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Sample records for restriction dna cutter

  1. Highlights of the DNA cutters: a short history of the restriction enzymes

    PubMed Central

    Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.; Murray, Noreen E.

    2014-01-01

    In the early 1950’s, ‘host-controlled variation in bacterial viruses’ was reported as a non-hereditary phenomenon: one cycle of viral growth on certain bacterial hosts affected the ability of progeny virus to grow on other hosts by either restricting or enlarging their host range. Unlike mutation, this change was reversible, and one cycle of growth in the previous host returned the virus to its original form. These simple observations heralded the discovery of the endonuclease and methyltransferase activities of what are now termed Type I, II, III and IV DNA restriction-modification systems. The Type II restriction enzymes (e.g. EcoRI) gave rise to recombinant DNA technology that has transformed molecular biology and medicine. This review traces the discovery of restriction enzymes and their continuing impact on molecular biology and medicine. PMID:24141096

  2. Almond brush module cutter

    SciTech Connect

    Zohns, M.A.; Jenkins, B.M.; Mehlschau, J.J.; Morrison, D.

    1983-06-01

    This paper addresses the design, construction, and evaluation of an almond brush module cutter. The module cutter is one link in a system which processes tree prunings for fuel and fiber. This system includes a modified cotton module builder, a module mover, the cutter, and a tub grinder. An economic analysis of the cutter is presented along with the problems involved in cutting brush modules.

  3. Effect of aging and dietary restriction on DNA repair

    SciTech Connect

    Weraarchakul, N.; Strong, R.; Wood, W.G.; Richardson, A.

    1989-03-01

    DNA repair was studied as a function of age in cells isolated from both the liver and the kidney of male Fischer F344 rats. DNA repair was measured by quantifying unscheduled DNA synthesis induced by UV irradiation. Unscheduled DNA synthesis decreased approximately 50% between the ages of 5 and 30 months in both hepatocytes and kidney cells. The age-related decline in unscheduled DNA synthesis in cells isolated from the liver and kidney was compared in rats fed ad libitum and rats fed a calorie-restricted diet; calorie restriction has been shown to increase the survival of rodents. The level of unscheduled DNA synthesis was significantly higher in hepatocytes and kidney cells isolated from the rats fed the restricted diet. Thus, calorie restriction appears to retard the age-related decline in DNA repair.

  4. Drag bit and cutters

    SciTech Connect

    Barr, J. D.

    1985-12-17

    A drill bit comprises a bit body having an operating end face. A plurality of self-sharpening cutters are mounted in the bit body and extend through the operating end face. The cutters have cutting faces adapted to engage an earth formation and cut the earth formation to a desired three-dimensional profile. The cutting faces define surfaces having back rake angles which decrease with distance from the profile. The individual cutting faces may be inwardly concave in a plane parallel to the intended direction of motion of the cutter in use.

  5. Cutter for Woven Materials

    NASA Technical Reports Server (NTRS)

    Hammons, J. M.; Keir, A. R.

    1983-01-01

    Simple tool makes accurate square cuts through strips of woven or felted materials, such as high temperature aramid fabric. Pressing handle on Guillotine Cutter forces razor blade through strip of material in slot, cutting strip off squarely.

  6. Selective microbial genomic DNA isolation using restriction endonucleases.

    PubMed

    Barnes, Helen E; Liu, Guohong; Weston, Christopher Q; King, Paula; Pham, Long K; Waltz, Shannon; Helzer, Kimberly T; Day, Laura; Sphar, Dan; Yamamoto, Robert T; Forsyth, R Allyn

    2014-01-01

    To improve the metagenomic analysis of complex microbiomes, we have repurposed restriction endonucleases as methyl specific DNA binding proteins. As an example, we use DpnI immobilized on magnetic beads. The ten minute extraction technique allows specific binding of genomes containing the DpnI Gm6ATC motif common in the genomic DNA of many bacteria including γ-proteobacteria. Using synthetic genome mixtures, we demonstrate 80% recovery of Escherichia coli genomic DNA even when only femtogram quantities are spiked into 10 µg of human DNA background. Binding is very specific with less than 0.5% of human DNA bound. Next Generation Sequencing of input and enriched synthetic mixtures results in over 100-fold enrichment of target genomes relative to human and plant DNA. We also show comparable enrichment when sequencing complex microbiomes such as those from creek water and human saliva. The technique can be broadened to other restriction enzymes allowing for the selective enrichment of trace and unculturable organisms from complex microbiomes and the stratification of organisms according to restriction enzyme enrichment. PMID:25279840

  7. Tubing Cutter is Activated Hydraulically

    NASA Technical Reports Server (NTRS)

    Mcsmith, D. G.; Richardson, J. I.

    1983-01-01

    Hydraulically-actuated tubing cutter severs tubing when operator squeezes handle grip. "Gooseneck" extension enables cutter to be used in areas where accessiblity is limited. Cutter has potential as flight-line tool and is useful in automobile and fire rescue work.

  8. Tubing cutter for tight spaces

    NASA Technical Reports Server (NTRS)

    Girala, A. S.

    1980-01-01

    Cutter requires few short swings of handle to rotate its cutting edge full 360 around tube. It will cut tubing installed in confined space that prevents free movement of conventional cutter. Cutter is snapped onto tube and held in place by spring-loaded clamp. Screw ratchet advances cutting wheel.

  9. Shaft drill bit with overlapping cutter arrangement

    SciTech Connect

    Cunningham, R.A.; Pessier, R.C.

    1981-02-03

    An earth boring drill bit for large diameter shafts has an improved cutter arrangement. The drill bit has a cutter support member with a number of cutters mounted to it for disintegrating the earth formation face. At least one inner cutter is mounted near the center for cutting the center area. A number of gage cutters are mounted at the periphery to cut the gage area of the shaft. A number of intermediate cutters are spaced between the inner and gage cutters. Each intermediate cutter overlaps onehalf of its width with an adjacent intermediate cutter.

  10. Adaptive DNA Computing Algorithm by Using PCR and Restriction Enzyme

    NASA Astrophysics Data System (ADS)

    Kon, Yuji; Yabe, Kaoru; Rajaee, Nordiana; Ono, Osamu

    In this paper, we introduce an adaptive DNA computing algorithm by using polymerase chain reaction (PCR) and restriction enzyme. The adaptive algorithm is designed based on Adleman-Lipton paradigm[3] of DNA computing. In this work, however, unlike the Adleman- Lipton architecture a cutting operation has been introduced to the algorithm and the mechanism in which the molecules used by computation were feedback to the next cycle devised. Moreover, the amplification by PCR is performed in the molecule used by feedback and the difference concentration arisen in the base sequence can be used again. By this operation the molecules which serve as a solution candidate can be reduced down and the optimal solution is carried out in the shortest path problem. The validity of the proposed adaptive algorithm is considered with the logical simulation and finally we go on to propose applying adaptive algorithm to the chemical experiment which used the actual DNA molecules for solving an optimal network problem.

  11. Separation of DNA restriction fragments using capillary electrophoresis

    SciTech Connect

    Chan, K.C.; Whang, Chenwen; Yeung, E.S. )

    1993-01-01

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

  12. Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq).

    PubMed

    Graham, Carly F; Glenn, Travis C; McArthur, Andrew G; Boreham, Douglas R; Kieran, Troy; Lance, Stacey; Manzon, Richard G; Martino, Jessica A; Pierson, Todd; Rogers, Sean M; Wilson, Joanna Y; Somers, Christopher M

    2015-11-01

    Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next-generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double-digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0-, 12-, 48- and 96-h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12-48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96-h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2- to 16-fold), and additional read-length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded. PMID:25783180

  13. Diamond cutters' grinders

    NASA Astrophysics Data System (ADS)

    Romanov, B. F.

    1985-03-01

    The development of diamond tool designs is determined by the development of the technology for the synthesis of artificial diamonds. The technology of syntehsizing artificial diamonds involves the production of mono and polycrystalline diamonds and composition diamond-containing materials. High strength and thermally stable monocrystalline diamonds brands AS30 to AS80 in a size of up to 800 micrometers, and polycrystalline diamonds: black diamonds, ballas (Synthetic Fiber) in a size up to 10mm, are manufactured. Production of single-layer and double-layer diamond plates used in cutting tools is organized. The raw materials base with the constant decrease in the use of natural diamonds is the basis for the development of the manufacture of a wide array of diamond tools. New areas of applications for tools using natural diamonds, such as diamond cutters for turning high-precision parts, straightening tools, hardness gages are outlined. Diamond cutters with natural diamonds are used to grind surfaces which have exceptionally high requirements with respect to the reflecting capacity and roughness.

  14. Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

    PubMed Central

    Shen, C J; Wiesehahn, G; Hearst, J E

    1976-01-01

    The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined. Images PMID:818625

  15. Open ended tubing cutters

    NASA Technical Reports Server (NTRS)

    Girala, A. S. (Inventor)

    1981-01-01

    A self clamping cutting tool which includes a handle attached to a C-shaped housing is described. Rotatably mounted within the housing is a C-shaped tool body carrying a set of clamping rolls, two support rolls, and an edged cutting roll (64). The support rolls are disposed to one side of the axis of a pipe and the cutting roll is disposed to the other side of a pipe axis so that these rolls contact a pipe at three circumferential points. Cutter advancing apparatus advance the cutting roll toward the support rollers. The support rolls and cutting roll are rotatable independently of the C-shaped housing. A one way ratchet mechanism disposed between the C-shaped housing and the C-shaped tool body permits operation by movement in one rotational direction about the pipe axis.

  16. A Time-Efficient and User-Friendly Method for Plasmid DNA Restriction Analysis.

    ERIC Educational Resources Information Center

    LaBanca, Frank; Berg, Claire M.

    1998-01-01

    Describes an experiment in which plasmid DNA is digested with restriction enzymes that cleave the plasmid either once or twice. The DNA is stained, loaded on a gel, electrophoresed, and viewed under normal laboratory conditions during electrophoresis. (DDR)

  17. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

    PubMed Central

    McClelland, M; Nelson, M; Raschke, E

    1994-01-01

    Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074

  18. Rapid restriction enzyme free detection of DNA methyltransferase activity based on DNA-templated silver nanoclusters.

    PubMed

    Kermani, Hanie Ahmadzade; Hosseini, Morteza; Dadmehr, Mehdi; Ganjali, Mohammad Reza

    2016-06-01

    DNA methylation has significant roles in gene regulation. DNA methyltransferase (MTase) enzyme characterizes DNA methylation and also induces an aberrant methylation pattern that is related to many diseases, especially cancers. Thus, it is required to develop a method to detect the DNA MTase activity. In this study, we developed a new sensitive and reliable method for methyltransferase activity assay by employing DNA-templated silver nanoclusters (DNA/Ag NCs) without using restriction enzymes. The Ag NCs have been utilized for the determination of M.SssI MTase activity and its inhibition. We designed an oligonucleotide probe which contained an inserted six-cytosine loop as Ag NCs formation template. The changes in fluorescence intensity were monitored to quantify the M.SssI activity. The fluorescence spectra showed a linear decrease in the range of 0.4 to 20 U/ml with a detection limit of 0.1 U/ml, which was significant compared with previous reports. The proposed method was applied successfully for demonstrating the Gentamicin effect as MTase inhibitor. The proposed method showed convenient reproducibility and sensitivity indicating its potential for the determination of methyltransferase activity. PMID:27052776

  19. Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

    PubMed Central

    Schalbetter, Stephanie A.; Mansoubi, Sahar; Chambers, Anna L.; Downs, Jessica A.; Baxter, Jonathan

    2015-01-01

    Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin. PMID:26240319

  20. Low cutter load raise head

    SciTech Connect

    Saxman, W.C.

    1981-03-31

    A raise head having a multiplicity of cutters for enlarging a pilot hole into a larger diameter hole by disintegrating the earth formations that surround the pilot hole is provided that will require lower cutter loads to penetrate the formations being bored by directing the rock fracture planes toward the pilot hole forcing the rock to yield with less input energy. The cutters are positioned on the raise head to provide an earth formation contact profile with a major portion of said earth formation contact profile extending outward and upward from said pilot hole. The included angle between the major portion of the earth formation contact profile and the axis of the pilot hole is less than 90/sup 0/.

  1. Exceptionally High Levels of Restriction Site Polymorphism in DNA near the Maize Adh1 Gene

    PubMed Central

    Johns, Mitrick A.; Strommer, Judith N.; Freeling, Michael

    1983-01-01

    Restriction maps have been prepared for the chromosomal region near seven biochemically and genetically distinct maize alcohol dehydrogenase-1 (Adh1) alleles using a small cDNA probe for Adh1. Five restriction sites spanning about 4 kb in and near the Adh1 transcription unit appear identical in all seven alleles. Outside this conserved region, variation in restriction site position is the rule. Six of the seven alleles are distinguishable, and the alleles appear to fall into four groups. The DNA flanking the 1S-type alleles seems to share no restriction site homology with the DNA near the 1F-type alleles. Several hypotheses are put forward to explain how such high levels of polymorphism could have arisen in a species that has been domesticated for only about 10,000 years. PMID:17246173

  2. Restriction endonuclease analysis of leukocyte mitochondrial DNA in Leber's optic atrophy.

    PubMed Central

    Holt, I J; Miller, D H; Harding, A E

    1988-01-01

    In order to test the hypothesis that Leber's optic atrophy may be caused by mutation of the mitochondrial (mt) genome, restriction fragment length polymorphism in leukocyte mt DNA was studied in 16 patients with Leber's optic atrophy, 28 of their unaffected matrilineal relatives, and 35 normal control subjects. No differences in restriction fragment patterns were observed between affected and unaffected individuals in the same maternal line, and there was no evidence of major deletion of mt DNA in patients. This study provides no positive evidence of mitochondrial inheritance in Leber's optic atrophy but does not exclude it. PMID:2905730

  3. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  4. New PDC cutters improve drilling efficiency

    SciTech Connect

    Mensa-Wilmot, G.

    1997-10-27

    New polycrystalline diamond compact (PDC) cutters increase penetration rates and cumulative footage through improved abrasion, impact, interface strength, thermal stability, and fatigue characteristics. Studies of formation characterization, vibration analysis, hydraulic layouts, and bit selection continue to improve and expand PDC bit applications. The paper discusses development philosophy, performance characteristics and requirements, Types A, B, and C cutters, and combinations.

  5. 40 CFR 1065.265 - Nonmethane cutter.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 34 2012-07-01 2012-07-01 false Nonmethane cutter. 1065.265 Section 1065.265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS... sample with purified air or oxygen (O2) upstream of the nonmethane cutter to optimize its...

  6. 40 CFR 1065.265 - Nonmethane cutter.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Nonmethane cutter. 1065.265 Section 1065.265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS... with purified air or oxygen (O2) upstream of the nonmethane cutter to optimize its performance....

  7. 40 CFR 1065.265 - Nonmethane cutter.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Nonmethane cutter. 1065.265 Section 1065.265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS... sample with purified air or oxygen (O2) upstream of the nonmethane cutter to optimize its...

  8. 40 CFR 1065.265 - Nonmethane cutter.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 34 2013-07-01 2013-07-01 false Nonmethane cutter. 1065.265 Section 1065.265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS... sample with purified air or oxygen (O2) upstream of the nonmethane cutter to optimize its...

  9. 40 CFR 1065.265 - Nonmethane cutter.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 33 2011-07-01 2011-07-01 false Nonmethane cutter. 1065.265 Section 1065.265 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR POLLUTION CONTROLS... with purified air or oxygen (O2) upstream of the nonmethane cutter to optimize its performance....

  10. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    PubMed Central

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

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

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  12. A restriction enzyme-powered autonomous DNA walking machine: its application for a highly sensitive electrochemiluminescence assay of DNA

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-12-01

    The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the nanostructure tracks and the Nt.AlwI-assisted cleavage of the overhang sequences in the presence of the walker are verified by using polyacrylamide gel electrophoresis analysis and cyclic voltammetry. The successive movement of the walker on the nanostructure tracks leads to continuous removal of massive ECL labels from the sensing electrode, which results in a significantly amplified suppression of the ECL emission for highly sensitive detection of sequence-specific DNA down to 0.19 pM. Results show that this DNA walking machine can also offer single-base mismatch discrimination capability. The successful application of the DNA walking machine for sequence-specific DNA detection can thus offer new opportunities for molecular machines in biosensing applications.The construction of a restriction enzyme (Nt.AlwI)-powered DNA walking machine and its application for highly sensitive detection of DNA are described. DNA nanostructure tracks containing four overhang sequences with electrochemiluminescence (ECL) labels and complementary to the walker (target DNA) are self-assembled on the sensing electrode. The walker hybridizes with the complementary sequences on the tracks and forms specific recognition sites for Nt.AlwI, which cleaves the overhang sequences, releases the ECL labels and enables directional movement of the walker along the tracks. The formation of the

  13. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism.

    PubMed

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  14. HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

    PubMed Central

    Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

    2016-01-01

    Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

  15. Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.

    PubMed

    Ambur, Ole Herman; Frye, Stephan A; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

    2012-01-01

    Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID

  16. Restriction and Sequence Alterations Affect DNA Uptake Sequence-Dependent Transformation in Neisseria meningitidis

    PubMed Central

    Ambur, Ole Herman; Frye, Stephan A.; Nilsen, Mariann; Hovland, Eirik; Tønjum, Tone

    2012-01-01

    Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination. PMID

  17. Machines and Cutters: Stellaris PC.

    PubMed

    Lai, Timothy Y Y

    2014-01-01

    The Stellaris PC (Bausch & Lomb) is a versatile platform that allows surgeons to perform both anterior segment and posterior segment surgeries. It was developed based on the Stellaris microincision cataract surgery system and can therefore perform <2-mm coaxial or bimanual microincision cataract surgery in combination with posterior segment surgery. Similar to Millenium, its predecessor, the Stellaris PC has an open architecture with modular design that allows products and instruments manufactured by other companies to be used with the system. Vitrectomy can be performed using the traditional 20-gauge system or with microincision 23- or 25-gauge transconjunctival sutureless vitrectomy systems. The special design of the vitreous cutter with increased port area, improved fluidics, and optimized port open time makes the Stellaris PC an efficient system for performing microincision vitrectomy surgery. This article will describe the design, features, and details of various components of the Stellaris PC, including machine controls, entry site alignment trocar/cannula system, fluidics, and high-speed vitreous cutter, as well as its unique illumination system. PMID:25196746

  18. Alignment of Escherichia coli K12 DNA sequences to a genomic restriction map.

    PubMed Central

    Rudd, K E; Miller, W; Ostell, J; Benson, D A

    1990-01-01

    We use the extensive published information describing the genome of Escherichia coli and new restriction map alignment software to align DNA sequence, genetic, and physical maps. Restriction map alignment software is used which considers restriction maps as strings analogous to DNA or protein sequences except that two values, enzyme name and DNA base address, are associated with each position on the string. The resulting alignments reveal a nearly linear relationship between the physical and genetic maps of the E. coli chromosome. Physical map comparisons with the 1976, 1980, and 1983 genetic maps demonstrate a better fit with the more recent maps. The results of these alignments are genomic kilobase coordinates, orientation and rank of the alignment that best fits the genetic data. A statistical measure based on extreme value distribution is applied to the alignments. Additional computer analyses allow us to estimate the accuracy of the published E. coli genomic restriction map, simulate rearrangements of the bacterial chromosome, and search for repetitive DNA. The procedures we used are general enough to be applicable to other genome mapping projects. PMID:2183179

  19. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

    SciTech Connect

    Horton, J. R.; Wang, H.; Mabuchi, M. Y.; Zhang, X.; Roberts, R. J.; Zheng, Y.; Wilson, G. G.; Cheng, X.

    2014-09-27

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

  20. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix

    DOE PAGESBeta

    Horton, J. R.; Wang, H.; Mabuchi, M. Y.; Zhang, X.; Roberts, R. J.; Zheng, Y.; Wilson, G. G.; Cheng, X.

    2014-09-27

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNAmore » molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.« less

  1. Restriction enzyme mapping of ribosomal DNA can distinguish between fasciolid (liver fluke) species.

    PubMed

    Blair, D; McManus, D P

    1989-10-01

    Recognition sites for nine different restriction endonucleases were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit in that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in restriction sites was noted in F. hepatica (individuals from 11 samples studied; hosts were sheep, cattle and laboratory animals; geographical origins. Australia, New Zealand, Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples; Indonesia and Malaysia). Only one sample of F. magna was available. One specimen of Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of F. gigantica. Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the non-transcribed spacer, even within individual worms. PMID:2552311

  2. Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement

    PubMed Central

    2013-01-01

    Background Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. Results Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. Conclusion Deletion of the cbeI gene removes a substantial barrier to routine DNA transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of

  3. Phenolic cutter for machining foam insulation

    NASA Technical Reports Server (NTRS)

    Blair, T. A.; Miller, A. C.; Price, B. W.; Stiles, W. S.

    1970-01-01

    Pre-pregged fiber glass is an efficient abrasive for machining polystyrene and polyurethane foams. It bonds easily to any cutter base made of aluminum, steel, or phenolic, is inexpensive, and is readily available.

  4. Bolt Cutter Blade's Imprint in Toolmarks Examination.

    PubMed

    Volkov, Nikolai; Finkelstein, Nir; Novoselsky, Yehuda; Tsach, Tsadok

    2015-11-01

    Bolt cutters are known as cutting tools which are used for cutting hard objects and materials, such as padlocks and bars. Bolt cutter blades leave their imprint on the cut objects. When receiving a cut object from a crime scene, forensic toolmarks examiners can determine whether the suspected cutting tool was used in a specific crime or not based on class characteristic marks and individual marks that the bolt cutter blades leave on the cut object. The paper presents preliminary results of a study on ten bolt cutters and suggests a quick preliminary examination-the comparison between the blade thickness and the width of the imprint left by the tool on the cut object. Based on the comparison result, if there is not a match, the examiner can eliminate the feasibility of the use of the suspected cutting tool in a specific crime. This examination simplifies and accelerates the comparison procedure. PMID:26257324

  5. Characterization of unrelated strains of Staphylococcus schleiferi by using ribosomal DNA fingerprinting, DNA restriction patterns, and plasmid profiles.

    PubMed Central

    Grattard, F; Etienne, J; Pozzetto, B; Tardy, F; Gaudin, O G; Fleurette, J

    1993-01-01

    The molecular characteristics of 31 unrelated strains of Staphylococcus schleiferi isolated from 13 hospitals between 1973 and 1991 were determined by ribosomal DNA fingerprinting by using a digoxigenin-labeled DNA probe, genomic DNA restriction patterns, and plasmid profiles. Only six strains harbored one or two plasmids. DNA restriction analysis, which was carried out with five endonucleases (EcoRI, HindIII, PstI, PvuII, and ClaI), did not allow us to discriminate between isolates. Ribotyping with HindIII, ClaI, or EcoRI enzymes generated six, seven, and nine distinct patterns, respectively. With the combination ClaI-EcoRI, 13 ribotypes were obtained among the 31 strains, suggesting a relative heterogeneity within the species. Moreover, all strains shared two or three common bands, according to the endonuclease used, which were relatively specific for S. schleiferi in comparison with the ribosomal banding patterns described for other coagulase-negative staphylococci. These results illustrate that ribotyping can be used for the epidemiological investigation of S. schleiferi isolates and possibly for taxonomic analysis in this species. Images PMID:8385149

  6. Jam-Resistant Cutters For Emergency Separation

    NASA Technical Reports Server (NTRS)

    Ordonez, Arturo C.; Yee, Ronald N.

    1990-01-01

    Pyrotechnic emergency-separation system includes shaped explosive charges that sever pair of hinges. System ensures reliable opening of escape hatch. Two pairs of cutters provided for each hinge so if one pair of cutters fails, other completes job. Pressure of explosions vented to prevent charge holders from fragmenting and forming sharp edges around open hatch. Exit slide deployed without tearing. Before detonation L-shaped retainers bear on hinge. After denonation, retainers fold outward to facilitate egress of severed hinges.

  7. Advantages of using the QIAshredder instead of restriction digestion to prepare DNA for droplet digital PCR

    PubMed Central

    Yukl, Steven A.; Kaiser, Philipp; Kim, Peggy; Li, Peilin; Wong, Joseph K.

    2016-01-01

    The viscosity of genomic DNA can interfere with digital PCR systems that partition samples into oil droplets or microfluidic wells. Restriction digestion may reduce the viscosity, but the process is labor-intensive, and the buffer can alter the conditions for PCR. DNA fragmentation using the QIAshredder (a biopolymer spin column) is faster, may result in more predictable and uniformly-sized fragments, and avoids the need for restriction buffers that can inhibit downstream PCR. In 10 separate head-to-head experiments comparing aliquots of DNA processed using the QIAshredder to those digested with RsaI or BsaJI prior to droplet digital PCR, we found that the copy numbers measured from the QIAshredded DNA tended to be greater than those measured from the digested DNA (average of 1.35-fold compared with BsaJI; P < 0.0001), even for inputs as high as 1.8 µg or dilution down to the single copy level. PMID:24724845

  8. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

    PubMed Central

    Horton, John R.; Nugent, Rebecca L.; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M.; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-01-01

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3′ to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5′ to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity. PMID:24604015

  9. Drill bit assembly for releasably retaining a drill bit cutter

    SciTech Connect

    Glowka, David A.; Raymond, David W.

    2002-01-01

    A drill bit assembly is provided for releasably retaining a polycrystalline diamond compact drill bit cutter. Two adjacent cavities formed in a drill bit body house, respectively, the disc-shaped drill bit cutter and a wedge-shaped cutter lock element with a removable fastener. The cutter lock element engages one flat surface of the cutter to retain the cutter in its cavity. The drill bit assembly thus enables the cutter to be locked against axial and/or rotational movement while still providing for easy removal of a worn or damaged cutter. The ability to adjust and replace cutters in the field reduces the effect of wear, helps maintains performance and improves drilling efficiency.

  10. Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism

    SciTech Connect

    Shields, P.G.; Bowman, E.D.; Weston, A.; Harris, C.C.; Sugimura, H.; Caporaso, N.E.; Petruzzelli, S.F. ); Trump, B.F. )

    1992-11-01

    Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the [sup 32]P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a US lung cancer case-control study, no association with lung cancer was found. 17 refs., 3 figs.

  11. Orion Parachute Riser Cutter Development

    NASA Technical Reports Server (NTRS)

    Oguz, Sirri; Salazar, Frank

    2011-01-01

    This paper presents the tests and analytical approach used on the development of a steel riser cutter for the CEV Parachute Assembly System (CPAS) used on the Orion crew module. Figure 1 shows the riser cutter and the steel riser bundle which consists of six individual cables. Due to the highly compressed schedule, initial unavailability of the riser material and the Orion Forward Bay mechanical constraints, JSC primarily relied on a combination of internal ballistics analysis and LS-DYNA simulation for this project. Various one dimensional internal ballistics codes that use standard equation of state and conservation of energy have commonly used in the development of CAD devices for initial first order estimates and as an enhancement to the test program. While these codes are very accurate for propellant performance prediction, they usually lack a fully defined kinematic model for dynamic predictions. A simple piston device can easily and accurately be modeled using an equation of motion. However, the accuracy of analytical models is greatly reduced on more complicated devices with complex external loads, nonlinear trajectories or unique unlocking features. A 3D finite element model of CAD device with all critical features included can vastly improve the analytical ballistic predictions when it is used as a supplement to the ballistic code. During this project, LS-DYNA structural 3D model was used to predict the riser resisting load that was needed for the ballistic code. A Lagrangian model with eroding elements shown in Figure 2 was used for the blade, steel riser and the anvil. The riser material failure strain was fine tuned by matching the dent depth on the anvil with the actual test data. LS-DYNA model was also utilized to optimize the blade tip design for the most efficient cut. In parallel, the propellant type and the amount were determined by using CADPROG internal ballistics code. Initial test results showed a good match with LS-DYNA and CADPROG

  12. Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme

    PubMed Central

    Csefalvay, Eva; Lapkouski, Mikalai; Guzanova, Alena; Csefalvay, Ladislav; Baikova, Tatsiana; Bialevich, Vitali; Shamayeva, Katsiaryna; Janscak, Pavel; Kuta Smatanova, Ivana; Panjikar, Santosh; Carey, Jannette; Weiserova, Marie; Ettrich, Rüdiger

    2015-01-01

    Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling. PMID:26039067

  13. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes.

    PubMed

    Chand, Mahesh K; Nirwan, Neha; Diffin, Fiona M; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D; Saikrishnan, Kayarat

    2015-11-01

    Production of endonucleolytic double-strand DNA breaks requires separate strand cleavage events. Although catalytic mechanisms for simple, dimeric endonucleases are known, there are many complex nuclease machines that are poorly understood. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide after convergent ATP-driven translocation. We report the 2.7-Å resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are located upstream of the direction of translocation, an observation inconsistent with simple nuclease-domain dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex in which the nuclease domains are distal. Sequencing of the products of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand-nicking events combine to produce DNA scission. PMID:26389736

  14. Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

    PubMed Central

    Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

    2015-01-01

    Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

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

    SciTech Connect

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

    1994-08-01

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

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

    PubMed Central

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

    1994-01-01

    We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci. Images Figure 1 Figure 2 PMID:7913584

  17. The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

    PubMed Central

    Gariano, Grazia Rosaria; Dell'Oste, Valentina; Bronzini, Matteo; Gatti, Deborah; Luganini, Anna; De Andrea, Marco; Gribaudo, Giorgio; Gariglio, Marisa; Landolfo, Santo

    2012-01-01

    Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between −54 and −43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor. PMID:22291595

  18. How quantum entanglement in DNA synchronizes double-strand breakage by type II restriction endonucleases.

    PubMed

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism. PMID:26682627

  19. Electrochemical biosensor modified with dsDNA monolayer for restriction enzyme activity determination.

    PubMed

    Zajda, Joanna; Górski, Łukasz; Malinowska, Elżbieta

    2016-06-01

    A simple and cost effective method for the determination of restriction endonuclease activity is presented. dsDNA immobilized at a gold electrode surface is used as the enzymatic substrate, and an external cationic redox probe is employed in voltammetric measurements for analytical signal generation. The assessment of enzyme activity is based on a decrease of a current signal derived from reduction of methylene blue which is present in the sample solution. For this reason, the covalent attachment of the label molecule is not required which significantly reduces costs of the analysis and simplifies the entire determination procedure. The influence of buffer components on utilized dsDNA/MCH monolayer stability and integrity is also verified. Electrochemical impedance spectroscopy measurements reveal that due to pinhole formation during enzyme activity measurement the presence of any surfactants should be avoided. Additionally, it is shown that the sensitivity of the electrochemical biosensor can be tuned by changing the restriction site location along the DNA length. Under optimal conditions the proposed biosensor exhibits a linear response toward PvuII activity within a range from 0.25 to 1.50 U/μL. PMID:26859430

  20. DNA Methylation Pattern in Overweight Women under an Energy-Restricted Diet Supplemented with Fish Oil

    PubMed Central

    do Amaral, Cátia Lira; Milagro, Fermín I.; Curi, Rui; Martínez, J. Alfredo

    2014-01-01

    Dietary factors modulate gene expression and are able to alter epigenetic signatures in peripheral blood mononuclear cells (PBMC). However, there are limited studies about the effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) on the epigenetic mechanisms that regulate gene expression. This research investigates the effects of n-3-rich fish oil supplementation on DNA methylation profile of several genes whose expression has been reported to be downregulated by n-3 PUFA in PBMC: CD36, FFAR3, CD14, PDK4, and FADS1. Young overweight women were supplemented with fish oil or control in a randomized 8-week intervention trial following a balanced diet with 30% energy restriction. Fatty acid receptor CD36 decreased DNA methylation at CpG +477 due to energy restriction. Hypocaloric diet-induced weight loss also reduced the methylation percentages of CpG sites located in CD14, PDK4, and FADS1. The methylation patterns of these genes were only slightly affected by the fish oil supplementation, being the most relevant to the attenuation of the weight loss-induced decrease in CD36 methylation after adjusting by baseline body weight. These results suggest that the n-3 PUFA-induced changes in the expression of these genes in PBMC are not mediated by DNA methylation, although other epigenetic mechanisms cannot be discarded. PMID:24579084

  1. 7 CFR 29.1164 - Cutters (C Group).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... injury. Grades, Grade Names, Minimum Specifications, and Tolerances C1L—Choice Quality Lemon Cutters Ripe.... Uniformity, 90 percent, injury tolerance, 5 percent. C2L—Fine Quality Lemon Cutters Ripe, open leaf structure... tolerance, 10 percent. C3L—Good Quality Lemon Cutters Ripe, open leaf structure, thin, oily, strong...

  2. 7 CFR 29.1164 - Cutters (C Group).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... injury. Grades, Grade Names, Minimum Specifications, and Tolerances C1L—Choice Quality Lemon Cutters Ripe.... Uniformity, 90 percent, injury tolerance, 5 percent. C2L—Fine Quality Lemon Cutters Ripe, open leaf structure... tolerance, 10 percent. C3L—Good Quality Lemon Cutters Ripe, open leaf structure, thin, oily, strong...

  3. 7 CFR 29.1164 - Cutters (C Group).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... injury. Grades, Grade Names, Minimum Specifications, and Tolerances C1L—Choice Quality Lemon Cutters Ripe.... Uniformity, 90 percent, injury tolerance, 5 percent. C2L—Fine Quality Lemon Cutters Ripe, open leaf structure... tolerance, 10 percent. C3L—Good Quality Lemon Cutters Ripe, open leaf structure, thin, oily, strong...

  4. 7 CFR 29.1164 - Cutters (C Group).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... injury. Grades, Grade Names, Minimum Specifications, and Tolerances C1L—Choice Quality Lemon Cutters Ripe.... Uniformity, 90 percent, injury tolerance, 5 percent. C2L—Fine Quality Lemon Cutters Ripe, open leaf structure... tolerance, 10 percent. C3L—Good Quality Lemon Cutters Ripe, open leaf structure, thin, oily, strong...

  5. 7 CFR 29.1164 - Cutters (C Group).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... injury. Grades, Grade Names, Minimum Specifications, and Tolerances C1L—Choice Quality Lemon Cutters Ripe.... Uniformity, 90 percent, injury tolerance, 5 percent. C2L—Fine Quality Lemon Cutters Ripe, open leaf structure... tolerance, 10 percent. C3L—Good Quality Lemon Cutters Ripe, open leaf structure, thin, oily, strong...

  6. Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

    PubMed Central

    Luo, Guan-Zheng; Wang, Fang; Weng, Xiaocheng; Chen, Kai; Hao, Ziyang; Yu, Miao; Deng, Xin; Liu, Jianzhao; He, Chuan

    2016-01-01

    Although extensively studied in prokaryotes, the prevalence and significance of DNA N6-methyladenine (6mA or m6dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts. PMID:27079427

  7. Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA

    SciTech Connect

    Horton, John R.; Borgaro, Janine G.; Griggs, Rose M.; Quimby, Aine; Guan, Shengxi; Zhang, Xing; Wilson, Geoffrey G.; Zheng, Yu; Zhu, Zhenyu; Cheng, Xiaodong

    2014-07-03

    AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves DNA containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ~ 70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ~ 22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

  8. Representative and efficient cloning of satellite DNAs based on PFGE pre-fractionation of restriction digests of genomic DNA.

    PubMed

    Burgtorf, C; Bünemann, H

    1994-06-01

    Using DNA from Drosophila hydei KUN-DH-33 cells we describe an efficient method for selective and representative cloning of complex mixtures of satellite DNAs from eukaryotic genomes. Effective separation of satellite DNA from the bulk of all other sequences it obtained by fractionation of high molecular weight DNA by PFGE after treating it with '6 bp' restriction enzymes. Since extended clusters of tandemly arranged, so called simple sequence, repeats are inert to cleavage by most '6 bp' restriction enzymes the DNA fraction recovered from the gel region > 50 kb is mainly a mixture of satellites. Efficient and representative cloning of this DNA is performed by sonication to an average size of 50-500 bp and ligation of the blunt ended DNA fragments into the Bluescript vector pBS. PMID:7963251

  9. Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF.

    PubMed

    Pogenberg, Vivian; Ogmundsdóttir, Margrét H; Bergsteinsdóttir, Kristín; Schepsky, Alexander; Phung, Bengt; Deineko, Viktor; Milewski, Morlin; Steingrímsson, Eiríkur; Wilmanns, Matthias

    2012-12-01

    Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer. PMID:23207919

  10. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

    PubMed

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  11. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    NASA Astrophysics Data System (ADS)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  12. Molecular discrimination of lactobacilli used as starter and probiotic cultures by amplified ribosomal DNA restriction analysis.

    PubMed

    Roy, D; Sirois, S; Vincent, D

    2001-04-01

    Lactic acid bacteria such as Lactobacillus helveticus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, L. acidophilus, and L. casei related taxa which are widely used as starter or probiotic cultures can be identified by amplified ribosomal DNA restriction analysis (ARDRA). The genetic discrimination of the related species belonging to these groups was first obtained by PCR amplifications by using group-specific or species-specific 16S rDNA primers. The numerical analysis of the ARDRA patterns obtained by using CfoI, HinfI, Tru9I, and ScrFI was an efficient typing tool for identification of species of the L. acidophilus and L. casei complex. ARDRA by using CfoI was a reliable method for differentiation of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. Finally, strains ATCC 393 and ATCC 15820 exhibited unique ARDRA patterns with CfoI and Tru9I restriction enzymes as compared with the other strains of L. casei, L. paracasei, and L. rhamnosus. PMID:11178730

  13. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    PubMed Central

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  14. Comparison of Mycoplasma arthritidis strains by enzyme-linked immunosorbent assay, immunoblotting, and DNA restriction analysis.

    PubMed Central

    Washburn, L R; Voelker, L L; Ehle, L J; Hirsch, S; Dutenhofer, C; Olson, K; Beck, B

    1995-01-01

    Twenty Mycoplasma arthritidis strains or isolates were compared by a combination of enzyme-linked immunosorbent assay by an antiserum adsorption technique, Western immunoblotting, and restriction analysis of chromosomal DNA. Antigenic markers that defined strains related to strains 158p10p9, PG6, and H606 were identified. In addition, restriction analysis allowed all 20 strains to be divided into six groups. Results of restriction analysis corresponded generally with antigenic similarities, although the former did not allow grouping with as fine a precision as the latter. However, intrastrain antigenic variability, which is common among many Mycoplasma species, including M. arthritidis, introduced a complicating factor into our attempts at antigenic analysis. While serologic and antigenic analyses remain useful, we recommend that they be used with caution and in combination with other techniques for identifying and characterizing new isolates and newly acquired strains. Combinations of these techniques have proven to be useful in our laboratory for quality control and for uncovering interesting relationships among strains subjected to animal passage and their less virulent antecedents and among strains originally classified as the same but obtained from different sources and maintained, sometimes for decades, in different laboratories. PMID:7494014

  15. Sharpening ball-nose mill cutters

    NASA Technical Reports Server (NTRS)

    Burch, C. F.

    1977-01-01

    Economical attachment allows faster, more precise grinding. Vibrationless and rigid relation between grinding wheel and cutter allows for extremely high finish and accurate grinding. Leveling device levels flutes with respect to toolholder rotation that generates ball-nose radius. Constant relief around entire profile of cutting edge produces longer tool life.

  16. Magical Thinking in Narratives of Adolescent Cutters

    ERIC Educational Resources Information Center

    Gregory, Robert J.; Mustata, Georgian T.

    2012-01-01

    Adolescents sometimes cut themselves to relieve distress; however, the mechanism is unknown. Previous studies have linked self-injury to deficits in processing emotions symbolically through language. To investigate expressive language of adolescent cutters, the authors analyzed 100 narratives posted on the Internet. Most narratives (n = 66)…

  17. Dome-shaped PDC cutters drill harder rock effectively

    SciTech Connect

    Moran, D.P. )

    1992-12-14

    This paper reports that rock mechanics and sonic travel time log data indicate that bits with convex-shaped polycrystalline diamond compact (PDC) cutters can drill harder rock formations than comparable bits with flat PDC cutters. The Dome-shaped cutters have drilled carbonate formations with sonic travel times as small as 50 [mu]sec/ft, compared to the standard cutoff of 75 [mu]sec/ft for flat PCD cutters. Recent field data from slim hole wells drilled in the Permian basin have shown successful applications of the 3/8-in. Dome cutter in the Grayburg dolomite with its sonic travel times as low as 50-55 [mu]sec/ft and compressive strengths significantly greater than the standard operating range for PDC bit applications. These field data indicate that the Dome cutters can successfully drill hard rock. The convex cutter shape as good impact resistance, cuttings removal, heat dissipation, and wear resistance.

  18. The Role of DNA Restriction-Modification Systems in the Biology of Bacillus anthracis

    PubMed Central

    Sitaraman, Ramakrishnan

    2016-01-01

    Restriction–modification (R–M) systems are widespread among prokaryotes and, depending on their type, may be viewed as selfish genetic elements that persist as toxin–antitoxin modules, or as cellular defense systems against phage infection that confer a selective advantage to the host bacterium. Studies in the last decade have made it amply clear that these two options do not exhaust the list of possible biological roles for R–M systems. Their presence in a cell may also have a bearing on other processes such as horizontal gene transfer and gene regulation. From genome sequencing and experimental data, we know that Bacillus anthracis encodes at least three methylation-dependent (typeIV) restriction endonucleases (RE), and an orphan DNA methyltransferase. In this article, we first present an outline of our current knowledge of R–M systems in B. anthracis. Based on available DNA sequence data, and on our current understanding of the functions of similar genes in other systems, we conclude with hypotheses on the possible roles of the three REs and the orphan DNA methyltransferase. PMID:26834729

  19. A new mtDNA mutation showing accumulation with time and restriction to skeletal muscle

    SciTech Connect

    Weber, K.; Wilson, J.N.; Taylor, L.

    1997-02-01

    We have identified a new mutation in mtDNA, involving tRNA{sup Leu(CUN)} in a patient manifesting an isolated skeletal myopathy. This heteroplasmic A{r_arrow}G transition at position 12320 affects the T{Psi}C loop at a conserved site and was not found in 120 controls. Analysis of cultured fibroblasts, white blood cells/platelets, and skeletal muscle showed that only skeletal muscle contained the mutation and that only this tissue demonstrated a biochemical defect of respiratory-chain activity. In a series of four muscle-biopsy specimens taken over a 12-year period, there was a gradual increase, from 70% to 90%, in the overall level of mutation, as well as a marked clinical deterioration. Single-fiber PCR confirmed that the proportion of mutant mtDNA was highest in cytochrome c oxidase-negative fibers. This study, which reports a mutation involving tRNA{sup Leu(CUN)}, demonstrates clearly that mtDNA point mutations can accumulate over time and may be restricted in their tissue distribution. Furthermore, clinical deterioration seemed to follow the increase in the level of mutation, although, interestingly, the appearance of fibers deficient in respiratory-chain activity showed a lag period. 32 refs., 4 figs., 1 tab.

  20. Identification of individual herbal drugs in tea mixtures using restriction analysis of ITS DNA and real-time PCR.

    PubMed

    Slanc, P; Ravnikar, M; Strukelj, B

    2006-11-01

    We have studied a sedative tea made of Valerianae radix (Valeriana officinalis L.), Lupuli strobuli (Humulus lupulus L.), Melissae folium (Melissa officinalis L.) and Menthae piperitae folium (Mentha piperita L.). In order to identify the constituent drugs a method was established involving amplification of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA on the basis of restriction analysis and real-time PCR. ITS regions of individual drugs were amplified and sequenced. Restriction analysis was performed with selected restriction endonucleases Nae I, PshA I and Xcm I. Real-time PCR was carried out, using primers specifically designed for each individual herbal drug. Real-time PCR proved to be a method for identifying individual herbal drugs in a tea mixture with a single DNA extraction in a single PCR run, since its limit of detection is lower than that for restriction analysis. PMID:17152982

  1. Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

    PubMed Central

    Kawai, J; Hirose, K; Fushiki, S; Hirotsune, S; Ozawa, N; Hara, A; Hayashizaki, Y; Watanabe, S

    1994-01-01

    Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue. Images PMID:7935456

  2. Phylogenomics of phrynosomatid lizards: conflicting signals from sequence capture versus restriction site associated DNA sequencing.

    PubMed

    Leaché, Adam D; Chavez, Andreas S; Jones, Leonard N; Grummer, Jared A; Gottscho, Andrew D; Linkem, Charles W

    2015-03-01

    Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both "recent" and "deep" timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

  3. Method of Rotational Turning With Multifaceted Cutters

    NASA Astrophysics Data System (ADS)

    Indakov, N.; Gordeev, Y.; Binchurov, A.

    2016-04-01

    The proposed method combines the elements of skew turning (in which a cut layer moves along the cutting edge) and rotary turning (in which the sections of the cutting edge are constantly renewed). This leads to better cooling and, hence, a longer tool life. The required cutting speed is ensured by selection of the required number of revolutions of the cutter; the rotation of the machined shaft corresponds to the rotary feed.

  4. Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes

    PubMed Central

    Tóth, Júlia; Bollins, Jack; Szczelkun, Mark D.

