumor Cell Growth Arrest Caused by Subchromosomal Transferable DNA Fragments from Chromosome 11  

Microsoft Academic Search

A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cell. As

Minoru Koi; Laura A. Johnson; Linda M. Kalikin; Peter F. R. Little; Yusuke Nakamura; Andrew P. Feinberg

1993-01-01

156

A WHEAT DNA FRAGMENT EXHIBITS REDUCED POLLEN TRANSMISSION IN TRANSGENIC MAIZE  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

157

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

1995-01-01

158

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

Microsoft Academic Search

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts

Peter Lindblad; Robert Haselkorn; Birgitta Bergman; Sandra A. Nierzwicki-Bauer

1989-01-01

159

The thermal stability of DNA fragments with tandem mismatches at a d(CXYG).d(CY'X'G) site  

Microsoft Academic Search

Temperature-Gradient Gel Electrophoresis (TGGE) was employed to determine the thermal stabilities of 28 DNA fragments, 373 bp long, with two adjacent mismatched base pairs, and eight DNAs with Watson- Crick base pairs at the same positions. Heteroduplex DNAs containing two adjacent mismatches were formed by melting and reannealing pairs of homo- logous 373 bp DNA fragments differing by two adjac-

Song-Hua Ke; Roger M. Wartell

1996-01-01

160

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

1996-01-01

161

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

PubMed

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 30 wt% of substituted slag aggregates, were performed. Although two of three slag-asphalt concretes generated 1.5-3.0 times larger amount of abrasion dust than the reference asphalt concrete did, it could not be explained only by abrasion resistance of slag. The enrichment of slag-derived fragments in abrasion dust, estimated on the basis of the peak intensity of quartz and heavy metal concentrations, had size dependency for all slag-asphalt concretes. Slag-derived fragments were enriched in abrasion dust particles with diameters of 150-1000 ?m. Enrichment factors were 1.4-2.1. In contrast, there was no enrichment in abrasion dust particles with diameter less than 75 ?m. This suggests that prior airborne-size fragmentation of substituted slag aggregates does not need to be considered for tested slag aggregates when environmental risks of abrasion dust of slag-asphalt pavement are assessed. PMID:21868161

Takahashi, Fumitake; Shimaoka, Takayuki; Gardner, Kevin; Kida, Akiko

2011-10-30

162

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

2008-11-26

163

Identification of healed terminal DNA fragments in linear minichromosomes of Schizosaccharomyces pombe.  

PubMed Central

The minichromosome Ch16 of the fission yeast Schizosaccharomyces pombe is derived from the centromeric region of chromosome III. We show that Ch16 and a shorter derivative, Ch12, made by gamma-ray cleavage, are linear molecules of 530 and 280 kilobases, respectively. Each minichromosome has two novel telomeres, as shown by genomic Southern hybridization with an S. pombe telomere probe. Comparison by hybridization of the minichromosomes and their chromosomal counterparts showed no signs of gross rearrangement. Cosmid clones covering the ends of the long arms of Ch16 and Ch12 were isolated, and subcloned fragments that contained the breakage sites were identified. They are apparently unique in the genome. By hybridization and Bal 31 digestion, the ends appear to consist of the broken-end sequences directly associated with short stretches (about 300 base pairs) of new DNA that hybridizes to a cloned S. pombe telomere. They do not contain the telomere-adjacent repeated sequences that are present in the normal chromosomes. The sizes of the short telomeric stretches are roughly the same as those of the normal chromosomes. Our results show that broken chromosomal ends in S. pombe can be healed by the de novo addition of the short telomeric repeats. The formation of Ch16 must have required two breakage-healing events, whereas a single cleavage-healing event in the long arm of Ch16 yielded Ch12. Images PMID:2830493

Matsumoto, T; Fukui, K; Niwa, O; Sugawara, N; Szostak, J W; Yanagida, M

1987-01-01

164

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

PubMed Central

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

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

2013-01-01

165

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; García, Nuria; Pääbo, Svante; Arsuaga, Juan-Luis; Meyer, Matthias

2013-01-01

166

Bioactive beads-mediated transformation of rice with large DNA fragments containing Aegilops tauschii genes.  

PubMed

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T(2) generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously. PMID:19214515

Wada, Naoki; Kajiyama, Shin'ichiro; Akiyama, Yukio; Kawakami, Shigeki; No, Daisuke; Uchiyama, Susumu; Otani, Motoyasu; Shimada, Takiko; Nose, Naoko; Suzuki, Go; Mukai, Yasuhiko; Fukui, Kiichi

2009-05-01

167

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

PubMed Central

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

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

2014-01-01

168

Pulsed-field electrophoresis of megabase-sized DNA.  

PubMed Central

Success in constructing a physical map of the human genome will depend on two capabilities: rapid resolution of very large DNA and identification of migration anomalies. To address these issues, a systematic exploration of pulsed-field electrophoresis conditions for separating multimegabase-sized DNA was undertaken. Conditions were found for first liberating and then separating DNA up to 6 megabases at higher field strengths and more rapidly than previously reported. In addition, some conditions for transversely pulsed fields produced mobility inversion, in which increased size was accompanied by faster rather than slower migration. Importantly, anomalous migration could be identified by the presence of lateral band spreading, in which the DNA band remained sharply defined but spread laterally while moving down the gel. These results have implications for both practical applications and theoretical models of pulsed-field electrophoresis. Images PMID:2038337

Gunderson, K; Chu, G

1991-01-01

169

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

PubMed

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

2004-10-01

170

Unzipping DNA by force: thermodynamics and finite size behaviour  

E-print Network

We discuss the thermodynamic behaviour near the force induced unzipping transition of a double stranded DNA in two different ensembles. The Y-fork is identified as the coexisting phases in the fixed distance ensemble. From finite size scaling of thermodynamic quantities like the extensibility, the length of the unzipped segment of a Y-fork, the phase diagram can be recovered. We suggest that such procedures could be used to obtain the thermodynamic phase diagram from experiments on finite length DNA.

Rajeev Kapri; Somendra M. Bhattacharjee

2005-11-22

171

Chloroplast DNA variation in Populus . I. Intraspecific restriction fragment diversity within Populus deltoides , P. nigra and P. maximowiczii  

Microsoft Academic Search

We examined intraspecific chloroplast (cp) DNA variation within Populus deltoides, P. nigra, and P. maximowiczii by restriction fragment analysis using 16 restriction endonucleases and six heterologous probes of cloned Petunia cpDNA fragments. All three Populus species showed intraspecific cpDNA variation, which was intra- and inter-varietal in P. deltoides, intervarietal in P. nigra, and origin-specific in P. maximowiczii. Two varieties of

O. P. Rajora; B. P. Dancik

1995-01-01

172

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

PubMed

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

2014-06-25

173

Cocrystal structure of an editing complex of Klenow fragment with DNA.  

PubMed

High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site. Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus. Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes. We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus. Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced. PMID:3194400

Freemont, P S; Friedman, J M; Beese, L S; Sanderson, M R; Steitz, T A

1988-12-01

174

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

2014-01-01

175

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

PubMed

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

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

2014-01-01

176

Polaprezinc, a gastroprotective agent: attenuation of monochloramine-evoked gastric DNA fragmentation.  

PubMed

We previously reported that NH2Cl induced extensive DNA fragmentation in gastric cells. Polaprezinc, a zinc-carnosine chelate compound, is reported to be a potent antioxidant in gastric mucosa. The present study was designed to examine whether polaprezinc could attenuate the NH3Cl-induced DNA damage. Gastric cell lines, MKN45, were exposed to NH2Cl in Ca(2+)-containing Hanks' balanced salt solution. DNA fragmentation was evaluated by photometric enzyme immunoassay for in vitro determination of cytoplasmic mono- and oligonucleosomes. Polaprezinc, L-carnosine, and zinc sulfate (ZnSO4) were added to the cell incubation medium to evaluate the inhibitory effect on the formation of cytoplasmic mono- and oligonucleosomes. Separately, the bleaching level of beta-carotene with the addition of each test solution was evaluated to confirm the inhibitory effect against hypochlorous acid. Polaprezinc or L-carnosine, but not ZnSO4, at a concentration of 0.001 mM, significantly attenuated the increased levels of cytoplasmic mono- and oligonucleosomes evoked by 0.001 mM NH2Cl. Polaprezinc and L-carnosine, but not ZnSO4, also inhibited NH2Cl-induced beta-carotene bleaching in the cell-free system. In conclusion, polaprezinc, especially its subportion L-carnosine, inhibited NH2Cl-evoked gastric epithelial DNA fragmentation, suggesting a role for this agent in preventing the progression of gastric epithelial injury induced by NH2Cl. PMID:10616765

Suzuki, H; Mori, M; Seto, K; Nagahashi, S; Kawaguchi, C; Morita, H; Suzuki, M; Miura, S; Yoneta, T; Ishii, H

1999-01-01

177

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

PubMed

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

2011-02-01

178

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

PubMed

Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis. PMID:24642253

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

2014-05-01

179

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

NASA Technical Reports Server (NTRS)

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

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

1995-01-01

180

In situ ligation simplified: using PCR fragments for detection of double-strand DNA breaks in tissue sections.  

PubMed

The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends. PMID:21057921

Didenko, Vladimir V

2011-01-01

181

IN SITU LIGATION SIMPLIFIED: USING PCR FRAGMENTS FOR DETECTION OF DOUBLE-STRAND DNA BREAKS IN TISSUE SECTIONS  

PubMed Central

The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends. PMID:21057921

Didenko, Vladimir V.

2012-01-01

182

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.

2014-01-01

183

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

PubMed Central

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

Sun, Mingyun; Lin, Jennifer S.

2012-01-01

184

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

NASA Technical Reports Server (NTRS)

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

Crawford, David A.

1997-01-01

185

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.

1995-12-31

186

Influence of hydrodynamic coupling on the electric linear dichroism of DNA fragments  

NASA Astrophysics Data System (ADS)

In the present work, we study the effect of translational-rotational hydrodynamic coupling on the stationary electric linear dichroism of DNA fragments. The theoretical resolution of the problem has, so far, been dealt with analytic methods valid only in the limit of low electric fields. In this work, we apply numerical methods that allow us to study the problem and also consider electric fields of arbitrary strength. We use the bent rod molecules model to describe DNA fragments with physical properties characterized by their electric charge, electric polarizability tensor, rotational diffusion tensor, and translation-rotation coupling diffusion tensor. The necessary orientational distribution function to calculate electric dichroism is obtained by solving the Fokker-Planck equation through the finite difference method. We analyze the different contributions due to electric polarizability and translational-rotational coupling to the electric dichroism.

Umazano, Juan P.; Bertolotto, Jorge A.

2011-03-01

187

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

PubMed Central

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

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

2015-01-01

188

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

PubMed

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

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

2013-11-01

189

Analysis of four tobacco mitochondrial DNA size classes.  

PubMed Central

Supercoiled mtDNAs were isolated from tobacco suspension culture cells and three of the smallest size classes (10.1, 20.2 and 30.3 kb) were characterized through denaturation, heteroduplex and restriction mapping. The 20.2 molecule was found to be a head-to-tail dimer of the 10.1 or X size class, while the 30.3 kb size class was found to contain two kinds of molecules, a head-to-tail trimer of X (X3) and a second molecule, ABC. X and ABC had a 118 +/- 35 bp region of homology, and both size classes shared a degree of homology with at least one other size class. Restriction maps of both the X and ABC molecules are presented and the possible origin and role of the many plant mtDNA size classes are discussed. Images PMID:6300773

Dale, R M; Wu, M; Kiernan, M C

1983-01-01

190

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.  

