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Sizing of DNA fragments by flow cytometry  

SciTech Connect

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

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



Sizing of DNA fragments by flow cytometry  

SciTech Connect

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

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



Automated DNA fragments recognition and sizing through AFM image processing  

Microsoft Academic Search

This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes

Elisa Ficarra; Luca Benini; Enrico Macii; Giampaolo Zuccheri



Sizing of single fluorescently stained DNA fragments by scanning microscopy  

PubMed Central

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

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



Distribution of DNA fragment sizes after irradiation with ions  

NASA Astrophysics Data System (ADS)

Ionizing radiation is responsible for production of double-strand breaks (DSBs) in a DNA structure. In contrast to sparsely ionizing radiation, densely ionizing radiation produces DSBs that are non-randomly distributed along the DNA molecule and can form clusters of various size. The paper discusses minimalistic models that describe observable patterns of fragment length in DNA segments irradiated with heavy ions and applies the formalism to interpret the recent experimental data collected by use of atomic force microscope (AFM).

Gudowska-Nowak, E.; Psonka-Anto?czyk, K.; Weron, K.; Elsässer, T.; Taucher-Scholz, G.



Automated DNA fragments recognition and sizing through AFM image processing.  


This paper presents an automated algorithm to determine DNA fragment size from atomic force microscope images and to extract the molecular profiles. The sizing of DNA fragments is a widely used procedure for investigating the physical properties of individual or protein-bound DNA molecules. Several atomic force microscope (AFM) real and computer-generated images were tested for different pixel and fragment sizes and for different background noises. The automated approach minimizes processing time with respect to manual and semi-automated DNA sizing. Moreover, the DNA molecule profile recognition can be used to perform further structural analysis. For computer-generated images, the root mean square error incurred by the automated algorithm in the length estimation is 0.6% for a 7.8 nm image pixel size and 0.34% for a 3.9 nm image pixel size. For AFM real images we obtain a distribution of lengths with a standard deviation of 2.3% of mean and a measured average length very close to the real one, with an error around 0.33%. PMID:16379368

Ficarra, Elisa; Benini, Luca; Macii, Enrico; Zuccheri, Giampaolo



Automated sizing of DNA fragments in atomic force microscope images  

Microsoft Academic Search

Current techniques used to measure lengths of DNA fragments in atomic force microscope (AFM) images require a user to operate\\u000a interactive software and execute tedious error-prone cursor selections. An algorithm is proposed which provides an automated\\u000a method for determining DNA fragment lengths from AFM images without interaction from the computer operator (e.g. cursor selections\\u000a or mouse clicks). The approach utilises

T. S. Spisz; Y. Fang; R. H. Reeves; C. K. Seymour; I. N. Bankman; J. H. Hoh



Analysis of DNA fragmentation using a dynamic size-sieving polymer solution in capillary electrophoresis.  


Various natural and induced processes cause DNA fragmentation. Examples of these processes include apoptosis, enzymatic digestion, free radical production from ionizing radiation, photoscission by laser radiation and thermal degradation. Slab gel electrophoresis has been used most often to monitor such DNA damage. We have investigated with capillary electrophoresis the use of a new size-sieving polymer solution, TreviSol-CE (TS-CE), to monitor the DNA fragments produced from a variety of degradation processes. This polymer solution provides high run-to-run migration time and peak width reproducibilities and high separation efficiency of double-stranded DNA fragments in the 500 to 7000 base pair size range. Analysis of apoptotic DNA fragments suggested the presence of multiple nucleosomes within each cell type investigated. For irradiated DNA standards, peak-width-at-half-height and peak area were used to monitor the progress of DNA fragmentation. For both apoptotic DNA and irradiated DNA standards, fine structural features of fragmentation were revealed. PMID:9210317

Siles, B A; Nackerdien, Z E; Collier, G B



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

SciTech Connect

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

Erixon, K.; Cedervall, B. [Karolinksa Institutet, Stockholm (Sweden)



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


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

Gough, E J; Gough, N M



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

PubMed Central

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

Gough, E J; Gough, N M



A wide-range, low-cost 150 bp ladder for sizing DNA fragments between 150 and 4500 bp.  


Agarose gel electrophoresis, a very routine procedure, requires molecular weight standards; these are usually manufactured from plasmid or viral DNA fragments, or more recently, from PCR products of defined sizes. We describe here the preparation of a molecular weight standard from a completely different DNA source - the uniquely organized genome of the beetle Tenebrio molitor. The standard can be used to accurately size DNAs between 150 and 4500 bp, a useful range of sizes for many agarose gel electrophoresis applications, including separation of PCR products and plasmid cloning targets. In addition, it is easy to prepare, inexpensive, and rivals the best of the commercial ladders. PMID:17309053

Barvish, Zeev; Davis, Claytus; Gitelman, Inna



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



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



Fragmentation of Genomic DNA using Microwave Irradiation  

PubMed Central

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

Yang, Yu; Hang, Jun



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.

Fernandez, Jose Luis; Cartelle, Monica; Muriel, Lourdes; Santiso, Rebeca; Tamayo, Maria; Goyanes, Vicente; Gosalvez, Jaime; Bou, German



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

Microsoft Academic Search

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

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



Apoptotic DNA fragmentation and tissue homeostasis  

Microsoft Academic Search

DNA fragmentation is a hallmark of apoptosis. The tightly controlled activation of the apoptosis-specific endonucleases provides an effective means to ensure the removal of unwanted DNA and the timely completion of apoptosis. Over the past several years, crucial progress has been made in identifying the long-awaited apoptotic endonucleases, and their importance in tissue homeostasis is beginning to unfold. Here, we

Jianhua Zhang; Ming Xu



Candida albicans- and Candida stellatoidea-specific DNA fragment.  


DNA was isolated from whole cells of Candida albicans and digested with MspI restriction enzyme. In addition to the expected large number of low-molecular-weight DNA pieces resulting from the digestion, multiple high-molecular-weight (greater than 3.0 kilobase pairs) fragments were generated by this enzyme, which cleaves DNA at CCGG sequences. Some of these fragments appeared highly repeated. An MspI fragment which was similar in size to one of the repeat elements (2.9 kilobase pairs) was cloned into the ClaI site of the plasmid vector pBR322 and replicated in a suitable Escherichia coli host strain. The candidal fragment was radiolabeled and used to probe Southern blots of DNA from several Candida species, various other fungi, and mouse and human cells. Only DNA from C. albicans and a strain of Candida stellatoidea was found to contain sequences of significant homology for hybridization. The cloned fragment may possibly be of use as a DNA probe for detection of the presence of C. albicans. PMID:2460494

Cutler, J E; Glee, P M; Horn, H L



Random DNA fragmentation with endonuclease V: application to DNA shuffling.  


The enzyme endonuclease V nicks uracil-containing DNA at the second or third phosphodiester bond 3' to uracil sites. I applied the enzyme to random fragmentation of DNA to revise the complex DNA shuffling protocol. The merit of using endonuclease V is that cleavage occurs at random sites and the length of the fragments can easily be adjusted by varying the concentration of dUTP in the polymerase chain reaction. Unlike the conventional method using DNase I, no partial digestion or gel separation of fragments is required. Therefore, labor is dramatically reduced and reproducibility ensured. I applied this method to recombine two truncated green fluorescent protein (GFP) genes and demonstrated successful DNA shuffling by the appearance of the fluorescent full-length GFP genes. PMID:12490730

Miyazaki, Kentaro



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

SciTech Connect

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

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



Short read DNA fragment anchoring algorithm  

PubMed Central

Background The emerging next-generation sequencing method based on PCR technology boosts genome sequencing speed considerably, the expense is also get decreased. It has been utilized to address a broad range of bioinformatics problems. Limited by reliable output sequence length of next-generation sequencing technologies, we are confined to study gene fragments with 30~50 bps in general and it is relatively shorter than traditional gene fragment length. Anchoring gene fragments in long reference sequence is an essential and prerequisite step for further assembly and analysis works. Due to the sheer number of fragments produced by next-generation sequencing technologies and the huge size of reference sequences, anchoring would rapidly becoming a computational bottleneck. Results and discussion We compared algorithm efficiency on BLAT, SOAP and EMBF. The efficiency is defined as the count of total output results divided by time consumed to retrieve them. The data show that our algorithm EMBF have 3~4 times efficiency advantage over SOAP, and at least 150 times over BLAT. Moreover, when the reference sequence size is increased, the efficiency of SOAP will get degraded as far as 30%, while EMBF have preferable increasing tendency. Conclusion In conclusion, we deem that EMBF is more suitable for short fragment anchoring problem where result completeness and accuracy is predominant and the reference sequences are relatively large.

Wang, Wendi; Zhang, Peiheng; Liu, Xinchun



DNA fragmentation in chicken spermatozoa during cryopreservation.  


Semen cryopreservation is fundamental both for the practice of artificial insemination, and for the conservation of genetic resources in cryobanks; nevertheless, there is still not an efficient standard freezing procedure assuring a steady and suitable level of fertility in fowl, and consequently there is no systematic use of frozen semen in the poultry industry. This study examined changes in motility (CASA), cell membrane integrity (Ethidium Bromide (EtBr) exclusion procedure and stress test) and DNA fragmentation (neutral comet assay) in fowl spermatozoa before, during and after cryopreservation and storage at -196 °C. An optimized comet assay for chicken semen was studied and applied to the analyses. Semen collected from 18 Mericanel della Brianza (local Italian breed) male chicken breeders was frozen in pellets and thawed in a water bath at 60 °C. Measurements were performed on fresh semen soon after dilution, after equilibration with 6% dimethylacetamide at 4 °C (processed semen) and after thawing. Sperm DNA damage occurred during cryopreservation of chicken semen and the proportion of spermatozoa with damaged DNA significantly increased from 6.2% in fresh and 6.4% in processed semen to 19.8% in frozen-thawed semen. The proportion of DNA in the comet tail of damaged spermatozoa was also significantly affected by cryopreservation, with an increase found from fresh (26.3%) to frozen-thawed (30.9%) sperm, whereas processed semen (30.1%) didn't show significant differences. The proportion of total membrane damaged spermatozoa (EtBr exclusion procedure) did not increase by 4 °C equilibration time, and greatly and significantly increased by cryopreservation; the values recorded in fresh, processed and frozen semen were 2.9, 5.6, and 66.7% respectively. As regards the proportion of damaged cells in the stress test, all values differed significantly (7.1% fresh semen, 11.7% processed semen, 63.7% frozen semen). Total motility was not affected by equilibration (52.1% fresh semen, 51.9% processed semen), whereas it decreased significantly after cryopreservation (19.8%). These results suggest a low sensitivity of frozen-thawed chicken spermatozoa to DNA fragmentation, therefore it should not be considered as a major cause of sperm injuries during cryopreservation. PMID:21396690

Gliozzi, T M; Zaniboni, L; Cerolini, S



Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.  


DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs. PMID:16061932

Müller, Kristian M; Stebel, Sabine C; Knall, Susanne; Zipf, Gregor; Bernauer, Hubert S; Arndt, Katja M



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


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

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



Protein fragment complementation in M.HhaI DNA methyltransferase  

Microsoft Academic Search

The 5mC DNA methyltransferase M.HhaI can be split into two individually inactive N- and C-terminal fragments that together can form an active enzyme in vivo capable of efficiently methylating DNA. This active fragment pair was identified by creating libraries of M.HhaI gene fragment pairs and then selecting for the pairs that code for an active 5mC methyltransferase. The site of

Wonchae Choe; Srinivasan Chandrasegaran; Marc Ostermeier



Folding super-sized DNA origami with scaffold strands from long-range PCR.  


A 26 kilobase single strand DNA fragment was obtained from long-range PCR amplification and subsequent enzymatic digestion, which we folded into a super-sized DNA origami nanostructure by using ?800 staple strands. PMID:22618197

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Huang, Qing; Fan, Chunhai



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.



Improving Enrichment of Circulating Fetal DNA for Genetic Testing: Size Fractionation Followed by Whole Gene Amplification  

PubMed Central

Objective Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Methods Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100–300, 500–700 and 1,500–2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of ?-globin and DYS1 were measured. Results Distribution of ?-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both ?-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100–300 bp) accounted for nearly 50% (39.76 ± 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 ± 6.49%) of ?-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100–300 bp fragments as template. Conclusions Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA.

Jorgez, Carolina J.; Bischoff, Farideh Z.



Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods  

PubMed Central

Background Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA.



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.



FDA Information on Gardasil – Presence of DNA Fragments ...  

Center for Biologics Evaluation and Research (CBER)

The FDA has recently received inquiries regarding the presence of human papillomavirus (HPV) DNA fragments in Gardasil and is aware that ... More results from


DNA fragmentation of spermatozoa and assisted reproduction technology  

Microsoft Academic Search

Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in

Ralf Henkel; Eva Kierspel; Marjam Hajimohammad; Thomas Stalf; Christiaan Hoogendijk; Claas Mehnert; Roelof Menkveld; Wolf-Bernhard Schill; Thinus F Kruger



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

PubMed Central

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

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



Adaptive weighted least squares method for the estimation of DNA fragment lengths from agarose gels.  


The size of DNA fragments is most frequently estimated from their electrophoretic mobilities. Agarose gels are used to estimate the size of DNA fragments ranging from a few hundred nucleotides to more than 20 kbp. The common practice when estimating the unknown fragment sizes is to plot the log of the size of molecular weight standards against their mobility and read the values of unknowns from this graph. However, due to perturbations in the gel, such plots often show pronounced curvature which may introduce significant subjectivity into the interpolation process. We present a new method "adaptive weighted least squares (AWLS)" based on the significance test to choose the order of polynomial. We compare this with the method introduced by Schaffer based on the modification of the Southern method. The results obtained by AWLS are significantly better than the method introduced by Schaffer. Different lanes are tested for consistency. PMID:11840520

Akbari, Akbar; Albregtsen, Fritz; Lingjaerde, Ole Christian



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

SciTech Connect

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

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



Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment  

Microsoft Academic Search

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3' end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1

Wilfried Wiese; Barbara Vornam; Elvira Krause; Helmut Kindl



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


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.



Clinical aspects of sperm DNA fragmentation detection and male infertility  

Microsoft Academic Search

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose

Donald P. Evenson; Regina Wixon



Selective Binding of anti-DNA Antibodies to Native dsDNA Fragments of Differing Sequence  

PubMed Central

Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs) has been shown. Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

Uccellini, Melissa B.; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A.



Rapid hierarchical assembly of medium-size DNA cassettes  

PubMed Central

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

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



Temporal Patterns of Nucleotide Misincorporations and DNA Fragmentation in Ancient DNA  

Microsoft Academic Search

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

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



In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization  

PubMed Central

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

Jiang, Xinglin; Yang, Jianming; Zhang, Haibo; Zou, Huibin; Wang, Cong; Xian, Mo



Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP  

Microsoft Academic Search

The 3.3-Å resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme

D. L. Ollis; P. Brick; R. Hamlin; N. G. Xuong; T. A. Steitz



Identification of viral proteins encoded by two DNA fragments of herpesvirus of turkeys (HVT)  

Microsoft Academic Search

Herpesvirus of turkeys (HVT) vaccine is used worldwide to immunize chickens against Marek's disease (MD). Polyclonal antiserum directed against one virus cross-reacts with proteins of the other, while only 5% homology at the DNA level was demonstrated between the two viruses. A partial library of HVT DNA fragments ranging from 1.5 to 13.5 kbp in size was constructed in pBR

H. Levy; T. Maray; I. Davidson; M. Malkinson; Y. Becker



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


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

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



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

Microsoft Academic Search

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

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



One-step isothermal assembly of DNA fragments.  


The One-Step Isothermal DNA Assembly method allows for the efficient assembly of DNA constructs using fragments up to several hundred kilobases in as little as 15 min. Applications of this method range from the addition of promoters to expression constructs to the assembly of bacterial genome fragments. The production of circularized DNA using this method also enables the direct transformation of target organisms, bypassing intermediate transformations for plasmid propagation in those species where expression could lead to toxicity and cell death. Variations of the method allow for specific cloning tasks to be performed, as well as the use of microarray slides as a source of DNA. The level of precision and simplicity of this method makes it a valuable tool for most cloning efforts and all levels of proficiency in molecular biology. PMID:23996438

Rodrigues, Rui T L; Bayer, Travis S



A new method for the determination of critical polyethylene glycol concentration for selective precipitation of DNA fragments.  


Separation strategies based on size-selective precipitation of DNA fragments with polyethylene glycol (PEG) have been used for achieving desired DNA interval in automated sample preparation for next-generation sequencing. By varying PEG concentration, DNA fragments of different sizes can be precipitated onto surfaces of carboxyl-coated paramagnetic particles selectively, and therefore, the desired DNA interval can be obtained. However, one of the crucial points in this approach is to determine the critical PEG concentration for DNA fragment of a certain size. The aim of this work was to develop a convenient and reliable method for accurately determining the critical PEG concentration. In our method, at a fixed concentration of sodium chloride (NaCl), recovered DNA samples obtained with different PEG concentrations were directly quantified, and their concentrations as a function of the PEG concentration were fitted by the logistic function. The critical PEG value was easily and accurately determined from the fitted logistic function. The repeatability and stability of the critical PEG value were assessed, showing an excellent reliability of the method. Based on this method, critical PEG values of different-size DNA fragments were determined at different NaCl concentrations. The effectiveness of the method was also demonstrated by selective precipitation of DNA fragments. PMID:23982329

He, Zhangyong; Zhu, Ying; Gu, Hongchen



Fiber optic system for rapid analysis of amplified DNA fragments  

NASA Astrophysics Data System (ADS)

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

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



Directed Transfer of Large DNA Fragments between Streptomyces Species  

Microsoft Academic Search

The biosynthesis of complex natural products in bacteria is invariably encoded within large gene clusters. Although this facilitates the cloning of such gene clusters, their heterologous expression in genetically ame- nable hosts remains a challenging problem, principally due to the difficulties associated with manipulating large DNA fragments. Here we describe a new method for the directed transfer of a gene




Short DNA fragments induce site specific recombination in mammalian cells  

Microsoft Academic Search

A defective hprt gene was corrected by homologous recombination in a lymphocyte cell line deficient in Hypoxanthine-phosphoribosyl-transferase activity (hprt). In a novel approach, only a fragment of a cDNA clone of the functional hprt gene was used to induce homologous recombination. The mutation that was corrected corresponds to a single base change in exon III of the hprt gene.

Katharina Hunger-Bertling; Petra Harrer; Wolf Bertling



Transformation of Escherichia coli by a specific DNA restriction fragment  

Microsoft Academic Search

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

Silvia M. Schweitzer; Hans Matzura



Secondary craters of Tycho: Size-frequency distributions and estimated fragment size–velocity relationships  

Microsoft Academic Search

We examined the size-frequency distribution and the spatial distribution of secondary craters around the lunar crater Tycho. Secondary crater diameters were found to range from 0.55 to 4.0 km, and their distance from Tycho to range from 130 to 370 km. The diameter and ejection velocity of the secondary-forming fragments were also estimated from the crater size and the distance

Naru Hirata; Akiko M. Nakamura



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


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

Valdés, Alberto; García-Cañas, Virginia; Cifuentes, Alejandro



The Sperm Chromatin Dispersion Test: A Simple Method for the Determination of Sperm DNA Fragmentation  

Microsoft Academic Search

Sperm DNA fragmentation is being increasingly rec- ognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm




Influence of PMMA shielding on DNA fragmentation induced in human fibroblasts by iron and titanium ions.  


In the framework of a collaborative project on the influence of the shielding on the biological effectiveness of space radiation, we studied DNA fragmentation induced by 1 GeV/nucleon iron ions and titanium ions with and without a 197-mm-thick polymethylmethacrylate (PMMA) shield in AG1522 human fibroblasts. Pulsed- and constant-field gel electrophoresis were used to analyze DNA fragmentation in the size range 1-5700 kbp. The results show that, mainly owing to a higher production of small fragments (1-23 kbp), titanium ions are more effective than iron ions at inducing DNA double-strand breaks (DSBs), their RBE being 2.4 and 1.5, respectively. The insertion of a PMMA shield decreases DNA breakage, with shielding protection factors (ratio of the unshielded/shielded cross sections for DSB production) of about 1.6 for iron ions and 2.1 for titanium ions. However, the DSB yield (no. of DSBs per unit mass per unit dose) is almost unaffected by the presence of the shield, and the relative contributions of the fragments in the different size ranges are almost the same with or without shielding. This indicates that, under our conditions, the effect of shielding is mainly to reduce the dose per unit incident fluence, leaving radiation quality practically unaffected. PMID:16187791

Dini, Valentina; Antonelli, Francesca; Belli, Mauro; Campa, Alessandro; Esposito, Giuseppe; Simone, Giustina; Sorrentino, Eugenio; Tabocchini, Maria Antonella



Dna fragmentation induced by protons and Fe-ions in human cells  

NASA Astrophysics Data System (ADS)

Protons and high energy heavy ions (HZE particles ) are the components of space radiation of major concern. Their biological consequences are expected to be related to DNA lesions. Since DNA double strand breaks (DSB) and other lesions produced by these charged particles occurs in a correlated manner along the particle track, the current working hypothesis is that spatial correlation of damage, that depends on the particle type and energy, affects its reparability. Several characteristic distances for damage correlation can be considered, related to various levels of chromatin organization. We have investigated, in AG1522 human fibroblasts, the DNA fragmentation patterns in the size range 23 kbp - 5.7 Mbp, characteristic of the loop structure of chromosomes, using calibrated Pulsed Field Gel electrophoresis (PFGE). We have compared various beams i.e., low -energy protons of 0.88 MeV (representative of stopping protons at the Bragg peak) and Fe ions of 1050 and 414 MeV/u, using -rays as reference. Irradiations with protons were performed at the Laboratori Naz ionali di Legnaro (Padova, Italy), those with 1050 MeV/u Fe ions at the Brookhaven National Laboratory (USA) and those with 414 MeV/u Fe ions at the National Institute for Radiological Sciences (Chiba, Japan). DSB yields were evaluated from the number of DNA fragments induced in the size range studied, at doses between 40 and 200 Gy. The results until now obtained show the general trend of a linear, or almost linear, increase of DSB with the dose. 1050 MeV/u Fe ions are the most effective particles in terms of both unit dose and unit fluence. The frequency distributions of fragments induced by the charged particles are shifted towards smaller sizes with respect to that induced by comparable doses of -rays. These distributions also suggest that at doses of the order of 100 Gy most of the fragments induced by protons have size > 23 kbp, while for Fe ions it is expected a significant amount of fragments smaller than that size, not revealed by the mentioned PFGE analysis. This is in agreement with recent experimental measurements of DNA fragments induced by 1050 MeV/u Fe ions in the size range 2 - 23 kbp. The occurrence of such small fragments (and even smaller) further increases the effectiveness for DSB induction of HZE particles compared to protons.

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


Amyloid-? peptide structure in aqueous solution varies with fragment size  

NASA Astrophysics Data System (ADS)

Various fragment sizes of the amyloid-? (A?) peptide have been utilized to mimic the properties of the full-length A? peptide in solution. Among these smaller fragments, A?16 and A?28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the A?16, A?28, and A?42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the A?16, A?28, and A?42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of A?42 cannot be completely captured by the A?16 and A?28 fragments. For example, the ?-sheet structures in the N-terminal region of A?16 and A?28 are either not present or the abundance is significantly decreased in A?42. The ?-helix and ?-sheet abundances in A?28 and A?42 show trends - to some extent - with the potential of mean forces but no such trend could be obtained for A?16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in A?28 but this trend is vice versa for A?42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length A?42 peptide cannot be completely mimicked by studying the A?16 and A?28 peptides.

Wise-Scira, Olivia; Xu, Liang; Kitahara, Taizo; Perry, George; Coskuner, Orkid



A novel antitumor compound, NC190, induces topoisomerase II-dependent DNA cleavage and DNA fragmentation  

Microsoft Academic Search

A novel benzophenazine derivative, NC-190, is a potent antitumor compound. NC-190 has been shown to inhibit the DNA strand-passing\\u000a activity of DNA topoisomerase II. We investigated further the mode of action of NC-190 against DNA topoisomerase II and DNA\\u000a fragmentation. NC-190 inhibited the decatenation activity of purified topoisomerase II, but had only a weak inhibitory effect\\u000a against topoisomerase I. A

Takehiro Yamagishi; Shiro Nakaike; Tomotake Ikeda; Hisao Ikeya; Susumu Otomo



High interindividual restriction fragment length and copy number of polymorphism of a TVRI family in moderate human DNA repeats  

SciTech Connect

The authors describe the selection of cloned human DNA sequences, with a copy number not exceeding 1000 copies per diploid genome, and their testing for interindividual restriction fragment lengths and copy number of polymorphism (RFLCP). As a result of the investigation a DNA clone was found (TVRI-6), about 2.8 kilobase-pairs in size, for which an unusually high level of interindividual RFLCP was discovered. The TVRI-6 sequence was obtained from a bank of Pst I restriction fragments of human placental nuclear DNA cloned in pBR 322. The bank was analyzed by hybridization of colonies with phosphorus 32-labelled human nuclear DNA.