    2015-01-01

    DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This ‘DNA sliding’ is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding. PMID:26538601

  5. A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

    PubMed Central

    Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

    2013-01-01

    Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

  6. 55. QUARRY TILE CUTTERS, SECOND FLOOR, NORTH WING. WORKERS PRESSED ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    55. QUARRY TILE CUTTERS, SECOND FLOOR, NORTH WING. WORKERS PRESSED THE CUTTERS INTO SLABS OF CLAY, LIFTED THEM ONTO DRYING BOARDS AND PRESSED THE PLUNGERS TO RELEASE THE CUT TILES. REPRODUCTIONS CUTTERS ARE NOT USED IN PRODUCTION. WOODEN FORMS FOR PRODUCING CLAY SLABS WITH ROLLING PINS REST AGAINST THE WALL. - Moravian Pottery & Tile Works, Southwest side of State Route 313 (Swamp Road), Northwest of East Court Street, Doylestown, Bucks County, PA

  7. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

  8. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  9. DpnA, a methylase for single-strand DNA in the Dpn II restriction system, and its biological function

    SciTech Connect

    Cerritelli, S.; Springhorn, S.S.; Lacks, S.A. )

    1989-12-01

    The two DNA-adenine methylases encoded by the Dpn II restriction gene cassette were purified, and their activities were compared on various DNA substrates. DpnA was able to methylate single-strand DNA and double-strand DNA, whereas DpnM methylated only double-strand DNA. Although both enzymes act at 5{prime}-GATC-3{prime} in DNA, DpnA can also methylate sequences altered in the guanine position, but at a lower rate. A deletion mutation in the dpnA gene was constructed and transferred to the chromosome. Transmission by way of the transformation pathway of methylated and unmethylated plasmids to dpnA mutant and wild-type recipients was examined. The mutant cells restricted unmethylated donor plasmic establishment much more strongly than did wild-type cells. In the wild type, the single strands of donor plasmid DNA that enter by the transformation pathway are apparently methylated by DpnA prior to conversion of the plasmid to a double-strand form, in which the plasmid would be susceptible to the Dpn II endonuclease. The biological function of DpnA may, therefore, be the enhancement of plasmid transfer to Dpn II-containing strains of Streptococcus pneumoniae.

  10. Effect of downhole assembly and cutter geometry on stability of drillstrings equipped with polycrystalline diamond compact (PDC) cutters

    SciTech Connect

    Elsayed, M.A.; Dareing, D.W.; Dupuy, C.A.

    1996-11-01

    Stability of drillstrings equipped with PDC cutters depends on many factors such as the design of the drillstring, design of the cutter, and formation type. In this paper, the authors show that the drill pipe plays a minor role in stability in comparison with that of the drill collars. They also show that the number of cutter blades affects the location of the stability pockets, while their spacing affects the size of these pockets. Using this data, a combination of drill collar and cutter design can be used to provide operating speeds resulting in maximum stability of the drillstring.

  11. Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates.

    PubMed

    Shyma, K P; Gupta, S K; Gupta, J P; Singh, Ajit; Chaudhari, S S; Singh, Veer

    2016-09-01

    The differences or similarities among different isolates of Trypanosoma evansi through endonuclease profile was identified in the present study. The repetitive nuclear DNA of T. evansi isolated from infected cattle, buffalo and equine blood was initially amplified by PCR using specific primers. A panel of restriction enzymes, EcoRI, Eco91l, HindIII and PstI were for complete digestion of PCR products. Agarose gel electrophoresis of digested product did not show cleavage fragments and only single DNA band of the original size was visible in the ethidium bromide stained agarose gel. This indicated that the 227 bp PCR product from repetitive sequence had no site-specific cleavage sites for the REs used in this study. No heterogeneity in the repetitive nuclear DNA restriction endonuclease profile among the different isolates was recorded. PMID:27605842

  12. Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem.

    PubMed

    Triga, D; Pamjav, H; Vellai, T; Fodor, A; Buzás, Z

    1999-06-01

    The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions. PMID:10380768

  13. Enhanced resolution of DNA restriction fragments: a procedure by two-dimensional electrophoresis and double-labeling.

    PubMed Central

    Yi, M; Au, L C; Ichikawa, N; Ts'o, P O

    1990-01-01

    A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an "x,y" coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 35P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map. We have utilized the difference map obtained from Escherichia coli strains HB101 and HB101 (lambda) genomic DNA to show the incorporation of one copy of phage lambda DNA without the use of a lambda DNA

  14. Association mapping of disease resistance traits in rainbow trout using restriction site associated DNA sequencing.

    PubMed

    Campbell, Nathan R; LaPatra, Scott E; Overturf, Ken; Towner, Richard; Narum, Shawn R

    2014-12-01

    Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site-associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs. PMID:25354781

  15. Association Mapping of Disease Resistance Traits in Rainbow Trout Using Restriction Site Associated DNA Sequencing

    PubMed Central

    Campbell, Nathan R.; LaPatra, Scott E.; Overturf, Ken; Towner, Richard; Narum, Shawn R.

    2014-01-01

    Recent advances in genotyping-by-sequencing have enabled genome-wide association studies in nonmodel species including those in aquaculture programs. As with other aquaculture species, rainbow trout and steelhead (Oncorhynchus mykiss) are susceptible to disease and outbreaks can lead to significant losses. Fish culturists have therefore been pursuing strategies to prevent losses to common pathogens such as Flavobacterium psychrophilum (the etiological agent for bacterial cold water disease [CWD]) and infectious hematopoietic necrosis virus (IHNV) by adjusting feed formulations, vaccine development, and selective breeding. However, discovery of genetic markers linked to disease resistance offers the potential to use marker-assisted selection to increase resistance and reduce outbreaks. For this study we sampled juvenile fish from 40 families from 2-yr classes that either survived or died after controlled exposure to either CWD or IHNV. Restriction site−associated DNA sequencing produced 4661 polymorphic single-nucleotide polymorphism loci after strict filtering. Genotypes from individual survivors and mortalities were then used to test for association between disease resistance and genotype at each locus using the program TASSEL. After we accounted for kinship and stratification of the samples, tests revealed 12 single-nucleotide polymorphism markers that were highly associated with resistance to CWD and 19 markers associated with resistance to IHNV. These markers are candidates for further investigation and are expected to be useful for marker assisted selection in future broodstock selection for various aquaculture programs. PMID:25354781

  16. Drill-bit with full offset cutter bodies

    SciTech Connect

    Frear, L.

    1985-11-12

    A rotary drag drill bit is seen wherein cutter bodies are rotatively connected to a main body structure at a fully offset position. The fully offset position is defined by a rotational axis of each cutter body, a longitudinal axis of the drill bit and end support points or positions of the cutter bodies. The rotational axes of the cutter bodies are perpendicular to the longitudinal axis of the drill bit. The end supports of the cutter body are each equal distance from any point on the longitudinal axis of the drill bit. The cutter bodies of essentially ellipsoidal configuration, being slightly thicker at a mid-portion thereof. Cutting elements are connected to flutes projecting above an outer surface of each cutter body. In a primary rotational direction of the drill string and drill bit, the rows abrade the bottom and side walls of a well bore as the cutter body attacks the earth formation as the drill bit is rotated. The impingement of the cutting elements of the cutter body on the earth formation imparts a secondary rotation to the cutter bodies, which secondary rotation is induced by the primary rotation. The secondary rotation allows the rows of cutting elements to engage the side wall of the bore and gauge the hole as well as abrading away material from the bottom of the well bore. A roller bearing assembly is provided for the cutter body to permit the secondary rotation, while a thrust bearing assembly assists the primary abrasive action imparted by the primary rotational movement of the rotary drill bit. A lubrication system is included in the main body structure of the drill bit wherein both the roller bearing assembly and thrust bearing assembly are lubricated.

  17. Reusable Hot-Wire Cable Cutter

    NASA Technical Reports Server (NTRS)

    Pauken, Michael T.; Steinkraus, Joel M.

    2010-01-01

    During the early development stage of balloon deployment systems for missions, nichrome wire cable cutters were often used in place of pyro-actuated cutters. Typically, a nichrome wire is wrapped around a bundle of polymer cables with a low melting point and connected to a relay-actuated electric circuit. The heat from the nichrome reduces the strength of the cable bundle, which quickly breaks under a mechanical load and can thus be used as a release mechanism for a deployment system. However, the use of hand-made heated nichrome wire for cutters is not very reliable. Often, the wrapped nichrome wire does not cut through the cable because it either pulls away from its power source or does not stay in contact with the cable being cut. Because nichrome is not readily soldered to copper wire, unreliable mechanical crimps are often made to connect the nichrome to an electric circuit. A self-contained device that is reusable and reliable was developed to sever cables for device release or deployment. The nichrome wire in this new device is housed within an enclosure to prevent it from being damaged by handling. The electric power leads are internally connected within the unit to the nichrome wire using a screw terminal connection. A bayonet plug, a quick and secure method of connecting the cutter to the power source, is used to connect the cutter to the power leads similar to those used in pyro-cutter devices. A small ceramic tube [0.25-in. wide 0.5-in. long (.6.4-mm wide 13-mm long)] houses a spiraled nichrome wire that is heated when a cable release action is required. The wire is formed into a spiral coil by wrapping it around a mandrel. It is then laid inside the ceramic tube so that it fits closely to the inner surface of the tube. The ceramic tube provides some thermal and electrical insulation so that most of the heat generated by the wire is directed toward the cable bundle in the center of the spiral. The ceramic tube is cemented into an aluminum block, which

  18. The comparative analysis of rocks' resistance to forward-slanting disc cutters and traditionally installed disc cutters

    NASA Astrophysics Data System (ADS)

    Zhang, Zhao-Huang; Fei, Sun; Liang, Meng

    2015-12-01

    At present, disc cutters of a full face rock tunnel boring machine are mostly mounted in the traditional way. Practical use in engineering projects reveals that this installation method not only heavily affects the operation life of disc cutters, but also increases the energy consumption of a full face rock tunnel boring machine. To straighten out this issue, therefore, a rock-breaking model is developed for disc cutters' movement after the research on the rock breaking of forward-slanting disc cutters. Equations of its displacement are established based on the analysis of velocity vector of a disc cutter's rock-breaking point. The functional relations then are brought forward between the displacement parameters of a rock-breaking point and its coordinate through the analysis of micro displacement of a rock-breaking point. Thus, the geometric equations of rock deformation are derived for the forward-slanting installation of disc cutters. With a linear relationship remaining between the acting force and its deformation either before or after the leap breaking, the constitutive relation of rock deformation can be expressed in the form of generalized Hooke law, hence the comparative analysis of the variation in the resistance of rock to the disc cutters mounted in the forward-slanting way with that in the traditional way. It is discovered that with the same penetration, strain of the rock in contact with forward-slanting disc cutters is apparently on the decline, in other words, the resistance of rock to disc cutters is reduced. Thus wear of disc cutters resulted from friction is lowered and energy consumption is correspondingly decreased. It will be useful for the development of installation and design theory of disc cutters, and significant for the breakthrough in the design of full face rock tunnel boring machine.

  19. The comparative analysis of rocks' resistance to forward-slanting disc cutters and traditionally installed disc cutters

    NASA Astrophysics Data System (ADS)

    Zhang, Zhao-Huang; Fei, Sun; Liang, Meng

    2016-08-01

    At present, disc cutters of a full face rock tunnel boring machine are mostly mounted in the traditional way. Practical use in engineering projects reveals that this installation method not only heavily affects the operation life of disc cutters, but also increases the energy consumption of a full face rock tunnel boring machine. To straighten out this issue, therefore, a rock-breaking model is developed for disc cutters' movement after the research on the rock breaking of forward-slanting disc cutters. Equations of its displacement are established based on the analysis of velocity vector of a disc cutter's rock-breaking point. The functional relations then are brought forward between the displacement parameters of a rock-breaking point and its coordinate through the analysis of micro displacement of a rock-breaking point. Thus, the geometric equations of rock deformation are derived for the forward-slanting installation of disc cutters. With a linear relationship remaining between the acting force and its deformation either before or after the leap breaking, the constitutive relation of rock deformation can be expressed in the form of generalized Hooke law, hence the comparative analysis of the variation in the resistance of rock to the disc cutters mounted in the forward-slanting way with that in the traditional way. It is discovered that with the same penetration, strain of the rock in contact with forward-slanting disc cutters is apparently on the decline, in other words, the resistance of rock to disc cutters is reduced. Thus wear of disc cutters resulted from friction is lowered and energy consumption is correspondingly decreased. It will be useful for the development of installation and design theory of disc cutters, and significant for the breakthrough in the design of full face rock tunnel boring machine.

  20. Anti-incendive coal cutter bits

    SciTech Connect

    Cheng, L.; Liebman, I.

    1981-11-09

    The invention is an improved cutter bit, useable with a material cutting mining machine, wherein the bit has an elongated shank portion which retains a cutting tip portion. The tip portion is enlarged in cross-section at its junction with the shank to partially mask the adjacent interface shank surface and thereby protect the shank from exposure at the point of impact. Two basic designs embody the invention. One is a mushroom shaped tip and the other is a dovetail tip/shank design.

  1. Fusion of GFP to the M.EcoKI DNA methyltransferase produces a new probe of Type I DNA restriction and modification enzymes

    SciTech Connect

    Chen, Kai; Roberts, Gareth A.; Stephanou, Augoustinos S.; Cooper, Laurie P.; White, John H.; Dryden, David T.F.

    2010-07-23

    Research highlights: {yields} Successful fusion of GFP to M.EcoKI DNA methyltransferase. {yields} GFP located at C-terminal of sequence specificity subunit does not later enzyme activity. {yields} FRET confirms structural model of M.EcoKI bound to DNA. -- Abstract: We describe the fusion of enhanced green fluorescent protein to the C-terminus of the HsdS DNA sequence-specificity subunit of the Type I DNA modification methyltransferase M.EcoKI. The fusion expresses well in vivo and assembles with the two HsdM modification subunits. The fusion protein functions as a sequence-specific DNA methyltransferase protecting DNA against digestion by the EcoKI restriction endonuclease. The purified enzyme shows Foerster resonance energy transfer to fluorescently-labelled DNA duplexes containing the target sequence and to fluorescently-labelled ocr protein, a DNA mimic that binds to the M.EcoKI enzyme. Distances determined from the energy transfer experiments corroborate the structural model of M.EcoKI.

  2. De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach

    PubMed Central

    2013-01-01

    Background Application of Single Nucleotide Polymorphism (SNP) marker technology as a tool in sunflower breeding programs offers enormous potential to improve sunflower genetics, and facilitate faster release of sunflower hybrids to the market place. Through a National Sunflower Association (NSA) funded initiative, we report on the process of SNP discovery through reductive genome sequencing and local assembly of six diverse sunflower inbred lines that represent oil as well as confection types. Results A combination of Restriction site Associated DNA Sequencing (RAD-Seq) protocols and Illumina paired-end sequencing chemistry generated high quality 89.4 M paired end reads from the six lines which represent 5.3 GB of the sequencing data. Raw reads from the sunflower line, RHA 464 were assembled de novo to serve as a framework reference genome. About 15.2 Mb of sunflower genome distributed over 42,267 contigs were obtained upon assembly of RHA 464 sequencing data, the contig lengths ranged from 200 to 950 bp with an N50 length of 393 bp. SNP calling was performed by aligning sequencing data from the six sunflower lines to the assembled reference RHA 464. On average, 1 SNP was located every 143 bp of the sunflower genome sequence. Based on several filtering criteria, a final set of 16,467 putative sequence variants with characteristics favorable for Illumina Infinium Genotyping Technology (IGT) were mined from the sequence data generated across six diverse sunflower lines. Conclusion Here we report the molecular and computational methodology involved in SNP development for a complex genome like sunflower lacking reference assembly, offering an attractive tool for molecular breeding purposes in sunflower. PMID:23947483

  3. MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5'-|CCNGG-3' sequences.

    PubMed

    Skowron, Marta A; Zebrowska, Joanna; Wegrzyn, Grzegorz; Skowron, Piotr M

    2016-02-01

    A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5'-CCNGG-3' sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5'-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5'-CCNGG-3' REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5'-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations. PMID:26280518

  4. Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

    PubMed Central

    Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

    2015-01-01

    To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding. PMID:26175625

  5. Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.).

    PubMed

    Urasaki, Naoya; Goeku, Satoko; Kaneshima, Risa; Takamine, Tomonori; Tarora, Kazuhiko; Takeuchi, Makoto; Moromizato, Chie; Yonamine, Kaname; Hosaka, Fumiko; Terakami, Shingo; Matsumura, Hideo; Yamamoto, Toshiya; Shoda, Moriyuki

    2015-06-01

    To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a 'piping-leaf-type' cultivar, 'Yugafu', and a 'spiny-tip-leaf-type' variety, 'Yonekura'. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the 'spiny-leaf type' as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding. PMID:26175625

  6. Sequence Capture versus Restriction Site Associated DNA Sequencing for Shallow Systematics.

    PubMed

    Harvey, Michael G; Smith, Brian Tilston; Glenn, Travis C; Faircloth, Brant C; Brumfield, Robb T

    2016-09-01

    Sequence capture and restriction site associated DNA sequencing (RAD-Seq) are two genomic enrichment strategies for applying next-generation sequencing technologies to systematics studies. At shallow timescales, such as within species, RAD-Seq has been widely adopted among researchers, although there has been little discussion of the potential limitations and benefits of RAD-Seq and sequence capture. We discuss a series of issues that may impact the utility of sequence capture and RAD-Seq data for shallow systematics in non-model species. We review prior studies that used both methods, and investigate differences between the methods by re-analyzing existing RAD-Seq and sequence capture data sets from a Neotropical bird (Xenops minutus). We suggest that the strengths of RAD-Seq data sets for shallow systematics are the wide dispersion of markers across the genome, the relative ease and cost of laboratory work, the deep coverage and read overlap at recovered loci, and the high overall information that results. Sequence capture's benefits include flexibility and repeatability in the genomic regions targeted, success using low-quality samples, more straightforward read orthology assessment, and higher per-locus information content. The utility of a method in systematics, however, rests not only on its performance within a study, but on the comparability of data sets and inferences with those of prior work. In RAD-Seq data sets, comparability is compromised by low overlap of orthologous markers across species and the sensitivity of genetic diversity in a data set to an interaction between the level of natural heterozygosity in the samples examined and the parameters used for orthology assessment. In contrast, sequence capture of conserved genomic regions permits interrogation of the same loci across divergent species, which is preferable for maintaining comparability among data sets and studies for the purpose of drawing general conclusions about the impact of

  7. Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

    PubMed Central

    Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

    2004-01-01

    The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the γ- and β-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). γ-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

  8. Measuring motion on DNA by the type I restriction endonuclease EcoR124I using triplex displacement

    PubMed Central

    Firman, Keith; Szczelkun, Mark D.

    2000-01-01

    The type I restriction enzyme EcoR124I cleaves DNA following extensive linear translocation dependent upon ATP hydrolysis. Using protein-directed displacement of a DNA triplex, we have determined the kinetics of one-dimensional motion without the necessity of measuring DNA or ATP hydrolysis. The triplex was pre-formed specifically on linear DNA, 4370 bp from an EcoR124I site, and then incubated with endonuclease. Upon ATP addition, a distinct lag phase was observed before the triplex-forming oligonucleotide was displaced with exponential kinetics. As the distance between type I and triplex sites was shortened, the lag time decreased whilst the displacement reaction remained exponential. This is indicative of processive DNA translocation followed by collision with the triplex and oligonucleotide displacement. A linear relationship between lag duration and inter-site distance gives a translocation velocity of 400 ± 32 bp/s at 20°C. Furthermore, the data can only be explained by bi-directional translocation. An endonuclease with only one of the two HsdR subunits responsible for motion could still catalyse translocation. The reaction is less processive, but can ‘reset’ in either direction whenever the DNA is released. PMID:10790375

  9. Software optimization for electrical conductivity imaging in polycrystalline diamond cutters

    SciTech Connect

    Bogdanov, G.; Ludwig, R.; Wiggins, J.; Bertagnolli, K.

    2014-02-18

    We previously reported on an electrical conductivity imaging instrument developed for measurements on polycrystalline diamond cutters. These cylindrical cutters for oil and gas drilling feature a thick polycrystalline diamond layer on a tungsten carbide substrate. The instrument uses electrical impedance tomography to profile the conductivity in the diamond table. Conductivity images must be acquired quickly, on the order of 5 sec per cutter, to be useful in the manufacturing process. This paper reports on successful efforts to optimize the conductivity reconstruction routine, porting major portions of it to NVIDIA GPUs, including a custom CUDA kernel for Jacobian computation.

  10. Capillary electrophoretic separation of DNA restriction fragments using dilute polymer solutions

    SciTech Connect

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

    1997-02-01

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

  11. Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease.

    PubMed

    Zhang, Can; Lou, Jing; Tu, Wenwen; Bao, Jianchun; Dai, Zhihui

    2015-01-21

    A universal and sensitive electrochemical biosensing platform for the detection and identification of DNA using CdSe quantum dots (CdSe QDs) as signal markers was designed. The detection mechanism was based on the specific recognition of MspI endonuclease combined with the signal amplification of gold nanoparticles (AuNPs). MspI endonuclease could recognize its specific sequence in the double-strand DNA (dsDNA) and cleave the dsDNA fragments linked with CdSe QDs from the electrode. The remaining attached CdSe QDs can be easily read out by square-wave voltammetry using an electrodeposited bismuth (Bi) film-modified glass carbon electrode. The concentrations of target DNA could be simultaneously detected by the signal of metal markers. Using mycobacterium tuberculosis (Mtb) DNA as a model, under the optimal conditions, the proposed biosensor could detect Mtb DNA down to 8.7 × 10(-15) M with a linear range of 5 orders of magnitude (from 1.0 × 10(-14) to 1.0 × 10(-9) M) and discriminate mismatched DNA with high selectivity. This strategy presented a universal and convenient biosensing platform for DNA assay, and its satisfactory performances make it a potential candidate for the early diagnosis of gene-related diseases. PMID:25408952

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

    PubMed

    Kamimura, K; Wakai, S; Sugio, T

    2001-01-01

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

  13. Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

    PubMed Central

    2011-01-01

    Background The TaqII enzyme is a member of the Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III. Results Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC). Conclusions The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical

  14. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    PubMed Central

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  15. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

    PubMed

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-12-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  16. 40 CFR 1065.365 - Nonmethane cutter penetration fractions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Hydrocarbon... a heated catalyst that removes nonmethane hydrocarbons from an exhaust sample stream before the FID analyzer measures the remaining hydrocarbon concentration. An ideal nonmethane cutter would have a...

  17. 40 CFR 1065.365 - Nonmethane cutter penetration fractions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Hydrocarbon... a heated catalyst that removes nonmethane hydrocarbons from an exhaust sample stream before the FID analyzer measures the remaining hydrocarbon concentration. An ideal nonmethane cutter would have a...

  18. 40 CFR 1065.365 - Nonmethane cutter penetration fractions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Hydrocarbon... a heated catalyst that removes nonmethane hydrocarbons from an exhaust sample stream before the FID analyzer measures the remaining hydrocarbon concentration. An ideal nonmethane cutter would have a...

  19. 40 CFR 1065.365 - Nonmethane cutter penetration fractions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Hydrocarbon... a heated catalyst that removes nonmethane hydrocarbons from an exhaust sample stream before the FID analyzer measures the remaining hydrocarbon concentration. An ideal nonmethane cutter would have a...

  20. 40 CFR 1065.365 - Nonmethane cutter penetration fractions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) AIR POLLUTION CONTROLS ENGINE-TESTING PROCEDURES Calibrations and Verifications Hydrocarbon... a heated catalyst that removes nonmethane hydrocarbons from an exhaust sample stream before the FID analyzer measures the remaining hydrocarbon concentration. An ideal nonmethane cutter would have a...

  1. 9. VIEW OF GEAR CUTTER. (Conrail 00018) Built 1913, was ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. VIEW OF GEAR CUTTER. (Conrail 00018) Built 1913, was a PRR piece. Gleason Works, Rochester, New York. - Juniata Shops, Machine Shop No. 1, East of Fourth Avenue at Third Street, Altoona, Blair County, PA

  2. A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting f...

  3. Evaluation of the applicability of amplified rDNA-restriction analysis (ARDRA) to identification of species of the genus Corynebacterium.

    PubMed

    Vaneechoutte, M; Riegel, P; de Briel, D; Monteil, H; Verschraegen, G; De Rouck, A; Claeys, G

    1995-10-01

    The 16S rRNA genes (rDNA) of 50 strains belonging to 26 different coryneform bacterial species and genomospecies and of the type strain of Rhodococcus equi were enzymatically amplified. Amplified rDNA restriction analysis (ARDRA) with the enzymes AluI, CfoI and RsaI was carried out. The combination of the ARDRA patterns obtained after restriction with these three different enzymes enabled the differentiation between the following species: Corynebacterium accolens (number of strains = 2), C. afermentans subsp. afermentans (2), C. afermentans subsp. lipophilum (2), C. amycolatum (3), CDC coryneform group ANF-1-like (1), CDC coryneform group ANF-3-like (1), C. cystitidis (1), C. diphtheriae (4), C. jeikeium (3), C. macginleyi (2), C. minutissimum (1), C. pilosum (1), C. pseudotuberculosis (2), C. renale (2), C. striatum (2), C. urealyticum (3), C. xerosis (1), CDC coryneform groups B-1 (2), B-3 (2), F-1, genomospecies 1 and 2 (6), G, genomospecies 1 (1) and G, genomospecies 2 (2). The following strains or species could not be differentiated from each other: C. pseudodiphtheriticum (2) from C. propinquum (former CDC coryneform group ANF-3) (2), CDC coryneform group F-1, genomospecies 1 (4) from genomospecies 2 (2) and C. jeikeium genomospecies A (1) from genomospecies C (2). ARDRA may represent a possible alternative for identification of coryneforms, since this technique enabled the identification of most coryneforms tested and since DNA extraction (i.e. cell lysis by boiling), amplification, restriction and electrophoresis can be carried out within 8 hours. This might allow quick identification of C. diphtheriae and other possible pathogens of the genus Corynebacterium. PMID:8584787

  4. Mitochondrial DNA variation in chinook salmon and chum salmon detected by restriction enzyme analysis of polymerase chain reaction products

    USGS Publications Warehouse

    Cronin, M.; Spearman, R.; Wilmot, R.; Patton, J.; Bickman, J.

    1993-01-01

    We analyze intraspecific mitochondrial DNA variation in chinook salmon from drainages in the Yukon River, the Kenai River, and Oregon and California rivers; and chum salmon from the Yukon River and vancouver Island, and Washington rivers. For each species, three different portions of the mtDNA molecule were amplified seperately using the polymerase chain reaction and then digested with at least 19 restrictions enzymes. Intraspecific sequence divergences between haplotypes were less than 0.01 base subsitution per nucleotide. Nine chum salmon haplotypes were identified. Yukon River chum salmon stocks displayed more haplotypes (8) occurred in all areas. Seven chinook salmon haplotypes were identified. Four haplotypes occurred in the Yukon and Kenai rviers and four occured in the Oregon/California, with only one haplotype shared between the regions. Sample sizes were too small to quantify the degree of stock seperation among drainages, but the patterns of variation that we observed suggest utility of the technique in genetic stock identification.

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

    PubMed Central

    Hauschild, Tomasz; Stepanović, Srdjan

    2008-01-01

    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. PMID:18832127

  6. Tuning the geometry of shape-restricted DNA molecules on the functionalized Si(1 1 1)

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaochun; Antonopoulos, Ioanna H.; Kumar, Sandip; Chen, Junghuei; Teplyakov, Andrew V.

    2009-11-01

    Designing a well-defined and stable interface between biomolecules and semiconductor surfaces is of great importance for current and future biosensing and bioelectronic applications. The well-characterized chemistry, stability, and easily tunable electronic properties of silicon substrate make it a practical platform for this type of interface. It has been established in our previous work that a robust, covalent attachment between thiol-DNA molecules of a pre-designed geometrical shape and a modified silicon surface can be achieved. This work focuses on using this binding model and altering the distance between the DNA molecules and silicon surface by strategically placing thiol linkers within the pre-determined geometric design of the rectangularly shaped DNA. The statistical analysis of the height profiles of DNA molecules attached to the surface, as determined by AFM, provides specific insight into how the construction of the DNA molecules affects the binding distance. A comparison between two thiol-DNA molecules with different numbers of thiol groups placed either within the rectangular shape or anchored to the free loop of the same geometric design suggest that the average distance of these molecules to the functionalized silicon surface can be changed by approximately 0.5 nm.

  7. Blackboard Electrophoresis: An Inexpensive Exercise on the Principles of DNA Restriction Analysis

    ERIC Educational Resources Information Center

    Costa, M. J.

    2007-01-01

    Undergraduates with little training on molecular biology may find the technical level of the typical introductory restriction laboratory too challenging and have problems with mastering the underlying concepts and processes. "Blackboard electrophoresis" is an active learning exercise, which focuses student attention on the sequences and principles…

  8. Evolutionary Relationships among Five Subspecies of MUS MUSCULUS Based on Restriction Enzyme Cleavage Patterns of Mitochondrial DNA

    PubMed Central

    Yonekawa, Hiromichi; Moriwaki, Kazuo; Gotoh, Osamu; Hayashi, Jun-Ichi; Watanabe, Junko; Miyashita, Nobumoto; Petras, Michael L.; Tagashira, Yusaku

    1981-01-01

    The intra- and intersubspecific genetic distances between five subspecies of Mus musculus were estimated from restriction enzyme cleavage patterns or maps of mitochondrial DNA (mtDNA). The European subspecies, M. m. domesticus and Asian subspecies, M. m. bactrianus, M. m. castaneus, M. m. molossinus and M. m. urbanus were examined. For each subspecies, except M. m. urbanus, at least two local races from widely separated localities were examined. Intrasubspecific heterogeneity was found in the mtDNA cleavage patterns of M. m. bactrianus and M. m. castaneus. M. m. molossinus and M. m. domesticus, however, revealed no intrasubspecific heterogeneity. Four of the subspecies had distinct cleavage patterns. The fifth, M. m. urbanus, had cleavage patterns identical to those of M. m. castaneus with several enzymes. Estimates of genetic distances between the various races and subspecies were obtained by comparing cleavage maps of the mtDNAs with various restriction enzymes. Nucleotide sequence divergences of mtDNA between local races were estimated to be less than 0.4% in M. m. bactrianus and less than 0.3% in M. m. castaneus. The times of divergence of both subspecies were calculated to be 0.1–0.2 x 106 years. These values suggest that the intrasubspecific divergence began some 0.1–0.2 x 106 years ago. On the other hand, nucleotide sequence divergences between European subspecies M. m. domesticus and Asian subspecies M. m. bactrianus and M. m. castaneus were 7.1% and 5.8%, respectively. The times of divergence were calculated to be 2.1–2.6 x 106 years. Further, the nucleotide sequence divergence and time of divergence between M. m. molossinus and the other two Asian subspecies were comparable to those between M. m. molossinus and M. m. domesticus (about 3% and 1 x 106 years, respectively). These results suggest that M. m. molossinus is situated in a unique evolutionary position among Asian subspecies. PMID:6277733

  9. Thrust and torque characteristics based on a new cutter-head load model

    NASA Astrophysics Data System (ADS)

    Liu, Jianqin; Ren, Jiabao; Guo, Wei

    2015-07-01

    Full face rock tunnel boring machine(TBM) has been widely used in hard rock tunnels, however, there are few published theory about cutter-head design, and the design criteria of cutter-head under complex geological is not clear yet. To deal with the complex relationship among geological parameters, cutter parameters, and operating parameters during tunneling processes, a cutter-head load model is established by using CSM(Colorado school of mines) prediction model. Force distribution on cutter-head under a certain geology is calculated with the new established load model, and result shows that inner cutters bear more force than outer cutters, combining with disc cutters abrasion; a general principle of disc cutters' layout design is proposed. Within the model, the relationship among rock uniaxial compressive strength(UCS), penetration and thrust on cutter-head are analyzed, and the results shows that with increasing penetration, cutter thrust increases, but the growth rate slows and higher penetration makes lower special energy(SE). Finally, a fitting mathematical model of ZT(ratio of cutter-head torque and thrust) and penetration is established, and verified by TB880E, which can be used to direct how to set thrust and torque on cutter-head. When penetration is small, the cutter-head thrust is the main limiting factor in tunneling; when the penetration is large, cutter-head torque is the major limiting factor in tunneling. Based on the new cutter-head load model, thrust and torque characteristics of TBM further are researched and a new way for cutter-head layout design and TBM tunneling operations is proposed.

  10. T Cells Detect Intracellular DNA but Fail to Induce Type I IFN Responses: Implications for Restriction of HIV Replication

    PubMed Central

    Kofod-Olsen, Emil; Holm, Christian K.; Melchjorsen, Jesper; Jensen, David G.; Hansen, Anne Louise; Jørgensen, Louise B.; Ostergaard, Lars; Tolstrup, Martin; Larsen, Carsten S.; Paludan, Søren R.; Jakobsen, Martin R.; Mogensen, Trine H.

    2014-01-01

    HIV infects key cell types of the immune system, most notably macrophages and CD4+ T cells. Whereas macrophages represent an important viral reservoir, activated CD4+ T cells are the most permissive cell types supporting high levels of viral replication. In recent years, it has been appreciated that the innate immune system plays an important role in controlling HIV replication, e.g. via interferon (IFN)-inducible restriction factors. Moreover, innate immune responses are involved in driving chronic immune activation and the pathogenesis of progressive immunodeficiency. Several pattern recognition receptors detecting HIV have been reported, including Toll-like receptor 7 and Retinoic-inducible gene-I, which detects viral RNA. Here we report that human primary T cells fail to induce strong IFN responses, despite the fact that this cell type does express key molecules involved in DNA signaling pathways. We demonstrate that the DNA sensor IFI16 migrates to sites of foreign DNA localization in the cytoplasm and recruits the signaling molecules stimulator of IFN genes and Tank-binding kinase, but this does not result in expression of IFN and IFN-stimulated genes. Importantly, we show that cytosolic DNA fails to affect HIV replication. However, exogenous treatment of activated T cells with type I IFN has the capacity to induce expression of IFN-stimulated genes and suppress HIV replication. Our data suggest the existence of an impaired DNA signaling machinery in T cells, which may prevent this cell type from activating cell-autonomous anti-HIV responses. This phenomenon could contribute to the high permissiveness of CD4+ T cells for HIV-1. PMID:24404168

  11. Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ν (POLN)*

    PubMed Central

    Takata, Kei-ichi; Tomida, Junya; Reh, Shelley; Swanhart, Lisa M.; Takata, Minoru; Hukriede, Neil A.; Wood, Richard D.

    2015-01-01

    DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase. PMID:26269593

  12. Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes.

    PubMed

    Kulkarni, Manasi; Nirwan, Neha; van Aelst, Kara; Szczelkun, Mark D; Saikrishnan, Kayarat

    2016-05-19

    Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes. PMID:26975655

  13. Structural insights into DNA sequence recognition by Type ISP restriction-modification enzymes

    PubMed Central

    Kulkarni, Manasi; Nirwan, Neha; van Aelst, Kara; Szczelkun, Mark D.; Saikrishnan, Kayarat

    2016-01-01

    Engineering restriction enzymes with new sequence specificity has been an unaccomplished challenge, presumably because of the complexity of target recognition. Here we report detailed analyses of target recognition by Type ISP restriction-modification enzymes. We determined the structure of the Type ISP enzyme LlaGI bound to its target and compared it with the previously reported structure of a close homologue that binds to a distinct target, LlaBIII. The comparison revealed that, although the two enzymes use almost a similar set of structural elements for target recognition, the residues that read the bases vary. Change in specificity resulted not only from appropriate substitution of amino acids that contacted the bases but also from new contacts made by positionally distinct residues directly or through a water bridge. Sequence analyses of 552 Type ISP enzymes showed that the structural elements involved in target recognition of LlaGI and LlaBIII were structurally well-conserved but sequentially less-conserved. In addition, the residue positions within these structural elements were under strong evolutionary constraint, highlighting the functional importance of these regions. The comparative study helped decipher a partial consensus code for target recognition by Type ISP enzymes. PMID:26975655

  14. Influence of aging and long-term caloric restriction on oxygen radical generation and oxidative DNA damage in rat liver mitochondria.

    PubMed

    López-Torres, Mónica; Gredilla, Ricardo; Sanz, Alberto; Barja, Gustavo

    2002-05-01

    The effect of long-term caloric restriction and aging on the rates of mitochondrial H2O2 production and oxygen consumption as well as on oxidative damage to nuclear (nDNA) and mitochondrial DNA (mtDNA) was studied in rat liver tissue. Long-term caloric restriction significantly decreased H2O2 production of rat liver mitochondria (47% reduction) and significantly reduced oxidative damage to mtDNA (46% reduction) with no changes in nDNA. The decrease in ROS production was located at complex I because it only took place with complex I-linked substrates (pyruvate/malate) but not with complex II-linked substrates (succinate). The mechanism responsible for that decrease in ROS production was not a decrease in mitochondrial oxygen consumption because it did not change after long-term restriction. Instead, the caloric restricted mitochondria released less ROS per unit electron flow, due to a decrease in the reduction degree of the complex I generator. On the other hand, increased ROS production with aging in state 3 was observed in succinate-supplemented mitochondria because old control animals were unable to suppress H2O2 production during the energy transition from state 4 to state 3. The levels of 8-oxodG in mtDNA increased with age in old animals and this increase was abolished by caloric restriction. These results support the idea that caloric restriction reduces the aging rate at least in part by decreasing the rate of mitochondrial ROS production and so, the rate of oxidative attack to biological macromolecules like mtDNA. PMID:11978489

  15. Superhard nanophase cutter materials for rock drilling applications

    SciTech Connect

    Voronov, O.; Tompa, G.; Sadangi, R.; Kear, B.; Wilson, C.; Yan, P.

    2000-06-23

    The Low Pressure-High Temperature (LPHT) System has been developed for sintering of nanophase cutter and anvil materials. Microstructured and nanostructured cutters were sintered and studied for rock drilling applications. The WC/Co anvils were sintered and used for development of High Pressure-High Temperature (HPHT) Systems. Binderless diamond and superhard nanophase cutter materials were manufactured with help of HPHT Systems. The diamond materials were studied for rock machining and drilling applications. Binderless Polycrystalline Diamonds (BPCD) have high thermal stability and can be used in geothermal drilling of hard rock formations. Nanophase Polycrystalline Diamonds (NPCD) are under study in precision machining of optical lenses. Triphasic Diamond/Carbide/Metal Composites (TDCC) will be commercialized in drilling and machining applications.

  16. Improving meat cutters' work: changes and effects following an intervention.

    PubMed

    Vogel, K; Karltun, J; Eklund, J; Engkvist, I-L

    2013-11-01

    Meat cutters face higher risks of injury and musculoskeletal problems than most other occupational groups. The aims of this paper were to describe ergonomics changes implemented in three meat cutting plants and to evaluate effects related to ergonomics on the individual meat cutters and their work. Data was collected by interviews, observations, document studies and a questionnaire (n = 247), as a post intervention study. The changes implemented consisted of reducing knife work to a maximum of 6 h per day and introducing a job rotation scheme with work periods of equal length. Tasks other than traditional meat cutting were added. A competence development plan for each meat cutter and easy adjustment of workplace height were introduced. The questionnaire showed a reduction in perceived physical work load. In general, the changes were perceived positively. Figures from the company showed a positive trend for injuries and sick leave. PMID:23647887

  17. Cleavage of a four-way DNA junction by a restriction enzyme spanning the point of strand exchange.

    PubMed Central

    Murchie, A I; Portugal, J; Lilley, D M

    1991-01-01

    The four-way DNA junction is believed to fold in the presence of metal ions into an X-shaped structure, in which there is pairwise coaxial stacking of helical arms. A restriction enzyme MboII has been used to probe this structure. A junction was constructed containing a recognition site for MboII in one helical arm, positioned such that stacking of arms would result in cleavage in a neighbouring arm. Strong cleavage was observed, at the sites expected on the basis of coaxial stacking. An additional cleavage was seen corresponding to the formation of an alternative stacking isomer, suggesting that the two isomeric forms are in dynamic equilibrium in solution. Images PMID:2001684

  18. Rock Failure and Crack Propagation Beneath Disc Cutters

    NASA Astrophysics Data System (ADS)

    Entacher, Martin; Schuller, E.; Galler, R.

    2015-07-01

    Analyses of rock failure mechanisms beneath disc cutters are presented. Full-scale cutting tests are conducted to assess the global energy input in comparison with rock chips and excavated volume. Small-scale cutting tests are subsequently used for macro- and microscopic analyses of rupture modes and crack propagation. A high spatial resolution allows to obtain pictures of crack networks in different rock types. It is shown that all specimens develop lateral cracks in sufficiently confined areas whereas median cracks typically develop in boundary regions. Regarding cutting forces, a hypothesis is proposed that associates sudden force drops accompanied by sudden sound emission with grain crushing in the proximity of the cutter tip.

  19. Tube cutter tool and method of use for coupon removal

    DOEpatents

    Nachbar, H.D.; Etten, M.P. Jr.; Kurowski, P.A.

    1997-05-06

    A tube cutter tool is insertable into a tube for cutting a coupon from a damaged site on the exterior of the tube. Prior to using the tool, the damaged site is first located from the interior of the tube using a multi-coil pancake eddy current test probe. The damaged site is then marked. A fiber optic probe is used to monitor the subsequent cutting procedure which is performed using a hole saw mounted on the tube cutter tool. Prior to completion of the cutting procedure, a drill in the center of the hole saw is drilled into the coupon to hold it in place. 4 figs.

  20. Tube cutter tool and method of use for coupon removal

    DOEpatents

    Nachbar, Henry D.; Etten, Jr., Marvin P.; Kurowski, Paul A.

    1997-01-01

    A tube cutter tool is insertable into a tube for cutting a coupon from a damaged site on the exterior of the tube. Prior to using the tool, the damaged site is first located from the interior of the tube using a multi-coil pancake eddy current test probe. The damaged site is then marked. A fiber optic probe is used to monitor the subsequent cutting procedure which is performed using a hole saw mounted on the tube cutter tool. Prior to completion of the cutting procedure, a drill in the center of the hole saw is drilled into the coupon to hold it in place.

  1. The Ser176 of T4 endonuclease IV is crucial for the restricted and polarized dC-specific cleavage of single-stranded DNA implicated in restriction of dC-containing DNA in host Escherichia coli

    PubMed Central

    Hirano, Nobutaka; Ohshima, Hiroyuki; Sakashita, Hidenori; Takahashi, Hideo

    2007-01-01

    Endonuclease (Endo) IV encoded by denB of bacteriophage T4 is an enzyme that cleaves single-stranded (ss) DNA in a dC-specific manner. Also the growth of dC-substituted T4 phage and host Escherichia coli cells is inhibited by denB expression presumably because of the inhibitory effect on replication of dC-containing DNA. Recently, we have demonstrated that an efficient cleavage by Endo IV occurs exclusively at the 5′-proximal dC (dC1) within a hexameric or an extended sequence consisting of dC residues at the 5′-proximal and the 3′-proximal positions (dCs tract), in which a third dC residue within the tract affects the polarized cleavage and cleavage rate. Here we isolate and characterize two denB mutants, denB(W88R) and denB(S176N). Both mutant alleles have lost the detrimental effect on the host cell. Endo IV(W88R) shows no enzymatic activity (<0.4% of that of wild-type Endo IV). On the other hand, Endo IV(S176N) retains cleavage activity (17.5% of that of wild-type Endo IV), but has lost the polarized and restricted cleavage of a dCs tract, indicating that the Ser176 residue of Endo IV is implicated in the polarized cleavage of a dCs tract which brings about a detrimental effect on the replication of dC-containing DNA. PMID:17913749

  2. Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease

    PubMed Central

    Shao, Chen; Wang, Chengliang; Zang, Jianye

    2014-01-01

    5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethyl­cytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington’s disease and Alzheimer’s disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethyl­cytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome. PMID:25195760

  3. Enzymatic cleavage of type II restriction endonucleases on the 2'-O-methyl nucleotide and phosphorothioate substituted DNA.

    PubMed

    Zhao, Guojie; Li, Jun; Tong, Zhaoxue; Zhao, Bin; Mu, Runqing; Guan, Yifu

    2013-01-01

    The effects of nucleotide analogue substitution on the cleavage efficiencies of type II restriction endonucleases have been investigated. Six restriction endonucleases (EcoRV, SpeI, XbaI, XhoI, PstI and SphI) were investigated respectively regarding their cleavage when substrates were substituted by 2'-O-methyl nucleotide (2'-OMeN) and phosphorothioate (PS). Substitutions were made in the recognition sequence and the two nucleotides flanking the recognition sequence for each endonuclease. The endonuclease cleavage efficiencies were determined using FRET-based assay. Results demonstrated a position-dependent inhibitory effect of substitution on the cleavage efficiency for all the six endonucleases. In general, the 2'-OMeN substitutions had greater impact than the PS substitutions on the enzymatic activities. Nucleotides of optimal substitutions for protection against RE cleavage were identified. Experimental results and conclusions in this study facilitate our insight into the DNA-protein interactions and the enzymatic cleavage mechanism, particularly for those whose detailed structure information is not available. In addition, the information could benefit the development of bioengineering and synthetic biology. PMID:24260216

  4. ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation.