PubMed

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes. PMID:1837283

Akli, S; Chelly, J; Lacorte, J M; Poenaru, L; Kahn, A

1991-09-01

191

HIV1 gp120 Produces DNA Fragmentation in the Cerebral Cortex of Rat  

Microsoft Academic Search

In the present experiments we have used morphological techniques to study the neuropathological profile of the brain of rats after intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120. Using brain cryostat sections (10 ?m) from rats treated with a single, daily dose of gp120 (100 ng\\/rat) given for 7 and 14 consecutive days, in situ DNA fragmentation was revealed in

G. Bagetta; M. T. Corasaniti; L. Berliocchi; M. Navarra; A. Finazziagro; G. Nistico

1995-01-01

192

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

PubMed

With the development of functional genomics, gene-knockout is becoming an important tool to elucidate gene functions in vivo. As a good model strain for archaeal genetics, Haloferax volcanii has received more attention. Although several genetic manipulation systems have been developed for some halophilic archaea, it is time-consuming because of the low percentage of positive clones during the second-recombination selection. These classical gene knockout methods are based on DNA recombination between the genomic homologous sequence and the circular suicide plasmid, which carries a pyrE selection marker and two DNA fragments homologous to the upstream and downstream fragments of the target gene. Many wild-type clones are obtained through a reverse recombination between the plasmid and genome in the classic gene knockout method. Therefore, it is necessary to develop an efficient gene knockout system to increase the positive clone percentage. Here we report an improved gene knockout method using a linear DNA cassette consisting of upstream and downstream homologous fragments, and the pyrE marker. Gene deletions were subsequently detected by colony PCR analysis. We determined the efficiency of our knockout method by deleting the xpb2 gene from the H. volcanii genome, with the percentage of positive clones higher than 50%. Our method provides an efficient gene knockout strategy for halophilic archaea. PMID:25881705

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

2015-04-20

193

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

2012-04-20

194

Pyroclasts and fragmentation: `misdirection' from size distributions for wall-rock particles (Invited)  

NASA Astrophysics Data System (ADS)

A principal tenet of explosive volcanology is that the size distribution of pyroclasts is a sensitive indicator of fragmentation mechanism and eruptive style. However lithic pyroclasts in many explosive eruptions are not fragmented by the explosion but instead are derived from collapse or erosion of the walls of the vent and shallow conduit. Under these conditions the size distribution of this component is often ‘inherited’ and reflects pre-existing properties of the wall rock. We demonstrate this for two basaltic eruptions of widely contrasting mass discharge rate and style. The basaltic Plinian eruption of Tarawera, New Zealand in 1886 lasted 6 hours and had a time averaged mass discharge rate of 10E+8 kg/s. Eight impulsive explosions of Halema`uma`u crater, Kilauea in 2008 had eruptions rates of 10E+3 to 10E+4 kg/s and durations of tens of seconds. In both cases the wall rock component of the ejecta was derived from failures of unstable vent walls. In the case of Halema`uma`u only the finer-grained part of this ‘primary’ clast population was incorporated into the eruption jet. The clue to inferring such processes for older eruptions is within in the grain size and componentry data, in the decoupling and independence of the wall-rock and juvenile clast populations. In both of our case studies, neither wall rock nor whole-deposit grain size characteristics can be used to constrain and compare the intensity of explosions, or define eruptive style.

Houghton, B. F.; Carey, R. J.; Swanson, D.

2010-12-01

195

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

PubMed Central

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

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

1984-01-01

196

EndoG Links Bnip3-Induced Mitochondrial Damage and Caspase-Independent DNA Fragmentation in Ischemic Cardiomyocytes  

PubMed Central

Mitochondrial dysfunction, caspase activation and caspase-dependent DNA fragmentation are involved in cell damage in many tissues. However, differentiated cardiomyocytes repress the expression of the canonical apoptotic pathway and their death during ischemia is caspase-independent. The atypical BH3-only protein Bnip3 is involved in the process leading to caspase-independent DNA fragmentation in cardiomyocytes. However, the pathway by which DNA degradation ensues following Bnip3 activation is not resolved. To identify the mechanism involved, we analyzed the interdependence of Bnip3, Nix and EndoG in mitochondrial damage and DNA fragmentation during experimental ischemia in neonatal rat ventricular cardiomyocytes. Our results show that the expression of EndoG and Bnip3 increases in the heart throughout development, while the caspase-dependent machinery is silenced. TUNEL-positive DNA damage, which depends on caspase activity in other cells, is caspase-independent in ischemic cardiomyocytes and ischemia-induced DNA high and low molecular weight fragmentation is blocked by repressing EndoG expression. Ischemia-induced EndoG translocation and DNA degradation are prevented by silencing the expression of Bnip3, but not Nix, or by overexpressing Bcl-xL. These data establish a link between Bnip3 and EndoG-dependent, TUNEL-positive, DNA fragmentation in ischemic cardiomyocytes in the absence of caspases, defining an alternative cell death pathway in postmitotic cells. PMID:21437288

Zhang, Jisheng; Ye, Junmei; Altafaj, Albert; Cardona, Maria; Bahi, Núria; Llovera, Marta; Cañas, Xavier; Cook, Stuart A.; Comella, Joan X.; Sanchis, Daniel

2011-01-01

197

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: scsa@brookings.net; Wixon, Regina [SCSA Diagnostics, 807 32nd Avenue, Brookings, SD 57007 (United States)

2005-09-01

198

Correlation between DNA restriction fragment length polymorphisms in Leptospira interrogans serovar pomona type kennewicki and host animal source.  

PubMed Central

Isolates (n = 147) of Leptospira interrogans serovar pomona type kennewicki from cattle, swine, horses, and wildlife were analyzed by DNA restriction endonuclease analysis. Restriction fragment length polymorphisms were identified in DNA digested with HpaII, and the restriction fragment length polymorphisms were correlated with the host animal source of the isolates. These results will be useful in understanding the epidemiology of serovar pomona infections in livestock. PMID:8789028

Bolin, C A; Zuerner, R L

1996-01-01

199

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

1994-01-01

200

Pyrrolamide DNA Gyrase Inhibitors: Fragment-Based Nuclear Magnetic Resonance Screening To Identify Antibacterial Agents  

PubMed Central

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 (IC50) 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

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

2012-01-01

201

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

PubMed

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

Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

2010-07-14

202

Paired-End Sequencing of Long-Range DNA Fragments for De Novo Assembly of Large, Complex Mammalian Genomes by Direct Intra-Molecule Ligation  

PubMed Central

Background The relatively short read lengths from next generation sequencing (NGS) technologies still pose a challenge for de novo assembly of complex mammal genomes. One important solution is to use paired-end (PE) sequence information experimentally obtained from long-range DNA fragments (>1 kb). Here, we characterize and extend a long-range PE library construction method based on direct intra-molecule ligation (or molecular linker-free circularization) for NGS. Results We found that the method performs stably for PE sequencing of 2- to 5- kb DNA fragments, and can be extended to 10–20 kb (and even in extremes, up to ?35 kb). We also characterized the impact of low quality input DNA on the method, and develop a whole-genome amplification (WGA) based protocol using limited input DNA (<1 µg). Using this PE dataset, we accurately assembled the YanHuang (YH) genome, the first sequenced Asian genome, into a scaffold N50 size of >2 Mb, which is over100-times greater than the initial size produced with only small insert PE reads(17 kb). In addition, we mapped two 7- to 8- kb insertions in the YH genome using the larger insert sizes of the long-range PE data. Conclusions In conclusion, we demonstrate here the effectiveness of this long-range PE sequencing method and its use for the de novo assembly of a large, complex genome using NGS short reads. PMID:23029438

Wu, Kui; Cai, Qingle; Wang, Yu; Lang, Yongshan; Cao, Hongzhi; Yang, Huangming; Wang, Jian; Zhang, Xiuqing

2012-01-01

203

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

PubMed Central

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

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

2012-01-01

204

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

PubMed Central

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

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

2014-01-01

205

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

PubMed

Spatial vegetation patterns are recognized as sources of valuable information that can be used to infer the state and functionality of semiarid ecosystems, particularly in the context of both climate and land use change. Recent studies have suggested that the patch-size distribution of vegetation in drylands can be described using power-law metrics, and that these scale-free distributions deviate from power-law linearity with characteristic scale lengths under the effects of increasing aridity or human disturbance, providing an early sign of desertification. These findings have been questioned by several modeling approaches, which have identified the presence of characteristic scale lengths on the patch-size distribution of semiarid periodic landscapes. We analyze the relationship between fragmentation of vegetation patterns and their patch-size distributions in semiarid landscapes showing different degree of periodicity (i.e., banding). Our assessment is based on the study of vegetation patterns derived from remote sensing in a series of semiarid Australian Mulga shrublands subjected to different disturbance levels. We use the patch-size probability density and cumulative probability distribution functions from both nondirectional and downslope analyses of the vegetation patterns. Our results indicate that the shape of the patch-size distribution of vegetation changes with the methodology of analysis applied and specific landscape traits, breaking the universal applicability of the power-law metrics. Characteristic scale lengths are detected in (quasi) periodic banded ecosystems when the methodology of analysis accounts for critical landscape anisotropies, using downslope transects in the direction of flow paths. In addition, a common signal of fragmentation is observed: the largest vegetation patches become increasingly less abundant under the effects of disturbance. This effect also explains deviations from power-law behavior in disturbed vegetation which originally showed scale-free patterns. Overall, our results emphasize the complexity of structure assessment in dryland ecosystems, while recognizing the usefulness of the patch-size distribution of vegetation for monitoring semiarid ecosystems, especially through the cumulative probability distributions, which showed high sensitivity to fragmentation of the vegetation patterns. We suggest that preserving large vegetation patches is a critical task for the maintenance of the ecosystem structure and functionality. PMID:22073660

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

2011-10-01

206

In vitro DNA fragmentation of mitochondrial DNA caused by single-stranded breakage related to macroplasmodial senescence of the true slime mold, Physarum polycephalum.  

PubMed

We found that mitochondrial DNA (mtDNA) isolated from Physarum polycephalum fragmented itself in weak ionic solutions. The mtDNA was dissolved in STE (saline Tris-EDTA: 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA), TE (10 mM Tris-HCl, 1 mM EDTA) and DW, and then electrophoresed in an agarose gel. The intact 86-kbp mtDNA band was seen in STE, but several novel bands appeared in TE and DW. In TE, two discrete bands appeared at 6.7-kbp (alpha-band) and 5.0-kbp (beta-band), whereas at least 17 discrete bands were observed in distilled water (DW). These fragmentation patterns were not stoichiometric, as seen when using restriction endonucleases, but were clearly different from the degradation of DNA caused by a physical shearing force or a contaminating nuclease. In this paper, we characterize this in vitro fragmentation of mtDNA from P. polycephalum. We located 19 fragments, including the alpha and beta fragments, on a mtDNA restriction map, and demonstrated that these cleavage sites were S1 nuclease-sensitive regions, which are single-stranded DNA regions such as nicks and gaps in the mtDNA. The alpha and beta fragments are derived from the region encoding ribosomal RNAs (rRNAs) and the ATP synthase (atpA) gene, while the other 17 fragments are not derived from any specific region, but the cleavage sites are located throughout the mtDNA molecule. In P. polycephalum, it is well known that the growth rate of macroplasmodia decreases with aging. Equal amounts of mtDNA from juvenile and aged macroplasmodia were electrophoresed and the frequency of the beta fragment in each sample was measured. The ratio of the beta band to the total signal including background was estimated to be 3.3-4.0% in juvenile macroplasmodia, whereas it increased to 8.3-28.2% in aged macroplasmodia. This result suggests that the in vitro fragmentation of mtDNA is associated with macroplasmodial senescence. The single-stranded breakage of mtDNA of P. polycephalum may accumulate with age. PMID:10743569

Abe, T; Takano, H; Sasaki, N; Mori, K; Kawano, S

2000-02-01

207

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

1994-09-15

208

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.

2010-12-01

209

Dynamics of Enzymatic Interactions During Short Flap Human Okazaki Fragment Processing by Two forms of Human DNA Polymerase ?  

PubMed Central

Lagging strand DNA replication requires the concerted actions of DNA polymerase ?, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol ?/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase ? were studied: Pol ?4 and Pol ?3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol ?3 exhibits very limited strand displacement activity in contrast to Pol ?4, and stalls on encounter with a 5’-blocking oligonucleotide. Pol ?4 and Pol ?3 exhibit different characteristics in the Pol ?/Fen1 reactions. While Pol ?3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol ?4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol ?3 and Pol ?4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol ?3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol ?3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol ? in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol ?3 has a role in lagging strand synthesis, and that both forms of Pol ? may participate in DNA replication in higher eukaryotic cells. PMID:24035200

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

2013-01-01

210

Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization.  