Rogaev, E.I.; Shapiro, Yu.A.



Modeling of DNA fragmentation induced in human fibroblasts by 56Fe ions  

NASA Astrophysics Data System (ADS)

In this work we have compared the pattern of DNA fragmentation (in the size range 1 5700 kbp) induced in human fibroblasts by ?-rays with that induced by Fe ions with an energy of 115 MeV/u; Fe ions are considered of biological significance for radiation protection issues during long term astronauts’ travels. The study has taken into account the comparison of the experimental fragmentation spectra, their analysis performed through the implementation of a phenomenological model, and Monte Carlo simulations performed with the PARTRAC code. The phenomenological method characterizes in an approximate but simple way the nonrandom nature of the experimental fragment distribution caused by high LET radiation; it has the advantage to take into account in a detailed way the background fragmentation. The PARTRAC simulations, on the other hand, thanks to the accurate representation of the chromatin geometry and of the physical and physico-chemical processes associated with the energy deposition by radiation, offer the possibility to compute the spectra and to compare them with the experimental data. We found a satisfactory agreement between the experimental data and the results obtained with PARTRAC, recently upgraded with the implementation of ions heavier than alpha particles; this agreement represents a first validation of the code. A relevant result is represented by the very high number of fragments that, according to the Monte Carlo simulations, are produced by iron ions in the size range <1 kbp: half of the total number of radiation induced fragments is given by fragments in this size range. This is consistent with another important result, i.e., the very high DSB correlation that is found through the GBS model in the lower part of the experimental size range (1 9 kbp) for iron ions. The consequence of this large production of fragments smaller than 1 kbp, undetected experimentally, is the large difference between the experimental and theoretical values of the RBE for DSB production, given respectively by 1.34 and 2.39.

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


Qualitative and quantitative analysis of DNA fragmentation using digital imaging.  


Apoptosis is an important and common pathway of cellular death. Differentiation from cellular necrosis and quantitation of apoptosis within the milieu of necrosis are analytical challenges. We describe the use of the RIT120 digital imaging software package for quantitative and qualitative analysis of apoptotic DNA ladders induced by a variety of agents, such as serum, tumor necrosis factor-alpha, transforming growth factor-beta1, and nitric oxide. Autoradiographs of DNA ladders are densitometrically scanned to yield a set of curves with peaks corresponding to specific DNA fragments, thereby allowing quantitative subtraction of concurrent DNA degradation from necrotic death. Integration of the areas specifically under the peaks yields a quantitative measure of apoptosis. We provide a useful, rapid, and objective means to quantitate apoptosis, using relatively inexpensive hardware and software. PMID:9245431

Vodovotz, Y; Hsing, A; Cook, J A; Miller, R W; Wink, D A; Ritt, D M; Mitchell, J B; Danielpour, D



DNA fragmentation in leukocytes following subacute lowdose nerve agent exposure  

Microsoft Academic Search

The objective of the present study was to determine levels of DNA fragmentation in blood leukocytes from guinea pigs by single-cell gel electrophoresis (comet assay) after exposure to the chemical warfare nerve agent (CWNA), soman, at doses ranging from 0.1 LD 50 to 0.4 LD 50, once per day for either 5 or 10 days. Post-exposure recovery periods ranged from

J. R. Moffett; R. A. Price; S. M. Anderson; M. L. Sipos; A. V. Moran; F. C. Tortella; J. R. Dave



Regulation of DNA fragmentation: the role of caspases and phosphorylation.  


DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation. PMID:21182594

Kitazumi, Ikuko; Tsukahara, Masayoshi



Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF  

Microsoft Academic Search

Ingestively masticated fragments were collected and sized via sieving. Different sizes of esopha- geal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were in- cubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (?) for onset of digestion of NDF was positively

W. C. Ellis; M. Mahlooji; C. E. Lascano; J. H. Matis



Application of automated DNA sizing technology for genotyping microsatellite loci  

SciTech Connect

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

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



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



From organic superconductors to DNA: Fragment orbital-based model  

NASA Astrophysics Data System (ADS)

A semi-empirical valence bond/Hartree-Fock (VB/HF) method is developed to calculate one- and two-electron interactions between molecular fragments in conducting supramolecular stacks. This fragment orbital-based formalism allows for consistent extraction of an effective Hamiltonian defined as a "frontier orbital" model. This Hamiltonian quantitatively describes transfer and electrostatic interactions between conducting electrons, while reducing the active space so dramatically that the electronic eigenstates of very large systems may be investigated. The capabilities of the VB/HF method are illustrated on two different supramolecular stacks involving a ?-? interacting fragment. In the first part of this study, the framework of the VB/HF method is used to evaluate the relative magnitude of the electronic interactions between conduction electrons in organic conductors and superconductors derived from Bechgaard salts. In the second part of this study, the VB/HF formalism is extended to derive an effective model for conduction holes along doped DNA double strands. Transferable intra- and intersite parameters were first evaluated from VB/HF calculations carried out on nucleoside pairs. From this interaction databank, the effective Hamiltonian of any type of nucleoside sequence can be defined. The thermalized charge distribution for a single hole delocalized along a DNA sequence containing 240 Watson-Crick pairs is then calculated and compared with the experimental yields of damage revealed by photocleavage experiments.

Castet, Frédéric; Ducasse, Laurent; Fritsch, Alain


In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level  

Microsoft Academic Search

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA

Yuichi Michikawa; Keisuke Sugahara; Tomo Suga; Yoshimi Ohtsuka; Kenichi Ishikawa; Atsuko Ishikawa; Naoko Shiomi; Tadahiro Shiomi; Mayumi Iwakawa; Takashi Imai



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.



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

NASA Astrophysics Data System (ADS)

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

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


The characterization of composite agarose/hydroxyethylcellulose matrices for the separation of DNA fragments using capillary electrophoresis.  


Mixtures of the polysaccharide derivatives, 19% hydroxyethylated SeaPrep agarose (SP-AG) and hydroxyethylcellulose (HEC), in aqueous buffer solutions are applied for the first time to the separation of DNA fragments using capillary electrophoresis (CE). These matrices form unique size-sieving networks that allow the separation of a wide size range of DNA fragments in a single analysis. Relative to their homogeneous counterparts, the composite separation matrices provide enhanced selectivity properties of DNA fragments, especially for fragments greater than 1000 base pairs (bp) in length. Additionally, the effects on separation performance of capillary temperature, the incorporation of a DNA intercalator, and applied field strength are demonstrated. Solution viscosity measurements of the homogeneous and composite matrix solutions were made in order to establish the entanglement threshold concentrations for the unique size-sieving solutions. The relatively low solution viscosities of the composite separation matrices allow reproducible replacement of the separation matrix between analyses. The mechanism of separation of DNA fragments for the composite matrices is proposed. PMID:9420156

Siles, B A; Anderson, D E; Buchanan, N S; Warder, M F



Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection.  


Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments. PMID:20307487

Protozanova, Ekaterina; Zhang, Meng; White, Eric J; Mollova, Emilia T; Broeck, Dirk Ten; Fridrikh, Sergey V; Cameron, Douglas B; Gilmanshin, Rudolf



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


Outside the magnetic field of the Earth, high energy heavy ions constitute a relevant part of the biologically significant dose to astronauts during the very long travels through space. The typical pattern of energy deposition in the matter by heavy ions on the microscopic scale is believed to produce spatially correlated damage in the DNA which is critical for radiobiological effects. We have investigated the influence of a lucite shielding on the initial production of very small DNA fragments in human fibroblasts irradiated with 1 GeV/u iron (Fe) ions. We also used gamma rays as reference radiation. Our results show: (1) a lower effect per incident ion when the shielding is used; (2) an higher DNA Double Strand Breaks (DSB) induction by Fe ions than by gamma rays in the size range 1-23 kbp; (3) a non-random DNA DSB induction by Fe ions. PMID:15880923

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



Sequence-specific modification of genomic DNA by small DNA fragments  

PubMed Central

Small DNA fragments have been used to modify endogenous genomic DNA in both human and mouse cells. This strategy for sequence-specific modification or genomic editing, known as small-fragment homologous replacement (SFHR), has yet to be characterized in terms of its underlying mechanisms. Genotypic and phenotypic analyses following SFHR have shown specific modification of disease-causing genetic loci associated with cystic fibrosis, ?-thalassemia, and Duchenne muscular dystrophy, suggesting that SFHR has potential as a therapeutic modality for the treatment of monogenic inherited disease.

Gruenert, Dieter C.; Bruscia, Emanuela; Novelli, Giuseppe; Colosimo, Alessia; Dallapiccola, Bruno; Sangiuolo, Federica; Goncz, Kaarin K.



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:; 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)



Homologous recombination of exogenous DNA fragments with genomic DNA in somatic cells of mice.  


We compared liposomes and empty viral capsids for their use as vehicles for DNA transfer into cells and animals. DNA binding capacity was high for liposomes, but DNase I protection of DNA bound to liposomes was only moderate in comparison to DNA incorporated into viral capsids. Cellular uptake of radiolabeled and physiologically active DNA was also compared. For animal studies we chose an endogenous retroposon as target gene. To identify recombinational events we replaced a part of this gene with an artificial sequence not present in the mouse genome. The recombination rate for DNA fragments transfected in Polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DNA transfer methods, mediated by positively charged liposomes (DOTMA) and by empty Polyoma viral capsids. PMID:1652361

Gareis, M; Harrer, P; Bertling, W M



Discriminatory 32P 3'-end labeling of restriction endonuclease co-digested DNA fragments.  


We present an improved method for selectively labeling specific DNA fragments in a mixture of restriction fragments. lambda DNA, used to develop the procedure, was digested with a combination of restriction enzymes and treated, at various incubation temperatures, with reverse transcriptase and one or more [alpha-32P]dNTP molecules. The labeled fragments were subjected to agarose gel electrophoresis and detected by autoradiography. We were able to direct the 3'-end labeling to precise subpopulations of DNA fragments, generated by multiple restriction enzyme digestions. We also show that labeling selectivity of DNA restriction fragments, by reverse transcriptase, is affected by the incubation temperature used during the labeling reaction. This paper describes an approach to 3'-end label select DNA fragments with reverse transcriptase and [alpha-32P]dNTPs. The procedure permits the bypassing of time-consuming isolation and purification steps required, by conventional radiolabeling techniques, to radiolabel specific DNA fragments. PMID:2543232

LeBlond, G F; Ts'o, P O




Microsoft Academic Search

The present study evaluates HPLC and CE experimental parameters, such as column packing properties, mobile phase pH, column temperature, and gel type on the separation of DNA fragments and heteroduplexes. The results of this study show that both HPLC and CE are useful techniques for the separation of DNA fragments and for the detection of DNA mutants.Not all HPLC columns

Haleem J. Issaq; Hongyu Xu; King C. Chan



DNA condensation and size effects of DNA condensation agent  

NASA Astrophysics Data System (ADS)

Based on the model of the strong correlation of counterions condensed on DNA molecule, by tailoring interaction potential, interduplex spacing and correlation spacing between condensed counterions on DNA molecule and interduplex spacing fluctuation strength, toroidal configuration, rod-like configuration and two-hole configurations are possible. The size effects of counterion structure on the toroidal structure can be detected by this model. The autocorrelation function of the tangent vectors is found as an effective way to detect the structure of toroidal conformations and the generic pathway of the process of DNA condensation. The generic pathway of all of the configurations involves an initial nucleation loop, and the next part of the DNA chain is folded on the top of the initial nucleation loop with different manners, in agreement with the recent experimental results.

Liu, Yan-Hui; Jiang, Chong-Ming; Guo, Xin-Miao; Tang, Yan-Lin; Hu, Lin



Fragmentation and labeling of probe DNA for whole-mount FISH in Drosophila.  


Good probes for whole-mount fluorescent in situ hybridization (FISH) must meet two criteria: The DNA fragments must be very small and they must be highly labeled. This article describes an effective labeling scheme that involves fragmenting the probe DNA and then adding a mixture of labeled and unlabeled nucleotides to the 3' ends using the enzyme terminal deoxynucleotidyl transferase (TdT). This method can be used to label a variety of DNA probes, regardless of their initial size (e.g., plasmid, cosmid, or P1 clones, polymerase chain reaction [PCR] products, or total genomic DNA). Short oligonucleotides may also be labeled in this way without digestion, because their small size allows them to diffuse through thick tissues. A potential advantage of end-labeling is that the modified nucleotides are not incorporated into the complementary probe sequence itself and may thus interfere less with hybridization. The free 3' tail may also make haptens more accessible to detection reagents. PMID:22135656

Dernburg, Abby F



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



DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution.  

PubMed Central

Computer simulations of the evolution of linear sequences have demonstrated the importance of recombination of blocks of sequence rather than point mutagenesis alone. Repeated cycles of point mutagenesis, recombination, and selection should allow in vitro molecular evolution of complex sequences, such as proteins. A method for the reassembly of genes from their random DNA fragments, resulting in in vitro recombination is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its original size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombination was obtained between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and incorporated into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed into a gene at defined positions based on homology. As an example, a library of chimeras of the human and murine genes for interleukin 1 beta has been prepared. Shuffling can also be used for the in vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombination over existing mutagenesis methods are likely to increase with the numbers of cycles of molecular evolution. Images

Stemmer, W P



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

Microsoft Academic Search

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




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

Microsoft Academic Search

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

Donald P.. Evenson; Regina Wixon



Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli.  

PubMed Central

Using currently available gene fusion techniques, we have constructed plasmids that direct the overproduction of the carboxyl-terminal two-thirds of DNA polymerase I, corresponding to the proteolytically derived "Klenow fragment." We have obtained overproduction amounting to several percent of the cellular protein using constructs in which expression is directed either from the lac promoter or from the leftward promoter of phage lambda. The polymerase fragment has been purified to homogeneity from such overproducing strains by a rapid three-stage purification procedure, yielding material capable of carrying out the same reactions (polymerization, 3' labeling, DNA sequence analysis) as the proteolytically derived fragment. The availability of such overproducing strains should greatly facilitate structural and mechanistic studies of DNA polymerase I. Moreover, the techniques we have described for the cloning and expression of a gene fragment should be generally applicable for the study of protein structure and function in other systems. Images

Joyce, C M; Grindley, N D



Regional localization of chromosome 3-specific DNA fragments by using a hybrid cell deletion mapping panel.  

PubMed Central

A series of human chromosome 3-specific DNA fragments isolated and characterized from a lamda phage genomic library were regionally localized on human chromosome 3. This was accomplished using filter hybridization blot analysis of a human chromosome 3 hybrid cell deletion mapping panel. Twenty-three new anonymous DNA fragments were assigned to one of four physical regions of chromosome 3. Seventeen DNA fragments were mapped to the long arm of chromosome 3, including one DNA fragment that demonstrated a restriction fragment length polymorphism (RFLP). Five DNA fragments were assigned to 3p14.2----pter, including one highly polymorphic fragment sublocalized at 3p25----pter by in situ hybridization. This DNA fragment is the second reported distal 3p polymorphic probe. One DNA fragment was localized to 3p14----p14.2. In addition, three fragments previously assigned to chromosome 3 were confirmed. Polymorphic DNA probes DNF15S2 (formerly D1S1) and D3S2 were mapped to 3p14.2----pter. The previous 3p25 in situ localization of the c-raf-1 oncogene was supported by deletion panel mapping. The physical localization of these twenty-three new DNA fragments has more than doubled the number of cloned DNA fragments assigned to chromosome 3. These and future regional assignments of DNA fragment probes will facilitate construction of both a physical and genetic linkage map of chromosome 3. They may also be useful in characterizing the chromosomal and molecular aberrations involved in small-cell lung cancer (SCLC), renal cell carcinoma, other malignancies, and the 3p14.2 common fragile site. Images p[446]-a Figure 2 Figure 3

Gerber, M J; Drabkin, H A; Firnhaber, C; Miller, Y E; Scoggin, C H; Smith, D I



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.

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



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

NASA Astrophysics Data System (ADS)

We carry out three-dimensional smoothed particle hydrodynamics simulations of spherical homogeneous SiO2 dust aggregates to investigate how the mass and the porosity of the aggregates affect their ability to survive an impact at various different collision velocities (between 1 and 27.5 m s-1). We explore how the threshold velocities for fragmentation vary with these parameters. Crucially, we find that the porosity plays a part of utmost importance in determining the outcome of collisions. In particular, we find that aggregates with filling factors ?37 per cent are significantly weakened and that the velocity regime in which the aggregates grow is reduced or even non-existent (instead, the aggregates either rebound off each other or break apart). At filling factors less than ?37 per cent we find that more porous objects are weaker but not as weak as highly compact objects with filling factors ?37 per cent. In addition, we find that (for a given aggregate density) collisions between very different mass objects have higher threshold velocities than those between very similar mass objects. We find that fragmentation velocities are higher than the typical values of 1 m s-1 and that growth can even occur for velocities as high as 27.5 m s-1. Therefore, while the growth of aggregates is more likely if collisions between different sized objects occurs or if the aggregates are porous with filling factor <37 per cent, it may also be hindered if the aggregates become too compact.

Meru, Farzana; Geretshauser, Ralf J.; Schäfer, Christoph; Speith, Roland; Kley, Wilhelm



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

NASA Astrophysics Data System (ADS)

We carry out three-dimensional smoothed particle hydrodynamics simulations of spherical homogeneous SiO2 dust aggregates to investigate how the mass and the porosity of the aggregates affect their ability to survive an impact at various different collision velocities (between 1 and 27.5 m s-1). We explore how the threshold velocities for fragmentation vary with these parameters. Crucially, we find that the porosity plays a part of utmost importance in determining the outcome of collisions. In particular, we find that aggregates with filling factors ?37 per cent are significantly weakened and that the velocity regime in which the aggregates grow is reduced or even non-existent (instead, the aggregates either rebound off each other or break apart). At filling factors less than ?37 per cent we find that more porous objects are weaker but not as weak as highly compact objects with filling factors ?37 per cent. In addition, we find that (for a given aggregate density) collisions between very different mass objects have higher threshold velocities than those between very similar mass objects. We find that fragmentation velocities are higher than the typical values of 1 m s-1 and that growth can even occur for velocities as high as 27.5 m s-1. Therefore, while the growth of aggregates is more likely if collisions between different sized objects occurs or if the aggregates are porous with filling factor <37 per cent, it may also be hindered if the aggregates become too compact.

Meru, Farzana; Geretshauser, Ralf J.; Schäfer, Christoph; Speith, Roland; Kley, Wilhelm



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



Autonomous replication of human chromosomal DNA fragments in human cells.  

PubMed Central

We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments. Images

Masukata, H; Satoh, H; Obuse, C; Okazaki, T



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


Reserve size and fragmentation alter community assembly, diversity, and dynamics.  


Abstract Researchers have disputed whether a single large habitat reserve will support more species than many small reserves. However, relatively little is known from a theoretical perspective about how reserve size affects competitive communities structured by spatial abiotic gradients. We investigate how reserve size affects theoretical communities whose assembly is governed by dispersal limitation, abiotic niche differentiation, and source-sink dynamics. Simulations were conducted with varying scales of dispersal across landscapes with variable environmental spatial autocorrelation. Landscapes were inhabited by simulated trees with seedling and adult stages. For a fixed total area in reserves, we found that small reserve systems increased the distance between environments dominated by different species, diminishing the effects of source-sink dynamics. As reserve size decreased, environmental limitations to community assembly became stronger, ? species richness decreased, and ? richness increased. When dispersal occurred across short distances, a large reserve strategy caused greater stochastic community variation, greater ? richness, and lower ? richness than in small reserve systems. We found that reserve size variation trades off between preserving different aspects of natural communities, including ? diversity versus ? diversity. Optimal reserve size will depend on the importance of source-sink dynamics and the value placed on different characteristics of natural communities. Anthropogenic changes to the size and separation of remnant habitats can have far-reaching effects on community structure and assembly. PMID:24107376

Lasky, Jesse R; Keitt, Timothy H



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.



[Fibronectin fragmentation unmasks the activity stimulating DNA and RNA biosynthesis in granulation tissue cells in vitro].  


Human blood plasma fibronectin decreased slightly the incorporation of precursors into nucleic acids of granulation tissue culture cells. A slight fragmentation of fibronectin, where the fragments with 180-200 kD molecular mass were developed, led to occurrence of the activity 2-fold stimulating the DNA synthesis. After more effective proteolysis using plasmin and trypsin the stimulating effect of fibronectin fragments on synthesis of nucleic acids maintained and constituted 165 +/- 12% and 127 +/- 7% for DNA and RNA, respectively. PMID:2437701

Zlatopol'ski?, A D; Za?denberg, M A; Berman, A E; Mazurov, V I; Karelin, A A


Direct haplotyping of kilobase-size DNA using carbon nanotube probes  

Microsoft Academic Search

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept

Adam T. Woolley; Chantal Guillemette; Chin Li Cheung; David E. Housman; Charles M. Lieber



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

Microsoft Academic Search

Summary  Size distributions of fragments of crushed rock in conveyor belts and of blasted rock in a muckpile obtained by sieving are\\u000a compared with the size distributions obtained by digital image analysis of photographs of the same materials taken on-site.\\u000a Several calculation methods are tested, based on the raw distribution of fragment areas and on the volume-transformed ones.\\u000a The influence of

J. A. Sanchidrián; P. Segarra; F. Ouchterlony; L. M. López



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




Free solution hydrodynamic separation of DNA fragments from 75 to 106,000 base pairs in a single run.  


Gel electrophoresis is commonly used to separate DNA, but narrow capillaries or microchannels desired for high throughput efficient separations are difficult to fill with gels. We report here that a narrow capillary can be used to hydrodynamically separate a wide size range of DNA fragments in a single run without the need for gels, wall coatings, or an electric field. We also demonstrate that attractive separation is possible in a few minutes and that the separated DNA can be collected into individual fractions that remain viable for amplification via the polymerase chain reaction. PMID:20014789

Wang, Xiayan; Veerappan, Vijaykumar; Cheng, Chang; Jiang, Xin; Allen, Randy D; Dasgupta, Purnendu K; Liu, Shaorong



Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay  

Microsoft Academic Search

BACKGROUND: Fluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused

María Tamayo; Rebeca Santiso; Jaime Gosalvez; Germán Bou; José Luis Fernández



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.

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



Clinical and legal significance of fragmentation of bullets in relation to size of wounds: retrospective analysis  

PubMed Central

Objective To examine the relation between fragmentation of bullets and size of wounds clinically and in the context of the Hague Declaration of 1899. Design Retrospective analysis of prospectively collected data on hospital admissions. Setting Hospitals of the International Committee of the Red Cross. Subjects 5215 people wounded by bullets in armed conflicts (5933 wounds). Main outcome measures Grade of wound computed from the Red Cross wound classification and presence of bullet fragments on radiography. Results Of the 347 wounds with fragmentation of bullets, 251 (72%) were large wounds (grade 2 or 3)—that is, those with a clinically detectable cavity. Of the 5586 wounds without fragmentation of bullets, 2915 (52.1%) were large wounds. Only 7.9% (251/3166) of large wounds were associated with fragmentation of bullets. Conclusions Fragmentation of bullets is associated with large wounds, but most large wounds do not contain bullet fragments. In addition, bullet fragments may occur in wounds that are not defined as large. Fragmentation of bullets is neither a necessary nor sufficient cause of large wounds, and surgeons should not diagnose extensive tissue damage because of the presence of fragments on radiography. Such findings also do not necessarily represent the use of bullets which contravene the law of war. Future legislation should take into account not only the construction of bullets but also their potential to transfer energy to the human body. Key messagesThe use of certain bullets has been prohibited in warWounds from bullets are caused by transfer of kinetic energy from the bullet to the tissuesThe relation between size of wound and fragmentation of bullets can be examined using the Red Cross wound classification system Fragments of bullets seen on radiographs of wounds sustained in wars do not necessarily represent large wounds or the use of illegal bulletsExisting legislation on the construction of bullets should be supplemented by legislation on how much energy is transferred to tissues

Coupland, Robin



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

Microsoft Academic Search

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

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



Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA  

Microsoft Academic Search

Fragments of phage T7 DNA have been cloned in Escherichia coli by using the plasmid pMB9. Such cloned fragments are able to recombine with infecting phages, thus providing a means to integrate the physical and genetic maps of T7 DNA. Approximately 65% of the T7 DNA molecule has been found in clones so far, and analysis of these clones has

J. L. Campbell; C. C. Richardson; F. W. Studier



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


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

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


Caspase-2 cleaves DNA fragmentation factor (DFF45)/inhibitor of caspase-activated DNase (ICAD).  