    PubMed

    Simons, Michelle; Diffin, Fiona M; Szczelkun, Mark D

    2014-10-29

    We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction-modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx- strain, consistent with DNA damage induced by unregulated motor activity. PMID:25260590

  5. DNA duplexes with reactive dialdehyde groups as novel reagents for cross-linking to restriction- modification enzymes.

    PubMed Central

    Brevnov, M G; Gritsenko, O M; Mikhailov, S N; Efimtseva, E V; Ermolinsky, B S; Van Aerschot, A; Herdewijn, P; Repyk, A V; Gromova, E S

    1997-01-01

    To create new, effective reagents for affinity modification of restriction-modification (R-M) enzymes, a regioselective method for reactive dialdehyde group incorporation into oligonucleotides, based on insertion of a 1-beta-D-galactopyranosylthymine residue, has been developed. We synthesized DNA duplex analogs of the substrates of the Eco RII and Mva I R-M enzymes that contained a galactose or periodate-oxidized galactose residue as single substituents either in the center of the Eco RII (Mva I) recognition site or in the flanking nucleotide sequence. The dependence of binding, cleavage and methylation of these substrate analogs on the modified sugar location in the duplex was determined. Cross-linking of the reagents to the enzymes under different conditions was examined. M. Eco RII covalent attachment to periodate-oxidized substrate analogs proceeded in a specific way and to a large extent depended on the location of the reactive dialdehyde group in the substrate. The yield of covalent attachment to a DNA duplex with a dialdehyde group in the flanking sequence with Eco RII or Mva I methylases was 9-20% and did not exceed 4% for R. Eco RII. PMID:9241245

  6. Design theory of full face rock tunnel boring machine transition cutter edge angle and its application

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaohuang; Meng, Liang; Sun, Fei

    2013-05-01

    At present, the inner cutters of a full face rock tunnel boring machine (TBM) and transition cutter edge angles are designed on the basis of indentation test or linear grooving test. The inner and outer edge angles of disc cutters are characterized as symmetric to each other with respect to the cutter edge plane. This design has some practical defects, such as severe eccentric wear and tipping, etc. In this paper, the current design theory of disc cutter edge angle is analyzed, and the characteristics of the rock-breaking movement of disc cutters are studied. The researching results show that the rotational motion of disc cutters with the cutter head gives rise to the difference between the interactions of inner rock and outer rock with the contact area of disc cutters, with shearing and extrusion on the inner rock and attrition on the outer rock. The wear of disc cutters at the contact area is unbalanced, among which the wear in the largest normal stress area is most apparent. Therefore, a three-dimensional model theory of rock breaking and an edge angle design theory of transition disc cutter are proposed to overcome the flaws of the currently used TBM cutter heads, such as short life span, camber wearing, tipping. And a corresponding equation is established. With reference to a specific construction case, the edge angle of the transition disc cutter has been designed based on the theory. The application of TBM in some practical project proves that the theory has obvious advantages in enhancing disc cutter life, decreasing replacement frequency, and making economic benefits. The proposed research provides a theoretical basis for the design of TBM three-dimensional disc cutters whose rock-breaking operation time can be effectively increased.

  7. Hepatic IGF1 DNA methylation is influenced by gender but not by intrauterine growth restriction in the young lamb.

    PubMed

    Carr, D J; Milne, J S; Aitken, R P; Adam, C L; Wallace, J M

    2015-12-01

    Intrauterine growth restriction (IUGR) and postnatal catch-up growth confer an increased risk of adult-onset disease. Overnourishment of adolescent ewes generates IUGR in ∼ 50% of lambs, which subsequently exhibit increased fractional growth rates. We investigated putative epigenetic changes underlying this early postnatal phenotype by quantifying gene-specific methylation at cytosine:guanine (CpG) dinucleotides. Hepatic DNA/RNA was extracted from IUGR [eight male (M)/nine female (F)] and normal birth weight (12 M/9 F) lambs. Polymerase chain reaction was performed using primers targeting CpG islands in 10 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2, H19, insulin receptor, growth hormone receptor, IGF receptors 1 and 2, and the glucocorticoid receptor. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 57 CpG sites. Messenger RNA (mRNA) expression of IGF system genes and plasma IGF1/insulin were determined. DNA methylation was independent of IUGR status but sexual dimorphism in IGF1 methylation was evident (MF (both P<0.001). IGF1 mRNA expression correlated negatively with IGF1 methylation (r=-0.507, P=0.002) and positively with plasma IGF1 (r=0.884, P<0.001). Carcass and empty body weights were greater in males (P=0.002-0.014) and this gender difference in early body conformation was mirrored by sexual dimorphism in hepatic IGF1 DNA methylation, mRNA expression and plasma IGF1 concentrations. PMID:26310177

  8. NEW HIGH STRENGTH AND FASTER DRILLING TSP DIAMOND CUTTERS

    SciTech Connect

    Robert Radtke

    2006-01-31

    The manufacture of thermally stable diamond (TSP) cutters for drill bits used in petroleum drilling requires the brazing of two dissimilar materials--TSP diamond and tungsten carbide. The ENDURUS{trademark} thermally stable diamond cutter developed by Technology International, Inc. exhibits (1) high attachment (shear) strength, exceeding 345 MPa (50,000 psi), (2) TSP diamond impact strength increased by 36%, (3) prevents TSP fracture when drilling hard rock, and (4) maintains a sharp edge when drilling hard and abrasive rock. A novel microwave brazing (MWB) method for joining dissimilar materials has been developed. A conventional braze filler metal is combined with microwave heating which minimizes thermal residual stress between materials with dissimilar coefficients of thermal expansion. The process results in preferential heating of the lower thermal expansion diamond material, thus providing the ability to match the thermal expansion of the dissimilar material pair. Methods for brazing with both conventional and exothermic braze filler metals have been developed. Finite element modeling (FEM) assisted in the fabrication of TSP cutters controllable thermal residual stress and high shear attachment strength. Further, a unique cutter design for absorbing shock, the densification of otherwise porous TSP diamond for increased mechanical strength, and diamond ion implantation for increased diamond fracture resistance resulted in successful drill bit tests.

  9. BRASS FOUNDRY ROOM SHOWING GATE CUTTERS USED TO REMOVE RUNNERS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    BRASS FOUNDRY ROOM SHOWING GATE CUTTERS USED TO REMOVE RUNNERS AND SPRUES FROM BRONZE CASTINGS TOO SOFT TO BE CLEANED IN TUMBLING MILLS. ALSO SHOWN ARE MOLD MACHINES AND THE SAND DELIVERY SYSTEM USED TO CREATE GREEN SAND MOLDS, POURED AT THE OTHER END OF THE GRAVITY CONVEYORS. - Stockham Pipe & Fittings Company, Brass Foundry, 4000 Tenth Avenue North, Birmingham, Jefferson County, AL

  10. 7 CFR 29.3152 - Lugs or Cutters (C Group).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Lugs or Cutters (C Group). 29.3152 Section 29.3152 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE COMMODITY STANDARDS AND STANDARD...

  11. Retail Meat Cutting I. Apprentice Meat Cutter Related Training. Revised.

    ERIC Educational Resources Information Center

    Johnson, Dale H., Ed.

    Intended as a first-year curriculum for apprentice meat cutters, this text focuses on retail meat cutting. Topics covered in the 24 chapters are background and purpose of apprenticeship, job preparation, general layout of the meat department, operational procedures, beef structure and evaluation, retail cuts and cooking methods, beef forequarter:…

  12. The Laser Cutter: A Terrific Addition to Your Tech Program

    ERIC Educational Resources Information Center

    Buxton, Richard

    2007-01-01

    A laser cutter has found a very welcome home in the technology program at Thomas Jefferson High School for Science and Technology. It has proven an easy-to-use major addition. Lasers come in different types, sizes and power ratings, which means several things must be taken into consideration when selecting the right one for the technology program.…

  13. Analysis on the Rock-Cutter Interaction Mechanism During the TBM Tunneling Process

    NASA Astrophysics Data System (ADS)

    Yang, Haiqing; Wang, He; Zhou, Xiaoping

    2016-03-01

    The accurate prediction of rock cutting forces of disc cutters is crucial for tunnel boring machine (TBM) design and construction. Disc cutter wear, which affects TBM penetration performance, has frequently been found at TBM sites. By considering the operating path and wear of the disc cutter, a new model is proposed for evaluating the cutting force and wear of the disc cutter in the tunneling process. The circular path adopted herein, which is the actual running path of the TBM disc cutter, shows that the lateral force of the disc cutter is asymmetric. The lateral forces on the sides of the disc cutter are clearly different. However, traditional solutions are obtained by assuming a linear path, where the later forces are viewed as equal. To simulate the interaction between the rock and disc cutter, a simple brittle damage model for rock mass is introduced here. Based on the explicit dynamic finite element method, the cutting force acting on the rock generated by a single disc cutter is simulated. It is shown that the lateral cutting force of the disc cutter strongly affects the wear extent of disc cutter. The wear mechanism is thus underestimated by the classical model, which was obtained by linear cutting tests. The simulation results are discussed and compared with other models, and these simulation results agree well with the results of present ones.

  14. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    ERIC Educational Resources Information Center

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  15. A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Smith, Maria W.; Ghindilis, Andrei L.; Seoudi, Ihab A.; Smith, Kenneth; Billharz, Rosalind; Simon, Holly M.

    2014-01-01

    PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified

  16. Voluntary Genital Ablations: Contrasting the Cutters and Their Clients

    PubMed Central

    Jackowich, Robyn A; Vale, Rachel; Vale, Kayla; Wassersug, Richard J; Johnson, Thomas W

    2014-01-01

    Introduction Some healthy males voluntarily seek castration without a recognized medical need. There are currently no standards of care for these individuals, which cause many of them to obtain surgery outside of a licensed medical setting. We seek to understand who performs these surgeries. Aim This study aims to characterize individuals who perform or assist in genital ablations outside of the healthcare system. Methods A cross-sectional Internet survey posted on eunuch.org received 2,871 responses. We identified individuals who had performed or assisted in human castrations (“cutters”; n = 98) and compared this group with all other survey respondents (n = 2,773), who had not assisted in castrations. Next we compared the cutters with the voluntary eunuchs. Lastly, because many of the cutters have themselves been castrated, we also divided the physically castrated population (n = 278) into cutters (n = 44) and noncutters (n = 234) and compared them. Main Outcome Measures Self-reported questionnaires were used to collect demographic information, gender identity and presentation, selected childhood experiences, and history of aggressive behaviors, self-harming behaviors, and hospitalization. Results Distinguishing characteristics of cutters included: (i) presenting themselves as very masculine, (ii) having had their longest sexual relationship with a man, (iii) growing up on a farm, (iv) witnessing animal castrations, (v) having a history of sexually inappropriate behavior, (vi) having been threatened with genital mutilation as a child, (vii) having a history of self-harm, (viii) being raised in a devoutly Christian household, (ix) having had an underground castration themselves, and (x) having body piercings and/or tattoos. Conclusions This study may help identify individuals who are at risk of performing illegal castrations. That information may help healthcare providers protect individuals with extreme castration ideations from injuring themselves or others

  17. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing

    PubMed Central

    2012-01-01

    Background Genetic mapping and QTL detection are powerful methodologies in plant improvement and breeding. Construction of a high-density and high-quality genetic map would be of great benefit in the production of superior grapes to meet human demand. High throughput and low cost of the recently developed next generation sequencing (NGS) technology have resulted in its wide application in genome research. Sequencing restriction-site associated DNA (RAD) might be an efficient strategy to simplify genotyping. Combining NGS with RAD has proven to be powerful for single nucleotide polymorphism (SNP) marker development. Results An F1 population of 100 individual plants was developed. In-silico digestion-site prediction was used to select an appropriate restriction enzyme for construction of a RAD sequencing library. Next generation RAD sequencing was applied to genotype the F1 population and its parents. Applying a cluster strategy for SNP modulation, a total of 1,814 high-quality SNP markers were developed: 1,121 of these were mapped to the female genetic map, 759 to the male map, and 1,646 to the integrated map. A comparison of the genetic maps to the published Vitis vinifera genome revealed both conservation and variations. Conclusions The applicability of next generation RAD sequencing for genotyping a grape F1 population was demonstrated, leading to the successful development of a genetic map with high density and quality using our designed SNP markers. Detailed analysis revealed that this newly developed genetic map can be used for a variety of genome investigations, such as QTL detection, sequence assembly and genome comparison. PMID:22908993

  18. The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

    PubMed Central

    Lo Cigno, Irene; De Andrea, Marco; Borgogna, Cinzia; Albertini, Silvia; Landini, Manuela M.; Peretti, Alberto; Johnson, Karen E.; Chandran, Bala; Landolfo, Santo

    2015-01-01

    ABSTRACT The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI

  19. Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

    PubMed Central

    Mandakovic, Dinka; Glasner, Benjamín; Maldonado, Jonathan; Aravena, Pamela; González, Mauricio; Cambiazo, Verónica; Pulgar, Rodrigo

    2016-01-01

    The gram negative facultative bacterium P. salmonis is the etiological agent of Salmonid Rickettsial Septicaemia (SRS), a severe disease that causes important economic losses in the global salmon farmer industry. Despite efforts to control this disease, the high frequency of new epizootic events indicate that the vaccine and antibiotics treatments have limited effectiveness, therefore the preventive and diagnostic approaches must be improved. A comparison of several methodologies for SRS diagnostic indicate differences in their specificity and its capacity to detect other bacteria coexisting with P. salmonis in culture media (contamination) and fish samples (coinfection), aspects relevant for research, vaccine development and clinical diagnostic. By computer-simulation analyses, we identified a group of restriction enzymes that generate unique P. salmonis 16S rDNA band patterns, distinguishable from all other bacteria. From this information, we designed and developed a PCR-RFLP (Polymerase Chain Reaction—Restriction Fragment Length Polymorphism) assay, which was validated using 16S rDNA universal primers and restriction enzyme PmaCI for the amplification and digestion, respectively. Experimental validation was performed by comparing the restriction pattern of P. salmonis with the restriction patterns generated by bacteria that cohabit with P. salmonis (fish bacterial isolates and culture media contaminants). Our results indicate that the restriction enzyme selection pipeline was suitable to design a more specific, sensible, faster and cheaper assay than the currently used P. salmonis detection methodologies. PMID:27242682

  20. Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.

    PubMed Central

    Jeltsch, A; Wenz, C; Stahl, F; Pingoud, A

    1996-01-01

    Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction. Images PMID:8890184

  1. Intrauterine growth restriction leads to changes in sulfur amino acid metabolism, but not global DNA methylation, in Yucatan miniature piglets.

    PubMed

    MacKay, Dylan S; Brophy, Julie D; McBreairty, Laura E; McGowan, Ross A; Bertolo, Robert F

    2012-09-01

    Intrauterine growth restriction (IUGR), in both animals and humans, has been linked to metabolic syndrome later in life. There has been recent evidence that perturbations in sulfur amino acid metabolism may be involved in this early programming phenomenon. Methionine is the precursor for cellular methylation reactions and for the synthesis of cysteine. It has been suggested that the mechanism behind the "fetal origins" of adult diseases may be epigenetic, involving DNA methylation. Because we have recently demonstrated the fetal origins phenomenon in Yucatan miniature swine, we hypothesized that sulfur amino acid metabolism is altered in IUGR piglets. In this study, metabolites and the activities of sulfur amino acid cycle enzymes were analyzed in liver samples of 3- to 5-day-old runt (IUGR: 0.85±0.13 kg) and large (1.36±0.21 kg) Yucatan miniature pig littermates (n=6 pairs). The IUGR piglets had significantly lower specific and total activities of betaine-homocysteine methyltransferase (BHMT) and cystathionine γ-lyase (CGL) than larger littermates (P<.05). Expression of CGL (but not BHMT) mRNA was also lower in IUGR piglets (P<.05). This low CGL reduced cysteine and taurine concentrations in IUGR pigs and led to an accumulation of hepatic cystathionine, with lower homocysteine concentrations. Methylation index and liver global DNA methylation were unaltered. Reduced prenatal growth in Yucatan miniature piglets impairs their remethylation capacity as well as their ability to remove cystathionine and synthesize cysteine and taurine, which could have important implications on long-term health outcomes of IUGR neonates. PMID:22137257

  2. Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference.

    PubMed

    Mastretta-Yanes, A; Arrigo, N; Alvarez, N; Jorgensen, T H; Piñero, D; Emerson, B C

    2015-01-01

    Restriction site-associated DNA sequencing (RADseq) provides researchers with the ability to record genetic polymorphism across thousands of loci for nonmodel organisms, potentially revolutionizing the field of molecular ecology. However, as with other genotyping methods, RADseq is prone to a number of sources of error that may have consequential effects for population genetic inferences, and these have received only limited attention in terms of the estimation and reporting of genotyping error rates. Here we use individual sample replicates, under the expectation of identical genotypes, to quantify genotyping error in the absence of a reference genome. We then use sample replicates to (i) optimize de novo assembly parameters within the program Stacks, by minimizing error and maximizing the retrieval of informative loci; and (ii) quantify error rates for loci, alleles and single-nucleotide polymorphisms. As an empirical example, we use a double-digest RAD data set of a nonmodel plant species, Berberis alpina, collected from high-altitude mountains in Mexico. PMID:24916682

  3. PCR-Restriction Enzyme Analysis for Detection of Candida DNA in Blood from Febrile Patients with Hematological Malignancies

    PubMed Central

    Morace, Giulia; Pagano, Livio; Sanguinetti, Maurizio; Posteraro, Brunella; Mele, Luca; Equitani, Francesco; D’Amore, Giuseppina; Leone, Giuseppe; Fadda, Giovanni

    1999-01-01

    Blood samples were drawn daily from 72 patients who had hematological malignancies, neutropenia, and fever and who had failed to respond to broad-spectrum antibiotics. Each sample was used for conventional fungal blood cultures and for detection and identification of Candida DNA by a PCR method with subsequent restriction enzyme analysis (REA) recently developed in our laboratory. The PCR method was able to detect five CFU of Candida spp. per ml of blood, and subsequent REA of the amplicons allowed the identification of the Candida species most commonly implicated in cases of candidiasis. Thirty-one patients were PCR-REA positive, and four of these patients were also culture positive. The ultimate diagnosis for 13 of these patients and 1 patient who was PCR-REA negative was disseminated candidiasis (confirmed by clinical data, multiple cultures, histology, autopsy, and/or ultrasonographic evidence of hepatosplenic candidiasis). The molecular method is significantly more sensitive than conventional fungal blood cultures and has a high negative predictive value (97.5%) for the development of disseminated candidiasis in neutropenic patients. PMID:10325339

  4. ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

    PubMed Central

    Simons, Michelle; Diffin, Fiona M.; Szczelkun, Mark D.

    2014-01-01

    We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx− strain, consistent with DNA damage induced by unregulated motor activity. PMID:25260590

  5. Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA.

    PubMed

    Villani, F; Moschetti, G; Blaiotta, G; Coppola, S

    1997-05-01

    Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified. Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains. PMID:9172399

  6. Genetic discrimination for three gynogenetic clones of silver carp Hypophthalmichthys molitrix, based on restriction endonuclease analysis of Nd5-Nd6 region of mitochondrial DNA

    NASA Astrophysics Data System (ADS)

    Zhou, Jianfeng; Ye, Yuzhen; Wu, Qingjiang

    2005-03-01

    Three artificial gynogenetic clones of silver carp were produced for the analysis of restriction enzyme digestion patterns of ND5-ND6 region from mtDNA of the clones. It is revealed that all intraclonal individuals shared completely the same digestion patterns but among interclonal individuals did not. The three clones were mixed and cultured in a pond together for two years, and restriction endonuclease digestion patterns of ND5 ND6 were used as genetic markers to assess the growth performance of each clone.

  7. STANDBY TOP AND BOTTOM ROTARY MILLING CUTTERS FOR TORIN LINE. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    STANDBY TOP AND BOTTOM ROTARY MILLING CUTTERS FOR TORIN LINE. SOME PRODUCT FROM THE #43 HOT ROLL IS PROCESSED ON THE TORIN LINE TO REMOVE OXIDIZED SURFACE MATERIAL. IN PRACTICE 15-20/1000 IS CUT FROM THE UPPER AND LOWER SURFACES OF THE STRIP AND RECYCLED TO THE CASTING SHOP. TORIN LINE ADDED AS PART OF 1981 EXPANSION PROGRAM. - American Brass Foundry, 70 Sayre Street, Buffalo, Erie County, NY

  8. SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

    PubMed

    Valdisser, Paula Arielle M R; Pappas, Georgios J; de Menezes, Ivandilson P P; Müller, Bárbara S F; Pereira, Wendell J; Narciso, Marcelo G; Brondani, Claudio; Souza, Thiago L P O; Borba, Tereza C O; Vianello, Rosana P

    2016-06-01

    Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel

  9. Prevention of chaff cutter injuries in rural India.

    PubMed

    Kumar, Adarsh; Singh, J K; Singh, Charanjit

    2013-01-01

    Chaff cutter is an extensively used machine in Indian rural households to chop fodder for feeding draft and mulch to animals. A survey was conducted in five villages of Ghaziabad district of Uttar Pradesh (a northern state of India) to determine the causal factors responsible for chaff cutter injuries. It was observed that major injuries were caused during children playing with the machine and workers feeding the fodder in to the chute. Further, a survey of chaff cutter manufacturers was conducted to determine the critical dimensions of the machine so that safety interventions could be developed. Based on the survey results and mechanism of injuries, three safety interventions were developed to prevent the injuries. These interventions can be retrofitted on old machines and can be incorporated in new machines as well. Experiments were conducted using different fodder crops to observe difficulty in chaff cutting with the safety interventions. It was observed that incorporation of the interventions had no effect on performance of chaff cutting operation. These were retrofitted on existing machines at different locations and the response was very positive. PMID:22551169

  10. Mapping with RAD (restriction-site associated DNA) markers to rapidly identify QTL for stem rust resistance in Lolium perenne.

    PubMed

    Pfender, W F; Saha, M C; Johnson, E A; Slabaugh, M B

    2011-05-01

    A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. A susceptible and a resistant plant were crossed to produce a pseudo-testcross population of 193 F(1) individuals. Markers were produced by the restriction-site associated DNA (RAD) process, which uses massively parallel and multiplexed sequencing of reduced-representation libraries. Additional simple sequence repeat (SSR) and sequence-tagged site (STS) markers were combined with the RAD markers to produce maps for the female (738 cM) and male (721 cM) parents. Stem rust phenotypes (number of pustules per plant) were determined in replicated greenhouse trials by inoculation with a field-collected, genetically heterogeneous population of urediniospores. The F(1) progeny displayed continuous distribution of phenotypes and transgressive segregation. We detected three resistance QTL. The most prominent QTL (qLpPg1) is located near 41 cM on linkage group (LG) 7 with a 2-LOD interval of 8 cM, and accounts for 30-38% of the stem rust phenotypic variance. QTL were detected also on LG1 (qLpPg2) and LG6 (qLpPg3), each accounting for approximately 10% of phenotypic variance. Alleles of loci closely linked to these QTL originated from the resistant parent for qLpPg1 and from both parents for qLpPg2 and qLpPg3. Observed quantitative nature of the resistance may be due to partial-resistance effects against all pathogen genotypes, or qualitative effects completely preventing infection by only some genotypes in the genetically mixed inoculum. RAD markers facilitated rapid construction of new genetic maps in this outcrossing species and will enable development of sequence-based markers linked to stem rust resistance in L. perenne. PMID:21344184

  11. DNA restriction fragment length polymorphism of HLA-DR2 haplotypes in normal individuals and in patients with rheumatoid arthritis.

    PubMed Central

    Singal, D P; Reid, B; Green, D; Bensen, W G; D'Souza, M

    1990-01-01

    A strong association between HLA-DR4 and rheumatoid arthritis (RA) has been found in a number of populations. In contrast, the incidence of DR2 is decreased in patients with RA, suggesting that this specificity may confer some protection against the disease. A number of subtypes of DR2 have been defined by serology, by responses in mixed lymphocyte culture reaction, and, more recently, by restriction fragment length polymorphism. These subtypes of DR2 are in linkage disequilibrium with different subspecificities of DQw1. It is thus likely that the distribution of these subtypic DR,DQ haplotypes in DR2 positive patients with RA may be important in understanding the genetic basis of susceptibility/resistance to RA. In this paper a study of the subtypes of DR2,DQw1 haplotypes in 18 patients with RA, who required sodium aurothiomalate as a disease remitting drug, and unrelated healthy individuals is reported. Three subtypes of DR2 haplotypes, DRw15 (Dw2),DQw1.2(DQw6), DRw15(Dw12),DQw1.12(DQw6), and DRw16(Dw21),DQw1, AZH (DQw5), were analysed with a cDNA probe for the DQ beta gene. The data show that DR2 positive patients with RA carried either the DRw15(Dw2),DQw6 or DRw15(Dw12),DQw6 haplotype. No patient with RA was positive for the DRw16(Dw21),DQw5 subspecificity. In contrast, six of 29 (21%) normal healthy DR2,DQw1 positive individuals carried the DRw16(Dw21),DQw5 haplotype. These data together with earlier results on the distribution of the DR4,DQw7 haplotype in patients with RA support the hypothesis that DQB1 chain polymorphism may be important in determining susceptibility to severe RA. Images PMID:1969727

  12. Construction of High-Density Linkage Maps of Populus deltoides × P. simonii Using Restriction-Site Associated DNA Sequencing

    PubMed Central

    Tong, Chunfa; Li, Huogen; Wang, Ying; Li, Xuran; Ou, Jiajia; Wang, Deyuan; Xu, Houxi; Ma, Chao; Lang, Xianye; Liu, Guangxin; Zhang, Bo; Shi, Jisen

    2016-01-01

    Although numerous linkage maps have been constructed in the genus Populus, they are typically sparse and thus have limited applications due to low throughput of traditional molecular markers. Restriction-site associated DNA sequencing (RADSeq) technology allows us to identify a large number of single nucleotide polymorphisms (SNP) across genomes of many individuals in a fast and cost-effective way, and makes it possible to construct high-density genetic linkage maps. We performed RADSeq for 299 progeny and their two parents in an F1 hybrid population generated by crossing the female Populus deltoides ‘I-69’ and male Populus simonii ‘L3’. A total of 2,545 high quality SNP markers were obtained and two parent-specific linkage maps were constructed. The female genetic map contained 1601 SNPs and 20 linkage groups, spanning 4,249.12 cM of the genome with an average distance of 2.69 cM between adjacent markers, while the male map consisted of 940 SNPs and also 20 linkage groups with a total length of 3,816.24 cM and an average marker interval distance of 4.15 cM. Finally, our analysis revealed that synteny and collinearity are highly conserved between the parental linkage maps and the reference genome of P. trichocarpa. We demonstrated that RAD sequencing is a powerful technique capable of rapidly generating a large number of SNPs for constructing genetic maps in outbred forest trees. The high-quality linkage maps constructed here provided reliable genetic resources to facilitate locating quantitative trait loci (QTLs) that control growth and wood quality traits in the hybrid population. PMID:26964097

  13. Differentiation of Mycobacterial Species by PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA Polymerase Gene (rpoB)

    PubMed Central

    Kim, Bum-Joon; Lee, Keun-Hwa; Park, Bo-Na; Kim, Seo-Jeong; Bai, Gill-Han; Kim, Sang-Jae; Kook, Yoon-Hoh

    2001-01-01

    PCR amplification-restriction analysis (PRA) of rpoB DNA (342 bp), which comprises the Rifr region, was used for the differential identification of 49 mycobacteria. The DNA had been used previously for the identification of mycobacterial species by comparative sequence analysis (B. J. Kim et al., J. Clin. Microbiol. 37:1714–1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and AccII), which were selected on the basis of rpoB DNA sequences, generated distinctive PRA patterns that allowed not only the reference strains but also the clinical isolates of mycobacteria to be distinguished. Both rapidly and slowly growing mycobacteria were distinctly differentiated by HaeIII digestion of the amplified rpoB DNA. By HindII digestion the Mycobacterium tuberculosis complex was distinguished from the other mycobacteria. Furthermore, six subspecies of Mycobacterium kansasii (subspecies I to VI) as well as the closely related Mycobacterium gastri, and other closely related species, were distinguished by simultaneous digestion of MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage specimens. These results suggest that PRA of rpoB DNA is a simple and feasible method not only for the differentiation of culture isolates but also for the rapid detection and identification of pathogenic mycobacteria in primary clinical specimens. PMID:11376042

  14. Mathematical Modeling And Design Optimization Of Plunge Shaving Cutter For Gears With Tooth Modifications

    NASA Astrophysics Data System (ADS)

    Chang, Shinn-Liang; Liu, Jia-Hung

    2009-10-01

    Gears are the most important components in transmission systems. Modifications of gear teeth can accommodate errors and deformations encountered in the manufacture, assembly, and operation of gear pairs. For plunge shaving gears with tooth modifications, the design criteria of cutter clearance manufactured by protuberance hob cutter is investigated. With this novel design, the cutter has better strength and stiffness to keep the shaved gear profile stable. With the analytical descriptions of crowned gear and hence plunge shaving cutter have been constructed so that the grinding wheel can be optimized to minimized the topographic error. Efficiency is greatly improved by avoiding the traditional trial and error method.

  15. Mathematical simulation of a profile cutter as a surface of revolution

    NASA Astrophysics Data System (ADS)

    Bubenchikov, A. M.; Kazakavitschyus, S. M.; Shcherbakov, N. R.

    2016-04-01

    Various types of cutters (spherical, toroidal, etc.) are used in surface processing of parts of a transmission mechanism. The cost of a special profile tool is somewhat higher than that of such cutters. But the increase in the cost of the tool is compensated by a significant reduction in the time of processing parts. The present paper deals with a mathematical model of a profile cutter surface (as a surface of revolution) for processing parts of a cylindrical transmission gear with an eccentrically cycloidal gearing (EC-gearing). A computer program for determining radii of the cutter's circular cross sections for a given set of axial displacements was created.

  16. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII.

    PubMed Central

    Summers, J

    1975-01-01

    Digestion of polyoma viral DNA with a restriction enzyme from Haemophilus aegyptius generates at least 22 unique fragments. The fragments have been characterized with respect to size and physical order on the polyoma genome, and the 5' to 3' orientation of the (+) and (-) strands has been determined. A method for specific radiolabeling of adjacent fragments was employed to establish the fragment order. This technique may be useful for ordering the fragments produced by digestion of complex DNAs. Images PMID:163927

  17. Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

    PubMed Central

    Redecker, D.; Thierfelder, H.; Walker, C.; Werner, D.

    1997-01-01

    A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation. PMID:16535592

  18. Comparative restriction endonuclease maps of proviral DNA of the primate type C simian sarcoma-associated virus and gibbon ape leukemia virus group.

    PubMed Central

    Trainor, C D; Wong-Staal, F; Reitz, M S

    1982-01-01

    Extrachromosomal DNA was purified from canine thymus cells acutely infected with different strains of infectious primate type C viruses of the woolly monkey (simian) sarcoma helper virus and gibbon ape leukemia virus group. All DNA preparations contained linear proviral molecules of 9.1 to 9.2 kilobases, at least some of which represent complete infectious proviral DNA. Cells infected with a replication-defective fibroblast-transforming sarcoma virus and its helper, a replication-competent nontransforming helper virus, also contained a 6.6- to 6.7-kilobase DNA. These proviral DNA molecules were digested with different restriction endonucleases, and the resultant fragments were oriented to the viral RNA by a combination of partial digestions, codigestion with more than one endonuclease, digestion of integrated proviral DNA, and hybridization with 3'- and 5'-specific viral probes. The 3'- and 5'-specific probes each hybridized to fragments from both ends of proviral DNA, indicating that, in common with those of other retroviruses, these proviruses contain a large terminal redundancy at both ends, each of which consists of sequences derived from both the 3' and 5' regions of the viral RNA. The proviral sequences are organized 3',5'-unique-3',5'. Four restriction enzymes (KpnI, SmaI, PstI, and SstI) recognized sites within the large terminal redundancies, and these sites were conserved within all the isolates tested. This suggests that both the 3' and 5' ends of the genomic RNA of these viruses are extremely closely related. In contrast, the restriction sites within the unique portion of the provirus were not strongly conserved within this group of viruses, even though they were related along most of their genomes. Whereas the 5' 60 to 70% of the RNA of these viruses was more closely related by liquid hybridization experiments than was the 3' 30 to 40%, restriction sites within this region were not preferentially conserved, suggesting that small sequence differences or

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

    PubMed

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

    1986-01-01

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

  20. Multiplex genotype determination at a DNA sequence polymorphism cluster in the human immunoglobulin heavy-chain region

    SciTech Connect

    Li, H.; Hood, L.

    1995-03-20

    We have developed a method for multilocus genotype determination. The method involves using restriction fragment length polymorphisms (RFLPs) for allele discrimination. If a polymorphism is not an RFLP, it is converted into an RFLP during the polymerase chain reaction (PCR). After amplification and restriction enzyme digestion, samples are analyzed by sequential gel loading during electrophoresis. The efficiency of this method was demonstrated by determining the genotypes of 108 semen samples at seven DNA sequence polymorphic sites identified in the human immunoglobulin heavy-chain variable region. It was shown that more than 1000 PCR products could be easily analyzed per day per investigator. To show the reliability of this method, some of the typing results were confirmed by DNA sequence analysis. By computer simulation, most (98%) polymorphisms were shown to be natural or convertible (by changing 1 bp close to or next to each polymorphic site) RFLPs for the commercially available 4-base cutters. 47 refs., 4 figs., 3 tabs.

  1. A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site

    NASA Technical Reports Server (NTRS)

    Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.

    1999-01-01

    To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

  2. A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease

    PubMed Central

    Bellamy, Stuart R.W.; Milsom, Susan E.; Kovacheva, Yana S.; Sessions, Richard B.; Halford, Stephen E.

    2007-01-01

    While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer. PMID:17870087

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  4. U.S. Coast Guard cutter personnel on Sweetbriar train their fire ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    U.S. Coast Guard cutter personnel on Sweetbriar train their fire hoses on a burning pleasure boat in an Alaskan harbor. A U.S. Coast Guard rigid-hull inflatable helps with the fire-fighting effort - U.S. Coast Guard Cutter SWEETBRIER, Cordova, Valdez-Cordova Census Area, AK

  5. Geometry and material choices govern hard-rock drilling performance of PDC drag cutters.

    SciTech Connect

    Wise, Jack LeRoy

    2005-06-01

    Sandia National Laboratories has partnered with industry on a multifaceted, baseline experimental study that supports the development of improved drag cutters for advanced drill bits. Different nonstandard cutter lots were produced and subjected to laboratory tests that evaluated the influence of selected design and processing parameters on cutter loads, wear, and durability pertinent to the penetration of hard rock with mechanical properties representative of formations encountered in geothermal or deep oil/gas drilling environments. The focus was on cutters incorporating ultrahard PDC (polycrystalline diamond compact) overlays (i.e., diamond tables) on tungsten-carbide substrates. Parameter variations included changes in cutter geometry, material composition, and processing conditions. Geometric variables were the diamond-table thickness, the cutting-edge profile, and the PDC/substrate interface configuration. Material and processing variables for the diamond table were, respectively, the diamond particle size and the sintering pressure applied during cutter fabrication. Complementary drop-impact, granite-log abrasion, linear cutting-force, and rotary-drilling tests examined the response of cutters from each lot. Substantial changes in behavior were observed from lot to lot, allowing the identification of features contributing major (factor of 10+) improvements in cutting performance for hard-rock applications. Recent field demonstrations highlight the advantages of employing enhanced cutter technology during challenging drilling operations.

  6. 40 CFR 35.3575 - Application of Federal cross-cutting authorities (cross-cutters).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... GRANTS AND OTHER FEDERAL ASSISTANCE STATE AND LOCAL ASSISTANCE Drinking Water State Revolving Funds § 35... assistance makes them applicable. A few cross-cutters apply by their own terms only to the State as the grant..., cross-cutter requirements apply in the following manner: (1) All projects for which a State...

  7. Do leaf-cutter ants orient their path-integrated, home vector with a magnetic compass?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaf-cutter ants Atta colombica forage over 250 m in structurally-complex, Neotropical rainforests that occlude sun or polarized light cues. Night foraging makes the use of celestial cues and landmarks all the more difficult. Typically leaf-cutter ants follow architecturally-modified, pheromonally-m...

  8. [A study on the vibration hazards due to using bush cutters (author's transl)].

    PubMed

    Futatsuka, M

    1979-05-01

    Bush cutters were introduced into Japanese forestry for cutting bush in 1957 or thereabout. Thereafter, they have been used widely not only in forestry but also in agriculture, public engineering, etc. In spite of the fact that vibration acceleration levels of the bush cutters are high, vibration hazards due to using these tools have not been studied. The reason lies in few professions using bush cutters exclusively. Moreover, the tool is only used seasonally from June to September. The author examined 641 bush cutters users in the State Forestry in southern Kyushu and compared the results with a sample of chain saw users matched by age, duration of use, and district. The prevalence rate of Raynaud's phenomenon is 0.4% at 4 years after beginning the use of bush cutters, 4.0% after 10 years and 9.2% after 18 years. The prevalence of Raynaud's phenomenon in chain saw users is twice as great as that in bush cutter users. Bush cutter users are attacked by Raynaud's phenomenon after about 1,000 hours' use of the tool. The difference is regarded to depend on the difference in the density of use and the difference in the levels of vibration acceleration. The vibration hazards due to using bush cutters were characterized by relatively mild complaints in the upper extremities, together with disturbances of peripheral circulation but not always with peripheral neuropathy. PMID:529563

  9. 40 CFR 35.3575 - Application of Federal cross-cutting authorities (cross-cutters).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Application of Federal cross-cutting authorities (cross-cutters). 35.3575 Section 35.3575 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY....3575 Application of Federal cross-cutting authorities (cross-cutters). (a) General. A number of...

  10. A new approach to application of end milling cutters of compound hard metal

    NASA Astrophysics Data System (ADS)

    Mokritsky, B. Ya; Morozova, A. V.; Pustovalov, D. A.

    2016-04-01

    The results of designing end milling cutters having a hard metal cutting point and a shank made of structural steel are shown. The shank and a cutting point are directed and connected. It allows reducing the requirements in expensive integral hard metal milling cutters and defining the field of rational application of integral and compound mills.

  11. STS-51-L Debris Aboard the USGS Cutter Dallas

    NASA Technical Reports Server (NTRS)

    1986-01-01

    On January 28, 1986, the Space Shuttle Challenger and her seven-member crew were lost when a ruptured O-ring in the right Solid Rocket Booster caused an explosion soon after launch. With the help of the U.S. Coast Guard and the U.S. Navy, search and recovery teams began retrieving pieces of the Shuttle from the Atlantic Ocean soon after the accident. Vessels brought the debris to the Trident Basin at Cape Canaveral Air Force Station, where they waited to be shipped to Kennedy Space Center for investigation. The USCG Cutter Dallas transported this fragment of exterior tiling.

  12. Trajectory Calculator for Finite-Radius Cutter on a Lathe

    NASA Technical Reports Server (NTRS)

    Savchenkov, Anatoliy; Strekalov, Dmitry; Yu, Nan

    2009-01-01

    A computer program calculates the two-dimensional trajectory (radial vs. axial position) of a finite-radius-of-curvature cutting tool on a lathe so as to cut a workpiece to a piecewise-continuous, analytically defined surface of revolution. (In the original intended application, the tool is a diamond cutter, and the workpiece is made of a crystalline material and is to be formed into an optical resonator disk.) The program also calculates an optimum cutting speed as F/L, where F is a material-dependent empirical factor and L is the effective instantaneous length of the cutting edge.

  13. Use of single-cutter data in the analysis of PDC bit designs

    SciTech Connect

    Glowka, D.A.

    1986-10-10

    A method is developed for predicting cutter forces, temperatures, and wear on PDC bits as well as integrated bit performance parameters such as weight-on-bit (WOB), drilling torque, and bit imbalance. A computer code called PDCWEAR has been developed to make this method available as a tool for general bit design. The method uses single-cutter data to provide a measure of rock drillability and employs theoretical considerations to account for interaction among closely spaced cutters on the bit. Experimental data are presented to establish the effects of cutter size and wearflat area on the forces that develop during rock cutting. Waterjet assistance is shown to significantly reduce cutting forces, thereby extending bit life and reducing WOB and torque requirements in hard rock. The effects of bit profile, cutter placement density, bit rotary speed, and wear mode on bit life and drilling performance are investigated. 21 refs., 34 figs., 4 tabs.

  14. Wear analysis of disc cutters of full face rock tunnel boring machine

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaohuang; Meng, Liang; Sun, Fei

    2014-11-01

    Wear is a major factor of disc cutters' failure. No current theory offers a standard for the prediction of disc cutter wear yet. In the field the wear prediction method commonly used is based on the excavation length of tunnel boring machine(TBM) to predict the disc cutter wear and its wear law, considering the location number of each disc cutter on the cutterhead(radius for installation); in theory, there is a prediction method of using arc wear coefficient. However, the preceding two methods have their own errors, with their accuracy being 40% or so and largely relying on the technicians' experience. Therefore, radial wear coefficient, axial wear coefficient and trajectory wear coefficient are defined on the basis of the operating characteristics of TBM. With reference to the installation and characteristics of disc cutters, those coefficients are modified according to penetration, which gives rise to the presentation of comprehensive axial wear coefficient, comprehensive radial wear coefficient and comprehensive trajectory wear coefficient. Calculation and determination of wear coefficients are made with consideration of data from a segment of TBM project(excavation length 173 m). The resulting wear coefficient values, after modification, are adopted to predict the disc cutter wear in the follow-up segment of the TBM project(excavation length of 5621 m). The prediction results show that the disc cutter wear predicted with comprehensive radial wear coefficient and comprehensive trajectory wear coefficient are not only accurate(accuracy 16.12%) but also highly congruous, whereas there is a larger deviation in the prediction with comprehensive axial wear coefficient(accuracy 41%, which is in agreement with the prediction of disc cutters' life in the field). This paper puts forth a new method concerning prediction of life span and wear of TBM disc cutters as well as timing for replacing disc cutters.

  15. Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by pulsed electron-electron double resonance spectroscopy.

    PubMed

    Gophane, Dnyaneshwar B; Endeward, Burkhard; Prisner, Thomas F; Sigurdsson, Snorri Th

    2014-11-24

    Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, (ExIm)U, was synthesized and incorporated into DNA oligonucleotides. (ExIm)U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base. Continuous-wave (CW) EPR measurements of (ExIm)U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label (Im)U, which has restricted mobility due to an intramolecular hydrogen bond. The X-band CW-EPR spectra of (ExIm)U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels (Im)U, (Ox)U, and (ExIm)U in duplex DNA showed a strong angular dependence for (Im)U, a medium dependence for (Ox)U, and no orientation effect for (ExIm)U. Thus, precise distances can be extracted from (ExIm)U without having to take orientational effects into account. PMID:25296640

  16. The beyond 12/23 restriction is imposed at the nicking and pairing steps of DNA cleavage during V(D)J recombination.

    PubMed

    Drejer-Teel, Anna H; Fugmann, Sebastian D; Schatz, David G

    2007-09-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jbeta substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  17. The Beyond 12/23 Restriction Is Imposed at the Nicking and Pairing Steps of DNA Cleavage during V(D)J Recombination▿

    PubMed Central

    Drejer-Teel, Anna H.; Fugmann, Sebastian D.; Schatz, David G.

    2007-01-01

    The beyond 12/23 (B12/23) rule ensures inclusion of a Dβ gene segment in the assembled T-cell receptor (TCR) β variable region exon and is manifest by a failure of direct Vβ-to-Jβ gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRβ gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRβ locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRβ locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jβ substrates and to some extent through poor synapsis of Vβ and Jβ substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled primarily by the RSS nonamer. The results demonstrate that different Jβ substrates are crippled at different steps of cleavage by distinct combinations of defects in the various DNA elements and strongly suggest that the DNA nicking step of V(D)J recombination can be rate limiting in vivo. PMID:17636023

  18. Stock Structure and Homing Fidelity in Gulf of Mexico Sturgeon (Acipenser Oxyrinchus Desotoi) Based on Restriction Fragment Length Polymorphism and Sequence Analyses of Mitochondrial DNA

    PubMed Central

    Stabile, J.; Waldman, J. R.; Parauka, F.; Wirgin, I.

    1996-01-01

    Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FL, (3) Choctawhatchee River, FL, and (4) Apalachicola, Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids. PMID:8889537

  19. Stock structure and homing fidelity in Gulf of Mexico sturgeon (Acipenser oxyrinchus desotoi) based on restriction fragment length polymorphism and sequence analyses of mitochondrial DNA.

