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Sample records for functional dna elements

  1. Defining functional DNA elements in the human genome.

    Science.gov (United States)

    Kellis, Manolis; Wold, Barbara; Snyder, Michael P; Bernstein, Bradley E; Kundaje, Anshul; Marinov, Georgi K; Ward, Lucas D; Birney, Ewan; Crawford, Gregory E; Dekker, Job; Dunham, Ian; Elnitski, Laura L; Farnham, Peggy J; Feingold, Elise A; Gerstein, Mark; Giddings, Morgan C; Gilbert, David M; Gingeras, Thomas R; Green, Eric D; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D; Myers, Richard M; Pazin, Michael J; Ren, Bing; Stamatoyannopoulos, John A; Weng, Zhiping; White, Kevin P; Hardison, Ross C

    2014-04-29

    With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease.

  2. Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Jason A Hilton

    Full Text Available Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision, up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption

  3. Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria.

    Science.gov (United States)

    Hilton, Jason A; Meeks, John C; Zehr, Jonathan P

    2016-01-01

    Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in

  4. Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

    Science.gov (United States)

    Vera, Daniel L; Madzima, Thelma F; Labonne, Jonathan D; Alam, Mohammad P; Hoffman, Gregg G; Girimurugan, S B; Zhang, Jinfeng; McGinnis, Karen M; Dennis, Jonathan H; Bass, Hank W

    2014-10-01

    The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5' and 3' ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

  5. The ENCODE (ENCyclopedia Of DNA Elements) Project.

    Science.gov (United States)

    2004-10-22

    The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (approximately 1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

  6. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir

    1998-01-01

    for a long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two......This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...... individual antiparallel DNA strands. Hydrogen bonds provide specificity that allows pairing between the complementary bases (A.T and G.C) in opposite strands. Base stacking occurs near the center of the DNA helix and provides a great deal of stability to the helix (in addition to hydrogen bonding). The sugar...

  7. Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping.

    Science.gov (United States)

    Bhullar, Simran; Chakravarthy, Suma; Advani, Sonia; Datta, Sudipta; Pental, Deepak; Burma, Pradeep Kumar

    2003-06-01

    The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence

  8. An integrated encyclopedia of DNA elements in the human genome.

    Science.gov (United States)

    2012-09-01

    The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research.

  9. Purification of functionalized DNA origami nanostructures.

    Science.gov (United States)

    Shaw, Alan; Benson, Erik; Högberg, Björn

    2015-05-26

    The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

  10. Transposable Elements: From DNA Parasites to Architects of Metazoan Evolution

    Directory of Open Access Journals (Sweden)

    Oliver Piskurek

    2012-07-01

    Full Text Available One of the most unexpected insights that followed from the completion of the human genome a decade ago was that more than half of our DNA is derived from transposable elements (TEs. Due to advances in high throughput sequencing technologies it is now clear that TEs comprise the largest molecular class within most metazoan genomes. TEs, once categorised as "junk DNA", are now known to influence genomic structure and function by increasing the coding and non-coding genetic repertoire of the host. In this way TEs are key elements that stimulate the evolution of metazoan genomes. This review highlights several lines of TE research including the horizontal transfer of TEs through host-parasite interactions, the vertical maintenance of TEs over long periods of evolutionary time, and the direct role that TEs have played in generating morphological novelty.

  11. Transposable elements: from DNA parasites to architects of metazoan evolution.

    Science.gov (United States)

    Piskurek, Oliver; Jackson, Daniel J

    2012-07-12

    One of the most unexpected insights that followed from the completion of the human genome a decade ago was that more than half of our DNA is derived from transposable elements (TEs). Due to advances in high throughput sequencing technologies it is now clear that TEs comprise the largest molecular class within most metazoan genomes. TEs, once categorised as "junk DNA", are now known to influence genomic structure and function by increasing the coding and non-coding genetic repertoire of the host. In this way TEs are key elements that stimulate the evolution of metazoan genomes. This review highlights several lines of TE research including the horizontal transfer of TEs through host-parasite interactions, the vertical maintenance of TEs over long periods of evolutionary time, and the direct role that TEs have played in generating morphological novelty.

  12. A user's guide to the encyclopedia of DNA elements (ENCODE).

    Science.gov (United States)

    2011-04-01

    The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome.

  13. Exploring functional elements and genomic variation in the noncoding genome

    NARCIS (Netherlands)

    van Heesch, S.A.A.C.

    2014-01-01

    Gene expression regulation is a delicate process that depends on multiple aspects including genome structure and transcription factor binding to DNA elements. The majority of our genome consists of noncoding DNA, which was shown to be crucial in providing the correct context for genome function. Alt

  14. Exploring functional elements and genomic variation in the noncoding genome

    NARCIS (Netherlands)

    van Heesch, S.A.A.C.|info:eu-repo/dai/nl/336463286

    2014-01-01

    Gene expression regulation is a delicate process that depends on multiple aspects including genome structure and transcription factor binding to DNA elements. The majority of our genome consists of noncoding DNA, which was shown to be crucial in providing the correct context for genome function. Alt

  15. Functionalizing Designer DNA Crystals

    Science.gov (United States)

    Chandrasekaran, Arun Richard

    Three-dimensional crystals have been self-assembled from a DNA tensegrity triangle via sticky end interaction. The tensegrity triangle is a rigid DNA motif containing three double helical edges connected pair-wise by three four-arm junctions. The symmetric triangle contains 3 unique strands combined in a 3:3:1 ratio: 3 crossover, 3 helical and 1 central. The length of the sticky end reported previously was two nucleotides (nt) (GA:TC) and the motif with 2-helical turns of DNA per edge diffracted to 4.9 A at beam line NSLS-X25 and to 4 A at beam line ID19 at APS. The purpose of these self-assembled DNA crystals is that they can be used as a framework for hosting external guests for use in crystallographic structure solving or the periodic positioning of molecules for nanoelectronics. This thesis describes strategies to improve the resolution and to incorporate guests into the 3D lattice. The first chapter describes the effect of varying sticky end lengths and the influence of 5'-phosphate addition on crystal formation and resolution. X-ray diffraction data from beam line NSLS-X25 revealed that the crystal resolution for 1-nt (G:C) sticky end was 3.4 A. Motifs with every possible combination of 1-nt and 2-nt sticky-ended phosphorylated strands were crystallized and X-ray data were collected. The position of the 5'-phosphate on either the crossover (strand 1), helical (strand 2), or central strand (3) had an impact on the resolution of the self-assembled crystals with the 1-nt 1P-2-3 system diffracting to 2.62 A at APS and 3.1 A at NSLS-X25. The second chapter describes the sequence-specific recognition of DNA motifs with triplex-forming oligonucleotides (TFOs). This study examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The TFO 5'-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine.oligopyrimidine binding site. As triplex formation involving cytidine

  16. Mobile DNA Elements: The Seeds of Organic Complexity on Earth.

    Science.gov (United States)

    Habibi, Laleh; Pedram, Mehrdad; AmirPhirozy, Akbar; Bonyadi, Khadijeh

    2015-10-01

    Mobile DNA or transposable elements (TEs) are genomic sequences capable of moving themselves independently into different parts of the genome. Viral invasion of eukaryotic genomes is assumed to be the main source of TEs. Selfish transposition of these elements could be a serious threat to the host cell, as they can insert themselves into the middle of coding genes and/or induce genomic instability. In response, through millions of years of evolution, cells have come up with various mechanisms such as genomic imprinting, DNA methylation, heterochromatin formation, and RNA interference to deactivate them. Interestingly, these processes have also greatly contributed to important cellular functions involved in cell differentiation, development, and differential gene expression. Propagation of TE copies during the course of evolution have resulted in increasing the genome size and providing proper space and flexibility in shaping the genome by creating new genes and establishing essential cellular structures such as heterochromatin, centromere, and telomeres. Yet, these elements are mostly labeled for playing a role in pathogenesis of human diseases. Here, we attempt to introduce TEs as factors necessary for making us human rather than just selfish sequences or obligatory guests invading our DNA.

  17. An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region.

    OpenAIRE

    Fiering, S; Kim, C. G.; Epner, E M; Groudine, M

    1993-01-01

    The human beta-globin locus control region (LCR) is a complex DNA regulatory element that controls the expression of the cis-linked beta-like globin genes located in the 55 kilobases 3' of the LCR. We have initiated the functional analysis of the LCR by homologous recombination in murine erythroleukemia cell somatic hybrids that carry a single copy of human chromosome 11 on which the beta-globin locus is situated. High-level expression of the human beta-globin gene normally occurs when these ...

  18. Interstitial Functionalization in elemental Si

    Science.gov (United States)

    Kiefer, Boris; Fohtung, Edwin

    Societies in the 21st century will face many challenges. Materials science and materials design will be essential to address and master some if not all of these challenges. Semiconductors are among the most important technological material classes. Properties such as electrical transport are strongly affected by defects and a central goal continues to be the reduction of defect densities as much as possible in these compounds. Here we present results of interstitial Fe doping in elemental Si using first-principles DFT calculations. The preliminary results show that Fe will only occupy octahedral interstitial sites. The analysis of the electronic structure shows that the compounds are ferromagnetic and that a bandgap opens as interstitial Fe concentrations decrease, with a possible intermittent semi-metallic phase. The formation energy for interstitial Fe is unfavorable, as expected, by ~1.5 eV but becomes favorable as the chemical potential of Fe increases. Therefore, we expect that biasing the system with an external electrical field will lead to the formation of these materials. Thus, our results show that interstitial defects can be beneficial for the design of functionalities that differ significantly from those of the host material.

  19. Functional Metamirrors Using Bianisotropic Elements

    Science.gov (United States)

    Asadchy, V. S.; Ra'di, Y.; Vehmas, J.; Tretyakov, S. A.

    2015-03-01

    Conventional mirrors obey the simple reflection law that a plane wave is reflected as a plane wave, at the same angle. To engineer spatial distributions of fields reflected from a mirror, one can either shape the reflector or position some phase-correcting elements on top of a mirror surface. Here we show, both theoretically and experimentally, that full-power reflection with general control over the reflected wave phase is possible with a single-layer array of deeply subwavelength inclusions. These proposed artificial surfaces, metamirrors, provide various functions of shaped or nonuniform reflectors without utilizing any mirror. This can be achieved only if the forward and backward scattering of the inclusions in the array can be engineered independently, and we prove that it is possible using electrically and magnetically polarizable inclusions. The proposed subwavelength inclusions possess desired reflecting properties at the operational frequency band, while at other frequencies the array is practically transparent. The metamirror concept leads to a variety of applications over the entire electromagnetic spectrum, such as optically transparent focusing antennas for satellites, multifrequency reflector antennas for radio astronomy, low-profile conformal antennas for telecommunications, and nanoreflectarray antennas for integrated optics.

  20. Systematic tissue-specific functional annotation of the human genome highlights immune-related DNA elements for late-onset Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Qiongshi Lu

    2017-07-01

    Full Text Available Continuing efforts from large international consortia have made genome-wide epigenomic and transcriptomic annotation data publicly available for a variety of cell and tissue types. However, synthesis of these datasets into effective summary metrics to characterize the functional non-coding genome remains a challenge. Here, we present GenoSkyline-Plus, an extension of our previous work through integration of an expanded set of epigenomic and transcriptomic annotations to produce high-resolution, single tissue annotations. After validating our annotations with a catalog of tissue-specific non-coding elements previously identified in the literature, we apply our method using data from 127 different cell and tissue types to present an atlas of heritability enrichment across 45 different GWAS traits. We show that broader organ system categories (e.g. immune system increase statistical power in identifying biologically relevant tissue types for complex diseases while annotations of individual cell types (e.g. monocytes or B-cells provide deeper insights into disease etiology. Additionally, we use our GenoSkyline-Plus annotations in an in-depth case study of late-onset Alzheimer's disease (LOAD. Our analyses suggest a strong connection between LOAD heritability and genetic variants contained in regions of the genome functional in monocytes. Furthermore, we show that LOAD shares a similar localization of SNPs to monocyte-functional regions with Parkinson's disease. Overall, we demonstrate that integrated genome annotations at the single tissue level provide a valuable tool for understanding the etiology of complex human diseases. Our GenoSkyline-Plus annotations are freely available at http://genocanyon.med.yale.edu/GenoSkyline.

  1. Alu Mobile Elements: From Junk DNA to Genomic Gems

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    Sami Dridi

    2012-01-01

    Full Text Available Alus, the short interspersed repeated sequences (SINEs, are retrotransposons that litter the human genomes and have long been considered junk DNA. However, recent findings that these mobile elements are transcribed, both as distinct RNA polymerase III transcripts and as a part of RNA polymerase II transcripts, suggest biological functions and refute the notion that Alus are biologically unimportant. Indeed, Alu RNAs have been shown to control mRNA processing at several levels, to have complex regulatory functions such as transcriptional repression and modulating alternative splicing and to cause a host of human genetic diseases. Alu RNAs embedded in Pol II transcripts can promote evolution and proteome diversity, which further indicates that these mobile retroelements are in fact genomic gems rather than genomic junks.

  2. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    Science.gov (United States)

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  3. An "in-out" strategy using gene targeting and FLP recombinase for the functional dissection of complex DNA regulatory elements: analysis of the beta-globin locus control region.

    Science.gov (United States)

    Fiering, S; Kim, C G; Epner, E M; Groudine, M

    1993-01-01

    The human beta-globin locus control region (LCR) is a complex DNA regulatory element that controls the expression of the cis-linked beta-like globin genes located in the 55 kilobases 3' of the LCR. We have initiated the functional analysis of the LCR by homologous recombination in murine erythroleukemia cell somatic hybrids that carry a single copy of human chromosome 11 on which the beta-globin locus is situated. High-level expression of the human beta-globin gene normally occurs when these hybrid cells are induced to differentiate. We have reported that the insertion of an expressed selectable marker gene (driven by the Friend virus enhancer/promoter) into the LCR disrupts the LCR-mediated regulation of globin transcription. In these cells, beta-globin is no longer expressed when the cells differentiate; instead, expression of the selectable marker gene increases significantly after differentiation. Since present techniques for homologous recombination require the insertion of a selectable marker, further progress in using homologous recombination to analyze the LCR depends on deletion of the selectable marker and demonstration that the locus functions normally after the insertion, expression, and deletion of the selectable marker. Here we show that after precise deletion of the selectable marker by using the FLP recombinase/FRT (FLP recombinase target) system, the locus functions as it did before the homologous recombination event. These studies demonstrate the feasibility of using homologous recombination to analyze the LCR in particular, and other complex cis-regulatory DNA elements in general, in their normal chromosomal context. Images Fig. 2 Fig. 3 Fig. 4 PMID:8378321

  4. Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.

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    de la Rica, Lorenzo; Stanley, Jatinder S; Branco, Miguel R

    2016-01-01

    DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.

  5. Mapping Fifteen Trace Elements in Human Seminal Plasma and Sperm DNA.

    Science.gov (United States)

    Ali, Sazan; Chaspoul, Florence; Anderson, Loundou; Bergé-Lefranc, David; Achard, Vincent; Perrin, Jeanne; Gallice, Philippe; Guichaoua, Marie

    2017-02-01

    Studies suggest a relationship between semen quality and the concentration of trace elements in serum or seminal plasma. However, trace elements may be linked to DNA and capable of altering the gene expression patterns. Thus, trace element interactions with DNA may contribute to the mechanisms for a trans-generational reproductive effect. We developed an analytical method to determine the amount of trace elements bound to the sperm DNA, and to estimate their affinity for the sperm DNA by the ratio: R = Log [metal concentration in the sperm DNA/metal concentration in seminal plasma]. We then analyzed the concentrations of 15 trace elements (Al, Cd, Cr, Cu, Hg, Mn, Mo, Ni, Pb, Ti, V, Zn, As, Sb, and Se) in the seminal plasma and the sperm DNA in 64 normal and 30 abnormal semen specimens with Inductively Coupled Plasma/Mass Spectrometry (ICP-MS). This study showed all trace elements were detected in the seminal plasma and only metals were detected in the sperm DNA. There was no correlation between the metals' concentrations in the seminal plasma and the sperm DNA. Al had the highest affinity for DNA followed by Pb and Cd. This strong affinity is consistent with the known mutagenic effects of these metals. The lowest affinity was observed for Zn and Ti. We observed a significant increase of Al linked to the sperm DNA of patients with oligozoospermia and teratozoospermia. Al's reproductive toxicity might be due to Al linked to DNA, by altering spermatogenesis and expression patterns of genes involved in the function of reproduction.

  6. DNA functionalization by dynamic chemistry

    Directory of Open Access Journals (Sweden)

    Zeynep Kanlidere

    2016-10-01

    Full Text Available Dynamic combinatorial chemistry (DCC is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80% was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

  7. Translating the ENCyclopedia Of DNA Elements Project findings to the clinic: ENCODE's implications for eye disease.

    Science.gov (United States)

    Sanfilippo, Paul G; Hewitt, Alex W

    2014-01-01

    Approximately 10 years after the Human Genome Project unravelled the sequence of our DNA, the ENCyclopedia Of DNA Elements (ENCODE) Project sought to interpret it. Data from the recently completed project have shed new light on the proportion of biologically active human DNA, assigning a biochemical role to much of the sequence previously considered to be 'junk'. Many of these newly catalogued functional elements represent epigenetic mechanisms involved in regulation of gene expression. Analogous to an Ishihara plate, a gene-coding region of DNA (target dots) only comes into context when the non-coding DNA (surrounding dots) is appreciated. In this review we provide an overview of the ENCODE project, discussing the significance of these data for ophthalmic research and eye disease. The novel insights afforded by the ENCODE project will in time allow for the development of new therapeutic strategies in the management of common blinding disorders.

  8. A surrogate approach to study the evolution of noncoding DNA elements that organize eukaryotic genomes.

    Science.gov (United States)

    Vermaak, Danielle; Bayes, Joshua J; Malik, Harmit S

    2009-01-01

    Comparative genomics provides a facile way to address issues of evolutionary constraint acting on different elements of the genome. However, several important DNA elements have not reaped the benefits of this new approach. Some have proved intractable to current day sequencing technology. These include centromeric and heterochromatic DNA, which are essential for chromosome segregation as well as gene regulation, but the highly repetitive nature of the DNA sequences in these regions make them difficult to assemble into longer contigs. Other sequences, like dosage compensation X chromosomal sites, origins of DNA replication, or heterochromatic sequences that encode piwi-associated RNAs, have proved difficult to study because they do not have recognizable DNA features that allow them to be described functionally or computationally. We have employed an alternate approach to the direct study of these DNA elements. By using proteins that specifically bind these noncoding DNAs as surrogates, we can indirectly assay the evolutionary constraints acting on these important DNA elements. We review the impact that such "surrogate strategies" have had on our understanding of the evolutionary constraints shaping centromeres, origins of DNA replication, and dosage compensation X chromosomal sites. These have begun to reveal that in contrast to the view that such structural DNA elements are either highly constrained (under purifying selection) or free to drift (under neutral evolution), some of them may instead be shaped by adaptive evolution and genetic conflicts (these are not mutually exclusive). These insights also help to explain why the same elements (e.g., centromeres and replication origins), which are so complex in some eukaryotic genomes, can be simple and well defined in other where similar conflicts do not exist.

  9. VEZF1 elements mediate protection from DNA methylation.

    Directory of Open Access Journals (Sweden)

    Jacqueline Dickson

    2010-01-01

    Full Text Available There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

  10. Ab initio work function of elemental metals

    DEFF Research Database (Denmark)

    Skriver, Hans Lomholt; Rosengaard, N. M.

    1992-01-01

    We have used a recently developed self-consistent Green’s-function technique based on tight-binding linear-muffin-tin-orbital theory to calculate the work function for the close-packed surfaces of 37 elemental metals. The results agree with the limited experimental data obtained from single...... crystals to within 15%, and they explain the smooth behavior of the polycrystalline data as a function of atomic number....

  11. New functionalities in abundant element oxides: ubiquitous element strategy.

    Science.gov (United States)

    Hosono, Hideo; Hayashi, Katsuro; Kamiya, Toshio; Atou, Toshiyuki; Susaki, Tomofumi

    2011-06-01

    While most ceramics are composed of ubiquitous elements (the ten most abundant elements within the Earth's crust), many advanced materials are based on rare elements. A 'rare-element crisis' is approaching owing to the imbalance between the limited supply of rare elements and the increasing demand. Therefore, we propose a 'ubiquitous element strategy' for materials research, which aims to apply abundant elements in a variety of innovative applications. Creation of innovative oxide materials and devices based on conventional ceramics is one specific challenge. This review describes the concept of ubiquitous element strategy and gives some highlights of our recent research on the synthesis of electronic, thermionic and structural materials using ubiquitous elements.

  12. Matrix elements from moments of correlation functions

    CERN Document Server

    Bouchard, Chris; Orginos, Kostas; Richards, David

    2016-01-01

    Momentum-space derivatives of matrix elements can be related to their coordinate-space moments through the Fourier transform. We derive these expressions as a function of momentum transfer $Q^2$ for asymptotic in/out states consisting of a single hadron. We calculate corrections to the finite volume moments by studying the spatial dependence of the lattice correlation functions. This method permits the computation of not only the values of matrix elements at momenta accessible on the lattice, but also the momentum-space derivatives, providing {\\it a priori} information about the $Q^2$ dependence of form factors. As a specific application we use the method, at a single lattice spacing and with unphysically heavy quarks, to directly obtain the slope of the isovector form factor at various $Q^2$, whence the isovector charge radius. The method has potential application in the calculation of any hadronic matrix element with momentum transfer, including those relevant to hadronic weak decays.

  13. Matrix elements from moments of correlation functions

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Chia Cheng [SLAC National Accelerator Lab., Menlo Park, CA (United States); Bouchard, Chris [College of William and Mary, Williamsburg, VA (United States); Orginos, Konstantinos [Thomas Jefferson National Accelerator Facility (TJNAF), Newport News, VA (United States); College of William and Mary, Williamsburg, VA (United States); Richards, David G. [Thomas Jefferson National Accelerator Facility (TJNAF), Newport News, VA (United States)

    2016-10-01

    Momentum-space derivatives of matrix elements can be related to their coordinate-space moments through the Fourier transform. We derive these expressions as a function of momentum transfer Q2 for asymptotic in/out states consisting of a single hadron. We calculate corrections to the finite volume moments by studying the spatial dependence of the lattice correlation functions. This method permits the computation of not only the values of matrix elements at momenta accessible on the lattice, but also the momentum-space derivatives, providing {\\it a priori} information about the Q2 dependence of form factors. As a specific application we use the method, at a single lattice spacing and with unphysically heavy quarks, to directly obtain the slope of the isovector form factor at various Q2, whence the isovector charge radius. The method has potential application in the calculation of any hadronic matrix element with momentum transfer, including those relevant to hadronic weak decays.

  14. Transposable DNA elements and life history traits: II. Transposition of P DNA elements in somatic cells reduces fitness, mating activity, and locomotion of Drosophila melanogaster.

    Science.gov (United States)

    Woodruff, R C; Thompson, J N; Barker, J S; Huai, H

    1999-01-01

    Some transposable DNA elements in higher organisms are active in somatic cells, as well as in germinal cells. What effect does the movement of DNA elements in somatic cells have on life history traits? It has previously been reported that somatically active P and mariner elements in Drosophila induce genetic damage and significantly reduce lifespan. In this study, we report that the movement of P elements in somatic cells also significantly reduces fitness, mating activity, and locomotion of Drosophila melanogaster. If other elements cause similar changes in life history traits, it is doubtful if transposable DNA elements remain active for long in somatic cells in natural populations.

  15. Membership Functions for Fuzzy Focal Elements

    Directory of Open Access Journals (Sweden)

    Porębski Sebastian

    2016-09-01

    Full Text Available The paper presents a study on data-driven diagnostic rules, which are easy to interpret by human experts. To this end, the Dempster-Shafer theory extended for fuzzy focal elements is used. Premises of the rules (fuzzy focal elements are provided by membership functions which shapes are changing according to input symptoms. The main aim of the present study is to evaluate common membership function shapes and to introduce a rule elimination algorithm. Proposed methods are first illustrated with the popular Iris data set. Next experiments with five medical benchmark databases are performed. Results of the experiments show that various membership function shapes provide different inference efficiency but the extracted rule sets are close to each other. Thus indications for determining rules with possible heuristic interpretation can be formulated.

  16. DNA structural elements required for ERCC1-XPF endonuclease activity

    NARCIS (Netherlands)

    W.L. de Laat (Wouter); E. Appeldoorn (Esther); J.H.J. Hoeijmakers (Jan); N.G.J. Jaspers (Nicolaas)

    1998-01-01

    textabstractThe heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the

  17. Controlling Function and Structure with DNA

    DEFF Research Database (Denmark)

    Tørring, Thomas

    2011-01-01

    In this thesis, the research on three different topics will be described. The overall area of the research is DNA nanotechnology, and the first chapter is therefore an introduction to DNA, and its advantages as a building material. The first research topic is the development of a new method...... investigated on a two dimensional DNA origami platform. This was done by incorporating functional groups on the surface of the origami, and reacting these with biotin analogues carrying the complementary functional groups. Successful reactions could then be observed using atomic force microscopy after addition...... of the protein streptavidin. While the implementation of chemical functionalities on origami can be achieved during automated DNA synthesis, this is laborious and costly. In a separate research project we aimed at improving the accessibility by applying an enzymatic labelling method. We demonstrated that the DNA...

  18. A brief review on the Human Encyclopedia of DNA Elements (ENCODE) project.

    Science.gov (United States)

    Qu, Hongzhu; Fang, Xiangdong

    2013-06-01

    The ENCyclopedia Of DNA Elements (ENCODE) project is an international research consortium that aims to identify all functional elements in the human genome sequence. The second phase of the project comprised 1640 datasets from 147 different cell types, yielding a set of 30 publications across several journals. These data revealed that 80.4% of the human genome displays some functionality in at least one cell type. Many of these regulatory elements are physically associated with one another and further form a network or three-dimensional conformation to affect gene expression. These elements are also related to sequence variants associated with diseases or traits. All these findings provide us new insights into the organization and regulation of genes and genome, and serve as an expansive resource for understanding human health and disease.

  19. Resurrection of DNA function in vivo from an extinct genome.

    Science.gov (United States)

    Pask, Andrew J; Behringer, Richard R; Renfree, Marilyn B

    2008-05-21

    There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  20. Resurrection of DNA function in vivo from an extinct genome.

    Directory of Open Access Journals (Sweden)

    Andrew J Pask

    Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  1. Characterization of "cis"-regulatory elements ("c"RE) associated with mammary gland function

    Science.gov (United States)

    The Bos taurus genome assembly has propelled dairy science into a new era; still, most of the information encoded in the genome has not yet been decoded. The human Encyclopedia of DNA Elements (ENCODE) project has spearheaded the identification and annotation of functional genomic elements in the hu...

  2. Functional DNA nanostructures for theranostic applications.

    Science.gov (United States)

    Pei, Hao; Zuo, Xiaolei; Zhu, Dan; Huang, Qing; Fan, Chunhai

    2014-02-18

    There has been tremendous interest in constructing nanostructures by exploiting the unparalleled ability of DNA molecules in self-assembly. We have seen the appearance of many fantastic, "art-like" DNA nanostructures in one, two, or three dimensions during the last two decades. More recently, much attention has been directed to the use of these elegant nanoobjects for applications in a wide range of areas. Among them, diagnosis and therapy (i.e., theranostics) are of particular interest given the biological nature of DNA. One of the major barricades for the biosensor design lies in the restricted target accessibility at the solid-water interface. DNA nanotechnology provides a convenient approach to well control the biomolecule-confined surface to increase the ability of molecular recognition at the biosensing interface. For example, tetrahedral DNA nanostructures with thiol modifications can be self-assembled at the gold surface with high reproducibility. Since DNA tetrahedra are highly rigid and well-defined structures with atomic precision and versatile functionality, they provide scaffolds for anchoring of a variety of biomolecular probes (DNA, aptamers, peptides, and proteins) for biosensing. Significantly, this DNA nanostructure-based biosensing platform greatly increases target accessibility and improves the sensitivity for various types of molecular targets (DNA, RNA, proteins, and small molecules) by several orders of magnitude. In an alternative approach, DNA nanostructures provide a framework for the development of dynamic nanosensors that can function inside the cell. DNA tetrahedra are found to be facilely cell permeable and can sense and image specific molecules in cells. More importantly, these DNA nanostructures can be efficient drug delivery nanocarriers. Since they are DNA molecules by themselves, they have shown excellent cellular biocompatibility with minimal cytotoxicity. As an example, DNA tetrahedra tailored with CpG oligonucleotide drugs have

  3. A DNA fingerprint probe from Mycosphaerella graminicola identifies an active transposable element

    NARCIS (Netherlands)

    Goodwin, S.B.; Cavaletto, J.R.; Waalwijk, C.; Kema, G.H.J.

    2001-01-01

    DNA fingerprinting has been used extensively to characterize populations of Mycosphaerella graminicola, the Septoria tritici blotch pathogen of wheat. The highly polymorphic DNA fingerprints of Mycosphaerella graminicola were assumed to reflect the action of transposable elements. However, there was

  4. TFIIIC bound DNA elements in nuclear organization and insulation.

    Science.gov (United States)

    Kirkland, Jacob G; Raab, Jesse R; Kamakaka, Rohinton T

    2013-01-01

    tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short interspersed nuclear elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer - blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vice versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. This article is part of a Special Issue entitled: Transcription by Odd Pols. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  6. Movable Genetic Elements: Detection of Changes in Maize DNA at the Shrunken Locus Due to the Intervention of Ds Elements

    Science.gov (United States)

    Burr, B.; Burr, F.A.

    1980-05-28

    This report describes our initial attempts at the molecular characterization of a maize controlling element. We have prepared a cDNA probe and used it to detect changes at a locus where Ds elements are found. Evidence of their presence are indicated by changes in the restriction patterns, but there is as yet no information on the physical nature of the controlling elements nor on the kinds of rearrangements they cause.

  7. A method for generating subtractive cDNA libraries retaining clones containing repetitive elements.

    OpenAIRE

    1997-01-01

    Here we describe a two-stepped photobiotin-based procedure to enrich a target (canine retinal) cDNA library for tissue specific clones without removing those containing repetitive ( SINE ) elements, despite the presence of these elements in the driver population. In a first hybridization excess SINE elements were hybridized to a driver (canine cerebellar) cDNA. In a second hybridization target cDNA was added to this reaction. The resulting cDNA library was enriched for retinal specific clones...

  8. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    OpenAIRE

    Mossi, R; Ferrari, E; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  9. Dead element replicating: degenerate R2 element replication and rDNA genomic turnover in the Bacillus rossius stick insect (Insecta: Phasmida.

    Directory of Open Access Journals (Sweden)

    Francesco Martoni

    Full Text Available R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5' end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s. Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun as well as a full-length but degenerate element (R2Brdeg. An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.

  10. Elements of the theory of functions

    CERN Document Server

    Knopp, Konrad

    2016-01-01

    Well-known book provides a clear, concise review of complex numbers and their geometric representation; linear functions and circular transformations; sets, sequences, and power series; analytic functions and conformal mapping; and elementary functions. 1952 edition.

  11. DANIO-CODE: Toward an Encyclopedia of DNA Elements in Zebrafish.

    Science.gov (United States)

    Tan, Haihan; Onichtchouk, Daria; Winata, Cecilia

    2016-02-01

    The zebrafish has emerged as a model organism for genomics studies. The symposium "Toward an encyclopedia of DNA elements in zebrafish" held in London in December 2014, was coorganized by Ferenc Müller and Fiona Wardle. This meeting is a follow-up of a similar previous workshop held 2 years earlier and represents a push toward the formalization of a community effort to annotate functional elements in the zebrafish genome. The meeting brought together zebrafish researchers, bioinformaticians, as well as members of established consortia, to exchange scientific findings and experience, as well as to discuss the initial steps toward the formation of a DANIO-CODE consortium. In this study, we provide the latest updates on the current progress of the consortium's efforts, opening up a broad invitation to researchers to join in and contribute to DANIO-CODE.

  12. Alu elements and DNA double-strand break repair

    OpenAIRE

    White, Travis B; Morales, Maria E.; Deininger, Prescott L.

    2015-01-01

    Alu elements represent one of the most common sources of homology and homeology in the human genome. Homeologous recombination between Alu elements represents a major form of genetic instability leading to deletions and duplications. Although these types of events have been studied extensively through genomic sequencing to assess the impact of Alu elements on disease mutations and genome evolution, the overall abundance of Alu elements in the genome often makes it difficult to assess the rele...

  13. A functional gene array for detection of bacterial virulence elements.

    Directory of Open Access Journals (Sweden)

    Crystal Jaing

    Full Text Available Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.

  14. The Numerical Integration of Discrete Functions on a Triangular Element

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    With the application of Hammer integral formulas of a continuousfunction on a triangular element, the numerical integral formulas of some discrete functions on the element are derived by means of decomposition and recombination of base functions. Hammer integral formulas are the special examples of those of the paper.

  15. Analysis of mammalian cis-regulatory DNA elements by homologous recombination.

    Science.gov (United States)

    Fiering, S; Bender, M A; Groudine, M

    1999-01-01

    The use of homologous recombination to modify and thereby functionally analyze cis-regulatory DNA elements in mammalian cells has become an important approach in mammalian gene expression research. We have emphasized the necessity of designing a system that allows the removal of selectable markers used in targeting and facilitates the further modification of the region under study. To perform these tasks, we presently favor making an initial HR-mediated replacement of the entire element under study with an active positive selectable marker in combination with either an inactive second positive selectable marker or an active negative selectable marker. The plug and socket system, in which an inactive selectable marker is complemented by HR, is the most dependable and well-characterized option for making secondary modifications. However, the double-replacement system has certain advantages, and the recently developed RMCE approach, which allows replacement of a negative selectable marker by site-specific recombinase-mediated insertion without using a positive selectable marker, will likely prove very valuable in future experiments. Each of the systems, or combinations thereof, should be considered in light of the specifics of any given experiment to select the most appropriate option. Although the emphasis of this article has been the analysis of cis-acting regulatory elements involved in transcription, these same approaches can be used to analyze other regulatory elements (e.g., origins of replication) and to make multiple subtle mutations in polypeptides.

  16. Elements and modulation of functional dynamics.

    Science.gov (United States)

    Gibbs, Alan C

    2014-10-09

    The existing structure-function paradigm of drug discovery has been evolving toward the essential incorporation of dynamics data. This new functional dynamics paradigm emphasizes conformational entropy as a driving force of protein function and intermolecular recognition. Conformational dynamics (a proxy of conformational entropy) impacts the degree of protein (dis)order and the constitution of the conformational ensemble, the mechanisms of allostery and drug resistance, and the free energy of ligand binding. Specific protein and ligand conformations facilitate favorable, reciprocal interactions. The number of protein and ligand conformers that exhibit favorable binding interactions will vary from system to system. All binding scenarios can modulate protein dynamics by various levels of enthalpic and entropic contribution, with significant influence on the functional dynamics of the system. Analysis and consideration of resulting changes of activity, signaling, catalysis, and subsequent phenotypic outcome are powerful motivations in the drug design process.

  17. Magnetic particle-based sandwich sensor with DNA-modified carbon nanotubes as recognition elements for detection of DNA hybridization.

    Science.gov (United States)

    Hu, Po; Huang, Cheng Zhi; Li, Yuan Fang; Ling, Jian; Liu, Yu Ling; Fei, Liang Run; Xie, Jian Ping

    2008-03-01

    In this contribution, we design a visual sensor for DNA hybridization with DNA probe-modified magnetic particles (MPs) and multiwalled carbon nanotubes (MWNTs) without involving a visual recognition element such as fluorescent/chemiluminescent reagents. It was found that DNA probe-modified MWNTs, which could be dispersed in aqueous medium and have strong light scattering signals under the excitation of a light beam in the UV-vis region, could connect with DNA probe-modified MPs together in the presence of perfectly complementary target DNA and form a sandwich structure. In a magnetic field, the formed MP-MWNT species can easily be removed from the solution, resulting in a decrease of light scattering signals. Thus, a magnetic particle-based sandwich sensor could be developed to detect DNA hybridization by measuring the light scattering signals with DNA-modified MWNTs as recognition elements. Experiments showed that the DNA-modified MPs sensor could be reused at least 17 times and was stable for more than 6 months.

  18. Chemical footprinting of structural and functional elements of dhfr oribeta during the CHOC 400 cell cycle.

    Science.gov (United States)

    Schroll, Alayne L; Heintz, Nicholas H

    2004-05-12

    Oribeta, an origin of replication 3' to Chinese hamster dihydrofolate reductase (dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO(4) in vitro and in living cells of three regions within dhfr oribeta which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT(23) dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO(4) modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT(23) repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT(23) repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO(4) in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oribeta adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT(23) repeat and DNA unwinding.

  19. Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element.

    OpenAIRE

    1991-01-01

    Diabetes mellitus is characterized by insulin deficiency and high plasma glucagon levels, which can be normalized by insulin replacement. It has previously been reported that glucagon gene expression is negatively regulated by insulin at the transcriptional level. By transfection studies, I have now localized a DNA control element that mediates insulin effects on glucagon gene transcription. This element also confers insulin responsiveness to a heterologous promoter. DNA-binding proteins that...

  20. Identification and functional analysis of a CDE/CHR element in the POLD1 promoter

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    DNA polymerase delta is encoded by the POLD1 gene,the transcription of which is strictly cell cycle-dependent.However,the means by which POLD1 transcription is regulated by the cell cycle mechanism is currently unknown.We discovered a novel element in the POLD1 promoter known as a CDE(cell cycle-dependent element)/CHR(cell cycle gene homology region) element.A series of luciferase reporter constructs containing various POLD1 promoter mutations were used to investigate the role of the CDE/CHR element in POLD1 transcription.When the CDE/CHR element was mutated,the promoter activity was up-regulated,and the cell-cycle related factors E2F1 and p21 stopped regulating the promoter.Furthermore,cell cycle-dependent changes in the promoter activity required the integrative CDE/CHR element.Electrophoretic mobility shift assay(EMSA) revealed the presence of at least three types of DNA/protein complexes binding to the CDE/CHR element.Our findings provide strong evidence that the CDE/CHR-like sequence is an active functional element in the POLD1 promoter,which is important for the cell cycle regulation of the POLD1 gene.

  1. Identification and functional analysis of a CDE/CHR element in the POLDI promoter

    Institute of Scientific and Technical Information of China (English)

    SONG NanMeng; ZHU XiaoYu; SHI Lei; AN Jing; WU YanWei; SANG JianLi

    2009-01-01

    Chinese Center for Disease Control and Prevention, Beijing 102206, China DNA polymerase delta is encoded by the POLD1 gene, the transcription of which is strictly cell cy-cle-dependent. However, the means by which POLD1 transcription is regulated by the cell cycle mechanism is currently unknown. We discovered a novel element in the POLD1 promoter known as a CDE(cell cycle-dependent element)lCHR(cell cycle gene homology region) element. A series of luci-ferase reporter constructs containing various POLD1 promoter mutations were used to investigate the role of the CDF_JCHR element in POLD1 transcription. When the CDE/CHR element was mutated, the promoter activity was up-regulated, and the cell-cycle related factors E2F1 and p21 stopped regulating the promoter. Furthermore, cell cycle-dependent changes in the promoter activity required the integra-tive CDE/CHR element. Electrophoretic mobility shift assay (EMSA) revealed the presence of at least three types of DNA/protein complexes binding to the CDE/CHR element. Our findings provide strong evidence that the CDE/CHR-like sequence is an active functional element in the POLD1 promoter, which is important for the cell cycle regulation of the POLD1 gene.

  2. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  3. Work function of elemental metals and its face dependence ...

    African Journals Online (AJOL)

    Journal of the Nigerian Association of Mathematical Physics. Journal Home ... Open Access DOWNLOAD FULL TEXT Subscription or Fee Access. Work function of elemental metals and its face dependence: Stabilized Jellium approach.

  4. Tunnelling matrix elements with Gutzwiller wave functions

    Energy Technology Data Exchange (ETDEWEB)

    Di Ciolo, Andrea; Tocchio, Luca F.; Gros, Claudius [Institut fuer Theoretische Physik, Goethe Universitaet Frankfurt, Frankfurt Am Main (Germany)

    2011-07-01

    We use a generalized Gutzwiller approach, in order to study projected particle (hole) excitations for superconducting systems and systems with antiferromagnetic (AFM) order. As in the standard Gutzwiller scheme the effects of the strong electronic correlations are given via the suppression of the site double occupancy; for our computations it is helpful to consider a lattice with a reservoir site unaffected by this suppression of the double occupancy. In this approach we obtain the probabilities for the tunnelling of a particle (hole) into the projected state. Our results are due only to the physical properties of the trial state and not to the choice of a specifical Hamiltonian: in this sense, they are model-independent but not universal, because they rely on the features of the chosen Gutzwiller wave function (projected Fermi Sea, BCS superconductor, AFM..) The accuracy and the reliability of our analytical approximation is tested using the Variational Monte Carlo. Possible comparisons with tunnelling experiments are discussed.

  5. Functional validation of a constitutive autonomous silencer element.

    Directory of Open Access Journals (Sweden)

    Heyuan Qi

    Full Text Available Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.

  6. Artificial ants deposit pheromone to search for regulatory DNA elements

    Directory of Open Access Journals (Sweden)

    Liu Yunlong

    2006-08-01

    Full Text Available Abstract Background Identification of transcription-factor binding motifs (DNA sequences can be formulated as a combinatorial problem, where an efficient algorithm is indispensable to predict the role of multiple binding motifs. An ant algorithm is a biology-inspired computational technique, through which a combinatorial problem is solved by mimicking the behavior of social insects such as ants. We developed a unique version of ant algorithms to select a set of binding motifs by considering a potential contribution of each of all random DNA sequences of 4- to 7-bp in length. Results Human chondrogenesis was used as a model system. The results revealed that the ant algorithm was able to identify biologically known binding motifs in chondrogenesis such as AP-1, NFκB, and sox9. Some of the predicted motifs were identical to those previously derived with the genetic algorithm. Unlike the genetic algorithm, however, the ant algorithm was able to evaluate a contribution of individual binding motifs as a spectrum of distributed information and predict core consensus motifs from a wider DNA pool. Conclusion The ant algorithm offers an efficient, reproducible procedure to predict a role of individual transcription-factor binding motifs using a unique definition of artificial ants.

  7. Epigenetic regulation of transcription and possible functions of mammalian short interspersed elements, SINEs.

    Science.gov (United States)

    Ichiyanagi, Kenji

    2013-01-01

    Short interspersed elements (SINEs) are a class of retrotransposons, which amplify their copy numbers in their host genomes by retrotransposition. More than a million copies of SINEs are present in a mammalian genome, constituting over 10% of the total genomic sequence. In contrast to the other two classes of retrotransposons, long interspersed elements (LINEs) and long terminal repeat (LTR) elements, SINEs are transcribed by RNA polymerase III. However, like LINEs and LTR elements, the SINE transcription is likely regulated by epigenetic mechanisms such as DNA methylation, at least for human Alu and mouse B1. Whereas SINEs and other transposable elements have long been thought as selfish or junk DNA, recent studies have revealed that they play functional roles at their genomic locations, for example, as distal enhancers, chromatin boundaries and binding sites of many transcription factors. These activities imply that SINE retrotransposition has shaped the regulatory network and chromatin landscape of their hosts. Whereas it is thought that the epigenetic mechanisms were originated as a host defense system against proliferation of parasitic elements, this review discusses a possibility that the same mechanisms are also used to regulate the SINE-derived functions.