To investigate the signal transduction pathway of caspase-2, cell permeable Tat-reverse-caspase-2 was constructed, characterized and utilized for biochemical and cellular studies. It could induce the cell death as early as 2h, and caspase-2-specific VDVADase activity but not other caspase activities including DEVDase and IETDase. Interestingly, nuclear DNA fragmentation occurred and consistently DNA fragmentation factor (DFF45)/Inhibitor of caspase-activated DNase (ICAD) was cleaved inside the cell as well as in vitro, suggesting a role of caspase-2 in nuclear DNA fragmentation. PMID:17945178

Dahal, Giri Raj; Karki, Pratap; Thapa, Arjun; Shahnawaz, Mohammad; Shin, Song Yub; Lee, Jung Sup; Cho, Byungyun; Park, Il-Seon



Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution  

Microsoft Academic Search

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone sub- sequently cleaved with piperidine.

Kristian M. Muller; Sabine C. Stebel; Susanne Knall; Gregor Zipf; Hubert S. Bernauer; Katja M. Arndt



A microfabricated device for sizing and sorting DNA molecules  

PubMed Central

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

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



Amphibian Activity, Movement Patterns, and Body Size in Fragmented Peat Bogs  

Microsoft Academic Search

I investigated the activity, direction of movement, and body size (snout-vent length) of am- phibians in both pristine and fragmented bogs of southeastern New Brunswick. I used drift-fences with pitfall traps to capture amphibians in six pristine bogs and six bogs undergoing peat mining (i.e, bog fragments) in 1997 and 1998. Results indicate that seasonal activity patterns of amphibians in




High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing  

PubMed Central

Background Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine. Methodology We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine. Principal Findings Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length. Conclusions With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.

Tsui, Nancy B. Y.; Jiang, Peiyong; Chow, Katherine C. K.; Su, Xiaoxi; Leung, Tak Y.; Sun, Hao; Chan, K. C. Allen; Chiu, Rossa W. K.; Lo, Y. M. Dennis



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

SciTech Connect

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 composed 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 {circ}OH, {circ}H, e{sub aq}, etc.; {circ}OH attack on sugar molecules leading to strand breaks; {circ}OH attack on bases; direct ionization of the sugar molecules leading to strand breaks; direct ionization of the bases. Our calculations predict significant clustering of damage both locally, over regions up to 40 hp 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. Such fragments have subsequently been detected experimentally and are reported in an accompanying paper 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. 27 refs., 7 figs.

Holley, W.R.; Chatterjee, A. [Lawrence Berkeley National Laboratory, Berkeley, CA (United States)



Synthesis of phosphorothioate-containing DNA fragments by a modified hydroxybenzotriazole phosphotriester approach.  

PubMed Central

The phosphorothioylating agent which was obtained by treating 2,5-dichlorophenyl phosphorodichloridothioate with 1-hydroxy-6-nitrobenzotriazole proved to be very effective for the synthesis in solution and on a solid support of phosphorothioate-containing DNA fragments.

Marugg, J E; van den Bergh, C; Tromp, M; van der Marel, G A; van Zoest, W J; van Boom, J H



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

Microsoft Academic Search

Bkm sequences, originally isolated from snake satellite DNA, are a component of eukaryote genomes with a preferential location on sex chromosomes. In the Ephestia genome, owing to the presence of only a few Bkm-positive BamHI restriction fragments and to extensive restriction fragment length polymorphisms between and within inbred strains, a genetic crossbreeding analysis was feasible. No sex linkage of Bkm

W. Traut; Accepted December



Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay  

Microsoft Academic Search

Meta-analyses were conducted to investigate the relationship of sperm DNA fragmentation on pregnancy outcome using in-vivo fertilization, IUI, routine IVF and ICSI. Couples with no known infertility problems were 7.0 times (CI 3.17, 17.7) more likely to achieve a pregnancy\\/delivery if the DNA fragmentation index (DFI) was <30% (n = 362, P = 0.0001) using in-vivo fertilization. Infertile couples using

Donald Evenson; Regina Wixon



Chromosomal localization and genomic organization of cloned repetitive DNA fragments in mosquitoes (Diptera: Culicidae)  

Microsoft Academic Search

The chromosomal localization and genomic organization of three cloned repetitive DNA fragments (viz., H-76, H-61, and H-19)\\u000a isolated from theAedes albopictus genome have been examined inAe. albopictus and six otherAedes species:Ae. aegypti, Ae. seatoi, Ae. flavopictus, Ae. polynesiensis, Ae. alcasidi andAe. katherinensis. The results fromin situ and Southern hybridization analyses show that the sequences homologous to cloned repetitive DNA fragments

A. Kumar; K. S. Rai



Differentiation and apoptosis without DNA fragmentation in cultured Schwann cells derived from wallerian-degenerated nerve.  


The Schwann cell cables provide particularly favorable sites for the growth of regenerating axonal sprouts. However, if they remain denervated, endoneurial fibrosis takes place with the Schwann cells atrophying and total Schwann cell number gradually decrease with time. Even when regenerating axonal sprouts invade into the cables, Schwann cells do not survive for long periods if they fail to make axonal contact. These observations strongly suggest the involvement of apoptosis in peripheral nerve degeneration and regeneration. So, we investigated the behavior of Schwann cells prepared from wallerian-degenerated adult rat sciatic nerve in vitro. The secondary cultured Schwann cells showed serial changes in morphology, mitotic activity and migratory activity as they do during Schwann cell cable formation in vivo. At the final stage of differentiation, the Schwann cells became rounded and detached from the flask with extensive blebbing. Electron micrographs clearly demonstrated typical cytoplasmic changes of apoptosis, but, nuclei of most of the cells retained their size and morphology with residual nucleolar structures. An agarose gel electrophoresis of DNA clearly demonstrated that there was not any DNA fragmentation up to 120 h after detachment. Results by in situ apoptosis detection assay did not show any DNA degradation despite the substantial decrease in Schwann cell number. In conclusion, during peripheral nerve degeneration and regeneration, supernumerary Schwann cells are removed by apoptosis, however, it lacks most of the nuclear events of usual apoptosis. PMID:14646482

Hirata, H; Hibasami, H; Yoshida, T; Morita, A; Ohkaya, S; Matsumoto, M; Sasaki, H; Uchida, A



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

NASA Astrophysics Data System (ADS)

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

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


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.



Electron impact ionization of size selected hydrogen clusters (H2)N: Ion fragment and neutral size distributions  

NASA Astrophysics Data System (ADS)

Clusters consisting of normal H2 molecules, produced in a free jet expansion, are size selected by diffraction from a transmission nanograting prior to electron impact ionization. For each neutral cluster (H2)N (N=2-40), the relative intensities of the ion fragments Hn+ are measured with a mass spectrometer. H3+ is found to be the most abundant fragment up to N=17. With a further increase in N, the abundances of H3+, H5+, H7+, and H9+ first increase and, after passing through a maximum, approach each other. At N=40, they are about the same and more than a factor of 2 and 3 larger than for H11+ and H13+, respectively. For a given neutral cluster size, the intensities of the ion fragments follow a Poisson distribution. The fragmentation probabilities are used to determine the neutral cluster size distribution produced in the expansion at a source temperature of 30.1 K and a source pressure of 1.50 bar. The distribution shows no clear evidence of a magic number N=13 as predicted by theory and found in experiments with pure para-H2 clusters. The ion fragment distributions are also used to extract information on the internal energy distribution of the H3+ ions produced in the reaction H2++H2-->H3++H, which is initiated upon ionization of the cluster. The internal energy is assumed to be rapidly equilibrated and to determine the number of molecules subsequently evaporated. The internal energy distribution found in this way is in good agreement with data obtained in an earlier independent merged beam scattering experiment.

Kornilov, Oleg; Toennies, J. Peter



Electron impact ionization of size selected hydrogen clusters (H{sub 2}){sub N}: Ion fragment and neutral size distributions  

SciTech Connect

Clusters consisting of normal H{sub 2} molecules, produced in a free jet expansion, are size selected by diffraction from a transmission nanograting prior to electron impact ionization. For each neutral cluster (H{sub 2}){sub N} (N=2-40), the relative intensities of the ion fragments H{sub n}{sup +} are measured with a mass spectrometer. H{sub 3}{sup +} is found to be the most abundant fragment up to N=17. With a further increase in N, the abundances of H{sub 3}{sup +}, H{sub 5}{sup +}, H{sub 7}{sup +}, and H{sub 9}{sup +} first increase and, after passing through a maximum, approach each other. At N=40, they are about the same and more than a factor of 2 and 3 larger than for H{sub 11}{sup +} and H{sub 13}{sup +}, respectively. For a given neutral cluster size, the intensities of the ion fragments follow a Poisson distribution. The fragmentation probabilities are used to determine the neutral cluster size distribution produced in the expansion at a source temperature of 30.1 K and a source pressure of 1.50 bar. The distribution shows no clear evidence of a magic number N=13 as predicted by theory and found in experiments with pure para-H{sub 2} clusters. The ion fragment distributions are also used to extract information on the internal energy distribution of the H{sub 3}{sup +} ions produced in the reaction H{sub 2}{sup +}+H{sub 2}{yields}H{sub 3}{sup +}+H, which is initiated upon ionization of the cluster. The internal energy is assumed to be rapidly equilibrated and to determine the number of molecules subsequently evaporated. The internal energy distribution found in this way is in good agreement with data obtained in an earlier independent merged beam scattering experiment.

Kornilov, Oleg; Toennies, J. Peter [Max-Planck-Institut fuer Dynamik und Selbstorganisation Bunsenstrasse 10, D-37073 Goettingen (Germany)



Processes involved in assisted reproduction technologies significantly increase sperm DNA fragmentation and phosphatidylserine translocation.  


Sperm preparation techniques in assisted reproduction technologies (ART) are potential generators of exogenous stresses that cause additional DNA damage. DNA fragmentation tests, such as the sperm chromatin structure assay, involve freezing sperm samples in the absence of cryoprotectant. Thermal, oxidative stress (OS) and freezing are detrimental to sperm DNA fragmentation and phosphatidylserine (PS) translocation. The primary aim of this study was to subject mature sperm to environmental insults that normally occur during ART. We tested the hypotheses that OS, thermal stress and freeze-thawing caused sperm nuclear and membrane damage and that a positive correlation exists between PS translocation and DNA fragmentation. Sperm DNA integrity deteriorates in semen samples from men with advancing age and a sperm concentration of <15 m ml(-1) . The significant increase in sperm DNA fragmentation at 37 °C after merely 1 h is important clinically as semen liquefaction and short-term sperm storage in an ART cycle involve incubating samples at this temperature. Freezing without a cryoprotectant significantly increases the level of sperm nuclear damage, so it is important not to freeze neat semen prior to DNA fragmentation testing. This study highlights the importance of minimising the production of exogenous stresses during sperm preparation in ART. PMID:23230887

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



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

NASA Astrophysics Data System (ADS)

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: n(l) = kl? exp (-l?), where n(l) represents the number of particles of diameter l, l is the normalized particle diameter, and k, ?, and ? 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 m': n(x, m) = C ?? n(x', m')p(?)dx' dm', where x' denotes spatial location along a linear axis, C is a constant, and integration is performed over distance from an origin to the sample location and mass limits from 0 to m. We show that the probability function that models the production of particles of different size from an initial mass and sorts that distribution, p(?), is related to mg, where g (noted as ? for fragmentation processes) is a free parameter that determines the location, breadth, and skewness of the distribution; g(?) must be greater than -1, and it increases from that value as the distribution matures with greater number of sequential steps in the fragmentation or transport process; ? is expected to be near -1 for "sudden" fragmentation mechanisms such as single-event explosions and transport mechanisms that are functionally dependent upon particle mass. This free parameter will be more positive for evolved fragmentation mechanisms such as ball milling and complex transport processes such as saltation. The SFT provides better fits to many types of volcanic ash samples than does the log normal curve. Modeling of the SFT shows its similarity to the log normal curve on size frequency histograms; it differs by its variable skewness controlled by ?. Skewed distributions are typical of many volcanic ash samples, and characterization of them by the SFT allows interpretation of eruptive and transport mechanisms.

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



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

Microsoft Academic Search

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: n(l)=klalpha exp(-lbeta), where n(l) represents the number of particles of diameter

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



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

PubMed Central

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

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



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.


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



Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil).  


We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC6 indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene. PMID:16738861

Yamada, Tetsuya; Takatsu, Yasumasa; Kasumi, Masakazu; Ichimura, Kazuo; van Doorn, Wouter G



Effects of ulipristal acetate on sperm DNA fragmentation during in vitro incubation.  


Abstract Objective Ulipristal acetate (UPA) acts as an emergency contraceptive by inhibiting ovulation. This study explores possible additional effects on the fragmentation of sperm DNA during in vitro incubation. Methods Motile spermatozoa from healthy donors were selected by swim-up and incubated under capacitating conditions in control medium or with UPA (1, 10, 100, 1,000 or 10,000 ng/ml). In some experiments, 200 ?M of H2O2 were added to induce oxidative stress. The sperm chromatin dispersion test was performed to analyse DNA integrity (400 cells; 1000×). Lipid peroxidation (thiobarbituric acid assay), induced-acrosome reaction (AR) and sperm vitality (Eosin Y) were also evaluated in spermatozoa exposed to UPA and/or H2O2. Results During sperm incubation, the percentage of fragmented DNA increased significantly, from 15.0 ± 1.3 to 41.0 ± 4.5% (p < 0.001). In the presence of UPA, DNA fragmentation decreased significantly (p < 0.05), in a dose-dependent manner. At 100 and 1000 ng/ml, UPA also counteracted the effect of H2O2 and prevented DNA fragmentation. No effect on sperm vitality, lipid peroxidation or induced-AR was found with any treatment. Conclusions During in vitro sperm capacitation DNA fragmentation increased but the latter was counteracted in the presence of UPA, which possibly acted as a scavenger of reactive oxygen species produced by spermatozoa. PMID:23879383

Munuce, María José; Cicaré, Juliana; Zumoffen, Carlos; Caille, Adriana; Ghersevich, Sergio; Bahamondes, Luis



Crystallographic Studies of Protein-Nucleic Acid Interaction: Catabolite Gene Activator Protein and the Large Fragment of DNA Polymerase I  

Microsoft Academic Search

Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor ‘Core’, 5S RNA fragment and its complex

T. A. Steitz; I. T. Weber; D. Ollis; P. Brick



A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo.  


We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo. PMID:2313033

Christova, R; Galcheva-Gargova, Z



Some technical parameters influencing the precision and accuracy of fragment size determination for RFLP's  

Microsoft Academic Search

Summary A comparison was carried out between 3 computer-assisted systems for the estimation of DNA fragment length: (1) the “Digitab” system (developed at our institute), (2) the FBI analysis system (FBI Quantico, USA) and (3) the BioImage system (Waters\\/Millipore, USA). Both the FBI system and the Biolmage system were found to be much more precise than the manual Digitab system.

C. Puers; P. Wiegand; B. Brinkmann



Cyclization of short DNA fragments and bending fluctuations of the double helix  

PubMed Central

Cloutier and Widom [Cloutier, T. E. & Widom, J. (2004) Mol. Cell 14, 355–362] recently reported that the cyclization efficiency of short DNA fragments, about 100 bp in length, exceeds theoretical expectations by three orders of magnitude. In an effort to resolve this discrepancy, we tried modifying the theory. We investigated how the distribution of the angles between adjacent base pairs of the double helix affects the cyclization efficiency. We found that only the incorporation of sharp kinks in the angle distribution provides the desired increase of the cyclization efficiency. We did not find a model, however, that fits all cyclization data for DNA fragments of different lengths. Therefore, we carefully reinvestigated the cyclization of 100-bp DNA fragments experimentally and found their cyclization efficiency to be in remarkable agreement with the traditional model of DNA bending. We also found an explanation for the discrepancy between our results and those of Cloutier and Widom.

Du, Quan; Smith, Chaim; Shiffeldrim, Nahum; Vologodskaia, Maria; Vologodskii, Alexander



Effects of the Geometric Constraints on the Size Distributions of Debris in Asteroidal Fragmentation  

NASA Astrophysics Data System (ADS)

It is commonly accepted that the formation of asteroid families is the consequence of catastrophic impacts on former {parent bodies} (K. Hirayama, Proc. Imp. Acad. Tokyo 9, 482-485, 1933). But to reproduce the puzzling steep size distributions of the currently known asteroid families has been, up to now, a task in which recent modeling techniques of fragmentation have typically failed. The role of geometric constraints in the production of fragments in asteroidal collisions is an issue that has been investigated in recent times only by Tanga et al. (Icarus 141, 65-78, 1999) and that might give some insight into the understanding of high-velocity collisional processes. Improvements to the approach by Tanga et al. are introduced in the present work in order to take into account in a more realistic way the different shapes that the largest remnants may have when formed in high-velocity collisional events involving spherical parent bodies. We also consider the case in which the parent body and the largest remnant are cubes and the fragments are (a) cubes and (b) parallelepipeds, instead of spheres. A somewhat uniform power-law behavior in the size distributions of the randomly generated fragments is found in the numerical simulations-not detected by Tanga et al.-and an analytical derivation of the upper limit to the corresponding exponent is given. Further improvements are introduced in the model in order to refine it and allow any fragment to develop any shape and to account for the fact that fragments form more or less at the same time, not sequentially. Finally, the results of the refined model are compared with the size distributions of the observed actual main belt asteroid families, and encouraging agreement is obtained in most cases.

Campo Bagatin, Adriano; Petit, Jean-Marc



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

Microsoft Academic Search

BACKGROUND: Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance), that calculates the relative abundance of genes in cDNA libraries. RESULTS: DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of

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



Simulation of DNA fragment distributions after irradiation with photons  

Microsoft Academic Search

The Monte Carlo track structure code PARTRAC has been further improved by implementing electron scattering cross-sections\\u000a for liquid water and by explicitly modelling the interaction of water radicals with DNA. The model of the genome inside a\\u000a human cell nucleus in its interphase is based on the atomic coordinates of the DNA double helix with an additional volume\\u000a for the

W. Friedland; Peter Jacob; Herwig G. Paretzke; Matteo Merzagora; Andrea Ottolenghi



Multimerization-cyclization of DNA fragments as a method of conformational analysis.  

PubMed Central

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.

Podtelezhnikov, A A; Mao, C; Seeman, N C; Vologodskii, A



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

SciTech Connect

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

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



Ionizing radiation-induced fragmentation of plasmid DNA Atomic force microscopy and biophysical modeling  

NASA Astrophysics Data System (ADS)

It is widely accepted that DNA double-strand breaks (DSBs) are closely correlated with radiation-induced cell killing and are the most critical lesions related to cellular endpoints like mutagenesis and transformation. High linear energy transfer (LET) radiation produces more severe and complex damage due to the fact that induced DSBs are not randomly distributed but clustered at different levels of DNA organization. In this paper, direct visualization of DSBs induced in a plasmid supercoiled DNA by low- and high-LET radiation is presented. Resulting DNA fragments distributions obtained by use of atomic force microscopy (AFM) are shown. Moreover, a biophysical model of spatially correlated DSBs formation in the framework of the Local Effect Model (LEM) is introduced and its predictions on DNA fragment formation are discussed.

Psonka, K.; Gudowska-Nowak, E.; Brons, S.; Elsässer, Th.; Heiss, M.; Taucher-Scholz, G.


Facile control of DNA-templated inorganic nanoshell size.  


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

Pu, Shengyan; Zinchenko, Anatoly; Murata, Shizuaki



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.

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



DNA fragmentation in developing lung fibroblasts exposed to Stachybotrys chartarum (atra) toxins.  


Stachybotrys chartarum (atra) is a toxic mold that grows on water-damaged cellulose-based materials. Research has revealed also that inhalation of S. chartarum spores caused marked changes in respiratory epithelium, especially to developing lungs. We analyzed the epigenetic potential of S. chartarum spore toxins on developing rat lung fibroblasts using single cell gel electrophoresis (comet assay). Isolated fetal lung fibroblasts were exposed to S. chartarum spore toxins for 15 min, 3, 14, or 24 hr and control cells were exposed to saline under the same conditions. Cells were embedded in agarose, electrophoresed under alkaline conditions and silver stained. DNA damage was assessed in terms of fragmentation as measured by comet tail length (DNA migration) and intensity (% DNA contained within head and tail). Upon visual inspection, control fibroblasts showed no DNA fragmentation whereas S. chartarum-treated cells had definable comets of various degrees depending upon the time-course. Analyses of the comets revealed that exposure to S. chartarum spore toxins for at least 15 min to 14 hr, induced increased DNA fragmentation in a time-dependent manner. The fact that exposure to toxins for 24 hr showed less damage suggested that developing lung fibroblasts may have the capability of repairing DNA fragmentation. PMID:17534970

McCrae, K C; Rand, T G; Shaw, R A; Mantsch, H H; Sowa, M G; Thliveris, J A; Scott, J E



Mitochondrial fission controls DNA fragmentation by regulating endonuclease G  

Microsoft Academic Search

Mitochondria constantly undergo fusion and fission that are necessary for the maintenance of organelle fidelity. However, growing evidence has shown that abnormal mitochondrial fusion and fission participate in the regulation of apoptosis. Mitochondrial fusion is able to inhibit apoptosis, whereas mitochondrial fission is involved in the initiation of apoptosis. It remains elusive as to whether mitochondrial fission can regulate DNA

Jincheng Li; Jing Zhou; Yanrui Li; Danian Qin; Peifeng Li



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.



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

NASA Astrophysics Data System (ADS)

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

Kato, Tomohiro; Hasegawa, Mikio


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

Microsoft Academic Search

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

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



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


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

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



FragIdent - Automatic identification and characterisation of cDNA-fragments  

PubMed Central

Background Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments. Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available. Results Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones. Conclusion We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at .

Seelow, Dominik; Goehler, Heike; Hoffmann, Katrin



Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis.  

PubMed Central

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules. Images

Stow, N D



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.



The Effect of Change in Population Size on DNA Polymorphism  

Microsoft Academic Search

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

Fumio Tajima


A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.  

PubMed Central

Specific, end-labeled DNA fragments can be simply and rapidly prepared using the polymerase chain reaction (PCR). Such fragments are suitable for use in DNase I protection footprint assays, chemical sequencing reactions, and for the production and analysis of paused RNA polymerase transcription complexes. Moreover, a general means of introducing a specific mutation at any position along the length of such PCR-generated fragments is described. These procedures, which can circumvent the need for large-scale phage or plasmid growths, preparative gel-electrophoresis and the screening of molecular clones, can facilitate the rapid study of sequence-specific interactions of proteins and DNA. A rapid means of removing excess oligonucleotide primers from completed PCRs is also described. Images

Higuchi, R; Krummel, B; Saiki, R K



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



Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.  

PubMed Central

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2. Figure 3. Figure 7.

Manning, F C; Blankenship, L J; Wise, J P; Xu, J; Bridgewater, L C; Patierno, S R



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

PubMed Central

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

Cheng, Xin; Ivessa, Andreas S.