    PubMed

    Stabile, J; Waldman, J R; Parauka, F; Wirgin, I

    1996-10-01

    Efforts have been proposed worldwide to restore sturgeon populations through the use of hatcheries to supplement natural reproduction and to reintroduce sturgeon where they have become extinct. We examined the population structure and inferred the extent of homing in the anadromous Gulf of Mexico (Gulf) sturgeon (Acipenser oxyrinchus desotoi). Restriction fragment length polymorphism and control region sequence analyses of mitochondrial DNA (mtDNA) were used to identify haplotypes of Gulf sturgeon specimens obtained from eight drainages spanning the subspecies' entire distribution from Louisiana to Florida. Significant differences in haplotype frequencies indicated substantial geographic structuring of populations. A minimum of four regional or river-specific populations were identified (from west to east): (1) Pearl River, LA and Pascagoula River, MS, (2) Escambia and Yellow rivers, FI, (3) Choctawbatchee River, FL and (4) Apalachicola Ochlockonee, and Suwannee rivers, FL. Estimates of maternally mediated gene flow between any pair of the four regional or river-specific stocks ranged between 0.15 to 1.2. Tandem repeats in the mtDNA control region of Gulf sturgeon were not perfectly conserved. This result, together with an absence of heteroplasmy and length variation in Gulf sturgeon mtDNA, indicates that the molecular mechanisms of mtDNA control region sequence evolution differ among acipenserids. PMID:8889537

  20. Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

    PubMed Central

    Callahan, Scott J.; Luyten, Yvette A.; Gupta, Yogesh K.; Wilson, Geoffrey G.; Roberts, Richard J.; Morgan, Richard D.; Aggarwal, Aneel K.

    2016-01-01

    The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others. PMID:27082731

  1. Intra- and Interspecific Variation of the Mitochondrial Genome in RATTUS NORVEGICUS and RATTUS RATTUS: Restriction Enzyme Analysis of Variant Mitochondrial DNA Molecules and Their Evolutionary Relationships

    PubMed Central

    Brown, Gregory G.; Simpson, Melvin V.

    1981-01-01

    Restriction endonuclease analysis has revealed extensive mtDNA polymorphism in two species of rats, Rattus rattus and Rattus norvegicus. Sequence divergence values for the eight detected R. norvegicus variants range from 0.2% to 1.8% and for the eight R. rattus variants, from 0.2% to 9.6%. Three of the most closely related R. norvegicus mtDNA's appear to differ by deletions/insertions of about 4 base pairs apiece. Restriction sites for seven enzymes have been mapped for 11 of these variants. The 31 intraspecific and 41 interspecific variant sites appear to be evenly distributed on the mtDNA molecule outside of the rRNA cistrons. The location of sites present in all the DNAs suggests that the rRNA genes and possibly the light strand origin of replication may be more highly evolutionarily conserved than other parts of the molecule. The sequence divergences among the mtDNAs of animals whose geographic origins are separated by major barriers, such as oceans, were significantly greater than those among animals found within large land masses, such as the continental United States. Dendrograms (phenograms), which have been constructed to depict the relationships among the various DNAs, indicate that East Asian members of the R. rattus species are more closely related to American rats of this species than to other Asian R. rattus animals from Sri Lanka. Moreover, it appears that R. norvegicus comprises a group taxonomically distinct from any of the R. rattus subspecies. PMID:6266911

  2. Propagation of restriction fragments from the mitochondrial DNA of Saccharomyces cerevisiae in E. coli by means of plasmid vectors.

    PubMed Central

    Berg, P E; Lewin, A; Christianson, T; Rabinowitz, M

    1979-01-01

    Some of the EcoRI fragments of yeast (Saccharomyces cerevisiae) mitochondrial DNA were cloned into E. coli using plasmid pMB9. The five smallest fragments in molecular weight appeared to be preferentially retained by E coli; partial fragments derived from larger mitochondrial DNA fragments were also found. One of the fragments, R7 (2.4 kb), may contain the OII gene. Cloned R7 DNA was stable under a variety of growth conditions, but showed some changes in molecular weight after transfer to different E. coli strains. Fragment R7 is transcribed in minicells, producing RNA that hybridizes specifically to mitochondrial DNA. Both DNA strands are transcribed, in contrast to the asymmetric transcription found in mitochondria. No new polypeptides were observed in minicells containing cloned fragment 7. Images PMID:379817

  3. Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

    PubMed Central

    Zarrin, Majid; Erfaninejad, Maryam

    2016-01-01

    Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085

  4. Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    ZARRIN, MAJID; GANJ, FARZANEH; FARAMARZI, SAMA

    2016-01-01

    The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species. PMID:27073635

  5. Study on the health hazards of scrap metal cutters.

    PubMed

    Ho, S F; Wong, P H; Kwok, S F

    1989-12-01

    Scrap metal cutters seemed to be left out in most preventive programmes as the workers were mainly contract workers. The health hazards of scrap metal cutting in 54 workers from a foundry and a ship breaking plant were evaluated. Environmental sampling showed lead levels ranging from 0.02 to 0.57 mg/m3 (threshold limit values is 0.15 mg/m3). Exposure to lead came mainly from the paint coat of the metals cut. Metal fume fever was not reported although their main complaints were cough and rhinitis. Skin burns at all stages of healing and residual scars were seen over hands, forearms and thighs. 96% of the cutters had blood lead levels exceeding 40 micrograms/100 ml with 10 workers exceeding 70 micrograms/100 ml. None had clinical evidence of lead poisoning. The study showed that scrap metal cutting is a hazardous industry associated with significant lead exposure. With proper medical supervision, the blood lead levels of this group of workers decreased illustrating the importance of identifying the hazard and implementing appropriate medical surveillance programmes. PMID:2635395

  6. Rapid prototyping of multiphase microfluidics with robotic cutters

    NASA Astrophysics Data System (ADS)

    Li, Zidong; Zhao, Zhengtuo; Lo, Joe Fu-jiou

    2014-03-01

    Microfluidic devices offer novel techniques to address biological and biomedical issues. Standard microfluidic fabrication uses photolithography to pattern channels on silicon wafers with high resolution. Even the relatively straightforward SU8 and soft lithography in microfluidics require investing and training in photolithography, which is also time consuming due to complicated thick resist procedures, including sensitive substrate pretreatment, coating, soft bake, expose, post-exposure bake, and developing steps. However, for applications where low resolution (>200 μm) and high turn-around (> 4 designs/day) prototyping are met with little or no lithography infrastructure, robotic cutters [1] offer flexible options for making glass and PDMS microfluidics. We describe the use of robotics cutters for designing microfluidic geometries, and compliment it with safe glass etching, with depths down to 60 μm. Soft lithography patterning of 200 μm thick PDMS membrane was also explored. Without high equipment investment and lengthy student training, both glass and PDMS microfluidics can be achieved in small facilities using this technique.

  7. Efficiency and daily work effort in sugar cane cutters.

    PubMed

    Spurr, G B; Barac-Nieto, M; Maksud, M G

    1977-05-01

    Productivity (metric tons (tonnes)/day), efficiency (kg cane cut/litre Vo2), and effort (percent Vo2 max sustained during an 8-hour workday) have been measured in 54 Colombian sugar cane cutters. In workers who sustained less than 40% Vo2 max during the workday, the effort expended was related to productivity (4 = 0-71) but efficiency and productivity were not significantly correlated. In 16 workers sustaining a greater than 40% Vo2 max during the workday, productivity and effort were not related and efficiency was significantly reduced. Subjects using less than 40% Vo2 max were divided into good, average and poor producers and compared with the men with low efficiencies. In general, these inefficient men had the anthropometric and physical fitness characteristics of low productivity workers (smaller stature, weight and Vo2 max). However, the frequency of good, average, and poor cutters in the inefficient group did not differ from that of the men expending less than 40% of their maximum effort nor was their average productivity different. No obvious reasons for the differences in efficiency and effort of these men were found. PMID:871445

  8. Representation of Clinical Nursing Protocols Using GEM II & GEM Cutter

    PubMed Central

    Koch, Karen A.; Woodcock, Michael W.; Harris, Marcelline R.

    2010-01-01

    The machineable representation and execution of clinical guidelines has been the focus of research efforts for some time, however there is less examination of whether the methods and techniques for guidelines are sufficient for clinical protocols. The objective of this study was to test the feasibility of using the Guideline Elements Model II (GEM II) and GEM Cutter for the representation of clinical protocols, specifically clinical protocols commonly used by nurses. After downloading the GEM Cutter 2.5, we decomposed a set of clinical protocols and analyzed the completeness in which elemental protocol data was represented. One of the most complicated of these protocols (extravasations of infused medication) is presented as an example. While GEM II adequately represents core elements of clinical protocols at the high level, it was not possible to adequately represent sequence and associated role based permissions via use of conditional criteria at branching and procedural levels. Functionality of the tool would also be enhanced with more robust terminology management and support for multi-authoring. PMID:21347008

  9. The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

    PubMed Central

    Serfiotis-Mitsa, Dimitra; Herbert, Andrew P.; Roberts, Gareth A.; Soares, Dinesh C.; White, John H.; Blakely, Garry W.; Uhrín, Dušan; Dryden, David T. F.

    2010-01-01

    Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic. PMID:20007596

  10. Long-Term Effects of Caloric Restriction or Exercise on DNA and RNA Oxidation Levels in White Blood Cells and Urine in Humans

    PubMed Central

    Hofer, Tim; Fontana, Luigi; Weiss, Edward P.; Villareal, Dennis; Malayappan, Bhaskar

    2008-01-01

    Abstract Excessive adiposity is associated with increased oxidative stress and accelerated aging. Weight loss induced by negative energy balance reduces markers of oxidation in experimental animals and humans. The long-term effects of weight loss induced by calorie restriction or increased energy expenditure induced by exercise on measures of oxidative stress and damage have not been studied in humans. The objective of the present study was to compare the effects of 20% caloric restriction or 20% exercise alone over 1 year on oxidative damage to DNA and RNA, as assessed through white blood cell and urine analyses. Eighteen men and women aged 50 to 60 years with a body mass index (BMI) between 23.5 to 29.9 kg/m2 were assigned to one of two conditions — 20% CR (n = 9) or 20% EX (n = 9) — which was designed to produce an identical energy deficit through increased energy expenditure. Compared to baseline, both interventions significantly reduced oxidative damage to both DNA (48.5% and 49.6% reduction for the CR and EX groups, respectively) and RNA (35.7% and 52.1% reduction for the CR and EX groups, respectively) measured in white blood cells. However, urinary levels of DNA and RNA oxidation products did not differ from baseline values following either 12-month intervention program. Data from the present study provide evidence that negative energy balances induced through either CR or EX result in substantial and similar improvements in markers of DNA and RNA damage to white blood cells, potentially by reducing systemic oxidative stress. PMID:18729811

  11. Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato.

    PubMed

    Shirasawa, Kenta; Hirakawa, Hideki; Isobe, Sachiko

    2016-04-01

    Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i)in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. PMID:26932983

  12. Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in tomato

    PubMed Central

    Shirasawa, Kenta; Hirakawa, Hideki; Isobe, Sachiko

    2016-01-01

    Double-digest restriction site-associated DNA sequencing (ddRAD-Seq) enables high-throughput genome-wide genotyping with next-generation sequencing technology. Consequently, this method has become popular in plant genetics and breeding. Although computational in silico prediction of restriction sites from the genome sequence is recognized as an effective approach for choosing the restriction enzymes to be used, few reports have evaluated the in silico predictions in actual experimental data. In this study, we designed and demonstrated a workflow for in silico and empirical ddRAD-Seq analysis in tomato, as follows: (i) in silico prediction of optimum restriction enzymes from the reference genome, (ii) verification of the prediction by actual ddRAD-Seq data of four restriction enzyme combinations, (iii) establishment of a computational data processing pipeline for high-confidence single nucleotide polymorphism (SNP) calling, and (iv) validation of SNP accuracy by construction of genetic linkage maps. The quality of SNPs based on de novo assembly reference of the ddRAD-Seq reads was comparable with that of SNPs obtained using the published reference genome of tomato. Comparisons of SNP calls in diverse tomato lines revealed that SNP density in the genome influenced the detectability of SNPs by ddRAD-Seq. In silico prediction prior to actual analysis contributed to optimization of the experimental conditions for ddRAD-Seq, e.g. choices of enzymes and plant materials. Following optimization, this ddRAD-Seq pipeline could help accelerate genetics, genomics, and molecular breeding in both model and non-model plants, including crops. PMID:26932983

  13. Rock deformation equations and application to the study on slantingly installed disc cutter

    NASA Astrophysics Data System (ADS)

    Zhang, Zhao-Huang; Meng, Liang; Sun, Fei

    2014-08-01

    At present the mechanical model of the interaction between a disc cutter and rock mainly concerns indentation experiment, linear cutting experiment and tunnel boring machine (TBM) on-site data. This is not in line with the actual rock-breaking movement of the disc cutter and impedes to some extent the research on the rock-breaking mechanism, wear mechanism and design theory. Therefore, our study focuses on the interaction between the slantingly installed disc cutter and rock, developing a model in accordance with the actual rock-breaking movement. Displacement equations are established through an analysis of the velocity vector at the rock-breaking point of the disc cutter blade; the functional relationship between the displacement parameters at the rock-breaking point and its rectangular coordinates is established through an analysis of micro-displacement vectors at the rock-breaking point, thus leading to the geometric equations of rock deformation caused by the slantingly installed disc cutter. Considering the basically linear relationship between the cutting force of disc cutters and the rock deformation before and after the leap break of rock, we express the constitutive relations of rock deformation as generalized Hooke's law and analyze the effect of the slanting installation angle of disc cutters on the rock-breaking force. This will, as we hope, make groundbreaking contributions to the development of the design theory and installation practice of TBM.

  14. Does Diatomaceous Earth Control Leaf-Cutter Ants (Hymenoptera: Formicidae) in the Eucalyptus Plantations?

    PubMed

    Ferreira-Filho, Pedro J; Wilcken, Carlos F; Neves, Daniela A; Pogetto, Mario H F A D; Carmo, Janaina B; Guerreiro, Julio C; Serrão, José E; Zanuncio, José C

    2015-06-01

    Genus Atta includes some of the most important Formicidae leaf cutter ants which cause extensive damage to the eucalyptus plantations. Atta sexdens rubropilosa Forel, one of the chief pests in Brazilian reforestation, can restrict and reduce forest productivity by its intense and constant leaf-cutting activities on plants at all stages. Therefore, the demand for new products to control A. sexdens rubropilosa indicates the study of the utilization of the dry powder formulation of diatomaceous earth (DE) against this pest in the eucalyptus cultivars. The study was conducted using 120 colonies of A. sexdens rubropilosa in Eucalyptus grandis Hill ex. Maiden x Eucalyptus urophylla Blake (Myrtaceae) (urograndis) stand. The randomized block experimental design was used with six treatments (1, 10, 25, and 50 g/m2 of DE, 6.0 g/m2 sulfluramid bait per square meter of loose soil, and the control) with five replications, each with four colonies of this ant. Diatomaceous earth was applied to the active A. sexdens rubropilosa ant holes, and the sulfluramid bait was applied in bulk in a localized manner. The control efficacy of A. sexdens rubropilosa with DE was low, showing values similar to that of the control, and, for this reason, it cannot be used to control this ant. The bait with sulfluramid showed higher efficacy than those of the other treatments. PMID:26470237

  15. Role of the single deaminase domain APOBEC3A in virus restriction, retrotransposition, DNA damage and cancer.

    PubMed

    Wang, Yaqiong; Schmitt, Kimberly; Guo, Kejun; Santiago, Mario L; Stephens, Edward B

    2016-01-01

    The apolipoprotein mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) proteins are a family of seven cytidine deaminases (A3A, A3B, A3C, A3D, A3F, A3G and A3H) that restrict certain viral infections. These innate defence factors are best known for their ability to restrict the replication of human immunodeficiency virus type 1 (HIV-1) lacking a functional Vif protein (HIV-1Δvif) through the deamination of cytidine residues to uridines during reverse transcription, ultimately leading to lethal G → A changes in the viral genome. The best studied of the A3 proteins has been APOBEC3G because of its potent activity against HIV-1Δvif. However, one member of this family, A3A, has biological properties that make it unique among the A3 proteins. In this review, we will focus on the structural and phylogenetic features of the human and non-human primate A3A proteins, their role in the restriction of retroviruses and other viruses, and current findings on other biological properties affected by this protein. PMID:26489798

  16. Improvement of Disc Cutter Performance by Water Jet Assistance

    NASA Astrophysics Data System (ADS)

    Ciccu, Raimondo; Grosso, Battista

    2014-03-01

    This article deals with the problem of assisting disc cutters by means of high-velocity jets of water, with the aim of increasing the excavation rate while improving the working conditions, with particular reference to wear. The results of an experimental research undertaken at the Waterjet Laboratory of the University of Cagliari on a medium-hard abrasive rock clearly show that a higher removal rate is achieved owing to the weakening action of a jet directed on one side of the disc, causing deeper penetration. This outcome is interpreted on the basis of the scale formation model, which explains why smaller scales are obtained on the water jet's side of the groove. Accordingly, it is suggested that the results can be further improved if the jet is directed ahead of the tool along the same path, since, in this way, larger scales can be produced on both sides.

  17. Desorption Mass Spectrometry for Nonvolatile Compounds Using an Ultrasonic Cutter

    NASA Astrophysics Data System (ADS)

    Habib, Ahsan; Ninomiya, Satoshi; Chen, Lee Chuin; Usmanov, Dilshadbek T.; Hiraoka, Kenzo

    2014-07-01

    In this work, desorption of nonvolatile analytes induced by friction was studied. The nonvolatile compounds deposited on the perfluoroalkoxy substrate were gently touched by an ultrasonic cutter oscillating with a frequency of 40 kHz. The desorbed molecules were ionized by a dielectric barrier discharge (DBD) ion source. Efficient desorption of samples such as drugs, pharmaceuticals, amino acids, and explosives was observed. The limits of detection for these compounds were about 1 ng. Many compounds were detected in their protonated forms without undergoing significant fragmentation. When the DBD was off, no ions for the neutral samples could be detected, meaning that only desorption along with little ionization took place by the present technique.

  18. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  19. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  20. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for...

  1. A controlled microfluidic electrochemical lab-on-a-chip for label-free diffusion-restricted DNA hybridization analysis.

    PubMed

    Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

    2015-02-15

    Lab-on-a-chip (LOC) devices for electrochemical analysis of DNA hybridization events offer a technology for real-time and label-free assessment of biomarkers at the point-of-care. Here, we present a microfluidic LOC, with 3 × 3 arrayed electrochemical sensors for the analysis of DNA hybridization events. A new dual layer microfluidic valved manipulation system is integrated providing controlled and automated capabilities for high throughput analysis. This feature improves the repeatability, accuracy, and overall sensing performance (Fig. 1). The electrochemical activity of the fabricated microfluidic device is validated and demonstrated repeatable and reversible Nernstian characteristics. System design required detailed analysis of energy storage and dissipation as our sensing modeling involves diffusion-related electrochemical impedance spectroscopy. The effect of DNA hybridization on the calculated charge transfer resistance and the diffusional resistance components is evaluated. We demonstrate a specific device with an average cross-reactivity value of 27.5%. The device yields semilogarithmic dose response and enables a theoretical detection limit of 1 nM of complementary ssDNA target. This limit is lower than our previously reported non-valved device by 74% due to on-chip valve integration providing controlled and accurate assay capabilities. PMID:25310492

  2. New polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with non-syndromic hearing loss

    SciTech Connect

    Mkaouar-Rebai, Emna Tlili, Abdelaziz; Masmoudi, Saber; Charfeddine, Ilhem; Fakhfakh, Faiza

    2008-05-09

    The 12S rRNA gene was shown to be a hot spot for aminoglycoside-induced and non-syndromic hearing loss since several deafness-associated mtDNA mutations were identified in this gene. Among them, we distinguished the A1555G, the C1494T and the T1095C mutations and C-insertion or deletion at position 961. One hundred Tunisian patients with non-syndromic hearing loss and 100 hearing individuals were analysed in this study. A PCR-RFLP analysis with HaeIII restriction enzyme showed the presence of the A1555G mutation in the 12S rRNA gene in only one out of the 100 patients. In addition, PCR-RFLP and radioactive PCR revealed the presence of a new HaeIII polymorphic restriction site in the same gene of 12S rRNA site in 4 patients with non-syndromic hearing loss. UVIDOC-008-XD analyses showed the presence of this new polymorphic restriction site with a variable heteroplasmic rates at position +1517 of the human mitochondrial genome. On the other hand, direct sequencing of the entire mitochondrial 12S rRNA gene in the 100 patients and in 100 hearing individuals revealed the presence of the A750G and A1438G polymorphisms and the absence of the C1494T, T1095C and 961insC mutations in all the tested individuals. Sequencing of the whole mitochondrial genome in the 4 patients showing the new HaeIII polymorphic restriction site revealed only the presence of the A8860G transition in the MT-ATP6 gene and the A4769G polymorphism in the ND2 gene.

  3. Restriction endonuclease fingerprinting by SSCP (REF), an efficient method of screening for mutations in long contiguous segments of DNA

    SciTech Connect

    Liu, O.; Sommer, S.S.

    1994-09-01

    Dideoxy fingerprinting is an efficient method of screening for the presence of mutations in short exons ({le}250 bp). Long contiguous segments can be screened by sequential ddF reactions. To screen long contiguous segments in a more rapid manner, REF has been developed. REF will be described in the context of a model system in exon H of the factor IX gene. A 1 kb segment is PCR amplified and digested with each of five groups of restriction endonucleases. The endonucleases are chosen such that, in each group, the average size of the fragments is about 150 bp. After digestion, the products are mixed, 5{prime} end-labeled with T4 polynucleotide kinase, boiled, and electrophoresed under nondenaturing conditions. Each lane screens 1 kb and contains 70 segments (7 fragments per digestion x 5 digestions x 2 strands). The matrices tested were 5.6% polyacrylamide (PA) and 7.5% GeneAmp{sup {trademark}} (GA) at temperatures of either 23{degrees}C (RT) or 8{degrees}C (LT). Point mutations resulted in the gain or loss of a restriction site in 21% of 24 test mutations. In addition, mutations could be detected if any of 5 restriction fragments with the same mutation (producing 10 denatured segments) displayed abnormal mobility (SSCP component). The average sensitivity per segment of the SSCP component for the 24 point mutations ranged from 49% for PA at RT to 68% with GA at LT. REF detected 96% of the mutations with PA at RT and 100% with GA at RT or LT. These latter two conditions detected 100% of a subsequent blinded sample that contained normal controls and 27 different mutations. A blinded analysis is in progress to determine the sensitivity of REF when the segment size is 2 kb.

  4. The study of responses to 'model' DNA breaks induced by restriction endonucleases in cells and cell-free systems: achievements and difficulties.

    PubMed

    Thacker, J

    1994-11-01

    The use of restriction endonucleases (RE) as a means of implicating DNA double-strand breaks (dsb) in cellular responses is reviewed. The introduction of RE into cells leads to many of the responses known to be characteristic of radiation damage--cell killing, chromosomal aberration, oncogenic transformation, gene mutation and amplification. Additionally, radiosensitive cell lines are hypersensitive to RE, including those from the human disorder ataxia-telangiectasia. However, quantitation of response and comparisons of the effectiveness of different RE are difficult, partly because of unknown activity and lifetime of RE in the cell. RE-induced dsb have also been used to reveal molecular mechanisms of repair and misrepair at specific sites in DNA. Dsb have been implicated in recombination processes including those leading to illegitimate rejoining (formation of deletions and rearrangements) at short sequence features in DNA. Also model dsb act as a signal to activate other cellular processes, which may influence or indirectly cause some responses, including cell death. In these signalling responses the detailed chemistry at the break site may not be very important, perhaps explaining why there is considerable overlap in responses to RE and to ionizing radiations. PMID:7983451

  5. Use of Plasmon Coupling to Reveal the Dynamics of DNA Bending andCleavage by Single EcoRV Restriction Enzymes

    SciTech Connect

    Reinhard, Bjorn; Sheikholeslami, Sassan; Mastroianni, Alexander; Alivisatos, A. Paul; Liphardt, Jan

    2006-09-06

    Pairs of Au nanoparticles have recently been proposed asplasmon rulers based on the dependence of their light scattering on theinterparticle distance. Preliminary work has suggested that plasmonrulers can be used to measure and monitor dynamic distance changes overthe 1 to 100nm length scale in biology. Here, we substantiate thatplasmon rulers can be used to effectively measure dynamical biophysicalprocesses by applying the ruler to a system that has been investigatedextensively using ensemble kinetic measurements: the cleavage of DNA bythe restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz wereobtained, and the end-to-end extension of up to 1000 individual dsDNAenzyme substrates could be monitored in parallel for hours. The singlemolecule cleavage trajectories acquired here agree well with valuesobtained in bulk through other methods, and confirm well-known featuresof the cleavage process, such as the fact that the DNA is bent prior tocleavage. New dynamical information is revealed as well, for instance,the degree of softening of the DNA just prior to cleavage. The unlimitedlife time, high temporal resolution, and high signal/noise make theplasmon ruler an excellent tool for studying macromolecular assembliesand conformational changes at the single molecule level.

  6. A physical match of a metallic chip found on a bolt cutters' blade.

    PubMed

    Finkelstein, Nir; Volkov, Nikolai; Novoselsky, Yehuda; Tsach, Tsadok

    2015-05-01

    Bolt cutters are known as devices which are used for cutting hard objects and rigid materials such as padlocks and bars. They are commonly used in instances of forced entries. In this case study, a bolt cutter was found in the car of two suspects in a grocery burglary. This study indicates how the presence of a small metallic chip found on a suspected bolt cutter can prove that the tool was used in the crime scene. During the initial examination, a metallic chip from the cut shackle padlock was found stuck to one of the bolt cutters' blades. By comparing the metallic chip's microscopic edge and the breaking (fracture) line of the padlock's shackle, a full physical match was noticed. We wish to report here how residue, even the smallest, can be used to link burglary tools to a crime scene with a high level of certainty. PMID:25716459

  7. Maintaining knife sharpness in industrial meat cutting: A matter of knife or meat cutter ability.

    PubMed

    Karltun, J; Vogel, K; Bergstrand, M; Eklund, J

    2016-09-01

    Knife sharpness is imperative in meat cutting. The aim of this study was to compare the impact of knife blade steel quality with meat cutters' individual ability to maintain the cutting edge sharp in an industrial production setting. Twelve meat cutters in two different companies using three different knives during normal production were studied in this quasi-experimental study. Methods included were measuring knife cutting force before and after knife use, time knives were used, ratings of sharpness and discomfort and interviews. Results showed that the meat cutters' skill of maintaining sharpness during work had a much larger effect on knife sharpness during work than the knife steel differences. The ability was also related to feelings of discomfort and to physical exertion. It was found that meat cutters using more knives were more likely to suffer from discomfort in the upper limbs, which is a risk for developing MSD. PMID:27184316

  8. 21 CFR 884.4150 - Bipolar endoscopic coagulator-cutter and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) Identification. A bipolar endoscopic coagulator-cutter is a device used to perform female sterilization and other...: For female tubal sterilization, and (ii) Instructions for use: (A) Destroy at least 2 centimeters...

  9. 21 CFR 884.4150 - Bipolar endoscopic coagulator-cutter and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) Identification. A bipolar endoscopic coagulator-cutter is a device used to perform female sterilization and other...: For female tubal sterilization, and (ii) Instructions for use: (A) Destroy at least 2 centimeters...

  10. 21 CFR 884.4150 - Bipolar endoscopic coagulator-cutter and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) Identification. A bipolar endoscopic coagulator-cutter is a device used to perform female sterilization and other...: For female tubal sterilization, and (ii) Instructions for use: (A) Destroy at least 2 centimeters...

  11. 21 CFR 884.4150 - Bipolar endoscopic coagulator-cutter and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) Identification. A bipolar endoscopic coagulator-cutter is a device used to perform female sterilization and other...: For female tubal sterilization, and (ii) Instructions for use: (A) Destroy at least 2 centimeters...

  12. Restriction maps of the regions coding for methicillin and tobramycin resistances on chromosomal DNA in methicillin-resistant staphylococci.

    PubMed Central

    Ubukata, K; Nonoguchi, R; Matsuhashi, M; Song, M D; Konno, M

    1989-01-01

    Chromosomal BamHI DNA fragments containing both the mecA gene encoding the penicillin-binding protein responsible for methicillin resistance and the aadD gene encoding 4',4"-adenylyltransferase responsible for tobramycin resistance were cloned from three methicillin- and tobramycin-resistant strains of Staphylococcus aureus and one strain of Staphylococcus epidermidis. Physical maps of the fragments were similar, suggesting their unique origin. Images PMID:2817861

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

  14. Protection against H1N1 influenza challenge by a DNA vaccine expressing H3/H1 subtype hemagglutinin combined with MHC class II-restricted epitopes

    PubMed Central

    2010-01-01

    Background Multiple subtypes of avian influenza viruses have crossed the species barrier to infect humans and have the potential to cause a pandemic. Therefore, new influenza vaccines to prevent the co-existence of multiple subtypes within a host and cross-species transmission of influenza are urgently needed. Methods Here we report a multi-epitope DNA vaccine targeted towards multiple subtypes of the influenza virus. The protective hemagglutinin (HA) antigens from H5/H7/H9 subtypes were screened for MHC II class-restricted epitopes overlapping with predicted B cell epitopes. We then constructed a DNA plasmid vaccine, pV-H3-EHA-H1, based on HA antigens from human influenza H3/H1 subtypes combined with the H5/H7/H9 subtype Th/B epitope box. Results Epitope-specific IFN-γ ELISpot responses were significantly higher in the multi-epitope DNA group than in other vaccine and control groups (P < 0.05). The multi-epitope group significantly enhanced Th2 cell responses as determined by cytokine assays. The survival rate of mice given the multi-epitope vaccine was the highest among the vaccine groups, but it was not significantly different compared to those given single antigen expressing pV-H1HA1 vaccine and dual antigen expressing pV-H3-H1 vaccine (P > 0.05). No measurable virus titers were detected in the lungs of the multi-epitope immunized group. The unique multi-epitope DNA vaccine enhanced virus-specific antibody and cellular immunity as well as conferred complete protection against lethal challenge with A/New Caledonia/20/99 (H1N1) influenza strain in mice. Conclusions This approach may be a promising strategy for developing a universal influenza vaccine to prevent multiple subtypes of influenza virus and to induce long-term protective immune against cross-species transmission. PMID:21134292

  15. [Molecular phylogeny of forest and field mice of the genus Apodemus (Muridae, Rodentia) based on the data on restriction analysis of total nuclear DNA].

    PubMed

    Chelomina, G N

    1998-09-01

    Based on restriction-fragment length polymorphism (RFLP) of total nuclear DNA (nDNA), analyses of phylogenetic relations and genetic similarity were performed in nine species of forest and field mice of the genus Apodemus. Genetic distances calculated for different species pairs ranged from 0.24 to 12.53%; i.e., the differences were 50-fold. The estimated evolutionary age of the genus Apodemus is approximately 12 million years. In general, the obtained data on genetic similarity and phylogenetic relationship allow us to differentiate at least three groups of species: (1) southern Paleoarctic (A. argenteus), (2) eastern (A. peninsulae, A. speciosus, and A. agrarius), and (3) western (A. sylvaticus, A. flavicollis, A. ponticus, A. uralensis, and A. fulvipectus) ones. The latter two groups are related to the northern Paleoarctic. Such a division into groups corresponds to characteristic features of karyotype organization and segmentation of satellite DNA (satDNA) of these species, as well as the nature of variation in isozymes and in a fragment of the enzyme-encoding sequence of cytochrome b gene isolated from the mitochondrial genome. Species groups (1) and (3) exhibited a high probability of a monophyletic origin (70 and 99%, respectively). Group (2) is unlikely to be monophyletic, and the genetic distances in it are significantly greater than those in group 3. A. argenteus is the most diverged, both phenogenetically and phylogenetically. The data are consistent with a new zoological classification, which assumes the division of the unified genus Apodemus into two taxa of generic rank and suggest that the southern Paleoarctic forest mouse should be regarded as a separate taxon of at least subgeneric rank. PMID:9879015

  16. Acute elevation by short-term dietary restriction or food deprivation of type I I-compound levels in rat liver DNA.

    PubMed

    Zhou, G D; Hernandez, N S; Randerath, E; Randerath, K

    1999-01-01

    Type I I-compounds are bulky endogenous DNA modifications detectable by 32P postlabeling that exhibit age, species, tissue, genotype, gender, and diet dependence. Their formation appears unrelated to oxidative stress. In fact, several lines of indirect evidence suggest that many type I I-compounds may represent normal functional DNA modifications. For example, long-term dietary restriction (DR), which retards the development of age-related diseases including cancer and extends median and maximum life spans, unexpectedly elicits significant increases rather than decreases in the levels of many I-compounds in different rodent tissues. Positive linear correlations have been observed between such levels and median life spans of the animals. In the present work we have investigated 1) whether elevation of I-compound levels does not depend on chronic DR, i.e., occurs after a short period of DR or fasting, and 2) whether I-compound levels return to control values after the animals are returned to unrestricted feeding after food deprivation. Female Fischer 344 rats (approx 140 g each) were randomized into three groups. Group I was fed a natural ingredient (Purina 5001) diet ad libitum (AL) throughout the study, Group 2 was switched to 60% of the AL amount (40% DR) at 0 hour, and Group 3 was given no food for up to 72 hours and then returned to AL feeding until the end of the experiment. Liver DNA of individual rats (n = 4) was isolated for I-compound analysis at 24, 72, and 240 hours. Restricted and food-deprived rats showed elevated levels of hepatic I-compounds, with fasting eliciting the highest levels. These effects were seen as early as the 24-hour time point. Refeeding after 72 hours of food deprivation restored the levels to control values, measured at 240 hours. Our observations are discussed in relation to carcinogenesis and tumor promotion. The almost instantaneous changes of endogenous DNA modifications showed their exquisite sensitivity to nutritional factors

  17. Novel DNA methylation profiles associated with key gene regulation and transcription pathways in blood and placenta of growth-restricted neonates.

    PubMed

    Hillman, Sara L; Finer, Sarah; Smart, Melissa C; Mathews, Chris; Lowe, Robert; Rakyan, Vardhman K; Hitman, Graham A; Williams, David J

    2015-01-01

    Fetal growth is determined by the feto-placental genome interacting with the maternal in utero environment. Failure of this interplay leads to poor placental development and fetal growth restriction (FGR), which is associated with future metabolic disease. We investigated whether whole genome methylation differences existed in umbilical cord blood and placenta, between gestational-matched, FGR, and appropriately grown (AGA) neonates. Using the Infinium HumanMethylation450 BeadChip®, we found that DNA from umbilical cord blood of FGR born at term (n = 19) had 839 differentially methylated positions (DMPs) that reached genome-wide significance compared with AGA (n = 18). Using gestational age as a continuous variable, we identified 76,249 DMPs in cord blood (adj. P < 0.05) of which 121 DMPs were common to the 839 DMPs and were still evident when comparing 12 FGR with 12 AGA [39.9 ± 1.2 vs. 40.0 ± 1.0 weeks (mean ± SD), respectively]. A total of 53 DMPs had a β methylation difference >10% and 25 genes were co-methylated more than twice within 1000 base pairs. Gene Ontology (GO) analysis of DMPs supported their involvement in gene regulation and transcription pathways related to organ development and metabolic function. A similar profile of DMPs was found across different cell types in the cord blood. At term, no DMPs between FGR and AGA placentae reached genome-wide significance, validated with an external dataset. GO analysis of 284 pre-term, placental DMPs associated with autophagy, oxidative stress and hormonal responses. Growth restricted neonates have distinct DNA methylation profiles in pre-term placenta and in cord blood at birth, which may predispose to future adult disease. PMID:25496377

  18. Novel DNA methylation profiles associated with key gene regulation and transcription pathways in blood and placenta of growth-restricted neonates

    PubMed Central

    Hillman, Sara L; Finer, Sarah; Smart, Melissa C; Mathews, Chris; Lowe, Robert; Rakyan, Vardhman K; Hitman, Graham A; Williams, David J

    2015-01-01

    Fetal growth is determined by the feto-placental genome interacting with the maternal in utero environment. Failure of this interplay leads to poor placental development and fetal growth restriction (FGR), which is associated with future metabolic disease. We investigated whether whole genome methylation differences existed in umbilical cord blood and placenta, between gestational-matched, FGR, and appropriately grown (AGA) neonates. Using the Infinium HumanMethylation450 BeadChip®, we found that DNA from umbilical cord blood of FGR born at term (n = 19) had 839 differentially methylated positions (DMPs) that reached genome-wide significance compared with AGA (n = 18). Using gestational age as a continuous variable, we identified 76,249 DMPs in cord blood (adj. P < 0.05) of which 121 DMPs were common to the 839 DMPs and were still evident when comparing 12 FGR with 12 AGA [39.9 ± 1.2 vs. 40.0 ± 1.0 weeks (mean ± SD), respectively]. A total of 53 DMPs had a β methylation difference >10% and 25 genes were co-methylated more than twice within 1000 base pairs. Gene Ontology (GO) analysis of DMPs supported their involvement in gene regulation and transcription pathways related to organ development and metabolic function. A similar profile of DMPs was found across different cell types in the cord blood. At term, no DMPs between FGR and AGA placentae reached genome-wide significance, validated with an external dataset. GO analysis of 284 pre-term, placental DMPs associated with autophagy, oxidative stress and hormonal responses. Growth restricted neonates have distinct DNA methylation profiles in pre-term placenta and in cord blood at birth, which may predispose to future adult disease. PMID:25496377

  19. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110 restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting.

    PubMed

    Thorne, N; Evans, J T; Smith, E G; Hawkey, P M; Gharbia, S; Arnold, C

    2007-10-01

    A rapid, simple and highly discriminatory DNA fingerprinting methodology which produces data that can be easily interpreted, compared and transported is the ultimate goal for studying the epidemiology of Mycobacterium tuberculosis. A novel TaqI fluorescent amplified fragment length polymorphism (fAFLP) approach to M. tuberculosis DNA fingerprinting that targeted the variable IS6110 marker was developed in this study. The new method was tested for specificity and reproducibility, and compared with the standard reference IS6110 restriction fragment length polymorphism (RFLP) method for a panel of 78 isolates. Clustering conflicts between the two methods were resolved using mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) data. Comparison with an in-silico digestion of strain H37Rv showed that fAFLP-detected fragments were highly specific in vitro. The reproducibility of repeated digestions of strain H37Rv was 100%. Clustering results obtained by fAFLP and RFLP were highly congruent, with fAFLP allocating 97% of RFLP-clustered isolates to the same eight clusters as RFLP. Two single-copy isolates that had been clustered by RFLP were not clustered by fAFLP, but the MIRU-VNTR patterns of these isolates were different, indicating that the RFLP data had falsely clustered these isolates. Analysis by fAFLP will allow rapid screening of isolates to confirm or refute epidemiological links, and thereby provide insights into the frequency, conservation and consequences of specific transposition events. PMID:17803750

  20. Innovative technology summary report: High-speed clamshell pipe cutter

    SciTech Connect

    1998-09-01

    The Hanford Site C Reactor Technology Demonstration Group demonstrated the High-Speed Clamshell Pipe Cutter technology, developed and marketed by Tri Tool Inc. (Rancho Cordova, California). The models demonstrated are portable, split-frame pipe lathes that require minimal radial and axial clearances for severing and/or beveling in-line pipe with ranges of 25 cm to 41 cm and 46 cm to 61 cm nominal diameter. The radial clearance requirement from the walls, floors, or adjacent pipes is 18 cm. The lathes were supplied with carbide insert conversion kits for the cutting bits for the high-speed technique that was demonstrated. Given site-specific factors, this demonstration showed the cost of the improved technology to be approximately 30% higher than the traditional (baseline) technology (oxyacetylene torch) cost of $14,400 for 10 cuts of contaminated 41-cm and 61-cm-diameter pipe at C Reactor. Actual cutting times were faster than the baseline technology; however, moving/staging the equipment took longer. Unlike the baseline torch, clamshell lathes do not involve applied heat, flames, or smoke and can be operated remotely, thereby helping personal exposures to be as low as reasonably achievable. The baseline technology was demonstrated at the C Reactor north and south water pipe tunnels August 19--22, 1997. The improved technology was demonstrated in the gas pipe tunnel December 15--19.

  1. Assessing the phylogeographic history of the montane caddisfly Thremma gallicum using mitochondrial and restriction-site-associated DNA (RAD) markers

    PubMed Central

    Macher, Jan-Niklas; Rozenberg, Andrey; Pauls, Steffen U; Tollrian, Ralph; Wagner, Rüdiger; Leese, Florian

    2015-01-01

    Repeated Quaternary glaciations have significantly shaped the present distribution and diversity of several European species in aquatic and terrestrial habitats. To study the phylogeography of freshwater invertebrates, patterns of intraspecific variation have been examined primarily using mitochondrial DNA markers that may yield results unrepresentative of the true species history. Here, population genetic parameters were inferred for a montane aquatic caddisfly, Thremma gallicum, by sequencing a 658-bp fragment of the mitochondrial CO1 gene, and 12,514 nuclear RAD loci. T. gallicum has a highly disjunct distribution in southern and central Europe, with known populations in the Cantabrian Mountains, Pyrenees, Massif Central, and Black Forest. Both datasets represented rangewide sampling of T. gallicum. For the CO1 dataset, this included 352 specimens from 26 populations, and for the RAD dataset, 17 specimens from eight populations. We tested 20 competing phylogeographic scenarios using approximate Bayesian computation (ABC) and estimated genetic diversity patterns. Support for phylogeographic scenarios and diversity estimates differed between datasets with the RAD data favouring a southern origin of extant populations and indicating the Cantabrian Mountains and Massif Central populations to represent highly diverse populations as compared with the Pyrenees and Black Forest populations. The CO1 data supported a vicariance scenario (north–south) and yielded inconsistent diversity estimates. Permutation tests suggest that a few hundred polymorphic RAD SNPs are necessary for reliable parameter estimates. Our results highlight the potential of RAD and ABC-based hypothesis testing to complement phylogeographic studies on non-model species. PMID:25691988

  2. The three-dimension model for the rock-breaking mechanism of disc cutter and analysis of rock-breaking forces

    NASA Astrophysics Data System (ADS)

    Zhang, Zhao-Huang; Sun, Fei

    2012-06-01

    To study the rock deformation with three-dimensional model under rolling forces of disc cutter, by carrying out the circular-grooving test with disc cutter rolling around on the rock, the rock mechanical behavior under rolling disc cutter is studied, the mechanical model of disc cutter rolling around the groove is established, and the theory of single-point and double-angle variables is proposed. Based on this theory, the physics equations and geometric equations of rock mechanical behavior under disc cutters of tunnel boring machine (TBM) are studied, and then the balance equations of interactive forces between disc cutter and rock are established. Accordingly, formulas about normal force, rolling force and side force of a disc cutter are derived, and their validity is studied by tests. Therefore, a new method and theory is proposed to study rock-breaking mechanism of disc cutters.

  3. Importance of Resultant Action of the Mining Machine Actuator for Stresses in Impact Zone of a Separate Cutter

    NASA Astrophysics Data System (ADS)

    Aksenov, V. V.; Beglyakov, V. Yu; Kazantsev, A. A.; Ganovichev, S. I.

    2016-04-01

    Two stress levels are considered in the general pattern of stress-strain state of the rock destroyed by the mining machine. The authors also ground the necessity of considering the interaction of all cutters of the actuating device when calculating the process of cutting with a separate cutter.

  4. Do leaf-cutter ants Atta colombica orient their path-integrated, home vector with a magnetic compass?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaf-cutter ants Atta colombica forage over 250 m in structurally-complex, Neotropical rainforests that occlude sun or polarized light cues. Night foraging makes the use of celestial cues and landmarks all the more difficult. We investigated the directional cues used by leaf-cutter ants to orient h...

  5. Contamination risk of the surgical team through ROBODOC's high-speed cutter.

    PubMed

    Nogler, M; Wimmer, C; Lass-Flörl, C; Mayr, E; Trobos, S; Gegenhuber, C

    2001-06-01

    During cutting of the femoral cavity in the ROBODOC procedure, an aerosol cloud of irrigation fluid, blood, and tissue debris was seen. This cloud potentially is contaminated with bacterial and viral vectors, which is an infection risk for the surgical team. A flat and a ball cutter were tested in four standard situations macroscopically with a colored solution. In a second experiment, the cutter was exposed to a fluid contaminated with Staphylococcus aureus, and bacterial room contamination was detected using standard cultures. The aerosol cloud was seen in a 6 x 3.6-m area. Extension and concentration varied, depending on the irrigation situation. ROBODOC's high-speed cutter produces an aerosol cloud in an area in which all members of the surgical team are affected. Sufficient protection is necessary for everyone in the operating room. PMID:11400889

  6. A new MOS mask cutter facility at Gemini/Cerro Tololo observatories

    NASA Astrophysics Data System (ADS)

    Wyman, Robert T.; Trancho, Gelys; Tighe, Roberto

    2010-07-01

    The installation and commissioning of a new laser cutter facility in La Serena, Chile is a cooperative effort between Gemini Observatory and the Cerro Tololo Inter-American Observatory. This system enables the cutting of aluminum and carbon fiber slit masks for three multi-object spectrographs operating in Chile: GMOS-S, Flamingos-2, and Goodman spectrograph. Selection of the new laser cutter tool was based on slit mask specifications developed for two materials. Prior to the commissioning all slit mask production was performed at Gemini's Northern base facility with a similar laser cutter system. The new facility supports two observatories and enhances the capabilities for both. This paper will discuss the observatory arrangement with respect to mask data tracking and handling. The laser system and facility will be discussed along with mask cutting performance, process development and manufacturing methods.