  8. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  9. Mining functional elements in messenger RNAs: overview, challenges, and perspectives

    Directory of Open Access Journals (Sweden)

    Firoz eAhmed

    2011-11-01

    Full Text Available Eukaryotic messenger RNA contains not only protein-coding regions but also a plethora of functional cis-elements that influence or coordinate a number of regulatory aspects of gene expression, such as mRNA stability, splicing forms, and translation rates. Understanding the rules that apply to each of these element types (e.g., whether the element is defined by primary or higher-order structure allows for the discovery of novel mechanisms of gene expression as well as the design of transcripts with controlled expression. Bioinformatics plays a major role in creating databases and finding non-evident patterns governing each type of eukaryotic functional element. Much of what we currently know about mRNA regulatory elements in eukaryotes is derived from microorganism and animal systems, with the particularities of plant systems lagging behind. In this review, we provide a general introduction to the most well-known eukaryotic mRNA regulatory motifs (splicing regulatory elements, internal ribosome entry sites, iron-responsive elements, AU-rich elements, zipcodes, and polyadenylation signals and describe available bioinformatics resources (databases and analysis tools to analyze eukaryotic transcripts in search of functional elements, focusing on recent trends in bioinformatics methods and tool development. We also discuss future directions in the development of better computational tools based upon current knowledge of these functional elements. Improved computational tools would advance our understanding of the processes underlying gene regulations. We encourage plant bioinformaticians to turn their attention to this subject to help identify novel mechanisms of gene expression regulation using RNA motifs that have potentially evolved or diverged in plant species.

  10. Identification of functional elements and regulatory circuits by Drosophila modENCODE.

    Science.gov (United States)

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L; Landolin, Jane M; Bristow, Christopher A; Ma, Lijia; Lin, Michael F; Washietl, Stefan; Arshinoff, Bradley I; Ay, Ferhat; Meyer, Patrick E; Robine, Nicolas; Washington, Nicole L; Di Stefano, Luisa; Berezikov, Eugene; Brown, Christopher D; Candeias, Rogerio; Carlson, Joseph W; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y; Will, Sebastian; Alekseyenko, Artyom A; Artieri, Carlo; Booth, Benjamin W; Brooks, Angela N; Dai, Qi; Davis, Carrie A; Duff, Michael O; Feng, Xin; Gorchakov, Andrey A; Gu, Tingting; Henikoff, Jorja G; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K; Riddle, Nicole C; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E; Schwartz, Yuri B; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E; Brent, Michael R; Cherbas, Lucy; Elgin, Sarah C R; Gingeras, Thomas R; Grossman, Robert; Hoskins, Roger A; Kaufman, Thomas C; Kent, William; Kuroda, Mitzi I; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J; Celniker, Susan E; Henikoff, Steven; Karpen, Gary H; Lai, Eric C; MacAlpine, David M; Stein, Lincoln D; White, Kevin P; Kellis, Manolis

    2010-12-24

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.

  11. Distinguishing Functional DNA Words; A Method for Measuring Clustering Levels

    Science.gov (United States)

    Moghaddasi, Hanieh; Khalifeh, Khosrow; Darooneh, Amir Hossein

    2017-01-01

    Functional DNA sub-sequences and genome elements are spatially clustered through the genome just as keywords in literary texts. Therefore, some of the methods for ranking words in texts can also be used to compare different DNA sub-sequences. In analogy with the literary texts, here we claim that the distribution of distances between the successive sub-sequences (words) is q-exponential which is the distribution function in non-extensive statistical mechanics. Thus the q-parameter can be used as a measure of words clustering levels. Here, we analyzed the distribution of distances between consecutive occurrences of 16 possible dinucleotides in human chromosomes to obtain their corresponding q-parameters. We found that CG as a biologically important two-letter word concerning its methylation, has the highest clustering level. This finding shows the predicting ability of the method in biology. We also proposed that chromosome 18 with the largest value of q-parameter for promoters of genes is more sensitive to dietary and lifestyle. We extended our study to compare the genome of some selected organisms and concluded that the clustering level of CGs increases in higher evolutionary organisms compared to lower ones. PMID:28128320

  12. Distinguishing Functional DNA Words; A Method for Measuring Clustering Levels

    Science.gov (United States)

    Moghaddasi, Hanieh; Khalifeh, Khosrow; Darooneh, Amir Hossein

    2017-01-01

    Functional DNA sub-sequences and genome elements are spatially clustered through the genome just as keywords in literary texts. Therefore, some of the methods for ranking words in texts can also be used to compare different DNA sub-sequences. In analogy with the literary texts, here we claim that the distribution of distances between the successive sub-sequences (words) is q-exponential which is the distribution function in non-extensive statistical mechanics. Thus the q-parameter can be used as a measure of words clustering levels. Here, we analyzed the distribution of distances between consecutive occurrences of 16 possible dinucleotides in human chromosomes to obtain their corresponding q-parameters. We found that CG as a biologically important two-letter word concerning its methylation, has the highest clustering level. This finding shows the predicting ability of the method in biology. We also proposed that chromosome 18 with the largest value of q-parameter for promoters of genes is more sensitive to dietary and lifestyle. We extended our study to compare the genome of some selected organisms and concluded that the clustering level of CGs increases in higher evolutionary organisms compared to lower ones.

  13. A Finite Circular Arch Element Based on Trigonometric Shape Functions

    Directory of Open Access Journals (Sweden)

    H. Saffari

    2007-01-01

    Full Text Available The curved-beam finite element formulation by trigonometric function for curvature is presented. Instead of displacement function, trigonometric function is introduced for curvature to avoid the shear and membrane locking phenomena. Element formulation is carried out in polar coordinates. The element with three nodal parameters is chosen on curvature. Then, curvature field in the element is interpolated as the conventional trigonometric functions. Shape functions are obtained as usual by matrix operations. To consider the boundary conditions, a transformation matrix between nodal curvature and nodal displacement vectors is introduced. The equilibrium equation is written by minimizing the total potential energy in terms of the displacement components. In such equilibrium equation, the locking phenomenon is eliminated. The interesting point in this method is that for most problems, it is sufficient to use only one element to obtain the solution. Four examples are presented in order to verify the element formulation and to show the accuracy and efficiency of the method. The results are compared with those of other concepts.

  14. Functions and applications of polypyridyl complexes in DNA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The syntheses and desymmetration of a series of novel polypyridyl ligands as well as their complexes, and their DNA-binding properties are reported in this review.The stresses are focused on the functions and potential applications of such complexes as DNA structural probe, DNA molecular light switches, anti-cancer drugs, and photoactivated cleavage agents.

  15. Plasma polymerized epoxide functional surfaces for DNA probe immobilization.

    Science.gov (United States)

    Chu, Li-Qiang; Knoll, Wolfgang; Förch, Renate

    2008-09-15

    The development of functional surfaces for the immobilization of DNA probe is crucial for a successful design of a DNA sensor. In this report, epoxide functional thin films were achieved simply by pulsed plasma polymerization (PP) of glycidyl methacrylate (GMA) at low duty cycle. The presence of epoxide groups in the resulting ppGMA films was confirmed by Fourier transform infrared spectroscopy. The ppGMA coatings were found to be resistant to the non-specific adsorption of DNA strands, while the epoxide groups obtained could react with amine-modified DNA probes in a mild basic environment without any activation steps. A DNA sensor was made, and was successfully employed to distinguish different DNA sequences with one base pair mismatch as seen by surface plasmon enhanced fluorescence spectroscopy (SPFS). The regeneration of the present DNA sensor was also discussed. This result suggests that surface modification with ppGMA films is very promising for the fabrication of various DNA sensors.

  16. Streching of (DNA/functional molecules) complex between electrodes towards DNA molecular wire

    Science.gov (United States)

    Kobayashi, Norihisa; Nishizawa, Makoto; Inoue, Shintarou; Nakamura, Kazuki

    2009-08-01

    DNA/functional molecules such as (Ru(bpy)32+ complex, conducting polymer etc.) complex was prepared to study molecular structure and I-V characteristics towards DNA molecular wire. For example, Ru(bpy)32+ was associated with duplex of DNA by not only electrostatic interaction but also intercalation in the aqueous solution. Singlemolecular structure of DNA/Ru(bpy)32+ complex was analyzed with AFM. We found a network structure of DNA/Ru(bpy)32+ complex on the mica substrate, which is similar to native DNA. The height of DNA/Ru(bpy)32+ complex on the mica substrate was ranging from 0.8 to 1.6 nm, which was higher than the naked DNA (0.5-1.0 nm). This indicates that single-molecular DNA/Ru(bpy)32+ complex also connects to each other to form network structure on a mica substrate. In order to stretch DNA complex between electrodes, we employed high frequency and high electric field stretching method proposed by Washizu et al. We stretched and immobilized DNA single molecules between a pair of electrodes and its structures were analyzed with AFM technique. The I-V characteristics of DNA single molecules between electrodes were improved by the association of functional molecules with DNA. The molecular structure and I-V characteristics of DNA complex were discussed.

  17. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    Science.gov (United States)

    Bu, Nan-Nan; Tang, Chun-Xia; He, Xi-Wen; Yin, Xue-Bo

    2011-07-21

    Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

  18. TFIIIC Bound DNA Elements in Nuclear Organization and Insulation

    OpenAIRE

    Kirkland, Jacob G.; Jesse R. Raab; Kamakaka, Rohinton T.

    2012-01-01

    tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subs...

  19. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Science.gov (United States)

    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

  20. Promiscuity of enhancer, coding and non-coding transcription functions in ultraconserved elements

    Directory of Open Access Journals (Sweden)

    Sanges Remo

    2010-03-01

    Full Text Available Abstract Background Ultraconserved elements (UCEs are highly constrained elements of mammalian genomes, whose functional role has not been completely elucidated yet. Previous studies have shown that some of them act as enhancers in mouse, while some others are expressed in both normal and cancer-derived human tissues. Only one UCE element so far was shown to present these two functions concomitantly, as had been observed in other isolated instances of single, non ultraconserved enhancer elements. Results We used a custom microarray to assess the levels of UCE transcription during mouse development and integrated these data with published microarray and next-generation sequencing datasets as well as with newly produced PCR validation experiments. We show that a large fraction of non-exonic UCEs is transcribed across all developmental stages examined from only one DNA strand. Although the nature of these transcripts remains a mistery, our meta-analysis of RNA-Seq datasets indicates that they are unlikely to be short RNAs and that some of them might encode nuclear transcripts. In the majority of cases this function overlaps with the already established enhancer function of these elements during mouse development. Utilizing several next-generation sequencing datasets, we were further able to show that the level of expression observed in non-exonic UCEs is significantly higher than in random regions of the genome and that this is also seen in other regions which act as enhancers. Conclusion Our data shows that the concurrent presence of enhancer and transcript function in non-exonic UCE elements is more widespread than previously shown. Moreover through our own experiments as well as the use of next-generation sequencing datasets, we were able to show that the RNAs encoded by non-exonic UCEs are likely to be long RNAs transcribed from only one DNA strand.

  1. Oxidative damages to DNA by indoor PM10s: Their relationships with trace element compositions

    Institute of Scientific and Technical Information of China (English)

    SHAO Longyi; ZHAO Houyin; T. P. JONES; LU Senlin; L. MEROLLA

    2005-01-01

    Plasmid DNA assay and ICP-MS analysis were conducted in order to investigate the bioreactivity of inhalable particles (PM10) and the relationship between bioreactivity and trace element compositions of PM10 in Beijing air. A total of four PM10 samples were carefully selected to represent the indoor and corresponding outdoor environments: one from urban smoker's home, two from nonsmoker's homes, and the other from the outdoor. In general, the oxidative damage by indoor PM10 was slightly higher than that of outdoor. Among the four sets of samples, the PM10 from the smoker's home hada lowest TD50 (toxic dose of PM10 causing 50% DNA to damage to plasmid DNA. The ICP-MS analysis combined with the DNA assay showed that the water-soluble zinc levels had better relationship with TD50 values than other elements, implying that water-soluble zinc might play an important role in the damage of DNA. It is concluded that the PM10 in smoker' s home had the highest level of water-soluble zinc as well as the lowest TD50 ( highest bioreactivity).Iron is considered to be one of the most bioreactive elements, but it will cause little damage to plasmid DNA, probably because iron is mainly in water-insoluble state in Beijing PM10.

  2. Evolutionarily conserved and conformationally constrained short peptides might serve as DNA recognition elements in intrinsically disordered regions.

    Science.gov (United States)

    Tayal, Nitish; Choudhary, Preeti; Pandit, Shashi B; Sandhu, Kuljeet Singh

    2014-06-01

    Despite recent advances, it is yet not clear how intrinsically disordered regions in proteins recognize their targets without any defined structures. Short linear motifs had been proposed to mediate molecular recognition by disordered regions; however, the underlying structural prerequisite remains elusive. Moreover, the role of short linear motifs in DNA recognition has not been studied. We report a repertoire of short evolutionarily Conserved Recognition Elements (CoREs) in long intrinsically disordered regions, which have very distinct amino-acid propensities from those of known motifs, and exhibit a strong tendency to retain their three-dimensional conformations compared to adjacent regions. The majority of CoREs directly interact with the DNA in the available 3D structures, which is further supported by literature evidence, analyses of ΔΔG values of DNA-binding energies and threading-based prediction of DNA binding potential. CoREs were enriched in cancer-associated missense mutations, further strengthening their functional nature. Significant enrichment of glycines in CoREs and the preference of glycyl ϕ-Ψ values within the left-handed bridge range in the l-disallowed region of the Ramachandran plot suggest that Gly-to-nonGly mutations within CoREs might alter the backbone conformation and consequently the function, a hypothesis that we reconciled using available mutation data. We conclude that CoREs might serve as bait for DNA recognition by long disordered regions and that certain mutations in these peptides can disrupt their DNA binding potential and consequently the protein function. We further hypothesize that the preferred conformations of CoREs and of glycyl residues therein might play an important role in DNA binding. The highly ordered nature of CoREs hints at a therapeutic strategy to inhibit malicious molecular interactions using small molecules mimicking CoRE conformations.

  3. Green's function and boundary elements of multifield materials

    CERN Document Server

    Qin, Qing-Hua

    2007-01-01

    Green's Function and Boundary Elements of Multifield Materials contains a comprehensive treatment of multifield materials under coupled thermal, magnetic, electric, and mechanical loads. Its easy-to-understand text clarifies some of the most advanced techniques for deriving Green's function and the related boundary element formulation of magnetoelectroelastic materials: Radon transform, potential function approach, Fourier transform. Our hope in preparing this book is to attract interested readers and researchers to a new field that continues to provide fascinating and technologically important challenges. You will benefit from the authors' thorough coverage of general principles for each topic, followed by detailed mathematical derivation and worked examples as well as tables and figures where appropriate. In-depth explanations of the concept of Green's function Coupled thermo-magneto-electro-elastic analysis Detailed mathematical derivation for Green's functions.

  4. Algebraic evaluation of matrix elements in the Laguerre function basis

    Science.gov (United States)

    McCoy, A. E.; Caprio, M. A.

    2016-02-01

    The Laguerre functions constitute one of the fundamental basis sets for calculations in atomic and molecular electron-structure theory, with applications in hadronic and nuclear theory as well. While similar in form to the Coulomb bound-state eigenfunctions (from the Schrödinger eigenproblem) or the Coulomb-Sturmian functions (from a related Sturm-Liouville problem), the Laguerre functions, unlike these former functions, constitute a complete, discrete, orthonormal set for square-integrable functions in three dimensions. We construct the SU(1, 1) × SO(3) dynamical algebra for the Laguerre functions and apply the ideas of factorization (or supersymmetric quantum mechanics) to derive shift operators for these functions. We use the resulting algebraic framework to derive analytic expressions for matrix elements of several basic radial operators (involving powers of the radial coordinate and radial derivative) in the Laguerre function basis. We illustrate how matrix elements for more general spherical tensor operators in three dimensional space, such as the gradient, may then be constructed from these radial matrix elements.

  5. Combing genomic DNA for structural and functional studies.

    Science.gov (United States)

    Schurra, Catherine; Bensimon, Aaron

    2009-01-01

    Molecular combing is a process whereby single DNA molecules bind by their extremities to a silanised surface and are then uniformly stretched and aligned by a receding air/water interface (1). This method, with a high resolution ranging from a few kilobases to megabases, has many applications in the field of molecular cytogenetics, allowing structural and functional analysis at the genome level. Here we describe protocols for preparing DNA for combing and for the use of fluorescent hybridisation (FH) applied to combed DNA to conduct physical mapping or genomic structural analysis. We also present the methodology for visualising and studying DNA replication using combed DNA.

  6. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  7. Functional aspects of baculovirus DNA photolyases

    NARCIS (Netherlands)

    Xu, F.

    2010-01-01

    Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects

  8. Functional aspects of baculovirus DNA photolyases

    NARCIS (Netherlands)

    Xu, F.

    2010-01-01

    Keywords: baculovirus, ChchNPV, CPD photolyase, phylogeny, UV resistance, DNA binding, localization, proteomics Baculoviruses are insect viruses that are applied as biological control agents due to adequate virulence, host specificity and safety for the environment. Solar light negatively affects

  9. Mechanisms and functions of DNA mismatch repair

    Institute of Scientific and Technical Information of China (English)

    Guo MinLi

    2008-01-01

    DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated dur-ing DNA replication and recombination. MMR also suppresses homeologous recombination and was recently shown to play a role in DNA damage signaling in eukaryotic cells. Escherichia coli MutS and MutL and their eukaryotic homo-logs, MutSα and MutLα, respectively, are key players in MMR-associated genome maintenance. Many other protein components that participate in various DNA metabolic pathways, such as PCNA and RPA, are also essential for MMR. Defects in MMR are associated with genome-wide instability, predisposition to certain types of cancer including he-reditary non-polyposis colorectal cancer, resistance to certain chemotherapeutic agents, and abnormalities in meiosis and sterility in mammalian systems.

  10. Telomerase RNA stem terminus element affects template boundary element function, telomere sequence, and shelterin binding.

    Science.gov (United States)

    Webb, Christopher J; Zakian, Virginia A

    2015-09-08

    The stem terminus element (STE), which was discovered 13 y ago in human telomerase RNA, is required for telomerase activity, yet its mode of action is unknown. We report that the Schizosaccharomyces pombe telomerase RNA, TER1 (telomerase RNA 1), also contains a STE, which is essential for telomere maintenance. Cells expressing a partial loss-of-function TER1 STE allele maintained short stable telomeres by a recombination-independent mechanism. Remarkably, the mutant telomere sequence was different from that of wild-type cells. Generation of the altered sequence is explained by reverse transcription into the template boundary element, demonstrating that the STE helps maintain template boundary element function. The altered telomeres bound less Pot1 (protection of telomeres 1) and Taz1 (telomere-associated in Schizosaccharomyces pombe 1) in vivo. Thus, the S. pombe STE, although distant from the template, ensures proper telomere sequence, which in turn promotes proper assembly of the shelterin complex.

  11. pENCODE: a plant encyclopedia of DNA elements.

    Science.gov (United States)

    Lane, Amanda K; Niederhuth, Chad E; Ji, Lexiang; Schmitz, Robert J

    2014-01-01

    ENCODE projects exist for many eukaryotes, including humans, but as of yet no defined project exists for plants. A plant ENCODE would be invaluable to the research community and could be more readily produced than its metazoan equivalents by capitalizing on the preexisting infrastructure provided from similar projects. Collecting and normalizing plant epigenomic data for a range of species will facilitate hypothesis generation, cross-species comparisons, annotation of genomes, and an understanding of epigenomic functions throughout plant evolution. Here, we discuss the need for such a project, outline the challenges it faces, and suggest ways forward to build a plant ENCODE.

  12. Radiation-induced changes in DNA methylation of repetitive elements in the mouse heart.

    Science.gov (United States)

    Koturbash, Igor; Miousse, Isabelle R; Sridharan, Vijayalakshmi; Nzabarushimana, Etienne; Skinner, Charles M; Melnyk, Stepan B; Pavliv, Oleksandra; Hauer-Jensen, Martin; Nelson, Gregory A; Boerma, Marjan

    2016-05-01

    DNA methylation is a key epigenetic mechanism, needed for proper control over the expression of genetic information and silencing of repetitive elements. Exposure to ionizing radiation, aside from its strong genotoxic potential, may also affect the methylation of DNA, within the repetitive elements, in particular. In this study, we exposed C57BL/6J male mice to low absorbed mean doses of two types of space radiation-proton (0.1 Gy, 150 MeV, dose rate 0.53 ± 0.08 Gy/min), and heavy iron ions ((56)Fe) (0.5 Gy, 600 MeV/n, dose rate 0.38 ± 0.06 Gy/min). Radiation-induced changes in cardiac DNA methylation associated with repetitive elements were detected. Specifically, modest hypomethylation of retrotransposon LINE-1 was observed at day 7 after irradiation with either protons or (56)Fe. This was followed by LINE-1, and other retrotransposons, ERV2 and SINE B1, as well as major satellite DNA hypermethylation at day 90 after irradiation with (56)Fe. These changes in DNA methylation were accompanied by alterations in the expression of DNA methylation machinery and affected the one-carbon metabolism pathway. Furthermore, loss of transposable elements expression was detected in the cardiac tissue at the 90-day time-point, paralleled by substantial accumulation of mRNA transcripts, associated with major satellites. Given that the one-carbon metabolism pathway can be modulated by dietary modifications, these findings suggest a potential strategy for the mitigation and, possibly, prevention of the negative effects exerted by ionizing radiation on the cardiovascular system. Additionally, we show that the methylation status and expression of repetitive elements may serve as early biomarkers of exposure to space radiation.

  13. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    Science.gov (United States)

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-04-07

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  14. A comparative encyclopedia of DNA elements in the mouse genome.

    Science.gov (United States)

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D; Shen, Yin; Pervouchine, Dmitri D; Djebali, Sarah; Thurman, Robert E; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K; Williams, Brian A; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M A; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D; Bansal, Mukul S; Kellis, Manolis; Keller, Cheryl A; Morrissey, Christapher S; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S; Cayting, Philip; Kawli, Trupti; Boyle, Alan P; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S; Cline, Melissa S; Erickson, Drew T; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A; Rosenbloom, Kate R; Lacerda de Sousa, Beatriz; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W James; Ramalho Santos, Miguel; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J; Wilken, Matthew S; Reh, Thomas A; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P; Neph, Shane; Humbert, Richard; Hansen, R Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E; Orkin, Stuart H; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J; Blobel, Gerd A; Cao, Xiaoyi; Zhong, Sheng; Wang, Ting; Good, Peter J; Lowdon, Rebecca F; Adams, Leslie B; Zhou, Xiao-Qiao; Pazin, Michael J; Feingold, Elise A; Wold, Barbara; Taylor, James; Mortazavi, Ali; Weissman, Sherman M; Stamatoyannopoulos, John A; Snyder, Michael P; Guigo, Roderic; Gingeras, Thomas R; Gilbert, David M; Hardison, Ross C; Beer, Michael A; Ren, Bing

    2014-11-20

    The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

  15. A Comparative Encyclopedia of DNA Elements in the Mouse Genome

    Science.gov (United States)

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D.; Shen, Yin; Pervouchine, Dmitri D.; Djebali, Sarah; Thurman, Bob; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K.; Williams, Brian A.; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M. A.; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T.; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D.; Bansal, Mukul S.; Keller, Cheryl A.; Morrissey, Christapher S.; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S.; Cayting, Philip; Kawli, Trupti; Boyle, Alan P.; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S.; Cline, Melissa S.; Erickson, Drew T.; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A.; Rosenbloom, Kate R.; de Sousa, Beatriz Lacerda; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W. James; Santos, Miguel Ramalho; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J.; Wilken, Matthew S.; Reh, Thomas A.; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P.; Neph, Shane; Humbert, Richard; Hansen, R. Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E.; Orkin, Stuart H.; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J.; Blobel, Gerd A.; Good, Peter J.; Lowdon, Rebecca F.; Adams, Leslie B.; Zhou, Xiao-Qiao; Pazin, Michael J.; Feingold, Elise A.; Wold, Barbara; Taylor, James; Kellis, Manolis; Mortazavi, Ali; Weissman, Sherman M.; Stamatoyannopoulos, John; Snyder, Michael P.; Guigo, Roderic; Gingeras, Thomas R.; Gilbert, David M.; Hardison, Ross C.; Beer, Michael A.; Ren, Bing

    2014-01-01

    Summary As the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases. PMID:25409824

  16. Finite element estimation of acoustical response functions in HID lamps

    Energy Technology Data Exchange (ETDEWEB)

    Baumann, Bernd; Wolff, Marcus [Department of Mechanical Engineering and Production, School of Engineering and Computer Science, Hamburg University of Applied Sciences, Berliner Tor 21, 20099 Hamburg (Germany); Hirsch, John; Antonis, Piet [Philips Lighting BV, Lightlabs, Mathildelaan 1, 5600 JM Eindhoven (Netherlands); Bhosle, Sounil [Universite de Toulouse (United States); Barrientos, Ricardo Valdivia, E-mail: bernd.baumann@haw-hamburg.d [National Nuclear Research Institute, Highway Mexico-Toluca s/n, La Marquesa, Ocoyoacac, CP 52750 (Mexico)

    2009-11-21

    High intensity discharge lamps can experience flickering and even destruction when operated at high frequency alternating current. The cause of these problems has been identified as acoustic resonances inside the lamp's arc tube. Here, a finite element approach for the calculation of the acoustic response function is described. The developed model does not include the plasma dynamics.

  17. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    Science.gov (United States)

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-01

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  18. Functional Nanofibers and Colloidal Gels: Key Elements to Enhance Functionality

    Science.gov (United States)

    Vogel, Nancy Amanda

    Nanomaterials bridge the gap between bulk materials and molecular structures and are known for their unique material properties and highly functional nature which make them attractive for a variety of potential applications, from energy storage and pollution sensors to agricultural and biomedical products. These potential applications, coupled with advances in nanotechnology, have generated considerable interest in nanostructure research. The work presented in this dissertation focuses on two such nanostructures, electrospun nanofibers and nanodiamond particles, with an overarching goal of tailoring the material behavior for a desired outcome. Our first research theme focuses on realizing the full potential of chitosan electrospinning by understanding the mechanism that enables fiber formation through cyclodextrin complexation as a function of solution properties, solvent types, and cyclodextrin content. We demonstrate that cyclodextrin addition not only enables chitosan fiber formation, but also extends the composition and solvent window for nanofiber synthesis while introducing a variety of mat topologies, including three-dimensional, self-supporting mats. These fiber formation improvements cannot be fully explained by conventional electrospinning parameters, but instead seem to be related to the molecular interactions between chitosan and cyclodextrin. Our second research theme entails the modification of highly water soluble, poly(vinyl alcohol) (PVA) nanofibers dissolution properties via atomic layer deposition (ALD) post treatments. In this work, we demonstrate that applying different thicknesses of aluminum oxide nano-coatings can improve the stability of PVA nanofibers in high humidity conditions and significantly decrease the solubility of electrospun PVA mats in water, from seconds to multiple weeks. Controlling mat dissolution allows for the unique opportunity to modulate small molecule, such as drug, release from nanofibers without altering the core

  19. NLO properties of functionalized DNA thin films

    Energy Technology Data Exchange (ETDEWEB)

    Krupka, Oksana [University d' Angers, Laboratoire POMA CNRS UMR 6136, France, 2 Bd. Lavoisier, 49045 (France)], E-mail: okrupka@mail.ru; El-ghayoury, Abdelkrim [University d' Angers, UFR Sciences, Laboratoire CIMMA UMR CNRS 6200, 2 Bd. Lavoisier, 49045 (France); Rau, Ileana; Sahraoui, Bouchta [University d' Angers, Laboratoire POMA CNRS UMR 6136, France, 2 Bd. Lavoisier, 49045 (France); Grote, James G. [Air Force Research Laboratory Materials and Manufacturing Directorate, Wright-Patterson Air Force Base, 3005 Hobson Way, Dayton, OH 45433-7707 (United States); Kajzar, Francois [University d' Angers, Laboratoire POMA CNRS UMR 6136, France, 2 Bd. Lavoisier, 49045 (France)

    2008-10-31

    In this paper we investigate the third-order nonlinear optical properties of spin deposited thin films of DNA-based complexes using the optical third harmonic generation (THG) technique at a fundamental wavelength of 1064 nm. We found that the third-order susceptibility, {chi}{sup (3)}(- 3{omega};{omega},{omega},{omega}), of DNA-based films was about one order of magnitude larger than that of our reference, a pure silica slab. In thin films doped with 5% of the chromophore disperse red 1 (DR1), a two order of magnitude larger value of {chi}{sup (3)}(- 3{omega};{omega},{omega},{omega}) was observed.

  20. High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

    OpenAIRE

    Ozdemir, Anil; Fisher-Aylor, Katherine I.; Pepke, Shirley; Samanta, Manoj; Dunipace, Leslie; McCue, Kenneth; Zeng, Lucy; Ogawa, Nobuo; Wold, Barbara J; Stathopoulos, Angelike

    2011-01-01

    Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identif...

  1. Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes.

    Science.gov (United States)

    Hernández-Hernández, Abrahan; Rincón-Arano, Héctor; Recillas-Targa, Félix; Ortiz, Rosario; Valdes-Quezada, Christian; Echeverría, Olga M; Benavente, Ricardo; Vázquez-Nin, Gerardo H

    2008-02-01

    The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC.

  2. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Science.gov (United States)

    Afek, Ariel; Cohen, Hila; Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B

    2015-08-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  3. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes.

    Directory of Open Access Journals (Sweden)

    Ariel Afek

    2015-08-01

    Full Text Available Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large

  4. Modeling of sensor function for piezoelectric bender elements

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    An analytical sandwich beam model for piezoelectric bender elements is derived based on the first-order shear deformation theory (FSDT), which assumes a single rotation angle for the whole cross-section and a quadratic distribution for coupled electric potential in piezoelectric layers. Shear coefficient is introduced to correct the effect of transverse shear strain on shear force and the electric displacement integration. Static and free vibration analyses of simply-supported bender elements are carried out for the sensor function. The results illustrate the high accuracy of the present model compared with the exact 2D solutions.

  5. Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells.

    Science.gov (United States)

    Yant, Stephen R; Kay, Mark A

    2003-12-01

    Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3' overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

  6. Radiation-induced changes in DNA methylation of repetitive elements in the mouse heart

    Energy Technology Data Exchange (ETDEWEB)

    Koturbash, Igor, E-mail: ikoturbash@uams.edu [Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Miousse, Isabelle R. [Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Sridharan, Vijayalakshmi [Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nzabarushimana, Etienne; Skinner, Charles M. [Department of Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Melnyk, Stepan B.; Pavliv, Oleksandra [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Hauer-Jensen, Martin [Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Surgical Service, Central Arkansas Veterans Healthcare System, Little Rock, AR 72205 (United States); Nelson, Gregory A. [Departments of Basic Sciences and Radiation Medicine, Loma Linda University, Loma Linda, CA 92354 (United States); Boerma, Marjan [Division of Radiation Health, Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2016-05-15

    Highlights: • Radiation-induced dynamic changes in cardiac DNA methylation were detected. • Early LINE-1 hypomethylation was followed by hypermethylation at a later time-point. • Radiation affected one-carbon metabolism in the heart tissue. • Irradiation resulted in accumulation of satellite DNA mRNA transcripts. - Abstract: DNA methylation is a key epigenetic mechanism, needed for proper control over the expression of genetic information and silencing of repetitive elements. Exposure to ionizing radiation, aside from its strong genotoxic potential, may also affect the methylation of DNA, within the repetitive elements, in particular. In this study, we exposed C57BL/6J male mice to low absorbed mean doses of two types of space radiation—proton (0.1 Gy, 150 MeV, dose rate 0.53 ± 0.08 Gy/min), and heavy iron ions ({sup 56}Fe) (0.5 Gy, 600 MeV/n, dose rate 0.38 ± 0.06 Gy/min). Radiation-induced changes in cardiac DNA methylation associated with repetitive elements were detected. Specifically, modest hypomethylation of retrotransposon LINE-1 was observed at day 7 after irradiation with either protons or {sup 56}Fe. This was followed by LINE-1, and other retrotransposons, ERV2 and SINE B1, as well as major satellite DNA hypermethylation at day 90 after irradiation with {sup 56}Fe. These changes in DNA methylation were accompanied by alterations in the expression of DNA methylation machinery and affected the one-carbon metabolism pathway. Furthermore, loss of transposable elements expression was detected in the cardiac tissue at the 90-day time-point, paralleled by substantial accumulation of mRNA transcripts, associated with major satellites. Given that the one-carbon metabolism pathway can be modulated by dietary modifications, these findings suggest a potential strategy for the mitigation and, possibly, prevention of the negative effects exerted by ionizing radiation on the cardiovascular system. Additionally, we show that the methylation status and

  7. Element orbitals for Kohn-Sham density functional theory

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Lin; Ying, Lexing

    2012-05-08

    We present a method to discretize the Kohn-Sham Hamiltonian matrix in the pseudopotential framework by a small set of basis functions automatically contracted from a uniform basis set such as planewaves. Each basis function is localized around an element, which is a small part of the global domain containing multiple atoms. We demonstrate that the resulting basis set achieves meV accuracy for 3D densely packed systems with a small number of basis functions per atom. The procedure is applicable to insulating and metallic systems.

  8. DNA elements reducing transcriptional gene silencing revealed by a novel screening strategy.

    Directory of Open Access Journals (Sweden)

    Naoki Kishimoto

    Full Text Available Transcriptional gene silencing (TGS--a phenomenon observed in endogenous genes/transgenes in eukaryotes--is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions-ASRs, based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume; the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements.

  9. Evaluation of effect of selected trace elements on dynamics of sperm DNA fragmentation.

    Science.gov (United States)

    Wdowiak, Artur; Bakalczuk, Grzegorz; Bakalczuk, Szymon

    2015-12-31

    Lead and cadmium can lead to negative effects on sperm chromatin DNA integrity. Copper, zinc and selenium are essential components of many enzymes which are important for reproduction. The aim of this research was to evaluate the influence of lead, cadmium, zinc, copper and selenium on the dynamics of semen DNA fragmentation. The present study concerned 85 fertile and 131 infertile men aged 25-35. DNA fragmentation in the samples was determined after 3 h, 6 h and 12 h. The Pb, Cd, Cu, Zn, and Se measurements were performed by the electrothermal-atomic absorption spectrometry method. We found that sperm DNA fragmentation was a dynamic process which was intensified with an increase in the level of lead in seminal plasma. The levels of lead and cadmium were higher in seminal plasma of infertile men, compared to fertile men. The levels of zinc, copper and selenium in seminal plasma were higher in men with proven fertility, compared to infertile men, and did not exert a significant effect on the dynamics of sperm DNA fragmentation. The level of cadmium had no significant effect on intensification of sperm DNA fragmentation in time. Reports in the literature which concern the effect of trace elements on human reproduction are equivocal. The present study confirmed an unfavourable effect, especially that of lead, on the dynamics of sperm DNA fragmentation; however, these studies need to be expanded and continued in the future.

  10. Basic functions of telecommunication channel elements for successful information transmission

    Directory of Open Access Journals (Sweden)

    Milorad S. Markagić

    2011-04-01

    the observed messages. Coder of messages generated by a message source should be transmitted to the recipient. For that purpose, an appropriate communication channel is used, with appropriate electrical signals as material bearers of the message. Definition of the code and the code system The set of combinations of digits that mirrors the elements of the set A is called a code. The established rule considers situations when each symbol from the set A is associated with the combination of elements of the set B. The function f defining this translation must be defined. This replacement is called a code replacement. Signal coder A coder performs signal transformation of coded messages to an electrical signal adapted for transmission via the transmission system. The most common signals are voltage transmission via cable connection or an electromagnetic field in the radio transmission. Modern systems for transferring discrete messages contain codecs and modems. Portable system A portable system is the medium for signal transmission from the source to the point of receipt. It can be wired and wireless. A wired transmission system is used in the stationary elements of communication systems. Wireless signal transmission is used in all conditions and it is more rational, efficient and economical. On their way through the transmission system, signals are subject to a variety of interferences. For a better insight into the interference impact, the source of interference is added to the whole system. Conclusion The model of the telecommunication channel is a complex system of a series of mutually dependent elements. Effectiveness of these elements is evaluated by the performances of the probability that the transfer of information through the channel will be successful. In a thus modeled telecommunication channel, regardless of the technical means used which is either a system or a circuit, the place and role of each element can be considered, which is the basis for consideration

  11. Self-assembly of DNA-functionalized colloids

    Directory of Open Access Journals (Sweden)

    P.E. Theodorakis

    2015-06-01

    Full Text Available Colloidal particles grafted with single-stranded DNA (ssDNA chains can self-assemble into a number of different crystalline structures, where hybridization of the ssDNA chains creates links between colloids stabilizing their structure. Depending on the geometry and the size of the particles, the grafting density of the ssDNA chains, and the length and choice of DNA sequences, a number of different crystalline structures can be fabricated. However, understanding how these factors contribute synergistically to the self-assembly process of DNA-functionalized nano- or micro-sized particles remains an intensive field of research. Moreover, the fabrication of long-range structures due to kinetic bottlenecks in the self-assembly are additional challenges. Here, we discuss the most recent advances from theory and experiment with particular focus put on recent simulation studies.

  12. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    黄承志; 李原芳; 黄新华; 范美坤

    2000-01-01

    The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  13. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The microarray of DNA probes with 5′-NH2 and 5′-Tex/3′-NH2 modified terminus on 10 m m carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is characterized in the present paper. It was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentration of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  14. Surface energy and work function of elemental metals

    DEFF Research Database (Denmark)

    Skriver, Hans Lomholt; Rosengaard, N. M.

    1992-01-01

    are in excellent agreement with a recent full-potential, all-electron, slab-supercell calculation of surface energies and work functions for the 4d metals. The present calculations explain the trend exhibited by the surface energies of the alkali, alkaline earth, divalent rare-earth, 3d, 4d, and 5d transition......We have performed an ab initio study of the surface energy and the work function for six close-packed surfaces of 40 elemental metals by means of a Green’s-function technique, based on the linear-muffin-tin-orbitals method within the tight-binding and atomic-sphere approximations. The results...... and noble metals, as derived from the surface tension of liquid metals. In addition, they give work functions which agree with the limited experimental data obtained from single crystals to within 15%, and explain the smooth behavior of the experimental work functions of polycrystalline samples...

  15. Functional self-assembled DNA nanostructures for molecular recognition

    Science.gov (United States)

    Zhang, Xiaojuan; Yadavalli, Vamsi K.

    2012-03-01

    Nucleic acids present a wonderful toolkit of structural motifs for nanoconstruction. Functional DNA nanostructures can enable protein recognition by the use of aptamers attached to a basic core shape formed by DNA self-assembly. Here, we present a facile, programmable strategy for the assembly of discrete aptamer-tagged DNA shapes and nanostructures that can function for molecular recognition and binding in an aqueous environment. These nanostructures, presented here to bind two different protein targets, are easily synthesized in large numbers, and are portable and stable over long periods of time. This construction modality can facilitate on-demand production of libraries of diverse shapes to recognize and bind proteins or catalyze reactions via functional nucleic acid tags.Nucleic acids present a wonderful toolkit of structural motifs for nanoconstruction. Functional DNA nanostructures can enable protein recognition by the use of aptamers attached to a basic core shape formed by DNA self-assembly. Here, we present a facile, programmable strategy for the assembly of discrete aptamer-tagged DNA shapes and nanostructures that can function for molecular recognition and binding in an aqueous environment. These nanostructures, presented here to bind two different protein targets, are easily synthesized in large numbers, and are portable and stable over long periods of time. This construction modality can facilitate on-demand production of libraries of diverse shapes to recognize and bind proteins or catalyze reactions via functional nucleic acid tags. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11711h

  16. Functional changes appropriate for determining mineral element requirements.

    Science.gov (United States)

    Lukaski, H C; Penland, J G

    1996-09-01

    One factor limiting efforts to determine human requirements for dietary intakes of mineral elements has been the unavailability of acceptable standards for evaluating the effects of marginal and mild deficiencies. Traditional approaches, such as growth, longevity, chemical balance and measurement of concentrations of minerals in plasma or serum and cellular components of the blood, have not been sensitive indicators of mineral nutriture. One alternative that has been shown to be responsive to graded dietary mineral intake is the evaluation of functional responses to specific challenges or stressors. Aberrant responses, either exaggerated or attenuated, to controlled stressors have been observed in a variety of physiological, psychological and immunological parameters when mineral intakes have been suboptimal by conventional standards, compared with adequate responses. In comparison to static biochemical approaches for assessment of mineral nutritional status, functional tests may be sensitive and responsive to alterations in mineral intake in adult humans. Dynamic functional measures complement static biochemical measures and reflect the net effect of deficiencies on integrated biological systems. The application of some of these types of dynamic evaluations of function may be a useful and productive approach for proposing mineral element intakes to optimize human health and biological function and performance.

  17. DNA-protein interaction at erythroid important regulatory elements of MEL cells by in vivo footprinting

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and β maj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and β maj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β -globin gene cluster participate in the regulation of developmental specificity.

  18. Origins, structures, and functions of circulating DNA in oncology.

    Science.gov (United States)

    Thierry, A R; El Messaoudi, S; Gahan, P B; Anker, P; Stroun, M

    2016-09-01

    While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many "kinds." This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.

  19. An amphipathic trans-acting phosphorothioate DNA element delivers uncharged PNA and PMO nucleic acid sequences in mammalian cells.

    Science.gov (United States)

    Jain, Harsh V; Beaucage, Serge L

    An innovative approach to the delivery of uncharged peptide nucleic acids (PNA) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA-polyA or PMO-polyA tail. Recognition of the polyA-tailed PNA or PMO oligomers by an amphipathic trans-acting polythymidylic thiophosphate triester element (dTtaPS) results in efficient internalization of those oligomers in several cell lines. Our findings indicate that cellular uptake of the oligomers occurs through an energy-dependent mechanism and macropinocytosis appears to be the predo-minant endocytic pathway used for internalization. The functionality of the internalized oligomers is demonstrated by alternate splicing of the pre-mRNA encoding luciferase in HeLa pLuc 705 cells. Amphipathic phosphorothioate DNA elements may represent a unique class of cellular transporters for robust delivery of uncharged nucleic acid sequences in live mammalian cells.