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


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

Cheng, Xin; Ivessa, Andreas S



Single-Molecule Analysis of Restriction DNA Fragments Using Fluorescence Correlation Spectroscopy  

Microsoft Academic Search

The cleavage of fluorescence-labeled M13DNA (7250 bp) usingHaeIII,HgaI,BsmAI, andBspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 × 10?15liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived

Masataka Kinjo; Goro Nishimura; Tomiyasu Koyama; Ülo Mets; Rudolf Rigler



The Restriction Fragment Map of Rat-Liver Mitochondrial DNA: A Reconsideration  

Microsoft Academic Search

1. Rat-liver mitochondrial DNA (mtDNA) contains at least 8 cleavage sites for the restriction endonuclease Eco RI, 6 for the restriction endonuclease Hind III, 2 for the restriction endonuclease Bam HI and 11 for the restriction endonuclease Hap II.\\u000a2. The physical map of the restriction fragments of Eco RI, Hind III, Bam HI and Hap II is constructed on

A. M. Kroon; G. Pepe; H. Bakker; M. Holtrop; J. E. Bollen; E. F. J. van Bruggen; P. Cantatore; P. Terpstra; C. Saccone



Induction of Tumor Suppressor p53 and DNA Fragmentation in Organotypic Hippocampal Cultures Following Excitotoxin Treatment  

Microsoft Academic Search

The p53 tumor suppressor gene encodes a cell cycle regulatory protein that is induced by DNA damage and has been implicated in apoptosis. To investigate whether excitotoxic cell death due to kainic acid (KA) and cell death due toN-methyl-d-aspartate (NMDA) share similar molecular mechanisms, we studied p53 expression and DNA fragmentation in organotypic hippocampal slice cultures following excitotoxin treatment. Cellular

Shahin Sakhi; Annadora Bruce; Ning Sun; Georges Tocco; Michel Baudry; Steven S. Schreiber



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


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

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



Early prediction of therapy response in patients with acute myeloid leukemia by nucleosomal DNA fragments  

PubMed Central

Background Elevated levels of nucleosomal DNA fragments can be detected in plasma and sera of patients with malignant diseases. Methods We investigated the course of nucleosomal DNA, thymidine kinase, lactate dehydrogenase and leukocytes in sera of 25 patients with acute myeloid leukemia during the first cycle of induction chemotherapy and tested their power to distinguish between patients with complete remission and those with no remission. Results Almost all patients showed strongly decreasing levels of nucleosomal DNA during the first week, in some cases after initial peaks. In overall analysis of variance, DNA levels could clearly distinguish between patients with complete remission, who had higher DNA values, and those with insufficient response (p = 0.017). The area under the curve of DNA values of days 2–4 after start of therapy (AUC 2–4) discriminated between both groups with a sensitivity of 56% at a specificity of 100%. Further, pretherapeutic levels and AUC 2–4 of nucleosomal DNA correlated significantly with blast reduction after 16 days. A tendency to higher levels in patients with complete response was also found for thymidine kinase, lactate dehydrogenase and leukocytes, however the difference did not reach the level of significance (p = 0.542, p = 0.260, and p = 0.144, respectively). Conclusion Our results indicate that nucleosomal DNA fragments are valuable markers for the early prediction of therapeutic efficacy in patients with acute myeloid leukemia.

Mueller, Susanne; Holdenrieder, Stefan; Stieber, Petra; Haferlach, Torsten; Schalhorn, Andreas; Braess, Jan; Nagel, Dorothea; Seidel, Dietrich



Ovarian Steroids Decrease DNA Fragmentation in the Serotonin Neurons of Non-Injured Rhesus Macaques  

Microsoft Academic Search

We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys were implanted

F B Lima; C L Bethea



Cytoplasmic DNAs and nuclear rDNA restriction fragment length polymorphisms in commercial witloof chicories  

Microsoft Academic Search

Restriction fragment length polymorphisms of cytoplasmic DNAs and nuclear rDNA were analyzed in several Cichorium intybus genotypes, comprising four white inbred lines, eight red witloof experimental lines, and a number of F1 hybrids derived from two white parents. Chloroplast and mitochondrial restriction patterns led to the distinction between two different cytoplasms, called I and II. Southern hybridization using a nuclear

A. Bellamy; C. Mathieu; F. Vedel; H. Bannerot



DNA restriction fragment length polymorphisms and heterozygosity in the human genome  

Microsoft Academic Search

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

David N. Cooper; Jörg Schmidtke



Preparation of DNA and RNA Fragments Containing Guanine N2-Thioalkyl Tethers  

PubMed Central

This unit describes procedures for preparation of deoxyguanosine and guanosine derivatives in which the guanine N2 contains a thiopropyl tether, protected as a tert-butyl disulfide. After incorporation into a DNA or RNA fragment, this tether allows site-specific crosslinking to a thiol of a protein or another nucleic acid.

Hou, Xiaorong; Wang, Gang; Gaffney, Barbara L.; Jones, Roger A.



Anatomical studies of DNA fragmentation in rat brain after systemic kainate administration  

Microsoft Academic Search

Rats treated systemically with kainate develop stereotyped epileptic seizures involving mainly limbic structures that may last for hours. This model of limbic status epilepticus has been widely studied using classical neuropathological techniques. We used in situ nick translation histochemistry to examine patterns of DNA fragmentation in this model. We found a stereotyped and reproducible pattern of neuronal populations that demonstrate




A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea  

Microsoft Academic Search

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans

Paula M. A. Lucchesi; Alberto E. Parma



Formation of Apoptotic Bodies Is Associated with Internucleosomal DNA Fragmentation during Drug-Induced Apoptosis  

Microsoft Academic Search

The onset of apoptosis is often coincident with internucleosomal DNA fragmentation or ladders which are considered a hallmark of the process. However, several studies have indicated that MOLT-4 human lymphoblastoid cells exposed to various agents, including VP16, display some apoptotic characteristics in the absence of either internucleosomal ladders or production of apoptotic bodies. The present study records that, in the

Daniel R. Catchpoole; Bernard W. Stewart



DNA restriction fragment analysis of the proopiomelanocortin gene in schizophrenia and bipolar disorders.  

PubMed Central

The method of DNA restriction fragment analysis using gene probes for the proopiomelanocortin (POMC) gene was employed to detect possible molecular variation in the POMC gene in schizophrenia and bipolar illness. No gross structural abnormalities in restriction fragments were observed with the set of restriction enzymes used. Two allelic restriction sites were observed giving rise to fragment length polymorphisms. One of these is a new polymorphism, not previously reported, which will be of value as a linkage marker. The associations between the two DNA polymorphisms that are closely linked to the POMC gene and both schizophrenia and bipolar disorder were investigated. No association was found, thus adding weight to the evidence that there are no alterations in the POMC gene in schizophrenia and bipolar illness. Images Fig. 1

Feder, J; Gurling, H M; Darby, J; Cavalli-Sforza, L L



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


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

Wang, Shanquan; He, Jianzhong



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


In traditional electrophoresis mobility shift assay (EMSA) a single (32)P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. An extension of this method uses a population of DNA restriction fragments derived from long genomic regions for the identification of fragments containing protein binding regions. Although the method allows simultaneous analysis of large fragments, it is relatively laborious and can be used to detect only fragments containing high affinity protein binding sites. Here we describe an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. With this strategy restriction fragments, derived from genomic DNA (<10 kb), containing high as well as low affinity protein binding regions may be easily identified. PMID:23436361

Kaer, Kristel; Speek, Mart



Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells.  


Concentration, motility and morphology are parameters commonly used to determine the fertilization potential of an ejaculate. These parameters give a general view on the quality of sperm but do not provide information about one of the most important components of the reproductive outcome: DNA. Either single or double DNA strand breaks can set the difference between fertile and infertile males. Sperm DNA fragmentation can be caused by intrinsic factors like abortive apoptosis, deficiencies in recombination, protamine imbalances or oxidative stress. Damage can also occur due to extrinsic factors such as storage temperatures, extenders, handling conditions, time after ejaculation, infections and reaction to medicines or post-testicular oxidative stress, among others. Two singular characteristics differentiate sperm from somatic cells: Protamination and absence of DNA repair. DNA repair in sperm is terminated as transcription and translation stops post-spermiogenesis, so these cells have no mechanism to repair the damage occurred during their transit through the epididymis and post-ejaculation. Oocytes and early embryos have been shown to repair sperm DNA damage, so the effect of sperm DNA fragmentation depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. In this contribution we review some of these issues. PMID:23203048

González-Marín, Clara; Gosálvez, Jaime; Roy, Rosa



A new PCR system for Agrobacterium tumefaciens detection based on amplification of T-DNA fragment.  


The design of the PCR system presented in this work is based on the knowledge of the molecular character of the crown gall disease. The virulence of Agrobacterium tumefaciens requires the presence of a big (up to 235,000 bp) plasmid Ti (pTi-tumour inducing plasmid). This plasmid carries the so-called T-DNA fragment (T-DNA-transferred DNA), which integrates into cell chromosomes of the infected plants and subsequently changes the plant morphology nad metabolism. In cannot be excluded that after T-DNA integration the presence of Agrobacterium is not necessary for the development of pathological changes. Thus, T-DNA is the only sign that must be present both in virulent bacteria and in infected plants in any stadium of the disease and even before the infection. This is why T-DNA was chosen as the target region for PCR amplification. Primers flanking a 220 bp fragment of one of the conservative regions responsible for Agrobacterium pathogenicity, namely tms2 gene coding for indolacetamide amidohydrolase (the second step of auxin biosynthesis) were designed as the optimal for PCR amplification. The PCR amplification reactions were performed for matrixes isolated from cultures of reference strains giving one predicted product for each sample. First attempts of T-DNA detection in infected soils and plants were performed. We hope that the presented new PCR system for Agrobacterium tumefaciens detection will help to fight the crown gall disease in the nearest future. PMID:9429288

Sachadyn, P; Kur, J



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


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

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



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

PubMed Central

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

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



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

BACKGROUND: In a traditional electrophoresis mobility shift assay (EMSA) a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE) in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb) for the identification of fragments

Kristel Kaer; Kert Mätlik; Madis Metsis



Regionspecific rates of molecular evolution: A fourfold reduction in the rate of accumulation of “Silent” mutations in transcribed versus nontranscribed regions of homologous DNA fragments derived from two closely related mouse species  

Microsoft Academic Search

Summary  We have sequenced homologous DNA fragments of 2.7 and 2.8 kbp derived from the closely related mouse speciesMus musculus domesticus (M. domesticus) andMus musculus musculus (M. musculus), respectively. These two species diverged approximately 1 million years ago. Each DNA fragment contains 1.35 kbp of the 3?\\u000a end of the constitutively expressed 2.2-kbpaprt (adenine phosphoribosyltransferase) gene and a similarly sized nontranscribed

Mitchell S. Turker; Gregory E. Cooper; Peggy L. Bishop



Nuclear Multifragmentation Time Scale and Fluctuations of the Largest Fragment Size  

NASA Astrophysics Data System (ADS)

Distributions of the largest fragment charge, Zmax?, in multifragmentation reactions around the Fermi energy can be decomposed into a sum of a Gaussian and a Gumbel distribution, whereas at much higher or lower energies one or the other distribution is asymptotically dominant. We demonstrate the same generic behavior for the largest cluster size in critical aggregation models for small systems, in or out of equilibrium, around the critical point. By analogy with the time-dependent irreversible aggregation model, we infer that Zmax? distributions are characteristic of the multifragmentation time scale, which is largely determined by the onset of radial expansion in this energy range.

Gruyer, D.; Frankland, J. D.; Botet, R.; P?oszajczak, M.; Bonnet, E.; Chbihi, A.; Ademard, G.; Boisjoli, M.; Borderie, B.; Bougault, R.; Guinet, D.; Lautesse, P.; Manduci, L.; Le Neindre, N.; Marini, P.; Paw?owski, P.; Rivet, M. F.; Rosato, E.; Spadaccini, G.; Vigilante, M.; Wieleczko, J. P.



Nuclear multifragmentation time scale and fluctuations of the largest fragment size.  


Distributions of the largest fragment charge, Zmax, in multifragmentation reactions around the Fermi energy can be decomposed into a sum of a Gaussian and a Gumbel distribution, whereas at much higher or lower energies one or the other distribution is asymptotically dominant. We demonstrate the same generic behavior for the largest cluster size in critical aggregation models for small systems, in or out of equilibrium, around the critical point. By analogy with the time-dependent irreversible aggregation model, we infer that Zmax distributions are characteristic of the multifragmentation time scale, which is largely determined by the onset of radial expansion in this energy range. PMID:23679716

Gruyer, D; Frankland, J D; Botet, R; P?oszajczak, M; Bonnet, E; Chbihi, A; Ademard, G; Boisjoli, M; Borderie, B; Bougault, R; Guinet, D; Lautesse, P; Manduci, L; Le Neindre, N; Marini, P; Paw?owski, P; Rivet, M F; Rosato, E; Spadaccini, G; Vigilante, M; Wieleczko, J P



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

PubMed Central

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

Kazlauskas, Darius; Venclovas, Ceslovas



High-speed electrophoretic separation of DNA fragments using a short capillary.  


Capillary electrophoresis using a replaceable gel buffer was applied to the separation of DNA fragments. A short effective length capillary (1-2 cm) at low electric field allowed the separation of a 20-1000 bp ladder in 1 min. Although similar separation speed was achieved with a longer capillary at high field, the resolution of larger fragments was degraded. The short effective length capillaries were able to separate the wildtype and mutant PCR products of the TGF-beta1 gene in under 45 s. PMID:9271135

Chan, K C; Muschik, G M; Issaq, H J



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

PubMed Central

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 spermatozoa by two treatments, (a) a scrotal heat treatment (42 degrees C, 30 min) and (b) irradiation with 137Cs gamma-rays (4 Gy, 1.25 Gy/min). The effects of the treatments were analyzed 21-25 days post heat stress or gamma-radiation. Postovulatory females mated either with treated or control males were sacrificed at Day 14 of pregnancy, and numbers of fetuses and resorptions were recorded. Results Both treatments decreased significantly implantation rates however, the proportion of fetuses/resorptions was only reduced in those females mated to males exposed to radiation, indicating a selection favoring fertilization of sperm with unfragmented DNA on the heat treatment group. To determine if DNA integrity is one of the keys of spermatozoa selection after postovulatory mating, we analyzed sperm DNA fragmentation by COMET assay in: a) sperm recovered from mouse epididymides; b) sperm recovered from three different regions of female uterine horns after mating; and c) sperm attached to the ZP after in vitro fertilization (IVF). Similar results were found for control and both treatments, COMET values decreased significantly during the transit from the uterine section close to the uterotubal junction to the oviduct, and in the spermatozoa attached to ZP. However, fertilization by IVF and intracytoplasmatic sperm injection (ICSI) showed that during sperm ZP-penetration, a stringent selection against fragmented-DNA sperm is carried out when the damage was induced by heat stress, but not when DNA fragmentation was induced by radiation. Conclusion Our results indicate that in postovulatory mating there is a preliminary general selection mechanism against spermatozoa with low motility and fragmented-DNA during the transport through the female reproductive tract and in the ZP binding, but the ability of the ZP to prevent fertilization by fragmented-DNA spermatozoa is achieved during sperm-ZP penetration, and depends on the source of damage.



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


Seminal proteins can be considered as factors that control fertilization. Clusterin is one such protein that has been implicated in many activities, including apoptosis inhibition, cell cycle control, DNA repair, and sperm maturation. In this study, the relationship between human secretory clusterin (sCLU) in seminal plasma with sperm parameters, protamine deficiency, and DNA fragmentation was investigated. Semen samples were collected from 63 Iranian men, and semen analysis was performed according to World Health Organization criteria and computer aided semen analysis (CASA). The concentration of sCLU in seminal plasma was measured by enzyme-linked immunosorbant assay (ELISA), protamine deficiency was determined by chromomycin A3 staining (CMA3 ), and sperm DNA fragmentation was checked by sperm chromatin dispersion (SCD) assay. The level of sCLU in seminal fluid of fertile patients was 48.3?±?38.6?ng/ml and in infertile patients was 15.5?±?9.7?ng/ml; this difference was significant (P?DNA fragmentation, and abnormal morphology. In conclusion, seminal clusterin can be considered as a marker for the quick assessment of semen quality in male infertility studies. Mol. Reprod. Dev. 80: 718-724, 2013. © 2013 Wiley Periodicals, Inc. PMID:23740886

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



Analysis of DNA fragmentation in mouse embryos exposed to an extremely low-frequency electromagnetic field.  


Effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on DNA damage in biological systems are still a matter of dispute. The aim of the present study was to investigate the possible effect of electromagnetic field exposure on DNA fragmentation in cells (blastomers) of mouse blastocysts. Eighty female NMRI mice were randomly divided into 2 groups of 40 animals each. The control group was left unexposed whereas the animals in the EMF-group were exposed to a 50-Hz EMF at 0.5 mT 4 h per day, 6 days a week for a duration of 2 weeks. After the 8(th) day of exposure, the female mice in both groups were superovulated (with injections of pregnant mare serum gonadotropin and human chorionic gonadotropin) and then mated overnight. At approximately 4 days after mating (102 h after the human chorionic gonadotropin treatment), blastocysts were obtained by flushing the uterus horns. The mean numbers of pregnant mice, blastocysts after flushing, blastomers within the blastocysts, and the DNA fragmentation index following staining in both groups were compared using statistical methods (SPSS, the Chi-square test, the Student's t-test and the Mann-Whitney U-test, P < 0.05). The results showed that the mean number of blastocysts after flushing was significantly decreased in the EMF-group compared to that of the control group (P < 0.03). The DNA fragmentation index was significantly increased in the EMF-group compared to control (10.53% vs. 7.14%; P < 0.001). However, there was no significant difference in the mean numbers of blastomers and numbers of pregnant mice between the EMF-exposed and control group. Our findings indicate that the EMF exposure in preimplantation stage could have detrimental effects on female mouse fertility and embryo development by decreasing the number of blastocysts and increasing the blastocysts DNA fragmentation. PMID:22047462

Borhani, Nasim; Rajaei, Farzad; Salehi, Zivar; Javadi, Amir



Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer  

Microsoft Academic Search

The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.97±0.15Å per base pair (bp) (mean±standard deviation) by imaging a 648-bp DNA fragment and 2.95±0.14Å per bp for

Albert Sanchez-Sevilla; Jean Thimonier; Monique Marilley; José Rocca-Serra; Jacques Barbet



Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from Celosia cristata in Escherichia coli  

Microsoft Academic Search

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

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



Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation  

Microsoft Academic Search

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be

Jeffrey Mullen; Gerhard Adam; Alan Blowers; Elizabeth Earle




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.

Didenko, Vladimir V.



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

SciTech Connect

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

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



Sperm ubiquitination and DNA fragmentation in men with occupational exposure and varicocele.  


Assessment of sperm ubiquitination and DNA fragmentation as sperm functional markers are proposed to complement routine semen analysis. This study focuses on the evaluation of these markers in infertile men with varicocele or exposed to occupational background. The results were compared with normozoospermic men. Semen parameters in both groups were lower than those in the control group. Ubiquitination median, as a marker for functionality of the ubiquitin-proteasome system, was also lower in both groups. The ubiquitination median showed a significant positive correlation with motility in both groups, while it showed only a negative correlation with sperm morphology in the varicocele group. DNA fragmentation showed a significant correlation with semen parameters, in total varicocele and also total exposure groups. In conclusion, significant difference of sperm ubiquitination between normal and study groups further validates that sperm ubiquitination as a potential molecular marker for sperm evaluation in addition to routine semen analysis in clinical laboratories. PMID:23594355

Hosseinpour, E; Shahverdi, A; Parivar, K; Sedighi Gilani, M A; Nasr-Esfahani, M H; Salman Yazdi, R; Sharbatoghli, M; Tavalaee, M; Chehrazi, M



The effect of CpG-rich DNA fragments on the development of hypertension in spontaneously hypertensive rats (SHR)  

Microsoft Academic Search

In this study we have investigated properties of blood plasma extracellular DNA (cell-free DNA, cfDNA) from patients with\\u000a essential arterial hypertension (AH). Concentration of cell-free DNA was basically the same as in healthy donors, however,\\u000a the content of the marker, CpG-rich cell-free DNA fragments (CpG-DNA) of the transcribed area of the ribosomal repeat (TArDNA,\\u000a CpG-DNA) was higher in AH patients.

N. N. Veiko; I. L. Konorova; M. E. Neverova; O. V. Fidelina; N. A. Mkrtumova; E. S. Ershova; M. S. Kon’kova; A. Yu. Postnov



Cloned fragment of human alphoid DNA: molecular marker of pericentromeric region of 18th chromosome  

SciTech Connect

Two recombinant plasmids were isolated from the collection of cloned human DNA fragments which contain sequences of alphoid DNA. It was shown using in situ hybridization on metaphase chromosomes that both cloned sequences hybridize preferentially with the region of pericentromeric heterochromatin of chromosome 18, less intensively with pericentric regions of chromosomes 2, 9, and 20, and are characterized by polymorphism according to number of copies in homologous chromosomes. These sequences may prove useful for cytogenetic analysis of chromosome reorganizations and study of polymorphism of regions of pericentromeric heterochromatin in human chromosomes.

Aleksandrov, I.A.; Yurov, Yu.B.; Mitkevich, S.P.; Gindilis, V.M.



Dynamics of sperm DNA fragmentation in domestic animals II. The stallion.  


The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage. PMID:17919715

López-Fernández, C; Crespo, F; Arroyo, F; Fernández, J L; Arana, P; Johnston, S D; Gosálvez, J



DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity  

PubMed Central

Background Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. Results Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk) gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection) were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. Conclusions We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.



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

PubMed Central

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

Lima, Fernanda B.; Bethea, Cynthia L.



GP7 can induce apoptotic DNA fragmentation of human leukemia cells through caspase-3-dependent and -independent pathways.  


GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy)amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin, is a promising anticancer drug of podophyllotoxin class. The primary effect of GP7 is the anticancer activity on transplanted mouse tumors and cultured tumor cells. However, its molecular mechanism of action is still obscure. In this study, we investigated the activity of GP7 to induce apoptosis in human leukemia HL-60 and Jurkat cells. Apoptosis was determined by detection of DNA fragmentation in agarose gel electrophoresis. GP7 induced apoptotic DNA fragmentation of HL-60 and Jurkat cells in time- and dose-dependent manner. We further investigated the activity of caspase-3 in GP7-induced apoptotic DNA fragmentation of HL-60 and Jurkat cells. GP7 also induced time- and dose-dependent caspase-3 activation in both cell lines, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. To determine the role of caspase-3 in GP7-induced apoptotic DNA fragmentation, we examined the effect of specific caspase-3 inhibitor, Ac-DEVD-CHO, on GP7-induced apoptotic DNA fragmentation. Ac-DEVD-CHO prevented GP7-induced caspase-3 activation in both HL-60 and Jurkat cells, however, it only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. We then employed L-carnitine to investigate the role of caspase-3 in GP7-induced apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation in both cell lines in a dose-dependent manner. Similar to Ac-DEVD-CHO, L-carnitine only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. These findings suggest that GP7 exerts an anti-leukemic effect by both caspase-3-dependent and -independent apoptotic signaling pathways. PMID:14654989

Qi, She-Ning; Yoshida, Akira; Wang, Zi-Ren; Ueda, Takanori



Arrangement of individual human ribosomal DNA fragments on stretched DNA fibers  

Microsoft Academic Search

We studied the arrangement of individual human ribosomal (r)DNA repeats by direct visualization of rDNA sequences. We used\\u000a high resolution fluorescence in situ hybridization on preparations of DNA fibers released from interphase nuclei of HeLa cells.\\u000a Probes from both the transcription unit and the intergenic spacer were used, and lengths of signals and of the gaps in between\\u000a were measured

C. Schöfer; Klara Weipoltshammer; Marlene Almeder; Franz Wachtler



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

Microsoft Academic Search

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

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



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

SciTech Connect

We have observed severe damage to films of DNA components (thymine, D-ribose, 2-deoxy-D-ribose, and thymidine) induced by 10 to 100 eV He{sup +} ions (2.5-25 eV/amu). The damage is attributed to the kinetic and potential energies, as well as the chemical reactivity of the He{sup +} projectiles. Hyperthermal He{sup +} ion impact on these films results in the complete destruction of the molecules via fragmentation, and direct and indirect (secondary fragment) reactive scattering, all of which leads to the desorption of abundant cation and anion fragments. The chemical composition of the fragments is identified, and the fragmentation patterns are compared to those produced by Ar{sup +} irradiation. While the lower mass of He{sup +} ions causes less efficient desorption of very heavy fragments, several reactive collisions are also observed, including hydrogen abstraction by incident He{sup +} from any of the molecules studied to yield desorbing HeH{sup +}. This process likely occurs via the formation of an intermediate molecular ion (He-H-R)*{sup +}, which decays to HeH{sup +}+R . Compared to Ar{sup +}, here a significant (x23) enhancement in H{sup +} desorption is observed during He{sup +} ion irradiation, which likely involves (a) the decay of the intermediate (He-H-R)*{sup +}, or desorbing HeH{sup +}, and (b) Auger or quasiresonant excitations of C, N, or O atom centers (or C-H, N-H, or O-H bonds) by the incident He{sup +} ion. The formation of several molecular cations, e.g., H{sub 3}O{sup +}, also requires hydrogen abstraction from its parent or adjacent molecules by initial cation fragments prior to desorption.

Deng Zongwu; Imhoff, Marjorie; Bald, Ilko; Illenberger, Eugen; Huels, Michael A. [Department of Nuclear Medicine and Radiobiology, Ion Reaction Laboratory, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4 (Canada)



Size, shape, and flexibility of proteins and DNA  

NASA Astrophysics Data System (ADS)

Size, shape, and flexibility are the important topological parameters which characterize the functional specificity and different types of interactions in proteins and DNA. The size of proteins and DNA, often measured by the radius of gyration (RG), are determined from the coordinates of their respective structures available in Protein Data Bank and Nucleic Acid Data Bank. The mean square radius of gyration obeys Flory's scaling law given by ~N2? where N is the number of amino acid residues/nucleotides. The scaling exponent ? reflects the different characteristic features of nonglobular proteins, natively unstructured proteins, and DNA. The asymmetry in the shapes of proteins and DNA are investigated using the asphericity (?) parameter and the shape parameter (S), calculated from the eigenvalues of the moment of inertia tensor. The distributions of ? and S show that most nonglobular proteins and DNA are aspherical and prolate (S>0). Natively unstructured proteins are comparatively spherically symmetrical having both prolate and oblate shapes. The flexibility of these molecules is characterized by the persistence length (lp). Persistence length for natively unstructured proteins is determined by fitting the distance distribution function P(r) to the wormlike chain (WLC) model in the limit of r>>RG. For nonglobular proteins and DNA, lp may be computed from the Benoit-Doty approximation for unperturbed radius of gyration of the WLC. The flexibilities of the proteins and DNA increases with the chain length. This is due to an increase in the nonlocal interactions with the increase in N, needed to minimize the conformational fluctuations in the native state. The persistence length of these proteins has not yet been measured directly. Analysis of the two-body contacts for the proteins reveals that the nonglobular proteins are less densely packed compared to the natively unstructured proteins with least side-chain side chain contacts even though side-chain backbone contacts predominate in the two types of proteins.

Rawat, Nidhi; Biswas, Parbati



Dynamics of relaxation and fragmentation in size-selected icosahedral Arn[NO-(v = 1)] clusters  

NASA Astrophysics Data System (ADS)

We study the vibrational relaxation and solvation dynamics in size-selected icosahedral Arn(NO-) at 300 K, where NO-(X3?-) is in v = 1 and n = 1-12, using a classical dynamics method and an interaction model consisting of detailed host-guest and host-host interactions. Two relaxation time scales are found: (i) the short-time (<200 ps), in which rate is nearly independent of cluster size, and (ii) the ns scale, in which a slow energy transfer process occurs between NO- vibration and argon modes at a rate (~108 s-1) decreasing slightly from n = 12 to 6 and rapidly from n = 5 to 1 (~106 s-1). In Ar12(NO-), less than one-quarter of the host atoms sampled evaporate, nearly 60% of evaporation occurring within 200 ps caused by rapid energy transfer from NO- at short time. The fraction of evaporation decreases nearly exponentially with increasing evaporation time, but ~16% of evaporation still occurs on a time scale longer than 1 ns. Evaporation from one hemisphere of Ar12(NO-) dominates the rest. Final cluster sizes commonly produced from the fragmentation of Ar12(NO-) are n = 6-11 (evaporation of 6-1 atoms) and n = 12 (no evaporation).

Shin, H. K.



Short-fragment Na-DNA dilute aqueous solutions: Fundamental length scales and screening  

NASA Astrophysics Data System (ADS)

Dielectric spectroscopy is used to investigate fundamental length scales of 146 bp short-fragment (nucleosomal) dilute Na-DNA solutions. Two relaxation modes are detected: the high- and the low-frequency mode. Dependence of the corresponding length scales on the DNA and on the (uni-valent) salt concentration is studied in detail, being different from the case of long, genomic DNA, investigated before. In low-added-salt regime, the length scale of the high-frequency mode scales as the average separation between DNAs, though it is smaller in absolute magnitude, whereas the length scale of the low-frequency mode is equal to the contour length of DNA. These fundamental length scales in low-added-salt regime do not depend on whether DNA is in a double-stranded or single-stranded form. On the other hand, with increasing added salt, the characteristic length scale of the low-frequency mode diminishes at low DNA concentrations probably due to dynamical formation of denaturation bubbles and/or fraying in the vicinity of DNA denaturation threshold.

Tomi?, S.; Dolanski Babi?, S.; Ivek, T.; Vuleti?, T.; Kr?a, S.; Livolant, F.; Podgornik, R.



Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer.  


The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.97 +/- 0.15 A per base pair (bp) (mean +/- standard deviation) by imaging a 648-bp DNA fragment and 2.95 +/- 0.14 A per bp for a 115-bp fragment. This confirms earlier observations suggesting that drying DNA fragments on mica in the presence of nickel induces limited conformational changes. At this point the exact nature of these conformational changes remains unknown. Simple hypotheses are the transconformation of stretches of the DNA molecules to the A-form of the double helix or alteration of the helix structure at the points of contact between DNA and mica. By contrast, in aqueous buffer, the measured helical rise was 3.14 +/- 0.15 A per bp for the 648-bp fragment and 3.17 +/- 0.13 A per bp for the 1115-bp fragment. Thus, measured helical rises do not depend on the fragment length and are significantly shorter than the 3.38 A per bp measured by crystallography, but close to the 3.18 A per bp found in NMR studies. These findings are discussed with respect to discrepancies in earlier results published in the literature. PMID:12213016

Sanchez-Sevilla, Albert; Thimonier, Jean; Marilley, Monique; Rocca-Serra, José; Barbet, Jacques



Patch occupancy, population size and reproductive success of a forest herb ( Primula elatior ) in a fragmented landscape  

Microsoft Academic Search

Forest fragmentation is expected to affect patch occupancy patterns, population size and population viability of plant populations through changes in both patch area and isolation. We tested the hypothesis that patch area has had a significant effect on patch occupancy and population size of Primula elatior, a common forest herbaceous plant species in Flanders, Belgium. The hypothesis that plants from

Hans Jacquemyn; Rein Brys; Martin Hermy



Purified Escherichia coli recA Protein Catalyzes Homologous Pairing of Superhelical DNA and Single-Stranded Fragments  

NASA Astrophysics Data System (ADS)

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages ? X174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.

Shibata, Takehiko; Dasgupta, Chanchal; Cunningham, Richard P.; Radding, Charles M.



‘Mitominis’: multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples  

Microsoft Academic Search

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the\\u000a two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300–400 bp\\u000a each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and\\u000a more stable approach using shortened

Cordula Eichmann; Walther Parson



Detection of JC Virus DNA Fragments but Not Proteins in Normal Brain Tissue  

PubMed Central

Objective Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the white matter affecting immunocompromised patients that results from the cytolytic destruction of glial cells by the human neurotropic JC virus (JCV). According to one model, during the course of immunosuppression, JCV departs from its latent state in the kidney and after entering the brain, productively infects and destroys oligodendrocytes. The goal of this study was to test the hypothesis that JCV may reside in a latent state in a specific region of the brains of immunocompetent (non-PML) individuals without any neurological conditions. Methods Gene amplification was performed together with immunohistochemistry to examine the presence of JCV DNA sequences and expression of its genome in five distinct regions of the brain from seven immunocompetent non-PML individuals. Results Although no viral proteins were expressed in any of these cases, fragments of the viral DNA were present in various regions of normal brain. Laser-capture microdissection showed the presence of JCV DNA in oligodendrocytes and astrocytes, but not in neurons. Interpretation The detection of fragments of viral DNA in non-PML brain suggests that JCV has full access to all regions of the brain in immunocompetent individuals. Thus, should the immune system become impaired, the passing and/or the resident virus may gain the opportunity to express its genome and initiate its lytic cycle in oligodendrocytes. The brain as a site of JCV latency is a possibility.

Perez-Liz, Georgina; Del Valle, Luis; Gentilella, Antonio; Croul, Sidney; Khalili, Kamel



Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: Size distributions and absence of nicks  

Microsoft Academic Search

Macronuclear DNAs from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from ~400 bp to ~20,000 bp. By EM, the number average molecular sizes for doublestranded DNA were 2,200 bp for Oxytricha sp.,

Marshal T. Swanton; John M. Heumann; David M. Prescott



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

Microsoft Academic Search

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

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



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

Microsoft Academic Search

Experimental stroke using a focal cere- bral ischemia and reperfusion (FCIR) model was in- duced in male Long-Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30-90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N-glycosylase (Fpg protein)-sensitive



Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay  

Microsoft Academic Search

ObjectiveTo investigate how moderate and\\/or high levels of DNA fragmentation (DFI), as measured by the sperm chromatin structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy.

Kjersten L Larson-Cook; John D Brannian; Keith A Hansen; Kay M Kasperson; Edward T Aamold; Donald P Evenson



Fragmentation of DNA components by hyperthermal heavy ion (Ar+ and Xe+) impact in the condensed phase  

NASA Astrophysics Data System (ADS)

The overriding environmental factor that presently limits human endeavors in space is exposure to heavy ion radiation. While knowledge of its damage to living tissue is essential for radiation protection and risk estimates for astronauts, very little data exists at the molecular level regarding the nascent DNA damage by the primary particle track, or by secondary species during subsequent reaction cascades. This persistent lack of a basic understanding of nascent damage induced by such low dose, high LET radiation, introduces unacceptable errors in radiation risk estimates (based mainly on extrapolation from high dose, low LET radiation), particularly for long term exposure. Mutagenic effects induced by heavy ion radiation to cells are largely due to DNA damage by secondary transient species, i.e. secondary ballistic ions, electrons and radicals generated along the ion tracks; the secondary ions have hyperthermal energies up to several 100 eV, which they will deposit within a few nm in the surrounding medium; thus their LET is very high, and yields lethal clustered DNA lesions. We present measurements of molecular damage induced in films of DNA components by ions with precisely such low energies (1-100 eV) and compare results to conventional electron impact measurements. Experiments are conducted in UHV using a mass selected low energy ion source, and a high-resolution quadrupole MS to monitor ion yields desorbing from molecular films. Among the major fragments, NH4 + is identified in the desorption mass spectra of irradiated films of Adenine, Guanine, Cytosine, indicating efficient deamination; in cells this results in pre-mutagenic lesions. Experiments with 5-amino-Uracil, and comparison to previous results on uracil and thymine show that deamination is a key step in the NH4 + fragment formation. For Adenine, we also observe formation of amine aducts in the films, viz. amination of Adenine, and global fragmentation in all ion impact mass spectra, attributed mainly to kinetic & potential ion scattering.[Funded by NSERC and the Canadian Space Agency].

Sarabipour, Sarvenaz; Sarvenaz Sarabipour, Ms; Michaud, Marc; Deng, Zongwu; Huels, Michael A.


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



One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome  

Microsoft Academic Search

We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of

Daniel G. Gibson; Gwynedd A. Benders; Kevin C. Axelrod; Jayshree Zaveri; Mikkel A. Algire; Monzia Moodie; Michael G. Montague; J. Craig Venter; Hamilton O. Smith; Clyde A. Hutchison



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.

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



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

SciTech Connect

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

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



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

Microsoft Academic Search

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

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



Structural and Thermodynamic Properties of Amyloid-? Peptides: Impact of Fragment Size  

NASA Astrophysics Data System (ADS)

Alzheimer's disease is a progressive neurodegenerative disease whose physiological characteristics include the accumulation of amyloid-containing deposits in the brain and consequent synapse and neuron loss. Unfortunately, most widely used drugs for the treatment can palliate the outer symptoms but cannot cure the disease itself. Hence, developing a new drug that can cure it. Most recently, the ``early aggregation and monomer'' hypothesis has become popular and a few drugs have been developed based on this hypothesis. Detailed understanding of the amyloid-? peptide structure can better help us to determine more effective treatment strategies; indeed, the structure of Amyloid has been studied extensively employing experimental and theoretical tools. Nevertheless, those studies have employed different fragment sizes of Amyloid and characterized its conformational nature in different media. Thus, the structural properties might be different from each other and provide a reason for the existing debates in the literature. Here, we performed all-atom MD simulations and present the structural and thermodynamic properties of A?1-16, A?1-28, and A?1-42 in the gas phase and in aqueous solution. Our studies show that the overall structures, secondary structures, and the calculated thermodynamic properties change with increasing peptide size. In addition, we find that the structural properties of those peptides are different from each other in the gas phase and in aqueous solution.

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



[Enhancer activity of DNA fragments from FXYD5-COX7A region of human chromosome 19].  


The enhancer activity of four previously identified within the one megabase region of human chromosome 19 DNA fragments was investigated. All four fragments had similar tissue-specific profile--maximum of enhancer activity was observed in HEK293 and minimum in HeLa cells. Enhancers obtained had pronounced specificity toward cytomegalovirus promoter compared with SV40 promoter. Functional dissection of one of the fragments (enhancer 14) demonstrated that only its inner 127 b.p. part possessed enhancer activity. The negative regulators, i.e. silencers or insulators are probably located in flanking regions of enhancer 14 and limit its effect on promoter. At the same time, enhancer activity of enhancer 14 depends on its orientation relative to promoter that isn't typical to majority of enhancer elements. Inner 127 b.p. fragment contains 11 transcription factor binding sites; 8 of them are factors that take part in immune system regulation. Enhancer 14 is located 500 b.p. upstream of transcription start site of TYROBP (DAP12) gene that codes for of T-killer cells activator protein and possibly functions as tissue-specific enhancer for this gene. PMID:21721257

Sorotokina, A N; Chernov, I P; Stukacheva, E A; Nikolaev, L G; Sverdlov, E D


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.



Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome.  


Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality. PMID:23940385

Simões, Renata; Feitosa, Weber Beringui; Siqueira, Adriano Felipe Perez; Nichi, Marcilio; Paula-Lopes, Fabíola Freitas; Marques, Mariana Groke; Peres, Maria Angélica; Barnabe, Valquíria Hyppolito; Visintin, José Antônio; Assumpção, Mayra Elena Ortiz



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



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


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

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



Genome size and the proportion of repeated nucleotide sequence DNA in plants  

Microsoft Academic Search

The reannealing kinetics of denatured DNA fragments from 23 species of higher plants have been studied, using hydroxylapatite chromatography to distinguish reannealed from single-stranded DNA. The 2C nuclear DNA contents of the species varied between 1.7 and 98 pg. The proportions of DNA in species with a nuclear DNA mass above 5 pg that reannealed with the kinetics of sequences

R. B. Flavell; M. D. Bennett; J. B. Smith; D. B. Smith



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

PubMed Central

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

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



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

PubMed Central

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

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



Malonaldehyde formation and DNA fragmentation: two independent sperm decays linked to reactive oxygen species.  


Malondialdehyde (MDA), a product involved in membrane lipid peroxidation, was dosed in the sperm of 163 patients who had consulted the clinic regarding hypofertility. We attempted to determine if there was correlation between MDA content, sperm World Health Organization parameters and DNA fragmentation that results mainly from reactive oxygen species assaults. We found that no correlation could be established; however MDA and sperm decondensation were shown to be significantly linked. The impact of membrane polyunsaturated fatty acids and the role of phospholipid hydroperoxide glutathione peroxidase are discussed. PMID:20331908

Montjean, Debbie; Ménézo, Yves; Benkhalifa, Moncef; Cohen, Marc; Belloc, Stephanie; Cohen-Bacrie, Paul; de Mouzon, Jacques



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



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

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



Increased Aneuploidy Rate in Sperm With Fragmented DNA as Determined by the Sperm Chromatin Dispersion (SCD) Test and FISH Analysis  

Microsoft Academic Search

Previous studies suggest that sperm DNA fragmen- tation may be associated with aneuploidy. However, currently available tests have not made it possible to simultaneously perform DNA fragmentation and chromosomal analyses on the same sperm cell. The recently introduced sperm chromatin dispersion (SCD) test allows users to determine this relationship. Semen samples from 16 males, including 4 fertile donors, 7 normozoospermic,




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



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

Microsoft Academic Search

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

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



The JAM Test and its daughter P-JAM: simple tests of DNA fragmentation to measure cell death and stasis  

Microsoft Academic Search

Cytotoxic T lymphocytes, and other death-inducing agents, have at least two different ways of killing their targets: drilling holes in the target cell membrane, or triggering the targets to commit suicide. The JAM Test is a method that measures the DNA fragmentation that accompanies cell suicide. We label target cells with radioactive DNA-precursor nucleotides and harvest them onto fiberglass filters,

David Usharauli; Ainhoa Perez-Diez; Polly Matzinger



Novel Approach to the Ligation of Single-Stranded DNA Fragments by T4 DNA Ligase—DNA Mobile Multiple-Restriction Fragments: “UNI-LINKERS” for Cloning of Genes  

Microsoft Academic Search

A group of uniquely designed single-stranded oligodeoxyribo-nucleotides that form hairpin loops were synthesized. These oligonucleotides can be ligated to other synthetic single-stranded fragents differing in length and design without the need for external annealing templates. A novel approach to building a limitless variety of mobile multiple-restriction DNA fragments termed “uni-linkers” and which are open only at one end, is described.

Fawzy Georges; Ravindra N. Chibbar; W. Jay Newsted; Friedrich Constabel



Relating fragmentation energy to grain-size, insights from triaxial glass deformation  

NASA Astrophysics Data System (ADS)

Natural glass can occur as an essential component to most volcanic rock types (e.g., pyroclastic vs. coherent), can form in all natural environments (submarine vs. subaerial, extrusive vs. intrusive), and can span nearly the complete spectrum of natural melt compositions. The formation of these glass-bearing volcanic rocks commonly occurs at elevated confining pressures and temperatures and under substantial deviatoric stresses. It is therefore surprising that the mechanical properties of volcanic glasses remain relatively unstudied. We have initiated an experimental program to recover the mechanical and rheological properties of synthetic and natural glasses as a function of confining pressure, strain rate, and temperature. Understanding of these properties will allow us to better constrain volcanic processes that ultimately lead to glass formation and the behaviour of the resulting materials under the dynamic conditions that they are exposed to. This experimental work documents the mechanical behaviour of synthetic glass (Pyrex) driven to failure under compressive triaxial stress. We perform room temperature experiments at confining pressures from 0.1 to 100 MPa with the intention of quantifying the storage and release of elastic energy in glasses. For all samples the fragmented material produced by the stress drop is recovered and its particle size distribution is analyzed and related to the energy release. Further we present preliminary results of a limited set of experiments on cores of natural and thermally treated, macroscopically homogenous portions of obsidian and discuss their significance in recovering volcanic processes.

Kolzenburg, S.; Russell, K.; Kennedy, L.



Effect of leukocytospermia and processing by discontinuous density gradient on sperm nuclear DNA fragmentation and mitochondrial activity  

Microsoft Academic Search

Purpose  To assess the effect of leukocytospermia and semen processing on sperm DNA and mitochondria.\\u000a \\u000a \\u000a \\u000a Methods  Twenty-two patients with and 41 without leukocytospermia were included. Sperm DNA fragmentation was assessed by the Comet\\u000a assay, and mitochondrial activity by a colorimetric method for active mitochondria. Semen was processed using Percoll, and\\u000a motility, DNA fragmentation, and mitochondrial activity were analyzed pre- and post-processing.\\u000a \\u000a \\u000a \\u000a Results  No

Roberta Maria Fariello; Paula Toni Del Giudice; Deborah Montagnini Spaine; Renato Fraietta; Ricardo Pimenta Bertolla; Agnaldo Pereira Cedenho



Brownian dynamics simulation of probe diffusion in DNA: effects of probe size, charge and DNA concentration.  


We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model. PMID:17023334

Dwyer, J D; Bloomfield, V A



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


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

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



Phenotypic Changes in Cyprinus carpiovar var. Jian Introduced by Sperm-Mediated Transgenesis of Rearranged Homologous DNA Fragments.  


Common carp, specifically the Jian variety (Cyprinus carpiovar var. Jian), is an important Chinese and global aquatic stock for commercial foodstuff. Homologous recombination of carp gene sequences has been widely used in population genetics to broadly screen for beneficial phenotypical variations, thus optimizing artificially engineered carp stocks with Jian variety and native stock varieties. Random rearrangement of homologous DNA fragments from parent specimens of C. carpiovar var. Jian were attained by digestion of genomic DNA with MspI followed by religation and redigestion with EcoR I to specifically rearrange homologous DNA fragments of myostatin and microsatellite genes. Based on known characteristics of myostatin gene function, growth pattern changes in resultant carp mutant varieties was expected. DNA fragments were introduced into metaphase-II oocytes, resulting in one to several dozen insertions of homologous fragments into the host genome by sperm-mediated transgenesis. Introduction of rearranged homologous DNA fragments often resulted in phenotypic changes in C. carpiovar var. Jian, including significant phenotypic changes linked to growth rate at 4 months. PMID:23824532

Cao, Zheming; Ding, Weidong; Ren, Hongtao



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:



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

PubMed Central

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 preparations of high concentrations of relatively small, sub-genome length DNA in preparations not treated with Eco81I. Furthermore, upon Eco81I digestion, the alkaline nuclease knockout bacmid also yielded substantially more sub genome 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; Vanarsdall, Adam L.; Rohrmann, George F.



Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.  


The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to control both the position and rate of mutation in degenerated region of the primers. The primers are integrated into newly synthesized plasmid DNA by primer extension reaction using Pfu DNA polymerase. After plasmid DNA template encoding wild-type sequence is eliminated from the reaction by DpnI digestion, the pool of mutagenized plasmids can then be used directly in screening procedures. PMID:15153637

Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth



DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)  

PubMed Central

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO1, leprosy remains endemic in many countries with approximately 250,000 new cases each year.2 The entire M. leprae genome has been mapped3,4 and many loci have been identified that have repeated segments of 2 or more base pairs (called micro- and minisatellites).5 Clinical strains of M. leprae may vary in the number of tandem repeated segments (short tandem repeats, STR) at many of these loci.5,6,7 Variable number tandem repeat (VNTR)5 analysis has been used to distinguish different strains of the leprosy bacilli. Some of the loci appear to be more stable than others, showing less variation in repeat numbers, while others seem to change more rapidly, sometimes in the same patient. While the variability of certain VNTRs has brought up questions regarding their suitability for strain typing7,8,9, the emerging data suggest that analyzing multiple loci, which are diverse in their stability, can be used as a valuable epidemiological tool. Multiple locus VNTR analysis (MLVA)10 has been used to study leprosy evolution and transmission in several countries including China11,12, Malawi8, the Philippines10,13, and Brazil14. MLVA involves multiple steps. First, bacterial DNA is extracted along with host tissue DNA from clinical biopsies or slit skin smears (SSS).10 The desired loci are then amplified from the extracted DNA via polymerase chain reaction (PCR). Fluorescently-labeled primers for 4-5 different loci are used per reaction, with 18 loci being amplified in a total of four reactions.10 The PCR products may be subjected to agarose gel electrophoresis to verify the presence of the desired DNA segments, and then submitted for fluorescent fragment length analysis (FLA) using capillary electrophoresis. DNA from armadillo passaged bacteria with a known number of repeat copies for each locus is used as a positive control. The FLA chromatograms are then examined using Peak Scanner software and fragment length is converted to number of VNTR copies (allele). Finally, the VNTR haplotypes are analyzed for patterns, and when combined with patient clinical data can be used to track distribution of strain types.

Jensen, Ronald W.; Rivest, Jason; Li, Wei; Vissa, Varalakshmi



Cloning and Mapping of Bam HI Endonuclease Fragments of DNA from the Transforming B95-8 Strain of Epstein-Barr Virus  

Microsoft Academic Search

DNA from the B95-8 strain of Epstein-Barr virus was cleaved into 29 different fragments by BamHI endonuclease (EC All of the fragments except the terminal fragments have been inserted into the pBR322 cloning vector and replicated in Escherichia coli. The location of each cloned DNA fragment in the viral genome has been determined, providing a more detailed physical map

James Skare; Jack L. Strominger



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.

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



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


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

Dastsooz, Hassan; Vahedi, Nazanin; Fardaei, Majid



Colloidal centrifugation of stallion semen results in a reduced rate of sperm DNA fragmentation.  


Stallion spermatozoa recovered and examined immediately after colloidal centrifugation resulted in a higher straight-line velocity (VSL) than sperm processed using direct conventional centrifugation (p = 0.000), but there was no differences in the progressive motility or sperm DNA fragmentation (SDF) as determined by the sperm chromatin dispersion assay. However, when centrifuged spermatozoa were incubated at 37 °C for 24 h to determine the rate of SDF (r-SDF), a lower r-SDF (p = 0.0011) was observed in those sperm recovered after colloidal separation (0.5 ± 0.1%/h) compared to direct (1.2 ± 0.4%/h) or no centrifugation (r-SDF = 1.2 ± 0.3%/h). These results confirm that colloidal separation of stallion spermatozoa results in prolonged sperm DNA longevity, but these differences were only apparent following a period of incubation and dynamic assessment. Consequently, we strongly recommend the use of the dynamic form of the SDF assay for evaluating centrifugation and/or other ex vivo procedures, as a single basal assessment of SDF may inadvertently result in a false-positive evaluation of DNA quality. PMID:22775967

Crespo, F; Gosalvez, J; Gutiérrez-Cepeda, L; Serres, C; Johnston, S D



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)



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

PubMed Central

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

Dastsooz, Hassan; Vahedi, Nazanin; Fardaei, Majid



A DNA prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge.  


The present study was undertaken to determine immune response and protection efficacy of a spike (S) protein fragment containing neutralizing epitopes (4F/4R) of turkey coronavirus (TCoV) by priming with DNA vaccine and boosting with the recombinant protein from the corresponding DNA vaccine gene segment. Turkeys were vaccinated by priming with either one dose (G1-750DP) or two doses (G3-750DDP) of 750?g DNA vaccine expressing 4F/4R S fragment and boosting with one dose of 200?g 4F/4R S fragment. One dose of 100?g DNA vaccine mixed with polyethyleneimine (PEI) and sodium hyaluronate (HA) followed by one dose of 750?g DNA vaccine and one dose of 200?g 4F/4R S fragment were given to the turkeys in group G2-100DPH. After infectious challenge by TCoV, clinical signs and TCoV detected by immunofluorescence antibody (IFA) assay were observed in less number of turkeys in vaccination groups than that in challenge control groups. TCoV viral RNA loads measured by quantitative real-time reverse transcription-PCR were lower in vaccinated turkeys than those in challenge control turkeys. The turkeys in G3-750DDP produced the highest level of TCoV S protein-specific antibody and virus neutralization (VN) titer. Comparing to the turkeys in G1-750DP, significantly less TCoV were detected by IFA in the turkeys in G2-100DPH receiving an extra dose of 100?g DNA mixed with PEI and HA. The results indicated that DNA-prime protein-boost DNA vaccination regimen targeting TCoV S fragment encompassing neutralizing epitopes induced humoral immune response and partially protected turkeys against infectious challenge by TCoV. PMID:23428360

Chen, Yi-Ning; Wu, Ching Ching; Yeo, Yoon; Xu, Peisheng; Lin, Tsang Long



One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid  

PubMed Central

A universal method to reconstitute sets of genes was developed. Owing to the intrinsic nature of the plasmid establishment mechanism in Bacillus subtilis, the assembly of five antibiotic resistance genes with a defined order and orientation was achieved. These five fragments and the plasmid have three-base protruding sequences at both ends. The protruding sequences are designed so that each fragment is ligated once in a row according to the pairing. Ligation by T4 DNA ligase in the presence of 150 mM NaCl and 10% polyethylene glycol at 37°C yielded high molecular tandem repeat linear form DNA. This multimeric form of DNA was preferentially used for plasmid establishment in B.subtilis. The method, referred to as Ordered Gene Assembly in B.subtilis (OGAB), allows for the design of multiple fragments with very high efficiency and great fidelity.

Tsuge, Kenji; Matsui, Kuniko; Itaya, Mitsuhiro



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




Visualization of elementary DNA replication units in human nuclei corresponding in size to DNA loop domains  

Microsoft Academic Search

Newly synthesized DNA in mammalian nuclei is concentrated in discrete nuclear granules called replication foci. These foci may be visualized using antibodies against 5-bromodeoxyuridine. In the early S-phase cells 100–250 foci are usually detected. On average, individual foci range between 0.5 and 2 µm in diameter and can be seen as clusters of more than ten average-sized (60–100 kb) synchronously

Nikolai Tomilin; Ljudmila Solovjeva; Raisa Krutilina; Claude Chamberland; Ronald Hancock; Baldev Vig



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

NASA Astrophysics Data System (ADS)

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

Bindeman, I.



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


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

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



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


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



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


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

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



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

SciTech Connect

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

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



Efficient replication bypass of size-expanded DNA base pairs in bacterial cells.  


Supersize me! Size-expanded DNA bases (xDNA) are able to encode natural DNA sequences in replication. In vitro experiments with a DNA polymerase show nucleotide incorporation opposite the xDNA bases with correct pairing. In vivo experiments using E. coli show that two xDNA bases (xA and xC, see picture) encode the correct replication partners. PMID:19444841

Delaney, James C; Gao, Jianmin; Liu, Haibo; Shrivastav, Nidhi; Essigmann, John M; Kool, Eric T



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.

Iglesias-Guimarais, Victoria; Gil-Guinon, Estel; Gabernet, Gisela; Garcia-Belinchon, Merce; Sanchez-Osuna, Maria; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.



Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease.  


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



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

Microsoft Academic Search

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,\\u000a 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\\u000a was seen in STE, but several novel bands appeared in TE

Takashi Abe; Hiroyoshi Takano; Narie Sasaki; Kimie Mori; Shigeyuki Kawano



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.



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

SciTech Connect

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

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



Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets  

Microsoft Academic Search

The present study tests the hypothesis that nitric oxide mediates the hypoxia-induced increase in expression of Bax and in DNA fragmentation in the cerebral cortex of newborn piglets, and that administration of N-nitro-l-arginine (NNLA), a nitric oxide synthase inhibitor, will prevent a change in hypoxia-induced expression of apoptotic genes and DNA damage. Piglets were assigned to normoxic, hypoxic, or NNLA-pretreated

Alan B Zubrow; Maria Delivoria-Papadopoulos; Qazi M Ashraf; Juan R Ballesteros; Karen I Fritz; Om P Mishra



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

Microsoft Academic Search

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

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



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

PubMed Central

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy- transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.



A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea.  


Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG. This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L. interrogans which crossreacts with equine cornea as proved Western-blotting. Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum. Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis. PMID:10587299

Lucchesi, P M; Parma, A E



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


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

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



Fragment charge difference method for estimating donor-acceptor electronic coupling: Application to DNA ?-stacks  

NASA Astrophysics Data System (ADS)

The purpose of this communication is two-fold. We introduce the fragment charge difference (FCD) method to estimate the electron transfer matrix element HDA between a donor D and an acceptor A, and we apply this method to several aspects of hole transfer electronic couplings in ?-stacks of DNA, including systems with several donor-acceptor sites. Within the two-state model, our scheme can be simplified to recover a convenient estimate of the electron transfer matrix element HDA=(1-?q2)1/2(E2-E1)/2 based on the vertical excitation energy E2-E1 and the charge difference ?q between donor and acceptor. For systems with strong charge separation, ?q>~0.95, one should resort to the FCD method. As favorable feature, we demonstrate the stability of the FCD approach for systems which require an approach beyond the two-state model. On the basis of ab initio calculations of various DNA related systems, we compared three approaches for estimating the electronic coupling: the minimum splitting method, the generalized Mulliken-Hush (GMH) scheme, and the FCD approach. We studied the sensitivity of FCD and GMH couplings to the donor-acceptor energy gap and found both schemes to be quite robust; they are applicable also in cases where donor and acceptor states are off resonance. In the application to ?-stacks of DNA, we demonstrated for the Watson-Crick pair dimer [(GC),(GC)] how structural changes considerably affect the coupling strength of electron hole transfer. For models of three Watson-Crick pairs, we showed that the two-state model significantly overestimates the hole transfer coupling whereas simultaneous treatment of several states leads to satisfactory results.

Voityuk, Alexander A.; Rösch, Notker



Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens).  


A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment-length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist. PMID:2895887

Quinn, T W; White, B N



Size and Structure of Replicating Mitochondrial DNA in Cultured Tobacco Cells  

Microsoft Academic Search

The BY-2 tobacco cell line was used to study the size and structure of replicating mitochondrial DNA (mtDNA). Approxi- mately 70 to 90% of the newly synthesized mtDNA did not migrate during pulsed-field gel electrophoresis. Moving pictures of the fluorescently labeled molecules showed that most of the immobile well-bound DNA was in structures larger than the size of the BY-2

Delene J. Oldenburg; Arnold J. Bendich



Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen.  


EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection. PMID:16833086

Sang, Fuming; Ren, Jicun



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



Coagulation and fragmentation: Universal steady-state particle-size distribution  

Microsoft Academic Search

A population balance model presented describes simultaneous coagulation and frag- mentation during shear-iaduced flocculation. Given sufficient time, a floc-size distribu- tion reaches steady state that reflects the balance between coagulation and fragmenta- tion. The model agrees with experimental data for the evolution of the average floc size. Higher shear shifts the steady-state size distribution to smaller sizes. When the steady-state

Patrick T. Spicer; Sotiris E. F'ratsinis



Theoretical investigation of finite size effects at DNA melting  

NASA Astrophysics Data System (ADS)

We investigated how the finiteness of the length of a sequence affects the phase transition that takes place at the DNA melting temperature. For this purpose, we modified the transfer integral method to adapt it to the calculation of both extensive (partition function, entropy, specific heat, etc.) and nonextensive (order parameter and average separation between paired bases) thermodynamic quantities of finite sequences with open boundary conditions, and applied the modified procedure to two different dynamical models. We characterized in some detail the three effects that take place when the length of the sequence is decreased, namely, (i) the decrease of the critical temperature, (ii) the decrease of the peak values of all quantities that diverge at the thermodynamic limit but remain finite for finite sequences, like the specific heat and the correlation length, and (iii) the broadening of the temperature range over which the transition affects the dynamics of the system. We also performed a finite size scaling analysis of the two models and showed that the singular part of the free energy can indeed be expressed in terms of a homogeneous function. However, Josephson’s identity is satisfied for none of the investigated models, so that the derivation of the characteristic exponents which appear, for example, in the expression of the specific heat requires some care.

Buyukdagli, Sahin; Joyeux, Marc



Genetic fingerprinting of grape plant (Vitis vinifera) using random amplified polymorphic DNA (RAPD) analysis and dynamic size-sieving capillary electrophoresis.  


Dynamic size-sieving capillary electrophoresis with laser-induced fluorescence detection (DSCE-LIF) was combined with random amplified polymorphic DNA (RAPD) analysis to demonstrate the feasibility of the genetic analysis of grape plant varieties and clones within a variety. Parameters of the genomic DNA extraction process, as well as those of the RAPD analysis, were optimized specifically for this application. Polymorphic DNA fragments were generated for four different grape plant varieties including Cabernet Franc, Cabernet Sauvignon, Merlot, and Chardonnay. Relative to slab gel electrophoresis (SGE) with ethidium bromide staining, DSCE-LIF provided superior separation efficiency and detection limits in the analysis of DNA polymorphic bands. Optimal DSCE-LIF analyses were achieved using a 10-fold RAPD sample dilution, hydrodynamic sample injection, and 100 ng/mL of YO-PRO-1 DNA intercalator in the dynamic size-sieving buffer solution. In addition, the reproducibility of both the DSCE-LIF and RAPD analyses were demonstrated. PMID:11312766

Siles, B A; O'Neil, K A; Fox, M A; Anderson, D E; Kuntz, A F; Ranganath, S C; Morris, A C



DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant.  

PubMed Central

A Lyt-2+, trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3H counts from target cells prelabeled with [3H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1+, ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery. Images

Schmid, D S; Tite, J P; Ruddle, N H



Plant Genome Size Measurement with DNA Image Cytometry  

Microsoft Academic Search

To test the reliability of DNA image cytometry for the measurement of nuclear DNA content in plant material, we conducted independent experiments in two laboratories using different image analysis instruments for densitometric measurement of nuclear DNA amount in Feulgen-stained squash preparations of root tips. The 2C nuclear DNA content of the nine species studied spanned a 100-fold range (approx. 0.3–33pg).

Barbara Vilhar; Johann Greilhuber; Jasna Dolenc Koce; Eva Maria Temsch; Marina Dermastia



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


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

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



Monoclonal antibody-based, selective isolation of DNA fragments containing an alkylated base to be quantified in defined gene sequences.  

PubMed Central

We have established a sensitive, monoclonal antibody (Mab)-based procedure permitting the selective enrichment of sequences containing the miscoding alkylation product O6-ethylguanine (O6-EtGua) from mammalian DNA. H5 rat hepatoma cells were reacted with the N-nitroso carcinogen N-ethyl-N-nitrosourea in vitro, to give overall levels of greater than or equal to 25 O6-EtGua residues per diploid genome (corresponding to O6-EtGua/guanine molar ratios of greater than or equal to 10(-8). For analysis, enzymatically restricted DNA from these cells is incubated with an antibody specific for O6-ethyl-2'-deoxyguanosine, the resulting Mab-DNA complexes are separated from (O6-EtGua)-free fragments by filtration through a nitrocellulose (NC) membrane, and the DNA is recovered from the filter-bound complexes quantitatively. The efficiency of Mab binding to DNA fragments containing O6-EtGua is constant over a range of O6-EtGua/guanine molar ratios between 10(-5) and 10(-8). (O6-EtGua)-containing restriction fragments encompassing known gene sequences (e.g., the immunoglobulin E heavy chain gene of H5 rat hepatoma cells used as a model in this study) are subsequently amplified by PCR and quantified by slot-blot hybridisation. The content and distribution of a specific carcinogen-DNA adduct in defined sequences of genomic DNA can thus be analyzed as well as the kinetics of intragenomic (toposelective) repair of any DNA lesion for which a suitable Mab is available.

Hochleitner, K; Thomale, J; Nikitin AYu; Rajewsky, M F



Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes.  


Carrot (Daucus carota L.) chromosomes are small and poorly differentiated in size and morphology. Here we demonstrate that fluorescent in situ hybridization (FISH) signals derived from arbitrary PCR probes can be used for chromosome identification in carrot. To prepare probes, we searched for nonpolymorphic products abundantly amplified with arbitrary decamer primers in a group of accessions representing carrot genetic diversity. As a result, 13 fragments ranging in size from 517 to 1758 bp were selected, sequenced, and used as probes for fluorescent in situ hybridization. Four of these probes produced clear and reproducible hybridization signals. The sequences showed similarity to a number of carrot BAC-end sequences, indicating their repetitive character. Three of them were similar to internal portions of gypsy and copia LTR retrotransposons previously identified in plants. Hybridization signals for the four probes were observed as dotted tracks on chromosomes, differing in distribution and intensity. Generally, they were present in pericentromeric and (or) interstitial localizations on chromosome arms. The use of the four probes allowed discrimination of chromosome pairs and construction of more detailed karyotypes and idiograms of carrot. PMID:22360760

Nowicka, Anna; Grzebelus, Ewa; Grzebelus, Dariusz



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


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

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



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

PubMed Central

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

Samarghandian, Saeed; Shabestari, Mahmoud M



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


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

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



Cloning and sequencing of RAPD fragments amplified from mitochondrial DNA of male-sterile and male-fertile cytoplasm of sugar beet (Beta vulgaris L.)  

Microsoft Academic Search

Mitochondrial DNA fragments of two nearly isogenic lines of sugar beet (Beta vulgaris L.) were amplified by RAPD analysis. A number of fragments, most of them unique to either the male-sterile or the male-fertile\\u000a cytoplasm, were selected for cloning and sequencing. One fragment was present in the PCR fingerprint pattern of both cytoplasms,\\u000a whereas five of the selected fragments were

M. Lorenz; A. Weihe; T. Börner



A Fuzzy Classifier to Taxonomically Group DNA Fragments within a Metagenome  

Microsoft Academic Search

Extracting microorganisms from their natural en- vironment has become a popular technique. These metagenomic fragments lack enough information that can mark them into taxonomic groups. In this paper, we implement a fuzzy k- means classifier to separate fragments into taxonomic groups present in a metagenomic data set. The fuzzy classifier is used to group shotgun sequence fragments as small as

Sara Nasser; Adrienne Breland; Frederick C. Harris Jr; Monica Nicolescu



Independence of fragment charge distributions of the size of heavy multifragmenting sources  

NASA Astrophysics Data System (ADS)

Charged product multiplicities and Z distributions were measured for single multifragmenting sources produced in collisions between 129Xe+natSn and 155Gd+238U at the same available energy per nucleon. Z distributions are found identical for both reactions while fragment multiplicities scale as the charge of the total systems. A complete dynamical simulation, in which multifragmentation originates in the spinodal decomposition of a finite piece of nuclear matter resulting from an incomplete fusion of projectile and target, well accounts for this experimental observation.

Rivet, M. F.; Bacri, Ch. O.; Borderie, B.; Frankland, J. D.; Assenard, M.; Auger, G.; Bocage, F.; Bougault, R.; Brou, R.; Buchet, Ph.; Chbihi, A.; Colin, J.; Dayras, R.; Demeyer, A.; Doré, D.; Durand, D.; Eudes, P.; Galichet, E.; Genouin-Duhamel, E.; Gerlic, E.; Germain, M.; Guinet, D.; Lautesse, P.; Laville, J. L.; Lecolley, J. F.; Le Fèvre, A.; Lefort, T.; Legrain, R.; Le Neindre, N.; Lopez, O.; Louvel, M.; Nalpas, L.; Nguyen, A. D.; Parlog, M.; Péter, J.; Plagnol, E.; Rahmani, A.; Reposeur, T.; Rosato, E.; Saint-Laurent, F.; Salou, S.; Squalli, M.; Steckmeyer, J. C.; Stern, M.; Tabacaru, G.; Tamain, B.; Tassan-Got, L.; Tirel, O.; Vintache, D.; Volant, C.; Wieleczko, J. P.; Guarnera, A.; Colonna, M.; Chomaz, P.



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

Microsoft Academic Search

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

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



DNA content and size of cell nuclei in the regenerating rat liver  

Microsoft Academic Search

The DNA content in single nuclei and the size of the nuclei were investigated in the intact and regenerating rat liver from 18 h to 21 days after partial hepatectomy. The results of the measurements show that the mean DNA content per nucleus in the intact rat liver is 6.5 pg, and that most nuclei are about equal in size

B. Fabianova; K. Kropacova; E. Misurova



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

SciTech Connect

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

Mathew, M.K.; Smith, C.L.; Cantor, C.R. (Columbia Univ., New York, NY (USA))



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


By use of the single-molecule observation, we count the number of DNA double-strand breaks caused by ?-ray irradiation with genome-sized DNA molecules (166 kbp). We find that P1, the number of double-strand breaks (DSBs) per base pair per unit Gy, is nearly inversely proportional to the DNA concentration above a certain threshold DNA concentration. The inverse relationship implies that the total number of DSBs remains essentially constant. We give a theoretical interpretation of our experimental results in terms of attack of reactive species upon DNA molecules, indicating the significance of the characteristics of genome-sized giant DNA as semiflexible polymers for the efficiency of DSBs. PMID:23656159

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



Carbon nanotube-enhanced separation of DNA fragments by a portable capillary electrophoresis system with contactless conductivity detection.  


It was demonstrated that separation of DNA fragments by a CE-contactless conductivity detection system (CE-CCD) could be enhanced with multiple-wall carbon nanotubes (MWCNs) as buffer additive. For HaeIII digest of PhiX174 DNA, optimized MWCN concentration was obtained when the MWCN was above its threshold concentration, at which MWCN could form a network in the buffer as pseudostationary phase to provide additional interaction sites. In the case of larger DNA, MWCN near or below its threshold concentration was enough to provide great improvement of the resolution, which was shown by the separation of the 2-Log DNA ladder. Furthermore, the buffer containing MWCN could provide a more stable baseline in the CE-CCD system, owing to less fluctuation of its conductivity. Compared with CE-UV, CE-CCD with MWCN could provide lower LODs as well as better resolution. PMID:16983641

Xu, Yan; Li, Sam Fong Yau



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

PubMed Central

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

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



Historic DNA reveals contemporary population structure results from anthropogenic effects, not pre-fragmentation patterns  

Microsoft Academic Search

Contemporary patterns of genetic structure among fragmented populations can either result from historic patterns or arise\\u000a from human-induced fragmentation. Use of historic samples collected prior to fragmentation allows for the origin of genetic\\u000a structure to be established and appropriate management steps to be determined. In this study, we compare historic and contemporary\\u000a levels of genetic diversity and structure of an

Lisa N. Tracy; Ian G. Jamieson



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

Microsoft Academic Search

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

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



Strain-rate effects on the strength and fragmentation size of rocks  

Microsoft Academic Search

Rock mass possesses a complex structural hierarchy. The size effect on the strength of geomedia is closely related to the structural hierarchy of rock mass. This paper demonstrates that the rock mass strength sensitivity to strain rate may be regarded as the result of competition between the coexisting thermally activated and macro-viscous mechanisms, which dominate at different ranges of strain

Chengzhi Qi; Mingyang Wang; Qihu Qian



Effective population sizes and migration rates in fragmented populations of an endangered insect (Coenagrion mercuriale: Odonata)  

Microsoft Academic Search

Summary 1. Effective population sizes ( N e ) and migration rates ( m ) are critical evolutionary parameters that impact on population survival and determine the relative influence of selection and genetic drift. While the parameter m is well-studied in animal populations, N e remains challenging to measure and consequently is only rarely estimated, particularly in insect taxa. 2.




Directly transforming PCR-amplified DNA fragments into plant cells is a versatile system that facilitates the transient expression assay.  


A circular plasmid containing a gene coding sequence has been broadly used for studying gene regulation in cells. However, to accommodate a quick screen plasmid construction and preparation can be time consuming. Here we report a PCR amplified dsDNA fragments (PCR-fragments) based transient expression system (PCR-TES) for suiting in the study of gene regulation in plant cells. Instead of transforming plasmids into plant cells, transient expression of PCR-fragments can be applicable. The transformation efficiency and expression property of PCR-fragments are comparable to transformation using plasmids. We analyzed the transformation efficiency in PCR-TES at transcription and protein levels. Our results indicate that the PCR-TES is as versatile as the conventional transformation system using plasmid DNA. Through reconstituting PYR1-mediated ABA signaling pathway in Arabidopsis mesophyll protoplasts, we were not only validating the practicality of PCR-TES but also screening potential candidates of CDPK family members which might be involved in the ABA signaling. Moreover, we determined that phosphorylation of ABF2 by CPK4 could be mediated by ABA-induced PYR1 and ABI1, demonstrating a crucial role of CDPKs in the ABA signaling. In summary, PCR-TES can be applicable to facilitate analyzing gene regulation and for the screen of putative regulatory molecules at the high throughput level in plant cells. PMID:23468926

Lu, Yuming; Chen, Xi; Wu, Yuxuan; Wang, Yanping; He, Yuqing; Wu, Yan



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

PubMed Central

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

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



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


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

Özdemirler Erata, Gül; Küçükgergin, Canan; Aktan, Gülsan; Kadioglu, Ates; Uysal, Müjdat; Koçak-Toker, Necla



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

PubMed Central

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 homologous 373 bp DNA fragments differing by two adjacent base pairs. Product DNAs were separated based on their thermal stability by parallel and perpendicular TGGE. The polyacrylamide gel contained 3.36 M urea and 19.2 % formamide to lower the DNA melting temperatures. The order of stability was determined in the sequence context d(CXYG).d(CY'X'G) where X.X' and Y.Y" represent the mismatched or Watson-Crick base pairs. The identity of the mismatched bases and their stacking interactions influence DNA stability. Mobility transition melting temperatures (T u) of the DNAs with adjacent mismatches were 1.0-3.6 degrees C (+/-0.2 degree C) lower than the homoduplex DNA with the d(CCAG).d(CTGG) sequence. Two adjacent G.A pairs, d(CGAG).d(CGAG), created a more stable DNA than DNAs with Watson-Crick A.T pairs at the same sites. The d(GA).d(GA) sequence is estimated to be 0.4 (+/-30%) kcal/mol more stable in free energy than d(AA).d(TT) base pairs. This result confirms the unusual stability of the d(GA).d(GA) sequence previously observed in DNA oligomers. All other DNAs with adjacent mismatched base pairs were less stable than Watson-Crick homoduplex DNAs. Their relative stabilities followed an order expected from previous results on single mismatches. Two homoduplex DNAs with identical nearest neighbor sequences but different next-nearest neighbor sequences had a small but reproducible difference in T u value. This result indicates that sequence dependent next neighbor stacking interactions influence DNA stability.

Ke, S H; Wartell, R M



Purified Escherichia coli recA Protein Catalyzes Homologous Pairing of Superhelical DNA and Single-Stranded Fragments  

Microsoft Academic Search

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl\\/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2%

Takehiko Shibata; Chanchal Dasgupta; Richard P. Cunningham; Charles M. Radding



High-Frequency Genetic Recombination and Reactivation of Orthopoxviruses from DNA Fragments Transfected into Leporipoxvirus-Infected Cells  

Microsoft Academic Search

and reactivation reactions could be used to delete nonessential regions of the vaccinia virus genome and to reconstruct vaccinia virus from overlapping DNA fragments. SFV-catalyzed recombination reactions need only two 18- to 20-bp homologies to target PCR amplicons to restriction enzyme-cut vaccinia virus vectors, and this reaction feature was used to rapidly clone and express a gene encoding fluorescent green

Xiao-Dan Yao; David H. Evans



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

Microsoft Academic Search

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

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



Transformation of Saccharomyces cerevisiae with plasmids containing fragments of yeast 2 ? DNA and a suppressor tRNA gene  

Microsoft Academic Search

Hybrid plasmids have been constructed containing segments of the yeast plasmid 2 µ DNA, the yeast ochre-suppressing SUP4.0 gene and the bacterial plasmid pBR322. Yeast transformation is detected with a host containing multiple ochre auxotrophic mutations. The transformed SUP4.0 gene is active and can promote growth in the absence of all the requirements. Plasmids containing different fragments of 2 µ

David Y. Thomas; Allen P. James



[Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate TLR-dependent signal pathway and supress apoptosis].  


Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter--MyD88--was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, ctDNA can significantly influence the functional activity ofMSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level. PMID:23350199

Kostiuk, S V; Malinovskaia, E M; Ermakov, A V; Smirnova, T D; Kameneva, L V; Chvartatskaia, O V; Loseva, P A; Ershova, E S; Liubchenko, L N; Ve?ko, N N


A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.  

PubMed Central

Saccharomyces cerevisiae centromeres contain a conserved region ranging from 111 to 119 base pairs (bp) in length, which is characterized by the three conserved DNA elements CDEI, CDEII, and CDEIII. We isolated a 125-bp CEN6 DNA fragment (named ML CEN6) containing only these conserved elements and assayed it completely separated from its chromosomal context on circular minichromosomes and on a large linear chromosome fragment. The results show that this 125-bp CEN6 DNA fragment is by itself sufficient for complete mitotic and meiotic centromere functions. Images

Cottarel, G; Shero, J H; Hieter, P; Hegemann, J H



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.



An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA.  


Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model. PMID:2720086

Antosiewicz, J; Porschke, D



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.

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



Land use vs. fragment size and isolation as determinants of small mammal composition and richness in Atlantic Forest remnants  

Microsoft Academic Search

The remaining Atlantic Forest fragments are structurally isolated by a matrix of pastures, plantations, or urban areas, and most remnants are small (<100ha). Island biogeography theory has been used to predict the effects of such fragmentation in the remaining fragments, but human activities and land use around fragments may be equally important. A related question is which aspects of land

Marcus V. Vieira; Natalie Olifiers; Ana C. Delciellos; Vanina Z. Antunes; Luis R. Bernardo; Carlos E. V. Grelle; Rui Cerqueira



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

NASA Astrophysics Data System (ADS)

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

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



The effect of chilled storage and cryopreservation on the sperm DNA fragmentation dynamics of a captive population of koalas.  


This experiment documented the incidence and variability of sperm characteristics found in freshly collected and ex vivo-manipulated semen samples from a population of disease-free captive koalas with a special emphasis on the dynamic aspects of DNA fragmentation. These changes were analyzed in light of the putative negative effect of iatrogenic damage after chilled storage and cryopreservation with respect to different semen extender compositions to maximize sperm longevity. Sperm DNA fragmentation (SDF) dynamics (SDF assessment after a varying period of time) was investigated with the sperm chromatin dispersion assay after either dilution in tris-citrate media and chilled preservation at 4°C for upward of 16 days or cryopreservation in either glycerol or dimethylacetamide (DMA) tris-citrate-based cryoprotectant media; corresponding data on progressive sperm motility, plasma membrane integrity, and the proportion of koala sperm with relaxed chromatin were also recorded. SDF analysis of the captive koala population revealed a low mean (± SEM) basal level of only 6.7% ± 0.9%. The percentage of progressive sperm motility, percentage of intact sperm plasma membranes, and the percentage of relaxed chromatin did not correlate significantly with that of basal SDF. Moreover, despite the absence of cysteine residues in marsupial protamines, koala spermatozoa showed remarkable stability in terms of their DNA integrity after the incubation of either fresh, chilled, or frozen-thawed semen samples; observations of progressive motility (P < .05) and plasma membrane integrity (P < .05) revealed that chilled koala spermatozoa declined after 4 days, whereas the incidence of relaxed chromatin increased after 8 days. Although koala SDF increased significantly (P < .05) with the period of chilled storage, these values remained less than 16% after 16 days storage and subsequent incubation at 35°C for a further 48 hours. Survivorship of prefreeze sperm DNA damage was not different when compared with sperm frozen in DMA or between sperm frozen in DMA or glycerol; however, spermatozoa frozen in glycerol showed a higher (P = .042) rate of DNA fragmentation than prefreeze spermatozoa. This result differed from that of observations of progressive motility, plasma membrane integrity, and relaxed chromatin, which were all adversely affected (P < .05) after cryopreservation in either glycerol or DMA; however, the postthaw characteristics of sperm cryopreserved in either glycerol or DMA were not different. After thawing, koala sperm chromatin tended to decondense; however, the incidence of sperm DNA fragmentation was not correlated with the incidence of sperm chromatin relaxation after glycerol (R = .2) or DMA (R = -.04) cryopreservation. PMID:22282436

Johnston, Stephen D; Zee, Yeng Peng; López-Fernández, Carmen; Gosálvez, Jamie



[New protein vectors based on an alpha-fetoprotein fragment for targeted DNA delivery into cancer cells].  


A human alpha-fetoprotein fragment (AFP) modified with oligocationic homologs of nuclear localization signal was used to construct new target cell-selective DNA-carrier proteins. The new recombinant vectors containing C- or N-terminal polynucleotide-binding domains are able to form stable complexes with single- or double-stranded oligonucleotides and plasmid DNA. Using flow cytometry and fluorescent microscopy, it was shown that such nucleoprotein complexes can be selectively internalized in target cells receptors superexpressing AFP receptors. The results obtained are important both for understanding mechanisms of formation of DNA-protein complexes and for studying their interaction with intracellular molecular targets. The new proteins can be used as a tool for the development of highly selective and efficacious gene-selective antitumour drugs. PMID:20408431

Tatarinova, O N; Gorokhovets, N V; Makarov, V A; Posypanova, G A; Serebriakova, M V; Pozmogova, G E



Is sperm DNA fragmentation a good marker for field AI bull fertility?  


This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success. PMID:22367070

Karoui, S; Díaz, C; González-Marín, C; Amenabar, M E; Serrano, M; Ugarte, E; Gosálvez, J; Roy, R; López-Fernández, C; Carabaño, M J



Proof of Principle In Vitro Study of a Prototype Ultrasound Technology to Size Stone Fragments During Ureteroscopy  

PubMed Central

Abstract Purpose Proof-of-principle in vitro experiments evaluated a prototype ultrasound technology to size kidney stone fragments. Materials and Methods Nineteen human stones were measured using manual calipers. A 10-MHz, 1/8? (10F) ultrasound transducer probe pinged each stone on a kidney tissue phantom submerged in water using two methods. In Method 1, the instrument was aligned such that the ultrasound pulse traveled through the stone. In Method 2, the instrument was aligned partially over the stone such that the ultrasound pulse traveled through water. Results For Method 1, the correlation between caliper- and ultrasound-determined stone size was r2?=?0.71 (P?size with good accuracy and precision. This technology may be possible to incorporate into ureteroscopy.

Teichman, Joel M.H.; Bailey, Michael R.



Origin of replication in chloroplast DNA of Euglena gracilis located close to the region of variable size  

PubMed Central

Chloroplast DNA (cpDNA), containing ˜10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I2 (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for ˜5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence. ImagesFig. 1.

Koller, Barbara; Delius, Hajo



Origin of replication in chloroplast DNA of Euglena gracilis located close to the region of variable size.  


Chloroplast DNA (cpDNA), containing 10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I(2) (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for 5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence. PMID:16453429

Koller, B; Delius, H



Electroporation of Alcaligenes eutrophus with (mega) plasmids and genomic DNA fragments.  

PubMed Central

Electroporation was used as a tool to explore the genetics of the heavy-metal-resistant strain Alcaligenes eutrophus CH34. A 12.9-kb A. eutrophus-Escherichia coli shuttle vector, pMOL850, was constructed to optimize electroporation conditions. This vector is derived from the E. coli plasmid pSUP202 and contains the replication region of the A. eutrophus megaplasmid pMOL28. Electroporation was used to transform A. eutrophus CH34 derivatives with megaplasmids (sizes up to 240 kb), and transformants were selected for resistance to heavy metals. Electroporation was also performed with endonuclease-digested genomic DNA. Transformation of markers affecting lysine biosynthesis (lysA194) and biosynthesis of the siderophore alcaligin E were observed. Transfer of the nonselected markers pheB332 and aro-333, linked to lysA194, confirmed the intervention of homologous recombination. However, during transformation of ale::Tn5-Tc, illegitimate recombination and transposition were also observed as an alternative for the inheritance of the Tn5-Tc markers. Images

Taghavi, S; van der Lelie, D; Mergeay, M



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



Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21. 3) by cDNA hybridization selection  

SciTech Connect

It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. 39 refs., 4 figs., 3 tabs.

Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. (Yale Univ. School of Medicine, New Haven, CT (United States))



Mitochondrial dna restriction fragment length polymorphisms in fusarium oxysporum f. sp. niveum  

Microsoft Academic Search

Mitochondrial DNA (mtDNA) extracts from 13 isolatesof Fusarium oxysporum f. sp.niveum, including 12 from widely separated geographic regions within the United States and representing the three races, and one\\u000a race 2 isolate from Israel, were examined for the presence of plasmid DNA and were also subjected to restriction endonucleases\\u000a analysis. None of the mtDNA from any isolate had a copurifying

D. H. kim; R. D. martyn; C. W. magill



Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria  

Microsoft Academic Search

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteo- bacteria ( and ), Bacteroidetes, and high-GC gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open

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



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


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

Zheng, Hao; Wu, Hongwei



The utility of mitochondrial DNA fragments for genetic identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China.  


Species-diagnostic anatomical characters of fleshflies are not known for most immature stages or even adults, and an existing key may be incomplete or difûcult for nonspecialists to use. The use of sarcophagids for PMI estimations has been greatly hampered by their highly similar morphological characters. DNA-based method can be used as a supplemental means of morphological method in identification of forensically important sarcophagid flies. However, relying solely on single DNA fragment for delimiting species is considered to be unreliable, especially when the fragment was small. Sequence data of selected regions of the cytochrome oxidase subunit two (COII) and 16S ribosomal RNA (16S rRNA) genes of the most important Chinese fleshfly taxa associated with cadavers are presented, which can be instrumental for implementation of the Chinese Sarcophagidae database. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into five species, which indicated the possibility of separation congeneric species with the short fragments. PMID:22543602

Guo, Y D; Cai, J F; Xiong, F; Wang, H J; Wen, J F; Li, J B; Chen, Y Q



Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.  

PubMed Central

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.

Jin, H; Ren, Z; Pozsgay, J M; Elkins, C; Whitby, P W; Morton, D J; Stull, T L



Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep  

Microsoft Academic Search

Objective: To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols.Design: Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique.Setting: Fertility clinic in a large university hospital.Animals: Five- to 6-month-old lambs.Intervention(s): Two-millimeter-thick slices of

Banu Demirci; Bruno Salle; Lucien Frappart; Michel Franck; Jean François Guerin; Jacqueline Lornage



Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids  

Microsoft Academic Search

A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the

Peter Zahm; Christine Hohmeyer; Klaus Geider



Size selective DNA transport through a nanoporous membrane in a PDMS microfluidic device.  


A microfluidic counter current dialysis device for size based purification of DNA is described. The device consists of two polydimethylsiloxane (PDMS) channels separated by a track etched polycarbonate membrane with a 50 nm pore size. Recovery of fluorescein across the membrane was compared with 10 and 80 nucleotide (nt) ssDNA to characterize the device. Recovery of all three analytes improved with decreasing flow rate. Size selectivity was observed. Greater than 2-fold selectivity between 10 nt and 80 nt ssDNA was observed at linear velocities less than 3mm s(-1). Increasing the ionic strength of the buffer increased transport across the membrane. Recovery of 80 nt ssDNA increased over 4-fold by adding 30 mM NaCl to the buffer. The effect was size dependent as 10 nt showed a smaller increase while the recovery of fluorescein was largely unaffected by increasing the ionic strength of the buffer. PMID:22262059

Sheng, Yixiao; Bowser, Michael T



High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis  

Microsoft Academic Search

We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3? recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end.

Ming-Cheng Luo; Carolyn Thomas; Frank M You; Joseph Hsiao; Shu Ouyang; C. Robin Buell; Marc Malandro; Patrick E McGuire; Olin D Anderson; Jan Dvorak



A fragment of chloroplast DNA was transferred horizontally, probably from non-eudicots, to mitochondrial genome of Phaseolus.  


The mitochondrial genomes of some Phaseolus species contain a fragment of chloroplast trnA gene intron, named pvs-trnA for its location within the Phaseolus vulgaris sterility sequence (pvs). The purpose of this study was to determine the type of transfer (intracellular or horizontal) that gave rise to pvs-trnA. Using a PCR approach we could not find the respective portion of the trnA gene as a part of pvs outside the Phaseolus genus. However, a BLAST search revealed longer fragments of trnA present in the mitochondrial genomes of some Citrus species, Helianthus annuus and Zea mays. Basing on the identity or near-identity between these mitochondrial sequences and their chloroplast counterparts we concluded that they had relocated from chloroplasts to mitochondria via recent, independent, intracellular DNA transfers. In contrast, pvs-trnA displayed a relatively higher sequence divergence when compared with its chloroplast counterpart from Phaseolus vulgaris. Alignment of pvs-trnA with corresponding trnA fragments from 35 plant species as well as phylogenetic analysis revealed that pvs-trnA grouped with non-eudicot sequences and was well separated from all Fabales sequences. In conclusion, we propose that pvs-trnA arose via horizontal transfer of a trnA intron fragment from chloroplast of a non-eudicot plant to Phaseolus mitochondria. This is the first example of horizontal transfer of a chloroplast sequence to the mitochondrial genome in higher plants. PMID:15803417

Woloszynska, Magdalena; Bocer, Tomasz; Mackiewicz, Pawel; Janska, Hanna



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


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



High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection.  


In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the phiX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the phiX174/HaeIII digest diluted from 500 microg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas (n=3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis. PMID:19211105

Xu, Zhongqi; Esumi, Toshiaki; Ikuta, Natsuki; Hirokawa, Takeshi



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

PubMed Central

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

Johansson, Par I; Windel?v, Nis A; Rasmussen, Lars S; S?rensen, Anne Marie; Ostrowski, Sisse R



Mitochondrial DNA of Clathrina clathrus (Calcarea, Calcinea): six linear chromosomes, fragmented rRNAs, tRNA editing, and a novel genetic code.  


Sponges (phylum Porifera) are a large and ancient group of morphologically simple but ecologically important aquatic animals. Although their body plan and lifestyle are relatively uniform, sponges show extensive molecular and genetic diversity. In particular, mitochondrial genomes from three of the four previously studied classes of Porifera (Demospongiae, Hexactinellida, and Homoscleromorpha) have distinct gene contents, genome organizations, and evolutionary rates. Here, we report the mitochondrial genome of Clathrina clathrus (Calcinea, Clathrinidae), a representative of the fourth poriferan class, the Calcarea, which proves to be the most unusual. Clathrina clathrus mitochondrial DNA (mtDNA) consists of six linear chromosomes 7.6-9.4 kb in size and encodes at least 37 genes: 13 protein codings, 2 ribosomal RNAs (rRNAs), and 24 transfer RNAs (tRNAs). Protein genes include atp9, which has now been found in all major sponge lineages, but no atp8. Our analyses further reveal the presence of a novel genetic code that involves unique reassignments of the UAG codons from termination to tyrosine and of the CGN codons from arginine to glycine. Clathrina clathrus mitochondrial rRNAs are encoded in three (srRNA) and ?6 (lrRNA) fragments distributed out of order and on several chromosomes. The encoded tRNAs contain multiple mismatches in the aminoacyl acceptor stems that are repaired posttranscriptionally by 3'-end RNA editing. Although our analysis does not resolve the phylogenetic position of calcareous sponges, likely due to their high rates of mitochondrial sequence evolution, it confirms mtDNA as a promising marker for population studies in this group. The combination of unusual mitochondrial features in C. clathrus redefines the extremes of mtDNA evolution in animals and further argues against the idea of a "typical animal mtDNA." PMID:23223758

Lavrov, Dennis V; Pett, Walker; Voigt, Oliver; Wörheide, Gert; Forget, Lise; Lang, B Franz; Kayal, Ehsan



'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.  


The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation. PMID:18369655

Eichmann, Cordula; Parson, Walther



Host range expansion of Autographa californica nuclear polyhedrosis virus (NPV) following recombination of a 0.6-kilobase-pair DNA fragment originating from Bombyx mori NPV.  

PubMed Central

We have isolated hybrid baculoviruses of Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica NPV (AcNPV) capable of replicating in both BmN (not susceptible to AcNPV) and SF-21 (not susceptible to BmNPV) cells (A. Kondo and S. Maeda, J. Virol. 65:3625-3632, 1991). Repeated backcross infection of one of these recombinant isolates with AcNPV generated eh-AcNPV, a virus with restriction endonuclease patterns of genomic DNA nearly identical to those of AcNPV but capable of replicating in both BmN and SF-21 cells, i.e., host range expanded. Expanded host range viruses were also isolated following cotransfection of AcNPV DNA with eh-AcNPV DNA cleaved with either HindIII or PstI. Subsequent cotransfection of AcNPV DNA with plasmids from an eh-AcNPV DNA fragment library identified an 11-kbp HindIII fragment that could expand the host range of AcNPV. Subcloning and cotransfection analyses localized a 572-bp SacI-HindIII fragment within this 11-kbp fragment which could alone expand the host range of AcNPV. Mapping and nucleotide sequencing analysis revealed that this fragment was identical to the corresponding 572-bp fragment (BmScH) of BmNPV. Furthermore, this fragment originated from the coding region of the putative DNA helicase gene. Cotransfection of AcNPV DNA with BmScH also generated a host range-expanded virus, eh2-AcNPV. These results indicated that the expanded host range characteristics of eh2-AcNPV were solely the result of recombination within the coding region of the putative DNA helicase gene. Images

Maeda, S; Kamita, S G; Kondo, A



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

PubMed Central

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

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



Investigating a Eukaryotic Genome: Cloning and Sequencing a Fragment of Yeast DNA  

NSDL National Science Digital Library

Getting students into the laboratory early in their college careers is quite important, and science educators can use this helpful resource to do just that. Created by Professor Sarah Elgin at Washington University, this lab exercise and guide introduces students to the molecular biology techniques used to clone a gene. Over the course of this activity, students sequence a small fragment of the yeast genome and then determine what genes, control elements, or repetitive sequences this fragment contains. The file contains pages for the instructor detailing how to conduct the activity and a selection of pages for students designed to test and interrogate what they have learned through this process of scientific inquiry.

Elgin, Sarah C.; Weston-Hafer, Kathleen



The Development of Reduced Size STR Amplicons as Tools for Analysis of Degraded DNA  

Microsoft Academic Search

New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This \\

John M. Butler; Yin Shen; Bruce R. McCord


The e ect of change in population size on DNA polymorphism  

Microsoft Academic Search

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

F. Tajima



Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA.  

PubMed Central

The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.

Williams, D W; Wilson, M J; Lewis, M A; Potts, A J



Identification of Pathogenic Leishmania Promastigotes by DNA: DNA Hybridization with Kinetoplast DNA Cloned into E. coli Plasmids.  

National Technical Information Service (NTIS)

We report the characterization of Leishmania (L. infantum, L. donovani, and L. major) kinetoplast DNA (kDNA) by the use of restriction endonuclease digestion patterns and Southern hybridizations. Overall, the sizes and fragment patterns of MspI restrictio...

J. M. Lawrie P. R. Jackson J. M. Stiteler W. T. Hockmeyer



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


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

Rana, Jagruti; Huang, Haidong



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


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

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



Molecular Identification of Bacteria from a Coculture by Denaturing Gradient Gel Electrophoresis of 16S Ribosomal DNA Fragments as a Tool for Isolation in Pure Cultures  

Microsoft Academic Search

Molecular information about the bacterial composition of a coculture capable of sulfate reduction after exposure to oxic and microoxic conditions was used to identify and subsequently to isolate the components of the mixture in pure culture. PCR amplification of 16S ribosomal DNA fragments from the coculture, analyzed by denaturing gradient gel electrophoresis, resulted in two distinct 16S ribosomal DNA bands,




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

Microsoft Academic Search

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

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



Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene?  

PubMed Central

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

Hauschild, Tomasz; Stepanovic, Srdjan



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



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


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

Kwok, J B; Tattersall, M H



The effect of plasmid DNA sizes and other factors on electrotransformation of Escherichia coli JM109  

Microsoft Academic Search

The plasmid size can be an important factor in electrotransformations. We have examined bacterial electroporation with a specific interest in the transformation of plasmids with different sizes of their molecules. We used plasmids pUC19, pBR322 and pPP4. Transformation efficiency drops with increasing size of the DNA. We achieved with plasmid pUC19 a 81% frequency of transformation. The optimal field strength

Monika Szostková; Dana Horáková



Biological activity of the spleen focus-forming virus is encoded by a molecularly cloned subgenomic fragment of spleen focus-forming virus DNA.  

PubMed Central

A biologically active subgenomic DNA fragment of a polycythemia-inducing strain of the replication-defective spleen focus-forming virus (SFFV) has been molecularly cloned. The SFFV DNA fragment includes 2.0 kilobase pairs (kbp) from the 3' end of SFFV, the long terminal repeat sequences of SFFV, and 0.4 kbp from the 5' end of SFFV. The fragment contains the previously described env-related gene of SFFV. All the properties associated with SFFV can be assigned to this SFFV DNA fragment by using a two-stage DNA transfection assay with infectious helper virus DNA. The virus recovered from the transfection assays can induce erythroblastosis, splenic foci, and polycythemia in infected mice. Fibroblast cultures transfected with the SFFV DNA fragment synthesize gp52, the known intracellular product of the env-related gene of SFFV. gp52 can also be detected in spleens from diseased mice infected with the virus recovered in the two-stage transfection. The results are consistent with the hypothesis that the env-related gene sequences of SFFV and their product gp52 are required for the initiation of SFFV-induced disease. Images

Linemeyer, D L; Ruscetti, S K; Scolnick, E M; Evans, L H; Duesberg, P H



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

PubMed Central

A molecular system of a nanometer-sized reel was developed from F1–ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9–6.0 pN) and the diameter of the wound loop (21.4–8.5?nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54?±?9?nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands.