  7. Simple and rapid fabrication of disposable carbon-based electrochemical cells using an electronic craft cutter for sensor and biosensor applications.

    PubMed

    Afonso, André S; Uliana, Carolina V; Martucci, Diego H; Faria, Ronaldo C

    2016-01-01

    This work describes the construction of an all-plastic disposable carbon-based electrochemical cell (DCell) using a simple procedure based on the use of a home cutter printer for prototyping and laminating. The cutter printer and adhesive vinyl films were used to produce three electrodes in an electrochemical cell layout, and a laminating process was then used to define the geometric area and insulate the electrodes. The DCell showed excellent performance in several applications including the determination of toxic metals in water samples, the immobilization of DNA and the detection of Salmonella. An unmodified DCell was applied for Pb and Cd detection in the range of 100-300 ng mL(-1) with a limit of detection of 50 and 39 ng mL(-1) for Cd and Pb, respectively. DNA was successfully immobilized on a DCell and used for studies of interaction between bisphenol A and DNA. The square wave voltammetry of a DNA modified DCell presented a guanine oxidation current 2.5 times greater after exposure of the electrode to bisphenol A and no current variation for the adenine moiety indicating that bisphenol A showed a preference for DNA interaction sites. A magneto-immunoassay was developed using a DCell for Salmonella detection in milk samples. The system presented a linear range from 100 to 700 cells mL(-1) with a limit of detection of 100 cells mL(-1) and good recovery values between 93% and 101% in milk samples, with no interference from Escherichia coli. Using the proposed method, hundreds of DCells can be assembled in less than two hours, at a material cost of less than US $0.02 per cell. The all-plastic disposable electrochemical cell developed was successfully applied as an electrochemical sensor and biosensor. The feasibility of the developed all-plastic disposable electrochemical cell was demonstrated in applications as both sensor and biosensor. PMID:26695279

  8. Prediction of dynamic cutting force and regenerative chatter stability in inserted cutters milling

    NASA Astrophysics Data System (ADS)

    Li, Zhongqun; Liu, Qiang; Yuan, Songmei; Huang, Kaisheng

    2013-05-01

    Currently, the modeling of cutting process mainly focuses on two aspects: one is the setup of the universal cutting force model that can be adapted to a broader cutting condition; the other is the setup of the exact cutting force model that can accurately reflect a true cutting process. However, there is little research on the prediction of chatter stablity in milling. Based on the generalized mathematical model of inserted cutters introduced by ENGIN, an improved geometrical, mechanical and dynamic model for the vast variety of inserted cutters widely used in engineering applications is presented, in which the average directional cutting force coefficients are obtained by means of a numerical approach, thus leading to an analytical determination of stability lobes diagram (SLD) on the axial depth of cut. A new kind of SLD on the radial depth of cut is also created to satisfy the special requirement of inserted cutter milling. The corresponding algorithms used for predicting cutting forces, vibrations, dimensional surface finish and stability lobes in inserted cutter milling under different cutting conditions are put forward. Thereafter, a dynamic simulation module of inserted cutter milling is implemented by using hybrid program of Matlab with Visual Basic. Verification tests are conducted on a vertical machine center for Aluminum alloy LC4 by using two different types of inserted cutters, and the effectiveness of the model and the algorithm is verified by the good agreement of simulation result with that of cutting tests under different cutting conditions. The proposed model can predict the cutting process accurately under a variety of cutting conditions, and a high efficient and chatter-free milling operation can be achieved by a cutting condition optimization in industry applications.

  9. Genotypic Characterization of Bradyrhizobium Strains Nodulating Endemic Woody Legumes of the Canary Islands by PCR-Restriction Fragment Length Polymorphism Analysis of Genes Encoding 16S rRNA (16S rDNA) and 16S-23S rDNA Intergenic Spacers, Repetitive Extragenic Palindromic PCR Genomic Fingerprinting, and Partial 16S rDNA Sequencing

    PubMed Central

    Vinuesa, Pablo; Rademaker, Jan L. W.; de Bruijn, Frans J.; Werner, Dietrich

    1998-01-01

    We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata. PMID:9603820

  10. Finger milling-cutter CNC generating hypoid pinion tooth surfaces based on modified-roll method and machining simulation

    NASA Astrophysics Data System (ADS)

    Li, Genggeng; Deng, Xiaozhong; Wei, Bingyang; Lei, Baozhen

    2011-05-01

    The two coordinate systems of cradle-type hypoid generator and free-form CNC machine tool by application disc milling-cutter to generate hypoid pinion tooth surfaces based on the modified-roll method were set up, respectively, and transformation principle and method for machine-tool settings between the two coordinate systems was studied. It was presented that finger milling-cutter was mounted on imagined disc milling-cutter and its motion was controlled directly by CNC shafts to replace disc milling-cutter blades effective cutting motion. Finger milling-cutter generation accomplished by ordered circular interpolation was determined, and interpolation center, starting and ending were worked out. Finally, a hypoid pinion was virtually machined by using CNC machining simulation software VERICUT.

  11. Finger milling-cutter CNC generating hypoid pinion tooth surfaces based on modified-roll method and machining simulation

    NASA Astrophysics Data System (ADS)

    Li, Genggeng; Deng, Xiaozhong; Wei, Bingyang; Lei, Baozhen

    2010-12-01

    The two coordinate systems of cradle-type hypoid generator and free-form CNC machine tool by application disc milling-cutter to generate hypoid pinion tooth surfaces based on the modified-roll method were set up, respectively, and transformation principle and method for machine-tool settings between the two coordinate systems was studied. It was presented that finger milling-cutter was mounted on imagined disc milling-cutter and its motion was controlled directly by CNC shafts to replace disc milling-cutter blades effective cutting motion. Finger milling-cutter generation accomplished by ordered circular interpolation was determined, and interpolation center, starting and ending were worked out. Finally, a hypoid pinion was virtually machined by using CNC machining simulation software VERICUT.

  12. Problem-Solving Test: Restriction Endonuclease Mapping

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  13. Molecular characterization by amplified ribosomal DNA restriction analysis and antimicrobial potential of endophytic fungi isolated from Luehea divaricata (Malvaceae) against plant pathogenic fungi and pathogenic bacteria.

    PubMed

    Bernardi-Wenzel, J; Garcia, A; Azevedo, J L; Pamphile, J A

    2013-01-01

    Luehea divaricata is an important plant in popular medicine; it is used for its depurative, anti-inflammatory, and other therapeutic activities. We evaluated the antimicrobial activity of endophytic fungi isolated from leaves of L. divaricata against phytopathogens and pathogenic bacteria, and characterized the isolates based on amplified ribosomal DNA restriction analysis (ARDRA). The in vitro antagonistic activity of these endophytes against the phytopathogen Alternaria alternata was assayed by dual culture technique. Based on this evaluation of antimicrobial activity, we extracted secondary metabolites from nine endophytic fungi by partitioning in ethyl acetate and methanol. These were tested against the phytopathogens A. alternata, Colletotrichum sp and Moniliophthora perniciosa, and against the human pathogenic bacteria Escherichia coli and Staphylococcus aureus. Molecular characterization by ARDRA technique was used for phylogenetic analysis, based on comparison with sequences in GenBank. The endophytes had varied effects on A. alternata. One isolate produced an inhibition halo against M. perniciosa and against E. coli. This antibiosis activity indicates a role in the protection of the plant against microbial pathogens in nature, with potential for pharmaceutical and agricultural applications. Based on ARDRA, the 13 isolates were grouped. We found three different haplotypes of Phomopsis sp, showing interspecific variability. It appears that examination of the microbial community associated with medicinal plants of tropical regions has potential as a useful strategy to look for species with biotechnological applications. PMID:24301768

  14. Restriction Site-Associated DNA Sequencing (RAD-seq) Reveals an Extraordinary Number of Transitions among Gecko Sex-Determining Systems.

    PubMed

    Gamble, Tony; Coryell, Jessi; Ezaz, Tariq; Lynch, Joshua; Scantlebury, Daniel P; Zarkower, David

    2015-05-01

    Sex chromosomes have evolved many times in animals and studying these replicate evolutionary "experiments" can help broaden our understanding of the general forces driving the origin and evolution of sex chromosomes. However this plan of study has been hindered by the inability to identify the sex chromosome systems in the large number of species with cryptic, homomorphic sex chromosomes. Restriction site-associated DNA sequencing (RAD-seq) is a critical enabling technology that can identify the sex chromosome systems in many species where traditional cytogenetic methods have failed. Using newly generated RAD-seq data from 12 gecko species, along with data from the literature, we reinterpret the evolution of sex-determining systems in lizards and snakes and test the hypothesis that sex chromosomes can routinely act as evolutionary traps. We uncovered between 17 and 25 transitions among gecko sex-determining systems. This is approximately one-half to two-thirds of the total number of transitions observed among all lizards and snakes. We find support for the hypothesis that sex chromosome systems can readily become trap-like and show that adding even a small number of species from understudied clades can greatly enhance hypothesis testing in a model-based phylogenetic framework. RAD-seq will undoubtedly prove useful in evaluating other species for male or female heterogamety, particularly the majority of fish, amphibian, and reptile species that lack visibly heteromorphic sex chromosomes, and will significantly accelerate the pace of biological discovery. PMID:25657328

  15. Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

    PubMed Central

    Guan, Le Luo; Hagen, Karen E.; Tannock, Gerald W.; Korver, Doug R.; Fasenko, Gaylene M.; Allison, Gwen E.

    2003-01-01

    The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 108 to 109 CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria. PMID:14602636

  16. A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

    PubMed

    Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H

    1989-11-01

    The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477

  17. Improved restriction landmark cDNA scanning and its application to global analysis of genes regulated by nerve growth factor in PC12 cells.

    PubMed

    Mayumi, K; Yaoi, T; Kawai, J; Kojima, S; Watanabe, S; Suzuki, H

    1998-07-30

    Restriction landmark cDNA scanning (RLCS) is a novel method by which more than 1000 genes can be simultaneously and quantitatively displayed as two-dimensional gel spots. Here we present an adaptation that allows an individual spot to correspond to a unique gene species without redundancy in more than two gel patterns. Using this improved RLCS, we examined global changes on the gene expression of PC12 cells before and after treatment with nerve growth factor. Among a total of 3000 spots, 21 (0.70%) and 91 (3.03%) spots newly appeared and became more intense with treatment. On the other hand, 15 (0.50%) and 44 (1.47%) spots disappeared, becoming less intense with treatment. These observations suggest that approx. 6% of the detected PC12 genes are up-(3.73%) or down-(1.97%) regulated when the cells differentiate to neuronal cells. In comparison with the results obtained using the expressed-sequence-tag approach, previously reported by Lee et al. (Proc. Natl. Acad. Sci. USA 92 (1995) 8303-8307), RLCS should be useful for quantitatively examining the global change of differentially expressed genes of various expression levels. PMID:9714711

  18. Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

    PubMed Central

    Hirano, Nobutaka; Ohshima, Hiroyuki; Takahashi, Hideo

    2006-01-01

    Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA. PMID:16971463

  19. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  20. 21 CFR 884.4150 - Bipolar endoscopic coagulator-cutter and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Bipolar endoscopic coagulator-cutter and accessories. 884.4150 Section 884.4150 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Obstetrical and Gynecological Surgical Devices § 884.4150...

  1. Methods of searching and analyzing the data of the gear tooth profile in gear cutter CAD

    NASA Astrophysics Data System (ADS)

    Pang, Xing-hua; Wang, Er-hua

    2011-05-01

    In gear-cutter CAD(Computer Aided Design), certain points belonging to series cutter loci which were generated by the simulation of the cutting process may form a gear-tooth profile. Finding out such points and analyzing them to predict the properties of a new gear is also a very important step which determines the succedent analyzing steps. However, the problem of abstracting these data from the loci rapidly and accurately remains to be solved. The two algorithms presented in this paper, concentric-arcs searching and revolving-scan searching, would provide theoretic basis for the on-line gear-tooth analysis. The former focuses on drawing concentric-arcs between the dedendum and addendum of the gear, the latter concentrates on drawing radial lines between two angles.Such arcs and lines will intersect one or more lines of the cutter profile.Correspongding screening criterions will greatly contribute to juge and identify useful intersections which may form the gear profile, and the gear-tooth shape analysis will be carried out as a result. Keyword: gear cutter; CAD; concentric-arcs searching; revolving-scan searching.

  2. Methods of searching and analyzing the data of the gear tooth profile in gear cutter CAD

    NASA Astrophysics Data System (ADS)

    Pang, Xing-Hua; Wang, Er-Hua

    2010-12-01

    In gear-cutter CAD(Computer Aided Design), certain points belonging to series cutter loci which were generated by the simulation of the cutting process may form a gear-tooth profile. Finding out such points and analyzing them to predict the properties of a new gear is also a very important step which determines the succedent analyzing steps. However, the problem of abstracting these data from the loci rapidly and accurately remains to be solved. The two algorithms presented in this paper, concentric-arcs searching and revolving-scan searching, would provide theoretic basis for the on-line gear-tooth analysis. The former focuses on drawing concentric-arcs between the dedendum and addendum of the gear, the latter concentrates on drawing radial lines between two angles.Such arcs and lines will intersect one or more lines of the cutter profile.Correspongding screening criterions will greatly contribute to juge and identify useful intersections which may form the gear profile, and the gear-tooth shape analysis will be carried out as a result. Keyword: gear cutter; CAD; concentric-arcs searching; revolving-scan searching.

  3. The evaluation of ergonomic risk factors among meat cutters working in Jabalpur, India

    PubMed Central

    Mukhopadhyay, Prabir; Khan, Amaltas

    2015-01-01

    Background: Manual meat cutters in India are at high risk of work-related musculoskeletal disorders (WMSDs) for a variety of reasons including holding awkward postures, repetitive forceful exertions, and inadequate rest. This is the first study of its kind to investigate the nature and magnitude of WMSDs among manual meat cutters in India. Objective: The aim of this study was to measure the ergonomic risk factors for WMSDs among adult male manual meat cutters working in Jabalpur, India. Methods: We used direct observation, activity analysis, questionnaires, interviews, photography, and video to measure the quantitative ergonomic risk factors. Results: Ovako working posture analysis indicated high scores (four for the back in peeling, six for the arms in cutting, and six for the arms during mincing tasks). Rapid entire body assessment method (REBA) scores were also high at 10/10 for deboning and mincing tasks, all associated with repetitive movements of the arms and awkward posture of the upper part of the body. Conclusions: The study indicates that most tasks for meat cutters fall in the high-risk category for occupational injury. Results suggest that ergonomic interventions that address retooling and workstation and process redesign would be useful in reducing the number of injuries. PMID:25658673

  4. Genetic Diversity of Mycobacterium africanum Clinical Isolates Based on IS6110-Restriction Fragment Length Polymorphism Analysis, Spoligotyping, and Variable Number of Tandem DNA Repeats

    PubMed Central

    Viana-Niero, Cristina; Gutierrez, Cristina; Sola, Christophe; Filliol, Ingrid; Boulahbal, Fadila; Vincent, Véronique; Rastogi, Nalin

    2001-01-01

    A collection of 105 clinical isolates originally identified as Mycobacterium africanum were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the M. tuberculosis complex, whereas genotypic characterization was based on IS6110-restriction fragment length polymorphism (IS6110-RFLP) analysis, IS1081-RFLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyping), variable number of tandem DNA repeats (VNTR), and the polymorphism of the oxyR, pncA, and mtp40 loci. The results obtained showed that a majority of M. africanum isolates were characterized by a specific spoligotyping pattern that was intermediate between those of M. tuberculosis and M. bovis, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respectively. A tentative M. africanum-specific spoligotyping signature appeared to be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as the polymorphism of oxyR and pncA, a total of 24 isolates were excluded from the final study (19 were identified as M. tuberculosis, 2 were identified as M. canetti, and 3 were identified as M. bovis). The remaining 81 M. africanum isolates were efficiently subtyped in three distinct subtypes (A1 to A3) by IS6110-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of the A2 subtype but showed significant variations for subtypes A1 and A3. A phylogenetic tree based on a selection of isolates representing the three subtypes using VNTR and spoligotyping alone or in combination confirmed the subtypes described as well as the heterogeneity of subtype A3. PMID:11136749

  5. Restriction site-associated DNA sequencing generates high-quality single nucleotide polymorphisms for assessing hybridization between bighead and silver carp in the United States and China.

    PubMed

    Lamer, James T; Sass, Greg G; Boone, Jason Q; Arbieva, Zarema H; Green, Stefan J; Epifanio, John M

    2014-01-01

    Bighead carp (Hypophthalmichthys nobilis) and silver carp (H. molitrix) are invasive species and listed as US federally injurious species under the Lacy Act. They have established populations in much of the Mississippi River Basin (MRB; Mississippi, Illinois, and Missouri rivers) and are capable of producing fertile hybrids and complex introgression. Characterizing the composition of this admixture requires a large set of high-quality, evolutionarily conserved, diagnostic genetic markers to aid in the identification and management of these species in the midst of morphological ambiguity. Restriction site-associated DNA (RAD) sequencing of 45 barcoded bighead and silver carp from the United States and China produced reads that were aligned to the silver carp transcriptome yielded 261 candidate single nucleotide polymorphisms (SNPs) with fixed allelic differences between the two species. We selected the highest quality 112 SNP loci for validation using 194 putative pure-species and F1 hybrids from the MRB and putative bighead carp and silver carp pure species from China (Amur, Pearl and Yangtze rivers). Fifty SNPs were omitted due to design/amplification failure or lack of diagnostic utility. A total of 57 species-diagnostic SNPs conserved between carp species in US and Chinese rivers were identified; 32 were annotated to functional gene loci. Twenty-seven of the 181 (15%) putative pure species were identified as hybrid backcrosses after validation, including three backcrosses from the Amur River, where hybridization has not been documented previously. The 57 SNPs identified through RAD sequencing provide a diagnostic tool to detect population admixture and to identify hybrid and pure-species Asian carps in the United States and China. PMID:23957862

  6. Comparison of Target-Capture and Restriction-Site Associated DNA Sequencing for Phylogenomics: A Test in Cardinalid Tanagers (Aves, Genus: Piranga).

    PubMed

    Manthey, Joseph D; Campillo, Luke C; Burns, Kevin J; Moyle, Robert G

    2016-07-01

    Restriction-site associated DNA sequencing (RAD-seq) and target capture of specific genomic regions, such as ultraconserved elements (UCEs), are emerging as two of the most popular methods for phylogenomics using reduced-representation genomic data sets. These two methods were designed to target different evolutionary timescales: RAD-seq was designed for population-genomic level questions and UCEs for deeper phylogenetics. The utility of both data sets to infer phylogenies across a variety of taxonomic levels has not been adequately compared within the same taxonomic system. Additionally, the effects of uninformative gene trees on species tree analyses (for target capture data) have not been explored. Here, we utilize RAD-seq and UCE data to infer a phylogeny of the bird genus Piranga The group has a range of divergence dates (0.5-6 myr), contains 11 recognized species, and lacks a resolved phylogeny. We compared two species tree methods for the RAD-seq data and six species tree methods for the UCE data. Additionally, in the UCE data, we analyzed a complete matrix as well as data sets with only highly informative loci. A complete matrix of 189 UCE loci with 10 or more parsimony informative (PI) sites, and an approximately 80% complete matrix of 1128 PI single-nucleotide polymorphisms (SNPs) (from RAD-seq) yield the same fully resolved phylogeny of Piranga We inferred non-monophyletic relationships of Piranga lutea individuals, with all other a priori species identified as monophyletic. Finally, we found that species tree analyses that included predominantly uninformative gene trees provided strong support for different topologies, with consistent phylogenetic results when limiting species tree analyses to highly informative loci or only using less informative loci with concatenation or methods meant for SNPs alone. PMID:26821912

  7. Restriction mapping of a YAC contig in the hemochromatosis gene region

    SciTech Connect

    Burt, M.J.; Smit, D.J.; Pyper, W.R.

    1994-09-01

    Hemochromatosis is a common inherited disorder of iron metabolism that can lead to cirrhosis, hepatocellular carcinoma, cardiomyopathy, diabetes and anthropathy. We have mapped the hemochromatosis gene to within 1 cM of HLA-A and the microsatellite D6S105, and our allele association studies have shown that D6S105 is the marker most closely associated with the hemochromatosis gene. We are currently constructing a YAC contig and restriction map of this region as part of a positional cloning strategy to identify the hemochromatosis gene. YACs containing HLA-A or D6S105 were selected, and fluorescent-in-situ-hybridization (FISH) was performed to confirm chromosomal location and exclude chimerism. YAC DNA was digested with a panel of rare cutters, separated by pulsed field gel electrophoresis, Southern blotted and probed with the vector arms to create restriction maps. YAC insert terminal ends were isolated using vectorette methodology. A contig extending 600 kb centromeric and 350 kb telomeric of HLA-A has been established. HLA-A, HLA-F and the microsatellite D6S265 have been positioned on this map. The contig does not yet overlap any D6S105 positive YACs but the telomeric end of the contig has been sequenced and is being used to identify additional YACs to bridge this interval. Restriction mapping of three D6S105 YACs has shown the presence of several CpG islands in this region. As these CpG islands are in close proximity to D6S105, they are being used to isolate coding sequences to determine whether any of these mark the position of the hemochromatosis gene.

  8. Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme

    PubMed Central

    Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

    2014-01-01

    The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5′-CCTGG-3′). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5′-ACTGGG-3′) complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C–DNA and EcoRII-N–DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C–DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs. PMID:24423868

  9. BamI, KpnI, and SalI restriction enzyme maps of the DNAs of herpes simplex virus strains Justin and F: occurrence of heterogeneities in defined regions of the viral DNA.

    PubMed Central

    Locker, H; Frenkel, N

    1979-01-01

    We present the locations of the cleavage sites for the BamI, KpnI, and SalI restriction endonucleases within the DNA molecules of herpes simplex virus type 1 (HSV-1) strains Justin and F. These restriction enzymes cleave the HSV-1 DNA at many sites, producing relatively small fragments which should prove useful in future studies of HSV-1 gene structure and function. The mapping data revealed the occurrence of heterogeneity within three regions of the viral genome including (i) the region spanning map coordinates 0.74--0.76, (ii) the ends of the large (L) DNA component, and (iii) the junction between the large (L) and the small (S) components. The heterogeneity in the ends of L and the S-L junctions of HSV-1 (Justin) and HSV-1 (F) DNAs was grossly similar to that previously reported to occur in the ends of L and the S-L junctions of the HSV-1 (KOS) DNA (M. J. Wagner and W. C. Summers, J. Virol. 27:374--387, 1978). Thus, cleavage of these regions with restriction endonucleases yielded sets of minor fragments differing in size by constant increments. However, the various strains of HSV-1 differed with respect to the numbers, size increments, and relative molarities of the various minor fragments, suggesting that the parameters of the heterogeneity are inherited in the structural makeup of the HSV-1 genome. The strain dependence of the pattern of heterogeneity can be most easily explained in terms of variable sizes of the terminally reiterated a sequence, contained in the DNA molecules of these three strains of HSV-1. Images PMID:228068

  10. Lung function, biological monitoring, and biological effect monitoring of gemstone cutters exposed to beryls

    PubMed Central

    Wegner, R.; Heinrich-Ramm, R.; Nowak, D.; Olma, K.; Poschadel, B.; Szadkowski, D.

    2000-01-01

    OBJECTIVES—Gemstone cutters are potentially exposed to various carcinogenic and fibrogenic metals such as chromium, nickel, aluminium, and beryllium, as well as to lead. Increased beryllium concentrations had been reported in the air of workplaces of beryl cutters in Idar-Oberstein, Germany. The aim of the survey was to study the excretion of beryllium in cutters and grinders with occupational exposure to beryls—for example, aquamarines and emeralds—to examine the prevalence of beryllium sensitisation with the beryllium lymphocyte transformation test (BeLT), to examine the prevalence of lung disease induced by beryllium, to describe the internal load of the respective metals relative to work process, and to screen for genotoxic effects in this particular profession.
METHODS—In a cross sectional investigation, 57 out of 100 gemstone cutters working in 12 factories in Idar-Oberstein with occupational exposure to beryls underwent medical examinations, a chest radiograph, lung function testing (spirometry, airway resistance with the interrupter technique), and biological monitoring, including measurements of aluminium, chromium, and nickel in urine as well as lead in blood. Beryllium in urine was measured with a newly developed direct electrothermal atomic absorption spectroscopy technique with a measurement limit of 0.06 µg/l. Also, cytogenetic tests (rates of micronuclei and sister chromatid exchange), and a BeLT were performed. Airborne concentrations of beryllium were measured in three factories. As no adequate local control group was available, the cutters were categorised into those with an exposure to beryls of >4 hours/week (group A) and ⩽4 hours/week (group B).
RESULTS—Clinical, radiological, or spirometric abnormalities indicating pneumoconiosis were detected in none of the gemstone cutters. Metal concentrations in biological material were far below the respective biological limit values, and beryllium in urine was only measurable in

  11. Restrictive cardiomyopathy

    MedlinePlus

    ... blood returns from the body (diastole). When the disease progresses, the heart may not pump blood strongly. The abnormal heart function can affect the lungs, liver, and other body systems. Restrictive cardiomyopathy may affect either or both of the ...

  12. Restrictive cardiomyopathy

    MedlinePlus

    ... blood returns from the body (diastole). When the disease progresses, the heart may not pump blood strongly. The abnormal heart function can affect the lungs, liver, and other body systems. Restrictive cardiomyopathy may affect ...

  13. Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

    PubMed

    Sennikov, Sergey Vital'evich; Shevchenko, Julia Alexandrovna; Kurilin, Vasilii Vasil'evich; Khantakova, Julia Nikolaevna; Lopatnikova, Julia Anatol'evna; Gavrilova, Elena Vasil'evna; Maksyutov, Rinat Amirovich; Bakulina, Anastasiya Yur'evna; Sidorov, Sergey Vasil'evich; Khristin, Alexander Alexandrovich; Maksyutov, Amir Zakievich

    2016-02-01

    Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro. PMID:26590947

  14. Detection and Validation of QTL Affecting Bacterial Cold Water Disease Resistance in Rainbow Trout Using Restriction-Site Associated DNA Sequencing

    PubMed Central

    Gao, Guangtu; Liu, Sixin; Hernandez, Alvaro G.; Rexroad, Caird E.

    2015-01-01

    Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellite markers in a genome scan, we previously detected significant and suggestive QTL affecting phenotypic variation in survival following challenge with Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout. In this study, we performed selective genotyping of SNPs from restriction-site associated DNA (RAD) sequence data from two pedigreed families (2009070 and 2009196) to validate the major QTL from the previous work and to detect new QTL. The use of RAD SNPs in the genome scans increased the number of mapped markers from ~300 to ~5,000 per family. The significant QTL detected in the microsatellites scan on chromosome Omy8 in family 2009070 was validated explaining up to 58% of the phenotypic variance in that family, and in addition, a second QTL was also detected on Omy8. Two novel QTL on Omy11 and 14 were also detected, and the previously suggestive QTL on Omy1, 7 and 25 were also validated in family 2009070. In family 2009196, the microsatellite significant QTL on Omy6 and 12 were validated and a new QTL on Omy8 was detected, but none of the previously detected suggestive QTL were validated. The two Omy8 QTL from family 2009070 and the Omy12 QTL from family 2009196 were found to be co-localized with handling and confinement stress response QTL that our group has previously identified in a separate pedigreed family. With the currently available data we cannot determine if the co-localized QTL are the result of genes with pleiotropic effects or a mere physical proximity on the same chromosome segment. The genetic markers linked to BCWD resistance QTL were used to query the scaffolds of the rainbow trout reference genome assembly and the QTL-positive scaffold sequences were found to include 100 positional candidate genes. Several of the candidate genes located on or near the two Omy8 QTL detected in family 2009070 suggest potential

  15. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  16. Proper nozzle location, bit profile, and cutter arrangement affect PDC-bit performance significantly

    SciTech Connect

    Garcia-Gavito, D.; Azar, J.J.

    1994-09-01

    During the past 20 years, the drilling industry has looked to new technology to halt the exponentially increasing costs of drilling oil, gas, and geothermal wells. This technology includes bit design innovations to improve overall drilling performance and reduce drilling costs. These innovations include development of drag bits that use PDC cutters, also called PDC bits, to drill long, continuous intervals of soft to medium-hard formations more economically than conventional three-cone roller-cone bits. The cost advantage is the result of higher rates of penetration (ROP's) and longer bit life obtained with the PDC bits. An experimental study comparing the effects of polycrystalline-diamond-compact (PDC)-bit design features on the dynamic pressure distribution at the bit/rock interface was conducted on a full-scale drilling rig. Results showed that nozzle location, bit profile, and cutter arrangement are significant factors in PDC-bit performance.

  17. Review of strategies to manage cutter roof failure in coal mines. Information circular/1994

    SciTech Connect

    Wuest, W.J.

    1994-01-01

    This U.S. Bureau of Mines report presents an overview of ground control considerations associated with cutter roof failure. Many of the relevant U.S. and international research studies since the late 1940s are reviewed, and summaries of recent design guides are included. Factors affecting cutter roof, such as underground stress field and rock structure around the entry, are described. Strata control techniques are also outlined. These techniques are divided into three sections: artificial support, mine structure design, and multiple-seam extraction. Other subjects covered in the report include monitoring and prediction, effect of geologic features, and mapping of adverse mining conditions. The emphasis of this report is on practical considerations; theoretical discussions are minimal.

  18. A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 V[sub H] gene

    SciTech Connect

    Van Es, J.H.; Aanstoot, H.; Gmelig-Meyling, F.H.J.; Derksen, R.H.W.M.; Logtenberg, T. )

    1992-09-15

    The authors report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The V[sub H] gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural antibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted V[sub H] genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations. 42 refs., 5 figs.

  19. Accuracy of tablet splitting: Comparison study between hand splitting and tablet cutter

    PubMed Central

    Habib, Walid A.; Alanizi, Abdulaziz S.; Abdelhamid, Magdi M.; Alanizi, Fars K.

    2013-01-01

    Background Tablet splitting is often used in pharmacy practice to adjust the administered doses. It is also used as a method of reducing medication costs. Objective To investigate the accuracy of tablet splitting by comparing hand splitting vs. a tablet cutter for a low dose drug tablet. Methods Salbutamol tablets (4 mg) were chosen as low dose tablets. A randomly selected equal number of tablets were split by hand and a tablet cutter, and the remaining tablets were kept whole. Weight variation and drug content were analysed for salbutamol in 0.1 N HCl using a validated spectrophotometric method. The percentages by which each whole tablet’s or half-tablet’s drug content and weight difference from sample mean values were compared with USP specification ranges for drug content. The %RSD was also calculated in order to determine whether the drugs met USP specification for %RSD. The tablets and half tablets were scanned using electron microscopy to show any visual differences arising from splitting. Results 27.5% of samples differed from sample mean values by a percentage that fell outside of USP specification for weight, of which 15% from the tablet cutter and 25% from those split by hand fell outside the specifications. All whole tablets and half tablets met the USP specifications for drug content but the variation of content between the two halves reached 21.3% of total content in case of hand splitting, and 7.13% only for the tablet cutter. The %RSDs for drug content and weight met the USP specification for whole salbutamol tablets and the half tablets which were split by tablet cutter. The halves which were split by hand fell outside the specification for %RSD (drug content = 6.43%, weight = 8.33%). The differences were visually clear in the electron microscope scans. Conclusion Drug content variation in half-tablets appeared to be attributable to weight variation occurring during the splitting process. This could have serious clinical consequences for

  20. TLR9 promotes tolerance by restricting survival of anergic anti-DNA B cells, yet is also required for their activation.

    PubMed

    Nickerson, Kevin M; Christensen, Sean R; Cullen, Jaime L; Meng, Wenzhao; Luning Prak, Eline T; Shlomchik, Mark J

    2013-02-15

    Nucleic acid-reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. In this study, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Fas(lpr) mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell-surface BCR expression. In contrast, whereas TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into Ab-forming cells via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells, yet is also required for their activation. PMID:23296704

  1. Caloric restriction.

    PubMed

    Speakman, John R; Mitchell, Sharon E

    2011-06-01

    Restricting the intake of calories has been practiced as a method for increasing both the length and quality of life for over 500 years. Experimental work confirming the success of this approach in animals has accumulated over the last 100 years. Lifelong caloric restriction (CR) may extend life by up to 50% in rodents, with progressively less impact the later in life it is started. This effect is matched by profound impacts on age related diseases including reduced risk of cancer, neurodegenerative disorders, autoimmune disease, cardiovascular disease and type II diabetes mellitus. The disposable soma theory of ageing suggests that CR evolved as a somatic protection response to enable animals to survive periods of food shortage. The shutdown of reproductive function during CR is consistent with this suggestion, but other features of the phenomenon are less consistent with this theory, and some have suggested that in rodents it may be mostly an artifact of domestication. CR induces profound effects on animals at all levels from the transcriptome to whole animal physiology and behavior. Animals under CR lose weight which is disproportionately contributed to by white adipose tissue. Generally animals on CR change their activity patterns so that they are more active prior to food delivery each day but total activity may be unchanged or reduced. Considerable debate has occurred over the effects of CR on resting metabolic rate (RMR). Total RMR declines, but as body mass and body composition also change it is unclear whether metabolism at the tissue level also declines, is unchanged or even increases. Body temperature universally decreases. Hunger is increased and does not seem to abate even with very long term restriction. Circulating adipokines are reduced reflecting the reduction in white adipose tissue (WAT) mass under restriction and there is a large reduction in circulating insulin and glucose levels. There are profound tissue level changes in metabolism with a

  2. Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences.

    PubMed Central

    Hughes, M S; Skuce, R A; Beck, L A; Neill, S D

    1993-01-01

    Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for "cording," and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identify over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense. Images PMID:7508456

  3. Performance evaluation of bolt-cutter system on first Taurus launch

    NASA Astrophysics Data System (ADS)

    Baban, F.; Williams, R.; Amimoto, S.; Hansen, W.; Bixler, T.

    1994-10-01

    In rapid response to the request of the Space Test and Experimentation Directorate in Space Launch Operations, a launch-critical experimental investigation was conducted to evaluate the performance of a particular bolt-cutter system for separating stages on the first Taurus launch. The tests were to examine the variation of tension preloading on the bolt system and to demonstrate the tolerable margin on this parameter for such launches with the new types of bolts since the preloading was known to vary as much as 12% from a preset value before launch. We planned and carried out the experiment, designed and assembled the fixture to properly simulate flight application, and developed diagnostics. Four bolt cutters were purchased from the manufacturer for these tests, and one was provided by the contractor. In addition to the obvious requirement to demonstrate the successful severing of bolts under varying preloads, ignition-wire current and timing of chisel impact on the bolt were monitored. An optical diagnostic was designed to determine the flyout velocity and kinetic energy of the broken pieces. These latter measurements will be useful in anchoring performance codes simulating and assessing the structural dynamics of the bolt-cutter function for future missions. The tests were conducted successfully and the bolts were severed successfully in all five tests. The preloads were successively lowered from 2,500 lb to 2,250, 2,000, 1,500, and 1,000 lb These tests contributed in a timely manner to the STEP launch decision and to launch mission assurance. They demonstrated important margin to the nominally set 3,200 lb. preload. The entire complicated experimental program from inception to completion was accomplished in less than three weeks.

  4. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation

    PubMed Central

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2016-01-01

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation. PMID:26527723

  5. Restriction-deletion polymorphism of region V of mitochondrial DNA in several populations of native inhabitants of Siberia and the Far East

    SciTech Connect

    Ivanova, A.V.; Kazakovtseva, M.A.; Bibe, B.P.; Karafet, T.M.; Osipova, L.P.; Voevoda, M.I.; Avksentyuk, A.V.; Astakhova, T.I.; Shields, G.F.

    1994-11-01

    The distribution of a deletion and of an Ava II site in region V of mitochondrial DNA (mtDNA) was studied in five populations of native inhabitants of the Asian part of Russia, including Chukchi, Asian Eskimos, Evenks, Buryats, and Northern Sel`kups. A deletion with a frequency of 6.3% was found only in Buryats; in Chukchi and Eskimos the Ava II site was not found. A maximal frequency of 11.3% was observed in Evenks. A comparison with published data was conducted; it revealed a gradient of decreasing frequency of the deletion from southeast Asia to the north, with its complete absence in the circumpolar regions. In the territory of northeast Asia, all three mitotypes are found, formed by a combination of two polymorphic markers of mtDNA region V, which were found earlier in humans in the Americas. The data obtained necessitates a more detailed analysis of the population polymorphism of mtDNA in this region of Asia. 32 refs., 1 fig., 3 tabs.

  6. Effects of DNA Extraction Procedures on Bacteroides Profiles in Fecal Samples From Various Animals Determined by Terminal Restriction Fragment Length Polymorphism Analysis

    EPA Science Inventory

    A major assumption in microbial source tracking is that some fecal bacteria are specific to a host animal, and thus provide unique microbial fingerprints that can be used to differentiate hosts. However, the DNA information obtained from a particular sample may be biased dependi...

  7. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation.

    PubMed

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2016-01-29

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation. PMID:26527723

  8. Cutter-Skidder Operator. Competency-Based Training Standards = Conducteur de Debusqueuse-Abatteur d'Arbes. Normes de formation professionnelle basees sur les aptitudes.

    ERIC Educational Resources Information Center

    Ontario Ministry of Skills Development, Toronto.

    This bilingual document contains standards that are based on industry-specific skills identified by labor and management representatives of the forest products industry, successful achievement of which results in issuance of a cutter operator, skidder operator, or cutter-skidder operator Canadian certificate of qualification. With French on all…

  9. In vitro induction of IgG anti-DNA antibody from high density B cells of systemic lupus erythematosus patients by an HLA DR-restricted T cell clone.

    PubMed Central

    Murakami, M; Kumagai, S; Sugita, M; Iwai, K; Imura, H

    1992-01-01

    An HLA-DR restricted T cell clone (26G11) which recognized a lymphoid cell-derived autoantigen associated with DR4 molecule was shown to induce not only autologous but also allogenic DR4+ B cells to produce large amounts of antibodies of the IgG and IgM classes. Using the helper activity of this clone, we investigated the mechanism of anti-DNA antibody production in DR-matched patients with systemic lupus erythematosus (SLE). When cultured with 26G11 cells, B cells from DR-matched normal control subjects produced large amounts of IgM anti-DNA antibody, but did not produce IgG anti-DNA antibody which is thought to have a pathological role in SLE. In contrast, B cells from DR-matched patients with active SLE spontaneously produced a fairly large amount of IgG anti-DNA antibody, and the production was augmented by the T cell clone. Little IgG anti-DNA antibody was produced by the B cells of patients with inactive SLE in either the presence or absence of T cell clone. We next fractionated B cells into low density B (LD-B) and high density B (HD-B) cells by centrifugation on discontinuous Percoll density gradients. IgG anti-DNA antibody was spontaneously produced by LD-B cells of active SLE patients but not by those either of inactive SLE patients or normal controls. On the other hand, although IgG anti-DNA antibody was not spontaneously produced by the HD-B cells of both active and inactive SLE patients, it could easily be induced by their culture with the T cell clone. Our results clearly show the existence of IgG anti-DNA antibody-producing B cells in the peripheral blood of SLE patients irrespective of their disease activity and suggest that autoreactive T cells may play a pathogenic role in SLE through the induction of autoantibody production. PMID:1424281

  10. A nano-cheese-cutter to directly measure interfacial adhesion of freestanding nano-fibers

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Najem, Johnny F.; Wong, Shing-Chung; Wan, Kai-tak

    2012-01-01

    A nano-cheese-cutter is fabricated to directly measure the adhesion between two freestanding nano-fibers. A single electrospun fiber is attached to the free end of an atomic force microscope cantilever, while a similar fiber is similarly prepared on a mica substrate in an orthogonal direction. External load is applied to deform the two fibers into complementary V-shapes, and the force measurement allows the elastic modulus to be determined. At a critical tensile load, "pull-off" occurs when the adhering fibers spontaneously detach from each other, yielding the interfacial adhesion energy. Loading-unloading cycles are performed to investigate repeated adhesion-detachment and surface degradation.

  11. Removal of a Penile Constriction Device with a Large Orthopedic Pin Cutter

    PubMed Central

    Wenzler, David; Fischer, Melissa

    2014-01-01

    Penile strangulation is an infrequent clinical condition that has widely been reported. It usually results following placement of a constriction device to enhance sexual stimulation. Early treatment is essential to avoid potential complications, including ischemic necrosis and autoamputation. We describe the use of a Large Orthopedic Pin Cutter to remove a metal penile constriction device in the Emergency Department (ED). This case report describes the relatively safe technique of using an instrument available in many hospitals that can be added to the physician's arsenal in the removal of metal constriction devices. PMID:24707434

  12. Development of a low cost, 3-DOF desktop laser cutter using 3D printer hardware

    NASA Astrophysics Data System (ADS)

    Jivraj, Jamil; Huang, Yize; Wong, Ronnie; Lu, Yi; Vuong, Barry; Ramjist, Joel; Gu, Xijia; Yang, Victor X. D.

    2015-03-01

    This paper presents the development of a compact, desktop laser-cutting system capable of cutting materials such as wood, metal and plastic. A re-commissioned beheaded MakerBot® Replicator 2X is turned into a 3-DOF laser cutter by way of integration with 800W (peak power) fiber laser. Special attention is paid to tear-down, modification and integration of the objective lens in place of the print head. Example cuts in wood and metal will be presented, as well as design of an exhaust system.