  20. Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Huang Jian-dong

    2011-04-01

    Full Text Available Abstract Background SXT is an integrating conjugative element (ICE originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo and single strand annealing protein (S065, SXT-Bet encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb. When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V

  1. Aptamer Selection Express: A Rapid Single-Step Selection of Double-stranded DNA Capture Elements

    Science.gov (United States)

    2009-08-21

    always the last word Approved for public release; distribution unlimited Sensitivity of Aptamers for Detecting Bacillus thuringiensis Spores and...for public release; distribution unlimited ALISA approach: Quantum Dot DCE Assay for Shiga Toxin Compared to FITC Antibody Assay 0.000 0.240 0.480...w ith Shiga Toxin STJ-9 w ith BSA Antibody w ith Shiga Toxin Antibody w ith BSA (Kiel et al, SPIE 5617: 382-387, 2004) DCE=DNA Capture Element

  2. The contribution of alu elements to mutagenic DNA double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Maria E Morales

    2015-03-01

    Full Text Available Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%, we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences. Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence

  3. Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

    Science.gov (United States)

    Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

    2004-12-01

    Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

  4. Functional interactions of DNA topoisomerases with a human replication origin.

    Science.gov (United States)

    Abdurashidova, Gulnara; Radulescu, Sorina; Sandoval, Oscar; Zahariev, Sotir; Danailov, Miltcho B; Demidovich, Alexander; Santamaria, Laura; Biamonti, Giuseppe; Riva, Silvano; Falaschi, Arturo

    2007-02-21

    The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

  5. Structure and Function Study of Phi29 DNA packaging motor

    Science.gov (United States)

    Fang, Huaming

    molecules were required to bind to one short dsDNA molecule. The inhibitive curve of Walker B mutant gp16 analyzed by binomial distribution model showed that one inactive mutant gp16 in the gp16 ring could block the function of the motor and the stoichiometry of gp16 was six. These findings facilitate our understanding of the molecular mechanism of viral DNA packaging: a novel viral DNA packaging model "push through a one-way valve" was proposed. In this model, the connector functioned as a valve to allow DNA to enter but prevented it from sliding out during DNA packaging; the six subunits in the gp16 ring acted sequentially to push DNA into the connector channel. ATP binding of gp16 induced a conformation change with a high affinity for dsDNA. Then, the ATP was hydrolyzed which resulted in the movement of subdomains in this individual gp16 subunit and DNA was pushed forward, followed by the double helix of dsDNA being brought forward to the adjacent subunit in the gp16 ring. The elucidation of the viral DNA packaging mechanism holds great potential for developing artificial motors for delivering drugs and other molecular cargos.

  6. Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements

    Directory of Open Access Journals (Sweden)

    Pareja Eduardo

    2006-03-01

    Full Text Available Abstract Background Mobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements. Results This global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates. Conclusion Our findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for

  7. LINE-1 retrotransposable element DNA accumulates in HIV-1-infected cells.

    Science.gov (United States)

    Jones, R Brad; Song, Haihan; Xu, Yang; Garrison, Keith E; Buzdin, Anton A; Anwar, Naveed; Hunter, Diana V; Mujib, Shariq; Mihajlovic, Vesna; Martin, Eric; Lee, Erika; Kuciak, Monika; Raposo, Rui André Saraiva; Bozorgzad, Ardalan; Meiklejohn, Duncan A; Ndhlovu, Lishomwa C; Nixon, Douglas F; Ostrowski, Mario A

    2013-12-01

    Type 1 long-interspersed nuclear elements (L1s) are autonomous retrotransposable elements that retain the potential for activity in the human genome but are suppressed by host factors. Retrotransposition of L1s into chromosomal DNA can lead to genomic instability, whereas reverse transcription of L1 in the cytosol has the potential to activate innate immune sensors. We hypothesized that HIV-1 infection would compromise cellular control of L1 elements, resulting in the induction of retrotransposition events. Here, we show that HIV-1 infection enhances L1 retrotransposition in Jurkat cells in a Vif- and Vpr-dependent manner. In primary CD4(+) cells, HIV-1 infection results in the accumulation of L1 DNA, at least the majority of which is extrachromosomal. These data expose an unrecognized interaction between HIV-1 and endogenous retrotransposable elements, which may have implications for the innate immune response to HIV-1 infection, as well as for HIV-1-induced genomic instability and cytopathicity.

  8. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Energy Technology Data Exchange (ETDEWEB)

    Arneodo, Alain, E-mail: alain.arneodo@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Vaillant, Cedric, E-mail: cedric.vaillant@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Audit, Benjamin, E-mail: benjamin.audit@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); Argoul, Francoise, E-mail: francoise.argoul@ens-lyon.f [Universite de Lyon, F-69000 Lyon (France); Laboratoire Joliot-Curie and Laboratoire de Physique, CNRS, Ecole Normale Superieure de Lyon, F-69007 Lyon (France); D' Aubenton-Carafa, Yves, E-mail: daubenton@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France); Thermes, Claude, E-mail: claude.thermes@cgm.cnrs-gif.f [Centre de Genetique Moleculaire, CNRS, Allee de la Terrasse, 91198 Gif-sur-Yvette (France)

    2011-02-15

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  9. Structure and function of the DNA ligases encoded by the mammalian LIG3 gene

    OpenAIRE

    Tomkinson, Alan E.; Sallmyr, Annahita

    2013-01-01

    Among the mammalian genes encoding DNA ligases (LIG), the LIG3 gene is unique in that it encodes multiple DNA ligase polypeptides with different cellular functions. Notably, this nuclear gene encodes the only mitochondrial DNA ligase and so is essential for this organelle. In the nucleus, there is significant functional redundancy between DNA ligase IIIα and DNA ligase I in excision repair. In addition, DNA ligase IIIα is essential for DNA replication in the absence of the replicative DNA lig...

  10. Specificity and function of Archaeal DNA replication initiator proteins

    DEFF Research Database (Denmark)

    Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina

    2013-01-01

    to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels...... the protein's structure rather than that of the DNA template....

  11. Beyond the dna: a prototype for functional genomics

    Energy Technology Data Exchange (ETDEWEB)

    Albala, J

    2000-03-02

    A prototype oligonucleotide ''functional chip'' has been developed to screen novel DNA repair proteins for their ability to bind or alter different forms of DNA. This chip has been developed as a functional genomics screen for analysis of protein-DNA interactions for novel proteins identified from the Human Genome Project The process of novel gene identification that has ensued as a consequence of available sequence information is remarkable. The challenge how lies in determining the function of newly identified gene products in a time-and cost-effective high-throughput manner. The functional chip is generated by the robotic application of DNA spotted in a microarray format onto a glass slide. Individual proteins are then analyzed against the different form of DNA bound to the slide. Several prototype functional chips were designed to contain various DNA fragments tethered to a glass slide for analysis of protein-DNA binding or enzymatic activity of known proteins. The technology has been developed to screen novel, putative DNA repair proteins for their ability to bind various types of DNA alone and in concert with protein partners. An additional scheme has been devised to screen putative repair enzymes for their ability to process different types of DNA molecules. Current methods to analyze gene expression primarily utilize either of two technologies. The oligonucleotide chip, pioneered by Fodor and co-workers and Affymetrix, Inc., consists of greater than 64,000 oligonucleotides attached in situ to a glass support. The oligonucleotide chip has been used primarily to identify specific mutations in a given gene by hybridization against a fluorescently-labeled substrate. The second method is the microarray, whereby DNA targets are systematically arranged on a glass slide and then hybridized with fluorescently-labeled complex targets for gene expression analysis (Jordan, 1998). By this technique, a large amount of information can be obtained

  12. Density Functional Modelling of Elastic Properties of Elemental Semiconductors

    Directory of Open Access Journals (Sweden)

    M. Verma

    2011-01-01

    Full Text Available The expressions for bulk modulus, its first and second pressure derivatives for elemental semiconductors are derived using the ab initio pseudopotential approach to the total crystal energy within the framework of local Density Functional formalism. The expression for the second pressure derivative of the bulk modulus for four-fold crystal structures are derived for the first time within the pseudopotential framework. The computed results for the semiconductors under study are very close to the available experimental data and will be useful in the study of equation of states.

  13. Elements of a function analytic approach to probability.

    Energy Technology Data Exchange (ETDEWEB)

    Ghanem, Roger Georges (University of Southern California, Los Angeles, CA); Red-Horse, John Robert

    2008-02-01

    We first provide a detailed motivation for using probability theory as a mathematical context in which to analyze engineering and scientific systems that possess uncertainties. We then present introductory notes on the function analytic approach to probabilistic analysis, emphasizing the connections to various classical deterministic mathematical analysis elements. Lastly, we describe how to use the approach as a means to augment deterministic analysis methods in a particular Hilbert space context, and thus enable a rigorous framework for commingling deterministic and probabilistic analysis tools in an application setting.

  14. Indirect readout of DNA sequence by p22 repressor: roles of DNA and protein functional groups in modulating DNA conformation.

    Science.gov (United States)

    Harris, Lydia-Ann; Watkins, Derrick; Williams, Loren Dean; Koudelka, Gerald B

    2013-01-09

    The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The "indirect readout" of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R-DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B' state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B' state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B'-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R-DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B' state and therefore play a key role in indirect readout by P22R.

  15. An 11bp region with stem formation potential is essential for de novo DNA methylation of the RPS element.

    Directory of Open Access Journals (Sweden)

    Matthew Gentry

    Full Text Available The initiation of DNA methylation in Arabidopsis is controlled by the RNA-directed DNA methylation (RdDM pathway that uses 24nt siRNAs to recruit de novo methyltransferase DRM2 to the target site. We previously described the REPETITIVE PETUNIA SEQUENCE (RPS fragment that acts as a hot spot for de novo methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate de novo methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced de novo methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that de novo methylation was not targeted to a specific DNA sequence element. These data suggest that de novo DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

  16. Functionalization of DNA Nanostructures for Cell Signaling Applications

    Science.gov (United States)

    Pedersen, Ronnie O.

    Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by

  17. Alu element-containing RNAs maintain nucleolar structure and function.

    Science.gov (United States)

    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

  18. DNA Modifications: Function and Applications in Normal and Disease States

    Directory of Open Access Journals (Sweden)

    Vichithra R. B. Liyanage

    2014-10-01

    Full Text Available Epigenetics refers to a variety of processes that have heritable effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are chemical modifications to DNA, histone, and non-histone chromosomal proteins, which establish a complex regulatory network that controls genome function. Methylation of DNA at the fifth position of cytosine in CpG dinucleotides (5-methylcytosine, 5mC, which is carried out by DNA methyltransferases, is commonly associated with gene silencing. However, high resolution mapping of DNA methylation has revealed that 5mC is enriched in exonic nucleosomes and at intron-exon junctions, suggesting a role of DNA methylation in the relationship between elongation and RNA splicing. Recent studies have increased our knowledge of another modification of DNA, 5-hydroxymethylcytosine (5hmC, which is a product of the ten-eleven translocation (TET proteins converting 5mC to 5hmC. In this review, we will highlight current studies on the role of 5mC and 5hmC in regulating gene expression (using some aspects of brain development as examples. Further the roles of these modifications in detection of pathological states (type 2 diabetes, Rett syndrome, fetal alcohol spectrum disorders and teratogen exposure will be discussed.

  19. From adjacent activation in E. coli and DNA cyclization to eukaryotic enhancers : the elements of a puzzle.

    Directory of Open Access Journals (Sweden)

    Michèle eAMOUYAL

    2014-11-01

    Full Text Available DNA cyclization, E. coli lac repressor binding to two spaced lac operators and repression enhancement can be successfully used for a better understanding of the conditions required for interaction between eukaryotic enhancers and the machinery of transcription initiation. Chronologically, the DNA looping model has first accounted for the properties initially defining enhancers, i.e. independence of action with distance or orientation with respect to the start of transcription. It has also predicted enhancer activity or its disruption at short distance (site orientation, alignment between promoter and enhancer sites, with high-order complexes of protein, or with transcription factor concentrations close or different from the w.t. situation. In another step, histones have been introduced into the model to further adapt it to eukaryotes. They in fact favor DNA cyclization in vitro. The resulting DNA compaction might explain the difference counted in base pairs in the distance of action between eukaryotic transcription enhancers and prokaryotic repression enhancers. The lac looping system provides a potential tool for analysis of this discrepancy and of chromatin state directly in situ. Furthermore, as predicted by the model, the contribution of operators O2 and O3 to repression of the lac operon clearly depends on the lac repressor level in the cell and is prevented in strains overproducing lac repressor. By extension, gene regulation especially that linked to cell fate, should also depend on transcription factor levels, providing a potential tool for cellular therapy. In parallel, a new function of the O1-O3 loop completes the picture of lac repression. The O1-O3 loop would at the same time ensure high efficiency of repression, inducibility through the low-affinity sites and limitation of the level of repressor through self-repression of the lac repressor. Last, the DNA looping model can be successfully adapted to the enhancer auxiliary elements

  20. In vitro selection of a single-stranded DNA molecular recognition element against atrazine.

    Science.gov (United States)

    Williams, Ryan M; Crihfield, Cassandra L; Gattu, Srikanth; Holland, Lisa A; Sooter, Letha J

    2014-08-18

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

  1. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    Directory of Open Access Journals (Sweden)

    Ryan M. Williams

    2014-08-01

    Full Text Available Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA molecular recognition elements (MRE were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples.

  2. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    Science.gov (United States)

    Williams, Ryan M.; Crihfield, Cassandra L.; Gattu, Srikanth; Holland, Lisa A.; Sooter, Letha J.

    2014-01-01

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples. PMID:25196435

  3. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    Science.gov (United States)

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  4. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  5. Concentration-dependent thermophoretic accumulation for the detection of DNA using DNA-functionalized nanoparticles.

    Science.gov (United States)

    Yu, Li-Hsien; Chen, Yih-Fan

    2015-03-03

    Thermophoresis is a phenomenon about the migration of particles along a temperature gradient and is sensitive to the properties of particles and the surrounding medium. While a few studies have investigated its mechanisms and effects on particle motion in recent years, the applications of thermophoresis in biosensing has not been well explored. In this study, we demonstrate a thermophoresis-based method for detecting DNA. We use DNA-functionalized gold nanoparticles and fluorescent DNA probes to capture target DNA in free solution, and we demonstrate that the hybridization between the specially designed capture probes and the target DNA significantly changes the thermophoretic properties of the fluorescent probes. As a result, the target DNA can be specifically detected in serum-containing buffers based on the spatial distribution of the fluorescent probes in a laser-induced temperature gradient. The optical setup consists of only a laser and an epifluorescence microscope, and the detection does not rely on any micro- or nanofabricated devices. In addition, because the detection is based on the thermophoretic motion of molecules in free solution, no capture probes need to be immobilized on a fixed surface before detection, and no channels or pumps are needed for washing away unbound molecules. The thermophoresis-based biosensing method is found to be simple and effective for detecting DNA.

  6. Deep investigation of Arabidopsis thaliana junk DNA reveals a continuum between repetitive elements and genomic dark matter.

    Science.gov (United States)

    Maumus, Florian; Quesneville, Hadi

    2014-01-01

    Eukaryotic genomes contain highly variable amounts of DNA with no apparent function. This so-called junk DNA is composed of two components: repeated and repeat-derived sequences (together referred to as the repeatome), and non-annotated sequences also known as genomic dark matter. Because of their high duplication rates as compared to other genomic features, transposable elements are predominant contributors to the repeatome and the products of their decay is thought to be a major source of genomic dark matter. Determining the origin and composition of junk DNA is thus important to help understanding genome evolution as well as host biology. In this study, we have used a combination of tools enabling to show that the repeatome from the small and reducing A. thaliana genome is significantly larger than previously thought. Furthermore, we present the concepts and results from a series of innovative approaches suggesting that a significant amount of the A. thaliana dark matter is of repetitive origin. As a tentative standard for the community, we propose a deep compendium annotation of the A. thaliana repeatome that may help addressing farther genome evolution as well as transcriptional and epigenetic regulation in this model plant.

  7. Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

    Science.gov (United States)

    Sone, Takefumi; Yahata, Kazuhide; Sasaki, Yukari; Hotta, Junko; Kishine, Hiroe; Chesnut, Jonathan D; Imamoto, Fumio

    2008-09-10

    Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

  8. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    Science.gov (United States)

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.

  9. Functional DNA: Teaching Infinite Series through Genetic Analogy

    Science.gov (United States)

    Kowalski, R. Travis

    2011-01-01

    This article presents an extended analogy that connects infinite sequences and series to the science of genetics, by identifying power series as "DNA for a function." This analogy allows standard topics such as convergence tests or Taylor approximations to be recast in a "forensic" light as mathematical analogs of genetic concepts such as DNA…

  10. Establishment and functions of DNA methylation in the germline

    DEFF Research Database (Denmark)

    Stewart-Morgan, Kathleen; Veselovska, Lenka; Kelsey, Gavin

    2016-01-01

    Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm...... the fate and function of gametic methylation and other epigenetic modifications after fertilization....

  11. Identification of functional elements and regulatory circuits by Drosophila modENCODE

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L.; Landolin, Jane M.; Bristow, Christopher A.; Ma, Lijia; Lin, Michael F.; Washietl, Stefan; Arshinoff, Bradley I.; Ay, Ferhat; Meyer, Patrick E.; Robine, Nicolas; Washington, Nicole L.; Stefano, Luisa Di; Berezikov, Eugene; Brown, Christopher D.; Candeias, Rogerio; Carlson, Joseph W.; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y.; Will, Sebastian; Alekseyenko, Artyom A.; Artieri, Carlo; Booth, Benjamin W.; Brooks, Angela N.; Dai, Qi; Davis, Carrie A.; Duff, Michael O.; Feng, Xin; Gorchakov, Andrey A.; Gu, Tingting; Henikoff, Jorja G.; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K.; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K.; Riddle, Nicole C.; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E.; Schwartz, Yuri B.; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H.; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E.; Brent, Michael R.; Cherbas, Lucy; Elgin, Sarah C. R.; Gingeras, Thomas R.; Grossman, Robert; Hoskins, Roger A.; Kaufman, Thomas C.; Kent, William; Kuroda, Mitzi I.; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W.; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R.; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J.; Celniker, Susan E.; Henikoff, Steven; Karpen, Gary H.; Lai, Eric C.; MacAlpine, David M.; Stein, Lincoln D.; White, Kevin P.; Kellis, Manolis

    2010-12-22

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions

  12. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

    Science.gov (United States)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  13. Population genetics and molecular evolution of DNA sequences in transposable elements. I. A simulation framework.

    Science.gov (United States)

    Kijima, T E; Innan, Hideki

    2013-11-01

    A population genetic simulation framework is developed to understand the behavior and molecular evolution of DNA sequences of transposable elements. Our model incorporates random transposition and excision of transposable element (TE) copies, two modes of selection against TEs, and degeneration of transpositional activity by point mutations. We first investigated the relationships between the behavior of the copy number of TEs and these parameters. Our results show that when selection is weak, the genome can maintain a relatively large number of TEs, but most of them are less active. In contrast, with strong selection, the genome can maintain only a limited number of TEs but the proportion of active copies is large. In such a case, there could be substantial fluctuations of the copy number over generations. We also explored how DNA sequences of TEs evolve through the simulations. In general, active copies form clusters around the original sequence, while less active copies have long branches specific to themselves, exhibiting a star-shaped phylogeny. It is demonstrated that the phylogeny of TE sequences could be informative to understand the dynamics of TE evolution.

  14. Chemical elemental distribution and soil DNA fingerprints provide the critical evidence in murder case investigation.

    Science.gov (United States)

    Concheri, Giuseppe; Bertoldi, Daniela; Polone, Elisa; Otto, Stefan; Larcher, Roberto; Squartini, Andrea

    2011-01-01

    The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

  15. Chemical elemental distribution and soil DNA fingerprints provide the critical evidence in murder case investigation.

    Directory of Open Access Journals (Sweden)

    Giuseppe Concheri

    Full Text Available BACKGROUND: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. METHODOLOGY/PRINCIPAL FINDINGS: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS and optical emission spectrometry (ICP-OES approaches, and b amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA. CONCLUSIONS: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

  16. Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome.

    Science.gov (United States)

    Iyama, Teruaki; Wilson, David M

    2016-01-16

    Cockayne syndrome (CS) is a premature aging disorder characterized by developmental defects, multisystem progressive degeneration and sensitivity to ultraviolet light. CS is divided into two primary complementation groups, A and B, with the CSA and CSB proteins presumably functioning in DNA repair and transcription. Using laser microirradiation and confocal microscopy, we characterized the nature and regulation of the CS protein response to oxidative DNA damage, double-strand breaks (DSBs), angelicin monoadducts and trioxsalen interstrand crosslinks (ICLs). Our data indicate that CSB recruitment is influenced by the type of DNA damage and is most rapid and robust as follows: ICLs>DSBs>monoadducts>oxidative lesions. Transcription inhibition reduced accumulation of CSB at sites of monoadducts and ICLs, but it did not affect recruitment to (although slightly affected retention at) oxidative damage. Inhibition of histone deacetylation altered the dynamics of CSB assembly, suggesting a role for chromatin status in the response to DNA damage, whereas the proteasome inhibitor MG132 had no effect. The C-terminus of CSB and, in particular, its ubiquitin-binding domain were critical to recruitment, while the N-terminus and a functional ATPase domain played a minor role at best in facilitating protein accumulation. Although the absence of CSA had no effect on CSB recruitment, CSA itself localized at sites of ICLs, DSBs and monoadducts but not at oxidative lesions. Our results reveal molecular components of the CS protein response and point to a major involvement of complex lesions in the pathology of CS.

  17. Molecular Design of Ionization-Induced Proton Switching Element Based on Fluorinated DNA Base Pair.

    Science.gov (United States)

    Tachikawa, Hiroto; Kawabata, Hiroshi

    2016-03-10

    To design theoretically the high-performance proton switching element based on DNA base pair, the effects of fluorine substitution on the rate of proton transfer (PT) in the DNA model base pair have been investigated by means of direct ab initio molecular dynamics (AIMD) method. The 2-aminopyridine dimer, (AP)2, was used as the model of the DNA base pair. One of the hydrogen atoms of the AP molecule in the dimer was substituted by a fluorine (F) atom, and the structures of the dimer, expressed by F-(AP)2, were fully optimized at the MP2/6-311++G(d,p) level. The direct AIMD calculations showed that the proton is transferred within the base pair after the vertical ionization. The rates of PT in F-(AP)2(+) were calculated and compared with that of (AP)2(+) without an F atom. It was found that PT rate is accelerated by the F-substitution. Also, the direction of PT between F-AP and AP molecules can be clearly controlled by the position of F-substitution (AP)2 in the dimer.

  18. Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

    Science.gov (United States)

    Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

    2000-12-01

    The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

  19. A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

    Directory of Open Access Journals (Sweden)

    Tar Viturawong

    2013-10-01

    Full Text Available Ultraconserved elements (UCEs have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

  20. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  1. Functional evolution of the p53 regulatory network through its target response elements

    Science.gov (United States)

    Jegga, Anil G.; Inga, Alberto; Menendez, Daniel; Aronow, Bruce J.; Resnick, Michael A.

    2008-01-01

    Transcriptional network evolution is central to the development of complex biological systems. Networks can evolve through variation of master regulators and/or by changes in regulation of genes within networks. To gain insight into meaningful evolutionary differences in large networks, it is essential to address the functional consequences of sequence differences in response elements (REs) targeted by transcription factors. Using a combination of custom bioinformatics and multispecies alignment of promoter regions, we investigated the functional evolution of REs in terms of responsiveness to the sequence-specific transcription factor p53, a tumor suppressor and master regulator of stress responses. We identified REs orthologous to known p53 targets in human and rodent cells or alternatively REs related to the established p53 consensus. The orthologous REs were assigned p53 transactivation capabilities based on rules determined from model systems, and a functional heat map was developed to visually summarize conservation of sequence and relative level of responsiveness to p53 for 47 REs in 14 species. Individual REs exhibited marked differences in transactivation potentials and widespread evolutionary turnover. Functional differences were often not predicted from consensus sequence evaluations. Of the established human p53 REs analyzed, 91% had sequence conservation in at least one nonprimate species compared with 67.5% for functional conservation. Surprisingly, there was almost no conservation of functional REs for genes involved in DNA metabolism or repair between humans and rodents, suggesting important differences in p53 stress responses and cancer development. PMID:18187580

  2. Enhancement of DNA vaccine-induced immune responses by a 72-bp element from SV40 enhancer

    Institute of Scientific and Technical Information of China (English)

    LI Hai-shan; XU Jian-qing; HONG Kun-xue; SHAO Yi-ming; LIU Yong; LI Ding-feng; ZHANG Ran-ran; TANG Hai-li; ZHANG Yu-wei; HUANG Wei; LIU Ying; PENG Hong

    2007-01-01

    Background Although DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed.Methods Vector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice.Results It was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells.gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner.Conclusions The 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

  3. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations

    NARCIS (Netherlands)

    Aanen, D.K.; Spelbrink, J.N.; Beekman, M.

    2014-01-01

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is r

  4. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations

    NARCIS (Netherlands)

    Aanen, D.K.; Spelbrink, J.N.; Beekman, M.

    2014-01-01

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is

  5. Amine-functionalized magnetic mesoporous silica nanoparticles for DNA separation

    Science.gov (United States)

    Sheng, Wei; Wei, Wei; Li, Junjian; Qi, Xiaoliang; Zuo, Gancheng; Chen, Qi; Pan, Xihao; Dong, Wei

    2016-11-01

    We report a modified approach for the functionalized magnetic mesoporous silica nanoparticles (MMSN) using polymer microspheres incorporated with magnetic nanoparticles in the presence of cetyltrimethylammonium bromide (CTAB) and the core-shell magnetic silica nanoparticles (MSN). These particles were functionalized with amino groups via the addition of aminosilane directly to the particle sol. We then evaluate their DNA separation abilities and find the capacity of DNA binding significantly increased (210.22 μg/mg) compared with normal magnetic silica spheres (138.44 μg/mg) by using an ultraviolet and visible spectrophotometer (UV). The morphologies, magnetic properties, particle size, pore size, core-shell structure and Zeta potential are characterized by Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), Transmission electron microscopy (TEM), Powder X-ray diffraction (XRD), and dynamic light scattering (DLS). This work demonstrates that our MMSN own an excellent potential application in bioseparation and drug delivery.

  6. Satellite DNA and Transposable Elements in Seabuckthorn (Hippophae rhamnoides), a Dioecious Plant with Small Y and Large X Chromosomes

    Science.gov (United States)

    Puterova, Janka; Razumova, Olga; Martinek, Tomas; Alexandrov, Oleg; Divashuk, Mikhail; Kubat, Zdenek; Hobza, Roman; Karlov, Gennady

    2017-01-01

    Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome. We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes. PMID:28057732

  7. DNA-functionalized solid state nanopore for biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Mussi, V; Fanzio, P; Repetto, L; Firpo, G; Valbusa, U [Nanomed Labs, Physics Department, University of Genova, Advanced Biotechnology Center, Largo R. Benzi, 10 Genova, 16132 (Italy); Scaruffi, P; Stigliani, S; Tonini, G P, E-mail: mussi@fisica.unige.it [Translational Pediatric Oncology, National Institute for Cancer Research (IST), Largo R. Benzi, 10 Genova, 16132 (Italy)

    2010-04-09

    The possible use of nanopores for single DNA molecules biosensing has been demonstrated, but much remains to do in order to develop advanced engineered devices with enhanced stability, and controlled geometry and surface properties. Here we present morphological and electrical characterization of solid state silicon nitride nanopores fabricated by focused ion beam direct milling and chemically functionalized by probe oligonucleotides, with the final aim of developing a versatile tool for biosensing and gene expression profiling.

  8. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  9. Positive and negative regulatory elements in the dnaA-dnaN-recF operon of Escherichia coli.

    Science.gov (United States)

    Pérez-Roger, I; García-Sogo, M; Navarro-Aviñó, J P; López-Acedo, C; Macián, F; Armengod, M E

    1991-01-01

    The recF gene of E coli lies within a cluster of genes which play essential roles in DNA replication; the gene order is dnaA dnaN recF gyrB. Each of these genes has its own promoters which, with the exception of dnaA promoters, reside entirely within the translated region of the respective preceding gene. In this report, we analyze the effect of the dnaA and dnaN promoters on recF expression by translational fusions between recF and the lacZ reporter gene. Our results indicate that recF is a distal gene of the dnaA operon, and support the previous proposal that dnaN and recF constitute a transcriptional unit under control of the dnaN promoters. They also suggest that dnaA, dnaN and recF are predominantly expressed from the same mRNA although transcriptional and/or post-transcriptional mechanisms should be specifically involved in lowering expression of the recF gene. Recently, we have localized 3 tandem transcription termination sites in the second half of the dnaN gene, downstream from the recF promoters. Neither of them shows the typical features of simple terminators and apparently they do not work in a minimal system of in vitro transcription. In this report, we present evidence that only one of them is dependent on the Rho protein. Although the operon structure allows coordinate expression of dnaA, dnaN and recF, the presence of internal promoters (the dnaN and recF promoters), which appear to be inducible by DNA damage, and intracistronic terminators, whose activity is inversely proportional to the efficiency of translation, permits expression of individual genes to be independently regulated in response to altered growth conditions.

  10. The ATRX cDNA is prone to bacterial IS10 element insertions that alter its structure.

    Science.gov (United States)

    Valle-García, David; Griffiths, Lyra M; Dyer, Michael A; Bernstein, Emily; Recillas-Targa, Félix

    2014-01-01

    The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

  11. DNA block copolymers: functional materials for nanoscience and biomedicine.

    Science.gov (United States)

    Schnitzler, Tobias; Herrmann, Andreas

    2012-09-18

    We live in a world full of synthetic materials, and the development of new technologies builds on the design and synthesis of new chemical structures, such as polymers. Synthetic macromolecules have changed the world and currently play a major role in all aspects of daily life. Due to their tailorable properties, these materials have fueled the invention of new techniques and goods, from the yogurt cup to the car seat belts. To fulfill the requirements of modern life, polymers and their composites have become increasingly complex. One strategy for altering polymer properties is to combine different polymer segments within one polymer, known as block copolymers. The microphase separation of the individual polymer components and the resulting formation of well defined nanosized domains provide a broad range of new materials with various properties. Block copolymers facilitated the development of innovative concepts in the fields of drug delivery, nanomedicine, organic electronics, and nanoscience. Block copolymers consist exclusively of organic polymers, but researchers are increasingly interested in materials that combine synthetic materials and biomacromolecules. Although many researchers have explored the combination of proteins with organic polymers, far fewer investigations have explored nucleic acid/polymer hybrids, known as DNA block copolymers (DBCs). DNA as a polymer block provides several advantages over other biopolymers. The availability of automated synthesis offers DNA segments with nucleotide precision, which facilitates the fabrication of hybrid materials with monodisperse biopolymer blocks. The directed functionalization of modified single-stranded DNA by Watson-Crick base-pairing is another key feature of DNA block copolymers. Furthermore, the appropriate selection of DNA sequence and organic polymer gives control over the material properties and their self-assembly into supramolecular structures. The introduction of a hydrophobic polymer into DBCs

  12. Functionalized nanopore-embedded electrodes for rapid DNA sequencing

    CERN Document Server

    He, Haiying; Pandey, Ravindra; Rocha, Alexandre Reily; Sanvito, Stefano; Grigoriev, Anton; Ahuja, Rajeev; Karna, Shashi P

    2007-01-01

    The determination of a patient's DNA sequence can, in principle, reveal an increased risk to fall ill with particular diseases [1,2] and help to design "personalized medicine" [3]. Moreover, statistical studies and comparison of genomes [4] of a large number of individuals are crucial for the analysis of mutations [5] and hereditary diseases, paving the way to preventive medicine [6]. DNA sequencing is, however, currently still a vastly time-consuming and very expensive task [4], consisting of pre-processing steps, the actual sequencing using the Sanger method, and post-processing in the form of data analysis [7]. Here we propose a new approach that relies on functionalized nanopore-embedded electrodes to achieve an unambiguous distinction of the four nucleic acid bases in the DNA sequencing process. This represents a significant improvement over previously studied designs [8,9] which cannot reliably distinguish all four bases of DNA. The transport properties of the setup investigated by us, employing state-o...

  13. The Saccharomyces cerevisiae Dna2 can function as a sole nuclease in the processing of Okazaki fragments in DNA replication.

    Science.gov (United States)

    Levikova, Maryna; Cejka, Petr

    2015-09-18

    During DNA replication, synthesis of the lagging strand occurs in stretches termed Okazaki fragments. Before adjacent fragments are ligated, any flaps resulting from the displacement of the 5' DNA end of the Okazaki fragment must be cleaved. Previously, Dna2 was implicated to function upstream of flap endonuclease 1 (Fen1 or Rad27) in the processing of long flaps bound by the replication protein A (RPA). Here we show that Dna2 efficiently cleaves long DNA flaps exactly at or directly adjacent to the base. A fraction of the flaps cleaved by Dna2 can be immediately ligated. When coupled with DNA replication, the flap processing activity of Dna2 leads to a nearly complete Okazaki fragment maturation at sub-nanomolar Dna2 concentrations. Our results indicate that a subsequent nucleolytic activity of Fen1 is not required in most cases. In contrast Dna2 is completely incapable to cleave short flaps. We show that also Dna2, like Fen1, interacts with proliferating cell nuclear antigen (PCNA). We propose a model where Dna2 alone is responsible for cleaving of RPA-bound long flaps, while Fen1 or exonuclease 1 (Exo1) cleave short flaps. Our results argue that Dna2 can function in a separate, rather than in a Fen1-dependent pathway.

  14. Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

    Science.gov (United States)

    Watanabe, Kohei; Koga, Hajime; Nakamura, Kodai; Fujita, Akiko; Hattori, Akimasa; Matsuda, Masaru; Koga, Akihiko

    2014-04-01

    DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.

  15. Cross-species functionality of pararetroviral elements driving ribosome shunting.

    Directory of Open Access Journals (Sweden)

    Mikhail M Pooggin

    Full Text Available BACKGROUND: Cauliflower mosaic virus (CaMV and Rice tungro bacilliform virus (RTBV belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (sORFs and a stable stem-loop structure. The shunt requires translation of a 5'-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism. We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland. We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.

  16. Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

    Science.gov (United States)

    Chappell, James; Jensen, Kirsten; Freemont, Paul S.

    2013-01-01

    A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

  17. Amine-functionalized magnetic mesoporous silica nanoparticles for DNA separation

    Energy Technology Data Exchange (ETDEWEB)

    Sheng, Wei; Wei, Wei; Li, Junjian; Qi, Xiaoliang; Zuo, Gancheng; Chen, Qi; Pan, Xihao; Dong, Wei, E-mail: weidong@njust.edu.cn

    2016-11-30

    Highlights: • Fe{sub 3}O{sub 4}@SiO{sub 2}@EDPS with uniform size and good dispersity is prepared. • We fabricated MMSN@EDPS with distinct core-shell–shell triple-layer composition. • DNA adsorption capacity of MMSN@EDPS is considerable. - Abstract: We report a modified approach for the functionalized magnetic mesoporous silica nanoparticles (MMSN) using polymer microspheres incorporated with magnetic nanoparticles in the presence of cetyltrimethylammonium bromide (CTAB) and the core-shell magnetic silica nanoparticles (MSN). These particles were functionalized with amino groups via the addition of aminosilane directly to the particle sol. We then evaluate their DNA separation abilities and find the capacity of DNA binding significantly increased (210.22 μg/mg) compared with normal magnetic silica spheres (138.44 μg/mg) by using an ultraviolet and visible spectrophotometer (UV). The morphologies, magnetic properties, particle size, pore size, core-shell structure and Zeta potential are characterized by Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), Transmission electron microscopy (TEM), Powder X-ray diffraction (XRD), and dynamic light scattering (DLS). This work demonstrates that our MMSN own an excellent potential application in bioseparation and drug delivery.

  18. Derivation of an Efficient Non-Prismatic Thin Curved Beam Element Using Basic Displacement Functions

    Directory of Open Access Journals (Sweden)

    Ahmad Shahba

    2012-01-01

    Full Text Available The efficiency and accuracy of the elements proposed by the Finite Element Method (FEM considerably depend on the interpolating functions, namely shape functions, used to formulate the displacement field within an element. In this paper, a new insight is proposed for derivation of elements from a mechanical point of view. Special functions namely Basic Displacement Functions (BDFs are introduced which hold pure structural foundations. Following basic principles of structural mechanics, it is shown that exact shape functions for non-prismatic thin curved beams could be derived in terms of BDFs. Performing a limiting study, it is observed that the new curved beam element successfully becomes the straight Euler-Bernoulli beam element. Carrying out numerical examples, it is shown that the element provides exact static deformations. Finally efficiency of the method in free vibration analysis is verified through several examples. The results are in good agreement with those in the literature.

  19. Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site*

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-01-01

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place. PMID:20061574

  20. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  1. Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Carolina Furtado

    Full Text Available The oxidative lesion 8-oxoguanine (8-oxoG is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1. This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1, the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/- (CD138 to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2O(2. Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2O(2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

  2. FARE-CAFE: a database of functional and regulatory elements of cancer-associated fusion events.

    Science.gov (United States)

    Korla, Praveen Kumar; Cheng, Jack; Huang, Chien-Hung; Tsai, Jeffrey J P; Liu, Yu-Hsuan; Kurubanjerdjit, Nilubon; Hsieh, Wen-Tsong; Chen, Huey-Yi; Ng, Ka-Lok

    2015-01-01

    Chromosomal translocation (CT) is of enormous clinical interest because this disorder is associated with various major solid tumors and leukemia. A tumor-specific fusion gene event may occur when a translocation joins two separate genes. Currently, various CT databases provide information about fusion genes and their genomic elements. However, no database of the roles of fusion genes, in terms of essential functional and regulatory elements in oncogenesis, is available. FARE-CAFE is a unique combination of CTs, fusion proteins, protein domains, domain-domain interactions, protein-protein interactions, transcription factors and microRNAs, with subsequent experimental information, which cannot be found in any other CT database. Genomic DNA information including, for example, manually collected exact locations of the first and second break points, sequences and karyotypes of fusion genes are included. FARE-CAFE will substantially facilitate the cancer biologist's mission of elucidating the pathogenesis of various types of cancer. This database will ultimately help to develop 'novel' therapeutic approaches. Database URL: http://ppi.bioinfo.asia.edu.tw/FARE-CAFE.

  3. Boosting functionality of synthetic DNA circuits with tailored deactivation.

    Science.gov (United States)

    Montagne, Kevin; Gines, Guillaume; Fujii, Teruo; Rondelez, Yannick

    2016-11-15

    Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network. This mechanism is based on programming deactivation laws using dedicated saturable pathways. We demonstrate this approach through the conversion of a single-node homoeostatic network into a bistable and reversible switch. Furthermore, we prove its generality by adding new functions to the library of reported man-made molecular devices: a system with three addressable bits of memory, and the first DNA-encoded excitable circuit. Specific saturable deactivation pathways thus greatly enrich the functional capability of a given circuit topology.

  4. Functional demonstration of adaptive immunity in zebrafish using DNA vaccination

    DEFF Research Database (Denmark)

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja

    studies have documented existence of a classical innate immune response, there is mainly indirect evidence of functional adaptive immunity. To address this aspect, groups of zebrafish were vaccinated with DNA-vaccines against the rhabdoviruses VHSV, IHNV and SVCV. Seven weeks later, the fish were...... challenged with SVCV by immersion. Despite some variability between replicate aquaria, there was a protective effect of the homologous vaccine and no effect of the heterologous vaccines. The results therefore confirm the existence of not only a well developed but also a fully functional adaptive immune......Due to the well characterized genome, overall highly synteny with the human genome and its suitability for functional genomics studies, the zebrafish is considered to be an ideal animal model for basic studies of mechanisms of diseases and immunity in vertebrates including humans. While several...

  5. Novel tools for the functional expression of metagenomic DNA.

    Science.gov (United States)

    Troeschel, Sonja Christina; Drepper, Thomas; Leggewie, Christian; Streit, Wolfgang R; Jaeger, Karl-Erich

    2010-01-01

    The functional expression of environmental genes in a particular host bacterium is hampered by various limitations including inefficient transcription of target genes as well as improper assembly of the corresponding enzymes. Therefore, the identification of novel enzymes from metagenomic libraries by activity-based screening requires efficient expression and screening systems. In the following chapter, we present two novel tools to improve the functional expression of metagenomic genes. (1) Comparative screenings of metagenomic libraries demonstrated that different enzymes were detected when phylogenetically distinct expression host strains were used. Thus, we have developed a strategy, which comprises library construction using a shuttle vector that allows comparative expression and screening of metagenomic DNA in Escherichia coli, Pseudomonas putida, and Bacillus subtilis. (2) Expression studies have revealed that functional expression of environmental genes in heterologous expression hosts is often limited by insufficient promoter recognition. Therefore, a method is described allowing to enhance the expression capacity of E. coli by using the transposon MuExpress. This recombinant transposon is able to insert randomly into environmental DNA fragments thereby facilitating gene expression from its two inducible promoters.

  6. The Daxx/Atrx Complex Protects Tandem Repetitive Elements during DNA Hypomethylation by Promoting H3K9 Trimethylation.

    Science.gov (United States)

    He, Quanyuan; Kim, Hyeung; Huang, Rui; Lu, Weisi; Tang, Mengfan; Shi, Fengtao; Yang, Dong; Zhang, Xiya; Huang, Junjiu; Liu, Dan; Songyang, Zhou

    2015-09-03

    In mammals, DNA methylation is essential for protecting repetitive sequences from aberrant transcription and recombination. In some developmental contexts (e.g., preimplantation embryos) DNA is hypomethylated but repetitive elements are not dysregulated, suggesting that alternative protection mechanisms exist. Here we explore the processes involved by investigating the role of the chromatin factors Daxx and Atrx. Using genome-wide binding and transcriptome analysis, we found that Daxx and Atrx have distinct chromatin-binding profiles and are co-enriched at tandem repetitive elements in wild-type mouse ESCs. Global DNA hypomethylation further promoted recruitment of the Daxx/Atrx complex to tandem repeat sequences, including retrotransposons and telomeres. Knockdown of Daxx/Atrx in cells with hypomethylated genomes exacerbated aberrant transcriptional de-repression of repeat elements and telomere dysfunction. Mechanistically, Daxx/Atrx-mediated repression seems to involve Suv39h recruitment and H3K9 trimethylation. Our data therefore suggest that Daxx and Atrx safeguard the genome by silencing repetitive elements when DNA methylation levels are low.

  7. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, Akari, E-mail: akari_yo@stu.musashino-u.ac.jp [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Kobayashi, Yume [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Tada, Shusuke [Department of Medical Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510 (Japan); Seki, Masayuki [Department of Biochemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai-shi, Miyagi 981-8558 (Japan); Enomoto, Takemi [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan)

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  8. A functional gene array for detection of bacterial virulence elements

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C

    2007-11-01

    We report our development of the first of a series of microarrays designed to detect pathogens with known mechanisms of virulence and antibiotic resistance. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples. To validate our approach, we developed a first generation array targeting genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for microorganism detection and discrimination, measured the required target concentration, and assessed tolerance for mismatches between probe and target sequences. Mismatch tolerance is a priority for this application, due to DNA sequence variability among members of gene families. Arrays were created using the NimbleGen Maskless Array Synthesizer at Lawrence Livermore National Laboratory. Purified genomic DNA from combinations of one or more of the four target organisms, pure cultures of four related organisms, and environmental aerosol samples with spiked-in genomic DNA were hybridized to the arrays. Based on the success of this prototype, we plan to design further arrays in this series, with the goal of detecting all known virulence and antibiotic resistance gene families in a greatly expanded set of organisms.

  9. The varicella-zoster virus ORFS/L (ORF0) gene is required for efficient viral replication and contains an element involved in DNA cleavage.

    Science.gov (United States)

    Kaufer, Benedikt B; Smejkal, Benjamin; Osterrieder, Nikolaus

    2010-11-01

    The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U(L)) and unique short (U(S)), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U(L) and U(S). VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U(L). An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U(L), directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5' region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5' terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U(L), we concluded that our results can provide an explanation for the almost exclusive orientation of the U(L) seen in packaged VZV DNA.

