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Sample records for dna synthesis clarification

  1. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  2. Biphasic DNA Synthesis in Spumaviruses

    PubMed Central

    Delelis, Olivier; Saïb, Ali; Sonigo, Pierre

    2003-01-01

    Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication. PMID:12829852

  3. Concepts in Biochemistry: Chemical Synthesis of DNA.

    ERIC Educational Resources Information Center

    Caruthers, Marvin H.

    1989-01-01

    Outlines the chemistry of the rapid synthesis of relatively large DNA fragments (100-200 monomers each) with yields exceeding 99 percent per coupling. DNA synthesis methodologies are outlined and a polymer-supported synthesis of DNA using deoxynucleoside phosphoramidites is described with structural formulas. (YP)

  4. Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

    PubMed Central

    Plevani, P; Chang, L M

    1977-01-01

    Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. PMID:325562

  5. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  6. Direct electrical detection of DNA synthesis

    PubMed Central

    Pourmand, Nader; Karhanek, Miloslav; Persson, Henrik H. J.; Webb, Chris D.; Lee, Thomas H.; Zahradníková, Alexandra; Davis, Ronald W.

    2006-01-01

    Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3′-hydroxyl group of the DNA molecule during phosphodiester bond formation. PMID:16614066

  7. Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

    PubMed

    Kwant, M M; van der Vliet, P C

    1980-09-11

    There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.

  8. Analytical approaches for clarification of DNA-double decker phthalocyanine binding mechanism: As an alternative anticancer chemotherapeutic.

    PubMed

    Bağda, Esra; Yabaş, Ebru; Bağda, Efkan

    2017-02-05

    In the present study a novel water soluble double-decker phthalocyanine was synthesized and calf thymus DNA interaction of the synthesized double-decker phthalocyanine was investigated. 5-(3-pyridyl)-1,3,4-oxadiazole substituted phthalonitrile 1 was prepared by a nucleophilic displacement reaction of 4-nitrophthalonitrile with 5-(3-pyridyl)-1,3,4-oxadiazole-2-thiol. Lutetium(III) double-decker phthalocyanine 2 was prepared by cyclotetramerization of compound 1. Water soluble lutetium(III) double-decker phthalocyanine 3 was prepared with quaternarization of compound 2. The synthesized double-decker phthalocyanine and calf thymus DNA interaction was investigated with UV-vis titrimetric methods, gel electrophoresis, and viscosity measurements. The fluorometric ethidium bromide replacement assay was conducted to clarify the binding mode of water soluble double-decker phthalocyanine. The thermodynamic parameters for interaction, K, ΔG(0), ΔH(0) and ΔS(0) were calculated between the temperature ranges of 25°C-75°C. To the best of our knowledge, this is the first study about a double-decker phthalocyanine and DNA interaction.

  9. Analytical approaches for clarification of DNA-double decker phthalocyanine binding mechanism: As an alternative anticancer chemotherapeutic

    NASA Astrophysics Data System (ADS)

    Bağda, Esra; Yabaş, Ebru; Bağda, Efkan

    2017-02-01

    In the present study a novel water soluble double-decker phthalocyanine was synthesized and calf thymus DNA interaction of the synthesized double-decker phthalocyanine was investigated. 5-(3-pyridyl)-1,3,4-oxadiazole substituted phthalonitrile 1 was prepared by a nucleophilic displacement reaction of 4-nitrophthalonitrile with 5-(3-pyridyl)-1,3,4-oxadiazole-2-thiol. Lutetium(III) double-decker phthalocyanine 2 was prepared by cyclotetramerization of compound 1. Water soluble lutetium(III) double-decker phthalocyanine 3 was prepared with quaternarization of compound 2. The synthesized double-decker phthalocyanine and calf thymus DNA interaction was investigated with UV-vis titrimetric methods, gel electrophoresis, and viscosity measurements. The fluorometric ethidium bromide replacement assay was conducted to clarify the binding mode of water soluble double-decker phthalocyanine. The thermodynamic parameters for interaction, K, ΔG0, ΔH0 and ΔS0 were calculated between the temperature ranges of 25 °C-75 °C. To the best of our knowledge, this is the first study about a double-decker phthalocyanine and DNA interaction.

  10. Thymidine analogues for tracking DNA synthesis.

    PubMed

    Cavanagh, Brenton L; Walker, Tom; Norazit, Anwar; Meedeniya, Adrian C B

    2011-09-15

    Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

  11. Chemically-enzymatic synthesis of photosensitive DNA.

    PubMed

    Westphal, Kinga; Zdrowowicz, Magdalena; Zylicz-Stachula, Agnieszka; Rak, Janusz

    2017-02-01

    The sensitizing propensity of radio-/photosensitizing nucleoside depends on DNA sequence surrounding a sensitizer. Therefore, in order to compare sensitizers with regard to their ability to induce a DNA damage one has to study the sequence dependence of damage yield. However, chemical synthesis of oligonucleotides labeled with sensitizing nucleosides is hindered due to the fact that a limited number of such nucleoside phosphoramidites are accessible. Here, we report on a chemically-enzymatic method, employing a DNA polymerase and ligase, that enables a modified nucleoside, in the form of its 5'-triphosphate, to be incorporated into DNA fragment in a pre-determined site. Using such a protocol two double-stranded DNA fragments - a long one, 75 base pairs (bp), and a short one, 30bp in length - were pin-point labeled with 5-bromodeoxyuridine. Four DNA polymerases together with DHPLC for the inspection of reaction progress were used to optimize the process under consideration. As an ultimate test showing that the product possessing an assumed nucleotide sequence was actually obtained, we irradiated the synthesized oligonucleotide with UVB photons and analyzed its photoreactivity with the LC-MS method. Our results prove that a general approach enabling precise labeling of DNA with any nucleoside modification processed by DNA polymerase and ligase has been worked out.

  12. Magnetic control of the DNA synthesis

    NASA Astrophysics Data System (ADS)

    Buchachenko, Anatoly L.; Orlov, Alexei P.; Kuznetsov, Dmitry A.; Breslavskaya, Natalia N.

    2013-10-01

    By using polymerases β loaded with isotopic ions 24Mg2+, 25Mg2+ and 26Mg2+ magnetic isotope effect was detected: 25Mg2+ ions with magnetic nuclei 25Mg suppress enzymatic activity by 2-3 times with respect to that of polymerases β loaded by 24Mg2+ and 26Mg2+ ions. No difference in enzymatic activity of polymerases β with 24Mg2+ and 26Mg2+ ions exists. The rate of DNA synthesis strongly depends on the magnetic field. The polymerase chain reaction is also suppressed by 25Mg2+ ions with respect to the ions with nonmagnetic nuclei. Magnetic control of the DNA synthesis may be used for medical purposes.

  13. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    PubMed Central

    Geuskens, M; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles. Images PMID:6262734

  14. DNA Compatible Multistep Synthesis and Applications to DNA Encoded Libraries.

    PubMed

    Satz, Alexander Lee; Cai, Jianping; Chen, Yi; Goodnow, Robert; Gruber, Felix; Kowalczyk, Agnieszka; Petersen, Ann; Naderi-Oboodi, Goli; Orzechowski, Lucja; Strebel, Quentin

    2015-08-19

    Complex mixtures of DNA encoded small molecules may be readily interrogated via high-throughput sequencing. These DNA encoded libraries (DELs) are commonly used to discover molecules that interact with pharmaceutically relevant proteins. The chemical diversity displayed by the library is key to successful discovery of potent, novel, and drug-like chemical matter. The small molecule moieties of DELs are generally synthesized though a multistep process, and each chemical step is accomplished while it is simultaneously attached to an encoding DNA oligomer. Hence, library chemical diversity is often limited to DNA compatible synthetic reactions. Herein, protocols for 24 reactions are provided that have been optimized for high-throughput production of DELs. These protocols detail the multistep synthesis of benzimidazoles, imidazolidinones, quinazolinones, isoindolinones, thiazoles, and imidazopyridines. Additionally, protocols are provided for a diverse range of useful chemical reactions including BOC deprotection (under pH neutral conditions), carbamylation, and Sonogashira coupling. Last, step-by-step protocols for synthesizing functionalized DELs from trichloronitropyrimidine and trichloropyrimidine scaffolds are detailed.

  15. The Logic of Values Clarification

    ERIC Educational Resources Information Center

    Kazepides, A. C.

    1977-01-01

    Traces the origin of the Values Clarification movement in education in Carl Roger's clien-centered therapy and exposes its unwarranted extreme ethical stance. Examines a model episode of values clarification and shows how the theoretical confusions of the Values Clarification proponents are reflected in their actual teaching strategies. (Editor/RK)

  16. Clarifications, Refutations, and Resolutions

    ERIC Educational Resources Information Center

    Rabinowitz, Jan C.; And Others

    1977-01-01

    The criticisms of Rabinowitz, Mandler, and Patterson offered by Broadbent and Broadbent (AA 527 085) are discussed. A clarification of some of the results supports the original view that item recognition often involves retrieval processes similar to those of free recall. Data are presented to refute Broadbents' suggestion that a recognition test…

  17. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    SciTech Connect

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  18. DNA sequencing by synthesis based on elongation delay detection

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2015-03-01

    The one of most important problem in modern genetics, biology and medicine is determination of the primary nucleotide sequence of the DNA of living organisms (DNA sequencing). This paper describes the label-free DNA sequencing approach, based on the observation of a discrete dynamics of DNA sequence elongation phase. The proposed DNA sequencing principle are studied by numerical simulation. The numerical model for proposed label-free DNA sequencing approach is based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) and dynamics of nucleotides incorporation to rising DNA strand. The estimates for number of copied DNA sequences for required probability of nucleotide incorporation event detection and correct DNA sequence determination was obtained. The proposed approach can be applied at all known DNA sequencing devices with "sequencing by synthesis" principle of operation.

  19. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    PubMed Central

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability. PMID:28298904

  20. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV.

    PubMed

    Tashjian, Tommy F; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M; Godoy, Veronica G

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  1. Inhibition of DNA synthesis in Meth A cells by chlorpromazine.

    PubMed

    Mizushima, T; Natori, S; Sekimizu, K

    1993-10-01

    We examined the influence of chlorpromazine, a phenothiazine derivative, on DNA synthesis in Meth A cells. Pulse-labelling experiments with [3H]thymidine showed that chlorpromazine inhibited DNA synthesis in cells cultured in vitro. The drug also inhibited DNA synthesis in isolated nuclei. Observation by fluorescence microscopy of fibroblastic cells stained with chlorpromazine indicated that the drug was localized in the cytoplasm and nuclear membranes, suggesting that it inhibited DNA synthesis in a manner dependent on the interaction of replication proteins with nuclear membranes. Meth A sarcomas growing in the endoderm of BALB/c mice regressed on intra-tumor injection of chlorpromazine, indicating that the drug has an anticancer action.

  2. Analytical Devices Based on Direct Synthesis of DNA on Paper.

    PubMed

    Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

    2016-01-05

    This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

  3. Design and Synthesis of Biaryl DNA-Encoded Libraries.

    PubMed

    Ding, Yun; Franklin, G Joseph; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Clark, Matthew A; Skinner, Steven R; Belyanskaya, Svetlana

    2016-10-10

    DNA-encoded library technology (ELT) is a powerful tool for the discovery of new small-molecule ligands to various protein targets. Here we report the design and synthesis of biaryl DNA-encoded libraries based on the scaffold of 5-formyl 3-iodobenzoic acid. Three reactions on DNA template, acylation, Suzuki-Miyaura coupling and reductive amination, were applied in the library synthesis. The three cycle library of 3.5 million diversity has delivered potent hits for phosphoinositide 3-kinase α (PI3Kα).

  4. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

    PubMed Central

    Aron, G M; Purifoy, D J; Schaffer, P A

    1975-01-01

    Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies. PMID:169388

  5. Nonuniform distribution of excision repair synthesis in nucleosome core DNA

    SciTech Connect

    Lan, S.Y.; Smerdon, M.J.

    1985-12-17

    We have studied the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. The differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to map the distribution of repair synthesis in these regions. Results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. The 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only internal locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

  6. COORDINATING DNA POLYMERASE TRAFFIC DURING HIGH AND LOW FIDELITY SYNTHESIS

    PubMed Central

    Sutton, Mark D.

    2009-01-01

    With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell’s genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS. PMID:19540941

  7. Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver

    SciTech Connect

    Lutz, W.K.; Buesser, M.T.; Sagelsdorff, P.

    1984-01-01

    In order to investigate the role of the stimulation of cell division for the initiation (and possibly promotion) of liver tumors by chemical carcinogens, the incorporation of radiolabelled thymidine into liver DNA was determined in male rats. Single doses of various levels of aflatoxin B1, benzidine and carbon tetrachloride (all known to be genotoxic via DNA binding) did not affect cell division, whereas several hepatocarcinogens known not to bind to DNA (alpha-HCH, clofibrate, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) gave rise to a dose-dependent stimulation of liver DNA synthesis within 24 h. An equation combining the influences of mitotic stimulation, expressed as dose required to double the control level of DNA synthesis, and DNA binding potency, expressed as the Covalent Binding Index, correlated well with the carcinogenic potency for both classes of hepatocarcinogens.

  8. Characterization of human translesion DNA synthesis across a UV-induced DNA lesion

    PubMed Central

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-01-01

    Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand. DOI: http://dx.doi.org/10.7554/eLife.19788.001 PMID:27770570

  9. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

    PubMed Central

    2015-01-01

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and

  10. Facile dimer synthesis for DNA-binding polyamide ligands.

    PubMed

    Wetzler, Modi; Wemmer, David E

    2010-08-06

    Pyrrole-imidazole polyamide ligands are highly sequence specific synthetic DNA-binding ligands that bind with high affinity. To counter the synthetic difficulties associated with coupling the electron-rich heterocyclic acids to the electron-deficient nucleophilic imidazole amine, a novel approach is described for synthesis of Fmoc-protected dimers for solid-phase peptide synthesis (SPPS). This method produces the dimers in high yields, is broadly applicable to other heterocyclic-containing polyamides, and results in improved ligand yields and synthesis times.

  11. Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases

    PubMed Central

    Zhao, Linlin; Washington, M. Todd

    2017-01-01

    DNA replication is constantly challenged by DNA lesions, noncanonical DNA structures and difficult-to-replicate DNA sequences. Two major strategies to rescue a stalled replication fork and to ensure continuous DNA synthesis are: (1) template switching and recombination-dependent DNA synthesis; and (2) translesion synthesis (TLS) using specialized DNA polymerases to perform nucleotide incorporation opposite DNA lesions. The former pathway is mainly error-free, and the latter is error-prone and a major source of mutagenesis. An accepted model of translesion synthesis involves DNA polymerase switching steps between a replicative DNA polymerase and one or more TLS DNA polymerases. The mechanisms that govern the selection and exchange of specialized DNA polymerases for a given DNA lesion are not well understood. In this review, recent studies concerning the mechanisms of selection and switching of DNA polymerases in eukaryotic systems are summarized. PMID:28075396

  12. Strand displacement synthesis by yeast DNA polymerase ε

    PubMed Central

    Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

    2016-01-01

    DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

  13. Towards the Batch Synthesis of Long DNA

    DTIC Science & Technology

    2002-10-01

    MISMATCHES In a series of papers,136 the SantaLucia NN model137 of Watson - Crick paired DNA thermodynamics was successfully extended to incorporate...generally indicate a- helix coding or structural motifs for DNA incorporation into chromatin. Trifonov, E. N., “3-,!10.5-, 200- and 400-base...double-stranded DNA , is well-described by Hearst’s “weakly bending rod” model with 3.4 Å rise/bp and 13 Å radius for the helix ; its persistence length39

  14. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  15. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  16. The control of lambda DNA terminase synthesis.

    PubMed Central

    Murialdo, H; Davidson, A; Chow, S; Gold, M

    1987-01-01

    Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques. Images PMID:3029667

  17. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer

    DTIC Science & Technology

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0238 TITLE: Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer PRINCIPAL...of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Oxygen-rich environments can create pro-mutagenic DNA lesions such as 8-oxoguanine (8-oxo-G) that can be misreplicated during translesion DNA synthesis

  18. Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2

    PubMed Central

    1993-01-01

    A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin- induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies. PMID:8458876

  19. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-05

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

  20. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

    PubMed

    Wilson, Marenda A; Kwon, YoungHo; Xu, Yuanyuan; Chung, Woo-Hyun; Chi, Peter; Niu, Hengyao; Mayle, Ryan; Chen, Xuefeng; Malkova, Anna; Sung, Patrick; Ira, Grzegorz

    2013-10-17

    During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

  1. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with ..gamma.. rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  2. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  3. Synchronization of mitochondrial DNA synthesis in Chinese hamster cells (line CHO) deprived of isoleucine.

    PubMed

    Ley, K D; Murphy, M M

    1973-08-01

    Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([(3)H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([(3)H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9-12 h after addition of isoleucine and virtually all [(3)H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G(1) because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

  4. Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

    PubMed

    Thomas, Baptiste; Lu, Xiaojie; Birmingham, William R; Huang, Kun; Both, Peter; Reyes Martinez, Juana Elizabeth; Young, Robert J; Davie, Christopher P; Flitsch, Sabine L

    2017-01-26

    DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry.

  5. Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

    DOEpatents

    Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

    2010-05-04

    A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

  6. HGF-induced DNA synthesis in hepatocytes is suppressed by p38.

    PubMed

    Aasrum, Monica; Brusevold, Ingvild J; Christoffersen, Thoralf; Thoresen, G Hege

    2016-12-01

    Previous studies in rat hepatocytes have shown that the MEK/ERK, PI3K/Akt and p38 pathways are all involved in the activation of DNA synthesis by EGF and that sustained activation of MEK/ERK is required. Here, we show that although HGF stimulated DNA synthesis and activated signaling in the same manner as EGF, the contribution of the signaling pathways to the induction of DNA synthesis differed. While HGF-induced DNA synthesis was dependent on MEK/ERK, with no significant contribution from PI3K/Akt, p38 suppressed HGF-induced DNA synthesis. The p38 inhibitor SB203580 increased HGF-induced DNA synthesis and enhanced the phosphorylation of ERK. In contrast, SB203580 decreased EGF-induced ERK phosphorylation. This suggests that p38 has distinct effects on DNA synthesis induced by EGF and HGF. Due to differential regulation of signaling through the MEK/ERK pathway, p38 acts as an enhancer of EGF-induced DNA synthesis and as a suppressor of HGF-induced DNA synthesis.

  7. Enzymatic synthesis of organic-polymer-grafted DNA.

    PubMed

    Baccaro, Anna; Marx, Andreas

    2010-01-04

    To create bioorganic hybrid materials, interdisciplinary work in the fields of chemistry, biology and materials science is conducted. DNA block copolymers are promising hybrid materials due to the combination of properties intrinsic to both the polymer and the nucleic acid blocks. Until now, the coupling of DNA and organic polymers has been exercised post-synthetically in solution or on solid support. Herein, we report the first enzyme-catalysed synthesis of DNA-organic polymer chimeras. For this purpose, four novel 2'-deoxyuridine triphosphates carrying polymer-like moieties linked to the nucleobase were synthesised. Linear polyethylene glycol monomethyl ethers of different sizes (1) and branched polyamido dendrons with varying terminal groups (2) were chosen as building blocks. We investigated the ability of DNA polymerases to accept the copolymers in comparison to the natural substrate and showed, through primer extensions, polymerase chain reactions and rolling circle amplification, that these building blocks could serve as a surrogate for the natural thymidine. By this method, DNA hybrid materials with high molecular weight, modification density, and defined structure are accessible.

  8. Cyclosporin A inhibits DNA synthesis by epidermal Langerhans cells.

    PubMed

    Haftek, M; Urabe, A; Kanitakis, J; Dusserre, N; Thivolet, J

    Cyclosporin A, a potent immunosuppressive drug currently used in organ transplant recipients, has been shown to exert in vitro a direct antiproliferative effect on a number of cell types present in the skin, including keratinocytes, fibroblasts, and endothelial cells. Although in vitro studies suggest that cyclosporin A may interfere with the functional capacities of epidermal Langerhans cells, there is no evidence that the treatment influences the distribution or number of Langerhans cells in vivo. We used a model of normal human skin graft to "nude" mice, which is free of the human systemic control mechanisms, for studies on the DNA synthesis of human Langerhans cells under the influence of cyclosporin A. The grafted animals were given daily subcutaneous (50 mg/kg) or intraperitoneal (5, 12.5, and 25 mg/kg) drug injections during three weeks, which resulted in mean blood levels comparable to those observed in treated patients with organ transplants or psoriasis, respectively. BrdU administered during the last week of the experiment was incorporated by all cells synthesizing DNA, including those passing through S-phase. Langerhans cells were detected on deparaffinized or frozen tissue sections of xenografts with anti-CD1a and anti-HLA DR monoclonal antibodies, and the number of BrdU-positive cells was determined by double labeling. Our results indicate that the Langerhans cell DNA synthesis is impaired by therapeutic levels of cyclosporin A.

  9. Selective inhibition of in vitro synthesis of cancer DNA by alkaloids of beta-carboline class.

    PubMed

    Beljanski, M; Beljanski, M S

    1982-01-01

    The high template in vitro activity of native DNA from cancerous mammalian and plant tissues, compared to DNA from healthy tissues, enabled us to select substances which selectively inhibit cancer DNA synthesis. Among them, alstonine, serpentine, sempervirine and flavopereirine, all alkaloids which belong to the Beta-carboline class, distinguish cancer DNA from healthy tissue DNA inhibit DNA in vitro synthesis when native DNA from different cancerous tissues or cells is used as template. They have practically no effect on DNA from healthy tissues. The inhibitory effect of alkaloids is due to their capacity to form an 'alkaloid-cancer DNA' complex which has been characterized by use of the Sephadex column. Evidence is presented showing that these alkaloids inhibit the initiation of DNA synthesis but not chain elongation. The stimulating action caused by carcinogens during cancer DNA in vitro synthesis may be prevented and reversed by alkaloids. Furthermore, the stimulating action of steroids during in vitro synthesis of hormone target tissue DNA might be neutralized by alkaloids. However, at relatively high doses, steroids reversibly compete with alkaloids for binding sites on breast cancer DNA. This is not observed with DNA from nonhormone target tissues.

  10. Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

    PubMed Central

    Krokan, H; Wist, E; Krokan, R H

    1981-01-01

    Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation. PMID:6795595

  11. FANCJ promotes DNA synthesis through G-quadruplex structures.

    PubMed

    Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

    2014-11-03

    Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ(-/-) cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.

  12. Membrane clarification of tea extracts.

    PubMed

    Subramanian, R; Kumar, Chandini S; Sharma, Pankaj

    2014-01-01

    The ready-to-drink (RTD) tea beverages are becoming increasingly popular owing to the health benefits associated with tea polyphenols, but instability due to development of haze and formation of tea cream is a common problem encountered in the product. Membrane technology provides a scope to produce natural, additive-free RTD teas while overcoming the major disadvantages associated with the conventional decreaming methods. Approaches employing membranes for the clarification of extracts from black and green tea have been discussed together with their relative advantages and limitations. The article also outlines the concerns to be addressed in the future attempts employing membrane technology.

  13. Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

    PubMed

    Harmon, C S; Lutz, D; Ducote, J

    1993-01-01

    We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

  14. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    PubMed

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  15. Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

    PubMed Central

    Hillebrand, G G; McCluskey, A H; Abbott, K A; Revich, G G; Beattie, K L

    1984-01-01

    A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme. Images PMID:6326053

  16. Study of stimulators of DNA synthesis in nerve tissue cells

    SciTech Connect

    Vitvitskii, V.N.

    1986-04-10

    Changes in proliferative activity in different regions of the brain during ontogenesis are connected with changes in the composition and properties of regulators of cell proliferation. Extracts of regions of the brain in which active cell division takes place in a given stage of development (cortex of 15- to 17-day-old embryos or cerebellum of 8- to 10-day-old rats) can stimulate the incorporation of labeled precursors into the brain cell DNA of both newborn and adult rats. Salting out at increasing ammonium sulfate concentrations, gel filtration on Sephadex, and isoelectric focusing led to the isolation of three fractions of stimulators of DNA synthesis: in acid, neutral, and alkaline pH regions. A method is described for obtaining purified preparations and for determining some physicochemical properties of the acid activator, which is a low-molecular-weight peptide capable of noticeably stimulating the incorporation of labeled precursors into the DNA of nerve tissue cells when added to an in vitro system in a concentration of the order of 1 ..mu..g/ml.

  17. Integrating Value Clarification with High School Biology

    ERIC Educational Resources Information Center

    Barman, Charles R.

    1975-01-01

    Reports on research to see if value clarification would affect student attitudes toward science and biology and improve achievement in a BSCS Yellow Version biology course. Results indicated higher achievement by the group exposed to value clarification but no significant difference in attitudes between this group and the control group. (BR)

  18. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  19. Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

    SciTech Connect

    Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

    1982-12-01

    Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

  20. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

    PubMed Central

    Takahashi, Shunsuke; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2014-01-01

    Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3′-5′exo–) was promoted by DNA stretching with buffer flow. PMID:24625741

  1. The effect of bleomycin on DNA synthesis in ataxia telangiectasia lymphoid cells

    SciTech Connect

    Cohen, M.M.; Simpson, S.J.

    1982-01-01

    Bleomycin, a radiomimetic glycopeptide, inhibits de novo DNA synthesis in ataxia telangiectasia lymphoblastoid B cells to a markedly lesser extent than in normal and xeroderma pigmentosum lymphoid cells. This observation is similar to that following ionizing radiation; however, the effect is slower following the chemical treatment. Recovery of the normal cells occurs 15-18 hours after treatment, whereas the ataxia telangiectasia lines do not attain normal levels of DNA synthesis during the entire 24-hour observation period. Similar differences were not observed following treatment with mitomycin C, a bifunctional alkylating agent, indicating a specific effect of bleomycin on DNA synthesis in ataxia telangiectasia cells. Following bleomycin treatment and preincubation with hydroxyurea, residual DNA synthesis in ataxia telangiectasia cells was similar to that in both normal and xeroderma pigmentosum lymphoid lines, suggesting that the capacity to repair the induced DNA lesion is present.

  2. Evaluation of DNA synthesis with carbon-11-labeled 4′-thiothymidine

    PubMed Central

    Toyohara, Jun

    2016-01-01

    In the cancer research field, the preferred method for evaluating the proliferative activity of cancer cells in vivo is to measure DNA synthesis rates. The cellular proliferation rate is one of the most important cancer characteristics, and represents the gold standard of pathological diagnosis. Positron emission tomography (PET) has been used to evaluate in vivo DNA synthetic activity through visualization of enhanced nucleoside metabolism. However, methods for the quantitative measurement of DNA synthesis rates have not been fully clarified. Several groups have been engaged in research on 4′-[methyl-11C]-thiothymidine (11C-4DST) in an effort to develop a PET tracer that allows quantitative measurement of in vivo DNA synthesis rates. This mini-review summarizes the results of recent studies of the in vivo measurement of cancer DNA synthesis rates using 11C-4DST. PMID:27721942

  3. Method and apparatus for synthesis of arrays of DNA probes

    DOEpatents

    Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.; Singh-Gasson, Sangeet; Green, Roland

    2002-04-23

    The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, which may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.

  4. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

    PubMed

    Ramsay, Nicola; Jemth, Ann-Sofie; Brown, Anthony; Crampton, Neal; Dear, Paul; Holliger, Philipp

    2010-04-14

    DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

  5. One-step synthesis of silver nanoparticles, nanorods, and nanowires on the surface of DNA network.

    PubMed

    Wei, Gang; Zhou, Hualan; Liu, Zhiguo; Song, Yonghai; Wang, Li; Sun, Lanlan; Li, Zhuang

    2005-05-12

    Here, we describe a one-step synthesis of silver nanoparticles, nanorods, and nanowires on DNA network surface in the absence of surfactant. Silver ions were first adsorbed onto the DNA network and then reduced in sodium borohydride solution. Silver nanoparticles, nanorods, and nanowires were formed by controlling the size of pores of the DNA network. The diameter of the silver nanoparticles and the aspect ratio of the silver nanorods and nanowires can be controlled by adjusting the DNA concentration and reduction time.

  6. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  7. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  8. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  9. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  10. Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

    PubMed

    Champeil, Elise; Paz, Manuel M; Lukasiewicz, Elaan; Kong, Wan S; Watson, Stephanie; Sapse, Anne-Marie

    2012-12-01

    We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

  11. Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

    PubMed Central

    Gold, B; Carrillo, N; Tewari, K K; Bogorad, L

    1987-01-01

    Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA. Images PMID:3025853

  12. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    PubMed

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  13. Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the Arenavirus Pichinde. Interim report

    SciTech Connect

    Friedlander, A.M.; Jahrling, P.B.; Merrill, P.; Tobery, S.

    1983-01-19

    Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early non-specific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose dependent inhibition of the DNA synthesis induced by macrophage growth factor/colony stimulating factor. At a multiplicity of inoculum of five there is a 75-95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pre-treated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by 3H-thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance.

  14. RecG Directs DNA Synthesis during Double-Strand Break Repair.

    PubMed

    Azeroglu, Benura; Mawer, Julia S P; Cockram, Charlotte A; White, Martin A; Hasan, A M Mahedi; Filatenkova, Milana; Leach, David R F

    2016-02-01

    Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

  15. Sugar-oligoamides: synthesis of DNA minor groove binders.

    PubMed

    Badía, Concepción; Souard, Florence; Vicent, Cristina

    2012-12-07

    Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-minor groove DNA interactions. Here we report an efficient solution-phase synthetic strategy to obtain two broad families of sugar-oligoamides. The first type, structure vector A (-Py[Me]-γ-Py-Ind), has a methyl group present as a substituent on the nitrogen of pyrrole B, connected to the C terminal of the oligoamide fragment. The second type, structure vector B (-Py[(CH(2))(11)OH]-γ-Py-Ind), has an alkyl chain present on the nitrogen of pyrrole B connected to the C terminal of the oligoamide fragment and has been designed to access to di- and multivalent sugar-oligoamides. By using sequential DIPC/HOBt coupling reactions, the oligoamide fragment -Py[R]-γ-Py-Ind has been constructed. The last coupling reaction between the anomeric amino sugar and the oligoamide fragment was carried out by activating the acid derivative as a BtO- ester, which has been performed by using TFFH. The isolated esters (BtO-Py[R]-γ-Py-Ind) were coupled with selected amino sugars using DIEA in DMF. The synthesis of two different selective model vectors (vector A (1) and vector B (2)) and two types of water-soluble sugar-oligoamide ligands, with vector A structure (compounds 3-7) and with vector B structure (compound 8), was carried out.

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

    SciTech Connect

    Dybvig, K.

    1981-01-01

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

  17. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    PubMed

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  18. Synthesis of DNA mimics representing HypNA-pPNA hetero-oligomers.

    PubMed

    Efimov, Vladimir A; Chakhmakhcheva, Oksana G

    2005-01-01

    The methods for the synthesis and purification of negatively charged peptide nucleic acid (PNA)-relative deoxyribonucleic acid (DNA) mimics containing alternating residues of phosphono peptide nucleic acid (pPNA) monomers and PNA-like monomers on the base of trans-4-hydroxy-L-proline are described. Examples of the chimeric oligomers hybridization with complementary DNA and ribonucleic acid fragments are demonstrated.

  19. Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study.

    PubMed Central

    Hardt, N; Pedrali-Noy, G; Focher, F; Spadari, S

    1981-01-01

    A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes. Images PLATE 1 PMID:6803764

  20. Autonomous Multistep Organic Synthesis in a Single Isothermal Solution Mediated by a DNA Walker

    PubMed Central

    He, Yu; Liu, David R.

    2010-01-01

    Multistep synthesis in the laboratory typically requires numerous reaction vessels, each containing a different set of reactants. In contrast, cells are capable of performing highly efficient and selective multistep biosynthesis under mild conditions with all reactants simultaneously present in solution. If the latter approach could be applied in the laboratory, it may improve the ease, speed, and efficiency of multistep reaction sequences. Here we show that a DNA mechanical device— a DNA walker moving along a DNA track— can be used to perform a series of amine acylation reactions in a single solution without any external intervention. The multistep products generated by this primitive ribosome mimetic are programmed by the sequence of the DNA track, are unrelated to the structure of DNA, and are formed with speeds and overall yields significantly greater than those previously achieved by multistep DNA-templated small-molecule synthesis. PMID:20935654

  1. Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II).

    PubMed Central

    Hansson, J; Wood, R D

    1989-01-01

    DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA. Images PMID:2554251

  2. Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

    PubMed Central

    Loewy, B; Marczynski, G T; Dingwall, A; Shapiro, L

    1990-01-01

    Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome. Images PMID:2211495

  3. The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  4. Effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and x-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to x rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between x-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I/sup -/, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  5. In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

    PubMed

    Saaem, Ishtiaq; Ma, Kuo-Sheng; Marchi, Alexandria N; LaBean, Thomas H; Tian, Jingdong

    2010-02-01

    Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

  6. Fractional synthesis rates of DNA and protein in rabbit skin are not correlated.

    PubMed

    Zhang, Xiao-jun; Chinkes, David L; Wu, Zhanpin; Martini, Wenjun Z; Wolfe, Robert R

    2004-09-01

    We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.

  7. Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

    PubMed Central

    Decker, R S; Yamaguchi, M; Possenti, R; DePamphilis, M L

    1986-01-01

    Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended

  8. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis.

    PubMed

    Levring, Trine B; Kongsbak, Martin; Rode, Anna K O; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte Menné; Geisler, Carsten

    2015-09-08

    Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

  9. Iron at the cell surface controls DNA synthesis in CCl 39 cells.

    PubMed

    Alcain, F J; Löw, H; Crane, F L

    1994-08-30

    Treatment of CCl 39 cells with the impermeable iron II chelator bathophenanthroline disulfonate (BPS) inhibits both DNA synthesis and transplasma membrane electron transport. The inhibition persists when the BPS is removed, and the extract from 10(6) cells contains up to 1.28 nmoles iron II chelated to BPS. The BPS iron II chelate itself is not inhibitory. Both DNA synthesis and electron transport are restored by addition of microM iron II or iron III compounds to extracted cells. Other impermeable chelators for iron II give similar inhibition, whereas the iron III-specific Tiron or copper-specific bathocuproine sulfonate do not inhibit. The inhibition differs from the permeable iron III chelator inhibition of ribonucleotide reductase, because inhibition of DNA synthesis by the permeable chelators is reversed when chelator is removed. The response to growth factors also differs, with no impermeable chelator inhibition on 10% fetal calf serum contrasting to inhibition by permeable chelators. DNA synthesis with both activation of tyrosine kinase with EGF plus insulin or by thrombin or ceruloplasmin led to protein kinase C activation as inhibited by the impermeable chelators. It is proposed that an iron available on the cell surface is required for DNA synthesis and plasma membrane electron transport.

  10. Inhibition of DNA synthesis in CCL 39 cells by impermeable iron chelators.

    PubMed

    Alcaín, F J; Löw, H; Crane, F L

    1997-02-01

    The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.

  11. The identification of translesion DNA synthesis regulators: inhibitors in the spotlight

    PubMed Central

    Bertolin, AP; Mansilla, SF; Gottifredi, V

    2015-01-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. PMID:26002196

  12. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  13. DNA-Based Synthesis and Assembly of Organized Iron Oxide Nanostructures

    NASA Astrophysics Data System (ADS)

    Khomutov, Gennady B.

    Organized bio-inorganic and hybrid bio-organic-inorganic nanostructures consisting of iron oxide nanoparticles and DNA complexes have been formed using methods based on biomineralization, interfacial and bulk phase assembly, ligand exchange and substitution, Langmuir-Blodgett technique, DNA templating and scaffolding. Interfacially formed planar DNA complexes with water-insoluble amphiphilic polycation or intercalator Langmuir monolayers were prepared and deposited on solid substrates to form immobilized DNA complexes. Those complexes were then used for the synthesis of organized DNA-based iron oxide nanostructures. Planar net-like and circular nanostructures of magnetic Fe3O4 nanoparticles were obtained via interaction of cationic colloid magnetite nanoparticles with preformed immobilized DNA/amphiphilic polycation complexes of net-like and toroidal morphologies. The processes of the generation of iron oxide nanoparticles in immobilized DNA complexes via redox synthesis with various iron sources of biological (ferritin) and artificial (FeCl3) nature have been studied. Bulk-phase complexes of magnetite nanoparticles with biomolecular ligands (DNA, spermine) were formed and studied. Novel nano-scale organized bio-inorganic nanostructures - free-floating sheet-like spermine/magnetite nanoparticle complexes and DNA/spermine/magnetite nanoparticle complexes were synthesized in bulk aqueous phase and the effect of DNA molecules on the structure of complexes was discovered.

  14. Liver nuclear DNA synthesis in mice following carbon tetrachloride administration or partial hepatectomy

    SciTech Connect

    Gans, J.H.; Korson, R.

    1984-02-01

    Long-term, continuous (twice per week) administration of CCl/sub 4/ to male mice resulted in a high incidence of liver nodules which appear to be resistant to the necrotizing effects of CCl/sub 4/ but showed no features of malignant neoplasia. Liver nuclear DNA synthesis was compared in mice given CCl/sub 4/ and in mice subjected to partial hepatectomy (PH). Mice were given by gavage corn oil or CCl/sub 4/ in corn oil for periods of 2 to 25 weeks and several mice were subjected to PH after 12 and 25 weeks of corn oil treatment. Mice were given (/sup 3/H)TdR during liver regeneration and newly synthesized liver nuclear DNA was isolated and separated by BND-cellulose chromatography. Greater than 85% of the labeled DNA from PH mice eluted from BND-cellulose columns as double-stranded (ds) DNA with single-stranded (ss) regions or ends and less than 15% as ds DNA. When mice were treated with CCl/sub 4/ for 8 weeks or longer a significantly greater portion of liver nuclear DNA eluted as ds DNA. Administration of HU and 5-FU with (/sup 3/H)TdR decreased (/sup 3/H)TdR incorporation into DNA to low levels incompatible with unscheduled DNA synthesis. Single doses of CCl/sub 4/ given to mice treated with corn oil for 2 to 12 weeks provided newly synthesized DNA which was primarily (>80%) ds DNA with ss regions or ends, but after 25 weeks of corn oil administration, a single dose of CCl/sub 4/ resulted in newly synthesized DNA with a greater proportion of ds DNA. The high labeling of ds DNA in mice treated with CCl/sub 4/ may have resulted from an alternate pathway of DNA synthesis catalyzed by the enzymes or enzyme complexes associated with semiconservative DNA synthesis or from proliferation of nonparenchymal cells with a rapid turn-over rate.

  15. Comparison of dna-copying fidelity during repair and semiconservative synthesis by radioactive precursor distribution analysis

    SciTech Connect

    Nemirovskii, L.E.; Vasil'ev, V.K.