PubMed

Nuclear DNA fragmentation and ultrastructural changes, indicative of myonuclear apoptosis, were examined in adult skeletal muscle in response to short-term immobilization. Adult rabbits were allocated to 2 days (n=5) or 6 days (n=5) of unilateral casting of the ankle in full plantar flexion or were used as untreated controls (n=2). Atrophy of the soleus muscle was apparent by significant reductions in wet mass of 15% and 26% after 2 days and 6 days of casting (P< or =0.05), respectively. Mean fibre cross-sectional area and myonuclear number per section were also lower (17% and 9.1%, respectively) after 6 days of casting, in comparison with contralateral control muscles (P< or =0.05). Electron-microscopic examination showed condensed chromatin and irregularly shaped myonuclei in muscles immobilized for either 2 days or 6 days. Myofibrillar disruption and abnormalities of the subsarcolemmal mitochondria were also apparent in the absence of inflammation or plasma membrane alterations in cast muscles. Longitudinal and transverse sections showed abundant in situ end-labelling of DNA strand breaks (TUNEL) after 2 days, with less after 6 days, of immobilization. Positive labelling corresponded to myonuclear locations within fibres, yet the number of TUNEL-positive nuclei indicated DNA fragmentation in additional cell types such as capillary endothelial cells or fibroblasts. The data indicate that the immobilization of slow-twitch skeletal muscle in a shortened position rapidly induces morphological alterations consistent with mitochondrial injury and apoptotic myonuclear elimination. PMID:11131134

Smith, H K; Maxwell, L; Martyn, J A; Bass, J J

2000-11-01

211

Association of Self-DNA Mediated TLR9-Related Gene, DNA Methyltransferase, and Cytokeratin Protein Expression Alterations in HT29-Cells to DNA Fragment Length and Methylation Status  

PubMed Central

To understand the biologic role of self-DNA bound to Toll-like Receptor 9 (TLR9), we assayed its effect on gene and methyltransferase expressions and cell differentiation in HT29 cells. HT29 cells were incubated separately with type-1 (normally methylated/nonfragmented), type-2 (normally methylated/fragmented), type-3 (hypermethylated/nonfragmented), or type-4 (hypermethylated/fragmented) self-DNAs. Expression levels of TLR9-signaling and proinflammatory cytokine-related genes were assayed by qRT-PCR. Methyltransferase activity and cell differentiation were examined by using DNA methyltransferase (DNMT1, -3A, -3B) and cytokeratin (CK) antibodies. Treatment with type-1 DNA resulted in significant increase in TLR9 expression. Type-2 treatment resulted in the overexpression of TLR9-related signaling molecules (MYD88A, TRAF6) and the IL8 gene. In the case of type-3 treatment, significant overexpression of NFkB, IRAK2, and IL8 as well as downregulation of TRAF6 was detected. Using type-4 DNA, TRAF6 and MYD88A gene expression was upregulated, while MYD88B, IRAK2, IL8, and TNFSF10 were all underexpressed. CK expression was significantly higher only after type-1 DNA treatment. DNMT3A expression could also be induced by type-1 DNA treatment. DNA structure may play a significant role in activation of the TLR9-dependent and even independent proinflammatory pathways. There may be a molecular link between TLR9 signaling and DNMT3A. The mode of self-DNA treatment may influence HT29 cell differentiation. PMID:24459426

F?ri, István; Sipos, Ferenc; Spisák, Sándor; Kiszner, Gerg?; Wichmann, Barnabás; Schöller, Andrea; Tulassay, Zsolt; M?zes, Györgyi; Molnár, Béla

2013-01-01

212

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

NASA Astrophysics Data System (ADS)

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

Bass, Andrew

2005-05-01

213

Sepcific fragmentation of DNA heteroduplex molecules of two bacteriophage lambda mutants with endonuclease Si from Aspergillus oryzae.  

PubMed Central

Heteroduplex DNA molecules of two bacteriophage mutants (lambda b2 and lambda i434ct68) were obtained by the method of molecular hybridization. These heteroduplexes possessed two types of loops formed as a result of: a) deletion in one of the DNA strands; and b) substitution of a DNA fragment for nonhomological one. The digestion of heteroduplexes with single-stranded specific nuclease SI from Aspergillus oryzae produced two fragments at 37 degrees C and three ones at 55 degrees C. The separation of fragments and determination of their molecular weight were carried out by means of electrophoresis in agarose. The molecular weights both measured and preliminarily calculated proved to be close. One of the fragments was identificated by its biological activity in CaCl2-dependent infectious system with helperphage. Images PMID:1272803

Ksënzenko, V N; Tikhomirova, L P; Matvienko, N I

1976-01-01

214

Colocalization of BAX and BCL2 in small intestine and kidney biopsies with different degrees of DNA fragmentation  

Microsoft Academic Search

Morphological changes associated with apoptosis are closely correlated with the expression of specific proteins. However, the cause-effect relationships between the expression of these proteins and DNA degradation are barely known. For studying expression of apoptosis-related proteins in relation to different degrees of DNA fragmentation, the small intestine with its spatially organized continuum of proliferation, differentiation and death is a very

A. P. Aschoff; U. Ott; R. Fünfstück; G. Stein; G. F. Jirikowski

1999-01-01

215

Genetic diversity of Dioscorea dumetorum (Kunth) Pax using Amplified Fragment Length Polymorphisms (AFLP) and cpDNA  

Microsoft Academic Search

We have utilized Amplified Fragment Length Polymorphisms (AFLP) in conjunction with chloroplast DNA (cpDNA) sequence data to study the genetic diversity in 53 accessions of Dioscorea dumetorum from six countries in West and Central Africa. Our results provide a comparison of the two marker systems with regards to their applicability to differentiate intraspecific genotypes and the grouping of the accessions

Mubo A. Sonibare; Robert Asiedu; Dirk C. Albach

2010-01-01

216

Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from chromosome 11  

SciTech Connect

A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cells. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the [beta]-globin and insulin genes. 34 refs., 5 figs.

Koi, M.; Johnson, L.A.; Kalikin, L.M.; Feinberg, A.P. (Univ. of Michigan, Ann Arbor (United States)); Little, P.F.R. (Imperial College of Science, Technology, and Medicine, London (United Kingdom)); Nakamura, Yusuke (Cancer Inst., Tokyo (Japan))

1993-04-16

217

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

PubMed

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

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

2014-06-01

218

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

2011-01-05

219

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

2013-01-01

220

Size does matter – effects of tropical rainforest fragmentation on the leaf litter ant community in Sabah, Malaysia  

Microsoft Academic Search

Primary tropical lowland rainforest in Sabah, Malaysia, has been largely reduced to small to medium-sized, often isolated, forest islands surrounded by a highly altered agricultural landscape. The biodiversity patterns of leaf litter ant communities were monitored in two forest fragments of differing size as well as in a contiguous forest over the course of two years. Species number and diversity

Carsten A. Brühl; Thomas Eltz; K. Eduard Linsenmair

2003-01-01

221

Storing DNA, 2D animationSite: DNA Interactive (www.dnai.org)  

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.

2008-10-06

222

Native fluorescence detection of nucleic acids and DNA restriction fragments in capillary electrophoresis  

SciTech Connect

A sensitive laser-induced fluorescence (LIF) detection scheme for native nucleic acids and DNA restriction fragments separated by capillary electrophoresis (CE) has been developed. The 275.4-nm line from an argon ion laser or the 248-nm line from a waveguide KrF laser is used to excite native fluorescence. Detection limits for guanosine and adenosine monophosphate (1.5 [times] 10[sup [minus]8] and 5 [times] 10[sup [minus]8] M, respectively) are up to 3 orders of magnitude lower than UV detection. Sensitivity for native fluorescence of DNA restriction fragments in gel-filled capillaries rivals that of UV absorption. The decrease in performance in gel-filled separations using LIF detection is caused by the high background associated with gel fluorescence, as well as gel quenching of the fluorescence emission. The development of gels exhibiting lower background fluorescence or off-column coupling should lead to significant improvements in sensitivity over UV detection. This novel and practical system enables, for the first time, the sensitive detection of nucleic-acid-containing compounds without the need for fluorescence labeling. 48 refs., 4 figs., 1 tab.

Milofsky, R.E.; Yeung, E.S. (Ames Laboratory, IA (United States))

1993-01-15

223

Isolation and characterization of human DNA fragments with nucleotide sequence homologies with the simian virus 40 regulatory region  

SciTech Connect

A recombinant library of human DNA sequences was screened with a segment of simian virus 40 (SV40) DNA that spans the viral origin of replication. One hundred and fifty phage were isolated that hybridized to this probe. Restriction enzyme and hybridization analyses indicated that these sequences were partially homologous to one another. Direct DNA sequencing of two such SV40-hybridizating segments indicated that this was not a highly conserved family of sequences, but rather a set of DNA fragments that contained repetitive regions of high guanine plus cytosine content. These sequences were not members of the previously described Alu family of repeats and hybridized to SV40 DNA more strongly than do Alu family members. The authors tested for the presence of several functional domains on two of these human DNA fragments. One SV40-hybridizing fragment, SVCR29, contained a sequence with enhanced the efficiency of thymidine kinase transformation in human cells by approximately 20-fold. This effect was seen in an orientation-independent manner when the sequence was present at the 3' end of the chicken thymidine kinase gene. They propose that this segment of DNA contains a sequence analogous to the 72-base-pair repeats of SV40. The existence of such an ''activator'' element in cellular DNA raises the possibility that families of these sequences may exist in the mammalian genome.

Conrad, S.E.; Botchan, M.R.

1982-08-01

224

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

Microsoft Academic Search

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

NIEVES VIZCAINO; AXEL CLOECKAERT; MICHEL S. ZYGMUNT; LUIS FERNANDEZ-LAGO

2001-01-01

225

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

PubMed Central

Trypanosoma brucei's mitochondrial genome, kinetoplast DNA (kDNA), is a giant network of catenated DNA rings. The network consists of a few thousand 1 kb minicircles and several dozen 23 kb maxicircles. Here we report that TbPIF5, one of T. brucei's six mitochondrial proteins related to Saccharomyces cerevisiae mitochondrial DNA helicase ScPIF1, is involved in minicircle lagging strand synthesis. Like its yeast homolog, TbPIF5 is a 5? to 3? DNA helicase. Together with other enzymes thought to be involved in Okazaki fragment processing, TbPIF5 localizes in vivo to the antipodal sites flanking the kDNA. Minicircles in wild type cells replicate unidirectionally as theta-structures and are unusual in that Okazaki fragments are not joined until after the progeny minicircles have segregated. We now report that overexpression of TbPIF5 causes premature removal of RNA primers and joining of Okazaki fragments on theta structures. Further elongation of the lagging strand is blocked, but the leading strand is completed and the minicircle progeny, one with a truncated H strand (ranging from 0.1 to 1 kb), are segregated. The minicircles with a truncated H strand electrophorese on an agarose gel as a smear. This replication defect is associated with kinetoplast shrinkage and eventual slowing of cell growth. We propose that TbPIF5 unwinds RNA primers after lagging strand synthesis, thus facilitating processing of Okazaki fragments. PMID:19779567

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

2009-01-01

226

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.

2009-01-01

227

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

PubMed Central

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

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

2014-01-01

228

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

SciTech Connect

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

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

2007-03-01

229

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)

2007-08-15

230

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

PubMed

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

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

1990-01-01

231

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

PubMed

We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(2)) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar(+) ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair. PMID:17764359

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

2007-08-01

232

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

PubMed

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

Danckaert, A; Chappey, C; Hazout, S

1991-10-01

233

Landscape division, splitting index, and effective mesh size: new measures of landscape fragmentation  

Microsoft Academic Search

Anthropogenic fragmentation of landscapes is known as a major reason for the loss of species in industrialized countries. Landscape fragmentation caused by roads, railway lines, extension of settlement areas, etc. ,f urther enhances the dispersion of pollutants and acoustic emissions and affects local climatic conditions, water balance, scenery, and land use. In this study, three new measures of fragmentation are

Jochen A. G. Jaeger

2000-01-01

234

Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*  

PubMed Central

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

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

2012-01-01

235

The effects of age on DNA fragmentation, chromatin packaging and conventional semen parameters in spermatozoa of oligoasthenoteratozoospermic patients  

Microsoft Academic Search

Purpose  To investigate the effects of male ageing on DNA fragmentation and chromatin packaging in the spermatozoa of oligoasthenoteratozoospermic\\u000a (OAT) patients.\\u000a \\u000a \\u000a \\u000a Methods  Sixty-one OAT patients and 49 men with proven fertility (controls) were included in the present study. DNA fragmentation was\\u000a detected by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labelling (TUNEL) assay, while chromatin packaging\\u000a was assessed by chromomycin A3 (CMA3) staining.\\u000a \\u000a \\u000a \\u000a Results  In

Konstantina Plastira; Pavlos Msaouel; Roxani Angelopoulou; Kyriaki Zanioti; Aris Plastiras; Alexios Pothos; Stamatis Bolaris; Nikolaos Paparisteidis; Dimitris Mantas

2007-01-01

236

The effects of male aging on semen quality, sperm DNA fragmentation and chromosomal abnormalities in an infertile population  

Microsoft Academic Search

Purpose  To investigate the effects of male aging on semen quality, DNA fragmentation and chromosomal abnormalities in the spermatozoa\\u000a of infertile patients and fertile men.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Semen samples of 140 infertile patients (24–76 years) and 50 men with proven fertility (25–65 years) were analyzed according\\u000a to WHO guidelines. DNA fragmentation was detected by TUNEL assay, while aneuploidy was assessed by FISH.\\u000a \\u000a \\u000a \\u000a \\u000a Results  In the patient

Sonia Brahem; Meriem Mehdi; Hatem Elghezal; Ali Saad

2011-01-01

237

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

PubMed

Isoniazid (INH) is usually administered to treat latent Mycobacterium tuberculosis (Mtb) infections and is used in combination therapy to treat active tuberculosis (TB). Unfortunately, resistance to this drug is hampering its clinical effectiveness. INH is a prodrug that must be activated by Mtb catalase-peroxidase (KatG) before it can inhibit InhA (Mtb enoyl-acyl-carrier-protein reductase). Isoniazid-resistant cases of TB found in clinical settings usually involve mutations in or deletion of katG, which abrogate INH activation. Compounds that inhibit InhA without requiring prior activation by KatG would not be affected by this resistance mechanism and hence would display continued potency against these drug-resistant isolates of Mtb. Virtual screening experiments versus InhA in the GO Fight Against Malaria (GO FAM) project were designed to discover new scaffolds that display base-stacking interactions with the NAD cofactor. GO FAM experiments included targets from other pathogens, including Mtb, when they had structural similarity to a malaria target. Eight of the 16 soluble compounds identified by docking against InhA plus visual inspection were modest inhibitors and did not require prior activation by KatG. The best two inhibitors discovered are both fragment-sized compounds and displayed Ki values of 54 and 59 ?M, respectively. Importantly, the novel inhibitors discovered have low structural similarity to known InhA inhibitors and thus help expand the number of chemotypes on which future medicinal chemistry efforts can be focused. These new fragment hits could eventually help advance the fight against INH-resistant Mtb strains, which pose a significant global health threat. PMID:25636146

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

2015-03-23

238

Increased circulating cell-free DNA levels and mtDNA fragments in interventional cardiologists occupationally exposed to low levels of ionizing radiation.  