  10. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

    Directory of Open Access Journals (Sweden)

    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  11. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

    Science.gov (United States)

    Li, Jinhuan; Shou, Jia; Guo, Ya; Tang, Yuanxiao; Wu, Yonghu; Jia, Zhilian; Zhai, Yanan; Chen, Zhifeng; Xu, Quan; Wu, Qiang

    2015-01-01

    The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters. PMID:25757625

  12. Hundreds of circular novel plasmids and DNA elements identified in a rat cecum metamobilome.

    Directory of Open Access Journals (Sweden)

    Tue Sparholt Jørgensen

    Full Text Available Metagenomic approaches are widespread in microbiological research, but so far, the knowledge on extrachromosomal DNA diversity and composition has largely remained dependant on cultivating host organisms. Even with the emergence of metagenomics, complete circular sequences are rarely identified, and have required manual curation. We propose a robust in silico procedure for identifying complete small plasmids in metagenomic datasets from whole genome shotgun sequencing. From one very pure and exhaustively sequenced metamobilome from rat cecum, we identified a total of 616 circular sequences, 160 of which were carrying a gene with plasmid replication domain. Further homology analyses indicated that the majority of these plasmid sequences are novel. We confirmed the circularity of the complete plasmid candidates using an inverse-type PCR approach on a subset of sequences with 95% success, confirming the existence and length of discrete sequences. The implication of these findings is a broadened understanding of the traits of circular elements in nature and the possibility of massive data mining in existing metagenomic datasets to discover novel pools of complete plasmids thus vastly expanding the current plasmid database.

  13. Fine-scale map of encyclopedia of DNA elements regions in the Korean population.

    Science.gov (United States)

    Yoo, Yeon-Kyeong; Ke, Xiayi; Hong, Sungwoo; Jang, Hye-Yoon; Park, Kyunghee; Kim, Sook; Ahn, TaeJin; Lee, Yeun-Du; Song, Okryeol; Rho, Na-Young; Lee, Moon Sue; Lee, Yeon-Su; Kim, Jaeheup; Kim, Young J; Yang, Jun-Mo; Song, Kyuyoung; Kimm, Kyuchan; Weir, Bruce; Cardon, Lon R; Lee, Jong-Eun; Hwang, Jung-Joo

    2006-09-01

    The International HapMap Project aims to generate detailed human genome variation maps by densely genotyping single-nucleotide polymorphisms (SNPs) in CEPH, Chinese, Japanese, and Yoruba samples. This will undoubtedly become an important facility for genetic studies of diseases and complex traits in the four populations. To address how the genetic information contained in such variation maps is transferable to other populations, the Korean government, industries, and academics have launched the Korean HapMap project to genotype high-density Encyclopedia of DNA Elements (ENCODE) regions in 90 Korean individuals. Here we show that the LD pattern, block structure, haplotype diversity, and recombination rate are highly concordant between Korean and the two HapMap Asian samples, particularly Japanese. The availability of information from both Chinese and Japanese samples helps to predict more accurately the possible performance of HapMap markers in Korean disease-gene studies. Tagging SNPs selected from the two HapMap Asian maps, especially the Japanese map, were shown to be very effective for Korean samples. These results demonstrate that the HapMap variation maps are robust in related populations and will serve as an important resource for the studies of the Korean population in particular.

  14. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations.

    Science.gov (United States)

    Aanen, Duur K; Spelbrink, Johannes N; Beekman, Madeleine

    2014-07-05

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection.

  15. The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

    Science.gov (United States)

    Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

    2013-01-01

    AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5′-NNCCAC-3′ and 5′-GCGMGN′N′-3′ (M:A or C; N and N′ form Watson–Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences. PMID:23709277

  16. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response.

    Science.gov (United States)

    Yoshimura, Akari; Kobayashi, Yume; Tada, Shusuke; Seki, Masayuki; Enomoto, Takemi

    2014-09-12

    WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH(-/-)) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  17. Main functional elements having defects of illegal residential buildings: The case of Cova da Moura district

    NARCIS (Netherlands)

    Vilhena, A.; Costa Branco De Oliveira Pedro, J.A.; Baptista Coelho, A.; Vasconcelos Paiva, J.

    2011-01-01

    The purpose of the paper is to describe the main functional elements with defects in illegal residential buildings. Three research questions are addressed: What are the main functional elements with defects in buildings and in dwellings? What are the main defects found? In which way these defects af

  18. Main functional elements having defects of illegal residential buildings: The case of Cova da Moura district

    NARCIS (Netherlands)

    Vilhena, A.; Costa Branco De Oliveira Pedro, J.A.; Baptista Coelho, A.; Vasconcelos Paiva, J.

    2011-01-01

    The purpose of the paper is to describe the main functional elements with defects in illegal residential buildings. Three research questions are addressed: What are the main functional elements with defects in buildings and in dwellings? What are the main defects found? In which way these defects

  19. DMPD: Activation of lymphokine genes in T cells: role of cis-acting DNA elements thatrespond to T cell activation signals. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 1492121 Activation of lymphokine genes in T cells: role of cis-acting DNA elements ...html) (.csml) Show Activation of lymphokine genes in T cells: role of cis-acting ...DNA elements thatrespond to T cell activation signals. PubmedID 1492121 Title Activation of lymphokine genes in T cells: role

  20. Evolution in the block: common elements of 5S rDNA organization and evolutionary patterns in distant fish genera.

    Science.gov (United States)

    Campo, Daniel; García-Vázquez, Eva

    2012-01-01

    The 5S rDNA is organized in the genome as tandemly repeated copies of a structural unit composed of a coding sequence plus a nontranscribed spacer (NTS). The coding region is highly conserved in the evolution, whereas the NTS vary in both length and sequence. It has been proposed that 5S rRNA genes are members of a gene family that have arisen through concerted evolution. In this study, we describe the molecular organization and evolution of the 5S rDNA in the genera Lepidorhombus and Scophthalmus (Scophthalmidae) and compared it with already known 5S rDNA of the very different genera Merluccius (Merluccidae) and Salmo (Salmoninae), to identify common structural elements or patterns for understanding 5S rDNA evolution in fish. High intra- and interspecific diversity within the 5S rDNA family in all the genera can be explained by a combination of duplications, deletions, and transposition events. Sequence blocks with high similarity in all the 5S rDNA members across species were identified for the four studied genera, with evidences of intense gene conversion within noncoding regions. We propose a model to explain the evolution of the 5S rDNA, in which the evolutionary units are blocks of nucleotides rather than the entire sequences or single nucleotides. This model implies a "two-speed" evolution: slow within blocks (homogenized by recombination) and fast within the gene family (diversified by duplications and deletions).

  1. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

    Science.gov (United States)

    Fazi, Amanda; Gobeski, Brianne; Foran, David

    2014-11-01

    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence.

  2. Rapid proliferation of repetitive palindromic elements in mtDNA of the endemic Baikalian sponge Lubomirskia baicalensis.

    Science.gov (United States)

    Lavrov, Dennis V

    2010-04-01

    Animal mitochondrial DNA (mtDNA) is a remarkably compact molecule largely because of the scarcity of noncoding "selfish" DNA. Recently, however, we found that mitochondrial genomes of several phylogenetically diverse species of demosponges contain small repetitive palindromic sequences, interspersed within intergenic regions and fused in protein and ribosomal RNA genes. Here, I report and analyze the proliferation of such elements in the mitochondrial genome of the endemic sponge of Lake Baikal Lubomirskia baicalensis. Because Baikal sponges are closely related to the circumglobally distributed freshwater sponge Ephydatia muelleri with which they shared a common ancestor approximately 3-10 Ma, both the rate of single nucleotide substitutions and the rate of palindromic repeat insertions can be calculated in this system. I found the rate of nucleotide substitutions in mtDNA of freshwater sponges to be extremely low (0.5-1.6 x 10(-9) per site per year), more similar to that in plants than bilaterian animals. By contrast, the per/nucleotide rate of insertions of repetitive elements is at least four times higher. This rapid rate of proliferation combined with the broad phylogenetic distribution of hairpin elements can make them a defining force in the evolution of mitochondrial genomes of demosponges.

  3. New element for optimizing the functioning of sediment traps

    Science.gov (United States)

    Schwindt, Sebastian; Franca, Mário; Schleiss, Anton

    2017-04-01

    Sediment traps protect urban areas against excessive sediment transport during hazardous floods and consist typically of a retention basin with an open sediment check dam at the downstream end. The design, as well as the morphological processes within the retention basin, were analyzed by several authors. With regard to open sediment check dams two types of triggering mechanisms for the initiation of sediment retention can be distinguished: (1) mechanical and (2) hydraulic clogging of the structure. Recent studies have shown that outlet structures combining both clogging principles may be considered to avoid undesired self-flushing. Further elements of check dams are conceivable, e.g. for retaining or conveying driftwood. This study analyses experimentally working principles and design criteria of standard elements of sediment traps. Furthermore, it introduces a new structural element to the sediment trap design with a guiding channel in the retention reservoir. Taking into account the natural shape of mountain rivers, the guiding channel has a trapezoidal cross-section shape and a rough but fixed bed. The effect of the guiding channel on sediment deposition pattern and re-mobilization are studied by means of physical model experiments with a standardized hydrograph and variable sediment supply. The results are evaluated by means of zenithal pictures and bedload transport rate, measured at the downstream end of the model. Major advantages of the combined use of both clogging principles include an improved control of the initiation of sediment deposition in order to allow for sediment transfer for small floods and a reduction of hazards related to self-flushing.

  4. Charge-Transfer Matrix Elements by FMO-LCMO Approach: Hole Transfer in DNA with Parameter Tuned Range-Separated DFT

    CERN Document Server

    Kitoh-Nishioka, Hirotaka

    2016-01-01

    A scheme for computing charge-transfer matrix elements with the linear combination of fragment molecular orbitals and the 'nonempirically tuned range-separated' density functional is presented. It takes account of the self-consistent orbital relaxation induced by environmental Coulomb field and the exchange interaction in fragment pairs at low computational scaling along the system size. The accuracy was confirmed numerically on benchmark systems of imidazole and furane homo-dimer cations. Applications to hole transfers in DNA nucleobase pairs and in a $\\pi$-stack adenine octomer highlight the effects of orbital relaxation.

  5. Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

    Directory of Open Access Journals (Sweden)

    Landsman David

    2006-09-01

    Full Text Available Abstract Background Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. Results We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM. The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. Conclusion Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances.

  6. New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases.

    Science.gov (United States)

    Zhang, Likui

    2017-09-15

    The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.

  7. Functional connection of didactical elements in teaching geography

    Directory of Open Access Journals (Sweden)

    Živković Ljiljana

    2010-01-01

    Full Text Available The aim of this work is to present complex relations within didactical triangle as well as review of the practical usage of relations of didactical polygon. Importance of subject of this work is, certainly, its constant actualization and irrepressible change which is happening within elements and in their mutual interaction. This kind of relation brings innovations and suffers changes from modern times. Starting from immediate ways of communication to closer and further perceive of their development, the subject gets dimension of expected and somewhat exciting. The theme ensued because of the need for logical separation of didactical elements and also to connect them through complex system of communication. Their imbues are coming to express daily while interaction is developing not only in historical period but in instantaneous stages. The media, today, gives grade to development of society and represent degree of progress. As such an important factor, the role of the media must be taken into consideration. Geographical achievement is represented by teaching methods of geography and in that way it represents significant media. Complicatedness of this theme also reflects in universality; geography has advanced as science and there is great expectation for methodical science to catch up with geography and to present new manners, ways of transferring knowledge. .

  8. Genetic and functional diversity of ubiquitous DNA viruses in selected Chinese agricultural soils

    Science.gov (United States)

    Han, Li-Li; Yu, Dan-Ting; Zhang, Li-Mei; Shen, Ju-Pei; He, Ji-Zheng

    2017-01-01

    Viral community structures in complex agricultural soils are largely unknown. Electron microscopy and viromic analyses were conducted on six typical Chinese agricultural soil samples. Tailed bacteriophages, spherical and filamentous viral particles were identified by the morphological analysis. Based on the metagenomic analysis, single-stranded DNA viruses represented the largest viral component in most of the soil habitats, while the double-stranded DNA viruses belonging to the Caudovirales order were predominanted in Jiangxi-maize soils. The majority of functional genes belonged to the subsystem “phages, prophages, transposable elements, and plasmids”. Non-metric multidimensional analysis of viral community showed that the environment medium type was the most important driving factor for the viral community structure. For the major viral groups detected in all samples (Microviridae and Caudovirales), the two groups gathered viruses from different sites and similar genetic composition, indicating that viral diversity was high on a local point but relatively limited on a global scale. This is a novel report of viral diversity in Chinese agricultural soils, and the abundance, taxonomic, and functional diversity of viruses that were observed in different types of soils will aid future soil virome studies and enhance our understanding of the ecological functions of soil viruses. PMID:28327667

  9. Two-dimensional finite element neutron diffusion analysis using hierarchic shape functions

    Energy Technology Data Exchange (ETDEWEB)

    Carpenter, D.C.

    1997-04-01

    Recent advances have been made in the use of p-type finite element method (FEM) for structural and fluid dynamics problems that hold promise for reactor physics problems. These advances include using hierarchic shape functions, element-by-element iterative solvers and more powerful mapping techniques. Use of the hierarchic shape functions allows greater flexibility and efficiency in implementing energy-dependent flux expansions and incorporating localized refinement of the solution space. The irregular matrices generated by the p-type FEM can be solved efficiently using element-by-element conjugate gradient iterative solvers. These solvers do not require storage of either the global or local stiffness matrices and can be highly vectorized. Mapping techniques based on blending function interpolation allow exact representation of curved boundaries using coarse element grids. These features were implemented in a developmental two-dimensional neutron diffusion program based on the use of hierarchic shape functions (FEM2DH). Several aspects in the effective use of p-type analysis were explored. Two choices of elemental preconditioning were examined--the proper selection of the polynomial shape functions and the proper number of functions to use. Of the five shape function polynomials tested, the integral Legendre functions were the most effective. The serendipity set of functions is preferable over the full tensor product set. Two global preconditioners were also examined--simple diagonal and incomplete Cholesky. The full effectiveness of the finite element methodology was demonstrated on a two-region, two-group cylindrical problem but solved in the x-y coordinate space, using a non-structured element grid. The exact, analytic eigenvalue solution was achieved with FEM2DH using various combinations of element grids and flux expansions.

  10. Cognitive-graphic method for constructing of hierarchical forms of basic functions of biquadratic finite element

    Science.gov (United States)

    Astionenko, I. O.; Litvinenko, O. I.; Osipova, N. V.; Tuluchenko, G. Ya.; Khomchenko, A. N.

    2016-10-01

    Recently the interpolation bases of the hierarchical type have been used for the problem solving of the approximation of multiple arguments functions (such as in the finite-element method). In this work the cognitive graphical method of constructing of the hierarchical form bases on the serendipity finite elements is suggested, which allowed to get the alternative bases on a biquadratic finite element from the serendipity family without internal knots' inclusion. The cognitive-graphic method allowed to improve the known interpolation procedure of Taylor and to get the modified elements with irregular arrangement of knots. The proposed procedures are universal and are spread in the area of finite-elements.

  11. Elements of mathematics functions of a real variable : elementary theory

    CERN Document Server

    Bourbaki, Nicolas

    2004-01-01

    This book is an English translation of the last French edition of Bourbaki’s Fonctions d'une Variable Réelle. The first chapter is devoted to derivatives, Taylor expansions, the finite increments theorem, convex functions. In the second chapter, primitives and integrals (on arbitrary intervals) are studied, as well as their dependence with respect to parameters. Classical functions (exponential, logarithmic, circular and inverse circular) are investigated in the third chapter. The fourth chapter gives a thorough treatment of differential equations (existence and unicity properties of solutions, approximate solutions, dependence on parameters) and of systems of linear differential equations. The local study of functions (comparison relations, asymptotic expansions) is treated in chapter V, with an appendix on Hardy fields. The theory of generalized Taylor expansions and the Euler-MacLaurin formula are presented in the sixth chapter, and applied in the last one to the study of the Gamma function on the real ...

  12. APPLICATION OF PENALTY FUNCTION METHOD IN ISOPARAMETRIC HYBRID FINITE ELEMENT ANALYSIS

    Institute of Scientific and Technical Information of China (English)

    CHEN Dao-zheng; JIAO Zhao-ping

    2005-01-01

    By the aid of the penalty function method, the equilibrium restriction conditions were introduced to the isoparametric hybrid finite element analysis, and the concrete application course of the penalty function method in three-dimensional isoparametric hybrid finite element was discussed. The separated penalty parameters method and the optimal hybrid element model with penalty balance were also presented.The penalty balance method can effectively refrain the parasitical stress on the premise of no additional degrees of freedom. The numeric experiment shows that the presented element not only is effective in improving greatly the numeric calculation precision of distorted grids but also has the universality.

  13. Analysis of LINE-1 Elements in DNA from Postmortem Brains of Individuals with Schizophrenia.

    Science.gov (United States)

    Doyle, Glenn A; Crist, Richard C; Karatas, Emre T; Hammond, Matthew J; Ewing, Adam D; Ferraro, Thomas N; Hahn, Chang-Gyu; Berrettini, Wade H

    2017-06-06

    Whereas some rare genetic variants convey high risk for schizophrenia (SZ), common alleles conveying even moderate risk remain elusive. Long interspersed element-1s (L1) are mobile retrotransposons comprising ~17% of the human genome. L1 retrotransposition can cause somatic mosaicism during neurodevelopment by insertional mutagenesis. We hypothesized that, compared to controls, patients diagnosed with schizophrenia (PDS) may have increased numbers of deleterious L1 insertions, perhaps occurring de novo, in brain-expressed genes of dorsolateral prefrontal cortex (DLPFC) neurons. Neuronal and non-neuronal nuclei were separated by fluorescence-activated cell sorting from postmortem DLPFC of 36 PDS and 26 age-matched controls. Genomic sequences flanking the 3'-side of L1s were amplified from neuronal DNA, and neuronal L1 libraries were sequenced. Aligned sequences were analyzed for L1 insertions using custom bioinformatics programs. Ontology and pathway analyses were done on lists of genes putatively disrupted by L1s in PDS and controls. Cellular or population allele frequencies of L1s were assessed by droplet digital PCR or Taqman genotyping. We observed a statistically significant increase in the proportion of intragenic novel L1s in DLPFC of PDS. We found over-representation of L1 insertions within the gene ontologies 'cell projection' and 'postsynaptic membrane' in the gene lists derived from PDS samples, but not from controls. Cellular allele frequencies of examined L1 insertions indicated heterozygosity in genomes of DLPFC cells. An L1 within ERI1 exoribonuclease family member 3 (ERI3) was found to associate with SZ. These results extend prior work documenting increased L1 genetic burden in the brains of PDS and also identify unique genes that may provide new insight into the pathophysiology of schizophrenia.Neuropsychopharmacology advance online publication, 5 July 2017; doi:10.1038/npp.2017.115.

  14. Squamous morules are functionally inert elements of premalignant endometrial neoplasia.

    Science.gov (United States)

    Lin, Ming-Chieh; Lomo, Lesley; Baak, Jan P A; Eng, Charis; Ince, Tan A; Crum, Christopher P; Mutter, George L

    2009-02-01

    Squamous morules are a common component of premalignant glandular lesions that are followed by glandular, rather than squamous, carcinomas. We tested the hypothesis that the appearance of glands associated with morules predicts cancer risk, and undertook molecular testing to determine the clonal and hormonal response properties of admixed squamous and glandular elements. A total of 66 patients with squamous morules in an index endometrial biopsy had follow-up clinical data (average follow-up: interval 31 months, 2.5 biopsies) showing development of carcinoma in 11% (7/66) of cases. The histological appearance of morule-associated glands in the index biopsy was significantly associated with this clinical outcome, with the majority (71%, 5/7) of cancer occurrences following an overtly premalignant lesion (endometrial intraepithelial neoplasia) with squamous morules. Eight endometrial intraepithelial neoplasias with squamous morules were examined by immunohistochemistry for estrogen and progesterone receptors and mitotic activity (Ki-67 antigen percent stained). Glandular components had abundant estrogen and progesterone receptors, and high levels of mitotic activity in all cases. In sharp contrast, all squamous morules were devoid of sex hormone receptors and had undetectable or extremely low-proliferation rates. When mutated, the same specific PTEN mutation was detected in squamous and glandular elements, indicating that both are of common lineage. The clinical and laboratory data are consistent with a model of morule biology in which squamous morules are a hormonally incompetent subpopulation of endometrial glandular lesions. Isolated morules might result from artifactual displacement from their native glandular context, or selective hormonally induced regression of the glandular but not squamous components over time. Subsequent cancer risk, as promoted by estrogens, is greatest when the glandular component has the appearance of endometrial intraepithelial

  15. Dynamical model for biological functions of DNA molecules

    Institute of Scientific and Technical Information of China (English)

    PANGXiao-fengI; YANGYao

    2004-01-01

    We proposed a dynamic model of DNA to study its nonlinear excitation and duplication and transcription in the basis of molecular structure and changes of conformation of DNA under influence of bioenergy.

  16. Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

    Science.gov (United States)

    Brusca, J S; Hale, M A; Carrasco, C D; Golden, J W

    1989-01-01

    The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element. Images PMID:2502534

  17. High-Density Noncovalent Functionalization of DNA by Electrostatic Interactions

    NARCIS (Netherlands)

    Chen, Wei; Gerasimov, Jennifer Y; Zhao, Pei; Liu, Kai; Herrmann, Andreas

    2015-01-01

    Preserving DNA hybridization in organic solvents could someday serve to significantly extend the applicability of DNA-based technologies. Here, we present a method that can be used to solubilize double-stranded DNA at high concentrations in organic media. This method requires first precipitating a

  18. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  19. Extensions of PDZ domains as important structural and functional elements

    OpenAIRE

    Wang, Conan K.; Pan, Lifeng; Chen, Jia; Zhang, Mingjie

    2010-01-01

    ‘Divide and conquer’ has been the guiding strategy for the study of protein structure and function. Proteins are divided into domains with each domain having a canonical structural definition depending on its type. In this review, we push forward with the interesting observation that many domains have regions outside of their canonical definition that affect their structure and function; we call these regions ‘extensions’. We focus on the highly abundant PDZ (PSD-95, DLG1 and ZO-1) domain. Us...

  20. Functional genomics reveals relationships between the retrovirus-like Ty1 element and its host Saccharomyces cerevisiae.

    Science.gov (United States)

    Griffith, Jacqulyn L; Coleman, Laura E; Raymond, Adam S; Goodson, Summer G; Pittard, William S; Tsui, Circe; Devine, Scott E

    2003-07-01

    Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.

  1. A transposable element within the Non-canonical telomerase RNA of Arabidopsis thaliana modulates telomerase in response to DNA damage [corrected].

    Directory of Open Access Journals (Sweden)

    Hengyi Xu

    2015-06-01

    Full Text Available Long noncoding RNAs (lncRNAs have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs. TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3' terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE. DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2 and without DRE (TER2Δ we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault.

  2. Sf-PHB2, A new transcription factor, Drives WSSV Ie1 Gene Expression via a 12-bp DNA Element

    Directory of Open Access Journals (Sweden)

    Ma Guoda

    2012-09-01

    Full Text Available Abstract Background The WSSV immediate early gene ie1 is highly expressed throughout viral infection cycle and may play a central role in initiating viral replication during infection. Results Here, a detailed characterization of the ie1 promoter was performed using deletion and mutation analyses to elucidate the role of the individual promoter motifs. Three results were obtained: 1 the ie1 promoter is a classical eukaryotic promoter that contains the initiator element (Inr and TATA box responsible for the basal promoter activity; 2 mutation or truncation of a predicted Sp1 site decreased the level of promoter activity by about 3-fold, indicating that the Sp1 site is an important cis-element of the promoter; and 3 truncation of a 12-bp sequence that resides at -78/-67 of the ie1 promoter decreased the level of promoter activity by about 14-fold, indicating that the 12-bp motif is a critical upstream element of the ie1 promoter for binding of a strong transcription factor to drive the ie1 gene expression in the cells. Further, the 12-bp DNA binding protein was purified from the nuclear proteins of Sf9 cells using DNA affinity chromatography, and was identified as a homologue of the prohibitin2 protein (named as Sf-PHB2 using mass spectrometry. Furthermore, the DNA binding activity of Sf-PHB2 was verified using a super shift analysis. Conclusion These results support that the Sf-PHB2 is a novel transcription factor that drives WSSV ie1 gene expression by binding to the 12-bp DNA element.

  3. Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

    Science.gov (United States)

    Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

    2016-12-13

    Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

  4. The electrokinetic characterization of gold nanoparticles, functionalized with cationic functional groups, and its' interaction with DNA.

    Science.gov (United States)

    Lazarus, Geraldine Genevive; Revaprasadu, Neerish; López-Viota, Julián; Singh, Moganavelli

    2014-09-01

    Gold nanoparticles have attracted strong biomedical interest for drug delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. However, gene transfection represents another important biological application. Considering that cellular barriers keep enclosed their secret to deliver genes using nanoparticles, an important step can be achieved by studying the functionalization of nanoparticles with DNA. In the present contribution the synthesis of nanoparticles consisting of a gold core coated with one or more layers of amino acid (l-lysine), and cationic polyelectrolytes (poly-ethyleneimine and poly-l-lysine) is reported. All nanoparticles were subjected to dynamic light scattering, electrophoretic mobility measurements, UV-vis optical spectrophotometry analysis and transmission electron microscopy imaging. In addition, the adsorption of DNA plasmid (pSGS) with linear and supercoiled configurations was studied for those gold nanoparticles under the most suitable surface modifications. Preliminary results showed that the gold nanoparticles functionalized with poly-ethyleneimine and poly-l-lysine, respectively, and bound to linear DNA configurations, present in absolute value a higher electrophoretic mobility irrespective of the pH of the media, compared to the supercoiled and nicked configuration. The findings from this study suggest that poly-ethyleneimine and poly-l-lysine functionalized gold nanoparticles are biocompatible and may be promising in the chemical design and future optimization of nanostructures for biomedical applications such as gene and drug delivery.

  5. Large-scale all-electron density functional theory calculations using an enriched finite element basis

    CERN Document Server

    Kanungo, Bikash

    2016-01-01

    We present a computationally efficient approach to perform large-scale all-electron density functional theory calculations by enriching the classical finite element basis with compactly supported atom-centered numerical basis functions that are constructed from the solution of the Kohn-Sham (KS) problem for single atoms. We term these numerical basis functions as enrichment functions, and the resultant basis as the enriched finite element basis. The enrichment functions are compactly supported through the use of smooth cutoff functions, which enhances the conditioning and maintains the locality of the basis. The integrals involved in the evaluation of the discrete KS Hamiltonian and overlap matrix in the enriched finite element basis are computed using an adaptive quadrature grid based on the characteristics of enrichment functions. Further, we propose an efficient scheme to invert the overlap matrix by using a block-wise matrix inversion in conjunction with special reduced-order quadrature rules to transform...

  6. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Science.gov (United States)

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  7. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    Directory of Open Access Journals (Sweden)

    Tonika Lam

    Full Text Available Class switch DNA recombination (CSR of the immunoglobulin heavy chain (IgH locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID expression and AID targeting to switch (S regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit and PKA-RIα (regulatory inhibitory subunit and uracil DNA glycosylase (Ung. 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198 or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR. 14-3-3 adaptors colocalized with AID and replication protein A (RPA in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr, an accessory protein of human immunodeficiency virus type-1 (HIV-1, which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  8. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    OpenAIRE

    Kuwabara, N; Uchida, H.

    1981-01-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]....

  9. CONVERGENCE OF SIMPLIFIED AND STABILIZED MIXED ELEMENT FORMATS BASED ON BUBBLE FUNCTION FOR THE STOKES PROBLEM

    Institute of Scientific and Technical Information of China (English)

    罗振东; 朱江

    2002-01-01

    Two simplified and stabilized mixed element formats for the Stokes problem are derived by bubble function, and their convergence,i.e,error analysis, are proved.These formats can save more freedom degrees than other usual formats.

  10. The Innovative Bike Conceptual Design by Using Modified Functional Element Design Method

    Directory of Open Access Journals (Sweden)

    Nien-Te Liu

    2016-11-01

    Full Text Available The purpose of the study is to propose a new design process by modifying functional element design approach which can commence a large amount of innovative concepts within a short period of time. Firstly, the original creative functional elements design method is analyzed and the drawbacks are discussed. Then, the modified is proposed and is divided into 6 steps. The creative functional element representations, generalization, specialization, and particularization are used in this method. Every step is described clearly, and users could design by following the process easily. In this paper, a clear and accurate design process is proposed based on the creative functional element design method. By following this method, a lot of innovative bicycles will be created quickly.

  11. Ion-selective electrodes in organic elemental and functional group analysis: a review

    Energy Technology Data Exchange (ETDEWEB)

    Selig, W.

    1977-11-08

    The literature on the use of ion-selective electrodes in organic elemental and functional group analysis is surveyed in some detail. The survey is complete through Chemical Abstracts, Vol. 83 (1975). 40 figures, 52 tables, 236 references.

  12. Nonlinear Strain Measures, Shape Functions and Beam Elements for Dynamics of Flexible Beams

    Energy Technology Data Exchange (ETDEWEB)

    Sharf, I. [University of Victoria, Department of Mechanical Engineering (Canada)

    1999-05-15

    In this paper, we examine several aspects of the development of an explicit geometrically nonlinear beam element. These are: (i) linearization of the displacement field; (ii) the effect of a commonly adopted approximation for the nonlinear Lagrangian strain; and (iii) use of different-order shape functions for discretization. The issue of rigid-body check for a nonlinear beam element is also considered. An approximate check is introduced for an element based on an (approximate) intermediate strain measure. Several numerical examples are presented to support the analysis. The paper concludes with a discussion on the use of explicit nonlinear beam elements for multibody dynamics simulation.

  13. Functional cooperation of the dnaE and dnaN gene products in Escherichia coli.

    Science.gov (United States)

    Kuwabara, N; Uchida, H

    1981-09-01

    A system was designed to isolate second-site intergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the alpha subunit of DNA polymerase III [McHenry, C. S. & Crow, W. (1979) J. Biol. Chem. 254, 1748-1753]. One such suppressor, named sueA77, was finely mapped and found to be located at 82 min on the E. coli chromosome, between dnaA and recF, and within the dnaN gene [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553]. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. Natl. Acad. Sci. USA 78, 5391-5395]. The sueA77 mutation was trans-dominant over its wild-type allele, and it suppressed different thermosensitive mutations of dnaE with different maximal permissive temperature. These properties were interpreted as providing genetic evidence for interaction of the dnaE and dnaN gene products in E. coli.

  14. Non-Maxwellian probability density function of fibers with lumped polarization mode dispersion elements.

    Science.gov (United States)

    Antonelli, Cristian; Mecozzi, Antonio

    2004-05-15

    We give an analytical expression for the probability density function of the differential group delay for a concatenation of Maxwellian fiber sections and an arbitrary number of lumped elements with constant and isotropically oriented birefringence. When the contribution of the average squared of the constant birefringence elements is a significant fraction of the total, we show that the outage probability can be significantly overestimated if the probability density function of the differential group delay is approximated by a Maxwellian distribution.

  15. Finite Element Analysis of Functionally Graded Material to Reduce Crazing in Transparent Armor

    Science.gov (United States)

    2015-09-01

    UNCLASSIFIED AD-E403 681 Technical Report ARMET-TR-14042 FINITE ELEMENT ANALYSIS OF FUNCTIONALLY GRADED MATERIAL TO REDUCE...prevent disclosure of its contents or reconstruction of the document. Do not return to the originator. UNCLASSIFIED REPORT DOCUMENTATION PAGE Form...September 2012 to April 2013 4. TITLE AND SUBTITLE FINITE ELEMENT ANALYSIS OF FUNCTIONALLY GRADED MATERIAL TO REDUCE CRAZING IN TRANSPARENT ARMOR

  16. Patch test function for axisymmetric element of conventional and couple stress theory

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The enhanced patch test proposed by Chen W J(2006) can be used to assess the convergence of the problem with non-homogeneous differential equations.Based on this theory,we establish the patch test function for axisymmetric elements of conventional and couple stress theories,and reach an important conclusion that the patch test function for axisymmetric elements cannot contain non-zero constant shear.

  17. Patch test function for axisymmetric element of conventional and couple stress theory

    Institute of Scientific and Technical Information of China (English)

    CHEN WanJi; ZHAO Jie; WANG JinZhi; JI Bian

    2009-01-01

    The enhanced patch test proposed by Chen W J (2006) can be used to assess the convergence of the problem with non-homogeneous differential equations. Based on this theory, we establish the patch test function for axisyrnrnetric elements of conventional and couple stress theories, and reach an im-portant conclusion that the patch test function for axisymrnetric elements cannot contain non-zero constant shear.

  18. Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.

    Science.gov (United States)

    Colinas, R J; Condit, R C; Paoletti, E

    1990-12-01

    Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.

  19. Heavy-ion radiation induces both activation of multiple endogenous transposable elements and alterations in DNA methylation in rice

    Science.gov (United States)

    Zhang, Meng; Sun, Yeqing; Li, Xishan; Xiaolin, Cui; Li, Xiang

    2012-07-01

    Space radiation represents a complex environmental condition in which several interacting factors such as electron, neutron, proton, heavy-ion are involved, which may provoke stress responses and jeopardize genome integrity. Given the inherent property of epigenetic modifications to respond to intrinsic aswell as external perturbations, it is conceivable that epigenetic markers like DNA methylation and transposition may undergo alterations in response to space radiation. Cytosine DNA methylation plays important roles in maintaining genome stability and controlling gene expression. A predominant means for Transposable elements (TEs) to cause genetic instability is via their transpositional activation. To find the detailed molecular characterization of the nature of genomic changes induced by space radiation, the seeds of rice were exposed to 0.02, 0.2, 1, 2 and 20 Gy dose of ^{12}C heavy-ion radiation, respectively. We found that extensive alteration in both DNA methylation and gene expression occurred in rice plants after different dose of heavy-ion radiation. Here we shown that heavy-ion radiation has induced transposition of mPing and Tos17 in rice, which belong to distinct classes including the miniature inverted terminal repeat TEs (MITEs) and long-terminal repeat (LTR) retrotransposons, respectively. mPing and Tos17 mobility were found to correlate with cytosine methylation alteration detected by MSAP and genetic variation detected by AFLP. The result showed that at least in some cases transposition of TEs was associated with cytosine demethylation within the elements. Our results implicate that the heavy-ion radiation represents a potent mutagenic agent that can cause genomic instabilities by eliciting transposition of endogenous TEs in rice. Keywords: Heavy-ion radiation, DNA methylation, Transposable elements, mPing, Tos17

  20. Functions and Malfunctions of Mammalian DNA-Cytosine Deaminases.

    Science.gov (United States)

    Siriwardena, Sachini U; Chen, Kang; Bhagwat, Ashok S

    2016-10-26

    The AID/APOBEC family enzymes convert cytosines in single-stranded DNA to uracils, causing base substitutions and strand breaks. They are induced by cytokines produced during the body's inflammatory response to infections, and they help combat infections through diverse mechanisms. AID is essential for the maturation of antibodies and causes mutations and deletions in antibody genes through somatic hypermutation (SHM) and class-switch recombination (CSR) processes. One member of the APOBEC family, APOBEC1, edits mRNA for a protein involved in lipid transport. Members of the APOBEC3 subfamily in humans (APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H) inhibit infections of viruses such as HIV-1, HBV, and HCV, and retrotransposition of endogenous retroelements through mutagenic and nonmutagenic mechanisms. There is emerging consensus that these enzymes can cause mutations in the cellular genome at replication forks or within transcription bubbles depending on the physiological state of the cell and the phase of the cell cycle during which they are expressed. We describe here the state of knowledge about the structures of these enzymes, regulation of their expression, and both the advantageous and deleterious consequences of their expression, including carcinogenesis. We highlight similarities among them and present a holistic view of their regulation and function.

  1. Simplex finite element analysis of viscous incompressible flow with penalty function formulation

    Science.gov (United States)

    Allaire, P. E.; Rosen, M. C.; Rice, J. G.

    1985-01-01

    Viscous flow calculations are important for the determination of separated flows, recirculating flows, secondary flows and so on. This paper presents a penalty function approach for the finite element analysis of steady incompressible viscous flow. A simplex element is used with linear velocity and constant pressure in contrast to other works which usually employ higher order elements. Simplex elements yield analytical expressions for the element matrices which in turn lead to efficient solutions. Earlier works have partially indicated how constraint and lock-up problems might be avoided for simplex elements. This paper extends the earlier works by indicating the approach in detail and verifying that it is successful for several applications not discussed in the literature so far. Solution times and accuracy considerations are discussed for Couette flow, plane Poiseuille flow, a driven cavity problem, and laminar and turbulent flow over a step.

  2. Natural frequencies of cracked functionally graded material plates by the extended finite element method

    CERN Document Server

    Natarajan, S; Bordas, S; Rabczuk, T; Kerfriden, P

    2011-01-01

    In this paper, the linear free flexural vibration of cracked functionally graded material plates is studied using the extended finite element method. A 4-noded quadrilateral plate bending element based on field and edge consistency requirement with 20 degrees of freedom per element is used for this study. The natural frequencies and mode shapes of simply supported and clamped square and rectangular plates are computed as a function of gradient index, crack length, crack orientation and crack location. The effect of thickness and influence of multiple cracks is also studied.

  3. Utilization of DNA as functional materials: preparation of filters containing DNA insolubilized with alginic acid gel.

    Science.gov (United States)

    Iwata, K; Sawadaishi, T; Nishimura, S I; Tokura, S; Nishi, N

    1996-02-01

    Thin films composed of DNA and alginic acid were prepared by casting their mixed solution on glass plate followed by coagulation with aqueous solution of calcium chloride. DNA could be conveniently insolubilized by this method. DNA in the films adsorbed intercalating materials, such as ethidium bromide. This phenomenon was successfully applied to the preparation of filters for the selective removal or accumulation of harmful intercalating pollutants.

  4. Birthweight, maternal weight trajectories and global DNA methylation of LINE-1 repetitive elements.

    Directory of Open Access Journals (Sweden)

    Karin B Michels

    Full Text Available Low birthweight, premature birth, intrauterine growth retardation, and maternal malnutrition have been related to an increased risk of cardiovascular disease, type 2 diabetes mellitus, obesity, and neuropsychiatric disorders later in life. Conversely, high birthweight has been linked to future risk of cancer. Global DNA methylation estimated by the methylation of repetitive sequences in the genome is an indicator of susceptibility to chronic diseases. We used data and biospecimens from an epigenetic birth cohort to explore the association between trajectories of fetal and maternal weight and LINE-1 methylation in 319 mother-child dyads. Newborns with low or high birthweight had significantly lower LINE-1 methylation levels in their cord blood compared to normal weight infants after adjusting for gestational age, sex of the child, maternal age at delivery, and maternal smoking during pregnancy (p = 0.007 and p = 0.036, respectively, but the magnitude of the difference was small. Infants born prematurely also had lower LINE-1 methylation levels in cord blood compared to term infants, and this difference, though small, was statistically significant (p = 0.004. We did not find important associations between maternal prepregnancy BMI or gestational weight gain and global methylation of the cord blood or fetal placental tissue. In conclusion, we found significant differences in cord blood LINE-1 methylation among newborns with low and high birthweight as well as among prematurely born infants. Future studies may elucidate whether chromosomal instabilities or other functional consequences of these changes contribute to the increased risk of chronic diseases among individuals with these characteristics.

  5. Co-evolution of transcriptional silencing proteins and the DNA elements specifying their assembly.

    Directory of Open Access Journals (Sweden)

    Oliver A Zill

    Full Text Available Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1 proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.

  6. Co-evolution of transcriptional silencing proteins and the DNA elements specifying their assembly.

    Science.gov (United States)

    Zill, Oliver A; Scannell, Devin; Teytelman, Leonid; Rine, Jasper

    2010-11-30

    Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1) proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.

  7. Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

    Science.gov (United States)

    Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

    2014-06-17

    Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

  8. Nucleic acid chemistry in the organic phase: from functionalized oligonucleotides to DNA side chain polymers.

    Science.gov (United States)

    Liu, Kai; Zheng, Lifei; Liu, Qing; de Vries, Jan Willem; Gerasimov, Jennifer Y; Herrmann, Andreas

    2014-10-08

    DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.

  9. Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

    Science.gov (United States)

    Martínez, Asunción; Osburne, Marcia S

    2013-01-01

    One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.

  10. Functions and Dynamics of DNA Repair Proteins in Mitosis and Meiosis

    NARCIS (Netherlands)

    E.J. Uringa

    2005-01-01

    textabstractMy PhD project encompassed studies on the functions of several different proteins, all involved in DNA repair, in somatic and germ-line cells. Hr6b and Rad18Sc are involved in a DNA repair mechanism called ‘Replicative Damage Bypass’ (RDB), and function as ubiquitin conjugating enzym

  11. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    DEFF Research Database (Denmark)

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders;

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  12. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function

    NARCIS (Netherlands)

    Biernat, M.A.; Eker, A.P.M.; Oers, van M.M.; Vlak, J.M.; Horst, van der G.T.J.; Chaves, I.

    2012-01-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors,

  13. Conformation of nanoconfined DNA as a function of ATP, AMP, CTP, Mg2+, and dye binding

    Science.gov (United States)

    Roushan, Maedeh; Riehn, Robert

    2014-03-01

    DNA molecules stretch in nanochannels with a channel cross-section of 100x100 nm2, thereby allowing analysis by observation of a fluorescent dye. The length and configuration of DNA can be directly observed, and the effect of different DNA-binding proteins on DNA configuration can be studied. Recently, we reported on the ability of T4 ligase to transiently manipulate DNA as a function of ATP and magnesium exposure. In this process we have extensively probed the interactions of dyes and enzyme co-factors with DNA under nanoconfinement. We find negligible effects if DNA is visualized using groove-binding dyes such as DAPI. However, if an intercalating dye (YOYO-1) is used, we find a significant shortening of the DNA in the presence of ATP that we attribute to an interaction of dye and ATP (as well as AMP and CTP). We did not record a noticeable effect due to Mg2+.

  14. Optical tweezers studies of viral DNA packaging: Motor function and DNA confinement in Bacteriophages phi29, lambda, and T4

    Science.gov (United States)

    Smith, Douglas

    2007-03-01

    In the assembly of many viruses a powerful molecular motor translocates the genome into a pre-assembled capsid. We use optical tweezers to directly measure translocation of a single DNA molecule into the viral capsid. Improved techniques allow us to measure initiation and early stages of packaging. With phi29 the DNA terminal protein was found to cause large variations in the starting point of packaging. Removal of this protein results in terminal initiation, permitting more accurate assessment of motor function and DNA confinement forces. We investigated the role of electrostatic repulsion by varying ionic screening of the DNA. The observed trends are in accord with those theoretically expected considering counter-ion competition; however the forces are larger than expected in comparison with recent theories and DNA ejection measurements. We have recently succeeded in extending our methods to study two other phages: lambda and T4. These systems have unique structural and functional features, presenting an opportunity for comparative studies in this family of molecular motors. Initial measurements show that lambda and T4 translocate DNA several times faster than the phi29 motor, but are more sensitive to applied load.