    1986-04-01

    The authors compare the fidelity of DNA copying during semiconservative and reparative synthesis under normal conditions and during cortisol-induced activation of free-radical processes, by examining the distribution of radioactivity among DNA pyrimidine isopliths. Radioactivity of nucleotide material in the isopliths was measured by counting in appropriate zones of the chromatograms in toluene scintillator. The investigation shows that injury to DNA of different organs, both directly and as a result of faulty repair, leads to shortening of the pyrimidine isopliths, i.e., to changes in the primary structure of DNA. These data help to explain the simultaneously cytostatic, carcinostatic, and mutagenic action of irradiation, cortisol and hydroxyurea.

  16. Role of mucosal prostaglandins and DNA synthesis in gastric cytoprotection by luminal epidermal growth factor.

    PubMed Central

    Konturek, S J; Brzozowski, T; Piastucki, I; Dembinski, A; Radecki, T; Dembinska-Kiec, A; Zmuda, A; Gregory, H

    1981-01-01

    This study compares the effect of epidermal growth factor and prostaglandins (PGE2 or PGI2), applied topically to gastric mucosa, on gastric secretion and formation of ASA-induced gastric ulcerations in rats. Epidermal growth factor given topically in non-antisecretory doses prevented dose-dependently the formation of ASA-induced ulcers without affecting prostaglandin generation but with a significant rise in DNA synthesis in the oxyntic mucosa. The anti-ulcer effect of topical prostaglandins was also accompanied by an increase in DNA synthesis. This study indicates that topical epidermal growth factor, like PGE2 or PGI2, is cytoprotective and that this cytoprotection is not mediated by the inhibition of gastric secretion or prostaglandin formation but related to the increase in DNA synthesis in oxyntic mucosa. PMID:7030877

  17. DNA synthesis in mouse brown adipose tissue is under. beta. -adrenergic control

    SciTech Connect

    Rehnmark, S.; Nedergaard, J. )

    1989-02-01

    The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of ({sup 3}H)thymidine. Cold exposure led to a large increase in the rate of ({sup 3}H)thymidine incorporation, reaching a maximum after 8 days, after which the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase ({sup 3}H)thymidine incorporation. When norepinephrine was injected in combination with the {alpha}-adrenergic antagonist phentolamine or with the {beta}-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a {beta}-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.

  18. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... have access to and use of information and data that is comparable to individuals without...

  19. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... have access to and use of information and data that is comparable to individuals without...

  20. Values Clarification: Essential for Leadership Learning

    ERIC Educational Resources Information Center

    Fritz, Mackenzie R.; Guthrie, Kathy L.

    2017-01-01

    Values clarification is a dynamic process in which people come to understand what they individually view as important in their lives by placing a name or label to what one values. This process commonly occurs during the traditional college years and is a critical component of leadership education. This qualitative study examined how junior-level…

  1. Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

    PubMed

    Salguero, Israel; Guarino, Estrella; Shepherd, Marianne E A; Deegan, Tom D; Havens, Courtney G; MacNeill, Stuart A; Walter, Johannes C; Kearsey, Stephen E

    2012-04-24

    Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.

  2. The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.

    PubMed

    Zhang, Huidong; Lee, Seung-Joo; Richardson, Charles C

    2012-07-06

    DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

  3. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    PubMed

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  4. Stimulation of adrenal DNA synthesis in cadmium-treated male rats

    SciTech Connect

    Nishiyama, S.; Nakamura, K.

    1984-07-01

    Cadmium chloride (CdCl2) at a dose of 1 mg/kg body wt was injected into male rats of the Wistar strain, weighing 250 g on the average, twice a day (12-hr intervals) for 7 consecutive days. DNA and RNA contents and (/sup 3/H)-thymidine and (/sup 3/H)-uridine incorporation into the acid-insoluble fraction significantly increased in the adrenals of rats treated with Cd for 2 and 7 consecutive days. Adrenal protein content and weight also significantly increased. These results indicate that continued treatment with Cd stimulates DNA and RNA synthesis in the adrenal cortex, which in turn results in the increase of the total protein contents of the adrenal gland and subsequently in the enlargement of the gland. Serum adrenocorticotrophin (ACTH) and insulin levels in Cd-treated rats were not higher than control levels, suggesting that the stimulation of DNA synthesis in the adrenals of Cd-treated rats is due to factor(s) other than serum ACTH and insulin. Treatment with Cd inhibited DNA synthesis in cultured adrenocortical cells at concentrations of 10(-4) to 10(-8) M, suggesting that Cd does not directly stimulate DNA synthesis in the adrenal gland in vivo. Although the adrenal gland became enlarged, the total adrenal corticosterone content decreased significantly. The decrease of total adrenal corticosterone content may be due to the fall in serum ACTH level of Cd-treated rats.

  5. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    SciTech Connect

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  6. EGFR Modulates DNA Synthesis and Repair through Tyr Phosphorylation of Histone H4

    PubMed Central

    Chou, Ruey-Hwang; Wang, Ying-Nai; Hsieh, Yi-Hsien; Li, Long-Yuan; Xia, Weiya; Chang, Wei-Chao; Chang, Ling-Chu; Cheng, Chien-Chia; Lai, Chien-Chen; Hsu, Jennifer L.; Chang, Wei-Jung; Chiang, Shu-Ya; Lee, Hong-Jen; Liao, Hsin-Wei; Chuang, Pei-Huan; Chen, Hui-Yu; Wang, Hung-Ling; Kuo, Sheng-Chu; Chen, Chung-Hsuan; Yu, Yung-Luen; Hung, Mien-Chie

    2014-01-01

    Summary Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical in DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 mono-methylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair. PMID:25073158

  7. Capture of a third Mg²⁺ is essential for catalyzing DNA synthesis.

    PubMed

    Gao, Yang; Yang, Wei

    2016-06-10

    It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase-DNA-deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.

  8. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    PubMed Central

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  9. Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

    PubMed

    Knobloch, Jürgen; Kunz, Werner; Grevelding, Christoph G

    2002-12-01

    The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

  10. Post-synthesis DNA Modifications Using a trans-Cyclooctene Click Handle

    PubMed Central

    Wang, Ke; Wang, Danzhu; Ji, Kaili; Chen, Weixuan; Zheng, Yueqin; Dai, Chaofeng

    2015-01-01

    Post-synthesis DNA modification is a very useful method for DNA functionalization. This is achieved by using a modified NTP, which has a handle for further modifications, replacing the corresponding natural NTP in polymerase-catalyzed DNA synthesis. Subsequently, the handle can be used for further functionalization after PCR, preferably through a very fast reaction. Herein we describe polymerase-mediated incorporation of trans-cyclooctene modified thymidine triphosphate (TCO-TTP). Subsequently, the trans-cyclooctene group was reacted with a tetrazine tethered to other functional groups through a very fast click reaction. The utility of this DNA functionalization method was demonstrated with the incorporation of a boronic acid group and a fluorophore. The same approach was also successfully used in modifying a known aptamer for fluorescent labelling applications. PMID:25407744

  11. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

  12. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  13. Studies on the mechanism of action of azinomycin B: definition of regioselectivity and sequence selectivity of DNA cross-link formation and clarification of the role of the naphthoate.

    PubMed

    Coleman, Robert S; Perez, Ronelito J; Burk, Christopher H; Navarro, Antonio

    2002-11-06

    Evaluation of the sequence selectivity, noncovalent association, and orientation of the DNA cross-linking agent azinomycin B on its duplex DNA receptor is described. A strong correlation between sequence nucleophilicity and cross-linking yield was observed, and steric effects due to the thymine C5-methyl group were identified. Detailed studies on the role of the azinomycin naphthoate using viscometry, fluorescence contact energy transfer, and DNA unwinding assays point to a nonintercalative binding mode for this group. A kinetic assay for agent regioselectivity was used to determine the orientation of binding and covalent cross-link formation.

  14. Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5'-flaps.

    PubMed

    Koc, Katrina N; Stodola, Joseph L; Burgers, Peter M; Galletto, Roberto

    2015-04-30

    The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3'-5' exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo(-) to carry out strand displacement synthesis and discovered that it is regulated by the 5'-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5'-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5'-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities.

  15. Porous silicon microparticles as an alternative support for solid phase DNA synthesis

    NASA Astrophysics Data System (ADS)

    McInnes, Steven; Graney, Sean; Khung, Yit-lung; Voelcker, Nicolas H.

    2006-01-01

    Current methods to produce short DNA strands (oligonucleotides) involve the stepwise coupling of phosphoramidites onto a solid support, typically controlled pore glass. The full-length oligonucleotide is then cleaved from the solid support using a suitable aqueous or organic base and the oligonucleotide is subsequently separated from the spent support. This final step, albeit seemingly easy, invariably leads to increased production costs due to increased synthesis time and reduced yields. This paper describes the preparation of a dissolvable support for DNA synthesis based on porous silicon (pSi). Initially it was thought that the pSi support would undergo dissolution by hydrolysis upon cleavage of the freshly synthesised oligonucleotide strands with ammonium hydroxide. The ability to dissolve the solid support after completion of the synthesis cycle would eliminate the separation step required in current DNA synthesis protocols, leading to simpler and faster synthesis as well as increased yields, however it was found that the functionalisation of the pSi imparted a stability that impeded the dissolution. This strategy may also find applications for drug delivery where the controlled release of carrier-immobilised short antisense DNA is desired. The approach taken involves the fabrication of porous silicon (pSi) microparticles and films. Subsequently, the pSi is oxidised and functionalised with a dimethoxytrityl protected propanediol to facilitate the stepwise solid phase synthesis of DNA oligonucleotides. The functionalisation of the pSi is monitored by diffuse reflectance infrared spectroscopy and the successful trityl labelling of the pSi is detected by UV-Vis spectroscopy after release of the dimethoxytrityl cation in the presence of trichloroacetic acid (TCA). Oligonucleotide yields can be quantified by UV-Vis spectroscopy.

  16. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    PubMed

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

  17. Synthesis and properties of 4′-ThioDNA: unexpected RNA-like behavior of 4′-ThioDNA

    PubMed Central

    Inoue, Naonori; Minakawa, Noriaki; Matsuda, Akira

    2006-01-01

    The synthesis and properties of fully modified 4′-thioDNAs, oligonucleotides consisting of 2′-deoxy-4′-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4′-thioDNA to 3′-exonuclease cleavage. Furthermore, we found that fully modified 4′-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4′-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4′-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4′-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V1, it was not only the physical properties but also this biological data suggested that a 4′-thioDNA duplex has an RNA-like structure. PMID:16855286

  18. Molecular Recognition of DNA. Synthesis of Novel Bases for Triple Helix Formation

    DTIC Science & Technology

    1991-01-01

    to the purine strand in the major groove of the Watson - Crick double helical DNA (TAT, C+GC triplets). Purine oligonucleotides bind antiparallel to...R&T Code 4135018 S MAy 05 199411 "Molecular Recognition of DNA . Synthesis of Novel Bases for Triple Helix Formation" Peter B. Dervan cv _California...035 T"IQA""D PART I A) Completed work (1988-91) Triple Helix Formation by Oligonucleotides on DNA Extended to the Physiological pH Range. T. J. Povsic

  19. Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

    SciTech Connect

    Gabrielson, E.W.; Gerwin, B.I.; Harris, C.C.; Roberts, A.B.; Sporn, M.B.; Lechner, J.F.

    1988-08-01

    Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.

  20. Evidence for cell surface control of macronuclear DNA synthesis in Stentor.

    PubMed

    de Terra, N

    1975-11-27

    In cell grafts, Stentor macronuclei associated with separate regions of cell surface can be made asynchronous with regard to morphology and DNA synthesis even though they demonstrably share a common endoplasm. These results suggest a mechanism for nuclear differentiation within a single cytoplasmic compartment, based on cell surface differences.

  1. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    PubMed

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  2. Synthesis and characterization of DNA nano-meso-microspheres as drug delivery carriers for intratumoral chemotherapy

    NASA Astrophysics Data System (ADS)

    Enriquez Schumacher, Iris Vanessa

    Conventional cancer chemotherapy results in systemic toxicity which severely limits effectiveness and often adversely affects patient quality of life. There is a need to find new drugs and delivery methods for less toxic therapy. Previous studies concerning DNA complexing with chemotherapy drugs suggest unique opportunities for DNA as a mesosphere drug carrier. The overall objective of this research was devoted to the synthesis and evaluation of novel DNA-drug nano-mesospheres designed for localized chemotherapy via intratumoral injection. My research presents DNA nano-meso-microspheres (DNA-MS) that were prepared using a modified steric stabilization method originally developed in this lab for the preparation of albumin MS. DNA-MS were prepared with glutaraldehyde covalent crosslinking (genipin crosslinking was attempted) through the DNA base pairs. In addition, novel crosslinking of DNA-MS was demonstrated using chromium, gadolinium, or iron cations through the DNA phosphate groups. Covalent and ionic crosslinked DNA-MS syntheses yielded smooth and spherical particle morphologies with multimodal size distributions. Optimized DNA-MS syntheses produced particles with narrow and normal size distributions in the 50nm to 5mum diameter size range. In aqueous dispersions approximately 200% swelling was observed with dispersion stability for more than 48 hours. Typical process conditions included a 1550rpm initial mixing speed and particle filtration through 20mum filters to facilitate preparation. DNA-MS were in situ loaded during synthesis for the first time with mitoxantrone, 5-fluorouracil, and methotrexate. DNA-MS drug incorporation was 12%(w/w) for mitoxantrone, 9%(w/w) for methotrexate, and 5%(w/w) for 5-fluorouracil. In vitro drug release into phosphate buffered saline was observed for over 35 days by minimum sink release testing. The effect of gadolinium crosslink concentration on mitoxantrone release was evaluated at molar equivalences in the range of 20% to

  3. Synthesis of the tellurium-derivatized phosphoramidites and their incorporation into DNA oligonucleotides.

    PubMed

    Jiang, Sibo; Sheng, Jia; Huang, Zhen

    2011-12-01

    In this unit, an efficient method for the synthesis of 2'-tellerium-modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine can be converted to the corresponding 2'-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2'-Te-nucleosides can be transformed into 3'-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification, and applications of oligonucleotides containing 2'-Te-U or 2'-Te-T are described in the protocol.

  4. DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

    PubMed Central

    Zarucki-Schulz, T; Jerez, C; Goldberg, G; Kung, H F; Huang, K H; Brot, N; Weissbach, H

    1979-01-01

    The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results. Images PMID:160561

  5. Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

    PubMed

    Karttunen, Heidi; Savas, Jeffrey N; McKinney, Caleb; Chen, Yu-Hung; Yates, John R; Hukkanen, Veijo; Huang, Tony T; Mohr, Ian

    2014-07-03

    DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

  6. Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

    PubMed Central

    Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

    2015-01-01

    SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

  7. The OPERA hypothesis: assumptions and clarifications.

    PubMed

    Patel, Aniruddh D

    2012-04-01

    Recent research suggests that musical training enhances the neural encoding of speech. Why would musical training have this effect? The OPERA hypothesis proposes an answer on the basis of the idea that musical training demands greater precision in certain aspects of auditory processing than does ordinary speech perception. This paper presents two assumptions underlying this idea, as well as two clarifications, and suggests directions for future research.

  8. Risk Communication, Values Clarification, and Vaccination Decisions.

    PubMed

    Witteman, Holly O; Chipenda Dansokho, Selma; Exe, Nicole; Dupuis, Audrey; Provencher, Thierry; Zikmund-Fisher, Brian J

    2015-10-01

    Many health-related decisions require choosing between two options, each with risks and benefits. When presented with such tradeoffs, people often make choices that fail to align with scientific evidence or with their own values. This study tested whether risk communication and values clarification methods could help parents and guardians make evidence-based, values-congruent decisions about children's influenza vaccinations. In 2013-2014 we conducted an online 2×2 factorial experiment in which a diverse sample of U.S. parents and guardians (n = 407) were randomly assigned to view either standard information about influenza vaccines or risk communication using absolute and incremental risk formats. Participants were then either presented or not presented with an interactive values clarification interface with constrained sliders and dynamic visual feedback. Participants randomized to the risk communication condition combined with the values clarification interface were more likely to indicate intentions to vaccinate (β = 2.10, t(399) = 2.63, p < 0.01). The effect was particularly notable among participants who had previously demonstrated less interest in having their children vaccinated against influenza (β = -2.14, t(399) = -2.06, p < 0.05). When assessing vaccination status reported by participants who agreed to participate in a follow-up study six months later (n = 116), vaccination intentions significantly predicted vaccination status (OR = 1.66, 95%CI (1.13, 2.44), p < 0.05) and rates of informed choice (OR = 1.51, 95%CI (1.07, 2.13), p < 0.012), although there were no direct effects of experimental factors on vaccination rates. Qualitative analysis suggested that logistical barriers impeded immunization rates. Risk communication and values clarification methods may contribute to increased vaccination intentions, which may, in turn, predict vaccination status if logistical barriers are also addressed.

  9. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  10. A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    PubMed Central

    Kosuri, Sriram; Eroshenko, Nikolai; LeProust, Emily; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M.

    2010-01-01

    Development of cheap, high-throughput, and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology1. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis2. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude3,4,5, yet efforts to scale their use have been largely unsuccessful due to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols, and enzymatic error correction to develop a highly parallel gene synthesis platform. We tested our platform by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ~35 kilo-basepairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ~2.5 megabases of DNA, which is at least 50 times larger than previously published attempts. PMID:21113165

  11. Translesion Synthesis of 2′-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases

    PubMed Central

    2016-01-01

    With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2′-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass. PMID:27760288

  12. Synthesis and evaluation of new spacers for use as dsDNA endcaps

    PubMed Central

    Ng, Pei-Sze; Laing, Brian M.; Balasundarum, Ganesan; Pingle, Maneesh; Friedman, Alan; Bergstrom, Donald E.

    2010-01-01

    A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. Endcaps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. Endcaps containing either a terthiophene or a naphthalene tetracarboxylic acid dimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A•T base pair. Spacers containing only methylene (-CH2-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together. PMID:20715857

  13. Synthesis and evaluation of new spacers for use as dsDNA end-caps.

    PubMed

    Ng, Pei-Sze; Laing, Brian M; Balasundarum, Ganesan; Pingle, Maneesh; Friedman, Alan; Bergstrom, Donald E

    2010-08-18

    A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl-protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. End-caps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. End-caps containing either a terthiophene or a naphthalene tetracarboxylic acid diimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A*T base pair. Spacers containing only methylene (-CH(2)-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

  14. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present.

    PubMed

    Chen, Cheng-Yao

    2014-01-01

    Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  15. Action of cytochalasin D on DNA synthesis in cells in culture

    SciTech Connect

    Glushankova, N.A.

    1986-10-01

    To solve the problem of the effect of changes in the actin cytoskeleton on DNA replication during the action of cytochalasins, the effect of long-term incubation of normal cells with cytochalasin D (CCD), which selectively destroys the microfilament system but does not affect transport of sugars, was investigated. Incorporation of labeled thymidine into mononuclear and binuclear cells in the presence of CCD and after its removal by rinsing also was studied separately. To investigate DNA synthesis the method of autoradiography with /sup 3/H-thymidine was used. A culture of mouse fibroblasts of the BALB/3T3 line and a secondary culture of fibroblasts obtained by trypsinization of mouse embryos (MEF) were used. On incubation of MEF and 3T3 cells, gradual inhibition of DNA synthesis is observed. The results obtained indicate that structural changes in the active cytoskeleton can abruptly and reversibly disturb passage of the normal cell through the cycle.

  16. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

    PubMed

    Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng; Wang, Yinsheng; Sabatini, Robert

    2015-12-01

    Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.

  17. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  18. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

    2013-06-25

    A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

  19. Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.

    PubMed

    Yuferov, V; Grandgenett, D P; Bondurant, M; Riggin, C; Tigges, M

    1978-07-24

    Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

  20. Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

    PubMed Central

    Chen, Youjun; Zhou, Yi-Hui; Neo, Dayna; Clement, Jean; Takata, Minoru; Takeda, Shunichi; Sale, Julian; Wright, Fred A.; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI). PMID:27907204

  1. Design and Synthesis of Triangulated DNA Origami Trusses.

    PubMed

    Matthies, Michael; Agarwal, Nayan P; Schmidt, Thorsten L

    2016-03-09

    DNA nanotechnology offers unique control over matter on the nanoscale. Here, we extend the DNA origami method to cover a range of wireframe truss structures composed of equilateral triangles, which use less material per volume than standard multiple-helix bundles. From a flat truss design, we folded tetrahedral, octahedral, or irregular dodecahedral trusses by exchanging few connector strands. Other than standard origami designs, the trusses can be folded in low-salt buffers that make them compatible with cell culture buffers. The structures also have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. Our graph routing program and a simple design pipeline will enable other laboratories to make use of this valuable and potent new construction principle for DNA-based nanoengineering.

  2. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    PubMed Central

    2011-01-01

    Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

  3. Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution

    NASA Astrophysics Data System (ADS)

    Christian, Thomas; Romano, Louis; Rueda, David

    2010-03-01

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule F"orster resonance energy transfer intensity drops in discrete steps to values consistent with single nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer-template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change following the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the pre-insertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  4. Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis.

    PubMed

    Qiao, Man; Luo, Dan; Kuang, Yi; Feng, Haiyan; Luo, Guangping; Liang, Peng

    2015-01-01

    p53 tumor-suppressor gene is a master transcription factor which controls cell cycle progression and apoptosis. killin was discovered as one of the p53 target genes implicated in S-phase control coupled to cell death. Due to its extreme proximity to pten tumor-suppressor gene on human chromosome 10, changes in epigenetic modification of killin have also been linked to Cowden syndrome as well as other human cancers. Previous studies revealed that Killin is a high-affinity DNA-binding protein with preference to single-stranded DNA, and it inhibits DNA synthesis in vitro and in vivo. Here, co-localization studies of RFP-Killin with either GFP-PCNA or endogenous single-stranded DNA binding protein RPA during S-phase show that Killin always adopts a mutually exclusive punctuated nuclear expression pattern with the 2 accessory proteins in DNA replication. In contrast, when cells are not in S-phase, RFP-Killin largely congregates in the nucleolus where rRNA transcription normally occurs. Both of these cell cycle specific localization patterns of RFP-Killin are stable under high salt condition, consistent with Killin being tightly associated with nucleic acids within cell nuclei. Together, these cell biological results provide a molecular basis for Killin in competitively inhibiting the formation of DNA replication forks during S-phase, as well as potentially negatively regulate RNA synthesis during other cell cycle phases.

  5. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    PubMed Central

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  6. The nexus of vitamin homeostasis and DNA synthesis and modification in mammalian brain.

    PubMed

    Spector, Reynold; Johanson, Conrad E

    2014-01-10

    The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function.

  7. The nexus of vitamin homeostasis and DNA synthesis and modification in mammalian brain

    PubMed Central

    2014-01-01

    The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function. PMID:24410751

  8. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  9. Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

    PubMed Central

    Kollek, R; Goulian, M

    1981-01-01

    The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma. Images PMID:6947222

  10. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

  11. Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

    SciTech Connect

    Kodadek, T.; Gamper, H.

    1988-05-03

    The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. They demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

  12. Endotoxin or cytokines attenuate ozone-induced DNA synthesis in rat nasal transitional epithelium

    SciTech Connect

    Hotchkiss, J.A.; Harkema, J.R. )

    1992-06-01

    Pretreatment of rats with endotoxin (E), a potent inducer of tumor necrosis factor alpha (TNF), and interleukin 1 beta (IL 1), or a combination of TNF and IL1, has been shown to increase levels of lung antioxidant enzymes and protect against pulmonary toxicity associated with hyperoxia. Inhalation of ozone (O3) induces cell injury, followed by increased DNA synthesis, cell proliferation, and secretory cell metaplasia in rat nasal transitional epithelium (NTE). This study was designed to test the effects of E, TNF, and IL1 pretreatment on acute O3-induced NTE cell injury as measured by changes in NTE cell DNA synthesis. Rats were exposed to either 0.8 ppm O3 or air for 6 hr in whole-body inhalation chambers. Immediately before exposure, rats in each group were injected intraperitoneally (ip) with either saline alone or saline containing E, TNF, IL1, or both TNF and IL1. Eighteen hours postexposure, rats were injected ip with bromodeoxyuridine to label cells undergoing DNA synthesis and were euthanized 2 hr later. NTE was processed for light microscopy and immunochemically stained to identify cells that had incorporated BrdU into nuclear DNA. The number of BrdU-labeled NTE nuclei per millimeter of basal lamina was quantitated. There were no significant differences in the number of BrdU-labeled NTE nuclei in air-exposed rats that were injected with E, TNF, IL1, or TNF/IL1 compared with those in saline-injected, air-exposed controls. Rats that were injected with saline and exposed to O3 had approximately 10 times the number of BrdU-labeled NTE nuclei than saline-injected, air-exposed control rats. O3 exposure also induced a significant increase in labeled nuclei in rats that were pretreated with TNF alone. In contrast, pretreatment with E, IL1, or TNF/IL1 attenuated the O3-induced increase in NTE DNA synthesis.

  13. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    PubMed

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

  14. Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

    PubMed

    Mercurio, Maria Emilia; Tomassi, Stefano; Gaglione, Maria; Russo, Rosita; Chambery, Angela; Lama, Stefania; Stiuso, Paola; Cosconati, Sandro; Novellino, Ettore; Di Maro, Salvatore; Messere, Anna

    2016-12-02

    Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

  15. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  16. 5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

    NASA Technical Reports Server (NTRS)

    Frank, Natia L.; Meade, Thomas J.

    2003-01-01

    Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

  17. [DNA reduplication cycle during chromosome polytenization in the salivary gland cells of Chironomus thummi larvae. III. The determination of the duration of the DNA synthesis period].

    PubMed

    Gundrina, L I; Sherudilo, A I; Mitina, R L

    1984-08-01

    The duration of DNA synthesis in the salivary gland cells of Chironomus thummi larvae of the IV instar was determined by means of autoradiography and cytophotometry. Cells of different levels of ploidy differ in the duration of their DNA synthesis period. The tS of 2(10)c and 2(11)c cells was equal to 17 and 22 hours, respectively. The doubling of DNA content of the chironomid salivary gland cells leads to a 1.3 time increase in the duration of S-phase.

  18. DNA Origami Rotaxanes: Tailored Synthesis and Controlled Structure Switching.

    PubMed

    Powell, John T; Akhuetie-Oni, Benjamin O; Zhang, Zhao; Lin, Chenxiang

    2016-09-12

    Mechanically interlocked supramolecular assemblies are appealing building blocks for creating functional nanodevices. Herein, we describe the multistep assembly of large DNA origami rotaxanes that are capable of programmable structural switching. We validated the topology and structural integrity of these rotaxanes by analyzing the intermediate and final products of various assembly routes by electrophoresis and electron microscopy. We further analyzed two structure-switching behaviors of our rotaxanes, which are both mediated by DNA hybridization. In the first mechanism, the translational motion of the macrocycle can be triggered or halted at either terminus. In the second mechanism, the macrocycle can be elongated after completion of the rotaxane assembly, giving rise to a unique structure that is otherwise difficult to access.

  19. Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

    PubMed

    Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo; Gu, Jianyou; Suwa, Yoshiaki; Pavlov, Youri I; Tahirov, Tahir H

    2016-05-06

    The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.

  20. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  1. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  2. Synthesis and DNA interaction of a mixed proflavine-phenanthroline Tröger base.

    PubMed

    Baldeyrou, Brigitte; Tardy, Christelle; Bailly, Christian; Colson, Pierre; Houssier, Claude; Charmantray, Franck; Demeunynck, Martine

    2002-04-01

    We report the synthesis of an asymmetric Tröger base containing the two well characterised DNA binding chromophores, proflavine and phenanthroline. The mode of interaction of the hybrid molecule was investigated by circular and linear dichroism experiments and a biochemical assay using DNA topoisomerase I. The data are compatible with a model in which the proflavine moiety intercalates between DNA base pairs and the phenanthroline ring occupies the DNA groove. DNase I cleavage experiments were carried out to investigate the sequence preference of the hybrid ligand and a well resolved footprint was detected at a site encompassing two adjacent 5'-GTC.5-GAC triplets. The sequence preference of the asymmetric molecule is compared to that of the symmetric analogues.

  3. DNA Dispose, but Subjects Decide. Learning and the Extended Synthesis.

    PubMed

    Lindholm, Markus

    Adaptation by means of natural selection depends on the ability of populations to maintain variation in heritable traits. According to the Modern Synthesis this variation is sustained by mutations and genetic drift. Epigenetics, evodevo, niche construction and cultural factors have more recently been shown to contribute to heritable variation, however, leading an increasing number of biologists to call for an extended view of speciation and evolution. An additional common feature across the animal kingdom is learning, defined as the ability to change behavior according to novel experiences or skills. Learning constitutes an additional source for phenotypic variation, and change in behavior may induce long lasting shifts in fitness, and hence favor evolutionary novelties. Based on published studies, I demonstrate how learning about food, mate choice and habitats has contributed substantially to speciation in the canonical story of Darwin's finches on the Galapagos Islands. Learning cannot be reduced to genetics, because it demands decisions, which requires a subject. Evolutionary novelties may hence emerge both from shifts in allelic frequencies and from shifts in learned, subject driven behavior. The existence of two principally different sources of variation also prevents the Modern Synthesis from self-referring explanations.

  4. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    NASA Astrophysics Data System (ADS)

    Shamaev, Alexei

    activated by UV-light. The mechanism of o-quinone release and intramolecular ET was studied in detail by methods of Ultrafast Transient Absortion Spectroscopy and supported by high-level quantum mechanical calculations. The binding properties of chiral intercalators based on PDHD to various DNA oligonucleotides were studied by various methods and DNA cleavage properties indicating strong binding and cleaving ability of the synthesized PDHDs. Also, a new method for synthesis of cyclohexa[e]pyrenes which possibly capable of intramolecular ET and electron transfer-oxidative stress (ET-OS) DNA cleavage was developed and partially accomplished.

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

    PubMed

    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.

  6. Clarification on the Clarification on the April 8th Memorandum from H.E.W.

    ERIC Educational Resources Information Center

    Journal of the National Association for Bilingual Education, 1976

    1976-01-01

    The clarification of the April 8th memorandum is that "the Lau remedies are minimum requirements and that in cases depending on student language dominance, grade level, and academic achievement, a bilingual program 'is' the remedy, and the only educationally sound way of ensuring effective participation in the instructional program." (NQ)

  7. On-Flow Synthesis of Co-Polymerizable Oligo-Microspheres and Application in ssDNA Amplification

    PubMed Central

    Lee, Se Hee; Lee, Jae Ha; Lee, Ho Won; Kim, Yang-Hoon; Jeong, Ok Chan; Ahn, Ji-Young

    2016-01-01

    We fabricated droplet-based microfluidic platform for copolymerizable microspheres with acrydite modified DNA probe. The copolymerizable 3-D polyacrylamide microspheres were successfully produced from microcontinuous-flow synthesis with on-channel solidification. DNA copolymerization activity, surface presentation and thermostability were assessed by using fluorescent labeled complementary probe. The binding performance was only visible on the surface area of oligo-microspheres. We show that the resulting oligo-microspheres can be directly integrated into a streamlined microsphere-PCR protocol for amplifying ssDNA. Our microspheres could be utilized as a potential material for ssDNA analysis such as DNA microarray and automatic DNA SELEX process. PMID:27447941

  8. Iron chelators hydroxyurea and bathophenanthroline disulfonate inhibit DNA synthesis by different pathways.

    PubMed

    Alcaín, F J; Löw, H; Crane, F L; Navas, P

    1994-09-01

    We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.

  9. [Synthesis of biflavones and their interaction with DNA].

    PubMed

    Zhang, Zun-Ting; Gao, Run-Li; Zhuang, Su-Kai

    2009-08-01

    To explore new biflavones, 7-hydroxy-8-hydroxymethyl-4'-methoxyisoflavone (1), (5, 7-dihydroxyflavone-8-yl)-(7'-hydroxy-4"-methoxyisoflavone-8'-yl)methane (2), bis(7-hydroxy-4'-methoxyflavone-8-yl) methane (3), bis(3', 5'-diisopropyl-7, 4'-dihydroxy-isoflavone-8-yl)methane (4), and bis(7-hydroxy-isoflavone-8-yl) methane (5) were designed and synthesized from chrysin, formononetin, 7, 4'-dihydroxy-3', 5'-diisopropyl-isoflavone and 7-hydroxy-isoflavone. Their structures were identified with IR, 1H NMR, 13C NMR and elemental analysis. The binding of 1-5 with DNA was studied with fluorescent spectroscopy. Compounds 2-5 showed higher binding affinity with DNA than 1. According to the Stern-Volmer equation, the binding constants of 2, 3 were determined at 35 degrees C and 25 degrees C respectively, they were Kq2 (25 degrees C) = 1.95 x 10(4) Lx mol(-1) and Kq2 (35 degrees C) = 1.67 x 10(4) L x mol(-1); Kq3 (25 degrees C) = 1.89 x 10(4) L x mol(-1) and Kq3 (35 degrees C) = 1.58 x 10(4) L x mol(-1). The quenching mechanism of 2, 3 was suggested as static quenching.

  10. [Synthesis of chrysin derivatives and their interaction with DNA].

    PubMed

    Zhang, Zun-Ting; Chen, Li-Li

    2007-05-01

    Using chrysin as a leading compound, intermediate 5, 7-dihydroxy-6, 8-bis (hydroxymethyl) flavone (1) was synthesized by hydroxymethylation. The intermediate reacted with different alcohols to afford 5, 7-dihydroxy-6, 8-bis ( methoxymethyl) flavone (2), 6, 8-bis (ethoxymethyl) -5, 7dihydroxyflavone (3), 6, 8-bis-(butoxymethyl)-5, 7-dihydroxyflavone (4), 6, 8-bis (pentyloxymethyl) -5,7-dihydroxy flavone (5) and 6, 8-bis-(ethoxymethyl) -5-hydroxy-7-methoxyflavone (6). These compounds were characterized by IR, 1H NMR, 13C NMR and element analysis. The crystal structure of 6 was determined by X-ray crystal diffraction. The interaction of the derivatives with CT-DNA was studied by fluorescent spectroscopy. According to the Stern-Volmer equation, the quenching constants of the compounds 1 - 4 were measured, separately, they were K(q1) = 9.71 x 10(3) L x mol(-1), K(q2) = 2.25 x 10(4) L x mol(-1), K(q3) = 1.03 x 10(4) L x mol(-1) and K(q4) = 7.96 x 10(3) L x mol(-1). Compounds 1-4 showed higher binding affinity with DNA than chrysin did. The results provided the experimental basis for developing a more effective flavonoid and worthing further thoroughly study.

  11. Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach

    PubMed Central

    Silverman, Janice Elaine Y.; Ciustea, Mihai; Shudofsky, Abigail M. Druck; Bender, Florent; Shoemaker, Robert H.; Ricciardi, Robert P.

    2008-01-01

    Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that i) screen for compounds that block vaccinia DNA synthesis, ii) eliminate trivial inhibitors, e.g. DNA intercalators, and iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2,222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola. PMID:18621425

  12. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  13. The Foundry: the DNA synthesis and construction Foundry at Imperial College

    PubMed Central

    Chambers, Stephen; Kitney, Richard; Freemont, Paul

    2016-01-01

    The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms. PMID:27284027

  14. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    PubMed

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  15. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    PubMed

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  16. Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.

    PubMed

    Korhonen, J M; Saïd, F A; Wong, A J; Kaplan, D R

    1999-12-24

    The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

  17. Iron reverses impermeable chelator inhibition of DNA synthesis in CCl 39 cells.

    PubMed

    Alcain, F J; Löw, H; Crane, F L

    1994-08-16

    Treatment of Chinese hamster lung fibroblasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.

  18. Iron Reverses Impermeable Chelator Inhibition of DNA Synthesis in CCl39 Cells

    NASA Astrophysics Data System (ADS)

    Alcain, Francisco J.; Low, Hans; Crane, Frederick L.

    1994-08-01

    Treatment of Chinese hamster lung fibro-blasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.

  19. Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus ▿

    PubMed Central

    Lentz, Thomas B.; Loeb, Daniel D.

    2011-01-01

    Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion. PMID:21900164

  20. Synthesis of linear polyethylenimine derivatives for DNA transfection.

    PubMed

    Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

    2003-01-01

    A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

  1. Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

    PubMed

    Narcross, Lauren; Bourgeois, Leanne; Fossati, Elena; Burton, Euan; Martin, Vincent J J

    2016-12-16

    The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

  2. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    PubMed

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal

    2014-11-01

    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

  3. Synthesis, Characterization, and DNA Binding Profile of a Macrocyclic β-Sheet Analogue of ARC Protein

    PubMed Central

    2015-01-01

    ARC repressor (apoptosis repressor with caspase recruitment domain) is a protein which binds selectively to a specific sequence of DNA. In humans, ARC is primarily expressed in striated muscle tissue, which normally does not undergo rapid cell turnover. This suggests that ARC may play a protective role in the prevention against Duchenne Muscular Dystrophy and several types of tumors. In this Letter we report the synthesis, characterization, and conformational analysis of a β-sheet ARC repressor mimetic, based on the amino acid sequence of the β-sheet domain in the ARC protein. The ability of this β-sheet macrocycle to bind to double-stranded DNA was also evaluated using spectroscopic methods. Our data show that the synthetic peptide has a defined conformation and is able to bind DNA with reasonable affinity. These initial results lay the groundwork for the design of novel β-sheets folded peptides as valuable substitutes of transcription factor proteins in drug therapy. PMID:26713108

  4. Inhibitor of DNA synthesis is present in normal chicken serum

    SciTech Connect

    Franklin, R.A.; Davila, D.R.; Westly, H.J.; Kelley, K.W.

    1986-03-05

    The authors have found that heat-inactivated serum (57/sup 0/C for 1 hour) from normal chickens reduces the proliferation of mitogen-stimulated chicken and murine splenocytes as well as some transformed mammalian lymphoblastoid cell lines. Greater than a 50% reduction in /sup 3/H-thymidine incorporation was observed when concanavalin A (Con A)-activated chicken splenocytes that were cultured in the presence of 10% autologous or heterologous serum were compared to mitogen-stimulated cells cultured in the absence of serum. Normal chicken serum (10%) also caused greater than 95% suppression of /sup 3/H-thymidine incorporation by bovine (EBL-1 and BL-3) and gibbon ape (MLA 144) transformed lymphoblastoid cell lines. The only cell line tested that was not inhibited by chicken serum was an IL-2-dependent, murine cell line. Chicken serum also inhibited both /sup 3/H-thymidine incorporation and IL-2 synthesis by Con A-activated murine splenocytes. Suppression was caused by actions other than cytotoxicity because viability of chicken splenocytes was unaffected by increasing levels of chicken serum. Furthermore, dialyzed serum retained its activity, which suggested that thymidine in the serum was not inhibiting uptake of radiolabeled thymidine. Suppressive activity was not due to adrenal glucocorticoids circulating in plasma because neither physiologic nor pharmacologic doses of corticosterone had inhibitory effects on mitogen-stimulated chicken splenocytes. These data demonstrate that an endogenous factor that is found in normal chicken serum inhibits proliferation of T-cells from chickens and mice as well as some transformed mammalian lymphoblastoid cell lines.