  13. Jumbo Cutter for Removal of A Bent Femoral Interlocking Nail: A Cost Effective Method.

    PubMed

    Dhanda, Manjeet Singh; Madan, Harnam Singh; Sharma, Sansar C; Ali, Nadeem; Bhat, Abedullah

    2015-06-01

    Closed diaphyseal femoral shaft fractures can be treated with multiple surgical options. It is more challenging to remove a bent nail than a broken one because it is difficult to retrieve the bent nail through the intramedullary canal. Various authors have published their techniques for removal of bent femoral interlocking nail. This article describes a simple technique using Jumbo cutter for sectioning and removal of bent interlocking nail. This technique will help orthopaedic surgeons to remove bent nail without using any specialised metal cutting instruments. PMID:26266173

  14. Jumbo Cutter for Removal of A Bent Femoral Interlocking Nail: A Cost Effective Method

    PubMed Central

    Dhanda, Manjeet Singh; Sharma, Sansar C; Ali, Nadeem; Bhat, Abedullah

    2015-01-01

    Closed diaphyseal femoral shaft fractures can be treated with multiple surgical options. It is more challenging to remove a bent nail than a broken one because it is difficult to retrieve the bent nail through the intramedullary canal. Various authors have published their techniques for removal of bent femoral interlocking nail. This article describes a simple technique using Jumbo cutter for sectioning and removal of bent interlocking nail. This technique will help orthopaedic surgeons to remove bent nail without using any specialised metal cutting instruments. PMID:26266173

  15. 2. Spar, bramble, and the larger cutters storis (W38) make ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. Spar, bramble, and the larger cutters storis (W38) make their way through arctic ice during the first transit of the northwest passage by a U.S. vessel. The lead 180 has a weight suspended over its starboard side. By swinging this weight back and forth across the centerline, the vessel can rock to free herself from ice. - U.S. Coast Guard Buoy Tenders, 180' Class, U.S. Coast Guard Headquarters, 2100 Second Street Southwest, Washington, District of Columbia, DC

  16. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes

    PubMed Central

    Wang, Shi; Lv, Jia; Zhang, Lingling; Dou, Jinzhuang; Sun, Yan; Li, Xue; Fu, Xiaoteng; Dou, Huaiqian; Mao, Junxia; Hu, Xiaoli; Bao, Zhenmin

    2015-01-01

    Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the ‘perfect solution’, and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost. PMID:26581575

  17. MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes.

    PubMed

    Wang, Shi; Lv, Jia; Zhang, Lingling; Dou, Jinzhuang; Sun, Yan; Li, Xue; Fu, Xiaoteng; Dou, Huaiqian; Mao, Junxia; Hu, Xiaoli; Bao, Zhenmin

    2015-11-01

    Characterization of dynamic DNA methylomes in diverse phylogenetic groups has attracted growing interest for a better understanding of the evolution of DNA methylation as well as its function and biological significance in eukaryotes. Sequencing-based methods are promising in fulfilling this task. However, none of the currently available methods offers the 'perfect solution', and they have limitations that prevent their application in the less studied phylogenetic groups. The recently discovered Mrr-like enzymes are appealing for new method development, owing to their ability to collect 32-bp methylated DNA fragments from the whole genome for high-throughput sequencing. Here, we have developed a simple and scalable DNA methylation profiling method (called MethylRAD) using Mrr-like enzymes. MethylRAD allows for de novo (reference-free) methylation analysis, extremely low DNA input (e.g. 1 ng) and adjustment of tag density, all of which are still unattainable for most widely used methylation profiling methods such as RRBS and MeDIP. We performed extensive analyses to validate the power and accuracy of our method in both model (plant Arabidopsis thaliana) and non-model (scallop Patinopecten yessoensis) species. We further demonstrated its great utility in identification of a gene (LPCAT1) that is potentially crucial for carotenoid accumulation in scallop adductor muscle. MethylRAD has several advantages over existing tools and fills a void in the current epigenomic toolkit by providing a universal tool that can be used for diverse research applications, e.g. from model to non-model species, from ordinary to precious samples and from small to large genomes, but at an affordable cost. PMID:26581575

  18. Plastibell Device Circumcision versus Bone Cutter Technique in terms of Operative Outcomes and Parent’s Satisfaction

    PubMed Central

    Mehmood, Tahir; Azam, Hammad; Tariq, Muhammad; Iqbal, Zafar; Mehmood, Hassan; Shah, Syed Asif Hussain

    2016-01-01

    Objective: To compare the rate of complications of Plastibell and bone cutter circumcision technique and recognition of top worries and satisfaction rate in the mind of parents before and after the procedure of Plastibell device (PD) circumcision in infants less than 6 months of age. Methods: It was a descriptive prospective study conducted at department of surgery Sheikh Zayed Hospital, Rahim Yar Khan. Two hundred parents of infants of less than six months of age were recruited for this study. Infants were divided into two equal groups. Group I included Plastibell circumcision technique and Group II included Bone Cutter Circumcision technique. Data was analyzed using SPSS Version 17. Independent sample t-test and chi-square test was used to compare quantitative and qualitative variables respectively. P-value <0.05 was taken as significant difference. Results: Total number of two hundred infants were included in this study. Most common worries of parents about Plastibell Device circumcision were; fear of fever (42.0%). Fear of pain and bleeding (66.0%). Plastibell Device method was associated with less operation time and bleeding as compared to bone cutter method (P-value <0.0001 and <0.0001 respectively). Incidence of complications other than bleeding and infection was 3.0% in bone cutter method and 1.0% in Plastibell device method. Pain score was significantly less in plastibell device group (p-value <0.0001). Post-operatively, 98% parents showed complete procedural satisfaction in Plastibell group versus 87% parents in bone cutter one month after surgery (P-value 0.003). About 4% parents in bone cutter method group showed cosmetic displeasure versus only 1% parents in plastibell device group. Conclusion: The study concluded that Plastibell Device circumcision is a safer technique for circumcision and is associated with highest level of parent’s satisfaction. PMID:27182237

  19. Distinction of deep versus superficial clinical and nonclinical isolates of Trichosporon beigelii by isoenzymes and restriction fragment length polymorphisms of rDNA generated by polymerase chain reaction.

    PubMed Central

    Kemker, B J; Lehmann, P F; Lee, J W; Walsh, T J

    1991-01-01

    Fifteen clinical and environmental strains of Trichosporon beigelii were analyzed for similarities by using morphological features, biochemical profiles based on carbon compound assimilation and uric acid utilization, isoenzyme electrophoresis, and restriction fragment length polymorphisms of a segment of genes coding for rRNA expanded with the polymerase chain reaction. The findings suggest that strains that cause invasive disease are distinct from the superficial and the nonclinical isolates and that isolates from the skin and mucosae represent a number of different organisms, including some environmental forms. The study shows that T. beigelii is a complex of genetically distinct organisms and that more than one type is found in clinical samples. Images PMID:1684798

  20. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity

    PubMed Central

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal ‘short-term stress memory’ with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. DOI: http://dx.doi.org/10.7554/eLife.13546.001 PMID:27242129

  1. Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

    PubMed

    Wibowo, Anjar; Becker, Claude; Marconi, Gianpiero; Durr, Julius; Price, Jonathan; Hagmann, Jorg; Papareddy, Ranjith; Putra, Hadi; Kageyama, Jorge; Becker, Jorg; Weigel, Detlef; Gutierrez-Marcos, Jose

    2016-01-01

    Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring. PMID:27242129

  2. The fungus gardens of leaf-cutter ants undergo a distinct physiological transition during biomass degradation

    SciTech Connect

    Huang, Eric L.; Aylward, Frank O.; Kim, Young-Mo; Webb-Robertson, Bobbie-Jo M.; Nicora, Carrie D.; Hu, Zeping; Metz, Thomas O.; Lipton, Mary S.; Smith, Richard D.; Currie, Cameron R.; Burnum-Johnson, Kristin E.

    2014-08-01

    Leaf-cutter ants are dominant herbivores in ecosystems throughout the Neotropics. Rather than directly consuming the fresh foliar biomass they harvest, these ants use it to cultivate specialized fungus gardens. Although recent investigations have shed light on how plant biomass is degraded in fungus gardens, the cycling of nutrients that takes place in these specialized microbial ecosystems is still not well understood. Here, using metametabolomics and metaproteomics techniques, we examine the dynamics of nutrient turnover and biosynthesis in these gardens. Our results reveal that numerous free amino acids and sugars are depleted throughout the process of biomass degradation, indicating that easily accessible nutrients from plant material are readily consumed by microbes in these ecosystems. Accumulation of cellobiose and lignin derivatives near the end of the degradation process is consistent with previous findings of cellulases and laccases produced by Leucoagaricus gongylophorus, the fungus cultivated by leaf-cutter ants. Our results also suggest that ureides may be an important source of nitrogen in fungus gardens, especially during nitrogen-limiting conditions. No free arginine was detected in our metametabolomics experiments despite evidence that the host ants cannot produce this amino acid, suggesting that biosynthesis of this metabolite may be tightly regulated in the fungus garden. These results provide new insights into the dynamics of nutrient cycling that underlie this important ant-fungus symbiosis.

  3. Laser forming cutting once quenched high-speed tool steel (HSTS) disk-shaped milling cutter

    NASA Astrophysics Data System (ADS)

    Ding, Zhihong; Liu, Yongzhen; Weng, Shiping

    1998-08-01

    Laser cutting technology has been applied to ordinary alloy steel circular sawblade, but it is very rarely used in quenched HSTS disk-shape milling-cutters due to the material particularity. In this paper, the authors systematically explain the advantages of this new technique, respecting the optimum design of HSTS disk-shape milling-cutter, the specific characteristics of laser forming cutting once for all, the technology testing, the analysis of structural performance of tooth and the small batch production for verifying. The article displays its advantages completely as follows: The design for a perfect tooth profile is not bound to the ordinary machining methods; The special laser technique does not lower the hardness on the tooth nose so that this process and needs no follow-up operational sequences, ensures the excellent dynamic-balance performance and operation properties, and prolongs the tools' service time; The new technique also has advantages of high efficiency and good economics. Therefore, this special laser cutting method, an integration of intensified heat-treatment and laser forming cutting once for all technology, will be regarded as a reform in HSTS tools Manufacturing field.

  4. Multiple paternity, relatedness and genetic diversity in Acromyrmex leaf-cutter ants

    PubMed Central

    Boomsma, J. J.; Fjerdingstad, E. J.; Frydenberg, J.

    1999-01-01

    Multiple queen-mating occurs in many social insects, but high degrees of multiple paternity have only been found in honeybees and some yellowjacket wasps. Here we report the first case of an ant species where multiple mating reduces relatedness among female offspring to values significantly lower than 0.5. Genetic analysis of a Panamanian population of the leaf-cutter ant Acromyrmex octospinosus showed that queens mate with at least 4 to 10 males. The detected (minimum) genetically effective paternity of nestmate females was 3.9 and estimates of mean relatedness among nestmate females were ca. 0.33. This implies that multiple queen-mating in Acromyrmex octospinosus reduces relatedness to 44% of the value in full-sib colonies (0.75), realizing 84% of the maximum reduction (to 0.25) that would be obtained with an infinite number of matings. Queens of Panamanian Acromyrmex octospinosus mate with more males than sympatric queens of Atta colombica, which is contrary to the positive relationship between queen-mating frequency and colony size found across more distantly related ant species. Possible selective forces that maintain high queen-mating frequencies in leaf-cutter ants are discussed.

  5. Influence of Corrosion on the Abrasion of Cutter Steels Used in TBM Tunnelling

    NASA Astrophysics Data System (ADS)

    Espallargas, N.; Jakobsen, P. D.; Langmaack, L.; Macias, F. J.

    2015-01-01

    Abrasion on tunnel boring machine (TBM) cutters may be critical in terms of project duration and costs. Several researchers are currently studying the degradation of TBM cutter tools used for excavating hard rock, soft ground and loose soil. So far, the primary focus of this research has been directed towards abrasive wear. Abrasive wear is a very common process in TBM excavation, but with a view to the environment in which the tools are working, corrosion may also exert an influence. This paper presents a selection of techniques that can be used to evaluate the influence of corrosion on abrasion on TBM excavation tools. It also presents the influence of corrosion on abrasive wear for some initial tests, with constant steel and geomaterial and varying properties of the excavation fluids (soil conditioners, anti-abrasion additives and water). The results indicate that the chloride content in the water media greatly influences the amount of wear, providing evidence of the influence of corrosion on the abrasion of the cutting tools. The presence of conditioning additives tailored to specific rock or soil conditions reduces wear. However, when chloride is present in the water, the additives minimise wear rates but fail to suppress corrosion of the cutting tools.

  6. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  7. APOBECs and Virus Restriction

    PubMed Central

    Harris, Reuben S.; Dudley, Jaquelin P.

    2015-01-01

    The APOBEC family of single-stranded DNA cytosine deaminases comprises a formidable arm of the vertebrate innate immune system. Pre-vertebrates express a single APOBEC, whereas some mammals produce as many as eleven enzymes. The APOBEC3 subfamily displays both copy number variation and polymorphisms, consistent with ongoing pathogenic pressures. These enzymes restrict the replication of many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. APOBEC1 and activation-induced cytosine deaminase (AID) have specialized functions in RNA editing and antibody gene diversification, respectively, whereas APOBEC2 and APOBEC4 appear to have different functions. Nevertheless, the APOBEC family protects against both periodic viral zoonoses as well as exogenous and endogenous parasite replication. This review highlights viral pathogens that are restricted by APOBEC enzymes, but manage to escape through unique mechanisms. The sensitivity of viruses that lack counterdefense measures highlights the need to develop APOBEC-enabling small molecules as a new class of anti-viral drugs. PMID:25818029

  8. Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse.

    PubMed

    Vu, Thanh H; Li, Tao; Hoffman, Andrew R

    2004-10-01

    Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes. PMID:15294879

  9. An Attachable Electromagnetic Energy Harvester Driven Wireless Sensing System Demonstrating Milling-Processes and Cutter-Wear/Breakage-Condition Monitoring

    PubMed Central

    Chung, Tien-Kan; Yeh, Po-Chen; Lee, Hao; Lin, Cheng-Mao; Tseng, Chia-Yung; Lo, Wen-Tuan; Wang, Chieh-Min; Wang, Wen-Chin; Tu, Chi-Jen; Tasi, Pei-Yuan; Chang, Jui-Wen

    2016-01-01

    An attachable electromagnetic-energy-harvester driven wireless vibration-sensing system for monitoring milling-processes and cutter-wear/breakage-conditions is demonstrated. The system includes an electromagnetic energy harvester, three single-axis Micro Electro-Mechanical Systems (MEMS) accelerometers, a wireless chip module, and corresponding circuits. The harvester consisting of magnets with a coil uses electromagnetic induction to harness mechanical energy produced by the rotating spindle in milling processes and consequently convert the harnessed energy to electrical output. The electrical output is rectified by the rectification circuit to power the accelerometers and wireless chip module. The harvester, circuits, accelerometer, and wireless chip are integrated as an energy-harvester driven wireless vibration-sensing system. Therefore, this completes a self-powered wireless vibration sensing system. For system testing, a numerical-controlled machining tool with various milling processes is used. According to the test results, the system is fully self-powered and able to successfully sense vibration in the milling processes. Furthermore, by analyzing the vibration signals (i.e., through analyzing the electrical outputs of the accelerometers), criteria are successfully established for the system for real-time accurate simulations of the milling-processes and cutter-conditions (such as cutter-wear conditions and cutter-breaking occurrence). Due to these results, our approach can be applied to most milling and other machining machines in factories to realize more smart machining technologies. PMID:26907297

  10. Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

    PubMed Central

    Aylward, Frank O.; Burnum-Johnson, Kristin E.; Tringe, Susannah G.; Teiling, Clotilde; Tremmel, Daniel M.; Moeller, Joseph A.; Scott, Jarrod J.; Barry, Kerrie W.; Piehowski, Paul D.; Nicora, Carrie D.; Malfatti, Stephanie A.; Monroe, Matthew E.; Purvine, Samuel O.; Goodwin, Lynne A.; Smith, Richard D.; Weinstock, George M.; Gerardo, Nicole M.; Suen, Garret; Lipton, Mary S.

    2013-01-01

    Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar. PMID:23584789

  11. An Attachable Electromagnetic Energy Harvester Driven Wireless Sensing System Demonstrating Milling-Processes and Cutter-Wear/Breakage-Condition Monitoring.

    PubMed

    Chung, Tien-Kan; Yeh, Po-Chen; Lee, Hao; Lin, Cheng-Mao; Tseng, Chia-Yung; Lo, Wen-Tuan; Wang, Chieh-Min; Wang, Wen-Chin; Tu, Chi-Jen; Tasi, Pei-Yuan; Chang, Jui-Wen

    2016-01-01

    An attachable electromagnetic-energy-harvester driven wireless vibration-sensing system for monitoring milling-processes and cutter-wear/breakage-conditions is demonstrated. The system includes an electromagnetic energy harvester, three single-axis Micro Electro-Mechanical Systems (MEMS) accelerometers, a wireless chip module, and corresponding circuits. The harvester consisting of magnets with a coil uses electromagnetic induction to harness mechanical energy produced by the rotating spindle in milling processes and consequently convert the harnessed energy to electrical output. The electrical output is rectified by the rectification circuit to power the accelerometers and wireless chip module. The harvester, circuits, accelerometer, and wireless chip are integrated as an energy-harvester driven wireless vibration-sensing system. Therefore, this completes a self-powered wireless vibration sensing system. For system testing, a numerical-controlled machining tool with various milling processes is used. According to the test results, the system is fully self-powered and able to successfully sense vibration in the milling processes. Furthermore, by analyzing the vibration signals (i.e., through analyzing the electrical outputs of the accelerometers), criteria are successfully established for the system for real-time accurate simulations of the milling-processes and cutter-conditions (such as cutter-wear conditions and cutter-breaking occurrence). Due to these results, our approach can be applied to most milling and other machining machines in factories to realize more smart machining technologies. PMID:26907297

  12. Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

    SciTech Connect

    Aylward, Frank O.; Burnum-Johnson, Kristin E.; Tringe, Susannah G.; Teiling, Clotilde; Tremmel, Daniel; Moeller, Joseph; Scott, Jarrod J.; Barry, Kerrie W.; Piehowski, Paul D.; Nicora, Carrie D.; Malfatti, Stephanie; Monroe, Matthew E.; Purvine, Samuel O.; Goodwin, Lynne A.; Smith, Richard D.; Weinstock, George; Gerardo, Nicole; Suen, Garret; Lipton, Mary S.; Currie, Cameron R.

    2013-06-12

    Plants represent a large reservoir of organic carbon comprised largely of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate fungus gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous symbiont that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and using genomic, metaproteomic, and phylogenetic tools we investigate its role in lignocellulose degradation in the fungus gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in fungus gardens, and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that may be playing an important but previously uncharacterized role in lignocellulose degradation. Our study provides a comprehensive analysis of plant biomass degradation in leaf-cutter ant fungus gardens and provides insight into the molecular dynamics underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.

  13. Dietary restrictions and cancer.

    PubMed Central

    Hart, R W; Turturro, A

    1997-01-01

    Dietary restriction (DR) alters a significant environmental factor in carcinogenesis, dietary intake, thus inhibiting both spontaneous and induced tumorigenesis. Potential mechanisms for the inhibition of spontaneous cancer may include the effects of DR to do the following: decrease body weight, which decreases cellular proliferation and increases apoptosis in a number of organs that increase and decrease with body size; decrease body temperature, thereby lowering the amount of endogenous DNA damage temperature generates; decrease oxidative damage, by increasing antioxidant damage defense systems; decrease, generally, cellular proliferation; and protect the fidelity of the genome by decreasing DNA damage, increasing DNA repair, and preventing aberrant gene expression. Potential mechanisms for reducing induced tumor incidence include lowering agent activation, changing agent disposition, decreasing the adducts most associated with agent toxicity, and inhibiting tumor progression through mechanisms similar to those that can effect spontaneous tumorigenesis. As a method to control a major source of environmental cancer, and as the major modulator of the agent induction of this disease, understanding how DR works may significantly contribute to the efforts to explain how diet impacts on development of cancer in the United States, and may suggest methods to reduce the adverse impacts of other environmental agents on the disease. PMID:9255593

  14. Bacterial community profiles on feathers during composting as determined by terminal restriction fragment length polymorphism analysis of 16S rDNA genes.

    PubMed

    Tiquia, S M; Ichida, J M; Keener, H M; Elwell, D L; Burtt, E H; Michel, F C

    2005-05-01

    Composting is one of the more economical and environmentally safe methods of recycling feather waste generated by the poultry industry, since 90% of the feather weight consists of crude keratin protein, and feathers contain 15% N. However, the keratin in waste feathers is resistant to biodegradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Two keratin-degrading bacteria isolated from plumage of wild songbirds and identified as Bacillus licheneformis (OWU 1411T) and Streptomyces sp. (OWU 1441) were inoculated into poultry feather composts (1.13 x 10(8) cfu g(-1) feathers) and co-composted with poultry litter and straw in 200-l compost vessels. Composting temperatures, as well as CO(2) and NH(3) evolution, were measured in these vessels to determine the effects of inoculation on the rate and extent of poultry feather decomposition during composting. Terminal restriction fragment length polymorphisms of 16S rRNA genes were used to follow changes in microbial community structure during composting. The results indicated that extensive carbon conversion occurred in both treatments (55.5 and 56.1%). The addition of the bacterial inocula did not enhance the rate of waste feather composting. The microbial community structure over time was very similar in inoculated and uninoculated waste feather composts. PMID:15614566

  15. Telomere Restriction Fragment (TRF) Analysis

    PubMed Central

    Mender, Ilgen; Shay, Jerry W.

    2016-01-01

    restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp. PMID:27500189

  16. Final Report: Very Low Friction Small Radius Dome Cutters for Percussion Bits - Phase II Development Efforts, April 1, 1997 - September 1, 1999

    SciTech Connect

    Pixton, David S.

    1999-07-15

    Phase II efforts to develop very low friction (polished) small radius cutters for drill bits are discussed. Key developments under this contract include: (1) improvements to robustness of polycrystalline diamond coatings enabling their use on sharper cutter shapes; (2) polishable coating materials which exhibit improved polish retention; and (3) a means of polishing a non-planar polycrystalline diamond surface economically. Field tests have shown acceptability of new small radius cutters, but have yet to show benefits of polishing. Further field tests are planned.

  17. Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis.

    PubMed

    Chandna, Piyush; Mallik, Sarita; Kuhad, Ramesh Chander

    2013-08-01

    An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2-6 bands ranging from 90-688 bp, HinfI generated 2-5 bands of 71-1,038 bp, and MspI 2-7 bands of 69-793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software "Diversity Database." HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing. PMID:23053087

  18. TBM performance prediction in Yucca Mountain welded tuff from linear cutter tests

    SciTech Connect

    Gertsch, R.; Ozdemir, L.; Gertsch, L.

    1992-11-01

    This paper discusses performance prediction which were developed for tunnel boring machines operating in welded tuff for the construction of the experimental study facility and the potential nuclear waste repository at Yucca Mountain. The predictions were based on test data obtained from an extensive series of linear cutting tests performed on samples of Topopah String welded tuff from the Yucca Mountain Project site. Using the cutter force, spacing, and penetration data from the experimental program, the thrust, torque, power, and rate of penetration were estimated for a 25 ft diameter tunnel boring machine (TBM) operating in welded tuff. The result show that the Topopah Spring welded tuff (TSw2) can be excavated at relatively high rates of advance with state-of-the-art TBMs. The result also show, however, that the TBM torque and power requirements will be higher than estimated based on rock physical properties and past tunneling experience in rock formations of similar strength.

  19. The Evolutionary Innovation of Nutritional Symbioses in Leaf-Cutter Ants

    PubMed Central

    Aylward, Frank O.; Currie, Cameron R.; Suen, Garret

    2012-01-01

    Fungus-growing ants gain access to nutrients stored in plant biomass through their association with a mutualistic fungus they grow for food. This 50 million-year-old obligate mutualism likely facilitated some of these species becoming dominant Neotropical herbivores that can achieve immense colony sizes. Recent culture-independent investigations have shed light on the conversion of plant biomass into nutrients within ant fungus gardens, revealing that this process involves both the fungal cultivar and a symbiotic community of bacteria including Enterobacter, Klebsiella, and Pantoea species. Moreover, the genome sequences of the leaf-cutter ants Atta cephalotes and Acromyrmex echinatior have provided key insights into how this symbiosis has shaped the evolution of these ants at a genetic level. Here we summarize the findings of recent research on the microbial community dynamics within fungus-growing ant fungus gardens and discuss their implications for this ancient symbiosis. PMID:26467948

  20. Hydraulic Resistance of Vitreous Cutters: The Impact of Blade Design and Cut Rate

    PubMed Central

    Rossi, Tommaso; Querzoli, Giorgio; Angelini, Giampiero; Malvasi, Carlo; Rossi, Alessandro; Morini, Mario; Esposito, Graziana; Micera, Alessandra; di Luca, Natale Mario; Ripandelli, Guido

    2016-01-01

    Purpose To measure the hydraulic resistance (HR) of vitreous cutters equipped with a Regular guillotine Blade (RB) or double edge blade (DEB) at cut rates comprised between 0 and 12,000 cuts per minute (CPM) and compare it with vitreous fragment size. This was an in vitro experimental study; in vivo HR measure and vitreous sampling. Methods HR, defined as aspiration pressure/flow rate, was measured in balanced salt solution (BSS; Alcon, Fort Worth, TX) (in vitro) and during pars plana vitrectomy of 20 consecutive patients aged 18 to 65, undergoing macular surgery. HR was recorded at increasing cut rates (500–6000 CPM for the RB and 500–12,000 CPM for the DEB; 5 mL/min flow). Vitreous samples were withdrawn and analyzed with Western and collagen type II and IX immunostaining to evaluate protein size. The main outcome measures were hydraulic resistance (mm Hg/ml/min [±SD]) and optic density for Western blot and immunostaining. Results RB and DEB showed identical HR in BSS between 0 and 3000 CPM. Above 3000 CPM, RB HR steadily increased, and was significantly higher than DEB HR. Vitreous HR was also similar for the two blades between 0 and 1500 CPM. Above 1500 CPM, RB offered a significantly higher resistance. Western blot and immunostaining of vitreous samples did not yield a significant difference in size, regardless of blade type and cut rate. Conclusions DEB is more efficient, offering a lower HR than RB over 1500 CPM in human vitreous. There is no viscosity reduction as a function of cut-rate between 1500 and 12,000 CPM, as HR does not vary. Translational Relevance Future vitreous cutters will benefit of a DEB; optimal cut rate needs to be defined, and the simple increase of cut rate does not provide benefits after a certain limit to be assessed. PMID:27441099

  1. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  2. Inhibitor of DNA Binding 4 (ID4) Is Highly Expressed in Human Melanoma Tissues and May Function to Restrict Normal Differentiation of Melanoma Cells

    PubMed Central

    Peretz, Yuval; Wu, Hong; Patel, Shayan; Bellacosa, Alfonso; Katz, Richard A.

    2015-01-01

    Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a

  3. Deletion of one nucleotide within the homonucleotide tract present in the hsdS gene alters the DNA sequence specificity of type I restriction-modification system NgoAV.

    PubMed

    Adamczyk-Poplawska, Monika; Lower, Michal; Piekarowicz, Andrzej

    2011-12-01

    As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdS(NgoAV1) and hsdS(NgoAV2). The specificity subunit, HsdS(NgoAV), the product of the hsdS(NgoAV1) gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3' end of the hsdS(NgoAV1) gene makes the NgoAV system a strong candidate for phase variation, i.e., stochastic addition or reduction in the guanine number. We have constructed mutants with 6 guanines instead of 7 and demonstrated that the deletion of a single nucleotide within the 3' end of the hsdS(NgoAV1) gene restored the fusion between the hsdS(NgoAV1) and hsdS(NgoAV2) genes. We have demonstrated that such a contraction of the homonucleotide tract may occur in vivo: in a Neisseria gonorrhoeae population, a minor subpopulation of cells appeared to have only 6 guanines at the 3' end of the hsdS(NgoAV1) gene. Escherichia coli cells carrying the fused gene and expressing the NgoAVΔ RM system were able to restrict λ phage at a level comparable to that for the wild-type NgoAV system. NgoAV recognizes the quasipalindromic interrupted sequence 5'-GCA(N(8))TGC-3' and methylates both strands. NgoAVΔ recognizes DNA sequences 5'-GCA(N(7))GTCA-3' and 5'-GCA(N(7))CTCA-3', although the latter sequence is methylated only on the complementary strand within the 5'-CTCA-3' region of the second recognition target sequence. PMID:21984785

  4. Deletion of One Nucleotide within the Homonucleotide Tract Present in the hsdS Gene Alters the DNA Sequence Specificity of Type I Restriction-Modification System NgoAV▿†

    PubMed Central

    Adamczyk-Poplawska, Monika; Lower, Michal; Piekarowicz, Andrzej

    2011-01-01

    As a result of a frameshift mutation, the hsdS locus of the NgoAV type IC restriction and modification (RM) system comprises two genes, hsdSNgoAV1 and hsdSNgoAV2. The specificity subunit, HsdSNgoAV, the product of the hsdSNgoAV1 gene, is a naturally truncated form of an archetypal specificity subunit (208 N-terminal amino acids instead of 410). The presence of a homonucleotide tract of seven guanines (poly[G]) at the 3′ end of the hsdSNgoAV1 gene makes the NgoAV system a strong candidate for phase variation, i.e., stochastic addition or reduction in the guanine number. We have constructed mutants with 6 guanines instead of 7 and demonstrated that the deletion of a single nucleotide within the 3′ end of the hsdSNgoAV1 gene restored the fusion between the hsdSNgoAV1 and hsdSNgoAV2 genes. We have demonstrated that such a contraction of the homonucleotide tract may occur in vivo: in a Neisseria gonorrhoeae population, a minor subpopulation of cells appeared to have only 6 guanines at the 3′ end of the hsdSNgoAV1 gene. Escherichia coli cells carrying the fused gene and expressing the NgoAVΔ RM system were able to restrict λ phage at a level comparable to that for the wild-type NgoAV system. NgoAV recognizes the quasipalindromic interrupted sequence 5′-GCA(N8)TGC-3′ and methylates both strands. NgoAVΔ recognizes DNA sequences 5′-GCA(N7)GTCA-3′ and 5′-GCA(N7)CTCA-3′, although the latter sequence is methylated only on the complementary strand within the 5′-CTCA-3′ region of the second recognition target sequence. PMID:21984785

  5. Restricting retrotransposons: a review.

    PubMed

    Goodier, John L

    2016-01-01

    Retrotransposons have generated about 40 % of the human genome. This review examines the strategies the cell has evolved to coexist with these genomic "parasites", focussing on the non-long terminal repeat retrotransposons of humans and mice. Some of the restriction factors for retrotransposition, including the APOBECs, MOV10, RNASEL, SAMHD1, TREX1, and ZAP, also limit replication of retroviruses, including HIV, and are part of the intrinsic immune system of the cell. Many of these proteins act in the cytoplasm to degrade retroelement RNA or inhibit its translation. Some factors act in the nucleus and involve DNA repair enzymes or epigenetic processes of DNA methylation and histone modification. RISC and piRNA pathway proteins protect the germline. Retrotransposon control is relaxed in some cell types, such as neurons in the brain, stem cells, and in certain types of disease and cancer, with implications for human health and disease. This review also considers potential pitfalls in interpreting retrotransposon-related data, as well as issues to consider for future research. PMID:27525044

  6. The effect of the Rim Cutter on cement pressurization and penetration on cemented acetabular fixation in total hip arthroplasty: an in vitro study.

    PubMed

    Smith, B N; Lee, A J C; Timperley, A J; Whitehouse, S L; Crawford, R W

    2010-01-01

    The Rim Cutter (Stryker Orthopedics, Mahwah, New Jersey) is a tool designed to cut a ledge inside the rim of the acetabulum, onto which a precisely trimmed, cemented, flanged cup can be fitted. The aim was to investigate the effect of the Rim Cutter on the intra-acetabular cement mantle pressure and the depth of cement penetration during cup insertion. The study had two parts. In the first part, hemi-pelvis models were fitted with pressure sensors. Pressure in the acetabulum was measured on insertion of a conventional cemented flanged cup with and without the use of a Rim Cutter to prepare the rim of the acetabulum. The second part assessed cement penetration when the same cups were inserted into a foam shell model. The shell was mounted in a jig and had holes drilled in it; the distance that cement penetrated into the holes was measured. A significant increase in cement pressure at the apex (p = 0.04) and the rim (p = 0.004) is seen when the Rim Cutter is used. Cement penetration in the Rim Cutter group was significantly increased at the rim of the acetabulum (p = 0.003). Insertion of a flanged cup after the acetabulum is prepared with the Rim Cutter leads to a significant increase in cement pressure and penetration during cup insertion in vitro when compared with conventional flanged cups. PMID:21053777

  7. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  8. In vitro analysis of a primary, major histocompatibility complex (MHC)-restricted, cytotoxic T-lymphocyte response to avian leukosis virus (ALV), using target cells expressing MHC class I cDNA inserted into a recombinant ALV vector.

    PubMed

    Thacker, E L; Fulton, J E; Hunt, H D

    1995-10-01

    The interaction between the major histocompatibility complex (MHC) and cytotoxic T lymphocytes (CTLs) is an important component of the host's resistance to viral infections and tumor formation. In this study, an avian leukosis virus (ALV) vector system, RCASBP, expressing MHC chicken class I (B-F) cDNA was used to develop target cells expressing the chicken class I glycoproteins complexed with ALV antigens on the cell surface. Peripheral blood from chickens inoculated with ALV was shown to contain antigen-specific, MHC-restricted, CD8+ effector CTLs, using a 51Cr release assay utilizing the RCASBP B-F target cells. The stimulated effector cells were also predominantly alpha beta T-cell receptor-positive (TCR2) T cells. The CTL response varied between two haplotypes of chickens which differed in their response to Rous sarcoma virus (RSV)-induced tumors. Chickens with the B21 haplotype which regress RSV-induced tumors showed maximal cytolytic activity, while chickens with the B13 haplotype which do not regress RSV-induced tumors had minimal to no cytolytic activity. In addition to assessing the CTL response to ALV, the creation of MHC-specific immortal target cell lines will be extremely useful in evaluating CTL responses to other viral disease in chickens. PMID:7666545

  9. Genome-Wide Mapping of Growth-Related Quantitative Trait Loci in Orange-Spotted Grouper (Epinephelus coioides) Using Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq).

    PubMed

    Yu, Hui; You, Xinxin; Li, Jia; Liu, Hankui; Meng, Zining; Xiao, Ling; Zhang, Haifa; Lin, Hao-Ran; Zhang, Yong; Shi, Qiong

    2016-01-01

    Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish. PMID:27058532

  10. Localization in the Tomato Genome of DNA Restriction Fragments Containing Sequences Homologous to the rRNA (45s), the Major Chlorophyll a/b Binding Polypeptide and the Ribulose Bisphosphate Carboxylase Genes

    PubMed Central

    Vallejos, C. E.; Tanksley, S. D.; Bernatzky, R.

    1986-01-01

    DNA restriction fragments containing sequences homologous to the ribosomal RNA (45s), the major chlorophyll a/b binding polypeptide (CAB) and the small subunit of ribulose bisphosphate carboxylase (RBCS) genes have been localized and mapped in the tomato nuclear genome by linkage analysis. Ribosomal RNA genes map to a single locus, R45s, which resides in a terminal position on the short arm of chromosome 2 and corresponds to the Nucleolar Organizer Region. The size of the 45s repeating unit is estimated to be approximately 9 kb in Lycopersicon esculentum and 11 kb in Lycopersicon pennellii. Five loci were found to contain CAB sequences. Two of the loci, Cab-1 (chromosome 2) and Cab-3 (chromosome 8), together accounted for more than 80% of the hybridization signal. These loci contain more than one CAB structural gene. The other three loci, Cab-2 (chromosome 8), Cab-4 (chromosome 7) and Cab-5 (chromosome 12), each account for <10% of the total signal and may contain only a single copy of the CAB structural sequence. Three loci were found to contain RBCS sequences. Rbcs-2 (chromosome 3) and Rbcs-3 (chromosome 2) were responsible for >80% of the signal, with the remainder being associated with Rbcs-1 (chromosome 2). Rbcs-2 and Rbcs-3 may contain more than one copy of the gene. PMID:17246311

  11. Genome-Wide Mapping of Growth-Related Quantitative Trait Loci in Orange-Spotted Grouper (Epinephelus coioides) Using Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq)

    PubMed Central

    Yu, Hui; You, Xinxin; Li, Jia; Liu, Hankui; Meng, Zining; Xiao, Ling; Zhang, Haifa; Lin, Hao-Ran; Zhang, Yong; Shi, Qiong

    2016-01-01

    Mapping of quantitative trait loci (QTL) is essential for the discovery of genetic structures that related to complex quantitative traits. In this study, we identified 264,072 raw SNPs (single-nucleotide polymorphisms) by double digest restriction site associated DNA sequencing (ddRADseq), and utilized 3029 of these SNPs to construct a genetic linkage map in orange-spotted grouper (Epinephelus coioides) using a regression mapping algorithm. The genetic map contained 24 linkage groups (LGs) spanning a total genetic distance of 1231.98 cM. Twenty-seven significant growth-related QTLs were identified. Furthermore, we identified 17 genes (fez2, alg3, ece2, arvcf, sla27a4, sgk223, camk2, prrc2b, mchr1, sardh, pappa, syk, tert, wdrcp91, ftz-f1, mate1 and notch1) including three (tert, ftz-f1 and notch1) that have been reported to be involved in fish growth. To summarize, we mapped growth-related QTLs in the orange-spotted grouper. These QTLs will be useful in marker-assisted selection (MAS) efforts to improve growth-related traits in this economically important fish. PMID:27058532

  12. ECHO-PHONOCARDIOGRAPHIC CHARACTERISTICS OF THE NORMALLY FUNCTIONING COOLEY-CUTTER MITRAL VALVE PROSTHESIS

    PubMed Central

    Pechacek, Leonard W.; Zarrabi, Ali; Massumkhani, Ali; Garcia, Efrain; De Castro, Carlos M.; Hall, Robert J.

    1979-01-01

    Echocardiographic and phonocardiographic records of 19 patients with a normally functioning Cooley-Cutter mitral valve were analyzed in order to provide quantitative baseline values for this prosthesis. The average duration between the second heart sound and peak opening of the valve (A2-OC interval) was 83 ± 4 (standard error of the mean) msec. The Q-CC interval (from the electrocardiographic Q wave to closure of the valve) was 71 ± 2 msec. Mean opening and closing velocities of the disc were similar (396 ± 11 mm/sec and 393 ± 12 mm/sec, respectively). Amplitude of disc excursion ranged from 6 to 9 mm, but this measurement was not possible in all patients due to the presence of spurious echoes. Early onset of prosthetic valve closure was a relatively common finding in patients with atrial fibrillation or with various forms of atrioventricular block, and frequently resulted in a variety of phonocardiographic alterations. Except for the A2-OC interval and amplitude of disc excursion, there was no significant correlation between valve size and echo-phonocardiographic measurements. Results of this study are compared with values previously reported for other types of caged disc valves, and the usefulness and limitations of echo-phonocardiographic assessment of prosthetic valve function are briefly discussed. Images PMID:15216292

  13. Tooth hardness increases with zinc-content in mandibles of young adult leaf-cutter ants.

    PubMed

    Schofield, Robert M S; Nesson, Michael H; Richardson, Kathleen A

    2002-12-01

    A wide variety of arthropods and members of other phyla have elevated concentrations of Zn, Mn, other heavy metals and halogens in their jaws, leg claws, and other "tools" for interacting with the environment. While measured Zn concentrations reach 25% of dry mass in scorpion stings, concentrations are often lower than this and the enriched structures are not heavily biomineralized like vertebrate teeth and the radula of mollusks. For this reason, the degree to which the inorganic components of these structures modify their mechanical properties is in question. Here we address this problem by measuring hardness during the development of Zn accumulations in ant mandibles. We found that Zn is incorporated into the mandibular teeth of leaf-cutter ants during early adult life, reaching concentrations of about 16% of dry mass. We show that the hardness of the mandibular teeth increases nearly three-fold as the adults age and that hardness correlates with Zn content ( r=0.91). We suggest that young adults rarely cut leaves partly because their mandibles are not yet rich in Zn. Zinc enrichment (along with enrichment by other heavy metals and halogens) may play an unrecognized role in the behavioral ecology and evolution of a wide variety of invertebrates. PMID:12536282

  14. TRM performance prediction in Yucca Mountain welded tuff from linear cutter tests

    SciTech Connect

    Gertsch, R.; Ozdemir, L.; Gertsch, L.

    1992-12-31

    Performance predictions were developed for tunnel boring machines operating in welded tuff for the construction of the experimental study facility and the potential nuclear waste repository at Yucca Mountain. The predictions were based on test data obtained from an extensive series of linear cutting tests performed on samples of Topopah Spring welded tuff from the Yucca Mountain Project site. Using the cutter force, spacing, and penetration data from the experimental program, the thrust, torque, power, and rate of penetration were estimated for a 25 ft diameter tunnel boring machine (TBM) operating in welded tuff. Guidelines were developed for the optimal design of the TBM cutterhead to achieve high production rates at the lowest possible excavation costs. The results show that the Topopah Spring welded tuff (TSw2) can be excavated at relatively high rates of advance with state-of-the-art TBMs. The results also show, however, that the TBM torque and power requirements will be higher than estimated based on rock physical properties and past tunneling experience in rock formations of similar strength.

  15. Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

    PubMed Central

    Aylward, Frank O; Burnum, Kristin E; Scott, Jarrod J; Suen, Garret; Tringe, Susannah G; Adams, Sandra M; Barry, Kerrie W; Nicora, Carrie D; Piehowski, Paul D; Purvine, Samuel O; Starrett, Gabriel J; Goodwin, Lynne A; Smith, Richard D; Lipton, Mary S; Currie, Cameron R

    2012-01-01

    Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans. PMID:22378535

  16. Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

    SciTech Connect

    Aylward, Frank O.; Burnum, Kristin E.; Scott, Jarrod J.; Suen, Garret; Tringe, Susannah G.; Adams, Sandra M.; Barry, Kerrie W.; Nicora, Carrie D.; Piehowski, Paul D.; Purvine, Samuel O.; Starrett, Gabriel J.; Goodwin, Lynne A.; Smith, Richard D.; Lipton, Mary S.; Currie, Cameron R.

    2012-09-01

    Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant Neotropical herbivores cultivate symbiotic fungus gardens on massive quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers in mature Atta colonies. Here we use metagenomic, and metaproteomic techniques to characterize the bacterial diversity and overall physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that, in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter, and Escherichia. We show that these bacterial communities possess genes commonly associated with lignocellulose degradation, and likely participate in the processing of plant biomass. Additionally, we demonstrate that bacteria in these environments encode a diverse suite of biosynthetic pathways, and that they may enrich the nitrogen-poor forage of the ants with B-vitamins, amino acids, and proteins. These results are consistent with the hypothesis that fungus gardens are highly-specialized fungus-bacteria communities that efficiently convert plant material into usable energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities to the ecology and evolution of herbivorous metazoans.

  17. Identification and characterization of LA08NC01 cosmids containing rare cutter AscI sites

    SciTech Connect

    Schertzer, M.; Wood, S.; Yaremko, M.L.

    1994-09-01

    LA08NC01 is a flow-sorted human chromosome 8 cosmid library that was constructed and arrayed at Los Alamos. We have used this library to produce a sub-library of those cosmids containing AscI (GGCGCGCC) sites, which are therefore AscI-linking clones. Two protocols have been employed to identify AscI sites. The first protocol relies upon restriction digestion for cloning into doubly digesting plasmids and thereby recovering an end clone. The second protocol relies upon sequence directly, by using a 12-mer NNNNGGCGCGCC as a DNA hybridization probe. Using these protocols we have identified and confirmed 44 cosmids that contain AscI sites. Our goal is to develop markers that are rich in information. Consequently, these cosmids have been screened for CA repeats, which provide a polymorphic STS. The region surrounding the AscI site has been sequenced to provide an identifier and developed as an STS site for those cosmids lacking a CA repeat. The sequence identifier has been used for sequence database library searches. We have identified 3 genes from 8p after screening the identifiers for 10 cosmids. In addition, we have found 2 additional AscI sites from the known genes SFTP2 and POLB. Identification of the AscI site adjacent to POLB required a chromosome walk of 2 steps. Many of these cosmids are rich in information since they are frequently polymorphic, contain STS sites, provide linking clones for PFGE mapping, and often encode genes that may be placed on expression maps. In conclusion, while the total number of identified cosmids is small, the majority of them are extremely rich in information.

  18. Restriction/modification polypeptides, polynucleotides, and methods

    SciTech Connect

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  19. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  20. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  1. 9 CFR 121.13 - Restricted experiments. 10

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... publication, “NIH Guidelines for Research Involving Recombinant DNA Molecules.” This document is available on.... (b) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate... medicine, or agriculture. (2) Experiments involving the deliberate formation of recombinant DNA...

  2. Energy supplementation and productivity of Guatemalan sugar-cane cutters: a longitudinal approach.

    PubMed

    Immink, M D; Blake, C C; Viteri, F E; Flores, R; Torún, B

    1986-06-01

    A long-term energy supplementation program was carried out to determine its effect on the productivity of agricultural workers in Guatemala. The program provided, free of charge, a low-energy (24 Kcal) and a high-energy (350 Kcal) bottled, orange-flavored soft drink to two groups of long-term resident sugar-cane cutters who worked on the same plantation, located in the Pacific Coast. Previous to, and periodically thereafter during implementation of the program, data relative to energy intake and anthropometry were collected. Through data obtained from payroll lists, a longitudinal series of average productivity (tons of sugar cane cut and loaded per day) covering 48 weeks of pre-supplementation, 90 weeks of supplementation and 21 weeks post-supplementation, was constructed. Control of the supplement consumption was daily observed. Random assignment of workers to the high-energy supplement (HES) and the low-energy (LES) groups, was not possible. Prior to supplementation both groups presented the same characteristics in terms of age, energy intake level, weight, height, tricipital adiposity and daily productivity. Little variation was found throughout the time the supplement was consumed by the HES Group. Energy intake of workers increased significantly in absolute terms in relation to the LES Group, except towards the end of the 28 months' supplementation period. Energy balance was maintained by workers throughout the study period. A time series of the difference in mean productivity of the two supplement groups (Yt) was modeled using the ARIMA techniques. No auto-regressive term was present in the Yt series. The ARIMA (0,0,1) model was fitted and expanded with different intervention components. None of the estimated parameters of the intervention components were statistically significant. It was therefore concluded that no abrupt, or gradual and sustained energy supplementation effect on productivity was present. PMID:3632204

  3. Frontier mutualism: coevolutionary patterns at the northern range limit of the leaf-cutter ant–fungus symbiosis

    PubMed Central

    Mueller, Ulrich G.; Mikheyev, Alexander S.; Solomon, Scott E.; Cooper, Michael

    2011-01-01

    Tropical leaf-cutter ants cultivate the fungus Attamyces bromatificus in a many-to-one, diffuse coevolutionary relationship where ant and fungal partners re-associate frequently over time. To evaluate whether ant–Attamyces coevolution is more specific (tighter) in peripheral populations, we characterized the host-specificities of Attamyces genotypes at their northern, subtropical range limits (southern USA, Mexico and Cuba). Population-genetic patterns of northern Attamyces reveal features that have so far not been observed in the diffusely coevolving, tropical ant–Attamyces associations. These unique features include (i) cases of one-to-one ant–Attamyces specialization that tighten coevolution at the northern frontier; (ii) distributions of genetically identical Attamyces clones over large areas (up to 81 000 km2, approx. the area of Ireland, Austria or Panama); (iii) admixture rates between Attamyces lineages that appear lower in northern than in tropical populations; and (iv) long-distance gene flow of Attamyces across a dispersal barrier for leaf-cutter ants (ocean between mainland North America and Cuba). The latter suggests that Attamyces fungi may occasionally disperse independently of the ants, contrary to the traditional assumption that Attamyces fungi depend entirely on leaf-cutter queens for dispersal. Peripheral populations in Argentina or at mid-elevation sites in the Andes may reveal additional regional variants in ant–Attamyces coevolution. Studies of such populations are most likely to inform models of coextinctions of obligate mutualistic partners that are doubly stressed by habitat marginality and by environmental change. PMID:21389026

  4. Ovine TRIM5α Can Restrict Visna/Maedi Virus

    PubMed Central

    Jáuregui, P.; Crespo, H.; Glaria, I.; Luján, L.; Contreras, A.; Rosati, S.; de Andrés, D.; Amorena, B.; Towers, G. J.