  15. Expression of 5 S rRNA genes linked to 35 S rDNA in plants, their epigenetic modification and regulatory element divergence

    Directory of Open Access Journals (Sweden)

    Garcia Sònia

    2012-06-01

    Full Text Available Abstract Background In plants, the 5 S rRNA genes usually occur as separate tandems (S-type arrangement or, less commonly, linked to 35 S rDNA units (L-type. The activity of linked genes remains unknown so far. We studied the homogeneity and expression of 5 S genes in several species from family Asteraceae known to contain linked 35 S-5 S units. Additionally, their methylation status was determined using bisulfite sequencing. Fluorescence in situ hybridization was applied to reveal the sub-nuclear positions of rDNA arrays. Results We found that homogenization of L-type units went to completion in most (4/6 but not all species. Two species contained major L-type and minor S-type units (termed Ls-type. The linked genes dominate 5 S rDNA expression while the separate tandems do not seem to be expressed. Members of tribe Anthemideae evolved functional variants of the polymerase III promoter in which a residing C-box element differs from the canonical angiosperm motif by as much as 30%. On this basis, a more relaxed consensus sequence of a plant C-box: (5’-RGSWTGGGTG-3’ is proposed. The 5 S paralogs display heavy DNA methylation similarly as to their unlinked counterparts. FISH revealed the close association of 35 S-5 S arrays with nucleolar periphery indicating that transcription of 5 S genes may occur in this territory. Conclusions We show that the unusual linked arrangement of 5 S genes, occurring in several plant species, is fully compatible with their expression and functionality. This extraordinary 5 S gene dynamics is manifested at different levels, such as variation in intrachromosomal positions, unit structure, epigenetic modification and considerable divergence of regulatory motifs.

  16. Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades

    Directory of Open Access Journals (Sweden)

    Frohme Marcus

    2009-10-01

    Full Text Available Abstract Background Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. Results To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Conclusion Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

  17. Tardigrade workbench: comparing stress-related proteins, sequence-similar and functional protein clusters as well as RNA elements in tardigrades.

    Science.gov (United States)

    Förster, Frank; Liang, Chunguang; Shkumatov, Alexander; Beisser, Daniela; Engelmann, Julia C; Schnölzer, Martina; Frohme, Marcus; Müller, Tobias; Schill, Ralph O; Dandekar, Thomas

    2009-10-12

    Tardigrades represent an animal phylum with extraordinary resistance to environmental stress. To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data. Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.

  18. DNA structure, binding mechanism and biology functions of polypyridyl complexes in biomedicine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    There is considerable research interest and vigorous debate about the DNA binding of polypyridyl complexes including the electron transfer involving DNA. In this review, based on the fluorescence quenching experiments, it was proposed that DNA might serve as a conductor. From the time-interval CD spectra, the different binding rates of D- and L-enantiomer to calf thymus DNA were observed. The factors influencing the DNA-binding of polypyridyl complexes, and the potential bio-functions of the complexes are also discussed.

  19. Toeplitz Matrices Whose Elements Are the Coefficients of Functions with Bounded Boundary Rotation

    Directory of Open Access Journals (Sweden)

    V. Radhika

    2016-01-01

    Full Text Available Let R denote the family of functions f(z=z+∑n=2∞anzn of bounded boundary rotation so that Ref′(z>0 in the open unit disk U={z:z<1}. We obtain sharp bounds for Toeplitz determinants whose elements are the coefficients of functions f∈R.

  20. Left-handed Z-DNA: structure and function

    Science.gov (United States)

    Herbert, A.; Rich, A.

    1999-01-01

    Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

  1. Functional amyloids as inhibitors of plasmid DNA replication

    Science.gov (United States)

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-del Álamo, María; Fernández-Tresguerres, M. Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  2. Glom Is a Novel Mitochondrial DNA Packaging Protein in Physarum polycephalum and Causes Intense Chromatin Condensation without Suppressing DNA Functions

    Science.gov (United States)

    Sasaki, Narie; Kuroiwa, Haruko; Nishitani, Chikako; Takano, Hiroyoshi; Higashiyama, Tetsuya; Kobayashi, Tamaki; Shirai, Yuki; Sakai, Atsushi; Kawano, Shigeyuki; Murakami-Murofushi, Kimiko; Kuroiwa, Tsuneyoshi

    2003-01-01

    Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions. PMID:12960433

  3. Higher-order adaptive finite-element methods for Kohn-Sham density functional theory

    CERN Document Server

    Motamarri, Phani; Leiter, Kenneth; Knap, Jaroslaw; Gavini, Vikram

    2012-01-01

    We present an efficient computational approach to perform real-space electronic structure calculations using an adaptive higher-order finite-element discretization of Kohn-Sham density-functional theory (DFT).To this end, we develop an \\emph{a priori} mesh adaption technique to construct a close to optimal finite-element discretization of the problem. We further propose an efficient solution strategy for solving the discrete eigenvalue problem by using spectral finite-elements in conjunction with Gauss-Lobatto quadrature, and a Chebyshev acceleration technique for computing the occupied eigenspace. Using the proposed solution procedure, we investigate the computational efficiency afforded by higher-order finite-element discretizations of the Kohn-Sham DFT problem. Our studies suggest that staggering computational savings---of the order of $1000-$fold---can be realized, for both all-electron and pseudopotential calculations, by using higher-order finite-element discretizations. On all the benchmark systems stu...

  4. Modelling Thermal Shock in Functionally Graded Plates with Finite Element Method

    Directory of Open Access Journals (Sweden)

    Vyacheslav N. Burlayenko

    2016-01-01

    Full Text Available Thermomechanical behavior and crack propagation in a functionally graded metal/ceramic plate undergoing thermal shock are analyzed by using the finite element method. A two-dimensional plane strain functionally graded finite element has been developed within the ABAQUS software environment for this purpose. An actual material gradation has been accomplished by sampling material quantities directly at the Gauss points of the element via programming appropriate user-defined subroutines. The virtual crack closure technique is used to model a crack growth under thermal loading. Contact possible between crack lips during the crack advance is taken into account in thermomechanical simulations as well. The paper shows that the presented finite element model can be applied to provide an insight into the thermomechanical respond and failure of the metal/ceramic plate.

  5. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Directory of Open Access Journals (Sweden)

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  6. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    Science.gov (United States)

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  7. Cooperativity between DNA Methyltransferases in the Maintenance Methylation of Repetitive Elements

    Science.gov (United States)

    Liang, Gangning; Chan, Matilda F.; Tomigahara, Yoshitaka; Tsai, Yvonne C.; Gonzales, Felicidad A.; Li, En; Laird, Peter W.; Jones, Peter A.

    2002-01-01

    We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells. PMID:11756544

  8. Viral and cellular SOS-regulated motor proteins: dsDNA translocation mechanisms with divergent functions.

    Science.gov (United States)

    Wolfe, Annie; Phipps, Kara; Weitao, Tao

    2014-01-01

    DNA damage attacks on bacterial cells have been known to activate the SOS response, a transcriptional response affecting chromosome replication, DNA recombination and repair, cell division and prophage induction. All these functions require double-stranded (ds) DNA translocation by ASCE hexameric motors. This review seeks to delineate the structural and functional characteristics of the SOS response and the SOS-regulated DNA translocases FtsK and RuvB with the phi29 bacteriophage packaging motor gp16 ATPase as a prototype to study bacterial motors. While gp16 ATPase, cellular FtsK and RuvB are similarly comprised of hexameric rings encircling dsDNA and functioning as ATP-driven DNA translocases, they utilize different mechanisms to accomplish separate functions, suggesting a convergent evolution of these motors. The gp16 ATPase and FtsK use a novel revolution mechanism, generating a power stroke between subunits through an entropy-DNA affinity switch and pushing dsDNA inward without rotation of DNA and the motor, whereas RuvB seems to employ a rotation mechanism that remains to be further characterized. While FtsK and RuvB perform essential tasks during the SOS response, their roles may be far more significant as SOS response is involved in antibiotic-inducible bacterial vesiculation and biofilm formation as well as the perspective of the bacteria-cancer evolutionary interaction.

  9. Association of hypomethylation of LINE-1 repetitive element in blood leukocyte DNA with an increased risk of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Jian-zhong DI; Xiao-dong HAN; Wen-ye GU; Yu WANG; Qi ZHENG; Pin ZHANG; Hui-min WU; Zhong-zheng ZHU

    2011-01-01

    Global DNA hypomethylation has been associated with increased risk for cancers of the colorectum,bladder,breast,head and neck,and testicular germ cells.The aim of this study was to examine whether global hypomethylation in blood leukocyte DNA is associated with the risk of hepatocellular carcinoma (HCC).A total of 315HCC cases and 356 age-,sex- and HBsAg status-matched controls were included.Global methylation in blood leukocyte DNA was estimated by analyzing long interspersed element-1 (LINE-1) repeats using bisulfite-polymerase chain reaction (PCR) and pyrosequencing.We observed that the median methylation level in HCC cases (percentage of 5-methylcytosine (5mC)=77.7%) was significantly lower than that in controls (79.5% 5mC) (P=0.004,Wilcoxon rank-sum test).The odds ratios (ORs) of HCC for individuals in the third,second,and first (lowest) quartiles of LINE-1methylation were 1.1 (95% confidence interval (CI) 0.7-1.8),1.4 (95% CI 0.8-2.2),and 2.6 (95% CI 1.7-4.1) (P for trend <0.001),respectively,compared to individuals in the fourth (highest) quartile.A 1.9-fold (95% CI 1.4-2.6) increased risk of HCC was observed among individuals with LINE-1 methylation below the median compared to individuals with higher (>median) LINE-1 methylation.Our results demonstrate for the first time that individuals with global hypomethylation measured in LINE-1 repeats in blood leukocyte DNA have an increased risk for HCC.Our data provide the evidence that global hypomethylation detected in the easily obtainable DNA source of blood leukocytes may help identify individuals at risk of HCC.

  10. RNA-directed DNA methylation: Mechanisms and functions

    KAUST Repository

    Mahfouz, Magdy M.

    2010-07-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

  11. DNA-carbon dots function as fluorescent vehicles for drug delivery.

    Science.gov (United States)

    Ding, Han; Du, Feiyue; Liu, Pengchang; Chen, Zhijun; Shen, Jiacong

    2015-04-01

    Carbon dots (CDs) are a new representative in the carbon-based material family, attracting tremendous interest in a large variety of fields, including biomedicine. In this report, we described a facile and green system for synthesizing DNA-CDs using genomic DNA isolated from Escherichia coli. DNA-CDs can be purified using a simple column centrifugation-based system. During DNA-CD synthesis, ribose was collapsed, accompanied by the release of nitrogen, and several new bonds (C-OH, N-O, and N-P) were formed, while the other covalent bonds of DNA were largely maintained. The presence of abundant chemical groups, such as amino or hydroxyl groups on DNA-CDs, may facilitate their future functionalization. These highly biocompatible DNA-CDs can serve as a new type of fluorescent vehicle for cell imaging and drug delivery studies. Our research may hasten the development of CDs for prominent future biomedical applications.

  12. A new method of heart sound signal analysis based on independent function element

    Directory of Open Access Journals (Sweden)

    Cheng Xie-feng

    2014-09-01

    Full Text Available In this paper, a new method is presented for heart sound signal processing in statistical domain. The multiple components obtained from the conventional linear transformation are possibly irrelevant, but usually do not possess the characteristics of statistical independence. First, the definition and obtaining method of independent function element are discussed; the method of signal decomposition and reconstruction based on the independent function element, not only inherits the advantages of linear transformation, but also has the capability of signal representation in the statistical domain. After that, the application of independent function element in heart sound signal analysis is analyzed in detail. The validity and practicability of the method are demonstrated through two experiments.

  13. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Pierre Caron

    2015-11-01

    Full Text Available DNA double-strand breaks (DSBs elicit the so-called DNA damage response (DDR, largely relying on ataxia telangiectasia mutated (ATM and DNA-dependent protein kinase (DNA-PKcs, two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

  14. [Mechanism of differential effect of low dose adaptogens on the functional activity of normal and transformed cellular elements in vitro].

    Science.gov (United States)

    Udintsev, S N; Shakhov, V P; Borovskoĭ, I G

    1991-01-01

    Influence of water solutions of chemically pure adaptogen--synthetic analog of Rhodiola Rosea extract phenol composition (SAR) on functional activity of hemopoietic and tumor cells of mice with Ehrlich ascite cancer was studied in vitro. The periodical character of SAR effects was shown to be different for both types of cells, and at 1 x 10(-2) and 1 x 10(-26) M concentrations simultaneous stimulation of blood marrow cells colony-forming activity and inhibition of the latter in tumor elements was revealed. Essential changes of reactions of both cell types after adding the DNA-dependent RNA polymerase blocker Actinomycin D permit to suggest SAR effects to be connected with drug influence on the membrane RNA of the target cells.

  15. Structural and Functional Regulation of DNA: Geometry, Topology and Methylation

    Science.gov (United States)

    Auclair, C.

    The work of Rosalind Franklin, then Watson and Crick [1], established the architecture of deoxyribose nucleic acid (DNA), carrier of all genetic information. The idea that DNA was structurally organised in the form of a double helix comprising two antiparallel and complementary polymer chains was one of the great scientific discoveries of the twentieth century. It revealed not only the way in which genetic information is stored, but also the mechanism by which the genetic code is read, and the way this code can be faultlessly copied from one cell to another during cell division.

  16. Reverse gyrase functions in genome integrity maintenance by protecting DNA breaks in vivo

    DEFF Research Database (Denmark)

    Han, Wenyuan; Feng, Xu; She, Qunxin

    2017-01-01

    Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophili...... genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo....

  17. Isolation and Characterization of Linear DNA Elements from the Mitochondria of Gaeumannomyces graminis†

    OpenAIRE

    Honeyman, Allen L.; Currier, Thomas C.

    1986-01-01

    Different Gaeumannomyces graminis strains of diverse geographic origin contain one or two small DNAs ranging in size from 7.2 to 10 kilobases. These DNAs exhibit different degrees of homology with each other. We have characterized these low-molecular-weight DNAs from one strain, Ha-01. These small DNAs, E1 and E2, are mitochondrial in origin and were isolated as linear molecules which exhibited an intrinsic difference in density from the high-molecular-weight DNA.

  18. Isolation and Characterization of Linear DNA Elements from the Mitochondria of Gaeumannomyces graminis.

    Science.gov (United States)

    Honeyman, A L; Currier, T C

    1986-10-01

    Different Gaeumannomyces graminis strains of diverse geographic origin contain one or two small DNAs ranging in size from 7.2 to 10 kilobases. These DNAs exhibit different degrees of homology with each other. We have characterized these low-molecular-weight DNAs from one strain, Ha-01. These small DNAs, E1 and E2, are mitochondrial in origin and were isolated as linear molecules which exhibited an intrinsic difference in density from the high-molecular-weight DNA.

  19. Non-Periodic Finite-Element Formulation of Orbital-Free Density Functional Theory

    Energy Technology Data Exchange (ETDEWEB)

    Gavini, V; Knap, J; Bhattacharya, K; Ortiz, M

    2006-10-06

    We propose an approach to perform orbital-free density functional theory calculations in a non-periodic setting using the finite-element method. We consider this a step towards constructing a seamless multi-scale approach for studying defects like vacancies, dislocations and cracks that require quantum mechanical resolution at the core and are sensitive to long range continuum stresses. In this paper, we describe a local real space variational formulation for orbital-free density functional theory, including the electrostatic terms and prove existence results. We prove the convergence of the finite-element approximation including numerical quadratures for our variational formulation. Finally, we demonstrate our method using examples.

  20. Higher-order adaptive finite-element methods for Kohn–Sham density functional theory

    Energy Technology Data Exchange (ETDEWEB)

    Motamarri, P. [Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States); Nowak, M.R. [Department of Electrical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States); Leiter, K.; Knap, J. [U.S. Army Research Labs, Aberdeen Proving Ground, Aberdeen, MD 21001 (United States); Gavini, V., E-mail: vikramg@umich.edu [Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48109 (United States)

    2013-11-15

    We present an efficient computational approach to perform real-space electronic structure calculations using an adaptive higher-order finite-element discretization of Kohn–Sham density-functional theory (DFT). To this end, we develop an a priori mesh-adaption technique to construct a close to optimal finite-element discretization of the problem. We further propose an efficient solution strategy for solving the discrete eigenvalue problem by using spectral finite-elements in conjunction with Gauss–Lobatto quadrature, and a Chebyshev acceleration technique for computing the occupied eigenspace. The proposed approach has been observed to provide a staggering 100–200-fold computational advantage over the solution of a generalized eigenvalue problem. Using the proposed solution procedure, we investigate the computational efficiency afforded by higher-order finite-element discretizations of the Kohn–Sham DFT problem. Our studies suggest that staggering computational savings—of the order of 1000-fold—relative to linear finite-elements can be realized, for both all-electron and local pseudopotential calculations, by using higher-order finite-element discretizations. On all the benchmark systems studied, we observe diminishing returns in computational savings beyond the sixth-order for accuracies commensurate with chemical accuracy, suggesting that the hexic spectral-element may be an optimal choice for the finite-element discretization of the Kohn–Sham DFT problem. A comparative study of the computational efficiency of the proposed higher-order finite-element discretizations suggests that the performance of finite-element basis is competing with the plane-wave discretization for non-periodic local pseudopotential calculations, and compares to the Gaussian basis for all-electron calculations to within an order of magnitude. Further, we demonstrate the capability of the proposed approach to compute the electronic structure of a metallic system containing 1688

  1. Global Analysis of DNA Methylation Variation in Adipose Tissue from Twins Reveals Links to Disease-Associated Variants in Distal Regulatory Elements

    Science.gov (United States)

    Grundberg, Elin; Meduri, Eshwar; Sandling, Johanna K.; Hedman, Åsa K.; Keildson, Sarah; Buil, Alfonso; Busche, Stephan; Yuan, Wei; Nisbet, James; Sekowska, Magdalena; Wilk, Alicja; Barrett, Amy; Small, Kerrin S.; Ge, Bing; Caron, Maxime; Shin, So-Youn; Ahmadi, Kourosh R.; Ainali, Chrysanthi; Barrett, Amy; Bataille, Veronique; Bell, Jordana T.; Buil, Alfonso; Deloukas, Panos; Dermitzakis, Emmanouil T.; Dimas, Antigone S.; Durbin, Richard; Glass, Daniel; Grundberg, Elin; Hassanali, Neelam; Hedman, Åsa K.; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lindgren, Cecilia M.; Lowe, Christopher E.; McCarthy, Mark I.; Meduri, Eshwar; di Meglio, Paola; Min, Josine L.; Montgomery, Stephen B.; Nestle, Frank O.; Nica, Alexandra C.; Nisbet, James; O’Rahilly, Stephen; Parts, Leopold; Potter, Simon; Sandling, Johanna; Sekowska, Magdalena; Shin, So-Youn; Small, Kerrin S.; Soranzo, Nicole; Spector, Tim D.; Surdulescu, Gabriela; Travers, Mary E.; Tsaprouni, Loukia; Tsoka, Sophia; Wilk, Alicja; Yang, Tsun-Po; Zondervan, Krina T.; Lathrop, Mark; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Bell, Jordana T.; Deloukas, Panos

    2013-01-01

    Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h2median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner. PMID:24183450

  2. Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

    Energy Technology Data Exchange (ETDEWEB)

    Stisova, Viktorie [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic); Goffinont, Stephane; Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire CNRS, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Davidkova, Marie, E-mail: davidkova@ujf.cas.c [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic)

    2010-08-15

    Signaling by estrogens, risk factors in breast cancer, is mediated through their binding to the estrogen receptor protein (ER), followed by the formation of a complex between ER and a DNA sequence, called estrogen response element (ERE). Anti-estrogens act as competitive inhibitors by blocking the signal transduction. We have studied in vitro the radiosensitivity of the complex between ERalpha, a subtype of this receptor, and a DNA fragment bearing ERE, as well as the influence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) on this radiosensitivity. We observe that the complex is destabilized upon irradiation with gamma rays in aerated aqueous solution. The analysis of the decrease of binding abilities of the two partners shows that destabilization is mainly due to the damage to the protein. The destabilization is reduced when irradiating in presence of tamoxifen and is increased in presence of estradiol. These effects are due to opposite influences of the ligands on the loss of binding ability of ER. The mechanism that can account for our results is: binding of estradiol or tamoxifen induces distinct structural changes of the ER ligand-binding domain that can trigger (by allostery) distinct structural changes of the ER DNA-binding domains and thus, can differently affect ER-ERE interaction.

  3. Benchmark investigation of diamondoid-functionalized electrodes for nanopore DNA sequencing

    Science.gov (United States)

    Sivaraman, Ganesh; Amorim, Rodrigo G.; Scheicher, Ralph H.; Fyta, Maria

    2016-10-01

    Small diamond-like particles, diamondoids, have been shown to effectively functionalize gold electrodes in order to sense DNA units passing between the nanopore-embedded electrodes. In this work, we present a comparative study of Au(111) electrodes functionalized with different derivatives of lower diamondoids. Focus is put on the electronic and transport properties of such electrodes for different DNA nucleotides placed within the electrode gap. The functionalization promotes a specific binding to DNA leading to different properties for the system, which provides a tool set to systematically improve the signal-to-noise ratio of the electronic measurements across the electrodes. Using quantum transport calculations, we compare the effectiveness of the different functionalized electrodes in distinguishing the four DNA nucleotides. Our results point to the most effective diamondoid functionalization of gold electrodes in view of biosensing applications.

  4. Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi Anemia

    Directory of Open Access Journals (Sweden)

    Robert M. Brosh

    2014-10-01

    Full Text Available The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA. FANCJ is linked to cancer suppression and DNA double strand break (DSB repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link (ICL repair and contributes to homologous recombination (HR. FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4 DNA structures that are known to affect cellular replication and transcription, and potentially plays a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity.

  5. Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities

    NARCIS (Netherlands)

    Uil, T.G.; Haisma, H.J.; Rots, Marianne

    2003-01-01

    Designer molecules that can specifically target pre-determined DNA sequences provide a means to modulate endogenous gene function. Different classes of sequence-specific DNA-binding agents have been developed, including triplex-forming molecules, synthetic polyamides and designer zinc finger protein

  6. Modeling shock waves using exponential interpolation functions with the Least-Squares Finite Element method

    Science.gov (United States)

    Smith, Bradford Scott, Jr.

    The hypothesis of this research is that exponential interpolation functions will approximate fluid properties at shock waves with less error than polynomial interpolation functions. Exponential interpolation functions are derived for the purpose of modeling sharp gradients. General equations for conservation of mass, momentum, and energy for an inviscid flow of a perfect gas are converted to finite element equations using the least-squares method. Boundary conditions and a mesh adaptation scheme are also presented. An oblique shock reflection problem is used as a benchmark to determine whether or not exponential interpolation provides any advantages over Lagrange polynomial interpolation. Using exponential interpolation in elements downstream of a shock and having edges coincident with the shock showed a slight reduction in the solution error. However there was very little qualitative difference between solutions using polynomial and exponential interpolation. Regardless of the type of interpolation used, the shocks were smeared and oscillations were present both upstream and downstream of the shock waves. When a mesh adaptation scheme was implemented, exponential elements adjacent to the shock waves became much smaller and the numerical solution diverged. Changing the exponential elements to polynomial elements yielded a convergent solution. There appears to be no significant advantage to using exponential interpolation in comparison to Lagrange polynomial interpolation.

  7. Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

    Science.gov (United States)

    Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

    2014-06-17

    CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single

  8. Identification of two new repetitive elements and chromosomal mapping of repetitive DNA sequences in the fish Gymnothorax unicolor (Anguilliformes: Muraenidae

    Directory of Open Access Journals (Sweden)

    E. Coluccia

    2011-05-01

    Full Text Available Muraenidae is a species-rich family, with relationships among genera and species and taxonomy that have not been completely clarified. Few cytogenetic studies have been conducted on this family, and all of them showed the same diploid chromosome number (2n=42 but with conspicuous karyotypic variation among species. The Mediterranean moray eel Gymnothorax unicolor was previously cytogenetically studied using classical techniques that allowed the characterization of its karyotype structure and the constitutive heterochromatin and argyrophilic nucleolar organizer regions (Ag-NORs distribution pattern. In the present study, we describe two new repetitive elements (called GuMboI and GuDdeI obtained from restricted genomic DNA of G. unicolor that were characterized by Southern blot and physically localized by in situ hybridization on metaphase chromosomes. As they are highly repetitive DNA sequences, they map in heterochromatic regions. However, while GuDdeI was localized in the centromeric regions, the GuMboI fraction was distributed on some centromeres and was co-localized with the nucleolus organizer region (NOR. Comparative analysis with other Mediterranean species such as Muraena helena pointed out that these DNA fractions are species-specific and could potentially be used for species discrimination. As a new contribution to the karyotype of this species, we found that the major ribosomal genes are localized on acrocentric chromosome 9 and that the telomeres of each chromosome are composed of a tandem repeat derived from a poly-TTAGGG DNA sequence, as it occurs in most vertebrate species. The results obtained add new information useful in comparative genomics at the chromosomal level and contribute to the cytogenetic knowledge regarding this fish family, which has not been extensively studied.

  9. Genetic and Functional Studies of Genes That Regulate DNA-Damage-Induced Cell Death

    Science.gov (United States)

    2005-11-01

    library analysis indicated that the BRCT domains from BRCA1, MDC1, BARD1, and DNA Ligase IV preferred distinct phosphoserine-containing peptides. In...Heterodimerization between single BRCT domains (e.g. XRCC1 and DNA Ligase III) has been reported (9, 10). In addition, BRCT domains of 53BP1 can...pS-[DE]-[DE]-E. DNA Ligase IV functions to join single-strand breaks in double- stranded DNA (28, 29). It plays a major role in V(D)J recombina- tion

  10. Band-limited Green's Functions for Quantitative Evaluation of Acoustic Emission Using the Finite Element Method

    Science.gov (United States)

    Leser, William P.; Yuan, Fuh-Gwo; Leser, William P.

    2013-01-01

    A method of numerically estimating dynamic Green's functions using the finite element method is proposed. These Green's functions are accurate in a limited frequency range dependent on the mesh size used to generate them. This range can often match or exceed the frequency sensitivity of the traditional acoustic emission sensors. An algorithm is also developed to characterize an acoustic emission source by obtaining information about its strength and temporal dependence. This information can then be used to reproduce the source in a finite element model for further analysis. Numerical examples are presented that demonstrate the ability of the band-limited Green's functions approach to determine the moment tensor coefficients of several reference signals to within seven percent, as well as accurately reproduce the source-time function.

  11. Biological Sensors Using DNA Functionalized Multiwalled Carbon Nanotubes

    Science.gov (United States)

    2009-10-01

    out using potassium ferro cyanide as the redox electrolyte. Cyclic voltammetry gave well-defined quasi-reversible voltammetric responses, flat and...performance studies have been carried out using potassium ferro cyanide as the redox electrolyte and DNA-MWNT film electrode and results have been discussed...for the synthesis of MWNT in large quantities using Mischmetal (Bharat Rare Earths Metals, India; composition:- Ce 50%, La 35%, Pr 8 %, Nd 5%, Fe

  12. DNA damage-dependent regulation of MyoD function

    OpenAIRE

    Simonatto, Marta

    1980-01-01

    Durante la rigenerazione muscolare, le cellule progenitrici sono esposte ad una serie di stimoli extracellulari che coordinano la loro attivazione, proliferazione e differenziamento. Al tempo stesso, tuttavia, l’ambiente rigenerativo, stimolando la sintesi del DNA e aumentando lo stato di ossidazione intracellulare, costituisce una fonte di stress genotossico, come mostriamo in vivo in questo lavoro. Uno studio svolto in precedenza nel nostro laboratorio ha identificato e ca...

  13. Hepatitis viruses exploitation of host DNA methyltransferases functions.

    Science.gov (United States)

    Pazienza, Valerio; Panebianco, Concetta; Andriulli, Angelo

    2016-08-01

    Hepatitis B virus (HBV), hepatitis C virus (HCV) and Delta (HDV) infections are a global health burden. With different routes of infection and biology, HBV, HCV and HDV are capable to induce liver cirrhosis and cancer by impinging on epigenetic mechanisms altering host cell's pathways. In the present manuscript, we reviewed the published studies taking into account the relationship between the hepatitis viruses and the DNA methyltransferases proteins.

  14. IMPORTANCE OF ACTIVATED CARBON'S OXYGEN SURFACE FUNCTIONAL GROUPS ON ELEMENTAL MERCURY ADSORPTION

    Science.gov (United States)

    The effect of varying physical and chemical properties of activated carbons on adsorption of elemental mercury [Hg(0)] was studied by treating two activated carbons to modify their surface functional groups and pore structures. Heat treatment (1200 K) in nitrogen (N2), air oxidat...

  15. Non-linear shape functions over time in the space-time finite element method

    Directory of Open Access Journals (Sweden)

    Kacprzyk Zbigniew

    2017-01-01

    Full Text Available This work presents a generalisation of the space-time finite element method proposed by Kączkowski in his seminal of 1970’s and early 1980’s works. Kączkowski used linear shape functions in time. The recurrence formula obtained by Kączkowski was conditionally stable. In this paper, non-linear shape functions in time are proposed.

  16. Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn-On DNA Probes.

    Science.gov (United States)

    Uno, Kakishi; Sasaki, Taeko; Sugimoto, Nagisa; Ito, Hideto; Nishihara, Taishi; Hagihara, Shinya; Higashiyama, Tetsuya; Sasaki, Narie; Sato, Yoshikatsu; Itami, Kenichiro

    2017-01-17

    Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure-property relationship study has revealed that the dialkylamino group at the 2-position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double-stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2-position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser-scanning microscope.

  17. DNA-Damage-Induced Type I Interferon Promotes Senescence and Inhibits Stem Cell Function

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    2015-05-01

    Full Text Available Expression of type I interferons (IFNs can be induced by DNA-damaging agents, but the mechanisms and significance of this regulation are not completely understood. We found that the transcription factor IRF3, activated in an ATM-IKKα/β-dependent manner, stimulates cell-autonomous IFN-β expression in response to double-stranded DNA breaks. Cells and tissues with accumulating DNA damage produce endogenous IFN-β and stimulate IFN signaling in vitro and in vivo. In turn, IFN acts to amplify DNA-damage responses, activate the p53 pathway, promote senescence, and inhibit stem cell function in response to telomere shortening. Inactivation of the IFN pathway abrogates the development of diverse progeric phenotypes and extends the lifespan of Terc knockout mice. These data identify DNA-damage-response-induced IFN signaling as a critical mechanism that links accumulating DNA damage with senescence and premature aging.

  18. Identification of scaffold/Matrix Attachment (S/MAR like DNA element from the gastrointestinal protozoan parasite Giardia lamblia

    Directory of Open Access Journals (Sweden)

    Gautam Pennathur

    2010-06-01

    Full Text Available Abstract Background Chromatin in the nucleus of all eukaryotes is organized into a system of loops and domains. These loops remain fastened at their bases to the fundamental framework of the nucleus, the matrix or the scaffold. The DNA sequences which anchor the bases of the chromatin loops to the matrix are known as Scaffold/Matrix Attachment Regions or S/MARs. Though S/MARs have been studied in yeast and higher eukaryotes and they have been found to be associated with gene organization and regulation of gene expression, they have not been reported in protists like Giardia. Several tools have been discovered and formulated to predict S/MARs from a genome of a higher eukaryote which take into account a number of features. However, the lack of a definitive consensus sequence in S/MARs and the randomness of the protozoan genome in general, make it a challenge to predict and identify such sequences from protists. Results Here, we have analysed the Giardia genome for the probable S/MARs predicted by the available computational tools; and then shown these sequences to be physically associated with the nuclear matrix. Our study also reflects that while no single computational tool is competent to predict such complex elements from protist genomes, a combination of tools followed by experimental verification is the only way to confirm the presence of these elements from these organisms. Conclusion This is the first report of S/MAR elements from the protozoan parasite Giardia lamblia. This initial work is expected to lay a framework for future studies relating to genome organization as well as gene regulatory elements in this parasite.

  19. Mycoplasma pneumoniae large DNA repetitive elements RepMP1 show type specific organization among strains.

    Directory of Open Access Journals (Sweden)

    Oxana Musatovova

    Full Text Available Mycoplasma pneumoniae is the smallest self-replicating bacterium with a streamlined genome of 0.81 Mb. Complete genome analysis revealed the presence of multiple copies of four large repetitive elements (designated RepMP1, RepMP2/3, RepMP4 and RepMP5 that are implicated in creating sequence variations among individual strains. Recently, we described RepMP1-associated sequence variations between reference strain M129 and clinical isolate S1 that involved three RepMP1-genes (i.e. mpn130, mpn137 and mpn138. Using PCR and sequencing we analyze 28 additional M. pneumoniae strains and demonstrate the existence of S1-like sequence variants in nine strains and M129-like variants in the remaining nineteen strains. We propose a series of recombination steps that facilitates transition from M129- to S1-like sequence variants. Next we examined the remaining RepMP1-genes and observed no other rearrangements related to the repeat element. The only other detected difference was varying numbers of the 21-nucleotide tandem repeats within mpn127, mpn137, mpn501 and mpn524. Furthermore, typing of strains through analysis of large RepMPs localized within the adhesin P1 operon revealed that sequence divergence involving RepMP1-genes mpn130, mpn137 and mpn138 is strictly type-specific. Once more our analysis confirmed existence of two highly conserved groups of M. pneumoniae strains.

  20. Elemental carbon exposure and lung function in school children from Mexico City.

    Science.gov (United States)

    Barraza-Villarreal, A; Escamilla-Nuñez, M C; Hernández-Cadena, L; Texcalac-Sangrador, J L; Sienra-Monge, J J; Del Río-Navarro, B E; Cortez-Lugo, M; Sly, P D; Romieu, I

    2011-09-01

    Though exposure to air pollution has a detrimental effect on respiratory health, few studies have examined the association between elemental carbon exposure and lung function among schoolchildren. The aim of the present study was to present the association between short-term elemental carbon exposure and lung function in schoolchildren from Mexico City. 55 asthmatic and 40 non-asthmatic children were followed for an average of 22 weeks. A spirometry test was performed every 15 days during follow-up. Portable air samplers collected particulate matter onto Teflon filters. Gravimetric analysis was conducted and elemental carbon was quantified using transmission densitometry. The association between the main variables was analysed using linear mixed effects models. The mean ± sd of elemental carbon light absorption was 92.7 ± 54.7 Mm(-1). An increase of one interquartile range in the 24-h average of elemental carbon (100.93 Mm(-1)) was associated with a significant negative impact on forced expiratory volume in 1 s (FEV(1)) (-62.0 (95% CI -123.3- -1.2) mL) and forced expiratory flow at 25-75% of forced vital capacity (FVC) (FEF(25-75%)) (-111 (95% CI -228.3- -4.1) mL) among asthmatic children, equal to 3.3% and 5.5%, respectively; and on FEV(1) (-95.0 (95% CI -182.3- -8.5) mL) and FVC (-105.0 (95% CI -197.0- -13.7) mL) among non-asthmatic children. Exposure to elemental carbon resulted in an important negative effect on lung function in atopic schoolchildren, regardless of asthma status.

  1. Function of ZFAND3 in the DNA Damage Response

    Science.gov (United States)

    2013-06-01

    by flow-cytometry. Figure 2. ZFAND3 silencing does not hypersensitize cells to HU. Sensitivity to HU was determined after depletion of ZFAND3 (using...proteins cause hypersensitivity to replication DDR Kinase-Dependent Regulation of DNA Repair Advanced Online Article. Cite this article as Cold Spring...heat inactivated fetal bovine serum (FBS), 50 U/ml penicillin , 50 mg/ml streptomycin, and 2 mM L-glutamine. Hut78 (Sézary Syndrome) cells (ATCC) were

  2. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  3. A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors.

    Science.gov (United States)

    Jaffrelo, Loic; Chabas, Sandrine; Reigadas, Sandrine; Pflieger, Aude; Wychowski, Czeslaw; Rumi, Julie; Ventura, Michel; Toulmé, Jean-Jacques; Staedel, Cathy

    2008-09-01

    We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5' end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.

  4. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    Science.gov (United States)

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  5. Replicating function of the RS1 element associated with Vibrio cholerae CTX phi prophage.

    Science.gov (United States)

    Campos, J; Fando, R; Silva, A; Rodriguez, B L; Benitez, J A

    1998-07-01

    The RS1 element associated with Vibrio cholerae CTX phi prophage was cloned from an E1 Tor biotype Vibrio cholerae strain. We used the recA- vaccine strain Peru-15, that lacks the target for RS-mediated site-specific integration, to show that RS1 promotes autonomous replication of a suicide vector. A linker insertion in the rstR open reading frame abolished autonomous replication in Peru-15 but not in a strain containing an RS1 in the chromosome. An AT-rich region containing cis-acting elements involved in autonomous replication was identified by deletion. This region was sufficient to support autonomous replication in a strain containing an RS1 in the chromosome. DNA sequence analysis of a region present in RS1 and not RS2 revealed the presence of putative binding sites for host proteins involved in plasmid replication. These results indicate that RS1 contains a replicon distinct from RS2 which could be involved in replicative recombination events associated with tandem amplification of the CTX element.

  6. A Fortran program to calculate the matrix elements of the Coulomb interaction involving hydrogenic wave functions

    Science.gov (United States)

    Sarkadi, L.

    2017-03-01

    The program MTRXCOUL [1] calculates the matrix elements of the Coulomb interaction between a charged particle and an atomic electron, ∫ ψf∗ (r) | R - r | - 1ψi(r) d r. Bound-free transitions are considered, and non-relativistic hydrogenic wave functions are used. In this revised version a bug discovered in the F3Y CPC Program Library (PL) subprogram [2] is fixed. Furthermore, the COULCC CPC PL subprogram [3] applied for the calculations of the radial wave functions of the free states and the Bessel functions is replaced by the CPC PL subprogram DCOUL [4].

  7. Correlation functions of scattering matrix elements in microwave cavities with strong absorption

    Energy Technology Data Exchange (ETDEWEB)

    Schaefer, R [Fachbereich Physik, Philipps-Universitaet Marburg, Renthof 5, D-35032 Marburg (Germany); Gorin, T [Theoretische Quantendynamik, Fakultaet fuer Physik, Universitaet Freiburg, Hermann-Herder-Str. 3, D-79104 Freiburg (Germany); Seligman, T H [Centro de Ciencias Fisicas, Universidad Nacional Autonoma de Mexico, Campus Morelos, CP 62251, Cuernavaca, Morelos (Mexico); Stoeckmann, H-J [Fachbereich Physik, Philipps-Universitaet Marburg, Renthof 5, D-35032 Marburg (Germany)

    2003-03-28

    The scattering matrix was measured for microwave cavities with two antennae. It was analysed in the regime of overlapping resonances. The theoretical description in terms of a statistical scattering matrix and the rescaled Breit-Wigner approximation has been applied to this regime. The experimental results for the auto-correlation function show that the absorption in the cavity walls yields an exponential decay. This behaviour can only be modelled using a large number of weakly coupled channels. In comparison to the auto-correlation functions, the cross-correlation functions of the diagonal S-matrix elements display a more pronounced difference between regular and chaotic systems.

  8. Correlation functions of scattering matrix elements in microwave cavities with strong absorption

    Science.gov (United States)

    Schäfer, R.; Gorin, T.; Seligman, T. H.; Stöckmann, H.-J.

    2003-03-01

    The scattering matrix was measured for microwave cavities with two antennae. It was analysed in the regime of overlapping resonances. The theoretical description in terms of a statistical scattering matrix and the rescaled Breit-Wigner approximation has been applied to this regime. The experimental results for the auto-correlation function show that the absorption in the cavity walls yields an exponential decay. This behaviour can only be modelled using a large number of weakly coupled channels. In comparison to the auto-correlation functions, the cross-correlation functions of the diagonal S-matrix elements display a more pronounced difference between regular and chaotic systems.

  9. Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

    Science.gov (United States)

    Scarpato, Margherita; Angelini, Claudia; Cocca, Ennio; Pallotta, Maria M; Morescalchi, Maria A; Capriglione, Teresa

    2015-09-01

    In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3' untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation.

  10. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    Directory of Open Access Journals (Sweden)

    Guillaume eRiviere

    2014-04-01

    Full Text Available DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster’s developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5’-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment.

  11. Genomic Organization of Repetitive DNA Elements and Its Implications for the Chromosomal Evolution of Channid Fishes (Actinopterygii, Perciformes)

    Science.gov (United States)

    Cioffi, Marcelo de Bello; Bertollo, Luiz Antonio Carlos; Villa, Mateo Andres; de Oliveira, Ezequiel Aguiar; Tanomtong, Alongklod; Yano, Cassia Fernanda; Supiwong, Weerayuth; Chaveerach, Arunrat

    2015-01-01

    Channid fishes, commonly referred to as “snakeheads”, are currently very important in Asian fishery and aquaculture due to the substantial decline in natural populations because of overexploitation. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. In this study, we analyzed the karyotype structure and the distribution of 7 repetitive DNA sequences in several Channa species from different Thailand river basins. The aim of this study was to investigate the chromosomal differentiation among species and populations to improve upon the knowledge of its biodiversity and evolutionary history. Rearrangements, such as pericentric inversions, fusions and polyploidization, appear to be important events during the karyotypic evolution of this genus, resulting in the chromosomal diversity observed among the distinct species and even among populations of the same species. In addition, such variability is also increased by the genomic dynamism of repetitive elements, particularly by the differential distribution and accumulation of rDNA sequences on chromosomes. This marked diversity is likely linked to the lifestyle of the snakehead fishes and their population fragmentation, as already identified for other fish species. The karyotypic features highlight the biodiversity of the channid fishes and justify a taxonomic revision of the genus Channa, as well as of the Channidae family as a whole, as some nominal species may actually constitute species complexes. PMID:26067030

  12. Genomic Organization of Repetitive DNA Elements and Its Implications for the Chromosomal Evolution of Channid Fishes (Actinopterygii, Perciformes.

    Directory of Open Access Journals (Sweden)

    Marcelo de Bello Cioffi

    Full Text Available Channid fishes, commonly referred to as "snakeheads", are currently very important in Asian fishery and aquaculture due to the substantial decline in natural populations because of overexploitation. A large degree of chromosomal variation has been found in this family, mainly through the use of conventional cytogenetic investigations. In this study, we analyzed the karyotype structure and the distribution of 7 repetitive DNA sequences in several Channa species from different Thailand river basins. The aim of this study was to investigate the chromosomal differentiation among species and populations to improve upon the knowledge of its biodiversity and evolutionary history. Rearrangements, such as pericentric inversions, fusions and polyploidization, appear to be important events during the karyotypic evolution of this genus, resulting in the chromosomal diversity observed among the distinct species and even among populations of the same species. In addition, such variability is also increased by the genomic dynamism of repetitive elements, particularly by the differential distribution and accumulation of rDNA sequences on chromosomes. This marked diversity is likely linked to the lifestyle of the snakehead fishes and their population fragmentation, as already identified for other fish species. The karyotypic features highlight the biodiversity of the channid fishes and justify a taxonomic revision of the genus Channa, as well as of the Channidae family as a whole, as some nominal species may actually constitute species complexes.

  13. The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Wiep Klaas Smits

    2010-11-01

    Full Text Available The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.

  14. The transcriptional regulator Rok binds A+T-rich DNA and is involved in repression of a mobile genetic element in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Wiep Klaas Smits

    2010-11-01

    Full Text Available The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria.

  15. Micro-optical elements functioning in non-visible spectral range

    Science.gov (United States)

    Wang, Qin; Zhang, Andy Z. Z.; Bergström, Andreas; Huo, Vicky Z. J.; Almqvist, Susanne; Kaplan, Wlodek; Andersson, Jan Y.