  5. In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

    Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons. PMID:1052527

  6. Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

    SciTech Connect

    Lovrinovic, Marina; Niemeyer, Christof M. . E-mail: christof.niemeyer@uni-dortmund.de

    2005-09-30

    We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

  7. Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds 1

    PubMed Central

    Callebaut, Alfons; Van Oostveldt, Patrick; Van Parijs, Roger

    1980-01-01

    Large doses of γ-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced. Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness. The results suggest that γ-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins. PMID:16661127

  8. Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli.

    PubMed

    Silverman, Adam P; Jiang, Qingfei; Goodman, Myron F; Kool, Eric T

    2007-12-04

    The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.

  9. Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis

    PubMed Central

    Freudenthal, Bret D.; Ramaswamy, S.; Hingorani, Manju M.; Washington, M. Todd

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a sliding clamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesis have been identified in genetic studies in yeast. One such mutant protein (encoded by the rev6-1 allele) is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To better understand how this substitution interferes with translesion synthesis, we have determined the X-ray crystal structure of the G178S PCNA mutant protein. This substitution has little effect on the structure of the domain in which the substitution occurs. Instead, significant, local structural changes are observed in the adjacent subunit. The most notable difference between mutant and wild-type structures is in a single, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutant protein structure, loop J adopts a very different conformation in which the atoms of the protein backbone have moved by as much as 6.5 Å from their positions in the wild-type structure. To better understand the functional consequences of this structural change, we have examined the ability of this mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady state kinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These results show that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis. PMID:19053247

  10. Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

    PubMed

    Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

    2014-10-20

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences

  11. DNA Binding and Recognition of a CC Mismatch in a DNA Duplex by Water-Soluble Peptidocalix[4]arenes: Synthesis and Applications.

    PubMed

    Alavijeh, Nahid S; Zadmard, Reza; Balalaie, Saeed; Alavijeh, Mohammad S; Soltani, Nima

    2016-10-07

    Water-soluble peptidocalix[4]arenes were synthesized by the introduction of arginine-rich narrow groove-binding residues at lower rims through solid-phase synthesis. The study of binding of these water-soluble bidentate ligands to well-matched and mismatched DNA duplexes by fluorescent titrations, ethidium bromide (EB) displacement assays, DNA-melting experiments, and circular dichroism (CD) analysis revealed a sequence-dependent groove-binding mechanism.

  12. Photoaugmentation in the hairless mouse: a study using ornithine decarboxylase activity and alteration of DNA synthesis as markers of epidermal response

    SciTech Connect

    Gange, R.W.; Mendelson, I.R.

    1981-01-01

    Photoaugmentation is the potentiation of UVB-induced cutaneous erythema by UV irradiation. We have examined other cutaneous responses to UVB irradiation-the 4 hr depression of DNA synthesis, the 48 hr stimulation of DNA synthesis, and the induction of ornithine decarboxylase (ODC), to determine whether these were also susceptible to augmentation by UVA, which does not cause these responses when administered alone. No photoaugmentation of DNA synthesis, stimulation or ODC induction occurred. The early depression of DNA synthesis was slightly augmented for this did not consistently reach significance.

  13. Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs.

    PubMed

    Lu, Haige; Krueger, Andrew T; Gao, Jianmin; Liu, Haibo; Kool, Eric T

    2010-06-21

    The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18-200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.

  14. Temporal and topographic changes in DNA synthesis after induced follicular atresia

    SciTech Connect

    Greenwald, G.S. )

    1989-07-01

    Hamsters were hypophysectomized on the morning of estrus (Day 1) and injected immediately with 30 IU pregnant mare's serum (PMS). This was followed on Day 4 by the injection of an antiserum to PMS (PMS-AS) that initiated follicular atresia (Time zero). From 0 to 72 h after PMS-AS, the animals were injected with (3H)thymidine and killed 4 h later. One ovary was saved for autoradiography and histology; from the other ovary, 5-10 large antral follicles were dissected and pooled, and incorporation into DNA was determined by scintillation counting. DNA synthesis dropped sharply between 12 and 18 h, coinciding with a fall in labeling index of the cumulus oophorus and thecal endothelial cells and a sharp fall in thecal vascularity. In contrast, for the mural granulosa cells bordering on the antral cavity, labeling index dropped sharply between 8 and 12 h when thecal vascularity was still high. The earliest sign of atresia was evident by 4 h in cumulus cells when, paradoxically, DNA synthesis was still high. It took 3 days for atresia of the antral follicles to progress to advanced stages, as evidenced by pseudo-pronuclei in the free floating ovum, further erosion of the mural granulosa, and minimal DNA/follicle. However, the theca still retained its histological integrity and contained no pyknotic cells. Although by 48 h the granulosal compartment was in disarray (DNA/follicle significantly different from earlier values), the egg was still viable, as judged by maximal fluorescence after the addition of fluoroscein diacetate.

  15. Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions

    PubMed Central

    Tsaalbi-Shtylik, Anastasia; Ferrás, Cristina; Pauw, Bea; Hendriks, Giel; Temviriyanukul, Piya; Carlée, Leone; Calléja, Fabienne; van Hees, Sandrine; Akagi, Jun-Ichi; Iwai, Shigenori; Hanaoka, Fumio; Jansen, Jacob G.

    2015-01-01

    In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa–Atr–Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects. PMID:25869665

  16. Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

    PubMed

    Nilforoushan, Arman; Furrer, Antonia; Wyss, Laura A; van Loon, Barbara; Sturla, Shana J

    2015-04-15

    Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

  17. Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination.

    PubMed

    Ho, Jenny; Huang, Yi; Danquah, Michael K; Wang, Huanting; Forde, Gareth M

    2010-03-18

    DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(D,L-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 microm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

  18. Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes

    SciTech Connect

    Yarosh, D.B.; Kibitel, J.T.; Green, L.A.; Spinowitz, A. )

    1991-07-01

    Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population.

  19. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    SciTech Connect

    Weinberger, Florian Mehrkens, Dennis Starbatty, Jutta Nicol, Philipp Eschenhagen, Thomas

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

  20. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    SciTech Connect

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J. )

    1991-05-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

  1. Synthesis and antiproliferative activity of some new DNA-targeted alkylating pyrroloquinolines.

    PubMed

    Ferlin, M G; Dalla Via, L; Gia, O M

    2004-02-15

    Two novel DNA-direct alkylating agents, consisting of aniline mustard linked to an angular 3H-pyrrolo[3,2-f]quinoline nucleus, were synthetized and assayed for their in vitro antiproliferative activity. Simple convergent synthesis, consisting of separate preparation of 9-chloro-3H-pyrrolo[3,2-f]quinoline and p-amino-aniline derivatives, and following their linkage by substitution reactions 8a, b and 10, yielded the corresponding diol derivatives 7b and 9. Biological properties were evaluated with respect to cell growth inhibition, ability to form cross-links with DNA, and capacity to give rise to a molecular complex with the macromolecule for 7b, 8b, 9 and 10.

  2. A chemical method for fast and sensitive detection of DNA synthesis in vivo.

    PubMed

    Salic, Adrian; Mitchison, Timothy J

    2008-02-19

    We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

  3. Novel designed enediynes: molecular design, chemical synthesis, mode of cycloaromatization and guanine-specific DNA cleavage.

    PubMed

    Toshima, K; Ohta, K; Kano, T; Nakamura, T; Nakata, M; Kinoshita, M; Matsumura, S

    1996-01-01

    The molecular design and chemical synthesis of novel enediyne molecules related to the neocarzinostatin chromophore (1), and their chemical and DNA cleaving properties are described. The 10-membered enediyne triols 16-18 were effectively synthesized from xylitol (10) in a short step, and found to be quite stable when handled at room temperature. The representative and acylated enediyne 16 was cycloaromatized by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in cyclohexa-1,4-diene-benzene to give the benzenoid product 21 through a radical pathway. On the other hand, the enediyne 16 was cycloaromatized by diethylamine in dimethyl sulfoxide-Tris-HCl, pH 8.5 buffer to afford another benzenoid product 22 as a diethylamine adduct through a polar pathway. Furthermore, the enediynes 16-18 were found to exhibit guanine-specific DNA cleavage under weakly basic conditions with no additive.

  4. Fabrication of polyurethane molecular stamps for the synthesis of DNA microarray

    NASA Astrophysics Data System (ADS)

    Liu, Zhengchun; He, Quanguo; Xiao, Pengfeng; He, Nongyao; Lu, Zuhong; Bo, Liang

    2001-10-01

    Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.

  5. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

    PubMed

    Taggart, David J; Dayeh, Daniel M; Fredrickson, Saul W; Suo, Zucai

    2014-10-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non

  6. Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA

    SciTech Connect

    Sega, G.A.; Generoso, E.E.; Brimer, P.A. )

    1990-01-01

    A study of meiotic and postmeiotic germ-cell-stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid-spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg. By using 14C-labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.

  7. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

    PubMed

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-03-29

    In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.

  8. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    PubMed

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  9. Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I

    PubMed Central

    Rideout, Marc C.; Raymond, Amy C.; Burgin, Alex B.

    2004-01-01

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein–DNA covalent complexes. Tdp1 hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and free tyrosine in vitro. Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds. Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells. Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions. Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3′-(4-methylumbelliferone)-phosphate. These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule. The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods. This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules. PMID:15333697

  10. Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

    PubMed Central

    Pagès, Vincent; Bresson, Anne; Acharya, Narottam; Prakash, Satya; Fuchs, Robert P.; Prakash, Louise

    2008-01-01

    In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases η or ζ or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities—a ubiquitin ligase that promotes Mms2–Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polζ. Using duplex plasmids carrying different site-specific DNA lesions—an abasic site, a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct—we show that Rad5 is needed for Polζ-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS. PMID:18757916

  11. Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

    NASA Astrophysics Data System (ADS)

    War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

    2017-02-01

    The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues.

  12. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  13. Promotion of role clarification in the Health Care Team Challenge.

    PubMed

    Hudson, C C; Gauvin, S; Tabanfar, R; Poffenroth, A M; Lee, J S; O'Riordan, A L

    2017-01-31

    Interprofessional collaboration has consistently been associated with positive client-care outcomes. Role clarification is one facet of interprofessional collaboration that is thought to be crucial for effective interprofessional team functioning. Given the positive outcomes associated with interprofessional collaboration, educators have begun to integrate formal interprofessional education events into healthcare curricula. The Health Care Team Challenge (HCTC) is a collaborative competition designed to promote interprofessional competencies among students in healthcare fields. The current study empirically investigated whether this event promoted role clarification among participants. Sixteen participants in five healthcare professions (occupational therapy, physiotherapy, clinical psychology, nursing, and medicine) completed two questionnaires to assess role clarification before and after participating in this event. Results indicate that participants' understanding of their own and other professions' roles improved after participating in this team activity. These results suggest that the HCTC is effective in promoting role clarification and collaboration among healthcare students.

  14. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  15. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  16. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  17. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  18. Depletion of Kupffer cells modulates ethanol-induced hepatocyte DNA synthesis in C57Bl/6 mice.

    PubMed

    Owumi, Solomon E; Corthals, Stacy M; Uwaifo, Anthony O; Kamendulis, Lisa M; Klaunig, James E

    2014-08-01

    Kupffer cells (KCs) are important in hepatic homeostasis and responses to xenobiotics. KCs are activated on interaction with endotoxin, releasing cytokines, and reactive oxygen species normally associated with increased gene expression, cellular growth, or hepatic injury. Ethanol-induced endotoxemia is one means of KC activation. We propose that KC depletion attenuates the effect of EtOH-induced endotoxemia to impact the hepatic growth response. Hepatic DNA synthesis was examined in KC competent (KC+) or KC-depleted (KC-) C57BL/6 mice fed EtOH-containing diet in the presence or absence of polyphenol-60 antioxidant. KC depletion was assessed by F4/80 antigen, and DNA synthesis was assessed by 5-bromo-2'-deoxyuridine incorporation. Tumor necrosis factor alpha (TNF-α) messenger RNA released was quantified by RT-PCR/electrophoresis. ERK1/2 phosphorylation was evaluated by Western blotting, and Nrf2 and CYP2E1protein were also assayed. Apoptosis and hepatic injury were examined by the Tunnel assay and hepatic transaminases in serum, respectively. Hepatic transaminases in serum (AST and ALT) were within normal range. Over 90% of KC was depleted by clodronate treatment. KC depletion decreased TNF-α mRNA release, ERK1/2 phosphorylation, and hepatocyte DNA synthesis. KC depletion is associated with increased numbers of apoptotic cells bodies in KC- mice. Antioxidant treatment decreased DNA synthesis, Nrf2, and CYP2E1 protein expression in EtOH-consuming mice. Our data indicate that upon ethanol exposure, KC participates in hepatic DNA synthesis and growth responses. Collectively, these observations suggest that KC depletion attenuates the downstream effect of ethanol-induced endotoxemia by reduced cytokine and reactive oxygen species production with its concomitant effect on MAPK-signaling pathway on hepatocyte DNA synthesis.

  19. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening.

    PubMed

    Ranall, Max V; Gabrielli, Brian G; Gonda, Thomas J

    2010-05-01

    Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.

  20. In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

    PubMed Central

    Hirota, Kouji; Tsuda, Masataka; Mohiuddin; Tsurimoto, Toshiki; Cohen, Isadora S.; Livneh, Zvi; Kobayashi, Kaori; Narita, Takeo; Nishihara, Kana; Murai, Junko; Iwai, Shigenori; Guilbaud, Guillaume; Sale, Julian E.; Takeda, Shunichi

    2016-01-01

    The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass. PMID:27185888

  1. Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

    SciTech Connect

    Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

    1985-07-01

    To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

  2. DNA Methyltransferase protein synthesis is reduced in CXXC finger protein 1-deficient embryonic stem cells.

    PubMed

    Butler, Jill S; Palam, Lakshmi R; Tate, Courtney M; Sanford, Jeremy R; Wek, Ronald C; Skalnik, David G

    2009-05-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.

  3. An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

    PubMed Central

    Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

    2012-01-01

    During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

  4. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    PubMed

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

  5. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis.

    PubMed

    Gilbert, M Thomas P; Binladen, Jonas; Miller, Webb; Wiuf, Carsten; Willerslev, Eske; Poinar, Hendrik; Carlson, John E; Leebens-Mack, James H; Schuster, Stephan C

    2007-01-01

    Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine-->thymine/guanine-->adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine-->adenine analogue modification, not the conventionally argued cytosine-->uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates.

  6. Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis.

    PubMed

    Smith, Kevin D; Fu, Michael A; Brown, Eric J

    2009-10-05

    The Tim (Timeless)-Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim-Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR-Chk1 pathway engagement. We now demonstrate that Tim-Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR-Chk1 pathway in Tim-Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim-Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim-Tipin dysfunction. Together, these experiments indicate that the Tim-Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR-Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

  7. Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo

    SciTech Connect

    Heintz, N.H.; Stillman, B.W.

    1988-05-01

    A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4/sup 0/C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases.

  8. [Clarification on publications concerning the synthesis of acetylsalicylic acid].

    PubMed

    Lafont, O

    1996-01-01

    Charles Frédéric Gerhardt (1816-1856) mentioned in his Traité de chimie Organique (1854) a publication, in French (realized in 1852 but published in 1853) entitled "Researches on anhydrous organic acids" in which, was reported the reaction of sodium salicylate with acetyl chloride. He thought that the reaction product was an acid anhydride, but obtained really crude acetylsalicylic acid. Later on, but also in 1853, a publication in german, by the same author related the same experiments. Surprisingly only the second publication has been mentioned in most of the historical studies on the subject. Acetyl salicylic acid was identified and synthesised in 1859 by von Gilm by another method and the product obtained by Gerhardt was identified to it in 1869.

  9. [Autoradiographic investigations on the effect of city smog extract on DNA synthesis and cell cycle of mammalian cells in vitro. I. Effect of city smog extract on DNA synthesis of kidney- and embryonic cells of the golden hamster in vitro (author's transl)].

    PubMed

    Krampitz, G; Seemayer, N

    1979-05-01

    We analysed the effect of city smog extract from Düsseldorf on DNA synthesis of mammalian cells in vitro. Airborne dust was extracted with aceton and thereafter transferred to dimethylsulfoxide. Dosage was calculated according to the benzo(a)pyrene content. We used logarithmically growing cultures of hamster kidney and embryonic cells. DNA synthesis was determined autoradiographically by incorporation of 3H-Thymidine. We found that city smog extract exerted a dose-dependent cytotoxic effect leading to a decrease of DNA synthesizing cells. High concentrations of city smog extract induced cell necrosis and suppressed DNA synthesis completely. Moderate doses of extract caused a dose-dependent, but temporary cessation of DNA synthesis. Cells resumed DNA synthesis after a certain delay. Low concentrations of city smog extract induced no detectable effects. Inhibition of DNA synthesis was evident already one hour after addition of extract. Therefore a direct effect on DNA metabolism could be supposed. Furthermore, exposed cultures demonstrated a delay in entrance of new cells into the DNA synthesis. Alterations in DNA synthesis could be of great importance for carcinogenesis, especially if we take in consideration the content of carcinogens in city smog extract.

  10. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  11. SYNTHESIS, IN VITRO METABOLISM, MUTAGENICITY, AND DNA-ADDUCTION OF NAPHTHO[1,2-E]PYRENE

    EPA Science Inventory

    SYNTHESIS, IN V1TRO METABOLISM, MUTAGENICITY , AND DNA-ADDUCnON OF NAPHTHO[l ,2-e ]PYRENE

    Literature data, although limited, underscore the contribution of C24HI4 polycyclic aromatic hydrocarbons to the biological activity of the extracts of complex environmental samples....

  12. UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey D.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer UAP56 is an important regulator of DNA synthesis in vascular smooth muscle cells. Black-Right-Pointing-Pointer UAP56 binds to Bcr. Black-Right-Pointing-Pointer Interaction between Bcr and UAP56 is critical for Bcr induced DNA synthesis. -- Abstract: Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPAR{gamma}. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

  13. The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds

    PubMed Central

    Hasinoff, Brian B.; Liang, Hong; Wu, Xing; Guziec, Lynn J.; Guziec, Frank S.; Marshall, Kyle; Yalowich, Jack C.

    2008-01-01

    Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n = 1 to 5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIα-mediated cleavage of DNA and inhibition of DNA topoisomerase IIα decatenation activities. The results suggest that the bisanthrapyrazoles with n = 2 to 5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents. PMID:18258442

  14. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  15. Quantification of histoautoradiographic evidence of DNA repair synthesis in the liver.

    PubMed

    Hochmann, J; Stambergová, H

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3H-thymidine incorporation in vivo into G phase hepatocytes. A description of the standard procedure for counting the grains as well as the mode of mathematical evaluation is presented. The results exhibited higher sensitivity than those in the investigation of the DNA repair synthesis by means of a scintillation counter using the method of detection of hydroxyurea-resistant incorporation of 3H-thymidine. Thus it was possible to simplify the investigation by lowering the number of evaluated cells. A suitable compromise between precision and laboriousness will probably be achieved by counting 20 hepatocytes per animal. In case that there are striking differences between the experimental and the control group, a qualitative conclusion may be drawn even without counting the grains.

  16. A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase eta.

    PubMed

    Kunz, Bernard A

    2008-10-01

    Eukaryotic DNA polymerase eta (Poleta) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Poleta has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Poleta is required for UV resistance in plants. Furthermore, the purified enzyme can perform TLS opposite a cyclobutane pyrimidine dimer and interacts with PCNA. Intriguingly, the biological activity of Poleta in a heterologous yeast assay depends on co-expression with Arabidopsis PCNA2 and Poleta sequences implicated in binding PCNA or ubiquitin. We suggest that interaction of Arabidopsis Poleta with ubiquitylated PCNA2 is required for TLS past UV photoproducts by Poleta.

  17. The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis

    PubMed Central

    Berman, Andrea J.; Akiyama, Benjamin M.; Stone, Michael D.; Cech, Thomas R.

    2011-01-01

    Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3′ end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation. PMID:22101935

  18. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

    PubMed Central

    Kim, S Y; Byrn, R; Groopman, J; Baltimore, D

    1989-01-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes. Images PMID:2760980

  19. Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

    PubMed

    Harmon, C S; Nevins, T D; Ducote, J; Lutz, D

    1997-01-01

    We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

  20. An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues

    PubMed Central

    Guo, Jia; Yu, Lin; Turro, Nicholas J.; Ju, Jingyue

    2010-01-01

    Conspectus The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine as well as uncovering the genomic contributions to diseases. The Sanger sequencing method—although enormously productive for more than 30 years—requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs. The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform. Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3′-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3′-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface

  1. Synthesis and Crystal Structure Study of 2’-Se-Adenosine-Derivatized DNA

    SciTech Connect

    Sheng, J.; Salon, J; Gan, J; Huang, Z

    2010-01-01

    The selenium derivatization of nucleic acids is a novel and promising strategy for 3D structure determination of nucleic acids. Selenium can serve as an excellent anomalous scattering center to solve the phase problem, which is one of the two major bottlenecks in macromolecule X-ray crystallography. The other major bottleneck is crystallization. It has been demonstrated that the incorporated selenium functionality at the 2'-positions of the nucleosides and nucleotides is stable and does not cause significant structure perturbation. Furthermore, it was observed that the 2'-Se-derivatization could facilitate crystallization of oligonucleotides with fast crystal growth and high diffraction quality. Herein, we describe a convenient synthesis of the 2'-Se-adenosine phosphoramidite, and report the first synthesis and X-ray crystal structure determination of the DNA containing the 2'-Se-A derivatization. The 3D structure of 2'-Se-A-DNA decamer [5'-GTACGCGT(2'-Se-A)C-3']{sub 2} was determined at 1.75 {angstrom} resolution, the 2'-Se-functionality points to the minor groove, and the Se-modified and native structures are virtually identical. Moreover, we have observed that the 2'-Se-A modification can greatly facilitate the crystal growth with high diffraction quality. In conjunction with the crystallization facilitation by the 2'-Se-U and 2'-Se-T, this novel observation on the 2'-Se-A functionality suggests that the 2'-Se moiety is sole responsible for the crystallization facilitation and the identity of nucleobases does not influence the crystal growth significantly.

  2. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

    PubMed

    Salmon, M; Walsh, D A; Koto, H; Barnes, P J; Chung, K F

    1999-09-01

    Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

  3. Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast Saccharomyces cerevisiae.

    PubMed

    Kochenova, O V; Soshkina, J V; Stepchenkova, E I; Inge-Vechtomov, S G; Shcherbakova, P V

    2011-01-01

    We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.

  4. 3-Phosphono-L-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase.

    PubMed

    Yang, Shiqiong; Froeyen, Mathy; Lescrinier, Eveline; Marlière, Philippe; Herdewijn, Piet

    2011-01-07

    A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of deoxyadenosine triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 μM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson-Crick rules.

  5. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells exposed to monochromatic uv wavelengths (252-313 nm) and photoreactivating light

    SciTech Connect

    Rosenstein, B.S.

    1982-06-01

    Exposure of ICR 2A frog cells to monochromatic uv wavelengths in the range 252-313 nm caused an inhibition of semiconservative DNA synthesis which was partially relieved in cells receiving a post irradiation treatment with photoreactivating light (>350 nm). Hence pyrimidine dimers acted as lesions blocking DNA synthesis in uv-irradiated cells based upon the specificity of photoreactivating enzyme for the light-dependent monomerization of dimers in DNA. Compared with the shorter wavelengths tested, however, this recovery of DNA synthesis was not as great in cells exposed to 302-nm radiation and was nearly absent in 313-nm-irradiated cells up to 12 hr after treatment. These results suggest that nondimer photoproducts also play an important role in causing DNA synthesis inhibition in cells exposed to wavelengths greater than 300 nm.

  6. Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis.

    PubMed

    Kint, Cyrielle; Verstraeten, Natalie; Hofkens, Johan; Fauvart, Maarten; Michiels, Jan

    2014-08-01

    Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

  7. Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: differential scanning calorimetric study.

    PubMed

    Florian, Jakub; Brabec, Viktor

    2012-02-06

    Differential scanning calorimetry (DSC) was used to measure the thermodynamic changes associated with translesion synthesis across major lesion induced in DNA by antitumor oxaliplatin [1,2-d(GG) intrastrand cross-link]. Insertion of matched nucleotides dC at the primer terminus (across unique 3'- or 5'-dG in the unplatinated template) and subsequent extensions resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite either platinated dG in the intrastrand cross-link formed in the template strand and subsequent extensions by one nucleotide resulted only in little changes in thermodynamics. A similar thermodynamic delay was observed for a control template primer containing a dG:dT mismatch across 3'- or 5'-dG in the template and subsequent Watson-Crick primer extensions. The thermodynamic scarcity generated by either the lesion or mismatches was not localized but extended to the 5'-downstream sites, which may be connected with the phenomenon termed "short-term memory" of replication errors retained by some DNA polymerases responding to DNA damages or mismatches. Interestingly, formation of the 1,2-d(GG) intrastrand cross-link of oxaliplatin altered the overall DSC profiles of the dG:dT mismatch template/primers only in a very small extent. While addition of matched nucleotide dC across either dG in the template strand was thermodynamically favored over the presence of a mismatched dT (ΔΔG(0)(310) was 7.6 or 6.8 kJ mol(-1), ΔΔH was 14 or 49 kJ mol(-1)), no such thermodynamic advantage was observed with the 1,2-d(GG) intrastrand cross-link of oxaliplatin at these positions (ΔΔG(0)(310) was 2.8 or -0.3 kJ mol(-1), ΔΔH was 4 or 9 kJ mol(-1)). The equilibrium thermodynamic data also provide insight into the processes associated with misincorporation of incorrect nucleotides during replication bypass across major cross-links of antitumor oxaliplatin. On the other hand, besides thermodynamic effects also kinetic

  8. REV1 restrains DNA polymerase zeta to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo.

    PubMed

    Szüts, Dávid; Marcus, Adam P; Himoto, Masayuki; Iwai, Shigenori; Sale, Julian E

    2008-12-01

    Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.

  9. Synthesis and characterization of DNA-quantum dot conjugates for the fluorescence ratiometric detection of unlabelled DNA.

    PubMed

    Page, Leah Elizabeth; Zhang, Xi; Tyrakowski, Christina Marie; Ho, Chiun-Teh; Snee, Preston Todd

    2016-11-21

    A quantum dot-based ratiometrically responsive fluorescent sensor for unlabeled single-stranded DNA (ssDNA) is reported. Several technical issues concerning the development of high yield ssDNA-QD conjugation chemistry were addressed. The DNA sensor was synthesized by conjugating methacrylic phosphoramidite-functional oligonucleotides to water-soluble cadmium zinc sulfide core/zinc sulfide shell quantum dots (CdZnS/ZnS QDs). Duplex DNA was formed when the QD-bound ssDNA was incubated with its complement. Next, titration with PicoGreen resulted in FRET energy transfer from the dot to the dsDNA intercalating dye. The resulting ratio of the dye to QD integrated emissions is a calibratable metric for label-free DNA detection with a LOD of 3.8 nmol.

  10. Synthesis, Acetylation, and Phosphorylation of Histone IV and Its Binding to DNA During Spermatogenesis in Trout*

    PubMed Central

    Louie, Andrew J.; Dixon, Gordon H.

    1972-01-01

    During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [3H]aminoacids and inorganic [32P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [3H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [3H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total 3H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an “obligatory” acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that ε-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH2-terminal region of histone IV to assume the correct conformation (in this case, an α-helix), and fit into the major groove of DNA. Deacetylation then “locks” histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known. Images PMID:4505675

  11. Glucocorticoid suppression of human lymphocyte DNA synthesis. Influence of phytohemagglutinin concentration

    SciTech Connect

    Segel, G.B.; Lukacher, A.; Gordon, B.R.; Lichtman, M.A.

    1980-04-01

    Glucocorticoids have been shown to suppress lectin-stimulated lymphocyte DNA synthesis in some studies, whereas in other studies, the hormones have had little effect. We have found that the position on the PHA dose-response curve that is studied is the most important determinant of whether cortisol inhibits /sup 3/H-thymidine incorporation into lymphocyte DNA. The proportion of monocytes in culture also influenced the cortisol effect, but it was quantitatively less important than PHA concentration. Cortisol (5 nM to 100 ..mu..M) had little effect on blastogenesis or thymidine incorporation into DNA in cultures that contained both a high concentration (14% +- 2 (S.E.)) of monocytes and a concentration of PHA (0.6 to 1.2 ..mu..g/ml) that produced maximal stimulation of mitogenesis. When monocytes were reduced from 14 to 1.4%, cortisol (5 ..mu..M) caused a 30% reduction in thymidine incorporation in cultures stimulated by 0.6 to 1.2 ..mu..g/ml PHA. Much greater cortisol suppression of thymidine incorporation occurred if the concentration of PHA was reduced. For example, reduction of the PHA concentration from 1.2 to 0.075 ..mu..g/ml resulted in an increase in suppression by 5 ..mu..M cortisol from 5 to 90% even in the presence of 14% monocytes. These data indicate that the suppressive effects of glucocorticoids on blastogenesis and thymidine incorporation in vitro depend principally on the concentration of PHA used to stimulate blastogenesis and secondarily on the proportion of monocytes in the culture system.

  12. DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae.

    PubMed

    Corrette-Bennett, Stephanie E; Borgeson, Claudia; Sommer, Debbie; Burgers, Peter M J; Lahue, Robert S

    2004-01-01

    Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis.

  13. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

    SciTech Connect

    Rosenstein, B.S.

    1984-11-01

    DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. In addition, cells were exposed to /sup 60/Co ..gamma.. rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in ..gamma..-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.

  14. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    PubMed

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  15. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  16. M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.

    PubMed

    Arion, D; Meijer, L

    1989-08-01

    Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.

  17. The Impact of a Classroom Values Clarification Program

    ERIC Educational Resources Information Center

    Logan, Donald; And Others

    1977-01-01

    The Benton High School values clarification program is an orderly sequence of structured group activities directed toward clarifying students' values and decisions as part of a one-semester elective sociology class open to all juniors and seniors. Evaluation indicates program participants moved toward a stronger belief in internal control in…

  18. 76 FR 38053 - Clarification of the Fireworks Approvals Policy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-29

    ... pertaining to application volume. We believe that this is a safety and data management issue. Furthermore, we... document PHMSA is restating our policy clarification in that we will issue firework classification... (75 FR 79085) clarifying its policy, consistent with the HMR, to issue firework...

  19. 76 FR 28031 - Clarification of Statement of Policy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... originally promulgated the Statement of Policy for Section 19 of the Federal Deposit Insurance Act (SOP) in December 1998. The FDIC, in 2007, issued a clarification to the SOP based on the 2006 amendment to Section... restating that previous change to the SOP in a slightly modified form, and addressing certain other...

  20. 76 FR 80831 - Clarification of Policy Regarding Approved Training Programs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ... Federal Aviation Administration 14 CFR Part 135 Clarification of Policy Regarding Approved Training... Notice that would require FAA inspectors to review 14 CFR part 135 approved training programs to identify and correct those programs which erroneously issued credit for previous training or checking....

  1. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

    PubMed Central

    Briggs, Adrian W.; Rios, Xavier; Chari, Raj; Yang, Luhan; Zhang, Feng; Mali, Prashant; Church, George M.

    2012-01-01

    DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical. PMID:22740649

  2. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  3. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

    PubMed Central

    Salmon, Michael; Walsh, David A; Huang, Tung-Jung; Barnes, Peter J; Leonard, Thomas B; Hay, Douglas W P; Chung, K Fan

    1999-01-01

    Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin-exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline-exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5-lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen-induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure. PMID:10455261

  4. Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

    NASA Astrophysics Data System (ADS)

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  5. Interacting RNA polymerase motors on a DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis.

    PubMed

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  6. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    PubMed

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  7. Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA.

    PubMed

    Robinson, Ian; Tung, Le D; Maenosono, Shinya; Wälti, Christoph; Thanh, Nguyen T K

    2010-12-01

    Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6±0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2±1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.

  8. Stimulation of DNA synthesis by ascorbate in cultures of articular chondrocytes

    SciTech Connect

    Krystal, G.; Morris, G.M.; Sokoloff, L.

    1982-03-01

    The addition of 0.2 mM Na L-ascorbate increased the incorporation of 3H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15-30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations greater than 0.2 mM in the presence of certain batches of serum.

  9. Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

    PubMed

    Vesole, D H; Goust, J M; Fett, J W; Fudenberg, H H

    1979-09-01

    Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.

  10. Dihydrochelerythrine and its derivatives: Synthesis and their application as potential G-quadruplex DNA stabilizing agents.

    PubMed

    Malhotra, Rajesh; Rarhi, Chhanda; Diveshkumar, K V; Barik, Rajib; D'cunha, Ruhee; Dhar, Pranab; Kundu, Mrinalkanti; Chattopadhyay, Subrata; Roy, Subho; Basu, Sourav; Pradeepkumar, P I; Hajra, Saumen

    2016-07-01

    A convenient route was envisaged toward the synthesis of dihydrochelerythrine (DHCHL), 4 by intramolecular Suzuki coupling of 2-bromo-N-(2-bromobenzyl)-naphthalen-1-amine derivative 5 via in situ generated arylborane. This compound was converted to (±)-6-acetonyldihydrochelerythrine (ADC), 3 which was then resolved by chiral prep-HPLC. Efficiency of DHCHL for the stabilization of promoter quadruplex DNA structures and a comparison study with the parent natural alkaloid chelerythrine (CHL), 1 was performed. A thorough investigation was carried out to assess the quadruplex binding affinity by using various biophysical and biochemical studies and the binding mode was explained by using molecular modeling and dynamics studies. Results clearly indicate that DHCHL is a strong G-quadruplex stabilizer with affinity similar to that of the parent alkaloid CHL. Compounds ADC and DHCHL were also screened against different human cancer cell lines. Among the cancer cells, (±)-ADC and its enantiomers showed varied (15-48%) inhibition against human colorectal cell line HCT116 and breast cancer cell line MDA-MB-231 albeit low enantio-specificity in the inhibitory effect; whereas DHCHL showed 30% inhibition against A431 cell line only, suggesting the compounds are indeed cancer tissue specific.

  11. Synthesis and investigation of the specific activity of the DNA-doxorubicin conjugates

    NASA Astrophysics Data System (ADS)

    Kokorev, A. V.; Zaborovskiy, A. V.; Kotlyarov, A. A.; Balykova, L. A.; Malkina, M. A.; Kargina, I. V.; Gromova, E. V.; Medvezhonkov, V. Yu; Gurevich, K. G.; Shchukin, S. A.; Pyataev, N. A.

    2017-01-01

    In the present work, the method of obtaining the conjugate of the anticancer chemotherapeutic agent doxorubicin to the exogenous double-stranded DNA of the sturgeons is proposed (the source: commercial drug “Derinat”). The optimal conditions for synthesis of conjugate (pH, temperature and the mass ratio of the components), ensuring the highest degree of binding the chemotherapeutic agent to a carrier, were picked out. Clearing the conjugate from the non-encapsulated chemotherapeutic agent was being made by ultrafiltration method. The investigation of the toxicity and specific antineoplastic activity of the synthesized complex was conducted. The performance of the drug toxicity were established on the intact mice in compliance with the accepted standards. The antineoplastic activity was evaluated upon the Tumor Growth Inhibition Index and Metastasis Inhibition Index on mice with the transplanted Lewis lung carcinoma (LLC). It was demonstrated that the conjugate toxicity is approximately lower that the one of the unconjugated doxorubicin (LD 50 was equal 14.6 mg/kg and 9.9 mg/kg for the conjugate and doxorubicin, respectively). The specific antineoplastic activity was investigated in equitoxic doses of the drug. It was established that the conjugate being administered in equitoxic doses possesses a stronger antineoplastic activity, than the water-soluble drug (maximum 35% more as to the tumor volume and 51% more as to the Tumor Growth Inhibition index).

  12. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro.

    PubMed

    Wahab-Wahlgren, Aida; Martinelle, Nina; Holst, Mikael; Jahnukainen, Kirsi; Parvinen, Martti; Söder, Olle

    2003-03-28

    Epidermal growth factor (EGF) superfamily of peptide growth factors (EGF-GFs) plays a role in male germ cell development, but the precise function is yet to be defined. The present study shows that EGF-GFs stimulate spermatogonial proliferation in vitro. The EGF-GF ligands, EGF, transforming growth factor-alpha and betacellulin all stimulated DNA synthesis in microdissected stage I segments of rat testis seminiferous tubules in vitro, as revealed by 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU) labeling. A fourfold increase over control of BrdU labeled cells, identified as spermatogonia, was seen after treatment with EGF. RT-PCR analysis revealed that the EGF receptors erbB1, erbB2, erbB3 and erbB4 were expressed at all stages of the spermatogenic wave, whereas differential expression was found in isolated Leydig, Sertoli and peritubular cells. The results show that EGF-GFs is spermatogonial growth factor(s) in vitro, although we have not discriminated between a direct action and an indirect effect via somatic cells. We suggest that EGF-GFs is involved in the paracrine control of spermatogenesis in vivo.

  13. Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.

    PubMed Central

    Blanco, L; Salas, M

    1985-01-01

    A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all phi 29 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA molecules had reached apparently full-length phi 29 DNA (approximately equal to 18,000 nucleotides). Ammonium ions strongly stimulated phi 29 DNA-protein p3 replication, the effect being due to stimulation of the initiation reaction. ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps. The results show that the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA. Images PMID:3863101

  14. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    PubMed

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

  15. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  16. Exploration of cellular DNA lesion, DNA-binding and biocidal ordeal of novel curcumin based Knoevenagel Schiff base complexes incorporating tryptophan: Synthesis and structural validation

    NASA Astrophysics Data System (ADS)

    Chandrasekar, Thiravidamani; Raman, Natarajan

    2016-07-01

    A few novel Schiff base transition metal complexes of general formula [MLCl] (where, L = Schiff base, obtained by the condensation reaction of Knoevenagel condensate of curcumin, L-tryptophan and M = Cu(II), Ni(II), Co(II), and Zn(II)), were prepared by stencil synthesis. They were typified using UV-vis, IR, EPR spectral techniques, micro analytical techniques, magnetic susceptibility and molar conductivity. Geometry of the metal complexes was examined and recognized as square planar. DNA binding and viscosity studies revealed that the metal(II) complexes powerfully bound via an intercalation mechanism with the calf thymus DNA. Gel-electrophoresis technique was used to investigate the DNA cleavage competence of the complexes and they establish to approve the cleavage of pBR322 DNA in presence of oxidant H2O2. This outcome inferred that the synthesized complexes showed better nuclease activity. Moreover, the complexes were monitored for antimicrobial activities. The results exposed that the synthesized compounds were forceful against all the microbes under exploration.