    2012-01-01

    The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections. PMID:22696640

  5. How restriction enzymes became the workhorses of molecular biology

    PubMed Central

    Roberts, Richard J.

    2005-01-01

    Restriction enzymes have proved to be invaluable for the physical mapping of DNA. They offer unparalleled opportunities for diagnosing DNA sequence content and are used in fields as disparate as criminal forensics and basic research. In fact, without restriction enzymes, the biotechnology industry would certainly not have flourished as it has. The first experiments demonstrating the utility of restriction enzymes were carried out by Danna and Nathans and reported in 1971. This pioneering study set the stage for the modern practice of molecular biology in which restriction enzymes are ubiquitous tools, although they are often taken for granted. PMID:15840723

  6. Alternating vs. direct current: A transient study of the U.S. Coast Guard's 270 foot medium endurance cutter's electrical distribution system

    NASA Astrophysics Data System (ADS)

    Hutton, Keoni Alexander

    While the United States Navy has conducted extensive research into the use of shipboard DC zonal electrical distribution systems (ZED), no project has analyzed the benefits for installation on a Coast Guard cutter which has a significantly different load profile than Navy warships. Simulink models of the existing 270' medium endurance cutter (WMEC) AC radial electrical distribution system and a proposed DC ZED system were created and tested with three transients. The result demonstrated a significant reduction in settling time and an increased robustness caused by the insulation provided by the introduction of power electronic converters. Beyond the transients, a DC ZED provides better standardized installation for any ship reducing construction costs and timelines, and simplifying training and support. Additionally, the DCZED increases a ship's innate survivability by reducing longitudinal cables that penetrate watertight bulkheads increasing a boundaries effectiveness. The Coast Guard would be best served by pushing for a zonal distribution system on all future cutter acquisitions.

  7. Comparison of standard 4-row versus 6-row 3-D linear cutter stapler in creation of gastrointestinal system anastomoses: a prospective randomized trial

    PubMed Central

    Sozutek, Alper; Colak, Tahsin; Dag, Ahmet; Olmez, Tolga

    2012-01-01

    OBJECTIVE: This prospective study was conducted to compare the clinical outcomes of a 6-row 3-D linear cutter with the standard 4-row linear cutter in patients who underwent elective gastrointestinal surgery anastomosis. METHOD: Patients who underwent elective open gastrointestinal surgery that included stapled anastomosis using a linear cutter (Proximate®, Ethicon Endo-Surgery, Cincinnati, OH) between January 2011 and May 2011 were included in the study. The patients were randomly assigned to two groups according to the linear cutter that was used in the surgery: the standard 4-row cutter (the S group) or the new 6-row cutter (the N group). The groups were compared based on the patient demographic data, the laboratory parameters, the preoperative diagnosis, the surgery performed, the operation time, intra- or postoperative complications, the time to oral tolerance and the length of the hospital stay. RESULTS: The S group included 11 male and nine female patients with a mean age of 65±12 (35-84) years, while the N group included 13 male and eight female patients with a mean age of 62±11 (46-79) years (p = 0.448, p = 0.443, respectively). Anastomotic line bleeding was observed in eight (40%) patients in the S group and in one (4.7%) patient in the N group (p = 0.006). Dehiscence of the anastomosis line was observed in two (10%) patients in the S group and none in the N group (p = 0.131). Anastomotic leakage developed in three (15%) patients in the S group and in one (4.7%) patient in the N group (p = 0.269). The mean hospital stay was 12.65±6.1 days in the S group and 9.52±2.9 days in the N group (p = 0.043). CONCLUSION: The 6-row 3-D linear cutter is a safe and easily applied instrument that can be used to create anastomoses in gastrointestinal surgery. The new stapler provides some usage benefits and is also superior to the standard linear cutter with regard to anastomotic line bleeding. PMID:23018300

  8. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  9. Leaf-cutter ant fungus gardens are biphasic mixed microbial bioreactors that convert plant biomass to polyols with biotechnological applications.

    PubMed

    Somera, Alexandre F; Lima, Adriel M; Dos Santos-Neto, Álvaro J; Lanças, Fernando M; Bacci, Maurício

    2015-07-01

    Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology. PMID:25911490

  10. Bos indicus-cross feedlot cattle with excitable temperaments have tougher meat and a higher incidence of borderline dark cutters.

    PubMed

    Voisinet, B D; Grandin, T; O'Connor, S F; Tatum, J D; Deesing, M J

    1997-08-01

    Temperament ratings based on a numerical scale (chute score) were assessed during weighing and handling of cattle at a feedlot. Breeds studied included Braford, Red Brangus and Simbrah. Cattle were fed to a constant fat thickness of 9 to 13 mm (target = 11 mm) over the 12th rib as determined by periodic ultrasound measurements. Cattle were slaughtered in a commercial slaughter plant and stunned by captive bolt. Temperament rating had a significant effect on the incidence of borderline dark cutters which were downgraded by a USDA grader (P = 0.01). Temperament score also had a significant effect on tenderness (P < 0.001) as evaluated by Warner-Bratzler Shear (WBS) force at day 14 post mortem. The calmest animals which stood still when restrained in a hydraulic squeeze chute had a mean WBS force of 2.86 ± 11 kg and cattle which became highly agitated and struggled violently during restraint averaged 3.63 ± 19 kg. Forty percent of these cattle had WBS force values which were over 3.9 kg which is a threshold value for acceptability in food service establishments. These data show that cattle with the most excitable temperament ratings produce carcasses with tougher meat and a higher incidence of borderline dark cutters than cattle with calm temperament ratings. PMID:22062320

  11. Leaf-Cutter Ant Fungus Gardens Are Biphasic Mixed Microbial Bioreactors That Convert Plant Biomass to Polyols with Biotechnological Applications

    PubMed Central

    Somera, Alexandre F.; Lima, Adriel M.; dos Santos-Neto, Álvaro J.; Lanças, Fernando M.

    2015-01-01

    Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology. PMID:25911490

  12. On the bi-dimensional variational decomposition applied to nonstationary vibration signals for rolling bearing crack detection in coal cutters

    NASA Astrophysics Data System (ADS)

    Jiang, Yu; Li, Zhixiong; Zhang, Chao; Hu, Chao; Peng, Z.

    2016-06-01

    This work aims to detect rolling bearing cracks using a variational approach. An original method that appropriately incorporates bi-dimensional variational mode decomposition (BVMD) into discriminant diffusion maps (DDM) is proposed to analyze the nonstationary vibration signals recorded from the cracked rolling bearings in coal cutters. The advantage of this variational decomposition based diffusion map (VDDM) method in comparison to the current DDM is that the intrinsic vibration mode of the crack can be filtered into a limited bandwidth in the frequency domain with an estimated central frequency, thus discarding the interference signal components in the vibration signals and significantly improving the crack detection performance. In addition, the VDDM is able to simultaneously process two-channel sensor signals to reduce information leakage. Experimental validation using rolling bearing crack vibration signals demonstrates that the VDDM separated the raw signals into four intrinsic modes, including one roller vibration mode, one roller cage vibration mode, one inner race vibration mode, and one outer race vibration mode. Hence, reliable fault features were extracted from the outer race vibration mode, and satisfactory crack identification performance was achieved. The comparison between the proposed VDDM and existing approaches indicated that the VDDM method was more efficient and reliable for crack detection in coal cutter rolling bearings. As an effective catalyst for rolling bearing crack detection, this newly proposed method is useful for practical applications.

  13. Performance prediction of mechanical excavators from linear cutter tests on Yucca Mountain welded tuffs; Yucca Mountain Site Characterization Project

    SciTech Connect

    Gertsch, R.; Ozdemir, L.

    1992-09-01

    The performances of mechanical excavators are predicted for excavations in welded tuff. Emphasis is given to tunnel boring machine evaluations based on linear cutting machine test data obtained on samples of Topopah Spring welded tuff. The tests involve measurement of forces as cutters are applied to the rock surface at certain spacing and penetrations. Two disc and two point-attack cutters representing currently available technology are thus evaluated. The performance predictions based on these direct experimental measurements are believed to be more accurate than any previous values for mechanical excavation of welded tuff. The calculations of performance are predicated on minimizing the amount of energy required to excavate the welded tuff. Specific energy decreases with increasing spacing and penetration, and reaches its lowest at the widest spacing and deepest penetration used in this test program. Using the force, spacing, and penetration data from this experimental program, the thrust, torque, power, and rate of penetration are calculated for several types of mechanical excavators. The results of this study show that the candidate excavators will require higher torque and power than heretofore estimated.

  14. An Empirical Determination of Tasks Essential to Successful Performance as a Meat Cutter. Determination of a Common Core of Basic Skills in Agribusiness and Natural Resources.

    ERIC Educational Resources Information Center

    Byrd, J. Rick; And Others

    To improve vocational educational programs in agriculture, occupational information on a common core of basic skills within the occupational area of the meat cutter is presented in the revised task inventory survey. The purpose of the occupational survey was to identify a common core of basic skills which are performed and are essential for…

  15. The Molecular Basis of Genetic Diversity among Cytoplasms of TRITICUM and AEGILOPS Species. II. on the Origin of Polyploid Wheat Cytoplasms as Suggested by Chloroplast DNA Restriction Fragment Patterns.

    PubMed

    Tsunewaki, K; Ogihara, Y

    1983-05-01

    In attempts to identify the phylogenetic donors of cytoplasm to Emmer-Dinkel and Timopheevi groups of wheat (Triticum), and the Aegilops kotschyi-Ae. variabilis complex, the restriction fragment patterns of chloroplast DNAs of representative species were compared with those of their putative diploid ancestors. The following seven restriction enzymes were used; BamHI, EcoRI, HindIII, KpnI, PstI, SmaI and XhoI. The restriction fragment patterns of an Emmer and a Dinkel (common) wheat were identical with those of Ae. longissima , and different from those of Ae. aucheri, Ae. bicornis, Ae. searsii, Ae. sharonensis, Ae. speltoides, and T. urartu by 4 to 12 fragments. The restriction fragment patterns of a Timopheevi wheat were identical with those of Ae. aucheri, and different from those of all other diploids by four to nine fragments. The restriction fragment patterns of Ae. variabilis were identical to those of Ae. bicornis and Ae. searsii , and different from those of all other species. Thus, we have concluded that Ae. longissima, Ae. aucheri and Ae. bicornis (or Ae. searsii) were the cytoplasm donors to the Emmer-Dinkel and the Timopheevi groups, and the Ae. kotschyi-Ae. variabilis complex, respectively. A diphyletic origin of Emmer and Timopheevi groups is supported by the present results. PMID:17246126

  16. QTL for resistance in Lolium perenne to a mixed population of Puccinia graminis subsp. graminicola: use of RAD (restriction site associated DNA) markers to rapidly populate a new linkage map

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. Susceptible and resistant plants were crossed to produce a pseudo-testcross population. Markers were produced by the Restriction-sit...

  17. Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

    PubMed Central

    Roberts, Gareth A.; Chen, Kai; Cooper, Laurie P.; White, John H.; Blakely, Garry W.; Dryden, David T. F.

    2012-01-01

    The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence. PMID:23002145

  18. DNA Electrophoresis on Nanopatterned surfaces

    NASA Astrophysics Data System (ADS)

    Luo, Haobin; Gersappe, Dilip

    2002-03-01

    Conventional electrophoretic methods for DNA separation use topological restriction such as the network point in a gel to separate DNA. Here, we present a new approach to DNA electrophoresis using a nanopatterned surface. We exploit the conformational differences that arise when chains of different length are adsorbed onto a flat nanopatterned surface. We use a MD simulation to determine conditions that will optimize separation and present guidelines for the design of nanopatterned surfaces that can separate DNA in a broad size range.

  19. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  20. Neuroaesthetics: range and restrictions.

    PubMed

    Chatterjee, Anjan

    2013-04-01

    Bullot & Reber (B&R) should be commended for highlighting tensions between scientific aesthetics and art history. The question of how each tradition can learn from the other is timely. While I am sympathetic to their views, their diagnosis of the problem appears exaggerated and their solution partial. They underestimate the reach of scientific aesthetics while failing to identify its inherent restrictions. PMID:23507092

  1. Laser Peening--Strengthening Metals to Improve Fatigue Lifetime and Retard Stress-Induced Corrosion Cracking in Gears, Bolts and Cutter

    SciTech Connect

    Hackel, L A; Chen, H-L

    2003-08-20

    Laser peening is an emerging modern process that impresses a compressive stress into the surfaces of metals. Treatment can reduce the rate of fatigue cracking and stress-corrosion-cracking in metals (such as gears, bolts and cutters) needed for tunnel boring and other construction & mining applications. Laser peening could also be used to form metals or alloys into a precise shape without yielding and leaving both sulfates in a crack resistant compressive state.

  2. RNA aptamer inhibitors of a restriction endonuclease

    PubMed Central

    Mondragón, Estefanía; Maher, L. James

    2015-01-01

    Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors. PMID:26184872

  3. The in vitro Biochemical Characterization of an HIV-1 Restriction Factor APOBEC3F: Importance of Loop 7 on Both CD1 and CD2 for DNA Binding and Deamination.

    PubMed

    Chen, Qihan; Xiao, Xiao; Wolfe, Aaron; Chen, Xiaojiang S

    2016-07-01

    APOBEC3F (A3F) is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of proteins that can deaminate cytosine (C) to uracil (U) on nucleic acids. A3F is one of the four APOBEC members with two Zn-coordinated homologous cytosine deaminase (CD) domains, with the others being A3G, A3D, and A3B. Here we report the in vitro characterization of DNA binding and deaminase activities using purified wild-type and various mutant proteins of A3F from an Escherichia coli expression system. We show that even though CD1 is catalytically inactive and CD2 is the active deaminase domain, presence of CD1 on the N-terminus of CD2 enhances the deaminase activity by over an order of magnitude. This enhancement of CD2 catalytic activity is mainly through the increase of substrate single-stranded (ss) DNA binding by the N-terminal CD1 domain. We further show that the loop 7 of both CD1 and CD2 of A3F plays an important role for ssDNA binding for each individual domain, as well as for the deaminase activity of CD2 domain in the full-length A3F. PMID:27063502

  4. Molecular recognition in the minor groove of the DNA helix. Studies on the synthesis of oligonucleotides and polynucleotides containing 3-deaza-2'-deoxyadenosine. Interaction of the oligonucleotides with the restriction endonuclease EcoRV.

    PubMed Central

    Cosstick, R; Li, X; Tuli, D K; Williams, D M; Connolly, B A; Newman, P C

    1990-01-01

    An improved procedure for the preparation of 3-deaza-2'-deoxyadenosine (d3CA) is described which is suitable for the synthesis of gram quantities of this analogue. Using phosphoramidite chemistry d3CA has been incorporated into the Eco RV restiction endonuclease recognition sequence (underlined) present in the self-complementary dodecamer d(GACGATATCGTC). The modified oligonucleotides have been thoroughly characterised by nucleoside composition analysis, circular dichroism and thermal melting studies. Studies with Eco RV show that incorporation of d3CA into either the central or outer dA-dT base-pair results in a substantial reduction in the rate of cleavage. The two-step conversion of d3CA to 3-deaza-2'-deoxyadenosine-5'-O-triphosphate (d3CATP) via the 5'-O-tosylate is also described. d3CATP is not a substrate in the poly[d(AT)].poly[d(AT)] primed polymerisation for either E. coli DNA polymerase I or Micrococcus luteus DNA polymerase. In a more detailed kinetic analysis d3CATP was shown to be a competitive inhibitor of E. coli DNA polymerase I with respect to dATP. PMID:2395641

  5. License restrictions at Barnwell

    SciTech Connect

    Autry, V.R.

    1991-12-31

    The State of South Carolina was delegated the authority by the US Nuclear Regulatory Commission to regulate the receipt, possession, use and disposal of radioactive material as an Agreement State. Since 1970, the state has been the principal regulatory authority for the Barnwell Low-Level Waste Disposal Facility operated by Chem-Nuclear Systems, Inc. The radioactive material license issued authorizing the receipt and disposal of low-level waste contains numerous restrictions to ensure environmental protection and compliance with shallow land disposal performance criteria. Low-level waste has evolved from minimally contaminated items to complex waste streams containing high concentrations of radionuclides and processing chemicals which necessitated these restrictions. Additionally, some waste with their specific radionuclides and concentration levels, many classified as low-level radioactive waste, are not appropriate for shallow land disposal unless additional precautions are taken. This paper will represent a number of these restrictions, the rationale for them, and how they are being dealt with at the Barnwell disposal facility.

  6. DNA Media Storage

    PubMed Central

    Bogard, Christy M.; Rouchka, Eric C.

    2010-01-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  7. DNA Media Storage.

    PubMed

    Bogard, Christy M; Rouchka, Eric C

    2007-09-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  8. The Mitochondrial Genome of the Leaf-Cutter Ant Atta laevigata: A Mitogenome with a Large Number of Intergenic Spacers

    PubMed Central

    Rodovalho, Cynara de Melo; Lyra, Mariana Lúcio; Ferro, Milene; Bacci, Maurício

    2014-01-01

    In this paper we describe the nearly complete mitochondrial genome of the leaf-cutter ant Atta laevigata, assembled using transcriptomic libraries from Sanger and Illumina next generation sequencing (NGS), and PCR products. This mitogenome was found to be very large (18,729 bp), given the presence of 30 non-coding intergenic spacers (IGS) spanning 3,808 bp. A portion of the putative control region remained unsequenced. The gene content and organization correspond to that inferred for the ancestral pancrustacea, except for two tRNA gene rearrangements that have been described previously in other ants. The IGS were highly variable in length and dispersed through the mitogenome. This pattern was also found for the other hymenopterans in particular for the monophyletic Apocrita. These spacers with unknown function may be valuable for characterizing genome evolution and distinguishing closely related species and individuals. NGS provided better coverage than Sanger sequencing, especially for tRNA and ribosomal subunit genes, thus facilitating efforts to fill in sequence gaps. The results obtained showed that data from transcriptomic libraries contain valuable information for assembling mitogenomes. The present data also provide a source of molecular markers that will be very important for improving our understanding of genomic evolutionary processes and phylogenetic relationships among hymenopterans. PMID:24828084

  9. Fatal Accident Circumstances and Epidemiology (FACE) report: Timber Cutter Killed by Falling Tree Limb, West Virginia, January 13, 1995

    SciTech Connect

    1995-02-13

    An accident was described in which a 49 year old male timber cutter (and co-owner) died 1 day after being struck on the head by a falling tree limb. The employer in this incident was a small logging company with five employees, two of whom were co-owners. The incident occurred on the second day in business for the company. Five workers were at a 25 acre site where hardwood trees were to be harvested. The victim had felled trees for most of the day at the time of the accident. As the taller poplar fell, it contacted a branch of a smaller poplar, forcing it backward until the larger tree cleared it. The force of the limb striking the helmet caused multiple trauma to his head. Recommendations include that tree fellers properly evaluate the area around timber to be felled so that potential hazards can be identified and eliminated, that employers should ensure that workers prepare adequate escape paths and move a safe distance from the base of the tree as it is falling, that employers ensure workers are assigned to separate work areas, and that a written safety program be developed and implemented.

  10. Natural enemies of Atta vollenweideri (Hymenoptera: Formicidae) leaf-cutter ants negatively affected by synthetic pesticides, chlorpyrifos and fipronil.

    PubMed

    Guillade, Andrea C; Folgarait, Patricia J

    2014-02-01

    In southern South America, Ada vollenweideri Forel (Hymenoptera: Formicidae) is a significant pest of several crops and forestry, also considered to reduce the carrying capacity of pastures. The most usual control method used in Latin America is the application of synthetic pesticides, mainly chlorpyrifos and fipronil. However, no studies have assessed the effects of these agrochemicals on natural enemies of ants. We aimed to evaluate the efficiency of these pesticides on leaf-cutter ants' control and to test their effect on phorid fly parasitoids. Chlorpyrifos failed to exert complete control over ant colonies in the field and was gravely detrimental to specific parasitoids, reducing their percentage of parasitism, pupal survivorship, and adult longevity. Fipronil, however, exerted complete control over the treated colonies. Laboratory tests using both pesticides, either on ants from foraging trails or on pupariae, showed that chlorpyrifos and fipronil decreased larval and pupal survivorship, as well as adult longevity of parasitoids, in comparison to controls. In conclusion, these pesticides will likely affect parasitoids with regard to their reproductive capacity, leading to the decreased levels of natural parasitism observed in the field after treatments. We discuss why neither pesticide should be taken into account for integrated pest management programs. PMID:24665691

  11. Type III restriction-modification enzymes: a historical perspective

    PubMed Central

    Rao, Desirazu N.; Dryden, David T. F.; Bheemanaik, Shivakumara

    2014-01-01

    Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis. PMID:23863841

  12. Type II restriction endonucleases cleave single-stranded DNAs in general.

    PubMed Central

    Nishigaki, K; Kaneko, Y; Wakuda, H; Husimi, Y; Tanaka, T

    1985-01-01

    Restriction endonucleases (13 out of 18 species used for the test) were certified to cleave single-stranded(ss)DNA. Such enzymes as AvaII, HaeII, DdeI, AluI, Sau3AI, AccII,TthHB8I and HapII were newly reported to cleave ssDNA. A model to account for the cleavage of ssDNA by restriction enzymes was proposed with supportive data. The essential part of the model was that restriction enzymes preferentially cleave transiently formed secondary structures (called canonical structures) in ssDNA composed of two recognition sequences with two fold rotational symmetry. This means that a restriction enzyme can cleave ssDNAs in general so far as the DNAs have the sequences of restriction sites for the enzyme, and that the rate of cleavage depends on the stabilities of canonical structures. Images PMID:2994012

  13. A 12 megabase restriction map at the cystic fibrosis locus.

    PubMed Central

    Fulton, T R; Bowcock, A M; Smith, D R; Daneshvar, L; Green, P; Cavalli-Sforza, L L; Donis-Keller, H

    1989-01-01

    We have constructed a physical map of the chromosomal region containing the cystic fibrosis locus using seven DNA markers and pulsed-field gel electrophoresis methods. The map includes cleavage sites for 8 rare-cutting restriction enzymes and spans over 12 megabases (Mb) of DNA, with one unlinked probe covering an additional 5 Mb. To our knowledge, this is the largest segment of human DNA which has been restriction-mapped to date. We can identify thirteen putative HTF islands spaced at intervals of 0.3-3.2 Mb. The region between loci D7S8 and MET, where the CF gene lies, includes 1.4-1.9 Mb of DNA. Images PMID:2911467

  14. A method to capture large DNA fragments from genomic DNA.

    PubMed

    Ball, Geneviève; Filloux, Alain; Voulhoux, Romé

    2014-01-01

    The gene capture technique is a powerful tool that allows the cloning of large DNA regions (up to 80 kb), such as entire genomic islands, without using restriction enzymes or DNA amplification. This technique takes advantage of the high recombinant capacity of the yeast. A "capture" vector containing both ends of the target DNA region must first be constructed. The target region is then captured by co-transformation and recombination in yeast between the "capture" vector and appropriate genomic DNA. The selected recombinant plasmid can be verified by sequencing and transferred in the bacteria for multiple applications. This chapter describes a protocol specifically adapted for Pseudomonas aeruginosa genomic DNA capture. PMID:24818928

  15. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    PubMed Central

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  16. Range Restriction and Attenuation Corrections.

    ERIC Educational Resources Information Center

    Mumford, Michael D.; Mendoza, Jorge L.

    The present paper reviews the techniques commonly used to correct an observed correlation coefficient for the simultaneous influence of attenuation and range restriction effects. It is noted that the procedure which is currently in use may be somewhat biased because it treats range restriction and attenuation as independent restrictive influences.…

  17. Distribution of contact loads over the flank-land of the cutter with a rounded cutting edge

    NASA Astrophysics Data System (ADS)

    Kozlov, V.; Gerasimov, A.; Kim, A.

    2016-04-01

    In this paper, contact conditions between a tool and a workpiece material for wear-simulating turning by a cutter with a sharp-cornered edge and with a rounded cutting edge are analysed. The results of the experimental study of specific contact load distribution over the artificial flank wear-land of the cutter in free orthogonal turning of the disk from titanium alloy (Ti6Al2Mo2Cr), ductile (63Cu) and brittle (57Cu1Al3Mn) brasses are described. Investigations were carried out by the method of ‘split cutter’ and by the method of the artificial flank-land of variable width. The experiments with a variable feed rate and a cutting speed show that in titanium alloy machining with a sharp-cornered cutting edge the highest normal contact load (σh max = 3400…2200 MPa) is observed immediately at the cutting edge, and the curve has a horizontal region with the length of 0.2… 0.6 mm. At a distance from the cutting edge, the value of specific normal contact load is dramatically reduced to 1100…500 MPa. The character of normal contact load for a rounded cutting edge is different -it is uniform, and its value is approximately 2 times smaller compared to machining with a sharp-cornered cutting edge. In author’s opinion it is connected with generation of a seizure zone in a chip formation region and explains the capacity of highly worn-out cutting tools for titanium alloys machining. The paper analyses the distribution of tangential contact loads over the flank land, which pattern differs considerably for machining with a sharp-cornered edge and with a rounded cutting edge. Abbreviation and symbols: m/s - meter per second (cutting speed v); mm/r - millimeter per revolution (feed rate f); MPa - mega Pascal (specific contact load as a stress σ or τ) hf - the width of the flank wear land (chamfer) of the cutting tool, flank wear land can be natural or artificial like the one in this paper [mm]; xh - distance from the cutting edge on the surface of the flank-land [mm

  18. Magnetic anisotropy and organization of nanoparticles in heads and antennae of neotropical leaf-cutter ants, Atta colombica

    NASA Astrophysics Data System (ADS)

    Alves, Odivaldo C.; Srygley, Robert B.; Riveros, Andre J.; Barbosa, Marcia A.; Esquivel, Darci M. S.; Wajnberg, Eliane

    2014-10-01

    Oriented magnetic nanoparticles have been suggested as a good candidate for a magnetic sensor in ants. Behavioural evidence for a magnetic compass in neotropical leaf-cutter ants, Atta colombica (Formicidae: Attini), motivated a study of the arrangement of magnetic particles in the ants’ four major body parts by measuring the angular dependence of the ferromagnetic resonance spectra at room temperature. Spectra of the thoraces and those of the abdomens showed no significant angular dependence, while those of the antennae and those of the heads exhibited a periodic dependence relative to the magnetic field. Fitting of the angular dependence of the resonant field resulted in an unexpected magnetic anisotropy with uniaxial symmetry. High values of the first order anisotropy constant were observed for the magnetic material in antennae (-2.9  ×  105 erg cm-3) and heads (-1  ×  106 erg cm-3) as compared to body parts of other social insects. In addition, the magnitude of the anisotropy in the heads was comparable to that observed in magnetite nanoparticles of 4-5 nm diameter. For the antennae, the mean angle of the particles’ easy magnetization axis (EA) was estimated to be 41° relative to the straightened antenna’s long axis. For the heads, EA was approximately 60° relative to the head’s axis running from midway between the spines to the clypeus. These physical characteristics indicate organized magnetic nanoparticles with a potential for directional sensitivity, which is an important feature of magnetic compasses.

  19. Test Plan for Evaluating Hammer and Fixed Cutter Grinders Using Multiple Varieties and Moistures of Biomass Feedstock

    SciTech Connect

    Not listed

    2007-07-01

    Biomass preprocessing is a critical operation in the preparation of feedstock for the front-end of a cellulosic ethanol biorefinery. Its purpose is to chop, grind, or otherwise format the biomass material into a suitable feedstock for optimum conversion to ethanol and other bioproducts. Without this operation, the natural size, bulk density, and flowability characteristics of harvested biomass would decrease the capacities and efficiencies of feedstock assembly unit operations and biorefinery conversion processes to the degree that programmatic cost targets could not be met. The preprocessing unit operation produces a bulk flowable material that 1) improves handling and conveying efficiencies throughout the feedstock assembly system and biorefinery 2) increases biomass surface areas for improved pretreatment efficiencies, 3) reduces particle sizes for improved feedstock uniformity and density, and 4) fractionates structural components for improved compositional quality. The Idaho National Laboratory (INL) is tasked with defining the overall efficiency/effectiveness of current commercial hammer and fixed cutter grinding systems and other connecting systems such as harvest and collection, storage, transportation, and handling for a wide variety of feedstock types used in bioethanol or syngas production. This test plan details tasks and activities for two separate full-scale grinding tests: Material Characterization Test and Machine Characterization Test. For the Material Characterization Test, a small amount (~5-7 tons each) of several feedstock varieties will be ground. This test will define the fractionation characteristics of the grinder that affect the bulk density, particle size distribution, and quality of the size reduced biomass resulting from different separation screen sizes. A specific screen size will be selected based on the characteristics of the ground material. The Machine Characterization Test will then use this selected screen to grind several 30

  20. Endoscopic Submucosal Dissection for Early Gastric Cancer using the Clutch Cutter: a large single-center experience

    PubMed Central

    Akahoshi, Kazuya; Motomura, Yasuaki; Kubokawa, Masaru; Gibo, Junya; Kinoshita, Nobukatsu; Osada, Shigeki; Tokumaru, Kayo; Hosokawa, Taizou; Tomoeda, Naru; Otsuka, Yoshihiro; Matsuo, Mie; Oya, Masafumi; Koga, Hidenobu; Nakamura, Kazuhiko

    2015-01-01

    Background and study aims: The Clutch Cutter (CC) was developed to reduce the risk of complications related to endoscopic submucosal dissection (ESD) using knives. The CC is able to grasp and coagulate and/or incise the targeted tissue using electrosurgical current, like a biopsy technique. The aim of this study was to evaluate the efficacy and safety of ESD using the CC (ESD-CC) for early gastric cancer (EGC). Patients and methods: From June 2007 to March 2014, 325 consecutive patients with a diagnosis of EGC were enrolled in this prospective study. They had all satisfied the Japanese gastric cancer treatment guidelines for ESD indication, namely confirmation by preliminary endoscopy, endoscopic ultrasound, and endoscopic biopsies. The CC was used for all steps of ESD (marking, circumferential marginal incision, submucosal dissection, and hemostatic treatment). The therapeutic efficacy and safety were assessed. Results: The en-bloc resection rate was 99.7 % (324/325) and the R0 resection rate was 95.3 % (310/325). The mean operating time was 97.2 minutes. Perforation during ESD-CC occurred in one case (0.3 %), which was managed with conservative medical treatment after endoscopic closure of the perforation. Post-ESD-CC bleeding occurred in 11 cases (3.4 %), which were successfully treated by endoscopic hemostatic treatment. The R0 resection rate was significantly low in tumors > 20 mm (88.9 %), and in the exclusion indication group (73.7 %). Significant differences were seen in the mean operating time, depending upon tumor size, histologic type, location, and indication criteria. Conclusions: ESD-CC is a technically efficient, safe, and easy method for resecting EGC. PMID:26528497

  1. Functional domains in Fok I restriction endonuclease.

    PubMed Central

    Li, L; Wu, L P; Chandrasegaran, S

    1992-01-01

    The PCR was used to alter transcriptional and translational signals surrounding the Flavobacterium okeanokoites restriction endonuclease (fokIR) gene, so as to achieve high expression in Escherichia coli. By changing the ribosome-binding site sequence preceding the fokIR gene to match the consensus E. coli signal and by placing a positive retroregulator stem-loop sequence downstream of the gene, Fok I yield was increased to 5-8% of total cellular protein. Fok I was purified to homogeneity with phosphocellulose, DEAE-Sephadex, and gel chromatography, yielding 50 mg of pure Fok I endonuclease per liter of culture medium. The recognition and cleavage domains of Fok I were analyzed by trypsin digestion. Fok I in the absence of a DNA substrate cleaves into a 58-kDa carboxyl-terminal and 8-kDa amino-terminal fragment. The 58-kDa fragment does not bind the DNA substrate. Fok I in the presence of a DNA substrate cleaves into a 41-kDa amino-terminal fragment and a 25-kDa carboxyl-terminal fragment. On further digestion, the 41-kDa fragment degrades into 30-kDa amino-terminal and 11-kDa carboxyl-terminal fragments. The cleaved fragments both bind DNA substrates, as does the 41-kDa fragment. Gel-mobility-shift assays indicate that all the protein contacts necessary for the sequence-specific recognition of DNA substrates are encoded within the 41-kDa fragment. Thus, the 41-kDa amino-terminal fragment constitutes the Fok I recognition domain. The 25-kDa fragment, purified by using a DEAE-Sephadex column, cleaves nonspecifically both methylated (pACYCfokIM) and nonmethylated (pTZ19R) DNA substrates in the presence of MgCl2. Thus, the 25-kDa carboxyl-terminal fragment constitutes the Fok I cleavage domain. Images PMID:1584761

  2. Effect of dietary protein restriction on liver transcription factors.

    PubMed Central

    Marten, N W; Sladek, F M; Straus, D S

    1996-01-01

    The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBP alpha and beta was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBP beta (liver-enriched transcriptional inhibitory protein, LIP), which is a protein dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3 alpha and beta DNA binding was increased and that HNF-3 gamma DNA

  3. Dietary restriction with and without caloric restriction for healthy aging

    PubMed Central

    Lee, Changhan; Longo, Valter

    2016-01-01

    Caloric restriction is the most effective and reproducible dietary intervention known to regulate aging and increase the healthy lifespan in various model organisms, ranging from the unicellular yeast to worms, flies, rodents, and primates. However, caloric restriction, which in most cases entails a 20–40% reduction of food consumption relative to normal intake, is a severe intervention that results in both beneficial and detrimental effects. Specific types of chronic, intermittent, or periodic dietary restrictions without chronic caloric restriction have instead the potential to provide a significant healthspan increase while minimizing adverse effects. Improved periodic or targeted dietary restriction regimens that uncouple the challenge of food deprivation from the beneficial effects will allow a safe intervention feasible for a major portion of the population. Here we focus on healthspan interventions that are not chronic or do not require calorie restriction. PMID:26918181

  4. Restrictive vs. non-restrictive composition: a magnetoencephalography study

    PubMed Central

    Leffel, Timothy; Lauter, Miriam; Westerlund, Masha; Pylkkänen, Liina

    2014-01-01

    Recent research on the brain mechanisms underlying language processing has implicated the left anterior temporal lobe (LATL) as a central region for the composition of simple phrases. Because these studies typically present their critical stimuli without contextual information, the sensitivity of LATL responses to contextual factors is unknown. In this magnetoencephalography (MEG) study, we employed a simple question-answer paradigm to manipulate whether a prenominal adjective or determiner is interpreted restrictively, i.e., as limiting the set of entities under discussion. Our results show that the LATL is sensitive to restriction, with restrictive composition eliciting higher responses than non-restrictive composition. However, this effect was only observed when the restricting element was a determiner, adjectival stimuli showing the opposite pattern, which we hypothesise to be driven by the special pragmatic properties of non-restrictive adjectives. Overall, our results demonstrate a robust sensitivity of the LATL to high level contextual and potentially also pragmatic factors. PMID:25379512

  5. AFLP: a new technique for DNA fingerprinting.

    PubMed Central

    Vos, P; Hogers, R; Bleeker, M; Reijans, M; van de Lee, T; Hornes, M; Frijters, A; Pot, J; Peleman, J; Kuiper, M

    1995-01-01

    A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity. Images PMID:7501463

  6. Fv-1 restriction and its effects on murine leukemia virus integration in vivo and in vitro.

    PubMed Central

    Pryciak, P M; Varmus, H E

    1992-01-01

    We have investigated the mechanisms by which alleles at the mouse Fv-1 locus restrict replication of murine leukemia viruses. Inhibition of productive infection is closely paralleled by reduced accumulation of integrated proviral DNA as well as by reduced levels of linear viral DNA in a cytoplasmic fraction. Nevertheless, viral DNA is present at nearly normal levels in a nuclear fraction, and total amounts of viral DNA are only mildly affected in restrictive infections, suggesting a block in integration to account for reduced levels of proviral DNA. However, integrase (IN)-dependent trimming of 3' ends of viral DNA occurs normally in vivo during restrictive infections, demonstrating that not all IN-mediated events are prevented in vivo. Furthermore, viral integration complexes present in nuclear extracts of infected restrictive cells are fully competent to integrate their DNA into a heterologous target in vitro. Thus, the Fv-1-dependent activity that restricts integration in vivo may be lost in vitro; alternatively, Fv-1 restriction may prevent a step required for integration in vivo that is bypassed in vitro. Images PMID:1326652

  7. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    SciTech Connect

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; Purvine, Samuel O.; Smith, Richard D.; Lipton, Mary S.; Burnum-Johnson, Kristin E.; Currie, Cameron R.

    2015-08-28

    Leaf-cutter ants are prolific and conspicuous Neotropical herbivores that derive energy from specialized fungus gardens they cultivate using foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain lignocellulases that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as it is foraged by the ants. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous lignocellulases likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three-quarters of all lignocellulases identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 23 lignocellulases enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.

  8. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  9. Comparison of restriction enzymes for pulsed-field gel electrophoresis typing of Moraxella catarrhalis.

    PubMed

    Marti, Sara; Puig, Carmen; Domenech, Arnau; Liñares, Josefina; Ardanuy, Carmen

    2013-07-01

    NotI, the most prevalent restriction enzyme used for typing Moraxella catarrhalis, failed to digest genomic DNA from respiratory samples. An improved pulsed-field gel electrophoresis (PFGE) methodology determined SpeI as the best choice for typing this bacterial species, with a good restriction of clinical samples and a good clustering correlation with NotI. PMID:23678064

  10. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    ERIC Educational Resources Information Center

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  11. Adult Competency Education Kit. Basic Skills in Speaking, Math, and Reading for Employment. Part Q. ACE Competency Based Job Descriptions: #91--Meat Cutter; #92--Shipping Clerk; #93--Long Haul Truck Driver; #94--Truck Driver--Light.

    ERIC Educational Resources Information Center

    San Mateo County Office of Education, Redwood City, CA. Career Preparation Centers.

    This fourteenth of fifteen sets of Adult Competency Education (ACE) Based Job Descriptions in the ACE kit contains job descriptions for Meat Cutter, Shipping Clerk, Long Haul Truck Driver, and Truck Driver--Light. Each begins with a fact sheet that includes this information: occupational title, D.O.T. code, ACE number, career ladder, D.O.T.…

  12. ALS - The cost cutter

    NASA Astrophysics Data System (ADS)

    Colucci, Frank

    1987-10-01

    The Advanced Launch System (ALS) development program will avail itself of existing technologies in the short term in order to produce an interim 'core' vehicle that may be operational by 1993; the full, booster-incorporating system objective will then be achieved in 1998. This programmatic 'decoupling' of booster and core vehicle development efforts will separate their funding peaks. The ALS program will cut costs by colocating manufacturing and launch facilities, using Al-Li alloys in booster primary structures, and aggressively applying 'paperless' CIM. The ALS launch vehicle configuration will be primarily determined by both payload requirements and flight frequency.

  13. Coaxial cable cutter

    DOEpatents

    Hall, Leslie C.; Hedges, Robert S.

    1990-04-10

    A cutting device is provided which is useful in trimming the jackets from semi-rigid coaxial cables and wire having a cutting bit and support attached to movable jaws. A thumbpiece is provided to actuate the opening of the jaws for receiving the cable to be trimmed, and a spring member is provided to actuate the closing of the jaws when thumbpiece is released. The cutting device utilizes one moving part during the cutting operation by using a rolling cut action. The nature of the jaws allows the cutting device to work in space having clearances less than 0.160 inches.

  14. Powered protrusion cutter

    DOEpatents

    Bzorgi, Fariborz M.

    2010-03-09

    An apparatus for clipping a protrusion of material is provided. The protrusion may, for example, be a bolt head, a nut, a rivet, a weld bead, or a temporary assembly alignment tab protruding from a substrate surface of assembled components. The apparatus typically includes a cleaver having a cleaving edge and a cutting blade having a cutting edge. Generally, a mounting structure configured to confine the cleaver and the cutting blade and permit a range of relative movement between the cleaving edge and the cutting edge is provided. Also typically included is a power device coupled to the cutting blade. The power device is configured to move the cutting edge toward the cleaving edge. In some embodiments the power device is activated by a momentary switch. A retraction device is also generally provided, where the retraction device is configured to move the cutting edge away from the cleaving edge.

  15. Postnatal nutritional restriction affects growth and immune function of piglets with intra-uterine growth restriction.

    PubMed

    Hu, Liang; Liu, Yan; Yan, Chuan; Peng, Xie; Xu, Qin; Xuan, Yue; Han, Fei; Tian, Gang; Fang, Zhengfeng; Lin, Yan; Xu, Shengyu; Zhang, Keying; Chen, Daiwen; Wu, De; Che, Lianqiang

    2015-07-14

    Postnatal rapid growth by excess intake of nutrients has been associated with an increased susceptibility to diseases in neonates with intra-uterine growth restricted (IUGR). The aim of the present study was to determine whether postnatal nutritional restriction could improve intestinal development and immune function of neonates with IUGR using piglets as model. A total of twelve pairs of normal-birth weight (NBW) and IUGR piglets (7 d old) were randomly assigned to receive adequate nutrient intake or restricted nutrient intake (RNI) by artificially liquid feeding for a period of 21 d. Blood samples and intestinal tissues were collected at necropsy and were analysed for morphology, digestive enzyme activities, immune cells and expression of innate immunity-related genes. The results indicated that both IUGR and postnatal nutritional restriction delayed the growth rate during the sucking period. Irrespective of nutrient intake, piglets with IUGR had a significantly lower villous height and crypt depth in the ileum than the NBW piglets. Moreover, IUGR decreased alkaline phosphatase activity while enhanced lactase activity in the jejunum and mRNA expressions of Toll-like receptor 9 (TLR-9) and DNA methyltransferase 1 (DNMT1) in the ileum of piglets. Irrespective of body weight, RNI significantly decreased the number and/or percentage of peripheral leucocytes, lymphocytes and monocytes of piglets, whereas the percentage of neutrophils and the ratio of CD4+ to CD8+ were increased. Furthermore, RNI markedly enhanced the mRNA expression of TLR-9 and DNMT1, but decreased the expression of NOD2 and TRAF-6 in the ileum of piglets. In summary, postnatal nutritional restriction led to abnormal cellular and innate immune response, as well as delayed the growth and intestinal development of IUGR piglets. PMID:26059215

  16. Using restriction mapping to teach basic skills in the molecular biology lab.

    PubMed

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A

    2007-05-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction mapping allows students to gain technical expertise while simultaneously exploring the utility of gel electrophoresis and restriction mapping. Because of its interpretive nature, this project also provides data suitable for a written report, and can thus be used to reinforce lessons on figure presentation and science writing skills. PMID:21591089

  17. Genome filtering using methylation-sensitive restriction enzymes with six-base pair recognition sites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The large fraction of repetitive DNA in many plant genomes has complicated all aspects of DNA sequencing and assembly, and thus techniques that enrich for genes and low-copy sequences have been employed to isolate gene space. Methyl sensitive restriction enzymes with six base pair recognition sites...

  18. Using Restriction Mapping to Teach Basic Skills in the Molecular Biology Lab

    ERIC Educational Resources Information Center

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A.

    2007-01-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction…

  19. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  20. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    NASA Technical Reports Server (NTRS)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  1. NaLaF{sub 4}:Pr{sup 3+},Yb{sup 3+}, an efficient blue to near infra-red quantum cutter

    SciTech Connect

    Guille, A.; Pereira, A.; Moine, B.

    2013-12-01

    In order to reduce the thermalization losses in solar cells, down-conversion of blue photons into near infra-red photons is a promising solution. In the present paper, we analyse the energy transfer processes between Pr{sup 3+} and Yb{sup 3+} in NaLaF{sub 4} and we show that an efficient quantum-cutting process occurs. Nevertheless, we also show that a back transfer from Yb{sup 3+} toward the {sup 1}G{sub 4} level of Pr{sup 3+} ion leading to emission beyond 1 μm reduces the potentiality of this material as a quantum cutter for Si solar cells.

  2. DNA repair

    SciTech Connect

    Friedberg, E.C.; Hanawalt, P.C. )

    1988-01-01

    Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.

  3. Type I restriction enzymes and their relatives

    PubMed Central

    Loenen, Wil A. M.; Dryden, David T. F.; Raleigh, Elisabeth A.; Wilson, Geoffrey G.

    2014-01-01

    Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction–modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes. PMID:24068554

  4. Identification of specific restriction fragments associated with a membrane subparticle from Bacillus subtilis.

    PubMed Central

    Sargent, M G; Bennett, M F; Burdett, I D

    1983-01-01

    When lysates of Bacillus subtilis were treated with restriction endonucleases EcoRI or HindIII, almost all of the DNA was released from the major plasma membrane fraction that was sedimentable at low speed. However, a very small part of the released DNA, when centrifuged at high speed, appeared to be bound to small membrane fragments. On agarose gels, this material, prepared with either enzyme, contained only a small number of restriction fragments, and the DNA in the sample hybridized with 11 to 12 EcoRI or HindIII fragments of chromosomal DNA. This DNA was used after nick-translation to screen Charon 4A clone banks for phages containing membrane-bound fragments. One of these was studied in detail. Only a part (about 5 kilobases) of the region present in this clone is important in binding the DNA to the membrane subparticle. Images PMID:6304013

  5. Comparison of genomes of malignant catarrhal fever-associated herpesviruses by restriction endonuclease analysis.

    PubMed

    Shih, L M; Zee, Y C; Castro, A E

    1989-01-01

    The restriction endonuclease DNA cleavage patterns of eight isolates of malignant catarrhal fever-associated herpesviruses were examined using the restriction endonucleases HindIII and EcoRI. The eight viruses could be assigned to two distinct groups. Virus isolates from a blue wildebeest, a sika deer and an ibex had restriction endonuclease DNA cleavage patterns that were in general similar to each other. The restriction pattern of these three viruses was distinct from the other five. Of these five, four were isolated from a greater kudu, a white tailed wildebeest, a white bearded wildebeest, and a cape hartebeest. The fifth isolate C500, was isolated from a domestic cow with malignant catarrhal fever. These five viruses had similar DNA cleavage patterns. PMID:2558629

  6. Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants

    PubMed Central

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel M.; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; Purvine, Samuel O.; Smith, Richard D.; Lipton, Mary S.; Burnum-Johnson, Kristin E.; Currie, Cameron R.

    2015-01-01

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass. PMID:26317212

  7. Enrichment and broad representation of plant biomass-degrading enzymes in the specialized hyphal swellings of Leucoagaricus gongylophorus, the fungal symbiont of leaf-cutter ants

    DOE PAGESBeta

    Aylward, Frank O.; Khadempour, Lily; Tremmel, Daniel M.; McDonald, Bradon R.; Nicora, Carrie D.; Wu, Si; Moore, Ronald J.; Orton, Daniel J.; Monroe, Matthew E.; Piehowski, Paul D.; et al

    2015-08-28

    Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plantmore » biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.« less

  8. Identification of intron/exon boundaries in genomic DNA by inverse PCR.

    PubMed

    Albertsen, H; Thliveris, A

    2001-05-01

    This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template. This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. PMID:18428300

  9. Some restrictions on the existence of second order limit language

    NASA Astrophysics Data System (ADS)

    Ahmad, Muhammad Azrin; Sarmin, Nor Haniza; Yusof, Yuhani; Fong, Wan Heng

    2015-10-01

    The cut and paste phenomenon on DNA molecules with the presence of restriction enzyme and appropriate ligase has led to the formalism of mathematical modelling of splicing system. A type of splicing system named Yusof-Goode splicing system is used to present the transparent behaviour of the DNA splicing process. The limit language that is defined as the leftover molecules after the system reaches its equilibrium point has been extended to a second order limit language. The non-existence of the second order limit language biologically has lead to this study by using mathematical approach. In this paper, the factors that restrict the formation of the second order limit language are discussed and are presented as lemmas and theorem using Y-G approach. In addition, the discussion focuses on Yusof- Goode splicing system with at most two initial strings and two rules with one cutting site and palindromic crossing site and recognition sites.

  10. Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system

    PubMed Central

    Morozova, Natalia; Sabantsev, Anton; Bogdanova, Ekaterina; Fedorova, Yana; Maikova, Anna; Vedyaykin, Alexey; Rodic, Andjela; Djordjevic, Marko; Khodorkovskii, Mikhail; Severinov, Konstantin

    2016-01-01

    Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level. PMID:26687717

  11. 48 CFR 225.7003-2 - Restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Other Statutory Restrictions on Foreign Acquisition 225.7003-2 Restrictions. The following restrictions...) Missile or space systems. (3) Ships. (4) Tank or automotive items. (5) Weapon systems. (6) Ammunition....

  12. 48 CFR 225.7003-2 - Restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Other Statutory Restrictions on Foreign Acquisition 225.7003-2 Restrictions. The following restrictions...) Missile or space systems. (3) Ships. (4) Tank or automotive items. (5) Weapon systems. (6) Ammunition....

  13. 48 CFR 225.7003-2 - Restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Other Statutory Restrictions on Foreign Acquisition 225.7003-2 Restrictions. The following restrictions...) Missile or space systems. (3) Ships. (4) Tank or automotive items. (5) Weapon systems. (6) Ammunition....

  14. 48 CFR 225.7003-2 - Restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Other Statutory Restrictions on Foreign Acquisition 225.7003-2 Restrictions. The following restrictions...) Missile or space systems. (3) Ships. (4) Tank or automotive items. (5) Weapon systems. (6) Ammunition....

  15. 48 CFR 225.7003-2 - Restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Other Statutory Restrictions on Foreign Acquisition 225.7003-2 Restrictions. The following restrictions...) Missile or space systems. (3) Ships. (4) Tank or automotive items. (5) Weapon systems. (6) Ammunition....

  16. A simple, general procedure for purifying restriction endonucleases.

    PubMed Central

    Bickle, T A; Pirrotta, V; Imber, R

    1977-01-01

    A simple, general method for purifying restriction endonucleases is described. The method employs precipitation of nucleic acids from crude extracts with polyethyleneimine followed by affinity chromatography on columns of heparin covalently linked to agarose. Most of the sixteen enzymes tested could be purified to a degree sufficient for DNA sequencing work by this method sometimes supplemented by at most one step of ion exchange chromatography. Images PMID:909783

  17. Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots

    SciTech Connect

    Gray, M.R. )

    1992-02-01

    Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hydridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, [beta][sub 2]-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFM's were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMP's.

  18. A simple, reliable, and fast protocol for thraustochytrid DNA extraction.

    PubMed

    Mo, C; Rinkevich, B

    2001-03-01

    DNA extraction of thraustochytrids, common marine unicellular organisms, is usually accomplished by either the cetyltrimethylammonium bromide (CTAB) or proteinase K protocols. A novel lysis buffer protocol for thraustochytrid total DNA extraction is described. The average isolated total DNA is 20 to 40 kb, and DNA samples are suitable for a variety of uses including 18S-ribosomal DNA polymerase chain reaction, restriction enzyme digestions, and amplified fragment length polymorphism analyses. The new protocol is also faster than the other protocols. PMID:14961371

  19. DNA Technology in the Classroom.

    ERIC Educational Resources Information Center

    Williamson, John H.; Campbell, A. Malcolm

    1997-01-01

    Presents a protocol that gives students hands-on experience in generating a meaningful physical map of a circular molecule of DNA. Topics include agarose gel electrophoresis, logic of restriction maps, extracting data from an agarose gel, managing data from gels, experimental protocol, loading gels, electrophoresis, photographing gels, collecting…

  20. Alphavirus Restriction by IFITM Proteins.

    PubMed

    Weston, Stuart; Czieso, Stephanie; White, Ian J; Smith, Sarah E; Wash, Rachael S; Diaz-Soria, Carmen; Kellam, Paul; Marsh, Mark

    2016-09-01

    Interferon inducible transmembrane proteins (IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV. PMID:27219333

  1. Generalized Pump-restriction Theorem

    SciTech Connect

    Sinitsyn, Nikolai A; Chernyak, Vladimir Y

    2008-01-01

    We formulate conditions under which periodic modulations of parameters on a finite graph with stochastic transitions among its nodes do not lead to overall pump currents through any given link. Our theorem unifies previously known results with the new ones and provides a universal approach to explore futher restrictions on stochastic pump effect in non-adiabatically driven systems with detailed balance.

  2. Pulsed-field gel electrophoresis for long-range restriction mapping.

    PubMed

    Gemmill, Robert M; Bolin, Richard; Albertsen, Hans; Tomkins, Jeff P; Wing, Rod A

    2002-02-01

    This unit describes procedures for generating long-range restriction maps of genomic DNA and for analysis of large insert clones. The basic protocol details restriction digestion of agarose-embedded DNA, PFGE separation, Southern transfer, and hybridization. Support protocols describe the preparation of high-molecular-weight genomic DNA samples in agarose blocks and in agarose microbeads, respectively. Additional support protocols describe the preparation of DNA size standards from l phage and two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe. An alternative method of preparing S. cerevisiae size standards using lithium dodecyl sulfate (LiDS) solubilization is provided. The final protocol details the preparation of BAC DNA suitable for digestion, mapping, and sequencing. PMID:18428330

  3. Restricted Diffusion in Biophysical Systems

    PubMed Central

    Cooper, Robert L.; Chang, David B.; Young, Allan C.; Martin, Carroll J.; Ancker-Johnson, Betsy

    1974-01-01

    The pulsed-gradient spin echo nuclear magnetic resonance (PGSENMR) technique was used to measure restricted diffusion of water in three types of animal tissue: human blood plasma and red cells; rat and rabbit heart; rat and rabbit liver. Characteristic lengths (L) for restriction of diffusion are estimated from dependence on the measuring time. Limitations on the range of observable restrictive lengths (1.5-15 μm) are discussed. The decrease in diffusivity due to 1 μm alumina powder (volume fraction = 0.18) in glycerin/water mixtures agrees with the Wang theory assuming spherical particles and no hydration. The characteristic length (L ≃ 4 μm) is larger than the particle size (1 μm) or separation (1.8 μm). Comparison of the diffusivities in tissues at short diffusion times with the Wang theory indicates some bound or trapped water. For packed red blood cells, a restriction (L ≃ 2.3 μm) was attributed tothe red cell membrane. A permeability p ≃ 0.014 cm/s may be estimated from the decrease in diffusivity. Average values of diffusivity ratio in heart were: 0.36 ± 0.02 for rat; and 0.26 ± 0.03 for rabbit; and in liver: 0.25 ± 0.01 for rat; 0.25 ± .04 for 10-day old rabbit; and 0.195 ± 0.03 for 2-yr old rabbit. A restriction (L ≃ 2.7 μm) in rat liver probably results from the mitochondria. PMID:4823458

  4. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  5. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  6. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  7. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as...

  8. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 5 2013-01-01 2013-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as...

  9. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 5 2012-01-01 2012-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as...

  10. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as...

  11. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Restricted cars. 215.203 Section 215.203 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts...

  12. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  13. Electroeluting DNA fragments.

    PubMed

    Zarzosa-Alvarez, Ana L; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L; Bermudez-Cruz, Rosa M

    2010-01-01

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation. PMID:20834225

  14. Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora

    PubMed Central

    Bastiaans, E.; Aanen, D. K.; Debets, A. J. M.; Hoekstra, R. F.; Lestrade, B.; Maas, M. F. P. M.

    2014-01-01

    The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a ‘Kalilo’ culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing. PMID:24864316

  15. Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora.

    PubMed

    Bastiaans, E; Aanen, D K; Debets, A J M; Hoekstra, R F; Lestrade, B; Maas, M F P M

    2014-07-01

    The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a 'Kalilo' culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing. PMID:24864316

  16. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  17. Effects of Long-Term Food Restriction Under Thermoneutral Conditions on Brown Adipose Tissue of Laboratory Mice.

    PubMed

    Elsukova, E I; Mizonova, O V; Medvedev, L N

    2015-09-01

    Long-term food restriction (3 weeks, 60% of normal consumption of control animals) was followed by an increase in DNA and protein content in the intercapsular brown fat of mice. As the animals were kept under thermoneutral conditions, these changes are thought to be a result of food restriction. PMID:26459485

  18. Overcoming Communication Restrictions in Collectives

    NASA Technical Reports Server (NTRS)

    Tumer, Kagan; Agogino, Adrian K.

    2004-01-01

    Many large distributed system are characterized by having a large number of components (eg., agents, neurons) whose actions and interactions determine a %orld utility which rates the performance of the overall system. Such collectives are often subject to communication restrictions, making it difficult for components which try to optimize their own private utilities, to take actions that also help optimize the world utility. In this article we address that coordination problem and derive four utility functions which present different compromises between how aligned a component s private utility is with the world utility and how readily that component can determine the actions that optimize its utility. The results show that the utility functions specifically derived to operate under communication restrictions outperform both traditional methods and previous collective-based methods by up to 75%.

  19. Temperature based Restricted Boltzmann Machines

    NASA Astrophysics Data System (ADS)

    Li, Guoqi; Deng, Lei; Xu, Yi; Wen, Changyun; Wang, Wei; Pei, Jing; Shi, Luping

    2016-01-01

    Restricted Boltzmann machines (RBMs), which apply graphical models to learning probability distribution over a set of inputs, have attracted much attention recently since being proposed as building blocks of multi-layer learning systems called deep belief networks (DBNs). Note that temperature is a key factor of the Boltzmann distribution that RBMs originate from. However, none of existing schemes have considered the impact of temperature in the graphical model of DBNs. In this work, we propose temperature based restricted Boltzmann machines (TRBMs) which reveals that temperature is an essential parameter controlling the selectivity of the firing neurons in the hidden layers. We theoretically prove that the effect of temperature can be adjusted by setting the parameter of the sharpness of the logistic function in the proposed TRBMs. The performance of RBMs can be improved by adjusting the temperature parameter of TRBMs. This work provides a comprehensive insights into the deep belief networks and deep learning architectures from a physical point of view.

  20. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  1. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  2. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  3. Ligand field density functional theory calculation of the 4f2→ 4f15d1 transitions in the quantum cutter Cs2KYF6:Pr3+.

    PubMed

    Ramanantoanina, Harry; Urland, Werner; Cimpoesu, Fanica; Daul, Claude

    2013-09-01

    Herein we present a Ligand Field Density Functional Theory (LFDFT) based methodology for the analysis of the 4f(n)→ 4f(n-1)5d(1) transitions in rare earth compounds and apply it for the characterization of the 4f(2)→ 4f(1)5d(1) transitions in the quantum cutter Cs2KYF6:Pr(3+) with the elpasolite structure type. The methodological advances are relevant for the analysis and prospection of materials acting as phosphors in light-emitting diodes. The positions of the zero-phonon energy corresponding to the states of the electron configurations 4f(2) and 4f(1)5d(1) are calculated, where the praseodymium ion may occupy either the Cs(+)-, K(+)- or the Y(3+)-site, and are compared with available experimental data. The theoretical results show that the occupation of the three undistorted sites allows a quantum-cutting process. However size effects due to the difference between the ionic radii of Pr(3+) and K(+) as well as Cs(+) lead to the distortion of the K(+)- and the Cs(+)-site, which finally exclude these sites for quantum-cutting. A detailed discussion about the origin of this distortion is also described. PMID:23846586

  4. Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

    DOE PAGESBeta

    Lewin, Gina R.; Johnson, Amanda L.; Soto, Rolando D. Moreira; Perry, Kailene; Book, Adam J.; Horn, Heidi A.; Pinto-Tomás, Adrián A.; Currie, Cameron R.

    2016-03-21

    Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using materialmore » from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within general containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. Lastly, a representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation.« less

  5. Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability

    PubMed Central

    Lewin, Gina R.; Johnson, Amanda L.; Soto, Rolando D. Moreira; Perry, Kailene; Book, Adam J.; Horn, Heidi A.; Pinto-Tomás, Adrián A.; Currie, Cameron R.

    2016-01-01

    Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation. PMID:26999749

  6. Sequential Indentation Tests to Investigate the Influence of Confining Stress on Rock Breakage by Tunnel Boring Machine Cutter in a Biaxial State

    NASA Astrophysics Data System (ADS)

    Liu, Jie; Cao, Ping; Han, Dongya

    2016-04-01

    The influence of confining stress on rock breakage by a tunnel boring machine cutter was investigated by conducting sequential indentation tests in a biaxial state. Combined with morphology measurements of breaking grooves and an analysis of surface and internal crack propagation between nicks, the effects of maximum confining stress and minimum stress on indentation efficiency, crack propagation and chip formation were investigated. Indentation tests and morphology measurements show that increasing a maximum confining stress will result in increased consumed energy in indentations, enlarged groove volumes and promoted indentation efficiency when the corresponding minimum confining stress is fixed. The energy consumed in indentations will increase with increase in minimum confining stress, however, because of the decreased groove volumes as the minimum confining stress increases, the efficiency will decrease. Observations of surface crack propagation show that more intensive fractures will be induced as the maximum confining stress increases, whereas the opposite occurs for an increase of minimum confining stress. An observation of the middle section, cracks and chips shows that as the maximum confining stress increases, chips tend to form in deeper parts when the minimum confining stress is fixed, whereas they tend to formed in shallower parts as the minimum confining stress increases when the maximum confining stress is fixed.

  7. Cellulose-Enriched Microbial Communities from Leaf-Cutter Ant (Atta colombica) Refuse Dumps Vary in Taxonomic Composition and Degradation Ability.

    PubMed

    Lewin, Gina R; Johnson, Amanda L; Soto, Rolando D Moreira; Perry, Kailene; Book, Adam J; Horn, Heidi A; Pinto-Tomás, Adrián A; Currie, Cameron R

    2016-01-01

    Deconstruction of the cellulose in plant cell walls is critical for carbon flow through ecosystems and for the production of sustainable cellulosic biofuels. Our understanding of cellulose deconstruction is largely limited to the study of microbes in isolation, but in nature, this process is driven by microbes within complex communities. In Neotropical forests, microbes in leaf-cutter ant refuse dumps are important for carbon turnover. These dumps consist of decaying plant material and a diverse bacterial community, as shown here by electron microscopy. To study the portion of the community capable of cellulose degradation, we performed enrichments on cellulose using material from five Atta colombica refuse dumps. The ability of enriched communities to degrade cellulose varied significantly across refuse dumps. 16S rRNA gene amplicon sequencing of enriched samples identified that the community structure correlated with refuse dump and with degradation ability. Overall, samples were dominated by Bacteroidetes, Gammaproteobacteria, and Betaproteobacteria. Half of abundant operational taxonomic units (OTUs) across samples were classified within genera containing known cellulose degraders, including Acidovorax, the most abundant OTU detected across samples, which was positively correlated with cellulolytic ability. A representative Acidovorax strain was isolated, but did not grow on cellulose alone. Phenotypic and compositional analyses of enrichment cultures, such as those presented here, help link community composition with cellulolytic ability and provide insight into the complexity of community-based cellulose degradation. PMID:26999749

  8. Application of water jet assisted drag bit and pick cutter for the cutting of coal measure rocks. Final technical report. [Tests of combination in different rocks

    SciTech Connect

    Ropchan, D.; Wang, F.D.; Wolgamott, J.

    1980-04-01

    A laboratory investigation was made of the effects of high pressure water jets on the cutting forces of drag bit cutters in sedimentary rocks. A hard and soft sandstone, shale and limestone were tested with commercially obtainable conical and plow type drag bits on the EMI linear cutting machine. About 1200 cuts were made at different bit penetration, jet orientation, and water pressure to determine the reduction of cutting forces on the bit from the use of the water jet. Both independent and interactive cutting was used. The greatest reduction in cutting forces were with both of the sandstones; the drag forces were reduced about 30 percent and the normal forces about 60 percent at 5000 psi water pressure with the nozzle behind the bit. The method was less effective in the shale, except at 10,000 psi water pressure the reduction in drag force was about 55 percent. Of the rocks tested, the limestone was least affected by the water jet. The cutting forces for the plow bit showed continuous change with wear so a machined conical bit was used for most of the testing. Tests with the plow bit did show a large reduction in cutting forces by using the water jet with worn bits. An economic analysis of equipping a drag bit tunnel boring machine indicated that the water jet system could reduce costs per foot in sandstone by up to 40 percent.

  9. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  10. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  11. UV damage in DNA promotes nucleosome unwrapping.

    PubMed

    Duan, Ming-Rui; Smerdon, Michael J

    2010-08-20

    The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased "intrinsic exposure" of nucleosome-associated DNA lesions in chromatin to repair proteins. PMID:20562439

  12. Conformation-selective methylation of geminivirus DNA.

    PubMed

    Paprotka, T; Deuschle, K; Metzler, V; Jeske, H

    2011-11-01

    Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially. PMID:21835804

  13. DNA Damage, Homology-Directed Repair, and DNA Methylation

    PubMed Central

    Angrisano, Tiziana; Morano, Annalisa; Lee, Bongyong; Pardo, Alba Di; Messina, Samantha; Iuliano, Rodolfo; Fusco, Alfredo; Santillo, Maria R; Muller, Mark T; Chiariotti, Lorenzo; Gottesman, Max E; Avvedimento, Enrico V

    2007-01-01

    To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments. PMID:17616978

  14. Recent advances in DNA assembly technologies.

    PubMed

    Chao, Ran; Yuan, Yongbo; Zhao, Huimin

    2014-06-01

    DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods. PMID:24903193

  15. Recent Advances in DNA Assembly Technologies

    PubMed Central

    Chao, Ran; Yuan, Yongbo; Zhao, Huimin

    2014-01-01

    DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods. PMID:24903193

  16. Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

    PubMed

    Bertini, L; Potenza, L; Zambonelli, A; Amicucci, A; Stocchi, V

    1998-07-15

    A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations. PMID:9682488

  17. A computer assisted method for the determination of restriction enzyme recognifion sites.

    PubMed Central

    Gingeras, T R; MIlazzo, J P; Roberts, R J

    1978-01-01

    A computer program has been developed which aids in the determination of restriction enzyme recognition sequences. This is achieved by cleaving DNAs of known sequence with a restriction endonuclease and comparing the fragmentation pattern with a computer-generated set of patterns. The feasibility of this approach has been tested using fragmentation patterns of 0X174 DNA produced by enzymes of both known and unknown specificity. Recognition sequences are predicted for two restriction endonucleases (BbvI and SfaNI) using this method. In addition, recognition sequences are predicted for two other new enzymes (PvuI and MstI) using another computer-assisted method. Images PMID:724510

  18. DNA templated magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Kinsella, Joseph M.

    Recent discoveries in nanoscience are predicted to potentially revolutionize future technologies in an extensive number of fields. These developments are contingent upon discovering new and often unconventional methods to synthesize and control nanoscale components. Nature provides several examples of working nanotechnology such as the use of programmed self assembly to build and deconstruct complex molecular systems. We have adopted a method to control the one dimensional assembly of magnetic nanoparticles using DNA as a scaffold molecule. With this method we have demonstrated the ability to organize 5 nm particles into chains that stretch up to ˜20 mum in length. One advantage of using DNA compared is the ability of the molecule to interact with other biomolecules. After assembling particles onto DNA we have been able to cleave the molecule into smaller fragments using restriction enzymes. Using ligase enzymes we have re-connected these fragments, coated with either gold or iron oxide, to form long one-dimensional arrangements of the two different types of nanoparticles on a single molecular guide. We have also created a sensitive magnetic field sensor by incorporating magnetic nanoparticle coated DNA strands with microfabricated electrodes. The IV characteristics of the aligned nanoparticles are dependant on the magnitude of an externally applied magnetic field. This transport phenomenon known as tunneling magnetoresistance (TMR) shows room temperature resistance of our devices over 80% for cobalt ferrite coated DNA when a field of 20 kOe is applied. In comparison, studies using two dimensional nanoparticle films of irox oxides xii only exhibit a 35% MR effect. Confinement into one dimension using the DNA guide produces a TMR mechanism which produces significant increases in magnetoresistance. This property can be utilized for applications in magnetic field sensing, data storage, and logic elements.

  19. Restricted Transport in Small Pores

    PubMed Central

    Anderson, John L.; Quinn, John A.

    1974-01-01

    The basic hydrodynamic equations governing transport in submicron pores are reexamined. Conditions necessary for a simplified, one-dimensional treatment of the diffusion/convection process are established. Steric restrictions and Brownian motion are incorporated directly into the resulting model. Currently available fluid mechanical results are used to evaluate an upper limit on hindered diffusion; this limit is valid for small particle-to-pore ratios. Extensions of the analysis are shown to depend on numerical solutions of the related hydrodynamic problem, that of asymmetrical particle motion in a bounded fluid. PMID:4813157

  20. Nucleotide sequence of the BsuRI restriction-modification system.

    PubMed Central

    Kiss, A; Posfai, G; Keller, C C; Venetianer, P; Roberts, R J

    1985-01-01

    The genes of the 5'-GGCC specific BsuRI restriction-modification system of Bacillus subtilis have been cloned and expressed in E. coli and their nucleotide sequence has been determined. The restriction and modification genes code for polypeptides with calculated molecular weights of 66,314 and 49,642, respectively. Both enzymes are coded by the same DNA strand. The restriction gene is upstream of the methylase gene and the coding regions are separated by 780 bp. Analysis of the RNA transcripts by S1-nuclease mapping indicates that the restriction and modification genes are transcribed from different promoters. Comparison of the amino acid sequences revealed no homology between the BsuRI restriction and modification enzymes. There are, however, regions of homology between the BsuRI methylase and two other GGCC specific modification enzymes, the BspRI and SPR methylases. Images PMID:2997708

  1. Long range restriction analysis of the bovine casein genes.

    PubMed Central

    Ferretti, L; Leone, P; Sgaramella, V

    1990-01-01

    Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes were demonstrated to be physically linked within a region of 300 kb, represented by two adjacent Xhol fragments in fibroblasts and by a single fragment in lymphocytes. A restriction map of the casein locus was derived and the order of the genes was shown to be kappa, alpha s2, beta, alpha s1. Images PMID:2263448

  2. 9 CFR 78.20 - General restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Bison Because of Brucellosis § 78.20 General restrictions. Bison may not be...

  3. 9 CFR 78.5 - General restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Cattle Because of Brucellosis § 78.5 General restrictions. Cattle may not be...

  4. 9 CFR 78.20 - General restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Bison Because of Brucellosis § 78.20 General restrictions. Bison may not be...

  5. 9 CFR 78.5 - General restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Cattle Because of Brucellosis § 78.5 General restrictions. Cattle may not be...

  6. Avoidant/Restrictive Food Intake Disorder

    MedlinePlus

    ... Professional Version Eating Disorders Definition of Eating Disorders Anorexia Nervosa Avoidant/Restrictive Food Intake Disorder Binge Eating ... they eat. Eating Disorders Definition of Eating Disorders Anorexia Nervosa Avoidant/Restrictive Food Intake Disorder Binge Eating ...

  7. Genetics Home Reference: familial restrictive cardiomyopathy

    MedlinePlus

    ... CARDIOMYOPATHY, FAMILIAL RESTRICTIVE, 3 Sources for This Page Elliott P, Andersson B, Arbustini E, Bilinska Z, Cecchi ... Sebire N, Ashworth M, Deanfield JE, McKenna WJ, Elliott PM. Idiopathic restrictive cardiomyopathy in children is caused ...

  8. Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes

    PubMed Central

    Fu, Yong-Bi; Peterson, Gregory W.; Dong, Yibo

    2016-01-01

    Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7–6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0–16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. PMID:26818077

  9. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    PubMed Central

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  10. Temperature based Restricted Boltzmann Machines

    PubMed Central

    Li, Guoqi; Deng, Lei; Xu, Yi; Wen, Changyun; Wang, Wei; Pei, Jing; Shi, Luping

    2016-01-01

    Restricted Boltzmann machines (RBMs), which apply graphical models to learning probability distribution over a set of inputs, have attracted much attention recently since being proposed as building blocks of multi-layer learning systems called deep belief networks (DBNs). Note that temperature is a key factor of the Boltzmann distribution that RBMs originate from. However, none of existing schemes have considered the impact of temperature in the graphical model of DBNs. In this work, we propose temperature based restricted Boltzmann machines (TRBMs) which reveals that temperature is an essential parameter controlling the selectivity of the firing neurons in the hidden layers. We theoretically prove that the effect of temperature can be adjusted by setting the parameter of the sharpness of the logistic function in the proposed TRBMs. The performance of RBMs can be improved by adjusting the temperature parameter of TRBMs. This work provides a comprehensive insights into the deep belief networks and deep learning architectures from a physical point of view. PMID:26758235

  11. 28 CFR 68.51 - Restricted access.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Restricted access. 68.51 Section 68.51... ALIENS, UNFAIR IMMIGRATION-RELATED EMPLOYMENT PRACTICES, AND DOCUMENT FRAUD § 68.51 Restricted access. On... be a restricted access portion of the record to contain any material in the record to which...

  12. 29 CFR 18.56 - Restricted access.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Restricted access. 18.56 Section 18.56 Labor Office of the... ADMINISTRATIVE LAW JUDGES General § 18.56 Restricted access. On his or her own motion, or on the motion of any party, the administrative law judge may direct that there be a restricted access portion of the...

  13. 29 CFR 18.56 - Restricted access.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 1 2011-07-01 2011-07-01 false Restricted access. 18.56 Section 18.56 Labor Office of the... ADMINISTRATIVE LAW JUDGES General § 18.56 Restricted access. On his or her own motion, or on the motion of any party, the administrative law judge may direct that there be a restricted access portion of the...

  14. 28 CFR 68.51 - Restricted access.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Restricted access. 68.51 Section 68.51... ALIENS, UNFAIR IMMIGRATION-RELATED EMPLOYMENT PRACTICES, AND DOCUMENT FRAUD § 68.51 Restricted access. On... be a restricted access portion of the record to contain any material in the record to which...

  15. Restriction Enzyme Mapping: A Simple Student Practical.

    ERIC Educational Resources Information Center

    Higgins, Stephen J.; And Others

    1990-01-01

    An experiment that uses the recombinant plasmid pX1108 to illustrate restriction mapping is described. The experiment involves three restriction enzymes and employs single and double restriction enzyme digestions. A list of needed materials, procedures, safety precautions, results, and discussion are included. (KR)

  16. Restricted Schur polynomials and finite N counting

    SciTech Connect

    Collins, Storm

    2009-01-15

    Restricted Schur polynomials have been posited as orthonormal operators for the change of basis from N=4 SYM to type IIB string theory. In this paper we briefly expound the relationship between the restricted Schur polynomials and the operators forwarded by Brown, Heslop, and Ramgoolam. We then briefly examine the finite N counting of the restricted Schur polynomials.

  17. 5 CFR 330.205 - Employment restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Employment restrictions. 330.205 Section 330.205 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS RECRUITMENT, SELECTION, AND PLACEMENT (GENERAL) Reemployment Priority List (RPL) § 330.205 Employment restrictions. (a) The restrictions in paragraph (b)...

  18. 47 CFR 1.1208 - Restricted proceedings.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 1 2012-10-01 2012-10-01 false Restricted proceedings. 1.1208 Section 1.1208 Telecommunication FEDERAL COMMUNICATIONS COMMISSION GENERAL PRACTICE AND PROCEDURE Grants by Random Selection Ex Parte Communications Restricted Proceedings § 1.1208 Restricted proceedings. Unless otherwise provided by the Commission or its...

  19. Microbial Populations Associated with Restricted Shell Eggs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Restricted shell eggs are found in all shell egg processing facilities. The manner with which companies handle them can vary greatly. In most cases, restricted eggs are diverted to further processing. Some facilities do not rewash eggs, choosing instead to include dirts in restricted eggs. Eggs ...

  20. 36 CFR 13.1176 - Speed restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Speed restrictions. 13.1176... Operating Restrictions § 13.1176 Speed restrictions. (a) From May 15 through September 30, in designated whale waters the following are prohibited: (1) Operating a motor vessel at more than 20 knots...

  1. 36 CFR 13.1176 - Speed restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 1 2011-07-01 2011-07-01 false Speed restrictions. 13.1176... Operating Restrictions § 13.1176 Speed restrictions. (a) From May 15 through September 30, in designated whale waters the following are prohibited: (1) Operating a motor vessel at more than 20 knots...

  2. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Restricted hazelnuts. 982.14 Section 982.14... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  3. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  4. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Restricted hazelnuts. 982.14 Section 982.14... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  5. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  6. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  7. Epidemiological typing of Moraxella catarrhalis by using DNA probes.

    PubMed Central

    Beaulieu, D; Scriver, S; Bergeron, M G; Low, D E; Parr, T R; Patterson, J E; Matlow, A; Roy, P H

    1993-01-01

    Small-fragment restriction enzyme analysis and DNA-DNA hybridization were used to compare 60 strains of Moraxella catarrhalis isolated from various geographic locations. Restriction enzyme analysis with HaeIII resulted in 46 different patterns, 7 of which were shared by more than one isolate. Hybridizations with two DNA probes resulted in 18 different patterns, 11 of which were shared by more than one isolate. Strains with the same restriction enzyme pattern always had the same hybridization pattern. However, of the 50 strains that shared the 11 hybridization patterns, 39 could be further differentiated by restriction enzyme analysis. We found that hybridization is a method that is specific for the epidemiological typing of M. catarrhalis, but because of limited sensitivity, combination with small-fragment restriction enzyme analysis may be necessary to better determine the relatedness of strains. Images PMID:8096219

  8. A rapid DNA digestion system.

    PubMed

    Fu, Lung-Ming; Lin, Che-Hsin

    2007-04-01

    This paper presents a novel microfluidic DNA digestion system incorporating a high performance micro-mixer. Through the appropriate control of fixed and periodic switching DC electric fields, electrokinetic forces are established to mix the DNA and restriction enzyme samples and to drive them through the reaction column of the device. The experimental and numerical results show that a mixing performance of 98% can be achieved within a mixing channel of length 1.6 mm when a 150 V/cm driving voltage and a 5 Hz switching frequency are applied. The relationship between the mixing performance, switching frequency, and main applied electric field is derived. It is found that the optimal switching frequency depends upon the magnitude of the main applied electric field. The successful digestion of lambda-DNA using Eco RI restriction enzyme is demonstrated. The DNA-enzyme reaction is completed within 15 min in the proposed microfluidic system, compared to 50 min in a conventional large-scale system. Hence, the current device provides a valuable tool for rapid lambda-DNA digestion, while its mixer system delivers a simple yet effective solution for mixing problems in the micro-total-analysis-systems field. PMID:17195107

  9. Gene expression in the DpnI and DpnII restriction enzyme systems of Streptococcus pneumoniae

    SciTech Connect

    Lacks, S.A.; Sabelnikov, A.G.; Chen, Jau-Der; Greenberg, B.

    1992-12-31

    Although a number of bacterial species are naturally transformable, that is, their cells are able to take up external DNA in substantial amounts and integrate it into the chromosome without artificial manipulation of the cell surface, Streptococcus pneumoniae, the first species in which this phenomenon was detected, remains a prototype of such transformation. Cells of S. pneumonias also contain potent restriction endonucleases able to severely restrict DNA introduced during viral infection. Our current understanding of the genetic basis of the complementary DpnI and DpnII restriction systems and of the biochemistry of their component enzymes are briefly reviewed. The manner in which these enzymes impinge on the transfer of chromosomal genes and of plasmeds will be examined in detail. It will be seen that far from acting against foreign DNA in general, the restriction systems seem to be designed to exclude only infecting viral DNA The presence of complementary restriction systems in different cells of S. pneumonias enhances their effectiveness in blocking viral infection and promoting species survival. This enhanced effectiveness requires the expression of alternative restriction systems. Therefore, the ability of the cells to transfer the restriction enzyme genes and to regulate their expression are important for survival of the species.

  10. Methionine restriction slows down senescence in human diploid fibroblasts

    PubMed Central

    Kozieł, Rafał; Ruckenstuhl, Christoph; Albertini, Eva; Neuhaus, Michael; Netzberger, Christine; Bust, Maria; Madeo, Frank; Wiesner, Rudolf J; Jansen-Dürr, Pidder

    2014-01-01

    Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly. PMID:25273919

  11. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  12. Polymorphic restriction patterns of ribosomal internal transcribed spacers in the biocontrol fungus Puccinia carduorum correlate with weed host origin.

    PubMed Central

    Berthier, Y T; Bruckart, W L; Chaboudez, P; Luster, D G

    1996-01-01

    The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified by PCR and used to develop genetic markers for isolates of Puccinia carduorum being evaluated for biological control of Carduus thoermeri (musk thistle). Unique patterns were produced upon restriction of ITS DNA amplified from four separate Puccinia spp. Restriction patterns of ITS DNA of isolates of P. carduorum from Carduus acanthoides and C. thoermeri were distinct from those of P. carduorum from Carduus tenuiflorus and Carduus pycnocephalus. By this technique, isolates of P. carduorum from four different weed hosts can be differentiated from other Puccinia spp. and separated into two host groups. PMID:8702298

  13. Restriction enzyme maps of the macropodid herpesvirus 2 genome.

    PubMed

    Johnson, M A; Whalley, J M

    1987-01-01

    The DNA of macropodid herpesvirus 2 (dorcopsis wallaby herpesvirus) was analysed using restriction enzymes and molecular cloning techniques. Sets of EcoRI and SalI clones were prepared which represented approximately 85 per cent of the genome, and these clones were used to map the DNA by double-digestion and hybridization experiments. Data on uncloned regions were obtained by analysing fragments excised from agarose gels, and terminal fragments were identified by exonuclease III digestion. The genome was shown to be approximately 135 kilobases (kb) long. It has a long unique sequence (95 kb) bounded by repeat sequences (5.5 kb) and joined at one end to a short unique sequence (15 kb) also bounded by repeat sequences (7.0 kb) in a manner similar to that of herpes simplex viruses 1 and 2. PMID:2889444

  14. Restriction enzyme analysis of the mitochondrial genome in mitochondrial myopathy.

    PubMed Central

    Poulton, J; Turnbull, D M; Mehta, A B; Wilson, J; Gardiner, R M

    1988-01-01

    The mitochondrial myopathies are a heterogeneous group of disorders some of which may be caused by mutations in the mitochondrial genome. Mitochondrial DNA from 10 patients with mitochondrial myopathy and their mothers was analysed using five restriction enzymes and 11 mitochondrial probes in bacteriophage M13. No abnormalities were found in seven out of the 10 patients. Polymorphisms which have not previously been reported were detected in three patients and two of their mothers. These results exclude the presence of deletions or insertions of greater than 60 bp in the region of the mitochondrial genome examined. Any causative mitochondrial DNA mutations in these disorders are therefore likely to be point mutations or small structural rearrangements. Images PMID:2903249

  15. Restrictive lung disease in pregnancy.

    PubMed

    King, T E

    1992-12-01

    Restrictive ventilatory defects characterized by a reduction in lung volumes and an increase in the ratio of forced expiratory volume in 1 second to forced vital capacity occur when lung expansion is limited because of alterations in the lung parenchyma or because of abnormalities in the pleura, chest wall, or neuromuscular apparatus. Few studies have examined pregnant women with carefully defined restrictive lung disorders. The majority of pulmonary diseases have their onset after the childbearing years. When present, most do not alter fertility. Further, these disorders are only a relative contraindication to pregnancy because both the fetus and mother are able to survive without a high risk of increased morbidity or mortality. The clinical course of sarcoidosis is generally not altered by pregnancy. Factors indicative of a poor prognosis in sarcoidosis and pregnancy include parenchymal lesions on chest radiography, advanced roentgenologic staging, advanced maternal age, low inflammatory activity, requirement for drugs other than corticosteroids, and the presence of extrapulmonary sarcoidosis. Pregnancy seldom has a significant effect on the course of the connective tissue diseases. In PSS with significant renal involvement, pregnancy has the potential for poor fetal prognosis and the risk of maternal death due to a lethal progression of renal failure. Worsening of SLE is uncommon in pregnancy, and prophylactic therapy is generally not necessary. Most women with LAM are advised to avoid pregnancy or the use of estrogens because of the concern that it will lead to worsening of their disease. The incidence of kyphoscoliosis in pregnancy is relatively high. Premature birth rates are higher than that in the normal population. The risk of progression of the abnormal curve in a scoliotic patient appears low. However, women with unstable scolioses at the time of pregnancy can demonstrate progression of the curve with the pregnancy. Respiratory complications during

  16. An Infinite Restricted Boltzmann Machine.

    PubMed

    Côté, Marc-Alexandre; Larochelle, Hugo

    2016-07-01

    We present a mathematical construction for the restricted Boltzmann machine (RBM) that does not require specifying the number of hidden units. In fact, the hidden layer size is adaptive and can grow during training. This is obtained by first extending the RBM to be sensitive to the ordering of its hidden units. Then, with a carefully chosen definition of the energy function, we show that the limit of infinitely many hidden units is well defined. As with RBM, approximate maximum likelihood training can be performed, resulting in an algorithm that naturally and adaptively adds trained hidden units during learning. We empirically study the behavior of this infinite RBM, showing that its performance is competitive to that of the RBM, while not requiring the tuning of a hidden layer size. PMID:27171012

  17. 10 CFR 1045.16 - Criteria for evaluation of restricted data and formerly restricted data information.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Criteria for evaluation of restricted data and formerly restricted data information. 1045.16 Section 1045.16 Energy DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NUCLEAR CLASSIFICATION AND DECLASSIFICATION Identification of Restricted Data and Formerly Restricted Data Information § 1045.16 Criteria...

  18. Identifying the "demon whale-biter": Patterns of scarring on large whales attributed to a cookie-cutter shark Isistius sp.

    PubMed

    Best, Peter B; Photopoulou, Theoni

    2016-01-01

    The presence of crater-like wounds on cetaceans and other large marine vertebrates and invertebrates has been attributed to various organisms. We review the evidence for the identity of the biting agent responsible for crater wounds on large whales, using data collected from sei (Balaenoptera borealis), fin (B. physalus), inshore and offshore Bryde's (B. brydeii sp) and sperm whales (Physeter macrocephalus) examined at the Donkergat whaling station, Saldanha Bay, South Africa between March and October 1963. We then analyse the intensity and trends in its predation on large whales. Despite the scarcity of local records, we conclude that a cookie-cutter shark Isistius sp is the most likely candidate. We make inferences about the trends in (1) total counts of unhealed bitemarks, and (2) the proportion of unhealed bitemarks that were recent. We use day of the year; reproductive class, social grouping or sex; depth interval and body length as candidate covariates. The models with highest support for total counts of unhealed bitemarks involve the day of the year in all species. Depth was an important predictor in all species except offshore Bryde's whales. Models for the proportion of recent bites were only informative for sei and fin whales. We conclude that temporal scarring patterns support what is currently hypothesized about the distribution and movements of these whale species, given that Isistius does not occur in the Antarctic and has an oceanic habitat. The incidence of fresh bites confirms the presence of Isistius in the region. The lower numbers of unhealed bites on medium-sized sperm whales suggests that this group spends more time outside the area in which bites are incurred, providing a clue to one of the biggest gaps in our understanding of the movements of mature and maturing sperm males. PMID:27055057

  19. Identifying the “demon whale-biter”: Patterns of scarring on large whales attributed to a cookie-cutter shark Isistius sp

    PubMed Central

    Photopoulou, Theoni

    2016-01-01

    The presence of crater-like wounds on cetaceans and other large marine vertebrates and invertebrates has been attributed to various organisms. We review the evidence for the identity of the biting agent responsible for crater wounds on large whales, using data collected from sei (Balaenoptera borealis), fin (B. physalus), inshore and offshore Bryde’s (B. brydeii sp) and sperm whales (Physeter macrocephalus) examined at the Donkergat whaling station, Saldanha Bay, South Africa between March and October 1963. We then analyse the intensity and trends in its predation on large whales. Despite the scarcity of local records, we conclude that a cookie-cutter shark Isistius sp is the most likely candidate. We make inferences about the trends in (1) total counts of unhealed bitemarks, and (2) the proportion of unhealed bitemarks that were recent. We use day of the year; reproductive class, social grouping or sex; depth interval and body length as candidate covariates. The models with highest support for total counts of unhealed bitemarks involve the day of the year in all species. Depth was an important predictor in all species except offshore Bryde’s whales. Models for the proportion of recent bites were only informative for sei and fin whales. We conclude that temporal scarring patterns support what is currently hypothesized about the distribution and movements of these whale species, given that Isistius does not occur in the Antarctic and has an oceanic habitat. The incidence of fresh bites confirms the presence of Isistius in the region. The lower numbers of unhealed bites on medium-sized sperm whales suggests that this group spends more time outside the area in which bites are incurred, providing a clue to one of the biggest gaps in our understanding of the movements of mature and maturing sperm males. PMID:27055057

  20. DNA ALTERATIONS

    EPA Science Inventory

    The exposure of an organism to genotoxic chemicals may induce a cascade of genetic events. nitially, structural alterations to DNA are formed. ext, the DNA damage is processed and subsequently expressed in mutant gene products. inally, diseases result from the genetic damage. he ...