    2010-05-01

    Nowadays novel micro-fabrication and wafer-based manufacturing approach allows realizing micro-optics in a way scientists have dreamt for generations, in particular, utilizing nano-imprint lithography as fabrication tooling enables greatly accelerating the micro-optics technology to its frontier. In this report, we present wafer-scale fabrication of various types of micro-optical elements based on photoresist, benzocyclobutene, photocurable imprint resist, and semiconductor materials by using thermal reflow, reactive ion etching, and imprint techniques. Especially, several concave or convex 3-dimensional micro-optical structures shaped by imprint method are detailed. These micro-optical elements can be monolithically or hybrid integrated onto optoelectronics devices, such as photodetectors and emitters as optical beam focuser, collimator, filter, or anti-reflectance elements. As application examples, polymer microlenses were integrated directly on the top of UV dual functional devices and quantum dot long wavelength infrared photodetectors, respectively.

  16. Hybrid graded element model for transient heat conduction in functionally graded materials

    Institute of Scientific and Technical Information of China (English)

    Lei-Lei Cao; Qing-Hua Qin; Ning Zhao

    2012-01-01

    This paper presents a hybrid graded element model for the transient heat conduction problem in functionally graded materials (FGMs).First,a Laplace transform approach is used to handle the time variable.Then,a fundamental solution in Laplace space for FGMs is constructed.Next,a hybrid graded element is formulated based on the obtained fundamental solution and a frame field.As a result,the graded properties of FGMs are naturally reflected by using the fundamental solution to interpolate the intra-element field.Further,Stefest's algorithm is employed to convert the results in Laplace space back into the time-space domain.Finally,the performance of the proposed method is assessed by several benchmark examples.The results demonstrate well the efficiency and accuracy of the proposed method.

  17. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

    Institute of Scientific and Technical Information of China (English)

    GAO Guanjun; LU Huiming; HUANG Lifen; HUA Yuejin

    2005-01-01

    PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. Radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. Radiodurans. The fragments containing complete pprI gene and 3'-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. Raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. Radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.

  18. Finite element analysis (FEA): applying an engineering method to functional morphology in anthropology and human biology.

    Science.gov (United States)

    Panagiotopoulou, O

    2009-01-01

    A fundamental research question for morphologists is how morphological variation in the skeleton relates to function. Traditional approaches have advanced our understanding of form-function relationships considerably but have limitations. Strain gauges can only record strains on a surface, and the geometry of the structure can limit where they can be bonded. Theoretical approaches, such as geometric abstractions, work well on problems with simple geometries and material properties but biological structures typically have neither of these. Finite element analysis (FEA) is a method that overcomes these problems by reducing a complex geometry into a finite number of elements with simple geometries. In addition, FEA allows strain to be modelled across the entire surface of the structure and throughout the internal structure. With advances in the processing power of computers, FEA has become more accessible and as such is becoming an increasingly popular tool to address questions about form-function relationships in development and evolution, as well as human biology generally. This paper provides an introduction to FEA including a review of the sequence of steps needed for the generation of biologically accurate finite element models that can be used for the testing of biological and functional morphology hypotheses.

  19. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Science.gov (United States)

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology.

  20. Genetic evidence for conserved non-coding element function across species--the ears have it

    Directory of Open Access Journals (Sweden)

    Eric E Turner

    2014-01-01

    Full Text Available Comparison of genomic sequences from diverse vertebrate species has revealed numerous highly conserved regions that do not appear to encode proteins or functional RNAs. Often these conserved non-coding elements, or CNEs, direct gene expression to specific tissues in transgenic models, demonstrating they have regulatory function. CNEs are frequently found near ‘developmental’ genes, particularly transcription factors, implying that these elements have essential regulatory roles in development. However, actual examples demonstrating CNE regulatory functions across species have been few, and recent loss-of-function studies of several CNEs in mice have shown relatively minor effects. In this Perspectives article, we discuss new findings in fancy rats and Highland cattle demonstrating that function of a CNE near the Hmx1 gene is crucial for normal external ear development and resembles loss-of function Hmx1 coding mutations in mice and humans. These findings provide important support for similar developmental roles of CNEs in divergent species, and reinforce the concept that CNEs should be examined systematically in the ongoing search for genetic causes of human developmental disorders in the era of genome-scale sequencing.

  1. Enriched Element-Free Galerkin Method for Fracture Analysis of Functionally Graded Piezoelectric Materials

    Directory of Open Access Journals (Sweden)

    Guang Wei Meng

    2015-01-01

    Full Text Available A new method using the enriched element-free Galerkin method (EEFGM to model functionally graded piezoelectric materials (FGPMs with cracks was presented. To improve the solution accuracy, extended terms were introduced into the approximation function of the conventional element-free Galerkin method (EFGM to describe the displacement and electric fields near the crack. Compared with the conventional EFGM, the new approach requires smaller domain to describe the crack-tip singular field. Additionally, the domain of the nodes was not affected by the crack. Therefore, the visibility method and the diffraction method were no longer needed. The mechanical response of FGPM was discussed, when its material parameters changed exponentially in a certain direction. The modified J-integrals for FGPM were deduced, whose results were compared with the results of the conventional EFGM and the analytical solution. Numerical example results illustrated that this method is feasible and precise.

  2. A novel human polycomb binding site acts as a functional polycomb response element in Drosophila.

    Directory of Open Access Journals (Sweden)

    Suresh Cuddapah

    Full Text Available Polycomb group (PcG proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs, which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.

  3. Intermediate elemental image reconstruction for refocused three-dimensional images in integral imaging by convolution with δ-function sequences

    Science.gov (United States)

    Yoo, Hoon; Jang, Jae-Young

    2017-10-01

    We propose a novel approach for intermediate elemental image reconstruction in integral imaging. To reconstruct intermediate elemental images, we introduce a null elemental image whose pixels are all zero. In the proposed method a number of null elemental images are inserted into a given elemental image array. The elemental image array with null elemental images is convolved with the δ-function sequence. The convolution result shows that the proposed method provides an efficient structure to expand an elemental image array. The resulting elemental image array from the proposed method can supply three-dimensional information for an object at a specific depth. In addition, the proposed method provides adjustable parameters, which can be utilized in design of integral imaging systems. The feasibility of the proposed method has been confirmed through preliminary experiments and theoretical analysis.

  4. Acoustic Transfer Functions Derived from Finite Element Modeling for Thermoacoustic Stability Predictions of Gas Turbine Engines

    OpenAIRE

    Black, Paul Randall

    2007-01-01

    Acoustic Transfer Functions Derived from Finite Element Modeling for Thermoacoustic Stability Predictions of Gas Turbine Engines Design and prediction of thermoacoustic instabilities is a major challenge in aerospace propulsion and the operation of power generating gas turbine engines. This is a complex problem in which multiple physical systems couple together. Traditionally, thermoacoustic models can be reduced to dominant physics which depend only on flame dynamics and acoustics. Th...

  5. Calculation of the matrix elements of the Coulomb interaction involving relativistic hydrogenic wave functions

    Science.gov (United States)

    Sarkadi, L.

    2017-03-01

    The program MTRDCOUL [1] calculates the matrix elements of the Coulomb interaction between a charged particle and an atomic electron, ∫ ψf∗ (r) ∣ R - r∣-1ψi(r) d r. Bound-free transitions are considered, and relativistic hydrogenic wave functions are used. In this revised version a bug discovered in the F3Y CPC Program Library subprogram [2] is fixed.

  6. Streptococcal group B integrative and mobilizable element IMESag-rpsI encodes a functional relaxase involved in its transfer

    Science.gov (United States)

    Lorenzo-Diaz, Fabian; Fernández-Lopez, Cris; Douarre, Pierre-Emmanuel; Baez-Ortega, Adrian; Flores, Carlos; Glaser, Philippe

    2016-01-01

    Streptococcus agalactiae or Group B Streptococcus (GBS) are opportunistic bacteria that can cause lethal sepsis in children and immuno-compromised patients. Their genome is a reservoir of mobile genetic elements that can be horizontally transferred. Among them, integrative and conjugative elements (ICEs) and the smaller integrative and mobilizable elements (IMEs) primarily reside in the bacterial chromosome, yet have the ability to be transferred between cells by conjugation. ICEs and IMEs are therefore a source of genetic variability that participates in the spread of antibiotic resistance. Although IMEs seem to be the most prevalent class of elements transferable by conjugation, they are poorly known. Here, we have studied a GBS-IME, termed IMESag-rpsI, which is widely distributed in GBS despite not carrying any apparent virulence trait. Analyses of 240 whole genomes showed that IMESag-rpsI is present in approximately 47% of the genomes, has a roughly constant size (approx. 9 kb) and is always integrated at a single location, the 3′-end of the gene encoding the ribosomal protein S9 (rpsI). Based on their genetic variation, several IMESag-rpsI types were defined (A–J) and classified in clonal complexes (CCs). CC1 was the most populated by IMESag-rpsI (more than 95%), mostly of type-A (71%). One CC1 strain (S. agalactiae HRC) was deep-sequenced to understand the rationale underlying type-A IMESag-rpsI enrichment in GBS. Thirteen open reading frames were identified, one of them encoding a protein (MobSag) belonging to the broadly distributed family of relaxases MOBV1. Protein MobSag was purified and, by a newly developed method, shown to cleave DNA at a specific dinucleotide. The S. agalactiae HRC-IMESag-rpsI is able to excise from the chromosome, as shown by the presence of circular intermediates, and it harbours a fully functional mobilization module. Further, the mobSag gene encoded by this mobile element is able to promote plasmid transfer among pneumococcal

  7. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  8. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    Science.gov (United States)

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-03-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

  9. RICD: A rice indica cDNA database resource for rice functional genomics

    Directory of Open Access Journals (Sweden)

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  10. DNA biosensors implemented on PNA-functionalized microstructured optical fibers Bragg gratings

    Science.gov (United States)

    Candiani, A.; Giannetti, S.; Cucinotta, A.; Bertucci, A.; Manicardi, A.; Konstantaki, M.; Margulis, W.; Pissadakis, S.; Corradini, R.; Selleri, S.

    2013-05-01

    A novel DNA sensing platform based on a Peptide Nucleic Acid - functionalized Microstructured Optical Fibers gratings has been demonstrated. The inner surface of different MOFs has been functionalized using PNA probes, OligoNucleotides mimic that are well suited for specific DNA target sequences detection. The hybrid sensing systems were tested for optical DNA detection of targets of relevance in biomedical application, using the cystic fibrosis gene mutation, and food-analysis, using the genomic DNA from genetic modified organism soy flour. After the solutions of DNA molecules has been infiltrated inside the fibers capillaries and hybridization has occurred, oligonucleotidefunctionalized gold nanoparticles were infiltrated and used to form a sandwich-like system to achieve signal amplification. Spectral measurements of the reflected signal reveal a clear wavelength shift of the reflected modes when the infiltrated complementary DNA matches with the PNA probes placed on the inner fiber surface. Measurements have also been made using the mismatched DNA solution for the c, containing a single nucleotide polymorphism, showing no significant changes in the reflected spectrum. Several experiments have been carried out demonstrating the reproducibility of the results and the high selectivity of the sensors, showing the simplicity and the potential of this approach.

  11. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  12. Tcf4 Regulates Synaptic Plasticity, DNA Methylation, and Memory Function

    Directory of Open Access Journals (Sweden)

    Andrew J. Kennedy

    2016-09-01

    Full Text Available Human haploinsufficiency of the transcription factor Tcf4 leads to a rare autism spectrum disorder called Pitt-Hopkins syndrome (PTHS, which is associated with severe language impairment and development delay. Here, we demonstrate that Tcf4 haploinsufficient mice have deficits in social interaction, ultrasonic vocalization, prepulse inhibition, and spatial and associative learning and memory. Despite learning deficits, Tcf4(+/− mice have enhanced long-term potentiation in the CA1 area of the hippocampus. In translationally oriented studies, we found that small-molecule HDAC inhibitors normalized hippocampal LTP and memory recall. A comprehensive set of next-generation sequencing experiments of hippocampal mRNA and methylated DNA isolated from Tcf4-deficient and WT mice before or shortly after experiential learning, with or without administration of vorinostat, identified “memory-associated” genes modulated by HDAC inhibition and dysregulated by Tcf4 haploinsufficiency. Finally, we observed that Hdac2 isoform-selective knockdown was sufficient to rescue memory deficits in Tcf4(+/− mice.

  13. RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.

    Science.gov (United States)

    van der Linden, Eddy; Sanchez, Humberto; Kinoshita, Eri; Kanaar, Roland; Wyman, Claire

    2009-04-01

    The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.

  14. DNA methylation and cognitive functioning in healthy older adults

    NARCIS (Netherlands)

    Schiepers, O.J.G.; Boxtel, van M.P.J.; Groot, R.H.M.; Jolles, J.; Kok, F.J.; Verhoef, P.; Durga, J.

    2012-01-01

    Long-term supplementation with folic acid may improve cognitive performance in older individuals. The relationship between folate status and cognitive performance might be mediated by changes in methylation capacity, as methylation reactions are important for normal functioning of the brain. Althoug

  15. Exploring function of conserved non-coding DNA in its chromosomal context

    Directory of Open Access Journals (Sweden)

    Delores J. Grant

    2015-11-01

    Full Text Available There is renewed interest in understanding expression of vertebrate genes in their chromosomal context because regulatory sequences that confer tissue-specific expression are often distributed over large distances along the DNA from the gene. One approach inserts a universal sensor/reporter-gene into the mouse or zebrafish genome to identify regulatory sequences in highly conserved non-coding DNA in the vicinity of the integrated reporter-gene. However detailed mechanisms of interaction of these regulatory elements among themselves and/or with the genes they influence remain elusive with the strategy. The inability to associate distant regulatory elements with the genes they regulate makes it difficult to examine the contribution of sequence changes in regulatory DNA to human disease. Such associations have been obtained in favorable circumstances by testing the regulatory potential of highly conserved non-coding DNA individually in small reporter-gene-containing plasmids. Alternative approaches use tiny fragments of chromosomes in Bacterial Artificial Chromosomes, BACs, where the gene of interest is tagged in vitro with a reporter/sensor gene and integrated into the germ-line of animals for expression. Mutational analysis of the BAC DNA identifies regulatory sequences. A recent approach inserts a sensor/reporter-gene into a BAC that is also truncated progressively from an end of genomic insert, and the end-deleted BAC carrying the sensor is then integrated into the genome of a developing animal for expression. The approach allows mechanisms of tissue-specific gene expression to be explored in much greater detail, although the chromosomal context of such mechanisms is limited to the length of the BAC. Here we discuss the relative strengths of the various approaches and explore how the integrated-sensor in the BACs method applied to a contig of BACs spanning a chromosomal region is likely to address mechanistic questions on interactions between

  16. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    Energy Technology Data Exchange (ETDEWEB)

    Jones, IM

    2002-01-09

    Exposure to ionizing radiation increases the incidence of cancer. However, predicting which individuals are at most risk from radiation exposure is a distant goal. Predictive ability is needed to guide policies that regulate radiation exposure and ensure that medical treatments have maximum benefit and minimum risk. Differences between people in susceptibility to radiation are largely based on their genotype, the genes inherited from their parents. Among the important genes are those that produce proteins that repair DNA damaged by radiation. Base Excision Repair (BER) proteins repair single strand breaks and oxidized bases in DNA. Double Strand Break Repair proteins repair broken chromosomes. Using technologies and information from the Human Genome Project, we have previously determined that the DNA sequence of DNA repair genes varies within the human population. An average of 3-4 different variants were found that affect the protein for each of 37 genes studied. The average frequency of these variants is 5%. Given the many genes in each DNA repair pathway and their many variants, technical ability to determine an individual's repair genotype greatly exceeds ability to interpret the information. A long-term goal is to relate DNA repair genotypes to health risk from radiation. This study focused on the BER pathway. The BER genes are known, variants of the genes have been identified at LLNL, and LLNL had recently developed an assay for BER function using white blood cells. The goal of this initial effort was to begin developing data that could be used to test the hypothesis that many different genotypes have similar DNA repair capacity phenotypes (function). Relationships between genotype and phenotype could then be used to group genotypes with similar function and ultimately test the association of groups of genotypes with health risk from radiation. Genotypes with reduced repair function are expected to increase risk of radiation-induced health effects. The

  17. Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Li Hui [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Haberzettl, Petra [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Albrecht, Catrin [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Hoehr, Doris [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Knaapen, Ad M. [Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), University of Maastricht (Netherlands); Borm, Paul J.A. [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Hogeschool Zuyd Heerlen (Netherlands); Schins, Roel P.F. [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany)]. E-mail: roel.schins@uni-duesseldorf.de

    2007-04-01

    Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism.

  18. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation

    OpenAIRE

    Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko

    2015-01-01

    A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX acc...

  19. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation

    OpenAIRE

    Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko

    2015-01-01

    A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ3-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ3-irradiation, including HP1β-, γ3H2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX ...

  20. Recent Advances in the Synthesis and Functions of Reconfigurable Interlocked DNA Nanostructures.

    Science.gov (United States)

    Lu, Chun-Hua; Cecconello, Alessandro; Willner, Itamar

    2016-04-27

    Interlocked circular DNA nanostructures, e.g., catenanes or rotaxanes, provide functional materials within the area of DNA nanotechnology. Specifically, the triggered reversible reconfiguration of the catenane or rotaxane structures provides a means to yield new DNA switches and to use them as dynamic scaffolds for controlling chemical functions and positioning functional cargoes. The synthesis of two-ring catenanes and their switchable reconfiguration by pH, metal ions, or fuel/anti-fuel stimuli are presented, and the functions of these systems, as pendulum or rotor devices or as switchable catalysts, are described. Also, the synthesis of three-, five-, and seven-ring catenanes is presented, and their switchable reconfiguration using fuel/anti-fuel strands is addressed. Implementation of the dynamically reconfigured catenane structures for the programmed organization of Au nanoparticle (NP) assemblies, which allows the plasmonic control of the fluorescence properties of Au NP/fluorophore loads associated with the scaffold, and for the operation of logic gates is discussed. Interlocked DNA rotaxanes and their different synthetic approaches are presented, and their switchable reconfiguration by means of fuel/anti-fuel strands or photonic stimuli is described. Specifically, the use of the rotaxane as a scaffold to organize Au NP assemblies, and the control of the fluorescence properties with Au NP/fluorophore hybrids loaded on the rotaxane scaffold, are introduced. The future prospectives and challenges in the field of interlocked DNA nanostructures and the possible applications are discussed.

  1. Matrix elements for sum of power-law potentials in quantum mechanic using generalized hypergeometric functions

    Directory of Open Access Journals (Sweden)

    Ma'zoozeh E. Abu-Amra

    2008-04-01

    Full Text Available In this paper we derive close form for the matrix elements for $hat H=-Delta +V$, where $V$ is a pure power-law potential. We use trial functions of the form $$ psi _n(r= sqrt{{frac{2eta ^{gamma/2}(gamma _n} {n!Gamma(gamma }}} r^{gamma - 1/2} e^{-frac{sqrt{eta }}{2}r^q} _pF_1 ( -n,a_2,ldots ,a_p;gamma;sqrt {eta } r^q, $$ for $eta, q,gamma >0$ to obtain the matrix elements for $hat H$. These formulas are then optimized with respect to variational parameters $eta ,q$ and $gamma $ to obtain accurate upper bounds for the given nonsolvable eigenvalue problem in quantum mechanics. Moreover, we write the matrix elements in terms of the generalized hypergeomtric functions. These results are generalization of those found earlier in [2], [8-16] for power-law potentials. Applications and comparisons with earlier work are presented.

  2. DNA methylation is a determinative element of photosynthesis gene expression in amyloplasts from liquid-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Ngernprasirtsiri, J; Kobayashi, H; Akazawa, T

    1990-10-01

    Transcriptional regulation has been shown to operate as a selective control mechanism of expression of photosynthetic genes in the nonphotosynthetic plastids, amyloplasts, of a white-wild cell line of sycamore (Acer pseudoplatanus L.). To elaborate the mechanisms governing the transcriptional regulation at the molecular level, we have examined the template activity of the amyloplast DNA compared to the chloroplast DNA by using the in vitro run-off transcription assay system with extracts of the two plastid types. The results of these assays clearly indicate that most of the amyloplast DNA regions do not serve as a template for the in vitro transcription regardless of the plastid extracts; this is in contrast to the chloroplast DNA which serves as an active template. It is highly likely that the template activity of amyloplast DNA per se is the modulating element of transcriptional regulation. Parallel experiments determining the DNA base content by HPLC analysis have shown that a variety of methylated bases, especially 5-methylcytosine, are localized in the DNA regions containing suppressed genes of the amyloplast genome. In sharp contrast, methylated bases were undetectable in the expressed gene regions of amyloplast and whole chloroplast genomes. The overall findings strongly support the notion that DNA methylation is involved in the selective suppression of photosynthetic genes in the nonphotosynthetic plastids of cultured sycamore cells.

  3. Evolutionary Dynamics of 5S rDNA and Recurrent Association of Transposable Elements in Electric Fish of the Family Gymnotidae (Gymnotiformes): The Case of Gymnotus mamiraua.

    Science.gov (United States)

    da Silva, Maelin; Barbosa, Patricia; Artoni, Roberto F; Feldberg, Eliana

    2016-01-01

    Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites. Isolation and cloning of the 5S rDNA sequences from G. mamiraua identified a fragment of a transposable element similar to the Tc1/mariner transposon associated with a non-transcribed spacer. Double fluorescence in situ hybridization analysis of this element and the 5S rDNA showed that they were colocalized on several chromosomes, in addition to acting as nonsyntenic markers on others. Our data show the association between these sequences and suggest that the Tc1 retrotransposon may be the agent that drives the spread of these 5S rDNA-like sequences in the G. mamiraua genome. © 2016 S. Karger AG, Basel.

  4. Study on the Explicit Formula of the Triangular Flat Shell Element Based on the Analytical Trial Functions for Anisotropy Material

    Directory of Open Access Journals (Sweden)

    Xiang-Rong Fu

    2013-01-01

    Full Text Available This paper presents a novel way to formulate the triangular flat shell element. The basic analytical solutions of membrane and bending plate problem for anisotropy material are studied separately. Combining with the conforming displacement along the sides and hybrid element strategy, the triangular flat shell elements based on the analytical trial functions (ATF for anisotropy material are formulated. By using the explicit integral formulae of the triangular element, the matrices used in proposed shell element are calculated efficiently. The benchmark examples showed the high accuracy and high efficiency.

  5. Multiscale finite element methods for high-contrast problems using local spectral basis functions

    KAUST Repository

    Efendiev, Yalchin

    2011-02-01

    In this paper we study multiscale finite element methods (MsFEMs) using spectral multiscale basis functions that are designed for high-contrast problems. Multiscale basis functions are constructed using eigenvectors of a carefully selected local spectral problem. This local spectral problem strongly depends on the choice of initial partition of unity functions. The resulting space enriches the initial multiscale space using eigenvectors of local spectral problem. The eigenvectors corresponding to small, asymptotically vanishing, eigenvalues detect important features of the solutions that are not captured by initial multiscale basis functions. Multiscale basis functions are constructed such that they span these eigenfunctions that correspond to small, asymptotically vanishing, eigenvalues. We present a convergence study that shows that the convergence rate (in energy norm) is proportional to (H/Λ*)1/2, where Λ* is proportional to the minimum of the eigenvalues that the corresponding eigenvectors are not included in the coarse space. Thus, we would like to reach to a larger eigenvalue with a smaller coarse space. This is accomplished with a careful choice of initial multiscale basis functions and the setup of the eigenvalue problems. Numerical results are presented to back-up our theoretical results and to show higher accuracy of MsFEMs with spectral multiscale basis functions. We also present a hierarchical construction of the eigenvectors that provides CPU savings. © 2010.

  6. Functionalization of Poly- (methyl methacrylate) (PMMA) as a substrate for DNA microarrays

    DEFF Research Database (Denmark)

    Fixe, A.F.; Dufva, Hans Martin; Telleman, Pieter

    2004-01-01

    amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from...... since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated......A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm(2). The availability of these primary...

  7. Regulatory mechanisms that prevent re-initiation of DNA replication can be locally modulated at origins by nearby sequence elements.

    Directory of Open Access Journals (Sweden)

    Christopher D Richardson

    2014-06-01

    Full Text Available Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs. We have mapped the two RIPs to ∼ 60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some

  8. Functions for patch test in finite element analysis of the Mindlin plate and the thin cylindrical shell

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Current patch test for Mindlin plate element only satisfies the zero shear deformation condition.The patch test of non-zero constant shear for Mindlin plate problem cannot be performed.For shell element, the patch test does not even exist.Based on the theory of enhanced patch test proposed by Chen W J (2006),the authors proposed the enhanced patch test function for Mindlin plate and thin cylindrical shell elements.This enhanced patch test function can be used to assess the convergence of the Mindlin plate and cylindrical thin shell elements.

  9. Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿ †

    Science.gov (United States)

    Palmeri, Diana; Carroll, Kyla Driscoll; Gonzalez-Lopez, Olga; Lukac, David M.

    2011-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is the etiologic agent of Kaposi's sarcoma (KS) and lymphoproliferative diseases. We previously demonstrated that the KSHV lytic switch protein Rta stimulates DNA binding of the cellular RBP-Jk/CSL protein, the nuclear component of the Notch pathway, on Rta target promoters. In the current study, we define the promoter requirements for formation of transcriptionally productive Rta/RBP-Jk/DNA complexes. We show that highly pure Rta footprints 7 copies of a previously undescribed repetitive element in the promoter of the essential KSHV Mta gene. We have termed this element the “CANT repeat.” CANT repeats are found on both strands of DNA and have a consensus sequence of ANTGTAACANT(A/T)(A/T)T. We demonstrate that Rta tetramers make high-affinity interactions (i.e., nM) with 64 bp of the Mta promoter but not single CANT units. The number of CANT repeats, their presence in palindromes, and their positions relative to the RBP-Jk binding site determine the optimal target for Rta stimulation of RBP-Jk DNA binding and formation of ternary Rta/RBP-Jk/DNA complexes. DNA binding and tetramerization mutants of Rta fail to stimulate RBP-Jk DNA binding. Our chromatin immunoprecipitation assays show that RBP-Jk DNA binding is broadly, but selectively, stimulated across the entire KSHV genome during reactivation. We propose a model in which tetramerization of Rta allows it to straddle RBP-Jk and contact repeat units on both sides of RBP-Jk. Our study integrates high-affinity Rta DNA binding with the requirement for a cellular transcription factor in Rta transactivation. PMID:21880753

  10. STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

    Science.gov (United States)

    Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

    2016-01-01

    Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

  11. DNA--a molecule in search of additional functions: recipient of pool wave emissions? A hypothesis.

    Science.gov (United States)

    Doerfler, Walter

    2010-09-01

    Almost the entire nucleotide sequence of human DNA is functionally unaccounted for, although large parts of the human genome are transcribed. The genes, as defined by current molecular biology, comprise about 1.5-2% of the DNA molecule. It is proposed that DNA encodes additional, hitherto unrecognized functions. In this discussion, the total information inside and outside the universe we live in is termed the pool or the sum total, known or unknown, of all laws, matter, energy, concepts and events. In a hypothetical model, a Gedankenexperiment, it is suggested that the total of all information emits pool waves of an unknown physical nature. They could be related to black energy or have completely different qualities. The designation pool waves should not imply any similarity to electromagnetism. Further, DNA is suggested to have the capability of interacting with the pool waves and thus permit humans - to some partly genetically determined and yet very limited extent - to perceive information from the pool. Pool emissions might be one of the forces that have been instrumental in and are still driving evolution from simple oligonucleotides to DNA with ever more complex recipient capacities. It will be a major challenge for researchers in the field to unravel these and less hypothetical undetected coding principles in DNA. It is uncertain whether the current trend to search the available DNA sequences with ever more refined computer technology on the basis of our present understanding of biology will detect unknown coding systems. For molecular medicine, research into the genetics of the most common human diseases could profit from the elucidation of presently still ephemeral codes in human DNA. Young scientists with a proven record of original research deserve support for the pursuit of unconventional ideas. This concept of granting priorities will be of the utmost importance in advancing the field beyond current concepts in molecular biology.

  12. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  13. Functional diversification of paralogous transcription factors via divergence in DNA binding site motif and in expression.

    Directory of Open Access Journals (Sweden)

    Larry N Singh

    Full Text Available BACKGROUND: Gene duplication is a major driver of evolutionary innovation as it allows for an organism to elaborate its existing biological functions via specialization or diversification of initially redundant gene paralogs. Gene function can diversify in several ways. Transcription factor gene paralogs in particular, can diversify either by changes in their tissue-specific expression pattern or by changes in the DNA binding site motif recognized by their protein product, which in turn alters their gene targets. The relationship between these two modes of functional diversification of transcription factor paralogs has not been previously investigated, and is essential for understanding adaptive evolution of transcription factor gene families. FINDINGS: Based on a large set of human paralogous transcription factor pairs, we show that when the DNA binding site motifs of transcription factor paralogs are similar, the expressions of the genes that encode the paralogs have diverged, so in general, at most one of the paralogs is highly expressed in a tissue. Moreover, paralogs with diverged DNA binding site motifs tend to be diverged in their function. Conversely, two paralogs that are highly expressed in a tissue tend to have dissimilar DNA binding site motifs. We have also found that in general, within a paralogous family, tissue-specific decrease in gene expression is more frequent than what is expected by chance. CONCLUSIONS: While previous investigations of paralogous gene diversification have only considered coding sequence divergence, by explicitly quantifying divergence in DNA binding site motif, our work presents a new paradigm for investigating functional diversification. Consistent with evolutionary expectation, our quantitative analysis suggests that paralogous transcription factors have survived extinction in part, either through diversification of their DNA binding site motifs or through alterations in their tissue-specific expression

  14. Uncoupling of satellite DNA and centromeric function in the genus Equus.

    Directory of Open Access Journals (Sweden)

    Francesca M Piras

    2010-02-01

    Full Text Available In a previous study, we showed that centromere repositioning, that is the shift along the chromosome of the centromeric function without DNA sequence rearrangement, has occurred frequently during the evolution of the genus Equus. In this work, the analysis of the chromosomal distribution of satellite tandem repeats in Equus caballus, E. asinus, E. grevyi, and E. burchelli highlighted two atypical features: 1 several centromeres, including the previously described evolutionary new centromeres (ENCs, seem to be devoid of satellite DNA, and 2 satellite repeats are often present at non-centromeric termini, probably corresponding to relics of ancestral now inactive centromeres. Immuno-FISH experiments using satellite DNA and antibodies against the kinetochore protein CENP-A demonstrated that satellite-less primary constrictions are actually endowed with centromeric function. The phylogenetic reconstruction of centromere repositioning events demonstrates that the acquisition of satellite DNA occurs after the formation of the centromere during evolution and that centromeres can function over millions of years and many generations without detectable satellite DNA. The rapidly evolving Equus species gave us the opportunity to identify different intermediate steps along the full maturation of ENCs.

  15. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Directory of Open Access Journals (Sweden)

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  16. Diamondoid-functionalized gold nanogaps as sensors for natural, mutated, and epigenetically modified DNA nucleotides.

    Science.gov (United States)

    Sivaraman, Ganesh; Amorim, Rodrigo G; Scheicher, Ralph H; Fyta, Maria

    2016-05-21

    Modified tiny hydrogen-terminated diamond structures, known as diamondoids, show a high efficiency in sensing DNA molecules. These diamond cages, as recently proposed, could offer functionalization possibilities for gold junction electrodes. In this investigation, we report on diamondoid-functionalized electrodes, showing that such a device would have a high potential in sensing and sequencing DNA. The smallest diamondoid including an amine modification was chosen for the functionalization. Here, we report on the quantum tunneling signals across diamondoid-functionalized Au(111) electrodes. Our work is based on quantum-transport calculations and predicts the expected signals arising from different DNA units within the break junctions. Different gating voltages are proposed in order to tune the sensitivity of the functionalized electrodes with respect to specific nucleotides. The relation of this sensitivity to the coupling or decoupling of the electrodes is discussed. Our results also shed light on the sensing capability of such a device in distinguishing the DNA nucleotides, in their natural and mutated forms.

  17. Construction of heteroduplex DNA and in vitro model for functional analysis of mismatch repair

    Institute of Scientific and Technical Information of China (English)

    WANG Yi; Clark Alan; WANG Jiaxun; SUN Menghong; SHI Daren

    2004-01-01

    Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZα Gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. Coli NR9162 (mutS-) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G·G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G·G is less than 20%. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary

  18. Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

    Institute of Scientific and Technical Information of China (English)

    Chun-Hui HOU; Jian HUANG; Ruo-Lan QIAN

    2004-01-01

    The developmental control of the human ε-globin gene expression is mediated by transcriptional regulatory elements in the 5' flanking DNA of this gene. A previously identified negative regulatory element (-3028 to -2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at -3004 bp, -2975 bp and -2948 bp were characterized. Electrophoresis mobility shift assay (EMSA)showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (-177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at-2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at -2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at -2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at -2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε-NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

  19. Equivalent Dynamic Stiffness Mapping technique for identifying nonlinear structural elements from frequency response functions

    Science.gov (United States)

    Wang, X.; Zheng, G. T.

    2016-02-01

    A simple and general Equivalent Dynamic Stiffness Mapping technique is proposed for identifying the parameters or the mathematical model of a nonlinear structural element with steady-state primary harmonic frequency response functions (FRFs). The Equivalent Dynamic Stiffness is defined as the complex ratio between the internal force and the displacement response of unknown element. Obtained with the test data of responses' frequencies and amplitudes, the real and imaginary part of Equivalent Dynamic Stiffness are plotted as discrete points in a three dimensional space over the displacement amplitude and the frequency, which are called the real and the imaginary Equivalent Dynamic Stiffness map, respectively. These points will form a repeatable surface as the Equivalent Dynamic stiffness is only a function of the corresponding data as derived in the paper. The mathematical model of the unknown element can then be obtained by surface-fitting these points with special functions selected by priori knowledge of the nonlinear type or with ordinary polynomials if the type of nonlinearity is not pre-known. An important merit of this technique is its capability of dealing with strong nonlinearities owning complicated frequency response behaviors such as jumps and breaks in resonance curves. In addition, this technique could also greatly simplify the test procedure. Besides there is no need to pre-identify the underlying linear parameters, the method uses the measured data of excitation forces and responses without requiring a strict control of the excitation force during the test. The proposed technique is demonstrated and validated with four classical single-degree-of-freedom (SDOF) numerical examples and one experimental example. An application of this technique for identification of nonlinearity from multiple-degree-of-freedom (MDOF) systems is also illustrated.

  20. A single whole-body low dose X-irradiation does not affect L1, B1 and IAP repeat element DNA methylation longitudinally.

    Directory of Open Access Journals (Sweden)

    Michelle R Newman

    Full Text Available The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1, B1 and Intracisternal-A-Particle (IAP repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17-19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen and a tissue fundamental to the aging process (liver. Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA

  1. The Function,Future and Support Elements of Farmer Cooperative Economy Organization

    Institute of Scientific and Technical Information of China (English)

    Jing; GAO; Peng; XIE

    2014-01-01

    The nature of farmer cooperative economy organization( known as FCEO) determines the fact that the economic effects of farmer cooperative economy organization are as important as its social effects. Many experts,however,now would only focus on its economic function, and either neglect or weaken its social influence. Therefore,this paper introduces the theoretical foundation of the farmer cooperative economy organization,and studies the nature of cooperative economics. Based on those typical cases,the future of cooperative organization and four supporting elements were put forward in this paper.

  2. Finite Element Quadrature of Regularized Discontinuous and Singular Level Set Functions in 3D Problems

    Directory of Open Access Journals (Sweden)

    Nicola Ponara

    2012-11-01

    Full Text Available Regularized Heaviside and Dirac delta function are used in several fields of computational physics and mechanics. Hence the issue of the quadrature of integrals of discontinuous and singular functions arises. In order to avoid ad-hoc quadrature procedures, regularization of the discontinuous and the singular fields is often carried out. In particular, weight functions of the signed distance with respect to the discontinuity interface are exploited. Tornberg and Engquist (Journal of Scientific Computing, 2003, 19: 527–552 proved that the use of compact support weight function is not suitable because it leads to errors that do not vanish for decreasing mesh size. They proposed the adoption of non-compact support weight functions. In the present contribution, the relationship between the Fourier transform of the weight functions and the accuracy of the regularization procedure is exploited. The proposed regularized approach was implemented in the eXtended Finite Element Method. As a three-dimensional example, we study a slender solid characterized by an inclined interface across which the displacement is discontinuous. The accuracy is evaluated for varying position of the discontinuity interfaces with respect to the underlying mesh. A procedure for the choice of the regularization parameters is proposed.

  3. Spherically symmetric volume elements as basis functions for image reconstructions in computed laminography.

    Science.gov (United States)

    Trampert, Patrick; Vogelgesang, Jonas; Schorr, Christian; Maisl, Michael; Bogachev, Sviatoslav; Marniok, Nico; Louis, Alfred; Dahmen, Tim; Slusallek, Philipp

    2017-03-21

    Laminography is a tomographic technique that allows three-dimensional imaging of flat and elongated objects that stretch beyond the extent of a reconstruction volume. Laminography images can be reconstructed using iterative algorithms based on the Kaczmarz method. This study aims to develop and demonstrate a new reconstruction algorithm that may provide superior image reconstruction quality for this challenged imaging application. The images are initially represented using the coefficients over basis functions, which are typically piecewise constant functions (voxels). By replacing voxels with spherically symmetric volume elements (blobs) based on the generalized Kaiser-Bessel window functions, the images are reconstructed using this new adapted version of the algebraic image reconstruction technique. Band-limiting properties of blob functions are beneficial particular in the case of noisy projections and with only a limited number of available projections. Study showed that using blob basis functions improved full-width-at-half-maximum resolution from 10.2±1.0 to 9.9±0.9 (p functions, especially if noisy data is expected.

  4. Functional elements in the minimal promoter of the human proton-coupled folate transporter

    Energy Technology Data Exchange (ETDEWEB)

    Stark, Michal; Gonen, Nitzan [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel); Assaraf, Yehuda G., E-mail: assaraf@tx.technion.ac.il [The Fred Wyszkowski Cancer Research Laboratory, Dept. of Biology, Technion-Israel Institute of Technology, Haifa 32000 (Israel)

    2009-10-09

    The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1 kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay. This analysis mapped the minimal promoter to 157 bp upstream to the first ATG. Crucial GC-box sites were identified within the minimal promoter and in its close vicinity which substantially contribute to promoter activity, as their disruption resulted in 94% loss of luciferase activity. We also identified upstream enhancer elements including YY1 and AP1 which, although distantly located, prominently transactivated the minimal promoter, as their inactivation resulted in 50% decrease in reporter activity. This is the first functional identification of the minimal PCFT promoter harboring crucial GC-box elements that markedly contribute to its transcriptional activation via putative interaction with distal YY1 and AP1 enhancer elements.

  5. The structure and function of the rous sarcoma virus RNA stability element.

    Science.gov (United States)

    Withers, Johanna B; Beemon, Karen L

    2011-11-01

    For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors.

  6. DNA

    Science.gov (United States)

    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)

  7. DNA element downstream of the κB site in the Lcn2 promoter is required for transcriptional activation by IκBζ and NF-κB p50.

    Science.gov (United States)

    Kohda, Akira; Yamazaki, Soh; Sumimoto, Hideki

    2014-08-01

    The nuclear protein IκBζ activates transcription of a subset of NF-κB-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. IκBζ functions as a coactivator via its interaction with NF-κB p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in IκBζ function have remained unknown. Here, we show that, although IκBζ by itself is unable to associate with the Lcn2 promoter, IκBζ interacts with the promoter via p50 binding to the NF-κB-binding site (κB site) and the interaction also requires the pyrimidine-rich site (CCCCTC) that localizes seven bases downstream of the κB site. The pyrimidine-rich site is also essential for IκBζ-mediated activation of the Lcn2 gene. Introduction of both sites into an IκBζ-independent gene culminates in IκBζ-p50-DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both IκBζ-DNA interaction and IκBζ-mediated gene activation. Thus, the pyrimidine-rich IκBζ-responsive site plays an essential role in productive interaction of IκBζ with the p50-DNA complex.

  8. Elemental and isotopic imaging to study biogeochemical functioning of intact soil micro-environments

    Science.gov (United States)

    Mueller, Carsten W.

    2017-04-01

    The complexity of soils extends from the ecosystem-scale to individual micro-aggregates, where nano-scale interactions between biota, organic matter (OM) and mineral particles are thought to control the long-term fate of soil carbon and nitrogen. It is known that such biogeochemical processes show disproportionally high reaction rates within nano- to micro-meter sized isolated zones ('hot spots') in comparison to surrounding areas. However, the majority of soil research is conducted on large bulk (> 1 g) samples, which are often significantly altered prior to analysis and analysed destructively. Thus it has previously been impossible to study elemental flows (e.g. C and N) between plants, microbes and soil in complex environments at the necessary spatial resolution within an intact soil system. By using nano-scale secondary ion mass spectrometry (NanoSIMS) in concert with other imaging techniques (e.g. scanning electron microscopy (SEM) and micro computed tomography (µCT)), classic analyses (isotopic and elemental analysis) and biochemical methods (e.g. GC-MS) it is possible to exhibit a more complete picture of soil processes at the micro-scale. I will present exemplarily results about the fate and distribution of organic C and N in complex micro-scale soil structures for a range of intact soil systems. Elemental imaging was used to study initial soil formation as an increase in the structural connectivity of micro-aggregates. Element distribution will be presented as a key to detect functional spatial patterns and biogeochemical hot spots in macro-aggregate functioning and development. In addition isotopic imaging will be demonstrated as a key to trace the fate of plant derived OM in the intact rhizosphere from the root to microbiota and mineral soil particles. Especially the use of stable isotope enrichment (e.g. 13CO2, 15NH4+) in conjunction with NanoSIMS allows to directly trace the fate of OM or nutrients in soils at the relevant scale (e.g. assimilate C

  9. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    Science.gov (United States)

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  10. Photoligation of self-assembled DNA constructs containing anthracene-functionalized 2'-amino-LNA monomers

    DEFF Research Database (Denmark)

    Pasternak, Karol; Pasternak, Anna; Gupta, Pankaj

    2011-01-01

    Efficient synthesis of a novel anthracene-functionalized 2'-amino-LNA phosphoramidite derivative is described together with its incorporation into oligodeoxynucleotides. Two DNA strands with the novel 2'-N-anthracenylmethyl-2'-amino-LNA monomers can be effectively cross-linked by photoligation...

  11. Functions of FUS/TLS From DNA Repair to Stress Response: Implications for ALS

    Directory of Open Access Journals (Sweden)

    Reddy Ranjith Kumar Sama

    2014-08-01

    Full Text Available Fused in sarcoma/translocated in liposarcoma (FUS/TLS or FUS is a multifunctional DNA-/RNA-binding protein that is involved in a variety of cellular functions including transcription, protein translation, RNA splicing, and transport. FUS was initially identified as a fusion oncoprotein, and thus, the early literature focused on the role of FUS in cancer. With the recent discoveries revealing the role of FUS in neurodegenerative diseases, namely amyotrophic lateral sclerosis and frontotemporal lobar degeneration, there has been a renewed interest in elucidating the normal functions of FUS. It is not clear which, if any, endogenous functions of FUS are involved in disease pathogenesis. Here, we review what is currently known regarding the normal functions of FUS with an emphasis on DNA damage repair, RNA processing, and cellular stress response. Further, we discuss how ALS-causing mutations can potentially alter the role of FUS in these pathways, thereby contributing to disease pathogenesis.