  17. Bloodstream form-specific up-regulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage.

    PubMed

    Sheader, Karen; te Vruchte, Daniëlle; Rudenko, Gloria

    2004-04-02

    The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage.

  18. Clickable Cγ-azido(methylene/butylene) peptide nucleic acids and their clicked fluorescent derivatives: synthesis, DNA hybridization properties, and cell penetration studies.

    PubMed

    Jain, Deepak R; Ganesh, Krishna N

    2014-07-18

    Synthesis, characterization, and DNA complementation studies of clickable C(γ)-substituted methylene (azm)/butylene (azb) azido PNAs show that these analogues enhance the stability of the derived PNA:DNA duplexes. The fluorescent PNA oligomers synthesized by their click reaction with propyne carboxyfluorescein are seen to accumulate around the nuclear membrane in 3T3 cells.

  19. Boron Clusters as a Platform for New Materials: Synthesis of Functionalized o-Carborane (C2 B10 H12 ) Derivatives Incorporating DNA Fragments.

    PubMed

    Janczak, Slawomir; Olejniczak, Agnieszka; Balabańska, Sandra; Chmielewski, Marcin K; Lupu, Marius; Viñas, Clara; Lesnikowski, Zbigniew J

    2015-10-19

    A synthetic strategy for functionalization of the three vertices of o-carborane and the attachment of the obtained triped to the solid support was developed. Further functionalization of the triped with short DNA sequences by automated DNA synthesis was achieved. The proposed methodology is a first example of boron cluster chemistry on a solid support opening new perspectives in boron cluster functionalization.

  20. Effects of 8-halo-7-deaza-2'-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation.

    PubMed

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2016-08-15

    8-OxodG (8-oxo-2'-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2'-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo(-)) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo(-)) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent.

  1. Phenomenology and the life sciences: Clarifications and complementarities.

    PubMed

    Sheets-Johnstone, Maxine

    2015-12-01

    This paper first clarifies phenomenology in ways essential to demonstrating its basic concern with Nature and its recognition of individual and cultural differences as well as commonalities. It furthermore clarifies phenomenological methodology in ways essential to understanding the methodology itself, its purpose, and its consequences. These clarifications show how phenomenology, by hewing to the dynamic realities of life itself and experiences of life itself, counters reductive thinking and "embodiments" of one kind and another. On the basis of these clarifications, the paper then turns to detailing conceptual complementarities between phenomenology and the life sciences, particularly highlighting studies in coordination dynamics. In doing so, it brings to light fundamental relationships such as those between mind and motion and between intrinsic dynamics and primal animation. It furthermore highlights the common concern with origins in both phenomenology and evolutionary biology: the history of how what is present is related to its inception in the past and to its transformations from past to present.

  2. Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12).

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

    Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity. Images PMID:6585787

  3. DNA synthesis in pulmonary alveolar macrophages and type II cells: effects of ozone exposure and treatment with alpha-difluoromethylornithine

    SciTech Connect

    Wright, E.S.; White, D.M.; Brady, A.N.; Li, L.C.; D'Arcy, J.B.; Smiler, K.L.

    1987-01-01

    An increase in the number of pulmonary alveolar macrophages (AM) can be induced by a number of toxic insults to the lung, including ozone, an important photochemical oxidant air pollutant. This increase could arise from an influx of monocytes from the vascular or interstitial compartments, or from proliferation of AM in situ. While proliferation of alveolar type II cells after oxidant exposure has been well documented, it is not clear whether AM are also capable of this response. Rats were exposed to air or to 0.12, 0.25, or 0.50 ppm ozone for 1, 2, 3, 7, or 14 d, 20 h/d. The labeling index in both AM and type II cells increased about 10-fold after 2 d of exposure to 0.25 and 0.50 ppm of ozone, but returned to control levels by the end of 1 wk of exposure. These changes closely paralleled the temporal and dose-response characteristics of changes in total lung DNA synthesis. alpha-Difluoromethylornithine (DFMO) administered to rats during a 2-d exposure to 0.50 ppm ozone did not inhibit the ozone-induced increase in labeling index in AM or type II cells, although evidence of inhibition of lung ornithine decarboxylase activity was obtained, and the ozone-induced increase in total lung DNA synthesis was inhibited by 23%. These results suggest that, like type II cells, AM are capable of entering the cell cycle and synthesizing new DNA in situ in response to short-term exposure to environmentally relevant doses of ozone, and that the ozone-induced stimulation of DNA synthesis in these cell types was refractory to inhibition by DFMO.

  4. Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres.

    PubMed

    Renier, A; Lévy, F; Pillière, F; Jaurand, M C

    1990-08-01

    Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

  5. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed Central

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein. Images PMID:6997508

  6. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  7. Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

    PubMed Central

    Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

    2013-01-01

    We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays. PMID:23673353

  8. Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

    PubMed Central

    Shammas, M. A.; Xia, S. J.; Reis, RJS.

    1997-01-01

    Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis. PMID:9258684

  9. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

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

  10. Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants

    PubMed Central

    Cairns, Tina M.; Craven, Rebecca C.

    2001-01-01

    The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle. PMID:11119594

  11. In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium

    SciTech Connect

    Harkema, J.R.; Hotchkiss, J.A. )

    1993-12-01

    Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. The authors did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls. In contrast, non-neutrophil-depleted rats instilled with endotoxin had a marked neutrophilic influx, but no significant differences in the number of nasal epithelial cells at 6 or 24 h, compared to controls. In addition, the BrdU-labeling index in neutrophil-sufficient rats was increased only 6 h after the last instillation, compared to controls.

  12. Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons.

    PubMed

    Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

    2014-05-01

    Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.

  13. Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells

    PubMed Central

    Borland, Kayla M.; AbdulSalam, Safnas F.; Solivio, Morwena J.; Burke, Matthew P.; Wolfkiel, Patrick R.; Lawson, Sean M.; Stockman, Courtney A.; Andersen, Joel M.; Smith, Skyler; Tolstolutskaya, Julia N.; Gurjar, Purujit N.; Bercz, Aron P.; Merino, Edward J.; Litosh, Vladislav A.

    2015-01-01

    Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5′-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork. PMID:25778768

  14. Excision of ultraviolet damage and the effect of irradiation on DNA synthesis in a strain of Bloom's syndrome fibroblasts

    SciTech Connect

    Henson, P.; Selsky, C.A.; Little, J.B.

    1981-03-01

    Researchers have studied repair of ultraviolet light-induced damage in a strain of Bloom's syndrome cells which we have shown to be defective in host cell reactivation of uv-irradiated herpes simplex virus. Excision repair was monitored by following loss of sensitivity of DNA in permeabilized cells to digestion by the Micrococcus luteus uv endonuclease preparation. The Bloom's syndrome fibroblasts apparently removed endonuclease-sensitive sites from the DNA slightly less efficiently than did normal strains. After 24 h, 38% of the sites remained in the Bloom's syndrome cells in comparison with 16% in normal fibroblasts. DNA newly synthesized in uv-irradiated Bloom's syndrome cells sedimented less far into alkaline sucrose gradients than did DNA from similarly treated normal cells. In other respects, including the effect of caffeine exposure, DNA synthesis in Bloom's syndrome cells was indistinguishable from that in normal cells. We were therefore able to detect only minor defects in the repair of uv-induced damage in Bloom's syndrome fibroblasts. This is consistent with the normal survival exhibited by these cells. The defect in excision repair may, however, be sufficient to allow the cellular repair capacity to become saturated at high infecting multiplicities of uv-irradiated herpes simplex virus.

  15. Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

    PubMed

    Fujino, Tomoko; Yasumoto, Ken-ichi; Yamazaki, Naomi; Hasome, Ai; Sogawa, Kazuhiro; Isobe, Hiroyuki

    2011-11-04

    A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins.

  16. Different manner of DNA synthesis in polyploidizations of meth-A and B16F10 cell lines.

    PubMed

    Fujikawa-Yamamoto, K; Zong, Z; Murakami, M; Odashima, S

    1997-10-01

    Polyploidization of Meth-A and B16-F10 cells by demecolcine was examined using flow cytometry (FCM). In the presence of demecolcine, both cell lines were polyploidized to more than 16c DNA content. A marked difference was observed in the durations of S phase of polyploidy. The S-phase duration of Meth-A cells was doubly increased with ploidy, but that of B16F10 cells remained constant. When the rate of DNA synthesis in the polyploidizing cells was examined through the BrdU-uptake experiments, it was confirmed that the level of DNA-synthesis rate was constant in Meth-A cells but increased in B16F10 cells. The cellular content of c-Myc protein in polyploidized cells was also examined using anti-c-Myc monoclonal antibody. The c-Myc level of Meth-A cells was constant regardless of the ploidy but that of B16F10 cells increased with ploidy. Thus, the c-Myc content seems to be related to the duration of S phase in polyploidy.

  17. Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: Testing of 24 compounds

    SciTech Connect

    Mirsalis, J.C.; Tyson, C.K.; Steinmetz, K.L.; Loh, E.K.; Hamilton, C.M.; Bakke, J.P. ); Spalding, J.W. )

    1989-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful for studying genotoxic hepatocarcinogens. In addition, measurement of S-phase synthesis (SPS) provides an indirect indicator of hepatocellular proliferation, which may be an important mechanism in rodent carcinogenesis. This assay was used to examine 24 chemicals for their ability to induce unscheduled DNA synthesis (UDS) or SPS in Fischer-344 rats or B6C3F1 mice following in vivo treatment. Hepatocytes were isolated by liver perfusion and incubated with {sup 3}H-thymidine following in vivo treatment by gavage. Chemicals chosen for testing were from the National Toxicology Program (NTP) genetic toxicology testing program and most were also evaluated in long-term animal studies conducted by the NTP. Dinitrotoluene and Michler's Ketone induced positive UDS response in rat, while N-nitrosodiethanolamine and selenium sulfide induced equivocal UDS results in mouse and rat, respectively. BCMEE, bromoform, chloroform, PBB, 1,1,2-trichloroethane, and trichloroethylene were all potent inducers of SPS in mouse liver, while C.I. Solvent Yellow 14, and 1,1,2,2-tetrachloroethane yielded equivocal SPS results in rat and mouse, respectively. These results indicate that most of the test compounds do not induced UDS in the liver; however, the significant S-phase response induced by many of these compounds, especially the halogenated solvents, may be an important mechanism in their hepatocarinogenicity.

  18. DNA-directed alkylating agents. 6. Synthesis and antitumor activity of DNA minor groove-targeted aniline mustard analogues of pibenzimol (Hoechst 33258)

    PubMed

    Gravatt, G L; Baguley, B C; Wilson, W R; Denny, W A

    1994-12-09

    A series of nitrogen mustard analogues of the DNA minor groove binding fluorophore pibenzimol (Hoechst 33258) have been synthesized and evaluated for antitumor activity. Conventional construction of the bisbenzimidazole ring system from the piperazinyl terminus, via two consecutive Pinner-type reactions, gave low yields of products contaminated with the 2-methyl analogue which proved difficult to separate. An alternative synthesis was developed, involving construction of the bisbenzimidazole from the mustard terminus, via Cu(2+)-promoted oxidative coupling of the mustard aldehydes with 3,4-diaminobenzonitrile to form the monobenzimidazoles, followed by a Pinner-type reaction and condensation with 4-(1-methyl-4-piperazinyl)-o-phenylenediamine. This process gives higher yields and pure products. The mustard analogues showed high hypersensitivity factors (IC50AA8/IC50 UV4), typical of DNA alkylating agents. There was a large increase in cytotoxicity (85-fold) across the homologous series which cannot be explained entirely by changes in mustard reactivity and may be related to altering orientation of the mustard with respect to the DNA resulting in different patterns of alkylation. Pibenzimol itself (which has been evaluated clinically as an anticancer drug) was inactive against P388 in vivo using a single-dose protocol, but the short-chain mustard homologues were highly effective, eliciting a proportion of long-term survivors.

  19. In Vitro Synthesis of Rous Sarcoma Virus-Specific RNA is Catalyzed by a DNA-Dependent RNA Polymerase

    PubMed Central

    Rymo, L.; Parsons, J. T.; Coffin, J. M.; Weissmann, C.

    1974-01-01

    Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [α-32P]triphosphates, Mn++, Mg++ and (NH4)2SO4. Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37°. About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. α-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II. PMID:4368801

  20. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

    PubMed Central

    Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

    2016-01-01

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

  1. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

    PubMed

    Fuller, Carl W; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J; Kasianowicz, John J; Davis, Randy; Roever, Stefan; Church, George M; Ju, Jingyue

    2016-05-10

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

  2. Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

    PubMed Central

    2011-01-01

    Background In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis. PMID:21324208

  3. Replication initiation and genome instability: a crossroads for DNA and RNA synthesis.

    PubMed

    Barlow, Jacqueline H; Nussenzweig, André

    2014-12-01

    Nuclear DNA replication requires the concerted action of hundreds of proteins to efficiently unwind and duplicate the entire genome while also retaining epigenetic regulatory information. Initiation of DNA replication is tightly regulated, rapidly firing thousands of origins once the conditions to promote rapid and faithful replication are in place, and defects in replication initiation lead to proliferation defects, genome instability, and a range of developmental abnormalities. Interestingly, DNA replication in metazoans initiates in actively transcribed DNA, meaning that replication initiation occurs in DNA that is co-occupied with tens of thousands of poised and active RNA polymerase complexes. Active transcription can induce genome instability, particularly during DNA replication, as RNA polymerases can induce torsional stress, formation of secondary structures, and act as a physical barrier to other enzymes involved in DNA metabolism. Here we discuss the challenges facing mammalian DNA replication, their impact on genome instability, and the development of cancer.

  4. Recent advances in small organic molecules as DNA intercalating agents: synthesis, activity, and modeling.

    PubMed

    Rescifina, Antonio; Zagni, Chiara; Varrica, Maria Giulia; Pistarà, Venerando; Corsaro, Antonino

    2014-03-03

    The interaction of small molecules with DNA plays an essential role in many biological processes. As DNA is often the target for majority of anticancer and antibiotic drugs, study about the interaction of drug and DNA has a key role in pharmacology. Moreover, understanding the interactions of small molecules with DNA is of prime significance in the rational design of more powerful and selective anticancer agents. Two of the most important and promising targets in cancer chemotherapy include DNA alkylating agents and DNA intercalators. For these last the DNA recognition is a critical step in their anti-tumor action and the intercalation is not only one kind of the interactions in DNA recognition but also a pivotal step of several clinically used anti-tumor drugs such as anthracyclines, acridines and anthraquinones. To push clinical cancer therapy, the discovery of new DNA intercalators has been considered a practical approach and a number of intercalators have been recently reported. The intercalative binding properties of such molecules can also be harnessed as diagnostic probes for DNA structure in addition to DNA-directed therapeutics. Moreover, the problem of intercalation site formation in the undistorted B-DNA of different length and sequence is matter of tremendous importance in molecular modeling studies and, nowadays, three models of DNA intercalation targets have been proposed that account for the binding features of intercalators. Finally, despite DNA being an important target for several drugs, most of the docking programs are validated only for proteins and their ligands. Therefore, a default protocol to identify DNA binding modes which uses a modified canonical DNA as receptor is needed.

  5. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents Against Lung Cancer

    DTIC Science & Technology

    2014-10-01

    effectively and accurately replicate crosslinked DNA lesions such as thymine dimers and cisplatinated DNA (19-21). A third group of DNA polymerases...lines correlates with resistance to chemotherapeutic agents such as cisplatin which damage DNA (38). Furthermore, pol  overexpression is a poor... cisplatinated GG adduct (33). In this model, dCTP is properly paired with the first templating nucleobase via conventional hydrogen bonds. In general, our

  6. Hypersensitivity and reduced inhibition of DNA synthesis in ataxia telangiectasia lymphoblasts treated with low levels of neocarzinostatin.

    PubMed

    Babilon, R W; Soprano, K J; Henderson, E E

    1985-07-01

    The effects of neocarzinostatin (NCS) on lymphoblastoid cell lines (LCLs) established from ataxia telangiectasia (A-T) were determined. A-T lymphoblasts were found to be hypersensitive to low levels of NCS as measured by cell growth and cell survival. On the other hand, A-T lymphoblasts failed to postpone DNA synthesis to the same degree as normal lymphoblasts following treatment with NCS. LCLs established from Nijmegen breakage syndrome (NBS) could be distinguished from ataxia and normal cell lines by their intermediate level of survival following exposure to NCS.

  7. Semi-automatic synthesis, antiproliferative activity and DNA-binding properties of new netropsin and bis-netropsin analogues.

    PubMed

    Szerszenowicz, Jakub; Drozdowska, Danuta

    2014-07-31

    A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the all new substances prepared in this investigation were fully characterized by NMR ((1)H, (13)C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322 confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.

  8. Induction of maturation of human B-cell lymphomas in vitro. Morphologic changes in relation to immunoglobulin and DNA synthesis.

    PubMed Central

    Beiske, K.; Ruud, E.; Drack, A.; Marton, P. F.; Godal, T.

    1984-01-01

    In vitro stimulation of cells from 8 non-Hodgkin's lymphomas comprising several histologic types with a tumor promotor (TPA) and with or without anti-immunoglobulins directed against the surface immunoglobulin of the tumor cells is reported. Morphologic transformation to immunoblastic and plasmablastic cells, but not to plasma cells, and induction of Ig and DNA synthesis were observed. A comparative analysis, including flow cytofluorometry, light microscopy combined with immunocytochemistry, and electron microscopy, suggests that the three events may not always be associated phenomena at the single-cell level even in monoclonal cell populations. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:6375389

  9. Synthesis, DNA binding, photo-induced DNA cleavage, cytotoxicity studies of a family of heavy rare earth complexes.

    PubMed

    Chen, Gong-Jun; Wang, Zhi-Gang; Qiao, Xin; Xu, Jing-Yuan; Tian, Jin-Lei; Yan, Shi-Ping

    2013-10-01

    As a continuing investigation of our previous studies about the influence of the different rare earth metal ions on the bioactivity, a family of heavy rare earth metal complexes, [RE(acac)3(dpq)] (RE=Tb (1), Dy (2), Ho (3), Er (4), Tm (5), Yb (6), Lu (7)) and [RE(acac)3(dppz)]·CH3OH (RE=Tb (8), Dy (9), Ho (10), Er (11), Tm (12), Yb (13), Lu (14) viz. acetylacetonate (acac), dipyrido[3,2-d:20,30-f]quinoxaline (dpq), dipyrido[3,2-a:20,30-c] phenazine (dppz)), has been synthesized and their biological activities were also investigated. On the irradiation with UV-A light of 365nm or ambient light, all complexes exhibit efficient DNA cleavage activity via the mechanistic pathway involving the formation of singlet oxygen and hydroxyl radical as the reactive species. In addition, the in vitro cytotoxicity of these complexes on HeLa cells has been examined by MTT assay, which indicate that these compounds have the potential to act as effective anticancer drugs. The results of the above biological experiments also reveal that the choice of different rare earth metal ions has little influence on the DNA binding, DNA cleavage and cytotoxicity.

  10. Photoinduced interactions of supramolecular ruthenium(II) complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage studies.

    PubMed

    Swavey, Shawn; DeBeer, Madeleine; Li, Kaiyu

    2015-04-06

    Two new bridging ligands have been synthesized by combining substituted benzaldehydes with phenanthrolinopyrrole (php), resulting in new polyazine bridging ligands. The ligands have been characterized by (1)H NMR, mass spectroscopy, and elemental analysis. These new ligands display π-π* transitions above 500 nm with modest molar absorptivities. Upon excitation at the ligand-centered charge-transfer transition, weak emission with a maximum wavelength of 612 nm is observed. When coordinated to two ruthenium(II) bis(bipyridyl) groups, the new bimetallic complexes generated give an overall 4+ charge. The electronic transitions of the bimetallic ruthenium(II) complexes display traditional π-π* transitions at 287 nm and metal-to-ligand charge-transfer transitions at 452 nm with molar absorptivities greater than 30000 M(-1) cm(-1). Oxidation of the ruthenium(II) metal centers to ruthenium(III) occurs at potentials above 1.4 V versus the Ag/AgCl reference electrode. Spectroscopic and electrochemical measurements indicate that the ruthenium(II) moieties behave independently. Both complexes are water-soluble and show the ability to photonick plasmid DNA when irradiated with low-energy light above 550 nm. In addition, one of the complexes, [Ru(bpy)2php]2Van(4+), shows the ability to linearize plasmid DNA and gives evidence, by gel electrophoresis, of photoinduced binding to plasmid DNA.

  11. Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms.

    PubMed

    Evans, Geraint W; Hohlbein, Johannes; Craggs, Timothy; Aigrain, Louise; Kapanidis, Achillefs N

    2015-07-13

    DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

  12. Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

    PubMed

    Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

    2016-03-15

    Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles.

  13. 76 FR 54497 - Compliance Testing Program Administrative Clarification to National Institute of Justice Standard...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-01

    ... Justice Standard-0101.06, Ballistic Resistance of Body Armor AGENCY: National Institute of Justice... administrative clarification of NIJ Standard 0101.06, ``Ballistic Resistance Body Armor'' (hereinafter,...

  14. SYNTHESIS OF THE FULLY PROTECTED PHOSPHORAMIDITE OF THE BENZENE-DNA ADDUCT, N2- (4-HYDROXYPHENYL)-2'-DEOXYGUANOSINE AND INCORPORATION OF THE LATER INTO DNA OLIGOMERS

    SciTech Connect

    Chenna, Ahmed; Gupta, Ramesh C.; Bonala, Radha R.; Johnson, Francis; Huang, Bo

    2008-06-09

    N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

  15. Synthesis of amino-rich silica-coated magnetic nanoparticles for the efficient capture of DNA for PCR.

    PubMed

    Bai, Yalong; Cui, Yan; Paoli, George C; Shi, Chunlei; Wang, Dapeng; Zhou, Min; Zhang, Lida; Shi, Xianming

    2016-09-01

    Magnetic separation has great advantages over traditional bio-separation methods and has become popular in the development of methods for the detection of bacterial pathogens, viruses, and transgenic crops. Functionalization of magnetic nanoparticles is a key factor for efficient capture of the target analytes. In this paper, we report the synthesis of amino-rich silica-coated magnetic nanoparticles using a one-pot method. This type of magnetic nanoparticle has a rough surface and a higher density of amino groups than the nanoparticles prepared by a post-modification method. Furthermore, the results of hydrochloric acid treatment indicated that the magnetic nanoparticles were stably coated. The developed amino-rich silica-coated magnetic nanoparticles were used to directly adsorb DNA. After magnetic separation and blocking, the magnetic nanoparticles and DNA complexes were used directly for the polymerase chain reaction (PCR), without onerous and time-consuming purification and elution steps. The results of real-time quantitative PCR showed that the nanoparticles with higher amino group density resulted in improved DNA capture efficiency. The results suggest that amino-rich silica-coated magnetic nanoparticles are of great potential for efficient bio-separation of DNA prior to detection by PCR.

  16. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

    PubMed

    Kupperman, E; Wen, W; Meinkoth, J L

    1993-08-01

    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

  17. Induction of pyrimidine dimers and unscheduled DNA synthesis in cultured mouse epithelial cells exposed to 254-nm- and u. v. -B radiation

    SciTech Connect

    Yotti, L.P.; Ley, R.D.

    1983-01-01

    The induction and fate of pyrimidine dimers and unscheduled DNA synthesis were measured in u.v.-irradiated primary, newborn SENCAR mouse epithelial cells. Unscheduled DNA synthesis was induced in a dose responsive manner by two u.v. sources, a germicidal lamp (254 nm) and an FS40 sunlamp (280--400 nm). Using the endonuclease-sensitive site assay to detect pyrimidine dimer production and excision, we examined the response of the newborn mouse cells to both u.v. sources. We were unable to detect the removal of pyrimidine dimers with either of the two sources of u.v. The speculation is made that primary, newborn mouse epidermal cells excise u.v.-induced pyrimidine dimers to an extent below the level of detection of the endonuclease-sensitive site assay but to an extent sufficient to induce unscheduled DNA synthesis.

  18. Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

    PubMed Central

    Kupperman, E; Wen, W; Meinkoth, J L

    1993-01-01

    Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase. Images PMID:8336696

  19. Influence of Nrf2 activators on subcellular skeletal muscle protein and DNA synthesis rates after 6 weeks of milk protein feeding in older adults.

    PubMed

    Konopka, Adam R; Laurin, Jaime L; Musci, Robert V; Wolff, Christopher A; Reid, Justin J; Biela, Laurie M; Zhang, Qian; Peelor, Fredrick F; Melby, Christopher L; Hamilton, Karyn L; Miller, Benjamin F

    2017-03-10

    In older adults, chronic oxidative and inflammatory stresses are associated with an impaired increase in skeletal muscle protein synthesis after acute anabolic stimuli. Conjugated linoleic acid (CLA) and Protandim have been shown to activate nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor for the antioxidant response element and anti-inflammatory pathways. This study tested the hypothesis that compared to a placebo control (CON), CLA and Protandim would increase skeletal muscle subcellular protein (myofibrillar, mitochondrial, cytoplasmic) and DNA synthesis in older adults after 6 weeks of milk protein feeding. CLA decreased oxidative stress and skeletal muscle oxidative damage with a trend to increase messenger RNA (mRNA) expression of a Nrf2 target, NAD(P)H dehydrogenase quinone 1 (NQO1). However, CLA did not influence other Nrf2 targets (heme oxygenase-1 (HO-1), glutathione peroxidase 1 (Gpx1)) or protein or DNA synthesis. Conversely, Protandim increased HO-1 protein content but not the mRNA expression of downstream Nrf2 targets, oxidative stress, or skeletal muscle oxidative damage. Rates of myofibrillar protein synthesis were maintained despite lower mitochondrial and cytoplasmic protein syntheses after Protandim versus CON. Similarly, DNA synthesis was non-significantly lower after Protandim compared to CON. After Protandim, the ratio of protein to DNA synthesis tended to be greater in the myofibrillar fraction and maintained in the mitochondrial and cytoplasmic fractions, emphasizing the importance of measuring both protein and DNA synthesis to gain insight into proteostasis. Overall, these data suggest that Protandim may enhance proteostatic mechanisms of skeletal muscle contractile proteins after 6 weeks of milk protein feeding in older adults.

  20. DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine.

    PubMed

    Raymond, M N; Foucault, G; Coffe, G; Pudles, J

    1986-04-01

    This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.

  1. Mitochondrial transcription terminator family members mTTF and mTerf5 have opposing roles in coordination of mtDNA synthesis.

    PubMed

    Jõers, Priit; Lewis, Samantha C; Fukuoh, Atsushi; Parhiala, Mikael; Ellilä, Simo; Holt, Ian J; Jacobs, Howard T

    2013-01-01

    All genomes require a system for avoidance or handling of collisions between the machineries of DNA replication and transcription. We have investigated the roles in this process of the mTERF (mitochondrial transcription termination factor) family members mTTF and mTerf5 in Drosophila melanogaster. The two mTTF binding sites in Drosophila mtDNA, which also bind mTerf5, were found to coincide with major sites of replication pausing. RNAi-mediated knockdown of either factor resulted in mtDNA depletion and developmental arrest. mTTF knockdown decreased site-specific replication pausing, but led to an increase in replication stalling and fork regression in broad zones around each mTTF binding site. Lagging-strand DNA synthesis was impaired, with extended RNA/DNA hybrid segments seen in replication intermediates. This was accompanied by the accumulation of recombination intermediates and nicked/broken mtDNA species. Conversely, mTerf5 knockdown led to enhanced replication pausing at mTTF binding sites, a decrease in fragile replication intermediates containing single-stranded segments, and the disappearance of species containing segments of RNA/DNA hybrid. These findings indicate an essential and previously undescribed role for proteins of the mTERF family in the integration of transcription and DNA replication, preventing unregulated collisions and facilitating productive interactions between the two machineries that are inferred to be essential for completion of lagging-strand DNA synthesis.

  2. Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

    PubMed Central

    Pearce, Kaycey; Cai, Diancai; Roberts, Adam C; Glanzman, David L

    2017-01-01

    Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.18299.001 PMID:28067617

  3. Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells.

    PubMed

    Wagshal, A B; Jegasothy, B V; Waksman, B H

    1978-01-01

    IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.

  4. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

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

  5. Mechanism of error-free DNA synthesis across N1-methyl-deoxyadenosine by human DNA polymerase-ι

    PubMed Central

    Jain, Rinku; Choudhury, Jayati Roy; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2017-01-01

    N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a “foothold” and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity. PMID:28272441

  6. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    PubMed Central

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  7. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

    PubMed

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; da Silva, Lúcia Patrícia Bezerra Gomes; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Ruiz, Ana Lucia Tasca Gois; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-06-09

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) M(-1) indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N- (4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

  8. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

    PubMed

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

    2015-11-17

    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

  9. Process cost and facility considerations in the selection of primary cell culture clarification technology.

    PubMed

    Felo, Michael; Christensen, Brandon; Higgins, John

    2013-01-01

    The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.

  10. Dissociation of tumor promoter-stimulated ornithine decarboxylase activity and DNA synthesis in mouse epidermis in vivo and in vitro by fluocinolone acetonide, a tumor-promotion inhibitor.

    PubMed Central

    Lichti, U; Slaga, T J; Ben, T; Patterson, E; Hennings, H; Yuspa, S H

    1977-01-01

    12-O-Tetradecanoyl phorbol-13-acetate (TPA), a tumor promoter, stimulates DNA synthesis in mouse epidermal cells in vivo and in vitro. This response appears to be mediated through polyamine metabolism because ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17)activity is markedly increased shortly after promoter exposure and this induction varies in magnitude according to dose and promoter potency of a series of phorbol esters. In vitro, exogenous putrescine (0.01-10 mM) results in a dose-related increase and prolongation of promoter-stimulated DNA DNA synthesis, a phenomenon noted in other systems of polyamine-mediated growth stimulation. The anti-inflammatory steroid fluocinolone acetonide (FA), an inhibitor of tumor promotion, prevents TPA stimulation of epidermal proliferation in vivo and in vitro. In vitro, FA most effectively prevents stimulation of DNA synthesis when applied is not required. Paradoxially, FA potentiates the increase in ornithine decarboxylase activity after TPA administeration both in vivo and in vitro. Furthermore, the inhibition of TPA-stimulated DNA synthesis by FA in vitro can be reversed by exogenous putrescine. These results suggestthat FA exerts its antipromotion effect by reducing the sensitivity of the cell to polyamines or by reducing intracellular polyamine levels. PMID:269443

  11. A large fragment approach to DNA synthesis: total synthesis of a gene for the protease inhibitor eglin c from the leech Hirudo medicinalis and its expression in E. coli.

    PubMed Central

    Rink, H; Liersch, M; Sieber, P; Meyer, F

    1984-01-01

    A DNA containing the coding sequence for the proteinase inhibitor protein, eglin c, from the leech Hirudo medicinalis has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 232 base-pair fragment containing initiation and termination codon signals with restriction enzyme recognition sites conveniently placed for cloning into a plasmid vector. Only six oligonucleotides from 34 to 61 bases in length, sharing pairwise stretches of complementary regions at their 3'-termini, were prepared by phosphotriester solid-phase synthesis. The oligomers were annealed pairwise and converted into double stranded DNA fragments by DNA polymerase I mediated repair synthesis. The fragments were assembled by ligation, and the synthetic gene was expressed in high yield in E. coli under the transcriptional control of the E. coli tryptophan promoter. The expression product was purified to homogeneity and was shown to have similar physicochemical and identical biological properties as the authentic protein isolated from the leech. Images PMID:6382168

  12. DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

    PubMed

    Smaill, J B; Fan, J Y; Denny, W A

    1998-12-01

    A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with

  13. Clarification process: Resolution of decision-problem conditions

    NASA Technical Reports Server (NTRS)

    Dieterly, D. L.

    1980-01-01

    A model of a general process which occurs in both decisionmaking and problem-solving tasks is presented. It is called the clarification model and is highly dependent on information flow. The model addresses the possible constraints of individual indifferences and experience in achieving success in resolving decision-problem conditions. As indicated, the application of the clarification process model is only necessary for certain classes of the basic decision-problem condition. With less complex decision problem conditions, certain phases of the model may be omitted. The model may be applied across a wide range of decision problem conditions. The model consists of two major components: (1) the five-phase prescriptive sequence (based on previous approaches to both concepts) and (2) the information manipulation function (which draws upon current ideas in the areas of information processing, computer programming, memory, and thinking). The two components are linked together to provide a structure that assists in understanding the process of resolving problems and making decisions.

  14. A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification.

    PubMed

    Sojitra, Uttam V; Nadar, Shamraja S; Rathod, Virendra K

    2016-12-15

    The major complications in fruit juice quality improvement are the presence of polysaccharides components in the form of disrupted fruit cell wall and cell materials. Hence, breakdown of cellulose along with pectin and starch is important for the juice processing. In this context, magnetic tri-enzyme nanobiocatalyst was prepared by simultaneously co-immobilizing three enzymes; α-amylase, pectinase and cellulase onto amino-functionalized magnetic nanoparticle by 60mM glutaraldehyde concentration with 10h cross-linking time for one pot juice clarification. The prepared nanobiocatalyst was characterized by FT-IR, SEM and XRD. The thermal (50-70°C) and pH (3-6) stability studies indicated more than two folds increment in half-life and enhanced tolerance to lower pH. The immobilized enzymes retained up to 75% of residual activity even after eight consecutive cycles of reuse. Finally, the clarification of apple, grapes and pineapple juices using magnetic tri-enzyme showed 41%, 46% and 53% respective reduction in turbidity till 150min treatment.

  15. Values clarification as a technique for family planning education.

    PubMed

    Toohey, J V; Valenzuela, G J

    1983-02-01

    A Spanish language family planning education program utilizing the dynamics of values clarification has been designed and implemented in the state of Oaxaca, Mexico. The design of the program features three basic personality identification activities to help individuals identify other dimensions of adult life expression than child rearing. In addition, a series of simple and precise scenarios specifically related to family planning are presented. Each scenario is accompanied by a set of valuing questions that direct the learner to respond to the scenario. The activity booklet is entitled, "Clarification De Valores En La Planificacion Familar." The booklet requires the learner to make responses to the learning materials. Responses are then used as a basis for inferring that people are comprehending and above all personalizing knowledge about themselves and their culture and family planning. The program is cross cultural and can be used in Spanish speaking communities in the U.S. Its English language form can be used with English speaking target populations. Statistical analysis of seven critical categories of the program indicated that the shifts in attitudes from pre-to post-values, whether positive or negative (desirable or undesirable), were not significant at the .05 level of confidence. It should, however, be noted that small shifts in the rate of natural increase, or rate of natural decrease for population growth can have a dramatic effect on population growth when multiplied by time.

  16. A High Performance Platform Based on cDNA Display for Efficient Synthesis of Protein Fusions and Accelerated Directed Evolution.

    PubMed

    Naimuddin, Mohammed; Kubo, Tai

    2016-02-08

    We describe a high performance platform based on cDNA display technology by developing a new modified puromycin linker-oligonucleotide. The linker consists of four major characteristics: a "ligation site" for hybridization and ligation of mRNA by T4 RNA ligase, a "puromycin arm" for covalent linkage of the protein, a "polyadenosine site" for a longer puromycin arm and purification of protein fusions (optional) using oligo-dT matrices, and a "reverse transcription site" for the formation of stable cDNA protein fusions whose cDNA is covalently linked to its encoded protein. The linker was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼ 50-95%) in various cell-free expression systems. Overall, this new cDNA display method provides 10-200 fold higher end-usage fusions than previous methods and benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30-40-fold enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial impact to accelerated drug discovery and proteome analysis.

  17. Effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with NBN mutations

    PubMed Central

    Nowak, Jerzy; Świątek-Kościelna, Bogna; Kałużna, Ewelina M.; Rembowska, Jolanta; Dzikiewicz-Krawczyk, Agnieszka; Zawada, Mariola

    2017-01-01

    Introduction The NBN gene product is part of the MRE11/RAD50/NBN complex, which plays an essential role in genomic stability. In the study we try to answer the question what is the effect of irradiation on DNA synthesis, NBN gene expression and chromosomal stability in cells with homozygous c.657-661del, and heterozygous c.657-661del, p.I171V and p.R215W NBN gene mutations. Material and methods Immortalized B-lymphocytes with NBN gene mutations were X-ray irradiated at doses of 1, 2, 5 and 8 Gy/min. Radioresistant DNA synthesis rate and the percentage of cells in phase S was analyzed by 3H thymidine and BrdU incorporation assays. NBN gene expression was quantified by real-time PCR with TaqMan fluorescent probe. Results Increasing the irradiation dose resulted in gradual decrease of 3H thymidine incorporation in all cells, but significantly only in homo- and heterozygous c.657-661del cells (p-values < 0.0001). After irradiation the relative expression of NBN was significantly higher in homozygous c.657-661del and heterozygous p.R215W cells as compared to heterozygous c.657-661del, p.I171V and control cells (p < 0.01). All cells with NBN gene mutations showed significantly higher total number of chromosomal aberrations per metaphase as compared to control cells, with the highest number of aberrations in homozygous c.657-661del cells (p < 0.001). Conclusions The results obtained indicate that homozygous c.657-661del mutation affects cell sensitivity to irradiation. Moreover, homozygous variant is associated with disturbance in the activation of cell cycle checkpoints and with defects in DNA repair. In turn, heterozygous c.657-661del, p.R215W and p.I171V mutations do not substantially alter the radiosensitivity. PMID:28261280

  18. Next Generation Gene Synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device

    PubMed Central

    Matzas, Mark; Stähler, Peer F.; Kefer, Nathalie; Siebelt, Nicole; Boisguérin, Valesca; Leonard, Jack T.; Keller, Andreas; Stähler, Cord F.; Häberle, Pamela; Gharizadeh, Baback; Babrzadeh, Farbod; Church, George

    2012-01-01

    The setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications. PMID:21113166

  19. Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies.

    PubMed

    Demirezen, Nihat; Tarınç, Derya; Polat, Duygu; Ceşme, Mustafa; Gölcü, Ayşegül; Tümer, Mehmet

    2012-08-01

    Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and (1)H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria. Antifungal activity against two different fungi has been evaluated and compared with the reference drug TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism.

  20. Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies

    NASA Astrophysics Data System (ADS)

    Demirezen, Nihat; Tarınç, Derya; Polat, Duygu; Çeşme, Mustafa; Gölcü, Ayşegül; Tümer, Mehmet

    Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and 1H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria. Antifungal activity against two different fungi has been evaluated and compared with the reference drug TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism.

  1. Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

    PubMed Central

    Stranges, P. Benjamin; Palla, Mirkó; Kalachikov, Sergey; Nivala, Jeff; Dorwart, Michael; Trans, Andrew; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Tao, Chuanjuan; Morozova, Irina; Li, Zengmin; Shi, Shundi; Aberra, Aman; Arnold, Cleoma; Yang, Alexander; Aguirre, Anne; Harada, Eric T.; Korenblum, Daniel; Pollard, James; Bhat, Ashwini; Gremyachinskiy, Dmitriy; Bibillo, Arek; Chen, Roger; Davis, Randy; Russo, James J.; Fuller, Carl W.; Roever, Stefan; Ju, Jingyue; Church, George M.

    2016-01-01

    Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform. PMID:27729524

  2. Implementation and Evaluation of a Values Clarification Activity for a Large Undergraduate Human Sexuality Course

    ERIC Educational Resources Information Center

    Lederer, Alyssa M.

    2016-01-01

    Values clarification is an important tool that helps individuals to clarify their beliefs about sexuality-related issues. This lesson plan provides instructions for a 1-hour values clarification activity for a large undergraduate human sexuality course that serves as an introduction to course content and tone, stimulates students' initial thinking…

  3. Strategies of Clarification in Judges' Use of Language: From the Written to the Spoken.

    ERIC Educational Resources Information Center

    Philips, Susan U.

    1985-01-01

    Reports on a study of judges' strategies in clarifying their verbal explanations of constitutional rights to criminal defendants. Identifies six clarification processes and compares them with other studies of clarification processes and with the properties of simplified registers, particularly speech addressed to first- and second-language…

  4. 23 CFR 636.507 - What subjects are prohibited in discussions, communications and clarifications with offerors?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ..., communications and clarifications with offerors? 636.507 Section 636.507 Highways FEDERAL HIGHWAY ADMINISTRATION..., communications and clarifications with offerors? You may not engage in conduct that: (a) Favors one offeror over another; (b) Reveals an offeror's technical solution, including unique technology, innovative and...

  5. 76 FR 18322 - Federal Acquisition Regulation; Clarification of Standard Form 26-Award/Contract

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-01

    ... Federal Acquisition Regulation; Clarification of Standard Form 26--Award/Contract AGENCIES: Department of... Acquisition Regulation (FAR) to include clarifications to Standard Form (SF) 26, Award/Contract. This revised... Standard Form (SF) 26, Award/Contract. FAR 53.214(a) prescribes the SF 26 for use in contracting...

  6. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  7. Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

    PubMed

    Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

    2016-10-01

    A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

  8. Gas-phase synthesis of solid state DNA nanoparticles stabilized by l-leucine.

    PubMed

    Raula, Janne; Hanzlíková, Martina; Rahikkala, Antti; Hautala, Juho; Kauppinen, Esko I; Urtti, Arto; Yliperttula, Marjo

    2013-02-28

    Aerosol flow reactor is used to generate solid-state nanoparticles in a one-step process that is based on drying of aerosol droplets in continuous flow. We investigated the applicability of aerosol flow reactor method to prepare solid state DNA nanoparticles. Precursor solutions of plasmid DNA with or without complexing agent (polyethylenimine), coating material (l-leucine) and mannitol (bulking material) were dispersed to nanosized droplets and instantly dried in laminar heat flow. Particle morphology, integrity and stability were studied by scanning electron microscopy. The stability of DNA was studied by gel electrophoresis. Plasmid DNA as such degraded in the aerosol flow process. Complexing agent protected DNA from degradation and coating material enabled production of dispersed, non-aggregated, nanoparticles. The resulting nanoparticles were spherical and their mean diameter ranged from 65 to 125nm. The nanoparticles were structurally stable at room temperature and their DNA content was about 10%. We present herein the proof of principle for the production of dispersed solid state nanoparticles with relevant size and intact plasmid DNA.

  9. DNA-targeting pyrroloquinoline-linked butenone and chalcones: synthesis and biological evaluation.

    PubMed

    Dalla Via, Lisa; Gia, Ornella; Chiarelotto, Gianfranco; Ferlin, Maria Grazia

    2009-07-01

    A series of conjugates of alpha,beta-unsaturated ketone systems, phenyl-butenone and diaryl-propenones (chalcones), with the tricyclic planar pyrroloquinoline nucleus were synthesised and evaluated for their anticancer properties. The aim was to target DNA by butenone and chalcones, and determine the occurrence of interactions with the macromolecule or related functional enzymes. The ability to inhibit cell growth was assayed on three human tumor cell lines, and the capacity to form molecular complexes with DNA was studied by linear flow dichroism (LD). The effect on the activity of the nuclear enzyme DNA topoisomerase II was also investigated. A noticeable cytotoxic effect was observed for all pyrroloquinoline-conjugated compounds 5 and 7a-c, particularly against human melanoma cell line JR8 (IC(50) 1.2-3.3 microM); the unconjugated chalcones (8a-c) and butenone had a lower or no effect at the tested concentrations. LD experiments confirmed the pyrroloquinoline nucleus as an efficacious carrier for intercalative complexation with DNA. The ability of pyrroloquinoline derivatives to intercalate between base pairs appears to inhibit the relaxation of supercoiled DNA by topoisomerase II, while they induce no significant DNA cleavage. Since the concentrations inhibiting the enzyme appear relatively high with respect to cytotoxicity, the effective intercalation could affect the activity of more DNA processing enzymes and these overall nuclear effects may induce cell death.

  10. Synthesis and characterization of azo-guanidine based alcoholic media naked eye DNA sensor

    PubMed Central

    Hashmat, Uzma; Yousaf, Muhammad; Lal, Bhajan; Ullah, Shafiq; Holder, Alvin A.; Badshah, Amin

    2016-01-01

    DNA sensing always has an open meadow of curiosity for biotechnologists and other researchers. Recently, in this field, we have introduced an emerging class of molecules containing azo and guanidine functionalities. In this study, we have synthesized three new compounds (UA1, UA6 and UA7) for potential application in DNA sensing in alcoholic medium. The synthesized materials were characterized by elemental analysis, FTIR, UV-visible, 1H NMR and 13C NMR spectroscopies. Their DNA sensing potential were investigated by UV-visible spectroscopy. The insight of interaction with DNA was further investigated by electrochemical (cyclic voltammetry) and hydrodynamic (viscosity) studies. The results showed that compounds have moderate DNA binding properties, with the binding constants range being 7.2 × 103, 2.4 × 103 and 0.2 × 103 M−1, for UA1, UA6 and UA7, respectively. Upon binding with DNA, there was a change in colour (a blue shift in the λmax value) which was observable with a naked eye. These results indicated the potential of synthesized compounds as DNA sensors with detection limit 1.8, 5.8 and 4.0 ng µl−1 for UA1, UA6 and UA7, respectively. PMID:28018613

  11. Mononuclear metal complexes with ciprofloxacin: Synthesis, characterization and DNA-binding properties.

    PubMed

    Psomas, George

    2008-09-01

    Five novel metal complexes of the quinolone antibacterial agent ciprofloxacin with Mn(2+), Fe(3+), Co(2+), Ni(2+) and MoO(2)(2+) have been prepared and characterized with physicochemical, spectroscopic and electrochemical techniques. In all these complexes, ciprofloxacin acts as a bidentate deprotonated ligand bound to the metal through the pyridone oxygen and one carboxylate oxygen. The central metal in each complex is six-coordinate and a slightly distorted octahedral geometry is proposed. The lowest energy model structures of the Mn(2+), Fe(3+) and MoO(2)(2+) complexes have been determined with molecular modeling calculations. The cyclic voltammograms of the complexes have been recorded in dmso solution and in 1/2 dmso/buffer (containing 150mM NaCl and 15mM trisodium citrate at pH 7.0) solution and the corresponding redox potentials have been estimated. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. Competitive studies with ethidium bromide (EB) have shown that the complexes exhibit the ability to displace the DNA-bound EB indicating that the complexes bind to DNA probably via intercalation in strong competition with EB for the intercalative binding site.

  12. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

    PubMed

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

    Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

  13. Synthesis of copper nanoparticles by electrolysis of DNA utilizing copper as sacrificial anode.

    PubMed

    Singh, Dinesh Pratap; Srivastava, Onkar Nath

    2007-06-01

    Copper nanoparticles have been synthesized by anodic oxidation through a simple electrolysis process employing de-oxy ribonucleic acid (DNA) as electrolyte. Platinum was taken as cathode and copper as anode. The applied voltage was 4 V and the electrolysis was performed for duration of 1 h. The copper nanoparticles were prepared in situ from the electron beam irradiation on residues of electrolyte consisting of DNA and copper particles: DNA (Cu) complexes. The size of the nanoparticles ranges between 5-50 nm. A tentative explanation has been given for the formation of copper nanoparticles.

  14. Inhibition of RNA and DNA synthesis in UV-irradiated normal human fibroblasts is correlated with pyrimidine (6-4) pyrimidone photoproduct formation.

    PubMed

    Petit Frère, C; Clingen, P H; Arlett, C F; Green, M H

    1996-07-05

    UV-irradiation of living cells results in an inhibition of RNA and DNA synthesis. The purpose of this study was to determine whether specific photoproducts or the total combined yield of lesions were responsible for these effects. Asynchronously dividing human fibroblasts from normal donors were irradiated with UVC (254 nm), broad spectrum UVB (290-320 + nm, Westinghouse FS20 lamp) or narrow spectrum UVB (310-315 nm, Philips TL01 lamp) at fluences which induce known yields of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts or Dewar isomers. DNA synthesis was approximately 3-4 times more sensitive to both UVC and UVB irradiation than RNA synthesis. The immediate inhibition of RNA and DNA synthesis was correlated with (6-4) rather than overall photoproduct formation suggesting that the (6-4) photoproduct is the mediator of these inhibitory effects. In support of this suggestion we found that photoreactivation of cells cultured from the marsupial, mouse Sminthopsis crassicaudata, resulted in removal of 70% of pyrimidine dimers from the overall genome, but had only a slight effect on the recovery of RNA synthesis.

  15. DNA synthesis during endomitosis is stimulated by insulin via the PI3K/Akt and TOR signaling pathways in the silk gland cells of Bombyx mori.

    PubMed

    Li, Yaofeng; Chen, Xiangyun; Tang, Xiaofang; Zhang, Chundong; Wang, La; Chen, Peng; Pan, Minhui; Lu, Cheng

    2015-03-18

    Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine (BrdU) labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase (PI3K)/Akt, the target of rapamycin (TOR) and the extracellular signal-regulated kinase (ERK) pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

  16. Synthesis of dihydromyricetin-manganese (II) complex and interaction with DNA

    NASA Astrophysics Data System (ADS)

    Guo, Qingquan; Yuan, Juan; Zeng, Jinhua; He, Xiangzhu; Li, Daguang

    2012-11-01

    Dihydromyricetin has many physiological functions and its metal complex could have better effects. DNA is very important in biological body, but little attention has been devoted to the relationship between dihydromyricetin-metal complex and the DNA. In this paper, dihydromyricetin-Mn (II) complex has been prepared and characterized using UV-vis absorption spectrophotometry, IR spectroscopy, elemental analysis, and thermal gravimetric analysis (TG-DTA Analysis). The interaction of dihydromyricetin-Mn (II) complex with DNA was investigated using UV-vis spectra, fluorescence measurements and viscosity measurements. The results indicate that the dihydromyricetin-manganese (II) complex can intercalate into the stacked base pairs of DNA with binding constant Kb = 5.64 × 104 M and compete with the strong intercalator ethidium bromide for the intercalative binding sites with Stern-Volmer quenching constant, Ksq = 1.16.

  17. Design, synthesis and biological activity of novel molecules designed to target PARP and DNA.

    PubMed

    Goodfellow, Elliot; Senhaji Mouhri, Zhor; Williams, Christopher; Jean-Claude, Bertrand J

    2017-02-01

    In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2-20 fold. The most selective dual PARP-DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

  18. A comparison of RNA with DNA in template-directed synthesis

    NASA Technical Reports Server (NTRS)

    Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

  19. Design, synthesis, cytotoxicities and DNA cleavage activities of dibenzoxepine and isoquinoline derivatives starting from dehydroabietylamine.

    PubMed

    Liu, Chao-Xiang; Lin, Zhong-Xiang; Zhou, Ai-Min

    2016-12-01

    A series of novel hexahydrodibenzoxepine and quinazoline derivatives were designed and synthesized starting from dehydroabietylamine. The cytotoxicities of the compounds against L02 and HepG2 cell lines were investigated. Meanwhile, the plasmid DNA (Escherichia coli) cleavage of several heterocyclic derivatives was studied. These compounds exhibit remarkable activities on plasmid DNA pBR322. Our study provides useful information for developing new and more potent antitumor agents.

  20. Design, synthesis, physicochemical studies, solvation, and DNA damage of quinoline-appended chalcone derivative: comprehensive spectroscopic approach toward drug discovery.

    PubMed

    Kumar, Himank; Chattopadhyay, Anjan; Prasath, R; Devaraji, Vinod; Joshi, Ritika; Bhavana, P; Saini, Praveen; Ghosh, Sujit Kumar

    2014-07-03

    The present study epitomizes the design, synthesis, photophysics, solvation, and interaction with calf-thymus DNA of a potential antitumor, anticancer quinoline-appended chalcone derivative, (E)-3-(anthracen-10-yl)-1-(6,8-dibromo-2-methylquinolin-3-yl)prop-2-en-1-one (ADMQ) using steady state absorption and fluorescence spectroscopy, molecular modeling, molecular docking, Fourier-transform infrared spectroscopy (FTIR), molecular dynamics (MD) simulation, and gel electrophoresis studies. ADMQ shows an unusual photophysical behavior in a variety of solvents of different polarity. The dual emission has been observed along with the formation of twisted intramolecular charge transfer (TICT) excited state. The radiationless deactivation of the TICT state is found to be promoted strongly by hydrogen bonding. Quantum mechanical (DFT, TDDFT, and ZINDO-CI) calculations show that the ADMQ is sort of molecular rotor which undergoes intramolecular twist followed by a complete charge transfer in the optimized excited state. FTIR studies reveals that ADMQ undergoes important structural change from its native structure to a β-hydroxy keto form in water at physiological pH. The concentration-dependent DNA cleavage has been identified in agarose gel DNA electrophoresis experiment and has been further supported by MD simulation. ADMQ forms hydrogen bond with the deoxyribose sugar attached with the nucleobase adenine DA-17 (chain A) and result in significant structural changes which potentially cleave DNA double helix. The compound does not exhibit any deleterious effect or toxicity to the E. coli strain in cytotoxicity studies. The consolidated spectroscopic research described herein can provide enormous information to open up new avenues for designing and synthesizing chalcone derivatives with low systematic toxicity for medicinal chemistry research.

  1. Synthesis of Site-Specific DNA–Protein Conjugates and Their Effects on DNA Replication

    PubMed Central

    2015-01-01

    DNA–protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA–protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  2. Scutellarin-graft cationic β-cyclodextrin-polyrotaxane: Synthesis, characterization and DNA condensation.

    PubMed

    Qin, Qi; Ma, Xue; Liao, Xiali; Yang, Bo

    2017-02-01

    As a prerequisite of gene delivery in living cells, DNA condensation has attracted more and more attention. In order to improve the efficiencies of polyamine-β-cyclodextrin-based cationic polyrotaxanes (PR-EDA and PR-DETA) as DNA condensation materials, we have designed and prepared two novel scutellarin-grafted cationic polyrotaxanes (PR-EDA-SCU and PR-DETA-SCU), in which scutellarins (SCU), the planar molecules, were conjugated on the cyclodextrin molecules of PR-EDA and PR-DETA. These materials were characterized by 1D and 2D NMR, XRD, TG and DSC. The electrophoresis assays showed that pDNA condensation efficiencies of PR-EDA and PR-DETA were better than that of PR-EDA and PR-DETA. The complexes of PR-EDA, PR-DETA, PR-EDA-SCU and PR-DETA-SCU with pDNA were further investigated by zeta potential and atomic force microscopy analysis. The results indicated that the planar structure of SCU played an important role in improvement of pDNA condensation efficiencies of PR-EDA-SCU and PR-DETA-SCU. The satisfactory pDNA condensation abilities of PR-EDA-SCU and PR-DETA-SCU could be helpful in designing non-viral gene delivery vectors to control gene expression and delivery.

  3. Microwave-assisted synthesis of highly water-soluble graphene towards electrical DNA sensor

    NASA Astrophysics Data System (ADS)

    Choi, Bong Gill; Park, Hoseok; Yang, Min Ho; Jung, Young Mee; Lee, Sang Yup; Hong, Won Hi; Park, Tae Jung

    2010-12-01

    Graphene sheets have the potential for practical applications in electrochemical devices, but their development has been impeded by critical problems with aggregation of graphene sheets. Here, we demonstrated a facile and bottom-up approach for fabrication of DNA sensor device using water-soluble sulfonated reduced graphene oxide (SRGO) sheets via microwave-assisted sulfonation (MAS), showing enhanced sensitivity, reliability, and low detection limit. Key to achieving these performances is the fabrication of the SRGOs, where the MAS method enabled SRGOs to be highly dispersed in water (10 mg mL-1) due to the acidic sulfonated groups generated within 3 min of the functionalization reaction. The water-soluble SRGO-DNA (SRGOD) hybrids prepared by electrostatic interactions between a flat single layer of graphene sheets and DNAs are suitable for fabrication of electrical DNA sensor devices because of the unique electrical characteristics of SRGODs. The high sensing performance of SRGOD sensors was demonstrated with detection ofDNA hybridization using complementary DNAs, single base mismatched DNAs, and noncomplementary DNAs, with results showing higher sensitivity and lower detection limit than those of reduced graphene oxide-based DNA sensors. Simple and easy fabrication of DNA sensor devices using SRGODs is expected to provide an effective way for electrical detection ofDNA hybridization using miniature sensors without the labor-intensive labeling of the sensor and complex measurement equipment.Graphene sheets have the potential for practical applications in electrochemical devices, but their development has been impeded by critical problems with aggregation of graphene sheets. Here, we demonstrated a facile and bottom-up approach for fabrication of DNA sensor device using water-soluble sulfonated reduced graphene oxide (SRGO) sheets via microwave-assisted sulfonation (MAS), showing enhanced sensitivity, reliability, and low detection limit. Key to achieving these

  4. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    PubMed

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-02-28

    Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

  5. Purification of a Factor from Human Placenta That Stimulates Capillary Endothelial Cell Protease Production, DNA Synthesis, and Migration

    NASA Astrophysics Data System (ADS)

    Moscatelli, David; Presta, Marco; Rifkin, Daniel B.

    1986-04-01

    A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.

  6. Simulated Screens of DNA Encoded Libraries: The Potential Influence of Chemical Synthesis Fidelity on Interpretation of Structure-Activity Relationships.

    PubMed

    Satz, Alexander L

    2016-07-11

    Simulated screening of DNA encoded libraries indicates that the presence of truncated byproducts complicates the relationship between library member enrichment and equilibrium association constant (these truncates result from incomplete chemical reactions during library synthesis). Further, simulations indicate that some patterns observed in reported experimental data may result from the presence of truncated byproducts in the library mixture and not structure-activity relationships. Potential experimental methods of minimizing the presence of truncates are assessed via simulation; the relationship between enrichment and equilibrium association constant for libraries of differing purities is investigated. Data aggregation techniques are demonstrated that allow for more accurate analysis of screening results, in particular when the screened library contains significant quantities of truncates.

  7. Optimization of enzymatic clarification of sapodilla juice: a statistical perspective.

    PubMed

    Jacob, Nicemol; Sukumaran, R K; Prema, P

    2008-12-01

    Response surface methodology (RSM) was employed to establish optimum conditions for enzymatic clarification of sapodilla juice. Polygalacturonase obtained from Streptomyces lydicus had been purified to homogeneity and was used for the treatment. The independent variables were temperature (30-45 degrees C), enzyme concentration (0.5-1.5 U), and treatment time (30-90 min), whose effects on viscosity and clarity of the juice were evaluated using a Box-Behnken design. Significant regression models describing the changes of viscosity and clarity with respect to the independent variables were obtained, with the coefficient of determination, R (2) , greater than 0.8. Based on response surfaces and contour plots, the optimum conditions for clarifying sapodilla juice were enzyme concentration 1.15 U, incubation temperature 34 degrees C, and incubation time 70 min.

  8. Beyond Values Clarification: Addressing Client Values in Clinical Behavior Analysis

    PubMed Central

    Bonow, Jordan T; Follette, William C

    2009-01-01

    Ethical principles of psychology, as exemplified in the American Psychological Association (APA) Code of Ethics (2002), provide impractical advice for addressing client values during psychotherapy. These principles seem to argue that each client's values should be respected and protected at all times, except in cases in which this would result in harm to the client or the general public. Although the code is appropriately designed as a defense against potential and actual abuses of professional power, this general proscription against directly targeting client values for change is based on an understanding of values that grants them special ontological status and has resulted in limited clinical interventions focusing solely on “values clarification.” With its strong foundation in a unified philosophical system, clinical behavior analysis offers a sophisticated alternative approach to values that both defines what they are and identifies when they can be ethically targeted to improve the lives of clients. PMID:22478514

  9. Herpes simplex virus 1 activates cdc2 to recruit topoisomerase II alpha for post-DNA synthesis expression of late genes.

    PubMed

    Advani, Sunil J; Weichselbaum, Ralph R; Roizman, Bernard

    2003-04-15

    A subset (gamma(2)) of late herpes simplex virus 1 genes depends on viral DNA synthesis for its expression. For optimal expression, a small number of these genes, exemplified by U(S)11, also requires two viral proteins, the alpha protein infected cell protein (ICP) 22 and the protein kinase U(L)13. Earlier we showed that U(L)13 and ICP22 mediate the stabilization of cdc2 and the replacement of its cellular partner, cyclin B, with the viral DNA polymerase processivity factor U(L)42. Here we report that cdc2 and its new partner, U(L)42, bind a phosphorylated form of topoisomerase II alpha. The posttranslational modification of topoisomerase II alpha and its interaction with cdc2-U(L)42 proteins depend on ICP22 in infected cells. Although topoisomerase II is required for viral DNA synthesis, ICP22 is not, indicating a second function for topoisomerase II alpha. The intricate manner in which the virus recruits topoisomerase II alpha for post-DNA synthesis expression of viral genes suggests that topoisomerase II alpha also is required for untangling concatemeric DNA progeny for optimal transcription of late genes.

  10. Modulation of in vitro DNA synthesis in the chicken ovarian granulosa cell follicular hierarchy by follicle-stimulating hormone and luteinizing hormone.

    PubMed

    McElroy, A P; Caldwell, D J; Proudman, J A; Hargis, B M

    2004-03-01

    Folliculogenesis in domestic hens appears to be controlled by numerous factors, particularly the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The involvement of LH in follicular steroidogenesis has been described in some detail; however, the specific role of FSH has remained elusive. In 3 experiments, the effects of ovine (o)- or chicken (c)-derived FSH (oFSH, cFSH) or LH (oLH, cLH) were evaluated on in vitro DNA synthesis [3H-thymidine (3H-TdR) incorporation], indicative of cellular proliferation, of granulosa cells from F1, F3, or F5-6 preovulatory follicles. In experiment 1, oFSH or cFSH stimulated (P < 0.05) and oLH or cLH decreased DNA synthesis by F1 granulosa cells. In experiment 2, oFSH resulted in concentration-related changes in DNA synthesis by F5-6 granulosa cells; however, no significant changes were observed in F1 or F3 granulosa cells. No effect of oLH was observed on granulosa cell proliferation from any of the follicles. Similar to oFSH, cFSH resulted in concentration-related increases in DNA synthesis in granulosa cells from F5-6 follicles with smaller magnitude changes in proliferation of F1 or F3 granulosa cells. Granulosa cells from F5-6 or F3 follicles had small increases in DNA synthesis in response to cLH. These data support the proposed role for FSH in granulosa cell proliferation, possibly contributing to follicle growth, and suggest that in vitro 3H-TdR incorporation by granulosa cells may provide a sensitive and selective bioassay for chicken gonadotropin preparations. Furthermore, data suggest that proliferative responsiveness of granulosa cells to FSH or LH may differ depending on position of follicles in the preovulatory hierarchy.

  11. Clarification of Orange Press Liquors by PVDF Hollow Fiber Membranes

    PubMed Central

    Simone, Silvia; Conidi, Carmela; Ursino, Claudia; Cassano, Alfredo; Figoli, Alberto

    2016-01-01

    Press liquors are typical by-products of the citrus juice processing characterized by a high content of organic compounds and associated problems of environmental impact, which imply high treatment costs. However, these wastes contain a great number of health promoting substances, including fibers, carotenoids and phenolic compounds (mainly flavonoids), whose recovery against waste-destruction technologies is very attractive for new business opportunities. In this work, the clarification of orange press liquor by using microfiltration (MF) membranes is studied as a preliminary step to obtain a permeate stream enriched in antioxidant compounds which can be further processed to produce extracts of nutraceutical and/or pharmaceutical interest. MF poly(vinylidene fluoride) (PVDF) hollow fibers were prepared by the dry/wet spinning technique. A series of fibers was produced from the same polymeric dope, in order to investigate the effect of selected spinning parameters, i.e., bore fluid composition and flowrate, on their properties. The morphology of the produced fibers was analyzed by Scanning Electron Microscopy (SEM). Fibers were further characterized for their mechanical properties, porosity, bubble point, pore size distribution and pure water permeability (PWP). Some of the produced fibers exhibited high permeability (pure water permeability ~530 L/m2·h·bar), coupled to good mechanical resistance and pore size in the range of MF membranes. These fibers were selected and used for the clarification of press liquor from orange peel processing. In optimized operating conditions, the selected fibers produced steady-state fluxes of about 41 L/m2·h with rejections towards polyphenols and total antioxidant activity of 4.1% and 1.4%, respectively. PMID:26805899

  12. Clarification of Orange Press Liquors by PVDF Hollow Fiber Membranes.

    PubMed

    Simone, Silvia; Conidi, Carmela; Ursino, Claudia; Cassano, Alfredo; Figoli, Alberto

    2016-01-20

    Press liquors are typical by-products of the citrus juice processing characterized by a high content of organic compounds and associated problems of environmental impact, which imply high treatment costs. However, these wastes contain a great number of health promoting substances, including fibers, carotenoids and phenolic compounds (mainly flavonoids), whose recovery against waste-destruction technologies is very attractive for new business opportunities. In this work, the clarification of orange press liquor by using microfiltration (MF) membranes is studied as a preliminary step to obtain a permeate stream enriched in antioxidant compounds which can be further processed to produce extracts of nutraceutical and/or pharmaceutical interest. MF poly(vinylidene fluoride) (PVDF) hollow fibers were prepared by the dry/wet spinning technique. A series of fibers was produced from the same polymeric dope, in order to investigate the effect of selected spinning parameters, i.e., bore fluid composition and flowrate, on their properties. The morphology of the produced fibers was analyzed by Scanning Electron Microscopy (SEM). Fibers were further characterized for their mechanical properties, porosity, bubble point, pore size distribution and pure water permeability (PWP). Some of the produced fibers exhibited high permeability (pure water permeability ~530 L/m²·h·bar), coupled to good mechanical resistance and pore size in the range of MF membranes. These fibers were selected and used for the clarification of press liquor from orange peel processing. In optimized operating conditions, the selected fibers produced steady-state fluxes of about 41 L/m²·h with rejections towards polyphenols and total antioxidant activity of 4.1% and 1.4%, respectively.

  13. A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site.

    PubMed

    Tanpure, Arun A; Srivatsan, Seergazhi G

    2011-11-04

    Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.

  14. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    PubMed

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-02

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer.

  15. Design and synthesis of N-benzoyl amino acid derivatives as DNA methylation inhibitors.

    PubMed

    Garella, Davide; Atlante, Sandra; Borretto, Emily; Cocco, Mattia; Giorgis, Marta; Costale, Annalisa; Stevanato, Livio; Miglio, Gianluca; Cencioni, Chiara; Fernández-de Gortari, Eli; Medina-Franco, José L; Spallotta, Francesco; Gaetano, Carlo; Bertinaria, Massimo

    2016-11-01

    The inhibition of human DNA Methyl Transferases (DNMT) is a novel promising approach to address the epigenetic dysregulation of gene expression in different diseases. Inspired by the validated virtual screening hit NSC137546, a series of N-benzoyl amino acid analogues was synthesized and obtained compounds were assessed for their ability to inhibit DNMT-dependent DNA methylation in vitro. The biological screening allowed the definition of a set of preliminary structure-activity relationships and the identification of compounds promising for further development. Among the synthesized compounds, L-glutamic acid derivatives 22, 23, and 24 showed the highest ability to prevent DNA methylation in a total cell lysate. Compound 22 inhibited DNMT1 and DNMT3A activity in a concentration-dependent manner in the micromolar range. In addition, compound 22 proved to be stable in human serum and it was thus selected as a starting point for further biological studies.

  16. Synthesis of new heterometallic macromolecules: Their DNA binding, cleavage activity and in vitro model electrochemotherapy study

    NASA Astrophysics Data System (ADS)

    Tabassum, Sartaj; Bhat, Irshad-ul-Haq; Arjmand, Farukh

    2009-12-01

    The homodinuclear C 16H 30N 8O 5Sn 2Cl 4 ( 1), heterotetranuclear C 16H 38N 8O 9Sn 2Cu 2Cl 8 ( 2) and C 16H 38N 8O 9Sn 2Mn 2Cl 8 ( 3) macrocyclic complexes were synthesized and characterized by elemental analysis, spectroscopic techniques and molar conductance measurements. The interaction studies of 1-3 with calf thymus DNA (CT-DNA) were carried out by UV-vis titration, fluorescence, cyclic voltammetry and viscosity measurements. These results were further authenticated by carrying out interaction studies of 1-3 with plasmid pBR322 DNA employing gel electrophoresis. To overcome the dose resistance, auto toxicity of the drugs, a model study based on electrochemotherapy (ECT) was carried out and the results were compared in the presence and in the absence of the applied electrical potential.

  17. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd

    2014-09-01

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N'-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by 1H and 13C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N2O2 from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed.

  18. Structural Insight into Processive Human Mitochondrial DNA Synthesis and Disease-Related Polymerase Mutations

    SciTech Connect

    Lee, Young-Sam; Kennedy, W. Dexter; Yin, Y. Whitney

    2010-09-07

    Human mitochondrial DNA polymerase (Pol {gamma}) is the sole replicase in mitochondria. Pol {gamma} is vulnerable to nonselective antiretroviral drugs and is increasingly associated with mutations found in patients with mitochondriopathies. We determined crystal structures of the human heterotrimeric Pol {gamma} holoenzyme and, separately, a variant of its processivity factor, Pol {gamma}B. The holoenzyme structure reveals an unexpected assembly of the mitochondrial DNA replicase where the catalytic subunit Pol {gamma}A interacts with its processivity factor primarily via a domain that is absent in all other DNA polymerases. This domain provides a structural module for supporting both the intrinsic processivity of the catalytic subunit alone and the enhanced processivity of holoenzyme. The Pol {gamma} structure also provides a context for interpreting the phenotypes of disease-related mutations in the polymerase and establishes a foundation for understanding the molecular basis of toxicity of anti-retroviral drugs targeting HIV reverse transcriptase.

  19. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    SciTech Connect

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd

    2014-09-03

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by {sup 1}H and {sup 13}C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N{sub 2}O{sub 2} from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed.

  20. The Chemical Synthesis of DNA/RNA: Our Gift to Science

    PubMed Central

    Caruthers, Marvin H.

    2013-01-01

    It is a great privilege to contribute to the Reflections essays. In my particular case, this essay has allowed me to weave some of my major scientific contributions into a tapestry held together by what I have learned from three colleagues (Robert Letsinger, Gobind Khorana, and George Rathmann) who molded my career at every important junction. To these individuals, I remain eternally grateful, as they always led by example and showed many of us how to break new ground in both science and biotechnology. Relative to my scientific career, I have focused primarily on two related areas. The first is methodologies we developed for chemically synthesizing DNA and RNA. Synthetic DNA and RNA continue to be an essential research tool for biologists, biochemists, and molecular biologists. The second is developing new approaches for solving important biological problems using synthetic DNA, RNA, and their analogs. PMID:23223445

  1. Synthesis, antioxidant, enzyme inhibition and DNA binding studies of novel N-benzylated derivatives of sulfonamide

    NASA Astrophysics Data System (ADS)

    Abbas, Aadil; Murtaza, Shahzad; Tahir, Muhammad Nawaz; Shamim, Saima; Sirajuddin, Muhammad; Rana, Usman Ali; Naseem, Khadija; Rafique, Hummera

    2016-08-01

    A series of novel N-benzylated derivatives of sulfonamide were synthesized and characterized by FT-IR, NMR and XRD analysis. The synthesized compounds were assayed for their biological potential. The biological studies involved antioxidant, enzyme inhibition, and DNA interaction studies. Antioxidant potential was investigated by Ferric Reducing Antioxidant Power assay (FRAP) and DPPH free radical scavenging method, the capacity of synthesized compounds to inhibit the enzyme's activity was assayed by using the well-known Elman method whereas DNA interaction studies were carried out with the help UV-Vis absorption titration method. Moreover, a direct correlation between enzyme inhibition activity and concentration of the compounds was observed both in experimental and molecular docking studies. DNA interaction studies of the synthesized compounds showed weak interaction.

  2. Synthesis of homogeneous glycopeptides and their utility as DNA condensing agents.

    PubMed

    Collard, W T; Evers, D L; McKenzie, D L; Rice, K G

    2000-01-12

    Two glycopeptides were synthesized by attaching purified glycosylamines (N-glycans) to a 20 amino acid peptide. Triantennary and Man9 Boc-tyrosinamide N-glycans were treated with trifluoroacetic acid to remove the Boc group and expose a tyrosinamide amine. The amine group was coupled with iodoacetic acid to produce N-iodoacetyl-oligosaccharides. These were reacted with the sulfhydryl group of a cysteine-containing peptide (CWK18), resulting in the formation of glycopeptides in good yield that were characterized by 1H NMR and ESIMS. Both glycopeptides were able to bind to plasmid DNA and form DNA condensates of approximately 110 nm mean diameter with zeta potential of +31 mV. The resulting homogeneous glycopeptide DNA condensates will be valuable as receptor-mediated gene-delivery agents.

  3. Synthesis of a new conjugated polymer for DNA alkylation and gene regulation.

    PubMed

    Nie, Chenyao; Zhu, Chunlei; Feng, Liheng; Lv, Fengting; Liu, Libing; Wang, Shu

    2013-06-12

    A new polyfluorene derivative containing pendent alkylating chlorambucil (PFP-Cbl) was synthesized and characterized. Under direct incubation with DNA in vitro, PFP-Cbl could undergo an efficient DNA alkylating reaction and induce DNA cross-linking. In vitro transcription and translation experiment exhibited that the PFP-Cbl significantly down-regulated the gene expression of luciferase reporter plasmid. The down-regulation of gene expression was also verified through the transfection experiment of p-EGFP plasmid, which showed decreased green fluorescent protein (GFP) in cells. Meanwhile, the self-luminous property of PFP-Cbl could make it able to trace the internalized PFP-Cbl and plasmid complexes resulted from cross-linking in cells by fluorescent microscopy. Combining the features of alkylating function, multivalent binding sites, and fluorescent characteristics, PFP-Cbl provides a new insight in the area of gene regulation and extends the new applications of conjugated polymers (CPs).

  4. DNA Based Micelles: Synthesis, Micellar Properties and Size-dependent Cell Permeability

    PubMed Central

    Liu, Haipeng; Zhu, Zhi; Kang, Huaizhi; Wu, Yanrong; Sefan, Kwame

    2012-01-01

    Functional nanomaterials based on molecular self-assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self-assemble into monodispersed, three dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while remain their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size-dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity, are all excellent features that make our DNA-micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems. PMID:20162643

  5. Gelatin-based nanoparticles as DNA delivery systems: Synthesis, physicochemical and biocompatible characterization.

    PubMed

    Morán, M C; Rosell, N; Ruano, G; Busquets, M A; Vinardell, M P

    2015-10-01

    The rapidly rising demand for therapeutic grade DNA molecules requires associated improvements in encapsulation and delivery technologies. One of the challenges for the efficient intracellular delivery of therapeutic biomolecules after their cell internalization by endocytosis is to manipulate the non-productive trafficking from endosomes to lysosomes, where degradation may occur. The combination of the endosomal acidity with the endosomolytic capability of the nanocarrier can increase the intracellular delivery of many drugs, genes and proteins, which, therefore, might enhance their therapeutic efficacy. Among the suitable compounds, the gelification properties of gelatin as well as the strong dependence of gelatin ionization with pH makes this compound an interesting candidate to be used to the effective intracellular delivery of active biomacromolecules. In the present work, gelatin (either high or low gel strength) and protamine sulfate has been selected to form particles by interaction of oppositely charged compounds. Particles in the absence of DNA (binary system) and in the presence of DNA (ternary system) have been prepared. The physicochemical characterization (particle size, polydispersity index and degree of DNA entrapment) have been evaluated. Cytotoxicity experiments have shown that the isolated systems and the resulting gelatin-based nanoparticles are essentially non-toxic. The pH-dependent hemolysis assay and the response of the nanoparticles co-incubated in buffers at defined pHs that mimic extracellular, early endosomal and late endo-lysosomal environments demonstrated that the nanoparticles tend to destabilize and DNA can be successfully released. It was found that, in addition to the imposed compositions, the gel strength of gelatin is a controlling parameter of the final properties of these nanoparticles. The results indicate that these gelatin-based nanoparticles have excellent properties as highly potent and non-toxic intracellular delivery

  6. Crosstalk between Translesion Synthesis, Fanconi Anemia Network, and Homologous Recombination Repair Pathways in Interstrand DNA Crosslink Repair and Development of Chemoresistance

    PubMed Central

    Haynes, Brittany; Saadat, Nadia; Myung, Brian; Shekhar, Malathy PV

    2014-01-01

    Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cell’s enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed. PMID:25795124

  7. Phenyl 1,2,3-Triazole-Thymidine Ligands Stabilize G-Quadruplex DNA, Inhibit DNA Synthesis and Potentially Reduce Tumor Cell Proliferation over 3′-Azido Deoxythymidine

    PubMed Central

    Mahesh Kumar, Jerald; Idris, Mohammed M.; Srinivas, Gunda; Vinay Kumar, Pallerla; Meghah, Vuppalapaty; Kavitha, Mitta; Reddy, Chada Raji; Mainkar, Prathama S.; Pal, Biswajit; Chandrasekar, Srivari; Nagesh, Narayana

    2013-01-01

    Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4–L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3′-Phenyl-1,2,3- triazole-thymidine (L2) and 3′-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). Tm of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (Kd≈10−7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for

  8. Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.

    PubMed

    Urrutia, R; McNiven, M A; Kachar, B

    1993-05-01

    We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.

  9. Emerging Issues in School, Family, & Community Connections. Annual Synthesis 2001

    ERIC Educational Resources Information Center

    Jordan, Catherine; Orozco, Evangelina; Averett, Amy

    2002-01-01

    This research synthesis is the first in a series that will examine key issues in the field of family and community connections with schools. The issues highlighted in this synthesis represent critical areas of work in family and community connections with schools where clarification, agreement, and further development are needed, as well as…

  10. Effect of ultraviolet B radiation and 100 Hz electromagnetic fields on proliferation and DNA synthesis of Jurkat cells.

    PubMed

    Nindl, G; Hughes, E F; Johnson, M T; Spandau, D F; Vesper, D N; Balcavage, W X

    2002-09-01

    The use of ultraviolet B light (UVB) has been proven to be highly effective for treatment of various inflammatory skin diseases, but UVB phototherapy is limited by its carcinogenic side effects. It is necessary to uncover effectors that augment UVB so that similar or improved efficacy can be obtained with lower UVB doses. We found that low frequency, low intensity electromagnetic fields (EMFs) can act as such an effector and synergistically inhibit T lymphocyte proliferation. We first characterized the effects of UVB on Jurkat cells, a model for cutaneous T lymphocytes, and determined UVB's dose dependent inhibition of cell proliferation and induction of apoptosis. Cells exposed to a sublethal UVB dose retained their sensitivity to UVB, but repetitive irradiation seemed to cause accumulation of delayed DNA damage. We then exposed cells to combinations of UVB plus EMFs and found that 100 Hz, 1 mT EMFs decrease DNA synthesis of UVB-activated Jurkat cells by 34 +/- 13% compared to UVB alone. The decrease is, however, most effective when relatively high UVB doses are employed. Since EMFs alone had only a very weak inhibitory effect (10 +/- 2%), the data suggest that EMFs augment the cell killing effects of UVB in a synergistic way. These findings could provide the basis for development of new and improved clinical phototherapy protocols.

  11. Synthesis, characterization, DNA binding, cleavage activity and cytotoxicity of copper(II) complexes.

    PubMed

    Li, Mei-Jin; Lan, Tao-Yu; Cao, Xiu-Hui; Yang, Huang-Hao; Shi, Yupeng; Yi, Changqing; Chen, Guo-Nan

    2014-02-21

    Three new mononuclear copper(II) complexes, [Cu(L2)](2+) (1), [Cu(acac)(L)](+) (2), and [Cu(acac-Cl)(L)](+) (3) (L = 2-(4-pyridine)oxazo[4,5-f]1,10-phenanthroline (4-PDOP); acac = acetylacetone; acac-Cl = 3-chloroacetylacetone), have been synthesized and characterized by elemental analysis, high resolution mass spectrometry (Q-TOF), and IR spectroscopy. Two of the complexes were structurally characterized by single-crystal X-ray diffraction techniques. Their interactions with DNA were studied by UV-vis absorption and emission spectra, viscosity, thermal melting, DNA unwinding assay and CD spectroscopy. The nucleolytic cleavage activity of the compounds was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of an oxidant (H2O2). Active oxygen intermediates such as hydroxyl radicals and hydrogen peroxide generated in the presence of L and complexes 1-3 may act as active species for the DNA scission. The cytotoxicity of the complexes against HepG2 cancer cells was also studied.

  12. Imidazolium tagged acridines: Synthesis, characterization and applications in DNA binding and anti-microbial activities

    NASA Astrophysics Data System (ADS)

    Raju, Gembali; Vishwanath, S.; Prasad, Archana; Patel, Basant K.; Prabusankar, Ganesan

    2016-03-01

    New water soluble 4,5-bis imidazolium tagged acridines have been synthesized and structurally characterized by multinuclear NMR and single crystal X-ray diffraction techniques. The DNA binding and anti-microbial activities of these acridine derivatives were investigated by fluorescence and far-UV circular dichroism studies.

  13. Translesion synthesis by human DNA polymerase eta across oxidative products of guanine.

    PubMed

    Kino, Katuhito; Ito, Nobutoshi; Sugasawa, Kaoru; Sugiyama, Hiroshi; Hanaoka, Fumio

    2004-01-01

    Guanine is the most oxidizable base among natural bases. 8-Oxoguanine (8-oxoG) is the typical oxidative product, but the amount of 8-oxoG does not directly reflect the strength of oxidative stress. Imidazolone, oxazolone and guanidinohydantoin are oxidative products of guanine and 8-oxoG. Here, we investigated enzymatic reactions with human DNA polymerase eta on these lesions.

  14. Synthesis, DNA binding and topoisomerase I inhibition activity of thiazacridine and imidazacridine derivatives.

    PubMed

    Lafayette, Elizabeth Almeida; Vitalino de Almeida, Sinara Mônica; Pitta, Marina Galdino da Rocha; Carneiro Beltrão, Eduardo Isidoro; da Silva, Teresinha Gonçalves; Olímpio de Moura, Ricardo; Pitta, Ivan da Rocha; de Carvalho, Luiz Bezerra; de Lima, Maria do Carmo Alves

    2013-12-06

    Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 10(4) to 6.01 × 10(4) M(-1). UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  15. Synthesis, DNA interaction, and cytotoxic activity of a novel proflavine-dithiazolidinone pharmacophore.

    PubMed

    Janovec, Ladislav; Sabolova, Danica; Kozurkova, Maria; Paulíkova, Helena; Kristian, Pavol; Ungvarský, Jan; Moravcíkova, Erika; Bajdichova, Maria; Podhradský, Dusan; Imrich, Jan

    2007-01-01

    Five novel proflavine-dithiazolidinone derivatives 4a-4e have been designed and synthesized by the reaction of dialkyl acridin-3,6-diyl dithioureas 3a-3e with methyl bromoacetate. The binding affinity of dithiazolidinone hydrochlorides 5a-5e with calf thymus DNA and plasmid (pUC19) DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence, and CD spectroscopy. The effects of 5a-5e on the thermal denaturation profiles of calf thymus DNA were also studied. From spectrophotometric and spectrofluorimetric titrations, the binding constants for the pUC19 DNA-drug complexes were determined (K = 6.2-2.2 x 104 M-1). In vitro cytotoxic activities of compounds 5a-5e toward murine leukemia cell line L1210 and human uterus carcinoma HeLa cells were also examined. 2',2' '-[(Acridin-3,6-diyl)diimino]-3',3' '-dipropyl-1,3-dithiazolidin-4-one hydrochloride (5b) showed the highest activity against these cells with IC50 values of 6.3 microM and 12.9 microM over the course of 72 h.

  16. Synthesis and antioxidant capacities of hydroxyl derivatives of cinnamoylphenethylamine in protecting DNA and scavenging radicals.

    PubMed

    Yang, Yang; Song, Zhi-Guang; Liu, Zai-Qun

    2011-04-01

    Cinnamoylphenethylamine (CNPA) derivatives including feruloylphenethylamine (FRPA), caffeoylphenethylamine (CFPA), cinnamoyltyramine (CNTA), feruloyltyramine (FRTA) and caffeoyltyramine (CFTA) were synthesized in order to investigate the influence of the number and position of hydroxyl group on Cu(2+)/glutathione (GSH) and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. The radical-scavenging properties of these CNPA derivatives were also evaluated by trapping 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) cationic radical (ABTS(+•)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and galvinoxyl radical. In addition, these CNPA derivatives were tested by linoleic acid (LH)-β-carotene-bleaching experiment. The chemical kinetic was employed to treat the results from AAPH-induced oxidation of DNA and gave the order of antioxidant ability as CFTA > CFPA > FRTA > FRPA. CFTA and CFPA also possessed high abilities to inhibit Cu²(+)/GSH-mediated degradation of DNA, whereas FRPA and FRTA can protect LH against the auto-oxidation efficiently. Finally, CFPA and FRPA exhibited high activity in trapping ABTS(+•), DPPH and galvinoxyl radicals. Therefore, the cinnamoyl group bearing ortho-dihydroxyl or hydroxyl with ortho-methoxyl benefited for CNPA derivatives to protect DNA, while hydroxyl in tyramine cannot enhance the radical-scavenging abilities of CNPA derivatives.

  17. Synthesis and spectroscopic DNA binding investigations of dibutyltin N-(5-chlorosalicylidene)-leucinate

    NASA Astrophysics Data System (ADS)

    Zhang, Shufang; Yuan, Hongyu; Tian, Laijin

    2017-02-01

    A new dibutyltin N-(5-Chlorosalicylidene)-leucinate (DNCL) was synthesized by the reaction of dibutyltin dichloride with in situ formed potassium N-(5-chlorosalicylidene)-L-isoleucinate 3 characterized by elemental analysis, IR, 1H NMR (1H, 13C and 119Sn) spectra. The interaction between DNCL and calf thymus deoxyribonucleic acid (ctDNA) in a pH 7.4 Tris-HCl buffer solution was investigated by UV-Vis spectroscopy, fluorescence spectroscopy, and viscosity measurements. It was found that DNCL molecules could intercalate into the base pairs of DNA, forming a DNCL-DNA complex with a binding constant of Kf = 5.75 × 105 L mol-1 (310 K). The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) and Gibbs free energy (ΔG) were calculated to be 1.16 × 105 J mol-1, 486.5 J K-1 mol -1 and -3.48 × 104 J mol-1 at 310 K, respectively. Hydrophobic interaction was the predominant intermolecular force in stabilizing the DNCL-DNA complex.

  18. Steroidal pyrimidines: Synthesis, characterization, molecular docking studies with DNA and in vitro cytotoxicity

    NASA Astrophysics Data System (ADS)

    Shamsuzzaman; Dar, Ayaz Mahmood; Yaseen, Zahid; Alam, Khursheed; Hussain, Altaf; Gatoo, Manzoor Ahmad

    2013-08-01

    A series of new steroid pyrimidines (7-9) were synthesized by reacting steroidal thiosemicarbazones (4-6) with diethyl malonate. The new compounds were characterized by IR, 1H NMR, 13C NMR, MS and analytical data. The interaction studies of compounds (7-9) with DNA were carried out by employing gel electrophoresis, UV-vis and fluorescence spectroscopy. The acting force between the compounds (7-9) and DNA was mainly hydrophobic while the other interactions like van der Waals, hydrogen bonding cannot be ruled out. The gel electrophoresis pattern also demonstrated that the compound 7 alone or in presence of Cu (II) causes the nicking of supercoiled pBR322 and it seems to follow the mechanistic pathway involving generation of hydroxyl radicals that are responsible for initiating DNA strand scission. The docking study of compounds (7-9) suggested that the intercalation of compounds in between the nucleotide base pairs might be due to the presence of pyrimidine moiety in steroid molecule. MTT assay was carried out to check the toxicity of new compounds (7-9) against the different human cancer as well as non-cancer cell lines A545, MCF-7, HeLa, HL-60, SW480, HepG2, HT-29, A549, 184B5, MCF10A, NL-20, HPC and HPLF. Apoptotic degradation of DNA in presence of steroidal pyrimidines (7-9) was analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).

  19. Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival

    PubMed Central

    Kuban, Wojciech; Vaisman, Alexandra; McDonald, John P.; Karata, Kiyonobu; Yang, Wei; Goodman, Myron F.; Woodgate, Roger

    2012-01-01

    Escherichia coli polymerase V (pol V/UmuD'2C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the “steric gate” of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C (Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C (Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD'C (Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions. PMID:22784977

  20. Synthesis and Functionalization of Gold Nanoparticles Using Chemically Modified ssDNA

    NASA Astrophysics Data System (ADS)

    Calabrese, P. G.

    In the first part of this thesis, methods for functionalizing spherical gold nanoparticles with nucleic acid binding ligands (aptamers) that target the VEGF receptor complex were developed. In order to provide a multiplexed labeling strategy for imaging the VEGF receptor complex in electron microscopy, gold nanoparticles of distinct sizes were conjugated to modified ssDNA aptamers that target the VEGF-A cytokine, the VEGFR-2 RTK receptor and a membrane associated co-receptor, Nrp-1. The modified ssDNA gold nanoparticle conjugates were applied to a human lung carcinoma cell line (A549) which has been shown to express each of these proteins and used as a model system for VEGF signaling. Binding constants for the modified aptamers were also determined using a fluorescence polarization anisotropy assay to determine KD and KOFF for the aptamers with their respective proteins. In the latter part of this thesis, a modied ssDNA SELEX protocol was also developed in order to evolve imidazole modied ssDNA sequences that assemble gold nanoparticles from Au3+ precursor ions in aqueous solution. Active sequences bound to nanoparticles were partitioned from inactive sequences based on density via ultracentrifugation through a discontinuous sucrose gradient. Colloidal gold solutions produced by the evolved pool had a distinct absorbance spectra and produced nanoparticles with a narrower distribution of sizes compared to colloidal gold solutions produced by the starting randomized pool of imidazole modified ssDNA. Sequencing data from the evolved pool shows that conserved 5 and 6 nt motifs were shared amongst many of the isolates, which indicates that these motifs could serve as chelation sites for gold atoms or help stabilize colloidal gold solutions in a base specific manner.

  1. Synthesis of thiazolidine-2,4-dione derivatives: anticancer, antimicrobial and DNA cleavage studies.

    PubMed

    Laxmi, S Vijaya; Anil, P; Rajitha, G; Rao, Asha Jyothi; Crooks, Peter A; Rajitha, B

    2016-10-01

    In the search of efficient anticancer agents, here, new 5-(4-alkylbenzyledene)thiazolidine-2,4-dione derivatives (5a-g) have been successfully synthesized and characterized and are evaluated for anticancer and antimicrobial activities using DNA cleavage studies. In vitro studies on anticancer activity of compound 5d (NSC: 768619/1) was done against the full panel of 60 human tumor cell lines. The five-level dose activity results revealed that, the compound 5d was active against all the cell lines, it has shown potential activity against leukemia SR (GI50: 2.04 μM), non-small cell lung cancer NCI-H522 (GI50: 1.36 μM), colon cancer COLO 205 (GI50: 1.64 μM), CNS cancer SF-539 (GI50: 1.87 μM), melanoma SK-MEL-2 (GI50: 1.64 μM), ovarian cancer OVCAR-3 (GI50: 1.87 μM), renal cancer RXF 393 (GI50: 1.15 μM), prostate cancer PC-3 (GI50: 1.90 μM), and breast cancer MDA-MB-468(GI50: 1.11 μM). DNA cleavage studies revealed that at 50 μg/mL concentration, partial DNA digestion was observed and when the concentration is increasing to threefold (150 μg/mL), complete linear DNA digestion and partial supercoiled DNA digestion was observed. Further antimicrobial studies indicate that all the synthesized compounds except compound 5a possess prominent activity against all the screened microbial species. This study throws a ray of light in the field of anticancer drugs.

  2. Templated synthesis of monodisperse mesoporous maghemite/silica microspheres for magnetic separation of genomic DNA

    NASA Astrophysics Data System (ADS)

    Chen, Feng; Shi, Ruobing; Xue, Yun; Chen, Lei; Wan, Qian-Hong

    2010-08-01

    A novel method is described for the preparation of superparamagnetic mesoporous maghemite (γ-Fe 2O 3)/silica (SiO 2) composite microspheres to allow rapid magnetic separation of DNA from biological samples. With magnetite (Fe 3O 4) and silica nanoparticles as starting materials, such microspheres were synthesized by the following two consecutive steps: (1) formation of monodispersed organic/inorganic hybrid microspheres through urea-formaldedyde (UF) polymerization and (2) removal of the organic template and phase transformation of Fe 3O 4 to γ-Fe 2O 3 by calcination at elevated temperatures. The as-synthesized particles obtained by heating at temperature 300 °C feature spherical shape and uniform particle size ( dparticle=1.72 μm), high saturation magnetization ( Ms=17.22 emu/g), superparamagnetism ( Mr/ Ms=0.023), high surface area ( SBET=240 m 2/g), and mesoporosity ( dpore=6.62 nm). The composite microsphere consists of interlocked amorphous SiO 2 nanoparticles, in which cubic γ-Fe 2O 3 nanocrystals are homogeneously dispersed and thermally stable against γ- to α-phase transformation at temperatures up to 600 °C. With the exposed iron oxide nanoparticles coated with a thin layer of silica shell, the magnetic microspheres were used as a solid-phase adsorbent for rapid extraction of genomic DNA from plant samples. The results show that the DNA templates isolated from pea and green pepper displayed single bands with molecular weights greater than 8 kb and A260/ A280 values of 1.60-1.72. The PCR amplification of a fragment encoding the endogenous chloroplast ndhB gene confirmed that the DNA templates obtained were inhibitor-free and amenable to sensitive amplification-based DNA technologies.

  3. (3H)-isoproterenol binding to subcellular fractions of mouse parotid: relationship to cyclic nucleotide formation and the stimulation of DNA synthesis.

    PubMed

    Durham, J P; Galanti, N

    1976-12-01

    (3H) Isoproterenol binding to subcellular fractions of mouse parotid: Relationship to cyclic nucleotide formation and the stimulation of DNA synthesis. (Unión the (3H) Isoproterenol a fracciones subcelulares de parótida de ratón y su relacón con la formacón de nucleótidos cíclicos y la estimulación de la síntesis de DNA). Arch. Biol. Med. Exper. 10: 105-114, 1976. Tritiated isoproterenol binds to all subcellular fractions of mouse parotid but 70% of the binding is to the nuclear fraction. Binding to other mouse tissues was less than to the parotid. The patterns of binding did not correlate with the distribution of adenylate cyclase, guanylate cyclase or catechol-O-methyl transferase among the fractions or tissues nor with the extent of response in stimulation of DNA synthesis among the tissues. Inhibition of (3H) Isoproterenol binding to parotid fractions by catecholamine analogs was studied. There was no correlation between their ability to inhibit binding and the ability of the analogs themselves to raise cyclic AMP levels or stimulate DNA synthesis.

  4. Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide (/sup 3/H)thymidine incorporation into macrophages

    SciTech Connect

    Nagao, S.; Ikegami, S.; Tanaka, A.

    1984-12-01

    Guinea pig peritoneal exudate macrophages actively incorporated (/sup 3/H)thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of (/sup 3/H)thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The (/sup 3/H)thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of (/sup 3/H)thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized cold thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by (/sup 3/H)thymidine. These results suggest that the suppression of (/sup 3/H)thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.

  5. The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element.

    PubMed Central

    Aiyar, A; Tyree, C; Sugden, B

    1998-01-01

    Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences. PMID:9799247

  6. Suppression of dexamethasone-stimulated DNA synthesis in an oncogene construct containing rat cell line by a DNA site-oriented ligand of poly-ADP-ribose polymerase: 6-amino-1,2-benzopyrone

    SciTech Connect

    Kirsten, E.; Bauer, P.I.; Kun, E. San Francisco State Univ., CA )

    1991-03-01

    The cellular inhibitory effects of 6-amino-1,2-benzopyrone (6-ABP), a DNA site-specific ligand of adenosine diphosphoribosyl transferase (ADPRT), were determined in a dexamethasone-sensitive EJ-ras gene construct containing cell line (14C cells). Dexamethansone in vitro transforms these cells to a tumorigenic phenotype and also stimulates cell replication. AT a nontoxic concentration 6-ABP treatment of intact cells for 4 days inhibits the dexamethasone-stimulated increment of cellular DNA content, depresses replicative DNA synthesis as assayed by thymidine incorporation to the level of cells that were not exposed to dexamethasone, and in permeabilized cells reduces the dexamethasone-stimulated increase of deoxyribonucleotide incorporation into DNA to the level of untreated cells. In situ pulse labeling of cells pretreated with 6-ABP indicated an inhibition of DNA synthesis at a stage prior to the formation of the 10-kb intermediate species. Neither dexamethasone nor the drug influenced the cellular quantity of ADPRT molecules, tested immunochemically.

  7. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    PubMed

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  8. Click Reaction on Solid Phase Enables High Fidelity Synthesis of Nucleobase-Modified DNA.

    PubMed

    Tolle, Fabian; Rosenthal, Malte; Pfeiffer, Franziska; Mayer, Günter

    2016-03-16

    The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer.

  9. Design and synthesis of coumarin-3-acylamino derivatives to scavenge radicals and to protect DNA.

    PubMed

    Yang, Yang; Liu, Qing-Wen; Shi, Ye; Song, Zhi-Guang; Jin, Ying-Hua; Liu, Zai-Qun

    2014-09-12

    In this study, a series of coumarin-3-acylamino derivatives containing phenethylamine moiety or tyramine moiety were synthesized and their antioxidant activities were evaluated by Cu(2+)/glutathione(GSH)-, ˙OH- and 2,2'-azobis(2-amidinopropane hydrochloride)(AAPH)-induced oxidation of DNA. It was found that both hydroxyl and ortho-methoxy groups at A ring, hydroxyl group at B ring and peptide bond can enhance the abilities of coumarin-3-acylamino derivatives to protect DNA against ˙OH- and AAPH-induced oxidation. Moreover, these coumarin-3-acylamino derivatives were employed to scavenge 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(+˙)). We found that tyramine moiety, hydroxyl and ortho-methoxy are the key groups to enhance the activities of antioxidants to quench ABTS(+˙). Therefore, tyramine linked with coumarin-3-carboxyl acid which containing hydroxyl and ortho-methoxy exhibited powerful antioxidant abilities.

  10. Synthesis, spectroscopic characterization, DNA interaction and antibacterial study of metal complexes of tetraazamacrocyclic Schiff base

    NASA Astrophysics Data System (ADS)

    Shakir, Mohammad; Khanam, Sadiqa; Firdaus, Farha; Latif, Abdul; Aatif, Mohammad; Al-Resayes, Saud I.

    The template condensation reaction between benzil and 3,4-diaminotoulene resulted mononuclear 12-membered tetraimine macrocyclic complexes of the type, [MLCl2] [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The synthesized complexes have been characterized on the basis of the results of elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz. FT-IR, 1H and 13C NMR, FAB mass, UV-vis and EPR. An octahedral geometry has been envisaged for all these complexes, while a distorted octahedral geometry has been noticed for Cu(II) complex. Low conductivity data of all these complexes suggest their non-ionic nature. The interactive studies of these complexes with calf thymus DNA showed that the complexes are avid binders of calf thymus DNA. The in vitro antibacterial studies of these complexes screened against pathogenic bacteria proved them as growth inhibiting agents.

  11. Synthesis, cytotoxic activities and DNA binding properties of β-carboline derivatives.

    PubMed

    Chen, Zhiyong; Cao, Rihui; Yu, Liang; Shi, Buxi; Sun, Jie; Guo, Liang; Ma, Qin; Yi, Wei; Song, Xiao; Song, Huacan

    2010-11-01

    In a continuing effort to develop novel β-carbolines endowed with better pharmacological profile, a series of water-soluble β-carbolines bearing a flexible amino side chain was designed and synthesized, and the cytotoxic activities in vitro of these compounds were evaluated. The N(9)-arylated alkyl substituted β-carbolines represented the most interesting cytotoxic agents, and compounds 4c and 4d were found to be the most potent compounds with IC(50) values lower than 10 μM against ten human tumor cell lines. The results confirmed that the N(9)-arylated alkyl substituents of β-carboline played a very important role in the modulation of the cytotoxic potencies. In addition, the interaction with DNA of these compounds was also investigated, these compounds were found to exhibit significant DNA binding affinity.

  12. Synthesis of Small-Molecule/DNA Hybrids through On-Bead Amide-Coupling Approach.

    PubMed

    Okochi, Kenji D; Monfregola, Luca; Dickerson, Sarah Michelle; McCaffrey, Ryan; Domaille, Dylan W; Yu, Chao; Hafenstine, Glenn R; Jin, Yinghua; Cha, Jennifer N; Kuchta, Robert D; Caruthers, Marvin; Zhang, Wei

    2017-03-20

    Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.

  13. Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

    PubMed Central

    Berthet, Nathalie; Crey-Desbiolles, Caroline; Kotera, Mitsuharu; Dumy, Pascal

    2009-01-01

    The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue. PMID:19586934

  14. The use of hairpin DNA duplexes as HIV-1 fusion inhibitors: synthesis, characterization, and activity evaluation.

    PubMed

    Xu, Liang; Jiang, Xifeng; Xu, Xiaoyu; Zheng, Baohua; Chen, Xueliang; Zhang, Tao; Gao, Fang; Cai, Lifeng; Cheng, Maosheng; Keliang Liu

    2014-07-23

    Discovery of new drugs for the treatment of AIDS that possess unique structures associated with novel mechanisms of action are of great importance due the rapidity with which drug-resistant HIV-1 strains evolve. Recently we reported on a novel class of DNA duplex-based HIV-1 fusion inhibitors modified with hydrophobic groups. The present study describes a new category of hairpin fusion inhibitor DNA duplexes bearing a 3 nucleotide loop located at either the hydrophobic or hydrophilic end. The new loop structures were designed to link 2 separate duplex-forming oligodeoxynucleotides (ODNs) to make helix-assembly easier and more thermally stable resulting in a more compact form of DNA duplex based HIV-1 fusion inhibitors. A series of new hairpin duplexes were tested for anti-HIV-1 cell-cell membrane fusion activity. In addition, Tm, CD, fluorescent resonance energy transfer assays, and molecular modeling analyses were carried out to define their structural activity relationships and possible mechanisms of action.

  15. Synthesis, spectral characterization, DNA interaction, radical scavenging and cytotoxicity studies of ruthenium(II) hydrazone complexes.

    PubMed

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-05-01

    Three new ruthenium(II) complexes with hydrazone ligands, furan-2-carboxylic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), furan-2-carboxylic acid [4-(ethyl-propyl-amino)-2-hydroxy-benzylidene]-hydrazide (HL(2)) and furan-2-carboxylic acid (3-ethoxy-2-hydroxy-benzylidene)-hydrazide (HL(3)) were synthesized and characterized by various spectro-analytical techniques. The hydrazone ligands act as a tridendate ligand with ONO as the donor sites and are preferably found in the enol form in all the complexes. The molecular structure of the ligands was determined by single crystal X-ray diffraction technique. The interaction of the ligands and the complexes with CT-DNA were evaluated by an absorption titration method which revealed that the compounds interact with CT-DNA through intercalation. Gel electrophoresis assay demonstrated the ability of the complexes to cleave the calf thymus DNA hydrolytically. Antioxidant studies showed that the ruthenium(II) complexes have a strong radical-scavenging properties. Further, the cytotoxic effect of the compounds examined on cancerous cell lines showed that the complexes exhibited substantial anticancer activity.

  16. Tolerance of base pair size and shape in postlesion DNA synthesis.

    PubMed

    Gahlon, Hailey L; Schweizer, W Bernd; Sturla, Shana J

    2013-05-01

    The influence of base pair size and shape on the fidelity of DNA polymerase-mediated extension past lesion-containing mispairs was examined. Primer extension analysis was performed with synthetic nucleosides paired opposite the pro-mutagenic DNA lesion O(6)-benzylguanine (O(6)-BnG). These data indicate that the error-prone DNA polymerase IV (Dpo4) inefficiently extended past the larger Peri:O(6)-BnG base pair, and in contrast, error-free extension was observed for the smaller BIM:O(6)-BnG base pair. Steady-state kinetic analysis revealed that Dpo4 catalytic efficiency was strongly influenced by the primer:template base pair. Compared to the C:G pair, a 1.9- and 79,000-fold reduction in Dpo4 efficiency was observed for terminal C:O(6)-BnG and BIM:G base pairs respectively. These results demonstrate the impact of geometrical size and shape on polymerase-mediated mispair extension.

  17. Synthesis, DNA Binding and Antitumor Evaluation of Styelsamine and Cystodytin Analogues

    PubMed Central

    Fong, Hugo K. H.; Copp, Brent R.

    2013-01-01

    A series of N-14 sidechain substituted analogues of styelsamine (pyrido[4,3,2-mn]acridine) and cystodytin (pyrido[4,3,2-mn]acridin-4-one) alkaloids have been prepared and evaluated for their DNA binding affinity and antiproliferative activity towards a panel of human tumor cell lines. Overall it was found that styelsamine analogues were stronger DNA binders, with the natural products styelsamines B and D having particularly high affinity (Kapp 5.33 × 106 and 3.64 × 106 M−1, respectively). In comparison, the cystodytin iminoquinone alkaloids showed lower affinity for DNA, but were typically just as active as styelsamine analogues at inhibiting proliferation of tumor cells in vitro. Sub-panel selectivity towards non-small cell lung, melanoma and renal cancer cell lines were observed for a number of the analogues. Correlation was observed between whole cell activity and clogP, with the most potent antiproliferative activity being observed for 3-phenylpropanamide analogues 37 and 41 (NCI panel average GI50 0.4 μM and 0.32 μM, respectively) with clogP ~4.0–4.5. PMID:23358307

  18. Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis.

    PubMed Central

    Klaver, B; Berkhout, B

    1994-01-01

    Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides. Images PMID:7510065

  19. Homodinuclear lanthanide complexes of phenylthiopropionic acid: synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity.

    PubMed

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H(2)O(2). The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  20. Regiospecific Photocyclization of Mono- and Bis-Styryl-Substituted N-Heterocycles: A Synthesis of DNA-Binding Benzo[c]quinolizinium Derivatives.

    PubMed

    Aliyeu, Tseimur M; Berdnikova, Daria V; Fedorova, Olga A; Gulakova, Elena N; Stremmel, Christopher; Ihmels, Heiko

    2016-10-07

    Regiospecific C-N photocyclization of mono- and bis-styryl-substituted N-heterocycles was investigated. We demonstrated that the C-N regiospecificity of the photoinduced electrocyclization is a general feature of ortho-styryl-substituted N-heterocycles comprising one and two nitrogen atoms. This phototransformation provides a straightforward synthesis of the pharmaceutically important benzo[c]quinolizinium cation and its aza-analogues. Noticeably, bis-styryl derivatives undergo only one-fold cyclization with the second styryl fragment remaining uninvolved in the cyclization process. Photocyclization products of monostyryl derivativatives intercalate into calf thymus DNA (ct DNA), whereas photocyclization products of bis-styryl derivativatives possess a mixed binding mechanism with ct DNA. The results can be used for development of novel DNA-targeting chemotherapeutics based on benzo[c]quinolizinium derivatives.

  1. 75 FR 1382 - Wireless Telecommunications Bureau Seeks Comment on Petition for Reconsideration or Clarification...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-11

    ... From the Federal Register Online via the Government Publishing Office FEDERAL COMMUNICATIONS COMMISSION Wireless Telecommunications Bureau Seeks Comment on Petition for Reconsideration or Clarification of the Commission's Declaratory Ruling Clarifying Provisions in Section 332(c)(7) of...

  2. Study on the development of pomegranate juice processing technology: clarification of pomegranate juice.

    PubMed

    Vardin, Hasan; Fenercioğlu, Hasan

    2003-10-01

    The effects of clarification agents and methods on pomegranate juice quality were studied. Fruits were pressed as whole, and extracted juice was clarified with gelatin, polyvinylypolypyrrolidone (PVPP) and natural sedimentation. In order to determine the effects of these clarifying techniques on product quality, pH, total phenolic substances (TPS), anthocyanin, turbidity, and total colour density tests were applied to each sample. In clarification, the main purpose was to reduce the amount of phenolic substances. Phenolic substances were controlled in each clarification method. The most effective method was the application of 1 g/L gelatine for clarification. Sensory evaluation also applied for clarified juices and gelatin application was selected by panelists to be superior.

  3. 76 FR 59769 - Clarification on the Division 1.1 Fireworks Approvals Policy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-27

    ... Pipeline and Hazardous Materials Safety Administration Clarification on the Division 1.1 Fireworks Approvals Policy AGENCY: Pipeline and Hazardous Materials Safety Administration (PHMSA), DOT. ACTION... approvals policy whereby the Office of Hazardous Materials Safety (OHMS), Approvals and Permits...

  4. 76 FR 77453 - Clarification of Policy Regarding Designated Aircraft Dispatcher Examiners; Reopening of Comment...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-13

    ... Federal Aviation Administration 14 CFR Part 183 Clarification of Policy Regarding Designated Aircraft Dispatcher Examiners; Reopening of Comment Period AGENCY: Federal Aviation Administration (FAA), Department... action reopens the comment period for guidance material that was published for comment in the...

  5. Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

    PubMed

    Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

    2014-08-20

    DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

  6. Activated levels of rRNA synthesis in fission yeast are driven by an intergenic rDNA region positioned over 2500 nucleotides upstream of the initiation site.

    PubMed Central

    Liu, Z; Zhao, A; Chen, L; Pape, L

    1997-01-01

    RNA polymerase I-catalyzed synthesis of the Schizosaccharomyces pombe approximately 37S pre-rRNAs was shown to be sensitive to regulatory sequences located several kilobases upstream of the initiation site for the rRNA gene. An in vitro transcription system for RNA polymerase I-catalyzed RNA synthesis was established that supports correct and activated transcription from templates bearing a full S. pombe rRNA gene promoter. A 780 bp region starting at -2560 significantly stimulates transcription of ac is-located rDNA promoter and competes with an rDNA promoter in trans, thus displaying some of the activities of rDNA transcriptional enhancers in vitro. Deletion of a 30 bp enhancer-homologous domain in this 780 bp far upstream region blocked its cis-stimulatory effect. The sequence of the S. pombe 3.5 kb intergenic spacer was determined and its organization differs from that of vertebrate, Drosophila, Acanthamoeba and plant intergenic rDNA spacers: it does not contain multiple, imperfect copies of the rRNA gene promoter nor repetitive elements of 140 bp, as are found in vertebrate rDNA enhancers. PMID:9016610

  7. Effects of microinjected photoreactivating enzyme on thymine dimer removal and DNA repair synthesis in normal human and xeroderma pigmentosum fibroblasts

    SciTech Connect

    Roza, L.; Vermeulen, W.; Bergen Henegouwen, J.B.; Eker, A.P.; Jaspers, N.G.; Lohman, P.H.; Hoeijmakers, J.H. )

    1990-03-15

    UV-induced thymine dimers (10 J/m2 of UV-C) were assayed in normal human and xeroderma pigmentosum (XP) fibroblasts with a monoclonal antibody against these dimers and quantitative fluorescence microscopy. In repair-proficient cells dimer-specific immunofluorescence gradually decreased with time, reaching about 25% of the initial fluorescence after 27 h. Rapid disappearance of dimers was observed in cells which had been microinjected with yeast photoreactivating enzyme prior to UV irradiation. This photoreactivation (PHR) was light dependent and (virtually) complete within 15 min of PHR illumination. In general, PHR of dimers strongly reduces UV-induced unscheduled DNA synthesis (UDS). However, when PHR was applied immediately after UV irradiation, UDS remained unchanged initially; the decrease set in only after 30 min. When PHR was performed 2 h after UV exposure, UDS dropped without delay. An explanation for this difference is preferential removal of some type(s) of nondimer lesions, which is responsible for the PHR-resistant UDS immediately following UV irradiation. After the rapid removal of these photoproducts, the bulk of UDS is due to dimer repair. From the rapid effect of dimer removal by PHR on UDS it can be deduced that the excision of dimers up to the repair synthesis step takes considerably less than 30 min. Also in XP fibroblasts of various complementation groups the effect of PHR was investigated. The immunochemical dimer assay showed rapid PHR-dependent removal comparable to that in normal cells. However, the decrease of (residual) UDS due to PHR was absent (in XP-D) or much delayed (in XP-A and -E) compared to normal cells. This supports the idea that in these XP cells preferential repair of nondimer lesions does occur, but at a much lower rate.

  8. Phylogenomically Guided Identification of Industrially Relevant GH1 β-Glucosidases through DNA Synthesis and Nanostructure-Initiator Mass Spectrometry

    PubMed Central

    2015-01-01

    Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput mass spectrometry and apply it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using nanostructure-initiator mass spectrometry (NIMS), generating over 10,000 data points. When combined with HPLC-based sugar profiling, we observed GH1 enzymes active over a broad temperature range and toward many different β-linked disaccharides. For some GH1s we also observed activity toward laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pretreatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% (v/v) [C2mim][OAc] over the course of a 24-h saccharification reaction. Using our unbiased approach, we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems. PMID:24984213

  9. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes.

    PubMed

    Astakhova, I Kira; Wengel, Jesper

    2013-01-14

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2'-O-propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid-phase copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy-transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40-110 nm), quenched fluorescence of single-stranded probes accompanied by up to 7.7-fold light-up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single-nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM).

  10. PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity.

    PubMed

    Pal, Suman; Singh, Vandana; Das, Prolay; Choudhury, Lokman H

    2013-06-01

    Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The notable features of this protocol are: mild reaction condition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized compounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage under physiological conditions.

  11. Clarification of vaccines: An overview of filter based technology trends and best practices.

    PubMed

    Besnard, Lise; Fabre, Virginie; Fettig, Michael; Gousseinov, Elina; Kawakami, Yasuhiro; Laroudie, Nicolas; Scanlan, Claire; Pattnaik, Priyabrata

    2016-01-01

    Vaccines are derived from a variety of sources including tissue extracts, bacterial cells, virus particles, recombinant mammalian, yeast and insect cell produced proteins and nucleic acids. The most common method of vaccine production is based on an initial fermentation process followed by purification. Production of vaccines is a complex process involving many different steps and processes. Selection of the appropriate purification method is critical to achieving desired purity of the final product. Clarification of vaccines is a critical step that strongly impacts product recovery and subsequent downstream purification. There are several technologies that can be applied for vaccine clarification. Selection of a harvesting method and equipment depends on the type of cells, product being harvested, and properties of the process fluids. These techniques include membrane filtration (microfiltration, tangential-flow filtration), centrifugation, and depth filtration (normal flow filtration). Historically vaccine harvest clarification was usually achieved by centrifugation followed by depth filtration. Recently membrane based technologies have gained prominence in vaccine clarification. The increasing use of single-use technologies in upstream processes necessitated a shift in harvest strategies. This review offers a comprehensive view on different membrane based technologies and their application in vaccine clarification, outlines the challenges involved and presents the current state of best practices in the clarification of vaccines.

  12. Helicobacter pylori lipopolysaccharide alters ECL cell DNA synthesis via a CD14 receptor and polyamine pathway in mastomys.

    PubMed

    Kidd, M; Tang, L H; Schmid, S; Lauffer, J; Louw, J A; Modlin, I M

    2000-01-01

    Chronic Helicobacter pylori infection is associated with alterations in gastric mucosal cell proliferation. Despite the recognition that bacterial lipopolysaccharide (LPS) is present in biologically active quantities in the gastric mucosa, the mechanisms by which it stimulates cells are largely unknown. We have previously established a gastric enterochromaffin-like (ECL) cell neoplasia model in the African rodent species Mastomys and identified that tumor ECL cell proliferation is associated with polyamine biosynthesis and ornithine decarboxylase (ODC) activity. In addition, we have shown that H. pylori LPS exhibits a specific mitogenic effect on naive ECL cells in vitro. The aim of this study was to evaluate whether H. pylori has a direct effect on tumor ECL cell proliferation in vitro and further to evaluate the possible molecular mechanisms for this effect. ECL cell neoplasia was generated in Mastomys by endogenous hypergastrinemia induced by H(2) blockade (loxtidine 1 g/kg/day) and tumor ECL cells prepared. The DNA synthesis in 24-hour cultured tumor cells was measured by bromodeoxyuridine uptake and ODC activity by (14)CO(2) formation from (14)C-ornithine. The putative LPS receptor, CD14, was evaluated by reverse-transcription polymerase chain reaction. Our results demonstrated: (1) H. pylori LPS (10(-12) to 10(-7) M) stimulated basal DNA synthesis (2.2-fold) with an estimated EC(50) of 10(-10) M; (2) this proliferative response correlated with an increase in ODC activity (1.4-fold, EC(50) approximately 10(-10) M) which could be inhibited by a specific ODC inhibitor, difluoromethyl ornithine, at 10(-9) M; (3) the CD14 receptor was identified in both naive and transformed ECL cells by reverse-transcription polymerase chain reaction, and (4) the effects of LPS were inhibited by blocking the CD14 receptor with its specific monoclonal antibody (1:100). Thus, H. pylori LPS appears to influence tumor ECL cell proliferation by activation of the intracellular

  13. Hydrothermal synthesis of copper based nanoparticles: antimicrobial screening and interaction with DNA.

    PubMed

    Giannousi, K; Lafazanis, K; Arvanitidis, J; Pantazaki, A; Dendrinou-Samara, C

    2014-04-01

    Copper based nanoparticles (Cu-based NPs) of different compositions and sizes have been hydrothermally synthesized by varying the reaction time in the presence of the biocompatible surfactants polyoxyethylene (20) sorbitan laurate (Tween 20) and polyethylene glycol 8000 (PEG 8000). Effective control of the above synthetic parameters gave rise to Cu, Cu2O and Cu/Cu2O NPs of 10-44 nm. The antibacterial activity of the NPs was screened against Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) and Gram-negative (Xanthomonas campestris, Escherichia coli) bacteria. The Cu-based NPs induce pDNA degradation in a dose-dependent manner as well as extensive ds CT-DNA degradation. Cu2O NPs of 16 nm and 12 nm exhibit the lowest IC50 values (2.13 μg/mL and 3.7 μg/mL) against B. cereus and B. subtilis, respectively. The agarose gel electrophoresis of ds CT-DNA treated with Cu2O NPs demonstrated degradation at high concentration. In lower concentrations, viscosity measurements indicated groove binding. In regard to the enhanced antibacterial effect and specificity of Cu2O NPs against the Gram-positive strains, the activity pathway was further explored and ROS production and lipid peroxidation verified. The released copper ions 5.15 mg/L in distilled water and 16.32 mg/L in nutrient medium, found below the critical value to inhibit bacterial growth and thus nanosized composition effect is predominant.

  14. Macrocyclic nickel(II) complexes: Synthesis, characterization, superoxide scavenging activity and DNA-binding

    NASA Astrophysics Data System (ADS)

    Ramadan, Abd El-Motaleb M.

    2012-05-01

    A new series of nickel(II) complexes with the tetraaza macrocyclic ligand have been synthesized as possible functional models for nickel-superoxide dismutase enzyme. The reaction of 5-amino-3-methyl-1-phenylpyrazole-4-carbaldehyde (AMPC) with itself in the presence of nickel(II) ion yields, the new macrocyclic cationic complex, [NiL(NO3)2], containing a ligand composed of the self-condensed AMPC (4 mol) bound to a single nickel(II) ion. A series of metathetical reactions have led to the isolation of a number of newly complexes of the types [NiL]X2; X = ClO4 and BF4, [NiLX2], X = Cl and Br (Scheme 1). Structures and characterizations of these complexes were achieved by several physicochemical methods namely, elemental analysis, magnetic moment, conductivity, and spectral (IR and UV-Vis) measurements. The electrochemical properties and thermal behaviors of these chelates were investigated by using cyclic voltammetry and thermogravimetric analysis (TGA and DTG) techniques. A distorted octahedral stereochemistry has been proposed for the six-coordinate nitrato, and halogeno complexes. For the four-coordinate, perchlorate and fluoroborate, complex species a square-planar geometry is proposed. The measured superoxide dismutase mimetic activities of the complexes indicated that they are potent NiSOD mimics and their activities are compared with those obtained previously for nickel(II) complexes. The probable mechanistic implications of the catalytic dismutation of O2rad - by the synthesized nickel(II) complexes are discussed. The DNA-binding properties of representative complexes [NiLCl2] and [NiL](PF4)2 have been investigated by the electronic absorption and fluorescence measurements. The results obtained suggest that these complexes bind to DNA via an intercalation binding mode and the binding affinity for DNA follows the order: [NiLCl2] □ [NiL](PF4)2.

  15. Chloroplast Division and DNA Synthesis in Light-grown Wheat Leaves 1

    PubMed Central

    Boffey, Stephen A.; Ellis, J. Raymond; Selldén, Gun; Leech, Rachel M.

    1979-01-01

    Light-grown 7-day-old wheat seedlings (Triticum aestivum, var. Maris Dove) showed an increase of 200% in plastids per cell between 1.7 and 4.5 centimeters from the leaf base. This increase was the result of divisions of young chloroplasts at various stages of development, and was well separated in distance, and therefore in time from the region of cell division in the basal meristem. [3H]Thymidine was incorporated into plastid DNA throughout the zone of plastid division, but not above it. Images PMID:16660998

  16. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

    SciTech Connect

    Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W. )

    1991-02-15

    The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) (interleukin 10 (IL-10)). cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

  17. Synthesis of the DNA-[Ru(tpy)(dppz)(CH(3)CN)](2+) conjugates and their photo cross-linking studies with the complementary DNA strand.

    PubMed

    Ossipov, Dimitri; Gohil, Suresh; Chattopadhyaya, Jyoti

    2002-11-13

    We here report our studies on the conjugation of photoreactive Ru(2+) complex to oligonucleotides (ODNs), which give a stable duplex with the complementary target DNA strand. These functionalized DNA duplexes bearing photoreactive Ru(2+) complex can be specifically photolyzed to give the reactive aqua derivative, [Ru(tpy)(dppz)(H(2)O)](2+)-ODN (tpy = 2,2':6',2' '-terpyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine), in situ, which successfully cross-links to give photoproduct(s) in the duplex form with the target complementary DNA strand. Thus, the stable precursor of the aquaruthenium complex, the monofunctional polypyridyl ruthenium complex [Ru(tpy)(dppz)(CH(3)CN)](2+), has been site-specifically tethered to ODN, for the first time, by both solid-phase synthesis and postsynthetic modifications. (i) In the first approach, pure 3'-[Ru(tpy)(dppz)(CH(3)CN)](2+)-ODN conjugate has been obtained in 42% overall yield (from the monomer blocks) by the automated solid-phase synthesis on a support labeled with [Ru(tpy)(dppz)Cl](+) complex with subsequent liberation of the crude conjugate from the support under mild conditions and displacement of the Cl(-) ligand by acetonitrile in the coordination sphere of the Ru(2+) label. (ii) In the second approach, the single-modified (3'- or 5'- or middle-modified) or 3',5'-bis-modified Ru(2+)-ODN conjugates were prepared in 28-50% yield by an amide bond formation between an active ester of the metal complex and the ODNs conjugated with an amino linker. The pure conjugates were characterized unambiguously by ultraviolet-visible (UV-vis) absorption spectroscopy, enzymatic digestion followed by HPLC quantitation, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry (MALDI-TOF as well as by ESI). [Ru(tpy)(dppz)(CH(3)CN)](2+)-ODNs form highly stabilized ODN.DNA duplexes compared to the unlabeled counterpart (DeltaT(m) varies from 8.4 to 23.6 degrees C) as a result of intercalation of the dppz moiety; they undergo clean and

  18. Synthesis, characterization and DNA-binding properties of La(III) complex of chrysin.

    PubMed

    Zeng, Yi-Bo; Yang, Nan; Liu, Wei-Sheng; Tang, Ning

    2003-11-01

    A novel La(III) complex of chrysin (5,7-dihydroxyflavone) was synthesized and characterized by UV, IR, 1H NMR, thermogravimetry/differential thermal analysis (TG/DTA) and elementary analyses. The interactions of the La(III) complex and chrysin with calf thymus DNA were investigated by spectrophotometric methods and viscosity measurements. The intrinsic binding constants of La(III) complex and chrysin are 1.29 x 10(6) and 5.44 x 10(5) M(-1), respectively. Experimental results indicated that La(III) complex and chrysin can both bind to DNA by intercalation modes, but the binding affinity of La(III) complex is much higher than that of chrysin. Comparative antitumor activities of La(III) complex and chrysin were tested by MTT and SRB methods. The results show that at the concentration of 10 microM for chrysin and La(III) complex, the inhibitory ratios of La(III) complex against the tested tumor cells were higher than those of chrysin.

  19. A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors.

    PubMed

    Strobel, Heike; Dugué, Laurence; Marlière, Philippe; Pochet, Sylvie

    2002-12-02

    We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.

  20. Synthesis, structural characterization, cytotoxic properties and DNA binding of a dinuclear copper(II) complex.

    PubMed

    Ferreira, B J M Leite; Brandão, P; Meireles, M; Martel, Fátima; Correia-Branco, Ana; Fernandes, Diana M; Santos, T M; Félix, V

    2016-08-01

    In this study a novel dinuclear copper(II) complex with adenine and phenanthroline has been synthesized and its structure determined by single crystal X-ray diffraction. In the dinuclear complex [Cu₂(μ-adenine)₂(phen)₂(H2O)2](NO3)4·0.5H2O (phen=1,10-phenanthroline) (1) the two Cu(II) centres exhibit a distorted square pyramidal coordination geometry linked by two nitrogen donors from adenine bridges leading to a Cu-Cu distance of 3.242(3)Å. Intramolecular and intermolecular π⋯π interactions as well as an H-bonding network were observed. The antitumor capacity of the complex has been tested in vitro against human cancer cell lines, cervical carcinoma (HeLa) and colorectal adenocarcinoma (Caco-2), by metabolic tests, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide as reagent. The complex 1 has remarkable low IC50 values of 0.87±0.06μM (HeLa) and 0.44±0.06μM (Caco-2), when compared with values for cisplatin against the same cell lines. The interaction of complex 1 with calf thymus DNA (CT DNA) was further investigated by absorption and fluorescence spectroscopic methods. A binding constant of 5.09×10(5)M(-1) was obtained from UV-vis absorption studies.

  1. Exploration of DAPI analogues: Synthesis, antitrypanosomal activity, DNA binding and fluorescence properties.

    PubMed

    Farahat, Abdelbasset A; Kumar, Arvind; Say, Martial; Wenzler, Tanja; Brun, Reto; Paul, Ananya; Wilson, W David; Boykin, David W

    2017-03-10

    The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or heteroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in vitro and in vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with high affinity, and exhibit superior in vitro antitrypanosomal activity to that of DAPI. Six new diamidines (5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in vivo activity to that of DAPI and four of these compounds provide 100% animal cure at a low dose of 4 × 5 mg/kg i.p. in T. b. rhodesiense infected mice. Generally, the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.

  2. Novel Pt(II) complexes containing pyrrole oxime; synthesis, characterization and DNA binding studies

    NASA Astrophysics Data System (ADS)

    Erdogan, Deniz Altunoz; Özalp-Yaman, Şeniz

    2014-05-01

    Since the discovery of anticancer activity and subsequent clinical success of cisplatin (cis-[PtCl2(NH3)2]), platinum-based compounds have since been widely synthesized and studied as potential chemotherapeutic agents. In this sense, three novel nuclease active Pt(II) complexes with general formula; [Pt(NH3)Cl(L)] (1), [Pt(L)2] (2), and K[PtCl2(L)] (3) in which L is 1-H-pyrrole-2-carbaldehyde oxime were synthesized. Characterization of complexes was performed by elemental analysis, FT-IR, 1H NMR and mass spectroscopy measurements. Interaction of complexes (1-3) with calf thymus deoxyribonucleic acid (ct-DNA) was investigated by using electrochemical, spectroelectrochemical methods and cleavage studies. The hyperchromic change in the electronic absorption spectrum of the Pt(II) complexes indicates an electrostatic interaction between the complexes and ct-DNA. Binding constant values between 4.42 × 103 and 5.09 × 103 M-1 and binding side size values between 2 and 3 base pairs were determined from cyclic voltammetry (CV) and differential pulse voltammetry (DPV) studies.

  3. Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effects of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes.

    PubMed

    Sjöblom, T; Parvinen, M; Lähdetie, J

    1995-10-01

    We have studied the effects of three known mutagens: vinblastine sulphate, mitomycin C and local irradiation of testes on the stage-specific DNA synthesis in the rat testis by using transillumination assisted microdissection of rat seminiferous tubules. It enables us to investigate the sensitivity of different types of spermatogonia and preleptotene spermatocytes to the genotoxic effects of these agents. According to our results, spermatogonia and preleptotene spermatocytes are quite resistant to the action of vinblastine at the treatment times and the doses used. After treatment with mitomycin C, type A2, A3 and A4 spermatogonia seem to be the first cell types affected, which shows itself as a reduction in the DNA synthesis at stages I, II-III, XIII-XIV of the epithelial cycle two and/or three days after the treatment. It also seems that they are mostly affected during the S-phase of their cell cycles. In addition, preleptotene spermatocytes are also sensitive to the action of mitomycin C when they are treated in the G1 phase of the cell cycle. The local irradiation of 3 Gy has severe effects on the spermatogonia of rat testis which can be seen already 18 h after the treatment and becomes more evident 42 and 66 h after the treatment as a reduction of DNA synthesis at stages XII-V. Type A spermatogonia (A1-A4) seem to be the most sensitive cell types to the action of irradiation. This study indicates that the novel method of stage-specific DNA synthesis in rat spermatogenesis allows detailed studies of sensitivities in differentiating spermatogonia to genotoxic agents.

  4. Phorbol ester and B cell-stimulatory factor synergize to induce B-chronic lymphocytic leukemia cells to simultaneous immunoglobulin secretion and DNA synthesis.

    PubMed

    Carlsson, M; Matsson, P; Rosén, A; Sundström, C; Tötterman, T H; Nilsson, K

    1988-11-01

    This paper discusses the response of two B cell-type chronic lymphocytic leukemia (B-CLL) clones, 173 and 183, to the phorbol ester TPA combined with a B cell-stimulatory factor (BSF) derived from a T helper cell hybridoma (MP6). Previous studies with 173 and 183 cells have consistently shown that TPA alone induces differentiation but no proliferation. However, when the two clones were exposed to TPA plus BSF-MP6, not only differentiation but also DNA synthesis was observed. Compared with TPA exposure alone, the fraction of cells with induced lymphoblastoid-plasmacytoid morphology increased and Ig secretion was enhanced. By a 1-hr TPA pulse followed by BSF-MP6, the DNA synthesis was further augmented, but less maturation was observed. T cell and monocyte removal, using cell sorting, showed that the DNA synthesis induced was independent of these cell types, also under serum-free conditions. Quantitation of several cell cycle-associated surface Ags showed that the 4F2, Ba, Bac-1, and cD23 Ags increased while the CD37 decreased in expression upon addition of BSF-MP6. We conclude that B-CLLs are inducible by TPA and BSF-MP6 not only to differentiation, but also to DNA synthesis even under serum-free conditions in vitro. The results furthermore suggest that the very low proliferation activity in B-CLL tumors in vivo may reflect a relative deficiency of proper growth and differentiation factors or a subnormal response of B-CLL cells to such factors.

  5. Lysophosphatidic acid induces both EGFR-dependent and EGFR-independent effects on DNA synthesis and migration in pancreatic and colorectal carcinoma cells.

    PubMed

    Tveteraas, Ingun Heiene; Aasrum, Monica; Brusevold, Ingvild Johnsen; Ødegård, John; Christoffersen, Thoralf; Sandnes, Dagny

    2016-02-01

    Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.

  6. Corrigendum to "Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate" [J. Mol. Struct. 1093 (2015) 135-143

    NASA Astrophysics Data System (ADS)

    Iqbal, Muhammad; Ali, Saqib; Tahir, Muhammad Nawaz; Muhammad, Niaz; Shah, Naseer Ali; Sohail, Manzar; Pandarinathan, Vedapriya

    2017-04-01

    The authors regret to inform that Scheme 1 in the article titled 'Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate' in vol. 1093 of the Journal of Molecular Structure is incorrect. The corrected scheme is as shown in this correction. This is purely a copy error. The error does not affect the conclusion in paper. The authors would like to apologize for any inconvenience caused.

  7. Metformin inhibition of mTORC1 activation, DNA synthesis and proliferation in pancreatic cancer cells: Dependence on glucose concentration and role of AMPK

    SciTech Connect

    Sinnett-Smith, James; Kisfalvi, Krisztina; Kui, Robert; Rozengurt, Enrique

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Metformin inhibits cancer cell growth but the mechanism(s) are not understood. Black-Right-Pointing-Pointer We show that the potency of metformin is sharply dependent on glucose in the medium. Black-Right-Pointing-Pointer AMPK activation was enhanced in cancer cells incubated in physiological glucose. Black-Right-Pointing-Pointer Reciprocally, metformin potently inhibited mTORC1, DNA synthesis and proliferation. Black-Right-Pointing-Pointer Metformin, at low concentrations, inhibited DNA synthesis through AMPK. -- Abstract: Metformin, a widely used anti-diabetic drug, is emerging as a potential anticancer agent but the mechanisms involved remain incompletely understood. Here, we demonstrate that the potency of metformin induced AMPK activation, as shown by the phosphorylation of its substrates acetyl-CoA carboxylase (ACC) at Ser{sup 79} and Raptor at Ser{sup 792}, was dramatically enhanced in human pancreatic ductal adenocarcinoma (PDAC) cells PANC-1 and MiaPaCa-2 cultured in medium containing physiological concentrations of glucose (5 mM), as compared with parallel cultures in medium with glucose at 25 mM. In physiological glucose, metformin inhibited mTORC1 activation, DNA synthesis and proliferation of PDAC cells stimulated by crosstalk between G protein-coupled receptors and insulin/IGF signaling systems, at concentrations (0.05-0.1 mM) that were 10-100-fold lower than those used in most previous reports. Using siRNA-mediated knockdown of the {alpha}{sub 1} and {alpha}{sub 2} catalytic subunits of AMPK, we demonstrated that metformin, at low concentrations, inhibited DNA synthesis through an AMPK-dependent mechanism. Our results emphasize the importance of using medium containing physiological concentrations of glucose to elucidate the anticancer mechanism of action of metformin in pancreatic cancer cells and other cancer cell types.

  8. Effect of various doses of chalone-containing alcohol precipitate from Ehrlich's ascites tumor on mitotic activity and DNA synthesis in that tumor

    SciTech Connect

    Matsak, N.Ya.; Romanov, Yu.A.; Antokhin, A.I.

    1987-06-01

    Experiments were carried out on noninbred male albino mice to assess the effects of varying doses of chalone-containing alcohol precipitates on mitotic activity and DNA synthesis in Ehrlich ascites tumor. Histological preparations of the tumor and small intestine were made by standard methods. Hydrolysis of the tumor in HCl was carried out before application of the nuclear photographic emulsion in order to prepare autoradiographs of the tumor. Tritium-labelled thymidine was injected as the radioactive marker.

  9. Isolation of a fraction from cauliflower mosaic virus-infected protoplasts which is active in the synthesis of (+) and (-) strand viral DNA and reverse transcription of primed RNA templates.

    PubMed Central

    Thomas, C M; Hull, R; Bryant, J A; Maule, A J

    1985-01-01

    Sub-cellular fractions, isolated from cauliflower mosaic virus (CaMV)-infected turnip protoplasts, are capable of synthesising CaMV DNA in vitro on an endogenous template and of reverse transcribing oligo dT-primed cowpea mosaic virus RNA. The activity was not detected in mock-inoculated protoplasts. In vitro-labelled DNA hybridized to single-stranded M13 clones complementary to the putative origins of (-) and (+) strand CaMV DNA synthesis and to restriction endonuclease fragments encompassing more than 90% of the CaMV genome. The synthesis of (-) and (+) strand DNA appeared asymmetric. The template(s) for in vitro CaMV DNA synthesis are in a partially nuclease-resistant form. Fractions capable of in vitro CaMV DNA synthesis contained CaMV RNA both heterogeneous and as discrete species; they also contained a range of different sizes of CaMV DNA. Several lines of evidence indicate that this range of in vitro-labelled CaMV DNA, extending from 0.6kb to 8.0kb in length, represents elongating (-) strand DNA. These are discussed in relation to their role as possible replicative intermediates. Images PMID:2409536

  10. N-Acetyl-seryl-aspartyl-lysyl-proline inhibits DNA synthesis in human mesangial cells via up-regulation of cell cycle modulators

    SciTech Connect

    Kanasaki, Keizo; Haneda, Masakazu; Sugimoto, Toshiro . E-mail: toshiro@belle.shiga-med.ac.jp; Shibuya, Kazuyuki; Isono, Motohide; Isshiki, Keiji; Araki, Shin-ichi; Uzu, Takashi; Kashiwagi, Atsunori; Koya, Daisuke

    2006-04-14

    N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) was originally reported as a natural inhibitor of the proliferation of stem cells. To elucidate whether Ac-SDKP inhibits the proliferation of human mesangial cells, we examined the effect of Ac-SDKP on fetal calf serum (FCS)- or platelet-derived growth factor (PDGF)-BB-induced DNA synthesis and a cell proliferation. Ac-SDKP inhibited PDGF-BB- or FCS-induced DNA synthesis without cellular toxicity. The protein expression of p53 and p27{sup kip1} was significantly increased by Ac-SDKP. Ac-SDKP also up-regulated the PDGF-BB-stimulated expression of p21{sup cip1} and suppressed PDGF-BB-induced cyclin D{sub 1} expression. In p53 knock-out human mesangial cells made with small interference RNA, the protein expression of p21{sup cip1} and p27{sup kip1} was also decreased and the inhibitory effect of Ac-SDKP on mesangial proliferation was completely abolished. Ac-SDKP increased the stability of p53 protein as demonstrated by pulse-chase experiment. These results suggest that p53 is the key mediator of Ac-SDKP-induced inhibition of DNA synthesis through the up-regulation of cell cycle modulators, highlighting a potential effect of Ac-SDKP on various progressive renal diseases.

  11. Effects of antiinflammatory agents on mouse skin tumor promotion, epidermal DNA synthesis, phorbol ester-induced cellular proliferation, and production of plasminogen activator.

    PubMed

    Viaje, A; Slaga, T J; Wigler, M; Weinstein, I B

    1977-05-01

    The antinflammatory ateroids fluocinoine acetonide, fluocinonide, and fluclorolone acetonide were found to be very effectiveinhibitory agents of mouse skin tumor promotion. These steroids also drastically inhibited epidermal DNA synthesis and epidermal cellular proliferation induced by a phorbal ester tumor promoter. In addition, these compounds were potent inhibitors, of plasminogen activator production in tumor cell cultures. The clinically used non-steroidal antiinflammatory agents oxyphenbutazone, indomethacin, and Seclazone also inhibite tumor promotion but were much less effective. Although these agents are useful against inflammatory disorders in general when given p.o., in our studies they had little effect on inflammation and epidermal cellular proliferation induced by a phorbol ester tumor promoter when given topically. The afore mentioned nonsteroidal antiinflammatory agents also had little effect on epidermal DNA synthesis. Oxyphenbutazone and indomethacin were less potent inhibitors of plasminogen activator production in tumor cells than were the antiinflammatory steroids, and Seclazone produced a negligible inhibition. There is, therefore, a general correlation in the potencies of a series of steroidal antiinflammatory agents for inhibition of tumor promotion and their ability to inhibit plasminogen activator production by tumor cell cultures and epidermal DNA synthesis.

  12. The epidermal growth factor-induced migration of rat liver epithelial cells is associated with a transient inhibition of DNA synthesis.

    PubMed

    Geimer, P; Bade, E G

    1991-10-01

    Epidermal growth factor (EGF) is a potent mitogen for most cultured cells and has previously been shown to induce the migration of rat liver epithelial cells. We have now demonstrated that under migration-inducing conditions EGF does not stimulate cell proliferation, but causes instead a transient inhibition of DNA synthesis. Analysis at the single-cell level by [3H]thymidine autoradiography indicated that in 40-50% of the EGF-treated cell population the entry into S phase is delayed. The simultaneous demonstration of migration tracks by laminin immunofluorescence revealed that the transient inhibition of DNA synthesis is not restricted to the migratory cells. The effect is also observed with the stationary subpopulation and appears, therefore, to be independent of the induction of migration. The independence of both processes was further supported by showing that induction of migration by EGF proceeds undisturbed in cells blocked in S phase by aphidicolin. These results indicated that for rat liver epithelial cells the induction of migration by EGF has priority over cell proliferation. The data also emphasize the need for a time-course analysis when studying factors that stimulate or inhibit DNA synthesis or cell proliferation.

  13. Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

    PubMed Central

    Hamilton, J A; Jessup, W; Brown, A J; Whitty, G

    2001-01-01

    Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo. PMID:11256965

  14. Irreversible repression of DNA synthesis in Fanconi anemia cells is alleviated by the product of a novel cyclin-related gene.

    PubMed Central

    Digweed, M; Günthert, U; Schneider, R; Seyschab, H; Friedl, R; Sperling, K

    1995-01-01

    Primary fibroblasts from patients with the genetic disease Fanconi anemia, which are hypersensitive to cross-linking agents, were used to screen a cDNA library for sequences involved in their abnormal cellular response to a cross-linking challenge. By using library partition and microinjection of in vitro-transcribed RNA, a cDNA clone, pSPHAR (S-phase response), which is able to correct the permanent repression of semiconservative DNA synthesis rates characteristic of these cells, was isolated. Wild-type SPHAR mRNA is expressed in all fibroblasts so far analyzed, including those of Fanconi anemia patients. Correction of the abnormal response in these cells appears therefore to be due to overexpression after cDNA transfer rather than to genetic complementation. The cDNA contains an open reading frame coding for a polypeptide of 7.5 kDa. Rabbit antiserum directed against a SPHAR peptide detects a protein of 7.9 kDa in Western blots (immunoblots) of whole-cell extracts from proliferating, but not resting, fibroblasts. The deduced amino acid sequence of SPHAR contains a motif found in the cyclins, and it is proposed that SPHAR acts within the injected cell by interfering with the cyclin-controlled maintenance of S phase. In agreement with this proposal, normal cells transfected with an antisense SPHAR expression vector have a significantly reduced rate of DNA synthesis during S phase and a prolonged G2 phase, reflecting the need for postreplicative DNA processing before entry into mitosis. PMID:7799938

  15. Kinetics of the inhibition and recovery of semiconservative DNA synthesis following the induction of non-dimer DNA damages by solar ultraviolet radiation in ICR 2A frog cells

    SciTech Connect

    Rosenstein, B.S.

    1985-01-01

    The purpose of the work presented in this paper was to investigate the kinetics of inhibition and recovery of DNA synthesis in cells following the solar UV induction of non-dimer DNA damages. This work was accomplished by exposing cells to the Mylar-filtered solar UV wavelengths produced by fluorescent sunlamps. The ICR 2A frog cell line was chosen for this study because it is highly proficient in enzymatic photoreactivation. Hence, it was possible to induce a relatively pure population of non-dimer DNA photoproducts by exposing the sunlamp-irradiated cells to photoreactivating light (PRL), which specifically eliminates most of the small yield of dimers induced by the solar UV irradiation.

  16. β-diketone-cobalt complexes inhibit DNA synthesis and induce S-phase arrest in rat C6 glioma cells.

    PubMed

    Zhang, Kaizhi; Zhao, Xingli; Liu, Junzhi; Fang, Xiangyang; Wang, Xuepeng; Wang, Xiaohong; Li, Rui

    2014-03-01

    β-diketone-cobalt complexes, a family of newly synthesized non-platinum metal compounds, exhibit potential antitumor activity; however, the antitumor mechanism is unclear. The current study investigated the mechanism by which β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation. It was found that β-diketone-cobalt complexes suppress rat C6 glioma cell viability in a dose-dependent manner (3.125-100 μg/ml). In rat C6 glioma cells, the IC50 value of β-diketone-cobalt complexes was 24.7±3.395 μg/ml and the IC10 value was 4.37±1.53 μg/ml, indicating a strong inhibitory effect. Further investigation suggested that β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation, which is associated with S-phase arrest and DNA synthesis inhibition. During this process, β-diketone-cobalt complexes decreased cyclin A expression and increased cyclin E and p21 expression. In addition, β-diketone-cobalt complexes exhibit a stronger antitumor capability than the antineoplastic agent, 5-fluorouracil.

  17. A DNA synthesis inhibitor is protective against proteotoxic stressors via modulation of fertility pathways in Caenorhabditis elegans

    PubMed Central

    Angeli, Suzanne; Klang, Ida; Sivapatham, Renuka; Mark, Karla; Zucker, David; Bhaumik, Dipa; Lithgow, Gordon J.; Andersen, Julie K.

    2013-01-01

    Loss of germline precursor cells in C. elegans has previously been shown to improve protein homeostasis and extend lifespan, possibly due to reallocation of resources to somatic cells. In contrast, mutants that are sterile simply due to loss of sperm or oocyte production have a normal lifespan, often leading to the conclusion that loss of reproduction per se may have minor effects on C. elegans. We have found that inhibiting reproduction in C. elegans via the DNA synthesis inhibitor 5-fluoro-2-deoxyuridine (FUdR) improves protein homeostasis, stress resistance, and healthspan in wild-type animals. We find that FUdR is dependent on oogenesis and oocytic maturation. The effects of FUdR are dependent on FEM pathways, which regulate initiation of spermatogenesis. Loss of FEM expression leads to feminized animals that maintain arrested oocytes and are refractory to the effects of FUdR. FUdR-dependence is restored by spermatogenic signals, which trigger oocytic maturation and ovulation. Further, loss of FEM-3, a novel protein required for spermatogenesis, is sufficient to improve aspects of proteostasis. These effects are independent of previously described germline signals, including the DAF-16/FOXO, DAF-12/VDR, and HSF-1 pathways. These findings suggest that genetic or chemical inhibition of oocyte production can improve protein homeostasis in C. elegans. PMID:24123581

  18. Microwave assisted synthesis, spectroscopic, electrochemical and DNA cleavage studies of lanthanide(III) complexes with coumarin based imines.

    PubMed

    Kapoor, Puja; Fahmi, Nighat; Singh, R V

    2011-12-01

    The present work stems from our interest in the synthesis, characterization and biological evaluation of lanthanide(III) complexes of a class of coumarin based imines which have been prepared by the interaction of hydrated lanthanide(III) chloride with the sodium salts of 3-acetylcoumarin thiosemicarbazone (ACTSZH) and 3-acetylcoumarin semicarbazone (ACSZH) in 1:3 molar ratio using thermal as well as microwave method. Characterization of the ligands as well as the metal complexes have been carried out by elemental analysis, melting point determinations, molecular weight determinations, magnetic moment, molar conductance, IR, (1)H NMR, (13)C NMR, electronic, EPR, X-ray powder diffraction and mass spectral studies. Spectral studies confirm ligands to be monofunctional bidentate and octahedral environment around metal ions. The redox behavior of one of the synthesized metal complex was investigated by cyclic voltammetry. Further, free ligands and their metal complexes have been screened for their antimicrobial as well as DNA cleavage activity. The results of these findings have been presented and discussed.

  19. Nitrated polycyclic aromatic hydrocarbons: potent bacterial mutagens and stimulators of DNA repair synthesis in cultured human cells.

    PubMed

    Campbell, J; Crumplin, G C; Garner, J V; Garner, R C; Martin, C N; Rutter, A

    1981-01-01

    Ten polycyclic aromatic hydrocarbons (PAHs), viz. anthracene pyrene, chrysene, perylene, fluoranthene, benzo[a]pyrene, benzo[a]pyrene, benz[a]anthracene, benzo[ghi]perylene, benzo[k]fluoranthene, have been nitrated using concentrated nitric acid and the crude nitrated mixture examined for biological activity. All the nitro PAHs examined were mutagenic to Salmonella typhimurium in the absence of a rat liver preparation. Addition of Aroclor-1254 induced liver had little effect on mutagenicity. Mutagenic potency differed for the various nitrated mixtures with nitrated pyrene and nitrated fluoranthene the most potent and nitrated anthracene the least potent. Both frame-shift and base-substitution mutations were induced by the nitrated PAHs. The nitrated PAHs were also able to induce DNA repair synthesis in cultured HeLa cells in the absence of liver, indicating that these cells have the necessary enzymes to activate nitro PAHs. Potency again varied from compound to compound with nitrated pyrene appearing to be the most active. Isolation of individual components from the crude nitrated mixtures has not been carried out in this study. In view of the possible wide-spread distribution of nitrated PAHs in the environment further work is required to assess the carcinogenic potency of these compounds which possibly pose a risk to man.

  20. Synthesis, characterization, DNA interactions and antiproliferative activity on glioblastoma of iminopyridine platinum(II) chelate complexes.

    PubMed

    Posadas, Inmaculada; Alonso-Moreno, Carlos; Bravo, Iván; Carrillo-Hermosilla, Fernando; Garzón, Andrés; Villaseca, Noemí; López-Solera, Isabel; Albaladejo, José; Ceña, Valentín

    2017-03-01

    A series of iminopyridine platinum chelate compounds has been prepared and characterized by NMR spectroscopy and single-crystal X-ray diffraction. The complexes were evaluated in C6 tumoral cells as an in vitro model for glioblastoma multiforme. The DNA-binding properties of these complexes were studied by UV-Vis absorption and fluorescence spectroscopy and Density Functional Theory calculations were performed in an effort to rationalize the observed properties at the molecular level. The most promising drug candidate displayed a similar potency in inducing cell death to the clinically used reference compound and showed significant inhibition of glioblastoma cell proliferation. Moreover, this compound had a safer profile than cisplatin on non-tumoral cells.

  1. Copper(I)-Phosphine Polypyridyl Complexes: Synthesis, Characterization, DNA/HSA Binding Study, and Antiproliferative Activity.

    PubMed

    Villarreal, Wilmer; Colina-Vegas, Legna; Visbal, Gonzalo; Corona, Oscar; Corrêa, Rodrigo S; Ellena, Javier; Cominetti, Marcia Regina; Batista, Alzir Azevedo; Navarro, Maribel

    2017-04-03

    A series of copper(I)-phosphine polypyridyl complexes have been investigated as potential antitumor agents. The complexes [Cu(PPh3)2dpq]NO3 (2), [Cu(PPh3)2dppz]NO3 (3), [Cu(PPh3)2dppa]NO3 (4), and [Cu(PPh3)2dppme]NO3 (5) were synthesized by the reaction of [Cu(PPh3)2NO3] with the respective planar ligand under mild conditions. These copper complexes were fully characterized by elemental analysis, molar conductivity, FAB-MS, and NMR, UV-vis, and IR spectroscopies. Interactions between these copper(I)-phosphine polypyridyl complexes and DNA have been investigated using various spectroscopic techniques and analytical methods, such as UV-vis titrations, thermal denaturation, circular dichroism, viscosity measurements, gel electrophoresis, and competitive fluorescent intercalator displacement assays. The results of our studies suggest that these copper(I) complexes interact with DNA in an intercalative way. Furthermore, their high protein binding affinities toward human serum albumin were determined by fluorescence studies. Additionally, cytotoxicity analyses of all complexes against several tumor cell lines (human breast, MCF-7; human lung, A549; and human prostate, DU-145) and non-tumor cell lines (Chinese hamster lung, V79-4; and human lung, MRC-5) were performed. The results revealed that copper(I)-phosphine polypyridyl complexes are more cytotoxic than the corresponding planar ligand and also showed to be more active than cisplatin. A good correlation was observed between the cytostatic activity and lipophilicity of the copper(I) complexes studied here.

  2. Synthesis, crystal structure and electrochemical and DNA binding studies of oxygen bridged-copper(II) carboxylate

    NASA Astrophysics Data System (ADS)

    Iqbal, Muhammad; Ali, Saqib; Tahir, Muhammad Nawaz; Muhammad, Niaz; Shah, Naseer Ali; Sohail, Manzar; Pandarinathan, Vedapriya

    2015-08-01

    A new binuclear O-bridged Cu(II) complex with 4-chlorophenyl acetate and 2,2‧-bipyridine has been synthesized and characterized using FT-IR, powder and single crystal XRD and electrochemical solution studies. The results revealed that the two penta-coordinated Cu(II) centers are linked by two carboxylate ligands in end-on bonding fashion. The coordination geometry is slightly distorted square pyramidal (SP) with bridging oxygen atoms occupying the apical position and other ligands lying in the equatorial plane. The striking difference in Cu-O bond distance of the bridging oxygen atom in the complex may be responsible for the SP geometry of Cu(II) ion. The complex gave rise to metal centered irreversible electro-activity where one electron Cu(II)/Cu(III) oxidation process and a single step two electron Cu(II)/Cu(0) reduction process was observed. The redox processes were found predominantly adsorption controlled. The values of diffusion coefficient and heterogeneous rate constant for oxidation process were 6.98 × 10-7 cm2 s-1 and 4.60 × 10-5 cm s-1 while the corresponding values for reduction were 5.30 × 10-8 cm2 s-1 and 5.41 × 10-6 cm s-1, respectively. The formal potential and charge transfer coefficient were also calculated. The DNA-binding ability was explored through cyclic voltammetry and UV-Visible spectroscopy. Diminution in the value of Do for oxidation indicated the binding of the complex with DNA corresponding to Kb = 8.58 × 104 M-1. UV-Visible spectroscopy yielded ε = 49 L mol-1 cm-1 and Kb = 2.96 × 104 M-1. The data of both techniques support each other. The self-induced redox activation of the complex, as indicated by cyclic voltammetry heralds its potential applications in redox catalysis and anticancer activity.

  3. Electron Microscopic Analysis of the Products of DNA Synthesis by DNA Polymerases from Calf Thymus and Herpes Simplex Virus Type I

    DTIC Science & Technology

    1988-10-03

    E . Coli single stranded binding (SSB) protein and ethidium bromide. The electron microscopic analysis of the replicative intermediates from the calf thymus DNA primase primed M13 DNA replication showed an average of 2.5 primers per M13 DNA circle. The measurement of the double stranded length from individual replicative intermediates by electron microscopy was within the accuracy of 10% standard deviation. The product length distribution obtained from the HSV-1 DNA polymerase catalyzed replication of M13 DNA primed with a specific pentadecamer and in the presence of E

  4. Peer-assisted learning: time for nomenclature clarification

    PubMed Central

    Olaussen, Alexander; Reddy, Priya; Irvine, Susan; Williams, Brett

    2016-01-01

    Background Peer-assisted learning (PAL) is used throughout all levels of healthcare education. Lack of formalised agreement on different PAL programmes may confuse the literature. Given the increasing interest in PAL as an education philosophy, the terms need clarification. The aim of this review is to 1) describe different PAL programmes, 2) clarify the terminology surrounding PAL, and 3) propose a simple pragmatic way of defining PAL programmes based on their design. Methods A review of current PAL programmes within the healthcare setting was conducted. Each programme was scrutinised based on two aspects: the relationship between student and teacher, and the student to teacher ratio. The studies were then shown to fit exclusively into the novel proposed classification. Results The 34 programmes found, demonstrate a wide variety in terms used. We established six terms, which exclusively applied to the programmes. The relationship between student and teacher was categorised as peer-to-peer or near-peer. The student to teacher ratio suited three groupings, named intuitively ‘Mentoring’ (1:1 or 1:2), ‘Tutoring’ (1:3–10), and ‘Didactic’ (1:>10). From this, six novel terms – all under the heading of PAL – are suggested: ‘Peer Mentoring’, ‘Peer Tutoring’, ‘Peer Didactic’, ‘Near-Peer Mentoring’, ‘Near-Peer Tutoring’, and ‘Near-Peer Didactic’. Conclusions We suggest herein a simple pragmatic terminology to overcome ambiguous terminology. Academically, clear terms will allow effective and efficient research, ensuring furthering of the educational philosophy. PMID:27415590

  5. Synthesis and properties of new DNA cleavage agents based on oxoruthenium(IV)

    SciTech Connect

    Gupta, N.; Grover, N.; Neyhart, G.A.; Singh, P.; Thorp, H.H. )

    1993-02-03

    New aquaruthenium(II) reagents that are capable of being oxidized to hydrooxoruthenium(III) and oxoruthenium(IV) have been prepared. Complexes based on Ru(tpy)(L)OH[sub 2][sup 2+] (L = [eta][sup 2]-tpt, phen, dppz, tmen; tpy = 2,2[prime]:6[prime],2[double prime]-terpyridine, tpt = 2,4,6-tripyridyltriazine, phen = 1,10-phenanthroline, dppz = dipyridophenazine, and tmen = N,N,N[prime],N[prime]-tetramethylethylenediamine) have been prepared and can all be reversibly oxidized to their Ru[sup IV]O forms, which are component DNA cleavage agents, as in Ru(phen)[sub 2](py)O[sup 2+]. In addition to Ru(tpy)([eta][sup 2]-tpt)OH[sub 2][sup 2+], the [eta][sup 3] complex of tpt, Ru(tpy)([eta][sup 3]-tpt)[sup 2+], can also be prepared under similar conditions. In the presence of Ag[sup +] ion, a novel Ru[sub 2]Ag complex can be isolated and has been crystallographically characterized. The complex [Ru(tpy)([eta][sup 3]-tpt)](ClO[sub 4])[sub 2][center dot]0.5AgClO[sub 4][center dot]0.5H[sub 2]O crystallizes in the monoclinic space group A2/A with a = 14.723 (5) [Angstrom], b = 26.061 (6) [Angstrom], c = 22.148 (6) [Angstrom], [beta] = 106.33 (3)[degrees], V = 8155 (5) [Angstrom][sup 3], Z = 4, R = 0.0807, and R[sub w] = 0.1156 for 2,923 reflections with I [ge] 2[sigma](I). The Ru(tpy)OH[sub 2][sup 2+] unit can also be attached to the tmen-AO[sup +] ligand, where a N,N[prime],N[prime]-trimethylethylenediamine function is appended via a (CH[sub 2])[sub 6] linker to the acridine orange intercalator. The Ru(tpy)(tmen-AO)OH[sub 2][sup 3+] complex is an effective cleavage agent, but only when oxidation is performed on the complex prebound to DNA. In homogeneous solution, electrochemically reversible access of only the Ru[sup III]OH form is possible, probably because of oxidation of the polymethylene linker. 25 refs., 11 figs., 9 tabs.

  6. Synthesis and characterization of low molecular weight polyethyleneimine-terminated Poly(β-amino ester) for highly efficient gene delivery of minicircle DNA.

    PubMed

    Zhao, Jing; Yang, Lei; Huang, Ping; Wang, ZhiYong; Tan, Yan; Liu, Hong; Pan, JiaJia; He, Cheng-Yi; Chen, Zhi-Ying

    2016-02-01

    Gene therapy has held great promise for treating specific acquired and inherited diseases. However, the lack of safe and efficient gene delivery systems remains as the major challenge. Poly(β-amino ester)s (PBAEs) have attracted much attention due to their outstanding properties in biosafety, DNA delivery efficiency and convenience in synthesis. In this paper, we reported the further enhancement of the PBAE functions by increasing its positive charge through conjugating with low molecular weight polyethylenimine (LPEI). The resulted LPEI-PBAE polymer was able to condense minicircle DNA (mcDNA) forming nanoparticles with a diameter of 50-200nm. Furthermore, as compared to parental PBAE and a commercial transfection reagent very common in laboratory application, the LPEI-PBAE demonstrated significantly higher transfection efficiency with little cytotoxicity. These results suggested LPEI-PBAEs are worthy of further optimization for gene therapy applications.

  7. Synthesis and Biological Evaluation of Indenoisoquinolines that Inhibit both Tyrosyl-DNA-Phosphodiesterase I (Tdp1) and Topoisomerase I (Top1)

    PubMed Central

    Conda-Sheridan, Martin; Narasimha Reddy, P. V.; Morrell, Andrew; Cobb, Brooklyn T.; Marchand, Christophe; Agama, Keli; Chergui, Adel; Renaud, Amélie; Stephen, Andrew G.; Pommier, Yves; Cushman, Mark

    2013-01-01

    Tyrosyl-DNA-phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationship. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 μM to 111 μM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean-graph midpoints ranged from 0.02 to 2.34 μM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents. PMID:23259865

  8. Cytotoxic activity of proflavine diureas: synthesis, antitumor, evaluation and DNA binding properties of 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas.

    PubMed

    Kozurková, Mária; Sabolová, Danica; Janovec, Ladislav; Mikes, Jaromír; Koval', Ján; Ungvarský, Ján; Stefanisinová, Miroslava; Fedorocko, Peter; Kristian, Pavol; Imrich, Ján

    2008-04-01

    The synthesis of novel 1',1''-(acridin-3,6-diyl)-3',3''-dialkyldiureas was reported. Their biological activity to inhibit cell proliferation was assessed by a MTT assay on two cell lines, HeLa and HCT-116, at micromolar concentration. 1',1''-(Acridin-3,6-diyl)-3',3''-dihexyldiurea hydrochloride was active on a HCT-116 cell line with an IC(50) value of 3.1 microM. The interaction of these compounds with calf thymus DNA was investigated by a variety of spectroscopic techniques including UV-vis, fluorescence and CD spectroscopy. From spectrofluorimetric titrations, binding constants for the DNA-drug complexes were determined (K=0.9-4.2x10(5) M(-1)). Antiproliferative activity of synthesized derivatives might be related to their intercalation into DNA.

  9. Synthesis of N-Methyl-2-trichloroacetylpyrrole-A Key Building Block in Peptides That Bind DNA: Micro-, Semimicro-, and Macro-Scale Organic Lab Experiments

    NASA Astrophysics Data System (ADS)

    Mosher, Michael D.; Verner, Erik J.; Oliver, Bradford J.; Hamlin, Daniel; Vietri, Nicholas; Palmer, Robert B.; Arnold, Tyrone V.; Natale, Nicholas R.

    1996-11-01

    The search for more efficient anticancer and antiviral agents has included the preparation and testing of a wide variety of molecules that bind DNA. Of these, particular attention has been devoted to the synthesis of analogues of DNA minor-groove binders such as netropsin and distamycin. These compounds have been shown to exhibit biological activity through their strong interactions with the DNA minor groove, and a relationship between their structure and biological activity has emerged (1). The repeating pyrrole unit contained in these compounds can be prepared from N-methylpyrrole via a noncatalyzed Friedel-Crafts acylation. The reaction has been adapted for use in the undergraduate organic chemistry laboratory on three different scales: microscale, semimicroscale, and macroscale.

  10. Synthesis, DNA cleavage and antimicrobial activity of 4-thiazolidinones-benzothiazole conjugates.

    PubMed

    Singh, Meenakshi; Gangwar, Mayank; Nath, Gopal; Singh, Sushil K

    2014-11-01

    Antimicrobial screening of several novel 4-thiazolidinones with benzothiazole moiety has been performed. These compounds were evaluated for antimicrobial activity against a panel of bacterial and fungal strains. The strains were treated with these benzothiazole derivatives at varying concentrations, and MIC's were calculated. Structures of these compounds have been determined by spectroscopic studies viz., FT-IR, 1H NMR, 13C NMR and elemental analysis. Significant antimicrobial activity was observed for some members of the series, and compounds viz. 3-(4-(benzo[d]thiazol-2-yl) phenyl-2-(4-methoxyphenyl)thiazolidin-4-one and 3-(4-(benzo[d]thiazol-2-yl)phenyl)-2-(4-hydroxy phenyl)thiazolidin-4-one were found to be the most active against E.coli and C. albicans with MIC values in the range of 15.6-125 microg/ml. Preliminary study of the structure-activity relationship revealed that electron donating groups associated with thiazolidine bearing benzothiazole rings had a great effect on the antimicrobial activity of these compounds and contributes positively for the action. DNA cleavage experiments gave valuable hints with supporting evidence for describing the mechanism of action and hence showed a good correlation between their calculated MIC's and its lethality.

  11. Nickel-diflunisal complexes: synthesis, characterization, in vitro antioxidant activity and interaction with DNA and albumins.

    PubMed

    Perontsis, Spyros; Hatzidimitriou, Antonios G; Papadopoulos, Athanasios N; Psomas, George

    2016-09-01

    The reaction of NiCl2 with the non-steroidal anti-inflammatory drug diflunisal (Hdifl) resulted in the formation of complex [Ni(difl-O)2(MeOH)4], 1. The co-existence of a N,N'-donor heterocyclic ligand 2,2'-dipyridylketone oxime (Hpko), 1,10-phenanthroline (phen), 2,2'-bipyridine (bipy) and 2,2'-bipyridylamine (bipyam) led to the formation of complexes [Ni(difl-O)2(Hpko-N,N')2], 2, [Ni(difl)2(phen)(MeOH)2], 3, [Ni(difl)2(bipy)(MeOH)2], 4 and [Ni(difl-O,O')2(bipyam)], 5, respectively. The complexes were characterized by physicochemical and spectroscopic techniques and the crystal structures of complexes 1 and 2 were determined by X-ray crystallography. The ability of the complexes to scavenge in vitro 1,1-diphenyl-picrylhydrazyl, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and hydroxyl radicals was investigated; the complexes were more active scavengers than free Hdifl. The interaction of the complexes with serum albumins was investigated by fluorescence emission spectroscopy and the binding constants of the compounds to the albumins were calculated. UV spectroscopy, cyclic voltammetry and viscosity measurements as well as fluorescence emission spectroscopy for the competitive studies of the complexes with ethidium bromide were employed so as to monitor the interaction of the compounds with calf-thymus DNA and revealed intercalation as the most possible mode of binding.

  12. Synthesis, anticancer activities, interaction with DNA and mitochondria of manganese complexes.

    PubMed

    Qiu-Yun, Chen; Dong-Fang, Zhou; Juan, Huang; Wen-Jie, Guo; Jing, Gao

    2010-11-01

    Two new complexes [(Etdpa)MnCl(2)] and [(Adpa)Mn(Cl)(H(2)O)] (Etdpa = ethyl bis(2-pyridylmethyl)amino-2-propionate; Adpa = bis(2-pyridylmethyl)amino-2-propionic acid) were synthesized and characterized by spectral methods. The crystal structure of [(Etdpa)MnCl(2)] shows that the Mn(II) atom is coordinated by three N atoms (N1, N2, N3), one oxygen atom (O1) of the ligand (Etdpa) and two chloride atoms (Cl1, Cl2), forming a distorted octahedral geometry. The binding interaction between ct-DNA and the synthesized complexes was relatively weak, but they can inhibit the induced swelling of Ca(2+)-loaded mitochondria in a dose-dependent manner. The [(Adpa)Mn(Cl)(H(2)O)] can cause the obvious decrease of mitochondria membrane potential. The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide) assay shows that the two Mn(II) complexes are more active against cancer cells. Especially [(Adpa)Mn(Cl)(H(2)O)] can inhibit the proliferation of glioma cells with IC(50) 9.5 μM. Experimental results indicate that the [(Adpa)Mn(Cl)(H(2)O)] could be a new potential antitumor complex to target the mitochondria.

  13. Synthesis, structural characterisation, bio-potential efficiency and DNA cleavage applications of nicotinamide metal complexes

    NASA Astrophysics Data System (ADS)

    Surendra Dilip, C.; Siva Kumar, V.; John Venison, S.; Vetha potheher, I.; Rajalaxmi (a) Subahashini, D.

    2013-05-01

    Mixed ligand complexes were synthesised using nicotinamide as the primary ligand and nitrite as the secondary ligand were characterised by FT-IR, UV-Vis, 1H NMR, TG-DTA-DTG, X-ray powder diffraction and physical analytical studies. From the molar conductance, magnetic moment and electronic spectral data of the synthesised complexes a general formula of [M(ONO)2(NA)2] where M = Co(II), Ni(II), Cu(II), Zn(II), Cd(II) and Hg(II) and [Cr2(ONO)6(NA)2] with a distorted octahedral structure were proposed. Thermal analyses show that the complexes lose molecules of hydration initially and subsequently expel anionic and organic ligands in continuous steps. The kinetic parameter values, such as, E*, ΔH*, ΔS* and ΔG* illustrate the spontaneous association of the metal and ligands in the formation of the complexes. The antimicrobial efficacy of the ligand and its complexes were examined by in vitro method against various pathogenic bacterial and fungal strains. The metal complexes were found to posses efficient antimicrobial properties compared to nicotinamide and a few of these complexes could turn out to be excellent models for the design of effective antibiotic drug substances. The intercalating interaction of Cu(II) complex with CT-DNA was inspected by absorption spectral and viscosity studies, thermal denaturation and electro-analytical experiments.

  14. Molecular Characterization of a Brucella Species Large DNA Fragment Deleted in Brucella abortus Strains: Evidence for a Locus Involved in the Synthesis of a Polysaccharide

    PubMed Central

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

    1999-01-01

    A Brucella melitensis 16M DNA fragment of 17,119 bp, which contains a large region deleted in B. abortus strains and DNA flanking one side of the deletion, has been characterized. In addition to the previously identified omp31 gene, 14 hypothetical genes have been identified in the B. melitensis fragment, most of them showing homology to genes involved in the synthesis of a polysaccharide. Considering that 10 of the 15 genes are missing in B. abortus and that all the polysaccharides described in the Brucella genus (lipopolysaccharide, native hapten, and polysaccharide B) have been detected in all the species, it seems likely that the genes described here might be part of a cluster for the synthesis of a novel Brucella polysaccharide. Several polysaccharides have been identified as important virulence factors, and the discovery of a novel polysaccharide in the brucellae which is probably not synthesized in B. abortus might be interesting for a better understanding of the pathogenicity and host preference differences observed between the Brucella species. However, the possibility that the genes described in this paper no longer encode the synthesis of a polysaccharide cannot be excluded. Brucellae belong to the alpha-2 subdivision of the class Proteobacteria, which includes other microorganisms living in association with eucaryotic cells, some of them synthesizing extracellular polysaccharides involved in the interaction with the host cell. The genes described in this paper might be a remnant of the common ancestor of the alpha-2 subdivision of the class Proteobacteria, and the brucellae might have lost such extracellular polysaccharide during evolution if it was not necessary for survival or for establishment of the infectious process. Nevertheless, further studies are necessary to identify the entire DNA fragment missing in B. abortus strains and to elucidate the mechanism responsible for such deletion, since only 9,948 bp of the deletion was present in the

  15. Cryptic protein priming sites in two different domains of duck hepatitis B virus reverse transcriptase for initiating DNA synthesis in vitro.

    PubMed

    Boregowda, Rajeev K; Lin, Li; Zhu, Qin; Tian, Fang; Hu, Jianming

    2011-08-01

    Initiation of reverse transcription in hepadnaviruses is accomplished by a unique protein-priming mechanism whereby a specific Y residue in the terminal protein (TP) domain of the viral reverse transcriptase (RT) acts as a primer to initiate DNA synthesis, which is carried out by the RT domain of the same protein. When separate TP and RT domains from the duck hepatitis B virus (DHBV) RT protein were tested in a trans-complementation assay in vitro, the RT domain could also serve, unexpectedly, as a protein primer for DNA synthesis, as could a TP mutant lacking the authentic primer Y (Y96) residue. Priming at these other, so-called cryptic, priming sites in both the RT and TP domains shared the same requirements as those at Y96. A mini RT protein with both the TP and RT domains linked in cis, as well as the full-length RT protein, could also initiate DNA synthesis using cryptic priming sites. The cryptic priming site(s) in TP was found to be S/T, while those in the RT domain were Y and S/T. As with the authentic TP Y96 priming site, the cryptic priming sites in the TP and RT domains could support DNA polymerization subsequent to the initial covalent linkage of the first nucleotide to the priming amino acid residue. These results provide new insights into the complex mechanisms of protein priming in hepadnaviruses, including the selection of the primer residue and the interactions between the TP and RT domains that is essential for protein priming.

  16. N-(2-chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with sup 32 P-end-labeled DNA

    SciTech Connect

    Church, K.M.; Wurdeman, R.L.; Zhang, Yi; Chen, Faxian; Gold, B. )

    1990-07-24

    The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in {sup 32}P-end-labeled restriction fragments with methidiumpropyl-EDTA{center dot}Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using {sup 32}P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. Linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion.

  17. Subnuclear localization, rates and effectiveness of UVC-induced unscheduled DNA synthesis visualized by fluorescence widefield, confocal and super-resolution microscopy.

    PubMed

    Pierzyńska-Mach, Agnieszka; Szczurek, Aleksander; Cella Zanacchi, Francesca; Pennacchietti, Francesca; Drukała, Justyna; Diaspro, Alberto; Cremer, Christoph; Darzynkiewicz, Zbigniew; Dobrucki, Jurek W

    2016-01-01

    Unscheduled DNA synthesis (UDS) is the final stage of the process of repair of DNA lesions induced by UVC. We detected UDS using a DNA precursor, 5-ethynyl-2'-deoxyuridine (EdU). Using wide-field, confocal and super-resolution fluorescence microscopy and normal human fibroblasts, derived from healthy subjects, we demonstrate that the sub-nuclear pattern of UDS detected via incorporation of EdU is different from that when BrdU is used as DNA precursor. EdU incorporation occurs evenly throughout chromatin, as opposed to just a few small and large repair foci detected by BrdU. We attribute this difference to the fact that BrdU antibody is of much larger size than EdU, and its accessibility to the incorporated precursor requires the presence of denatured sections of DNA. It appears that under the standard conditions of immunocytochemical detection of BrdU only fragments of DNA of various length are being denatured. We argue that, compared with BrdU, the UDS pattern visualized by EdU constitutes a more faithful representation of sub-nuclear distribution of the final stage of nucleotide excision repair induced by UVC. Using the optimized integrated EdU detection procedure we also measured the relative amount of the DNA precursor incorporated by cells during UDS following exposure to various doses of UVC. Also described is the high degree of heterogeneity in terms of the UVC-induced EdU incorporation per cell, presumably reflecting various DNA repair efficiencies or differences in the level of endogenous dT competing with EdU within a population of normal human fibroblasts.

  18. A model for triplet mutation formation based on error-prone translesional DNA synthesis opposite UV photolesions.

    PubMed

    Ikehata, Hironobu; Ono, Tetsuya; Tanaka, Kiyoji; Todo, Takeshi

    2007-05-01

    A triplet mutation is defined as multiple base substitutions or frameshifts within a three-nucleotide sequence which includes a dipyrimidine sequence. Triplet mutations have recently been identified as a new type of UV-specific mutation, although the mechanism of their formation is unknown. A total of 163 triplet mutations were identified through an extensive search of previously published data on UV-induced mutations, including mutations from skin, skin cancer, and cultured mammalian cells. Seven common patterns of sequence changes were found: Type I, NTC-->TTT; Type IIa, NCC-->PyTT or PyCT (Py, pyrimidine); Type IIb, TCC-->PuTT or PuCT (Pu, purine); Type III, NCC-->NAT or NTA; Type IV, NTT-->AAT; Type Va, NCT-->NTX; and Type Vb, PuCT-->XTT (N and X, independent anonymous bases). Furthermore, it is suggested that the type of UV lesion responsible for each of these triplet mutation classes are (a) pyrimidine(6-4)pyrimidone photoproducts for Types I, IIb, III, IV and Vb, (b) cyclobutane pyrimidine dimers for Type Va, and (c) Dewar valence isomers for Types IIa and IIb. These estimations are based primarily on results from previous studies using photolyases specific for each type of UV lesion. A model is proposed to explain the formation of each type of triplet mutation, based on error-prone translesional DNA synthesis opposite UV-specific photolesions. The model is largely consistent with the 'A-rule', and predicts error-prone insertions not only opposite photolesions but also opposite the undamaged template base one-nucleotide downstream from the lesions.

  19. Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination

    SciTech Connect

    Kaepyaho, K.; Lauharanta, J.; Jaenne, J.

    1983-08-01

    Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: (/sup 3/H)thymidine incorporation decreased by 80% and (/sup 14/C)leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of (/sup 3/H)histidine/(/sup 14/C)leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

  20. Pseudomonas sp. xylanase for clarification of Mausambi and Orange fruit juice

    NASA Astrophysics Data System (ADS)

    Sharma, Pawan Kumar; Chand, Duni

    2012-07-01

    Xylanase can be usd for many Industrial applications and juice clarification is one of them. Pseudomonas sp. xylanase was used for fruit juice clarification in free State. Maximum amount of juice clarification was in case of Mausambi juice was observed at 40 C∞ and 52 hours, in case of free enzyme treated juice there is 46.9% increase in clarity and 1.7 fold increase in reducing sugars of the juice and enzyme dose was optimized as 8U with maximum flow rate of 6 ml/min at this dose. In case of orange juice in free enzyme treated juice maximum clarity was observed at 40 C∞ and 52 hours, juice was found to be 42.14 % clear with increase of 1.9 fold of reducing sugars, enzyme dose optimized was 8.06U with maximum flow rate of 0.86 ml/min.

  1. The multimerization state of retroviral RNA is modulated by ammonium ions and affects HIV-1 full-length cDNA synthesis in vitro.

    PubMed Central

    Weiss, S; Häusl, G; Famulok, M; König, B

    1993-01-01

    Genomic human immunodeficiency virus type 1 (HIV-1) RNA fragments containing the dimer linkage structure (DLS) can be dimerized and multimerized in the presence of NH4+ and in the absence of any other cation and any viral or cellular protein. This effect strongly supports the notion that dimerization and multimerization of genomic RNA occurs via purine-quartet formation in quadruple helical RNA structures. The efficiency of RNA dimerization and multimerization in the presence of ammonium ions is about 400 fold increased as compared to alkali metal ions such as potassium. Dimerized retroviral RNA representing a pseudodiploid genome could account for genetic recombination within the virion and during reverse transcription. Application of a novel South-Northern-Blotting procedure with biotinylated RNA and digoxigenin-labelled cDNA in vitro reveals that efficient human- and bovine tRNA(Lys3) primed full-length cDNA-synthesis only takes place with a predominantly monomerized RNA template. Dimerization and multimerization of the RNA significantly reduces full-length cDNA-synthesis. This suggests that monomerization of the dimerized RNA, effected by deionization in vitro, is essential for efficient retroviral reverse transcription in vivo. Images PMID:8177734

  2. Class-specific regulation of anti-DNA antibody synthesis and the age-associated changes in (NZB x NZW)F1 hybrid mice.

    PubMed

    Sekigawa, I; Okada, T; Noguchi, K; Ueda, G; Hirose, S; Sato, H; Shirai, T

    1987-05-01

    Investigations of regulatory helper and suppressor T cells in the in vitro anti-DNA antibody synthesis in NZB x NZW (B/W) F1 hybrid mice were initiated by the development of an in vitro system in which G10-passed B cells from B/W F1 mice were cocultured with mitomycin C-treated T cells in the presence of Con A and either in the presence or in the absence of LPS. It was revealed that each IgG and IgM anti-DNA antibody synthesis was under the regulation of separate L3T4+ helper and Ly-2+ suppressor T cells. The function of these class-specific regulatory T cells was age-dependent. Although the helper effect of L3T4+ T cells on IgG antibody synthesis increased, the effect of L3T4+ T cells on IgM antibody production decreased in B/W F1 mice with aging. The IgG anti-DNA antibody production in the cocultures of L3T4+ T cells and B cells was suppressed by addition of Ly-2+ T cells from young but not aged B/W F1 mice, whereas the production of IgM anti-DNA antibodies was suppressed by Ly-2+ T cells from aged but not young B/W F1 mice. We also found that although IgM anti-DNA antibody-producing B cells were already present in 2-mo-old mice, B cells producing IgG antibodies under the influence of L3T4+ T cells appeared in mice at 7 mo of age. These data clearly indicate that separate class-specific regulatory T cells are involved in the production of IgM and IgG anti-DNA antibodies and that the total serum level of the antibodies is reflected by both their age-associated changes and the generation of antibody-forming B cells in B/W F1 mice.

  3. AzaHx, a novel fluorescent, DNA minor groove and G·C recognition element: Synthesis and DNA binding properties of a p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (azaHx-PI) polyamide.

    PubMed

    Satam, Vijay; Babu, Balaji; Patil, Pravin; Brien, Kimberly A; Olson, Kevin; Savagian, Mia; Lee, Megan; Mepham, Andrew; Jobe, Laura Beth; Bingham, John P; Pett, Luke; Wang, Shuo; Ferrara, Maddi; Bruce, Chrystal D; Wilson, W David; Lee, Moses; Hartley, John A; Kiakos, Konstantinos

    2015-09-01

    The design, synthesis, and DNA binding properties of azaHx-PI or p-anisyl-4-aza-benzimidazole-pyrrole-imidazole (5) are described. AzaHx, 2-(p-anisyl)-4-aza-benzimidazole-5-carboxamide, is a novel, fluorescent DNA recognition element, derived from Hoechst 33258 to recognize G·C base pairs. Supported by theoretical data, the results from DNase I footprinting, CD, ΔT(M), and SPR studies provided evidence that an azaHx/IP pairing, formed from antiparallel stacking of two azaHx-PI molecules in a side-by-side manner in the minor groove, selectively recognized a C-G doublet. AzaHx-PI was found to target 5'-ACGCGT-3', the Mlu1 Cell Cycle Box (MCB) promoter sequence with specificity and significant affinity (K(eq) 4.0±0.2×10(7) M(-1)).

  4. Increasing customer service behaviors using manager-delivered task clarification and social praise.

    PubMed

    Rice, Anna; Austin, John; Gravina, Nicole

    2009-01-01

    This project assessed an intervention to improve employee customer service behaviors (correct greetings and closing behaviors). A combination of task clarification and manager-delivered social praise resulted in increased correct greeting from 11.5% to 66% and correct closing from 8% to 70%. The effect was maintained at a 48-week follow-up for employees who were present during the initial study period, but not for more recently hired employees. The results suggest that task clarification combined with manager-delivered social praise is an effective way to improve employee customer service behaviors.

  5. In situ DNA-templated synthesis of silver nanoclusters for ultrasensitive and label-free electrochemical detection of microRNA.

    PubMed

    Yang, Cuiyun; Shi, Kai; Dou, Baoting; Xiang, Yun; Chai, Yaqin; Yuan, Ruo

    2015-01-21

    On the basis of the use of silver nanoclusters (AgNCs) in situ synthesized by cytosine (C)-rich loop DNA templates as signal amplification labels, the development of a label-free and highly sensitive method for electrochemical detection of microRNA (miRNA-199a) is described. The target miRNA-199a hybridizes with the partial dsDNA probes to initiate the target-assisted polymerization nicking reaction (TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction (HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets.

  6. Regulation of initiation of DNA synthesis in Chinese hamster cells. I. Production of stable, reversible G1-arrested populations in suspension culture.

    PubMed

    Tobey, R A; Ley, K D

    1970-07-01

    Suspension cultures of Chinese hamster cells (line CHO) were grown to stationary phase (approximately 8-9 x 10(5) cells/ml) in F-10 medium. Cells remained viable (95%) for at least 80 hr in stationary phase, and essentially all of the cells were in G(1) Upon resuspension or dilution with fresh medium, the cells were induced to resume traverse of the life cycle in in synchrony, and the patterns of DNA synthesis and division were similar to those observed in cultures prepared by mitotic selection. Immediately after dilution, the rates of synthesis of RNA and protein increased threefold. This system provides a simple technique for production of large quantities of highly synchronized cells and may ultimately provide information on the biochemical mechanisms regulating cell-cycle traverse.

  7. μ-Opioid Receptor-Induced Ca2+ Mobilization and Astroglial Development: Morphine Inhibits DNA Synthesis and Stimulates Cellular Hypertrophy through a Ca2+-Dependent Mechanism

    PubMed Central

    Hauser, Kurt F.; Stiene-Martin, Anne; Mattson, Mark P.; Elde, Robert P.; Ryan, S. Eric; Godleske, Chrystal C.

    2015-01-01

    Morphine, a preferential μ opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca2+-dependent mechanism, few studies have examined whether Ca2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca2+ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ opioid receptors and Ca2+ mobilization in morphine-induced astrocyte development. Morphine (1 μM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca2+-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca2+ ([Ca2+]o), or in unmodified media containing Ca2+ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or L-glutamate (100 μM) for 0–72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca2+ ([Ca2+]i) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine’s actions were mimicked by the selective μ agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca2+]i in developing astroglia. At normal [Ca2+]o, morphine attenuated DNA synthesis by increasing [Ca2+]i; low [Ca2+]o (0.3 mM) blocked this effect, while treatment with Ca2+ ionophore or glutamate mimicked morphine’s actions. At extremely low [Ca2+]o (<0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase

  8. Synthesis and evaluation of DNA-targeted spatially separated bis(aniline mustards) as potential alkylating agents with enhanced DNA cross-linking capability.

    PubMed

    Gourdie, T A; Prakash, A S; Wakelin, L P; Woodgate, P D; Denny, W A

    1991-01-01

    DNA-targeted separated bis-mustards were synthesized by attaching aniline mono-mustards at the 4- and 9-positions of the DNA-intercalating ligand 9-aminoacridine-4-carboxamide, with the intention of improving the low cross-link to monoadduct ratio found with most alkylating agents. The geometry of these compounds requires that, when the acridine binds to DNA by intercalation, one alkylating moiety is delivered to each DNA groove. Gel electrophoretic studies show that only one arm of these compounds (probably that attached to the 9-position) alkylates DNA, such alkylation occurring specifically in the major groove at the N7 of guanines. Cell-line studies confirm that the mode of cytotoxicity of these compounds (unlike that of untargeted aniline bis-mustards of comparable reactivity) is due to bulky DNA monoadduct formation. It is concluded that more information is required about the exact orientation of the initial monoadducts before ligands with specific DNA cross-linking ability can be designed.

  9. 77 FR 54910 - Media Bureau Seeks Comment on TiVo's Request for Clarification and Waiver of the Commission's...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-09-06

    ... COMMISSION Media Bureau Seeks Comment on TiVo's Request for Clarification and Waiver of the Commission's... document, the Media Bureau seeks comment on a petition for waiver and clarification of the Commission's... standard'' in the rule. TiVo seeks a waiver of 76.640(b)(4)(iii) for TiVo boxes that cable operators...

  10. Using Task Clarification, Goal Setting, and Feedback to Decrease Table Busing Times in a Franchise Pizza Restaurant

    ERIC Educational Resources Information Center

    Amigo, Seth; Smith, Andrew; Ludwig, Timothy

    2008-01-01

    The current study investigated the effects of task-clarification, and manager verbal and graphic feedback on employee busing times at a pizza restaurant. Using an ABC design, task-clarification was provided in a memo, which described the process, priority, and goal time of busing. The busing time decreased slightly, from an average of 315 seconds…

  11. Stimulation of α₁-adrenoceptor or angiotensin type 1 receptor enhances DNA synthesis in human-induced pluripotent stem cells via Gq-coupled receptor-dependent signaling pathways.

    PubMed

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2013-08-15

    Stimulation of either α₁-adrenoceptor or angiotensin type 1 receptor (AT₁ receptor) induces proliferation of mouse induced pluripotent stem (iPS) cells. Both α₁-adrenoceptor and AT₁ receptor are guanine nucleotide-binding protein q polypeptide (Gq)-coupled receptors. However, it is not fully understood whether stimulation of these Gq-coupled receptors exert a similar effect in human iPS cells, i.e. proliferation of human iPS cells. In this study, we evaluated the involvement of α₁-adrenoceptor and AT₁ receptor in the DNA synthesis of human iPS cells. Treatment with either l-phenylephrine (a selective α₁-adrenoceptor agonist) or angiotensin II (Ang II) significantly increased DNA synthesis in human iPS cells. Enhanced DNA synthesis was significantly inhibited by pretreatment with protein kinase C (PKC) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitor, or phosphatidylinositol-3 phosphate kinase (PI3K) inhibitor. Treatment with either l-phenylephrine or Ang II significantly increased Akt and p44/42 MAPK phosphorylation. Short interfering RNA (siRNA) directed against Gq significantly inhibited DNA synthesis and phosphorylation of Akt and p44/42 MAPK enhanced by l-phenylephrine or Ang II. These results suggest that stimulation of α₁-adrenoceptor or AT₁ receptor may enhance DNA synthesis in human iPS cells via Gq-coupled receptor-dependent signaling pathways.

  12. Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK, DNA Synthesis and Proliferation in Pancreatic Cancer Cells.

    PubMed

    Ming, Ming; Sinnett-Smith, James; Wang, Jia; Soares, Heloisa P; Young, Steven H; Eibl, Guido; Rozengurt, Enrique

    2014-01-01

    Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3-6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser79. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr389 and S6 at Ser240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α1 and α2 catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.

  13. Dissociation of centrosome replication events from cycles of DNA synthesis and mitotic division in hydroxyurea-arrested Chinese hamster ovary cells

    PubMed Central

    1995-01-01

    Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-1. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence micros-copy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle-dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re- initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events. PMID:7790366

  14. Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

    PubMed Central

    McIlroy, J; Chen, D; Wjasow, C; Michaeli, T; Backer, J M

    1997-01-01

    We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway. PMID:8972205

  15. Coupling of the thrombin receptor to G12 may account for selective effects of thrombin on gene expression and DNA synthesis in 1321N1 astrocytoma cells.

    PubMed Central

    Post, G R; Collins, L R; Kennedy, E D; Moskowitz, S A; Aragay, A M; Goldstein, D; Brown, J H

    1996-01-01

    In 1321N1 astrocytoma cells, thrombin, but not carbachol, induces AP-1-mediated gene expression and DNA synthesis. To understand the divergent effects of these G protein-coupled receptor agonists on cellular responses, we examined Gq-dependent signaling events induced by thrombin receptor and muscarinic acetylcholine receptor stimulation. Thrombin and carbachol induce comparable changes in phosphoinositide and phosphatidylcholine hydrolysis, mobilization of intracellular Ca2+, diglyceride generation, and redistribution of protein kinase C; thus, activation of these Gq-signaling pathways appears to be insufficient for gene expression and mitogenesis. Thrombin increases Ras and mitogen-activated protein kinase activation to a greater extent than carbachol in 1321N1 cells. The effects of thrombin are not mediated through Gi, since ribosylation of Gi/Go proteins by pertussis toxin does not prevent thrombin-induced gene expression or thrombin-stimulated DNA synthesis. We recently reported that the pertussis toxin-insensitive G12 protein is required for thrombin-induced DNA synthesis. We demonstrate here, using transfection of receptors and G proteins in COS-7 cells, that G alpha 12 selectively couples the thrombin receptor to AP-1-mediated gene expression. This does not appear to result from increased mitogen-activated protein kinase activity but may reflect activation of a tyrosine kinase pathway. We suggest that preferential coupling of the thrombin receptor to G12 accounts for the selective ability of thrombin to stimulate Ras, mitogen-activated protein kinase, gene expression, and mitogenesis in 1321N1 cells. Images PMID:8930892

  16. Dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis induced in male mouse germ cells by glycidamide, a metabolite of acrylamide.

    PubMed

    Generoso, W M; Sega, G A; Lockhart, A M; Hughes, L A; Cain, K T; Cacheiro, N L; Shelby, M D

    1996-12-20

    The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

  17. The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercury on RNA synthesis in isolated nuclei.

    PubMed

    Frenkel, G D; Ducote, J

    1987-10-01

    Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.

  18. Synthesis and multi-spectroscopic DNA binding study of 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives of fatty acid

    NASA Astrophysics Data System (ADS)

    Hassan, Mohammad F.; Rauf, Abdul

    2016-01-01

    A facile and convenient synthesis of a series of fatty acid derivatives of 1,3,4-oxadiazole and 1,3,4-thiadiazole has been described. The key step of this protocol is the cyclization of acyl thiosemicarbazides via iodobenzene diacetate and methanesulfonic acid under mild conditions. The newly synthesized compounds were characterized by FT-IR, 1HNMR, 13CNMR and mass spectral study. The binding affinity of 5-(pentadecyl)-N-propenyl-1,3,4-oxadiazol-2-amine (3a) and 5-(heptadecyl)-2-amino-1,3,4-thiadiazole (6a) with CT-DNA has been evaluated by UV, fluorescence, Circular Dichroism (CD) and thermal denaturation studies. It has been found that these small and planer heteroaromatic compounds are capable of binding to the minor groove region of DNA.

  19. Synthesis and multi-spectroscopic DNA binding study of 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives of fatty acid.

    PubMed

    Hassan, Mohammad F; Rauf, Abdul

    2016-01-15

    A facile and convenient synthesis of a series of fatty acid derivatives of 1,3,4-oxadiazole and 1,3,4-thiadiazole has been described. The key step of this protocol is the cyclization of acyl thiosemicarbazides via iodobenzene diacetate and methanesulfonic acid under mild conditions. The newly synthesized compounds were characterized by FT-IR, (1)HNMR, (13)CNMR and mass spectral study. The binding affinity of 5-(pentadecyl)-N-propenyl-1,3,4-oxadiazol-2-amine (3a) and 5-(heptadecyl)-2-amino-1,3,4-thiadiazole (6a) with CT-DNA has been evaluated by UV, fluorescence, Circular Dichroism (CD) and thermal denaturation studies. It has been found that these small and planer heteroaromatic compounds are capable of binding to the minor groove region of DNA.

  20. Computational Study of the Force Dependence of Phosphoryl Transfer during DNA Synthesis by a High Fidelity Polymerase

    NASA Astrophysics Data System (ADS)

    Venkatramani, Ravindra; Radhakrishnan, Ravi

    2008-02-01

    High fidelity polymerases are efficient catalysts of phosphodiester bond formation during DNA replication or repair. We interpret molecular dynamics simulations of a polymerase bound to its substrate DNA and incoming nucleotide using a quasiharmonic model to study the effect of external forces applied to the bound DNA on the kinetics of phosphoryl transfer. The origin of the force dependence is shown to be an intriguing coupling between slow, delocalized polymerase-DNA modes and fast catalytic site motions. Using noncognate DNA substrates we show that the force dependence is context specific.