PubMed

Circulating cell-free DNA (ccf-DNA) and mtDNA (ccf-mtDNA) have often been used as indicators of cell death and tissue damage in acute and chronic disorders, but little is known about changes in ccf-DNA and ccf-mtDNA concentrations following radiation exposure. The aim of the study was to investigate the impact of chronic low-dose radiation exposure on serum ccf-DNA levels and ccf-mtDNA fragments (mtDNA-79 and mtDNA-230) of interventional cardiologists working in high-volume cardiac catheterization laboratory to assess their possible role as useful radiation biomarkers. We enrolled 50 interventional cardiologists (26 males; age?=?48.4?±?10 years) and 50 age- and gender-matched unexposed controls (27 males; age?=?47.6?±?8.3 years). Quant-iT™ dsDNA High-Sensitivity assay was used to measure circulating ccf-DNA isolated from serum samples. Quantitative analysis of mtDNA fragments was performed by real-time PCR. No significant relationships were found between ccf-DNA and ccf-mtDNA, and age, gender, smoking, or other clinical parameters. Ccf-DNA levels (44.2?±?31.1 vs. 30.6?±?19.2 ng/ml, P?=?0.013), ccf-mtDNA-79 (2.6?±?2.1 vs. 1.1?±?0.8, P < 0.01), and ccf-mtDNA-230 copies (2.0?±?1.8 vs. 1.04?±?0.9, P?=?0.02) were significantly higher in interventional cardiologists compared with the non-exposed group. In a subset (n?=?15) of interventional cardiologists with a reliable reconstruction of cumulative professional exposure (59.7?±?48.4 mSv; range: 1.4-182 mS), ccf-DNA (53.2?±?41.3 vs. 36.4?±?22.9 and 32.2?±?20.5, P?=?0.08), mtDNA-79 (2.4?±?2.1 vs. 2.03?±?1.7 and 1.09?±?0.82, P?=?0.05), and mtDNA-230 (2.0?±?2.2 vs. 1.5?±?1.4 and 1.04?±?0.9, P?=?0.09) tended to be significantly increased in high-exposure subjects compared with both low-exposure interventional cardiologists and controls. Our results provide evidence for a possible role of circulating DNA as a relevant biomarker of cellular damage induced by exposure to chronic low-dose radiation. Environ. Mol. Mutagen. 56:293-300, 2015. © 2014 Wiley Periodicals, Inc. PMID:25327629

Borghini, Andrea; Mercuri, Antonella; Turchi, Stefano; Chiesa, Maria Rosa; Piccaluga, Emanuela; Andreassi, Maria Grazia

2015-04-01

239

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

NASA Astrophysics Data System (ADS)

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

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

2012-02-01

240

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

2012-11-01

241

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

PubMed Central

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

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

2013-01-01

242

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

2014-01-01

243

Screening and characterization of sex-specific DNA fragments in the freshwater fish matrinchã, Brycon amazonicus (Teleostei: Characiformes: Characidae).  

PubMed

The matrinchã Brycon amazonicus, a commercially important freshwater fish resource, has no heteromorphic sex chromosomes so far described. In the present study, we performed a screening of sex-associated DNA markers in this species, through the use of a random amplified polymorphic DNA (RAPD) assay and a genomic DNA restriction digestion analysis. DNA digestions evidenced no differences between sexes. Sixty-six random primers were used in pooled and individual DNA samples of males and females, and the analysis of the RAPD fingerprints revealed one female sex-associated band. Cloning and sequencing of this band led to the identification of two distinct DNA segments. While one of the isolated fragments showed a significant identity with a described protein gene (phosphatidylinositol glycan anchor biosynthesis, class W), the other fragment, composed of 535 bp, corresponds to a novel DNA marker. Further experiments were performed with this second DNA fragment in order to verify its sex-specificity. Data on dot blot hybridization, using total DNA of both sexes, confirmed its female-specificity in B. amazonicus. A primer set was designed based on its sequence data and used in PCR with DNA samples of this species, leading to diagnose the animals' sexes with a 100 % overall accuracy through a sequence characterized amplified region approach. No amplification results were found for two other species of the genus--B. orbignyanus and B. lundii. The obtained data can lead to the hypothesis that B. amazonicus may present heteromorphic sex chromosomes that should be in an early phase of differentiation. PMID:22527611

da Silva, Eder Marques; Wong, Marina Sek Lien; Martins, Cesar; Wasko, Adriane Pinto

2012-10-01

244

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

PubMed

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

2005-09-01

245

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 (pat-1.).  

PubMed

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 (pat-1) conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 (pat-1) locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700-800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster. PMID:24177891

Klein-Lankhorst, R M; Salentijn, E M; Dirkse, W G; Arens-de Reuver, M; Stiekema, W J

1994-10-01

246

DNA fragmentation is increased in non-GABAergic neurons in bipolar disorder but not in schizophrenia  

PubMed Central

Apoptosis is thought to contribute to neuronal loss in bipolar disorder and schizophrenia, although empiric evidence in support of this idea has been lacking. In this study, we investigated whether or not apoptosis is associated with GABAergic interneurons in the anterior cingulate cortex in schizophrenia (n = 14) and bipolar disorder (n = 14) when compared to normal controls (n = 14). A double-labeling technique using the Klenow method of in situ end-labeling (ISEL) of single-stranded DNA breaks was combined with an in situ hybridization localization of mRNA for the 67 kiloDalton (kDa) isoform of glutamate decarboxylase (GAD67) and applied to the anterior cingulate cortex of 14 normal controls, 14 schizophrenics, and 14 patients with bipolar disorder matched for age and postmortem interval. An increase in Klenow-positive, GAD67-negative nuclei was observed in layer V/VI of patients with bipolar disorder, but not schizophrenics. Klenow-positive cells that were also positive for GAD67 mRNA did not show differences in either patient group. Conclusions: This is the first demonstration that there is more DNA fragmentation in cells showing no detectable GAD67 mRNA in patients with bipolar disorder than in schizophrenics or controls. These findings suggest that non-GABAergic cells may be selectively vulnerable to oxidative stress in patients with bipolar disorder. PMID:17442540

Buttner, Ned; Bhattacharyya, Sujoy; Walsh, John; Benes, Francine M.

2007-01-01

247

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.  

PubMed

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries. PMID:10735310

Neumann, M; Herten, D P; Dietrich, A; Wolfrum, J; Sauer, M

2000-02-25

248

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

PubMed

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

2014-08-01

249

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua.  

PubMed

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories. PMID:14653790

Kjølner, S; Såstad, S M; Taberlet, P; Brochmann, C

2004-01-01

250

A comparison of morphology, pathogenicity and restriction fragment patterns of mitochondrial DNA among isolates of Phytophthora porri Foister  

Microsoft Academic Search

Thirteen strains ofPhytophthora porri from five different hosts were compared with respect to their morphology, cardinal temperatures for growth, pathogenicity to leek and cabbage and restriction fragment patterns of mitochondrial DNA. Morphology of vegetative growth was rather similar in most isolates. Those characters which differed among isolates showed overlapping variability and could not be used to distinguish groups, with the

Arthur W. A. M. De Cock; Afra Neuvel; Günther Bahnweg; Johanna C. J. M. De Cock; Hermann H. Prell

1992-01-01

251

DNA Fragmentation Follows Delayed Neuronal Death in CA1 Neurons Exposed to Transient Global Ischemia in the Rat  

Microsoft Academic Search

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of

Carol K. Petito; Jorge Torres-Munoz; Brenda Roberts; John-Paul Olarte; Thaddeus S. Nowak; William A. Pulsinelli

1997-01-01

252

Does the marine biotoxin okadaic acid cause DNA fragmentation in the blue mussel and the pacific oyster?  

PubMed

Two bivalve species of global economic importance: the blue mussel, Mytilus edulis and the pacific oyster, Crassostrea gigas were exposed in vivo, to the diarrhoetic shellfish toxin okadaic acid (OA), and impacts on DNA fragmentation were measured. Shellfish were exposed using two different regimes, the first was a single (24 h) exposure of 2.5 nM OA (?0.1 ?g/shellfish) and algal feed at the beginning of the trial (T0), after which shellfish were only fed algae. The second was daily exposure of shellfish to two different concentrations of OA mixed with the algal feed over 7 days; 1.2 nM OA (?0.05 ?g OA/shellfish/day) and 50 nM OA (?2 ?g OA/shellfish/day). Haemolymph and hepatopancreas cells were extracted following 1, 3 and 7 days exposure. Cell viability was measured using the trypan blue exclusion assay and remained above 85% for both cell types. DNA fragmentation was examined using the single-cell gel electrophoresis (comet) assay. A significant increase in DNA fragmentation was observed in the two cell types from both species relative to the controls. This increase was greater in the pacific oyster at the higher toxin concentration. However, there was no difference in the proportion of damage measured between the two cell types, and a classic dose response was not observed, increasing toxin concentration did not correspond to increased DNA fragmentation. PMID:25440785

McCarthy, Moira; O'Halloran, John; O'Brien, Nora M; van Pelt, Frank F N A M

2014-10-01

253

Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low-and High-Molecular-Weight  

E-print Network

Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low- and High-Molecular-Weight separation by CE in HEC solutions is strongly influenced by the average HEC molecular weight as well of the effects of a mixture of low- and high-molecular weight HEC polymers, over a range of concentrations

Barron, Annelise E.

254

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

2014-01-01

255

Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.  

PubMed

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

2014-01-01

256

Use of cloned DNA fragments for event-specific quantification of genetically modified organisms in pure and mixed food products  

Microsoft Academic Search

An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique

Isabel Taverniers; Pieter Windels; Erik Van Bockstaele; Marc De Loose

2001-01-01

257

Early DNA Sequencing  

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. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents early DNA sequencing through a series of illustrations of the processes involved.

258

Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high penicillin producing strains of Penicillium chrysogenum  

Microsoft Academic Search

The isopenicillin N synthase (pcbC) and acyl-CoA:6-APA acyltransferase (penDE) genes of Penicillium chrysogenum were located in a 19.5-kb DNA fragment that had been previously cloned in phage vector EMBL3. This 19.5-kb DNA fragment was mapped with several endonucleases, and the (pcbC) and penDE genes were located by hybridization with probes corresponding to internal fragments of each gene. A low penicillin

Jose L. Barredo; Bruno Díez; Emilio Alvarez; Juan F. Martín

1989-01-01

259

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells.  

PubMed

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death. PMID:11259492

Jiang, D; Jha, N; Boonplueang, R; Andersen, J K

2001-03-01

260

A modular assembly cloning technique (aided by the BIOF software tool) for seamless and error-free assembly of long DNA fragments  

PubMed Central

Background Molecular cloning of DNA fragments >5 kbp is still a complex task. When no genomic DNA library is available for the species of interest, and direct PCR amplification of the desired DNA fragment is unsuccessful or results in an incorrect sequence, molecular cloning of a PCR-amplified region of the target sequence and assembly of the cloned parts by restriction and ligation is an option. Assembled components of such DNA fragments can be connected together by ligating the compatible overhangs produced by different restriction endonucleases. However, designing the corresponding cloning scheme can be a complex task that requires a software tool to generate a list of potential connection sites. Findings The BIOF program presented here analyzes DNA fragments for all available restriction enzymes and provides a list of potential sites for ligation of DNA fragments with compatible overhangs. The cloning scheme, which is called modular assembly cloning (MAC), is aided by the BIOF program. MAC was tested on a practical dataset, namely, two non-coding fragments of the translation elongation factor 1 alpha gene from Chinese hamster ovary cells. The individual fragment lengths exceeded 5 kbp, and direct PCR amplification produced no amplicons. However, separation of the target fragments into smaller regions, with downstream assembly of the cloned modules, resulted in both target DNA fragments being obtained with few subsequent steps. Conclusions Implementation of the MAC software tool and the experimental approach adopted here has great potential for simplifying the molecular cloning of long DNA fragments. This approach may be used to generate long artificial DNA fragments such as in vitro spliced cDNAs. PMID:22709633

2012-01-01

261

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

2014-05-01

262

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

PubMed

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

2014-01-01

263

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

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

2013-01-01

264

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

PubMed

The DNA of Novikoff hepatoma ascites cells was found to contain a 3.6-megadalton EcoRI restriction fragment, referred to as EcoRI fragment A (Parker et al., 1979). C0t analyses demonstrated an enrichment of fragment A sequences in Novikoff hepatoma genome relative to normal rat liver DNA. This fragment was cloned in lambda gtWES to determine its molecular structure and sequence organization. The DNA from a positive clone was labeled by nick translation and hybridized to a Southern blot of EcoRI digested Novikoff DNA. Distinct hybrids formed with the region corresponding to fragment A. The greater degree of hybridization to the nucleolar fraction suggested a nucleolar enrichment of fragment A. Fragment A has a PstI site approximately 300 base pairs from one terminus which was used to generate mono-5'-32P-labeled fragments. The larger PStI subfragment, 5500 base pairs, labeled at a single terminus, was used to evolve a restriction enzyme map. The 300 base pair fragment was partially sequenced, revealing the presence of a repetitive sequence "island", TT(GTCT)8(GAAT)5G-. C0t analysis, utilizing the purified clone as a probe, confirmed the enrichment of fragment A sequences in the tumor relative to the normal rat liver control. PMID:6260140

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

1981-02-17

265

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

PubMed Central

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

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

2014-01-01

266

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

1995-06-30

267

DNA Fragments in the Blood Plasma of Cancer Patients: Quantitations and Evidence for Their Origin from Apoptotic and Necrotic Cells1  

Microsoft Academic Search

Increased levels of DNA fragments have frequently been found in the blood plasma of cancer patients. Published data suggest that only a fraction of the DNA in blood plasma is derived from cancer cells. How- ever, it is not known how much of the circulating DNA is from cancer or from noncancer cells. By quantitative methylation-specific PCR of the promoter

Sabine Jahr; Hannes Hentze; Sabine Englisch; Dieter Hardt; Frank O. Fackelmayer; Rolf-Dieter Hesch; Rolf Knippers

2001-01-01

268

Delivery of plasmid DNA encoding bone morphogenetic protein-2 with a biodegradable branched polycationic polymer in a critical-size rat cranial defect model.  

PubMed

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

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

2011-03-01

269

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

PubMed Central

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

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

2011-01-01

270

[Effect of N-stearoylethanolamine on the DNA fragmentation intensity in tumour and extratumoral tissues of the human adrenal cortex].  

PubMed

The effect of different concentrations of N-stearoylethanolamine (NSE 18:0) on fragmentation of DNA in the tumoural and extratumour tissues of the adrenal glands in vitro was studied. In this work the following types of tissue were investigated: extratumoural tissue from patients with hormonally active tumours, benign tumour tissue (hormonally active and hormonally inactive), tissue of malignant tumours and hyperplasic tissue of the adrenal glands (Itsenko-Cushing disease). It has been established that the NSE increases the intensity of DNA fragmentation only in the tissue of hormonally inactive tumours. Benign hormonally active tumours, malignant tumours and hyperplastic tissue of the adrenal glands were resistant to the NSE. The possible mechanisms of resistance to the drug are discussed. PMID:22946300

Levchuk, N I; Pushkar'ov, V M; Kovzun, O I; Mykosha, O S; Hula, N M; Tron'ko, M D

2012-01-01

271

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

PubMed

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent for a swine disease affecting the pig industry worldwide. Infection with PRRSV leads to reproductive complications, respiratory illness, and weak immunity to secondary infections. To better control PRRSV infection, novel approaches for generating control measures are critically needed. Here, in vitro Gibson assembly (GA) of viral genomic cDNA fragments was tested for its use as a quick and simple method to recover infectious PRRSV in cell culture. GA involves the activities of T5-exonuclease, Phusion polymerase, and Taq ligase to join overlapping cDNA fragments in an isothermal condition. Four overlapping cDNA fragments covering the entire PRRSV genome and one vector fragment were used to create a plasmid capable of expressing the PRRSV genome. The assembled product was used to transfect a co-culture of 293T and MARC-145 cells. Supernatants from the transfected cells were then passaged onto MARC-145 cells to rescue infectious virus particles. Verification and characterization of the recovered virus confirmed that the GA protocol generated infectious PRRSV that had similar characteristics to the parental virus. This approach was then tested for the generation of a chimeric virus. By replacing one of the four genomic fragments with that of another virus strain, a chimeric virus was successfully recovered via GA. In conclusion, this study describes for the first time the use of GA as a simple, yet powerful tool for generating infectious PRRSV needed for studying PRRSV biology and developing novel vaccines. PMID:25300804

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

2015-01-01

272

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

1997-01-01

273

An estimate of the comet Shoemaker-Levy 9 fragment sizes from the debris field  

NASA Technical Reports Server (NTRS)

The impacts of Comet Shoemaker-Levy 9 left spots on Jupiter with diameters on the order of tens of thousands of kilometers, which have the appearance of debris fields strewn upon the Jovian cloud tops. In this note we employ a measurement of the optical depth of the debris at the impact site of fragment G to estimate mass in the debris field and lower limits to the G fragment mass of 4 x 10(exp12) - 4 x 10(exp 13) g and diameter of 0.1 - 0.3 km.

Knacke, R. F.; A'Hearn, M. F.

1994-01-01

274

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

PubMed Central

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

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

1985-01-01

275

Induction of apoptosis associated with chromosomal DNA fragmentation and caspase-3 activation in leukemia L1210 cells by TiO2 nanoparticles.  

PubMed

We investigated the effects of nanosized TiO2 particles on the death of mouse leukemia L1210 cells. TiO2 particles suppressed proliferation and induced cell death, as measured by lactate dehydrogenase (LDH) release into the culture medium. Chromatin condensation, which is typical of the initiation of cell death, was observed in approximately 14% cells cultured with titanium dioxide (TiO2) particles for 12 h. Furthermore, giant DNA fragments of approximately 2 Mbp and high-molecular-weight DNA fragments between 100 kbp and 1 Mbp were observed in cells cultured for 18 h with TiO2 particles. These giant and high-molecular-weight DNA fragments were further degraded into smaller DNA fragments, appearing as DNA ladders. Corresponding to the generation of DNA fragments, caspase-3 activity increased in cells treated with TiO2 particles. TiO2 particle-induced LDH release was not inhibited by cytochalasin D, an inhibitor of endocytosis. These results suggest that nanosized TiO2 particles can induce apoptosis associated with DNA fragmentation and caspase-3 activation and that TiO2 particle-induced apoptosis is not caused by endocytosis but is associated with contact of the particles with the cell surface. PMID:23849803

Takaki, Keiko; Higuchi, Yoshihiro; Hashii, Minako; Ogino, Chiaki; Shimizu, Nobuaki

2014-01-01

276

The effect of sperm DNA fragmentation on live birth rate after IVF or ICSI: a systematic review and meta-analysis.  

PubMed

A systematic review and meta-analysis was conducted to evaluate the relationship between the extent of sperm DNA damage and live birth rate (LBR) per couple and the influence of the method of fertilization on treatment outcome. Searches were conducted on MEDLINE, EMBASE and Cochrane Library. Six studies were eligible for inclusion in the meta-analysis. Overall, LBR increased signficantly in couples with low sperm DNA fragmentation compared with those with high sperm DNA fragmentation (RR 1.17, 95% CI 1.07 to 1.28; P = 0.0005). After IVF and intracytoplasmic sperm injection (ICSI), men with low sperm DNA fragmentation had significantly higher LBR (RR 1.27, 95% CI 1.05 to 1.52; P = 0.01) and (RR 1.11, 95% CI 1.00 to 1.23, P = 0.04), respectively. A sensitivity analysis showed no statistically significant difference in LBR between low and high sperm DNA fragmentation when ICSI treatment was used (RR 1.08, 95% CI 0.39 to 2.96; P = 0.88). High sperm DNA fragmentation in couples undergoing assisted reproduction techniques is associated with lower LBR. Well-designed randomized studies are required to assess the role of ICSI over IVF in the treatment of men with high sperm DNA fragmentation. PMID:25530036

Osman, A; Alsomait, H; Seshadri, S; El-Toukhy, T; Khalaf, Y

2015-02-01

277

Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome.  

PubMed Central

The penultimate EcoRI fragments from the left and right ends of the vaccinia virus genomes were cloned in phage lambda. Heteroduplex analysis and comparison of restriction fragments indicated that the inverted terminal repetition extended 780 base pairs (bp) beyond the EcoRI site or about 9,800 bp from each end of the genome. Detailed physical, transcriptional, and translational maps of the 6,600-bp left penultimate EcoRI fragment were prepared so as to extend previous maps of the 9,000-bp terminal EcoRI fragment. Polypeptides with molecular weights of 6,000 (6K polypeptide), 13,000, 19,000, 21,000, and 60,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA (made in the presence of cycloheximide) or early RNA (made in the presence of cytosine arabinoside) and selected by hybridization to immobilized recombinant DNA. A 22K polypeptide was detected as a translation product of late RNA that hybridized to this DNA fragment. A variety of biochemical procedures were used to size and map the mRNA's. Of the five messages that hybridized to this 6,600-bp EcoRI fragment, only the one for the 21K polypeptide was encoded within the inverted terminal repetition and hybridized to the rightward-reading DNA strand. (Three additional early polypeptides were encoded within the first 9,000 bp of the inverted terminal repetition.) The remaining early polypeptides were encoded within the unique portion of the penultimate EcoRI fragment and were transcribed from the leftward-reading strand. Additional high-molecular-weight early RNAs of unknown function were also detected; however, there was no evidence indicating that mature mRNA's were spliced. Images PMID:6270347

Wittek, R; Cooper, J A; Moss, B

1981-01-01

278

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

PubMed

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

1990-01-01

279

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

2013-11-15

280

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.

2013-12-01

281

Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring  

PubMed Central

Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. Methods PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. Results DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. Conclusion Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method. PMID:15035672

Sharma, Rakesh; Kline, Richard

2004-01-01

282

Chromosome size and DNA values in sundews ( Droseraceae )  

Microsoft Academic Search

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

K. ROTIIFELS; M. Heimburger

1968-01-01

283

Molecular-Sized DNA or RNA Sequencing Machine  

Cancer.gov

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

284

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

285

Magnetic activated cell sorting: an effective method for reduction of sperm DNA fragmentation in varicocele men prior to assisted reproductive techniques.  

PubMed

Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P < 0.05). It can be concluded that MACs technique is a simple, noninvasive, technique that can efficiently reduce DNA fragmentation in infertile varicocele patients prior to ART. PMID:25209213

Degheidy, T; Abdelfattah, H; Seif, A; Albuz, F K; Gazi, S; Abbas, S

2014-09-11

286

Phosphorylation regulates proteasomal-mediated degradation and solubility of TAR DNA binding protein-43 C-terminal fragments  

PubMed Central

Background Inclusions of TAR DNA binding protein-43 (TDP-43) are the defining histopathological feature of several neurodegenerative diseases collectively referred to as TDP-43 proteinopathies. These diseases are characterized by the presence of cellular aggregates composed of abnormally phosphorylated, N-terminally truncated and ubiquitinated TDP-43 in the spinal cord and/or brain. Recent studies indicate that C-terminal fragments of TDP-43 are aggregation-prone and induce cytotoxicity. However, little is known regarding the pathways responsible for the degradation of these fragments and how their phosphorylation contributes to the pathogenesis of disease. Results Herein, we established a human neuroblastoma cell line (M17D3) that conditionally expresses an enhanced green fluorescent protein (GFP)-tagged caspase-cleaved C-terminal TDP-43 fragment (GFP-TDP220-414). We report that expression of this fragment within cells leads to a time-dependent formation of inclusions that are immunoreactive for both ubiquitin and phosphorylated TDP-43, thus recapitulating pathological hallmarks of TDP-43 proteinopathies. Phosphorylation of GFP-TDP220-414 renders it resistant to degradation and enhances its accumulation into insoluble aggregates. Nonetheless, GFP-TDP220-414 inclusions are reversible and can be cleared through the ubiquitin proteasome system. Moreover, both Hsp70 and Hsp90 bind to GFP-TDP220-414 and regulate its degradation. Conclusions Our data indicates that inclusions formed from TDP-43 C-terminal fragments are reversible. Given that TDP-43 inclusions have been shown to confer toxicity, our findings have important therapeutic implications and suggest that modulating the phosphorylation state of TDP-43 C-terminal fragments may be a promising therapeutic strategy to clear TDP-43 inclusions. PMID:20804554

2010-01-01

287

A localized transition in the size variation of circular DNA in nanoslits  

NASA Astrophysics Data System (ADS)

We observe a localized transition in the size variation of circular DNA between strong and moderate regimes of nanofluidic slitlike confinement. We applied a rigorous statistical analysis to our recent experimental measurements of DNA size for linear and circular topologies in nanoslits with depths around 2p, where p is the DNA persistence length [E. A. Strychalski, J. Geist, M. Gaitan, L. E. Locascio, S. M. Stavis. Macromolecules, 45, 1602-1611 (2012)]. Our empirical approach revealed a localized transition between confinement regimes for circular DNA at a nanoslit depth of 3p but detected no such transition for linear DNA with a similar contour length. These results provide the first indication of the localized influence of polymer topology on size variation across changing nanoslit depths. Improved understanding of differences in polymer behavior due to topology in this controversial system is of fundamental importance in polymer science and will inform new nanofluidic methods for biopolymer analysis.

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

2013-03-01

288

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

Czury?o, Edward A.; Hellweg, Thomas; Eimer, Wolfgang; Da?browska, Renata

1997-01-01

289

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

E-print Network

, Chourrout D (1996) Rapid one tube DNA extraction for reliable PCR detection of fish poly- morphic markers JM (1993) Characterisation of (GT)n and (CT)n microsatellites in two insect species: Apis mellifera, 271Ð272. Taylor EB (1998) Microsatellites isolated from the threespine stick- leback Gasterosteus

Provan, Jim

290

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

PubMed

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

Ryerson, D. E.; Heath, M. C.

1996-03-01

291

Estimating Dataset Size Requirements for Classifying DNA Microarray Data  

Microsoft Academic Search

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

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

2003-01-01

292

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

E-print Network

The behavior of long DNA molecules in a cell-sized confined space was investigated. We prepared water-in-oil droplets covered by phospholipids, which mimic the inner space of a cell, following the encapsulation of DNA molecules with unfolded coil and folded globule conformations. Microscopic observation revealed that the adsorption of coiled DNA onto the membrane surface depended on the size of the vesicular space. Globular DNA showed a cell-size-dependent unfolding transition after adsorption on the membrane. Furthermore, when DNA interacted with a two-phase membrane surface, DNA selectively adsorbed on the membrane phase, such as an ordered or disordered phase, depending on its conformation. We discuss the mechanism of these trends by considering the free energy of DNA together with a polyamine in the solution. The free energy of our model was consistent with the present experimental data. The cooperative interaction of DNA and polyamines with a membrane surface leads to the size-dependent behavior of molec...

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

2015-01-01

293

Surface salt bridges modulate the DNA site size of bacterial histone-like HU proteins  

PubMed Central

Bacterial histone-like DNA-binding proteins are best known for their role in compacting the genomic DNA. Of these proteins, HU is ubiquitous and highly conserved across the eubacterial kingdom. Using the HBsu (Bacillus subtilis-encoded HU homologue) as a model, we explore here the molecular basis for the ability of some HU homologues to engage a longer approx. 35 bp DNA site as opposed to the much shorter sites reported for other homologues. Using electrophoretic mobility-shift assays, we show that the DNA site size for HBsu is approx. 10–13 bp and that a specific surface salt bridge limits the DNA site size for HBsu. Surface exposure of the highly conserved Lys3, achieved by substitution of its salt-bridging partner Asp26 with Ala, leads to enhanced DNA compaction by HBsu-D26A (where D26A stands for the mutant Asp26?Ala), consistent with the interaction of Lys3 with the ends of a 25 bp duplex. Both HBsu and HBsu-D26A bend DNA, as demonstrated by their equivalent ability to promote ligase-mediated DNA cyclization, indicating that residues involved in mediating DNA kinks are unaltered in the mutant protein. We suggest that Lys3 is important for DNA wrapping due to its position at a distance from the DNA kinks where it can exert optimal leverage on flanking DNA and that participation of Lys3 in a surface salt bridge competes for its interaction with DNA phosphates, thereby reducing the occluded site size. PMID:15845027

Kamau, Edwin; Tsihlis, Nick D.; Simmons, L. Alice; Grove, Anne

2005-01-01

294

A novel fluorescent biosensor for detection of target DNA fragment from the transgene cauliflower mosaic virus 35S promoter.  

PubMed

In this paper, we reported a convenient fluorescence method for the detection of genetically modified organisms (GMOs). As it is known that the cauliflower mosaic virus (CaMV) 35S promoter is widely used in most transgenic plants (Schnurr and Guerra, 2000), we thus design a simple method based on the detection of a section target DNA (DNA-T) from the transgene CaMV 35S promoter. In this method, the full-length guanine-rich single-strand sequences were split into fragments (Probe 1 and 2) and each part of the fragment possesses two GGG repeats. In the presence of K(+) ion and berberine, if a complementary target DNA of the CaMV 35S promoter was introduced to hybridize with Probe 1 and 2, a G-quadruplex-berberine complex was thus formed and generated a strong fluorescence signal. The generation of fluorescence signal indicates the presence of CaMV 35S promoter. This method is able to identify and quantify Genetically Modified Organisms (GMOs), and it shows wide linear ranges from 5.0×10(-9) to 9.0×10(-7) mol/L with a detection limit of 2.0×10(-9) mol/L. PMID:22959013

Qiu, Bin; Zhang, Ya-shan; Lin, Yi-bing; Lu, Yu-Jing; Lin, Zhen-yu; Wong, Kwok-Yin; Chen, Guo-nan

2013-03-15

295

Effect of different gold nanoparticle sizes to build an electrical detection DNA between nanogap electrodes  

Microsoft Academic Search

An experimental study for identifying DNA strands immobilized in the different particle sizes of gold nanoparticle (AuNP) between nanogap electrodes is presented. The nanogap-based electrics chip demonstrates a significant electrical property with a target DNA (TDNA) concentration of 10pM in the different particle sizes of AuNP to be analyzed in order to make the assay useful for an integrated lab-on-a-chip

Tien-Li Chang; Ya-Wei Lee; Chun-Chi Chen; Fu-Hsiang Ko

2007-01-01

296

Spent fuel waste form characteristics: Grain and fragment size statistical dependence for dissolution response  

SciTech Connect

The Yucca Mountain Project of the US Department of Energy is investigating the suitability of the unsaturated zone at Yucca Mountain, NV, for a high-level nuclear waste repository. All of the nuclear waste will be enclosed in a container package. Most of the nuclear waste will be in the form of fractured UO{sub 2} spent fuel pellets in Zircaloy-clad rods from electric power reactors. If failure of both the container and its enclosed clad rods occurs, then the fragments of the fractured UO{sub 2} spent fuel will be exposed to their surroundings. Even though the surroundings are an unsaturated zone, a possibility of water transport exists, and consequently, UO{sub 2} spent fuel dissolution may occur. A repository requirement imposes a limit on the nuclide release per year during a 10,000 year period; thus the short term dissolution response from fragmented fuel pellet surfaces in any given year must be understood. This requirement necessitates that both experimental and analytical activities be directed toward predicting the relatively short term dissolution response of UO{sub 2} spent fuel. The short term dissolution response involves gap nuclides, grain boundary nuclides, and grain volume nuclides. Analytical expressions are developed that describe the combined geometrical influences of grain boundary nuclides and grain volume nuclides on the dissolution rate of spent fuel. 7 refs., 1 fig.

Stout, R.B.; Leider, H.; Weed, H.; Nguyen, S.; McKenzie, W.; Prussin, S. [Lawrence Livermore National Lab., CA (USA); Wilson, C.N.; Gray, W.J. [Pacific Northwest Lab., Richland, WA (USA)

1991-04-01

297

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

2013-01-01

298

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

1992-01-01

299

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

2011-01-01

300

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

Microsoft Academic Search

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

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

1999-01-01

301

Early-replicating DNA from mosquito cells is associated with a distinct EcoRI fragment  

Microsoft Academic Search

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteristic features of single-stranded DNA to methotrexate-resistant Aedes albopictus cells. The Mtx-5011-256 cells contained approximately 1000 copies of a 200kb amplicon containing the dihydrofolate reductase locus, which likely contained one or more replication origins. When Mtx-5011-256 cells

Zhong Hui Wang; Ann M Fallon

1999-01-01

302

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

NASA Astrophysics Data System (ADS)

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

Brancolini, Giorgia; Di Felice, Rosa

2011-05-01

303

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

PubMed

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection. PMID:21639482

Brancolini, Giorgia; Di Felice, Rosa

2011-05-28

304

A new quasi-interpenetrating network formed by poly(N-acryloyl-tris-(hydroxymethyl)aminomethane and polyvinylpyrrolidone: separation matrix for double-stranded DNA and single-stranded DNA fragments by capillary electrophoresis with UV detection.  

PubMed

The preparation of a new separation matrix, quasi-interpenetrating networks (quasi-IPNs) formed by poly(N-acryloyl-Tris) (poly(tris-A)) and PVP, and its application for dsDNA and ssDNA fragments separation by CE with UV detection, are presented. This new quasi-IPN exhibited high sieving performance, good dynamic coating ability, and low viscosity. Single-base resolutions of dsDNA fragments (Rs = 0.92 for 123/124 bp) and ssDNA fragments (Rs = 0.65 for 123/124 base, Rs = 0.48 for 309/310 base) were achieved by using the quasi-IPN of poly(tris-A)/PVP (2% + 2%) solution in a 31 cm effective length linear polyacrylamide (LPA)-coated column. Single-base separation of dsDNA fragments (Rs = 0.92 for 123/124 bp) was also obtained within 28 min in a 46.7 cm effective length bare column at higher 160 V/cm electric field strength by using the same quasi-IPN solution. The RSD of the migration time measured for each DNA fragments was less than 1.5% in the bare column for nine continuous runs. The effects of temperature and electric field strength on the DNA separation were also investigated. PMID:16586410

Wang, Qian; Xu, Xu; Dai, Lixin

2006-05-01

305

Histone Hl-DNA interaction. Influence of phosphorylation on the interaction of histone Hl with linear fragmented DNA.  

PubMed Central

By measuring the fluorescence polarization of fluorescent histone H1 derivatives complexed with DNA, binding of the histone to DNA was studied as a function of ionic strength in the solution prior to and after the H1 phosphorylation on Ser-37 residue. Fluorescent labels were covalently linked either specifically to Tyr-72 residues or unspecifically to lysine residues in the H1 polypeptide chain. The values of the corresponding rotational relaxation times showed that at low ionic strength all the segments of the H1 molecule were immobilized on binding to DNA. The gradual increasing NaC1 concentration in the solution of H1-DNA complex was accompanied at first by additional retardation of the histone mobility in the complex, and then by progressive release of histone H1 from from the complex which was completed at 0.5-0.6 M NaC1 irrespective of phosphorylation. tat the same time the phosphorylation of histone H1 led to removal of the central and, presumably, N-terminal regions of H1 from DNA. PMID:194228

Glotov, B O; Nikolaev, L G; Kurochkin, S N; Severin, E S

1977-01-01

306

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

PubMed

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

2013-05-01

307

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.  

PubMed Central

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene. Images PMID:1990269

You, Y; Aufderheide, K; Morand, J; Rodkey, K; Forney, J

1991-01-01

308

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

Siviková, Katarína; Dianovsky, Ján; Holecková, Beáta

2011-01-01

309

Constructing a DNA ladder Range for Lambda Phage by multiplex PCR  

PubMed Central

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

Gopalakrishnan, R; Joseph, S; Sellappa, S

2010-01-01

310

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

Microsoft Academic Search

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

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

2006-01-01

311

Gel electrophoresis, 2D animationSite: DNA Interactive (www.dnai.org)  

NSDL National Science Digital Library

In the early days of DNA manipulation, DNA fragments were laboriously separated by gravity. In the 1970s, the powerful tool of DNA gel electrophoresis was developed. This process uses electricity to separate DNA fragments by size as they migrate through a gel matrix.

2008-10-06

312

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

PubMed

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

2014-10-15

313

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

2012-01-01

314

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

Veselý, Pavel; Bureš, Petr; Šmarda, Petr; Pavlí?ek, Tomáš

2012-01-01

315

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.

2003-01-01

316

Multi-Scale Particle Size Distributions of Mars, Moon and Itokawa based on a time-maturation dependent fragmentation model  

NASA Astrophysics Data System (ADS)

We present the development of a soil evolution framework and multiscale modelling of the surface of Mars, Moon and Itokawa thus providing an atlas of extra-terrestrial Particle Size Distributions (PSD). These PSDs are profoundly based on a tailoring method which interconnects several datasets from different sites captured by the various missions. The final integrated product is then fully justified through a soil evolution analysis model mathematically constructed via fundamental physical principles (Charalambous, 2013). The construction of the PSD takes into account the macroscale fresh primary impacts and their products, the mesoscale distributions obtained by the in-situ data of surface missions (Golombek et al., 1997, 2012) and finally the microscopic scale distributions provided by Curiosity and Phoenix Lander (Pike, 2011). The distribution naturally extends at the magnitudinal scales at which current data does not exist due to the lack of scientific instruments capturing the populations at these data absent scales. The extension is based on the model distribution (Charalambous, 2013) which takes as parameters known values of material specific probabilities of fragmentation and grinding limits. Additionally, the establishment of a closed-form statistical distribution provides a quantitative description of the soil's structure. Consequently, reverse engineering of the model distribution allows the synthesis of soil that faithfully represents the particle population at the studied sites (Charalambous, 2011). Such representation essentially delivers a virtual soil environment to work with for numerous applications. A specific application demonstrated here will be the information that can directly be extracted for the successful drilling probability as a function of distance in an effort to aid the HP3 instrument of the 2016 Insight Mission to Mars. Pike, W. T., et al. "Quantification of the dry history of the Martian soil inferred from in situ microscopy." Geophysical Research Letters 38.24 (2011). C. A. Charalambous and W. T. Pike (2013). 'Evolution of Particle Size Distributions in Fragmentation Over Time' Abstract Submitted to the AGU 46th Fall Meeting. Charalambous, C., Pike, W. T., Goetz, W., Hecht, M. H., & Staufer, U. (2011, December). 'A Digital Martian Soil based on In-Situ Data.' In AGU Fall Meeting Abstracts (Vol. 1, p. 1669). Golombek, M., & Rapp, D. (1997). 'Size-frequency distributions of rocks on Mars and Earth analog sites: Implications for future landed missions.' Journal of Geophysical Research, 102(E2), 4117-4129. Golombek, M., Huertas, A., Kipp, D., & Calef, F. (2012). 'Detection and characterization of rocks and rock size-frequency distributions at the final four Mars Science Laboratory landing sites.' Mars, 7, 1-22.

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

2013-12-01

317

The influence of polaron size on the conductivity of poly-DNA  

E-print Network

The velocity of polaron migration in the long poly-DNA chain (~40 base pairs) in an applied electric field has been studied within a polaron model. We found that the polaron velocity strongly depends on the polaron size. A small polaron shows a slow propagation and strong tolerance to the electric field, while a large polaron is much faster and less stable with increasing electric field. Moreover, the conductance of the DNA molecule within the polaron model is found to be sensitive to structural disorders in the DNA geometry, but that dependence diminishes with increasing temperature.

Julia A. Berashevich; Adam D. Bookatz; Tapash Chakraborty

2007-09-07

318

Internucleosomal DNA fragmentation in wild emmer wheat is catalyzed by S1-type endonucleases translocated to the nucleus upon induction of cell death.  

PubMed

Leaves of cereal plants display nucleosomal fragmentation of DNA attributed to the action of nucleases induced during program cell death (PCD). Yet, the specific nuclease activity responsible for generating double strand DNA breaks (DSBs) that lead to DNA fragmentation has not been fully described. Here, we characterized a Ca(2+)/Mg(2+)-dependent S1-type endonuclease activity in leaves of wild emmer wheat (Triticum dicoccoides Köern.) capable of introducing DSBs as demonstrated by the conversion of supercoiled plasmid DNA into a linear duplex DNA. In-gel nuclease assay revealed a nuclease of about 35kDa capable of degrading both single stranded DNA and RNA. We further showed that the endonuclease activity can be purified on Concanavalin A and treatment with peptide-N-glycosidase F (PNGase F) did not abolish its activity. Furthermore, ConA-associated endonuclease was capable of generating nucleosomal DNA fragmentation in tobacco nuclei. Since S1-type endonucleases lack canonical nuclear localization signal it was necessary to determine their subcellular localization. To this end, a cDNA encoding for a putative 34kDa S1-type nuclease, designated TaS1-like (TaS1L) was synthesized based on available sequence data of Triticum aestivum and fused with RFP. Introduction into protoplasts showed that TaS1L-RFP is cytoplasmic 24h post transformation but gradually turn nuclear at 48h concomitantly with induction of cell death. Our results suggest that DNA fragmentation occurring in leaves of wild emmer wheat may be attributed to S1-type endonuclease(s) that reside in the cytoplasm but translocate to the nucleus upon induction of cell death. PMID:25497371

Granot, Gila; Morgenstern, Yaakov; Khan, Asif; Givaty Rapp, Yemima; Pesok, Anat; Nevo, Eviatar; Grafi, Gideon

2015-03-01

319

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

2009-01-01

320

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

1993-01-01

321

Structure and expression of DNA transferred to tobacco via transformation of protoplasts with Ti-plasmid DNA: co-transfer of T-DNA and non T-DNA sequences  

Microsoft Academic Search

The T-DNA structure and organization in tissues obtained via transformation of tobacco protoplasts with Ti-plasmid DNA was found to be completely different from the T-DNA introduced via Agrobacterium tumefaciens. It is often fragmented. Overlapping copies of T-DNA, having various sizes, as well as separated fragments of T-DNA were detected. The border sequences of 23 basepairs (bp), flanking the T-region in

Frans A. Krens; Ruud M. W. Mans; Truus M. S. Slogteren; J. Harry C. Hoge; George J. Wullems; Robbert A. Schilperoort

1985-01-01

322

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.

1985-01-01

323

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms  

Microsoft Academic Search

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear\\u000a but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a\\u000a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed\\u000a intrageneric

Mitsuyasu Hasebe; Toshio Ando; Kunio Iwatsuki

1998-01-01

324

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

2010-01-01

325

Analysis of conflicting experimental studies of DNA size in nanofluidic slits  

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

326

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.

2013-01-01

327

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

PubMed

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; Almeida, Rodrigo P P

2013-03-01

328

An analysis of population genetic structure and species history of Drosophila melanogaster and Drosophila simulans using restriction fragment length polymorphisms of mitochondrial DNA  

Microsoft Academic Search

Animal mitochondrial DNA (mtDNA) has several features that give it great utility in the study of geographic structure of natural populations. Its small size and covalently closed circular conformation make it easy to purify. Strict maternal inheritance and homoplasmy makes the effective copy number of mtDNA as little as 1\\/4 that of nuclear loci; this renders populational complements of mtDNA

Lawrence Richard Hale

1989-01-01

329

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

2011-01-01

330

Early DNA sequencing, 2D animationSite: DNA Interactive (www.dnai.org)  

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.

2008-10-06

331

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.

1993-01-01

332

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

2005-11-16

333

Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle.  

PubMed

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaflets (DNA amounts per organelle. The approach is based on plastid purification from homogenates of moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4',6-diamidino-2-phenylindole) was used to estimate numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 x 10(-2) pg DNA for plastids of 1-->8 microm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves differing in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value. PMID:19911199

Rauwolf, Uwe; Golczyk, Hieronim; Greiner, Stephan; Herrmann, Reinhold G

2010-01-01

334

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

335

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

NASA Astrophysics Data System (ADS)

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

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

2015-01-01

336

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

1999-01-01

337

Trypanosoma evansi: Genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates  

Microsoft Academic Search

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar (‘type A’) whereas 2 isolates differed substantially (‘type B’). Type

Daniel K. Masiga; Kariuki Ndung’u; Alison Tweedie; Andrew Tait; C. Michael R. Turner

2006-01-01

338

DNA isolated from Mycobacterium leprae: genome size, base ratio, and homology with other related bacteria as determined by optical DNA-DNA reassociation.  

PubMed Central

DNA derived from Mycobacterium leprae (grown in armadillos) was isolated, purified, and analyzed spectrophotometrically. The genome size and the guanine-plus-cytosine content of M. leprae were 1.3 x 10(9) and 55.8%, respectively. Among selected strains of mycobacterial, nocardial, and corynebacterial species, Corynebacterium sp. 2628 LB, isolated from a human leprosy patient, showed the highest DNA homology with M. leprae. Of the DNAs derived from mycobacteria, those of M. tuberculosis and M. scrofulaceum showed a comparatively high reassociation with the DNMA of M. liprae. PMID:6801025

Imaeda, T; Kirchheimer, W F; Barksdale, L

1982-01-01

339

Genetic diversity in the Block 2 region of the merozoite surface protein 1 (MSP-1) of Plasmodium falciparum: Additional complexity and selection and convergence in fragment size polymorphism  

Microsoft Academic Search

Fragment size in the Block 2 repetitive region of merozoite surface protein 1 (MSP1) has commonly been used as a molecular marker in studies of malaria transmission dynamics and host immunity in Plasmodium falciparum malaria. In this study, we further explore the genetic variation in MSP-1 Block 2 underlying potential problems faced while studying the immune responses elicited by this

S. L. Takala; A. A. Escalante; O. H. Branch; S. Kariuki; S. Biswas; S. C. Chaiyaroj; A. A. Lal

2006-01-01

340

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, sizeexpanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. Themost relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMOLUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

Fuentes-Cabrera, Miguel A [ORNL; Orozco, Modesto [Institut de Recerca Biomedica, Parc Cientific de Barcelona, Barcelona, Spain; Luque, Javier [Universitat de Barcelona; Sumpter, Bobby G [ORNL; Blas, Jose [Universidad de Castilla-La Mancha; Ordejon, Pablo J [ORNL; Huertas, Oscar [Universitat de Barcelona; Tabares, Carolina [Universitat de Barcelona

2011-01-01

341

DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping*  

PubMed Central

Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresis†; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3?-terminus and seven from the right-hand 3?-terminus of bacteriophage ? DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3?-terminus and five nucleotides from the right-hand terminus of bacteriophage ?80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed. Images PMID:10793670

Jay, Ernest; Bambara, Robert; Padmanabhan, R.; Wu, Ray

1974-01-01

342

Multiplex analysis of DNA  

DOEpatents

This invention features vectors and a method for sequencing DNA. The method includes the steps of: a) ligating the DNA into a vector comprising a tag sequence, the tag sequence includes at least 15 bases, wherein the tag sequence will not hybridize to the DNA under stringent hybridization conditions and is unique in the vector, to form a hybrid vector, b) treating the hybrid vector in a plurality of vessels to produce fragments comprising the tag sequence, wherein the fragments differ in length and terminate at a fixed known base or bases, wherein the fixed known base or bases differs in each vessel, c) separating the fragments from each vessel according to their size, d) hybridizing the fragments with an oligonucleotide able to hybridize specifically with the tag sequence, and e) detecting the pattern of hybridization of the tag sequence, wherein the pattern reflects the nucleotide sequence of the DNA.

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

1992-01-01

343

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.  

PubMed

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction. PMID:10330695

Marzais, B; Vorsanova, S G; Roizes, G; Yurov, Y B

1999-01-01

344

Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes.  

PubMed

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56-150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles. PMID:21417671

Carstens, Myrra G; van der Maaden, Koen; van der Velden, Daniel; Ottenhoff, Tom H M; Melief, Cornelis J; Ossendorp, Ferry; Bouwstra, Joke A; Jiskoot, Wim

2011-12-01

345

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

PubMed

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

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

2011-12-01

346

5-Hydroxypyrimidine deoxynucleoside triphosphates are more efficiently incorporated into DNA by exonuclease-free Klenow fragment than 8-oxopurine deoxynucleoside triphosphates.  

PubMed Central

Recent studies with 8-oxodeoxyguanosine triphosphate (8-oxodGTP) have suggested that incorporation of oxidized nucleotides from the precursor pool into DNA may have deleterious effects. Here we show that 5-hydroxydeoxycytosine triphosphate (5-OHdCTP) and 5-hydroxydeoxyuridine triphosphate (5-OHdUTP) are more efficient substrates than 8-oxodGTP for Escherichia coli DNA polymerase I Klenow fragment lacking proofreading activity, while 8-oxodeoxyadenosine triphosphate (8-oxodGTP, 5-OHdCTP can mispair with dA in DNA but with lower efficiency. Since the 5-hydroxypyrimidines are present in normal and oxidized cellular DNA in amounts similar to the 8-oxopurines, these data suggest that enzymatic mechanisms might exist for removing them from the DNA precursor pools. Images PMID:7937115

Purmal, A A; Kow, Y W; Wallace, S S

1994-01-01

347

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

Microsoft Academic Search

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

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

1997-01-01

348

Rapid Identification of Nocardia farcinica Clinical Isolates by a PCR Assay Targeting a 314-Base-Pair Species-Specific DNA Fragment  

PubMed Central

Nocardia farcinica is the most clinically significant species within the Nocardia asteroides complex. Differentiation of N. farcinica from other members of N. asteroides complex is important because this species characteristically demonstrates resistance to several extended-spectrum antimicrobial agents. Traditional phenotypic characterization of this species is time- and labor-intensive and often leads to misidentification in the clinical microbiology laboratory. We previously observed a 409-bp product for all strains of N. farcinica by using randomly amplified polymorphic DNA analysis with the primer DKU49. In this investigation, the 409-bp fragment was sequenced and then used to design a specific primer pair, Nf1 (16-mer) and Nf2 (16-mer), complementary to the 409-bp fragment. PCR amplification of genomic DNA from 28 N. farcinica isolates with Nf1 and Nf2 generated a single intense 314-bp fragment. The specificity of the assay with these primers was verified, since there were no PCR amplification products observed from heterologous nocardial species (n = 59) or other related bacterial genera (n = 41). Restriction enzyme digestion using CfoI and direct sequencing of the 314-bp fragment further confirmed the specificity of the assay for N. farcinica. This highly sensitive and specific PCR assay provides a rapid (within 1 day of obtaining DNA) method for identification of this medically important emerging pathogen. Rapid diagnosis of N. farcinica infection may allow for earlier initiation of effective therapy, thus improving patient outcome. PMID:15297512

Brown, June M.; Pham, Kim N.; McNeil, Michael M.; Lasker, Brent A.

2004-01-01

349

Factors controlling particle size during nebulization of DNA-polycation complexes.  

PubMed

Pulmonary gene therapy has the potential to treat or cure respiratory diseases such as cystic fibrosis. Much work has focused on the delivery of genes to the lung using viral vectors with varying degrees of success. Viral vectors are problematic and undesirable for use in the lung because they can provoke an acute immune response. This study has focused on the characterization of nonviral, polymer-based gene vectors for use with nebulizers. Calf thymus DNA has been used as a model, and was complexed with each of the three polycations; 22 kDa linear polyethyleneimine, 25 kDa branched polyethyleneimine, and 29.5 kDa polylysine using water, glucose solution, and phosphate-buffered saline (PBS) as carrier liquids. Fourier transform infrared spectroscopy has shown that the DNA retains the B form during the complex formation. The complexes prepared at N:P ratios of 10, have been nebulized using a vibrating plate nebulizer and the particle size and Zeta potentials measured before and after nebulization. The particle size distributions of the DNA complexes prepared in water and glucose solution were unimodal before and after nebulization with a small increase in particle size following nebulization. Choice of complexing polymer is shown to have only a small effect on particle size with the dominant effect coming from the ionic character of the dispersion fluid. Complexes prepared in PBS, although originally unimodal, showed pronounced agglomeration on nebulization. With all polymers in water or glucose solution, the Zeta potential increases after nebulization, but with PBS as the carrier liquid the potential falls and is clearly associated with the observed agglomeration. Gel electrophoresis shows that the complexing polymers protect the DNA through the nebulization process in all cases. PMID:17894533

Lynch, J; Behan, N; Birkinshaw, Colin

2007-01-01

350

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)

2013-05-07

351

Reaction of protein chloramines with DNA and nucleosides: evidence for the formation of radicals, protein-DNA cross-links and DNA fragmentation.  

PubMed Central

Stimulated phagocyte cells produce the oxidant HOCl, via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is important in bacterial cell killing, but excessive or misplaced generation can damage the host tissue and may lead to the development of certain diseases such as cancer. The role of HOCl in the oxidation of isolated proteins, DNA and their components has been investigated extensively, but little work has been performed on the protein-DNA (nucleosome) complexes present in eukaryotic cell nuclei. Neither the selectivity of damage in such complexes nor the possibility of transfer of damage from the protein to DNA or vice versa, has been studied. In the present study, kinetic modelling has been employed to predict that reaction occurs predominantly with the protein and not with the DNA in the nucleosome, using molar HOCl excesses of up to 200-fold. With 50-200-fold excesses, 50-80% of the HOCl is predicted to react with histone lysine and histidine residues to yield chloramines. The yield and stability of such chloramines predicted by these modelling studies agrees well with experimental data. Decomposition of these species gives protein-derived, nitrogen-centred radicals, probably on the lysine side chains, as characterized by the EPR and spin-trapping experiments. It is shown that isolated lysine, histidine, peptide and protein chloramines can react with plasmid DNA to cause strand breaks. The protection against such damage afforded by the radical scavengers Trolox (a water-soluble alpha-tocopherol derivative) and 5,5-dimethyl-1-pyrroline-N-oxide suggests a radical-mediated process. The EPR experiments and product analyses have also provided evidence for the rapid addition of protein radicals, formed on chloramine decomposition, to pyrimidine nucleosides to give nucleobase radicals. Further evidence for the formation of such covalent cross-links has been obtained from experiments performed using (3)H-lysine and (14)C-histidine chloramines. These results are consistent with the predictions of the kinetic model and suggest that histones are major targets for HOCl in the nucleosome. Furthermore, the resulting protein chloramines and the radicals derived from them may act as contributing agents in HOCl-mediated DNA oxidation. PMID:12010123

Hawkins, Clare L; Pattison, David I; Davies, Michael J

2002-01-01

352

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

PubMed

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

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

2014-02-01

353

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

2006-01-01

354

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)

2013-11-21

355

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

NASA Astrophysics Data System (ADS)

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 108 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.

2013-11-01

356

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

PubMed

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

2012-01-01

357

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.  

PubMed Central

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein. Images Fig. 2. Fig. 3. Fig. 5. PMID:1372802

Nilsson, B; Grossberger, D; Nilsson Ekdahl, K; Riegert, P; Becherer, D J; Nilsson, U R; Lambris, J D

1992-01-01

358

Investigation of the plastoquinone pool size and fluorescence quenching in thylakoid membranes and Photosystem II (PS II) membrane fragments  

Microsoft Academic Search

The efficiency of oxidized endogenous plastoquinone-9 (PQ-9) as a non-photochemical quencher of chlorophyll fluorescence has\\u000a been analyzed in spinach thylakoids and PS II membrane fragments isolated by Triton X-100 fractionation of grana stacks. The\\u000a following results were obtained: (a) After subjection of PS II membrane fragments to ultrasonic treatment in the presence\\u000a of PQ-9, the area over the induction curve

Jens Kurreck; René Schödel; Gernot Renger

2000-01-01

359

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

PubMed

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

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

2013-11-01

360

Analysis of plastid and mitochondrial DNA insertions in the nucleus (NUPTs and NUMTs) of six plant species: size, relative age and chromosomal localization.  

PubMed

We analysed the size, relative age and chromosomal localization of nuclear sequences of plastid and mitochondrial origin (NUPTs-nuclear plastid DNA and NUMTs-nuclear mitochondrial DNA) in six completely sequenced plant species. We found that the largest insertions showed lower divergence from organelle DNA than shorter insertions in all species, indicating their recent origin. The largest NUPT and NUMT insertions were localized in the vicinity of the centromeres in the small genomes of Arabidopsis and rice. They were also present in other chromosomal regions in the large genomes of soybean and maize. Localization of NUPTs and NUMTs correlated positively with distribution of transposable elements (TEs) in Arabidopsis and sorghum, negatively in grapevine and soybean, and did not correlate in rice or maize. We propose a model where new plastid and mitochondrial DNA sequences are inserted close to centromeres and are later fragmented by TE insertions and reshuffled away from the centromere or removed by ectopic recombination. The mode and tempo of TE dynamism determines the turnover of NUPTs and NUMTs resulting in their species-specific chromosomal distributions. PMID:23715017

Michalovova, M; Vyskot, B; Kejnovsky, E

2013-10-01

361

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))

1993-01-01

362

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)

1993-06-01

363

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.

1995-01-01

364

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

2008-01-01

365

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)

2010-05-15

366

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

Microsoft Academic Search

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

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

1998-01-01

367

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

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

2001-01-01

368

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

PubMed Central

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

2013-01-01

369

Genetic markers in ribosomal DNA for the identification of members of the genus Anisakis (Nematoda: Ascaridoidea) defined by polymerase-chain-reaction-based restriction fragment length polymorphism  

Microsoft Academic Search

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction

S D'Amelio; K. D Mathiopoulos; C. P Santos; O. N Pugachev; S. C Webb; M Picanço; L Paggi

2000-01-01

370

In situ detection of fragmented DNA (tunel assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: A cautionary note  

Microsoft Academic Search

Detection of DNA fragments in situ using the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay is increasingly applied to investigate active cell death (apoptosis). We studied the specificity of the assay in well-defined models of apoptosis and necrosis as well as in postmortem autolysis in rat liver. During involution of liver hyperplasia, which follows stopping treatment with

Bettina Grasl-Kraupp; Branislav Ruttkay-Nedecky; Helga Koudelka; Krystyna Bukowska; Wilfried Bursch; Rolf Schulte-Hermann

1995-01-01

371

Phylogenetic relationships of Thiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity\\u000a of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein.\\u000a DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the\\u000a other sample, which probably correspond to the dominant

Gerard Muyzer; Andreas Teske; Carl O. Wirsen; Holger W. Jannasch

1995-01-01

372

When free transposon ends are used in the reaction, the target DNA is fragmented and the transferred strand of the transposon end  

E-print Network

The advent of next-generation sequencing has made possible genome analysis at previously unattainable depth sequencing. Regardless of the instrument, one of the bottlenecks for next-generation sequencing is the amount frag- ment (Fig. 1a).The size distribution of the fragments can be controlled At present, next-generation

Cai, Long

373

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

2003-01-01

374

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

2004-01-01

375

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. PMID:25401071

2014-01-01

376

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

PubMed Central

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445–448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique. PMID:16349533

Clerc, Agathe; Manceau, Charles; Nesme, Xavier

1998-01-01

377

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

2002-07-01

378

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

PubMed

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

2014-01-01

379

Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants. Evaluation by stable isotopic enrichment.  

PubMed

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed. PMID:1903521

Hutchens, T W; Henry, J F; Yip, T T; Hachey, D L; Schanler, R J; Motil, K J; Garza, C

1991-03-01

380

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

PubMed

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

1992-02-01

381

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

2012-01-01

382

DNA DNA DNA (d)DNA DNA DNA  

E-print Network

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

Hagiya, Masami

383

Mitochondrial DNA polymorphism in Japanese  

Microsoft Academic Search

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

Satoshi Horai; Ei Matsunaga

1986-01-01

384

Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies  

PubMed Central

Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519–539, E969–983, E1063–1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. PMID:19816594

Wang, Yong; Qian, Pei-Yuan

2009-01-01

385

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.

386

Nucleotide sequence of a 2 kbp BamH I fragment of Vicia faba chloroplast DNA containing the genes for threonine, glutamic acid and tyrosine transfer RNAs.  

PubMed Central

The entire nucleotide sequence of a 2014 bp BamH I fragment from broad bean (Vicia faba) chloroplast DNA containing the genes for tRNAThr (trnT), tRNAGlu (trnE) and tRNATyr (trnY) has been determined. The tRNAGlu and tRNATyr genes are separated by only 60 bp and are probably part of the same transcriptional unit. The tRNAThr gene is located on the complementary strand, 876 bp away from the tRNAGlu gene. This fragment also contains an open reading frame of 82 codons, as well as a series of AT-rich, direct and inverted repeats. PMID:6330696

Kuntz, M; Weil, J H; Steinmetz, A

1984-01-01

387

Application of a time-dependent coalescence process for inferring the history of population size changes from DNA sequence data.  

PubMed

Distribution of pairwise differences of nucleotides from data on a sample of DNA sequences from a given segment of the genome has been used in the past to draw inferences about the past history of population size changes. However, all earlier methods assume a given model of population size changes (such as sudden expansion), parameters of which (e.g., time and amplitude of expansion) are fitted to the observed distributions of nucleotide differences among pairwise comparisons of all DNA sequences in the sample. Our theory indicates that for any time-dependent population size, N(tau) (in which time tau is counted backward from present), a time-dependent coalescence process yields the distribution, p(tau), of the time of coalescence between two DNA sequences randomly drawn from the population. Prediction of p(tau) and N(tau) requires the use of a reverse Laplace transform known to be unstable. Nevertheless, simulated data obtained from three models of monotone population change (stepwise, exponential, and logistic) indicate that the pattern of a past population size change leaves its signature on the pattern of DNA polymorphism. Application of the theory to the published mtDNA sequences indicates that the current mtDNA sequence variation is not inconsistent with a logistic growth of the human population. PMID:9576903

Polanski, A; Kimmel, M; Chakraborty, R

1998-05-12

388

Application of a time-dependent coalescence process for inferring the history of population size changes from DNA sequence data  

PubMed Central

Distribution of pairwise differences of nucleotides from data on a sample of DNA sequences from a given segment of the genome has been used in the past to draw inferences about the past history of population size changes. However, all earlier methods assume a given model of population size changes (such as sudden expansion), parameters of which (e.g., time and amplitude of expansion) are fitted to the observed distributions of nucleotide differences among pairwise comparisons of all DNA sequences in the sample. Our theory indicates that for any time-dependent population size, N(?) (in which time ? is counted backward from present), a time-dependent coalescence process yields the distribution, p(?), of the time of coalescence between two DNA sequences randomly drawn from the population. Prediction of p(?) and N(?) requires the use of a reverse Laplace transform known to be unstable. Nevertheless, simulated data obtained from three models of monotone population change (stepwise, exponential, and logistic) indicate that the pattern of a past population size change leaves its signature on the pattern of DNA polymorphism. Application of the theory to the published mtDNA sequences indicates that the current mtDNA sequence variation is not inconsistent with a logistic growth of the human population. PMID:9576903

Polanski, Andrzej; Kimmel, Marek; Chakraborty, Ranajit

1998-01-01

389

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

2001-01-01

390

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

PubMed

Sperm DNA fragmentation (SDF) has been proposed to be one of the main markers regarding male infertility. A prospective study was performed to assess primarily whether sperm DNA damage has any impact on embryological data and secondarily on pregnancy rates. This prospective study evaluated the sperm DNA damage in fresh ejaculated sperm samples from couples undergoing IVF/ICSI treatments, using the improved SCD method, known as Halosperm(®) . The results were evaluated by performing statistical analysis with the statistical package of SPSS v17. A total of 156 fresh semen samples derived from 156 couples undergoing 156 IVF/ICSI cycles. From the 156 couples, 139 finally reached the embryo transfer (ET) procedure. Overall, SDF did not correlate with embryological data, while ongoing pregnancy rate/ET was 21.6%. SDF only correlated with sperm characteristics. After the categorisation of SDF (?35% and >35%), according to the specifi