  12. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  13. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

    Science.gov (United States)

    Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

    2014-01-01

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

  14. A density-functional-theory-based finite element model to study the mechanical properties of zigzag phosphorene nanotubes

    Science.gov (United States)

    Ansari, R.; Shahnazari, A.; Rouhi, S.

    2017-04-01

    In this paper, the density functional theory calculations are used to obtain the elastic properties of zigzag phosphorene nanotubes. Besides, based on the similarity between phosphorene nanotubes and a space-frame structure, a three-dimensional finite element model is proposed in which the atomic bonds are simulated by beam elements. The results of density functional theory are employed to compute the properties of the beam elements. Finally, using the proposed finite element model, the elastic modulus of the zigzag phosphorene nanotubes is computed. It is shown that phosphorene nanotubes with larger radii have larger Young's modulus. Comparing the results of finite element model with those of density functional theory, it is concluded that the proposed model can predict the elastic modulus of phosphorene nanotubes with a good accuracy.

  15. The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach.

    Science.gov (United States)

    Gonzalez, Diego; Kozdon, Jennifer B; McAdams, Harley H; Shapiro, Lucy; Collier, Justine

    2014-04-01

    DNA methylation is involved in a diversity of processes in bacteria, including maintenance of genome integrity and regulation of gene expression. Here, using Caulobacter crescentus as a model, we exploit genome-wide experimental methods to uncover the functions of CcrM, a DNA methyltransferase conserved in most Alphaproteobacteria. Using single molecule sequencing, we provide evidence that most CcrM target motifs (GANTC) switch from a fully methylated to a hemi-methylated state when they are replicated, and back to a fully methylated state at the onset of cell division. We show that DNA methylation by CcrM is not required for the control of the initiation of chromosome replication or for DNA mismatch repair. By contrast, our transcriptome analysis shows that >10% of the genes are misexpressed in cells lacking or constitutively over-expressing CcrM. Strikingly, GANTC methylation is needed for the efficient transcription of dozens of genes that are essential for cell cycle progression, in particular for DNA metabolism and cell division. Many of them are controlled by promoters methylated by CcrM and co-regulated by other global cell cycle regulators, demonstrating an extensive cross talk between DNA methylation and the complex regulatory network that controls the cell cycle of C. crescentus and, presumably, of many other Alphaproteobacteria.

  16. New induction relations for homogeneous functions in Jucys-Murphy elements

    CERN Document Server

    Feray, Valentin

    2010-01-01

    The problem of computing the class expansion of some symmetric function evaluated in Jucys-Murphy elements appears in different contexts, for instance in the computation of matrix integrals. Recently, M. Lassalle gave a unified algebraic method to obtain some induction relations on the coefficients in this kind of expansion. In this paper, we give a simple purely combinatorial proof of its result. Using the same type of argument, we also obtain new simpler formulas. We also prove an analogous formula in the double class algebra and use it to prove a conjecture of S. Matsumoto on the subleading term of orthogonal Weingarten function. Finally, we formulate a conjecture for a continuous interpolation between the two problems.

  17. Microscopic insight into the DNA condensation process of a zwitterion-functionalized polycation.

    Science.gov (United States)

    Sun, Hui; Zhou, Li; Chen, Xiaolu; Han, Xia; Wang, Rui; Liu, Honglai

    2016-11-01

    Zwitterion-functionalized polycations are ideal gene carriers with long circulation, high cellular uptaking and low cell viability. However, the trade-off between the DNA condensation efficiency and the cell viability must be addressed. The purpose of this study is to provide a microscopic insight into the DNA condensation process and to explore the effect of a zwitterionic block of zwitterion-functionalized polycation, which is of great significance in designing novel gene delivery systems. Poly[2-(dimethylamino)ethyl methacrylate-b-(sulfobetaine methacrylate)] (PDMAEMA-b-PSBMA) copolymers were synthesized and used as the model systems. Different from the conventional concept that the PSBMA zwitterionic block act only as the "stealthy" groups, the subtle differences in physical and colloidal characteristics between the polycation/DNA polyplexes show that the PSBMA segment is capable of wrapping DNA attributed to the quaternary ammonium cations, without compromising the DNA condensation capability. On the other hand, the incorporation of PSBMA block reduces the surface charge of the polyplexes, which substantially result in the inefficient transfection and the reduced cytotoxicity.

  18. Mitochondrial DNA and Functional Investigations into the Radiosensitivity of Four Mouse Strains

    Directory of Open Access Journals (Sweden)

    Steven B. Zhang

    2014-01-01

    Full Text Available We investigated whether genetic radiosensitivity-related changes in mtDNA/nDNA ratios are significant to mitochondrial function and if a material effect on mtDNA content and function exists. BALB/c (radiosensitive, C57BL/6 (radioresistant, and F1 hybrid mouse strains were exposed to total body irradiation. Hepatic genomic DNA was extracted, and mitochondria were isolated. Mitochondrial oxygen consumption, ROS, and calcium-induced mitochondrial swelling were measured. Radiation influenced strain-specific survival in vivo. F1 hybrid survival was influenced by maternal input. Changes in mitochondrial content corresponded to survival in vivo among the 4 strains. Calcium-induced mitochondrial swelling was strain dependent. Isolated mitochondria from BALB/c mice were significantly more sensitive to calcium overload than mitochondria from C57BL/6 mice. Maternal input partially influenced the recovery effect of radiation on calcium-induced mitochondrial swelling in F1 hybrids; the hybrid with a radiosensitive maternal lineage exhibited a lower rate of recovery. Hybrids had a survival rate that was biased toward maternal input. mtDNA content and mitochondrial permeability transition pores (MPTP measured in these strains before irradiation reflected a dominant input from the parent. After irradiation, the MPTP opened sooner in radiosensitive and hybrid strains, likely triggering intrinsic apoptotic pathways. These findings have important implications for translation into predictors of radiation sensitivity/resistance.

  19. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

    Science.gov (United States)

    Samanta, Anirban; Medintz, Igor L.

    2016-04-01

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  20. Adsorption enhancement of elemental mercury onto sulphur-functionalized silica gel adsorbents.

    Science.gov (United States)

    Johari, Khairiraihanna; Saman, Norasikin; Mat, Hanapi

    2014-01-01

    In this study, elemental mercury (EM) adsorbents were synthesized using tetraethyl orthosilicate (TEOS) and 3-mercaptopropyl trimethoxysilane as silica precursors. The synthesized silica gel (SG)-TEOS was further functionalized through impregnation with elemental sulphur and carbon disulphide (CS2). The SG adsorbents were then characterized by using scanning electron microscope, Fourier transform infra-red spectrophotometer, nitrogen adsorption/desorption, and energy-dispersive X-ray diffractometer. The EM adsorption of the SG adsorbents was determined using fabricated fixed-bed adsorber. The EM adsorption results showed that the sulphur-functionalized SG adsorbents had a greater Hgo breakthrough adsorption capacity, confirming that the presence of sulphur in silica matrices can improve Hgo adsorption performance due to their high affinity towards mercury. The highest Hgo adsorption capacity was observed for SG-TEOS(CS2) (82.62 microg/g), which was approximately 2.9 times higher than SG-TEOS (28.47 microg/g). The rate of Hgo adsorption was observed higher for sulphur-impregnated adsorbents, and decreased with the increase in the bed temperatures.

  1. Structure/Function Analysis of DNA-glycosylases That Repair Oxidized Purines and Pyrimidines and the Influence of Surrounding DNA Sequence on Their Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Wallace, Susan S.

    2005-08-22

    The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner.

  2. High-density DNA functionalization by a combination of Cu-catalyzed and cu-free click chemistry.

    Science.gov (United States)

    Gutsmiedl, Katrin; Fazio, Danila; Carell, Thomas

    2010-06-18

    We report the regioselective Cu-free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3-Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5-disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu-free and Cu-catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high-density functionalization of DNA is simple, modular, and efficient.

  3. The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks.

    Directory of Open Access Journals (Sweden)

    Se-Jin Lee

    2010-01-01

    Full Text Available WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

  4. An algorithm for computing an element of the Clarke generalized Jacobian of a difference of max-type functions

    CERN Document Server

    Horta, Ana

    2011-01-01

    We show that the algorithm for computing an element of the Clarke generalized Jacobian of a max-type function proposed by Zheng-da Huang and Guo-chun Ma can be extended to a much wider class of functions representable as a difference of max-type functions.

  5. Multiple Functions of Nuclear DNA Helicase Ⅱ (RNA helicase A) in Nucleic Acid Metabolism

    Institute of Scientific and Technical Information of China (English)

    Suisheng ZHANG; Frank GROSSE

    2004-01-01

    Nuclear DNA helicase Ⅱ(NDH Ⅱ),or RNA helicase A(RHA),was initially discovered in mammals by conventional protein purification methods.Molecular cloning identified apparent sequence homologies between NDH Ⅱ and a Drosophila protein named maleless(MLE),the latter being essential for the Drosophila X-chromosome dosage compensation.Increasing amounts of evidence suggest that NDH Ⅱ is involved in multiple aspects of cellular and viral DNA and RNA metabolism.Moreover the functions of NDH Ⅱ may have potential clinical implications related to viral infection,autoimmune diseases,or even tumorigenesis.

  6. Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

    DEFF Research Database (Denmark)

    Dufva, Martin; Petersen, Jesper; Poulsen, Lena

    2009-01-01

    DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need...... to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing...

  7. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Liu Lanxia; Bai Yuanyuan; Song Chunni; Zhu Dunwan; Song Liping; Zhang Hailing; Dong Xia; Leng Xigang, E-mail: lengxg@bme.org.c [Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Laboratory of Bioengineering (China)

    2010-06-15

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1{beta}, IL-6, IL-10, IL-12, and TNF-{alpha}). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 {mu}g/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  8. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    Science.gov (United States)

    Liu, Lanxia; Bai, Yuanyuan; Song, Chunni; Zhu, Dunwan; Song, Liping; Zhang, Hailing; Dong, Xia; Leng, Xigang

    2010-06-01

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1β, IL-6, IL-10, IL-12, and TNF-α). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 μg/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  9. Three-dimensional finite element analysis of Eustachian tube function under normal and pathological conditions.

    Science.gov (United States)

    Sheer, F J; Swarts, J D; Ghadiali, S N

    2012-06-01

    A primary etiological factor underlying chronic middle ear disease is an inability to open the collapsible Eustachian tube (ET). However, the structure-function relationships responsible for ET dysfunction in patient populations at risk for developing otitis media (OM) are not known. In this study, three-dimensional (3D) finite element (FE) modeling techniques were used to investigate how changes in biomechanical and anatomical properties influence opening phenomena in three populations: normal adults, young children and infants with cleft palate. Histological data was used to create anatomically accurate models and FE techniques were used to simulate tissue deformation and ET opening. Lumen dilation was quantified using a computational fluid dynamic (CFD) technique and a sensitivity analysis was performed to ascertain the relative importance of the different anatomical and tissue mechanical properties. Results for adults suggest that ET function is highly sensitive to tensor veli palatini muscle (TVPM) forces and to periluminal mucosal tissue (PMT) elasticity. Young children and cleft palate subjects exhibited reduced sensitivity to TVPM forces while changes in PMT stiffness continued to have a significant impact on ET function. These results suggest that reducing PMT stiffness might be an effective way to restore ET function in these populations. Varying TVPM force vector relationships via changes in hamulus location had no effect on ET opening in young children and cleft palate subjects but did alter force transmission to the ET lumen during conditions of elevated adhesion. These models have therefore provided important new insights into the biomechanical mechanisms responsible for ET dysfunction.

  10. 3D finite element model of the chinchilla ear for characterizing middle ear functions.

    Science.gov (United States)

    Wang, Xuelin; Gan, Rong Z

    2016-10-01

    Chinchilla is a commonly used animal model for research of sound transmission through the ear. Experimental measurements of the middle ear transfer function in chinchillas have shown that the middle ear cavity greatly affects the tympanic membrane (TM) and stapes footplate (FP) displacements. However, there is no finite element (FE) model of the chinchilla ear available in the literature to characterize the middle ear functions with the anatomical features of the chinchilla ear. This paper reports a recently completed 3D FE model of the chinchilla ear based on X-ray micro-computed tomography images of a chinchilla bulla. The model consisted of the ear canal, TM, middle ear ossicles and suspensory ligaments, and the middle ear cavity. Two boundary conditions of the middle ear cavity wall were simulated in the model as the rigid structure and the partially flexible surface, and the acoustic-mechanical coupled analysis was conducted with these two conditions to characterize the middle ear function. The model results were compared with experimental measurements reported in the literature including the TM and FP displacements and the middle ear input admittance in chinchilla ear. An application of this model was presented to identify the acoustic role of the middle ear septa-a unique feature of chinchilla middle ear cavity. This study provides the first 3D FE model of the chinchilla ear for characterizing the middle ear functions through the acoustic-mechanical coupled FE analysis.

  11. Australopithecus anamensis: a finite-element approach to studying the functional adaptations of extinct hominins.

    Science.gov (United States)

    Macho, Gabriele A; Shimizu, Daisuke; Jiang, Yong; Spears, Iain R

    2005-04-01

    Australopithecus anamensis is the stem species of all later hominins and exhibits the suite of characters traditionally associated with hominins, i.e., bipedal locomotion when on the ground, canine reduction, and thick-enameled teeth. The functional consequences of its thick enamel are, however, unclear. Without appropriate structural reinforcement, these thick-enameled teeth may be prone to failure. This article investigates the mechanical behavior of A. anamensis enamel and represents the first in a series that will attempt to determine the functional adaptations of hominin teeth. First, the microstructural arrangement of enamel prisms in A. anamensis teeth was reconstructed using recently developed software and was compared with that of extant hominoids. Second, a finite-element model of a block of enamel containing one cycle of prism deviation was reconstructed for Homo, Pan, Gorilla, and A. anamensis and the behavior of these tissues under compressive stress was determined. Despite similarities in enamel microstructure between A. anamensis and the African great apes, the structural arrangement of prismatic enamel in A. anamensis appears to be more effective in load dissipation under these compressive loads. The findings may imply that this hominin species was well adapted to puncture crushing and are in some respects contrary to expectations based on macromorphology of teeth. Taking together, information obtained from both finite-element analyses and dental macroanatomy leads us to suggest that A. anamensis was probably adapted for habitually consuming a hard-tough diet. However, additional tests are needed to understand the functional adaptations of A. anamensis teeth fully.

  12. Synthesis and Characterisation of Copper(II Complexes with Tridentate NNO Functionalized Ligand: Density Function Theory Study, DNA Binding Mechanism, Optical Properties, and Biological Application

    Directory of Open Access Journals (Sweden)

    Madhumita Hazra

    2014-01-01

    Full Text Available The photo physical properties of two mononuclear pentacoordinated copper(II complexes formulated as [Cu(L(Cl(H2O] (1 and [Cu(L(Br(H2O] (2 HL = (1-[(3-methyl-pyridine-2-ylimino-methyl]-naphthalen-2-ol were synthesized and characterized by elemental, physicochemical, and spectroscopic methods. The density function theory calculations are used to investigate the electronic structures and the electronic properties of ligand and complex. The interactions of copper(II complexes towards calf thymus DNA were examined with the help of absorption, viscosity, and fluorescence spectroscopic techniques at pH 7.40. All spectroscopy's result indicates that complexes show good binding activity to calf thymus DNA through groove binding. The optical absorption and fluorescence emission properties of microwires were characterized by fluorescence microscope. From a spectroscopic viewpoint, all compounds strongly emit green light in the solid state. The microscopy investigation suggested that microwires exhibited optical waveguide behaviour which are applicable as fluorescent nanomaterials and can be used as building blocks for miniaturized photonic devices. Antibacterial study reveals that complexes are better antimicrobial agents than free Schiff base due to bacterial cell penetration by chelation. Moreover, the antioxidant study of the ligand and complexes is evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH free-radical assays, which demonstrate that the complexes are of higher antioxidant activity than free ligand.

  13. Density functional calculations of large systems containing heavy elements by means of the regionalization algorithm

    Institute of Scientific and Technical Information of China (English)

    HU Xiangqian; LI Lemin

    2004-01-01

    The regionalized computational method is extended to the non-relativistic, scalar and 2-component relativistic density functional calculation of large systems containing transition series or heavy main-group metal elements. A large system is divided into several regions which can be considered as relatively independent quantum mechanical subsystems. Taking into account the Coulomb and exchange-correlation potentials as well as the Pauli repulsion exerted by the other subsystems, the Kohn-Sham equation related to subsystem K can be written as: (FK+FKP)CK =SKCKεK K=A,B,C,…,where FK,CK,SK,εK are the Fock matrix, the matrix of combination coefficients of orbitals, the overlap matrix of basis sets and the energy eigenvalue matrix, respectively. The matrix FKP reflects the Pauli repulsion from the other subsystems.FK may be non-relativistic, scalar or 2-component relativistic Fock matrix determined by the theoretical method related to the density functional calculations. The other matrices are mated with FK. Solving the Kohn-Sham equation for every subsystem and combining the results from the subsystem calculations, the electronic structural information of the whole system is derived. The density functional calculation of several molecules containing transition metal Ni or heavy main-group metal Tl or Bi is performed by the afore-mentioned regionalization algorithm. The obtained results for each molecule are compared with those from the density functional calculation of that molecule in its entirety in order to check the feasibility of the regionalization algorithm. It is found that with sufficiently large regional basis set in the subsystem calculation the accuracy of the results calculated by the regionalization algorithm is essentially the same as that from the calculation of the molecule in its entirety. With proper smaller regional basis sets the accuracy of the results calculated with the regionalization algorithm can still match the actual accuracy of the

  14. Chemical grafting of a DNA intercalator probe onto functional iron oxide nanoparticles: a physicochemical study.

    Science.gov (United States)

    Bouffier, Laurent; Yiu, Humphrey H P; Rosseinsky, Matthew J

    2011-05-17

    Spherical magnetite nanoparticles (MNPs, ∼ 24 nm in diameter) were sequentially functionalized with trimethoxysilylpropyldiethylenetriamine (TMSPDT) and a synthetic DNA intercalator, namely, 9-chloro-4H-pyrido[4,3,2-kl]acridin-4-one (PyAcr), in order to promote DNA interaction. The designed synthetic pathway allowed control of the chemical grafting efficiency to access MNPs either partially or fully functionalized with the intercalator moiety. The newly prepared nanomaterials were characterized by a range of physicochemical techniques: FTIR, TEM, PXRD, and TGA. The data were consistent with a full surface coverage by immobilized silylpropyldiethylenetriamine (SPDT) molecules, which corresponds to ∼22,300 SPDT molecules per MNP and a subsequent (4740-2940) PyAcr after the chemical grafting step (i.e., ∼ 2.4 PyAcr/nm(2)). A greater amount of PyAcr (30,600) was immobilized by the alternative strategy of binding a fully prefunctionalized shell to the MNPs with up to 16.1 PyAcr/nm(2). We found that the extent of PyAcr functionalization strongly affects the resulting properties and, particularly, the colloidal stability as well as the surface charge estimated by ζ-potential measurement. The intercalator grafting generates a negative charge contribution which counterbalances the positive charge of the single SPDT shell. The DNA binding capability was measured by titration assay and increases from 15 to 21.5 μg of DNA per mg of MNPs after PyAcr grafting (14-20% yield) but then drops to only ∼2 μg for the fully functionalized MNPs. This highlights that even if the size of the MNPs is obviously a determining factor to promote surface DNA interaction, it is not the only limiting parameter, as the mode of binding and the interfacial charge density are essential to improve loading capability.

  15. Synthesis of a drug delivery vehicle for cancer treatment utilizing DNA-functionalized gold nanoparticles

    Science.gov (United States)

    Brann, Tyler

    The treatment of cancer with chemotherapeutic agents has made great strides in the last few decades but still introduces major systemic side effects. The potent drugs needed to kill cancer cells often cause irreparable damage to otherwise healthy organs leading to further morbidity and mortality. A therapy with intrinsic selective properties and/or an inducible activation has the potential to change the way cancer can be treated. Gold nanoparticles (GNPs) are biocompatible and chemically versatile tools that can be readily functionalized to serve as molecular vehicles. The ability of these particles to strongly absorb light with wavelengths in the therapeutic window combined with the heating effect of surface plasmon resonance makes them uniquely suited for noninvasive heating in biologic applications. Specially designed DNA aptamers have shown their ability to serve as drug carriers through intercalation as well as directly acting as therapeutic agents. By combining these separate molecules a multifaceted drug delivery vehicle can be created with great potential as a selective and controllable treatment for cancer. Oligonucleotide-coated GNPs have been created using spherical GNPs but little work has been reported using gold nanoplates in this way. Using the Diasynth method gold nanoplates were produced to absorb strongly in the therapeutic near infrared (nIR) window. These particles were functionalized with two DNA oligonucleotides: one serving as an intercalation site for doxorubicin, and another, AS1411, serving directly as an anticancer targeting/therapeutic agent. These functional particles were fully synthesized and processed along with confirmation of DNA functionalization and doxorubicin intercalation. Doxorubicin is released via denaturation of the DNA structure into which doxorubicin is intercalated upon the heating of the gold nanoplate well above the DNA melting temperature. This temperature increase, due to light stimulation of surface plasmon

  16. New function of exonuclease and highly sensitive label-free colorimetric DNA detection.

    Science.gov (United States)

    Li, Hongbo; Wang, Suqin; Wu, Zaisheng; Xu, Jianguo; Shen, Guoli; Yu, Ruqin

    2016-03-15

    Enzymatic manipulation and modulation of nucleic acids are a central part of cellular function, protection, and reproduction, while rapid and accurate detection of ultralow amount of nucleic acids remains a major challenge in molecular biology research and clinic diagnosis of genetic diseases. Herein, we reported that exonuclease III can degrade the G-quadruplex structure, indicating the new exonuclease's function. Basing on the function of exonuclease III, a novel G-quadruplex-hemin DNAzyme-based colorimetric detection of tumor suppressor gene p53 was successfully developed. Although only one oligonucleotide probe was involved, the sensing strategy could suppress the optical background and achieve an efficient G-quadruplex-hemin DNAzyme-based signal amplification. Specifically, a label-free functional nucleic acid probe (called THzyme probe) was designed via introducing target DNA probe-contained hairpin structure into G-quadruplex DNAzyme. Even if this probe can fold into G-quadruplex structure in the presence of hemin very different from the double-stranded DNA, it is easily degraded by exonuclease III. Thus, no change in UV-vis absorption intensity is detected in the absence of target DNA. However, the hybridization of target DNA can protect the integrity and catalytic activity of THzyme probe, producing the DNAzyme-amplified colorimetric signal. As a result, the p53 gene was able to be detected down to 1.0 pM (final concentration in the signal-generating solution: 50.0 fM) and mismatched target DNAs were easily distinguished. It is expected that this simple sensing methodology for DNA detection can find its utility in point-of-care applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Charge-Transfer Excited States in Aqueous DNA: Insights from Many-Body Green's Function Theory

    Science.gov (United States)

    Yin, Huabing; Ma, Yuchen; Mu, Jinglin; Liu, Chengbu; Rohlfing, Michael

    2014-06-01

    Charge-transfer (CT) excited states play an important role in the excited-state dynamics of DNA in aqueous solution. However, there is still much controversy on their energies. By ab initio many-body Green's function theory, together with classical molecular dynamics simulations, we confirm the existence of CT states at the lower energy side of the optical absorption maximum in aqueous DNA as observed in experiments. We find that the hydration shell can exert strong effects (˜1 eV) on both the electronic structure and CT states of DNA molecules through dipole electric fields. In this case, the solvent cannot be simply regarded as a macroscopic screening medium as usual. The influence of base stacking and base pairing on the CT states is also discussed.

  18. Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant%Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant

    Institute of Scientific and Technical Information of China (English)

    Zhang, Zhihong; Li, Guijuan; Yan, Fufeng; Zhang, Zhonghou; Fang, Shaoming

    2012-01-01

    In order to exploit the applications ofpolypyrrole (PPy) derivatives in biosensors and bioelectronics, the different immobilization mechanisms of biomolecules onto differently functionalized conducting PPy films are investigated. Pyrrole and pyrrole derivatives with carboxyl and amino groups were copolymerized with ω-(N-pyrrolyl)-octylthiol self-assembled on Au surface by the method of the chemical polymerization to form a layer of the copolymer film, i.e., poly[pyrrole-co-(N-pyrrolyl)-caproic acid] (poly(Py-co-PyCA)) and poly[pyrrole-co-(N-pyrrolyl)-hexylamine] (poly(Py-co-PyHA)), in which the carboxyl groups in poly(Py-co-PyCA) were activated to the ester groups. Based on the structure characteristics, the immobilization/hybridization of DNA molecules on PPy, poly(Py-co-PyCA) and poly(Py-co-PyHA) were surveyed by cyclic voltammograms measurements. For differently functionalized copolymers, the immobilization mechanisms of DNA are various. Besides the electrochemical properties of the composite electrodes of PPy and its copolymers being detected before and after bovine serum albumin (BSA) adsorption, the kinetic process of protein binding was determined by surface plasmon resonance of spectroscopy. Since few BSA molecules could anchor onto the PPy and its copolymers surfaces, it suggests this kind of conducting polymers can be applied as the protein-resistant material.

  19. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation

    Directory of Open Access Journals (Sweden)

    Kathleen H Wood

    2016-05-01

    Full Text Available DNA methylation is an epigenetic mark that is essential for many biological processes and is linked to diseases such as cancer. Methylation is usually associated with transcriptional silencing, but new research has challenged this model. Both transcriptional activation and repression have recently been found to be associated with DNA methylation in a context-specific manner. How DNA methylation patterns are interpreted into different functional output remains poorly understood. One mechanism involves the protein ‘readers’ of methylation, which includes the methyl-CpG binding domain (MBD family of proteins. This review examines the molecular and biological functions of MBD2, which binds to CpG methylation and is an integral part of the nucleosome remodeling and histone deacetylation (NuRD complex. MBD2 has been linked to immune system function and tumorigenesis, yet little is known about its functions in vivo. Recent studies have found the MBD2 protein is ubiquitously expressed, with relatively high levels in the lung, liver and colon. Mbd2 null mice surprisingly show relatively mild phenotypes compared to mice with loss of function of other MBD proteins. This evidence has previously been interpreted as functional redundancy between the MBD proteins. Here we examine and contextualize research that suggests MBD2 has unique properties and functions among the MBD proteins. These functions translate to recently described roles in the development and differentiation of multiple cell lineages, including pluripotent stem cells and various cell types of the immune system, as well as in tumorigenesis. We also consider possible models for the dynamic interactions between MBD2 and NuRD in different tissues in vivo. The functions of MBD2 may have direct therapeutic implications for several areas of human disease, including autoimmune conditions and cancer, in addition to providing insights into the actions of NuRD and chromatin regulation.

  20. An improved method for TAL effectors DNA-binding sites prediction reveals functional convergence in TAL repertoires of Xanthomonas oryzae strains.

    Directory of Open Access Journals (Sweden)

    Alvaro L Pérez-Quintero

    Full Text Available Transcription Activators-Like Effectors (TALEs belong to a family of virulence proteins from the Xanthomonas genus of bacterial plant pathogens that are translocated into the plant cell. In the nucleus, TALEs act as transcription factors inducing the expression of susceptibility genes. A code for TALE-DNA binding specificity and high-resolution three-dimensional structures of TALE-DNA complexes were recently reported. Accurate prediction of TAL Effector Binding Elements (EBEs is essential to elucidate the biological functions of the many sequenced TALEs as well as for robust design of artificial TALE DNA-binding domains in biotechnological applications. In this work a program with improved EBE prediction performances was developed using an updated specificity matrix and a position weight correction function to account for the matching pattern observed in a validation set of TALE-DNA interactions. To gain a systems perspective on the large TALE repertoires from X. oryzae strains, this program was used to predict rice gene targets for 99 sequenced family members. Integrating predictions and available expression data in a TALE-gene network revealed multiple candidate transcriptional targets for many TALEs as well as several possible instances of functional convergence among TALEs.

  1. Cloning, expression, and functional characterization of the equine herpesvirus 1 DNA polymerase and its accessory subunit.

    Science.gov (United States)

    Loregian, Arianna; Case, Alessandro; Cancellotti, Enrico; Valente, Carlo; Marsden, Howard S; Palù, Giorgio

    2006-07-01

    We report the expression and characterization of the putative catalytic subunit (pORF30) and accessory protein (pORF18) of equine herpesvirus 1 DNA polymerase, which are encoded by open reading frames 30 and 18 and are homologous to herpes simplex virus type 1 UL30 and UL42, respectively. In vitro transcription-translation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitro-expressed pORF30 possessed basal DNA polymerase activity that was stimulated by pORF18, as measured by DNA polymerase assays in vitro. Purified baculovirus-expressed pORF30 exhibited DNA polymerase activity similar to that of the in vitro-expressed protein, and baculovirus-expressed pORF18 could stimulate both nucleotide incorporation and long-chain DNA synthesis by pORF30 in a dose- and time-dependent manner. The salt optima for activity of both pORF30 and the holoenzyme were substantially different from those for other herpesvirus DNA polymerases. As demonstrated by yeast two-hybrid assays, pORF30 and pORF18 could physically interact, most likely with a 1:1 stoichiometry. Finally, by mutational analysis of the 1,220-residue pORF30, we demonstrated that the extreme C terminus of pORF30 is important for physical and functional interaction with the accessory protein, as reported for UL30 and other herpesvirus DNA polymerases. In addition, a C-proximal region of pORF30, corresponding to residues 1114 to 1172, is involved in binding to, and stimulation by, pORF18. Taken together, the results indicate that pORF30 and pORF18 are the equine herpesvirus 1 counterparts of herpes simplex virus type 1 UL30 and UL42 and share many, but not all, of their characteristics.

  2. Z-DNA and its biological function%Z-DNA及其生物学功能

    Institute of Scientific and Technical Information of China (English)

    汤雅男; 杨攀; 胡成钰

    2009-01-01

    Z-DNA是一种处于高能状态、不稳定的DNA分子构象.形成Z-DNA的原因有很多:首先,转录过程中,移动的RNA聚合酶在模板DNA的5'端产生负超螺旋扭曲力,导致Z-DNA的形成;其次,含有d(GC)_n序列的核酸分子在高浓度的NaCl、[Co(NH_3)_6]~(2+)盐溶液中也能够形成Z-DNA;最后,化学修饰也可以使DNA产生稳定的Z-DNA.Z-DNA是在体外首先发现的,但随着研究的不断深入,发现Z-DNA在体内也广泛存在并可能具有功能的多样性,包括参与基因表达调控、染色体断裂、基因重组、抗病毒、病毒发生等生物学过程.%Z-DNA is an instable and high energy DNA conformation in vivo and it can be formed in many conditions.Firstly,when RNA polymerase is moving on a DNA template,transcription can result in negative supercoiling behind the polymerase,thus facilitating Z-DNA conformation at permissive regions.Secondly,the Z-DNA-conformation which is formed by d(GC)_n sequence can also be stabled in high-salt solution of NaCl or [Co(NH_3)_6]~(2+) and lastly,the Z-DNA can be formed by covalent modifications.Though Z-DNA was firstly discovered in vitro,recently many evidences revealed that Z-DNA was widely existed and may possessed diversity of function in vivo,including gene expression and regulation,chromosomal breaks,recombination,antivirus defense and virus generation and so on.

  3. Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking

    Energy Technology Data Exchange (ETDEWEB)

    Usachenko, S.I. [Univ. of California, Davis, CA (United States); Bradbury, E.M. [Los Alamos National Lab., NM (United States). Life Science Div.]|[Univ. of California, Davis, CA (United States)

    1998-12-31

    The magnitude of the problem of understanding the structure/function relationships of eukaryotic chromosomes can be appreciated from the fact that the human diploid genome contains more than 2 meters of DNA packaged into 46 chromosomes, each at metaphase being several microns in length. Each chromatid of a chromosome contains a single DNA molecule several centimeters in length. In addition to the DNA, chromosomes contain an equal weight of histones and an equal weight of non-histone chromosomal proteins. These histones are the major chromosomal structural proteins. The non-histone chromosomal proteins are involved in the DNA processes of transcription and replication, in chromosome organization and in nuclear architecture. Polytene chromosomes with their bands and interbands and puffs of active genetic loci provide visual evidence for long range order as do the bands and interbands of mammalian metaphase chromosomes. The gentle removal of histones and all but the most tightly bound 2--3% of non-histone proteins from metaphase chromosomes revealed by electron microscopy a residual protein scaffold constraining a halo of DNA loops extending out from the scaffold.

  4. mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

    Directory of Open Access Journals (Sweden)

    Riikka H. Hämäläinen

    2015-06-01

    Full Text Available mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.

  5. Structure-function analysis of ribonucleotide bypass by B family DNA replicases

    Energy Technology Data Exchange (ETDEWEB)

    Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.; Pedersen, Lars C.; Kunkel, Thomas A. [NIH

    2013-11-01

    Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.

  6. Changes in the infrared microspectroscopic characteristics of DNA caused by cationic elements, different base richness and single-stranded form.

    Directory of Open Access Journals (Sweden)

    Maria Luiza S Mello

    Full Text Available BACKGROUND: The infrared (IR analysis of dried samples of DNA and DNA-polypeptide complexes is still scarce. Here we have studied the FT-IR profiles of these components to further the understanding of the FT-IR signatures of chromatin and cell nuclei. METHODOLOGY/PRINCIPAL FINDINGS: Calf thymus and salmon testis DNA, and complexes of histone H1, protamine, poly-L-lysine and poly-L-arginine (histone-mimic macromolecules with DNA were analyzed in an IR microspectroscope equipped with an attenuated total reflection diamond objective and Grams software. Conditions including polypeptides bound to the DNA, DNA base composition, and single-stranded form were found to differently affect the vibrational characteristics of the chemical groups (especially, PO(2(- in the nucleic acid. The antisymmetric stretching (ν(as of the DNA PO(2(- was greater than the symmetric stretching (ν(s of these groups and increased in the polypeptide-DNA complexes. A shift of the ν(as of the DNA PO(2(- to a lower frequency and an increased intensity of this vibration were induced especially by lysine-rich histones. Lysine richness additionally contributed to an increase in the vibrational stretching of the amide I group. Even in simple molecules such as inorganic phosphates, the vibrational characteristics of the phosphate anions were differently affected by different cations. As a result of the optimization of the DNA conformation by binding to arginine-rich polypeptides, enhancements of the vibrational characteristics in the FT-IR fingerprint could be detected. Although different profiles were obtained for the DNA with different base compositions, this situation was no longer verified in the polypeptide-DNA complexes and most likely in isolated chromatin or cell nuclei. However, the ν(as PO(2(-/ν(s PO(2(- ratio could discriminate DNA with different base compositions and DNA in a single-stranded form. CONCLUSIONS/SIGNIFICANCE: FT-IR spectral profiles are a valuable tool

  7. Effect of duct obstruction on structure, elemental composition, and function of rat submandibular glands

    Energy Technology Data Exchange (ETDEWEB)

    Sagstroem, S.S.; Sagulin, G.B.; Roomans, G.M. (Univ. of Uppsala (Sweden))

    1989-06-01

    Obstruction of salivary glands occurs in association with a number of pathological conditions. It has been suggested that the major changes found in the salivary glands of patients with cystic fibrosis are due to obstruction of the excretory duct by viscous mucus. In the present study, the effect of excretory duct obstruction on structure, elemental composition and function of rat submandibular gland was investigated. Obstruction was effected by infusion of a fast-hardening protein emulsion in the main excretory duct. After 1 week, and more pronounced after 2 weeks of obstruction the number of granular duct cells had decreased in the obstructed gland. X-ray microanalysis showed an increase in Mg, Ca and K, and a decrease in Na levels in the acinar cells, compared to normal glands. The contralateral glands apparently underwent compensatory hypertrophy and showed a similar pattern of changes in elemental composition. The composition of pilocarpine-induced submandibular saliva was neither in the obstructed nor in the contralateral gland significantly different from that in control glands. However, the flow rate was somewhat lower. Hence, increase in cellular Ca levels in submandibular gland acinar cells in cystic fibrosis could be secondary to duct obstruction, but the present study does not support the hypothesis that duct obstruction would result in changes in the composition of saliva.

  8. Cytogenetic variation of repetitive DNA elements in Hoplias malabaricus (Characiformes - Erythrinidae) from white, black and clear water rivers of the Amazon basin.

    Science.gov (United States)

    Santos, Fabíola Araújo Dos; Marques, Diego Ferreira; Terencio, Maria Leandra; Feldberg, Eliana; Rodrigues, Luís Reginaldo R

    2016-03-01

    Hoplias malabaricus is a common fish species occurring in white, black and clear water rivers of the Amazon basin. Its large distribution across distinct aquatic environments can pose stressful conditions for dispersal and creates possibilities for the emergence of local adaptive profiles. We investigated the chromosomal localization of repetitive DNA markers (constitutive heterochromatin, rDNA and the transposable element REX-3) in populations from the Amazonas river (white water), the Negro river (black water) and the Tapajós river (clear water), in order to address the variation/association of cytogenomic features and environmental conditions. We found a conserved karyotypic macrostructure with a diploid number of 40 chromosomes (20 metacentrics + 20 submetacentrics) in all the samples. Heteromorphism in pair 14 was detected as evidence for the initial differentiation of an XX/XY system. Minor differences detected in the amount of repetitive DNA markers are interpreted as possible signatures of local adaptations to distinct aquatic environments.

  9. Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

    Science.gov (United States)

    Martinez, E; Wahli, W

    1989-12-01

    The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

  10. Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene.

    Science.gov (United States)

    Maxson, J M; Woodson, W R

    1996-07-01

    Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.

  11. Elemental and compound semiconductor surface chemistry: Intelligent interfacial design facilitated through novel functionalization and deposition strategies

    Science.gov (United States)

    Porter, Lon Alan, Jr.

    The fundamental understanding of silicon surface chemistry is an essential tool for silicon's continued dominance of the semiconductor industry in the years to come. By tapping into the vast library of organic functionalities, the synthesis of organic monolayers may be utilized to prepare interfaces, tailored to a myriad of applications ranging from silicon VLSI device optimization and MEMS to physiological implants and chemical sensors. Efforts in our lab to form stable organic monolayers on porous silicon through direct silicon-carbon linkages have resulted in several efficient functionalization methods. In the first chapter of this thesis a comprehensive review of these methods, and many others is presented. The following chapter and the appendix serve to demonstrate both potential applications and studies aimed at developing a fundamental understanding of the chemistry behind the organic functionalization of silicon surfaces. The remainder of this thesis attempts to demonstrate new methods of metal deposition onto both elemental and compound semiconductor surfaces. Currently, there is considerable interest in producing patterned metallic structures with reduced dimensions for use in technologies such as ULSI device fabrication, MEMS, and arrayed nanosensors, without sacrificing throughput or cost effectiveness. Research in our laboratory has focused on the preparation of precious metal thin films on semiconductor substrates via electroless deposition. Continuous metallic films form spontaneously under ambient conditions, in the absence of a fluoride source or an externally applied current. In order to apply this metallization method toward the development of useful technologies, patterning utilizing photolithography, microcontact printing, and scanning probe nanolithography has been demonstrated.

  12. Finite element analysis on longitudinal and radial functionally graded femoral prosthesis.

    Science.gov (United States)

    Oshkour, Azim Ataollahi; Abu Osman, N A; Davoodi, M M; Yau, Y H; Tarlochan, F; Wan Abas, W A B; Bayat, M

    2013-12-01

    This study focused on developing a 3D finite element model of functionally graded femoral prostheses to decrease stress shielding and to improve total hip replacement performance. The mechanical properties of the modeled functionally graded femoral prostheses were adjusted in the sagittal and transverse planes by changing the volume fraction gradient exponent. Prostheses with material changes in the sagittal and transverse planes were considered longitudinal and radial prostheses, respectively. The effects of cemented and noncemented implantation methods were also considered in this study. Strain energy and von Mises stresses were determined at the femoral proximal metaphysis and interfaces of the implanted femur components, respectively. Results demonstrated that the strain energy increased proportionally with increasing volume fraction gradient exponent, whereas the interface stresses decreased on the prostheses surfaces. A limited increase was also observed at the surfaces of the bone and cement. The periprosthetic femur with a noncemented prosthesis exhibited higher strain energy than with a cemented prosthesis. Radial prostheses implantation displayed more strain energy than longitudinal prostheses implantation in the femoral proximal part. Functionally graded materials also increased strain energy and exhibited promising potentials as substitutes of conventional materials to decrease stress shielding and to enhance total hip replacement lifespan.

  13. Three-Dimensional Finite Element Modeling of Thermomechanical Problems in Functionally Graded Hydroxyapatite/Titanium Plate

    Directory of Open Access Journals (Sweden)

    S. N. S. Jamaludin

    2014-01-01

    Full Text Available The composition of hydroxyapatite (HA as the ceramic phase and titanium (Ti as the metallic phase in HA/Ti functionally graded materials (FGMs shows an excellent combination of high biocompatibility and high mechanical properties in a structure. Because the gradation of these properties is one of the factors that affects the response of the functionally graded (FG plates, this paper is presented to show the domination of the grading parameter on the displacement and stress distribution of the plates. A three-dimensional (3D thermomechanical model of a 20-node brick quadratic element is used in the simulation of the thermoelastic behaviors of HA/Ti FG plates subjected to constant and functional thermal, mechanical, and thermomechanical loadings. The convergence properties of the present results are examined thoroughly in order to assess the accuracy of the theory applied and to compare them with the established research results. Instead of the grading parameter, this study reveals that the loading field distribution can be another factor that reflects the thermoelastic properties of the HA/Ti FG plates. The FG structure is found to be able to withstand the thermal stresses while preserving the high toughness properties and thus shows its ability to operate at high temperature.

  14. Numerical simulation of thermal fracture in functionally graded materials using element-free Galerkin method

    Indian Academy of Sciences (India)

    SAHIL GARG; MOHIT PANT

    2017-03-01

    In the present work, element-free Galerkin method (EFGM) has been extended and implemented to simulate thermal fracture in functionally graded materials. The thermo-elastic fracture problem is decoupled into two separate parts. Initially, the temperature distribution over the domain is obtained by solving the heat transfer problem. The temperature field so obtained is then employed as input for the mechanical problem to determine the displacement and stress fields. The crack surfaces are modelled as non-insulated boundaries; hence the temperature field remains undisturbed by the presence of crack. A modified conservative M-integral technique has been used in order to extract the stress intensity factors for the simulated problems. The present analysisshows that the results obtained by EFGM are in good agreement with those available in the literature.

  15. High-resolution interrogation of functional elements in the noncoding genome.

    Science.gov (United States)

    Sanjana, Neville E; Wright, Jason; Zheng, Kaijie; Shalem, Ophir; Fontanillas, Pierre; Joung, Julia; Cheng, Christine; Regev, Aviv; Zhang, Feng

    2016-09-30

    The noncoding genome affects gene regulation and disease, yet we lack tools for rapid identification and manipulation of noncoding elements. We developed a CRISPR screen using ~18,000 single guide RNAs targeting >700 kilobases surrounding the genes NF1, NF2, and CUL3, which are involved in BRAF inhibitor resistance in melanoma. We find that noncoding locations that modulate drug resistance also harbor predictive hallmarks of noncoding function. With a subset of regions at the CUL3 locus, we demonstrate that engineered mutations alter transcription factor occupancy and long-range and local epigenetic environments, implicating these sites in gene regulation and chemotherapeutic resistance. Through our expansion of the potential of pooled CRISPR screens, we provide tools for genomic discovery and for elucidating biologically relevant mechanisms of gene regulation. Copyright © 2016, American Association for the Advancement of Science.