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



Fragmentation and plasmid strand breaks in pure and gold-doped DNA irradiated by beams of fast hydrogen atoms  

NASA Astrophysics Data System (ADS)

The results of an investigation into the damage caused to dry plasmid DNA after irradiation by fast (keV) hydrogen atoms are presented. Agarose gel electrophoresis was used to assess single and double strand break yields as a function of dose in dry DNA samples deposited on a mica substrate. Damage levels were observed to increase with beam energy. Strand break yields demonstrated a considerable dependence on sample structure and the method of sample preparation. Additionally, the effect of high-Z nanoparticles on damage levels was investigated by irradiating DNA samples containing controlled amounts of gold nanoparticles. In contrast to previous (photonic) studies, no enhancement of strand break yields was observed with the particles showing a slight radioprotective effect. A model of DNA damage as a function of dose has been constructed in terms of the probability for the creation of single and double strand breaks, per unit ion flux. This model provides quantitative conclusions about the effects of both gold nanoparticles and the different buffers used in performing the assays and, in addition, infers the proportion of multiply damaged fragments.

Wyer, J. A.; Butterworth, K. T.; Hirst, D. G.; Latimer, C. J.; Montenegro, E. C.; Shah, M. B.; Currell, F. J.



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


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

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



Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments.  

PubMed Central

Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E. coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion of 9.5% marker yield is described.

Kramer, W; Schughart, K; Fritz, H J



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


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

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



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

Microsoft Academic Search

Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT (8-oxo-dGTPase) of Esch- erichia coli hydrolyze noncanonical DNA precursors to prevent incorporation of base analogs into DNA. Previous studies reported dramatic AT3CG mutagenesis in mutT mutants, suggesting a considerable density of 8-oxo-G in DNA that should cause frequent excision and chromosomal fragmentation, irreparable in the absence of RecBCD-catalyzed repair and

Ella Rotman; Andrei Kuzminov



Optimization of single-molecule fluorescence burst detection of ds-DNA: Application to capillary electrophoresis separations of 100-1000 basepair fragments  

Microsoft Academic Search

Methods for optimizing the dye labeling, laser excitation, and data analysis for single-molecule fluorescence burst detection of ds-DNA have been developed and then validated through capillary electrophoresis (CE) separations of 100â1000 basepair (bp) DNA. Confocal microscopy is used to observe fluorescence bursts from individual DNA fragments labeled with the intercalation dye TO6 as they pass through the â¼2-μm-diameter focused laser

Brian B. Haab; Richard A. Mathies



Appearance of 1?2 Mbp Giant DNA Fragments as an Early Common Response Leading to Cell Death Induced by Various Substances That Cause Oxidative Stress  

Microsoft Academic Search

The effects of oxidative stress on double strand DNA breakage were examined in T-24 human bladder tumor cells using various active oxygen producing agents such as hydrogen peroxide (H2O2), bleomycin (BLM), neocarzinostatin (NCS), and x-ray irradiation. Analysis of the DNA by pulsed-field gel electrophoresis (PFGE) revealed that discrete giant DNA fragments of 1–2 Mbp and 200–800 kbp had accumulated in

Yoshihiro Higuchi; Shigeru Matsukawa



[Long-fragment DNA of blood plasma as one of the criteria of individual sensitivity to emotional stress and to cerebral ischemia].  


Intravenous injection ofpolyethylenoxide WSR-301 reducing hydrodynamic blood resistance (Toms effect) improves gas exchange in the lungs and halved lethality of the animals with cerebral ischemia. The aim of the study was to establish whether free plasma DNA influences blood gases and lethality of the animals with brain ischemia. Common carotid arteries were ligated for 15 min in intact stressed and tested in the open field Wistar male rats, then some of the rats received intravenous solution of homologous long-fragment DNA (20x10(-6) g/ml of blood). Cerebral circulation, acid-base equilibrium, paO2, paCO2, asymptotic blood viscosity, plasmic concentration and length of DNA fragments in plasma, lethality and neurological status of the survivors were studied. It was found that long fragments of rat DNA show hydrodynamic Toms effect. In normal passive rats sensitive to cerebral ischemia part of plasm DNA is fragmented, gas composition and blood viscosity of blood is worse (p < 0.05) than in active animals. There is a direct correlation between the level of long-fragment DNA in plasm and paO2 (r = 0.55) and inverse--with paCO2 (r = -0.84). Intravenous injection of long-fragment DNA improved the course and reduced lethality of brain ischemia 2-3-fold. Thus, qualitative and quantitative characteristics of plasma circulating DNA are responsible for differences in blood gases in rats differently tolerable to cerebral ischemia and can serve as one of the criteria of individual sensitivity to it being essential in pathogenesis of ischemic stroke. PMID:17002040

Gannushkina, I V; Konorova, I L; Ve?ko, N N


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

NASA Astrophysics Data System (ADS)

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

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


Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments  

Microsoft Academic Search

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its

Fredrik Dahl; Mats Gullberg; Johan Stenberg; Ulf Landegren; Mats Nilsson



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

PubMed Central

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

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



Adding resolution to ordinal level relationships of tapeworms (Platyhelminthes: Cestoda) with large fragments of mtDNA.  


The construction of a stable phylogeny for the Cestoda, indicating the interrelationships of recognised orders and other major lineages, has proceeded iteratively since the group first received attention from phylogenetic systematists. Molecular analyses using nuclear ribosomal RNA gene fragments from the small (ssrDNA) and large (lsrDNA) subunits have been used to test competing evolutionary scenarios based on morphological data but could not arbitrate between some key conflicting hypotheses. To the ribosomal data, we have added a contiguous fragment of mitochondrial (mt) genome data (mtDNA) of partial nad1-trnN-trnP-trnI-trnK-nad3-trnS-trnW-cox1-trnT-rrnL-trnC-partial rrnS, spanning 4034-4447 bp, where new data for this region were generated for 18 species. Bayesian analysis of mtDNA and rDNA as nucleotides, and where appropriate as amino acids, demonstrated that these two classes of genes provide complementary signal across the phylogeny. In all analyses, except when using mt amino acids only, the Gyrocotylidea is sister group to all other Cestoda (Nephroposticophora), and Amphilinidea forms the sister group to the Eucestoda. However, an earliest-diverging position of Amphilinidea is strongly supported in the mt amino acid analysis. Amphilinidea exhibit a unique tRNA arrangement (nad1-trnI-trnL2-trnP-trnK-trnV-trnA-trnN-nad3), whereas Gyrocotylidea shares that of the derived lineages, providing additional evidence of the uniqueness of amphilinid genes and genomes. The addition of mtDNA to the rDNA genes supported the Caryophyllidea as the sister group to (Spathebothriidea+remaining Eucestoda), a hypothesis consistently supported by morphology. This relationship suggests a history of step-wise evolutionary transitions from simple monozoic, unsegmented tapeworms to the more familiar polyzoic, externally segmented (strobilate) forms. All our data partitions recovered Haplobothriidea as the sister group to Diphyllobothriidae. The sister-group relationship between Diphyllidea and Trypanorhyncha, as previously established using rDNA, is not supported by the mt data, although it is supported by the combined mt and rDNA analysis. With regards to the more derived taxa, in all except the mt amino acid analysis, the following topology is supported: (Bothriocephalidea (Litobothriidea (Lecanicephalidea (Rhinebothriidea (Tetraphyllidea, (Acanthobothrium, Proteocephalidea), (Nippotaeniidea, Mesocestoididae, Tetrabothriidea, Cyclophyllidea)))))), where the Tetraphyllidea are paraphyletic. Evidence from the mt data provides strong (nucleotides) to moderate (amino acids) support for Tetraphyllidea forming a group to the inclusion of Proteocephalidea, with the latter consistently forming the sister group to Acanthobothrium. The interrelationships among Nippotaeniidea, Mesocestoididae, Tetrabothriidea and Cyclophyllidea remain ambiguous and require further systematic attention. Mitochondrial and nuclear rDNA data provide conflicting signal for certain parts of the cestode tree. In some cases mt data offer results in line with morphological evidence, such as the interrelationships of the early divergent lineages. Also, Tetraphyllidea, although remaining paraphyletic with the inclusion of the Proteocephalidea, does not include the most derived cestodes; a result which has consistently been obtained with rDNA. PMID:22406529

Waeschenbach, Andrea; Webster, B L; Littlewood, D T J



DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer  

PubMed Central

Objective The present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens. Methods Morphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Sat?) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival. Results We identified a significant association between LINE1 DNA-hypomethylation and patient age (p = 0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r = 0.47; p<0.001) and the proliferation index (r = 0.36; p<0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and = 0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival. Conclusions In this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.

Zeimet, Alain G.; Fiegl, Heidi; Goebel, Georg; Kopp, Francis; Allasia, Claude; Reimer, Daniel; Steppan, Ilona; Mueller-Holzner, Elisabeth; Ehrlich, Melanie; Marth, Christian



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.

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



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


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

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


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

PubMed Central

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

Clasen, Jessica L.; Suttle, Curtis A.



Identification of freshwater Phycodnaviridae and their potential phytoplankton hosts, using DNA pol sequence fragments and a genetic-distance analysis.  


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

Clasen, Jessica L; Suttle, Curtis A



[Isolation of fibronectin peptide fragments of various sizes and biological activity using affinity chromatography on an immobilized plasmin].  


Plasmin, immobilized on Sepharose, was used for isolation of human blood plasma fibronectin fragments obtained after proteolysis. Under definite conditions the major part of the fibronectin fragments, liberated during proteolysis, remained to be bound to plasmin-Sepharose. As shown by electrophoretic analysis, the fraction of fragments bound to plasmin-Sepharose constituted mainly "heavy" (greater than or equal to 120 kD) peptides and one "light" (29 kD) peptide, while only "light" fragments (less than or equal to 45 kD) were detected in the free unbound fraction. These unbound to plasmin-Sepharose fibronectin fragments were found to stimulate proliferation of human embryonal fibroblasts in cell culture, whereas the plasmin-Sepharose bound peptides did not exhibit any effects on proliferation. PMID:2534245

Chubukina, A N; Zlatopol'ski?, A D; Za?denberg, M A; Bychkova, V V


Conformational properties of DNA fragments containing GAC trinucleotide repeats associated with skeletal displasias  

Microsoft Academic Search

The human gene for cartilage oligomeric matrix protein contains five tandem repeats of the GAC trinucleotide. Its expansion by one repeat causes multiple epiphyseal dysplasia, while expansion by two repeats or, remarkably, deletion of one repeat causes pseudoachondroplasia. Here we used CD spectroscopy, PAGE and UV absorption spectroscopy to compare conformational properties of the DNA strands containing four, five, six

Michaela Vorlícková; Iva Kejnovská; Marcela Tumová; Jaroslav Kypr



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

Microsoft Academic Search

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

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



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

PubMed Central

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

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



Cloning and characterization of a DNA fragment that confers sulfonamide resistance in a serogroup B, serotype 15 strain of Neisseria meningitidis.  

PubMed Central

By cloning studies and complementation experiments, the sulfonamide resistance gene of a serogroup B and serotype 15 (B:15) strain of Neisseria meningitidis was localized to a 1.2-kb chromosomal SspI fragment expressing a drug-resistant dihydropteroate synthase. The fragment hybridized to DNA from both resistant and susceptible strains, suggesting that the resistance gene is a variant of the normal gene for dihydropteroate synthase. Images

Kristiansen, B E; Radstrom, P; Jenkins, A; Ask, E; Facinelli, B; Skold, O



Comparative genomics of DNA fragments from six Antarctic marine planktonic bacteria.  


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

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



Trapping of Megabase-Sized DNA Molecules during Agarose Gel Electrophoresis  

Microsoft Academic Search

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated lambda -ladders, sheared during gel electrophoresis at a given field,

Sergio Gurrieri; Steven B. Smith; Carlos Bustamante



Fabrication of mono-sized magnetic anion exchange beads for plasmid DNA purification  

Microsoft Academic Search

Mono-sized magnetic polyglycidyl methacrylate (PGMA) microspheres cross-linked by divinylbenzene were fabricated by the dispersion polymerization and penetration–deposition method. The magnetic beads were nonporous, spherical, and mono-sized (1.0?m). Moreover, it was superparamagnetic with a saturation magnetization of 16.5emu\\/g. The prepared magnetic beads were then functionalized with high-density amino groups and used as anion exchangers to capture plasmid DNA from concentrated bacterial

Hai-Ping Zhang; Shu Bai; Liang Xu; Yan Sun



Quantification of Fetal and Total Circulatory DNA in Maternal Plasma Samples Before and After Size Fractionation by Agarose Gel Electrophoresis  

Microsoft Academic Search

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and ? 900 bp) after size fractionation by agarose gel electrophoresis. DNA

I. Hromadnikova; L. Zejskova; J. Doucha; D. Codl



Adding Zn 2+ induces DNA fragmentation and cell condensation in cultured human Chang liver cells  

Microsoft Academic Search

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting\\u000a multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects,\\u000a such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo

Ramanujam Paramanantham; Kwok-Hung Sit; Boon-Huat Bay



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

Microsoft Academic Search

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

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



Inheritance and restriction fragment length polymorphism of chloroplast DNA in the genus Coffea L  

Microsoft Academic Search

CpDNA variation among 52 tree samples belonging to 25 different taxa of Coffea and two species of Psilanthus was assessed by RFLP analysis on both the total chloroplast genome and the atpB-rbcL intergenic region. Twelve variable characters were distinguished allowing the identification of 12 different plastomes. The low sequence divergence observed might suggest that Coffea is a young genus. The

P. Lashermes; J. Cros; M. C. Combes; P. Trouslot; F. Anthony; S. Hamon; A. Charrier



Investigation of the transfection capability of cloned tandemly-repeated chicken anaemia virus DNA fragments.  


Chicken anaemia virus (CAV) is an icosahedral virus, 25 nm in diameter, which, on the basis of its circular single-stranded DNA genome, has recently been classified in the family, Circoviridae. We have investigated whether infectious, monomeric CAV DNA from recombinant plasmids containing tandemly-repeated CAV replicative form (RF) DNAs, following transfection, was generated by homologous recombination or a replicational release mechanism involving rolling circle replication (RCR) of DNA. Experiments designed to locate the virus strand origin of RCR and/or sites of recombination were performed by sequence analyses of hybrid viruses generated after transfection with cloned tandemly-repeated RFs specified by the sequence-distinct Cux-1 and 26P4 isolates. Positive transfection results obtained from 2 recombinant plasmid constructs were shown to have resulted from homologous recombination occurring at different sites within the RF sequence. Three of 5 hybrid viruses analysed were "circularised" within the same 105 bp sequence, that contains four 19bp repeats and with which promoter/enhancer activity has been associated. This region may represent a novel origin or recombination hot-spot within the CAV genome. A distinctive cruciform-loop structure within the non-coding region was shown to contain an S1 nuclease-sensitive site, detected in CAV RF and in recombinant plasmids containing RF inserts. PMID:8856031

Todd, D; Creelan, J L; Meehan, B M; McNulty, M S



Prolonged moderate-intensity aerobic exercise does not alter apoptotic signaling and DNA fragmentation in human skeletal muscle.  


Apoptosis in skeletal muscle plays an important role in age- and disease-related tissue dysfunction. Physical activity can influence apoptotic signaling; however, this process has not been well studied in human skeletal muscle. The purpose of this study was to perform a comprehensive analysis of apoptosis-related proteins/enzymes, DNA fragmentation, and oxidative stress in skeletal muscle of humans during an acute bout of prolonged moderate-intensity exercise. Eight healthy, recreationally active individuals (age 20.8 +/- 0.5 yr, Vo(2peak) 51.2 +/- 0.9 ml . kg(-1) . min(-1), BMI 21.5 +/- 0.8 kg/m(2)) exercised on a cycle ergometer at approximately 60% Vo(2peak) for 2 h. Muscle biopsies were obtained at rest as well as at 60 and 120 min of exercise. Although exercise was associated with a significant whole body and muscle metabolic response, there were no significant changes in the content of antiapoptotic (ARC, Bcl-2, Hsp70, XIAP) and proapoptotic (AIF, Bax, Smac) proteins, activity of proteolytic enzymes (caspase-3, caspase-8, caspase-9), DNA fragmentation, or TUNEL-positive nuclei in skeletal muscle. Furthermore, the protein levels of several antioxidant enzymes (catalase, CuZnSOD, MnSOD), concentrations of GSH and GSSG, and degree of ROS generation in skeletal muscle were not altered by exercise. Fiber type-specific analysis also revealed that ARC (P < 0.001) and Hsp70 (P < 0.05) protein were significantly higher in type I compared with type IIA and type IIAX/X fibers; however, protein levels were not affected by exercise. These findings suggest that a single bout of prolonged moderate-intensity aerobic exercise is not sufficient to alter apoptotic signaling in skeletal muscle of healthy humans. PMID:19996388

Quadrilatero, Joe; Bombardier, Eric; Norris, Sarah M; Talanian, Jason L; Palmer, Matthew S; Logan, Heather M; Tupling, A Russell; Heigenhauser, George J F; Spriet, Lawrence L



Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment).  


To investigate the interactions that determine DNA polymerase accuracy, we have measured the fidelity of 26 mutants with amino acid substitutions in the polymerase domain of a 3'-5'-exonuclease-deficient Klenow fragment. Most of these mutant polymerases synthesized DNA with an apparent fidelity similar to that of the wild-type control, suggesting that fidelity at the polymerase active site depends on highly specific enzyme-substrate interactions and is not easily perturbed. In addition to the previously studied Y766A mutator, four novel base substitution mutators were identified; they are R668A, R682A, E710A, and N845A. Each of these five mutator alleles results from substitution of a highly conserved amino acid side chain located on the exposed surface of the polymerase cleft near the polymerase active site. Analysis of base substitution errors at four template positions indicated that each of the five mutator polymerases has its own characteristic error specificity, suggesting that the Arg-668, Arg-682, Glu-710, Tyr-766, and Asn-845 side chains may contribute to polymerase fidelity in a variety of different ways. We separated the contributions of the nucleotide insertion and mismatch extension steps by using a novel fidelity assay that scores base substitution errors during synthesis to fill a single nucleotide gap (and hence does not require mismatch extension) and by measuring the rates of polymerase-catalyzed mismatch extension reactions. The R682A, E710A, Y766A, and N845A mutations cause decreased fidelity at the nucleotide insertion step, whereas R668A results in lower fidelity in both nucleotide insertion and mismatch extension. Relative to wild type, several Klenow fragment mutants showed substantially more discrimination against extension of a T.G mismatch under the conditions of the fidelity assay, providing one explanation for the anti-mutator phenotypes of mutants such as R754A and Q849A. PMID:9915846

Minnick, D T; Bebenek, K; Osheroff, W P; Turner, R M; Astatke, M; Liu, L; Kunkel, T A; Joyce, C M



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


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



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



Bean Dwarf mosaic geminivirus movement proteins recognize DNA in a form- and size-specific manner.  


Plant viral movement proteins mediate the cell-to-cell movement of nucleic acids. This involves either a direct interaction between the viral movement protein and the nucleic acid or an indirect interaction involving host factors. The bipartite geminiviruses possess two movement proteins, BV1 and BC1, that coordinate movement of viral DNA across nuclear and plasmodesmal boundaries, respectively. Here, we demonstrate that both BV1 and BC1 interact directly with DNA and, in addition, that they have the unique property to recognize DNA on the basis of form and size rather than sequence. This is a novel feature for plant virus movement proteins and raises the possibility that BV1 and BC1 may be determinants of genome size in the bipartite geminiviruses. PMID:9778251

Rojas, M R; Noueiry, A O; Lucas, W J; Gilbertson, R L



Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3.  


Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway. PMID:12941599

Lewis, Aurélia E; Wong, Benjamin C Y; Langman, Michael J S; Eggo, Margaret C



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


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

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



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

Microsoft Academic Search

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

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



Protein-mediated DNA loops: effects of protein bridge size and kinks.  


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 of a protein bridge and/or the presence of a kink at half DNA length. The DNA is modeled by the wormlike 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 kBT to 13 kBT when the loop is closed by a protein of r=10 length. It further decreases 5 kBT to when the loop has a kink of 120 degrees at half-length. PMID:16485969

Douarche, Nicolas; Cocco, Simona



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


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

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



Chromosomal localization of a highly repeated Eco RI DNA fragment in Megoura viciae (Homoptera, Aphididae) by nick translation and fluorescence in situ hybridization  

Microsoft Academic Search

To investigate the genome of the aphidMegoura viciae at molecular level, we have studied total DNA by agarose gel electrophoresis after cleavage with different restriction endonucleases.EcoRI digestion produced a highly repeated DNA fragment, about 600 bp long. The contribution of thisEcoRI element to the total genome ofM. viciae was estimated at about 6% by means of densitometric scanning of agarose

Davide Bizzaro; Gian Carlo Manicardi; Umberto Bianchi



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


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

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



Detection of genetically modified maize DNA fragments in the intestinal contents of pigs fed StarLink CBH351.  


We tried to detect DNA fragments derived from maize in the intestinal contents of pigs fed genetically modified (GM) StarLink CBH351 maize (SL) or non-GM maize. Intestinal contents of 8 SL and 8 non-GM maize-fed pigs were collected at slaughter, and the genes of the recombinant cry9C and the maize intrinsic zein (Zel) were assayed by polymerase chain reaction (PCR) 3 times with a total of 4 primer pairs of different expected lengths. The cry9C gene (either 103 or 170 bp) was detected in the rectal contents (with a frequency of 25-37.5%) and in the cecal contents (25-50%) of the pigs fed SL. In a similar fashion, the zein (Zel) gene (either 242 or 329 bp) was detected in the rectal contents (with a frequency of 31.3%) and in the cecal contents (25-37.5%) of pigs fed on SL non-GM maize. These results suggested that ingested DNA was not totally degraded, but is present in a form detectable by PCR. PMID:12678298

Chowdhury, E H; Mikami, O; Nakajima, Y; Hino, A; Kuribara, H; Suga, K; Hanazumi, M; Yomemochi, C



Purification and characterization of a proteolytic active fragment of DNA topoisomerase I from the brine shrimp Artemia franciscana (Crustacea Anostraca).  

PubMed Central

The ATP-independent type I topoisomerase from the crustacean Artemia franciscana was purified to near-homogeneity. Its activity was measured by an assay that uses the formation of an enzyme-cleaved DNA complex in the presence of the specific inhibitor camptothecin. The purification procedure is reported. Purified topoisomerase is a single-subunit enzyme with a molecular mass of 63 kDa. Immunoblot performed on the different steps of purification shows that the purified 63 kDa peptide is a proteolytic fragment of a protein with a molecular mass of 110 kDa. Similarly to the other purified eukaryotic topoisomerases, the crustacean enzyme does not require a bivalent cation for activity, but is stimulated in the presence of 10 mM-MgCl2; moreover, it can relax both negative and positive superhelical turns. The enzyme activity is strongly inhibited by the antitumour drug camptothecin. The enzyme inhibition is related to the stabilization of the cleavable complex between topoisomerase I and DNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7.

Badaracco, G; Landsberger, N; Benfante, R



Rapid radiation events in the family Ursidae indicated by likelihood phylogenetic estimation from multiple fragments of mtDNA.  


The bear family (Ursidae) presents a number of phylogenetic ambiguities as the evolutionary relationships of the six youngest members (ursine bears) are largely unresolved. Recent mitochondrial DNA analyses have produced conflicting results with respect to the phylogeny of ursine bears. In an attempt to resolve these issues, we obtained 1916 nucleotides of mitochondrial DNA sequence data from six gene segments for all eight bear species and conducted maximum likelihood and maximum parsimony analyses on all fragments separately and combined. All six single-region gene trees gave different phylogenetic estimates; however, only for control region data was this significantly incongruent with the results from the combined data. The optimal phylogeny for the combined data set suggests that the giant panda is most basal followed by the spectacled bear. The sloth bear is the basal ursine bear, and there is weak support for a sister taxon relationship of the American and Asiatic black bears. The sun bear is sister taxon to the youngest clade containing brown bears and polar bears. Statistical analyses of alternate hypotheses revealed a lack of strong support for many of the relationships. We suggest that the difficulties surrounding the resolution of the evolutionary relationships of the Ursidae are linked to the existence of sequential rapid radiation events in bear evolution. Thus, unresolved branching orders during these time periods may represent an accurate representation of the evolutionary history of bear species. PMID:10508542

Waits, L P; Sullivan, J; O'Brien, S J; Ward, R H



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


Wild apples (Malus sp.) have been a major food source in the northern Alpine region since prehistory and their use is well understood. The onset of deliberate fruit tree growing in the area is, however, less clear. It is generally assumed that horticulture was practised in Roman times, but it might be ev