  16. Across-Platform Imputation of DNA Methylation Levels Incorporating Nonlocal Information Using Penalized Functional Regression.

    Science.gov (United States)

    Zhang, Guosheng; Huang, Kuan-Chieh; Xu, Zheng; Tzeng, Jung-Ying; Conneely, Karen N; Guan, Weihua; Kang, Jian; Li, Yun

    2016-05-01

    DNA methylation is a key epigenetic mark involved in both normal development and disease progression. Recent advances in high-throughput technologies have enabled genome-wide profiling of DNA methylation. However, DNA methylation profiling often employs different designs and platforms with varying resolution, which hinders joint analysis of methylation data from multiple platforms. In this study, we propose a penalized functional regression model to impute missing methylation data. By incorporating functional predictors, our model utilizes information from nonlocal probes to improve imputation quality. Here, we compared the performance of our functional model to linear regression and the best single probe surrogate in real data and via simulations. Specifically, we applied different imputation approaches to an acute myeloid leukemia dataset consisting of 194 samples and our method showed higher imputation accuracy, manifested, for example, by a 94% relative increase in information content and up to 86% more CpG sites passing post-imputation filtering. Our simulated association study further demonstrated that our method substantially improves the statistical power to identify trait-associated methylation loci. These findings indicate that the penalized functional regression model is a convenient and valuable imputation tool for methylation data, and it can boost statistical power in downstream epigenome-wide association study (EWAS).

  17. A closed parameterization of DNA-damage by charged particles as a function of energy

    CERN Document Server

    D, Frank Van den Heuvel Ph

    2013-01-01

    Purpose: To present a closed formalism calculating charged particle radiation damage induced in DNA, based on a simplified molecular model. The formalism is valid for all types of charged particles and due to its closed nature is suited to provide fast conversion of dose to DNA-damage. Methods: The induction of complex DNA--damaged is modelled using the standard scattering theory with a simplified effective potential. This leads to a proposal to use the Breit-Wigner expression to model the probability of the complex damage inelastic scatter as a function of kinetic energy of the scattered particle. A microscopic phenomenological Monte Carlo code is used to predict the damage to a DNA molecule embedded in a cell. The model is fit to the result of the simulation for four particles: electrons, protons, alpha--particles, and Carbon ions. The model is then used to predict the damage in a cell as a function of kinetic energy. Finally, a framework is proposed and implemented to provide data that can be assessed expe...

  18. Coumestan inhibits radical-induced oxidation of DNA: is hydroxyl a necessary functional group?

    Science.gov (United States)

    Xi, Gao-Lei; Liu, Zai-Qun

    2014-06-18

    Coumestan is a natural tetracycle with a C═C bond shared by a coumarin moiety and a benzofuran moiety. In addition to the function of the hydroxyl group on the antioxidant activity of coumestan, it is worth exploring the influence of the oxygen-abundant scaffold on the antioxidant activity as well. In this work, seven coumestans containing electron-withdrawing and electron-donating groups were synthesized to evaluate the abilities to trap 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(•+)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), and galvinoxyl radical, respectively, and to inhibit the oxidations of DNA mediated by (•)OH, Cu(2+)/glutathione (GSH), and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), respectively. It was found that all of the coumestans used herein can quench the aforementioned radicals and can inhibit (•)OH-, Cu(2+)/GSH-, and AAPH-induced oxidations of DNA. In particular, substituent-free coumestan exhibits higher ability to quench DPPH and to inhibit AAPH-induced oxidation of DNA than Trolox. In addition, nonsubstituted coumestan shows a similar ability to inhibit (•)OH- and Cu(2+)/GSH-induced oxidations of DNA relative to that of Trolox. The antioxidant effectiveness of the coumestan can be attributed to the lactone in the coumarin moiety and, therefore, a hydroxyl group may not be a necessary functional group for coumestan to be an antioxidant.

  19. Protein export elements from Lactococcus lactis

    NARCIS (Netherlands)

    Perez-Martinez, Gaspar; Kok, Jan; Venema, Gerhardus; Dijl, Jan Maarten van; Smith, Hilda; Bron, Sierd

    1992-01-01

    Broad-host-range plasmids carrying α-amylase or β-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the

  20. Protein export elements from Lactococcus lactis

    NARCIS (Netherlands)

    Perez-Martinez, Gaspar; Kok, Jan; Venema, Gerhardus; Dijl, Jan Maarten van; Smith, Hilda; Bron, Sierd

    Broad-host-range plasmids carrying α-amylase or β-lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the

  1. Properties of non-coding DNA and identification of putative cis-regulatory elements in Theileria parva

    Directory of Open Access Journals (Sweden)

    Guo Xiang

    2008-12-01

    Full Text Available Abstract Background Parasites in the genus Theileria cause lymphoproliferative diseases in cattle, resulting in enormous socio-economic losses. The availability of the genome sequences and annotation for T. parva and T. annulata has facilitated the study of parasite biology and their relationship with host cell transformation and tropism. However, the mechanism of transcriptional regulation in this genus, which may be key to understanding fundamental aspects of its parasitology, remains poorly understood. In this study, we analyze the evolution of non-coding sequences in the Theileria genome and identify conserved sequence elements that may be involved in gene regulation of these parasitic species. Results Intergenic regions and introns in Theileria are short, and their length distributions are considerably right-skewed. Intergenic regions flanked by genes in 5'-5' orientation tend to be longer and slightly more AT-rich than those flanked by two stop codons; intergenic regions flanked by genes in 3'-5' orientation have intermediate values of length and AT composition. Intron position is negatively correlated with intron length, and positively correlated with GC content. Using stringent criteria, we identified a set of high-quality orthologous non-coding sequences between T. parva and T. annulata, and determined the distribution of selective constraints across regions, which are shown to be higher close to translation start sites. A positive correlation between constraint and length in both intergenic regions and introns suggests a tight control over length expansion of non-coding regions. Genome-wide searches for functional elements revealed several conserved motifs in intergenic regions of Theileria genomes. Two such motifs are preferentially located within the first 60 base pairs upstream of transcription start sites in T. parva, are preferentially associated with specific protein functional categories, and have significant similarity to know

  2. Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

    Science.gov (United States)

    Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R; Jauch, Ralf

    2014-10-01

    Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

  3. Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution

    Directory of Open Access Journals (Sweden)

    Purves Joanne

    2012-09-01

    Full Text Available Abstract Background Staphylococcus aureus Repeat (STAR elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis.

  4. Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution.

    Science.gov (United States)

    Purves, Joanne; Blades, Matthew; Arafat, Yasrab; Malik, Salman A; Bayliss, Christopher D; Morrissey, Julie A

    2012-09-28

    Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis.

  5. Modeling structure-function relationships in synthetic DNA sequences using attribute grammars.

    Directory of Open Access Journals (Sweden)

    Yizhi Cai

    2009-10-01

    Full Text Available Recognizing that certain biological functions can be associated with specific DNA sequences has led various fields of biology to adopt the notion of the genetic part. This concept provides a finer level of granularity than the traditional notion of the gene. However, a method of formally relating how a set of parts relates to a function has not yet emerged. Synthetic biology both demands such a formalism and provides an ideal setting for testing hypotheses about relationships between DNA sequences and phenotypes beyond the gene-centric methods used in genetics. Attribute grammars are used in computer science to translate the text of a program source code into the computational operations it represents. By associating attributes with parts, modifying the value of these attributes using rules that describe the structure of DNA sequences, and using a multi-pass compilation process, it is possible to translate DNA sequences into molecular interaction network models. These capabilities are illustrated by simple example grammars expressing how gene expression rates are dependent upon single or multiple parts. The translation process is validated by systematically generating, translating, and simulating the phenotype of all the sequences in the design space generated by a small library of genetic parts. Attribute grammars represent a flexible framework connecting parts with models of biological function. They will be instrumental for building mathematical models of libraries of genetic constructs synthesized to characterize the function of genetic parts. This formalism is also expected to provide a solid foundation for the development of computer assisted design applications for synthetic biology.

  6. Kinetic control of the coverage of oil droplets by DNA-functionalized colloids

    OpenAIRE

    Joshi, Darshana; Bargteil, Dylan; Caciagli, Alessio; Burelbach, Jerome; Xing, Zhongyang; Nunes, André S.; Pinto, Diogo E. P.; Araújo, Nuno A. M.; Brujic, Jasna; Eiser, Erika

    2016-01-01

    We report a study of reversible adsorption of DNA-coated colloids on complementary functionalized oil droplets. We show that it is possible to control the surface coverage of oil droplets by colloidal particles, by exploiting the fact that during slow adsorption, compositional arrest takes place well before structural arrest occurs. As a consequence, we can prepare colloid-coated oil droplets with a `frozen' degree of loading, but with fully ergodic colloidal dynamics on the droplets. We illu...

  7. E. coli chaperones DnaK, Hsp33 and Spy inhibit bacterial functional amyloid assembly.

    Science.gov (United States)

    Evans, Margery L; Schmidt, Jens C; Ilbert, Marianne; Doyle, Shannon M; Quan, Shu; Bardwell, James C A; Jakob, Ursula; Wickner, Sue; Chapman, Matthew R

    2011-01-01

    Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homologue in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.

  8. Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples.

    Science.gov (United States)

    Patterson, M J; Sutton, R E; Forrest, I; Sharrock, R; Lane, M; Kaufmann, A; O'Donnell, R; Edmondson, R J; Wilson, B T; Curtin, N J

    2014-07-08

    Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells. A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells. From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1. HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.

  9. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  10. Design of new generation femoral prostheses using functionally graded materials: a finite element analysis.

    Science.gov (United States)

    Oshkour, A A; Abu Osman, N A; Yau, Y H; Tarlochan, F; Abas, W A B Wan

    2013-01-01

    This study aimed to develop a three-dimensional finite element model of a functionally graded femoral prosthesis. The model consisted of a femoral prosthesis created from functionally graded materials (FGMs), cement, and femur. The hip prosthesis was composed of FGMs made of titanium alloy, chrome-cobalt, and hydroxyapatite at volume fraction gradient exponents of 0, 1, and 5, respectively. The stress was measured on the femoral prosthesis, cement, and femur. Stress on the neck of the femoral prosthesis was not sensitive to the properties of the constituent material. However, stress on the stem and cement decreased proportionally as the volume fraction gradient exponent of the FGM increased. Meanwhile, stress became uniform on the cement mantle layer. In addition, stress on the femur in the proximal part increased and a high surface area of the femoral part was involved in absorbing the stress. As such, the stress-shielding area decreased. The results obtained in this study are significant in the design and longevity of new prosthetic devices because FGMs offer the potential to achieve stress distribution that more closely resembles that of the natural bone in the femur.

  11. Function of the vegetative elements in contemporaneous interpretation of the architectonic work

    Directory of Open Access Journals (Sweden)

    Markéta Krejčí

    2008-01-01

    Full Text Available The creative process during which a vegetation element finds itself in the position of a fundamental part of material design of the building can be found in the field of modern architectural production. Along with other building materials, it has its own task to participate in the composition of architectural space. This represents an authentic approach of the current production where the do­mi­na­ting position of the vegetation material determines the unique character of the final work. In these exis­ting factors of practice, the vegetation material is treated, according to the three branches of purposefulness after Friedrich Schinkel, as fundamental part of structural composition of the architectural work. In considered cases, when the vegetation material is removed, the building loses its functional qualities or basic value of expression. Studied cases have proved the existing application of vegetation motives and their combinations the garden art has worked with for centuries. However, mo­dern architectural production adapts them to fields of application that are completely new. It develops these original sources of inspiration that lead modern architecture to totally inventive and new results. The above-mentioned factors are the subject of this paper the purpose of which is to provide basic determination of real applicability of the green mass in the materials applied in the building construction and give examples of current leading finished examples. On the one hand, ar­chi­tec­tu­ral objects restore, with the application of vegetation elements, natural form of greenery in urban interior on the individual level of human dimension. On the other hand, with their help, when siting a project in open space you can also prevent building of a totalitarian wall in the form of a building mass. Thus contextuality of the executed project is achieved in relation to its surroundings. In the presented architectural initiatives the vegetation

  12. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  13. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  14. ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

    DEFF Research Database (Denmark)

    Zhao, A.; Gray, F. C; MacNeill, S. A.

    2006-01-01

    DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile...... Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

  15. A static analysis of three-dimensional functionally graded beams through hierarchical one-dimensional finite elements

    Energy Technology Data Exchange (ETDEWEB)

    Giunta, G.; Belouettar, S. [Centre de Recherche Public Henri Tudor, 29, av. John F. Kennedy, L-1855, Luxembourg-Kirchberg, Luxembourg (Belgium)

    2015-03-10

    In this paper, the static response of three-dimensional beams made of functionally graded materials is investigated through a family of hierarchical one-dimensional finite elements. A wide variety of elements is proposed differing by the kinematic formulation and the number of nodes per elements along the beam axis. Elements’ stiffness matrix and load vector are derived in a unified nuclear form that does not depend upon the a priori expansion order over the cross-section nor the finite element approximation along the beam axis. Results are validated towards three-dimensional finite element models as well as equivalent Navier-type analytical solutions. The numerical investigations show that accurate and efficient solutions (when compared with full three-dimensional FEM solutions) can be obtained by the proposed family of hierarchical one-dimensional elements’ family.

  16. Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

    Full Text Available Abstract Background Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available. Results The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield, a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage λ genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence. Conclusion This study supports the conclusion that physics-based methods can offer a completely complementary

  17. Critical functions of Rpa3/Ssb3 in S-phase DNA damage responses in fission yeast.

    Directory of Open Access Journals (Sweden)

    Santiago Cavero

    2010-09-01

    Full Text Available Replication Protein A (RPA is a heterotrimeric, single-stranded DNA (ssDNA-binding complex required for DNA replication and repair, homologous recombination, DNA damage checkpoint signaling, and telomere maintenance. Whilst the larger RPA subunits, Rpa1 and Rpa2, have essential interactions with ssDNA, the molecular functions of the smallest subunit Rpa3 are unknown. Here, we investigate the Rpa3 ortholog Ssb3 in Schizosaccharomyces pombe and find that it is dispensable for cell viability, checkpoint signaling, RPA foci formation, and meiosis. However, increased spontaneous Rad11Rpa1 and Rad22Rad52 nuclear foci in ssb3Δ cells indicate genome maintenance defects. Moreover, Ssb3 is required for resistance to genotoxins that disrupt DNA replication. Genetic interaction studies indicate that Ssb3 has a close functional relationship with the Mms1-Mms22 protein complex, which is required for survival after DNA damage in S-phase, and with the mitotic functions of Mus81-Eme1 Holliday junction resolvase that is required for recovery from replication fork collapse. From these studies we propose that Ssb3 plays a critical role in mediating RPA functions that are required for repair or tolerance of DNA lesions in S-phase. Rpa3 orthologs in humans and other species may have a similar function.

  18. The HIV-1 integrase α4-helix involved in LTR-DNA recognition is also a highly antigenic peptide element.

    Directory of Open Access Journals (Sweden)

    Sandy Azzi

    Full Text Available Monoclonal antibodies (MAbas constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159 formed by residues 147 to 175 of the HIV-1 integrase (IN, we obtained a monoclonal antibody (MAba4 recognizing an epitope lying in the N-terminal portion of K159 (residues 147-166 of IN. The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4 was mostly a random coil in solution, while in the CCD (catalytic core domain crystal, the homologous segment displayed an amphipathic helix structure (α4-helix at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats and inhibitors (5CITEP, for instance, significantly impaired the binding of K156 to the antibody. Moreover, we found that in competition ELISAs, the processed and unprocessed LTR oligonucleotides interfered with the binding of MAba4 to IN and K156, confirming that the IN α4-helix uses common residues to interact with the DNA target and the MAba4 antibody. This also explains why, in our standard in vitro concerted integration assays, MAba4 strongly impaired the IN enzymatic activity.

  19. Protein ruthenation and DNA alkylation: chlorambucil-functionalized RAPTA complexes and their anticancer activity.

    Science.gov (United States)

    Nazarov, Alexey A; Meier, Samuel M; Zava, Olivier; Nosova, Yulia N; Milaeva, Elena R; Hartinger, Christian G; Dyson, Paul J

    2015-02-28

    Chemotherapeutics for the treatment of tumorigenic conditions that feature novel modes of action are highly sought after to overcome the limitations of current chemotherapies. Herein, we report the conjugation of the alkylating agent chlorambucil to the RAPTA scaffold, a well-established pharmacophore. While chlorambucil is known to alkylate DNA, the RAPTA complexes are known to coordinate to amino acid side chains of proteins. Therefore, such a molecule combines DNA and protein targeting properties in a single molecule. Several chlorambucil-tethered RAPTA derivatives were prepared and tested for their cytotoxicity, stability in water and reactivity to protein and DNA substrates. The anticancer activity of the complexes is widely driven by the cytotoxicity of the chlorambucil moiety. However, especially in the cisplatin-resistant A2780R cells, the chlorambucil-functionalized RAPTA derivatives are in general more cytotoxic than chlorambucil and also a mixture of chlorambucil and the parent organoruthenium RAPTA compound. In a proof-of-principle experiment, the cross-linking of DNA and protein fragments by a chlorambucil-RAPTA derivative was observed.

  20. Molecular Dynamics Studies of Self-Assembling Biomolecules and DNA-functionalized Gold Nanoparticles

    Science.gov (United States)

    Cho, Vince Y.

    This thesis is organized as following. In Chapter 2, we use fully atomistic MD simulations to study the conformation of DNA molecules that link gold nanoparticles to form nanoparticle superlattice crystals. In Chapter 3, we study the self-assembly of peptide amphiphiles (PAs) into a cylindrical micelle fiber by using CGMD simulations. Compared to fully atomistic MD simulations, CGMD simulations prove to be computationally cost-efficient and reasonably accurate for exploring self-assembly, and are used in all subsequent chapters. In Chapter 4, we apply CGMD methods to study the self-assembly of small molecule-DNA hybrid (SMDH) building blocks into well-defined cage-like dimers, and reveal the role of kinetics and thermodynamics in this process. In Chapter 5, we extend the CGMD model for this system and find that the assembly of SMDHs can be fine-tuned by changing parameters. In Chapter 6, we explore superlattice crystal structures of DNA-functionalized gold nanoparticles (DNA-AuNP) with the CGMD model and compare the hybridization.

  1. The effects of proliferation and DNA damage on hematopoietic stem cell function determine aging.

    Science.gov (United States)

    Khurana, Satish

    2016-07-01

    In most of the mammalian tissues, homeostasis as well as injury repair depend upon a small number of resident adult stem cells. The decline in tissue/organ function in aged organisms has been directly linked with poorly functioning stem cells. Altered function of hematopoietic stem cells (HSCs) is at the center of an aging hematopoietic system, a tissue with high cellular turnover. Poorly engrafting, myeloid-biased HSCs with higher levels of DNA damage accumulation are the hallmark features of an aged hematopoietic system. These cells show a higher proliferation rate than their younger counterparts. It was proposed that quiescence of these cells over long period of time leads to accumulation of DNA damage, eventually resulting in poor function/pathological conditions in hematopoietic system. However, various mouse models with premature aging phenotype also show highly proliferative HSCs. This review examines the evidence that links proliferation of HSCs with aging, which leads to functional changes in the hematopoietic system. Developmental Dynamics 245:739-750, 2016. © 2016 Wiley Periodicals, Inc.

  2. "Braingame Brian": Toward an Executive Function Training Program with Game Elements for Children with ADHD and Cognitive Control Problems.

    Science.gov (United States)

    Prins, Pier J M; Brink, Esther Ten; Dovis, Sebastiaan; Ponsioen, Albert; Geurts, Hilde M; de Vries, Marieke; van der Oord, Saskia

    2013-02-01

    In the area of childhood attention-deficit hyperactivity disorder, there is an urgent need for new, innovative, and child-focused treatments. A computerized executive functioning training with game elements aimed at enhancing self-control was developed. The first results are promising, and the next steps involve replication with larger samples, evaluating transfer of training effects to daily life, and enhancing motivation through more gaming elements.

  3. Analysis of delayed convergence in the three-noded Timoshenko beam element using the function space approach

    Indian Academy of Sciences (India)

    Somenath Mukherjee; Gangan Prathap

    2002-10-01

    Despite satisfying only completeness and continuity requirements, elements often perform erroneously in a certain class of problems, called the locking situations, where they display spurious stress oscillations and enhanced stiffness properties. The function space approach that effectively substantiates the postulates of the field consistency paradigm is an efficient tool to reveal the fundamental cause of locking phenomena, and propose methods to eliminate this pathological problem. In this paper, we review the delayed convergence behaviour of three-noded Timoshenko beam elements using the rigorous function space approach. Explicit, closed form algebraic results for the element strains, stresses and errors have been derived using this method. The performance of the field-inconsistent three-noded Timoshenko beam element is compared with that of the field-inconsistent twonoded beam element. It is demonstrated that while the field-inconsistent two-noded linear element is prone to shear locking, the field-inconsistent three-noded element is not very vulnerable to this pathological problem, despite the resulting shear oscillations.

  4. Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis.

    Directory of Open Access Journals (Sweden)

    Sabine Jurado

    2010-10-01

    Full Text Available Zn²(+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+-finger protein (ASCIZ; also known as ATMIN and ZNF822 was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

  5. An 8-Node Shell Element for Nonlinear Analysis of Shells Using the Refined Combination of Membrane and Shear Interpolation Functions

    Directory of Open Access Journals (Sweden)

    Woo-Young Jung

    2013-01-01

    Full Text Available An improved 8-node shell finite element applicable for the geometrically linear and nonlinear analyses of plates and shells is presented. Based on previous first-order shear deformation theory, the finite element model is further improved by the combined use of assumed natural strains and different sets of collocation points for the interpolation of the different strain components. The influence of the shell element with various conditions such as locations, number of enhanced membranes, and shear interpolation is also identified. By using assumed natural strain method with proper interpolation functions, the present shell element generates neither membrane nor shear locking behavior even when full integration is used in the formulation. Furthermore, to characterize the efficiency of these modifications of the 8-node shell finite elements, numerical studies are carried out for the geometrically linear and non-linear analysis of plates and shells. In comparison to some other shell elements, numerical examples for the methodology indicate that the modified element described locking-free behavior and better performance. More specifically, the numerical examples of annular plate presented herein show good validity, efficiency, and accuracy to the developed nonlinear shell element.

  6. Fabrication and functionalization of PCB gold electrodes suitable for DNA-based electrochemical sensing.

    Science.gov (United States)

    Salvo, P; Henry, O Y F; Dhaenens, K; Acero Sanchez, J L; Gielen, A; Werne Solnestam, B; Lundeberg, J; O'Sullivan, C K; Vanfleteren, J

    2014-01-01

    The request of high specificity and selectivity sensors suitable for mass production is a constant demand in medical research. For applications in point-of-care diagnostics and therapy, there is a high demand for low cost and rapid sensing platforms. This paper describes the fabrication and functionalization of gold electrodes arrays for the detection of deoxyribonucleic acid (DNA) in printed circuit board (PCB) technology. The process can be implemented to produce efficiently a large number of biosensors. We report an electrolytic plating procedure to fabricate low-density gold microarrays on PCB suitable for electrochemical DNA detection in research fields such as cancer diagnostics or pharmacogenetics, where biosensors are usually targeted to detect a small number of genes. PCB technology allows producing high precision, fast and low cost microelectrodes. The surface of the microarray is functionalized with self-assembled monolayers of mercaptoundodecanoic acid or thiolated DNA. The PCB microarray is tested by cyclic voltammetry in presence of 5 mM of the redox probe K3Fe(CN6) in 0.1 M KCl. The voltammograms prove the correct immobilization of both the alkanethiol systems. The sensor is tested for detecting relevant markers for breast cancer. Results for 5 nM of the target TACSTD1 against the complementary TACSTD1 and non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A probes show a remarkable detection limit of 0.05 nM and a high specificity.

  7. Functional analysis of Borrelia burgdorferi uvrA in DNA damage protection

    Science.gov (United States)

    Sambir, Mariya; Ivanova, Larisa B.; Bryksin, Anton V.; Godfrey, Henry P.; Cabello, Felipe C.

    2012-01-01

    Bacterial pathogens face constant challenges from DNA-damaging agents generated by host phagocytes. Although Borrelia burgdorferi appears to have many fewer DNA repair enzymes than pathogens with larger genomes, it does contain homologues of uvrA and uvrB (subunits A and B of excinuclease ABC). As a first step to exploring the physiologic function of uvrABbu and its possible role in survival in the host in the face of DNA damaging agents, a partially deleted uvrA mutant was isolated by targeted inactivation. While growth of this mutant was markedly inhibited by UV irradiation, mitomycin C (MMC) and hydrogen peroxide at doses which lacked effect on wild-type B. burgdorferi, its response to pH 6.0 – 6.8 and reactive nitrogen intermediates was similar to that of the wild-type parental strain. The sensitivity of the inactivation mutant to UV irradiation, MMC and peroxide was complemented by an extrachromosomal copy of uvrABbu. We conclude that uvrABbu is functional in B. burgdorferi. PMID:21272060

  8. The Foldback-like element Galileo belongs to the P superfamily of DNA transposons and is widespread within the Drosophila genus.

    Science.gov (United States)

    Marzo, Mar; Puig, Marta; Ruiz, Alfredo

    2008-02-26

    Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs.

  9. The relationship between mitochondrial DNA copy number and stallion sperm function.

    Science.gov (United States)

    Darr, Christa R; Moraes, Luis E; Connon, Richard E; Love, Charles C; Teague, Sheila; Varner, Dickson D; Meyers, Stuart A

    2017-05-01

    Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by

  10. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    Science.gov (United States)

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  11. RAD50, an SMC family member with multiple roles in DNA break repair: How does ATP affect function?

    NARCIS (Netherlands)

    E. Kinoshita (Eri); E. van der Linden (Eddy); H. Sanchez (Humberto); C. Wyman (Claire)

    2009-01-01

    textabstractThe protein complex including Mre11, Rad50, and Nbs1 (MRN) functions in DNA double-strand break repair to recognize and process DNA ends as well as signal for cell cycle arrest. Amino acid sequence similarity and overall architecture make Rad50 a member of the structural maintenance of c

  12. The repertoire of DNA-binding transcription factors in prokaryotes: functional and evolutionary lessons.

    Science.gov (United States)

    Perez-Rueda, Ernesto; Martinez-Nuñez, Mario Alberto

    2012-01-01

    The capabilities of organisms to contend with environmental changes depend on their genes and their ability to regulate their expression. DNA-binding transcription factors (TFs) play a central role in this process, because they regulate gene expression positively and/or negatively, depending on the operator context and ligand-binding status. In this review, we summarise recent findings regarding the function and evolution of TFs in prokaryotes. We consider the abundance of TFs in bacteria and archaea, the role of DNA-binding domains and their partner domains, and the effects of duplication events in the evolution of regulatory networks. Finally, a comprehensive picture for how regulatory networks have evolved in prokaryotes is provided.

  13. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

    KAUST Repository

    Börjesson, Karl

    2010-09-28

    We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption. © 2010 American Chemical Society.

  14. A 7872 cDNA microarray and its use in bovine functional genomics.

    Science.gov (United States)

    Everts, Robin E; Band, Mark R; Liu, Z Lewis; Kumar, Charu G; Liu, Lei; Loor, Juan J; Oliveira, Rosane; Lewin, Harris A

    2005-05-15

    The strategy used to create and annotate a 7872 cDNA microarray from cattle placenta and spleen cDNA sequences is described. This microarray contains approximately 6300 unique genes, as determined by BLASTN and TBLASTX similarity search against the human and mouse UniGene and draft human genome sequence databases (build 34). Sequences on the array were annotated with gene ontology (GO) terms, thereby facilitating data analysis and interpretation. A total of 3244 genes were annotated with GO terms. The array is rich in sequences encoding transcription factors, signal transducers and cell cycle regulators. Current research being conducted with this array is described, and an overview of planned improvements in our microarray platform for cattle functional genomics is presented.

  15. [Duplication of DNA--a mechanism for the development of new functionality of genes].

    Science.gov (United States)

    Maślanka, Roman; Zadrąg-Tęcza, Renata

    2015-01-01

    The amplification of DNA is considered as a mechanism for rapid evolution of organisms. Duplication can be especially advantageous in the case of changing environmental conditions. Whole genome duplication maintains the proper balance between gene expression. This seems to be the main reason why WGD is more favorable than duplication of the fragments of DNA. The polyploidy status disappear as a result of the loss of the majority of duplicated genes. The preservation of duplicated genes is associated with the development of their new functions. Polyploidization is often noted for plants. However due to sequencing technique, the duplications episodes are more frequently reports also for the other systematic taxa, including animals. The occurrence of ancient genome duplication is also considered for yeast Saccharomyces cerevisiae. The existence of two active copies of ribosomal protein genes can be a confirmation of this process. Development of the fermentation process might be one of the probable causes of the yeast genome duplication.

  16. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    Science.gov (United States)

    Conde, J.; de la Fuente, J. M.; Baptista, P. V.

    2010-12-01

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression—inhibition of both transcription and translation.

  17. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Conde, J; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); De la Fuente, J M, E-mail: pmvb@fct.unl.pt [Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza (Spain)

    2010-12-17

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression-inhibition of both transcription and translation.

  18. A novel cis-acting element from the 3′UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression

    Science.gov (United States)

    Melanson, Brian D.; Cabrita, Miguel A.; Bose, Reetesh; Hamill, Jeffrey D.; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A.; Zhao, Tong T.; Holcik, Martin; McKay, Bruce C.

    2013-01-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3′ untranslated region (3′UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3′UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3′UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3′UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3′UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked. PMID:23605047

  19. A novel cis-acting element from the 3'UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression.

    Science.gov (United States)

    Melanson, Brian D; Cabrita, Miguel A; Bose, Reetesh; Hamill, Jeffrey D; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A; Zhao, Tong T; Holcik, Martin; McKay, Bruce C

    2013-06-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.

  20. Comparison of various functionally graded femoral prostheses by finite element analysis.

    Science.gov (United States)

    Oshkour, Azim Ataollahi; Talebi, Hossein; Shirazi, Seyed Farid Seyed; Bayat, Mehdi; Yau, Yat Huang; Tarlochan, Faris; Abu Osman, Noor Azuan

    2014-01-01

    This study is focused on finite element analysis of a model comprising femur into which a femoral component of a total hip replacement was implanted. The considered prosthesis is fabricated from a functionally graded material (FGM) comprising a layer of a titanium alloy bonded to a layer of hydroxyapatite. The elastic modulus of the FGM was adjusted in the radial, longitudinal, and longitudinal-radial directions by altering the volume fraction gradient exponent. Four cases were studied, involving two different methods of anchoring the prosthesis to the spongy bone and two cases of applied loading. The results revealed that the FG prostheses provoked more SED to the bone. The FG prostheses carried less stress, while more stress was induced to the bone and cement. Meanwhile, less shear interface stress was stimulated to the prosthesis-bone interface in the noncemented FG prostheses. The cement-bone interface carried more stress compared to the prosthesis-cement interface. Stair climbing induced more harmful effects to the implanted femur components compared to the normal walking by causing more stress. Therefore, stress shielding, developed stresses, and interface stresses in the THR components could be adjusted through the controlling stiffness of the FG prosthesis by managing volume fraction gradient exponent.

  1. Thermal-mechanical study of functionally graded dental implants with the finite element method.

    Science.gov (United States)

    Wang, F; Lee, H P; Lu, C

    2007-01-01

    This article investigates the thermal-mechanical performance of hydroxyapatite/titanium (HA/Ti) functionally graded (FG) dental implants with the three-dimensional finite element method. The stresses induced by occlusal force for the present HA/Ti FG implant are calculated to compare with the corresponding stresses for the titanium dental implant. Thermal-mechanical effect of temperature variation due to daily oral activity is also studied. The HA/Ti FG dental implant performance is evaluated against the maximum von Mises stress, which is the general performance indicator, the first principal/tensile stress for mechanical failure of implant-bone-bond and the third principal/compressive stress for bone absorption. Simulation results indicate that under the influence of occlusal force only, the FG implants with different HA fraction along the implant length perform almost equally well, while the titanium implant sustains much higher von Mises stress. However, when thermal stress is also considered, the FG implant having HA fraction exponential index of m = 2 with temperature decrease of 20 degrees C yields the highest first principal and von Mises stresses among all the FG and titanium implants.

  2. Comparison of Various Functionally Graded Femoral Prostheses by Finite Element Analysis

    Directory of Open Access Journals (Sweden)

    Azim Ataollahi Oshkour

    2014-01-01

    Full Text Available This study is focused on finite element analysis of a model comprising femur into which a femoral component of a total hip replacement was implanted. The considered prosthesis is fabricated from a functionally graded material (FGM comprising a layer of a titanium alloy bonded to a layer of hydroxyapatite. The elastic modulus of the FGM was adjusted in the radial, longitudinal, and longitudinal-radial directions by altering the volume fraction gradient exponent. Four cases were studied, involving two different methods of anchoring the prosthesis to the spongy bone and two cases of applied loading. The results revealed that the FG prostheses provoked more SED to the bone. The FG prostheses carried less stress, while more stress was induced to the bone and cement. Meanwhile, less shear interface stress was stimulated to the prosthesis-bone interface in the noncemented FG prostheses. The cement-bone interface carried more stress compared to the prosthesis-cement interface. Stair climbing induced more harmful effects to the implanted femur components compared to the normal walking by causing more stress. Therefore, stress shielding, developed stresses, and interface stresses in the THR components could be adjusted through the controlling stiffness of the FG prosthesis by managing volume fraction gradient exponent.

  3. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  4. Bio-bar-code functionalized magnetic nanoparticle label for ultrasensitive flow injection chemiluminescence detection of DNA hybridization.

    Science.gov (United States)

    Bi, Sai; Zhou, Hong; Zhang, Shusheng

    2009-10-07

    A signal amplification strategy based on bio-bar-code functionalized magnetic nanoparticles as labels holds promise to improve the sensitivity and detection limit of the detection of DNA hybridization and single-nucleotide polymorphisms by flow injection chemiluminescence assays.

  5. Doping Level of Boron-Doped Diamond Electrodes Controls the Grafting Density of Functional Groups for DNA Assays.

    Science.gov (United States)

    Švorc, Ĺubomír; Jambrec, Daliborka; Vojs, Marian; Barwe, Stefan; Clausmeyer, Jan; Michniak, Pavol; Marton, Marián; Schuhmann, Wolfgang

    2015-09-02

    The impact of different doping levels of boron-doped diamond on the surface functionalization was investigated by means of electrochemical reduction of aryldiazonium salts. The grafting efficiency of 4-nitrophenyl groups increased with the boron levels (B/C ratio from 0 to 20,000 ppm). Controlled grafting of nitrophenyldiazonium was used to adjust the amount of immobilized single-stranded DNA strands at the surface and further on the hybridization yield in dependence on the boron doping level. The grafted nitro functions were electrochemically reduced to the amine moieties. Subsequent functionalization with a succinic acid introduced carboxyl groups for subsequent binding of an amino-terminated DNA probe. DNA hybridization significantly depends on the probe density which is in turn dependent on the boron doping level. The proposed approach opens new insights for the design and control of doped diamond surface functionalization for the construction of DNA hybridization assays.

  6. Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast

    National Research Council Canada - National Science Library

    Riesmeier, J.W; Willmitzer, L; Frommer, W.B

    1992-01-01

    ...‐symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression...

  7. T-T mismatch-driven biosensor using triple functional DNA-protein conjugates for facile detection of Hg2+.

    Science.gov (United States)

    Wang, Ruoyu; Zhou, Xiaohong; Shi, Hanchang; Luo, Yi

    2016-04-15

    We report herein a T-T mismatch-driven biosensor using triple functional DNA-protein conjugates for facile detection of mercury ions (Hg(2+)) based on evanescent wave fluorescence excitation. Fluorescein-labeled DNA strands and streptavidin molecules were conjugated using heterobifunctional crosslinkers, and the obtained conjugates were named as "Hg(2+) dependent conjugates, HDCs". Initially hybridized with quencher-labeled DNA (Q-DNA) strands, HDCs showed low evanescent wave-induced fluorescence emission signals; however, in the presence of Hg(2+), the DNA moieties of HDCs tended to form hairpin structures stabilized by T-T mismatches, releasing Q-DNA strands, which was accompanied by increases in the fluorescent signals. The novel detection strategy enables the fluorescent detection of mercury ions with high specificity and a low detection limit of 1.06 nM in a facile way.

  8. First-principles photoemission spectroscopy of DNA and RNA nucleobases from Koopmans-compliant functionals

    CERN Document Server

    Nguyen, Ngoc Linh; Ferretti, Andrea; Marzari, Nicola

    2016-01-01

    The need to interpret ultraviolet photoemission data strongly motivates the refinement of first-principles techniques able to accurately predict spectral properties. In this work we employ Koopmans-compliant functionals, constructed to enforce piecewise linearity in approximate density functionals, to calculate the structural and electronic properties of DNA and RNA nucleobases. Our results show that not only ionization potentials and electron affinities are accurately predicted with mean absolute errors < 0.1 eV, but also that calculated photoemission spectra are in excellent agreement with experimental ultraviolet photoemission spectra. In particular, the role and contribution of different tautomers to the photoemission spectra are highlighted and discussed in detail. The structural properties of nucleobases are also investigated, showing an improved description with respect to local and semilocal density-functional theory. Methodologically, our results further consolidate the role of Koopmans-compliant ...

  9. Functional cDNA expression cloning: Pushing it to the limit

    Science.gov (United States)

    OKAYAMA, Hiroto

    2012-01-01

    The 1970s and the following decade are the era of the birth and early development of recombinant DNA technologies, which have entirely revolutionized the modern life science by providing tools that enable us to know the structures of genes and genomes and to dissect their components and understand their functions at the molecular and submolecular levels. One major objective of the life sciences is to achieve molecular and chemical understandings of the functions of genes and their encoded proteins, which are responsible for the manifestation of all biological phenomena in organisms. In the early 1980s, I developed, together with Paul Berg, a new technique that enables the cloning of full-length complementary DNAs (cDNAs) on the basis of their functional expression in a given cell of interest. I review the development, application and future implications in the life sciences of this gene-cloning technique. PMID:22450538

  10. Large-scale all-electron density functional theory calculations using an enriched finite-element basis

    Science.gov (United States)

    Kanungo, Bikash; Gavini, Vikram

    2017-01-01

    We present a computationally efficient approach to perform large-scale all-electron density functional theory calculations by enriching the classical finite element basis with compactly supported atom-centered numerical basis functions that are constructed from the solution of the Kohn-Sham (KS) problem for single atoms. We term these numerical basis functions as enrichment functions, and the resultant basis as the enriched finite element basis. The compact support for the enrichment functions is obtained by using smooth cutoff functions, which enhances the conditioning and maintains the locality of the enriched finite element basis. The integrals involved in the evaluation of the discrete KS Hamiltonian and overlap matrix in the enriched finite element basis are computed using an adaptive quadrature grid that is constructed based on the characteristics of enrichment functions. Further, we propose an efficient scheme to invert the overlap matrix by using a blockwise matrix inversion in conjunction with special reduced-order quadrature rules, which is required to transform the discrete Kohn-Sham problem to a standard eigenvalue problem. Finally, we solve the resulting standard eigenvalue problem, in each self-consistent field iteration, by using a Chebyshev polynomial based filtering technique to compute the relevant eigenspectrum. We demonstrate the accuracy, efficiency, and parallel scalability of the proposed method on semiconducting and heavy-metallic systems of various sizes, with the largest system containing 8694 electrons. We obtain accuracies in the ground-state energies that are ˜1 mHa with reference ground-state energies employing classical finite element as well as Gaussian basis sets. Using the proposed formulation based on enriched finite element basis, for accuracies commensurate with chemical accuracy, we observe a staggering 50 -300 -fold reduction in the overall computational time when compared to classical finite element basis. Further, we find a

  11. A FINITE-ELEMENTS APPROACH TO THE STUDY OF FUNCTIONAL ARCHITECTURE IN SKELETAL-MUSCLE

    NARCIS (Netherlands)

    OTTEN, E; HULLIGER, M

    1994-01-01

    A mathematical model that simulates the mechanical processes inside a skeletal muscle under various conditions of muscle recruitment was formulated. The model is based on the finite-elements approach and simulates both contractile and passive elastic elements. Apart from the classic strategy of solv

  12. A FINITE-ELEMENTS APPROACH TO THE STUDY OF FUNCTIONAL ARCHITECTURE IN SKELETAL-MUSCLE

    NARCIS (Netherlands)

    OTTEN, E; HULLIGER, M

    1994-01-01

    A mathematical model that simulates the mechanical processes inside a skeletal muscle under various conditions of muscle recruitment was formulated. The model is based on the finite-elements approach and simulates both contractile and passive elastic elements. Apart from the classic strategy of solv

  13. Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

    Science.gov (United States)

    Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

    2014-01-01

    The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date.

  14. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp. Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    Directory of Open Access Journals (Sweden)

    Caroline Chénard

    2016-06-01

    Full Text Available Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages.

  15. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B

    Science.gov (United States)

    Chénard, Caroline; Wirth, Jennifer F.

    2016-01-01

    ABSTRACT   Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. PMID:27302758

  16. ESL Based Cylindrical Shell Elements with Hierarchical Shape Functions for Laminated Composite Shells

    Directory of Open Access Journals (Sweden)

    Jae S. Ahn

    2015-01-01

    Full Text Available We introduce higher-order cylindrical shell element based on ESL (equivalent single-layer theory for the analysis of laminated composite shells. The proposed elements are formulated by the dimensional reduction technique from three-dimensional solid to two-dimensional cylindrical surface with plane stress assumption. It allows the first-order shear deformation and considers anisotropic materials due to fiber orientation. The element displacement approximation is established by the integrals of Legendre polynomials with hierarchical concept to ensure the C0-continuity at the interface between adjacent elements as well as C1-continuity at the interface between adjacent layers. For geometry mapping, cylindrical coordinate is adopted to implement the exact mapping of curved shell configuration with a constant curvature with respect to any direction in the plane. The verification and characteristics of the proposed element are investigated through the analyses of three cylindrical shell problems with different shapes, loadings, and boundary conditions.

  17. Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function.

    Directory of Open Access Journals (Sweden)

    Sugith Babu Badugu

    Full Text Available The eukaryotic Meiotic Recombination protein 11 (Mre11 plays pivotal roles in the DNA damage response (DDR. Specifically, Mre11 senses and signals DNA double strand breaks (DSB and facilitates their repair through effector proteins belonging to either homologous recombination (HR or non-homologous end joining (NHEJ repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11 that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11. Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N. PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.

  18. Identification of Plasmodium falciparum DNA Repair Protein Mre11 with an Evolutionarily Conserved Nuclease Function.

    Science.gov (United States)

    Badugu, Sugith Babu; Nabi, Shaik Abdul; Vaidyam, Pratap; Laskar, Shyamasree; Bhattacharyya, Sunanda; Bhattacharyya, Mrinal Kanti

    2015-01-01

    The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR)